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diff --git a/75389-0.txt b/75389-0.txt new file mode 100644 index 0000000..b4594b1 --- /dev/null +++ b/75389-0.txt @@ -0,0 +1,27953 @@ + +*** START OF THE PROJECT GUTENBERG EBOOK 75389 *** + + + + + +Transcriber’s Notes: + + Underscores “_” before and after a word or phrase indicate _italics_ in + the original text. + Equal signs “=” before and after a word or phrase indicate =bold= in + the original text. + A single underscore after a symbol indicates a subscript. + Small capitals have been converted to SOLID capitals. + Illustrations have been moved so they do not break up paragraphs. + Antiquated spellings have been preserved. + Typographical and punctuation errors have been silently corrected. + The “CORRECTIONS FOR VOL. III.” listed at the end of the book have + already been applied to the text by the transcriber. + + + + + PRINCIPLES AND PRACTICE OF + AGRICULTURAL ANALYSIS. + + A MANUAL FOR THE EXAMINATION OF SOILS, + FERTILIZERS, AND AGRICULTURAL PRODUCTS. + + FOR THE USE OF ANALYSTS, TEACHERS, AND + STUDENTS OF AGRICULTURAL CHEMISTRY. + + VOLUME III. + + AGRICULTURAL PRODUCTS. + + BY HARVEY W. WILEY, + CHEMIST OF THE U. S. DEPARTMENT OF AGRICULTURE. + + EASTON, PA. + CHEMICAL PUBLISHING CO. + 1897. + + COPYRIGHT, 1897, + BY HARVEY W. WILEY. + + + + +PREFACE TO VOLUME THIRD. + + +The concluding volume of the Principles and Practice of Agricultural +Analysis has been written in harmony with the plan adopted at the +commencement of the first volume. In it an effort has been made to +place the analyst or student _en rapport_ with all the best methods of +studying the composition of agricultural products. During the progress +of the work the author has frequently been asked why some special +method in each case has not been designated as the proper one to be +used. To do this would be a radical departure from the fundamental +idea of the work; _viz._, to rely on the good judgment and experience +of the chemist. It is not likely that the author’s judgment in such +matters is better than that of the analyst using the book, and, except +for beginning students pursuing a course of laboratory instruction, a +biased judgment is little better than none at all. For student’s work +in the laboratory or classroom it is probable that a volume of selected +methods based on the present work may be prepared later on, but this +possible future need has not been allowed to change the purpose of +the author as expressed in the preface of the second volume “to +present to the busy worker a broad view of a great subject.” For the +courtesy and patience of the publishers, for the uniformly commendatory +notices of the reviewers of volumes one and two, and for the personal +encouraging expressions of his professional brethren the author is +sincerely grateful. He finds in this cordial reception of his book a +grateful compensation for long years of labor. The plates of the first +edition of the three volumes have been destroyed in order to insure a +re-writing of the second edition when it shall be demanded, in order +to keep it abreast of the rapid progress in the field of agricultural +chemical analysis. + +WASHINGTON, D. C., + +Beginning of January, 1897. + + + + +TABLE OF CONTENTS OF VOLUME THIRD. + + + PART FIRST. + SAMPLING, DRYING, INCINERATION AND EXTRACTIONS. + + _Introduction_, pp. 1-3.—Methods of study; + Scope of the work; Limitations of work; General + manipulations. + + _Methods of Sampling_, pp. 3-13.—Vegetable + substances; Animal substances; Preserving samples; + Collecting samples; Grinding samples; Grinding + apparatus. + + _Drying Organic Bodies_, pp. 13-36.—Volatile + bodies; Drying ovens; Air baths; Drying in vacuum; + Electric drying ovens; Steam coil apparatus; Drying + in hydrogen; Drying in tubes; Drying viscous liquids; + General principles of drying. + + _Incineration_, pp. 36-40.—Principles of + incineration; Products of combustion; Purpose + and conduct of incineration; German ash method; + Courtonne’s muffle. + + _Extraction of Organic Bodies_, pp. + 40-57.—Object of extraction; Solvents; Methods of + extraction; Extraction by digestion; Extraction + by percolation; Apparatus for extraction; Knorr’s + extraction apparatus; Soxhlet’s extraction apparatus; + Compact extraction apparatus; Recovery of solvents; + Authorities cited in Part First. + + PART SECOND. + SUGARS AND STARCHES. + + _Introduction_, pp. 58-62.—Carbohydrates; + Nomenclature; Preparation of pure sugar; + Classification of methods of analysis. + + _Analysis by Density of Solution_, pp. + 63-72.—Principles of the method; Pyknometers; + Calculating volume of pyknometers; Hydrostatic + balance; Areometric method; Correction for + temperature; Brix hydrometer; Comparison of brix and + baumé degrees; Errors due to impurities. + + _Estimation of Sugars with Polarized Light_, + pp. 74-120.—Optical properties of sugars; + Polarized light; Nicol prism; Polariscope; Kinds + of polariscopes; Character of light; Description + of polarizing instruments: Laurent polariscope; + Polariscope lamps; Soleil-Ventzke polariscope; Half + Shadow polariscope; Triple field polariscope; Setting + the polariscope; Control observation tube; Quartz + plates; Correcting quartz plates; Application + of quartz plates; Sugar flasks; Preparing sugar + solutions for polarization; Alumina cream; Errors + due to lead solutions; Double polarization; + Mercuric compounds; Bone-black; Inversion of sugar; + Clerget’s method; Influence of strength of solution; + Calculation of results; Method of Lindet; Use of + invertase; Activity of invertase; Inversion by yeast; + Determination of sucrose; Determination of raffinose; + Specific rotatory power; Calculating specific + rotatory power; Variations in specific rotatory + power; Gyrodynatic data; Birotation. + + _Chemical Methods of Estimating Sugar_, pp. + 120-149.—General principles; Classification of + methods; Reduction of mercuric salts; Sachsse’s + solution; Volumetric copper methods; Action of copper + solution on dextrose; Fehling’s solution; List of + copper solutions; Volumetric laboratory method; + Filtering tubes; Correction of errors; Permanganate + process; Modified permanganate method; Specific + gravity of cuprous oxid; Soldaini’s process; Relation + of reducing sugar to quantity of suboxid; Factors for + different sugars; Pavy’s process; Peska’s process; + Method of Allein and Gaud; Method of Gerrard; + Sidersky’s modification; Titration of excess of + copper. + + _Gravimetric Copper Methods_, pp. + 149-170.—General principles; Laboratory copper + method; Halle method; Allihn’s method; Meissl’s + method; Determination of invert sugar; Estimation of + milk sugar; Determination of maltose; Preparation of + levulose; Estimation of levulose. + + _Miscellaneous Methods of Sugar Analysis_, + pp. 171-196; Phenylhydrazin; Molecular weights of + carbohydrates; Birotation; Estimation of pentosans; + Determination of furfurol; Method of Tollens; + Method of Stone; Method of Chalmot; Method of Krug; + Precipitation with pyrogalol; Precipitation with + phloroglucin; Fermentation methods; Estimating + alcohol; Estimating carbon dioxid; Precipitation + with earthy bases; Barium saccharate; Strontium + saccharate; Calcium saccharate; Qualitive tests; + Optical tests; Cobaltous nitrate test; The Dextrose + group; Tests for levulose; Tests for galactose; Tests + for invert sugar; Compounds with phenylhydrazin; + Detection of sugars by means of furfurol; Bacterial + action on sugars. + + _Determination of Starch_, pp. + 196-226.—Constitution of starch; Separation of + starch; Methods of separation; Separation with + diastase; Separation in an autoclave; Principles + of analysis; Estimation of water; Estimation of + ash; Estimation of nitrogen; Hydrolysis with acids; + Factors for calculation; Polarization of starch; + Solution at high pressure; Method of Hibbard; + Precipitation with barium hydroxid; Disturbing bodies + in starch determinations; Colorimetric estimation + of starch; Fixation of iodin; Identification of + starches; Vogel’s table; Muter’s table; Blyth’s + classification; Preparation of starches for the + microscope; Mounting in canada balsam; Description of + typical starches; Authorities cited in Part Second. + + PART THIRD. + SEPARATION AND DETERMINATION OF CARBOHYDRATES IN + CRUDE OR MANUFACTURED AGRICULTURAL PRODUCTS. + + _Sugars in Vegetable Juices_, pp. + 227-253.—Introduction; Sugar in the sap of trees; + Sugar in sugar canes; Weighing pipettes; Gravimeter; + Reducing sugars in juices; Preservation of juices; + Direct estimation of sugar; Cutting or shredding + canes; Methods of analysis; Drying and extracting; + Examination of bagasse; Fiber in canes; Sugar beets; + Estimation of sugar in sugar beets; Machines for + pulping beets; Instantaneous diffusion; Pellet’s + process; Alcohol digestion; Extraction with alcohol; + Determination of sugar in mother beets; Determination + of sugars without weighing; Continuous observation tube. + + _Analysis of Sirups and Massecuites_, pp. + 254-264.—Specific gravity; Determination of water; + Determination of ash; Determination of reducing + sugars; Estimation of minute quantities of invert + sugar; Soldaini’s gravimetric method; Weighing the + copper as oxid; Analyses for factory control. + + _Separation of Carbohydrates in Mixtures_, pp. + 264-292.—Occurrence of sugars; Optical methods; + Optical neutrality of invert sugar; Separation of + sucrose and invert sugar; Separation of sucrose and + raffinose; Determination of levulose; Formula for + calculating levulose; Separation of sucrose from + dextrose; Estimation of lactose in milk; Error due + to volume of precipitate; Separation of sucrose, + levulose and dextrose; Sieben’s method; Wiechmann’s + method; Copper carbonate method; Winter’s process; + Separation with lead oxid; Analysis of commercial + glucose and grape sugar; Fermentation method; + Oxidation method; Removal of dextrose by copper + acetate; Separation of dextrin with alcohol. + + _Carbohydrates in Milk_, pp. 293-298.—Copper + tartrate method; The official method; The copper + cyanid process; Separation of sugars in evaporated + milks; Method of Bigelow and McElroy. + + _Separation and Determination of Starch and + Fiber_, pp. 298-306.—Occurrence; Separation of + starch; Dry amyliferous bodies; Indirect method + of determining water; Removal of oils and sugars; + Preparation of diastase; Estimation of starch in + potatoes; Constitution of cellulose; Fiber in + cellulose; Official method; Separation of cellulose: + Solubility of cellulose; Qualitive reactions for + cellulose; Rare carbohydrates; Authorities cited in + Part Third. + + PART FOURTH. + FATS AND OILS. + + _General Principles_, pp. 309-316.—Nomenclature; + Composition; Principal glycerids; Presses for extraction; + Solvents; Freeing extracts of petroleum; Freeing fats of + moisture; Sampling and drying for analysis; Estimation of + water. + + _Physical Properties of Fats and Oils_, pp. + 317-350.—Specific gravity; Balance for determining + specific gravity; Expression of specific gravity; + Coefficient of expansion of oils; Densities of common + fats and oils; Melting point; Determination in + capillary tube; Determination by spheroidal state; + Solidifying point; Temperature of crystallization; + Refractive power; Refractive index; Abbe’s + refractometer; Pulfrich’s refractometer; Refractive + indices of common oils; Oleorefractometer; + Butyrorefractometer; Range of application of the + butyrorefractometer; Viscosity; Torsion viscosimeter; + Microscopic appearance; Preparation of fat crystals; + Observation of fat crystals with polarized light; + Spectroscopic examination of oils; Critical + temperature; Polarization; Turbidity temperature. + + _Chemical Properties of Fats and Oils_, pp. + 351-406.—Solubility in alcohol; Coloration produced + by oxidants; Nitric acid coloration; Phosphomolybdic + acid coloration; Picric acid coloration; Silver + nitrate coloration; Stannic bromid coloration; + Auric chlorid coloration; Thermal reactions; Heat + of sulfuric saponification; Maumené’s process; + Method of Richmond; Relative maumené figure; Heat + of bromination; Method of Hehner and Mitchell; + Author’s method; Haloid addition numbers; Hübl + number; Character of chemical reaction; Solution in + carbon tetrachlorid; Estimation of the iodin number; + Use of iodin monochlorid; Preservation of the hübl + reagent; Bromin addition number; Method of Hehner; + Halogen absorption by fat acids; Saponification; + Saponification in an open dish; Saponification under + pressure; Saponification in the cold; Saponification + value; Saponification equivalent; Acetyl value; + Determination of volatile fat acids; Removal of the + alcohol; Determination of soluble and insoluble fat + acids; Formulas for calculation; Determination of + free fat acids; Identification of oils and fats; + Nature of fat acids; Separation of glycerids; + Separation with lime; Separation with lead salts; + Separation of arachidic acid; Detection of peanut + oil; Bechi’s test; Milliau’s test; Detection of + sesamé oil; Sulfur chlorid reaction; Detection of + cholesterin and phytosterin; Absorption of oxygen; + Elaidin reactions; Authorities cited in Part Fourth. + + PART FIFTH. + SEPARATION AND ESTIMATION OF BODIES + CONTAINING NITROGEN. + _Introduction and Definitions_, pp. + 410-418.—Nature of nitrogenous bodies; + Classification of proteids; Albuminoids; Other forms + of nitrogen; Occurrence of nitrates. + + _Qualitive Tests for Nitrogenous Bodies_, + pp. 418-422.—Nitric acid; Amid nitrogen; Ammoniacal + nitrogen; Proteid nitrogen; Qualitive tests for albumni; + Qualitive tests for peptones and albuminates; Action + of polarized light on albumins; Alkaloidal nitrogen. + + _Estimation of Nitrogenous Bodies in Agricultural + Products_, pp. 423-432.—Total nitrogen; + Ammoniacal nitrogen; Amid nitrogen; Sachsse’s method; + Preparation of asparagin; Estimation of asparagin + and glutamin; Cholin and betain; Lecithin; Factors + for calculating results; Estimation of alkaloidal + nitrogen. + + _Separation of Proteid Bodies in Vegetable + Products_, pp. 432-448.—Preliminary treatment; + Character of proteids; Separation of gluten; + Extraction with water; Action of water on composition + of proteids; Extraction with dilute salt solution; + Separation of bodies soluble in water; Separation of + the globulins; Proteids soluble in dilute alcohol; + Solvent action of acids and alkalies; Method of + extraction; Methods of drying separated proteids; + Determination of ash; Determination of carbon and + hydrogen; Estimation of nitrogen; Determination of + sulfur; Dialysis. + + _Separation and Estimation of Nitrogenous Bodies + in Animal Products_, pp. 448-462.—Preparation of + sample; Extraction of muscular tissues; Composition + of meat extracts; Analysis of meat extracts; Use + of phosphotungstic acid; Separation of albumoses + and peptones; Estimation of gelatin; Estimation + of nitrogen in flesh bases; Treatment of residue + insoluble in alcohol; Pancreas peptone; Albumose + peptone; Authorities cited in Part Fifth. + + PART SIXTH. + DAIRY PRODUCTS. + _Milk_, pp. 464-512.—Composition of milk; + Alterability of milk; Effects of boiling on milk; + Micro-organisms of milk; Sampling milk; Scovell’s + milk sampler; Preserving milk for analysis; Freezing + point; Electric conductivity; Viscosity; Acidity + and alkalinity; Determination of acidity; Opacity; + Creamometry; Specific gravity; Lactometry; Quévenne + lactometer; Lactometer of the New York Board of + Health; Density of sour milk; Density of milk + serum; Total solids; Formulas for calculating total + solids; Determination of ash; Estimation of fat; Fat + globules; Number of fat globules; Counting globules; + Classification of methods of analysis; Dry extraction + methods; Official methods; Variations of extraction + methods; Gypsum method; Estimation of fat in malted + milk; Comparison of fat methods; Wet extraction + methods; Solution in acid; Solution in alkali; + Method of Short; Method of Thörner; Liebermann’s + method; Densimetric methods; Areometric methods; + Lactobutyrometer; Volumetric methods of fat analysis; + Method of Patrick; The lactocrite; Modification of + Lindström; Babcock’s method; Method of Leffmann and + Beam; Method of Gerber; Proteid bodies in milk; + Estimation of total proteid matter; Copper sulfate + as a reagent; Precipitation by ammonium sulfate; + Precipitation by tannic acid; Separation of casein from + albumin; Estimation of casein; Factors for + calculation; Separation of casein; Separation of + casein with carbon dioxid; Separation of albumin; + Separation of globulin; Precipitants of milk + proteids; Precipitation by dialysis; Carbohydrates in + milk; Dextrinoid body in milk; Amyloid bodies in milk. + + _Butter_, pp. 512-523.—General principles + of analysis; Appearance of melted butter; + Microscopic examination; Refractive power; + Estimation of water, fat, casein, ash and salt; + Volatile and soluble acids; Relative proportion of + glycerids; Saponification value; Reichert number; + Reichert-Meissl method; Elimination of sulfurous + acid; Errors due to poor glass; Molecular weight of + butter; Substitutes for and adulterants of butter; + Butter colors. + + _Cheese and Koumiss_, pp. 524-536.—Composition + of cheese; Manufacture of cheese; Official methods + of analysis; Process of Mueller; Separation of fat + from cheese; Filled cheese; Separation of nitrogenous + bodies; Preparation of koumiss; Determination of + carbon dioxid; Acidity; Estimation of alcohol; + Proteids in koumiss; Separation by porous porcelain; + Separation by precipitation with alum; Separation + with mercury salts; Determination of water and ash; + Composition of koumiss; Authorities cited in Part Sixth. + + PART SEVENTH. + MISCELLANEOUS AGRICULTURAL PRODUCTS. + _Cereals and Cereal Foods_, pp. 541-545.— + Classification; General methods of analysis; + Composition and analysis of bread; Determination + of alum in bread; Chemical changes produced by baking. + + _Fodders, Grasses, and Ensilage_, pp. + 545-547.—General principles of analysis; Organic + acids in ensilage; Changes due to fermentation; + Alcohol in ensilage; Comparative values of dry fodder + and ensilage. + + _Flesh Products_, pp. 547-555.—Names of meats; + Sampling; General methods of analysis; Examination + of nitrogenous bodies; Fractional analysis of meats; + Starch in meats; Detection of horse flesh. + + _Methods of Digestion_, pp. 555-564.—Artificial + digestion; Amylolytic ferments; Aliphalytic ferments; + Proteolytic ferments; Pepsin and pancreatin; + Digestion in pancreas extract; Artificial digestion + of cheese; Natural digestion; Digestibility of + pentosans. + + _Preserved Meats_, pp. 565-566.—Methods of + examination; Estimation of fat; Meat preservatives. + + _Determination of Nutritive Values_, pp. + 566-576.—Nutritive value of foods; Comparative value + of food constituents; Nutritive ratio; Calorimetric + analysis of foods; Combustion in oxygen; Bomb + calorimeter; Manipulation and calculation; Computing + the calories of combustion; Calorimetric equivalents; + Distinction between butter and oleomargarin. + + _Fruits, Melons and Vegetables_, pp. 577-582.— + Preparation of samples; Separation of carbohydrates; + Examination of the fresh matter; Examination of fruit + and vegetable juices; Separation of pectin; + Determination of free acid; Composition of fruits; + Composition of ash of fruits; Dried fruits; Zinc in + evaporated fruits; Composition of melons. + + _Tea and Coffee_, pp. 582-588.—Special points + in analysis; Estimation of caffein; Iodin method; + Spencer’s method; Separation of chlorophyll; + Determination of proteid nitrogen; Carbohydrates of + coffee; Estimation of galactan; Revised factors for + pentosans; Use of roentgen rays. + + _Tannins and Allied Bodies_, pp. 588-596.— + Occurrence and composition; Detection and estimation; + Precipitation with metallic salts; The gelatin method; + The hide powder method; Permanganate gelatin method; + Permanganate hide powder method; Preparation of infusion. + + _Tobacco_, pp. 596-610.—Fermented and + unfermented tobacco; Acid and basic constituents; + Composition of ash; Composition of tobacco; + Estimation of water; Estimation of nitric acid; + Estimation of sulfuric and hydrochloric acids; + Estimation of oxalic, malic and citric acids; + Estimation of acetic acid; Estimation of pectic acid; + Estimation of tannic acid; Estimation of starch and + sugar; Estimation of ammonia; Estimation of nicotin; + Polarization method of Popovici; Estimation of amid + nitrogen; Fractional extraction; Burning qualities; + Artificial smoker. + + _Fermented Beverages_, pp. 610-641.— + Description; Important constituents; Specific + gravity; Determination of alcohol; Distilling + apparatus; Specific gravity of the distillate; + Hydrostatic plummet; Calculating results; Table + giving percentage of alcohol by weight and volume; + Determination of percentage of alcohol by means of + vapor temperature; Improved ebullioscope; Indirect + determination of extract; Determination of total + acids; Determination in a vacuum; Estimation of + water; Total acidity; Volatile acids; Tartaric acid; + Tartaric, malic and succinic acids; Polarizing bodies + in fermented beverages; Reducing sugars; Polarization + of wines and beers; Application of analytical + methods; Estimation of carbohydrates; Determination + of glycerol; Coloring matters; Determination of ash; + Determination of potash; Sulfurous acid; Salicylic + acid; Detection of gum and dextrin; Determination of + nitrogen; Substitutes for hops; Bouquet of fermented + and distilled liquors; Authorities cited in Part + Seventh; Index. + + + + +ILLUSTRATIONS TO VOLUME THIRD. + + + Page. + Figure 1. Mill for grinding dry samples 7 + ” 2. Comminutor for green samples 9 + ” 3. Rasp for sugar beets 10 + ” 4. Dreef grinding apparatus 11 + ” 5. Water jacket drying oven 14 + ” 6. Thermostat for Steam-Bath 15 + ” 7. Spencer’s drying oven 17 + ” 8. Electric vacuum drying oven 19 + ” 9. Steam coil drying oven 21 + ” 10. Carr’s vacuum drying oven 22 + ” 10. (Bis.) vacuum oven open 23 + ” 11. Apparatus for drying in a current of hydrogen 25 + ” 12. Caldwell’s hydrogen drying apparatus 27 + ” 13. Liebig’s ente 28 + ” 14. Drying apparatus used at the Halle Station 29 + ” 15. Wrampelmayer’s oven 30 + ” 16. Ulsch drying oven 31 + ” 17. Courtoune muffle 39 + ” 18. Knorr’s extraction apparatus 45 + ” 19. Extraction flask 46 + ” 20. Extraction tube 46 + ” 21. Extraction siphon tube 46 + ” 22. Soxhlet extraction apparatus 48 + ” 23. Compact condensing apparatus 49 + ” 24. Improved compact extraction apparatus 51 + ” 25. Knorr’s apparatus for recovering solvents 54 + ” 26. Apparatus for recovering solvents from open dishes 55 + ” 27. Common forms of pyknometers 63 + ” 28. Bath for pyknometers 66 + ” 29. Aereometers, pyknometers and hydrostatic balance 68 + ” 30. Hydrostatic balance 69 + ” 31. Course of rays of light in a nicol 77 + ” 32. Theory of the nicol 78 + ” 33. Laurent lamp 83 + ” 34. Lamp for producing constant monochromatic flame 85 + ” 35. Field of vision of a Laurent polariscope 86 + ” 36. Laurent polariscope 88 + ” 37. Tint polariscope 89 + ” 38. Double compensating shadow polariscope 91 + ” 39. Triple shadow polariscope 92 + ” 40. Apparatus for producing a triple shadow 92 + ” 41. Control observation tube 95 + ” 42. Apparatus for the volumetric estimation of + reducing sugars 131 + ” 43. Apparatus for the electrolytic deposition of copper 151 + ” 44. Apparatus for filtering copper suboxid 154 + ” 45. Apparatus for reducing copper suboxid 154 + ” 46. Distilling apparatus for pentoses 179 + ” 47. Autoclave for starch analysis 199 + ” 47. (Bis). Maercker’s hydrolyzing apparatus for starch 204 + ” 48. Maranta starch × 350 } + ” 49. Potato starch × 350 } + ” 50. Ginger starch × 350 } + ” 51. Sago starch × 350 } + ” 52. Pea starch × 350 } + ” 53. Bean starch × 350 } + ” 54. Wheat starch × 350 } + ” 55. Barley starch × 350 } to face 220 + ” 56. Rye starch × 350 } + ” 57. Oat starch × 350 } + ” 58. Indian corn starch × 350 } + ” 59. Rice starch × 350 } + ” 60. Cassava starch × 150 } + ” 61. Indian corn starch × 150 } + ” 62. Laboratory cane mill 230 + ” 63. Weighing pipette 231 + ” 64. Gird’s gravimeter 233 + ” 65. Machine for cutting canes 236 + ” 66. Cane cutting mill 237 + ” 67. Apparatus for pulping beets 243 + ” 68. Apparatus for cold diffusion 245 + ” 69. Sickel-Soxhlet extractor 247 + ” 70. Scheibler’s extraction tube 248 + ” 71. Battery for alcoholic digestion 250 + ” 72. Rasp for sampling mother beets 251 + ” 73. Hand press for beet analysis 251 + ” 74. Perforating rasp 252 + ” 75. Tube for continuous observation 253 + ” 75. (Bis). Chandler and Rickett’s Polariscope 266 + ” 76. Apparatus for polarimetric observations at + low temperatures 267 + ” 77. Construction of desiccating tube 268 + ” 78. Apparatus for polarizing at high temperatures 269 + ” 79. Oil press 312 + ” 80. Apparatus for fractional distillation of + petroleum ether 314 + ” 81. Section showing construction of a funnel for + hot filtration 316 + ” 82. Balance and Westphal sinker 318 + ” 83. Melting point tubes 322 + ” 84. Apparatus for the determination of melting point 324 + ” 85. Apparatus for determining crystallizing point 327 + ” 86. Abbe’s refractometer 329 + ” 87. Charging position of refractometer 330 + ” 88. Prism of Pulfrich’s refractometer 331 + ” 89. Pulfrich’s new refractometer 332 + ” 90. Heating apparatus for Pulfrich’s refractometer 333 + ” 91. Spectrometer attachment 333 + ” 92. Oleorefractometer 335 + ” 93. Section showing construction of oleorefractometer 335 + ” 94. Butyrorefractometer 339 + ” 95. Doolittle’s viscosimeter 343 + ” 96. Lard crystals × 65 } + ” 97. Refined lard crystals × 65 } to face 348 + ” 98. Apparatus for determining rise of temperature with + sulfuric acid 358 + ” 99. Apparatus for determining heat of bromination 362 + ” 100. Olein tube 374 + ” 101. Apparatus for saponifying under pressure 380 + ” 102. Apparatus for the distillation of volatile acids 388 + ” 103. Apparatus for amid nitrogen 425 + ” 104. Sachsse’s eudiometer 425 + ” 105. Dialyzing apparatus 447 + ” 106. Scovell’s milk sampling tube 470 + ” 107. Lactoscope, lactometer, and creamometer 474 + ” 108. Areometric fat apparatus 493 + ” 109. Babcock’s butyrometer and acid measure 500 + ” 110. Gerber’s butyrometers 502 + ” 111. Gerber’s centrifugal 503 + ” 112. Thermometer for butyrorefractometer 515 + ” 113. Apparatus for determining carbon dioxid in koumiss 533 + ” 114. Cuts of mutton 548 + ” 115. Cuts of beef 548 + ” 116. Cuts of pork 548 + ” 117. Bath for artificial digestion 559 + ” 118. Bag for collecting feces 563 + ” 119. Fecal bag attachment 563 + ” 120. Hempel and Atwater’s calorimeter 570 + ” 121. Apparatus for acetic acid 603 + ” 122. Apparatus for smoking 610 + ” 123. Metal distilling apparatus 613 + ” 124. Distilling apparatus 614 + ” 125. Improved ebullioscope 623 + + + + +VOLUME THIRD. + +AGRICULTURAL PRODUCTS. + + + + +PART FIRST. + +SAMPLING, DRYING, INCINERATION AND EXTRACTIONS. + + +=1. Introduction.=—The analyst may approach the examination of +agricultural products from various directions. In the first place +he may desire to know their proximate and ultimate constitution +irrespective of their relations to the soil or to the food of man and +beast. Secondly, his study of these products may have reference solely +to the determination of the more valuable plant foods which they have +extracted from the soil and air. Lastly, he may approach his task from +a hygienic or economic standpoint for the purposes of determining the +wholesomeness or the nutritive and economic values of the products +of the field, orchard, or garden. In each case the object of the +investigation will have a considerable influence on the method of the +examination. + +It will be the purpose of the present volume to discuss fully the +principles of all the standard processes of analysis and the best +practice thereof, to the end that the investigator or analyst, whatever +may be the design of his work, may find satisfactory directions for +prosecuting it. As in the previous volumes, it should be understood +that these pages are written largely for the teacher and the analyst +already skilled in the principles of analytical chemistry. Much is +therefore left to the individual judgment and experience of the worker, +to whom it is hoped a judicious choice of approved processes may be +made possible. + +=2. Scope Of the Work.=—Under the term agricultural products is +included a large number of classes of bodies of most different +constitution. In general they are the products of vegetable and animal +metabolism. First of all come the vegetable products, fruits, grains +and grasses. These may be presented in their natural state, as cereals, +green fruits and fodders, or after a certain preparation, as starches, +sugars and flours. They may also be met with in even more advanced +stages of change, as cooked foods, alcohols and secondary organic +acids, such as vinegar. In general, by the term agricultural products +is meant not only the direct products of the farm, orchard and forest, +but also the modified products thereof and the results of manufacture +applied to the raw materials. Thus, not only the grain and straw of +wheat are proper materials for agricultural analysis, but also flour +and bran, bread and cakes made therefrom. In the case of maize and +barley, the manufactured products may extend much further, for not only +do we find starch and malt, but also alcohol and beer falling within +the scope of our work. In respect of animal products, the agricultural +analyst may be called on to investigate the subject of leather and +tanning; to determine the composition of meat, milk and butter; to +pass upon the character of lard, oleomargarine, and, in general, to +determine as fully as possible the course of animal food in all its +changes between the field, the packing house and the kitchen. + +=3. Limitations of Work.=—It is evident from the preceding paragraph, +that in order to keep the magnitude of this volume within the limits +fixed for a single volume the text must be rigidly confined to the +fundamental principles and practice of agricultural analysis. The +interesting region of pharmacy and allied branches, in respect of +plant analysis, can find no description here, and in those branches +of technical chemistry, where the materials of elaboration are the +products of the field only a superficial view can be given. The main +purpose and motive of this volume must relate closely to the more +purely agricultural processes. + +=4. General Manipulations.=—There are certain analytical operations +which are more or less of a general nature, that is, they are of +general application without reference to the character of the material +at hand. Among these may be mentioned the determination of moisture +and of ash, and the estimation of matters soluble in ether, alcohol and +other solvents. These processes will be first described. Preliminary to +these analytical steps it is of the utmost importance that the material +be properly prepared for examination. In general, this is accomplished +by drying the samples until they can be ground or crushed to a fine +powder, the attrition being continued until all the particles are made +to pass a sieve of a given fineness. The best sieve for this purpose +is one having circular apertures half a millimeter in diameter. Some +products, both vegetable and animal, require to be reduced to as fine +a state as possible without drying. In such instances, passing the +product through a sieve is obviously impracticable. Special grinding +and disintegrating machines are made for these purposes and they will +be described further on. + +There are some agricultural products which have to be prepared for +examination in special ways and these methods will be given in +connection with the processes for analyzing the bodies referred +to. Nearly all the bodies, however, with which the analyst will be +concerned, can be prepared for examination by the general methods about +to be described. + +=5. Preparation of the Sample.= (_a_) _Vegetable Substances._—For all +processes of analysis not executed on the fresh sample, substances of +a vegetable nature should, if in a fresh state, be dried as rapidly +as possible to prevent fermentative changes. It is often of interest +to determine the percentage of moisture in the fresh sample. For this +purpose a representative portion of the sample should be rapidly +reduced to as fine a condition as possible. To accomplish this it +should be passed through a shredding machine, or cut by scissors or a +knife into fine pieces. A few grams of the shredded material are dried +in a flat-bottomed dish at progressively increasing temperatures, +beginning at about 60° and ending at from 100° to 110°. The latter +temperature should be continued for only a short time. The principle of +this process is based upon the fact that if the temperature be raised +too high at first, some of the moisture in the interior cells of the +vegetable substance can be occluded by the too rapid desiccation of +the exterior layers which would take place at a high temperature. The +special processes for determining moisture will be given in another +place. + +The rest of the sample should be partly dried at a lower temperature +or air-dried. In the case of fodders and most cattle foods the samples +come to the analyst in a naturally air-dried state. When grasses are +harvested at a time near their maturity they are sun-dried in the +meadows before placing in the stack or barn. Such sun-dried samples are +already in a state fit for grinding. Green grasses and fodders should +be dried in the sun, or in a bath at a low temperature from 50° to +60° until all danger of fermentative action is over, and then air- or +sun-dried in the usual way. + +Seeds and cereals usually reach the analyst in a condition suited to +grinding without further preliminary preparation. Fruits and vegetables +present greater difficulties. Containing larger quantities of water, +and often considerable amounts of sugar, they are dried with greater +difficulty. The principles which should guide all processes of drying +are those already mentioned, _viz._, to secure a sufficient degree of +desiccation to permit of fine grinding and at a temperature high enough +to prevent fermentative action, and yet not sufficiently high to cause +any marked changes in the constituents of the vegetable organism. + +(_b_) _Animal Substances._—The difficulties connected with the +preliminary treatment of animal substances are far greater than those +just mentioned. Such samples are composed of widely differing tissues, +blood, bone, tendon, muscle and adipose matters, and all the complex +components of the animal organism are to be considered. The whole +animal may be presented for analysis, in which case the different parts +composing it should be separated and weighed as exactly as possible. +Where only definite parts are to be examined it is best to separate the +muscle, bone, and fat as well as may be, before attempting to reduce +the whole to a fine powder. The soft portions of the sample are to be +ground as finely as possible in a meat or sausage cutter. The bones +are crushed in some appropriate manner, and thus prepared for further +examination. Where the flesh and softer portions are to be dried and +finely ground, the presence of fat often renders the process almost +impossible. In such cases the fat must be at least partially removed +by petroleum or other solvent. In practically fat-free samples the +material, after grinding in a meat cutter, can be partially dried at +low temperatures from 60° to 75°, and afterwards ground in much the +same manner as is practiced with vegetable substances. + +As is the case with the preliminary treatment of vegetable matters, +it is impossible to give any general directions of universal +applicability. The tact and experience of the analyst in all these +cases are better than any dicta of the books. In some instances, as +will appear further on, definite directions for given substances can be +given, but in all cases the general principles of procedure are on the +lines already indicated. + +=6. Preserving Samples.=—In most cases, as is indicated in the +foregoing paragraphs, the sample may be dried before grinding to such a +degree as to prevent danger from fermentation or decay. The fine-ground +samples are usually preserved in glass-stoppered bottles, carefully +marked or numbered. In some cases it is advisable to sterilize the +bottles after stoppering, by subjecting them to a temperature of 100° +for some time. In the case of cereals assurance should be had that the +samples do not contain the eggs of any of the pests that often destroy +these products. As a rule, samples should be kept for a time after the +completion of the analytical work, and this is especially true in all +cases where there is any prospect of dispute or litigation. In general +it may be said, that samples should be destroyed only when they are +spoiled, or when storage room is exhausted. + +=7. Collecting Samples.=—When possible, the analyst should be his +own collector. There is often as much danger from data obtained on +non-representative samples as from imperfect manipulation. When +personal supervision is not possible, the sample when received, +should be accompanied by an intelligible description of the method of +taking it, and of what it represents. In all cases the object of the +examination must be kept steadily in view. Where comparisons are to be +made the methods of collecting must be rigidly the same. + +The processes of analysis, as conducted with agricultural products, +are tedious and difficult. The absolutely definite conditions that +attend the analysis of mineral substances, are mostly lacking. The +simple determinations of carbon, hydrogen, nitrogen and sulfur, +which are required in the usual processes of organic analysis, are +simplicity itself when compared with the operations which have to be +performed on agricultural products to determine their character and +their value as food and raiment. We have to do here with matters on +which the sustenance, health and prosperity of the human race are more +intimately concerned than with any other of the sciences. This fact +also emphasizes the necessity for care in collecting the materials on +which the work is to be performed. + +=8. Grinding Samples.=—In order to properly conduct the processes of +agricultural analysis it is important to have the sample finely ground. +This arises both from the fact that such a sample is apt to contain an +average content of the various complex substances of which the material +under examination is composed, and because the analytical processes can +be conducted with greater success upon the finely divided matter. In +mineral analysis it is customary to grind the sample to an impalpable +powder in an agate mortar. With agricultural products, however, such +a degree of fineness is difficult to attain, and moreover, is not +necessary. There is a great difference of opinion among analysts +respecting the degree of fineness desirable. In some cases we must be +content with a sample which will pass a sieve with a millimeter mesh; +in fact it may be found impossible, on account of the stickiness of the +material, to sift it at all. In such cases a thorough trituration, so +as to form a homogeneous mass will have to be accepted as sufficient. +Where bodies can be reduced to a powder however, it is best to pass +them through a sieve with circular perforations half a millimeter in +diameter. A finer degree of subdivision than this is rarely necessary. + +=9. The Grinding Apparatus.=—The simplest form of apparatus for +reducing samples for analysis to a condition suited to passing a fine +sieve is a mortar. Where only a few samples are to be prepared and in +small quantities, it will not be necessary to provide anything further. +After the sample is well disintegrated it is poured on the sieve and +all that can pass is shaken or brushed through. The sieve is provided +with a receptacle, into which it fits closely, to avoid loss of any +particles which may be reduced to a dust. The top of the sieve, when +shaken, may also be covered if there be any tendency to loss from dust. +Any residue failing to pass the sieve is returned to the mortar and +the process thus repeated until all the material has been secured in +the receiver. The particles more difficult of pulverization are often +different in structure from the more easily pulverized portions, and +the sifted matter must always be carefully mixed before the subsample +is taken for examination. Often the materials, or portions thereof, +will contain particles tough and resistant to the pestle, but the +operator must have patience and persistence, for it is highly necessary +to accurate work that the whole sample be reduced to proper size. + +[Illustration: FIGURE 1. MILL FOR GRINDING DRY SAMPLES.] + +Where many samples are to be prepared, or in large quantities, mills +should take the place of mortars. For properly air-dried vegetable +substances, some form of mill used in grinding drugs may be employed. +Grinding surfaces of chilled corrugated steel are to be preferred. +The essential features of such a mill are that it be made of the best +material, properly tempered, and that the parts be easily separated +for convenience in cleaning. The grinding surfaces must also be so +constructed and adjusted as to secure the proper degree of fineness. +In fig. 1 is shown a mill of rather simple construction, which has +long been in satisfactory use in this laboratory. Small mills may +be operated by hand power, but when they are to be used constantly +steam power should be provided. In addition to the removal of nearly +all the moisture by air-drying there are many oleaginous seeds which +cannot be finely ground until their oil has been removed. For this +purpose the grinding surfaces of the mill are opened so that the seeds +are only coarsely broken in passing through. The fragments are then +digested with light petroleum in a large flask, furnished with a reflux +condenser. After digestion the fragments are again passed through the +mill adjusted to break them into finer particles. + +The alternate grinding and digestion are thus continued until the +pulverization is complete. On a small specially prepared sample the +total content of oil is separately determined. + +Fresh animal tissues are best prepared for preliminary treatment by +passing through a sausage mill. The partially homogeneous mass thus +secured should be dried at a low temperature and reground as finely as +possible. Where much fat is present it may be necessary to extract it +as mentioned above, in the case of oleaginous seeds. In such cases both +the moisture and fat in the original material should be determined on +small specially prepared samples with as great accuracy as possible. +Bones, hoofs, horns, hair and hides present special difficulties in +preparation, which the analyst will have to overcome with such skill +and ingenuity as he may possess. + +The analyst will find many specially prepared animal foods already in +a fairly homogeneous form, such as potted and canned meats, infant +and invalid foods, and the like. Even with these substances, however, +a preliminary grinding and mixing will be found of advantage before +undertaking the analytical work. Many cases will arise which are +apparently entirely without the classification given above. But even in +such instances the analyst should not be without resources. Frequently +some dry inert substance may be mixed with the material in definite +quantities, whereby it is rendered more easily prepared. Perhaps no +case will be presented where persistent and judicious efforts to secure +a fairly homogeneous sample for analysis will be wholly unavailing. + +[Illustration: FIGURE 2. COMMINUTOR FOR GREEN SAMPLES.] + +In the case of green vegetable matters which require to be reduced +rapidly to a fine state of subdivision in order to secure even a +fairly good sample some special provision must be made. This is +the case with stalks of maize and sugar cane, root crops, such as +potatoes and beets, and green fodders, such as clover and grasses. +The chopping of these bodies into fine fodders by hand is slow and +often impracticable. The particles rapidly lose moisture and it is +important to secure them promptly as in the preparation of beet pulp +for polarization. For general use we have found the apparatus shown +in fig. 2 quite satisfactory in this laboratory. It consists of a +series of staggered circular saws carried on an axis and geared to be +driven at a high velocity, in the case mentioned, 1,400 revolutions +per minute. The green material is fed against the revolving saws by +the toothed gear-work shown, and is thus reduced to a very fine pulp, +which is received in the box below. Stalks of maize, green fodders, +sugar canes, beets and other fresh vegetable matters are by this +process reduced to a fine homogeneous pulp, suited for sampling and for +analytical operations. Such pulped material can also be spread in a +fine layer and dried rapidly at a low temperature, thus avoiding danger +of fermentative changes when it is desired to secure the materials in +a dry condition or to preserve them for future examination. Samples of +sorghum cane, thus pulped and dried, have been preserved for many years +with their sugar content unchanged. + +[Illustration: FIGURE 3. RASP FOR SUGAR BEETS.] + +Such a machine is also useful in preparing vegetable matter for the +separation of its juices in presses. Samples of sugar cane, sugar +beets, apples and other bodies of like nature can thus be prepared to +secure their juices for chemical examination. Such an apparatus we +have found is fully as useful and indispensable in an agricultural +laboratory as a drug mill for air-dried materials. + +It is often desirable in the preparation of roots for sugar analysis +to secure them in a completely disintegrated state, that is with the +cellular tissues practically all broken. Such a pulped material can +be treated with water and the sugar juices it contains thus at once +distributed to all parts of the liquid mass. The operation is known +as instantaneous diffusion. The pulp of the vegetable matter is thus +introduced into the measuring flask along with the juices and the +content of sugar can be easily determined. Several forms of apparatus +have been devised for this purpose, one of which is shown in fig. +3. This process, originally devised by Pellet, has come into quite +general use in the determination of the sugar content of beets.[1] +It is observed that it can be applied to other tubers, such as the +turnip, potato, artichoke, etc. It is desirable, therefore, that +an agricultural laboratory be equipped with at least three kinds +of grinding machines; _viz._, first, the common drug mill used for +grinding seeds, air-dried fodders, and the like; second, a pulping +machine like the system of staggered saws above described for the +purpose of reducing green vegetable matter to a fine state of +subdivision, or one like the pellet rasp for tubers; third, a mill for +general use such as is employed for making sausages from soft animal +tissues. + +[Illustration: FIGURE 4. DREEF GRINDING APPARATUS.] + +=10. Grinding Apparatus at Halle Station.=—The machine used at the +Halle station for grinding samples for analysis is shown in Fig. 4.[2] +It is so adjusted as to have both the upper and lower grinding surfaces +in motion. The power is transmitted through the pulley D, which is +fixed to an axis carrying also the inner grinding attachment B. Through +C₂, C₃, C₄, and C₁, the reverse motion is transmitted to the outer +grinder A. By means of the lever E the two grinding surfaces can be +separated when the mill is to be cleaned. The dree mill above described +is especially useful for grinding malt, dry brewers’ grains, cereals +for starch determinations and similar dry bodies. It is not suited to +grinding oily seeds and moist samples. These, according to the Halle +methods, are rubbed up in a mortar until of a size suited to analysis, +and samples such as moist residues, wet cereals, mashes, beet cuttings, +silage, etc., are dried before grinding. If it be desired to avoid the +loss of acids which may have been formed during fermentation, about ten +grams of magnesia should be thoroughly incorporated with each kilogram +of the material before drying. + +=11. Preliminary Treatment of Fish.=—The method used by Atwater in +preparing fish for analysis is given below.[3] The same process may +also be found applicable in the preparation of other animal tissues. +The specimens, when received at the laboratory, are at once weighed. +The flesh is then separated from the refuse and both are weighed. There +is always a slight loss in the separation, due to evaporation and to +slimy and fatty matters and small fragments of the tissues which adhere +to the hands and the utensils employed in preparing the sample. Perfect +separation of the flesh from the other parts of the fish is difficult, +but the loss resulting from imperfect separation is small. The skin +of the fish, although it has considerable nutritive value, should be +separated with the other refuse. + +The partial drying of the flesh for securing samples for analytical +work is accomplished by chopping it as finely as possible and +subjecting from fifty to one hundred grams of it for a day to a +temperature of 96° in an atmosphere of hydrogen. After cooling and +allowing to stand in the open air for twelve hours, the sample is again +weighed, and then ground to a fine powder and made to pass a sieve +with a half millimeter mesh. If the samples be very fat they cannot +be ground to pass so fine a sieve. In such a case a coarser sieve may +be used or the sample reduced to as fine and homogeneous a state as +possible, and bottled without sifting. + +The reason for drying in hydrogen is to prevent oxidation of the fats. +As will be seen further on, however, such bodies can be quickly and +accurately dried at low temperatures in a vacuum, and thus all danger +of oxidation be avoided. In fact, the preliminary drying of all animal +and vegetable tissues, where oxidation is to be feared, can be safely +accomplished in a partial vacuum by methods to be described in another +place. In order to be able to calculate the data of the analysis to the +original fresh state of the substance, a portion of the fresh material +should have its water quantitively determined as accurately as possible. + + +DRYING ORGANIC BODIES. + +=12. Volatile Bodies.=—In agricultural analysis it becomes necessary +to determine the percentage of bodies present in any given sample +which is volatile at any fixed temperature. The temperature reached by +boiling water is the one which is usually selected. It is true that +this temperature varies with the altitude and within somewhat narrow +limits at the same altitude, due to variations in barometric pressure. +As the air pressure to which any given body is subjected, however, is a +factor in the determination of its volatile contents, it will be seen +that within the altitudes at which chemical laboratories are found, +the variations in volatile content will not be important. This arises +from the fact that while water boils at a lower temperature, as the +height above the sea level increases, the corresponding diminished +air pressure permits a more ready escape of volatile matter. As a +consequence, a body dried to constant weight at sea level, where the +temperature of boiling water is 100°, will show the same percentage +of volatile matter as if dried at an altitude where water boils at +99°. When, therefore, it is desirable to determine the volatile matter +in a sample approximately at 100°, it is better to direct that it be +done in a space surrounded by steam at the natural pressure rather +than at exactly 100°, a temperature somewhat difficult to constantly +maintain. However, where it is directed or desired to dry to constant +weight exactly at 100°, it can be accomplished by means of an air-bath +or by a water-jacketed-bath under pressure, or to which enough solid +matter is added to raise the boiling-point to 100°. It is not often, +however, that it is worth while to make any special efforts to secure a +temperature of 100°. When bodies are to be dried at temperatures above +100°, such as 105°, 110°, and so on, an air-bath is the most convenient +means of securing the desired end. The different kinds of apparatus to +be employed will be described in succeeding paragraphs. + +=13. Drying at the Temperature of Boiling Water.=—The best apparatus +for this process is so constructed as to have an interior space +entirely surrounded with boiling water or steam, with the exception +of the door by which entrance is gained thereto. The metal parts of +the apparatus are constructed of copper, and to keep a constant level +of water and avoid the danger of evaporating all the liquid, it is +advisable to have a reflux condenser attached to the apparatus. It is +also well to secure entrance to the interior drying oven, not only by +the door, but also by small circular openings, which serve both to +hold a thermometer and to permit of the aspiration of a slow stream +of dry air through the apparatus during the progress of desiccation. +The gaseous bodies formed by the volatilization of the water and other +matters are thus carried out of the drying box and the process thereby +accelerated. The bath should be heated by a burner so arranged as to +distribute the flame as evenly as possible over the base. A single +lamp, while it will boil the water in the center, will not keep it at +the boiling-point on the sides. The temperature of the interior of the +bath will not therefore reach 100°. The interior of the oven should be +coated with a non-detachable carbon paint to promote the radiation of +the heat from its walls, as well as to protect the parts from oxidation +where acid fumes are produced during desiccation. Instead of a reflux +condenser a constant water level may be maintained in the bath by means +of a mariotte bottle or other similar device. + +[Illustration: FIGURE 5. WATER-JACKETED DRYING OVEN.] + +When a bath of this kind is arranged for use with a partial vacuum, it +should be made cylindrical in shape, with conical ends, as shown in +fig. 5, in order to bear well the pressure to which it is subjected. +Among the many forms of steam-baths offered, the analyst will have +but little difficulty in selecting one suited to his work. To avoid +radiation the exterior of the apparatus should be covered with a +non-conducting material. + +[Illustration: FIGURE 6. THERMOSTAT FOR STEAM-BATH.] + +=14. Drying In a Closed Water Oven.=—When it is desired to keep the +temperature of a drying oven exactly at 100° instead of at the heat of +boiling water, a closed water oven with a thermostat is to be employed. +The oven should be so constructed as to secure a free circulation of +the water about the inner space. Since as a rule the water between the +walls of the apparatus will be subjected to a slight pressure, these +walls should be made strong, or the cylindrical form of apparatus +should be used. The thermostat used by the Halle Station is shown in +Fig. 6.[4] A =⋃= shaped tube, with a bulb on one arm and a lateral +smaller tube sealed on the other, is partly filled with mercury and +connected by rubber tubes on the right with the gas supply, and on +the left with the burner. The end carrying the bulb is connected +directly by a rubber and metal tube with the water space of the oven. +This device is provided with a valve which is left open until the +temperature of the drying space reaches about 95°. The tube conducting +the gas is held in the long arm of the =⋃= by means of a cork through +which it passes air-tight and yet is loose enough to permit of its +being moved. Its lower end is provided with a long ▲ shaped slit. +When the valve leading to the water space is closed and the water +reaches the boiling point, the pressure of the vapor depresses the +mercury in the bulb arm of the =⋃= and raises it in the other. As the +mercury rises it closes the wider opening of the ▲ shaped slit, thus +diminishing the flow of gas to the burner. By moving the gas entry +tube up or down a position is easily found in which the temperature of +the drying space, as shown by the thermometer, is kept accurately and +constantly at 100°. + +In a bath arranged in this way a steam condenser is not necessary. +Since, however, in laboratories which are not at a higher altitude than +1,000 feet the boiling-point of water is nearly 100°, it does not seem +necessary to go to so much trouble to secure the exact temperature +named. There could be no practical difference in the percentage of +moisture determined at 100°, and at the boiling-point of water at a +temperature not more than 1° lower. + +=15. Drying in an Air-Bath.=—In drying a substance in a medium of hot +air surrounded by steam, as has been described, the process is, in +reality, one of drying in air. The apparatus usually meant by the term +air-bath, however, has its drying space heated directly by a lamp, or +indirectly by a stratum of hot air occupying the place of steam in the +oven already described. The simplest form of the apparatus is a metal +box, usually copper, heated from below by a lamp. In the jacketed forms +the currents of hot air produced directly or indirectly by the lamp +are conducted around the inner drying oven, thus securing a more even +temperature. The bodies to be dried are held on perforated metal or +asbestos shelves in appropriate dishes, and the temperature to which +they are subjected is determined by a thermometer, the bulb of which is +brought as near as possible to the contents of the dish. One advantage +of the air-bath is in being able to secure almost any desired +temperature from that of the room to one of 150° or even higher. Its +chief disadvantage lies in the difficulty of securing and maintaining +an even temperature throughout all parts of the apparatus. Radiation +from the sides of the drying oven should be prevented by a covering of +asbestos or other non-combustible and non-conducting substance. The +burner employed should be a broad one and give as even a distribution +of the heat as possible over the bottom of the apparatus. + +[Illustration: FIGURE 7. SPENCER’S DRYING OVEN.] + +=16. Spencer’s Air-Drying Oven.=—In order to secure an even +distribution of the heat in the desiccating space of the oven, Spencer +has devised an apparatus, shown in the figure, in which the temperature +is maintained evenly throughout the apparatus by means of a fan.[5] +The oven has a double bottom, the space between the two bottoms being +filled with air. The sides are also double, the space between being +filled with plaster. The fan is driven by a toy engine connected with +the compressed air service or other convenient method. Thermometers +placed in different parts of the apparatus, while in use, show a +rigidly even heat at all points so long as the fan is kept in motion. +The actual temperature desired can be controlled by a gas regulator. +This form of apparatus is well suited to drying a large number of +samples at once. Portions of liquids and viscous masses may also be +dried by enclosing them in bulbs and connecting with a vacuum. + +Spencer’s oven can also be used to advantage in drying viscous +liquids in a partial vacuum. For this purpose the flask A, Fig. 7, +containing the substance is made with a round bottom to resist the +atmospheric pressure. Its capacity is conveniently from 150 to 200 +cubic centimeters. It is closed with a rubber stopper carrying a trap, +H Hʹ, to keep the evaporated water from falling back. The details of +the construction of the trap H are shown at the right of the figure. +The vapors enter at the lateral orifice, just above the bulb, while the +condensed water falls back into the bulb instead of into the flask A. +A series of flasks can be used at once connected through the stopcocks +G with the circular tube E leading to the vacuum. A water pump easily +exhausts the apparatus, maintaining a vacuum of about twenty-seven +inches. The hot air in the oven is kept in motion by the fan B, thus +ensuring an even temperature in every part. The flask A may be partly +filled with sand or pumice stone before the addition of the samples +to be dried, and the weight of water lost is determined by weighing +A before and after desiccation. If it be desired to introduce a slow +current of dry air or some inert gas into A, it is easily accomplished +by passing a small tube, connected with the dry air or gas supply, +through the rubber stopper and extending it into the flask as far as +possible without coming into contact with the contents. + +=17. Drying Under Diminished Air Pressure.=—The temperature at which +any given body loses its volatile products is conditioned largely +by the pressure to which it is subjected. At an air pressure of 760 +millimeters of mercury, water boils at 100° but it is volatilized +at all temperatures. As the pressure diminishes the temperature at +which a body loses water at a given rate falls. This is a fact of +importance to be considered in drying many agricultural products. This +is especially true of those containing oils and sugars, nearly the +whole number. Invert sugar especially is apt to suffer profound changes +at a temperature of 100°, the levulose it contains undergoing partial +decomposition. Oils are prone to oxidation and partial decomposition at +high temperatures in the presence of oxygen. + +In drying in a partial vacuum therefore a double advantage is secured, +that of a lower temperature of desiccation and in presence of less +oxygen. It is not necessary to have a complete vacuum. There are few +organic products which cannot be completely deprived of their volatile +matters at a temperature of from 70° to 80° in a partial vacuum in +which the air pressure has been diminished to about one-quarter of its +normal force. + +[Illustration: FIGURE 8. ELECTRIC VACUUM DRYING OVEN.] + +=18. Electric Drying-Bath.=—The heat of an electric current can be +conveniently used for drying in a partial vacuum by means of the +simple device illustrated in Fig. 8. In ordering a heater of this kind +the voltage of the current should be stated. The current in use in this +laboratory has a voltage of about 120, and is installed on the three +wire principle. It is well to use a rheostat with the heater in order +to control the temperature within the bell jar. The ground rim of the +bell jar rests on a rubber disk placed on a thick ground glass or a +metal plate, making an air-tight connection. A disk of asbestos serves +to separate the heater from the dish containing the sample, in order to +avoid too high a temperature. + +=19. Steam Coil Apparatus.=—For drying at the temperature of +superheated steam, it is convenient to use an apparatus furnished with +layers or coils of steam pipes. The drying may be accomplished either +in the air or in a vacuum. In this laboratory a large drying oven, +having three shelves of brass steam-tubes and sides of non-conducting +material, is employed with great advantage. The series of heating +pipes is so arranged as to be used one at a time or collectively. Each +series is furnished with a separate steam valve, and is provided with +a trap to control the escape of the condensed vapors. In the bottom of +the apparatus are apertures through which air can enter, which after +passing through the interior of the oven escapes through a ventilator +at the top. With a pressure of forty pounds of steam to the square inch +and a free circulation of air, the temperature on the first shelf of +the apparatus is about 98°; on the second from 103° to 104°, and on +the third about 100°. The vessels containing the bodies to be dried +are not placed directly on the brass steam pipes, but the latter are +first covered with thick perforated paper or asbestos. For drying +large numbers of samples, or large quantities of one sample, such an +apparatus is almost indispensable to an agricultural laboratory. + +[Illustration: FIGURE 9. STEAM COIL DRYING OVEN.] + +A smaller apparatus is shown in Fig. 9. The heating part G is made of +a small brass tube arranged near the bottom in a horizontal coil and +continued about the sides in a perpendicular coil. Bodies placed on the +horizontal shelf are thus entirely surrounded by the heating surfaces +except at the top.[6] The steam pipe S is connected with the supply +by the usual method, and the escape of the condensation is controlled +either by a valve or trap in the usual way. The whole apparatus is +covered by a bell jar B, resting on a heavy cast-iron plate P, through +which also the ends of the brass coil pass. The upper surface of the +iron plate may be planed, or a planed groove may be cut into it, to +secure the edge of the bell jar. When the air is to be exhausted from +the apparatus, a rubber washer should be placed under the rim of the +bell jar. The latter piece of apparatus may either be closed, as shown +in the figure, by a rubber stopper, or it is better, though not shown, +to have a stopper with three holes. One tube passes just through the +stopper and is connected with the vacuum; the second passes to the +bottom of the apparatus and serves to introduce a slow stream of dry +air or of an inert gas during the desiccation. The third hole is for +a thermometer. When no movement of the residual gas in the apparatus +is secured, a dish containing strong sulfuric acid S’ is placed on the +iron plate and under the horizontal coil, as is shown in the figure. +The sulfuric acid so placed does not reach the boiling-point of water, +and serves to absorb the aqueous vapors from the residual air in the +bell jar. By controlling the steam supply the desiccation of a sample +can be secured in the apparatus at any desired temperature within the +limit of the temperature of steam at the pressure used. Where no steam +service is at hand a strong glass flask may be used as a boiler, in +which case the trap end of the coil must be left open. The vacuum may +be supplied by an air or bunsen pump. When a vacuum is not used an +atmosphere of dry hydrogen may be supplied through H. + +[Illustration: FIGURE 10. CARR’S VACUUM DRYING OVEN.] + +[Illustration: FIGURE 10. (BIS) VACUUM OVEN OPEN.] + +=20. Carr’s Vacuum Oven.=—A convenient drying oven has been devised in +this laboratory by Carr.[7] It is made of a large tube, preferably +of brass. The tube may be from six to nine inches in diameter and +from twelve to fifteen inches long. One end is closed air-tight by +a brass end-piece attached by a screw, or brazed. The other end is +detachable and is made air-tight by ground surfaces and a soft washer. +In the figure this movable end-piece is shown attached by screw-nuts, +but experience has shown that these are not necessary. On the upper +longitudinal surfaces are apertures for the insertion of a vacuum gauge +and for attachment to a vacuum apparatus. + +In the figure the thermometer and aperture for introducing dry air or +an inert gas are shown in the movable end disk, but they would be more +conveniently placed in the fixed end. The oven is heated below by a gas +burner, which conveniently should be as long as the oven. The heat is +not allowed to strike the brass cylinder directly, but the latter is +protected by a piece of asbestos paper. + +The temperature inside of the oven can be easily kept practically +constant by means of a gas regulator, not shown in the figure, or +by a little attention to the lamp. For a vacuum of twenty inches a +temperature of about 80° should be maintained. When the vacuum is more +complete a lower temperature can be employed. This apparatus is simple +in construction, strong, cheap, and highly satisfactory in use. + +=21. Drying in Hydrogen.=—In some of the processes of agricultural +analysis it becomes important to dry the sample in hydrogen or other +inert gas. This may be accomplished by introducing the dry gas desired +into some form of the apparatus already described. The drying may +either be accomplished in an atmosphere of hydrogen practically at rest +or in a more limited quantity of the gas in motion. The latter method +is to be preferred by reason of its greater rapidity. The analyst +has at his command many forms of apparatus designed for the purpose +mentioned above. It will be sufficient here to describe only two, +devised particularly for agricultural purposes. + +The first one of these, designed by the author, was intended especially +for drying the samples of fodders for analysis according to the methods +of the Association of Agricultural Chemists.[8] + +[Illustration: FIGURE 11. APPARATUS FOR DRYING IN A CURRENT OF +HYDROGEN.] + +For the purpose of drying materials contained in flasks and tubes +in a current of hydrogen the apparatus shown in Fig. 11 is used. +This apparatus consists of a circular box, B, conveniently made of +galvanized iron, having a movable cover, S, fitted for the introduction +of steam into the interior of the apparatus. Condensed steam escapes at +W. A stream of perfectly pure and dry hydrogen enters at H, passes up +through the material to be dried, down through the bulb V, containing +sulfuric acid, and follows the direction of the arrows through the rest +of the apparatus. The stream of hydrogen is thus completely dried by +passing through bulbs containing sulfuric acid, on the way from one +piece of the apparatus to the other. A, represents a flask such as is +used, with the extraction apparatus described. The apparatus which we +have used will hold eight tubes or flasks at a time, and thus a single +stream of hydrogen is made to do duty eight times in drying eight +separate samples. The great advantage of the apparatus is in the fact +that the stream of hydrogen must pass over and through the substance +to be dried. In order to prevent any sulfuric acid from being carried +forward into the next tube the bulb K, above the sulfuric acid, may be +filled with solid pieces of soda or potash. + +This apparatus has been in use for a long time and no accidents from +sulfuric acid being carried forward have occurred, and there is no +danger, provided the stream of hydrogen is kept running at a slow +rate. If, however, by any accident the stream of hydrogen should be +admitted with great rapidity, particles of the sulfuric acid might be +carried forward and spoil the next sample. To avoid any such accident +as this the proposal to introduce the potash bulb has been made. The +apparatus works with perfect satisfaction, and it is believed that when +properly adjusted check weighings can be made by weighing the bulbs, +showing their increase in weight, which will give the volatile matter, +and weighing the flasks or tubes, which will show the loss of weight. +The only chance for error in weighing the bulbs is that some of the +volatile matter may be material which is not dissolved in sulfuric +acid, and is thus carried on and out of the apparatus. The blackening +of the sulfuric acid in the bulbs, in the drying of all forms of +organic matter, shows that the loss in weight of such bodies is not +due to water alone, but also to organic volatile substances, which are +capable of being decomposed by the sulfuric acid, thus blackening it. + +=22. Caldwell’s Hydrogen Drying-Bath.=—An excellent device for drying +in hydrogen has been described by Caldwell.[9] A vessel of copper or +other suitable material serves to hold the tubes containing the samples +to be dried. It should be about twenty-four centimeters long, fifteen +high, and eight wide. This vessel is contained in another made of the +same material and of the dimensions shown in the figure. On one side +the edge of this containing vessel may not be more than one centimeter +high and the bath should rest against it. The other side is made higher +to form a support for the drying tubes as indicated. + +[Illustration: FIGURE 12. CALDWELL’S HYDROGEN DRYING APPARATUS.] + +The tube containing the substance _a d_ is made of glass and may be +closed by the ground stoppers _c b_ or the tube stoppers _e f_. At _a_ +it carries a perforated platinum disk for holding the filtering felt. +The tube should be about thirteen centimeters long and have an internal +diameter of about twenty millimeters. With its stoppers it should weigh +only a little over thirty grams. The asbestos felt should not be thick +enough to prevent the free passage of gas. Passing diagonally through +the bath are metal tubes, preferably made of copper, and of such a +size as just to receive the glass drying tubes. If these be a little +loose they should be made tight by wrapping them with a narrow coil of +paper at either end of the tubular receptacle. The entrance of cold +air between the glass tube and its metal holder is thus prevented, and +the glass tube is held firmly in position. The glass tube should be +weighed with its two solid stoppers. Afterwards the sample, about two +grams, is placed on the asbestos felt and the stoppers replaced and +the whole reweighed. The exact weight of the sample is thus obtained. +The solid stoppers are then removed and the tube stoppers inserted. +The lower end of the tube is then connected with the supply of dry +hydrogen. The upper tube stopper is connected by a rubber tube with +a small bottle containing sulfuric acid through which the escaping +hydrogen is made to bubble. A double purpose is thus secured; moisture +is kept from entering the drying tube and the rate at which the +hydrogen is passing is easily noted. After the drying is completed the +solid stoppers are again inserted, the tube cooled in a desiccator and +weighed. The loss of weight is entered as water. The tube containing +the sample can afterwards be put into an extractor and treated with +ether or petroleum in the manner hereafter described. This apparatus +requires more hydrogen than the one previously described, but it is +rather simple in construction, is easily controlled, and has given +satisfactory results. + +[Illustration: FIGURE 13. LIEBIG’S ENTE.] + +=23. Drying in Liebig’s Tubes.=—In drying samples, especially of +fodders, the method practiced at the Halle Station is to place them +in drying tubes, the form of which is shown in Fig. 13. A stream of +illuminating gas, previously dried by passing over sulfuric acid and +calcium chlorid, is directed through the tubes.[10] Many of these tubes +can be used at once, arranged as shown in Fig. 14. When the air is all +driven out the stream of gas can be ignited so as to regulate the flow +properly by the size of the flame. The tubes are held in drying ovens, +as shown in the figure, the temperature of which should be kept at +105°-107°. The drying should be continued for eight or ten hours. At +the end of this time the gas in the tube is to be expelled by a stream +of dry air and the tubes cooled in a desiccator and weighed. There are +few advantages in this method not possessed by the processes already +described. The samples, moreover, are not left in a condition for +further examination, either by incineration or extraction. + +[Illustration: FIGURE 14. DRYING APPARATUS USED AT THE HALLE STATION.] + +=24. Wrampelmayer’s Drying Oven.=—The apparatus used at the Wageningen +Station, in Holland, for drying agricultural samples, was devised by +Wrampelmayer and is shown in Fig. 15. The oven is so constructed as +to permit of drying in a stream of inert gas. Illuminating gas is +let into the drying space of the oven through the tube A B. At B the +entering gas is heated by the same lamp which boils the liquid in the +water space of the apparatus. The hot gas is dried in the calcium +chlorid tube c and then passes into the oven at D. At E it leaves the +apparatus and is thence conducted to the lamp F, used for heating the +bath. The lamp should be closed by a wire gauze diaphragm to prevent +any possible explosion by reason of any admixture with the air in the +oven. The condensation of the aqueous vapors is effected by means of +the condenser G. In the drying space is a small shelf holder, which, +by means of the hook H, can be removed from the apparatus. The drying +space is closed from the upper part of the apparatus, which contains no +water by the cover J, resting on a support K. This rim is covered with +a rubber gasket L, by means of which the cover J can be fastened with a +bayonet latch air-tight. This fastening is shown at N. Being closed in +this way the part of the cylindrical oven above the cover may be left +entirely open. Instead of the rather elaborate method of closing the +bath, some simple and equally effective device might be used. The cover +J is best made with double metallic walls enclosing an asbestos packing. + +[Illustration: FIGURE 15. WRAMPELMAYER’S OVEN.] + +It is evident that this oven could be used with an atmosphere of carbon +dioxid or of air, provided the gas for heating were derived from a +separate source and the tube between E and F broken. In a drying oven +designed by the author, the movable top is made with double walls and +the space between is joined to the steam chamber by means of a flexible +metallic tube, thus entirely surrounding the drying space with steam. + +=25. The Ulsch Drying Oven.=—A convenient drying oven is described +by Ulsch which varies from the ordinary form of a water-jacketed +drying apparatus in having a series of drying tubes inserted in the +water-steam space. + +[Illustration: FIGURE 16. ULSCH DRYING OVEN.] + +The arrangement of the oven is shown in the accompanying figure. The +water space is filled only to about one-third of its height. When the +heat is applied the cock _c_ is left open until the steam has driven +out all the air. It is then closed and the temperature of the bath is +then regulated by the manometer _e_, connected with the bath by _d_. +The bottom of the manometer cylinder contains enough mercury to always +keep sealed the end of the manometer tube. The rest of the space is +filled with water. At the top the manometer tube is expanded into a +small bulb which serves as a gas regulator, as shown in the figure. The +gas is admitted also by a small hole above the mercury in the bulb, +so that when the end of the gas inlet tube is sealed enough gas still +passes through to keep the lamp burning. With a mercury pressure of +thirty centimeters the temperature of the bath will be about 105°. The +walls of the bath should be made strong enough to bear the pressure +corresponding to this degree. The drying can be accomplished either in +the cubical drying box _a_ or in the drying tubes made of thin copper +and disposed as shown in the figure. The natural draft is shown by the +arrows. The substance is held in boats placed in the tube as indicated. +The air in traversing the tube is brought almost to the temperature of +the water-steam space in which the tube lies. The natural current of +hot air can easily be replaced by a stream of dry illuminating or other +inert gas. + +=26. Drying Viscous Liquids.=—In the case of cane juices, milk, +and similar substances, the paper coil method may be used.[11] The +manipulation is conducted as follows: A strip of filtering paper from +five to eight centimeters wide and forty centimeters in length, is +rolled into a loose coil and dried at the temperature of boiling water +for two hours, placed in a dry glass-stoppered weighing tube, cooled +in a desiccator and weighed. The stoppered weighing tube prevents the +absorption of hygroscopic moisture. About three cubic centimeters of +the viscous or semi-viscous liquid are placed in a flat dish covered +by a plate of thin glass and weighed. The coil is then placed on end +in the dish, and the greater part of the liquid is at once absorbed. +The proportions between the coil and the amount of liquid should be +such that the coil will not be saturated more than two-thirds of its +length. It is then removed and placed dry end down in a steam-bath +and dried two hours. The dish, covered by the same plate of glass, +is again weighed, the loss in weight representing the quantity of +liquid absorbed by the coil. After drying for the time specified the +coil is again placed in the hot weighing tube, cooled and its weight +ascertained. The increase represents the solid matter in the sample +taken. This method has been somewhat modified by Josse, who directs +that it be conducted as follows:[12] Filter-paper is cut into strips +from one to two centimeters wide and three meters long. The strips are +crimped so they will not lie too closely together and then wrapped +into coils. These coils can absorb about ten cubic centimeters of +liquid. One of them is placed in a flat dish about two centimeters +high and seven in diameter, and dried as described, covered, cooled +and weighed. There are next placed in the dish and weighed one or two +grams of the massecuite, molasses, etc., which are to be dried and the +dish again weighed and the total weight of the matter added, determined +by deducting the weight of the dish and cover. About eight cubic +centimeters of water are added, the material dissolved with gentle +warming, the coil placed in the dish, and the whole dried for two +hours. The cover is then replaced and the whole cooled in a desiccator +and weighed. The increase in weight represents the dry matter in the +sample taken. + +The above method of solution of a viscous sample in order to divide +it evenly for desiccation is based on the principle of the method +first proposed by the author and Broadbent for drying honeys and other +viscous liquids.[13] In this process the sample of honey, molasses, or +other viscous liquid is weighed in a flat dish, dissolved in eighty +per cent alcohol, and then a weighed quantity of pure dry sand added, +sufficient to fill the dish three-quarters full. The alcoholic solution +of the viscous liquid is evenly distributed throughout the mass of sand +by capillary attraction, and thus easily and rapidly dried when placed +on the bath. + +Pumice stone, on account of its great porosity, is also an excellent +medium for the distribution of a viscous liquid in aiding the process +of desiccation. The method has been worked out in great detail in +this laboratory by Carr and Sanborn,[14] and most excellent results +obtained. Round aluminum dishes two centimeters high and from eight to +ten centimeters in diameter are conveniently used for this process. +The pumice stone is dried and broken into fragments the size of a pea +before use. + +=27. General Principles of Drying Samples.=—It would be a needless +waste of space to go into further details of devices for desiccation. A +sufficient number has been given to fully illustrate all the principles +involved. In general, it may be said that drying in the open air at a +temperature not exceeding that of boiling water can be safely practiced +with the majority of samples. For instance, we have found practically +no change in this laboratory in the composition of cereals dried in +the air and in an inert gas. The desiccation should in all cases be +accomplished as speedily as possible. To this end the atmosphere in +contact with the sample should be dry and kept in motion. An oven +surrounded by boiling water and steam is to be preferred to one heated +by air. Constancy of temperature is quite as important as its degree +and this steadiness is most easily secured by steam at atmospheric +pressure. Where higher temperatures than 100° are desired the steam +must be under pressure, or the boiling-point of the water may be raised +by adding salt or other soluble matters. A bath of paraffin or calcium +chlorid may also be used or a sand or air-bath may be employed. The +analyst must not forget, however, that inorganic matters are prone to +change at temperatures above 100°, even in an inert atmosphere, and +higher temperatures must be used with extreme caution. + +Drying in partial vacuum and in a slowly changing atmosphere may +be practiced with all bodies and must be employed with some. The +simple form of apparatus already described will be found useful for +this purpose. At a vacuum of twenty inches or more, even unstable +organic agricultural products are in little danger of oxidation. In +the introduction of a dry gas, therefore, air will be found as a +rule entirely satisfactory. In the smaller form of vacuum apparatus +described, however, there is no objection to the employment of hydrogen +or of carbon dioxid. The gas entering the apparatus should be dried +by passing over calcium chlorid or by bubbling through sulfuric acid. +In this laboratory the vacuum is provided by an air-pump connected +with a large exhaust cylinder. This cylinder is connected by a system +of pipes to all the working desks. The chief objection to this system +is the unsteadiness of the pressure. When only a few are using the +vacuum apparatus for filtering or other purposes the vacuum will stand +at about twenty inches. When no one is using it the vacuum will rise +to twenty-eight or twenty-nine inches. At other times, when in general +use, it may fall to fifteen inches. Where a constant vacuum is desired +for drying, therefore, it is advisable to connect the apparatus with a +special aspirator which will give a pressure practically constant. + +The dishes containing the sample should be low and flat, exposing as +large a surface as possible. For viscous liquids it will be found +advisable to previously fill the dishes with pumice stone or other +inert absorbent material to increase the surface exposed. + +The special methods of drying milk, sirup, honeys, and like bodies, +will be described in the paragraphs devoted to these substances. + +In drying agricultural products, not only water but all other matters +volatile at the temperature employed are expelled. It is only necessary +to conduct the products of volatilization through sulfuric acid to +demonstrate the fact that organic bodies are given off. In the case +mentioned the sulfuric acid will be speedily changed to a brown and +even black color by these bodies. It is incontestable, however, that +in most cases the essential oils and other volatile matters thus +escaping are not large in quantity and could not appreciably affect +the percentage composition of the sample. In such cases the whole of +the loss on drying is entered in the note book as water. There are +evidently many products, however, where a considerable percentage of +the volatile products is not water. The percentage of essential oils, +which have a lower boiling-point than water, can be determined in a +separate sample and this deducted from the total loss on drying will +give the water. + +Simple as it seems, the determination of water in agricultural products +often presents peculiar difficulties and taxes to the utmost the +patience and skill of the analyst. Having set forth the substantial +principles of the process and indicated its more important methods, +there is left for the worker in the laboratory the choice of processes +already described, or, in special cases, the device of new ones and +adaption of old ones to meet the requirements of necessity. + + +INCINERATION. + +=28. Determination of Ash.=—The principle to be kept in view in the +preparation of the ash of agricultural products is to conduct the +incineration at as low a temperature as possible to secure a complete +combustion. The danger of too high a temperature is two-fold. In the +first place some of the mineral constituents constantly present in the +ash, notably, some of the salts of potassium and sodium are volatile +at high temperatures and thus escape detection. In the second place, +some parts of the ash are rather easily fusible and in the melted state +occlude particles of unburned organic matter, and thus protect them +from complete oxidation. Both of these dangers are avoided, and an ash +practically free of carbon obtained, by conducting the combustion at +the lowest possible temperature capable of securing the oxidation of +the carbonaceous matter. + +=29. Products Of Combustion.=—The most important product of combustion, +from the present point of view, is the mineral residue obtained. The +organic matter of the sample undergoes decomposition in various ways, +depending chiefly on its nature. Complex volatile compounds are formed +first largely of an acid nature. The residual carbon is oxidized to +carbon dioxid and the hydrogen to water. The relative proportions +of these bodies formed, in any given case, depend on the conditions +of combustion. With a low temperature and a slow supply of oxygen, +the proportion of volatile organic compounds is increased. At a high +temperature, and in a surplus of oxygen, the proportions of water and +carbon dioxid are greater. At the present time, however, our attention +is to be directed exclusively to the mineral residue; the organic +products of combustion belonging to the domain of organic chemistry. As +has already been intimated, the ash of agricultural samples consists +of the mineral matters derived from the tissues, together with any +accidental mineral impurities which may be present, some unburned +carbon, and the sulfur, phosphorus, chlorin, nitrogen, etc., existing +previously in combination with the mineral bases. The organic sulfur +and phosphorus may also undergo complete or partial oxidation during +incineration and be found in the ash. Unless special precautions be +taken, however, a portion of the organic sulfur and phosphorus may +escape as volatile compounds during the combustion.[15] The organic +nitrogen is probably completely lost, at most, only traces of it being +oxidized during the combustion in such a way as to combine with a +mineral base. The rare mineral elements that are taken up by plants +will also be found in the ash. Here the analyst would look for copper, +boron, zinc, manganese, and the other elements which, when existing +in the soil, are apt to be found in the tissues of the plants, not, +perhaps, as organic or essential compounds, but as concomitants of the +other mineral foods absorbed by growing vegetation. This fact is often +of importance in toxicological and hygienic examinations of foods. +For instance, traces of copper or of boron in the ash of a prescribed +food would not be evidence of the use of copper or borax salts as +preservatives unless it could be shown that the soil on which the food +in question was grown was free of these bodies. + +This fact manifestly applies only to those cases where mere traces +of these rare bodies are in question. The presence of considerable +quantities of them, enough to be inimical to health, could only be +attributed to artificial means. + +=30. Purpose and Conduct of Incineration.=—In burning a sample of an +agricultural product the analyst may desire to secure either a large +sample of ash for analytical purposes as already described or to +determine the actual percentage of ash. The first purpose is secured +in many ways. In the preparation of ash for manurial purposes, for +instance, little care is exercised either to prevent volatilization of +mineral matters or to avoid the occurrence of a considerable quantity +of carbon in the sample. With this operation we have, at present, +nothing whatever to do. In preparing a sample of ash for chemical +analysis it is important, where a sufficient quantity of the sample +can be obtained, to use as large a quantity of it as convenient. +While it is true that very good results may be secured on very small +samples, it is always advisable to have a good supply of the material +at hand. Since the materials burned have only from one to three per +cent of ash, a kilogram of them will supply only from ten to thirty +grams. To supply all needful quantities of material and replace the +losses due to accident, whenever possible at least twenty grams of the +ash should be prepared. The combustion can be carried on in platinum +dishes with all bodies free of metallic oxids capable of injuring the +platinum. Otherwise porcelain or clay dishes may be employed. As a +rule the combustion is best conducted in a muffle at a low red heat. +With substances very rich in fusible ash, as for instance the cereals, +it is advisable to first char them, extract the greater part of the +ash with water, and afterwards burn the residual carbon. The aqueous +extract can then be added to the residue of combustion and evaporated +to dryness at the temperature of boiling water. During the combustion +the contents of the dish should not be disturbed until the carbon is as +completely burned out as possible. The naturally porous condition in +which the mass is left during the burning is best suited to the entire +oxidation of the carbon. At the end however, it may become necessary to +bring the superficial particles of unburned carbon into direct contact +with the bottom of the dish by stirring its contents. In most instances +very good results may be obtained by burning the ash in an open dish +without the aid of a muffle. In this case a lamp should be used with +diffuse flame covering as evenly as possible the bottom of the dish and +thus securing a uniform temperature. The carbon, when once in active +combustion, will as a rule be consumed, and an ash reasonably pure be +obtained. + +The second purpose held in view by the analyst is to determine the +actual content of ash in a sample. For this purpose only a small +quantity of the material should be used, generally from two to ten +grams. The combustion should be conducted in flat-bottomed, shallow +dishes, and at a low temperature. In many cases the residue, after +determining the moisture, can be at once subjected to incineration, +and thus an important saving of time be secured. A muffle, with gentle +draft, will be found most useful for securing a white ash. The term, +white ash, is sometimes a deceptive one. In samples containing iron or +manganese, the ash may be practically free of carbon and yet be highly +colored. The point at which the combustion is to be considered as +finished therefore should be at the time the carbon has disappeared +rather than when no coloration exists. In general the methods of +incineration are the same for all substances, but some cases may arise +in which special processes must be employed. Some analysts prefer to +saturate the substance before incineration with sulfuric acid, securing +thus a sulfated ash. This is practiced especially with molasses. In +such cases the ash obtained is free of carbon dioxid and roughly the +difference in weight is compensated for by deducting one-tenth of +the weight of the ash when comparison is to be made with ordinary +carbonated ash. Naturally this process could not be used when sulfuric +acid is to be determined in the product. + +[Illustration: FIGURE 17. COURTOUNE MUFFLE.] + +=31. German Ash Method.=—The method pursued at the Halle Station for +securing the percentage of ash in a sample is as follows:[16] Five +grams of the air-dried sample are incinerated in a platinum dish and +the ash ignited until it has assumed a white, or at least a bright +gray tint. As soon as combustible gases are emitted at the beginning +of the incineration they are ignited and allowed to burn as long as +possible. It is advisable to hasten the oxidation by stirring the mass +with a piece of platinum wire. If the ash should become agglomerated, +as sometimes happens with rich food materials, it must be separated by +attrition. The ash, when cooled on a desiccator, is to be weighed. When +great exactness is required, it is advised, as set forth in a former +paragraph, to first carbonize the mass and then extract the soluble +ash with hot water before completing the oxidation. When the latter is +complete and the dish cooled the aqueous extract is added, evaporated +to dryness and the incineration completed. + +=32. Courtonne’s Muffle.=—The ordinary arrangement of a muffle, as in +assaying, may be conveniently used in incineration. A special muffle +arrangement has been prepared by Courtonne which not only permits of +the burning of a large number of samples at once, but also effects a +considerable saving in gas. The muffle as shown in Fig. 17, is made in +two stages, and the floor projects in front of the furnace, forming a +convenient hearth. The incineration is commenced on the upper stage, +where the temperature is low, and finished on the lower one at a higher +heat. The furnace is so arranged as to permit the flame of the burning +gas to entirely surround the muffle. The draft and temperature within +the muffle are controlled by the fire-clay door shown resting on the +table. + + +TREATMENT WITH SOLVENTS. + +=33. Object Of Treatment.=—The next step, in the analytical work, after +sampling, drying, and incinerating, is the treatment of the sample with +solvents. The object of this work is to separate the material under +examination into distinct classes of bodies distinguished from each +other by their solubilities. It is not the purpose of this section +to describe the various bodies which may be separated in this way, +especially from vegetable products. For this description the reader may +consult the standard works on plant analysis.[17] + +The chief object of a strictly agricultural examination of a field +or garden product is to determine its food value. This purpose can +be accomplished without entering into a minute separation of nearly +allied bodies. For example, in the case of carbohydrates it will be +sufficient as a rule, to separate them into four classes. In the first +class will be found those soluble in water as the ordinary sugars. In +the second group will be found those which, while not easily soluble in +water, are readily rendered so by treatment with certain ferments or by +hydrolysis with an acid. The starches are types of this class. In the +third place are found those bodies which resist the usual processes of +hydrolysis either with an acid or alkali, and therefore remain in the +residue as fiber. Cellulose is a type of these bodies. In the fourth +class are included those bodies which on hydrolysis with an acid yield +furfurol on distillation, and therefore belong to the type containing +five atoms of carbon or some multiple thereof in their molecule. For +ordinary agricultural purpose the separation is not even as complete as +is represented above. + +What is true of the carbohydrates applies equally well to the fats and +to other groups. Especially in the analysis of cereals and of cattle +foods, the treatment with solvents is confined to the use in successive +order of ether or petroleum, alcohol, dilute acids, and alkalies, the +latter at a boiling temperature. The general method of treatment with +these solvents will be the subject of the following paragraphs. + +=34. Extraction of the Fats and Oils.=—Two solvents are in general use +for the extraction of fats and oils; _viz._, ethylic ether and a light +petroleum. The former is the more common reagent. Before use it should +be made as pure as possible by washing first with water, afterwards +removing the water by lime or calcium chlorid, and then completing +the drying by treatment with metallic sodium. The petroleum spirit +used should be purified by several fractional distillations until it +has nearly a constant boiling-point of from 45° to 50°. The detailed +methods of preparing these reagents will be given in another place. For +rigid scientific determinations the petroleum is to be preferred to the +ether. It is equally as good a solvent for fats and oils and is almost +inert in respect to other vegetable constituents. Ether, on the other +hand, dissolves chlorophyll and its partial oxidation products, resins, +alkaloids and the like. The extract obtained by ether is therefore less +likely to be a pure fat than that secured by petroleum. For purposes of +comparison, however, the ether should be employed, inasmuch as it has +been used almost exclusively in analytical operations in the past. + +=35. Methods Of Extraction.=—The simplest method for accomplishing +the extraction of fat from a sample consists in treating it with +successive portions of the solvent in an open dish or a closed flask. +This process is actually employed in some analytical operations, as, +for instance, in the determination of fat in milk. Experience has +shown, however, that a portion of the substance soluble, for instance, +in ether, passes very slowly into solution, so that a treatment such as +that just described would have to be long continued to secure maximum +results. The quantity of solvent required would thus become very large +and in the case of ether would entail a great expense. For the greater +number of analytical operations, therefore, some device is employed for +using the same solvent continually. The methods of extraction therefore +fall into two general classes; _viz._, extraction by digestion and +extraction by percolation. This classification holds good also for +other solvents besides ether and petroleum. In general, the principles +and practice of extraction described for ether may serve equally well +for alcohol, acetone and other common solvents. + +=36. Extraction by Digestion.=—In the use of ether or petroleum the +sample is covered with an excess of the solvent and allowed to remain +for some time in contact therewith. The soluble portions of the +sample diffuse into the reagent. The speed of diffusion is promoted +by stirring the mixtures. The operation may be conducted in an open +dish or a flask. Inasmuch as the residue is, as a rule, to be dried +and weighed, an open dish is to be preferred. To avoid loss of reagent +and to prevent filling a working room with very dangerous gases, the +temperature of digestion should be kept below the boiling-point of the +solvent. The greater part of the soluble matter will be extracted with +three or four successive applications of the reagent, but, as intimated +above, the last portions of the soluble material are extracted with +difficulty by this process. In pouring off the solvent care must be +exercised to avoid loss of particles of the sample suspended therein. +To this end it is best to pour the solvent through a filter. For the +extraction of large quantities of material for the purpose of securing +the extract for future examination, or simply to remove it, the +digestion process is usually employed. This excess of solvent required +is easily recovered by subsequent distillation and used again. The +method is rarely used for the quantitive estimation of the extract, +the process of continuous percolation being more convenient and more +exact. + +=37. Extraction by Percolation.=—In this method the solvent employed +is poured on the top of the material to be extracted and allowed to +pass through it usually by gravitation alone, sometimes with the help +of a filter-pump. The principle of the process is essentially that of +washing precipitates. + +Two distinct forms of apparatus are in use for this process. In +the first kind the solvent is poured over the material and after +percolation is secured by distillation in another apparatus. In the +second kind the solvent is secured after percolation in a flask where +it is at once subjected to distillation. The vapors of the solvent +are conducted by appropriate means to a condenser placed above the +sample. After condensation the solvent is returned to the upper part of +the sample. The percolation thus becomes continuous and a very small +quantity of the solvent may thus be made to extract a comparatively +large amount of material. This process is particularly applicable +to the quantitive determination of the extract. After distillation +and drying the latter may be weighed in the flask in which it was +received or the sample may be dried and weighed in the vessel in which +it is held both before and after extraction. One great advantage of +the continuous extraction method lies in the fact that when it is +once properly started it goes on without further attention from the +analyst save an occasional examination of the flow of water through +the condenser and of the rate of the distillation. For this reason the +process may be continued for many hours without any notable loss of +time. The vapor of the solvent in passing to the condenser may pass +through a tube out of contact with the material to be extracted or it +may pass directly around the tube holding the sample. In the former +case the advantage is secured of conducting the extraction at a higher +temperature, but there is danger of boiling the solvent in contact with +the material and thus permitting the loss of a portion of the sample. + +=38. Apparatus Used for Extractions.=—For extraction by digestion, as +has already been said, an open dish may be used. When large quantities +of material are under treatment, heavy flasks, holding from five to +ten liters, will be found convenient. In these cases a condenser can +be attached to the flask and the extraction conducted at the boiling +temperature of the solvent. During the process of extraction it is +advisable to shake the flask frequently. By proceeding in this way the +greater part of the solvent matter will be removed after three or four +successive treatments. + +In extraction by percolation various forms of apparatus are employed. +The ordinary percolators of the manufacturing pharmacist may be used +for the larger operations, while the more elaborate forms of continuous +extractors will be found most convenient for quantitive work. In each +case the analyst must choose that process and form of apparatus best +suited to the purpose in view. In the next paragraphs will be described +some of the more common forms of apparatus in use. + +=39. Knorr’s Extraction Apparatus.=—The apparatus which has been +chiefly used in this laboratory for the past few years is shown in +the accompanying figure.[18] The principle of the construction of the +apparatus lies in the complete suppression of stoppers and in sealing +the only joint of the device with mercury. + +The construction and operation of the apparatus will be understood by a +brief description of its parts. + +A is the flask containing the solvent, W a steam bath made by cutting +off the top of a bottle, inverting it and conducting the steam into one +of the tubes shown in the stopper while the condensed water runs out of +the other. The top of the bath is covered with a number of concentric +copper rings, so that the opening may be made of any desirable size. B +represents the condenser, which is a long glass tube on which a number +of bulbs has been blown, and which is attached to the hood for holding +the material to be extracted, as represented at Bʹ, making a solid +glass union. Before joining the tube at Bʹ the rubber stopper which is +to hold it into the outside condenser of B is slipped on, or the rubber +stopper may be cut into its center and slipped over the tube after the +union is made. In case alcohol is to be used for the solvent, requiring +a higher temperature, the flask holding the solvent is placed entirely +within the steam-bath, as represented at Aʹ. + +[Illustration: FIGURE 18. KNORR’S EXTRACTION APPARATUS.] + +[Illustration: FIGURE 19. EXTRACTION FLASK.] + +[Illustration: FIGURE 20. EXTRACTION TUBE.] + +[Illustration: FIGURE 21. EXTRACTION SIPHON TUBE.] + +A more detailed description of the different parts of the apparatus +can be seen by consulting Figs. 19, 20, and 21. In A, Fig. 19, is +represented a section of the flask which holds the solvent, showing +how the sides of the hood containing the matters to be extracted pass +over the neck of the flask, and showing at S a small siphon inserted +in the space between the neck of the flask and the walls of the hood +for the purpose of removing any solvent that may accumulate in this +space. A view of the flask itself is shown at Aʹ. It is made by taking +an ordinary flask, softening it about the neck and pressing the neck +in so as to form a cup, as indicated at Aʹ, to hold the mercury which +seals the union of the flask with the condenser. The flask is held in +position by passing a rubber band below it, which is attached to two +glass nipples, _b_, blown onto the containing vessel, as shown in Fig. +18. The material to be extracted may be contained in an ordinary tube, +as shown in Fig. 20, which may be made from a test tube drawn out, as +indicated in the figure, having a perforated platinum disk sealed in +at D. The containing tube rests upon the edges of the flask containing +the solvent by means of nipples shown at _t_. If a siphon tube is to +be used, one of the most convenient forms is shown in Fig. 21, in +which the siphon lies entirely within the extracting tube, thus being +protected from breakage. By means of this apparatus the extractions +can be carried on with a very small quantity of solvent, there being +scarcely any leakage, even with the most volatile solvents, such as +ether and petroleum. The apparatus is always ready for use, no corks +are to be extracted, and no ground glass joints to be fitted. + +=40. Soxhlet’s Extraction Apparatus.=—A form of continuous extraction +apparatus has been proposed by Soxhlet which permits the passage of +the vapors of the solvent into the condenser by a separate tube and +the return of the condensed solvent after having stood in contact +with the sample, to the evaporating flask by a siphon. The advantage +of this process lies in freeing the sample entirely from the rise of +temperature due to contact with the vapors of the solvent, and in the +second place in the complete saturation of the sample with the solvent +before siphoning. The sample is conveniently held in a cylinder of +extracted filter-paper open above and closed below. This is placed +in the large tube between the evaporating flask and the condenser. +The sample should not fill the paper holder, and if disposed to float +in the solvent, should be held down with a plug of asbestos fiber +or of glass wool. The extract may be transferred, by dissolving in +the solvent, from the flask to a drying dish, or it may be dried and +weighed in the flask where first received. + +[Illustration: FIGURE 22. SOXHLET EXTRACTION APPARATUS.] + +There are many forms of apparatus of this kind, one of which is shown +in Fig. 22, but a more extended description of them is not necessary. +The disadvantages of this process as compared with Knorr’s, are quite +apparent. The connections with the evaporating flask and condenser are +made with cork stoppers, which must be previously thoroughly extracted +with ether and alcohol. These corks soon become dry and hard and +difficult to use. The joints are likely to leak, and grave dangers +of explosion arise from the vapors of the solvents escaping into the +working room. Moreover, it is an advantage to have the sample warmed +by the vapors of the solvent during the progress of the extraction, +provided the liquid in direct contact with the sample does not boil +with sufficient vigor to cause loss. + +The use of extraction apparatus with ground glass joints is also +unsatisfactory. By reason of unequal expansion and contraction these +joints often are not tight. They are also liable to break and thus +bring danger and loss of time. + +=41. Compact Extraction Apparatus.=—In order to bring the extraction +apparatus into a more compact form, the following described device has +been successfully used in this laboratory.[19] The condenser employed +is made of metal and is found entirely within the tube holding the +solvent. + +This form of condenser is shown in Fig. 23, in which the tube E serves +to introduce the cold water to the bottom of the condensing device. +The tube D serves to carry away the waste water. The tube F serves +for the introduction of the solvent by means of a small funnel. When +the solvent is introduced and has boiled for a short time, the tube +F should be closed. In each of the double conical sections of the +condenser a circular disk B is found, which causes the water flowing +from A upward to pass against the metallic surfaces of the condenser. + +A section of the double conical condenser is shown in the upper right +hand corner. It is provided with two small hooks _hh_, soldered on the +lower surface, by means of which the crucible G can be hung with a +platinum wire. The condenser is best made smooth and circular in form. + +The crucible G, which holds the material to be extracted, can be made +of platinum, but for sake of economy also of porcelain. The bottom +of the porcelain crucible is left open excepting a small shelf, as +indicated, which supports a perforated disk of platinum on which an +asbestos film is placed. + +[Illustration: FIGURE 23. COMPACT CONDENSING APPARATUS.] + +The whole apparatus is of such size as to be easily contained in the +large test-tube T. + +The mouth of the test-tube is ground so as to fit as smoothly as +possible to the ground-brass plate of the metallic condenser P. + +In case it is desired to weigh the extract it may be done directly by +weighing it in the test-tube T after drying in the usual way at the +end of the extraction; or a glass flask H, made to fit freely into the +test-tube, may be used, in which case a little mercury is poured into +the bottom of the tube to seal the space between H and T. To prevent +spirting of the substance in H, or projecting any of the extracted +material without or against the bottom of the crucible G, the funnel +represented by the dotted lines in the right hand section may be used. + +Heat may be applied to the test-tube either by hot water, or steam, or +by a bunsen, which permits of the flame being turned down to minimum +proportions without danger of burning back. When the test-tube alone +is used it is advisable to first put into it some fragments of pumice +stone, particles of platinum foil, or a spoonful of shot, to prevent +bumping of the liquid when the lamp is used as the source of heat. + +Any air which the apparatus contains is pushed out through F when the +boiling begins, the tube F not being closed until the vapor of the +liquid has reached its maximum height. With cold water in the condenser +the vapor of ether very rarely reaches above the lower compartment and +the vapor of alcohol rarely above the second. + +When the plate P is accurately turned so as to fit the ground surface +of the mouth of T, it is found that ten cubic centimeters of anhydrous +ether or alcohol are sufficient to make a complete extraction, and +there is not much loss of solvent in six hours. The thickness of the +asbestos film in G, or its fineness, is so adjusted as to prevent too +rapid filtration so that the solvent may just cover the material to +be extracted, or, after the material is placed in a crucible, a plug +of extracted glass wool may be placed above it for the purpose of +distributing the solvent evenly over the surface of the material to be +extracted and of preventing the escape of fine particles. + +[Illustration: FIGURE 24. IMPROVED COMPACT EXTRACTION APPARATUS.] + +In very warm weather the apparatus may be arranged as shown in figure +24. The bath for holding the extraction tubes is made in two parts, +K and Kʹ. The bath K has a false bottom shown in the dotted line O, +perforated to receive the ends of the extraction tubes and which holds +them in place and prevents them from touching the true bottom, where +they might be unequally heated by the lamp. The upper bath Kʹ has a +perforated bottom, partly closed with rubber-cloth diaphragms Gʹ Nʹ Hʹ. +The extraction tubes passing through this bath, water-tight, permit +broken ice or ice-water to be held about their tops, and thus secure +a complete condensation of the vapors of the solvent which in warm +weather might escape the metal condenser. In practice care must be +taken to avoid enveloping too much of the upper part of the extraction +tube with the ice-water, otherwise the vapors of the solvent will be +chiefly condensed on the sides of the extraction tube and will not be +returned through the sample. It is not often that the upper bath is +needed, and then only with ether, never with alcohol. This apparatus +has proved especially useful with alcohol, using, as suggested, +glycerol in the bath. The details of its further construction and +arrangement are shown in the figure. The extraction tubes are most +conveniently arranged in a battery of four, one current of cold water +passing in at A and out at B, serving for all. The bath is supported +on legs long enough to allow the lamp plenty of room. The details of +the condenser M are shown in Bʹ, Aʹ, T, Fʹ, and Lʹ. Instead of a gooch +Lʹ for holding the sample a glass tube R, with a perforated platinum +disk Q, may be used. The water line in the bath is shown by W. This +apparatus may be made very cheaply and without greatly impairing its +efficiency by using a plain concentric condenser and leaving off the +upper bath Kʹ. + +=42. Solvents Employed.=—It has already been intimated that the chief +solvents employed in the extraction of agricultural samples are ether +or petroleum and aqueous alcohol. The ether used should be free of +alcohol and water, the petroleum should be subjected to fractional +distillation to free it of the parts of very high and very low boiling +points, and the alcohol as a rule should contain about twenty per cent +of water. + +There are many instances, however, where other solvents should be used. +The use of aqueous alcohol is sometimes preceded by that of alcohol of +greater strength or practically free of water. For the extraction of +soluble carbohydrates (sugars) cold or tepid water is employed, the +temperature of which is not allowed to rise high enough to act upon +starch granules. For the solution of the starch itself an acid solvent +is used at a boiling temperature, whereby the starch molecules undergo +hydrolysis and form dextrin or soluble sugars (maltose, dextrose). By +this process also the carbohydrates, whose molecules contain five, or +some multiple thereof, atoms of carbon form soluble sugars of which +xylose and arabinose are types. The solvent action of acids followed +by treatment with dilute alkalies at a boiling temperature, completes +practically the solution of all the carbohydrate bodies, save cellulose +and nearly related compounds. The starch carbohydrates are further +dissolved by the action of certain ferments such as diastase. + +Dilute solutions of mineral salts exert a specific solvent action on +certain nitrogenous compounds and serve to help separate the albuminoid +bodies into definite groups. + +Under the proper headings the uses of these principal solvents will be +described, but a complete discussion of their action, especially on +samples of a vegetable origin, should be looked for in works on plant +analysis.[20] + +The application of acids and alkalies for the extraction of +carbohydrates, insoluble in water and alcohol, will be described, +in the paragraphs devoted to the analysis of fodders and cereals. +The extraction of these matters, made soluble by ferments, will be +discussed in the pages devoted to starch and artificial digestion. +It is thus seen that the general preliminary treatment of a sample +preparatory to specific methods of examination is confined to drying, +extraction with ether and alcohol, and incineration. + +=43. Recovery of the Solvent.=—In using such solvents as ether, +chloroform, and others of high value, it is desirable often to recover +the solvent. Various forms of apparatus are employed for this purpose, +arranged in such a way as both to secure the solvent and to leave the +residue in an accessible condition, or in a form suited to weighing +in quantitive work. When the extractions are made according to the +improved method of Knorr, the flask containing the extract may be at +once connected with the apparatus shown in figure 25.[21] A represents +the flask containing the solvent to be recovered, W the steam-bath, +B the condenser sealed by mercury, M and R the flask receiving the +products of condensation. It will be found economical to save ether, +alcohol, and chloroform even when only a few cubic centimeters remain +after the extraction is complete. In the figure the neck of the flask +A is represented as narrower than it really is. The open end of the +connecting tube, which is sealed on A by mercury, should be the same +size as the tube connecting with the condenser in the extraction +apparatus. + +[Illustration: FIGURE 25.—KNORR’S APPARATUS FOR RECEIVING SOLVENTS.] + +[Illustration: FIGURE 26. APPARATUS FOR RECOVERING SOLVENTS FROM OPEN +DISHES.] + +It often happens that materials which are dissolved by the ordinary +solvents in use are to be collected in open dishes in order that +their properties may be studied. At the same time large quantities +of solvents must be used, and it is desirable to have some method of +recovering them. The device shown in Fig. 26 has been found to work +excellently well for this purpose.[22] It consists of a steam-bath, W, +and a bottle, B, with the bottom cut off, resting on an iron dish, P, +containing a small quantity of mercury, enough to seal the bottom of +the bottle. The dish containing the solvent is placed on the mercury, +and the bottle placed down over it, forming a tight joint. On the +application of steam the solvent escapes into the condenser, C, and is +collected as a liquid in the flask A. In very volatile solvents the +flask A may be surrounded with ice, or ice-cold water passed through +the condenser. When an additional quantity of the solvent is to be +added to the dish for the purpose of evaporating it is poured into the +funnel F, and the stopcock H opened, which allows the material to run +into the dish in B without removing the bottle. In this way many liters +of the solvent may be evaporated in any one dish, and the total amount +of extract obtained together. At the last the bottle B is removed, and +the extract which is found in the dish is ready for further operations. + + +AUTHORITIES CITED IN PART FIRST. + +[1] Sidersky: Traité d’Analyse des Matières Sucrées, p. 311. + +[2] Die Agricultur-Chemische Versuchs-Station, Halle a/S., S. 34. (Read +Dreef instead of Dree.) + +[3] Report of Commissioner of Fish and Fisheries, 1888, p. 686. + +[4] Vid. op. cit. 2, p. 14. + +[5] Journal of the American Chemical Society, Vol. 15, p. 83. + +[6] Chemical Division, U. S. Department of Agriculture, Bulletin No. +28, p. 101. + +[7] Not yet described in any publication. Presented at 12th annual +meeting of the Association of Agricultural Chemists, Aug. 7th, 1895. + +[8] Vid. op. cit. 6, p. 100. + +[9] Cornell University Agricultural Experiment Station, Bulletin 12. + +[10] (bis. p. 28). Vid. op. cit. 2, p. 15. + +[11] Bulletin No. 13, Chemical Division, U. S. Department of +Agriculture, Part First pp. 85-6. + +[12] Bulletin de 1’ Association des Chimistes de Sucrerie, 1893, p. 656. + +[13] Chemical News, Vol. 52, p. 280. + +[14] Presented to 12th Annual Convention of the Association of Official +Agricultural Chemists, Sept. 7th, 1895. + +[15] Vid. Volume First, p. 411. + +[16] Vid. op. cit. 2, p. 17. + +[17] Dragendorff, Plant Analysis. + +[18] Vid. op. cit. 6, p. 96. + +[19] Journal of Analytical and Applied Chemistry, Vol. 7, p. 65, and +Journal of the American Chemical Society, March 1893. + +[20] Vid. op. cit. 16. + +[21] Vid. op. cit. 6, p. 99. + +[22] Vid. op. cit. 6, p. 103. + + + + +PART SECOND. + +SUGARS AND STARCHES. + + +=44. Introduction.=—Carbohydrates, of which sugars and starches are +the chief representatives, form the great mass of the results of +vegetable metabolism. The first functions of the chlorophyll cells of +the young plant are the condensation of carbon dioxid and water. The +simplest form of the condensation is formaldehyd, CH₂O. There is no +convincing evidence, however, that this is the product resulting from +the functional activity of the chlorophyll cells. The first evidence +of the condensation is found in more complex molecules; _viz._, those +having six atoms of carbon. It is not the purpose of this work to +discuss the physiology of this process, but the interested student can +easily find access to the literature of the subject.[23] When a sample +of a vegetable nature reaches the analyst he finds by far the largest +part of its substance composed of these products of condensation of the +carbon dioxid and water. The sugars, starches, pentosans, lignoses, and +celluloses all have this common origin. Of many air-dried plants these +bodies form more than eighty per cent. + +In green plants the sugars exist chiefly in the sap. In plants cut +green and quickly dried by artificial means the sugars are found +in a solid state. They also exist in the solid state naturally in +certain sacchariferous seeds. Many sugar-bearing plants when allowed +to dry spontaneously lose all or the greater part of their sugar by +fermentation. This is true of sugar cane, sorghum, maize stalks, and +the like. The starches are found deposited chiefly in tubers, roots or +seeds. In the potato the starch is in the tuber, in cassava the tuber +holding the starch is also a root, in maize, rice and other cereals the +starch is in the seeds. The wood-fibers; _viz._, pentosans, lignose, +cellulose, etc., form the framework and support of the plant structure. +Of all these carbohydrate bodies the most important as foods are the +sugars and starches, but a certain degree of digestibility cannot be +denied to other carbohydrate bodies with the possible exception of +pure cellulose. In the following paragraphs the general principles of +determining the sugars and starches will be given and afterwards the +special processes of extracting these bodies from vegetable substances +preparatory to quantitive determination. + +=45. Nomenclature.=—In speaking of sugars it has been thought best +to retain for the present the old nomenclature in order to avoid +confusion. The terms dextrose, levulose, sucrose, etc., will therefore +be given their commonly accepted significations. + +A more scientific nomenclature has recently been proposed by Fischer, +in which glucose is used as the equivalent of dextrose and fructose +as the proper name for levulose. All sugars are further classified +by Fischer into groups according to the number of carbon atoms found +in the molecule. We have thus trioses, tetroses, pentoses, hexoses, +etc. Such a sugar as sucrose is called hexobiose by reason of the fact +that it appears to be formed of two molecules of hexose sugars. For a +similar reason raffinose would belong to the hexotriose group.[24] + +Again, the two great classes of sugars as determined by the structure +of the molecule are termed aldoses and ketoses according to their +relationship to the aldehyd or ketone bodies. + +Since sugars may be optically twinned, that is composed of equal +molecules of right and left-handed polarizing matter it may happen that +apparently the same body may deflect the plane of polarization to the +right, to the left, or show perfect neutrality. + +Natural sugars, as a rule, are optically active, but synthetic sugars +being optically twinned are apt to be neutral to polarized light. + +To designate the original optical properties of the body therefore the +symbols _d_, _l_, and _i_, meaning dextrogyratory, levogyratory, and +inactive, respectively, are prefixed to the name. Thus we may have _d_, +_l_, or _i_ glucose, _d_, _l_, or _i_ fructose, and so on. + +The sugars that are of interest here belong altogether to the pentose +and hexose groups; _viz._, C₅H₁₀O₅ and C₆H₁₂O₆, respectively. Of the +hexobioses, sucrose, maltose, and lactose are the most important, +and of the hexotrioses, raffinose. In this manual, unless otherwise +stated, the term dextrose corresponds to _d_ glucose, and levulose to +_d_ fructose. In this connection, however, it should be noted that the +levulose of nature, or that which is formed by the hydrolysis of inulin +or sucrose is not identical in its optical properties with the _l_ +fructose of Fischer. + +=46. Preparation of Pure Sugar.=—In using the polariscope or in testing +solutions for the chemical analysis of samples, the analyst will be +required to keep always on hand some pure sugar. Several methods of +preparing pure sugar have been proposed. The finest granulated sugar +of commerce is almost pure. In securing samples for examination those +should be selected which have had a minimum treatment with bluing in +manufacture. The best quality of granulated sugar when pulverized, +washed with ninety-five per cent and then with absolute alcohol and +dried over sulfuric acid at a temperature not exceeding 50° will be +found nearly pure. Such a sugar will, as a rule, not contain more +than one-tenth per cent of impurities, and can be safely used for all +analytical purposes. It is assumed in the above that the granulated +sugar is made from sugar cane. + +Granulated beet sugars may contain raffinose and so may show a +polarization in excess of 100. This sugar may be purified by dissolving +seventy parts by weight in thirty parts of water. The sugar is +precipitated by adding slowly an equal volume of ninety-six per cent +alcohol with constant stirring, the temperature of the mixture being +kept at 60°. While still warm the supernatant liquor is decanted +and the precipitated sugar washed by decantation several times with +strong warm alcohol. The sugar, on a filter, is finally washed with +absolute alcohol and dried in a thin layer over sulfuric acid at from +35° to 40°. By this process any raffinose which the sugar may have +contained is completely removed by the warm alcohol. Since beet sugar +is gradually coming into use in this country it is safer to follow the +above method with all samples.[25] In former times it was customary +to prepare pure sugar from the whitest crystals of rock candy. These +crystals are powdered, dissolved in water, filtered, precipitated with +alcohol, washed and dried in the manner described above. + +=47. Classification of Methods.=—In the quantitive determination of +pure sugar the various processes employed may all be grouped into three +classes. In the first class are included all those which deduce the +percentage of sugar present from the specific gravity of its aqueous +solution. The accuracy of this process depends on the purity of the +material, the proper control of the temperature, and the reliability +of the instruments employed. The results are obtained either directly +from the scale of the instruments employed or are calculated from the +arbitrary or specific gravity numbers observed. It is evident that +any impurity in the solution would serve to introduce an error of a +magnitude depending on the percentage of impurity and the deviation of +the density from that of sugar. The different classes of sugars, having +different densities in solution, give also different readings on the +instruments employed. It is evident, therefore, that a series of tables +of percentages corresponding to the specific gravities of the solutions +of different sugars would be necessary for exact work. Practically, +however, the sugar which is most abundant, _viz._, sucrose, may be +taken as a representative of the others and for rapid control work the +densimetric method is highly useful. + +In the second class of methods are grouped all those processes which +depend upon the property of sugar solutions to rotate the plane of +polarized light. Natural sugars all have this property and if their +solutions be found neutral to polarized light it is because they +contain sugars of opposite polarizing powers of equal intensity. Some +sugars turn the polarized plane to the right and others to the left, +and the degree of rotation in each case depends, at equal temperatures +and densities of the solutions, on the percentages of sugars present. +In order that the optical examination of a sugar may give correct +results the solution must be of a known density and free of other +bodies capable of affecting the plane of polarized light. In the +following paragraphs an attempt will be made to give in sufficient +detail the methods of practice of these different processes in so +far as they are of interest to the agricultural analyst. The number +of variations, however, in these processes is so great as to make +the attempt to fully discuss them here impracticable. The searcher +for additional details should consult the standard works on sugar +analysis.[26] + +In the third class of methods are included those which are of a +chemical nature based either on the reducing power which sugar +solutions exercise on certain metallic salts, upon the formation of +certain crystalline and insoluble compounds with other bodies or +upon fermentation. Under proper conditions solutions of sugar reduce +solutions of certain metallic salts, throwing out either the metal +itself or a low oxid thereof. In alkaline solutions of mercury and +copper, sugars exercise a reducing action, throwing out in the one case +metallic mercury and in the other cuprous oxid. With phenylhydrazin, +sugars form definite crystalline compounds, quite insoluble, which +can be collected, dried and weighed. There is a large number of other +chemical reactions with sugars such as their union with the earthy +bases, color reactions with alkalies, oxidation products with acids, +and so on, which are of great use qualitively and in technological +processes, but these are of little value in quantitive determinations. + + +THE DETERMINATION OF THE PERCENTAGE OF SUGAR BY THE DENSITY OF ITS +SOLUTION. + +=48. Principles of the Method.=—This method of analysis is applied +almost exclusively to the examination of one kind of sugar, _viz._, the +common sugar of commerce. This sugar is derived chiefly from sugar cane +and sugar beets and is known chemically as sucrose or saccharose. The +method is accurate only when applied to solutions of pure sucrose which +contain no other bodies. It is evident however, that other bodies in +solution can be determined by the same process, so that the principle +of the method is broadly applicable to the analyses of any body +whatever in a liquid state or in solution. Gases, liquids and solids, +in solution, can all be determined by densimetric methods. + +Broadly stated the principle of the method consists in determining the +specific gravity of the liquid or solution, and thereafter taking the +percentage of the body in solution from the corresponding specific +gravity in a table. These tables are carefully prepared by gravimetric +determinations of the bodies in solution of known densities, varying by +small amounts and calculation of the percentages for the intervening +increments or decrements of density. This tabulation is accomplished at +definite temperatures and the process of analysis secured thereby is +rapid and accurate, with pure or nearly pure solutions. + +=49. Determination of Density.=—While not strictly correct from a +physical point of view, the terms density and specific gravity are here +used synonymously and refer to a direct comparison of the weights of +equal volumes of pure water and of the solution in question, at the +temperature named. When not otherwise stated, the temperature of the +solution is assumed to be 15°.5. + +[Illustration: FIGURE 27. COMMON FORMS OF PYKNOMETERS.] + +The simplest method of determining the density of a solution is to +get the weight of a definite volume thereof. This is conveniently +accomplished by the use of a pyknometer. A pyknometer is any vessel +capable of holding a definite volume of a liquid in a form suited +to weighing. It may be a simple flask with a narrow neck distinctly +marked, or a flask with a ground perforated stopper, which, when +inserted, secures always the same volume of liquid contents. A very +common form of pyknometer is one in which the central stopper carries a +thermometer and the constancy of volume is secured by a side tubulure +of very small or even capillary dimensions, which is closed by a ground +glass cap. + +The apparatus may not even be of flask form, but assume a quite +different shape as in Sprengel’s tube. Pyknometers are often made to +hold an even number of cubic centimeters, but the only advantage of +this is in the ease of calculation which it secures. As a rule, it +will be found necessary to calibrate even these, and then the apparent +advantage will be easily lost. A flask which is graduated to hold fifty +cubic centimeters, may, in a few years, change its volume at least +slightly, due to molecular changes in the glass. Some of the different +forms of pyknometers are shown in the accompanying figures. + +In use the pyknometer should be filled with pure water of the desired +temperature and weighed. From the total weight the tare of the flask +and stopper, weighed clean and dry, is to be deducted. The remainder +is the weight of the volume of water of the temperature noted, which +the pyknometer holds. The weight of the solution under examination is +taken in the same way and at the same temperature, and thus a direct +comparison between the two liquids is secured. + + _Example._—Let the weight of the pyknometer be 15.2985 grams. + and its weight with pure water at 15°.5 be 26.9327 ” + Then the weight of water is 11.6342 ” + The weight filled with the sugar solution is 28.3263 ” + Then the weight of the sugar solution is 13.0278 ” + +The specific gravity of the sugar solution is therefore, 13.0278 ÷ +11.6342 = 1.1198. + +For strictly accurate results the weight must be corrected for the +volume of air displaced, or in other words, be reduced to weights in +vacuo. This however is unnecessary for the ordinary operations of +agricultural analysis. + +If the volume of the pyknometer be desired, it can be calculated from +the weight of pure water which it holds, one cubic centimeter of pure +water weighing one gram at 4°. + +The weights of one cubic centimeter of water at each degree of +temperature from 1° to 40°, are given in the following table: + + TABLE SHOWING WEIGHTS OF ONE CUBIC CENTIMETER OF + PURE WATER AT TEMPERATURES VARYING FROM 1° TO 40°. + + Weight, Weight, + Temperature. Gram. Temperature. Gram. + + 0° 0.999871 21° 0.998047 + 1° 0.999928 22° 0.997826 + 2° 0.999969 23° 0.997601 + 3° 0.999991 24° 0.997367 + 4° 1.000000 25° 0.997120 + 5° 0.999990 26° 0.996866 + 6° 0.999970 27° 0.996603 + 7° 0.999933 28° 0.998331 + 8° 0.999886 29° 0.995051 + 9° 0.999824 30° 0.995765 + 10° 0.999747 31° 0.995401 + 11° 0.999655 32° 0.995087 + 12° 0.999549 33° 0.994765 + 13° 0.999430 34° 0.994436 + 14° 0.999299 35° 0.994098 + 15° 0.999160 36° 0.993720 + 16° 0.999002 37° 0.993370 + 17° 0.998841 38° 0.993030 + 18° 0.998654 39° 0.992680 + 19° 0.998460 40° 0.992330 + 20° 0.998259 + +From the table and the weight of water found, the volume of the +pyknometer is easily calculated. + +_Example._—Let the weight of water found be 11.72892 grams, and the +temperature 20°. Then the volume of the flask is equal to 11.72892 ÷ +0.998259, _viz._, 11.95 cubic centimeters. + +=50. Use of Pyknometer at High Temperatures.=—It is often found +desirable to determine the density of a liquid at temperatures above +that of the laboratory, _e. g._, at the boiling-point of water. This is +easily accomplished by following the directions given below: + +_Weight of Flask._—Use a small pyknometer of from twenty-five to thirty +cubic centimeters capacity. The stopper should be beveled to a fine +edge on top and the lower end should be slightly concave to avoid any +trapping of air. The flask is to be thoroughly washed with hot water, +alcohol and ether, and then dried for some time at 100°. After cooling +in a desiccator the weight of the flask and stopper is accurately +determined.[27] + +[Illustration: FIGURE 28. BATH FOR PYKNOMETERS.] + +_Weight of Water._—The flask in an appropriate holder, Fig. 28, +conveniently made of galvanized iron, is filled with freshly boiled and +hot distilled water and placed in a bath of pure, very hot distilled +water, in such a way that it is entirely surrounded by the liquid with +the exception of the top. + +The water of the bath is kept in brisk ebullition for thirty minutes, +any evaporation from the flask being replaced by the addition of +boiling distilled water. The stopper should be kept for a few minutes +before use in hot distilled water and is then inserted, the flask +removed, wiped dry, and, after it is nearly cooled to room temperature, +placed in the balance and weighed when balance temperature is reached. +A convenient size of holder will enable the analyst to use eight or ten +flasks at once. The temperature at which water boils in each locality +may also be determined; but unless at very high altitudes, or on days +of unusual barometric disturbance the variations will not be great, and +will not appreciably affect the results. + +=51. Alternate Method of Estimating the Weight of Water in +Flasks.=—Formulas for calculating the volume _V_, in cubic centimeters, +of a glass vessel from the weight _P_ of water at the temperature _t_ +contained therein, and the volume _Vʹ_ at any other temperature _t’_ +are given by Landolt and Börnstein.[28] They are as follows: + + _p_ + _V_ = _P_ --- + _d_ + + _p_ + _Vʹ_ = _P_ ---- [1 + _γ_(_tʹ_- _t_)] + _d_ + +_p_ = weight (in brass weights) of one cubic centimeter H₂O in vacuo. +This is so nearly one gram that it will not affect the result in the +fifth place of decimals and may therefore be disregarded. Hence the +formula stands: + + 1 + _Vʹ_ = _P_ ---- [1 + _γ_(_tʹ_-_t_)]; in which + _d_ + +_d_ = density of water at temperature _t_. + +_γ_ = 0.000025, the cubical expansion coefficient of glass. + +From this volume the weight of the water may be readily obtained by +referring to tables 13, 14 and 15_a_ in Landolt and Börnstein’s book. + +=52. Example Showing Determination of Specific Gravity of a Fat.=—The +flask is emptied of its water, rinsed with alcohol and ether, and dried +again for a few minutes at 100°. It is then filled with the dry, hot, +fresh-filtered fat, which should be entirely free from air bubbles. + +The stoppered flask is then replaced in the water-bath, kept for thirty +minutes at the temperature of boiling water, removed, and treated as +above. The weight of fat having been determined, the specific gravity +is obtained by dividing it by the weight of water previously found. + + _Example._ + Grams. + Weight of flask, dry 10.0197 + Weight of flask, plus water 37.3412 + Weight of water 27.3215 + Weight of flask, plus fat 34.6111 + Weight of fat 24.5914 + + Specific gravity = 24.5914 ÷ 27.3215 = 0.90008. + +The weight of the flask dry and empty and the weight of water at 99° to +100° contained therein may be used constantly if great care be taken in +handling and cleaning the apparatus. + + _Example._ + Grams. + Weight of flask, dry and empty 10.0028 + Weight of flask after three weeks’ use 10.0030 + +[Illustration: FIGURE 29. AEREOMETERS, PYKNOMETERS, AND HYDROSTATIC +BALANCE.] + +=53. Determination of Density by the Hydrostatic Balance.=—While +the pyknometer is useful in control work and in fixing standards of +comparison, it is not used extensively in practical work. Quicker +methods of determination are desired in such work, and these are +found in the use of other forms of apparatus. A convenient method of +operation consists in determining the weight of a sinker, whose exact +weights in air and in pure water of a definite temperature, have been +previously determined. The instrument devised by Mohr and modified by +Westphal, is based upon that principle, and is extensively used in +practical work. The construction of this apparatus and also that of the +pyknometers and areometers is shown in the illustrations, figures 29 +and 30. + +[Illustration: FIGURE 30. HYDROSTATIC BALANCE.] + +The weight of the sinker is so adjusted that the index of the balance +arm marks zero when the sinker is wholly immersed in pure water at +the standard temperature. The density of a solution of sugar at the +same temperature, is then determined by placing the rider-weights on +the divided arm of the balance, until the index again marks zero. The +density can then be read directly from the position of the weights in +the arm of the balance or calculated therefrom. + +=54. The Areometric Method.=—The most rapid method of determining +the density of a solution and the one in most common use, is based +on the distance to which a heavy bulb with a slender graduated stem +will sink therein. An instrument of this kind is called an areometer. +Many forms of this instrument are employed but they all depend on the +same principle and differ only in the manner of graduation. The one of +widest application has the stem graduated in such a manner as to give +directly the specific gravity of the solution in which it is placed. + +Others are made with a special graduation giving directly the +percentage of solid matter in the solution. These instruments can be +used only for the special purposes for which they are constructed. +Other forms are provided with an arbitrary graduation, the numbers +of which by appropriate tables can be converted into expressions of +specific gravity or of per cents of dissolved matters. It is not +practicable to give here, a discussion of the principles of the +construction of areometers.[29] The two which are commonly used, are the +baumé hydrometer and the balling or brix spindle. + +In the baumé instrument the zero of the scale is fixed at the point +marked by the surface of distilled water at 15°, and the point to which +it sinks in pure monohydrated sulfuric acid at the same temperature is +marked 66, corresponding to a specific gravity of 1.8427. + +The specific gravity corresponding to any degree of the scale, may be +calculated in the absence of a table giving it, by the following formula + + 144.3 + _P_ = -----------. + 144.3 - _d_ + +In this formula _P_ is the density and _d_ the degree of the scale.[30] +In former times the baumé instruments were graduated with a solution +of common salt and a different formula was employed for calculating +specific gravity, but these older instruments are no longer in common +use. + +The following table shows the specific gravities of solutions +corresponding to baumé degrees from 1° to 75° consecutively[31]: + + Degree Specific Degree Specific Degree Specific Degree Specific + baumé gravity baumé gravity baumé gravity baumé gravity + 0 1.0000 19 1.1516 38 1.3574 57 1.6527 + 1 1.0069 20 1.1608 39 1.3703 58 1.6719 + 2 1.0140 21 1.1702 40 1.3834 59 1.6915 + 3 1.0212 22 1.1798 41 1.3968 60 1.7115 + 4 1.0285 23 1.1895 42 1.4104 61 1.7321 + 5 1.0358 24 1.1994 43 1.4244 62 1.7531 + 6 1.0433 25 1.2095 44 1.4386 63 1.7748 + 7 1.0509 26 1.2197 45 1.4530 64 1.7968 + 8 1.0586 27 1.2301 46 1.4678 65 1.8194 + 9 1.0665 28 1.2407 47 1.4829 66 1.8427 + 10 1.0744 29 1.2514 48 1.4983 67 1.8665 + 11 1.0825 30 1.2624 49 1.5140 68 1.8909 + 12 1.0906 31 1.2735 50 1.5301 69 1.9161 + 13 1.0989 32 1.2849 51 1.5465 70 1.9418 + 14 1.1074 33 1.2964 52 1.5632 71 1.9683 + 15 1.1159 34 1.3081 53 1.5802 72 1.9955 + 16 1.1246 35 1.3201 54 1.5978 73 2.0235 + 17 1.1335 36 1.3323 55 1.6157 74 2.0523 + 18 1.1424 37 1.3447 56 1.6340 75 2.0819 + +=55. Correction for Temperature.=—The baumé hydrometer should be used +at the temperature for which it is graduated, usually 15°. In this +country the mean temperature of our working rooms is above 15°. The +liquid in the hydrometer flask should therefore be cooled to a trifle +below 15°, or kept in a bath exactly at 15° while the observation is +made. When this is not convenient, the observation may be made at any +temperature, and the reading corrected as follows: When the temperature +is above 15° multiply the difference between the observed temperature +and fifteen, by 0.0471 and add the product to the observed reading of +the baumé hydrometer; when the temperature on the other hand, is below +fifteen, the corresponding product is subtracted.[32] + +=56. The Balling or Brix Hydrometer.=—The object of the balling or +brix instrument is to give in direct percentages the solid matter in +solution. It is evident that for this purpose the instrument must be +graduated for a particular kind of material, since ten per cent of +sugar in solution, might have a very different specific gravity from a +similar quantity of another body. Instruments of this kind graduated +for pure sugar, find a large use in technical sugar analysis. To +attain a greater accuracy and avoid an instrument with too long a +stem, the brix hydrometers are made in sets. A convenient arrangement +is to have a set of three graduated as follows; one from 0° to 30°, one +from 25° to 50°, and one from 45° to 85°. When the percentage of solid +matter dissolved is over seventy the readings of the scale are not very +reliable. + +=57. Correction for Temperature.=—The brix as the baumé scale is +graduated at a fixed temperature. This temperature is usually 17°.5. +The following table shows the corrections to be applied to the scale +reading when made at any other temperature:[33] + + PER CENT OF SUGAR IN SOLUTION. + + 0. 5. 10. 15. 20. 25. 30. 35. 40. 50. 60. 70. 75. + Temp. _To be subtracted from the degree read._ + 0° 0.17 0.30 0.41 0.52 0.62 0.72 0.82 0.92 0.98 1.11 1.22 1.25 1.29 + 5° 0.23 0.30 0.37 0.44 0.52 0.59 0.65 0.72 0.75 0.80 0.88 0.91 0.94 + 10° 0.20 0.26 0.29 0.33 0.36 0.39 0.42 0.45 0.48 0.50 0.54 0.58 0.61 + 11° 0.18 0.23 0.26 0.28 0.31 0.34 0.36 0.39 0.41 0.43 0.47 0.50 0.53 + 12° 0.16 0.20 0.22 0.24 0.26 0.29 0.31 0.33 0.34 0.36 0.40 0.42 0.46 + 13° 0.14 0.18 0.19 0.21 0.22 0.24 0.26 0.27 0.28 0.29 0.33 0.35 0.39 + 14° 0.12 0.15 0.16 0.17 0.18 0.19 0.21 0.22 0.22 0.23 0.26 0.28 0.32 + 15° 0.09 0.11 0.12 0.14 0.14 0.15 0.16 0.16 0.17 0.17 0.19 0.21 0.25 + 16° 0.06 0.07 0.08 0.09 0.10 0.10 0.11 0.12 0.12 0.12 0.14 0.16 0.18 + 17° 0.02 0.02 0.03 0.03 0.03 0.04 0.04 0.04 0.04 0.04 0.05 0.05 0.06 + + _To be added to the degree read._ + 18° 0.02 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.02 + 19° 0.06 0.08 0.08 0.09 0.09 0.10 0.10 0.10 0.10 0.10 0.10 0.08 0.06 + 20° 0.11 0.14 0.15 0.17 0.17 0.18 0.18 0.18 0.19 0.19 0.18 0.15 0.11 + 21° 0.16 0.20 0.22 0.24 0.24 0.25 0.25 0.25 0.26 0.26 0.25 0.22 0.18 + 22° 0.21 0.26 0.28 0.31 0.31 0.32 0.32 0.32 0.33 0.34 0.32 0.29 0.25 + 23° 0.27 0.32 0.35 0.37 0.38 0.39 0.39 0.39 0.40 0.42 0.39 0.36 0.33 + 24° 0.32 0.38 0.41 0.43 0.44 0.46 0.46 0.47 0.47 0.50 0.46 0.43 0.40 + 25° 0.37 0.44 0.47 0.49 0.51 0.53 0.54 0.55 0.55 0.58 0.54 0.51 0.48 + 26° 0.43 0.50 0.54 0.56 0.58 0.60 0.61 0.62 0.62 0.66 0.62 0.58 0.55 + 27° 0.49 0.57 0.61 0.63 0.65 0.68 0.68 0.69 0.70 0.74 0.70 0.65 0.62 + 28° 0.56 0.64 0.68 0.70 0.72 0.76 0.76 0.78 0.78 0.82 0.78 0.72 0.70 + 29° 0.63 0.71 0.75 0.78 0.79 0.84 0.84 0.86 0.86 0.90 0.88 0.80 0.78 + 30° 0.70 0.78 0.82 0.87 0.87 0.92 0.92 0.94 0.94 0.98 0.94 0.88 0.86 + 35° 1.10 1.17 1.22 1.24 1.30 1.32 1.33 1.35 1.36 1.39 1.34 1.27 1.25 + 40° 1.50 1.61 1.67 1.71 1.73 1.79 1.79 1.80 1.82 1.83 1.78 1.69 1.65 + 50° ---- 2.65 2.71 2.74 2.78 2.80 2.80 2.80 2.80 2.79 2.70 2.56 2.51 + 60° ---- 3.87 3.88 3.88 3.88 3.88 3.88 3.88 3.90 3.82 3.70 3.43 3.41 + 70° ---- ---- 5.18 5.20 5.14 5.13 5.10 5.08 5.06 4.90 4.72 4.47 4.35 + 80° ---- ---- 6.62 6.59 6.54 6.16 6.38 6.30 6.26 6.06 5.82 5.50 5.33 + +According to observations of Gerlach, the correction for temperature +varies with the concentration of the solution and the range of +temperature as shown in the table. + +=58. Comparison of Brix and Baumé Degrees.=—The following table +shows the degree baumé and the specific gravity of a sugar solution +for each degree brix (per cent of sugar in solution) from zero to +ninety-five:[34] + + Degree Degree Specific Degree Degree Specific + brix. baumé. gravity. brix. baumé. gravity. + 1.0 0.6 1.00388 37.0 20.7 1.16413 + 2.0 1.1 1.00779 38.0 21.2 1.16920 + 3.0 1.7 1.01173 39.0 21.8 1.17430 + 4.0 2.3 1.01570 40.0 22.3 1.17943 + 5.0 2.8 1.01970 41.0 22.9 1.18460 + 6.0 3.4 1.02373 42.0 23.4 1.18981 + 7.0 4.0 1.02779 43.0 24.0 1.19505 + 8.0 4.5 1.03187 44.0 24.5 1.20033 + 9.0 5.1 1.03599 45.0 25.0 1.20565 + 10.0 5.7 1.04014 46.0 25.6 1.21100 + 11.0 6.2 1.04431 47.0 26.1 1.21639 + 12.0 6.8 1.04852 48.0 26.6 1.22182 + 13.0 7.4 1.05276 49.0 27.2 1.22128 + 14.0 7.9 1.05703 50.0 27.7 1.23278 + 15.0 8.5 1.06133 51.0 28.2 1.23832 + 16.0 9.0 1.06566 52.0 28.8 1.24390 + 17.0 9.6 1.07002 53.0 29.3 1.24951 + 18.0 10.1 1.07441 54.0 29.8 1.25517 + 19.0 10.7 1.07884 55.0 30.4 1.26086 + 20.0 11.3 1.08329 56.0 30.9 1.26658 + 21.0 11.8 1.08778 57.0 31.4 1.27235 + 22.0 12.4 1.09231 58.0 31.9 1.27816 + 23.0 13.0 1.09686 59.0 32.5 1.28400 + 24.0 13.5 1.10145 60.0 33.0 1.28989 + 25.0 14.1 1.10607 61.0 33.5 1.29581 + 26.0 14.6 1.11072 62.0 34.0 1.30177 + 27.0 15.2 1.11541 63.0 34.5 1.30177 + 28.0 15.7 1.12013 64.0 35.1 1.31381 + 29.0 16.3 1.12488 65.0 35.6 1.31989 + 30.0 16.8 1.12967 66.0 36.1 1.32601 + 31.0 17.4 1.13449 67.0 36.6 1.33217 + 32.0 18.0 1.13934 68.0 37.1 1.33836 + 33.0 18.5 1.14423 69.0 37.6 1.34460 + 34.0 19.1 1.14915 70.0 38.1 1.35088 + 35.0 19.6 1.15411 71.0 38.6 1.35720 + 36.0 20.1 1.15911 72.0 39.1 1.36355 + 73.0 39.6 1.36995 85.0 45.5 1.44986 + 74.0 40.1 1.37639 86.0 46.0 1.45678 + 75.0 40.6 1.38287 87.0 46.5 1.46374 + 76.0 41.1 1.38939 88.0 47.0 1.47074 + 77.0 41.6 1.39595 89.0 47.5 1.47778 + 78.0 42.1 1.40254 90.0 49.9 1.48486 + 79.0 42.6 1.40918 91.0 48.4 1.49199 + 80.0 43.1 1.41586 92.0 48.9 1.49915 + 81.0 43.6 1.42258 93.0 49.3 1.50635 + 82.0 44.1 1.42934 94.0 49.8 1.51359 + 83.0 44.6 1.43614 95.0 50.3 1.52087 + 84.0 45.1 1.44298 + +=59. Error Due to Impurities.=—The fact that equal per cents of solid +bodies in solution affect the specific gravity in different degrees +has already been noted. The specific gravities of the solutions of the +common sugars, however, are so nearly the same for equal per cents of +solid matter in solution as to render the use of a brix hydrometer +quite general for technical purpose. For the mineral salts which often +occur in sugar solutions the case is quite different. A twenty per cent +solution of cane sugar at 17°.5 has a specific gravity 1.08329 and of +dextrose 1.08310, practically identical. But a solution of calcium +acetate of similar strength has a specific gravity of 1.0874; of sodium +sulfate 1.0807, and of potassium nitrate 1.1359. This latter number +would correspond to a sugar content of nearly twenty-seven per cent. +The brix scale can, therefore, be regarded as giving only approximately +the percentage of solid matter in sugar solutions and, while useful in +technical work, should never be relied upon for exact analytical data. + + +THE DETERMINATION OF SUGAR WITH POLARIZED LIGHT. + +=60. Optical Properties of Natural Sugars.=—The solutions of all +natural sugars have the property of deflecting the plane of polarized +light and the degree of deflection corresponds to the quantity of sugar +in solution. By measuring the amplitude of the rotation produced the +percentage of sugar in the solution can be determined. In order to +secure accuracy in the determinations it is necessary that only one +kind of sugar be present, or, if more than one, that the quantities +of all but one be determined by other means, and the disturbances +produced thereby in the total rotation be properly arranged. In point +of fact the process in practice is applied chiefly to cane and milk +sugars, both of which occur in nature in an approximately pure state. +The process is also useful in determining cane sugar when mixed with +other kinds, by reason of the fact that this sugar after hydrolysis +by treatment with a weak acid for a long or a strong acid for a short +time, definitely changes its rotating power. Since, by the same +treatment, the rotating power of other sugars which may be present is +only slightly altered, the total disturbance produced is approximately +due to the inversion of the cane sugar. + +Dextrose and maltose arising from the hydrolysis of starch may also +be determined with a fair degree of accuracy by their deportment +with polarized light. When a solution of natural sugars shows +negative results when examined with polarized light, it is due to an +admixture of two or more sugars of opposite polarizing powers in such +proportions as to produce neutrality. This condition often occurs +in the examination of honeys or in submitting artificial sugars to +polarimetric observations. In the latter case the neutrality is caused +by the tendency manifested by artificially produced sugars to form twin +compounds of optically opposite qualities. + +The instrument used for measuring the degree of deflection produced in +a plane of polarized light is called a polariscope, polarimeter, or +optical saccharimeter. For a theoretical discussion of the principles +of polarization and the application of these principles in the +construction of polariscopes, the reader is referred to the standard +works on optics and the construction of optical instruments.[35] For +the purposes of this work a description of the instruments commonly +employed and the methods of using them will be sufficient. + +=61. Polarized Light.=—When a ray of light has been repeatedly +reflected from bright surfaces or when it passes through certain +crystalline bodies it acquires peculiar properties and is said to be +polarized. + +Polarization is therefore a term applied to a phenomenon of light, in +which the vibrations of the ether are supposed to be restricted to a +particular form of an ellipse whose axes remain fixed in direction. If +the ellipse become a straight line it is called plane polarization. +This well-known phenomenon is most easily produced by a nicol prism, +consisting of a cut crystal of calcium carbonate (Iceland spar). This +rhombohedral crystal, the natural ends of which form angles of 71° and +109°, respectively, with the opposite edges of its principal section, +is prepared as follows: + +The ends of the crystals are ground until the angles just mentioned +become 68° and 112°. The crystal is then divided diagonally at right +angles with the planes of the ends and with the principal section, and +after the new surfaces are polished they are joined again by canada +balsam. The principal section of this prism passes through the shorter +diagonal of the two rhombic ends. If now a ray of light fall on one +of the ends of this prism, parallel with the edge of its longer side, +it suffers double refraction, and each ray is plane polarized, the +one at right angles with the other. That part of the entering ray of +light which is most refracted is called the ordinary and the other the +extraordinary ray. The refractive index of the film of balsam being +intermediate between those of the rays, permits the total reflection of +the ordinary ray, which, passing to the blackened sides of the prism, +is absorbed. The extraordinary ray passes the film of balsam without +deviation and emerges from the prism in a direction parallel with the +incident ray, having, however, only half of its luminous intensity. + +Two such prisms, properly mounted, furnish the essential parts of a +polarizing apparatus. They are called the polarizer and the analyzer, +respectively. + +If now the plane of vibration in each prism be regarded as coincident +with its principal section, the following phenomena are observed: +If the prisms are so placed that the principal sections lie in the +prolongation of the same plane, then the extraordinary polarized ray +from the polarizer passes into the analyzer, which practically may +be regarded in this position as a continuation of the same prism. +It happens, therefore, that the extraordinary polarized ray passes +through the analyzer exactly as it did through the polarizer, and is +not reflected by the film of balsam, but emerges from the analyzer +in seemingly the same condition as from the polarizer. If now the +analyzer be rotated 180°, bringing the principal section again in the +same plane, the same phenomenon is observed. But if the rotation be in +either direction only 90°, then the polarized ray from the first prism, +incident on the second, deports itself exactly as the ordinary ray, +and on meeting the film of balsam is totally reflected. The field of +vision, therefore, is perfectly dark. + +In all other inclinations of the planes of the principal sections of +the two prisms the ray incident in the analyzer is separated into +two, an ordinary and extraordinary, varying in luminous intensity in +proportion to the square of the cosine of the angle of the two planes. + +[Illustration: FIGURE 31. COURSE OF RAYS OF LIGHT IN A NICOL.] + +Thus, by gradually turning the analyzer, the field of vision passes +slowly from maximum luminosity to complete obscurity. The expression +crossed nicols refers to the latter condition of the field of vision. + +=62. Description of the Prism.=—In a nicol made as described above, +Fig. 31, suppose a ray of light parallel with the longer side of the +prism be incident to the end _a_ _b_ at _m_. By the double refracting +power of the spar the ray is divided into two, which traverse the first +half of the prism. The two rays are polarized at right angles to one +another. The less refracted ray when it strikes the film of Canada +balsam passes through it without interference. The more refracted ray +strikes the balsam at _o_ at such an angle as to be totally reflected +and made to pass out of the prism in the direction _o r_. If the prism +be blackened at the surface the ray will be entirely absorbed. The +other ray passes on through the other half of the prism and emerges in +the direction of _qs_. It is evident that the emergent light from a +nicol has only half the illuminating power possessed by the immergent +rays. + +The polarized plane of light from the nicol just described may be +regarded as passing also into a second nicol of essentially the same +construction as the first. + +This second nicol, called the analyzer, is so constructed as to +revolve freely about its longitudinal axis, and is attached to a +graduated circle in such a way that the degree of rotation can be +accurately read. If the planes of polarization of the two nicols are +coincident when prolonged, the ray of light passing from the first +nicol will pass through the second practically unchanged in character +or intensity. If, however, the analyzing nicol be turned until the +plane of polarization is at right angles to that of the polarizer the +immergent ray will suffer refraction in such a manner as to be totally +reflected when reaching the film of balsam and will be thus entirely +lost. In making a complete revolution of the analyzer, therefore, two +positions of maximum intensity of light and two of darkness will be +observed. In intermediate positions the ray immergent to the analyzer +will be separated as in the first instance into two rays _g p_ varying +intensities, one of which will be always totally reflected. + +[Illustration: FIGURE 32. THEORY OF THE NICOL.] + +In Fig. 32 is given a more detailed illustration of the action of the +rays of light. The film of balsam is represented as enlarged and of +the thickness _bb_. Draw the perpendiculars represented by the dotted +lines _n_₁ _n_ʹ₁, _n_₂ _n_ʹ₂, _n_₃ _n_ʹ₃ and _n_₄ _n_ʹ₄. In passing +into the prism at _m_ both refracted rays are bent towards the normal +_m n_ʹ₁. The degree of deflection depends on the refractive index of +the two rays 1.52 and 1.66 respectively. The refractive index of the +extraordinary ray in calcspar being 1.52, and in Canada balsam 1.54, +it suffers but little disturbance in passing from one to the other. +On the other hand the balsam, being considerably less refractive +for the ordinary ray than the calcspar, causes that ray to diverge +outwards from the normal _o n_ʹ₂, and to such a degree as to suffer +total reflection. The critical angle, that is the angle at which a ray +issuing from a more refractive into a less refractive medium, emerges +just parallel to the bounding surfaces, depends on the relative index +of refraction. In the case under consideration the ratio for balsam +and spar is 1.54/1.66 = 0.928 = sin 68°. Therefore the limiting value +of _m o_ _n_₃ so that _m o_ may just emerge in the direction _od_ is +68°. If now _mo_ were parallel to _o d_ the angle _m o n_, would be +just 68°, being opposite _b a d_. which has been ground to 68° in the +construction of the prism. But in passing into the prism, _m o_ is +refracted so that the angle _m o n_₃ is greater than _b a d_. It is +therefore always certain that by grinding _b a d_ to 68° the ordinary +ray _m o_ will be with certainty entirely thrown out in every case. In +respect of the analyzing nicol the following additional observations +will be found useful. In all uniaxial crystals there are two directions +at right angles to each other, one of greatest and one of least +resistance to the propagation of luminous vibrations. These planes are +in the direction of the principal axis and at right angles thereto. +Only light vibrating in these two directions can be transmitted through +calcspar; and all incident light propagated by vibrations in a plane +at any other angle to the principal section is resolved into two +such component rays. But the velocities of transmission in the two +directions are unequal, that is, the refractive index of the spar for +the two rays is different. If the analyzing nicol be so adjusted as to +receive the emergent light from the polarizer when the corresponding +planes of the two prisms are coincident when extended, the emergent +extraordinary ray falling into a plane of the same resistance as +that it had just left is propagated through the second nicol with +the same velocity that it passed the first one. It is therefore +similarly refracted. If, however, the two prisms be so arranged that +corresponding planes cross then the extraordinary ray falls into a +plane which it traverses with greater velocity than it had before and +is accordingly refracted and takes the course which ends in total +reflection at the film of balsam. No light therefore can pass through +the prism in that position. If any other substance, as for instance +a solution of sugar, capable of rotating a plane of polarized light, +be interposed between the two nicols the effect produced is the same +as if the analyzer had been turned to a corresponding degree. When +the analyzer is turned to that degree the corresponding planes again +coincide and the light passes. This is the principle on which the +construction of all polarizing instruments is based.[36] + +=63. The Polariscope.=—A polariscope for the examination of solutions +of sugar consists essentially of a prism for polarizing the light, +called a nicol, a tube of definite length for holding the sugar +solution, a second nicol made movable on its axis for adjustment to +the degree of rotation and a graduated arc for measuring it. Instead +of having the second nicol movable, many instruments have an adjusting +wedge of quartz of opposite polarizing power to the sugar, by means of +which the displacement produced on the polarized plane is corrected. +A graduated scale and vernier serve to measure the movement of the +wedges and give in certain conditions the desired reading of the +percentage of sugar present. Among the multitude of instruments which +have been devised for analytical purposes, only three will be found +in common use, and the scope of this volume will not allow space +for a description of a greater number. For a practical discussion +of the principles of polarization and their application to optical +saccharimetry, the reader may conveniently refer to the excellent +manuals of Sidersky, Tucker, Landolt, and Wiechmann.[37] + +=64. Kinds of Polariscopes.=—The simplest form of a polarizing +apparatus consists of two nicol prisms, one of which, _viz._, the +analyzer, is capable of rotation about its long axis. The prolongation +of this axis is continuous with that of the other prism, _viz._, the +polarizer. The two prisms are sufficiently removed from each other to +allow of the interposition of the polarizing body whose rotatory power +is to be measured. + +For purposes of description three kinds of polarimeters may be +mentioned. + +1. _Instruments in which the deviation of the plane of polarization is +measured by turning the analyzer about its axis._ + +Instruments of this kind conform to the simple type first +mentioned, and are _coeteris paribus_ the best. The Laurent, Wild, +Landolt-Lippich, etc., belong to this class. + +2. _Instruments in which both nicols are fixed and the direction of the +plane of polarized light corrected by the interposition of a wedge of a +solid polarizing body._ + +Belonging to this class are the apparatus of Soleil, Duboscq, +Scheibler, and the compensating apparatus of Schmidt and Haensch. + +3. _Apparatus in which the analyzer is set at a constant angle with +the polarizer, and the compensation secured by varying the length or +concentration of the interposed polarizing liquid._ + +The apparatus of Trannin belongs to this class. + +=65. Appearance of Field of Vision.=—Polarimeters are also classified +in respect of the appearance of the field of vision. + +1. _Tint Instruments._—The field of vision in these instruments in +every position of the nicols, except that on which the plane of +vibration of the polarized light is coincident with the three principal +sections, is composed of two semi-disks of different colors. + +2. _Shadow Instruments._—The field of vision in this class of +polarimeters in all except neutral positions, is composed of two +semi-disks, one dark and one yellow. As the neutral position is +approximated the two disks gradually assume a light yellow color, and +when neutrality is reached they appear to be equally colored. + +The Laurent, Schmidt and Haensch shadow and Landolt-Lippich +instruments, are of this class. + +3. _Striated Instruments._—In this class the field of vision is +striated. The lines may be tinted as in Wild’s polaristrobometer or +black, as in the Duboscq and Trannin instruments. The neutral position +is indicated either by the disappearance of the striae (Wild) or by the +phenomenon of their becoming continuous. (Duboscq, Trannin.) + +=66. Character of Light Used.=—Polariscopes may be further divided into +two classes, based on the kind of light employed. + +1. _Instruments which Use Ordinary White Light._—(Oil lamp, etc.) +Scheibler, Schmidt and Haensch. + +2. _Instruments Employing Monochromatic Light._—(Sodium flame, etc.) +Laurent, Landolt-Lippich, etc. + +=67. Interchangeable Instruments.=—Some of the instruments in common +use are arranged to be used either with ordinary lamp or gas light, or +with a monochromatic flame. Laurent’s polarimeter is one of this kind. +The compensating instruments also may have the field of vision arranged +for tints or shadows. Theoretically the best instrument would be one in +which the light is purely monochromatic, the field of vision a shadow, +and the compensation secured by the rotation of the second nicol. + +The accuracy of an instrument depends, however, on the skill and care +with which it is constructed and used. With quartz wedges properly +ground and mounted, and with ordinary white light, polariscopes may be +obtained which give readings as accurate as can be desired. + +Since many persons are more or less affected with color-blindness, the +shadow are to be preferred to the tint fields of vision. + +For practical use in sugar analysis the white light is much more +convenient than the monochromatic light. + +For purposes of general investigation the polarimeters built on +the model of the laurent are to be preferred to all others. Such +instruments are not only provided with a scale which shows the +percentage of sucrose in a solution, but also with a scale and vernier +by means of which the angular rotation which the plane of vibration has +suffered, can be accurately measured in more than one-quarter of the +circle. + + +DESCRIPTION OF POLARIZING INSTRUMENTS. + +=68. Rotation Instruments.=—This instrument has already been described +as one in which the extent of deviation in the plane of polarized light +caused by the intervention of an optically active substance is measured +by rotating one of the nicols about its axis and measuring the degree +of this rotation by a vernier on a graduated arc. + +In Germany these instruments are called _polaristrobometers_, and +in France _polarimètrés_. In England and this country the term +_polariscope_ or _polarimeter_ is applied without discrimination to all +kinds of optical saccharimeters. + +The polariscope of Mitscherlich was one of the earliest in use. It has +now been entirely superseded by more modern and accurate instruments. + +=69. The Laurent Instrument.=—A polariscope adapted by Laurent to +the use of monochromatic yellow light is almost exclusively used in +France and to a considerable extent in this country. In case a worker +is confined to the use of a single instrument, the one just mentioned +is to be recommended as the best suited to general work. It has the +second nicol, called the analyzer, movable and the degree of rotation +produced is secured in angular terms directly on a divided circle. The +scale is graduated both in angular measurements and in per cents of +sugar for a definite degree of concentration of the solution and length +of observation tube. The normal solution in the laurent instrument +contains 16.19 grams of pure sugar in 100 true cubic centimeters, and +the length of the observation tube is 200 millimeters. Both the angular +rotation and the direct percentage of sugar can be read at the same +time. Great accuracy can be secured by making the readings in each +of the four quadrants. The light is rendered yellow monochromatic by +bringing into the flames of a double bunsen, spoons made of platinum +wire, which carry fragments of fused sodium chlorid. + +[Illustration: FIGURE 33. LAURENT LAMP.] + +=70. The Laurent Burner.=—The theory of the illumination of the laurent +burner is illustrated by the accompanying Fig. 33. The lamp consists +essentially of two bunsens, surmounted by a chimney.[38] A curved +spoon made of platinum gauze serves to hold the fused particles of +sodium chlorid which are used to produce the yellow light. The spoon +is shown at G, held by the arm F, fastened by the key P. The interior +intense flame B B is surrounded by an exterior less highly colored +flame A A. It is important that the optical axis of the polariscope +be directed accurately upon the disk B, which is the most intense +part of the illumination. The point of the spoon carrying the salt +should be coincident with the prolongation of the lamp TT, so that it +just strikes the edge of the blue flame. Care should be taken not to +press the spoons into the interior of the flame as by so doing the +intensity of the illumination is very much diminished. Great care must +be observed in the position of the spoon G, and the platinum arm F +being flexible, the operator with a little patience, will be enabled +to properly place the spoon by bending it. Moreover, if the spoon be +pressed too far into the flame, the melted particles of salt collecting +in the bottom of G may drop into the lamp and occlude the orifices +through which the gas enters. The light of the yellow flame produced by +the lamp may be further purified by passing through a prism filled with +a solution of potassium dichromate, or better, a homogeneous disk cut +from a crystal of that salt. + +Since the flame produced by the above device is not perfectly constant, +being more intense at the moment of introducing a fresh portion of +the fused salt, the author has used a lamp designed to furnish an +absolutely constant flame.[39] This device which is shown in Fig. 34, +is based on the principle of adding constantly a fresh portion of the +salt to the flame. The flame is thus kept perfectly uniform in its +intensity. + +The lamp consists essentially of two wheels with platinum gauze +perimeters and platinum wire spokes, driven by a clock-work D, and +mounted by the supports AAʹ as shown in the figure. The sodium salt, +chlorid or bromid, in dilute solution, is placed in the porcelain +crucibles F, supported by BBʹ as indicated in the figure, to such a +depth that the rims of the platinum wheels dip beneath the surface as +they revolve. The salt is volatilized by the lamp E. By means of the +crossed bands the wheels are made to revolve in opposite directions +as indicated by the arrows. The solution of the salt which is taken +up by the platinum net-work of the rim of the wheel, thus has time +to become perfectly dry before it enters the flame and the sputtering +which a moist salt would produce is avoided. At every instant, by this +arrangement, a minute fresh portion of salt is introduced into the +flame with the result of making a perfectly uniform light which can +be used for hours without any perceptible variation. The mechanism of +the apparatus is so simple that no further description is necessary. +The polariscope should be so directed toward the flame as to bring +into the field of vision its most luminous part. The platinum wheels +are adjustable and should be so arranged as to produce between them an +unbroken yellow flame. The wheels are eight centimeters in diameter and +are driven at a rate to make one revolution in from six to ten minutes. + +[Illustration: FIGURE 34. LAMP FOR PRODUCING CONSTANT MONOCHROMATIC +FLAME.] + +=71. Construction of Laurent’s Apparatus.=—The shadow polariscope +invented by Laurent is constructed as follows: The polarizer is a +special nicol which is not fixed in its position, but is so arranged +as to be turned through a small arc about its axis. By this device, +the quantity of light passing through it can be regulated, and the +apparatus is thus useful with colored solutions which are not easily +cleared by any of the common bleaching agents. The greater the quantity +of light admitted, however, the less delicate is the reading of the +shadow produced. The plane of polarized light emergent from this prism, +falls on a disk of glass half covered by a thin lamina of quartz which +thus divides the field of vision into halves. It is this semi-disk of +quartz which is the distinguishing feature of the apparatus.[40] The +polarized light thus passes without hindrance the half field of vision +which is covered by the glass only, but can not pass the quartz plate +unless its axis is set in a certain way. The field of vision may be +thus half dark, or both halves may be equally illuminated or equally +dark according to the position of the nicol analyzer which is freely +movable about its axis and carries a vernier and reading glass over +a graduated circle. The field of vision in the laurent may have any +of the following forms.[41] Let the polarizer be first so adjusted +that the plane of polarization of the transmitted pencil of light is +parallel to the axis of the plate lying in the direction A B. The two +halves of the field of vision will then appear equally illuminated in +every position of the analyzer. But if the polarizing nicol be inclined +to AB at an angle a, the plane of polarization of the rays passing +through the quartz plate will undergo deviation through an equal angle +in the opposite direction. + +[Illustration: FIGURE 35. FIELD OF VISION OF A LAURENT POLARISCOPE.] + +It happens from this, that when in the uncovered half of the field, the +plane of polarization has the direction AC, in the other half it will +have the direction ACʹ. When the analyzer is rotated, if its plane of +polarization lie in the direction cc, the rays polarized parallel to AC +will be completely extinguished and the corresponding half of the field +will be dark. The opposite happens when the plane of polarization lies +in the direction of cʹcʹ. When one-half of the field is thus obscured, +the other suffers only a partial diminution in the intensity of its +illumination. When the middle position bb is reached in the rotation of +the analyzer, the illumination of the two halves is uniform, and this +is the point at which the zero of the scale is reached. The slightest +rotation of the analyzer to the right or left of this neutral point +will cause a shadow to appear on one of the halves of the field, which +by an oscillatory movement of the analyzer, seems to leap from side +to side. The smaller the angle _a_ or BAC, the more delicate will be +the shading and the more accurate the observation. This angle being +adjustable by the mechanism already described, should be made as small +as will permit the admission of the quantity of light requisite for +accurate observation. + +The various pieces composing the polariscope are arranged in the +following positions, beginning on the right of Fig. 36, and passing to +the left, where the observer is seated.[42] + +1. The lamp VV, TT, AA, or the wheel burner: + +2. The lens B for condensing the rays and rendering them parallel: + +3. The tube I, blackened inside to carry the lens: + +4. A thin lamina E, cut from a crystal of potassium bichromate, serving +to render the sodium light more monochromatic: When the saccharine +liquids under examination are colored the crystal of bichromate is +removed before the observation is made. + +5. The polarizer R, which is rotatable through a small angle by the +lever K: + +6. The lever JK for rotating the tube containing the polarizer: This is +operated by the rod X extending to the left. + +7. Diaphragm D, half covered with a lamina of quartz. + +8. Trough L for holding the observation tube: In the large instrument +shown in the figure, it is more than half a meter in length and +arranged to hold an observation tube 500 millimeters long. + +9. Disk C, carrying divided circle and arbitrary sugar scale: + +10. Mirror M, to throw the light of the lamp on the vernier of the +scale: + +[Illustration: FIGURE 36. LAURENT POLARISCOPE.] + +11. Reading glass N, carried on the same radius as the mirror and used +to magnify and read the scale: + +12. Device F, to regulate the zero of the instrument: + +13. Tube H, carrying a nicol analyzer and ocular O for defining the +field of vision: This tube is rotated by the radial arm G, carrying the +mirror and reading glass. + +=72 Manipulation.=—The lamp having been adjusted, the instrument, in a +dark room, is so directed that the most luminous spot of the flame is +in the line of vision. An observation tube filled with water is placed +in the trough and the zero of the vernier is placed accurately on the +zero of the scale. The even tint of the field of vision is then secured +by adjusting the apparatus by the device number 12. + +=73 The Soleil-Ventzke Polariscope.=—A form of polariscope giving a +colored field of vision was in use in this country almost exclusively +until within ten years, and is still largely employed. There are +many forms of tint instruments, but the one almost exclusively used +here is that mentioned. A full description of their construction and +manipulation is given by Tucker.[43] By the introduction of a third +rotating nicol in front of the lens next to the lamp, the sensitive +tint at which the reading is made can be kept at a maximum delicacy. +These instruments are capable of rendering very reliable service, +especially in the hands of those who have a delicate perception of +color. They are inferior, however, to the shadow instruments in +delicacy, and are more trying to the eye of the observer. The shadow +instruments therefore, especially those making use of an ordinary +kerosene lamp, have practically driven the tint polariscopes out of use. + +The general arrangement of a tint instrument as modified by Scheibler +is shown in Fig. 37. + +[Illustration: FIGURE 37. TINT POLARISCOPE.] + +Beginning on the right of the figures, its optical parts are as +follows: A is a nicol which, with the quartz plate B, forms the +apparatus for producing the light rose neutral tint. The proper +degree of rotation of these two parts is secured by means of the +button L attached to the rod carrying the ratchet wheel as shown. The +polarizing nicol is at C, and D is a quartz disk, one-half of which is +right-handed and the other left-handed. At G is another quartz plate +belonging to the analyzing part of the apparatus. This, together with +the fixed quartz wedge F, and the movable quartz wedge E, constitute +the compensating apparatus of the instrument whereby the deviation +produced in the plane of polarized light by the solution in the tube is +restored. + +Next to the compensating apparatus is the analyzing nicol which in +this instrument is fixed in a certain place, _viz._, the zero of the +scale. The analyzer and the telescope for observing the field of vision +are carried in the tube HJ. The movable quartz wedge has a scale which +is read with a telescope K, provided with a mirror inclined at an angle +of 45°, just over the scale and serving to illuminate it. The quartz +wedges are also provided with a movement by which the zero point of +the scale can be adjusted. A kerosene lamp with two flat wicks is the +best source of illumination and the instrument should be used in a dark +room and the light of the lamp, save that which passes through the +polariscope, be suppressed by a shade. The sensitive or transition tint +is produced by that position of the regulating apparatus which gives +a field of view of such a nature that a given small movement of the +quartz compensating wedge gives the greatest contrast in color between +the halves of the field of vision. For most eyes a faint rose-purple +tint, as nearly colorless as possible, possesses this quality. A slight +movement of the quartz wedge by means of the screw head M will, with +this tint, produce on one side a faint green and on the other a pink +color, which are in strong contrast. The neutral point is reached by so +adjusting the quartz wedge as to give to both halves of the field the +same faint rose-purple tint. + +=74. The Shadow Polariscope for Lamp Light.=—This form of instrument +is now in general use for saccharimetric purposes. It possesses on +the one hand, the advantages of those instruments using monochromatic +light, and on the other, the ease of manipulation possessed by the tint +polariscopes. It differs from the tint instrument in dispensing with +the nicol and quartz plate used to regulate the sensitive tint, and +in having its polarizing nicol peculiarly constructed in harmony with +the optical principles of the jellet-corny prism. The more improved +forms of the apparatus have a double quartz wedge compensation. The +two wedges are of opposite optical properties, and serve to make +the observations more accurate by mutual correction. The optical +arrangement of the different parts of such a polariscope is shown in +the following figure. + +The lenses for concentrating the rays of light and rendering them +parallel are contained in the tube N. At O is placed the modified +polarizing nicol. The two compensating quartz wedges are moved by the +milled screw-heads EG. The rest of the optical apparatus is arranged +as described under the tint polariscope. For practical purposes, only +one of the wedges is employed, but for all accurate work the readings +should be made with both wedges and thus every possible source of error +eliminated. + +[Illustration: FIGURE 38. DOUBLE COMPENSATING SHADOW POLARISCOPE.] + +=75. The Triple Shadow Instrument.=—When properly made, all the +instruments which have been mentioned, are capable of giving accurate +results if used in harmony with the directions given. In the use +of polariscopes having colored fields of vision a delicate sense +of distinguishing between related tints is necessary to good work. +Color-blind observers could not successfully use such apparatus. In +the shadow instruments it is only necessary to distinguish between the +halves of a field of vision unequally illuminated and to reduce this +inequality to zero. A neutral field is thus secured of one intensity +of illumination and of only one color, usually yellow. Such a field of +vision permits of the easy discrimination between the intensity of the +coloration of its two halves, and is consequently not trying to the eye +of the observer, and allows of great accuracy of discrimination. This +field of vision has lately been still further improved by dividing it +into three parts instead of two. An instrument of this kind, Fig. 39, +in use in this laboratory, permits a delicacy of reading not possessed +by any other instrument used for sugar analysis, and approaching that +of the standard Landolt-Lippich apparatus, used by us for research +work and for determining the rotation of quartz plates and testing the +accuracy of other polariscopes. + +[Illustration: FIGURE 39. TRIPLE SHADOW POLARISCOPE.] + +The triple shadow is secured by interposing in front of the polarizing +nicol two small nicols as indicated in Fig. 40. The end views in +different positions of the polarizer are shown in the lower part of the +diagrams. + +[Illustration: FIGURE 40. APPARATUS FOR PRODUCING A TRIPLE SHADOW.] + +Instead of the comparison of the intensity of the illumination being +made on the halves of the field of vision it is made by comparing +the segments of the halves with a central band, which also changes +in intensity synchronously with the two segments, but in an opposite +direction. + + +THE ANALYTICAL PROCESS. + +=76. General Principles.=—Having described the instruments chiefly +employed in the optical examination of sugar solutions, the next step +is to apply them to the analytical work. A common set of directions for +use will be found applicable to all instruments with such modifications +only as are required by peculiarities of construction. With the +best made instruments it is always advisable to have some method of +controlling the accuracy of the observation. The simplest way of doing +this is to test the apparatus by standard quartz plates. These plates +are made from right-handed polarizing quartz crystal ground into plates +of definite thickness and accurately tested by standard instruments. +Theoretically such quartz plates deflect the plane of polarized light +in a degree proportionate to their thickness, but practically some +small deviations from the rule are found. With a source of light of the +same tint, and at a constant temperature, such plates become a safe +test for the accuracy of the graduation of polariscopes. They are more +convenient for use than pure sugar solutions of known strength which +are the final standards in all disputed cases. These quartz plates +are conveniently mounted in tubes of the same size as those holding +the sugar solution, and thus fit accurately into the trough of the +polariscope, the optical axis of which passes through their center. +The quartz plate when used for setting the scale of a polariscope +should be placed always in the same position. In some plates slight +differences of reading may be noticed on rotating the tubes holding +them. Theoretically, such differences should not exist, but in practice +they are sometimes found. The temperature of observation should also +be noted, and if not that at which the value of the plate was fixed a +proper correction should be made. + +=77. Setting the Polariscope.=—While mention has been made of several +forms of apparatus in the preceding paragraphs, those in common use +are limited to a very small number. In this country quite a number of +color instruments may still be found, together with a few laurents, +and a constantly increasing number of shadow instruments for use with +lamp light. The following description of setting the polariscope is +especially adapted to the last named instrument, but the principles of +adjustment are equally applicable to all. + +The scale of the instrument is first so adjusted by means of the +adjusting screws provided with each instrument, as to bring the zero +of the vernier and that of the scale exactly together. The telescope +or ocular is then adjusted until the sharp line separating the halves +of the field of vision is brought into focus. This being accomplished +an observation tube filled with pure water is placed in the apparatus +and the telescope again adjusted to bring the dividing line of the +field into focus. The beginner especially, should repeatedly study +this adjustment and be impressed with the fact that only in a sharply +defined field are practical observations of any worth. The importance +of having all the lenses perfect and all the cover glasses without a +flaw may be fully appreciated when it is remembered that the polarized +ray, already deprived of half its original luminous power, must pass +through several centimeters of crystallized calcium carbonate, and +half a dozen disks of glass and quartz, and as many lenses before +reaching the eye of the observer. Only with the greatest care and +neatness is it possible to secure the required degree of illumination. +The zero point having been well studied and accurately adjusted, the +scale of the instrument may be tried with a series of quartz plates of +known polarizing power at the temperature of the observation. In the +apparatus with double quartz wedge compensation, it will be noticed +that the marks on one scale are black and on the other red. The +black is the working and the red the control scale. To operate this +instrument, the red scale is placed exactly at the zero point. The +black scale is also placed at zero, and if the field of vision is not +neutral, it is made so by the micrometer screw with which the black +scale is provided. In a right-handed solution, the red scale is left +at zero and the black one moved to the right until neutrality in the +field of vision is reached and the reading is taken. The observation +tube containing the sugar solution is taken out and the red scale +moved until the field of vision is again neutral and the reading of +the red scale taken. The two readings should agree. Any failure in the +agreement shows some fault either in adjusting the apparatus or in its +construction, or some error in manipulation. + +The double compensating shadow instruments are more readily tested +for accuracy in all parts of the scale than those of any other +construction. The two compensating wedges are cut with the greatest +care, one from a left-handed and the other from a right-handed +perfectly homogeneous quartz crystal. Since faults in these wedges are +due either to lack of parallelism of surface, or of perpendicularity +to the optical axis of the crystal, and since these faults of +crystallization or construction must be in a very limited degree common +they would not coincide once in many thousand times in the two wedges. +This is easily shown by the theory of probabilities. If, therefore, the +two readings made at any point, should not agree, it must be due either +to a fault in one of the wedges, or to a fault in reading or a lack of +adjustment, as has been mentioned. In such cases the readings should be +retaken and the errors are usually easily discovered. + +=78. Control Observation Tube.=—Instead of using quartz plates of known +values for testing the accuracy of the scale, an observation tube may +be used, the length of which can be varied at the pleasure of the +observer. + +The construction of a tube of this kind is shown in Fig. 40. The tube +B is movable telescopically in A by means of the ratchet wheel shown. +It is closed at D water-tight by a glass disk. The tube B fits as +accurately into A as is possible to permit of free movement, and any +liquid which may infilter between its outer surface and the inner +surface of A is prevented from gaining exit by the washer C, which +fits both tubes water-tight. The ratchet which moves B in A carries +a millimeter scale and vernier N whereby the exact thickness of the +liquid solution between the surfaces of the glass disks D and E can be +always determined. + +[Illustration: FIGURE 41. CONTROL OBSERVATION TUBE.] + +By this device the length of liquid under observation can be accurately +read to a tenth of a millimeter. The cover glass E is held in position +by any one of the devices in common use for this purpose in the case in +question, by a bayonet fastening. The funnel T, communicating directly +with the interior of A, serves to hold the solution, there being always +enough of it to fill the tube when D is removed to the maximum distance +from C, which is usually a little more than 200 millimeters. + +Let the control tube be adjusted to 200 millimeters and filled with a +solution of pure sugar, which reads 100 per cent or degrees in a 200 +millimeter tube. Since the degree of rotation is, other things being +equal, proportional to the length of the column of polarizing solution, +it follows that if the tube B be moved inward until the distance +between D and C is 100 millimeters, the scale should read 50° or per +cent. By adjusting the length of the distance between B and C it is +easily seen that every part of the scale can be accurately tested. + +The tube should be filled by removing the funnel and closing the +orifice with a screw cap which comes with the apparatus. The cap E +is then removed and the tube filled in the ordinary manner. This +precaution is practiced to avoid carrying air bubbles into the tube +when filled directly through the funnel. With a little care, however, +this danger may be avoided, or should air bubbles enter they can be +easily removed by inclining the tube. + +In case the solution used be not strictly pure it may still be employed +for testing the scale. Suppose, for instance, that a solution made up +in the usual way, has been made from a sample containing only 99.4 per +cent of sugar. Then in order to have this solution read 100° on the +scale the tube should be set at 201.2 millimeters, according to the +formula + + 200 × 100 + --------- = 201.2. + 99.4 + +By a similar calculation the position of the tube for reading any +desired degree on the scale can be determined. The importance of +controlling all parts of the scale in compensating instruments is +emphasized by the fact that a variation of only 0.016 millimeter in the +thickness of the compensating wedge will cause a change of one degree +in the reading of the instrument. + +=79. Setting the Polariscope with Quartz Plates.=—Pure sugar is +not always at the command of the analyst, and it is more convenient +practically to adjust the instrument by means of quartz plates, the +sugar values of which have been previously tested for the character +of the light used. Assuming the homogeneity of a plate of quartz, the +degree of deflection which it imparts to a plane of polarized light +depends on the quality of the light, the thickness of the plate, and +the temperature. + +In respect of the quality of light, red polarized rays are least, and +violet most deflected. The degree of rotation produced with any ray, +at a given temperature, is directly proportional to the thickness of +the plate. Temperature affects the rotating power of a quartz plate +in a degree highly significant from a scientific point of view and +not wholly negligible for practical purposes. The rotating power of a +quartz plate increases with the temperature and the variation may be +determined by the formula given below:[44] + +The formula is applicable for temperatures between 0° and 100°. Its +values are expressed in degrees of angular measure which can be +converted into degrees of the sugar scale by appropriate factors: + +_Formula.—a_ᵗ = _a_°(1 + 0.000146_t_); + +in which _a_° = polarization in angular degrees at 0°, _t_ the +temperature of observation and _a_ᵗ the rotation desired. + +_Example._—A quartz plate which has an angular rotation of 33° at 0° +will have a rotation at 20° of 33°.09834. + +_a_ᵗ = 33(1 + 0.000146 × 20) = 33.09834. + +Since in instruments using the ventzke scale one degree of the sugar +scale is equal to 0.3467 degree angular measure, the sugar value of the +quartz plate mentioned is equal to 95.47 percent; 33.09834 ÷ 0.3467 = +95.47. + +The sugar value of this plate at 0° is 95.18 per cent; 33 ÷ 0.3467 = +95.18. + +=80. Tables for Correcting Quartz Plates.=—Instead of calculating the +variation in quartz plates for each temperature of observation, it is +recommended by the Bureau of Internal Revenue of the Treasury, to use +control quartz plates the values of which at any given temperature, are +found on a card which accompanies each one.[45] The variations given, +are from temperatures between 10° and 35°. Three control plates are +provided with each instrument used by the Bureau, for polarimetric +work in the custom houses, or in ascertaining bounties to be paid on +the production of domestic sugars. For example, the case of a sugar +which polarizes 80°.5 may be cited. One of the control plates nearest +to this number, is found to have at the temperature of observation, a +polarization of 91°.4, the reading being made in each case at 25°. On +consulting the card which accompanies the control plate, it is seen +that its value at the temperature mentioned, is 91°.7. The reading of +the instrument is therefore too low by three-tenths of a degree, and +this quantity should be added to the observed polarization, making it +80°.8. In this method of correcting the reading for temperature, it is +assumed that the compensating wedges of the instrument, are free of +error at the points of observation. The plates used for the purpose +above, are all standardized in the office of weights and measures of +the Coast and Geodetic Survey, before delivery to the analysts. + +=81. Applicability of Quartz Plates.=—Quartz plates which are correctly +set for one instrument or kind of light, should be equally accurate +for the sugar scales of all instruments, using the same sugar factor. +In other words a quartz plate which reads 99° on a scheibler color +polariscope, should give the same reading on the sugar scale of a +shadow compensating or a monochromatic direct reading apparatus using +26.048 grams of sugar. + +The most useful quartz plates for sugar analysis, are those which give +the readings at points between 80° and 96°, which cover the limits of +ordinary commercial sugars. For molasses the plates should read from +45° to 55°. For sugar juices of the cane and beet, the most convenient +graduation would be from 10° to 20°, but plates of this value would be +too thin for practical work and are not in use. When quartz plates are +to be used for control purposes, they should be purchased from reliable +manufacturers, or better, tested directly against pure sugar solutions +by the observer. + +In practice we have found quartz plates as a rule, true to their +markings. + +=82. The Sugar Flask.=—Sugar solutions are prepared for polarization in +flasks graduated to hold fifty or one hundred cubic centimeters. For +scientific work a flask is marked to hold 100 grams of distilled water +at 4°. The weights are all to be reduced to a vacuum standard. One +flask having been marked in this way, others may be compared directly +therewith by means of pure mercury. For this purpose the flasks must be +perfectly dry and the mercury pure, leaving no stain on the sides of +the flask. The glass must also be strong enough to undergo no change in +shape from the weight of mercury used. + +For sugar work the true 100 gram flask is not usually employed, but +one graduated by weighing at 17°.5. These flasks are graduated by +first weighing them perfectly dry, filling with distilled water and +again weighing fifty and fifty-five, or 100 and 110 grams of water at +the temperature named. Since the volume of water at 17°.5 is greater +than at 4° the sugar flask in ordinary use has a greater volume by +about 0.25 cubic centimeter than the true flask. The observer should +always secure a statement from the dealer in respect of the volume +of the flask used in testing the scale of the polariscope purchased. +In the graduation of a flask in true cubic centimeters, when brass +weights are used it will be necessary to correct the weight of each +gram of water by adding to it one milligram, which is almost exactly +the weight of the volume of air displaced by one gram of water in the +circumstances named. If the flask be first counterbalanced and it be +desired to mark it at 100 cubic centimeters the sum of the weights +placed in the opposite pan should be 100 - 0.100 = 99.900 grams. While +this is not a rigidly exact correction it will be sufficient for all +practical purposes. A liter of dry air weighs 1.29366 grams; and 100 +cubic centimeters of water would therefore displace 0.129 gram of air. +But the brass weights also displace a volume of air which when deducted +reduces the correction to be made for the water to nearly the one +named. For convenience in inverting sugar solutions the flasks used in +practical work are graduated at fifty and fifty-five and 100 and 110 +cubic centimeters respectively. + +=83. Preparing Sugar Solutions for Polarization.=—If sugar samples +were always pure the percentage of sugar in a given solution could be +directly determined by immediate polarization. Such cases, however, +are rarely met in practice. In the majority of cases the sample is +not only to be brought into solution but is also to be decolorized +and rendered limpid by some one of the methods to be described. A +perfectly limpid liquid is of the highest importance to secure correct +observations. With a cloudy solution the field of vision is obscured, +the dividing line of the two halves, or the double line in the triple +field, becomes blurred or invisible and the intensity of illumination +is diminished. A colored liquid which is bright is far more easy to +polarize than a colorless liquid which is turbid. In fact, it is only +rarely in sugar work that samples will be found which require any +special decolorizing treatment other than that which is received in +applying the reagents which serve to make the solutions limpid. In the +following paragraphs the approved methods of clarifying sugar solutions +preparatory to observation in the polariscope will be described. + +=84. Alumina Cream.=—The hydrate of alumina, commonly known as alumina +cream, is always to be preferred as a clarifying agent in all cases +where it can be successfully applied.[46] It is a substance that acts +wholly in a mechanical way and therefore leaves the sugars in solution +unchanged, carrying out only suspended matters. In the preparation +of this reagent a solution of alum is treated with ammonia in slight +excess, the aluminum hydroxid produced washed on a filter or by +decantation until neutral in reaction. The hydroxid is suspended +in pure water in proportions to produce a creamy liquid. Although +apparently very bulky, the actual space occupied by the amount of dry +hydroxid added in a few cubic centimeters is so small as to produce no +disturbing effect of importance on the volume of the sugar solution. +The cream thus prepared is shaken just before using and from one to +five cubic centimeters of it, according to the degree of turbidity of +the saccharine solution, are added before the volume in the flask is +completed to the mark. After filling the flask to the mark the ball of +the thumb is placed over the mouth and the contents well shaken and +allowed to stand for a few moments before filtering. + +The alumina cream is well suited to use with solutions of commercial +sugars of not too low a grade and of most honeys and high grade +sirups. It is usually not powerful enough to clarify beet and cane +juices, molasses and massecuites. + +=85. Basic Lead Acetate.=—A solution of basic lead acetate is an +invaluable aid to the sugar analyst in the preparation of samples for +polarimetric observation. It acts as a clarifying agent by throwing +out of solution certain organic compounds and by uniting with the +organic acids in solution forms an additional quantity of precipitate, +and these precipitates act also mechanically in removing suspended +matters from solution. The action of this reagent is therefore much +more vigorous than that of alumina cream. Coloring matters are often +precipitated and removed by treatment with lead acetate. It happens +therefore that there are few samples of saccharine bodies whose +solutions cannot be sufficiently clarified by lead acetate to permit of +polarimetric observation. + +The reagent is most frequently employed of the following strength:[47] +Boil for half an hour in one and a half liters of water 464 grams of +lead acetate and 264 grams of litharge with frequent stirring. When +cool, dilute with water to two liters, allow to stand until clear, and +decant the solution. The specific gravity of this solution is about +1.267. + +In a solution of basic lead acetate of unknown strength the percentage +of lead acetate may be determined from its specific gravity by the +following table:[48] + + PERCENTAGE OF LEAD ACETATE CORRESPONDING TO + DIFFERENT SPECIFIC GRAVITIES AT 15°. + + Percentage of Percentage of + Specific gravity. lead acetate. Specific gravity. lead acetate. + 1.0127 2 1.2040 28 + 1.0255 4 1.2211 30 + 1.0386 6 1.2395 32 + 1.0520 8 1.2579 34 + 1.0654 10 1.2768 36 + 1.0796 12 1.2966 38 + 1.0939 14 1.3163 40 + 1.1084 16 1.3376 42 + 1.1234 18 1.3588 44 + 1.1384 20 1.3810 46 + 1.1544 22 1.4041 48 + 1.1704 24 1.4271 50 + 1.1869 26 + +=86. Errors Due to use of Lead Solutions.=—In the use of lead solutions +there is danger of errors intruding into the results of the work. These +errors are due to various sources. Lead subacetate solution, when +used with low grade products, or sugar juices, or sirups from beets +and canes, precipitates albuminous matters and also the organic acids +present. The bulk occupied by these combined precipitates is often of +considerable magnitude, so that on completing the volume in the flask +the actual sugar solution present is less than indicated. The resulting +condensation tends to give too high a polarimetric reading. With purer +samples this error is of no consequence, but especially with low grade +sirups and molasses it is a disturbing factor, which must be considered. + +One of the best methods of correcting it has been proposed by +Scheibler.[49] To 100 cubic centimeters of a solution of the sample, +ten of lead solution are added, and after shaking and filtering +the polarimetric reading is taken. Another quantity of 100 cubic +centimeters of the solution with ten of lead is diluted to 220 cubic +centimeters, shaken, filtered, and polarized. Double the second +reading, subtract it from the first, multiply the difference by 2.2, +and deduct the product from the first reading. The remainder is the +correct polarization. + +The process just described is for the usual work with beet juices and +sirups. For cane juices measured by the graduated pipette, hereafter to +be described, and for weighed samples of molasses and massecuites, the +following method of calculation is pursued.[50] To the sample dissolved +in water, add a measured portion of the lead subacetate solution, make +its volume 100 cubic centimeters and observe the polarimetric reading. +Prepare a second solution in the same way and make the volume double +that of the first and again take the polarimetric reading. Multiply the +second reading by two, subtract the product from the first reading and +multiply the remainder by two, and subtract the product from the first +reading. + + _Example._—First polarization 30.0 + Second polarization 14.9 + Then 30 - (2 × 14.9 = 29.8) = 0.2 + 0.2 × 2 = 0.4 + and 30 - 0.4 = 29.6 + +The corrected reading therefore shows that the sample contained 29.6 +per cent of sugar. + +=87. Error Due to Action of Lead Subacetate on Levulose.=—In the use of +lead subacetate solution not only is there danger of error due to the +causes just described, but also to a more serious one, arising from the +chemical interaction of the clarifying agent and levulose.[51] + +Lead subacetate forms a chemical union with levulose and the resulting +compound has a different rotatory power from the left-handed sugar in +an uncombined state. By adding a sufficient quantity of subacetate +solution, the left-handed rotation of levulose may be greatly +diminished if not entirely destroyed. In this case the dextrose, which +with levulose forms inverted sugar, serves to increase the apparent +right rotation due to the sucrose in solution. The reading of the +scale is therefore higher than would be given by the sucrose alone. +If the lead subacetate could be added in just the proportion to make +the invert sugar neutral to polarized light, its use would render the +analysis more accurate; but such a case could only arise accidentally. +To correct the error, after clarification, the compound of levulose and +lead may be decomposed by the addition of acetic acid according to the +method of Spencer. In this case the true content of sucrose can only be +obtained by the method of inversion proposed by Clerget, which will be +described in another paragraph. + +=88. Clarification with Mercuric Compounds.=—Where the disturbing +bodies in a solution are chiefly of an albuminoid nature, one of the +best methods of securing clarification is by the use of a solution +containing an acid mercuric compound.[52] In the case of milk this +method is to be preferred to all others. Albuminoid bodies themselves, +have the property of deflecting the plane of polarization, as a rule, +to the left, and therefore, should be completely removed from solutions +containing right-handed sugars such as lactose. For this purpose the +mercuric compound is more efficient than any other. It is prepared and +used as follows.[53] Dissolve mercury in double its weight of strong +nitric acid and dilute the solution with an equal volume of water. One +cubic centimeter of this solution is sufficient to clarify fifty times +its volume of milk. + +=89. Decolorization by Means of Bone-Black.=—Where the means already +described fail to make a solution sufficiently colorless to permit of +the passage of a ray of polarized light, recourse should be had to a +decolorizing agent. The most efficient of these is bone-black. For +laboratory work it is finely ground and should be dry if added to an +already measured solution. When moist it should be added to the flask +before the volume is completed, and a correction made for the volume of +the dry char employed. Bone-black has the power of absorbing a certain +quantity of sugar, and for this reason as little of it should be +employed as is sufficient to secure the end in view. If not more than +one gram of the char be used for 100 cubic centimeters of solution, +the error is not important commercially. The error may be avoided by +placing the char on the filter and rejecting the first half of the +filtered solution. The char becomes saturated with the first portion +of the solution, and does not absorb any sugar from the second. This +method, however, does not secure so complete a decolorization as is +effected by adding the black directly to the solution and allowing to +stand for some time with frequent shaking. + +=90. Remarks on Analytical Process.=—Since large weights of sugar are +taken for polarization, a balance which will weigh accurately to one +milligram may be used. In commercial work the weighing is made in a +counterpoised dish with a prominent lip, by means of which the sample +can be directed into the mouth of the flask after partial solution. +Where the air in the working room is still, an uncovered balance is +most convenient. With a little practice the analyst will be able to +dissolve and transfer the sample from the dish to the flask without +danger of loss. The source of light used in polarizing should be in +another room, and admitted by a circular opening in the partition. In a +close polarizing room, which results from the darkening of the windows, +the temperature will rapidly rise if a lamp be present, endangering +notably the accuracy of the work, and also interfering with the comfort +of the observer. The greatest neatness must be practiced in all stages +of the work, and especially the trough of the polariscope must be kept +from injury which may arise from the leaking of the observation tubes. +Dust and dirt of all kinds must be carefully excluded from the lenses, +prisms, wedges and plates of the instrument. + +=91. Determination of Sucrose by Inversion.=—In the foregoing +paragraphs directions have been given for the estimation of sugar +(sucrose) by its optical properties. It has been assumed so far, that +no other disturbing bodies have been present, save those which could be +removed by the clarifying agents described. The case is different when +two or more sugars are present, each of which has a specific relation +to polarized light. In such cases some method must be used for the +optical determination of sucrose, which is independent of the influence +of the other polarizing bodies, or else recourse must be had to other +methods of analysis. The conversion of the sucrose present into invert +sugar by the action of an acid or a ferment, affords an opportunity for +the estimation of sucrose in mixed sugars, by purely optical methods. +This process rests upon the principle that by the action of a dilute +acid for a short time, or of a ferment for a long time, the sucrose +is completely changed, while other sugars present are not sensibly +affected. Neither of these assumptions is rigidly correct but each is +practically applicable. + +The sucrose by this process of hydrolysis is converted into an equal +mixture of levulose and dextrose. The former, at room temperatures, has +the higher specific rotating power, and the deflection of the plane of +polarization in a solution of inverted sugar is therefore to the left. +The levorotatory power of invert sugar varies with the temperature, +and this arises from the optical properties of the levulose. The +influence of temperature on the rotating power of other sugars, is not +imperceptible in all cases, but in practice is negligible. + +This method of analysis is invaluable in control work in factories, +in the customs and in agricultural laboratories. Since the rotating +power of levulose diminishes as the temperature rises, an accurate +thermometric observation must accompany each polarimetric reading. At +about 88° the rotatory powers of dextrose and levulose are equal, and a +solution of pure invert sugar examined at that degree, is found to be +neutral to polarized light. + +=92. Clerget’s Method of Inversion.=—The classical method of Clerget +for the determination of cane sugar by double polarization before and +after inversion, was first described in a memoir presented to the +Society of Encouragement for National Industry on the 14th of October, +1846. The following description of the original method is taken from a +reprint of the proceedings of that Society, dated Nov. 1846: + +Clerget points out first the observation of Mitscherlich regarding +the influence of temperature on the rotatory power of invert sugar, +and calls attention to the detailed experiments he has made which +resulted in the determination of the laws of the variation. From these +studies he was able to construct a table of corrections, applicable in +the analysis of all saccharine substances in which the cane sugar is +polarized before and after inversion. The basis of the law rests upon +the observation that a solution of pure sugar, polarizing 100° on the +sugar scale, before inversion, will polarize 44° to the left after +inversion at a temperature of zero. The quantity of sugar operated upon +by Clerget amounted to 16.471 grams in 100 cubic centimeters of liquid. +On the instrument employed by him this quantity of sugar in 100 cubic +centimeters gave a reading of 100° to the right on the sugar scale +when contained in a tube twenty centimeters in length. The process of +inversion carried on by Clerget is as follows: + +The sugar solution is placed in a flask, marked on the neck at 100 +and 110 cubic centimeters; or if smaller quantities are used, in a +flask marked on the neck at fifty and fifty-five cubic centimeters. +The flask is filled with the sugar solution to the first mark and +then a sufficient quantity of strong hydrochloric acid added to bring +the volume of the liquid to the second mark. The mouth of the flask +is then closed with the thumb and its contents thoroughly mixed by +shaking. A thermometer is placed in the flask which is set in a +water-bath in such a way that the water comes just above the level of +the liquid in the neck of the flask. The water is heated in such a +manner as to bring the temperature of the contents of the flask, as +determined by the thermometer, exactly to 68° and at such a rate as to +require fifteen minutes to reach this result. At the end of fifteen +minutes the temperature having reached 68° the flask is removed and +placed at once in another water-bath at the temperature of the room, +to which temperature the contents of the flask are cooled as rapidly +as possible. To make the polarimetric observation a tube twenty-two +centimeters in length is filled with the inverted sugar solution by +means of a tubulure in its center, which serves not only the purpose of +filling the tube but also afterwards to carry the thermometer, by means +of which the temperature of observation can be taken. If the sugar +solution be turbid, or contain any lead chlorid due to the previous use +of basic lead acetate in clarification, it should be filtered before +being introduced into the observation tube. This tube being one-tenth +longer than the original compensates for the dilution caused by the +addition of the hydrochloric acid in inversion. + +When reading, the bulb of the thermometer should be withdrawn far +enough to permit the free passage of the ray of light and the exact +temperature of the solution noted. + +The above outline of Clerget’s method of inversion is given in order +that the analyst may compare it with any of the variations which he may +find in other works. The chief points to which attention is called, +are, first, the fact that only a little over sixteen grams of sugar are +used for ten cubic centimeters of strong hydrochloric acid, and second, +that the time of heating is exactly fifteen minutes, during which time +the contents of the flask should be raised from room temperature to +exactly 68°. + +From the above it is seen that the process of Clerget, as originally +described, can be applied directly to all instruments, using +approximately sixteen grams of sugar in 100 cubic centimeters. +Experience has also shown that even when larger quantities of sugar +are employed, as for instance, approximately twenty-six grams, +the inversion is effected with practical completeness in the same +circumstances. It is advised, therefore, that in all analytical +processes, in which cane sugar is to be determined by the process of +inversion with an acid, the original directions of Clerget be followed +as strictly as possible. Experience has shown that no one of the +variations proposed for Clerget’s original method has any practical +advantage and the analyst is especially cautioned against those +methods of inversion in which the temperature is continued at 68° for +fifteen minutes or in which it is allowed to go above that degree. + +=93. Influence of Strength of Solution and Time of Heating on the +Inversion of Sucrose.=—As has been intimated, the strength of a sugar +solution and the time of heating with hydrochloric acid are factors +that must be considered in determining a formula for the calculation +of sucrose by inversion. The Clerget formula holds good only for the +conditions specified and these conditions must be rigidly adhered to +in order to secure the proper results. This matter has been thoroughly +studied by Bornträger, who also gives a nearly complete bibliography +of the subject.[54] As a result of his investigations it seems +well established that the original Clerget formula is practically +correct for the conditions indicated, Bornträger modifying it only +by substituting in the formula 143.66 for 144. This is so nearly the +same as the Clerget factor that it is not advisable to substitute it +therefor. If, however, the inverted sugar solution be diluted to double +its volume before polarization the factor proposed by Landolt, _viz._, +142.4, gives more nearly accurate results. If the hydrochloric acid be +neutralized before polarization by an alkaline body, the character of +the salt which is formed also influences, to a greater or less extent, +the specific rotatory power of the solution. Hydrochloric acid itself +also influences the rotation to a certain degree.[55] + +=94. Calculation of Results.=—The percentage of sucrose in a solution +which has been polarized before and after inversion is calculated by an +appropriate formula from the data obtained or is taken directly from +tables. These tables are too long to insert here, and in point of fact +the calculation can be made from the formula almost as quickly as the +result can be taken from a table. + +Two factors are commonly used in the calculations, one based on +the supposition that a sugar solution polarizing 100° to the right +will, after inversion, give a reading of 44° to the left, at zero +temperature. In the second formula in common use the polarization to +the left in the circumstances mentioned above is assumed to be 42.4, a +number reached by Landolt after a long series of experiments.[56] The +principle of the calculation of the percentage of sucrose is based upon +the original observation of Clerget to the effect that the algebraic +difference of the two readings, divided by 144, less half of the +temperature, will give the percentage of sucrose desired. The formula +by which this is obtained is + + _a_ - _b_ + _S_ = -----------. + _K_ - _t_ + ---- + 2 + +In this formula _a_ is the polarization on the sugar scale before +inversion, _b_ the polarization after inversion, _K_ the constant +representing the algebraic difference of the two polarizations of +pure sugar at 0° and _t_ the temperature of the observation. To _K_ +may be assigned the values 144 or 142.4, the one in more common use. +In case the polarization, after inversion, is to the left, which is +more commonly the case, the sum of the two readings is taken for +_a_ - (-_b_) = _a_ + _b_; when both polarizations are to the right +or left the difference is taken. S is the percentage of sucrose desired. + + _Example.—_Let the polarization before inversion be +95 + and after inversion -26 + and the temperature 20° + + 95 + 26 + Then _S_= -------- = 121 ÷ 134 = 90.6. + 144 - 10 + +Substituting the value 142.4 for _K_, the result of the calculation is +91.4. + +In high grade sugars, therefore, the difference in the results secured +by taking the two values of _K_ amounts to about 1 per cent of sucrose. + +For a further discussion of the theory and practice of inversion the +reader is referred to the articles of Herles, Herzfeld, and Wohl.[57] + +=95. Method Of Lindet.=—Courtonne recommends the method of Lindet for +securing the inversion instead of the method of Clerget.[58] Modified +by Courtonne, the method is as follows: + +Make two or three times the normal weight of sugar dissolved in water +to a volume of 200 or 300 cubic centimeters, as the case may be. After +thoroughly mixing proceed as follows: + +_First, to Obtain the Polarization Direct._—Place fifty or 100 cubic +centimeters of the prepared solution in a flask marked at fifty and +fifty-five or at 100 and 110 cubic centimeters, add a sufficient +quantity of lead acetate to secure a complete clarification, make +the volume to fifty-five or 110 cubic centimeters, shake thoroughly, +filter, and polarize in a 220 millimeter tube. + +_Second, to Obtain the Rotation after Inversion._—Place twenty cubic +centimeters of the original solution, in a flask marked at fifty cubic +centimeters, containing five grams of powdered zinc. The flask should +be placed in boiling water. Add, little by little so as to avoid a too +rapid evolution of hydrogen, ten cubic centimeters of hydrochloric +acid made of equal parts of the strongest acid and water. After the +operation is terminated, cool to the temperature of the room, make +the volume to fifty cubic centimeters, polarize, and determine the +rotation. The volume occupied by the zinc which is not dissolved, will +be about one-half cubic centimeter, hence the deviation should be +multiplied by the factor 2.475 in order to get the true deviation which +would have been produced by the pure liquor. We have then: + + _A_ = the deviation direct. + _B_ = the deviation after inversion. + _C_ = the algebraic difference of the deviations. + +The amount of sucrose, therefore, would be calculated by the formula of +Creydt,[59] + + _C_ - 0.493_A_ + _X_ = --------------; + 0.827 + + +for raffinose the formula would be + + _A_ - _S_ + _Y_ = ---------, + 1.57 + +in which _S_ is the deviation due to the sucrose present. The solutions +inverted in the manner described are absolutely colorless. There is no +need of employing bone-black to secure the saccharimetric reading nor +does it present any uncertainty. It is thought by Courtonne that this +method will soon take the place of the method of Clerget on account of +the advantages above mentioned. The method will be somewhat improved by +adopting the following suggestions: + +1. Instead of allowing any arbitrary number for the volume of the +undissolved zinc, decant the liquid, after inversion, into another +flask and wash repeatedly with hot water until all trace of sugar is +removed from the flask in which the inversion took place. + +2. Instead of polarizing in a 200 millimeter tube make the observation +in a 500 millimeter tube, which will permit of the reading being made +without any correction whatever. + +=96. Inversion by Means of Invertase.=—Instead of using acids for the +inversion of cane sugar the hydrolysis can be easily effected by means +of a ferment derived from yeast. A complete history of the literature +and characteristics of this ferment, together with a study of its +properties and the various methods of preparing it, has been given by +O’Sullivan and Tompson.[60] In the preparation of invertase, the method +found most effective is the following: + +The yeast is allowed to liquify for at least a month in a fairly warm +room without stirring. At the end of this time the surface is removed +and any supernatant liquid poured away. The lower sedimentary part is +thrown on a quick-acting filter and allowed to drain for two days. +To the filtrate, alcohol of specific gravity 0.87 is gradually added +to the extent of one and a half times its volume, with continued and +vigorous stirring. The process of adding the alcohol and stirring +should require about half an hour, after which the mixture is allowed +to stand for twenty-four hours to allow the precipitated invertase +to settle. The supernatant liquid is poured away and the precipitate +washed several times on successive days by decantation with alcohol of +0.92 specific gravity. When the washings become nearly colorless the +precipitate is thrown on a filter, allowed to drain, and immediately +removed and mixed with a large bulk of alcohol of 0.92 specific +gravity. The precipitate is again collected, mixed thoroughly with its +own bulk of water, and some alcohol of 0.97 specific gravity, allowed +to stand for a few hours and thrown on a filter. The filtrate contains +the invertase. + +=97. Determination of Activity of Invertase.=—The activity of a +solution of invertase, prepared as above, is measured by the number +of minutes required for it to reduce to zero the optical power of a +solution of 100 times its weight of cane sugar at a temperature of +15°.5. In order to facilitate the action of the invertase, a trace of +sulfuric acid is added to the solution. The manipulation is as follows: + +Fifty grams of sucrose are dissolved in water and made to a volume of +nearly a quarter of a liter and placed in a bath maintained at 15°.5. +Half a gram of the invertase is added, the time noted, the solution +immediately made up to a quarter of a liter and well shaken. The +contents of the flask are poured rapidly into five beakers; the actual +quantity in each beaker is not necessarily the same. To each of these +beakers, in succession, are added the following amounts of decinormal +sulfuric acid, _viz._, one-tenth, three-tenths, six-tenths, one, and +one and four-tenths cubic centimeters. After an hour a small quantity +of the solution is taken from beaker No. 3 and the reaction of the +invertase stopped by adding a few drops of strong potassium hydroxid +and the time of adding this reagent noted. This solution is then read +in the polariscope and the percentage of sugar inverted is calculated +from the formula C₁₂H₂₂O₁₁ + H₂O = C₆H₁₂O₆ + C₆H₁₂O₆. + +The calculation of the amount of cane sugar inverted is based on +the formula, (38.4 - _d_) ÷ 0.518 = _p_. In this formula _d_ equals +the divisions of the sugar scale read on the polariscope; _p_ the +percentage of cane sugar inverted; 38.4 the reading on the sugar scale +of the original sugar solution and 51.8 the total number of divisions +of the cane sugar scale that the polariscope reading would fall through +if all the sugar were inverted. The observation tubes used in the +polarization are only 100 millimeters in length. After stopping the +action of the invertase with potassium hydroxid the solution is allowed +to stand for some time before polarization inasmuch as the dextrose +formed appears to assume the state of birotation and some time is +required for it to reach its normal rotatory power. If the invertase +be used in the alcoholic solution a sufficient quantity should be +added to be equivalent to 0.01 of the sucrose present. The time which +the contents of beaker No. 3 will take to reach optical activity is +calculated in a manner described by O’Sullivan and Tompson, but too +long to be inserted here.[61] The five beakers mentioned above are +examined in succession and the amount of sulfuric acid best suited to +the maximum inversion thus determined. This quantity is then used in +subsequent hydrolyses with the given sample of invertase. + +The action of invertase on sucrose is very rapid at the first and +becomes very much slower towards the end. At a temperature of 15°.5 it +is advisable to let the solution stand for forty-eight hours in order +to be sure that complete inversion has taken place. For this reason +the method by inversion by means of invertase is one of no great +practical importance, but it may often be useful to the analyst when +the employment of an acid is inadmissible. + +=98. Inversion by Yeast.=—Owing to the difficulty of preparing +invertase, O’Sullivan and Thompson[62] propose to use yeast as the +hydrolytic agent, as first suggested by Kjeldahl. It is shown that in +the use of yeast it is not necessary to employ thymol or any other +antiseptic. The method of procedure is as follows: The cane sugar +solution of usual strength should not be alkaline, but, if possible, +should be exactly neutral. If there be any ferment suspected, the +temperature should be momentarily raised to 80° to destroy its +activity. The polariscopic reading of the solution is then taken at +15°.5 and the amount of copper reduced by the solution should also be +determined. + +Fifty cubic centimeters of the solution are poured into a beaker and +raised to a temperature of 55° in a constant temperature bath. Some +brewers yeast amounting to about one-tenth of the total amount of sugar +to be inverted, pressed in a towel, is thrown into the hot solution and +the whole stirred until mixture is complete. The solution is left for +four hours in the water-bath, at the end of this time it is cooled to +15°.5, a little freshly precipitated aluminum hydroxid added, and the +volume made to 100 cubic centimeters. A portion of this solution is +filtered and its polariscopic reading observed. The solution is then +left till the next day, when another polariscopic reading is taken in +order to prove that inversion is complete. The copper reducing power is +also determined. The method of calculating the results is the same as +when invertase is used. The following formulas are employed. + +_a_ = the number of divisions indicated by the polariscopic reading for +a 200 millimeter tube: + +_aʹ_ = the same number after inversion: + +_m_ = the number of the divisions of the polariscopic scale which 200 +millimeters of the sugar solution containing one gram of cane sugar +per 100, alter at 15°.5 on being inverted: In the case of the ventzke +polarimeter scale, one gram of cane sugar in 100 cubic centimeters, +indicates +3.84 divisions and after inversion it gives -1.34 div. In +experiments of this kind, therefore, _m_ = 5.18. + +_P_ = the weight of cane sugar present in 100 cubic centimeters of the +original solution: + +The formula employed then is + + _a_ - 2_a_ʹ + _P_ = -----------. + _m_ + +For the copper reduction data the following are used: + +_G_ = the weight of 100 cubic centimeters of the original solution: + +_Gʹ;_ = the same for the inverted solution: Allowance must be made here +both for the dilution and for the 5 per cent increase of the inverted +sugar, but the latter number is so small that it need not be calculated +accurately. + +_w_ = the weight of the original solution used for the estimation: + +_wʹ_ = the same factor for the inverted solution: + +_k_ = the weight of cupric oxid reduced by _w_: + +_kʹ_ = the same factor for _wʹ_: + +_p_ = the weight of cane sugar present in 100 cubic centimeters of the +original solution: The formula to be employed then is + + _Gʹ kʹ G k_ + _p_ = 0.4308(2 ------ - -----). + _wʹ w_ + +This method has been applied to the estimation of cane sugar in +molasses, apple juices and other substances. It is recommended by the +authors as a simple and accurate means of estimating sucrose in all +solutions containing it. The methods of making the copper reductions +will be given hereafter. + +=99. Application of the Process.=—In practice the process of inversion +is used chiefly in the analysis of molasses and low grade massecuites. +In approximately pure sugars the direct polarization is sufficiently +accurate for all practical purposes. In molasses resulting from the +manufacture of beet sugar are often found considerable quantities of +raffinose, and the inversion process has been adapted to that character +of samples. In molasses, in sugar cane factories, the disturbing +factors are chiefly invert sugars and gums. The processes used for +molasses will be given in another paragraph. In certain determinations +of lactose the process of inversion is also practiced, but in this +case the lactose is converted into dextrose and galactose, and the +factors of calculation are altogether different. The process has also +been adapted by McElroy and Bigelow to the determination of sucrose in +presence of lactose, and this method will be described further on. In +general the process of inversion is applicable to the determination of +sucrose in all mixtures of other optically active bodies, which are not +affected by the methods of inversion employed. + +=100. Determination of Sucrose and Raffinose.=—Raffinose is a sugar +which often occurs in beets, and is found chiefly in the molasses after +the chief part of the sucrose has been removed by crystallization. It +is also found in many seeds, notably in those of the cotton plant. In a +pure solution of sucrose and raffinose, both sugars may be determined +by the inversion method of Creydt.[63] The inversion is effected by +means of hydrochloric acid in the manner described by Clerget. The +following formulas are calculated for a temperature of observation +of 20°, and the readings should be made as near that temperature as +possible. + + _C_ - 0.493_A_ + (1) _S_ = -------------- + 0.827 + + _A_ - _S_ 6 + (2) _R_ = --------- = 1.017_A_ - ------. + 1.57 1.298 + +In these formulas _S_ and _R_ are the respective per cents of sucrose +and raffinose desired, _A_ the polarization in sugar degrees before +inversion, _B_ the polarization after inversion read at 20°, and _C_ +is the algebraic difference between _A_ and _B_. It must be understood +that these formulas are applicable only to a solution containing no +other optically active substances, save sucrose and raffinose. + +=101. Specific Rotatory Power.=—In order to compare among themselves +the rotations produced on a plane of polarized light by different +optically active bodies in solution, it is convenient to refer them all +to an assumed standard. The degree of rotation which the body would +show in this condition, is found by calculation, since, in reality, +the conditions assumed are never found in practice. In the case of +sugars and other optically active bodies, the standard of comparison +is called the specific rotatory power. This factor in any given case, +is the angular rotation which would be produced by any given substance +in a pure anhydrous state if it were one decimeter in length and of a +specific gravity equal to water. These are conditions which evidently +do not exist in the case of sugars, since crystalline sugar particles +have no polarizing power, and it would be impossible to pass a ray +of light through an amorphous sugar column of the length specified. +The specific rotatory power is therefore to be regarded as a purely +theoretical factor, calculated from the actual data obtained by the +examination of the solution of any given substance. If the length +of the observation tube in decimeters be represented by _l_, the +percentage of the polarizing body in 100 grams by _p_, and the specific +gravity of the solution by _d_, and the observed angle of rotation by +_a_, then the factor is calculated from the formula: + + _a_. 100 + [_a_]_{Dj} = ---------------. + _p_. _d_. _l_. + + +The symbols Dj refer to the character of light employed, D indicating +the monochromatic sodium flame, and j the transition tint from white +light. + +If the weight of the polarizing body _c_ be given or known for 100 +cubic centimeters of the solution the formula becomes + + _a_. 100 + [_a_]_{Dj} = ----------. + _c_. _l_. + +The latter formula is the one easier of application since it is only +necessary in applying it to dissolve a given weight of the active body +in an appropriate solvent and to complete the volume of the solution +exactly to 100 cubic centimeters. It is therefore unnecessary in this +case to determine the specific gravity. + +=102. Formulas for Calculating Specific Rotatory Power.=—In order +to determine the specific rotatory power (gyrodynat[64]) of a given +substance it is necessary to know the specific gravity and percentage +composition or concentration of its solution, and to examine it with +monochromatic polarized light in an instrument by which the angular +rotation can be measured. The gyrodynat of any body changes with its +degree of concentration, in some cases with the temperature, and +always with the color of the light. With the red rays the gyrodynat +is least and itprogressively increases as the violet end of the +spectrum is approached. In practice the yellow ray of the spectrum +has been found most convenient for use, and in the case of sugars +the gyrodynat is always expressed either in terms of this ray or if +made with color compensating instruments in terms of the sensitive or +transition tint. In the one case the symbol used is (_a_)_{D} and in +the other (_a_)_{j}. From this statement it follows that (_a_)_{D} is +always numerically less than (_a_)_{j}. Unless otherwise specified +the gyrodynat of a body is to be considered as determined by yellow +monochromatic light, and therefore corresponds to _a__{D}.[65] + +=103. Variations in Specific Rotatory Power.=—The gyrodynat of any +optically active body varies with the nature of the solvent, the +strength of the solution, and the temperature.[66] + +Since water is the only solvent of importance in determining the +gyrodynat of sugars it will not be necessary here to discuss the +influence of the nature of the solvent. In respect of the strength of +the solution it has been established that in the case of cane sugar the +gyrodynat decreases while with dextrose it increases with the degree +of concentration. The influence of temperature on the gyrodynat of +common sugars is not of great importance save in the case of levulose, +where it is the most important factor, the gyrodynat rapidly increasing +as the temperature falls. It is of course understood that the above +remarks do not apply to the increase or decrease in the volume of a +solution at changed temperatures. This influence of temperature is +universally proportional to the change of volume in all cases, and +this volumetric change is completely eliminated when the polarizations +are made at the temperatures at which the solutions are completed to +standard volumes. + +=104. Gyrodynatic Data for Common Sugars.=—In the case of cane sugar +the gyrodynat for twenty-five grams of sugar in 100 grams of solution +at 20° is [_a_]_{D} = 66°.37. This is about the degree of concentration +of the solutions employed in the shadow lamplight polariscopes. For +seventeen grams of sugar in 100 grams of solution the number is +[_a_]_{D} = 66°.49. This is approximately the degree of concentration +for the laurent instrument. + +For any degree of concentration according to Tollens the gyrodynat may +be computed by the following formula: [_a_]_{D} = 66°.386 + 0.015035_p_ +- 0.0003986_p_², in which _p_ is the number of grams of sugar in 100 +grams of the solution.[67] In the table constructed by Schmitt the data +obtained are as follows: + + In 100 parts by weight Specific Rotation _a_ + of solution. gravity Concentration for 100 mm. + Sugar _p_. Water _q_. at 20° C. _d_. _c_ = _pd_. at 20° C. [_a_]_{D}. + 64.9775 35.0225 1.31650 85.5432 56°.134 65°.620 + 54.9643 45.0357 1.25732 69.1076 45°.533 65°.919 + 39.9777 60.0223 1.17664 47.0392 31°.174 66°.272 + 25.0019 74.9981 1.10367 27.5938 18°.335 66°.441 + 16.9926 83.0074 1.06777 18.1442 12°.064 66°.488 + 9.9997 90.0003 1.03820 10.3817 6°.912 66°.574 + 4.9975 95.0025 1.01787 5.0868 3°.388 66°.609 + 1.9986 98.0014 1.00607 2.0107 1°.343 66°.802 + +=105. Bi-Rotation.=—Some sugars in fresh solution show a gyrodynat +much higher than the normal, sometimes lower. The former phenomenon +is called bi- the latter semi-rotation. Dextrose shows birotation in +a marked degree, also maltose and lactose. After standing for a few +hours, or immediately on boiling, solutions of these sugars assume +their normal state of rotation. The addition of a small quantity of +ammonia also causes the birotation to disappear.[68] This phenomenon +is doubtless due to a certain molecular taxis, which remains after +solution is apparently complete. The groups of molecules thus held in +place have a certain rotatory power of their own and this is superadded +to that of the normal solution. After a time, under the stress of the +action of the solvent, these groups are broken up and the solution then +assumes its normal condition. + +=106. Gyrodynat of Dextrose.=—The gyrodynat of dextrose, as has already +been mentioned, increases with the degree of concentration, thus +showing a property directly opposite that of sucrose. + +The general formula for the anhydrous sugar is [_a_]_{D} = 52.°718 + +0.017087_p_ + 0.0004271_p_². In this formula _p_ represents the grams +of dextrose in 100 grams of the solution. In a ten per cent solution +the gyrodynat of dextrose is therefore nearly exactly [_a_]_{D}20° = +53°. As calculated by Tollens the gyrodynats corresponding to several +degrees of concentration are shown in the following table: + + _p_ = grams in 100 [_a_]_{D}20° calculated for + grams of solution. anhydrous dextrose. + 7.6819 52°.89 + 9.2994 52°.94 + 9.3712 52°.94 + 10.0614 52°.96 + 10.6279 52°.98 + 12.9508 53°.05 + 18.6211 53°.25 + 31.6139 53°.83 + 40.7432 54°.34 + 43.9883 54°.54 + 53.0231 55°.17 + 82.6111 57°.80 + +=107. Gyrodynats of Other Sugars.=—Of the other sugars it will be +sufficient to mention only levulose, maltose, lactose, and raffinose. +For complete tables of gyrodynatic powers the standard books on +carbohydrates may be consulted.[69] + +The gyrodynat of levulose is not definitely established. At 14° the +number is nearly expressed by [_a_]_{D}14° = -93°.7. + +Invert sugar, which should consist of exactly equal molecules of +dextrose and levulose, has a gyrodynat expressed by the formula +[_a_]_{D}0° = -27°.9, with a concentration equivalent to 17.21 grams of +sugar in 100 cubic centimeters. The gyrodynat decreases with increase +of temperature, according to the formula [_a_]_{D}_t_° = -(27°.9 +- 0.32_t_°). According to this formula the solution is neutral to +polarized light at 87°.2, and this corresponds closely to the data of +experiment. + +Maltose, in a ten per cent solution at 20°, shows a gyrodynat of +[_a_]_{D}20° = 138°.3. + +The general formula for other degrees of concentration is [_a_]_{D} = +140°.375 - 0.01837_p_ - 0.095_t_, in which _p_ represents the number +of grams in 100 grams of the solution and _t_ the temperature of +observation. + +In the case of lactose [_a_]_{D} = 52°.53, and this number does not +appear to be greatly influenced by the degree of concentration; but is +somewhat diminished by a rising temperature. + +The gyrodynat of raffinose in a ten per cent solution is [_a_]_{D} = +104°.5. + + +CHEMICAL METHODS OF ESTIMATING SUGARS. + +=108. General Principles.=—The methods for the chemical estimation of +sugars in common use depend on the reducing actions exerted on certain +metallic salts, whereby the metal itself or some oxid thereof, is +obtained. The reaction is either volumetric or the resulting oxid or +metal may be weighed. The common method is, therefore, resolved into +two distinct processes, and each of these is carried out in several +ways. Not all sugars have the faculty of exerting a reducing action +on highly oxidized metallic salts and the most common of them all, +_viz._, sucrose is practically without action. This sugar, however, by +simple hydrolysis, becomes reducing, but the two components into which +it is resolved by hydrolytic action do not reduce metallic salts in +the same proportion. Moreover, in all cases the reducing power of a +sugar solution is largely dependent on its degree of concentration, and +this factor must always be taken into consideration. Salts of copper +and mercury are most usually selected to measure the reducing power +of a sugar and in point of fact copper salts are almost universally +used. Copper sulfate and carbonate are the salts usually employed, and +of these the sulfate far more frequently, but after conversion into +tartrate. Practically, therefore, the study of the reducing action of +sugar as an analytical method will be confined almost exclusively to +the determination of its action on copper tartrate. + +Direct gravimetric methods are also practiced to a limited extent in +the determination of sugars as in the use of the formation of sucrates +of the alkaline earths and of the combinations which certain sugars +form with phenylhydrazin. Within a few years this last named reaction +has assumed a marked degree of importance as an analytical method. +The most practical treatment of this section, therefore, for the +limited space which can be given it, will be the study of the reducing +action of sugars, both from a volumetric and gravimetric point of +view, followed by a description of the best approved methods of the +direct precipitation of sugars by such reagents as barium hydroxid and +phenylhydrazin. + + +VOLUMETRIC METHODS. + +=109. Classification.=—Among the volumetric methods will be given those +which are in common use or such as have been approved by the practice +of analysts. Since the use of mercuric salts is now practiced to a +limited extent, only a brief study of that process will be attempted. +With the copper methods a somewhat extended description will be given +of those depending on the use of copper sulfate, and a briefer account +of the copper carbonate process. + +In the copper sulfate method two distinct divisions must be noted, +_viz._, first an indirect process depending first upon the reduction +of the copper to a suboxid, the subsequent action of this body on iron +salts, measured finally by titration with potassium permanganate; and +second, a direct process determined either by the disappearance of the +blue color from the copper solution, or by the absence of copper from a +drop of the solution withdrawn and tested with potassium ferrocyanid. +This last mentioned reaction is one which is found in common use. The +volumetric methods are not, as a rule, as accurate as the gravimetric, +depending on weighing the resultant metal, but they are far more rapid +and well suited to technical control determinations. + +=110. Reduction of Mercuric Salts.=—The method of determining sugar by +its action on mercuric salts, is due to Knapp.[70] The method is based +on the observation that dextrose and other allied sugars, will reduce +an alkaline solution of mercuric cyanid, and that the mercury will +appear in a metallic state. + +The mercuric liquor is prepared by adding to a solution of ten grams of +mercuric cyanid, 100 cubic centimeters of a solution of caustic soda of +1.145 specific gravity, and making the volume to one liter with water. +The solution of sugar to be titrated, should be as nearly as possible +of one per cent strength. + +To 100 cubic centimeters of the boiling solution, the sugar solution is +added in small portions from a burette and in such a way as to keep the +whole mass in gentle ebullition. + +To determine when all the mercuric salt has been decomposed, a drop of +the clear boiling liquid is removed and brought into contact with a +drop of stannous chlorid solution on a white surface. A brownish black +coloration or precipitate will indicate that the mercury is not all +precipitated. Fresh portions of the sugar must then be added, until no +further indication of the presence of mercury is noted. The approximate +quantity of sugar solution required to precipitate the mercury having +thus been determined, the process is repeated by adding rapidly, nearly +the quantity of sugar solution required, and then only a few drops at a +time, until the reduction is complete. + +One hundred cubic centimeters of the mercuric cyanid solution prepared +as directed above, will be completely reduced by + + 202 milligrams of dextrose, + 200 ” ” invert sugar, + 198 ” ” levulose, + 308 ” ” maltose, + 311 ” ” lactose. + +By reason of the unpleasant odor of the boiling mercuric cyanid when +in presence of a reducing agent, the process should be conducted in a +well ventilated fume chamber. With a little practice the process is +capable of rapid execution, and gives reasonably accurate results. + +=111. Sachsse’s Solution.=—The solution of mercuric salts proposed +by Sachsse, is made by dissolving eighteen grams of mercuric iodid +in twenty-five cubic centimeters of an aqueous solution of potassium +iodid. To this solution are added 200 cubic centimeters of potash lye, +containing eighty grams of caustic potash. After mixing the solution, +the volume is completed to one liter. The sugar solutions used to +reduce this mixture, should be more dilute than those employed with +the mercuric cyanid, and should not be over one-half per cent in +strength. The end of the reduction is determined as already described. +After a preliminary trial, nearly all the sugar necessary to complete +reduction, should be added at once, and the end of the reduction then +determined by the addition of successive small quantities. One hundred +cubic centimeters of the mercuric iodid solution prepared as directed +above, require the following quantities of sugar to effect a complete +reduction: + + 325 milligrams of dextrose, + 269 ” ” invert sugar, + 213 ” ” levulose, + 491 ” ” maltose, + 387 ” ” lactose. + +By reason of the great difference between the reducing power of +dextrose and levulose in this solution, it has been used in combination +with the copper reduction method, to be described, to determine the +relative proportion of dextrose and levulose in a mixture.[71] + +It is now known that copper solutions require slightly different +quantities of dextrose, levulose, or invert sugar to effect complete +reduction, but the variations are not great and in the calculation +above mentioned, it may be assumed that these differences do not exist. + +Instead of using stannous chlorid as an indicator, the end of the +reaction may be determined as follows: A disk of filtering paper is +placed over a small beaker containing some ammonium sulfid. A drop of +the clear hot solution is placed on this disk, and if salts of mercury +be still present a dark stain will be produced; or a drop of the +ammonium sulfid may be brought near the moist spot formed by the drop +of mercury salt. An alkaline solution of zinc oxid may also be used. + +The methods depending on the use of mercuric salts have, of late, been +supplanted by better processes, and space will not be given here to +their further discussion. + +=112. The Volumetric Copper Methods.=—The general principle on which +these methods depend, is found in the fact that certain sugars, +notably, dextrose, (glucose), levulose, (fructose), maltose and +lactose, have the property of reducing an alkaline solution of copper +to a lower state of combination, in which the copper is separated as +cuprous oxid. The end of the reaction is either determined by the +disappearance of the blue color of the solution, or by the reaction +produced by a drop of the hot filtered solution, when placed in contact +with a drop of potassium ferrocyanid acidified with acetic. + +The copper salt which is found to give the most delicate and reliable +reaction, is the tartrate. The number of volumetric processes proposed +and which are in use, is very great, and an attempt even to enumerate +all of these can not be made in this volume. A few of the most reliable +and best attested methods will be given, representing if possible, the +best practice in this and other countries. The rate of reduction of +the copper salt to suboxid, is influenced by the rate of mixing with +the sugar solutions, the temperature, the composition of the copper +solution and the strength of the sugar solution. + +The degree of reduction is also modified by the rate at which the +sugar solution is added, and by the degree and duration of heating, +and all these variables together, make the volumetric methods somewhat +difficult and their data, to a certain extent, discordant. By reason, +however, of the ease with which they are applied and the speed of their +execution, they are invaluable for approximately correct work and for +use in technical control. + +=113. Historical.=—It is not the purpose in this paragraph to trace +the development of the copper reduction method for the determination +of reducing sugars, but only to refer to the beginning of the exact +analytical application of it. + +Peligot, as early as 1844, made a report to the Society for the +Encouragement of National Industry on methods proposed by Barreswil +and Fromherz for the quantitive estimation of sugar by means of copper +solution.[72] These methods were based on the property of certain +sugars to reduce alkaline copper solution to a state of cuprous oxid +first announced by Trommer.[73] This was followed by a paper by Falck +on the quantitive determination of sugar in urine.[74] + +In 1848 the methods, which have been proposed, were critically examined +by Fehling, and from the date of his paper the determination of sugar +by the copper method may be regarded as resting on a scientific +basis.[75] + +Since the date mentioned the principal improvements in the process +have been in changing the composition of the copper solution in order +to render it more stable, which has been accomplished by varying the +proportions of copper sulfate, alkali and tartaric acid. For the better +keeping of the solution the method of preserving the copper sulfate +and the alkaline tartrates in separate flasks and only mixing them +at the time of use has been found very efficacious.[76] For testing +for the end of the reaction by means of an acetic acid solution of +potassium ferrocyanid the filtering tube suggested by the author, the +use of which will be described further on, has proved quite useful. +Pavy has suggested that by the addition of ammonia to the copper +solution the precipitated suboxid may be kept in solution and the end +of the reaction thus easily distinguished by the disappearance of the +blue color.[77] Allen has improved on this method by covering the hot +mixture with a layer of paraffin oil whereby any oxidation of the +suboxid is prevented.[78] + +The introduction and development of the gravimetric process depending +on securing the reduced copper oxid in a metallic state as developed by +Allihn, Soxhlet, and others, completes the resumé of this brief sketch +of the rise and development of the process. + +=114. Action of Alkaline Copper Solution on Dextrose.=—The action to +which dextrose and other reducing sugars are subjected in the presence +of a hot alkaline copper solution is two-fold in its nature. In the +first place there is an oxidation of the sugar which is transformed +into tartronic, formic and oxalic acids, the two latter in very small +quantities. At the same time another part of the sugar is attacked +directly by the alkali and changed to complex products among which +have been detected lactic, oxyphenic and oxalic acids, also two bodies +isomeric with dioxyphenolpropionic acid. When the sugar is in large +excess melassic and glucic acids have also been detected. The glucic +acid may be regarded as being formed by simple dehydration but becomes +at once resolved into pyrocatechin and gluconic acid according to +the reaction C₁₂H₁₈O₉ = C₆H₆O₂ + C₅H₁₂O₇. The gluconic acid also is +decomposed and gives birth to lactic and glyceric acids according to +the formula C₆H₁₂O₇ = C₃H₆O₃ + C₃H₆O₄. The glyceric acid also in the +presence of a base is changed into lactic and oxalic acids. Between +lactic acid and pyrocatechin, existing in a free state, there is +produced a double reciprocal etherification in virtue of which there +arise two ethers isomeric with hydrocaffeic acid, C₉H₁₀O₄. One of these +bodies is an acid and corresponds to the constitution + + CH₃ + / + O ── CH + / \ + C₆H₄ CO₂H (2) + \ + OH (2) + +and the other is of an alcoholic nature corresponding to the formula + + CO₂ ── CHOH ── CH₃ (1) + / + C₆H₄ + \ + OH₂ (2) + +Of all these products only oxyphenic and lactic acids and their ethers +and oxalic acid remain unchanged and they can be isolated. All the +others are transformed in an acid state and they can only be detected +by operating in the presence of metallic oxids capable of precipitating +them at the time of their formation.[79] + +=115. Fehling’s Solution.=—The copper solution which has been most used +in the determination of reducing sugars is the one proposed by Fehling +as a working modification of the original reagent used by Trommer.[80] + +Following is the formula for the preparation of the fehling solution: + + Pure crystallized copper sulfate CuSO₄.5H₂O, 34.64 grams: + Potassium tartrate, 150.00 ” + Sodium hydroxid, 90.00 ” + +The copper sulfate is dissolved in water and the potassium tartrate in +the aqueous solution of the sodium hydroxid which should have a volume +of about 700 cubic centimeters. The two solutions are mixed and the +volume completed to a liter. Each cubic centimeter of this solution +will be reduced by five milligrams of dextrose, equivalent to four and +a half milligrams of sucrose. + +The reaction which takes place is represented by the following +molecular proportions: + + C₆H₁₂O₆ = 10CuSO₄.5H₂O + Dextrose. Copper sulfate. + 180 2494 + +Fehling’s solution is delicate in its reactions but does not keep well, +depositing cuprous oxid on standing especially in a warm place exposed +to light. The fehling liquor was soon modified in its constitution by +substituting 173 grams of the double sodium and potassium tartrate for +the neutral potassium tartrate first used, and, in fact, the original +fehling reagent contained forty grams of copper sulfate instead of the +quantity mentioned above. Other proportions of the ingredients are also +given by many authors as fehling solution. + +=116. Comparison of Copper Solutions for Oxidizing Sugars.=—For the +convenience of analysts there is given below a tabular comparison +of the different forms of fehling liquor which have been proposed +for oxidizing sugars. The table is based on a similar one prepared +by Tollens and Rodewald, amended and completed by Horton.[81] The +solutions are arranged alphabetically according to authors’ names: + + 1. _Allihn_: + + 34.6 grams copper sulfate, solution made up to half a liter; 173 + grams potassium-sodium tartrate; 125 grams potassium hydroxid + (equivalent to 89.2 grams sodium hydroxid) solution made up to + half a liter. + + 2. _A. H. Allen_: + + 34.64 grams copper sulfate, solution made up to 500 cubic + centimeters; 180 grams potassium-sodium tartrate; 70 grams sodium + hydroxid (not less than 97° NaOH), solution made up to half a + liter. + + 3. _Bödeker_: + + 34.65 grams copper sulfate; 173 grams potassium-sodium tartrate; + 480 cubic centimeters sodium hydroxid solution, 1.14 specific + gravity; 67.3 grams sodium hydroxid; fill to one liter; 0.180 gram + grape sugar reduces according to Bödeker, 36.1 cubic centimeters + of the copper solution = 0.397 gram copper oxid. The same quantity + of milk sugar reduces, however, only twenty-seven cubic + centimeters copper solution = 0.298 copper oxid. + + 4. _Boussingault_: + + 40 grams copper sulfate; 160 grams potassium tartrate; 130 grams + sodium hydroxid. + + 5. _Dietzsch_: + + 34.65 grams copper sulfate; 150 grams potassium-sodium tartrate; + 250 grams sodium hydroxid solution, 1.20 specific gravity; 150 + grams glycerol. + + 6. _Fleischer_: + + 69.278 grams copper sulfate dissolved in about half a liter of + water, add to this 200 grams tartaric acid; fill to one liter with + concentrated sodium hydroxid solution; twenty cubic centimeters + copper solution = forty cubic centimeters sugar solution, that + contain in every cubic centimeter five milligrams grape sugar. + + 7. _Fehling_: + + 40 grams copper sulfate; 160 grams di-potassium tartrate = 600-700 + cubic centimeters sodium hydroxid solution, 1.12 specific gravity, + or from 54.6 to 63.7 grams sodium hydroxid, fill to 1154.4 cubic + centimeters. + + 8. _Gorup-Besanez_: + + 34.65 grams copper sulfate; 173 grams potassium-sodium tartrate; + 480 cubic centimeters sodium hydroxid solution, 1.14 specific + gravity; equal 67.3 sodium hydroxid. Fill to one liter. + + 9. _Grimaux_: + + 40 grams copper sulfate; 160 grams potassium-sodium tartrate; + 600-700 cubic centimeters sodium hydroxid solution, 1.20 specific + gravity, equal to 92.5-107.9 grams sodium hydroxid. Fill to + 1154.4 cubic centimeters. Ten cubic centimeters of this solution + are completely decolorized by 0.050 gram glucose. + + 10. _Holdefleis_: + + 34.632 grams copper sulfate in one liter of water; 125 grams + potassium hydroxid, equivalent to 89.2 grams sodium hydroxid; 173 + grams potassium-sodium tartrate. Fill to one liter. + + 11. _Hoppe-Seyler_: + + 34.65 grams copper sulfate; 173 grams potassium-sodium tartrate; + 600-700 cubic centimeters sodium hydroxid solution, 1.12 specific + gravity; equal to 63.0-73.5 grams potassium hydroxid. Fill to + one liter. One cubic centimeter is reduced by exactly 0.005 gram + grape sugar. + + 12. _Krocker_: + + 6.28 grams copper sulfate; 34.6 grams potassium-sodium tartrate; + 100 cubic centimeters sodium hydroxid solution, 1.14 specific + gravity. Fill to 200 cubic centimeters. In 100 cubic centimeters + of this solution is contained 0.314 gram copper sulfate, which is + reduced by 0.050 grape sugar. + + 13. _Liebermann_: + + 4 grams copper sulfate; 20 grams potassium-sodium tartrate; 70 + grams sodium hydroxid solution, 1.12 specific gravity. Fill to + 115.5 cubic centimeters. + + 14. _Löwe_: + + 15 grams copper sulfate; 60 grams glycerol; 80 cubic centimeters + sodium hydroxid, 1.34 specific gravity; 160 cubic centimeters + water. Fill to half a liter. + + 15. _Mohr_: + + 34.64 copper sulfate; 150 grams di-potassium tartrate; 600-700 + cubic centimeters sodium hydroxid solution, 1.12 specific + gravity, equal to 70.5-82.3 grams sodium hydroxid. Fill to one + liter. + + 16. _Märcker_: + + 35 grams copper sulfate, solution made up to one liter: 175 + grams potassium-sodium tartrate; 125 grams potassium hydroxid, + equivalent to 89.2 grams sodium hydroxid, solution made up to one + liter. + + 17. _Maumenè_: + + 375 grams copper sulfate; 188 grams potassium-sodium tartrate; + 166 grams potassium hydroxid. Fill to nine liters. + + 18. _Monier_: + + 40 grams copper sulfate; 3 grams stannic chlorid; 80 grams cream + of tartar; 130 grams sodium hydroxid. Fill to one liter. + + 19. _Neubauer and Vogel_: + + 34.639 grams copper sulfate; 173 grams potassium-sodium tartrate; + 500-600 grams sodium hydroxid solution, 1.12 specific gravity. + Fill to one liter. + + 20. _Pasteur_: + + 40 grams copper sulfate; 105 grams tartaric acid; 80 grams + potassium hydroxid; 130 grams sodium hydroxid. + + 21. _Possoz_: + + 40 grams copper sulfate; 300 grams potassium-sodium tartrate; + 29 grams sodium hydroxid; 159 grams sodium bicarbonate, allow + to stand six months before use. Fill to one liter. One cubic + centimeter equals 0.0577 gram dextrose. One cubic centimeter + equals 0.0548 gram cane sugar. + + 22. _Rüth_: + + 34.64 grams copper sulfate; 143 grams potassium-sodium tartrate; + 600-700 cubic centimeters sodium hydroxid solution, 1.12 specific + gravity. Fill to one liter. + + 23. _Rodewald and Tollens_: + + 34.639 grams copper sulfate, solution made up to half a liter; + 173 grams potassium-sodium tartrate; 60 grams sodium hydroxid, + solution made up to half a liter. + + 24. _Schorlemmer_: + + 34.64 grams copper sulfate; 200 grams potassium-sodium tartrate; + 600-700 cubic centimeters sodium hydroxid solution, 1.20 specific + gravity. Fill to one liter. + + 25. _Soxhlet_: + + 34.639 grams copper sulfate, solution made up to half a liter. + 173 grams potassium-sodium tartrate; 51.6 grams sodium hydroxid, + solution made up to half a liter. + + 26. _Soldaini_: + + 3.464 grams copper sulfate; 297 grams potassium bicarbonate. Fill + to one liter. + + 27. _Violette_: + + 34.64 grams copper sulfate; 187.0 potassium-sodium tartrate; 78.0 + sodium hydroxid made up to one liter. Ten cubic centimeters equal + 0.050 gram dextrose. Ten cubic centimeters equal 0.0475 gram cane + sugar. + +=117. Volumetric Method used in this Laboratory.=—The alkaline copper +solution preferred in this laboratory has the composition proposed by +Violette. The copper sulfate and alkaline tartrate solutions are kept +in separate vessels and mixed in proper proportions immediately before +use, and diluted with about three volumes of water. The reduction is +accomplished in a long test tube at least twenty-five centimeters in +length, and from thirty-five to forty millimeters in diameter. + +The sugar solution employed should contain approximately one per cent +of reducing sugar. If it should have a greater content it should be +reduced with water to approximately the one named. If it have a less +content, it should be evaporated in a vacuum at a low temperature +until it reaches the strength mentioned above. A preliminary test +will indicate almost the exact quantity of the sugar solution to be +added to secure a complete reduction of the copper. This having been +determined the whole quantity should be added at once to the boiling +copper solution, the test tube held in the open flame of a lamp +giving a large circular flame and the contents of the tube kept in +brisk ebullition for just two minutes. The lamp is withdrawn and the +precipitated suboxid allowed to settle. If a distinct blue color remain +an additional quantity of the sugar solution is added and again boiled +for two minutes. When the blue coloration is no longer distinct, the +presence or absence of copper is determined by aspirating a drop or +two of the hot solution with the apparatus described below. This clear +filtered liquor is then brought into contact with a drop of potassium +ferrocyanid solution acidulated with acetic. The production of a brown +precipitate or color indicates that some copper is still present, +in which case an additional quantity of the sugar solution is added +and the operation continued as described above until after the last +addition of sugar solution no coloration is produced. + +=118. The Filtering Tube.=—The filtering tube used in the above +operation is made of a long piece of narrow glass tubing with thick +walls. The length of the tube should be from forty to forty-five +centimeters. One end of the tube being softened in the flame is pressed +against a block of wood so as to form a flange. Over this flange is +tied a piece of fine linen.[82] + +Instead of using a linen diaphragm the tube is greatly improved, as +suggested by Knorr, by sealing into the end of the tube while hot a +perforated platinum disk. Before using, the tube is dipped into a +vessel containing some suspended asbestos felt and by aspiration a thin +felt of asbestos is formed over the outer surface of the platinum disk. +By inverting the tube the water which has entered during aspiration is +removed. The tube thus prepared is dipped into the boiling solution in +the test tube above described and aspiration continued until a drop of +the liquor has entered the tube. It is then removed from the boiling +solution, the asbestos felt wiped off with a clean towel, and the drop +of liquor in the tube blown through the openings in the platinum disk +and brought into contact with a drop of potassium ferrocyanid in the +usual way. In this way a drop of the liquor is secured without any +danger of a reoxidation of the copper which may sometimes take place on +cooling. + +[Illustration: FIGURE 42. APPARATUS FOR THE VOLUMETRIC ESTIMATION OF +REDUCING SUGARS.] + +The careful analyst by working in this way with the volumetric method +is able to secure highly accurate results. The apparatus used is shown +in the accompanying illustration. + +=119. Suppression of the Error Caused by the Action of the Alkali on +Reducing Sugars.=—Three methods are proposed by Gaud for correcting +or suppressing the error due to the action of the alkali upon +reducing sugars. In the first place, the common method followed may +be employed, depending upon the use of an alkaline copper solution of +known composition and the employment of a reducing sugar solution of +a strength varying between one-half and one per cent. The error which +is introduced into such a reaction is a constant one and the solution +having been tested once for all against pure sugar is capable of giving +fairly accurate results. + +In the second place, a table may be constructed in which the error +is determined for sugar solutions for varying strengths, _viz._, +from one-tenth of one per cent to ten per cent. If _y_ represent the +error and _x_ the exact percentage of reducing sugar present then the +correction may be made by the following formula; + + _y_ = -0.00004801_x_ + 0.02876359_x_². + +In order to use this formula in practice the percentage of reducing +sugar obtained by the actual analysis must be introduced and may be +represented by θ. The formula for correction then becomes 0.02876_x_² - +1.000048_x_ + θ = 0; whence the value of _x_ is easily computed. + +In the third place, the error may be eliminated by substituting for +an alkali which acts upon the glucose one which does not, _viz._, +ammonia. At the temperature of boiling water ammonia does not have any +decomposing effect upon reducing sugars. It is important, however, +that the reduction take place in an inert atmosphere in order to +avoid the oxidation of the dissolved cuprous oxid and the temperature +need not be carried beyond 80°. The end of the reaction can be easily +distinguished in this case by the disappearance of the blue color. When +one reaction is finished the copper may be completely reoxidized by +conducting through it a current of air or oxygen for half an hour, when +an additional quantity of ammonia may be added to supply any that may +have evaporated, and a new reduction accomplished with exactly the same +quantity of copper as was used in the first. The solution used by Gaud +contains 36.65 grams of crystallized copper sulfate dissolved in water +and the volume completed to one liter with ordinary aqueous ammonia.[83] + +=120. Permanganate Process for the Estimation of Reducing +Sugars.=—Dextrose, invert sugar, and other reducing sugars can also be +determined with a fair degree of accuracy by an indirect volumetric +process, in which a standard solution of potassium permanganate is +used as the final reagent.[84] The principle of the process is based +upon the observation that two molecules of dextrose reduce from an +alkaline cupric tartrate solution five molecules of cuprous oxid. +The five molecules of cuprous oxid thus precipitated when added to an +acid solution of ferric sulfate, will change five molecules of the +ferric sulfate to ten molecules of ferrous sulfate. The reaction is +illustrated by the following equation: + + { 5Cu₂O } + { 5Fe₂(SO₄)₃ } + { 5H₂SO₄ } = { 10CuSO₄ } + {715 parts} { 2000 parts } {490 parts} {1595 parts} + + + { 10FeSO₄ } + { 5H₂O } + {1520 parts} {90 parts} + +The ten molecules of ferrous sulfate formed as indicated in the above +reaction, are reoxidized to ferric sulfate by a set solution of +potassium permanganate. This reaction is illustrated by the equation +given below: + + { 10FeSO₄ } + { K₂Mn₂O₈ } + { 5H₂SO₄ } = { 5Fe₂(SO₄)₂ } + {1520 parts} {316.2 parts} {784 parts} { 2000 parts } + + + { 2MnSO₄ } + { K₂SO₄ } + { 8H₂O } + {302 parts} {174.2 parts} {144 parts}. + +By the study of the above equations it is seen that two molecules +of dextrose or other similar reducing sugar, are equivalent to one +molecule of potassium permanganate, as is shown by the following +equations: + + { 2C₆H₁₂O₆ } = { 5Cu₂O } = { 10FeSO₄ } = { K₂Mn₂O₈ } + { 360 parts } {715 parts} {1520 parts} {316.2 parts} + +It is thus seen that 316.2 parts by weight of potassium permanganate +are equivalent to 360 parts by weight of dextrose; or one part of +permanganate corresponds to 1.1385 parts by weight of dextrose. If, +therefore, the amount of permanganate required in the above reaction to +restore the iron to the ferric condition, be multiplied by the factor +mentioned above, the quotient will represent in weight the amount of +dextrose which enters into the reaction. The standard solution of +potassium permanganate should contain 4.392 grams of the salt in a +liter. One cubic centimeter of this solution is equivalent to five +milligrams of dextrose. + +=121. Manipulation.=—The saccharine solution whose strength is to be +determined should contain approximately about one per cent of sugar. +Of this solution ten cubic centimeters are placed in a porcelain dish +together with a considerable excess of fehling solution. When no +sucrose is present, the mixture may be heated to the temperature of +boiling water and kept at that temperature for a few minutes until +all the reducing sugar is oxidized. There should be enough of the +copper solution used to maintain a strong blue coloration at the end +of the reaction. A greater uniformity of results will be secured by +using in all cases a considerable excess of the copper solution. When +sucrose or other non-reducing sugars are present, the temperature of +the reaction should not be allowed to exceed 80° and the heating may +be continued somewhat longer. At this temperature the copper solution +is absolutely without action on sucrose. The precipitated suboxid is +allowed to settle, the supernatant liquid poured off through a filter +and the suboxid washed thoroughly a number of times by decantation with +hot water, the washings being poured through the filter. This process +of washing is greatly facilitated by decanting the supernatant liquid +from the porcelain dish first into a beaker and from this into a third +beaker and so on until no suboxid is carried off. Finally the wash +water is poured through a filter-paper bringing as little as possible +of the suboxid onto the paper. The suboxid on the filter-paper and in +the beakers is next dissolved in a solution of ferric sulfate made +strongly acid with sulfuric; or in a sulfuric acid solution of ammonia +ferric sulfate which is more easily obtained free from impurities than +the ferric sulfate. When all is dissolved from the beakers the solution +is poured upon the suboxid which still remains in the porcelain dish. +When the solution is complete it is washed into a half liter flask and +all the vessels which contain the suboxid are also thoroughly washed +and the wash waters added to the same flask. The whole is rendered +strongly acid with sulfuric and made up to a volume of half a liter. + +The process carried out as directed, when tested against pure sugar, +gives good results, not varying from the actual content of the sugar +by more than one-tenth per cent below or three-tenths above the true +content. The distinct pink coloration imparted to the solution by the +permanganate solution as soon as the iron is all oxidized to the ferric +state marks sharply the end of the reaction. In this respect this +process is very much to be preferred to the usual volumetric processes +depending upon the coloration produced with potassium ferrocyanid +by a copper salt for distinguishing the end of the reaction. It is +less convenient than the ordinary volumetric process by reason of the +somewhat tedious method of washing the precipitated cuprous oxid. When +a large number of analyses is to be made, however, the whole can be +washed with no more expenditure of time than is required for a single +sample. One analyst can, in this way, easily attend to fifty or a +hundred determinations at a time. + +In the application of the permanganate method to the analysis of the +juices of sugar cane and sorghum it is directed to take 100 cubic +centimeters of the expressed juice and clarify by the addition of +twenty-five cubic centimeters of basic lead acetate, diluted with +water, containing enough of the lead acetate, however, to produce a +complete clarification. It is not necessary to remove the excess of +lead from the filtrate before the determination. Ten cubic centimeters +of the filtrate correspond to eight cubic centimeters of the original +juice. For percentage calculation the specific gravity of the original +juice must be known. Before the addition of the alkaline copper +solution, from fifty to seventy-five cubic centimeters of water should +be added to the clarified sugar juice and the amount of fehling +solution used in each case should be from fifty to seventy-five cubic +centimeters. The heating at 75° should be continued for half an hour +in order to insure complete reduction and oxidation of the sugar. The +sucrose can also be estimated in the same juices by inverting five +cubic centimeters of the clarified juice with five cubic centimeters +of dilute hydrochloric acid, by heating for an hour at a temperature +not above 90°. Before adding the acid for inversion, about 100 cubic +centimeters of water should be poured over the five cubic centimeters +of sugar solution. The washing of the suboxid and the estimation of the +amount reduced are accomplished in the manner above described. + +This method has been extensively used in this laboratory and with very +satisfactory results. The only practical objection which can be urged +to it is in the time required for filtering. This fault is easily +remedied by adopting the method of filtering through asbestos felt +described in the next paragraph. + +For the sake of uniformity, however, the copper solution should +be boiled for a few minutes before the addition of the sugar in +order to expel all oxygen, the sugar solutions should be made with +recently boiled water and the precipitation of the suboxid should be +accomplished by heating for just thirty minutes at 75°. At the end of +this time an equal volume of cold, recently boiled, water should be +added and the filtration at once accomplished. + +=122. Modified Permanganate Method.=—The permanganate method as used +by Ewell, in this laboratory, is conducted as follows: After the +precipitate is obtained, according to the directions given in the +methods described, it is thoroughly washed with hot, recently boiled +water, on a gooch. The asbestos, with as much of the precipitate as +possible, is transferred to the beaker in which the precipitation was +made, beaten up with from twenty-five to thirty cubic centimeters +of hot, recently boiled water, and from fifty to seventy-five cubic +centimeters of a saturated solution of ferric sulfate in twenty-five +per cent sulfuric acid are added to the beaker and then poured through +the crucible to dissolve the cuprous oxid remaining therein. If the +precipitate be first beaten up with water as directed, so that no +large lumps of it remain, there is no difficulty in dissolving the +oxid in the ferric salt; while if any lumps of the oxid be allowed to +remain there is great difficulty. After the solution is obtained, it is +titrated with a solution of potassium permanganate of such a strength +that each cubic centimeter is equivalent to 0.01 gram of copper. + +In triplicate determinations made by this method the precipitates +obtained required after solution in the ferric salt, 28.7, 28.9, 28.6 +cubic centimeters of potassium permanganate solution, respectively. +For the quantities taken this was equivalent to an average percentage +of reducing sugars of 4.19. The percentage obtained by the gravimetric +method was 4.26. + +The method seems to be sufficiently accurate for all ordinary purposes +and is extremely rapid. + +The permanganate solution used should be standardized by means +of metallic iron, but in ordinary work it is also recommended to +standardize by check determinations of reducing sugars in the same +sample by the gravimetric method. + +=123. Determination of Reducing Sugar by the Specific Gravity of the +Cuprous Oxid.=—Gaud proposes to determine the percentage of reducing +sugar from the specific gravity of the cuprous oxid. The manipulation +is carried out as follows: + +In a porcelain dish are placed fifty cubic centimeters of the alkaline +copper solution and an equal quantity of water and the mixture +maintained in ebullition for two or three minutes. The dish is then +placed on a boiling water-bath and twenty-five cubic centimeters of a +reducing sugar of approximately one per cent strength added at once. +The reduction is thus secured at a temperature below 100°, which is an +important consideration in securing the minimum decomposing effect of +the alkali upon the sugar. The dish is kept upon the water-bath for +about ten minutes when the reduction is complete and the supernatant +liquor should still be intensely blue. The precipitate is washed by +decantation with boiling water, taking care to avoid the loss of any +of the cuprous oxid. The washing is continued until the wash waters +are neutral to phenolphthalein. The cuprous oxid is then washed into a +pyknometer of from twenty to twenty-five cubic centimeters capacity, +the exact content of which has been previously determined at zero. It +is filled with boiling water, the stopper inserted, and after cooling +the flask is weighed. Let _P_ be the weight of the pyknometer plus the +liquid and the precipitate, the total volume of which is equal to the +capacity of the flask at the temperature at which it was filled, that +is _V_ₜ = _V_₀ [1 + 3β(_t_-_t_₀)]. + +This formula is essentially that given in paragraph =51=, for +calculating the volume of a pyknometer at any temperature, substituting +for 3β, γ the cubical expansion of glass, _viz_., 0.000025. + +The specific gravity of the dry cuprous oxid is Δ = 5.881 and let the +specific gravity of water at the temperature of filling, which can be +taken from any of the tables of the density of water, be _d_. The total +weight _p_ of the precipitated suboxid may then be calculated by the +following formula: + + + _P_-_V_ₜ_d_ + _P_ = -----------. + 1 - _d_ + ---- + Δ + +The density of water at 99°, which is about the mean temperature of +boiling water for laboratories in general, is 0.95934, and this may be +taken as the weight of one cubic centimeter for purposes of calculation +in the formula above. + +In order to obtain exact results, it is important that the weight +_P_ be reduced to a vacuum. The weight of cuprous oxid not varying +proportionally to the weight of reducing sugar, it is necessary to +prepare a table showing the principal numerical values of the two, in +order to be able to calculate easily all the possible values, either +directly from the table or by appropriate interpolations. Following are +the chief values which are necessary for the calculation: + + Milligrams Milligrams Milligrams Milligrams + cuprous oxid. dextrose. cuprous oxid. dextrose. + + 10 5.413 100 46.221 + 20 9.761 200 91.047 + 30 14.197 300 138.842 + 50 23.036 400 188.928 + +It is claimed by the author that the above method is both simple and +rapid and can be applied with an error of not more than one-thousandth +if the corrections for temperature and pressure be rigorously +applied.[85] + +=124. The Copper Carbonate Process.=—While the copper solutions which +have been mentioned in previous paragraphs have only a slight action +on sucrose and dextrin yet on prolonged boiling even these bodies show +a reducing effect due probably to a preliminary change in the sugar +molecules whereby products analogous to dextrose or invert sugar are +formed. In order to secure a reagent, to which the sugar not reducing +alkaline copper solutions might be more resistant Soldaini has proposed +to employ a liquor containing the copper as carbonate instead of +as tartrate.[86] This solution is prepared by adding to a solution +of forty grams of copper sulfate one of equal strength of sodium +carbonate. The resulting copper carbonate and hydroxid are collected on +a filter, washed with cold water, and dried. The reaction which takes +place is represented by the following formula: + + 2CuSO₄ + 2Na₂CO₃ + H₂O = CuCO₃ + CuO₂H₂ + 2Na₂SO₄ + CO₂. + +The dry precipitate obtained, which will weigh about fifteen grams, is +placed in a large flask with about 420 grams of potassium bicarbonate +and 1400 cubic centimeters of water. The contents of the flask are +heated on a steam-bath for several hours with occasional stirring +until the evolution of carbon dioxid has ceased. During this time the +liquid is kept at the same volume by the addition of water, or by +attaching a reflux condenser to the flask. The potassium and copper +compounds at the end of this time will be found dissolved and the +resulting liquor will have a deep blue color. After filtration the +solution is boiled for a few minutes and cooled to room temperature. +The volume is then completed to two liters. A more direct method of +preparing the solution, and one quite as effective, consists in adding +the solution of the copper sulfate directly to the hot solution of +potassium bicarbonate and heating and shaking the mixture until the +copper carbonate formed is dissolved. After filtering the volume is +made as above. The proportions of reagents employed are placed by +Preuss at 15.8 grams of crystallized copper sulfate and 594 grams of +potassium bicarbonate.[87] The soldaini reagent is extremely sensitive +and is capable of detecting as little as half a milligram of invert +sugar. The presence of sucrose makes the reagent more delicate, and it +is especially useful in determining the invert sugar arising during the +progress of manufacture by the action of heat and melassigenic bodies +on sucrose. + +=125. The Analytical Process.=—As in the case of fehling solution a +great many methods of conducting the analysis with the soldaini reagent +have been proposed. The general principle of all these processes is +the one already described for the alkaline copper tartrate solution, +_viz._, the addition of the reducing sugar solution to the boiling +reagent, and the determination of the end of the reaction by the +disappearance of the copper.[88] + +Practically, however, these methods have had no general application, +and the use of the soldaini reagent has been confined chiefly to the +determination of invert sugar in presence of a large excess of sucrose. +For this purpose the sugar solution is not added until the blue color +of the reagent has been destroyed, but on the other hand, the reagent +has been used in excess, and the cuprous oxid formed collected and +weighed as metallic copper. The weight of the metallic copper found, +multiplied by the factor 0.3546, gives the weight of invert sugar in +the volume of the sugar solution used. According to Preuss, the factor +is not a constant one, but varies with the quantity of invert sugar +present, as is seen in the formula _y_ = 2.2868 + 3.3_x_ + 0.0041_x_², +in which _x_ = the invert sugar, and _y_ the metallic copper.[89] + +=126. Tenth Normal Copper Carbonate Solution.=—In the study of some +of the solutions of copper carbonate, proposed for practical work, +Ettore Soldaini was impressed with the difficulty of dissolving so +large a quantity of carbonate in the solvent employed.[90] The solution +recommended by Bodenbender and Scheller,[91] in which forty grams +of the crystallized copper sulfate were used, failed to disclose +an equivalent amount of copper in the reagent ready for use. For +this reason a tenth-normal copper solution is prepared by Soldaini +containing the equivalent of 3.464 grams of copper sulfate in one +liter. The reagent is easily prepared by adding slowly the dissolved +or finely powdered copper salt to a solution of 297 grams of potassium +bicarbonate, and after complete solution of the copper carbonate +formed, completing the volume to one liter. With this reagent as little +as one-quarter of a milligram of reducing sugar can be easily detected. +For the quantitive estimation of sugar a solution of the above strength +is to be preferred to the other forms of the soldaini reagent by reason +of the ease of direct comparison with standard fehling solutions. + +The analytical process is conducted with the tenth-normal solution, +prepared by Soldaini and described above, as follows: Place 100 cubic +centimeters of the reagent in each of several porcelain dishes heat +to boiling, and add little by little the sugar solution to one dish +until the blue color has disappeared. Having thus determined nearly the +exact quantity of sugar solution required for the copper in 100 cubic +centimeters of the reagent the whole of the sugar solution is added at +once, varying slightly the amounts added to each dish. The boiling is +continued for fifteen minutes, and the contents of the dishes poured +on filters. That filtrate which contains neither copper nor sugar +represents the exact quantity of sugar solution which contained fifty +milligrams of dextrose. + +=127. Relation of Reducing Sugar to Quantity of Copper Suboxid +Obtained.=—The relation of the quantity of copper reduced to the amount +of sugar oxidized by the copper carbonate solution has been determined +by Ost, and the utility of the process thereby increased.[92] The +solution used should have the following composition: 23.5 grams of +crystallized copper sulfate, 250 grams of potassium carbonate, and 100 +grams of potassium bicarbonate in one liter. Without an indicator the +end reaction is distinctly marked by the passage of the blue color into +a colorless solution. Ost affirms that this solution is preferable +to any form of fehling liquor because it can be kept indefinitely +unchanged; it attacks sucrose far less strongly, and an equal quantity +of sugar precipitates nearly double the quantity of copper. The boiling +requires a longer time, as a rule ten minutes, but this is a matter of +no importance, when the other advantages are taken into consideration. +The relations of the different sugars to the quantity of copper +precipitated are given in the table in the next paragraph. + +=128. Factor for Different Sugars.=—For pure dextrose the relation +between sugar and copper reduced has been determined by Ost, and the +data are given in the table below. The data were obtained by adding +to fifty cubic centimeters of the copper solution twenty-five cubic +centimeters of sugar solutions of varying strength and collecting, +washing, and reducing the cuprous oxid obtained in a current of +hydrogen in a glass tube by the method described further on. + +The boiling in all cases was continued, just ten minutes, although a +slight variation from the standard time did not produce so great a +difference as with fehling reagent. In the case of dextrose, when fifty +milligrams were used with fifty cubic centimeters of the solution, +the milligrams of copper obtained after six, ten and twenty minutes’ +boiling were 164.6, 165.5, and 166.9 respectively.[93] + +The data differ considerably from those obtained by Herzfeld, but +in his experiments the boiling was continued only for five minutes, +and this is not long enough to secure the proper reduction of the +copper.[94] + + TABLE SHOWING THE QUANTITY OF COPPER REDUCED BY DIFFERENT SUGARS. + + Copper. Invert Sugar. Dextrose. Levulose. Galactose. Arabinose. + Milli- Milli- Milli- Milli- Milli- Milli- + grams. grams. grams. grams. grams. grams. + 50 15.2 15.6 14.7 17.4 17.0 + 55 16.6 17.0 16.1 19.1 18.6 + 60 18.0 18.5 17.5 20.8 20.3 + 65 19.4 19.9 18.9 22.5 21.9 + 70 20.8 21.4 20.3 24.2 23.5 + 75 22.3 22.9 21.7 25.9 25.1 + 80 23.7 24.4 23.0 27.7 26.7 + 85 25.2 25.8 24.3 29.3 28.3 + 90 26.6 27.3 25.7 31.1 29.9 + 95 28.1 28.8 27.1 32.8 31.5 + 100 29.5 30.3 28.5 34.5 33.1 + 105 31.0 31.8 29.9 36.2 34.7 + 110 32.4 33.3 31.2 38.0 36.3 + 115 33.9 34.8 32.6 39.7 37.9 + 120 35.3 36.3 34.0 41.4 39.5 + 125 36.8 37.8 35.4 43.1 41.1 + 130 38.2 39.3 36.8 44.8 42.8 + 135 39.7 40.8 38.2 46.5 44.4 + 140 41.1 42.3 39.6 48.3 46.0 + 145 42.6 43.8 41.0 50.0 47.6 + 150 44.0 45.3 42.5 51.8 49.3 + 155 45.5 46.8 43.9 53.6 50.9 + 160 47.0 48.3 45.3 55.4 52.6 + 165 48.5 49.8 46.7 57.2 54.3 + 170 50.0 51.4 48.1 59.0 55.9 + 175 51.5 52.9 49.5 60.8 57.5 + 180 53.0 54.5 51.0 62.7 59.2 + 185 54.5 56.0 52.5 64.5 60.9 + 190 56.0 57.6 54.0 66.4 62.7 + 195 57.5 59.2 55.5 68.3 64.4 + 200 59.1 60.8 57.0 70.3 66.2 + 205 60.7 62.4 58.6 72.3 68.0 + 210 62.4 64.1 60.2 74.3 69.8 + 215 64.1 65.8 61.8 76.3 71.6 + 220 65.8 67.5 63.5 78.3 73.5 + 225 67.5 69.2 65.2 80.3 75.4 + 230 69.3 70.9 66.9 82.4 77.3 + 235 71.1 72.7 68.7 84.5 79.3 + 240 72.9 74.5 70.6 86.6 81.3 + 245 74.8 76.4 72.5 88.9 83.4 + 250 76.7 78.4 74.4 91.2 85.5 + 255 78.6 80.5 76.5 93.5 87.6 + 260 80.5 82.8 78.8 95.9 89.8 + 265 82.5 85.1 81.1 98.3 92.2 + 270 84.7 87.5 83.5 100.7 94.6 + 275 87.1 89.9 85.9 103.3 97.1 + 280 89.7 92.4 88.6 106.1 99.6 + 285 92.3 94.9 91.3 109.0 102.3 + 290 95.1 97.6 94.2 112.0 105.1 + 295 98.0 100.4 97.2 115.1 107.9 + 298 100.0 102.5 99.0 117.0 109.5 + + +VOLUMETRIC METHODS BASED UPON THE USE OF AN AMMONIACAL COPPER SOLUTION. + +=129. Pavy’s Process.=—The well-known solubility of cuprous oxid in +ammonia led Pavy to adopt a copper reagent containing ammonia in the +volumetric determination of reducing sugars.[95] In Pavy’s process an +alkaline copper solution is employed made up in the usual way, to which +a sufficient quantity of ammonia is added to hold in solution all the +copper when precipitated as cuprous oxid. The solution used by Pavy has +the following composition: One liter contains + + Crystallized copper sulfate 34.65 grams + Potassium-sodium tartrate 173.00 ” + Caustic potash 160.00 ” + +For use 120 cubic centimeters of the above reagent are mixed with 300 +of ammonia of specific gravity 0.88, and the volume completed to one +liter with distilled water. Twenty cubic centimeters of this reagent +are equivalent to ten milligrams of dextrose or invert sugar when added +in a one per cent solution. + +In the use of ammoniacal copper solution, care must be taken that +all the liquids employed be entirely free of oxygen and that the +contents of the flask in which the reduction takes place be in some way +excluded from contact with the air. Pavy secured this by conducting +the reduction in a flask closed with a stopper carrying two holes; +one of these served for the introduction of the burette carrying the +sugar solution and the other carried a tube dipping into a water seal +by means of a slit rubber tube, which would permit of the exit of the +vapors of steam and ammonia, but prevent the regurgitation of the water +into the flask. + +The complete decoloration of the copper solution marks the end of the +reaction. The usual precautions in regard to the length of the time of +boiling must be observed. + +It is easy to see that in the Pavy process the quantity of ammonia in +the solution is rapidly diminished during the boiling and this has led +to the suggestion of other methods to exclude the air. Among these +have been recommended the introduction of a current of hydrogen or +carbon dioxid. One of the best methods of procedure is that proposed +by Allen, who recommends covering the copper solution by a layer of +paraffin oil (kerosene).[96] + +=130. Process Of Peska.=—Peska has also independently made use of +Allen’s method of covering the solutions with a layer of paraffin oil +and finds it reliable.[97] The copper reagent employed by him has the +following composition: + + Crystallized copper sulfate 6.927 grams + Ammonia, twenty-five per cent strength 160.00 cc. + +The copper sulfate is dissolved in water, the ammonia added, and +the volume completed to half a liter with distilled water. A second +solution containing half a liter is made by dissolving 34.5 grams +of potassium-sodium tartrate and ten grams of sodium hydroxid and +completing the cool solution to half a liter with distilled water. +In all cases the water used in making up the above solutions must be +freshly boiled to exclude the air. + +For the titration, fifty cubic centimeters of each of the above +solutions are taken, mixed and covered with a layer of paraffin oil +half a centimeter in depth. The reduction is not accomplished at a +boiling temperature, but at from 80° to 85°. The manipulation is +conducted as follows: + +The mixed solutions are placed in a beaker, covered with oil, and +heated to 80°. The temperature is measured by a thermometer which also +serves as a stirring rod. The sugar solution is run down the sides +of the beaker from a burette of such a shape as to be protected from +the heat. After each addition of the sugar solution the mixture is +carefully stirred, keeping the temperature at from 80° to 85°. The +first titration is made to determine approximately the quantity of +sugar solution necessary to decolorize the copper. This done, the +actual titration is accomplished by adding at once the total amount +of sugar solution necessary to decolorize, less about one cubic +centimeter. Any sugar solution adhering to the side of the beaker +is washed down by distilled water, the contents of the beaker well +stirred, and the temperature kept at 85° for two minutes. The rest of +the sugar solution is then added in quantities of one-tenth of a cubic +centimeter until the decoloration is completed. The total time of the +final titration should not exceed five minutes. The sugar solution +should be as nearly as possible of one per cent strength. If a lower +degree of strength be employed a larger quantity of the sugar is +necessary to reduce a given quantity of copper. + +In the case of dextrose, when a one per cent solution is used, eight +and two-tenths cubic centimeters, corresponding to 80.2 milligrams of +dextrose, are required to reduce 100 cubic centimeters of the mixed +reagent. On the other hand, when the sugar solution is diluted to +one-tenth of a per cent strength 82.1 milligrams are required. + +With invert sugar slightly larger quantities are necessary, the +reducing power being as 94.9 to 100 as compared with dextrose. With +a one per cent strength of invert sugar it is found that eighty-four +milligrams are required to reduce 100 cubic centimeters of the mixed +reagent and when the strength of the invert sugar is reduced to +one-tenth per cent 87.03 milligrams are required. + +=131. Method Of Allein and Gaud.=—Allein and Gaud have proposed a +further modification of the ammonia process which consists essentially +in the suppression of rochelle salt and fixed caustic alkali and the +entire substitution therefor of ammonia. Ammonia acts with much less +vigor upon sugars than the caustic alkalies, and it is therefore +claimed that the decomposition of the sugar due to the alkali is +reduced to a minimum when ammonia is employed.[98] The copper solution +is made as follows: + +Dissolve 8.7916 grams of electrolytic copper in ninety-three grams +of concentrated sulfuric acid diluted with an equal volume of water. +Complete the resulting solution to one liter with concentrated ammonia. +Ten cubic centimeters of this solution are equal to fifty grams of +dextrose. + +It is recommended that the reduction be accomplished in an atmosphere +of hydrogen, but it is apparent that the use of kerosene is permissible +in this case, and on account of its greater simplicity it is to be +recommended as the best means of excluding the oxygen. The reduction is +accomplished at a temperature of about 80°. + +It is also proposed to reoxidize the copper by substituting a current +of air for the hydrogen at the end of the reaction, and thus use the +same copper a number of times. The danger of loss of ammonia, and the +difficulty of determining when the oxidation is complete, render this +regeneration of the reagent undesirable. + +=132. Method of Gerrard.=—The method of Gerrard does not depend upon +the use of ammonia, but the principle involved is the same, _viz._, the +holding of the separated cuprous oxid in solution and the determination +of the end of the reaction by the disappearance of the blue color. +As first proposed by Gerrard, the copper sulfate solution is made of +double the strength usually employed and to each 100 cubic centimeters +thereof, before use, three and three-tenths grams of potassium cyanid +are added. This is sufficient to hold the precipitated cuprous oxid in +solution.[99] + +The original method of Gerrard is found difficult of execution and the +author, in conjunction with Allen, has lately modified it and reduced +it to a practical working basis.[100] + +In the new method the ordinary fehling solution is employed and it +is prepared for use in the following way: Ten cubic centimeters of +the fehling solution, or half that quantity of each of the component +parts kept in separate bottles, are placed in a porcelain dish with +forty cubic centimeters of water and brought to the boiling-point. To +the boiling liquid is added, from a pipette, a five per cent solution +of potassium cyanid until the blue color just disappears, or only a +very faint tint of blue remains, avoiding any excess of the cyanid. A +second portion of the fehling solution equal to that first employed +is added, and to the boiling mixture the solution of sugar is added, +from a burette, until the blue color disappears. The contents of the +dish should be kept boiling during the addition of the sugar solution. +The volume used will contain fifty milligrams of dextrose. The sugar +solution should be of such a strength as to contain no more than half a +per cent of reducing sugar. + +The principle of the preparation of the solution may be stated as +follows: If to a solution of copper sulfate, potassium be added until +the blue color disappears, a double cyanid of copper and potassium +cyanid is formed according to the following reaction: CuSO₄ + 4KCN = +Cu(CN)₂.2KCN + K₂SO₄. This double cyanid is a salt of considerable +stability. It is not decomposed by alkalies, hydrogen or ammonium, +sulfid. With mineral acids it gives a whitish, curdy precipitate. With +fehling solution the same double cyanid is formed as that described +above. If, however, fehling solution be present in excess of the amount +necessary to form the double cyanid of copper, this excess can be used +in the oxidation of reducing sugar and the colorless condition of the +solution will be restored as soon as the excess of the fehling is +destroyed. The double cyanid holds in solution the cuprous oxid formed +and thus complete decoloration is secured. + +=133. Sidersky’s Modification of Soldaini’s Process.=—In all cases +where the sugar solutions are not too highly colored, Sidersky finds +that the method of reduction in a large test tube, as practiced by +Violette, is applicable with the copper carbonate solution.[101] For +more exact work it is preferred to determine the quantity of copper +reduced by an indirect volumetric method. The sugar solution, properly +clarified and the lead removed if subacetates have been used, is made +of such a volume as to contain less than one per cent of reducing +sugars. In a flask or large test tube are placed 100 cubic centimeters +of the copper solution, which is boiled for a short time and the sugar +solution added, little by little, from a pipette, at such a rate as +not to stop the ebullition. The boiling is continued for five minutes +after the last addition of the sugar. The vessel is taken from the +flame and 100 cubic centimeters of cold water added, the whole brought +on an asbestos felt and the cuprous oxid washed with hot water until +the alkaline reaction has disappeared. The residual cuprous oxid +is dissolved in a measured quantity of set sulfuric acid, semi- or +fifth-normal, a few particles of potassium chlorate added, and the +mixture boiled to convert any cuprous into cupric sulfate. The reaction +is represented by the following formula: + + 3Cu₂O + 6H₂SO₄ + KClO₃ = 6CuSO₄ + KCl + 6H₂O. + +The residual sulfuric acid is titrated with a set alkali in excess, +ammonia being preferred. + +The solution of ammonia is made by diluting 200 cubic centimeters of +commercial aqua ammonia with 800 of water. Its strength is determined +by adding a little copper sulfate solution as indicator and then +the set solution of sulfuric acid until the blue color disappears. +The copper sulfate secured from the cuprous sulfate as described +above is cooled, and a quantity of the ammonia, equal to twenty-five +cubic centimeters of the set sulfuric acid, added. The excess of the +ammonia is then determined by titration with the sulfuric acid, the +disappearance of the blue color being the indication of the end of the +reaction. The number of cubic centimeters of the set sulfuric acid +required to saturate the ammonia represents the equivalent of cuprous +oxid originally present. One cubic centimeter of normal sulfuric acid +is equivalent to 0.0317 gram of metallic copper. + +To determine the weight of invert sugar oxidized, multiply the weight +of copper, calculated as above described, by the factor 0.3546.[102] +For a general application of this method of analysis the relative +quantities of copper reduced by different quantities of sugar must be +taken into consideration. + +While, as has already been stated, the copper carbonate process has +heretofore been applied chiefly to the detection of invert sugar, it +has merits which justify the expectation that it may some time supplant +the fehling liquor both for volumetric and gravimetric work. Large +volumes of the reagent can be prepared at once and without danger of +subsequent change. The action of the reagent on the hexobioses and +trioses is far less vigorous than that of the alkaline copper tartrate, +and the end reactions for volumetric work are, at least, as easily +determined in the one case as the other. + +=134. Method Depending on Titration of Excess of Copper.=—Instead of +measuring the quantity of copper reduced, either by its disappearance +or by reducing the cuprous oxid to a metallic state, Politis has +proposed a method of analysis depending on the titration of the +residual copper.[103] The reagents employed are: + +(1) A copper solution containing 24.95 grams of crystallized copper +sulfate, 140 grams of sodium and potassium tartrate, and twenty-five +grams of sodium hydroxid in one liter: + +(2) A solution of sodium thiosulfate containing 24.8 grams of the salt +in one liter: + +(3) A solution of potassium iodid containing 12.7 grams of iodin in one +liter. + +The reaction is represented by the formula + + 2CuCl₂ + 4KI = Cu₂I₂ + 4KCl + I₂. + +The analytical process is carried out as follows: In a 100 cubic +centimeter flask are boiled fifty cubic centimeters of the copper +solution, ten cubic centimeters of about one-tenth per cent reducing +sugar solution are added, the boiling continued for five minutes, +the flask filled to the mark with boiling water and its contents +filtered. Fifty cubic centimeters of the hot filtrate are cooled, +slightly acidified, potassium iodid solution added in slight excess; +and the iodin set free determined by titration with sodium thiosulfate. +The quantity of iodin obtained corresponds to the unreduced copper +remaining after treatment with the reducing sugar. The number of cubic +centimeters of thiosulfate used subtracted from twenty-five will give +the number of cubic centimeters of the copper solution which would be +reduced by five cubic centimeters of the sugar solution used. + +_Example._—In the proportions given above it was found that eleven +cubic centimeters of thiosulfate were required to saturate the iodin +set free. Then 25 - 11 = 14 cubic centimeters of copper solution +reduced by five cubic centimeters of the sugar solution. Since one +cubic centimeter of the copper solution is reduced by 0.0036 gram of +dextrose the total dextrose in the five cubic centimeters = 0.0036 × 5 += 0.0180 gram. + +The above method does not seem to have any practical advantage over +those based on noting the disappearance of the copper and is given only +to illustrate the principle of the process. While the titration of the +iodin by sodium thiosulfate is easily accomplished in the absence of +organic matter, it becomes difficult, as shown by Ewell, when organic +matters are present, as they always are in the oxidation of a sugar +solution. Ewell has therefore proposed to determine the residual copper +by a standard solution of potassium cyanid, but the method has not yet +been developed.[104] + + +GRAVIMETRIC COPPER METHODS. + +=135. General Principles.=—In the preceding pages the principles +of the volumetric methods of sugar analysis by means of alkaline +copper solution have been set forth. They depend either on the total +decomposition of the copper solution employed by the reducing sugar, +or else on the collection and titration of the cuprous oxid formed in +the reaction. In the gravimetric methods the general principle of the +process rests upon the collection of the cuprous oxid formed and its +reduction to metallic copper, the weight of which serves as a starting +point in the calculations of the weight of reducing sugar, which has +been oxidized in the solution. + +The factors which affect the weight of copper obtained are essentially +those which influence the results in the volumetric method. The +composition of the copper solution, the temperature at which the +reduction is accomplished, the time of heating, the strength of the +sugar solution and the details of the manipulation, all affect more or +less the quantity of copper obtained. As in the volumetric method also, +the kind of reducing sugar must be taken in consideration, dextrose, +levulose, invert sugar, maltose and other sugars having each a definite +factor for reduction in given conditions. It follows, therefore, that +only those results are of value which are obtained under definite +conditions, rigidly controlled. + +=136. Gravimetric Methods of the Department of Agriculture +Laboratory.=—The process used in this laboratory is based essentially +on the methods of Maercker, Behrend, Morgen, Meissl, Hiller and +Allihn.[105] Where dextrose alone is present, the table of factors +proposed by Allihn is used and also the copper solution corresponding +thereto. + +For pure invert sugar, the tables and solutions of Meissl are used. For +invert sugar in the presence of sucrose, the table and process proposed +by Hiller are used. + +[Illustration: FIGURE 43. APPARATUS FOR THE ELECTROLYTIC DEPOSITION OF +COPPER.] + +The reduction of the copper solution and the electrolytic deposition of +the copper are accomplished as follows: + +The copper and alkali solutions are kept in separate bottles. After +mixing the equivalent volume of the two solutions in a beaker, heat is +applied and the mixture boiled. To the boiling liquid the proper volume +of the cold sugar solution is added. This must always be less than the +amount required for complete reduction. The solution is again brought +into ebullition and kept boiling exactly two minutes. A two-minute +sand glass is conveniently used to determine the time of boiling. At +the end of this time an equal volume of freshly boiled cold water is +added, and the supernatant liquor at once passed through a gooch under +pressure. The residual cuprous oxid is covered with boiling water and +washed by decantation until the wash water is no longer alkaline. It +is more convenient to wash in such a way that, at the end, the greater +part of the cuprous oxid is in the gooch. The felt and cuprous oxid are +then returned to the beaker in which the reduction is made. The gooch +is moistened with nitric acid to dissolve any adhering oxid and then +is washed into the beaker. Enough nitric acid is added to bring all +the oxid into solution, an excess being avoided, and a small amount +of water added. The mixture is again passed under pressure through +a gooch having a thin felt, to remove the asbestos and the filtrate +collected in a flask of about 150 cubic centimeters capacity. The +washing is continued until the gooch is free of copper, when the volume +of the filtrate should be about 100 cubic centimeters. The liquid is +transferred to a platinum dish holding about 175 cubic centimeters and +the flask washed with about twenty-five cubic centimeters of water. +From three to five cubic centimeters of strong sulfuric acid are added +and the copper deposited by an electric current. + +=137. Precipitating the Copper.=—When no more nitric acid is used than +indicated in the previous paragraph, it will not be necessary to remove +it by evaporation. The platinum dishes containing the solutions of the +cuprous oxid are arranged as shown in the figure for the precipitation +of the copper by the electric current. Each of the supporting stands +has its base covered with sheet-copper, on which the platinum dishes +rest. The uprights are made of heavy glass rods and carry the supports +for the platinum cylinders which dip into the copper solutions. The +current used is from the city service and is brought in through the +lamp shown at the right of the figure. This current has a voltage of +about 120. After passing the lamp it is conducted through the regulator +shown at the right, a glass tube closed below by a stopper carrying a +piece of platinum foil, and above by one holding a glass tube, in the +lower end of which is sealed a piece of sheet platinum connected, +through the glass tube, with the lamp. The regulating tube contains +dilute sulfuric acid. The strength of current desired is secured by +adjusting the movable pole. A battery of this kind easily secures the +precipitation of sixteen samples at once, but only twelve are shown +in the figure. The practice here is to start the operation at the +time of leaving the laboratory in the afternoon. The next morning +the deposition of the copper will be found complete. The wiring of +the apparatus is shown in the figure. The wire from the regulator is +connected with the base of the first stand, and thence passes through +the horizontal support to the base of the second, and so on. The return +to the lamp is accomplished by means of the upper wire. This plan of +arranging the apparatus has been used for two years, and with perfect +satisfaction. + +Where a street current is not available, the following directions +may be followed: Use four gravity cells, such as are employed in +telegraphic work, for generating the current. This will be strong +enough for one sample and by working longer for two. Connect the +platinum dish with the zinc pole of the battery. The current is allowed +to pass until all the copper is deposited. Where a larger number of +samples is to be treated at once, the size of the battery must be +correspondingly increased. + +=138. Method Used at the Halle Station.=—The method used at the Halle +station is the same as that originally described by Maercker for +dextrose.[106] The copper solution employed is the same as in the allihn +method, _viz._, 34.64 grams of copper sulfate in 500 cubic centimeters, +and 173 grams of rochelle salt and 125 grams of potassium hydroxid in +the same quantity of water. In a porcelain dish are placed thirty cubic +centimeters of copper solution and an equal quantity of the alkali, +sixty cubic centimeters of water added and the mixture boiled. To the +solution, in lively ebullition, are added twenty-five cubic centimeters +of the dextrose solution to be examined which must not contain more +than one per cent of sugar. The mixture is again boiled and the +separated cuprous oxid immediately poured into the filter and washed +with hot water, until the disappearance of an alkaline reaction. For +filtering, a glass tube is employed, provided with a platinum disk, +and resembling in every respect similar tubes used for the extraction +of substances with ether and alcohol. The arrangement of the filtering +apparatus is shown in Fig. 44. In the Halle method it is recommended +that the tubes be prepared by introducing a platinum cone in place of +the platinum disk and filling it with asbestos felt, pressing the felt +tightly against the sides of the glass tube and making the asbestos +fully one centimeter in thickness. This is a much less convenient +method of working than the one described above. After filtration and +washing, the cuprous oxid is washed with ether and alcohol and dried +for an hour at 110°, and finally reduced to metallic copper in a stream +of pure dry hydrogen, heat being applied by means of a small flame. The +apparatus for the reduction of the cuprous oxid is shown in Fig. 45. +The metallic copper, after cooling and weighing, is dissolved in nitric +acid, the tube washed with water, ether and alcohol, and again dried, +when it is ready for use a second time. The percentage of dextrose is +calculated from the milligrams of copper found by Allihn’s table. + +[Illustration: FIGURE 44. APPARATUS FOR FILTERING COPPER SUBOXID.] + +[Illustration: FIGURE 45. APPARATUS FOR REDUCING COPPER SUBOXID.] + +=139. Tables for Use in the Gravimetric Determination of Reducing +Sugars.=—The value of a table for computing the percentage of a +reducing sugar present in a solution, is based on the accuracy with +which the directions for the determination are followed. The solution +must be of the proper strength and made in the way directed. The degree +of dilution prescribed must be scrupulously preserved and the methods +of boiling during reduction and washing the reduced copper, followed. +The quantity of copper obtained by the use of different alkaline copper +solutions and of sugar solutions of a strength different from that +allowed by the fixed limits, is not a safe factor for computation. +It must be understood, therefore, that in the use of the tables the +directions which are given are to be followed in every particular. + +=140. Allihn’s Gravimetric Method for the Determination of +Dextrose.=—_Reagents_: + + I. 34.639 grams of CuSO₄.5H₂O, dissolved in water and diluted to + half a liter: + + II. 173 grams of rochelle salts } dissolved in water and diluted + 125 grams of KOH, } to half a liter. + +_Manipulation_: Place thirty cubic centimeters of the copper solution +(I), thirty cubic centimeters of the alkaline tartrate solution (II), +and sixty cubic centimeters of water in a beaker and heat to boiling. +Add twenty-five cubic centimeters of the solution of the material to +be examined, which must be so prepared as not to contain more than one +per cent of dextrose, and boil for two minutes. Filter immediately +after adding an equal volume of recently boiled cold water and obtain +the weight of copper by one of the gravimetric methods given. The +corresponding weight of dextrose is found by the following table: + + ALLIHN’S TABLE FOR THE DETERMINATION OF DEXTROSE. + + (A) = Milligrams of copper. + (B) = Milligrams of dextrose. + + (A) (B) (A) (B) (A) (B) (A) (B) (A) (B) + 10 6.1 46 23.9 82 41.8 118 60.1 154 78.6 + 11 6.6 47 24.4 83 42.3 119 60.6 155 79.1 + 12 7.1 48 24.9 84 42.8 120 61.1 156 79.6 + 13 7.6 49 25.4 85 43.4 121 61.6 157 80.1 + 14 8.1 50 25.9 86 43.9 122 62.1 158 80.7 + 15 8.6 51 26.4 87 44.4 123 62.6 159 81.2 + 16 9.0 52 26.9 88 44.9 124 63.1 160 81.7 + 17 9.5 53 27.4 89 45.4 125 63.7 161 82.2 + 18 10.0 54 27.9 90 45.9 126 64.2 162 82.7 + 19 10.5 55 28.4 91 46.4 127 64.7 163 83.3 + 20 11.0 56 28.8 92 46.9 128 65.2 164 83.8 + 21 11.5 57 29.3 93 47.4 129 65.7 165 84.3 + 22 12.0 58 29.8 94 47.9 130 66.2 166 84.8 + 23 12.5 59 30.3 95 48.4 131 66.7 167 85.3 + 24 13.0 60 30.8 96 48.9 132 67.2 168 85.9 + 25 13.5 61 31.3 97 49.4 133 67.7 169 86.4 + 26 14.0 62 31.8 98 49.9 134 68.2 170 86.9 + 27 14.5 63 32.3 99 50.4 135 68.8 171 87.4 + 28 15.0 64 32.8 100 50.9 136 69.3 172 87.9 + 29 15.5 65 33.3 101 51.4 137 69.8 173 88.5 + 30 16.0 66 33.8 102 51.9 138 70.3 174 89.0 + 31 16.5 67 34.3 103 52.4 139 70.8 175 89.5 + 32 17.0 68 34.8 104 52.9 140 71.3 176 90.0 + 33 17.5 69 35.3 105 53.5 141 71.8 177 90.5 + 34 18.0 70 35.8 106 54.0 142 72.3 178 91.1 + 35 18.5 71 36.3 107 54.5 143 72.9 179 91.6 + 36 18.9 72 36.8 108 55.0 144 73.4 180 92.1 + 37 19.4 73 37.3 109 55.5 145 73.9 181 92.6 + 38 19.9 74 37.8 110 56.0 146 74.4 182 93.1 + 39 20.4 75 38.3 111 56.5 147 74.9 183 93.7 + 40 20.9 76 38.8 112 57.0 148 75.5 184 94.2 + 41 21.4 77 39.3 113 57.5 149 76.0 185 94.7 + 42 21.9 78 39.8 114 58.0 150 76.5 186 95.2 + 43 22.4 79 40.3 115 58.6 151 77.0 187 95.7 + 44 22.9 80 40.8 116 59.1 152 77.5 188 96.3 + 45 23.4 81 41.3 117 59.6 153 78.1 189 96.8 + + (A) (B) (A) (B) (A) (B) (A) (B) (A) (B) + 190 97.3 233 120.1 276 143.3 319 167.0 362 191.1 + 191 97.8 234 120.7 277 143.9 320 167.5 363 191.7 + 192 98.4 235 121.2 278 144.4 321 168.1 364 192.3 + 193 98.9 236 121.7 279 145.0 322 168.6 365 192.9 + 194 99.4 237 122.3 280 145.5 323 169.2 366 193.4 + 195 100.0 238 122.8 281 146.1 324 169.7 367 194.0 + 196 100.5 239 123.4 282 146.6 325 170.3 368 194.6 + 197 101.0 240 123.9 283 147.2 326 170.9 369 195.1 + 198 101.5 241 124.4 284 147.7 327 171.4 370 195.7 + 199 102.0 242 125.0 285 148.3 328 172.0 371 196.3 + 200 102.6 243 125.5 286 148.8 329 172.5 372 196.8 + 201 103.1 244 126.0 287 149.5 330 173.1 373 197.4 + 202 103.7 245 126.6 288 149.4 331 173.7 374 198.0 + 203 104.2 246 127.1 289 150.9 332 174.2 375 198.6 + 204 104.7 247 127.6 290 151.0 333 174.8 376 199.1 + 205 105.3 248 128.1 291 151.6 334 175.3 377 199.7 + 206 105.8 249 128.7 292 152.1 335 175.9 378 200.3 + 207 106.3 250 129.2 293 152.7 336 176.5 379 200.8 + 208 106.8 251 129.7 294 153.2 337 177.0 380 201.4 + 209 107.4 252 130.3 295 153.8 338 177.6 381 202.0 + 210 107.9 253 130.8 296 154.3 339 178.1 382 202.5 + 211 108.4 254 131.4 297 154.9 340 178.7 383 203.1 + 212 109.0 255 131.9 298 155.4 341 179.3 384 203.7 + 213 109.5 256 132.4 299 156.0 342 179.8 385 204.3 + 214 110.0 257 133.0 300 156.5 343 180.4 386 204.8 + 215 110.6 258 133.5 301 157.1 344 180.9 387 205.4 + 216 111.1 259 134.1 302 157.6 345 181.5 388 206.0 + 217 111.6 260 134.6 303 158.2 346 182.1 389 206.5 + 218 112.1 261 135.1 304 158.7 347 182.6 390 207.1 + 219 112.7 262 135.7 305 159.3 348 183.2 391 207.7 + 220 113.2 263 136.2 306 159.8 349 183.7 392 208.3 + 221 113.7 264 136.8 307 160.4 350 184.3 393 208.8 + 222 114.3 265 137.3 308 160.9 351 184.9 394 209.4 + 223 114.8 266 137.8 309 161.5 352 185.4 395 210.0 + 224 115.3 267 138.4 310 162.0 353 186.0 396 210.6 + 225 115.9 268 138.9 311 162.6 354 186.6 397 211.2 + 226 116.4 269 139.5 312 163.1 355 187.2 398 211.7 + 227 116.9 270 140.0 313 163.7 356 187.7 399 212.3 + 228 117.4 271 140.6 314 164.2 357 188.3 400 212.9 + 229 118.0 272 141.1 315 164.8 358 188.9 401 213.5 + 230 118.5 273 141.7 316 165.3 359 189.4 402 214.1 + 231 119.0 274 142.2 317 165.9 360 190.0 403 214.6 + 232 119.6 275 142.8 318 166.4 361 190.6 404 215.2 + + (A) (B) (A) (B) (A) (B) (A) (B) (A) (B) + 405 215.8 417 222.8 429 229.8 441 236.9 453 244.0 + 406 216.4 418 223.3 430 230.4 442 237.5 454 244.6 + 407 217.0 419 223.9 431 231.0 443 238.1 455 245.2 + 408 217.5 420 224.5 432 231.6 444 238.7 456 245.7 + 409 218.1 421 225.1 433 232.2 445 239.3 457 246.3 + 410 218.7 422 225.7 434 232.8 446 239.8 458 246.9 + 411 219.3 423 226.3 435 233.4 447 240.4 459 247.5 + 412 219.9 424 226.9 436 233.9 448 241.0 460 248.1 + 413 220.4 425 227.5 437 234.5 449 241.6 461 248.7 + 414 221.0 426 228.0 438 235.1 450 242.2 462 249.3 + 415 221.6 427 228.6 439 235.7 451 242.8 463 249.9 + 416 222.2 428 229.2 440 236.3 452 243.4 + +=141. Meissl’s Table for Invert Sugar.=—Invert sugar is usually the +product of the hydrolysis of sucrose. The following table is to be used +when the hydrolysis is complete, _i_. _e_., when no sucrose is left +in the solution. The solution of copper sulfate and of the alkaline +tartrate are made up as follows: 34.64 grams of copper sulfate in half +a liter, and 173 grams of rochelle salt and 51.6 grams sodium hydroxid +in the same volume. The quantity of sugar solution used must not +contain more than 245 nor less than ninety milligrams of invert sugar. + +In the determination twenty-five cubic centimeters of the copper +solution and an equal volume of the alkaline tartrate are mixed and +boiled, the proper amount of sugar solution added to secure a quantity +of invertose within the limits named, the volume completed to 100 +cubic centimeters with boiling water, and the mixture kept in lively +ebullition for two minutes. An equal volume of recently boiled cold +water is added and the cuprous oxid at once separated by filtration on +asbestos under pressure, and washed free of alkali with boiling water. +The metallic copper is secured by one of the methods already described. + + TABLE FOR INVERT SUGAR BY MEISSL AND WIEN.[107] + + (A) = Milligrams of copper. + (B) = Milligrams of invert sugar. + + (A) (B) (A) (B) (A) (B) (A) (B) + 90 46.9 133 69.7 176 93.0 219 117.0 + 91 47.4 134 70.3 177 93.5 220 117.5 + 92 47.9 135 70.8 178 94.1 221 118.1 + 93 48.4 136 71.3 179 94.6 222 118.7 + 94 48.9 137 71.9 180 95.2 223 119.2 + 95 49.5 138 72.4 181 95.7 224 119.8 + 96 50.0 139 72.9 182 96.2 225 120.4 + 97 50.5 140 73.5 183 96.8 226 120.9 + 98 51.1 141 74.0 184 97.3 227 121.5 + 99 51.6 142 74.5 185 97.8 228 122.1 + 100 52.1 143 75.1 186 98.4 229 122.6 + 101 52.7 144 75.6 187 99.0 230 123.2 + 102 53.2 145 76.1 188 99.5 231 123.8 + 103 53.7 146 76.7 189 100.1 232 124.3 + 104 54.3 147 77.2 190 100.6 233 124.9 + 105 54.8 148 77.8 191 101.2 234 125.5 + 106 55.3 149 78.3 192 101.7 235 126.0 + 107 55.9 150 78.9 193 102.3 236 126.6 + 108 56.4 151 79.4 194 102.9 237 127.2 + 109 56.9 152 80.0 195 103.4 238 127.8 + 110 57.5 153 80.5 196 104.0 239 128.3 + 111 58.0 154 81.0 197 104.6 240 128.9 + 112 58.5 155 81.6 198 105.1 241 129.5 + 113 59.1 156 82.1 199 105.7 242 130.0 + 114 59.6 157 82.7 200 106.3 243 130.6 + 115 60.1 158 83.2 201 106.8 244 131.2 + 116 60.7 159 83.8 202 107.4 245 131.8 + 117 61.2 160 84.3 203 107.9 246 132.3 + 118 61.7 161 84.8 204 108.5 247 132.9 + 119 62.3 162 85.4 205 109.1 248 133.5 + 120 62.8 163 85.9 206 109.6 249 134.1 + 121 63.3 164 86.5 207 110.2 250 134.6 + 122 63.9 165 87.0 208 110.8 251 135.2 + 123 64.4 166 87.6 209 111.3 252 135.8 + 124 64.9 167 88.1 210 111.9 253 136.3 + 125 65.5 168 88.6 211 112.5 254 136.9 + 126 66.0 169 89.2 212 113.0 255 137.5 + 127 66.5 170 89.7 213 113.6 256 138.1 + 128 67.1 171 90.3 214 114.2 257 138.6 + 129 67.6 172 90.8 215 114.7 258 139.2 + 130 68.1 173 91.4 216 115.3 259 139.8 + 131 68.7 174 91.9 217 115.8 260 140.4 + 132 69.2 175 92.4 218 116.4 261 140.9 + + (A) (B) (A) (B) (A) (B) (A) (B) + 262 141.5 305 166.8 348 192.6 391 219.3 + 263 142.1 306 167.3 349 193.2 392 219.9 + 264 142.7 307 167.9 350 193.8 393 220.5 + 265 143.2 308 168.5 351 194.4 394 221.2 + 266 143.8 309 169.1 352 195.0 395 221.8 + 267 144.4 310 169.7 353 195.6 396 222.4 + 268 144.9 311 170.3 354 196.2 397 223.1 + 269 145.5 312 170.9 355 196.8 398 223.7 + 270 146.1 313 171.5 356 197.4 399 224.3 + 271 146.7 314 172.1 357 198.0 400 224.9 + 272 147.2 315 172.7 358 198.6 401 225.7 + 273 147.8 316 173.3 359 199.2 402 226.4 + 274 148.4 317 173.9 360 199.8 403 227.1 + 275 149.0 318 174.5 361 200.4 404 227.8 + 276 149.5 319 175.1 362 201.1 405 228.6 + 277 150.1 320 175.6 363 201.7 406 229.3 + 278 150.7 321 176.2 364 202.3 407 230.0 + 279 151.3 322 176.8 365 203.0 408 230.7 + 280 151.9 323 177.4 366 203.6 409 231.4 + 281 152.5 324 178.0 367 204.2 410 232.1 + 282 153.1 325 178.6 368 204.8 411 232.8 + 283 153.7 326 179.2 369 205.5 412 233.5 + 284 154.3 327 178.8 370 206.1 413 234.3 + 285 154.9 328 180.4 371 206.7 414 235.0 + 286 155.5 329 181.0 372 207.3 415 235.7 + 287 156.1 330 181.6 373 208.0 416 236.4 + 288 156.7 331 182.2 374 208.6 417 237.1 + 289 157.2 332 182.8 375 209.2 418 237.8 + 290 157.8 333 183.5 376 209.9 419 238.5 + 291 158.4 334 184.1 377 210.5 420 239.2 + 292 159.0 335 184.7 378 211.1 421 239.9 + 293 159.6 336 185.4 379 211.7 422 240.6 + 294 160.2 337 186.0 380 212.4 423 241.3 + 295 160.8 338 186.6 381 213.0 424 242.0 + 296 161.4 339 187.2 382 213.6 425 242.7 + 297 162.0 340 187.8 383 214.3 426 243.4 + 298 162.6 341 188.4 384 214.9 427 244.1 + 299 163.2 342 189.0 385 215.5 428 244.9 + 300 163.8 343 189.6 386 216.1 429 245.6 + 301 164.4 344 190.2 387 216.8 430 246.3 + 302 165.0 345 190.8 388 217.4 + 303 165.6 346 191.4 389 218.0 + 304 166.2 347 192.0 390 218.7 + +=142. Table for the Determination of Invert Sugar (Reducing Sugars) +in the Presence of Sucrose.=—The method adopted by the Association of +Official Agricultural Chemists is essentially that proposed by Meissl +and Hiller.[108] Prepare a solution of the material to be examined in +such a manner that it contains twenty grams of the mixed sugars in +one hundred cubic centimeters, after clarification and the removal of +the excess of lead. Prepare a series of solutions in large test tubes +by adding one, two, three, four, five etc. cubic centimeters of this +solution to each tube successively. Add five cubic centimeters of the +mixed copper reagent to each, heat to boiling, boil two minutes and +filter. Note the volume of sugar solution which gives the filtrate +lightest in tint, but still distinctly blue. Place twenty times this +volume of the sugar solution in a 100 cubic centimeter flask, dilute to +the mark, and mix well. Use fifty cubic centimeters of the solution for +the determination, which is conducted as already described, until the +weight of copper is obtained. For the calculation of the results use +the following formulas and table of factors of Meissl and Hiller:[109] + + Let Cu = the weight of the copper obtained; + P = the polarization of the sample; + W = the weight of the sample in the fifty cubic + centimeters of the solution used for determination; + F = the factor obtained from the table for conversion + of copper to invert sugar; + Cu + ---- = approximate absolute weight of invert sugar = Z; + 2 + + 100 + Z × ----- = approximate per cent of invert sugar = _y_; + W + + 100P + ------- = R, relative number for sucrose; + P + _y_ + + 100 - R = I, relative number for invert sugar; + + Cu + ---- = per cent of invert sugar. + W + +Z indicates the vertical column, and the ratio of R to I, the +horizontal column of the table, which are to be used for the purpose of +finding the factor (F) for calculating copper to invert sugar. + +_Example_:—The polarization of a sugar is 86.4, and 3.256 grams of it +(W) are equivalent to 0.290 gram of copper. Then: + + Cu 0.290 + ---- = ----- = 0.145 = Z + 2 2 + + 100 100 + Z × ----- = 0.145 × ------ = 4.45 = _y_ + W 3.256 + + 100P 8640 + -------- = ------------ = 95.1 = R + P + _y_ 86.4 + 4.45 + + 100 - R = 100 - 95.1 = 4.9 = I + + R : I = 95.1 : 4.9 + +By consulting the table it will be seen that the vertical column headed +I = 150 is nearest to Z, 145, the horizontal column headed 95: 5 is +nearest to the ratio of R to I, 95.1: 4.9. Where these columns meet we +find the factor 51.2, which enters into the final calculation: + + CuF .290 × 51.2 + ----- = ------------- = 4.56 the true per cent of invert sugar. + W 3.256 + + MEISSL AND HILLER’S FACTORS FOR THE DETERMINATION OF + MORE THAN ONE PER CENT OF INVERT SUGAR. + + Ratio of Approximate absolute weight of invert sugar = _Z_. + sucrose I = I = I = I = I = I = I = + to invert 200 175 150 125 100 75 50 + sugar = mg. mg. mg. mg. mg. mg. mg. + R : I. + 0 : 100 56.4 55.4 54.5 53.8 53.2 53.0 53.0 + 10 : 90 56.3 55.3 54.4 53.8 53.2 52.9 52.9 + 20 : 80 56.2 55.2 54.3 53.7 53.2 52.7 52.7 + 30 : 70 56.1 55.1 54.2 53.7 53.2 52.6 52.6 + 40 : 60 55.9 55.0 54.1 53.6 53.1 52.5 52.4 + 50 : 50 55.7 54.9 54.0 53.5 53.1 52.3 52.2 + 60 : 40 55.6 54.7 53.8 53.2 52.8 52.1 51.9 + 70 : 30 55.5 54.5 53.5 52.9 52.5 51.9 51.6 + 80 : 20 55.4 54.3 53.3 52.7 52.2 51.7 51.3 + 90 : 10 54.6 53.6 53.1 52.6 52.1 51.6 51.2 + 91 : 9 54.1 53.6 52.6 52.1 51.6 51.2 50.7 + 92 : 8 53.6 53.1 52.1 51.6 51.2 50.7 50.3 + 93 : 7 53.6 53.1 52.1 51.2 50.7 50.3 49.8 + 94 : 6 53.1 52.6 51.6 50.7 50.3 49.8 48.9 + 95 : 5 52.6 52.1 51.2 50.3 49.4 48.9 48.5 + 96 : 4 52.1 51.2 50.7 49.8 48.9 47.7 46.9 + 97 : 3 50.7 50.3 49.8 48.9 47.7 46.2 45.1 + 98 : 2 49.9 48.9 48.5 47.3 45.8 43.3 40.0 + 99 : 1 47.7 47.3 46.5 45.1 43.3 41.2 38.1 + +=143. Table for the Estimation of Milk Sugar.=—The solutions to be used +for this table are the same as those employed in the preceding table +for the estimation of invert sugar. The milk sugar is supposed to be in +a pure form in solution before beginning the analysis. The method to be +employed for milk will be given in the part devoted to dairy products. + +In the conduct of the work twenty-five cubic centimeters of the copper +solution are mixed with an equal quantity of the alkaline tartrate +mixture, and from twenty to one hundred cubic centimeters of the sugar +solution added, according to its concentration. This solution should +not contain less than seventy nor more than 306 milligrams of lactose. +The volume is completed to 150 cubic centimeters with boiling water and +kept in lively ebullition for six minutes. The rest of the operation is +conducted in the manner already described. From the weight of copper +obtained the quantity of milk sugar is determined by inspecting the +table. It is recommended to use such a weight of milk sugar as will +give about 200 milligrams of copper. + + TABLE FOR DETERMINING MILK SUGAR. + + (A) = Milligrams of copper. + (B) = Milligrams of milk sugar. + + (A) (B) (A) (B) (A) (B) (A) (B) + 100 71.6 120 86.4 140 101.3 160 116.4 + 101 72.4 121 87.2 141 102.0 161 117.1 + 102 73.1 122 87.9 142 102.8 162 117.9 + 103 73.8 123 88.7 143 103.5 163 118.6 + 104 74.6 124 89.4 144 104.3 164 119.4 + 105 75.3 125 90.1 145 105.1 165 120.2 + 106 76.1 126 90.9 146 105.8 166 120.9 + 107 76.8 127 91.6 147 106.6 167 121.7 + 108 77.6 128 92.4 148 107.3 168 122.4 + 109 78.3 129 93.1 149 108.1 169 123.2 + 110 79.0 130 93.8 150 108.8 170 123.9 + 111 79.8 131 94.6 151 109.6 171 124.7 + 112 80.5 132 95.3 152 110.3 172 125.5 + 113 81.3 133 96.1 153 111.1 173 126.2 + 114 82.0 134 96.9 154 111.9 174 127.0 + 115 82.7 135 97.6 155 112.6 175 127.8 + 116 83.5 136 98.3 156 113.4 176 128.5 + 117 84.2 137 99.1 157 114.1 177 129.3 + 118 85.0 138 99.8 158 114.9 178 130.1 + 119 85.7 139 100.5 159 115.6 179 130.8 + + (A) (B) (A) (B) (A) (B) (A) (B) + 180 131.6 223 164.2 266 197.2 309 231.4 + 181 132.4 224 164.9 267 198.0 310 232.2 + 182 133.1 225 165.7 268 198.8 311 232.9 + 183 133.9 226 166.4 269 199.5 312 233.7 + 184 134.7 227 167.2 270 200.3 313 234.5 + 185 135.4 228 167.9 271 201.1 314 235.3 + 186 136.2 229 168.6 272 201.9 315 236.1 + 187 137.0 230 169.4 273 202.7 316 236.8 + 188 137.7 231 170.1 274 203.5 317 237.6 + 189 138.5 232 170.9 275 204.3 318 238.4 + 190 139.3 233 171.6 276 205.1 319 239.2 + 191 140.0 234 172.4 277 205.9 320 240.0 + 192 140.8 235 173.1 278 206.7 321 240.7 + 193 141.6 236 173.9 279 207.5 322 241.5 + 194 142.3 237 174.6 280 208.3 323 242.3 + 195 143.1 238 175.4 281 209.1 324 243.1 + 196 143.9 239 176.2 282 209.9 325 243.9 + 197 144.6 240 176.9 283 210.7 326 244.6 + 198 145.4 241 177.7 284 211.5 327 245.4 + 199 146.2 242 178.5 285 212.3 328 246.2 + 200 146.9 243 179.3 286 213.1 329 247.0 + 201 147.7 244 180.1 287 213.9 330 247.7 + 202 148.5 245 180.8 288 214.7 331 248.5 + 203 149.2 246 181.6 289 215.5 332 249.2 + 204 150.0 247 182.4 290 216.3 333 250.0 + 205 150.7 248 183.2 291 217.1 334 250.8 + 206 151.5 249 184.0 292 217.9 335 251.6 + 207 152.2 250 184.8 293 218.7 336 252.5 + 208 153.0 251 185.5 294 219.5 337 253.3 + 209 153.7 252 186.3 295 220.3 338 254.1 + 210 154.5 253 187.1 296 221.1 339 254.9 + 211 155.2 254 187.9 297 221.9 340 255.7 + 212 156.0 255 188.7 298 222.7 341 256.5 + 213 156.7 256 189.4 299 223.5 342 257.4 + 214 157.5 257 190.2 300 224.4 343 258.2 + 215 158.2 258 191.0 301 225.2 344 259.0 + 216 159.0 259 191.8 302 225.9 345 259.8 + 217 159.7 260 192.5 303 226.7 346 260.6 + 218 160.4 261 193.3 304 227.5 347 261.4 + 219 161.2 262 194.1 305 228.3 348 262.3 + 220 161.9 263 194.9 306 229.1 349 263.1 + 221 162.7 264 195.7 307 229.8 350 263.9 + 222 163.4 265 196.4 308 230.6 351 264.7 + + (A) (B) (A) (B) (A) (B) (A) (B) + 352 265.5 365 276.2 377 286.5 389 296.8 + 353 266.3 366 277.1 378 287.4 390 297.7 + 354 267.2 367 277.9 379 288.2 391 298.5 + 355 268.0 368 278.8 380 289.1 392 299.4 + 356 268.8 369 279.6 381 289.9 393 300.3 + 357 269.6 370 280.5 382 290.8 394 301.1 + 358 270.4 371 281.4 383 291.7 395 302.0 + 359 271.2 372 282.2 384 292.5 396 302.8 + 360 272.1 373 283.1 385 293.4 397 303.7 + 361 272.9 374 283.9 386 294.2 398 304.6 + 362 273.7 375 284.8 387 295.1 399 305.4 + 363 274.5 376 285.7 388 296.0 400 306.3 + 364 275.3 + +=144. Table for the Determination of Maltose.=—The copper and alkaline +solutions employed for the oxidation of maltose are the same as those +used for invert and milk sugars. + +In the manipulation twenty-five cubic centimeters each of the copper +and alkali solutions are mixed and boiled and an equal volume of the +maltose solution added, which should not contain more than one per +cent of the sugar. The boiling is continued for four minutes, an equal +volume of cold recently boiled water added, the cuprous oxid separated +by filtration and the metallic copper obtained in the manner already +described. The weight of maltose oxidized is then ascertained from the +table. + + _Example._ Weight of impure maltose taken, ten grams to a liter: + Quantity used, twenty-five cubic centimeters: + Weight of copper obtained 268 milligrams: + Weight of maltose oxidized 237 milligrams: + Weight of impure maltose taken 250 milligrams: + Percentage of maltose in sample 94.8. + + TABLE FOR MALTOSE. + + (A) = Milligrams of copper. + (B) = Milligrams of maltose. + + (A) (B) (A) (B) (A) (B) (A) (B) + 30 25.3 35 29.6 40 33.9 45 38.3 + 31 26.1 36 30.5 41 34.8 46 39.1 + 32 27.0 37 31.3 42 35.7 47 40.0 + 33 27.9 38 32.2 43 36.5 48 40.9 + 34 28.7 39 33.1 44 37.4 49 41.8 + + (A) (B) (A) (B) (A) (B) (A) (B) + 50 42.6 94 81.2 138 120.6 182 160.1 + 51 43.5 95 82.1 139 121.5 183 160.9 + 52 44.4 96 83.0 140 122.4 184 161.8 + 53 45.2 97 83.9 141 123.3 185 162.7 + 54 46.1 98 84.8 142 124.2 186 163.6 + 55 47.0 99 85.7 143 125.1 187 164.5 + 56 47.8 100 86.6 144 126.0 188 165.4 + 57 48.7 101 87.5 145 126.9 189 166.3 + 58 49.6 102 88.4 146 127.8 190 167.2 + 59 50.4 103 89.2 147 128.7 191 168.1 + 60 51.3 104 90.1 148 129.6 192 169.0 + 61 52.2 105 91.0 149 130.5 193 169.8 + 62 53.1 106 91.9 150 131.4 194 170.7 + 63 53.9 107 92.8 151 132.3 195 171.6 + 64 54.8 108 93.7 152 133.2 196 172.5 + 65 55.7 109 94.6 153 134.1 197 173.4 + 66 56.6 110 95.5 154 135.0 198 174.3 + 67 57.4 111 96.4 155 135.9 199 175.2 + 68 58.3 112 97.3 156 136.8 200 176.1 + 69 59.2 113 98.1 157 137.7 201 177.0 + 70 60.1 114 99.0 158 138.6 202 177.9 + 71 61.0 115 99.9 159 139.5 203 178.7 + 72 61.8 116 100.8 160 140.4 204 179.6 + 73 62.7 117 101.7 161 141.3 205 180.5 + 74 63.6 118 102.6 162 142.2 206 181.4 + 75 64.5 119 103.5 163 143.1 207 182.3 + 76 65.4 120 104.4 164 144.0 208 183.2 + 77 66.2 121 105.3 165 144.9 209 184.1 + 78 67.1 122 106.2 166 145.8 210 185.0 + 79 68.0 123 107.1 167 146.7 211 185.9 + 80 68.9 124 108.0 168 147.6 212 186.8 + 81 69.7 125 108.9 169 148.5 213 187.7 + 82 70.6 126 109.8 170 149.4 214 188.6 + 83 71.5 127 110.7 171 150.3 215 189.5 + 84 72.4 128 111.6 172 151.2 216 190.4 + 85 73.2 129 112.5 173 152.0 217 191.2 + 86 74.1 130 113.4 174 152.9 218 192.1 + 87 75.0 131 114.3 175 153.8 219 193.0 + 88 75.9 132 115.2 176 154.7 220 193.9 + 89 76.8 133 116.1 177 155.6 221 194.8 + 90 77.7 134 117.0 178 156.5 222 195.7 + 91 78.6 135 117.9 179 157.4 223 196.6 + 92 79.5 136 118.8 180 158.3 224 197.5 + 93 80.3 137 119.7 181 159.2 225 198.4 + + (A) (B) (A) (B) (A) (B) (A) (B) + 226 199.3 245 216.3 264 233.4 283 250.4 + 227 200.2 246 217.2 265 234.3 284 251.3 + 228 201.1 247 218.1 266 235.2 285 252.2 + 229 202.0 248 219.0 267 236.1 286 253.1 + 230 202.9 249 219.9 268 237.0 287 254.0 + 231 203.8 250 220.8 269 237.9 288 254.9 + 232 204.7 251 221.7 270 238.8 289 255.8 + 233 205.6 252 222.6 271 239.7 290 256.6 + 234 206.5 253 223.5 272 240.6 291 257.5 + 235 207.4 254 224.4 273 241.5 292 258.4 + 236 208.3 255 225.3 274 242.4 293 259.3 + 237 209.1 256 226.2 275 243.3 294 260.2 + 238 210.0 257 227.1 276 244.2 295 261.1 + 239 210.9 258 228.0 277 245.1 296 262.0 + 240 211.8 259 228.9 278 246.0 297 262.8 + 241 212.7 260 229.8 279 246.9 298 263.7 + 242 213.6 261 230.7 280 247.8 299 264.6 + 243 214.5 262 231.6 281 248.7 300 265.5 + 244 215.4 263 232.5 282 249.6 + +=145. Preparation of Levulose.=—It is not often that levulose, unmixed +with other reducing sugars, is brought to the attention of the analyst. +It probably does not exist in the unmixed state in any agricultural +product. The easiest method of preparing it is by the hydrolysis of +inulin. A nearly pure levulose has also lately been placed on the +market under the name of diabetin. It is prepared from invert sugar. + +Inulin is prepared from dahlia bulbs by boiling the pulp with water +and a trace of calcium carbonate. The extract is concentrated to +a sirup and subjected to a freezing temperature to promote the +crystallization of the inulin. The separated product is subjected to +the above operations several times until it is pure and colorless. It +is then washed with alcohol and ether and is reduced to a fine powder. +Before the repeated treatment with water it is advisable to clarify +the solution with lead subacetate. The lead is afterwards removed by +hydrogen sulfid and the resultant acetic acid neutralized with calcium +carbonate. + +By the action of hot dilute acids inulin is rapidly converted into +levulose. + +Levulose may also be prepared from invert sugar, but in this case it +is difficult to free it from traces of dextrose. The most successful +method consists in forming a lime compound with the invert sugar and +separating the lime levulosate and dextrosate by their difference +in solubility. The levulose salt is much less soluble than the +corresponding compound of dextrose. In the manufacture of levulose +from beet molasses, the latter is dissolved in six times its weight of +water and inverted with a quantity of hydrochloric acid, proportioned +to the quantity of ash present in the sample. After inversion the +mixture is cooled to zero and the levulose precipitated by adding +fine-ground lime. The dextrose and coloring matters in these conditions +are not thrown down. The precipitated lime levulosate is separated by +filtration and washed with ice-cold water. The lime salt is afterwards +beaten to a cream with water and decomposed by carbon dioxid. The +levulose, after filtration, is concentrated to the crystallizing +point.[110] + +=146. Estimation of Levulose.=—Levulose, when free of any admixture +with other reducing sugars, may be determined by the copper method +with the use of the subjoined table, prepared by Lehmann.[111] The +copper solution is the same as that used for invert sugar, _viz._, +69.278 grams of pure copper sulfate in one liter. The alkali solution +is prepared by dissolving 346 grams of rochelle salt and 250 grams of +sodium hydroxid in water and completing the volume to one liter. + +_Manipulation._—Twenty-five cubic centimeters of each solution +are mixed with fifty of water and boiled. To the boiling mixture +twenty-five cubic centimeters of the levulose solution are added, which +must not contain more than one per cent of the sugar. The boiling is +then continued for fifteen minutes, and the cuprous oxid collected, +washed and reduced to the metallic state in the usual way. The quantity +of levulose is then determined by inspection from the table given +below. Other methods of determining levulose in mixtures will be given +further on. + + TABLE FOR THE ESTIMATION OF LEVULOSE. + + (A) = Milligrams of copper. + (B) = Milligrams of levulose. + + (A) (B) (A) (B) (A) (B) (A) (B) + 20 7.15 62 31.66 104 56.85 146 82.81 + 21 7.78 63 32.25 105 57.46 147 83.43 + 22 8.41 64 32.84 106 58.07 148 84.06 + 23 9.04 65 33.43 107 58.68 149 84.68 + 24 9.67 66 34.02 108 59.30 150 85.31 + 25 10.30 67 34.62 109 59.91 151 85.93 + 26 10.81 68 35.21 110 60.52 152 86.55 + 27 11.33 69 35.81 111 61.13 153 87.16 + 28 11.84 70 36.40 112 61.74 154 87.88 + 29 12.36 71 37.00 113 62.36 155 88.40 + 30 12.87 72 37.59 114 62.97 156 89.05 + 31 13.46 73 38.19 115 63.58 157 89.69 + 32 14.05 74 38.78 116 64.21 158 90.34 + 33 14.64 75 39.38 117 64.84 159 90.98 + 34 15.23 76 39.98 118 65.46 160 91.63 + 35 15.82 77 40.58 119 66.09 161 92.26 + 36 16.40 78 41.17 120 66.72 162 92.90 + 37 16.99 79 41.77 121 67.32 163 93.53 + 38 17.57 80 42.37 122 67.92 164 94.17 + 39 18.16 81 42.97 123 68.53 165 94.80 + 40 18.74 82 43.57 124 69.13 166 95.44 + 41 19.32 83 44.16 125 69.73 167 96.08 + 42 19.91 84 44.76 126 70.35 168 96.77 + 43 20.49 85 45.36 127 70.96 169 97.33 + 44 21.08 86 45.96 128 71.58 170 97.99 + 45 21.66 87 46.57 129 72.19 171 98.63 + 46 22.25 88 47.17 130 72.81 172 99.27 + 47 22.83 89 47.78 131 73.43 173 99.90 + 48 23.42 90 48.38 132 74.05 174 100.54 + 49 24.00 91 48.98 133 74.67 175 101.18 + 50 24.59 92 49.58 134 75.29 176 101.82 + 51 25.18 93 50.18 135 75.91 177 102.46 + 52 25.76 94 50.78 136 76.53 178 103.11 + 53 26.35 95 51.38 137 77.15 179 103.75 + 54 26.93 96 51.98 138 77.77 180 104.39 + 55 27.52 97 52.58 139 78.39 181 105.04 + 56 28.11 98 53.19 140 79.01 182 105.68 + 57 28.70 99 53.79 141 79.64 183 106.33 + 58 29.30 100 54.39 142 80.28 184 106.97 + 59 29.89 101 55.00 143 80.91 185 107.62 + 60 30.48 102 55.62 144 81.55 186 108.27 + 61 31.07 103 56.23 145 82.18 187 108.92 + + (A) (B) (A) (B) (A) (B) (A) (B) + 188 109.56 232 138.57 276 168.68 320 199.97 + 189 110.21 233 139.25 277 169.37 321 200.71 + 190 110.86 234 139.18 278 170.06 322 201.44 + 191 111.50 235 140.59 279 170.75 323 202.18 + 192 112.14 236 141.27 280 171.44 324 202.91 + 193 112.78 237 141.94 281 172.14 325 203.65 + 194 113.42 238 142.62 282 172.85 326 204.39 + 195 114.06 239 143.29 283 173.55 327 205.13 + 196 114.72 240 143.97 284 174.26 328 205.88 + 197 115.38 241 144.65 285 174.96 329 206.62 + 198 116.04 242 145.32 286 175.67 330 207.36 + 199 116.70 243 146.00 287 176.39 331 208.10 + 200 117.36 244 146.67 288 177.10 332 208.83 + 201 118.02 245 147.35 289 177.82 333 209.57 + 202 118.68 246 148.03 290 178.53 334 210.30 + 203 119.33 247 148.71 291 179.24 335 211.04 + 204 119.99 248 149.40 292 179.95 336 211.78 + 205 120.65 249 150.08 293 180.65 337 212.52 + 206 121.30 250 150.76 294 181.63 338 213.25 + 207 121.96 251 151.44 295 182.07 339 213.99 + 208 122.61 252 152.12 296 182.78 340 214.73 + 209 123.27 253 152.81 297 183.49 341 215.48 + 210 123.92 254 153.49 298 184.21 342 216.23 + 211 124.58 255 154.17 299 184.92 343 216.97 + 212 125.24 256 154.91 300 185.63 344 217.72 + 213 125.90 257 155.65 301 186.35 345 218.47 + 214 126.56 258 156.40 302 187.06 346 219.21 + 215 127.22 259 157.14 303 187.78 347 219.97 + 216 127.85 260 157.88 304 188.49 348 220.71 + 217 128.48 261 158.49 305 189.21 349 221.46 + 218 129.10 262 159.09 306 189.93 350 222.21 + 219 129.73 263 159.70 307 190.65 351 222.96 + 220 130.36 264 160.30 308 191.37 352 223.72 + 221 131.07 265 160.91 309 192.09 353 224.47 + 222 131.77 266 161.63 310 192.81 354 225.23 + 223 132.48 267 162.35 311 193.53 355 225.98 + 224 133.18 268 163.07 312 194.25 356 226.74 + 225 133.89 269 163.79 313 194.97 357 227.49 + 226 134.56 270 164.51 314 195.69 358 228.25 + 227 135.23 271 165.21 315 196.41 359 229.00 + 228 135.89 272 165.90 316 197.12 360 229.76 + 229 136.89 273 166.60 317 197.83 361 230.52 + 230 137.23 274 167.29 318 198.55 362 231.28 + 231 137.90 275 167.99 319 199.26 363 232.05 + + (A) (B) (A) (B) (A) (B) (A) (B) + 364 232.81 370 237.39 376 241.87 382 246.25 + 365 233.57 371 238.16 377 242.51 383 247.17 + 366 234.33 372 238.93 378 243.15 384 248.08 + 367 235.10 373 239.69 379 243.79 385 248.99 + 368 235.86 374 240.46 380 244.43 + 369 236.63 375 241.23 381 245.34 + +=147. Precipitation of Sugars with Phenylhydrazin=.—The combination of +phenylhydrazin with aldehyds and ketones was first studied by Fischer, +and the near relationship of these bodies to sugar soon led to the +investigation of the compounds formed thereby with this reagent.[112] +Reducing sugars form with phenylhydrazin insoluble crystalline bodies, +to which the name osazones has been given. The reaction which takes +place is a double one and is represented by the following formulas: + + Dextrose. Phenylhydrazin. Dextrose-phenylhydrazone. + + C₆H₁₂O₆ + C₆H₅NH.NH₂ = C₆H₁₂O₅.N.NHC₆H₅ + H₂O + and C₆H₁₂O₅.N.NHC₆H₅ + C₆H₅NH.NH₂ = + Phenyldextrosazone. + C₆H₁₀O₄(N.NHC₆H₅)₂ + 2H₂O. + +The dextrosazone is commonly called glucosazone. The osazones formed +with the commonly occurring reducing sugars are crystalline, stable, +insoluble bodies which can be easily separated from any attending +impurities and identified by their melting points. Glucosazone melts at +205°, lactosazone at 200° and maltosazone at 206°. + +The osazones are precipitated in the following way: The reducing sugar, +in about ten per cent solution, is treated with an excess of the +acetate of phenylhydrazin in acetic acid and warmed to from 75° to 85°. +In a short time the separation is complete and the yellow precipitate +formed is washed, dried and weighed. The sugar can be recovered from +the osazone by decomposing it with strong hydrochloric acid by means +of which the phenylhydrazin is displaced and a body, osone, is formed, +which by treatment with zinc dust and acetic acid, is reduced to the +original sugar. The reactions which take place are represented by the +following equations:[113] + + Glucososone. + C₆H₁₀O₄(N.NH.C₆H₅)₂ + 2H₂O = C₆H₁₀O₆ + 2C₆H₅N₂H₂ + + Dextrose (Glucose). + C₆H₁₀O₆ + H₂ = C₆H₁₂O₆. + +For the complete precipitation of dextrose as osazone Lintner and +Kröber show that the solution of dextrose should not contain more than +one gram in 100 cubic centimeters. Twenty cubic centimeters containing +0.2 gram dextrose should be used for the precipitation.[114] To this +solution should be added one gram of phenylhydrazin and one gram of +fifty per cent acetic acid. The solution is then to be warmed for about +two hours and the precipitate washed with from sixty to eighty cubic +centimeters of hot water and dried for three hours at 105°. One part +of the osazone is equivalent to one part of dextrose when maltose and +dextrin are absent. When these are present the proportion is one part +of osazone to 1.04 of dextrose. Where levulose is precipitated instead +of dextrose 1.43 parts of the osazone are equal to one part of the +sugar. + +Sucrose is scarcely at all precipitated as osazone until inverted. + +After inversion and precipitation as above, 1.33 parts osazone are +equal to one part of sucrose. + +The reaction with phenylhydrazin has not been much used for quantitive +estimations of sugars, but it has been found especially useful in +identifying and separating reducing sugars. It is altogether probable, +however, that in the near future phenylhydrazin will become a common +reagent for sugar work. + +Maquenne has studied the action of phenylhydrazin on sugars and +considers that this reaction offers the only known means of +precipitating these bodies from solutions where they are found mixed +with other substances.[115] The osazones, which are thus obtained, are +usually very slightly soluble in the ordinary reagents, for which +reason it is easy to obtain them pure when there is at the disposition +of the analyst a sufficient quantity of the material. But if the sugar +to be studied is rare and if it contain, moreover, several distinct +reducing bodies, the task is more delicate. It is easy then to confound +several osazones which have almost identical points of fusion; +for example, glucosazone with galactosazone. Finally, it becomes +impossible by the employment of phenylhydrazin to distinguish glucose, +dextrose or mannose from levulose alone or mixed with its isomers. +Indeed, these three sugars give, with the acetate of phenylhydrazin +the same phenylglucosazone which melts at about 205°. It is noticed +that the weights of osazones which are precipitated when different +sugars are heated for the same time with the same quantity of the +phenylhydrazin, vary within extremely wide limits. It is constant for +each kind of sugar if the conditions under which the precipitation +is made are rigorously the same. There is then, in the weight of the +osazones produced, a new characteristic of particular value. The +following numbers have been obtained by heating for one hour at 100°, +one gram of sugar with 100 cubic centimeters of water and five cubic +centimeters of a solution containing forty grams of phenylhydrazin +and forty grams of acetic acid per hundred. After cooling the liquid, +the osazones are received upon a weighed filter, washed with 100 +cubic centimeters of water, dried at 110° and weighed. The weights of +osazones obtained are given in the following table: + + Weight of the osazones. + Character of the sugar. gram. + + Sorbine, crystallized 0.82 + Levulose ” 0.70 + Xylose ” 0.40 + Glucose, anhydrous 0.32 + Arabinose, crystallized 0.27 + Galactose ” 0.23 + Rhamnose ” 0.15 + Lactose ” 0.11 + Maltose ” 0.11 + +With solutions twice as dilute as those above, the relative conditions +are still more sensible, and the different sugars arrange themselves in +the same order, with the exception of levulose, which shows a slight +advantage over sorbine and acquires the first rank. From the above +determinations, it is shown that levulose and sorbine give vastly +greater quantities of osazones, under given conditions, than the other +reducing sugars. It would be easy, therefore, to distinguish them by +this reaction and to recognize their presence also even in very complex +mixtures, where the polarimetric examination alone would furnish only +uncertain indications. + +It is remarkable that these two sugars are the only ones among the +isomers or the homologues of dextrose, actually known, which possess +the functions of an acetone. They are not, however, easily confounded, +since the glucosazone forms beautiful needles which are ordinarily +visible to the naked eye, while the sorbinosazone is still oily and +when heated never gives perfectly distinct crystals. + +This method also enables us to distinguish between dextrose and +galactose, of which the osazone is well crystallized and melts at +almost the same temperature as the phenylglucosazone. Finally, it +is observed that the reducing sugars give less of osazones than the +sugars which are not capable of hydrolysis, and consequently differ +in their inversion products. It is specially noticed in this study of +the polyglucoses (bioses, trioses), that this new method of employing +the phenylhydrazin appears very advantageous. It is sufficient to +compare the weights of the osazones to that which is given under the +same conditions by a known glucose, in order to have a very certain +verification of the probabilities of the result of the chemical or +optical examination of the mixture which is under study. All the +polyglucoses which have been examined from this point of view give +very decided results. The numbers which follow have reference to one +gram of sugar completely inverted by dilute sulfuric acid, dissolved +in 100 cubic centimeters of water, and treated with two grams of +phenylhydrazin, the same quantity of acetic acid, and five grams of +crystallized sodium acetate. All these solutions have been compared +with the artificial mixtures and corresponding glucoses, with the same +quantities of the same reagents. The following are the results of the +examination: + + Weight of the osazone. + Character of the sugar. gram. + + 1 {Saccharose, ordinary 0.71 + {Glucose and levulose (.526 g each) 0.73 + + 2 {Maltose 0.55 + {Glucose (1.052 g) 0.58 + + 3 {Raffinose, crystallized 0.48 + {Levulose, glucose and galactose (.333 g each) 0.53 + + 4 {Lactose, crystallized 0.38 + {Glucose and galactose (.500 g each) 0.39 + +It is noticed that the agreement for each saccharose is as +satisfactory as possible. Numbers obtained with the products of +inversion are always a little low by reason of the destructive action +of sulfuric acid, and in particular, upon levulose. This is, moreover, +quite sensible when the product has to be heated for a long time with +sulfuric acid in order to secure a complete inversion. It is evident +from the data cited from the papers of Fischer, Maquenne, and others, +that the determination of sugars by this method is not a very difficult +analytical process and may, in the near future, become of great +practical importance. + +=148. Molecular Weights of Carbohydrates.=—In the examination of +carbohydrates the determination of the molecular weights is often of +the highest analytical value. + +The uncertainty in respect of the true molecular weights of the +carbohydrates is gradually disappearing by reason of the insight into +the composition of these bodies, which recently discovered physical +relations have permitted. + +Raoult, many years ago,[116] proposed a method of determining molecular +weights which is particularly applicable to carbohydrates soluble in +water. + +The principle of Raoult’s discovery may be stated as follows: The +depression of the freezing point of a liquid, caused by the presence of +a dissolved liquid or solid, is proportionate to the absolute amount of +substance dissolved and inversely proportionate to its molecular weight. + +The following formulas may be used in computing results: + +_C_ = observed depression of freezing point: + +_P_ = weight of anhydrous substance in 100 grams: + + _C_ + --- = _A_ = depression produced by one gram substance in 100 grams: + _P_ + +_K_ = depression produced by dissolving in 100 cubic centimeters a +number of grams of the substance corresponding to its molecular weight: + +_M_ = molecular weight: + + _C_ + Then we have, _K_ = ---- × _M_. + _P_ + +_K_ is a quantity varying with the nature of the solvent but with +the same solvent remaining sensibly constant for numerous groups of +compounds. + +The value of + + _C_ + _A_ ---- + _P_ + +can be determined by experiment. The molecular weight can therefore be +calculated from the formula + + _K_ + _M_ = ----. + _A_ + +With organic compounds in water the value of _K_ is almost constant. + +Brown and Morris[117] report results of their work in extending Raoult’s +investigations of the molecular weight of the carbohydrates. The +process is carried on as follows: + +A solution of the carbohydrate is prepared containing a known weight +of the substance in 100 cubic centimeters of water. About 120 cubic +centimeters of the solution are introduced into a thin beaker of about +400 capacity. This beaker is closed with a stopper with three holes. +Through one of these a glass rod for stirring the solution is inserted. +The second perforation carries a delicate thermometer graduated to +0°.05. The temperature is read with a telescope. The beaker is placed +in a mixture of ice and brine at a temperature from 2° to 3° below +the freezing point of the solution. The solution is cooled until its +temperature is from 0°.5 to 1° below the point of congelation. Through +the third aperture in the stopper a small lump of ice taken from a +frozen portion of the same solution, is dropped, causing at once the +freezing process to begin. The liquid is briskly stirred and as the +congelation goes on the temperature rises and finally becomes constant. +The reading is then taken. The depression in the freezing point, +controlled by the strength of the solution, should never be more than +from 1° to 2°. + +The molecular weights may also be determined by the boiling points of +their solutions as indicated by the author,[118] Beckmann,[119] Hite, +Orndorff and Cameron.[120] + +The method applied to some of the more important carbohydrates gave the +following results: + + DEXTROSE. + + Calculated for C₆H₁₂O₆. Found. + _M_ = 180 _M_ = 180.2 + + SUCROSE. + + Calculated for C₁₂H₂₂O₁₁. Found. + _M_ = 342 _M_ = 337.5 + + INVERTOSE (DEXTROSE AND LEVULOSE). + + Calculated for C₆H₁₂O₆. Found. + _M_ = 180 _M_ = 174.3 + + MALTOSE. + + Calculated for C₁₂H₂₂O₁₁. Found. + _M_ = 342 _M_ = 322 + + LACTOSE. + + Calculated for C₁₂H₂₂O₁₁. Found. + _M_ = 342 _M_ = 345 + + ARABINOSE. + + Calculated for C₅H₁₀O₅. Found. + _M_ = 150 _M_ = 150.3 + + RAFFINOSE. + Calculated for + C₁₈H₃₂O₁₆.5H₂O. Found. + _M_ = 594 _M_ = 528 + +=149. Birotation.=—As is well known, dextrose exhibits in fresh +solutions the phenomenon of birotation. The authors supposed that +this phenomenon might have some relation to the size of the molecule. +They, therefore, determined the molecular volume of freshly dissolved +dextrose by the method of Raoult and found _M_ = 180. The high rotatory +power of recently dissolved dextrose is therefore not due to any +variation in the size of its molecule. + +The mathematical theory of birotation is given by Müller as +follows.[121] In proportion as the unstable modification _A_ is +transformed into the stable modification _B_, the rotation will vary. +Let ρ = the specific rotatory power of _B_ and _a_ρ = that of _A_, both +in the anhydrous state. Let now _p_ grams of the substance be dissolved +in _V_ cubic centimeters of solvent and observed in a tube _l_ +decimeters in length. The time from making the solution is represented +by θ. The angle of rotation α is read at the time θ. Let _x_ = the mass +of _A_, and _y_ = that of _B_, and the equation is derived. + + _a_ρ_xl_ ρ_yl_ + α = --------- + ------: + _V_ _V_ + + But _x_ + _y_ = _p_ + + ρ_l_ + whence α = [(_a_ - 1)_x_ + _p_] ----. + _V_ + +If now there be introduced into the calculation the final angle of +rotation αₙ, which can be determined with great exactness; we have + + _p_ρ_l_ (_a_ - 1)_x_ + αₙ = ------- and consequently α = αₙ[1 + ------------], + _V_ _p_ + + (_a_ - 1)_x_ α + whence ------------- = --- - 1. + _p_ αₙ + +This equation gives the quantity _x_ of the unstable matter which is +transformed into the stable modification in the time θ. + +It must be admitted that the quantity _dx_ which is changed during the +infinitely small time _d_θ is proportional to the mass _x_ which still +exists at the moment θ, whence _dx_ = -Cʹ_xd_θ where Cʹ represents a +constant positive factor. From this is derived the equation + + _dx_ + ----- = -Cʹ_d_θ. + _x_ + +Integrating and calling _x_ the quantity of matter changed to the +stable form at the moment θ, corresponding to a rotation α₀, we have + + 1 _x_₀ + Cʹ = ------- log. nap. ----, and taking into consideration + θ - θ₀ _x_ + +the equation given above, and substituting common for superior +logarithms we get + + 1 α₀ - αₙ + C = --------- log. ---------. + θ - θ₀ α - αₙ + +Experience has shown that such a constant C really exists, and +its value can be easily calculated from the data of Parcus and +Tollens.[122] The mean value of C from these data is 0.0301 for +arabinose; 0.0201 for xylose; 0.0393 for rhamnose; 0.0202 for fucose; +0.00927 for galactose; 0.00405 for lactose; 0.00524 for maltose, and +for dextrose, 0.00348 at 11° to 13° and 0.00398 from 13° to 15°. The +constant C as is well known, increases as the temperature is raised. + +The constant C, at a given temperature, measures the progress of the +phenomenon of the change from the unstable to the stable state. It will +be noticed that among the sugars possessing multirotation properties +the pentoses possess a much higher speed of transformation than the +others. + +=150. Estimation of Pentose Sugars and Pentosans as Furfurol.=—The +production of furfurol by distilling carbohydrates with an acid has +already been mentioned. Tollens and his associates have shown that with +pentose sugars, and carbohydrate bodies yielding them, the production +of furfurol is quantitive. + +The production and estimation of furfurol have been systematically +studied by Krug, to whose paper the reader is referred for the complete +literature of the subject.[123] The essential principles of the +operation are based on the conversion of the pentoses into furfurol by +distilling with a strong acid, and the subsequent precipitation and +estimation of the furfurol formed in the first part of the reaction. + +The best method of conducting the distillation is as follows: + +Five grams of the pentose substance are placed in a flask of about a +quarter liter capacity, with 100 cubic centimeters of hydrochloric acid +of 1.06 specific gravity. The arrangement of the apparatus is shown in +Fig. 46. The flame of the lamp is so regulated as to secure about two +cubic centimeters of distillate per minute. + +[Illustration: FIGURE 46. DISTILLING APPARATUS FOR PENTOSES.] + +The distillate is received in a graduated cylinder and as soon +as thirty cubic centimeters are collected, an equal quantity of +hydrochloric acid, of the strength noted, is added to the distilling +flask, allowing it to flow in slowly so as not to stop the ebullition. +The process is continued until a drop of the distillate gives no +sensible reaction for furfurol when tested with anilin acetate. The +test is applied as follows: Place a drop of the distillate on a piece +of filter paper moistened with anilin acetate. The presence of furfurol +will be disclosed by the production of a brilliant red color. Usually +about three hours are consumed in the distillation, during which time a +little less than 400 cubic centimeters of distillate is obtained. The +distillate is neutralized with solid sodium carbonate and, in order +to have always the same quantity of common salt present, 10.2 grams +of sodium chlorid are added for each fifty cubic centimeters of water +necessary to make the total volume to half a liter.[124] + +The reactions with pentosans probably consist in first splitting up of +the molecule into a pentose and the subsequent conversion of the latter +into furfurol according to the following equations: + + (C₅H₈O₄)ₙ + (H₂O)ₙ = (C₅H₁₀O₅)ₙ + Pentosan. Water. Pentose. + + and + + (C₅H₁₀O₅)ₙ = (C₅H₄O₂)ₙ + (3H₂O)ₙ. + Pentose. Furfurol. Water. + +=151. Determination of Furfurol.=—The quantity of furfurol obtained by +the process mentioned above may be determined in several ways. + +_As Furfuramid._—When ammonia is added to a saturated solution of +furfurol, furfuramid, (C₅H₄O)₃N₂, is formed. In order to secure the +precipitate it is necessary that the furfurol be highly concentrated +and this can only be accomplished by a tedious fractional distillation. +This method, therefore, has little practical value. + +_As Furfurolhydrazone._—Furfurol is precipitated almost quantitively, +even from dilute solutions, by phenylhydrazin. The reaction is +represented by the equation: + + C₆H₈N₂ + C₅H₄O₂ = C₁₁H₁₀N₂O + H₂O. + Phenylhydrazin. Furfurol. Furfurolhydrazone. Water. + +=152. Volumetric Methods.=—Tollens and Günther have proposed +a volumetric method which is carried out as follows:[125] The +distillation is accomplished in the manner described. The distillate +is placed in a large beaker, neutralized with sodium carbonate and +acidified with a few drops of acetic. Phenylhydrazin solution of +known strength is run in until a drop of the liquid, after thorough +mixing, shows no reaction for furfurol with anilin acetate. The +reagent is prepared by dissolving five grams of pure phenylhydrazin and +three of glacial acetic acid in distilled water, and diluting to 100 +cubic centimeters. The solution is set by dissolving from two-tenths +to three-tenths gram of pure furfurol in half a liter of water and +titrating with the phenylhydrazin as indicated above. The quantity of +the pentose used has a great influence on the result. + +With nearly a gram of arabinose about fifty per cent of furfurol were +obtained while when nearly five grams were used only about forty-six +per cent of furfurol were found. With xylose a similar variation was +found, the percentage of furfurol, decreasing as the quantity of +pentose increased. The method, therefore, gives only approximately +accurate results. + +=153. Method of Stone.=—Another volumetric method proposed by Stone +is based on the detection of an excess of phenylhydrazin by its +reducing action on the fehling reagent.[126] A standard solution of +phenylhydrazin is prepared by dissolving one gram of the hydrochlorate +and three grams of sodium acetate in water and completing the volume +of the liquor to 100 cubic centimeters. This solution contains 1.494 +milligrams of phenylhydrazin in each cubic centimeter, theoretically +equivalent to 1.328 milligrams of furfurol. The reagent is set by +titrating against a known weight of furfurol. Pure furfurol may be +prepared by treating the crude article with sulfuric acid and potassium +dichromate, and subjecting the product to fractional distillation. +The distillate is treated with ammonia and the furfuramid formed is +purified by recrystallizing from alcohol and drying over sulfuric acid. +One gram of this furfuramid is dissolved in dilute acetic acid and +the volume completed to one liter with water.[127] The phenylhydrazin +solution being unstable, is to be prepared at the time of use. + +The titration is conducted as follows: Twenty-five cubic centimeters of +the distillate obtained from a pentose body, by the method described +above, are diluted with an equal volume of water, a certain quantity +of the phenylhydrazin solution added to the mixture from a burette and +the whole heated quickly to boiling. The flask is rapidly cooled and a +portion of its contents poured on a filter. The filtrate should have +a pale yellow color and be perfectly clear. If it become turbid on +standing, it should be refiltered. Two cubic centimeters of the clear +filtrate are boiled with double the quantity of the fehling reagent. +If phenylhydrazin be present, the color of the mixture will change +from blue to green. By repeating the work, with varying quantities of +phenylhydrazin, a point will soon be reached showing the end of the +reaction in a manner entirely analogous to that observed in volumetric +sugar analysis. + +In practice the volumetric methods have given place to the more exact +gravimetric methods described below. + +=154. Gravimetric Methods.=—The distillation is carried on and the +volume of the distillate completed to half a liter as described above. +Chalmot and Tollens then proceed as follows:[128] Ten cubic centimeters +of a solution of phenylhydrazin acetate, containing in 100 cubic +centimeters twelve grams of the phenylhydrazin and seven and a half +grams of glacial acetic acid dissolved and filtered, are added to the +distillate and the mixture stirred with an appropriate mechanism for +half an hour. The furfurolhydrazone at the end of this time will have +separated as small reddish-brown crystals. The mixture is then thrown +onto an asbestos filter and the liquid separated with suction. The +suction should be very gradually applied so as not to clog the felt. +The precipitate adhering to the beaker is washed into the filter with +100 cubic centimeters of water. The precipitate is dried at about 60° +and weighed. As a check the hydrazone may be dissolved in hot alcohol, +the filter well washed, dried and again weighed. To obtain the weight +of furfurol the weight of hydrazone found is multiplied by 0.516 and +0.025 added to compensate for the amount which was held in solution +or removed by washing. Less than one per cent of pentose can not be +determined by this method since that amount is equalled by the known +losses during the manipulation. + +_Factor._—To convert the furfurol found into pentoses, the following +factors are used: + + Per cent furfurol Multiply for + obtained from Multiply for Multiply for penta-glucoses + five grams of arabinose by. xylose by. by. + pentoses. + + 2.5 per cent or less 1.90 1.70 1.67 + 5.0 ” ” ” more 2.04 1.90 1.92 + +=155. Method Of Krug.=—In conducting the determination of furfurol, +according to the method of Chalmont and Tollens just noticed, Krug +observed that the filtrate, after standing for some time, yielded a +second precipitate of furfurol hydrazone. Great difficulty was also +experienced in collecting the precipitate upon the filter on account +of the persistency with which it stuck to the sides of the vessel +in which the precipitation took place.[129] In order to avoid these +two objections, Krug modified the method as described below and this +modified method is now exclusively used in this laboratory. + +After the precipitation of the furfurol hydrazone, it is stirred +vigorously, by means of an appropriate mechanical stirrer, for at +least half an hour and then allowed to rest for twenty-four hours. On +filtering after that length of time the filtrate remains perfectly +clear and no further precipitation takes place. After the filtration is +complete and the beaker and filtering tube well washed, no attempt is +made to detach the part of the filtrate adhering to the beaker but the +whole of the precipitate, both that upon the filter and that adhering +to the sides of the beaker, is dissolved in strong alcohol, from thirty +to forty cubic centimeters being used. The alcoholic solution is +collected in a small weighed flask, the alcohol evaporated at a gentle +heat and the last traces of water removed by heating to 60° and blowing +a current of dry air through the flask. After weighing the precipitate +of furfurol hydrazone, obtained as above, the calculation of the weight +of pentose bodies is accomplished by means of the usual factors. + +=156. Precipitation of Furfurol with Pyrogalol.=—Furfurol is thrown +out of solution in combination with certain phenol bodies by heating +together in an acid solution. Hotter has proposed a method for the +determination of furfurol based on the above fact.[130] The furfurol +is obtained by distillation in the manner already described and +hydrochloric acid is added if necessary to secure twelve per cent of +that body in a given volume. The furfurol is thrown out of an aliquot +portion by heating with an excess of pyrogalol in closed tubes for +about two hours at 110°. The reaction takes place in two stages, +represented by the following equations: + + C₅H₄O₂ + C₆H₆O₃ = C₁₁H₁₀O₅ + + and + + 2C₁₁H₁₀O₅ = C₂₂H₁₈O₉ + H₂O. + +The aliquot part of the distillate used should not contain more than +one-tenth of a gram of furfurol. The precipitate formed in this way is +collected on an asbestos felt, dried at 103° and weighed. The weight +obtained divided by 1.974 gives the corresponding amount of furfurol. +There is some difficulty experienced in loosening the precipitate from +the sides of the tubes in which the heating takes place, but this +defect can be overcome by heating in covered beakers in an autoclave. + +=157. Precipitation with Phloroglucin.=—Instead of using pyrogalol for +the precipitating reagent phloroglucin may be employed. The method of +procedure proposed by Councler for this purpose is given below.[131] +The furfurol is prepared by distillation in the usual way. The volume +of the distillate obtained is completed to half a liter with twelve per +cent hydrochloric acid, and an aliquot portion, varying in volume with +the percentage of furfurol is withdrawn for precipitation. This portion +is placed in a glass-stoppered flask with about twice the quantity of +finely powdered phloroglucin necessary to combine with the furfurol +present. The contents of the flask are well shaken and allowed to stand +fifteen hours. The precipitate is collected on an asbestos filter, +washed free of chlorin, dried at the temperature of boiling water and +weighed. + +The theoretical quantity of precipitate corresponding to one part of +furfurol, _viz._, 2.22 parts, is never obtained since the precipitate +is not wholly insoluble in water. The actual proportions between +the precipitate and the original furfurol vary with the amount of +precipitate obtained. + +When the weight of the precipitate is 200 milligrams and over, 2.12 +parts correspond to one part of furfurol. When the weight of the +precipitate varies from fifty to 100 milligrams, the ratio is as 2.05:1 +and when only about twenty-five milligrams of precipitate are obtained +the ratio is as 1.98:1. + +The quantity of pentose bodies corresponding to the furfurol is +calculated from the factors given by Tollens in a preceding paragraph. + +The reaction which takes place with furfurol and phloroglucin is simply +a condensation of the reagents with the separation of water. It is very +nearly represented by the following formula: + + 2C₅H₄O₂ + C₆H₆O₃ = C₁₆H₁₂O₆ + H₂O. + Furfurol. Phloroglucin Condensation Water. + product. + +It has been shown by Welbel and Zeisel,[132] that in the presence of +twelve per cent of hydrochloric acid phloroglucin itself is condensed +into dark insoluble compounds. When three molecules of furfurol and two +molecules of phloroglucin are present, the bodies are both condensed +and separated by continued action. When from one and a quarter to three +parts of phloroglucin by weight are used to one part of furfurol, the +weight of the precipitate obtained under constant conditions may serve +sufficiently well for the calculation of the furfurol. The precipitates +contain chlorin, which they give up even in the cold, to water. For +these reasons the analytical data obtained by the method of Councler, +given above, are apt to be misleading. It is probable also that +similar conditions may to a certain extent prevail in the separation +of furfurol with phenylhydrazin, and further investigation in this +direction is desirable. For the present the very best method that can +be recommended for the estimation of pentoses and pentosans is the +conversion thereof into furfurol and the separation of the compound +with phenylhydrazin acetate. + +=158. Estimation of Sugars by Fermentation.=—When a solution of +a hexose sugar is subjected to the action of certain ferments a +decomposition of the molecule takes place with the production of +carbon dioxid and various alcohols and organic acids. Under the action +of the ferment of yeast _Saccharomyces cerevisiae_ the sugar yields +theoretically only carbon dioxid and ethyl alcohol, as represented by +the equation: + + C₆H₁₂O₆ = 2C₂H₆O + 2CO₂. + +The theoretical quantities of alcohol and carbon dioxid obtained +according to this equation are 51.11 percent of alcohol and 48.89 per +cent of carbon dioxid. + +When the yeast ferment acts on cane sugar the latter first suffers +inversion, and the molecules of dextrose and levulose produced are +subsequently converted into alcohol and carbon dioxid as represented +below: + + C₁₂H₂₂O₁₁ + H₂O = 2C₆H₁₂O₆ + + 2C₆H₁₂O₆ = 4C₂H₆O + 4CO₂. + +Cane sugar, plus the water of hydrolysis, will yield theoretically 53.8 +per cent of alcohol and 51.5 per cent of carbon dioxid. + +In practice the theoretical proportions of alcohol and carbon dioxid +are not obtained because of the difficulty of excluding other +fermentative action, resulting in the formation especially of succinic +acid and glycerol. Moreover, a part of the sugar is consumed by the +yeast cells to secure their proper growth and development. In all +only about ninety-five per cent of the sugar can be safely assumed as +entering into the production of alcohol. About 48.5 per cent of alcohol +are all that may be expected of the weight of dextrose or invert sugar +used. Only sugars containing three molecules of carbon or some multiple +thereof are fermentable. Thus the trioses, hexoses, nonoses, etc., are +susceptible of fermentation, while the tetroses, pentoses, etc., are +not. + +=159. Estimating Alcohol.=—In the determination of sugar by +fermentation, a rather dilute solution not exceeding ten per cent +should be used. A quantity of pure yeast, equivalent to four or five +per cent of the sugar used, is added, and the contents of the vessel, +after being well shaken, exposed to a temperature of from 25° to 30° +until the fermentation has ceased, which will be usually in from +twenty-four to thirty-six hours. The alcohol is then determined in the +residue by the methods given hereafter. + +The weight of the alcohol obtained multiplied by 100 and divided by +48.5, will give the weight of the hexose reducing sugar which has been +fermented. Ninety-five parts of sucrose will give 100 parts of invert +sugar. + +_Example._—Let the weight of alcohol obtained be 0.625 gram. Then 0.625 +× 100 ÷ 48.5 = 1.289 grams, the weight of the hexose, which has been +fermented; 1.289 grams of dextrose or levulose correspond to 1.225 of +sucrose. + +=160. Estimating Carbon Dioxid.=—The sugar may also be determined +by estimating the amount of carbon dioxid produced during the +fermentation. For this purpose the mixture of sugar solution and yeast, +prepared as above mentioned, is placed in a flask whose stopper carries +two tubes, one of which introduces air free of carbon dioxid into the +contents of the flask, and the other conducts the evolved carbon dioxid +into the absorption bulbs. In passing to the absorption bulbs the +carbon dioxid is freed of moisture by passing through another set of +bulbs filled with strong sulfuric acid. During the fermentation, the +carbon dioxid is forced through the bulbs by the pressure produced, or +better, a slow current of air is aspirated through the whole apparatus. +The aspiration is continued after the fermentation, has ceased, until +all the carbon dioxid is expelled. Towards the end, the contents of +the flask may be heated to near the boiling-point. The increase of +the weight of the potash bulbs will give the weight of carbon dioxid +obtained. A hexose reducing sugar will yield about 46.5 per cent of its +weight of carbon dioxid. The calculation is made as suggested for the +alcohol process. + +The fermentation process has little practical value save in determining +sucrose in presence of lactose, as will be described in another place. + +=161. Precipitation of Sugars in Combination with the Earthy +Bases.=—The sugars combine in varying proportions with the oxids +and hydroxids of calcium, strontium and barium. Sucrose especially, +furnishes definite crystalline aggregates with these bases in such +a way as to form the groundwork of several technical processes in +the separation of that substance from its normally and abnormally +associated compounds. These processes have little use as analytical +methods, but are of great value, as mentioned, from a technical point +of view. + +=162. Barium Saccharate.=—This compound is formed by mixing the aqueous +solutions of barium hydroxid and sugar. The saccharate separates +in bright crystalline plates or needles from the warm solution, +as C₁₂H₂₂O₁₁BaO. One part of this precipitate is soluble in about +forty-five parts of water, both at 15° and 100°. + +=163. Strontium Saccharates.=—Both the mono- and distrontium +saccharates are known, _viz._, C₁₂H₂₂O₁₁SrO + 5H₂O and C₁₂H₂₂O₁₁2SrO. + +The monosalt may be easily secured by adding a few of its crystals to a +mixture of sugar and strontium hydroxid solutions. + +The disaccharate is precipitated as a granular substance when from two +to three molecules of strontium hydroxid are added to a boiling sugar +solution. The reaction is extensively used in separating the sugar from +beet molasses. + +=164. Calcium Saccharates.=—Three calcium saccharates are known in +which one molecule of sugar is combined with one, two and three +molecules of lime respectively. + +The monosaccharate is obtained by mixing the sugar and lime in the +proper proportion and precipitating by adding alcohol. + +The precipitate is partly granular and partly jelly-like, and is +soluble in cold water. The dicalcium compound is obtained in the same +way and has similar properties. Both, on boiling, with water, form the +trisaccharate and free sugar. + +The tricalcium saccharate is the most important of these compounds, and +may be obtained directly by mixing freshly burned and finely ground +lime (CaO) with a very cold dilute solution of sugar. + +The compound crystallizes with three or four molecules of water. +When precipitated as described above, however, it has a granular, +nearly amorphous structure, and the process is frequently used in the +separation of sugar from beet molasses. + +In the laboratory but little success has been had in using even the +barium hydroxid as a chemical reagent, and therefore the reactions +mentioned above are of little value for analytical purposes. In +separating sugar from vegetable fibers and seeds, however, the +treatment with strontium hydroxid is especially valuable the sugar +being subsequently recovered in a free state by breaking up the +saccharate with carbon dioxid. The technical use of these reactions +also is of great importance in the beet sugar industry. + +=165. Qualitive Tests for the Different Sugars.=—The analyst will +often be aided in examining an unknown substance by the application +of qualitive tests, which will disclose to him the nature of the +saccharine bodies with which he has to deal. + +=166. Optical Test for Sucrose.=—The simplest test for the presence of +sucrose is made with the polariscope. A small quantity of the sample +under examination is dissolved in water, clarified by any of the usual +methods, best with alumina cream, and polarized. A portion of the +liquor is diluted with one-tenth its volume of strong hydrochloric acid +and heated to just 68°, consuming about fifteen minutes time in the +operation. The mixture is quickly cooled and again polarized in a tube +one-tenth longer than before used; or the same tube may be used and the +observed reading of the scale increased by one-tenth. If sucrose be +present the second reading will be much lower than the first, or may +even be to the left. + +=167. Cobaltous Nitrate Test for Sucrose.=—Sucrose in solution may be +distinguished from other sugars by the amethyst violet color which it +imparts to a solution of cobaltous nitrate. This reaction was first +described by Reich, in 1856, but has only lately been worked out in +detail. The test is applied as follows: + +To about fifteen cubic centimeters of the sugar solution add five cubic +centimeters of a five per cent solution of cobaltous nitrate. After +thoroughly mixing the two solutions, add two cubic centimeters of a +fifty per cent solution of sodium hydroxid. Pure sucrose gives by this +treatment an amethyst violet color, which is permanent. Pure dextrose +gives a turquoise blue color which soon passes into a light green. When +the two sugars are mixed the coloration produced by the sucrose is the +predominant one, and one part of sucrose in nine parts of dextrose can +be distinguished. If the sucrose be mixed with impurities such as gum +arabic or dextrin, they should be precipitated by alcohol or basic lead +acetate, before the application of the test. Dextrin may be thrown +out by treatment of the solution with barium hydroxid and ammoniacal +lead acetate. The reaction may also be applied to the detection of +cane sugar in wines, after they are thoroughly decolorized by means of +lead acetate and bone-black. The presence of added sucrose to milk, +either in the fresh or condensed state, may also be detected after the +disturbing matters are thrown out with lead acetate. The presence of +sucrose in honey may also be detected by this process. The reaction +has been tried in this laboratory with very satisfactory results. The +amethyst violet coloration with sucrose is practically permanent. On +boiling the color is made slightly bluish, but is restored to the +original tint on cooling. Dextrose gives at first a fine blue color +which in the course of two hours passes into a pale green. A slight +flocculent precipitate is noticed in the tube containing the dextrose. +Maltose and lactose act very much as dextrose, but in the end do not +give so fine a green color. If the solutions containing dextrose, +lactose and maltose be boiled, the original color is destroyed and a +yellow-green color takes its place. The reaction is one which promises +to be of considerable practical value to analysts, as it may be applied +for the qualitive detection of sucrose in seeds and other vegetable +products.[133] + +=168. The Dextrose Group.=—In case the carbohydrate in question shows a +right-handed rotation and the absence of sucrose is established by the +polariscopic observation described above, the presence of the dextrose +group may be determined by the following test.[134] + +Five grams of the carbohydrate are oxidized by boiling with from twenty +to thirty cubic centimeters of nitric acid of 1.15 specific gravity, +and then at gentle heat evaporated to dryness with stirring. If much +mucic acid be present, as will be the case if the original matter +contained lactose some water is added and the mixture well stirred, +and again evaporated to dryness to expel all nitric acid. The residue +should be of a brown color. The mass is again mixed with a little +water and the acid reaction neutralized by rubbing with fine-ground +potassium carbonate. The carbonate should be added in slight excess and +acetic acid added to the alkaline mixture, which is concentrated by +evaporation and allowed to stand a few days. At the end of this time +potassium saccharate has formed and is separated from the mother liquid +by pouring on a porous porcelain plate. The residue is collected, +dissolved in a little water and again allowed to crystallize, when it +is collected on a porous plate, as before, and washed by means of an +atomizer with a little aqueous spray until it is pure white and free of +any oxalic acid. The residual acid potassium saccharate may be weighed +after drying and then converted into the silver salt. The potash salt +for this purpose is dissolved in water, neutralized with ammonia and +precipitated with a solution of silver nitrate. The precipitate is +well stirred, collected on a gooch and washed and dried in a dark +place. It contains 50.94 percent of silver. All sugars which contain +the dextrose group yield silver saccharate when treated as above +described. Inulin, sorbose, arabinose and galactose yield no saccharic +acid under this treatment, and thus it is shown that they contain +no dextrose group. Milk sugar, maltose, the dextrins, raffinose and +sucrose yield saccharic acid when treated as above and therefore all +contain the dextrose group. + +=169. Levulose.=—The levulose group of sugars, wherever it occurs, when +oxidized with nitric acid, gives rise to tartaric, racemic, glycolic +and oxalic acids, which are not characteristic, being produced also +by the oxidation of other carbohydrates. A more distinguishing test +is afforded by the color reactions produced with resorcin.[135] The +reagent is prepared by dissolving half a gram of resorcin in thirty +cubic centimeters each of water and strong hydrochloric acid. To the +sugar solution under examination an equal volume of strong hydrochloric +acid is added, and then a few drops of the reagent. The mixture is +gently warmed, and in the presence of levulose develops a fire-red +color. Dextrose, lactose, mannose and the pentoses do not give the +coloration, but it is produced by sorbose in a striking degree, and +also by sucrose and raffinose since these sugars contain the levulose +group. + +=170. Galactose.=—The galactose which arises from the hydrolysis of +milk sugar is readily recognized by the mucic acid which it gives on +oxidation with nitric acid.[136] The analytical work is conducted +as follows: The body containing galactose or galactan is placed in +a beaker with about sixty cubic centimeters of nitric acid of 1.15 +specific gravity for each five grams of the sample used. The beaker +is placed on a steam-bath and heated, with frequent stirring, until +two-thirds of the nitric acid have been evaporated. The residual +mixture is allowed to stand over night and the following morning is +treated with ten cubic centimeters of water, allowed to stand for +twenty-four hours, filtered through a gooch, and the collected matter +washed with twenty-five cubic centimeters of water, dried at 100° and +weighed. The mucic acid collected in this way will amount to about +thirty-seven per cent of the milk sugar or seventy-five per cent of the +galactose oxidized. Raffinose yields under similar treatment, about +twenty-three per cent of mucic acid, which proves that the galactose +group is contained in that sugar. Raffinose, therefore, is composed of +one molecule each of dextrose, levulose, and galactose. + +=171. Invert Sugar.=—The presence of a trace of invert sugar +accompanying sucrose can be determined by Soldaini’s solution, +paragraph =124=, or by boiling with methyl blue.[137] Methyl blue is +the hydrochlorate of an ammonium base, which, under the influence of +a reducing agent, loses two atoms of hydrogen and becomes a colorless +compound. The test for invert sugar is made as follows: The reagent +is prepared by dissolving one gram of methyl blue in water. If the +sugar solution is not clear, twenty grams of the sugar are dissolved +in water clarified by lead subacetate, the volume completed to 100 +cubic centimeters, and the solid matters separated by filtration. The +filtrate is made slightly alkaline with sodium carbonate to remove the +lead. A few drops of soda lye solution are then added and the mixture +thrown on a filter. To twenty-five cubic centimeters of the filtrate a +drop of the methyl blue solution is added, and a portion of the liquor +heated in a test tube over the naked flame. If, after boiling for one +minute, the coloration disappear, the sample contains at least 0.01 +per cent of invert sugar; if the solution remain blue it contains none +at all or less than 0.01 per cent. The test may also be made with the +dilute copper carbonate solution of Ost described further on. + +=172. Compounds with Phenylhydrazin.=—Many sugars may also be +qualitively distinguished by the character of their compounds with +phenylhydrazin. In general, it may be said that those sugars which +reduce fehling solution form definite crystalline compounds with the +reagent named. If a moderately dilute hot solution of a reducing sugar +be brought into contact with phenylhydrazin acetate, a crystalline +osazone is separated. The reaction takes place between one molecule of +the sugar and two molecules of the hydrazin compound, according to the +following formula: + + C₆H₁₂O₆ + 2C₆H₅N₂H₃ = C₁₈H₂₂N₄O₄ + 2H₂O + H₂. + +The hydrogen does not escape but combines in the nascent state with the +excess of phenylhydrazin to form anilin and ammonia. + +The precipitation is accomplished as follows: + +One part by weight of the sugar, two parts of phenylhydrazin +hydrochlorate, and three parts of sodium acetate are dissolved in +twenty parts of water and gradually heated on the water-bath. + +The osazone slowly separates in a crystalline form and it is freed from +the mother liquor by filtration, and purified by solution in alcohol +and recrystallization. The crystals are composed of yellow needles, +which are difficultly soluble in water and more easily in hot alcohol. +The crystals are not decomposed by a dilute acid but are destroyed by +the action of strong acids. + +_Dextrosazone._—The crystals melt at from 204° to 205°, reduce fehling +liquor, and dissolved in glacial acetic acid are slightly left rotating. + +_Levulosazone._—This body has the same properties as the dextrose +compound. + +_Maltosazone._—This substance melts at 206° with decomposition. It is +left rotating. Its structure is represented by the formula C₂₄H₃₂N₄O₉. + +_Galactosazone._—This substance, C₁₈H₂₂N₄O₄, has the same centesimal +composition as the corresponding bodies produced from dextrose and +levulose. It is distinguished from these compounds, however, by its low +melting point, _viz._, 193°. + +The above comprise all the phenylosazones which are important from +the present point of view. Sucrose, by inversion, furnishes a mixture +of dextros- and levulosazones when treated with phenylhydrazin, while +starch and dextrin yield the dextros- or maltosazone when hydrolyzed. +Lactose yields a mixture of dextros- and galactosazones when hydrolyzed +and treated as above described. + +The reactions with phenylhydrazin are approximately quantitive and it +is possible that methods of exact determination may be based on them in +the near future.[138] + +=173. Other Qualitive Tests for Sugars.=—The analyst may sometimes +desire a more extended test of qualitive reactions than those given +above. The changes of color noticed on heating with alkalies may +often be of advantage in discriminating between different sugars. The +formation of definite compounds with the earthy and other mineral bases +may also be used for qualitive determinations. One of the most delicate +qualitive tests is found in the production of furfurol and this will be +described in the following paragraphs. + +=174. Detection of Sugars and Other Carbohydrates by Means of +Furfurol.=—The production of furfurol (furfuraldehyd) as noted in +paragraph =150=, is also used quantitively for the determination of +pentose sugars and pentosans. + +Furfurol was first obtained from bran (_furfur_), whence its name, +by treating this substance with sulfuric acid, diluted with three +volumes of water, and subjecting the mixture to distillation. Its +percentage composition is represented by the symbol C₅H₄O₂, and its +characteristics as an aldehyd by the molecular structure C₄H₃O,C-HO. + +Carbohydrates in general, when treated as described above for bran, +yield furfurol, but only in a moderate quantity, with the exception of +the pentoses. + +Mylius has shown[139] that Pettenkofer’s reaction for choleic acid is +due to the furfurol arising from the cane sugar employed, which, with +the gall acid, produces the beautiful red-blue colors characteristic of +the reaction. + +Von Udránszky[140] describes methods for detecting traces of +carbohydrates by the furfurol reaction, which admit of extreme +delicacy. The solution of furfurol in water, at first proposed by +Mylius, is to be used and it should not contain more than two and +two-tenths per cent, while a solution containing five-tenths per cent +furfurol is found to be most convenient. The furfurol, before using, +should be purified by distillation, and, as a rule, only a single drop +of the solution used for the color reaction. + +The furfurol reaction proposed by Schiff[141] appears to be well suited +for the detection of carbohydrates. It is made as follows: + +Xylidin is mixed with an equal volume of glacial acetic acid and the +solution treated with some alcohol. Strips of filter paper are then +dipped in the solution and dried. When these strips of prepared paper +are brought in contact with the most minute portion of furfurol, +furoxylidin is formed, C₄H₃OCH(C₈H₈NH₂)₂, producing a beautiful red +color. In practice, a small portion of the substance, supposed to +contain a carbohydrate, is placed in a test tube and heated with a +slight excess of concentrated sulfuric acid. The prepared paper is then +placed over the mouth of the test tube so as to be brought into contact +with the escaping vapors of furfurol. + +The furfurol reaction with α-naphthol for some purposes, especially +the detection of sugar in urine, is more delicate than the one just +described. This reaction was first described by Molisch[142], who, +however, did not understand its real nature. + +The process is carried on as follows: The dilute solution should +contain not to exceed from 0.05 to one-tenth per cent of carbohydrates. +If stronger, it should be diluted. Place one drop of the liquid in a +test tube with two drops of fifteen per cent alcoholic solution of +α-naphthol, add carefully one-half cubic centimeter of concentrated +sulfuric acid, allowing it to flow under the mixture. The appearance +of a violet ring over a greenish fringe indicates the presence of a +carbohydrate. If the substance under examination contain more than a +trace of nitrogenous matter, this must be removed before the tests +above described are applied. + +If the liquids be mixed by shaking when the violet ring is seen, a +carmine tint with a trace of blue is produced. If this be examined with +a spectroscope, a small absorption band will be found between D and E, +and from F outward the whole spectrum will be observed. One drop of +dextrose solution containing 0.05 per cent of sugar gives a distinct +reaction by this process. It can be used, therefore, to detect the +presence of as little as 0.028 milligram of grape sugar. This test has +been found exceedingly delicate in this laboratory, and sufficiently +satisfactory without the spectroscopic adjunct. + +The furfurol reaction is useful in detecting the presence of minute +traces of carbohydrates but is of little value in discriminating +between the different classes of these bodies. + +It is not practical here to go into greater detail in the description +of qualitive reactions. The analyst, desiring further information, +should consult the standard works on sugar chemistry.[143] + +=175. Detection of Sugars by Bacterial Action.=—Many forms of bacteria +manifest a selective action towards sugars and this property may in the +future become the basis of a qualitive and even quantitive test for +sugars and other carbohydrates. Our present knowledge of the subject +is due almost exclusively to the researches of Smith, conducted at the +Department of Agriculture.[144] Dextrose is the sugar first and most +vigorously attacked by bacterial action, and by proper precautions the +whole of the dextrose may be removed from mixtures with sucrose and +lactose. + +The development of other forms of micro-organisms which will have the +faculty of attacking other and special forms of carbohydrates is to be +looked for with confident assurance of success. + + +DETERMINATION OF STARCH. + +=176. Constitution of Starch.=—The molecule of starch is without doubt +formed by the condensation of a large number of hexose bodies. On +account of its great insolubility its molecular weight has not been +determined with any degree of accuracy. Its formula may be expressed +either as (C₆H₁₀O₅)ₙ or (C₁₂H₂₀O₁₀)ₙ. It is insoluble in cold water and +other common solvents and does not pass into solution in any reagent +without undergoing a change of structure. In hot water it forms a paste +and when heated under pressure with water it undergoes a partial change +and becomes soluble. Heated with acids or subjected to the action of +certain ferments it suffers hydrolysis and is transformed into dextrin, +maltose and dextrose. In analytical work an attempt is usually made to +transform the starch entirely into dextrose, the quantity of which is +then determined by some of the processes already given. All starches +possess the property of giving an intensely blue color with iodin and +this reaction serves to detect the most minute quantity of the material. + +Starch grains derived from different sources are distinguished by +differences in size and appearance. In most cases a careful examination +of the starch particles will reveal their origin.[145] The greatest +part of the cereal grains is composed of starch, the percentage ranging +from sixty to eighty. Rice has the greatest percentage of starch in +its composition of any substance. Certain root crops are also rich +in starch, such as the potato, artichoke and cassava. Starch appears +as one of the first products of vegetable metabolism, according to +some authorities, preceding the formation of sugars. By reason of its +greater complexity, however, it is more probable that the production +of simple sugars precedes the formation of the more complex molecule. +Starch granules are probably used as a food by the plant in the +building of more complex structures and the excess of this food is +stored in the seeds and in tubers. + +=177. Separation of Starch Particles.=—Advantage is taken of the +insolubility of the starch particles to secure their separation from +the other vegetable structures with which they are associated. The +substances containing starch are reduced to a pulp as fine as possible, +and this pulp being placed in a fine cloth the starch particles are +washed through the cloth with water. The milky filtrate carrying the +starch is collected in an appropriate holder and, after some time, the +particles subside. They may then be collected and dried. While this +process is the one used commercially in the manufacture of starch, it +can only give approximate data respecting the actual quantity of starch +in a given weight of the sample. It is not quite possible by this +method to get all the starch separated from the rest of the vegetable +matter, and particles of foreign substances, such as cellulose and +albuminoid matters, may pass through the filter cloth and be found +with the deposited granules. It follows from this that the quantitive +determination of the starch in a given sample by any direct method is +only approximately exact. + +=178. Methods of Separation.=—Hot acids cannot be safely employed +to dissolve starch from its natural concomitants because other +carbohydrate bodies become soluble under similar conditions. In such +cases the natural sugars which are present should be removed by cold +water and the starch dissolved from the residue by a diastatic ferment. +Instead of this the sugars may be determined in a separate portion +of the pulped material and the starch, together with the sugars, +determined, and the quantity of sugar found deducted from the final +result. + +In these cases the final determinations are made on the sugars, after +inverting the sucrose, and proceeding as directed for invert sugars +in paragraph =141=. The starch, after separation with diastase, is +converted into dextrose by one of the methods to be given and the +resulting dextrose determined by one of the approved methods. + +=179. Separation with Diastase.=—Diastase or malt extract at a +temperature of about 65° rapidly renders starch soluble. Cereals, +potato meal and other starch-holding bodies are dried, first at a low +temperature, and extracted with ether or petroleum to remove fat. The +material is then rubbed up with water, boiled, cooled to 65°, and +treated with malt extract (diastase) prepared as given below. One +kilogram of ground green malt is mixed with one liter of glycerol +and an equal quantity of water, and allowed to stand, with frequent +shaking, for eight days. After that time the mixture is filtered, +first through a small filter press and afterwards through paper. In +case no filter press is at hand the mixture may be pressed in a bag +and the liquor obtained, filtered. Malt extract obtained in this way +will keep its diastatic properties for a long time. In its use, blank +determinations must be made of the dextrose produced by treating equal +portions of it with hydrochloric acid. For three grams of starchy +material twenty-five cubic centimeters of the malt solution should be +used and the mixture kept at 65° for two hours.[146] + +=180. Method in Use at the Halle Station.=—The method of separating +starch from cereals, potatoes and other starch-holding materials, +employed at the Halle station, is essentially the same as already +described.[147] + +The malt extract used is prepared immediately beforehand, inasmuch +as no preservative is added to it. It can be quickly prepared by +digesting, for a short time at not above 50°, 100 grams of finely +ground dried malt with one liter of water and separating the extract by +filtration. This extract will keep only a few hours. + +The material in which the starch is to be determined is dried and +extracted with ether. From two to four grams of the extracted +material, according to the amount of starch which it contains, are +boiled for half an hour with 100 cubic centimeters of water, cooled to +65°, treated with ten cubic centimeters of malt extract and kept at +the temperature named for half an hour. It is then again boiled for +fifteen minutes, cooled to the temperature mentioned and again treated +with malt extract as above. Two treatments with malt extract are +usually sufficient to bring all the starch into solution. Finally it +is again boiled and the volume completed to 250 cubic centimeters and +thrown upon a filter. Two hundred cubic centimeters of the filtrate are +converted into dextrose by boiling with hydrochloric acid, and the rest +of the analysis is conducted in the usual manner. The dextrose value of +the quantity of malt extract used must be determined upon a separate +portion thereof, and the quantity of dextrose found deducted from the +total amount obtained in the analysis. + +[Illustration: FIGURE 47. AUTOCLAVE FOR STARCH ANALYSIS.] + +=181. Separation by Hydrolysis with Water at High +Temperatures.=—Instead of dissolving the starch with diastase, it may +be brought into solution by heating with water under pressure. The +former method employed of heating in sealed flasks has been entirely +superceded by heating in an autoclave. The materials are best held in +metal beakers furnished with a cover which prevents loss from boiling +if the pressure should be removed too rapidly after the completion +of the operation. The autoclave is a strong metal vessel capable of +resisting the pressure of several atmospheres. It is furnished with a +pressure gauge C and a safety valve D, as shown in the figure. The top +is securely screwed on by means of a wrench, shown at the right hand +side. In the figure a portion of the case is represented cut away to +show the arrangement of the metal beakers inside. + +In the method of Reinke, as practiced at the Halle station, and in +this laboratory, about three grams of the starchy substance are placed +in each of the metal beakers with twenty-five cubic centimeters of +a one per cent lactic acid solution and thirty cubic centimeters of +water. The contents of the beaker are thoroughly mixed and they are +then heated for two and a half hours in the autoclave, at a pressure +of three and a half atmospheres. The addition of the lactic acid is +for the purpose of protecting any sugar which may be present from +decomposition at the high pressure and temperature employed. After +the completion of the heating, the autoclave is allowed to cool, the +cover is removed and the beakers taken out and their contents washed +with hot water into quarter liter flasks. After cooling, the volume +is completed with cold water, and after standing for half an hour, +with frequent shaking, the contents of the flasks are filtered and +200 cubic centimeters of the filtrate in each case converted into +dextrose with hydrochloric acid in the usual way. In order to obtain +agreeing results, it is highly necessary that the substance before +treatment should be ground to a fine powder. The addition of the lactic +acid, as practiced in the reinke method, tends to give somewhat high +results, due probably to the hydrolytic action of the acid on the +fiber present. When starchy bodies are heated in the autoclave for the +determination of their starch by polarimetric methods, or for ordinary +determinations, the use of lactic acid should be omitted. + +_Example._—The following data indicate the methods of calculation to +be followed in the determination of the percentage of starch in the +material by diastatic hydrolysis: Three grams of a barley were inverted +by diastase, as directed above, the volume of the solution made a +quarter of a liter, filtered, 200 cubic centimeters of the filtrate +converted into dextrose by hydrochloric acid, the volume completed to +half a liter with water and fifty cubic centimeters thereof oxidized +by the alkaline copper solution in the usual way. The amount of copper +obtained was 331 milligrams, corresponding to 174 milligrams of +dextrose. The amount of malt extract used in hydrolyzing the barley +mentioned above, was ten cubic centimeters. The diastatic solution +inverted with hydrochloric acid and treated as indicated above, yielded +191 milligrams of copper, corresponding to ninety-eight milligrams of +dextrose in ten cubic centimeters of the malt extract. The quantity of +malt extract represented in the final determination of copper, however, +was only one and six-tenths cubic centimeters. We then have: + + Total dextrose 174 milligrams + Dextrose in one and six-tenths cubic centimeters + malt extract 16 milligrams + Dextrose corresponding to 240 milligrams of + barley 158 milligrams + +Calculated on the proportion that dextrose is to starch, as ten is to +nine, this is equivalent to 142 milligrams of starch. The percentage +of starch in the original substance, therefore, was equivalent to 142 +multiplied by 100, divided by 240, _viz._, 59.17. + +=182. Principles of the Methods of Determination.=—In the approximately +pure state in which starch exists in the trade, it may be determined +by conversion into dextrose and estimating the latter by one of the +methods given. It is probable that there is no known method by which +starch can be entirely converted into dextrose, and all the methods of +hydrolysis, when used for quantitive purposes, must be standardized, +not by the theoretical quantity of dextrose which a given weight of +pure starch should yield, but by the actual quantity obtained. Starch +is not largely converted into dextrose by any of the diastatic ferments +which produce principally maltose and dextrins. Recourse must therefore +be had to strong acids. In practice, hydrochloric is the one usually +employed. By the action of a hot mineral acid, not only is starch +converted into dextrose, but also the dextrose found is subjected to +changes. In such cases an opposing action seems to be exerted by the +hydrolytic agent, a part of the dextrose formed suffering a partial +condensation, and thus assuming a state of higher molecular weight, +approaching the constitution of the dextrins. Another part of the +dextrose may also suffer oxidation and thus disappear entirely in +respect of the further steps in starch analysis. + +In such cases, the best the analyst can do is to conduct the hydrolysis +in as nearly as possible constant conditions, and to assume that the +percentage of dextrose present at a given time bears a constant ratio +to the quantity of starch hydrolyzed. In reality almost all the starch +appears finally as dextrose, and by proceeding on the assumption noted +above a fairly satisfactory accounting may be made of the remainder. + +Starch being insoluble, it cannot be determined directly by its +rotatory power. When heated for a few hours in contact with water at a +high pressure, starch becomes soluble, and in this state has a fairly +constant gyrodynat, _viz._, [_a_]_{D} = 197°. + +Starch is also rendered soluble by rubbing it in a mortar for about ten +minutes with an excess of strong hydrochloric acid, and in this way +a quick approximate idea may be obtained of the percentage present. +Starch prepared in this manner, however, has a strong reducing power on +metallic salts, showing that a part of it has already, even in so short +a time, assumed the state of maltose or dextrose. The gyrodynat of +pure anhydrous starch in such conditions varies from [_a_]_{D} = 197° +to [_a_]_{D} = 194°. Starch is also rendered soluble by boiling with +salicylic acid, whereby a solution is obtained having a gyrodynat of +[_a_]_{D} = 200°(circa). The methods of procedure for the analysis of +starch will be set forth in detail in the following paragraphs. + +=183. Estimation of Water.=—In prepared or commercial starches the +water may be determined by heating in a partial vacuum. The temperature +at first should be low, not exceeding 60°. After drying for an hour +at that heat the temperature may be gradually increased. The last +traces of water come off from starch with difficulty, and the final +temperature may be carried a little beyond 100° without danger of +decomposition. + +Ost recommends the use of an atmosphere of hydrogen or illuminating +gas.[148] One and a half grams of the finely powdered sample are +placed in the drying tube described in paragraph =23=, and heated in +a stream of dry hydrogen. The temperature at first is kept at about +60° for several hours and is then gradually increased to 120°. Ost +states that even at 150° the sample preserves its pure white color, +but so high a temperature is not necessary. Maercker, at the Halle +station, makes use of the same process, but employs illuminating gas +instead of hydrogen. The importance of beginning the desiccation at +a low temperature arises from the fact that at a higher temperature, +before the greater part of the water is driven off, the starch will +suffer a partial fusion and form a paste which is very difficult to +dry. The dried sample must be kept in a stoppered vessel to prevent the +absorption of hygroscopic moisture. + +=184. Estimation of Ash.=—When the drying is accomplished in a flat +platinum dish, the same sample may serve for incineration. Otherwise +the incineration may be accomplished in another portion of the sample +by following directions already given.[149] + +=185. Nitrogen.=—Even very pure samples of starch may contain a little +nitrogen which is most conveniently determined by moist combustion.[150] + +As a rule, in commercial starches of good quality, the quantity of pure +starch may be considered to be the remainder after subtracting the sum +of the weights of water, ash and nitrogen multiplied by 6.25, from the +original weight of the sample taken. + + _Example_:— + + Per cent of moisture found 12.85 + ” ” ” ash found 0.08 + ” ” ” nitrogen × 6.25 0.27 + ----- + Sum 13.20 + Per cent of pure starch in sample 86.80 + +Samples of starch usually contain also traces of fat and fiber, and +these when present in weighable quantities, should be determined and +proper deductions made. + +=186. Hydrolysis with Acids.=—The acids commonly chosen for hydrolyzing +starch are sulfuric and hydrochloric. The former has the advantage of +being more easily removed from the finished product but the latter +performs the work with less damage to the sugars formed. For commercial +purposes sulfuric and for analytical practice hydrochloric acids are +commonly employed. + +[Illustration: FIGURE 47 (BIS). MAERCKER’S HYDROLYZING APPARATUS FOR +STARCH.] + +The best process for analytical purposes is the one proposed by +Sachsse.[151] In this method the starch is heated with the hydrolyzing +mixture in the proportion of three grams to 200 cubic centimeters +of water and twenty of hydrochloric acid of 1.125 specific gravity, +containing five and six-tenths grams of the pure gas. The heating is +continued for three hours on a steam-bath. Maercker recommends, instead +of the above procedure, heating for two hours at gentle ebullition in +an oil-bath. In this method three grams of the starch are reduced to +paste with 200 cubic centimeters of water, and then boiled for two +hours with fifteen cubic centimeters of hydrochloric acid of 1.125 +specific gravity. The erlenmeyers in which the hydrolysis takes place +are heated in an oil-bath and are provided with reflux condensers made +of long glass tubes on which some bulbs have been blown, as shown in +the accompanying figure. In all cases after hydrolysis the solution +is neutralized, made to a standard volume and an aliquot part, after +filtration, diluted to contain an amount of dextrose suited to the use +of the table by Allihn for calculating the percentage of sugar. In +diluting the solution preparatory to the estimation of dextrose, it is +well to remember that nine parts of starch will furnish theoretically +ten parts of dextrose. Since three grams of the sample are used, +containing approximately eighty-five per cent of starch, the quantity +of dextrose present is a little less than three grams. The solution +should therefore contain not less than 300 cubic centimeters. + +=187. Factor for Calculating Starch from the Dextrose Obtained.=—If all +the starch could be converted into dextrose without loss, the quantity +of it could be easily calculated theoretically on the supposition that +the formula of starch is (C₆H₁₀O₅)ₙ. The factor by this assumption is, +starch = dextrose × 0.90. If the starch have the formula assigned to it +by Nägeli, _viz._, C₃₆H₆₂O₃₁ the formula becomes, starch = dextrose × +0.918. + +Ost prefers to work by Sachsse’s method and to use the factor 0.925 to +convert the dextrose into starch.[152] + +In view of all the facts in the case it appears that the analyst will +reach nearly correct results by converting the starch into dextrose +by heating for three hours at 100° with hydrochloric acid or for two +hours at gentle ebullition as directed above, determining the resultant +dextrose and multiplying the weight thereof by 0.92. + +=188. Polarization of Starch.=—Starch may be prepared for polarization +by dissolving it in cold hydrochloric acid. The process as carried +out by Effront is as follows.[153] Five grams of starch are rubbed +with twenty cubic centimeters of cold concentrated hydrochloric +acid for nearly ten minutes or until the solution is quite clear. +The volume is completed to 200 cubic centimeters with water and the +solution polarized. By this process there is always produced a notable +quantity of reducing sugars, and for this reason it must be admitted +that a portion of the starch has suffered complete hydrolysis. Ost +therefore recommends the use of an acid of 1.17 specific gravity, +and the gyrodynat of the soluble starch thus produced is found to +vary from [_a_]_{D} = 196°.3 to 196°.7. When acid of 1.20 specific +gravity is employed the gyrodynat falls to [_a_]_{D} = 194.2.[154] For +approximately correct work the solution with the weaker hydrochloric +acid and subsequent polarization is to be recommended as the most rapid +method for starch determination. + +It will be of interest to add the observation that the gyrodynat of +maltose has lately been redetermined by Ost, who finds it to be +[_a_]_{D}²⁰ ° = 137°.04 ± 0.19.[155] + +=189. Solutions of Starch at High Pressure.=—Starch may also be brought +into a condition suited to polarization by dissolving in water at a +high temperature and pressure. The solution is accomplished in an +autoclave as described in =181=. + +From two to three grams of starch are used and from eighty to ninety +cubic centimeters of water. The starch is first reduced to a pasty +state by heating with the water and, when evenly distributed throughout +the flask, is rendered soluble by heating from three to five hours in +an autoclave at from two to three atmospheres. The material is entirely +without action on an alkaline copper solution. After heating, the +volume of the solution is completed to 100 cubic centimeters and it is +then polarized. The gyrodynat of starch dissolved in this way varies +from [_a_]_{D} = 196°.5 to 197°.[156] + +Starch is prepared by Baudry for polarization by boiling with salicylic +acid.[157] The gyrodynat of starch dissolved in this way is [_a_]_{D} = +200°.25. + +=190. Polarization after Solution in Dilute Nitric Acid.=—Guichard +recommends saccharification with ten per cent nitric acid (ten cubic +centimeters strong acid, ninety cubic centimeters water).[158] This +treatment, even after prolonged boiling, gives only a light straw color +to the solution which does not interfere with its polarization with a +laurent instrument. + +In working on cereals four grams of the finely ground material, in +which the bran and flour are intimately mixed, are used. + +The material is placed in a flask of about 500 cubic centimeters +capacity, with 100 cubic centimeters of the dilute acid. The flask is +closed with a stopper carrying a reflux condenser. After boiling for +an hour the contents of the flask are filtered and examined in the +saccharimeter. The dextrose formed is determined by the polarimetric +data and the quantity of starch transformed calculated from the +dextrose. The following formula is used: + + _av_ × 25 × 0.016 + _A_ = ------------------. + 2 × 52.8 + +In this formula _a_ = the rotation in angular degrees, _v_ = the volume +of the liquid and _A_ = the starch transformed. + +In this method no account is taken of the sucrose and other sugars +which are present in cereals. In the case of sucrose the left-handed +sugar produced by treatment with nitric acid would diminish the +rotation to the right and thus introduce an error. On the other hand +the dextrose formed from the fiber of the bran would be calculated as +starch. If these two errors should be compensating the method might +prove practical. + +=191. Rapid Estimation Of Starch.=—For the rapid estimation of starch +in cereals, cattle foods and brewery refuse, Hibbard recommends a +method which is carried out as follows: + +The malt extract is prepared by covering ground, dry malt with water +containing from fifteen to twenty per cent of alcohol. The object of +adding alcohol is to preserve the filtered extract. It exercises a +slight retarding effect on the action of the diastase, but prevents the +malt extract from fermenting. After standing for a few hours in contact +with the malt, the liquid is separated by filtration and is then ready +for use. The substance in which the starch is to be determined should +be dry enough to be finely pulverized, but previous extraction with +ether is omitted. Enough of the material to contain at least half a +gram of starch is placed in a flask with fifty cubic centimeters of +water and from one to two cubic centimeters of malt extract added. The +mixture is at once heated to boiling with frequent shaking to prevent +the formation of clots. The addition of the diastase before boiling is +to aid in preventing the formation of lumps. After boiling a minute +the mixture is cooled to 60° and from two to three cubic centimeters +of the malt extract added. It is then slowly heated until it again +boils, consuming about fifteen minutes, when, after cooling, it is +tested with iodin for starch. If a blue color be produced the operation +above described is repeated until it fails to reappear. The mixture +is then made up to a standard volume, thrown on a linen filter and an +aliquot part of the filtrate, representing from 200 to 300 milligrams +of starch, is boiled with five cubic centimeters of hydrochloric acid, +of thirty per cent strength, for half an hour. The total volume of the +liquid before boiling should be completed to sixty cubic centimeters. +By the method above described, it is claimed that the determination of +starch in a cereal or similar substance can be completed within two +hours. The chief amount of time saved is in the heating with the malt +extract, which instead of being continued for two hours, as usually +directed, can be accomplished in thirty minutes.[159] + +=192. Precipitation of Starch with Barium Hydroxid.=—The tendency of +carbohydrate bodies to unite with the earthy bases has been utilized by +Asboth as a basis for the quantitive determination of starch.[160] + +About three grams of the finely ground sample containing the starch, +or one gram of pure starch, are rubbed up in a mortar with water and +the detached starch remaining suspended in the wash water is poured +off. This operation is repeated until all the starch is removed. In +difficult cases hot water may be used. The starch thus separated is +heated in a quarter liter flask to the boiling point to reduce it to +the condition of paste. When the paste is cold it is treated with fifty +cubic centimeters of the barium hydroxid solution, the flask closed and +well shaken for two minutes. The volume is then completed to the mark +with forty-five per cent alcohol, the flask well shaken and allowed +to stand. In a short time the barium-starch compound separates and +settles. Fifty cubic centimeters of the clear supernatant liquor are +removed with a pipette, or the liquor may be passed through a filter +and the quantity mentioned removed for titration of the residual barium +hydroxid after the addition of a few drops of phenolphthalein solution. + +The quantity of barium hydroxid remaining, deducted from the original +quantity, gives the amount which has entered into composition with +the starch; the composition of the molecule being BaOC₂₄H₄₀O₂₀, which +contains 19.10 per cent of barium oxid and 80.90 per cent of starch. + +The set solution of barium hydroxid must be preserved from contact with +the carbon dioxid of the air. The burette should be directly attached +to the bottle holding the set solution, by any of the usual appliances, +and the air entering the bottle must be deprived of carbon dioxid. The +water used in the work must be also free of air, and this is secured by +boiling immediately before use. + +_Example._—A sample of flour selected for the analysis weighed +3.212 grams. The starch was separated and reduced to paste in the +manner described above. Thirty and four-tenths cubic centimeters of +tenth-normal hydrochloric acid were exactly neutralized by ten cubic +centimeters of the barium hydroxid solution. After treatment as above +described, fifty cubic centimeters of the clear liquor, corresponding +to ten cubic centimeters of the added barium hydroxid, required 19.05 +cubic centimeters of tenth-normal hydrochloric acid. Then 30.4 - 19.05 += 11.35, and 11.35 x 5 = 56.75, which number corresponds to the total +titration of the residual barium hydroxid in terms of tenth-normal +hydrochloric acid. This number multiplied by 0.0324, _viz._, starch +corresponding to one equivalent of barium, gave 1.8387 grams of starch +or 57.24 per cent of the weight of flour employed. + +The barium hydroxid method has been given a thorough trial in this +laboratory and the results have been unsatisfactory when applied to +cereals. The principle of the process, however, appears to be sound, +and with a proper variation of working details, it may become practical. + +=193. Disturbing Bodies in Starch Determinations.=—Stone has made a +comparison of the standard methods of starch determinations, and the +results of his work show that in the case of pure starch all of the +standard methods give approximately correct figures. For instance, in +the case of a pure potato starch, the following data were obtained: + +By inversion with hydrochloric acid, 85.75 per cent; by inversion with +oxalic and nitric acids, 85.75 per cent; by solution in salicylic acid, +85.47 per cent; and by precipitation with barium hydroxid, 85.58 per +cent.[161] + +When these methods are used, however, for the determination of starch +in its original state, the widest variations are secured. Stone shows +that these variations are due chiefly to the inverting effect of the +reagents employed upon the pentosans present. In experiments made with +pure xylan obtained from wheat straw, the methods employed gave from +44.73 to 67.16 per cent of material, which would be calculated by +the usual methods as starch. Stone also shows that the pentosans are +practically unaffected by the action of diastase or malt extract. Pure +xylan treated with diastase, under the condition in which starch is +converted into maltose and other soluble carbohydrates, fails to give +any subsequent reaction whatever with alkaline copper solution. In +all cases, therefore, where starch occurs in conjunction with pentose +bodies, it is necessary to separate it by diastatic action before +applying any of the methods of conversion of the starch into dextrose +or its precipitation by barium hydroxid. + +=194. Colorimetric Estimation of Starch.=—The production of the +intensely blue color which starch gives with iodin has been used not +only as the basis of a qualitive method, but also of many attempts at +quantitive determination. These attempts have, as a rule, been attended +with very unsatisfactory results, due both to the extraordinary +delicacy of the reaction and to the fact that starches of different +origin do not always give exactly the same intensity of tint when +present in the same quantity. At the present it must be admitted that +little should be expected of any quantitive colorimetric test. + +In case such a test is desired the procedure described by Dennstedt +and Voigtländer may be followed.[162] A weighed quantity of the +starch-holding material, containing approximately half a gram of +starch, is placed in a two liter flask and boiled with a liter of +water. After cooling, the volume is completed to two liters and +the starch allowed to subside. Five cubic centimeters of the clear +supernatant liquor are placed in a graduated cylinder holding 100, and +marked in half cubic centimeters. One drop of a solution of iodin in +potassium iodid is added and the volume completed to the mark. A half +gram of pure starch is treated in the same way and different measured +portions of the solution treated as above until the color of the first +cylinder is matched. From the quantity of pure starch in the matched +cylinder the quantity in the sample is determined. The test should be +made in duplicate or triplicate. If a violet color be produced instead +of a blue, it may be remedied by treating the sample with alcohol +before the starch granules are dissolved. + +=195. Fixation of Iodin.=—In addition to forming a distinctive blue +color with iodin, starches have the power of fixing considerable +quantities of that substance. The starches of the cereals have this +power in a higher degree than those derived from potatoes. In presence +of a large excess of iodin the starches of rice and wheat have a +maximum iodin-fixing power of about nineteen per cent of their weight. +When only enough of iodin is employed to enter into combination +the percentage absorbed varies from nine to fifteen per cent. The +absorption of iodin by starches is a matter of importance from a +general chemical standpoint, but as at present determined has but +little analytical value. It is evident, however, that this absorption +must take place according to definite chemical quantities and the +researches of investigators may in the future discover some definite +quantitive method of measuring it.[163] + +=196. Identification of Starches of Different Origin.=—It is often +important, especially in cases of suspected adulteration, to determine +the origin of the starch granules. For this purpose the microscope +is the sole resort. In many cases it is easy to determine the origin +of the starch by the size or the shape and marking of the grains. In +mixtures of more than one kind of starch the distinguishing features of +the several starches can be clearly made out in most instances. There +are, however, many instances where it is impossible to discriminate +by reason of the fact that the characteristics of starch granules +vary even in the same substance and from year to year with varying +conditions of culture. + +In many cases the illustrations of the forms and characteristics of +starch granules which are found in books are misleading and no reliance +can be placed on any illustrations which are not either photographs or +drawings made directly from them. In the microscopic study of starches +the analyst will be greatly helped by the following descriptions of the +characteristic appearance of the granules and the classifications based +thereon.[164] + +=197. Vogel’s Table of the Different Starches and Arrowroots of +Commerce.=—_A._ Granules simple, bounded by rounded surfaces. + + I. Nucleus central, layers concentric. + _a._ Mostly round, or from the side, lens-shaped. + 1. Large granules 0.0396-0.0528 mm, _rye starch_: + 2. Large granules 0.0352-0.0396 mm, _wheat starch_: + 3. Large granules 0.0264 mm, _barley starch_. + + _b._ Egg-shaped, oval, kidney-shaped: Hilum often long + and ragged: + 1. Large granules 0.032-0.079 mm, _leguminous starches_. + + II. Nucleus eccentric, layers plainly eccentric or meniscus-shaped. + _a._ Granules not at all or only slightly flattened: + 1. Nucleus mostly at the smaller end; 0.06-0.10 mm, + _potato starch_: + 2. Nucleus mostly at the broader end or towards the + middle in simple granules; 0.022-0.060 mm, _maranta + starch_. + + _b._ Granules more or less strongly flattened. + 1. Many drawn out to a short point at one end. + _a._ At most 0.060 mm long, _curcuma starch_: + _b._ As much as 0.132 mm long, _canna starch_: + 2. Many lengthened to bean-shaped, disk-shaped, or + flattened; nucleus near the broader end; 0.044-0.075 mm, + _banana starch_: + 3. Many strongly kidney-shaped; nucleus near the edge; + 0.048-0.056 mm, _sisyrinchium starch_: + 4. Egg-shaped; at one end reduced to a wedge, at the other + enlarged; nucleus at smaller end; 0.05-0.07 mm, + _yam starch_: + +_B._ Granules simple or compound, single granules or parts of granules, +either bounded entirely by plain surfaces, many-angled, or by partly +round surfaces. + + I. Granules entirely angular. + 1. Many with prominent nucleus: At most 0.0066 mm, _rice starch_: + 2. Without a nucleus: The largest 0.0088 mm, _millet starch_: + + II. Among the many-angled also rounded forms. + _a._ No drum-shaped forms present, angular form predominating. + 1. Without nucleus or depression very small; 0.0044 mm, + _oat starch_: + 2. With nucleus or depression; 0.0132-0.0220 mm. + _a._ Nucleus or its depression considerably rounded; + here and there the granules united into differently + formed groups; _buckwheat starch_: + _b._ Nucleus mostly radiate or star-shaped; all the + granules free; _maize_ (_corn_) _starch_: + + _b._ More or less numerous kettledrum and sugar-loaf like forms. + 1. Very numerous eccentric layers; the largest granules + 0.022-0.0352 mm, _batata_ (sweet potato) _starch_: + 2. Without layers or rings; 0.008-0.022 mm. + _a._ In the kettledrum-shaped granules the nucleal + depression mostly widened on the flattened + side; 0.008-0.022 mm, _cassava starch_: + _b._ Depression wanting or not enlarged. + _aa._ Nucleus small, eccentric; 0.008-0.016 mm, + _pachyrhizus starch_: + _bb._ Nucleus small, central, or wanting. + _aaa._ Many irregular angular forms; + 0.008-0.0176 mm, _sechium starch_: + _bbb._ But few angular forms; some with + radiate, nucleal fissure; 0.008-0.0176 mm, + _chestnut starch_. + +_C._ Granules simple and compound; predominant forms, oval, with +eccentric nucleus and numerous layers; the compound granule made up +of a large granule and one or more relatively small kettledrum-shaped +ones; 0.025-0.066 mm, _sago starch_. + +=198. Muter’s Table for the Detection of Starches when Magnified about +230 Diameters.= + +[All measurements are given in decimals of an inch.] + +_Group I_: All more or less oval in shape and having both hilum and +rings visible. + + -------------+----------------+-------------+----------------------- + Name. | Shape. | Normal | Remarks. + | |measurements.| + -------------+----------------+-------------+----------------------- + Tous les mois|Oval, with flat | 0.00370 |Hilum annular, near one + | ends | to 0.00185 | end and incomplete + | | | rings. + | | | + Potato |Oval | 0.00270 |Hilum annular, rings + | | to 0.00148 | incomplete, shape and + | | | size very variable. + | | | + Bermuda |Sack-shaped | 0.00148 |Hilum distinct annular, + arrowroot | | to 0.00129 | shape variable, rings + | | | faint. + | | | + St. Vincent |Oval-oblong | 0.00148 |Hilum semi-lunar, rings + arrowroot | | to 0.00129 | faint, shape not very + | | | variable. + | | | + Natal |Broadly ovate | 0.00148 |Hilum annular, in + arrowroot | | to 0.00129 | center and well marked + | | | complete rings. + | | | + Galangal |Skittle-shaped |About 0.00135|Hilum elongated, very + | | | faint incomplete + | | | rings. + | | | + Calumba |Broadly | ” 0.00185|Hilum semi-lunar, faint + | pear-shaped | | but complete rings, + | | | shape variable. + | | | + Orris root |Elongated-oblong| ” 0.00092|Hilum faint, shape + | | | characteristic. + | | | + Turmeric |Oval-oblong, | ” 0.00148|Very strongly marked + | conical | | incomplete rings. + | | | + Ginger |Shortly conical,| ” 0.00148|Hilum and rings + | with rounded | | scarcely visible, + | angles. | | shape variable but + | | | characteristic. + -------------+----------------+-------------+------------------------- + +_Group II_: With strongly developed hilum more or less stellate. + + -------------+--------------+-------------+------------------------- + Name. | Shape. | Normal | Remarks. + | |measurements.| + -------------+--------------+-------------+------------------------- + Bean |Oval-oblong |About 0.00135|Fairly uniform. + | | | + Pea |Like bean | 0.00111 |Very variable in size, + | | to 0.00074 | with granules under + | | | 0.00111 preponderating. + | | | + Lentil |Like bean |About 0.00111|Hilum, a long depression + | | | seldom radiate. + | | | + Nutmeg |Rounded | ” 0.00055|The small size and + | | | rounded form + | | | distinctive. + | | | + Dari |Elongated | ” 0.00074|Irregular appearance and + | hexagon | | great convexity + | | | distinctive. + | | | + Maize |Round and | ” 0.00074|The rounded angles of the + | polygonal | | polygonalgranules + | | | distinctive. + -------------+--------------+-------------+------------------------- + +_Group III_: Hilum and rings practically invisible. + + -------------+--------------+-------------+------------------------- + Name. | Shape. | Normal | Remarks. + | |measurements.| + -------------+--------------+-------------+------------------------- + Wheat |Circular and | 0.00185 |Very variable in size and + | flat | to 0.00009 | very dull polarization + | | | in water. + | | | + Barley |Slightly |About 0.00073|The majority measuring + | angular | | about 0.00373 + | circles | | distinctive, and a few + | | | four times this size. + | | | + Rye |Like barley | 0.00148 |Small granules, quite + | | to 0.00009 | round, and here and + | | | there cracked. + | | | + Jalap |Like wheat | |Polarizes brightly in + | | | water. + | | | + Rhubarb | do. | 0.00055 |Polarizes between jalap + | | to 0.00033 | and wheat, and runs + | | | smaller and more convex. + -------------+--------------+-------------+------------------------- + Senega |Like wheat | 0.00148- | + | | 0.00009 | + Bayberry | do. | 0.00074- | Measurements the + | | 0.00011 | only guide. + Sumbul | do. | 0.00074- | + | | 0.00009 | + -------------+--------------+-------------+------------------------- + Chestnut |Very variable | 0.00090- |Variable form, and small + | | 0.00009 | but regular size, + | | | distinctive. + | | | + Acorn |Round-oval |About 0.00074|Small and uniform size, + | | | distinctive. + | | | + Calabar bean |Oval-oblong | 0.00296 |Large size and shape + | | to 0.00180 | characteristic. + | | | + Licorice |Elongated-oval|About 0.00018|Small size and shape + | | | distinctive. + | | | + Hellebore |Perfectly | 0.00037 |Small, regular size and + (green or | rotund | to 0.00009 | rotundity, distinctive. + black) | | | + | | | + Hellebore |Irregular | 0.00055 |Irregular shape and faint + (white) | | to 0.00009 | central depression, + | | | distinctive. + -------------+--------------+-------------+------------------------- + +_Group IV_: More or less truncated at one end. + + -------------+--------------+-------------+------------------------ + Name. Shape. | Normal | Remarks. + | |measurements.| + -------------+--------------+-------------+------------------------ + Cassia |Round | 0.00111 |Round or muller shaped + | | to 0.00018 | granules and faint + | | | circular hilum. + | | | + Cinnamon |Like cassia | 0.00074 |More frequently truncated + | | to 0.00009 | than cassia, and + | | | smaller. + | | | + Sago (raw) |Oval-ovate | 0.00260 |Has circular hilum at + | | to 0.00111 | convex end and rings + | | | faintly visible. + | | | + Sago | ” | 0.00260 |Has a large oval or + (prepared) | | to 0.00111 | circular depression, + | | | covering one-third + | | | nearly of each granule. + | | | + Tapioca |Roundish | 0.00074 |A little over fifty per + | | to 0.00055 | cent truncated by one + | | | facet, and a pearly + | | | hilum. + | | | + Arum |Like tapioca |About 0.00056|Smaller than tapioca and + | | | truncated by two facets. + | | | + Belladonna | do. | |Not distinguishable + | | | from tapioca. + | | | + Colchicum | do. |About 0.00074|Larger than tapioca, + | | | and contains many + | | | more truncated + | | | granules. + | | | + Scammony | do. | ” 0.00045|Smaller than tapioca, + | | | more irregular, and + | | | hilum not visible. + | | | + Cancella |Very variable | 0.00033- |Very variable, form and + | | 0.00022 | small size the only + | | | points. + | | | + Podophyllum |Like tapioca |About 0.00040|Like scammony, but has + | | | visible hilum in most + | | | of the granules. + | | | + Aconite | do. | ” 0.00037|Like tapioca, but half + | | | the size. + -------------+--------------+-------------+------------------------ + +_Group V_: All granules more or less polygonal. + + -------------+--------------+-------------+------------------------- + Name. | Shape. | Normal | Remarks. + | |measurements.| + -------------+--------------+-------------+------------------------- + Tacca |Poly- or | 0.00075 |Distinguished from maize + | hexagonal | to 0.00037 | by its sharp angles. + | | | + Oat |Polygonal |About 0.00037|Larger than rice and + | | | hilum visible in some + | | | granules. + | | | + Rice | do. | 0.00030- |Measurement using + | | 0.00020 | one-eighth or + | | | one-twelfth inch power, + | | | and then hilum visible. + | | | + Pepper | do. | 0.00020- | Do. + | | 0.00002 | + Ipecacuanha | do. |About 0.00018|Some round and truncated + | | | granules, adhering in + | | | groups of three. + -------------+--------------+-------------+------------------------- + +=199. Blyth’s Classification.=—Blyth gives the following scheme for the +identification of starch granules by their microscopic appearance.[165] + +_Division I.—Starches showing a play of colors with polarized light and +selenite plate_: + +The hilum and concentric rings are clearly visible, and all the starch +granules, oval or ovate. Canna arrowroot, potato, arrowroot, calumba, +orris root, ginger, galangal and turmeric belong to this division. + +_Division II.—Starches showing no iridescence, or scarcely any, when +examined by polarized light and selenite_: + +Class I.—The concentric rings are all but invisible, and the hilum +stellate. The bean, pea, maize, lentil, dari and nutmeg starches are in +this class. + +Class II.—Starches which have both the concentric rings and hilum +invisible in the majority of granules: this important class includes +wheat, barley, rye, chestnut, acorn, and many starches in medicinal +plants. + +Class III.—All the granules are truncated at one end. This class +includes sago, tapioca and arum, several drugs and cinnamon and cassia. + +Class IV.—In this class all the granules are angular in form and it +includes oats, tacca, rice, pepper and ipecacuanha. + +=200. Preparation of Starches for Microscopical Examination.=—The +approximately pure starches of commerce may be prepared for microscopic +examination by rubbing them up with water and mounting some of the +suspended particles by one of the methods to be described below. + +In grains, seeds and nuts the starch is separated by grinding with +water and working through fine linen. The starch which is worked +through is allowed to subside, again beaten up with water if necessary +and the process continued until the grains are separated sufficiently +for microscopic examination. A little potash or soda lye may be used, +if necessary, to separate the granules from albuminous and other +adhering matter. The analyst should have a collection of samples of all +common starches of known origin for purposes of comparison. + +The granules are mounted for examination by plain light in a medium +of glycerol and camphor water. When polarized light is used the +mounting should be in Canada balsam.[166] The reader can find excellent +photomicrographs of the more common starches in Griffith’s book.[167] + +=201. Appearance in Balsam with Polarized Light.=—Mounted in balsam +the starches are scarcely visible under any form of illumination +with ordinary light, the index of refraction of the granules and the +balsam being so nearly alike. When, however, polarized light is used +the effect is a striking one. It is very easy to distinguish all the +characteristics, except the rings, the center of the cross being at the +nucleus of the granule. + +With the selenite plate a play of colors is produced, which is peculiar +to some of the starches and forms the basis of Blyth’s classification. + +=202. Description Of Typical Starches.=—The more commonly occurring +starches are described by Richardson as they appear under the +microscope magnified about 350 diameters.[168] + +The illustrations, with the exception of the cassava starch, and the +maize starch accompanying it were drawn by the late Dr. Geo. Marx from +photographs made by Richardson in this laboratory. The two samples +excepted were photographed for the author by Dr. G. L. Spencer. + +_Maranta Starch._—Of the same type as the potato starch are the various +arrowroots, the only one of which commonly met with in this country +being the Bermuda, the starch of the rhizome of _Maranta arundinacea_, +and the starch of turmeric. + +The granules are usually not so varied in size or shape as those of +the potato, averaging about 0.07 millimeter in length as may be seen +in Fig. 48. They are about the same size as the average of the potato, +but are not often found with the same maximum or minimum magnitude, +which circumstance, together with the fact that the end at which the +nucleus appears is broader in the maranta and more pointed in the +potato, enables one to distinguish the two starches without difficulty. +With polarized light the results are similar to those seen with potato +starch, and this is a ready means of distinguishing the two varieties, +by displaying in a striking way the form of the granule and position of +the hilum. + +_Potato Starch._—The starch grains of the potato are very variable in +size, being found from 0.05 to 0.10 millimeter in length, and in shape +from oval and allied forms to irregular and even round in the smallest. +These variations are illustrated in Fig. 49, but the frequency of the +smaller granules is not as evident as in some other cases. The layers +are visible in some granules with great distinctness and in others +hardly at all, being rather more prominent in the starch as obtained +from a freshly cut surface. The rings are more distinct, too, near +the hilum or nucleus, which in this, as in all tuberous starches, is +eccentric, shading off toward the broader or more expanded portion +of the granule. The hilum appears as a shadowy depression, and with +polarized light its position is well marked by the junction of the arms +of the cross. With polarized light and a selenite plate a beautiful +play of colors is obtained. The smaller granules, which are nearly +round, may readily be confused with other starches, but their presence +serves at once to distinguish this from maranta or Bermuda arrowroot +starch. Rarely compound granules are found composed of two or three +single ones each with its own nucleus. + +_Ginger Starch._—This starch is of the same class as those from the +potato and maranta and several others which are of underground origin. +In outline the granules are not oval like those named, but more +rectangular, having more obtuse angles in the larger ones and being +cylindrical or circular in outline in the smaller, as indicated in Fig. +50. They average nearly the same size as maranta starch, but are much +more variable, both in size and form. The rings are scarcely visible +even with the most favorable illuminations. + +_Sago Starch._—This exists in two modifications in the market; as raw +and as prepared sago. In the prepared condition it is characterized by +a larger circular depression in the center of most of the granules. The +rings are not visible. They are mostly circular in form or approaching +it, and vary from 0.025 to 0.065 millimeter in diameter, as indicated +in Fig. 51. + +[Illustration: FIG. 48. MARANTA STARCH × 350.] + +[Illustration: FIG. 49. POTATO STARCH × 350.] + +[Illustration: FIG. 50. GINGER STARCH × 350.] + +[Illustration: FIG. 51. SAGO STARCH × 350.] + +[Illustration: FIG. 52. PEA STARCH × 350.] + +[Illustration: FIG. 53. BEAN STARCH × 350. + +DRAWN BY GEO. MARX. + +A. Hoen & Co., Lithocaustic] + +[Illustration: FIG. 54. WHEAT STARCH × 350.] + +[Illustration: FIG. 55. BARLEY STARCH × 350.] + +[Illustration: FIG. 56. RYE STARCH × 350.] + +[Illustration: FIG. 57. OAT STARCH × 350.] + +[Illustration: FIG. 58. INDIAN CORN STARCH × 350.] + +[Illustration: FIG. 59. RICE STARCH × 350. + +DRAWN BY Geo. MARX. + +A. Hoen & Co., Lithocaustic] + +[Illustration: FIG. 60. CASSAVA STARCH × 150. + +PLAIN ILLUMINATION.] + +[Illustration: FIG. 61. INDIAN CORN STARCH × 150. + +PLAIN ILLUMINATION. + +A. Hoen & Co., Lithocaustic] + +_Pea and Bean Starches._—These starches produce but a slight effect +with polarized light. The rings are scarcely visible, and the hilum is +stellate or much cracked along a median line, the bean more so than the +pea, the latter resembling fresh dough kneaded again into the center +as in making rolls, and the former the shape assumed by the same after +baking. The grains of both are somewhat variable in size, ranging from +0.025 to 0.10 millimeter in length, as shown in Figs. 52 and 53. + +_Wheat Starch_ grains are quite variable in size, varying from 0.05 +to 0.010 millimeter in diameter. They belong to the same class as +barley and rye, the hilum being invisible and the rings not prominent. +The granules are circular disks in form, and there are now and then +contorted depressions resembling those in pea starch. They are the +least regular of the three starches named and do not polarize actively. +The typical forms of these granules are shown in Fig. 54. + +_Barley Starch_ is quite similar to that of wheat, but the grains do +not vary so much in size, averaging 0.05 millimeter. They have rings +which are much more distinct, and very small granules adhering to the +largest in bud-like forms, as seen in Fig. 55. + +_Rye Starch_ is more variable in size, many of the granules not +exceeding 0.02 millimeter, while the largest reach 0.06 to 0.07 +millimeter. It lacks distinctive characteristics entirely, and is the +most simple in form of all the starches. Fig. 56 shows the appearance +of the granules under the microscope. + +_Oat Starch_ is unique, being composed of large compound masses of +polyhedral granules from 0.12 to 0.02 millimeter in length, the single +granules averaging 0.02 to 0.015 millimeter. It does not polarize +actively, and displays neither rings nor hilum. The illustration, Fig. +57, shows its nature with accuracy. + +_Indian Corn Starch._—The granules of maize starch are largely of +the same size, from 0.02 to 0.03 millimeter in diameter, with now +and then a few which are much smaller. They are mostly circular in +shape or rather polyhedral, with rounded angles, as shown in Figs. 58 +and 61. They form very brilliant objects with polarized light, but +with ordinary illumination show but the faintest sign of rings and a +well-developed hilum, at times star-shaped, and at others more like a +circular depression. + +_Rice Starch_ is very similar to that of maize, and is easily confused +with it, the grains being about the same size. The grain, however, +is distinguished from it by its polygonal form, and its well defined +angles, as indicated in Fig. 59. The hilum is more prominent and more +often stellate or linear. Several granules are at times united. + +_Cassava Starch._—This variety of starch is obtained from the root of +the sweet cassava, which grows in great profusion in Florida. It is +compared with maize starch in Figs. 60 and 61. In the illustration the +granules are represented as magnified 150 diameters. The grains of the +cassava starch measure about 0.012 millimeter in diameter and resemble +very nearly maize starch, except that they have greater evenness of +outline.[169] + +For further descriptions of starch grains the reader is referred to the +work of Griffith, already cited. + +These descriptions, it will be seen, do not agree entirely with those +of some other authors, but they are based on a somewhat extensive +experience. + +There are peculiarities of size, shape and appearance of starch +granules, which must be allowed for, and the necessity for every +investigator to compare a starch which he is desirous of identifying +with authentic specimens, must always be recognized. + + +AUTHORITIES CITED IN PART SECOND. + +[23] Vines, Vegetable Physiology. + +[24] Berichte der deutschen chemischen Gesellschaft, Band 23, S. 2136; +Stone, Agricultural Science Vol. 6, p. 180. Page 59, eighth line from +bottom insert “original” before “optical.” Page 60, second line from +top, read _d_ instead of _l_ fructose. + +[25] Herles, Zeitschrift des Vereins für die Rübenzucker-Industrie, +1890. S. 217. + +[26] Tucker; Wiechmann; Sidersky; von Lippman; Tollens and Spencer. + +[27] Bulletin No. 28, Department of Agriculture, Division of Chemistry, +p. 197. + +[28] Physikalisch-Chemische Tabellen, S. 42. + +[29] Tucker’s Manual of Sugar Analysis, pp. 100 et seq. + +[30] Vid. op. cit. supra, p. 108. + +[31] Op. cit. supra, p. 109. + +[32] Op. cit. supra, p. 110. + +[33] Op. cit. supra, p. 114. + +[34] Spencer’s Handbook for Sugar Manufacturers, p. 92. + +[35] Landolt’s Handbook of the Polariscope, pp. 95 et seq. + +[36] Robb, vid. op. cit. supra, p. 8. + +[37] Spencer’s Handbook for Sugar Manufacturers, pp. 22 et seq. +Tucker’s Manual of Sugar Analysis, pp. 120 et seq. + +[38] Sidersky; Traité d’Analyse des Matières Sucrées, p. 104. + +[39] Journal of the American Chemical Society, 1893. Vol. 15, p. 121. + +[40] Comptes rendus, 1879. Seance du 20th Octobre 1879; Dingler’s +polytechniches Journal, Band 223, S. 608. + +[41] Landolt’s Handbook of the Polariscope. p. 120. + +[42] Sidersky; Traité d’Analyse des Matières Sucrées, p. 97. + +[43] Manual of Sugar Analysis, pp. 143 et seq. + +[44] Landolt und Börnstein, Physikalisch-Chemische Tabellen. S. 460. + +[45] Bulletin No. 31. Department of Agriculture, Division of Chemistry, +p. 232. + +[46] Zeitschrift des Vereins für die Rübenzucker-Industrie. 1870, S. +223. + +[47] Tucker’s Manual of Sugar Analysis, p. 164. + +[48] (bis). Gerlach, Spencer’s Handbook for Sugar Manufacturers, p. 91. + +[49] Vid. op. cit. supra, p. 45. + +[50] Vid. loc. et op. cit. supra. + +[51] Gill; Journal of the Chemical Society, Vol. 24, 1871, p. 91. + +[52] Wiley; American Chemical Journal, Vol. 6, p. 289. + +[53] Vid. op. cit. supra, p. 301. + +[54] Zeitschrift des Vereins für die Rübenzucker-Industrie, 1890. S. +876. + +[55] Weber and McPherson; Journal of the American Chemical Society. +Vol. 17, p. 320; Bulletin No. 43. Department of Agriculture, Division +of Chemistry, p. 126. + +[56] Zeitschrift des Vereins für die Rübenzucker-Industrie, 1888, S. 51. + +[57] Vid. op. cit. supra, Ss, 699 und 763; 1890. S. 217. + +[58] Bulletin de l’Association des Chimistes de Sucrerie et de +Distillerie, May, 1890, p. 431. + +[59] Neue Zeitschrift für Rübenzucker-Industrie, Band 19, S. 71. + +[60] Journal of the Chemical Society, Transactions, Vol. 57, pp. 834, +et seq. + +[61] Op. cit. supra, p. 866. + +[62] Op. cit. supra, 1891, p. 46. + +[63] Neue Zeitschrift für Rübenzucker-Industrie, Band 19, S. 71. + +[64] From γῦρος and δῦνᾶτός (δύνᾶμις). + +[65] Landolt’s Handbook of the Polariscope, p. 125. + +[66] Vid. op. cit. supra, pp. 48 et seq. + +[67] Berichte der deutschen chemischen Gesellschaft, 1877, S. 1403. + +[68] Die landwirtschaftlichen Versuchs-Stationen, Band 40, S. 307. + +[69] Spencer’s Handbook for Sugar Manufacturers, p. 80; Landolt’s +Handbook of the Polariscope, p. 216; Tollens’ Handbuch der +Kohlenhydrate. + +[70] Annalen der Chemie and Pharmacie, May, 1870. + +[71] Tucker’s Manual of Sugar Analysis, p. 208. + +[72] Rapport fait a la Société d’Encouragement d’Agriculture; Journal +de Pharmacie et de Chimie, 1844. 3d serie, Tome 6, p. 301. + +[73] Annalen der Chemie und Pharmacie, Band 39, S. 361. + +[74] Jahrbücher für praktische Heilkunde, 1845, S. 509. + +[75] Archives für Physiologische Heilkunde, 1848, Band 7, S 64. + +[76] Rodewald and Tollens; Berichte der deutschen chemischen +Gesellschaft, Band 11, S. 2076. + +[77] Chemical News, Vol. 39, p. 77. + +[78] The Analyst, Vol. 19, p. 181. + +[79] Gaud; Bulletin de l’Association des Chimistes de Sucrerie et de +Distillerie, Apr. 1895, p. 629; Comptes rendus, 1894, Tome 119, p. 604. + +[80] Annalen der Chemie und Pharmacie, B. 72, S. 106. + +[81] Journal of Analytical and Applied Chemistry, Vol. 4, p. 370. + +[82] Wiley; Bulletin de l’Association des Chimistes de Sucrerie et de +Distillerie, April, 1884. + +[83] Vid. op. cit. supra, 1895, p. 642; Comptes rendus, Tome 119, 1894, +p. 650. + +[84] Annual Report, United States Department of Agriculture, 1879, +p. 65; Zeitschrift für Analytische Chemie, Band 12, S. 296; Mohr +Titrirmethode, sechste auflage, S. 508. + +[85] Comptes rendus, 1894, Tome 119, p. 478. + +[86] Gazetta Chimica Italiana, Tome 6, p. 322. + +[87] Sidersky; Traité d’Analyse des Matières Sucrées, p. 148. + +[88] Vid. op. cit. supra, p. 149. + +[89] Neue Zeitschrift für die Rübenzucker-Industrie, Band 22, S. 220. + +[90] Zeitschrift des Vereins für Rübenzucker-Industrie, 1889, S. 933. + +[91] Vid. op. cit. supra, 1887, S. 147. + +[92] Berichte der deutschen chemischen Gesellschaft, Band 23, No. 14, +S. 3003; Zeitschrift des Vereins für die Rübenzucker-Industrie, 1891, +S. 97. + +[93] Ost; vid. op. et loc. cit. supra. + +[94] Zeitschrift des Vereins für die Rübenzucker-Industrie, 1890, S. +187. + +[95] Chemical News, Vol. 39, p. 77. + +[96] The Analyst, 1894, p. 181. + +[97] Chemical News, Vol. 71, p. 235. + +[98] Journal de Pharmacie et de Chimie, 1894, Tome 30, p. 305. + +[99] Pharmaceutical Journal, (3), 23, p. 208. + +[100] Vid. op. cit. supra, (3), 25, p. 913. + +[101] Sidersky; Bulletin de l’Association des Chimistes, Juillet, 1886 +et Sept. 1888. + +[102] Bodenbender and Scheller; Zeitschrift des Vereins für die +Rübenzucker-Industrie, 1887, S. 138. + +[103] Vid. op. cit. supra, 1889, S. 935. + +[104] Ewell; Manuscript communication to author. + +[105] Journal für praktische Chemie, 1880, Band 22, 46; Handbuch der +Spiritusfabrication, 1890, S. 79; Zeitschrift des Vereins für die +Rübenzucker-Industrie, 1879, S. 1050; _Ibid_, 1883, S. 769; _Ibid_, +1889, S. 734. + +[106] Handbuch der Spiritusfabrication, 1890, 79. + +[107] Wein; Tabellen zur quantitativen Bestimmung der Zuckerarten, S. +13. (The caption for the table on page 159 should read as on page 160.) + +[108] Zeitschrift des Vereins für die Rübenzucker-Industrie, 1889, S. +735. + +[109] Bulletin No. 43, Department of Agriculture, Division of +Chemistry, p. 209. + +[110] Chemiker-Zeitung, 1893, S. 548. + +[111] Wein; Tabellen zur quantitativen Bestimmung der Zuckerarten, S. +35. + +[112] Berichte der deutschen chemischen Gesellschaft, Band 16, S. 661. + +[113] Vid. op. cit. supra, Band 22, S. 87. + +[114] Chemisches Centralblatt, 1895, Band 2, S. 66. + +[115] Comptes rendus; Tome 112, No. 15, p. 799. + +[116] Vid. op. cit. supra, Tome 94, p. 1517. + +[117] Journal of the Chemical Society, June, 1888, p. 610. (In the +formulas for lactose and arabinose read H₂₂ and H₁₀ respectively.) + +[118] American Chemical Journal, Vol. 11, No. 7, p. 469. + +[119] Chemisches Centralblatt, 1889, No. 7. + +[120] American Chemical Journal, Vol. 17, No. 7, pp. 507, 517. + +[121] Comptes rendus, Tome 118, p. 426. + +[122] Justus Liebig’s Annalen der Chemie, 1890. Band 257, S. 160. + +[123] Journal of Analytical and Applied Chemistry, Vol. 7, pp. 68 et +seq. + +[124] Flint and Tollens; Berichte der deutschen chemischen +Gesellschaft, Band 25, S. 2912. + +[125] Vid. op. cit. supra, Band 23, S. 1751. (Read Günther.) + +[126] Journal of Analytical and Applied Chemistry, Vol. 5, p. 421. + +[127] Vid. op. cit. supra, p. 426. + +[128] Berichte der deutschen chemischen Gesellschaft, Band 24, S. 3575. + +[129] Journal of Analytical and Applied Chemistry, Vol. 7, p. 74. + +[130] Chemiker-Zeitung, Band 17, 1743. + +[131] Vid. op. cit. supra, Band 18, N. 51, S. 966. + +[132] Monatshefte für Chemie, Band 16, S. 283; Berichte der deutschen +chemischen Gesellschaft, Referate Band 28, S. 629. + +[133] Papasogli; Bulletin de l’Association des Chimistes de Sucrerie et +de Distillerie, Juillet 1895, p. 68. + +[134] Gans und Tollens; Zeitschrift des Vereins für die +Rübenzucker-Industrie, Band 38, S. 1126. + +[135] Berichte der deutschen chemischen Gesellschaft, 20, S. 181; +Zeitschrift des Vereins für die Rübenzucker-Industrie, 1891, S. 895. + +[136] Zeitschrift des Vereins für die Rübenzucker-Industrie, 1891, S. +891. + +[137] Chemiker-Zeitung, 1888, No. 2; Zeitschrift des Vereins für die +Rübenzucker-Industrie, 1888, S. 347. + +[138] Fischer; Berichte der deutschen chemischen Gesellschaft, Band 20, +S. 821; Band 21, Ss. 988; 2631. + +[139] Zeitschrift für physiologische Chemie, Band 11, S. 492. + +[140] Vid. op. cit. supra, Band 12, No. 4, Ss. 355 et seq; No. 5, Ss. +377 et seq. + +[141] Berichte der deutschen chemischen Gesellschaft, Band 20, S. 540. + +[142] Sitzungsberichte der Mathematisch-Naturwissenschaften in Wien, +Band 93, Heft 2, S. 912. + +[143] Tollens; Handbuch der Kohlenhydrate; von Lippmann, Chemie der +Zuckerarten. + +[144] Wilder Quarter-Century Book, 1893; Abdruck aus dem Centralblatt +für Bakteriologie und Parasitenkunde, Band 18, 1895, No. 1; American +Journal of Medical Sciences, Sept., 1895. + +[145] Griffiths, Principal Starches used as Food; Nägeli’s Beiträge zur +näheren Kenntniss der Stärkegruppe. + +[146] Zeitschrift für Physiologische Chemie, Band 12, Ss. 75-78. + +[147] Maercker; Handbuch der Spiritusfabrikation, 1890, S. 90. + +[148] Chemiker-Zeitung, Band 19, S. 1501. + +[149] Paragraphs =28-32=, this volume. + +[150] Vol. 2, p. 204. + +[151] Chemisches Centralblatt, 1877, Band 8, S. 732. + +[152] Chemiker-Zeitung, Band 19, S. 1501. + +[153] Vid. op. cit. supra, S. 1502; Moniteur Scientifique, 1887, p. 538. + +[154] Chemiker-Zeitung, Band 19, S. 1502. + +[155] Vid. op. cit. supra, 1895, S. 1727. + +[156] Chemiker-Zeitung, Band 19, S. 1502. + +[157] Jahresberichte der Agrikulturchemie, 1892, S. 664. + +[158] Journal de Pharmacie et de Chimie, 5ᵉ, Série, Tome 25, p. 394. + +[159] Journal of the American Chemical Society, Vol. 17, p. 64. + +[160] Repertorium der Analytischen Chemie, 1887, S. 299. + +[161] Journal of the American Chemical Society, Vol. 16, p. 726. + +[162] Förschungs Berichte über Lebensmittel, Hamburg; Abs., The +Analyst, Vol. 20, p. 210. + +[163] Rouvier; Comptes rendus, Tome 107, pp. 272, 278; Tome 111, pp. +64, 186; Tome 120, p. 1179. + +[164] Bulletin 13, Department of Agriculture, Division of Chemistry, +pp. 154 et. seq. + +[165] Foods, Their Composition and Analysis, p. 139. + +[166] Richardson, Vid. op. cit. 142, p. 158. + +[167] Principal Starches used as Food, Cirencester, Baily & Son, Market +Place. + +[168] Vid. op. cit. 142, pp. 158 et seq. + +[169] Bulletin 44, Department of Agriculture, Division of Chemistry, +p. 14. + + + + +PART THIRD. + +PROCESSES FOR DETECTING AND DETERMINING SUGARS AND STARCHES AND OTHER +CARBOHYDRATES IN CRUDE OR MANUFACTURED AGRICULTURAL PRODUCTS. + + +=203. Introduction.=—In the preceding part directions have been given +for the estimation of sugars and starches in approximately pure forms. +In the present part will be described the most approved methods of +separating these bodies and other carbohydrates from crude agricultural +products and for their chemical examination. In many respects the +processes which in a small way are used for preparing samples for +analysis are employed on a large scale for technical and manufacturing +purposes. It is evident, however, that the following paragraphs must be +confined strictly to the analytical side of the question inasmuch as +anything more than mere references to technical processes would lead +into wide digressions. + +In the case of sugars the analyst is for the most part quite as much in +need of reliable methods of extraction and preparation as of processes +for analysis. With starches the matter is more simple and the chief +methods of separating them for examination were necessarily described +in the previous part. + +Sugars in fresh plants exist almost entirely in solution. This is true +of all the great sources of the sugar of commerce, _viz._, the palm, +the maple, the sugar beet and sugar cane. This statement is also true +of fruits and the natural nectar of flowers. By natural or artificial +drying the sugar may be reduced to the solid or semisolid state as in +the cases of raisins and honey. In certain seeds, deficient in water, +sugars may possibly exist in a solid state naturally, as may be the +case with sucrose in the peanut and raffinose in cotton seed. + +Starches on the other hand when soluble, are probably not true +starches, but they partake more or less of a dextrinoid nature. Fine +starch particles occur abundantly in the juices of some plants, +as for instance sorghum, where they are associated with sugar and +can be obtained from the expressed juice by subsidence. But even +in such a case it is not certain that the starch enters into the +general circulation. It is more likely formed locally by biochemical +condensation of its constituents. Starches in a soluble or semisoluble +state are transported, as a rule, to the tubers or seeds of plants +where they are accumulated in large quantities as a reserve food for +future growth. For a study of the plant metabolism whereby starch is +produced and for its histological and physiological properties the +reader may consult the standard authorities on vegetable physiology.[170] + +=204. Sugar in the Sap of Trees.=—Many trees at certain seasons of the +year, carry large quantities of sugar in their sap. Among these the +maple and sugar palm are preeminent. The sap is secured by cutting a +pocket into the side of the tree or by boring into it and allowing the +sap to run into an appropriate receptacle through a spile. The content +of sugar in the sap of the maple and palm varies greatly. In some cases +it falls as low as one and a half and in others rises to as much as +six or seven per cent.[171] In most cases the sugar in the maple sap is +pure sucrose, but towards the end of the flowing season it may undergo +changes of a viscous nature due to fermentation, or inversion, forming +traces of invert sugar. In this country the sap of the maple may flow +freely on any warm day in winter, but the sugar season proper begins +about February 15th in Southern Ohio and Indiana, and about March 25th +in Vermont. It lasts from six weeks to two months. The sap flows best +during moderately warm, still days, after a light freeze. + +In addition to sugar the maple sap contains a trace of albuminoid +matters and some malic acid combined with lime. As a rule it can be +subjected to polarization without preliminary clarification. + +=205. Determination of Sugar in Saps.=—In most cases the sap may be +directly polarized in a 200 millimeter tube. Its specific gravity is +obtained by a spindle or pyknometer, and the percentage of sugars taken +directly from the table on page 73, the degree brix corresponding to +the sugar percentage. + +On polarizing, the sugar percentage is calculated as follows: + +Multiply the specific gravity of the sap by 100 and divide the product +by 26.048. Divide the direct reading of the sap on the sugar scale by +the quotient obtained above, and the quotient thus obtained will be the +correct percentage of sugar in the original solution. + +The formula is applicable for those instruments in which 26.048 grams +represent the normal quantity of sugar which in 100 cubic centimeters +reads 100 divisions on the scale. When other factors are used they +should be substituted for 26.048 in the above formula. + +The principle of the calculation is based on the weight of the sap +which is contained in 100 cubic centimeters, and this is evidently +obtained by multiplying 100 by the specific gravity of the sap. Since +26.048 is the normal quantity of sugar in that volume of the solution +the quotient of the actual weight divided by that factor shows how many +times too great the observed polarization is. The simple division of +the polariscope reading by this factor gives the correct reading. + +_Example_: Let the specific gravity of the sap be 1.015 and the +observed polarization be 15.0. Then the true percentage of sugar in the +sap is found by the equation: + +101.5 : 26.048 = 15.0 : _x_. + +Whence _x_ = 3.85 = percentage of sugar in the sap. + +The process outlined above is not applicable when a clarifying reagent +such as lead subacetate or alumina cream must be used. But even in +these cases it will not be found necessary to weigh the sap. A sugar +flask graduated at 100 and 110 cubic centimeters is used and filled to +the first mark with the sap, the specific gravity of which is known. +The clarifying reagent is added, the volume completed to the second +mark with water, and the contents of the flask well shaken and thrown +on a dry filter. The observation tube, which should be 220 millimeters +in length, is then filled with the clear filtrate and the rest of the +process is as described above. A 200 millimeter tube may also be used +in this case and the observed reading increased by one-tenth. + +[Illustration: FIG. 62. LABORATORY CANE MILL.] + +[Illustration: FIG. 63. WEIGHING PIPETTE.] + +=206. Estimation of Sugar in the Sap of Sugar Cane and Sorghum.=—In +bodies like sugar cane and sorghum the sap containing the sugar will +not flow as in the cases of the maple and sugar palm. The simplest way +of securing the sap of the bodies named is to subject them to pressure +between rolls. A convenient method of obtaining the sap or juice is +by passing the cane through a small three-roll mill indicated in the +figure. Small mills of this kind have been used in this division for +many years and with entire satisfaction. Small canes, such as sorghum, +may be milled one at a time, or even two or three when they are very +small. In the case of large canes, it is necessary that they be split +and only half of them used at once. The mill should not be crowded by +the feed in such a way as to endanger it or make it too difficult for +the laborer to turn. From fifty to sixty per cent of the weight of a +cane in juice may be obtained by passing it through one of these small +mills. Experience has shown that there is a little difference between +the juice as first expressed and the residual sap remaining in the +bagasse, but the juice first expressed may be used for analysis for +control purposes as a fair representative of all that the cane contains. + +To determine the percentage of juice expressed, the canes may be +weighed before passing through the mill and the juice collected. Its +weight divided by the weight of the original cane will give the per +cent of the juice expressed, calculated on the whole cane. Instead of +weighing the juice the bagasse may also be collected and weighed; but +on account of the rapidity with which it dries the operation should be +accomplished without delay. The expressed juice is clarified with lead +subacetate, filtered and polarized in the manner described in former +paragraphs. Instead of weighing the juice, its specific gravity may +be taken by an accurate spindle and the volume of it, equivalent to a +given weight, measured from a sucrose pipette.[172] + +A sucrose pipette for cane juice has a graduation on the upper part +of the stem which enables the operator to deliver double the normal +weight for the polariscope used, after having determined the density of +the juice by means of a spindle. A graduation of from 5° to 25° of the +brix spindle will be sufficient for all variations in the density of +the juice, or one covering a range of from 10° to 20° will suffice for +most instances. The greater the density of the juice the less volume +of it will be required for the weight mentioned. For general use, the +sucrose pipette is graduated on the stem to deliver from forty-eight +to 50.5 cubic centimeters, the graduations being in terms of the brix +spindle. The graduation of the stem of this instrument is shown in the +accompanying figure. In the use of the pipette it is only necessary +to fill it to the degree on the stem corresponding to the degree brix +found in the preliminary trial. + +The quantities of juice corresponding to each degree and fractional +degree of the brix spindle are given in the following table; calculated +for the normal weight 26.048 grams for the ventzke and for 16.19 grams +for the laurent scale. The measured quantities of juice are placed in +a 100 cubic centimeter sugar flask, treated with the proper quantity +of lead subacetate, the volume completed to the mark, and the juice +filtered and polarized in a 200 millimeter tube. The reading of the +polariscope is divided by two for the factor 26.048 and by three for +the factor 16.19. + + TABLE FOR USE OF SUCROSE PIPETTES. + + Cubic centimeters Cubic centimeters + of juice for 26.048 of juice for 16.19 + Degrees factor. Divide Degrees factor. Divide + brix. reading by two. brix. reading by three. + + 5.0 51.1 5.0 47.6 + 5.4 51.0 5.7 47.5 + 5.7 50.9 6.3 47.4 + 6.4 50.8 6.8 47.3 + 6.9 50.7 7.3 47.2 + 7.4 50.6 7.8 47.1 + 7.9 50.5 8.3 47.0 + 8.4 50.4 8.9 46.9 + 8.9 50.3 9.5 46.8 + 9.4 50.2 10.0 46.7 + 9.9 50.1 10.5 46.6 + 10.4 50.0 11.0 46.5 + 10.9 49.9 11.6 46.4 + 11.4 49.8 12.1 46.3 + 11.9 49.7 12.7 46.2 + 12.4 49.6 13.3 46.1 + 12.9 49.5 13.8 46.0 + 13.4 49.4 14.3 45.9 + 13.9 49.3 14.8 45.8 + 14.4 49.2 15.3 45.7 + 14.9 49.1 15.9 45.6 + 15.4 49.0 16.4 45.5 + 15.9 48.9 17.0 45.4 + 16.4 48.8 17.5 45.3 + 16.9 48.7 18.0 45.2 + 17.4 48.6 18.6 45.1 + 17.9 48.5 19.1 45.0 + 18.4 48.4 19.7 44.9 + 18.9 48.3 20.2 44.8 + 19.4 48.2 + 19.9 48.1 + +In ordering sucrose pipettes the factor for which they are to be +graduated should be stated. + +It is evident also that with the help of the foregoing table the +measurements may be made by means of a burette. For instance, if the +degree brix is found to be 19.9, 48.1 cubic centimeters are to be used. +This quantity can be easily run from a burette. In order to make the +pipette more convenient it has been customary in this laboratory, as +practiced by Carr, to attach a glass tube with a stopcock by means of +a rubber tube to the upper part of the pipette, whereby the exact +level of the juice in the stem of the pipette can be easily set at any +required mark. + +In the polarization of dilute solutions, such as are found in the +saps and juices referred to above, it must not be forgotten that the +gyrodynat of the sucrose is increased as the density of the solution +is diminished. This change introduces a slight error into the work +which is of no consequence from a technical point of view, but +becomes a matter which must be considered in exact determinations. To +avoid the annoyance of calculating the gyrodynat for every degree of +concentration, tables have been constructed by Schmitz and Crampton by +means of which the actual percentage of sugar, corresponding to any +degree of polarization, is determined by inspection. These tables may +be used when extremely accurate work is required.[173] + +[Illustration: FIGURE 64. GIRD’S GRAVIMETER.] + +=207. Measuring Sugar Juices with a Gravimeter.=—A convenient method of +weighing sugar juices is the gravimetric process designed by Gird.[174] +The apparatus is fully illustrated by Fig. 64. The hydrometer F has a +weight of 26.048 grams and its stem is also graduated in degrees brix. +The juice is poured into the cylinder A and allowed to stand until air +bubbles have escaped. In filling A the finger is held over the orifice +G so that the siphon tube B is completely filled, the air escaping at +the vent C. After the tube is filled the finger is withdrawn from G and +all the liquid which will run out at G allowed to escape. The sugar +flask D is now brought under G and the hydrometer F allowed to descend +into A. The hydrometer will displace exactly its own weight of liquid. +For convenience of reading, the index E may be used which is set five +degrees above the surface of the liquid in A. The number of degrees +brix read by E is then diminished by five. The hydrometer has been +improved since the description given by the addition of a thermometer +which, in addition to carrying a graduation in degrees, also shows the +correction to be made upon the degree brix for each degree read. It is +evident that the hydrometer may be made of any weight, and thus the +delivery of any desired amount of juice be secured. + +=208. Determination of Reducing Bodies in Cane Juices.=—Sucrose in +cane juices is constantly accompanied with reducing sugars, or other +bodies which have a similar action on fehling liquor, which interfere +to a considerable degree with the practical manufacture of sugar. It is +important to determine with a moderate degree of accuracy the quantity +of these bodies. These sugars or reducing bodies are of a peculiar +nature. The author pointed out many years ago that these reducing +bodies were without action on polarized light, and for this reason +proposed the name anoptose as one characteristic of their nature.[175] It +is also found that these bodies do not yield theoretically the quantity +of alcohol which a true sugar of the hexose type would give.[176] It is +entirely probable, therefore, that they are quite different in their +nature from many of the commonly known sugars. On account of the +difficulty of separating these bodies in a pure state their actual +copper reducing power is not known. For practical purposes, however, it +is assumed to be the same as that of dextrose or invert sugar and the +percentage of these bodies present is calculated on that assumption. +In the determination of these sugars or reducing bodies, the quantity +weighed may be determined by an apparatus entirely similar to the +sucrose pipette just described above. The quantity of juice used should +be diluted as a rule to such a degree as not to contain more than one +per cent of the reducing bodies. For the best work, the juices should +be clarified with lead subacetate and the excess of lead removed with +sodium carbonate. For technical control work in sugar factories, this +process may be omitted as in such cases rapidity of work is a matter +of considerable importance and the approximate estimation of the total +quantity of reducing bodies is all that is desired. + +For volumetric work, the solution of copper and the method of +manipulation described in paragraph =117= are most conveniently used. + +=209. Preservation of Sugar Juices for Analysis.=—Lead subacetate not +only clarifies the juices of canes and thus permits of their more exact +analytical examination, but also exercises preservative effects which +enable it to be used as a preserving agent and thus greatly diminish +the amount of work necessary in the technical control of a sugar +factory. Instead, therefore, of the analyst being compelled to make an +examination of every sample of the juice, aliquot portions representing +the different quantities can be preserved and one analysis made for +all. This method has been thoroughly investigated by Edson, who also +finds that the errors, which may be introduced by the use of the lead +subacetate in the analytical work, may be entirely avoided by using the +normal lead acetate.[177] + +In the use of the normal lead acetate, much less acetic acid is +required in the polariscopic work than when the subacetate is used. +The normal lead acetate is not so good a clarifying agent as the +subacetate, but its efficiency in this respect is increased by the +addition of a little acetic acid. In its use, it is not necessary to +remove the lead, even for the determination of the reducing bodies. + +For further details in regard to the technical determination of +reducing bodies, special works may be consulted.[178] + +=210. Direct Determination of Sugars in Canes.=—The methods, which +have just been described, of securing the juices of cane by pressure +and of determining the sugars therein, do not give the actual +percentage of sugar in the cane. An approximate result may be secured +by assuming that the cane is composed of ninety parts of juice and +ten parts of cellular tissues and other insoluble matters. This +assumption is approximately true in most cases, but there are often +conditions arising which render the data calculated on the above +assumption misleading. In any particular case in order to be certain +that the correct percentage of sugar is secured it will be necessary +to determine the fiber in the cane. This is an analytical process of +considerable labor and especially so on account of the difficulty of +securing samples which represent the average composition of the cane. +The fibrous structure of the canes, the hardness of their external +covering and the toughness of their nodes or joints render the +sampling extremely difficult. Moreover, the content of sugar varies +in different parts of the cane. The parts nearest the ground are, +as a rule, richer than the upper joints and this is especially true +of sugar cane. In order, therefore, to get a fair sample, even of a +single cane, all parts of it must be considered. Several methods of the +direct determination of sugar in canes have been proposed and will be +described below. + +[Illustration: FIGURE 65. MACHINE FOR CUTTING CANES.] + +=211. Methods of Cutting or Shredding the Cane for Analytical +Purposes.=—A simple method of cutting canes into small pieces which +will permit of an even sampling is very much to be desired. The +cutting apparatus shown in Fig. 65 has been long in use in this +laboratory. The canes by it are cut into thin slices, but the cutting +edge of the knife being perpendicular to the length of the cane renders +the use of the instrument somewhat laborious and unsatisfactory. A +considerable time is required to cut a single cane and the slices which +are formed should be received in a vessel which will protect them as +much as possible from evaporation during the process of the work. +Instead of the apparatus above a small cane cutting machine arranged +with four knives on a revolving disk maybe used. The apparatus is shown +in Fig. 66. The cane is fed against the knives through the hole shown +in the open front of the apparatus and the knives thus strike the cane +obliquely.[179] The knives can be set in the revolving disk at any +desired position so as to cut the canes into chips as fine as may be +desired. The cossettes furnished by this method may be sampled directly +for the extraction of the sugar. In the case of the cossettes from +both instruments described above a finer subdivision may be secured by +passing them through a sausage cutter. + +[Illustration: FIGURE 66. CANE CUTTING MILL.] + +The best method for shredding canes, however, is to pass them through +the apparatus described on page 9. That machine gives an extremely +fine, moist mass, which is of uniform nature and capable of being +directly sampled. + +=212. Methods of Determination.=—Even the finely divided material +obtained by the machine just described is not suited to give an +instantaneous diffusion for polarization as is done by the finely +ground beet pulp to be described further on. For the determination of +sugar a proper weight of the cossettes or pulp obtained as described +above, taken after thorough mixing, is placed in a flask graduated +properly and treated with water.[180] + +The flask in which the mixture takes place should be marked to +compensate for the volume of the fiber of the cane. When the normal +weight of cane is taken for the ventzke scale, _viz._, 26.048, the +flask should be graduated at 102.6 cubic centimeters. If double the +normal weight be taken, the flask should be graduated at 205.2 cubic +centimeters. This graduation is based on the assumption of the presence +of fiber amounting to ten per cent of the weight of the cossettes. The +fiber is so nearly the density of the juice obtained as to be regarded +as one gram equal to one cubic centimeter. The flask is at first filled +almost full of water and then warmed to near the boiling point for an +hour with frequent shaking. It is then filled to a little above the +mark, the contents well mixed and warmed for ten minutes more with +frequent shaking. After cooling, the volume is made up to the mark, +well shaken and poured upon a filter. The filtrate is collected in a +sugar flask marked at fifty and fifty-five cubic centimeters. When +filled to the first mark a proper quantity of lead subacetate is added, +the volume completed to the second mark with water, the contents of the +flask well shaken, poured upon a filter and the filtrate polarized in +the usual way. + +The reducing sugar is determined in an aliquot part of the filtrate by +one of the alkaline copper methods. + +=213. Determination by Drying and Extraction.=—Instead of the diffusion +and polarization method just described, the fine pulp obtained may +be dried, the dried residue ground in a drug mill and extracted with +aqueous alcohol or with water. + +To facilitate the calculation when this method is employed, the water +content of a small portion of the well sampled pulp is determined. The +rest of the pulp is dried, first for a few hours at a temperature not +above 60° or 70°, and then at the temperature of boiling water, either +in the open air or a partial vacuum, until all the water is driven off. +The dried residue can then be preserved in well stoppered bottles for +the determination of sugar at any convenient period. The finely ground +dried residue for this purpose is placed in an extraction apparatus +and thoroughly exhausted with eighty per cent alcohol. The extract is +dried and weighed, giving the total weight of all sugars present. After +weighing, the extract is dissolved in water, made up to a definite +volume and the reducing sugars determined in an aliquot portion thereof +by the usual methods. The weight of reducing sugars found, calculated +for the whole extract deducted from the total weight of this extract +will give the weight of the sucrose in the sample. From this number the +content of sugar in the original cane is determined from the percentage +of water found in the original sample. + +_Example._—In a sample of finely pulped canes the content of water is +found to be 76.5 per cent. The thoroughly dried pulp is ground and +extracted with aqueous alcohol. Five grams give two and five-tenths +grams of the extract. The extract is dissolved in water, made up to +a definite volume and the reducing sugars determined in an aliquot +part and calculated for the whole, amounting to 150 milligrams. The +extract is therefore composed of 2.35 grams of sucrose and 0.15 gram +of reducing sugars. The calculation is now made to the original sample +which contained 76.5 per cent of water and 23.5 per cent of dry matter, +as follows: + + 5 : _x_ :: 23.5 : 100, whence _x_ = 21.27, + +the weight of the original material corresponding to five grams of the +dry substance. The original composition of the sample is therefore +expressed by the following numbers: + + Per cent. + Sucrose 11.1 + Reducing sugars 0.7 + Water 76.5 + Fiber (insoluble matter) 11.7 + +=214. Examination of the Bagasse.=—The method just described for the +examination of canes may be also applied to the analysis of bagasses, +with the changes made necessary by the increased percentage of fiber +therein. On account of the large surface exposed by the bagasse, the +sampling, shredding and weighing should be accomplished as speedily as +possible to avoid loss of moisture. + +The optical examination of bagasses is rendered difficult by reason of +the uneven pressure to which the canes are subjected. With fairly good +milling in technical work the bagasses will have at least thirty per +cent of fiber. The method for the polariscopic examination is therefore +based upon that assumption, but the volume of the solution must be +changed for varying percentages of fiber in the bagasse. On account of +the smaller percentage of sugar, it is convenient to take double or +three times the normal weight of the bagasse for examination. Since +large sugar flasks are not commonly to be had the diffusion of the +bagasse may be conducted in a quarter liter flask. In a quarter liter +flask place 52.096 grams of the finely shredded bagasse, very nearly +fill the flask with water and extract the sugar as described for canes +in the foregoing paragraphs. In the weight of bagasse used there will +be, in round numbers, fifteen grams of fiber. When the volume of water +is completed to the mark the actual content of liquid in the flask will +therefore be only 235 cubic centimeters. Fifty cubic centimeters of the +filtrate are placed in a sugar flask marked at fifty and fifty-five +cubic centimeters, the proper quantity of lead subacetate solution +added, the volume completed to the upper mark, the contents of the +flask well shaken, filtered and polarized in a 200 millimeter tube. Let +the reading obtained be four degrees and increase this by one-tenth for +the increased volume of solution above fifty cubic centimeters. The +true reading is therefore four degrees and four-tenths. This reading, +however, must be corrected, because the original volume instead of +being 200 cubic centimeters, is 235 cubic centimeters. The actual +percentage of sugar in the sample examined is obtained by the following +proportion: + + 200 : 235 = 4.4 : _x_. + +The correct reading is therefore 5°.2, the percentage of sugar in the +sample examined. + +The results obtained by the method just described may vary somewhat +from the true percentage by reason of the variation of the content +of fiber in the bagasse. It is, however, sufficiently accurate for +technical control in sugar factories and on account of its rapidity +of execution is to be preferred for this purpose. More accurate +results would be obtained by drying the bagasse, and proceeding with +the examination in a manner entirely analogous to that described for +the extraction of sugar from dried canes by aqueous alcohol. In both +instances the reducing sugar is determined in the manner already +mentioned. + +=215. Determination of Fiber in Cane.=—In estimating the content +of sugar in canes by the analysis of the expressed juices, it is +important to make frequent determinations of the fiber for the purpose +of obtaining correct data for calculation. In periods of excessive +drought, or when the canes are quite mature, the relative content of +fiber is increased, while, on the other hand, in case of immature +canes, or during excessive rainfalls, it is diminished. The chief +difficulty in determining the content of fiber in canes is found in +securing a representative sample. On account of the hard and fibrous +nature of the envelope and of their nodular tissues, canes are reduced +to a fine pulp with great difficulty by the apparatus in ordinary +use. A fairly homogeneous pulp, however, may be obtained by means of +the shredder described on page 9. The canes having been shredded as +finely as possible, a weighed quantity is placed in any convenient +extraction apparatus and thoroughly exhausted with hot water. The +treatment with hot water should be continued until a few drops of the +extract evaporated on a watch glass will leave no sensible residue. +The residual fiber is dried to constant weight at the temperature of +boiling water, cooled in a desiccator and rapidly weighed and the +percentage of fiber calculated from the data obtained. On account of +the great difficulty of securing a homogeneous pulp, even with the best +shredding machines, the determination should be made in duplicate or +triplicate and the mean of the results entered as the percentage of +fiber. The term fiber as used in this sense, must not be confounded +with the same term employed in the analysis of fodders and feeding +stuffs. In the latter case the term is applied to the residue left +after the successive treatment of the material with boiling, dilute +acid and alkali. The analysis of canes for feeding purposes is +conducted in the general manner hereinafter described for fodders. + +=216. Estimation of Sugar in Sugar Beets.=—The methods employed for +the determination of the sugar content of beets are analogous to those +used for canes, with such variations in the method of extraction as are +made possible and necessary by the difference in the nature of these +sacchariferous plants. The sugar beet is more free of fiber and the +hard and knotty substances composing the joints of plants are entirely +absent from their composition. For this reason they are readily +reduced to a fine pulp, from which the sugar is easily extracted. +The analytical processes are also greatly simplified by the complete +absence of reducing sugars from the juices of healthy beets. The only +sugar aside from sucrose which is present in these juices is raffinose, +and this is not found in healthy beets, except when they have been +injured by frost or long keeping. In practical work, therefore, the +determination of sucrose completes the analysis in so far as sugars are +concerned. Four methods of procedure will illustrate all the principles +of the various processes employed. + +=217. Estimation of Sucrose in the Expressed Juice.=—In the first +method the beets are reduced by any good shredding machine, to a fine +pulp, which is placed in a press and the juice expressed. In this +liquor, after clarification with lead subacetate, the sucrose is +determined by the polariscope. The methods of measuring, clarifying and +polarizing are the same as those described for saccharine juices in +paragraphs =83-85=. The mean percentage of juice in the sugar beet is +ninety-five. The corrected polariscopic reading obtained multiplied by +0.95 will give the percentage of sugar in the beet. + +_Example._—The solids in a sample of beet juice, as measured by a brix +spindle, are 17.5 per cent. Double the normal weight of the juice is +measured from a sucrose pipette, placed in a sugar flask, clarified, +the volume completed to 100 cubic centimeters, the contents of the +flask well shaken and filtered. The polariscopic reading obtained is +29°.0. Then (29.0 ÷ 2) × 0.95 = 13.8 = percentage of sucrose in the +beet. + +=218. Instantaneous Diffusion.=—In the second process employed for +determining the sugar content of beets, the principle involved depends +on the use of a pulp so finely divided as to permit of the almost +instant diffusion of the sugar present throughout the added liquid. +This diffusion takes place even in the cold and the process thus +presents a convenient and rapid method for the accurate determination +of the percentage of sugar in beets. The pulping is accomplished by +means of the machine described on page 10, or the one shown in Fig. 67. +The beet is pressed against the rapidly revolving rasp by means of the +grooved movable block and the finely divided pulp is received in the +box below. These machines afford a pulp which is impalpable and which +readily permits an almost instantaneous diffusion of its sugar content. + +[Illustration: FIG. 67. APPARATUS FOR PULPING BEETS.] + +=219. Pellet’s Method of Cold Diffusion.=—The impalpable pulp having +been obtained, by one of the processes described, the content of sugar +therein is determined as follows:[181] + +A normal or double normal quantity of the pulp is quickly weighed, +to avoid evaporation, in a sugar dish with an appropriate lip, and +washed into the flask, which should be graduated, as shown in Fig. 68, +to allow for the volume of the fiber or marc of the beet. Since the +beet pulp contains, on an average, four per cent of marc, the volume +which is occupied thereby is assumed to be a little more than one cubic +centimeter. Since it is advisable to have as large a volume of water +as convenient, it is the practice of Pellet to wash the pulp into a +flask graduated at 201.35 cubic centimeters. If a 200 cubic centimeter +flask be used, the weight of the pulp should be 25.87 instead of +26.048 grams. After the pulp is washed into the flask, about six cubic +centimeters of lead subacetate of 30° baumé are added, together with a +little ether, to remove the foam. The flask is now gently shaken and +water added to the mark and the contents thoroughly shaken. If the pulp +is practically perfect, the filtration and polarization may follow +immediately. The filter into which the contents of the flask are poured +should be large enough to hold the whole quantity. It is recommended +to add a drop or two of strong acetic acid just before completing +the volume of the liquid in the flask to the mark. The polarization +should be made in a 400 millimeter tube, which will give directly the +percentage of sugar present. It is not necessary to heat the solution +in order to insure complete diffusion, but the temperature at which the +operation is conducted should be the ordinary one of the laboratory. +In case the pulp is not as fine as should be, the flask should be +allowed to stand for half an hour after filling, before filtration. +An insufficient amount of lead subacetate may permit some rotatory +bodies other than sugar to pass into solution, and care should be +taken to have always the proper quantity of clarifying material added. +The presence of these rotating bodies, mostly of a pectic nature, +may be shown by extracting the pulp first with cold water until all +the sugar is removed, and afterwards with boiling water. The liquor +obtained from the last precipitation will show a decided right-handed +rotation, unless first treated with lead subacetate, in which case +the polarization will be zero. A very extended experience with the +instantaneous cold aqueous diffusion has shown that the results +obtained thereby are quite as reliable as those given by hot alcoholic +or aqueous digestion. + +=220. Flask for Cold Diffusion and Alcohol Digestion.=—For convenience +in washing the pulp into the sugar flask, the latter is made with +an enlarged mouth as shown in Fig. 68. The dish holding the weighed +quantity of pulp is held with the lip in the mouth of the flask, and +the pulp washed in by means of a jet of water furnished from a pressure +bottle or washing flask. The flask shown is graduated for the normal +weight of pulp, _viz._, 26.048 grams. The marking is on the constricted +neck and extends from 100 to 101.3 cubic centimeters. This permits +of making the proper allowance for the volume occupied by the marc +or fiber, but this is unnecessary for the usual character of control +analyses. In the case of healthy, fresh beets, the volume occupied +by the marc is nearly one and three-tenths cubic centimeters for the +normal polariscopic weight of 26.048 grams of pulp. For the laurent +instrument this volume is nearly one cubic centimeter. + +[Illustration: FIGURE 68. APPARATUS FOR COLD DIFFUSION.] + +=221. Extraction with Alcohol.=—The third method of determining sugar +in beets is by alcoholic extraction. The principle of the method is +based on the fact that aqueous alcohol of not more than eighty per +cent strength will extract all the sugar from the pulp, but will not +dissolve the pectic and other rotatory bodies, which, in solution, +are capable of disturbing the rotatory power of the sugar present. +It is also further to be observed that the rotatory power of pure +sucrose, in an aqueous alcoholic solution, is not sensibly different +from that which is observed in a purely aqueous liquid. The pulp, +which is to be extracted, should be in as fine a state of subdivision +as convenient, and the process may be carried on in any of the forms +of extraction apparatus already described, or in the apparatus shown +in Fig. 69. The extraction tube, of the ordinary forms of apparatus, +however, is scarcely large enough to hold the required amount of pulp, +and therefore special tubes and forms of apparatus have been devised +for this method of procedure. In weighing the pulp for extraction, +a quarter, half, or the exact amount required for the polariscope +employed, should be used. If the tubes are of sufficient size the full +weight may be taken, _viz._, 26.048 or 16.19 grams for the instruments +in ordinary use. Since the pulp contains a large quantity of water, +the extraction could be commenced with alcohol of standard strength, +_viz._, about ninety-five per cent. The volume of alcohol employed +should be such as will secure a strength of from seventy to eighty +per cent when mixed with the water contained in the pulp. The flask +receiving the extract should be kept in continuous ebullition and the +process may be regarded as complete in about one hour, when the pulp +has been properly prepared. The method of extracting beet pulp with +alcohol is due to Scheibler, and in its present form the process is +conducted according to the methods described by Scheibler, Sickel, and +Soxhlet.[182] + +[Illustration: FIG. 69. SICKEL-SOXHLET EXTRACTOR.] + +If the pulp be obtained by any other means than that of a fine rasp, +the extraction of the sugar by the aqueous alcohol takes a long time, +and even a second extraction may be necessary. It is convenient to use +as a flask for holding the solvent, one already graduated at 100 or 110 +cubic centimeters. A flask especially constructed for this purpose, has +a constricted neck on which the graduations are made, and a wide mouth +serving to attach it to the extracting apparatus, as shown in Fig. +68. When the extract is obtained in this way, it is not necessary to +transfer it to a new flask before preparing it for polarization. When +the extraction is complete, the source of heat is removed, and when all +the alcohol is collected in the flask, the latter is removed from the +extraction apparatus, cooled to room temperature, a sufficient quantity +of lead subacetate added, the flask well shaken, the volume completed +to the mark with water, again well shaken and the contents of the +flask thrown upon the filter. It is important to avoid loss of alcohol +during filtration. For this purpose it is best to have a folded filter +and to cover the funnel immediately after pouring the contents of the +flask upon the filter paper, with a second larger funnel. The stem of +the funnel carrying the filter paper, should dip well into the flask +receiving the filtrate. As in other cases of filtering sugar juices for +polarization, the first portions of the filtrate received should be +rejected. The percentage of sugar is obtained in the filtrate in the +usual way. + +Where a weight of pulp equal to the normal factor of the polariscope +employed is used, and the extract collected in a 100 cubic centimeter +flask, the percentage of sugar is directly obtained by making the +reading in a 200 millimeter tube. With other weights of pulp, or other +sizes of flask, the length of the observation tube may be changed or +the reading obtained corrected by multiplication or division by an +appropriate factor. A battery of sickel-soxhlet extractors is shown in +Fig. 69.[183] + +[Illustration: FIG. 70.] + +=222. Scheibler’s Extraction Tube.=—In order to secure a speedy +extraction of large quantities of pulp, Scheibler recommends the use of +the extraction tube shown in Fig. 70.[184] The apparatus is composed of +three concentric glass cylinders. The outer and middle cylinders are +sealed together at the top, and the inner one is movable and carries +a perforated diaphragm below, for filtering purposes. Near the top it +is provided with small circular openings, whereby the alcoholic vapors +may gain access to the condenser (not shown). The middle cylinder is +provided with two series of apertures, through the higher of which the +vapor of alcohol passes to the condenser, while the alcohol which has +passed through the pulp and collected between the inner and middle +cylinders, flows back through the lower into the flask (not shown) +containing the boiling alcohol. + +The middle cylinder is provided with a curved bottom to prevent the +filtering end of the inner tube from resting too tightly against it. + +The tube containing the pulp is thus protected from the direct heat +of the alcoholic vapors during the progress of extraction by a thin +cushion of liquid alcohol. + +=223. Alcoholic Digestion.=—The fourth method of determining sugar in +beet pulp, is by means of digestion with hot alcohol. The principle +of this method is precisely the same as that which is involved in +aqueous diffusion in the cold. The diffusion, however, in the case of +the alcohol, is not instantaneous, but is secured by maintaining the +mixture of the pulp and alcohol for some time at or near the boiling +point. The methods of preparing the pulp, weighing it and introducing +it into the digestion flask are precisely those used for aqueous +digestion, but in the present case a somewhat coarser pulp may be +employed. The method is commonly known as the rapp-degener process.[185] + +Any convenient method of heating the alcohol may be used. In this +laboratory the flasks are held on a false bottom in a bath composed of +two parts of glycerol and one of water. One side of the bath holder +is made of glass, as shown in Fig. 71, in order to keep the flasks in +view. In order to avoid the loss of alcohol, the digestion flask should +be provided with a reflux condenser, or be attached to an ordinary +condenser, which will reduce the vapors of alcohol again to a liquid. +Unless the weather be very warm, the reflux condenser may consist of +a glass tube of rather wide bore and at least one meter in length, as +shown in Fig. 71. A slight loss of alcohol during the digestion is of +little consequence. A convenient method of procedure is the following. + +Double the quantity of the beet pulp required for the ventzke +polariscope, _viz._, 52.096 grams, weighed in a lipped metal dish, +is washed, by means of alcohol, into a flask marked at 202.6 cubic +centimeters, and the flask filled two-thirds with ninety-five per +cent alcohol and well shaken. Afterwards, a proper quantity of lead +subacetate is added, and then sufficient alcohol to complete the volume +to the mark. The flask is then attached to the condenser, placed in +a water-glycerol bath and heated to a temperature of 75° for about +forty-five minutes. At the end of this time, the flask is removed from +the bath and condenser, cooled quickly with water, alcohol added to +the mark and well shaken. The filtration should be accomplished with +precautions, to avoid the loss of alcohol mentioned in paragraph =221=. +The filtrate is examined in the polariscope in a 200 millimeter tube, +and the reading obtained gives directly the percentage of sugar in the +sample examined. Half the quantity of pulp mentioned, in a 101.3 cubic +centimeter flask, may also be used. A convenient form of arranging a +battery of flasks is shown in the accompanying figure. + +[Illustration: FIG. 71. BATTERY FOR ALCOHOLIC DIGESTION.] + +=224. Determination of Sugar in Mother Beets.=—In selecting mother +beets for seed production, it is necessary to secure only those of a +high sugar content. This is accomplished by boring a hole about two and +a half centimeters in diameter obliquely through the beet by means of +the apparatus shown in Fig. 72. + +The beet is not injured for seed production by this process, and the +pulp obtained is used for the determination of sugar. The juice is +expressed by means of the small hand press shown in Fig. 73. Since only +a small quantity of juice is obtained, it is advisable to prepare it +for polarization in a sugar flask marked at fifty cubic centimeters. +The density of the juice, by reason of its small volume, is easiest +obtained by the hydrostatic balance, as described in paragraph =53=. +In lieu of this, the juice may be quickly weighed in a counterbalanced +dish on a balance giving results accurate to within one milligram. The +rest of the analytical process is similar to that already described. + +[Illustration: FIG. 72. RASP FOR SAMPLING MOTHER BEETS.] + +[Illustration: FIG. 73. HAND PRESS FOR BEET ANALYSIS.] + +[Illustration: FIG. 74.] + +=225. Aqueous Diffusion.=—The process of instantaneous aqueous +diffusion may also be applied to the examination of mother beets. For +this purpose the beets are perforated by a rasp, devised by Keil, shown +lying on the floor in Fig. 72, the characteristics of which are shown +in Fig. 74. The conical end of the rasp is roughened in such a way +as to reduce the beet to an impalpable pulp. This end is fastened by +a bayonet fastening to the cylindrical carrier or arm in such a way +that, by means of a groove in the conical end of the rasp, the pulp is +introduced into the cylinder. The cylinder is provided with a small +piston by means of which the pulp can be withdrawn when the cylindrical +portion of the rasp is detached from the driving machinery. It is +important that the rasp be driven at a high rate of speed, _viz._, from +1500 to 2000 revolutions a minute. The sample of pulp at this rate of +revolution is taken almost instantly, and with skilled manipulators the +whole operation of taking a sample, removing the rasp by means of its +bayonet fastenings, withdrawing the sample of pulp and replacing the +rasp ready for another operation does not consume more than from ten +to twenty seconds. From three to four samples may thus be taken in a +minute. The samples of pulp as taken are dropped into numbered dishes +corresponding to the numbers on the beets. One-quarter of the normal +weight for the polariscope is used for the analysis. The pulp is placed +in a fifty cubic centimeter flask, water and lead subacetate added, the +flask well shaken, filled to the mark with water, again well shaken, +the contents thrown on the filter, and the filtrate polarized in a 400 +millimeter tube, giving the direct percentage of sugar. For practical +purposes the percentage of marc in the beet may be neglected. If the +polarization take place in a 200 millimeter tube the number obtained +should be multiplied by two for the content of sugar. + +In numbering sugar beets which are to be analyzed for seed production, +it is found that a small perforated tin tag bearing a number may be +safely affixed to the beet by means of a tack. It is not safe to use +paper tags as they may become illegible by becoming wet before the +sorting of the beets is completed. Where from 1000 to 2000 beets +are to be examined in a day, the number of the beets and the dishes +corresponding thereto must be carefully controlled to avoid confusion +and mistakes. + +=226. Determination of Sugars without Weighing.=—An ingenious device +for the rapid analysis of mother beets is based upon the use of a +machine which cuts from the beet a core of given dimensions and this +core is subsequently reduced to a pulp which is treated with cold water +and polarized in the manner described above. The cutting knives of the +sampler can be adjusted to take a core of any desired size. Since the +beets used for analysis have essentially the same specific gravity, the +cores thus taken weigh sensibly the same and the whole core is used +for the analysis, thus doing away with the necessity of weighing. The +core obtained is reduced to a pulp in a small machine so adjusted as +to permit the whole of the pulp, when prepared, to be washed directly +into the sugar flask. By the use of this machine a very large number of +analyses can be made in a single day, and this is highly important in +the selection of mother beets, for often 50,000 or 100,000 analyses are +to be made in a short time. + +[Illustration: FIG. 75. TUBE FOR CONTINUOUS OBSERVATION.] + +=227. Continuous Diffusion Tube.=—To avoid the delay occasioned by +filling and emptying observation tubes in polariscopic work, where +large numbers of analyses of canes and beets are to be made, Pellet +has devised a continuous diffusion tube, by means of which a solution, +which has just been observed, is rapidly and completely displaced by a +fresh solution. This tube, improved by Spencer, is shown in Fig. 75. +The fresh solution is poured in at the funnel, displacing completely +the old solution which flows out through the tube at the other end. +The observer watches the field vision and is able to tell when the +old solution is completely displaced by the clearing of the field, at +which time the reading of the new solution can be quickly made. When +solutions are all ready for examination an expert observer can easily +read, by the aid of this device, from four to five of them in a minute. + +=228. Analysis of Sirups and Massecuites.=—The general principles which +control the analysis of sirups and massecuites are the same whether +these products be derived from canes or beets. In the case of the +products of canes, the sirups or massecuites contain chiefly sucrose, +invert sugar, and other copper reducing bodies, inorganic matters and +water. In the case of products derived from sugar beets the contents +are chiefly sucrose, inorganic matters, a trace of invert sugar, +raffinose and water. The principles of the determination of these +various constituents have already been described. + +=229. Specific Gravity.=—The specific gravity of sirups and molasses +can be determined by the spindle in the usual way, but in the case of +molasses which is quite dense, the spindle method is not reliable. It +is better, therefore, both in molasses and massecuites, to determine +the density by dilution. For this purpose, as described by Spencer, +a definite weight of material, from 200 to 250 grams, is dissolved +in water and the volume of the solution completed to half a liter. A +portion of the solution is then placed in a cylinder and the quantity +of total solids contained therein determined in the usual way by a brix +or specific gravity hydrometer. In case 250 grams of the material be +used the calculation of the brix degree for the original material is +conducted according to the following formula: + + G × _B_ × _V_ + _x_ = -------------. + _W_ + +In the above formula _x_ is the required brix degree, _V_ the volume +of the solution, _B_ the observed brix degree of the solution, and G +the corresponding specific gravity obtained from the table on page 73. +When only small quantities of the material are at hand the hydrostatic +balance (=53=) should be employed. For this purpose twenty-five grams +of the material are dissolved in water and the volume of the solution +made up to 100 cubic centimeters. The sinker of the hydrostatic balance +is placed in the solution and equilibrium secured by placing the +weights upon the arm of the balance in the usual manner. Since the arm +of the balance is graduated to give, by direct reading, the specific +gravity, the density can be obtained at once. + +_Example._—Let the position of the weights or riders upon the balance +arm be as follows: + + (1) at point of suspension of the bob = 1.000 + (3) at mark 7 on beam = 0.07 + (4) at mark 9 on beam = 0.009 + Specific gravity = 1.079 + +The nearest brix degree corresponding to this specific gravity (=58=) +is 19. The total weight of the solution is equal to 100 × 1.079, +_viz._, 107.9 grams. Since the solution contains nineteen per cent of +solid matter as determined by the hydrostatic balance, the total weight +of solid matter therein is 107.9 × 19 ÷ 100 = 20.5 grams. The total per +cent. of solid matter in the original sample is therefore 20.5 ÷ 25 × +100 = 82 and the specific gravity corresponding thereto (page 74) is +1.42934. + +The specific gravity of a massecuite may also be determined in +pyknometers especially constructed for this purpose.[186] + +=230. Determination Of Water.=—The accurate determination of water +in sirups and massecuites is a matter of considerable difficulty. +The principles of conducting the process (=26=), applicable also to +the determination of water in honeys and other viscous liquids, are +as follows: In all cases where invert sugar is present the drying +should be conducted at a temperature not exceeding 75° or 80°. In +dense molasses and massecuites a weighed quantity should be dissolved +and made up to a definite volume and an aliquot portion taken for +the determination. In order to secure complete desiccation at a low +temperature, the drying should be accomplished in partial vacuum (pages +22, 23). The process of desiccation should be conducted in shallow, +flat-bottom dishes which may be conveniently and cheaply made of +aluminum and the process is hastened by filling the dish previously +with thoroughly dried fragments of pumice stone. When the sample does +not contain any invert sugar the desiccation can be safely accomplished +at the temperature of boiling water. Drying should be continued in all +cases until practically constant weight is obtained. + +=231. Determination Of Ash.=—Ash is an important constituent of the +sirups, molasses, and massecuites from canes and exists in very much +larger quantities in the same products from beets. The ash may be +determined directly by careful incineration, but it is customary to add +a few drops of sulfuric acid, sufficient to combine with all the bases +present and be in slight excess. The presence of sulfuric acid is of +some advantage in the beginning of the carbonization and renders the +process somewhat easier of accomplishment. When sulfuric acid is used, +the weight of ash obtained must be diminished by one-tenth to allow for +the increased weight obtained by the conversion of the carbonates into +sulfates. In general, the principles and methods described on pages +36-40 are to be employed. + +=232. Determination of Reducing Sugars in Sirups, Molasses, and +Massecuites.=—The quantity of reducing sugars in the products derived +from the sugar beet, as a rule, is insignificant. In the products from +sugar cane there are large quantities of reducing matters which, in +general, are determined by any of the standard methods already given. +It has been shown by the author[187] that the juices of healthy sugar +canes contain a small quantity of invert sugar, but this statement has +been contradicted by Bloufret.[188] It is certain, however, that the +reducing bodies derived from the products of manufacture of sugar cane +and sorghum deport themselves in a manner somewhat different from pure +invert sugar. In the absence of definite information in respect of the +constitution of these bodies, the methods applicable to dextrose and +invert sugar may be applied. + +Since the paragraphs relating to these processes were printed some +important improvements in the preparation of the alkaline copper +solutions have been made. The copper carbonate solution, as has already +been said, is peculiarly suited to the determination of reducing sugars +in the presence of sucrose and the modified forms of this solution, +and the methods of employing them with invert sugar, dextrose, +levulose, and maltose, are described below. + +=233. Estimation of Minute Quantities of Invert Sugar in Mixtures.=—The +method of Hiller and Meissl, paragraph =142=, may be used for the +estimation of small quantities of invert sugar in mixtures. A modified +form of Soldaini’s reagent is, however, to be preferred for this +purpose. Ost has proposed and tested a copper carbonate solution for +the purpose mentioned which gives reliable results.[189] The solution +has the following composition: + + One liter contains 3.6 grams crystallized copper sulfate. + 250.0 ” potassium carbonate. + 100.0 ” hydrogen potassium sulfate. + +This reagent undergoes no change when kept for a long while, especially +in large vessels. Even in smaller vessels it can be kept for a year or +more without undergoing any change. + +The method of analysis is the same as that described in paragraph +=128=, with the exception that the boiling is continued for only five +minutes instead of ten, and the quantities of the copper and sugar +solutions used are doubled, being 100 and fifty cubic centimeters +respectively. In no case must the solution used contain more than +thirty-eight milligrams of invert sugar. The quantity of sucrose in the +mixture is obtained by polarization (=94=). Ost has also recalculated +the reduction values of the common sugars for the strong copper +carbonate solution, and the numbers obtained are slightly different +from those given on page 142.[190] + +For different percentages of invert sugar in mixtures of sucrose, the +quantities of invert sugar are calculated from the number of milligrams +of copper obtained by the following table: + + (A) = Milligrams of copper obtained. + (B) = Pure invert sugar. + (C) = Invert sugar. + (D) = Sucrose. + + MILLIGRAMS OF INVERT SUGAR IN MIXTURES OF + 5(C) 2(C) 1.5(C) 1.0(C) 0.8 (C) 0.6(C) 0.5(C) + (A) (B) 95(D) 98(D) 98.5(D) 99.0(D) 99.2 (D) 99.4(D) 99.5(D) + 88 37.9 37.1 36.0 35.4 34.7 34.2 33.9 33.6 + 85 36.3 35.5 34.5 34.0 33.4 32.9 32.5 32.2 + 80 33.9 33.0 33.2 31.7 31.2 30.7 30.2 29.9 + 75 31.6 30.7 30.0 29.5 29.0 28.5 28.1 27.7 + 70 29.4 28.5 27.8 27.4 26.8 26.4 25.9 25.6 + 65 27.3 26.3 25.7 25.3 24.7 24.3 23.8 23.5 + 60 25.2 24.2 23.6 23.2 22.6 22.2 21.8 21.5 + 55 23.1 22.1 21.6 21.2 20.6 20.2 19.8 19.6 + 50 21.2 20.1 19.6 19.2 18.6 18.3 17.9 17.7 + 45 19.3 18.2 17.6 17.2 16.7 16.3 16.0 15.8 + 40 17.3 16.3 15.7 15.3 14.8 14.5 14.2 14.0 + 35 15.4 14.5 13.8 13.4 13.0 12.7 12.5 12.3 + 30 13.5 12.6 12.0 11.6 11.2 11.0 10.8 10.6 + 25 11.5 10.8 10.3 10.0 9.5 9.3 9.1 9.0 + 20 9.6 9.1 8.6 8.3 7.9 7.7 7.5 7.3 + 15 7.7 7.3 6.9 6.7 6.3 6.1 5.8 5.6 + 10 5.8 5.4 5.1 5.0 4.7 4.5 4.2 3.9 + + MILLIGRAMS OF INVERT SUGAR IN MIXTURES OF + 0.4(C) 0.3(C) 0.2(C) 0.1(C) 0.05(C) 0.02(C) + (A) 99.6(D) 99.7(D) 99.8(D) 99.9(D) 99.95(D) 99.98(D) + 88 33.3 + 85 32.0 31.8 + 80 29.7 29.5 + 75 27.4 27.2 + 70 25.3 25.0 + 65 23.2 22.8 + 60 21.2 20.8 20.4 + 55 19.3 18.9 18.5 + 50 17.4 17.0 16.7 + 45 15.6 15.3 14.9 + 40 13.8 13.5 13.2 + 35 12.1 11.9 11.5 10.3 + 30 10.4 10.2 9.9 8.8 + 25 8.8 8.6 8.2 7.3 + 20 7.1 6.9 6.6 5.8 4.9 + 15 5.4 5.2 5.0 4.4 3.7 2.0 + 10 3.8 3.5 3.4 3.0 2.5 1.7 + +=234. Soldaini’s Method Adapted to Gravimetric Work.=—By reason of +their better keeping qualities and because of their less energetic +action on non-reducing sugars, copper carbonate solutions are to be +preferred to the alkaline copper tartrate solutions for gravimetric +determinations of reducing sugars in cane juices and sugar house +products, provided the difficulties which attend the manipulation +can be removed. Ost has succeeded in securing perfectly satisfactory +results with copper carbonate solution by slightly varying the +composition thereof and continuing the boiling, for the reduction of +the copper, ten minutes.[191] The copper solution is made as follows: + + 17.5 grams crystallized copper sulfate. + 250.0 ” potassium carbonate. + 100.0 ” ” bicarbonate. + +The above ingredients are dissolved in water and the volume of +the solution completed to one liter. The object of the potassium +bicarbonate is to secure in the solution an excess of carbon dioxid +and thus prevent the deposition of basic copper carbonate on keeping. +The manipulation is conducted as follows: + +One hundred cubic centimeters of the copper solution are mixed with +half that quantity of the sugar solution in a large erlenmeyer, which +is placed upon a wire gauze, heated quickly to boiling and kept in +ebullition just ten minutes. The sugar solution should contain not +less than eighty nor more than 150 milligrams of the reducing sugar, +and the quantity of the solution representing this should be diluted +to fifty cubic centimeters before mixing with the copper solution. +After boiling, the contents of the erlenmeyer are quickly cooled and +filtered with suction through an asbestos filter and the whole of the +copper suboxid washed into the filter tube. This precipitated suboxid +is washed once with a little potassium carbonate solution then with hot +water and finally with alcohol, well dried, heated to redness, and the +copper oxid obtained reduced to metallic copper in an atmosphere of +hydrogen entirely free of arsenic. From the weight of metallic copper +obtained the quantity of sugar which has been oxidized is calculated +from the tables below. + +It is evident that the process given above may be varied so as to +conform to the practice observed in this laboratory of cooling the +boiling solution sufficiently at once by adding to it an equal volume +of recently boiled, cold water, collecting the precipitated copper +suboxid in a gooch, and, after washing it, securing solution in nitric +acid and the precipitation of the copper by electrolysis. + + TABLE SHOWING MILLIGRAMS DEXTROSE, + LEVULOSE AND INVERT SUGAR OXIDIZED, + CORRESPONDING TO MILLIGRAMS OF + COPPER REDUCED. + + Copper. Dextrose. Levulose. Invert. + + 435 152.3 145.9 147.5 + 430 149.8 143.4 145.3 + 425 147.3 140.9 143.1 + 420 144.8 138.4 140.8 + 415 142.3 135.9 138.5 + 410 139.8 133.5 136.2 + 405 137.3 131.1 133.9 + 400 134.9 128.7 131.6 + 395 132.5 126.4 129.3 + 390 130.1 124.1 127.0 + 385 127.8 121.8 124.8 + 380 125.5 119.5 122.6 + 375 123.3 117.2 120.4 + 370 121.1 115.0 118.2 + 365 119.0 112.8 116.0 + 360 116.9 110.6 113.9 + 355 114.8 108.5 111.8 + 350 112.8 106.4 109.8 + 345 110.8 104.3 107.8 + 340 108.8 102.3 105.8 + 335 106.8 100.3 103.8 + 330 104.9 98.4 101.8 + 325 103.0 96.5 99.9 + 320 101.1 94.6 98.0 + 315 99.2 92.8 96.2 + 310 97.4 91.0 94.4 + 305 95.6 89.2 92.6 + 300 93.8 87.5 90.9 + 295 92.0 85.8 89.2 + 290 90.2 84.1 87.5 + 285 88.4 82.4 85.8 + 280 86.7 80.8 84.1 + 275 85.0 79.2 82.4 + 270 83.3 77.6 80.7 + 265 81.5 76.1 79.1 + 260 79.8 74.6 77.5 + 255 78.1 73.1 75.9 + 250 76.5 71.6 74.3 + 245 74.9 70.1 72.7 + 240 73.3 68.6 71.1 + 235 71.7 67.2 69.5 + 230 70.1 65.7 68.0 + 225 68.5 64.3 66.5 + 220 66.9 62.8 65.0 + 215 65.3 61.4 63.5 + 210 63.8 59.9 62.0 + 205 62.2 58.5 60.5 + 200 60.7 57.0 59.0 + 195 59.1 55.6 57.5 + 190 57.6 54.1 56.0 + 185 56.0 52.7 54.5 + 180 54.5 51.2 53.1 + 175 53.0 49.8 51.6 + 170 51.5 48.4 50.2 + 165 50.0 46.9 48.7 + 160 48.5 45.5 47.3 + 155 47.0 44.1 45.8 + 150 45.5 42.7 44.4 + 145 44.0 41.3 42.9 + 140 42.5 39.9 41.5 + 135 41.0 38.5 40.1 + 130 39.6 37.1 38.6 + 125 38.1 35.7 37.2 + 120 36.7 34.3 35.8 + 115 35.2 32.9 34.3 + 110 33.7 31.6 32.9 + 105 32.2 30.3 31.4 + 100 30.7 29.0 30.0 + 95 29.2 27.7 28.5 + 90 27.8 26.4 27.1 + 85 26.3 25.1 25.6 + 80 24.8 23.8 24.2 + 75 23.3 21.5 22.8 + 70 21.8 20.2 21.4 + + CORRESPONDING TABLE FOR MALTOSE. + + Milligrams Milligrams + Milligrams maltose maltose + copper anhydrid hydrate + obtained. oxidized. oxidized. + + 435 263.7 277.6 + 430 259.3 273.0 + 425 255.0 268.4 + 420 250.9 264.1 + 415 247.0 260.0 + 410 243.2 256.0 + 405 339.4 252.0 + 400 235.6 248.0 + 395 231.9 244.1 + 390 228.2 240.2 + 385 224.6 236.4 + 380 221.1 232.7 + 375 217.7 229.1 + 370 214.4 225.6 + 365 211.1 222.2 + 360 207.9 218.8 + 355 204.7 215.4 + 350 201.5 212.1 + 345 198.3 208.7 + 340 195.2 205.4 + 335 192.0 202.1 + 330 188.8 198.8 + 325 185.7 195.4 + 320 182.5 192.1 + 315 179.4 188.8 + 310 176.3 185.6 + 305 173.3 182.4 + 300 170.3 179.2 + 295 167.3 176.1 + 290 164.4 173.0 + 285 161.4 169.9 + 280 158.5 166.8 + 275 155.5 163.7 + 270 152.6 160.7 + 265 149.7 157.6 + 260 146.8 154.6 + 255 143.9 151.5 + 250 141.1 148.5 + 245 138.2 145.5 + 240 135.4 142.5 + 235 132.5 139.5 + 230 129.7 136.5 + 225 126.8 133.5 + 220 124.0 130.6 + 215 121.2 127.6 + 210 118.4 124.7 + 205 115.7 121.8 + 200 112.9 118.9 + 195 110.2 116.0 + 190 107.4 113.1 + 185 104.7 110.2 + 180 101.9 107.3 + 175 99.2 104.4 + 170 96.4 101.5 + 165 93.7 98.6 + 160 90.9 95.7 + 155 88.2 92.8 + 150 85.4 89.9 + 145 82.6 87.0 + 140 79.9 84.1 + 135 77.1 81.2 + 130 74.4 78.3 + 125 71.6 75.4 + 120 68.9 72.5 + 115 66.1 69.6 + 110 63.4 66.7 + 105 60.6 63.8 + 100 57.9 60.9 + 95 55.1 58.0 + 90 52.3 55.1 + 85 49.6 52.2 + 80 46.8 59.3 + 75 44.1 56.4 + 70 41.4 53.5 + +=235. Weighing the Copper as Oxid.=—In the usual methods of the +determination of reducing bodies, the percentage is calculated either +volumetrically from the quantity of the sugar solution required to +decolorize a given volume of the alkaline copper solution, or the +reduced copper suboxid is brought into a metallic state by heating in +an atmosphere of hydrogen or by electrolytic deposition. A quicker +method of procedure is found in completing the oxidation of the cupric +oxid by heating to low redness in a current of air.[192] For this +determination the precipitation of the cuprous oxid and its filtration +are made in the usual manner. The cuprous oxid is collected in a +filtering tube, made by drawing out to proper dimensions a piece of +combustion tube, and has a length of about twelve centimeters in all. +The unchanged part of the tube is about eight centimeters in length +and twelve millimeters in diameter. It is filled by first putting in +a plug of glass wool and covering this with an asbestos felt on top +of which another plug of glass wool is placed. After the cuprous oxid +is collected in the tube it is washed with boiling water, alcohol and +ether. The rubber tube connecting it with the suction is of sufficient +length to permit the tube being taken in one hand and brought into +a horizontal position over a bunsen. The tube is gradually heated, +rotating it meanwhile, until any residual moisture, alcohol or ether, +is driven off from the filtering material. The layer of glass wool +holding the cuprous oxid is gradually brought into the flame and as +the oxidation begins the material will be seen to glow. The heating is +continued for some time after the glowing has ceased, in all for three +or four minutes, the tube and the copper oxid which it contains being +brought to a low redness. The current of air passing over the red-hot +material in this time oxidizes it completely. The filtering tube, +before use, must be ignited and weighed in exactly the same manner as +described above. The heat is so applied as not to endanger the rubber +tube attached to one end of the filtering tube nor to burn the fingers +of the operator as he turns the tube during the heating. After complete +oxidation the tube is cooled in a desiccator and weighed, the increase +of weight giving the copper oxid. For the atomic weights, 63.3 copper +and 15.96 oxygen, one gram of copper oxid is equivalent to 0.79864 +gram of copper, and for the weights 63.17 copper and 15.96 oxygen, one +gram of copper oxid equals 0.79831 gram of copper. From the amount of +metallic copper calculated by one of these factors, the reducing sugar +is determined by the tables already given. + +=236. Estimation of Dry Substance, Polarization and Apparent Purity for +Factory Control.=—For technical purposes the methods of determining +the above factors, proposed by Weisberg and applicable to concentrated +sirups, massecuites, and molasses, may be used.[193] Five times the half +normal quantity of the material, _viz._, 65.12 grams, are placed in a +quarter liter flask, dissolved in water and the flask filled to the +mark. In the well shaken mixture, which is allowed to stand long enough +to be free of air, the degree brix is estimated by an accurate spindle. +For example, in the case of molasses, let the number obtained be 18.8. + +Fifty cubic centimeters of the solution are poured into a 100 cubic +centimeter flask, the proper quantity of lead subacetate added, the +flask filled to the mark with water, its contents filtered, and the +filtrate polarized in a 200 millimeter tube. Let the number obtained +on polarization be 22°.1. This number may be used in two ways. If +it be multiplied by two the polarization of the original sample is +obtained; in this case, _viz._, 44°.2. In the second place, if 44.2 be +multiplied by 0.26048 and this product divided by the specific gravity +corresponding to 18°.8. _viz._, 1.078, the quotient 10.68 is secured +representing the polarization or per cent of sugar contained in the +solution of which the degree brix was 18.8°. From the numbers 18.8 and +10.68 the apparent purity of the solution, 56.8, is calculated, _viz._, +10.68 × 100 ÷ by 18.8. The original product as calculated above gives a +polarization of 44.2 and this number multiplied by 100 and divided by +56.8 gives 77.8, or the apparent percentage of dry matter. The original +sample of molasses, therefore, had the following composition: + + Degree brix (total solids) 77.8 per cent. + Sucrose 44.2 ” + Solids, not sucrose 33.6 ” + Apparent purity 56.8 ” + +It is seen from the above that with a single weighing and a single +polarization, and within from ten to fifteen minutes, all needful +data in respect of the proper treatment of molasses for the practical +control and direction of a factory can be obtained. + +In case a laurent polariscope is used, five times the normal weight, +_viz._, eighty-one grams of the raw material are used and the process +conducted as above. + + +SUCROSE, DEXTROSE, INVERT SUGAR, LEVULOSE, MALTOSE, RAFFINOSE, DEXTRIN +AND LACTOSE IN MIXTURES. + +=237. Occurrence.=—Sucrose and invert sugar are found together in many +commercial products, especially in raw sugars and molasses made from +sugar cane, and in these products sucrose is usually predominant. They +also form the principal saccharine contents of honey, the invert sugar, +in this case, being the chief ingredient. + +In commercial grape sugar, made from starch, dextrose is the important +constituent, while in the hydrolysis of starch by a diastatic ferment, +maltose is principally produced. In the manufacture of commercial +glucose by the saccharification of starch with sulfuric acid, dextrin, +maltose, and dextrose are the dominant products, while in the similar +substance midzu ame, maltose and dextrose are chiefly found, and only a +small quantity of dextrose.[194] In honeys derived from the exudations +of coniferous trees are found also polarizing bodies not enumerated +above and presumably of a pentose character.[195] In evaporated milks +are usually found large quantities of sucrose in addition to the +natural sugar therein contained. These mixtures of carbohydrates often +present problems of great difficulty to the analyst, and the following +paragraphs will be devoted to an elucidation of the best approved +methods of solving them. + + +OPTICAL METHODS. + +=238. Sucrose and Invert Sugar.=—The chemical methods of procedure to +be followed in the case of a sample containing both sucrose and invert +sugar have been given in sufficient detail in preceding paragraphs +(=124, 171=). When, however, it is desirable to study further the +composition of the mixture, important changes in the method are +rendered imperative. While the estimation of the sucrose and the total +invert sugar, or the sum of the dextrose and levulose, is easy of +accomplishment the separate determination of the dextrose and levulose +is not so readily secured. In the latter case the total quantity of the +two sugars may be determined, and after the destruction or removal of +one of them the other be estimated in the usual way; or in the mixture +the levulose can be determined by the variation in its gyrodynat, +caused by changes of temperature. + +=239. Optical Neutrality of Invert Sugar.=—The gyrodynat of levulose +decreases as the temperature rises (=107=) and at or near a temperature +of 87°.2, it becomes equal to that of dextrose, and, therefore, pure +invert sugar composed of equal molecules of levulose and dextrose is +optically neutral to polarized light at that temperature. On this fact +Chandler and Ricketts have based a method of analysis which excludes +any interference in polarization due to invert sugar.[196] To secure +the polarization at approximately a temperature of 87°, a water-bath +is placed between the nicols of an ordinary polariscope in such a way +as to hold a tubulated observation tube in the optical axis of the +instrument. The ends of the bath, in the prolongation of this axis, are +provided with clear glass disks. The space between the cover glasses +of the observation tube and the glass disks of the bath is occupied by +the water of the bath. When this is kept at a constant temperature it +does not interfere with the reading. The observation tube may be of +glass, but preferably is constructed of metal plated with platinum on +the inside. For the most exact work the length of the observation tube, +at 87°, is determined by measurement or calculation. The bath is heated +with alcohol lamps or other convenient means. The arrangement of the +apparatus is shown in Fig. 75. + +In a mixture of sucrose and invert sugar any rotation of the plane +of polarized light at 87° is due to the sucrose alone. In a mixture +of dextrose and sucrose the polarization is determined, and, after +inversion, again determined at 87°. The latter number is due to +dextrose alone, and the difference between the two gives the rotation +due to sucrose. + +[Illustration: FIG. 75.—CHANDLER AND RICKETTS’ POLARISCOPE.] + +=240. Sucrose and Raffinose.=—In raw sugars made from beet molasses +considerable quantities of raffinose are found. The method of inversion +and polarization in such cases is described in paragraph =100=. In +making the inversion by the method proposed by Lindet (=95=), and +conducting the polarization on a laurent instrument, a slightly +different formula, given below, is used; _viz._: + + _C_ - 0.4891_A_ + _S_ = --------------- + 0.810 + + _A_ - _S_ + and _R_ = ----------, + 1.54 + + + +in which the several letters refer to the same factors as are indicated +by them in the formula of Creydt. In the application of the formula +just given the normal weight of the mixed raw sugars used is 16.2 +grams.[197] + +=241. Optical Determination of Levulose.=—The determination of levulose +by optical methods alone is made possible by reason of the fact that +the gyrodynats of the sugars with which it is associated are not +sensibly affected by changes of temperature. The principle of the +process, as developed by the author, rests on the ascertainment of the +change in the gyrodynat of levulose when its rotation is observed at +widely separated temperatures.[198] The observation tube employed for +reading at low temperatures is provided with desiccating end tubes, +which prevent the deposition of moisture on the cover glasses. The +relations of this device to the optical parts of the apparatus are +illustrated in Fig. 76. + +[Illustration: FIG. 76.—APPARATUS FOR POLARIMETRIC OBSERVATIONS AT LOW +TEMPERATURES.] + +The protecting tubes are made of hard rubber and the desiccation is +secured by surrounding the space between the rubber and the perforated +metal axis with fragments of potash or calcium chlorid. + +The details of the construction are shown in a horizontal section +through the center of the observation tube in Fig. 77. In this figure +the observation tube, made of glass or metal, is represented by _i_, +the metal jacket, open at the top in the =V= shape as described, by +_k_. The observation tube is closed by the heavy disk _b_ made of +non-polarizing glass. This disk is pressed against the end of the +observation tube by the rubber washer _a_, when the drying system about +to be described is screwed on to _k_. The apparatus for keeping the +cover glass dry is contained in the hard rubber tube _m_ and consists +of a perforated cylinder of brass _e_, supported at one end by the +perforated disk _c_ and at the outer ends by the arms _d_. It is closed +by a cover glass of non-polarizing glass _s_ and can be screwed on to +the system _h_ at _n_. The space _p_ is filled with coarse fragments +of caustic soda, potash, or calcium chlorid by removing the cover +glass _s_. The perforated disk _c_ prevents any of the fragments from +entering the axis of observation. When the cover glass _s_ is replaced, +it just touches the free end of the perforated metal tube preventing +any of the fragments of the drying material from falling into the +center at the outer end. When this drying tube is placed in position, +the contents of the observation tube _i_ can be kept at the temperature +of zero for an indefinite time without the deposition of a particle of +moisture either upon the glass _b_ or _s_. + +[Illustration: FIG. 77.—CONSTRUCTION OF DESICATING TUBE.] + +For determining the rotation at a high temperature the apparatus of +Chandler and Ricketts (=238=) may be used or the following device: +The polarizing apparatus shown above, Fig. 76, may be used after the +=V= shape box is removed from the stand, which is so constructed as +to receive a large box covered with asbestos felt an inch thick. The +observation tube is held within this box in the same way as in the one +just described so that the hot water extends not only the entire length +of the tube but also covers the cover glasses. In both cases the cover +glasses are made of heavier glass and are much larger in diameter than +found in the ordinary tubes for polariscopes. The protecting cylinders +of hard rubber are not needed at high temperatures but can be left on +without detriment. + +The illustration, Fig. 78, shows the arrangement of the apparatus with +a silver tube in position, which can be filled and emptied without +removing it. + +[Illustration: FIG. 78.—APPARATUS FOR POLARIZING AT HIGH TEMPERATURES.] + +In practice the water is heated with a jet of steam and an even +temperature is secured by a mechanical stirrer kept slowly in motion. +With such a box it is easy to maintain a temperature for several +hours which will not vary more than half a degree. The temperature +for reading the hot solutions was fixed at 88°, this being nearly the +temperature at which a mixture of equal molecules of levulose and +dextrose is optically inactive. In every case the sugar solutions were +made up to the standard volume at the temperatures at which they were +to be read and thus the variations due to expansion or contraction +were avoided. When solutions are read at a high temperature, they must +be made with freshly boiled water so as to avoid the evolution of air +bubbles which may otherwise obscure the field of vision. + +By means of the apparatus described it is easy for the analyst to make +a polarimetric reading at any temperature desired. In all cases the +observation tube should be left at least a half an hour and sometimes +longer in contact with the temperature control media before the reading +is made. + +The appearance of the field of vision is usually a pretty fair index +of the point of time at which a constant temperature is established +throughout all parts of the system. Any variation in temperature +produces a distortion of the field of vision while a constant fixed +temperature will disclose the field of vision in its true shape and +distinctness of outline. + +_Principles of the Calculation._—If 26.048 grams of pure sucrose be +dissolved in water and the volume made 100 cubic centimeters, it will +produce an angular rotation of 34°.68 when examined in a 200 millimeter +tube with polarized sodium monochromatic light. Upon the cane sugar +scale of an accurately graduated shadow instrument the reading will be +100 divisions corresponding to 100 per cent of pure sucrose. + +In the complete inversion of the cane sugar the reaction which takes +place is represented by the following formula: + + — + + C₁₂H₂₂O₁₁ + H₂0 = C₆H₁₂O₆ + C₆H₁₂O₆. + +The minus and plus signs indicate that the resulting invert sugar is +a mixture of equal parts of levulose (_d_ fructose) and dextrose (_d_ +glucose). We are not concerned here with the fact that a complete +inversion of cane sugar is a matter of great difficulty nor with the +danger which is always experienced of destroying a part of one of the +products of inversion. They are matters which may cause a variation +in the analytical data afterward, but do not affect the principles on +which the process is based. + +In the inversion of 26.048 grams of cane sugar there are therefore +produced 13.71 grams of levulose and 13.71 grams of dextrose or, in +all, 27.42 grams of the mixed sugars. + +The angular rotation which would be produced by 13.71 grams of +dextrose in a volume of 100 cubic centimeters and through a column 200 +millimeters in length is, with sodium light, 14°.53 equivalent to 41.89 +divisions of the cane sugar scale. The specific rotatory power of a +dextrose solution of the density given is almost exactly 53, and this +number is used in the calculations. + +In a mixture of the two sugars under the conditions mentioned and at a +temperature of 0° the angular rotation observed is -15°.15 equivalent +to 43.37 divisions of the cane sugar scale. + +The + rotation due to the dextrose is 14°.53. Therefore the total +negative rotation due to levulose at 0° is 15°.15 + 14°.53 = +29°.68. Hence the gyrodynat of levulose at 0° and in the degree of +concentration noted is readily calculated from the formula + + 29.68 × 100 + [α]°_{D} = - ------------ = -108.24. + 2 × 13.71 + +Since at 88° (_circa_) the mixture of levulose and dextrose is neutral +to polarized light, it follows that at that temperature the specific +rotatory power of levulose is equal to that of dextrose, _viz._, 53°. + + [α]⁸⁸ °_{D} = - 53°. + +The total variation in the specific rotatory power of levulose, between +zero and 88°, is 55°.24. The variation for each degree of temperature, +therefore, of the specific rotatory power of levulose is equal to +55.24 divided by 88, which is equal to 0.628. From these data it is +easy to calculate the specific rotatory power of levulose for any +given temperature. For instance, let it be required to determine the +gyrodynat of levulose at a temperature of 20°. It will be found equal +to 108.24 - 0.628 × 20 = 95.68. The required rotatory power is then +[_a_]²⁰ °_{D} = -95°.68. + +In these calculations the influence of the presence of hydrochloric +acid upon the rotatory power of the levulose is neglected. + +Since the variation in angular rotation in the mixture at different +temperatures is due almost wholly to the change in this property of the +levulose it follows that the variation for each degree of temperature +and each per cent of levulose can be calculated. Careful experiments +have shown that the variation in the rotatory power of levulose between +0° and 88° is represented by a straight line. For 13.71 grams per +100 cubic centimeters the variation for each degree of temperature +is equal to 43.37 ÷ 88 = 0.49 divisions on the cane sugar scale, or +15.15 ÷ 88 = 0°.1722 angular measure. If 13.71 grams of levulose in +100 cubic centimeters produce the deviations mentioned for each degree +of temperature, one gram would give the deviation obtained by the +following calculations: + +For the cane sugar scale 0.49 ÷ 13.71 = 0°.0357 and for angular +rotation 0.1722 ÷ 13.71 = 0.01256. + +The above data afford a simple formula for calculating the percentage +of levulose present from the variation observed in polarizing a +solution containing levulose, provided that the quantity of levulose +present is approximately fourteen grams per 100 cubic centimeters. + +_Example._—Suppose in a given case the difference of reading between +a solution containing an unknown quantity of levulose at 0° and 88° +is equal to thirty divisions of the cane sugar scale. What weight of +levulose is present? We have already seen that one gram in 100 cubic +centimeters produces a variation of 0.0357 division for 1°. For 88° +this would amount to 3.1416 divisions. The total weight of levulose +present is therefore 30 ÷ 3.1416 = 9.549 grams. In the case given +26.048 grams of honey were taken for the examination. The percentage of +levulose was therefore 9.549 × 100 ÷ 26.048 = 36.66 per cent. + +If it be inconvenient to determine the polarimetric observations at +temperatures so widely separated as 0° and 88° the interval may be +made less. In the above case if the readings had been made at 20° and +70° the total variation would have been only ⁵⁰/⁸⁸ of the one given, +_viz._, 17.05 divisions of the cane sugar scale. The calculation would +then have proceeded as follows: + +0.0357 × 50 = 1.785. + +Then, 17.05 ÷ 1.785 = 9.552 grams of levulose, from which the actual +percentage of levulose can be calculated as above. + +With honeys the operation is to be conducted as follows: + +Since honeys contain approximately twenty per cent of water and in the +dry substance have approximately forty-five per cent of levulose, about +38.50 grams of the honey should be taken to get approximately 13.8 +grams of levulose. + +In the actual determination the calculations may be based on +the factors above noted, but without respect to the degree of +concentration. If half the quantity of dextrose noted be present its +specific rotatory power is only reduced to about 52°.75, and this will +make but little difference in the results. In the case of honey 13.024 +grams of the sample are conveniently used in the examination, half the +normal weight for the ventzke sugar scale. The error, however, due to +difference in concentration is quite compensated for by the ease of +clarification and manipulation. Alumina cream alone is used in the +clarification, thus avoiding the danger of heating the solution to a +high temperature in the presence of an excess of lead acetate. + +An interesting fact is observed in cooling solutions of honey to +0°. The maximum left hand rotation is not reached as soon as the +temperature reaches 0° but only after it has been kept at that +temperature for two or three hours. The line representing the change in +rotatory power in solutions of honey between 10° and 88° is practically +straight but from 10° to 0°, if measured by the readings taken without +delay, it is decidedly curved; the reading being less at first than +it is afterwards. After three hours the 0° becomes sensibly constant +and then the whole line is nearly straight, but still with a slight +deficiency in the reading at the 0°. For this reason the computations +should be based on readings between 10° and 88° rather than on a +number covering the whole range of temperature. Nevertheless, if the +solution be kept at 10° for three hours before the final reading is +taken, no error of any practical magnitude is introduced. + +The calculations given above, for the cane sugar scale, can also be +made in an exactly similar manner for angular rotation. The angular +variation produced by one gram of levulose for 1° of temperature +is 0°.01256. For 88° this would become 1°.10528. Suppose the total +observed angular deviation in a given case between 0° and 88° to be +10°.404, then the weight of levulose present is 10.404 ÷ 1.10528 = +9.413 grams. + +In the case mentioned 26.048 grams of honey were taken for the +examination. The percentage of levulose present, therefore, was 9.413 × +100 ÷ 26.048 = 36.13. + +=241. General Formula for the Calculation of Percentage of +Levulose.=—Let _K_ = deviation in divisions of the cane sugar scale or +in angular rotation produced by one gram of levulose for 1° temperature. + +Let _T_ and _tʹ_ = temperatures at which observations are made. + +Let _R_ = observed deviation in rotation. + +Let _W_ = weight of levulose obtained. + +Let _L_ = per cent of levulose required. + + _R_ + Then _L_ = --------------- ÷ _W_. + _K_(_T_ - _tʹ_) + +In most genuine honeys the value of _R_ between 0° and 88° is +approximately thirty divisions of the cane sugar scale or 10° angular +measure for 26.048 grams in 100 cubic centimeters, read in a 200 +millimeter tube, or, for 13.024 grams in 100 cubic centimeters read in +a 400 millimeter tube. + +The method of analysis outlined above has been applied in the +examination of a large number of honeys with most satisfactory results. +It can also be applied with equal facility to other substances +containing levulose. + +=242. Sucrose and Dextrose.=—In mixtures these two sugars are easily +determined by optical processes, provided no other bodies sensibly +affecting the plane of polarized light be present. The total deviation +due to both sugars is determined in the usual way. The percentage +of sucrose is afterwards found by the inversion method (=92=). The +rotation, in the first instance due to the sucrose, is calculated from +the amount of this body found by inversion, and the residual rotation +is caused by the dextrose. The percentage of dextrose is easily +calculated by a simple proportion into which the numbers expressing the +gyrodynats of sucrose and dextrose enter. When the readings are made on +a ventzke scale the calculations are made as follows: + + Weight of sample used 26.048 grams. + First polarization 88°.5 + Polarization after inversion 10°.5 + Temperature 20°.0 + Percentage of sucrose 58.4 + Rotation due to dextrose 30°.1 + +Percentage of dextrose: + + 66.5 : 53 = _x_ : 30.1; whence _x_ = 37.8. + +The sample examined therefore contains 58.4 per cent of sucrose and +37.8 per cent of dextrose. + +It is evident that the method just described is also applicable when +maltose, dextrin, or any other sugar or polarizing body, not sensibly +affected by the process of inversion to which the sucrose is subjected, +is substituted for dextrose. When, however, more than two optically +active bodies are present the purely polariscopic process is not +applicable. In such cases the chemical or the combined chemical and +optical methods described further on can be employed. + +=243. Lactose in Milk.=—By reason of its definite gyrodynat lactose +in milk is quickly and accurately determined by optical methods, when +proper clarifying reagents are used to free the fluid of fat and +nitrogenous substances. Soluble albuminoids have definite levogyratory +powers and, if not entirely removed, serve to diminish the rotation due +to the lactose. + +Milk casein precipitated by magnesium sulfate has the following +gyrodynatic numbers assigned to it:[199] + + Dissolved in water [_a_]_{D} = -80° + ” ” very dilute solution [_a_]_{D} = -87°. + ” ” dilute sodium hydroxid solution [_a_]_{D} = -76°. + ” ” strong potassium hydroxid solution [_a_]_{D} = -91°. + +The hydrates of albumen have rotation powers which vary from [_a_]_{D} += -71°.40 to [_a_]_{D} = -79°-05. From the chaotic state of knowledge +concerning the specific rotating power of the various albumens, it is +impossible to assign any number which will bear the test of criticism. +For the present, however, this number may be fixed at [_a_]_{D} = -70° +for the albumens which remain in solution in the liquids polarized for +milk sugar.[200] + +Many reagents have been prepared for the removal of the disturbing +bodies from milk in order to make its polarization possible. Among +the precipitants which have been used in this laboratory may be +mentioned:[201] + +(1) Saturated solution basic lead acetate, specific gravity 1.97: + +(2) Nitric acid solution of mercuric nitrate diluted with an equal +volume of water: (=88.=) + +(3) Acetic acid, specific gravity 1.040, containing twenty-nine per +cent acetic acid: + +(4) Nitric acid, specific gravity 1.197, containing thirty per cent +nitric acid: + +(5) Sulfuric acid, specific gravity 1.255, containing thirty-one per +cent sulfuric acid: + +(6) Saturated solution of sodium chlorid: + +(7) Saturated solution of magnesium sulfate: + +(8) Solution of mercuric iodid in acetic acid, formula; potassium +iodid, 33.2 grams; mercuric chlorid, 13.5 grams; strong acetic acid, +20.0 cubic centimeters; water 640 cubic centimeters. + +Alcohol, ether, and many solutions of mineral salts, hydrochloric and +other acids are also used as precipitants for albumen, but none of them +presents any advantages. + +Experience has shown that the best results in polariscopic work are +secured by the use of either the mercuric iodid or the acid mercuric +nitrate for clarifying the milk. The latter reagent should be used +in quantities of about three cubic centimeters for each 100 of milk. +It is evident when it is desired to determine the residual nitrogen +in solution, the former reagent must be employed. The quantity of +albuminoid matter left in solution after clarification with mercurial +salts is so minute as to exert no sensible effect on the rotation of +the plane of polarized light produced by the lactose. + +For purposes of calculation the gyrodynat of lactose in the ordinary +conditions of temperature and concentration may be represented by +[_a_]_{D} = 52°.5 (=107=). + +_Polarization._—The proper weight of milk is placed in a sugar flask, +diluted with water, clarified with the mercuric salt, the volume +completed to the mark, and the contents shaken and poured on a filter. +The filtrate is polarized in tubes of convenient length. The observed +rotation may be expressed either in degrees of angular measurement or +of the sugar scale. The weight of milk used may be two or three times +that of the normal weight calculated for the instrument employed. +Instead of weighing the milk a corresponding volume determined by its +specific gravity may be delivered from a burette-pipette (p. 231). +For the laurent polariscope three times, and for the half-shadow +instruments for lamplight, twice the normal weight of milk should be +used. For approximately sixty cubic centimeters of milk the flask +should be marked at 105 cubic centimeters in compensation for the +volume of precipitated solids or the reading obtained from a 100 cubic +centimeter flask, decreased by one-twentieth. + +For the laurent instrument the normal weight of lactose is determined +by the following proportions: + +Gyrodynat of sucrose, 66.5: lactose: 52.5 = _x_: 16.19. + +Whence _x_ = 20.51, that is, the number of grams of pure lactose in 100 +cubic centimeters required to read 100 divisions of the sugar scale of +the instrument. + +For the ventzke scale the normal quantity of lactose required to read +100 divisions is found from the following equation: + +66.4: 52.5 = _x_: 26.048 + +Whence _x_ = 32.74. + +In the one case three times the normal weight of milk is 61.53 and in +the other twice the normal weight, 65.48 grams. + +=244. Error due to Volume of Precipitate.=—Vieth states that the +volume allowed for the precipitated solids in the original process, +_viz._, two and four-tenths cubic centimeters, is not sufficiently +large.[202] In such cases it is quite difficult to decide on any +arbitrary correction based on the supposed quantities of fat and +albuminoids present. A better method than to try to compensate for +any arbitrary volume is to remove entirely the disturbing cause or +eliminate it by indirect means. To wash the precipitate free of sugar +without increasing the bulk of the filtrate unduly would be extremely +difficult and tend, moreover, to bring some of the precipitated matters +again into solution. It is better, therefore, to eliminate the error by +double dilution and polarization (=86=). The principle of this method +is based on the fact, that, within limits not sensibly affecting the +gyrodynat by reason of different densities, the polarizations of two +solutions of the same substance are inversely proportional to their +volumes. + +For convenience, it is recommended that the volumes of the samples in +each instance be 100 and 200 cubic centimeters, respectively, in which +case the true reading is obtained by the simple formula given in the +latter part of =86=. + +In this laboratory the double dilution method of determining the volume +of the precipitate is conducted as follows:[203] + +In each of two flasks marked at 100 and 200 cubic centimeters, +respectively, are placed 65.52 grams of milk, four cubic centimeters +of mercuric nitrate added, the volume completed to the mark and the +contents of the flask well shaken. + +After filtering, the polarization is made in a 400 millimeter tube +by means of the triple shadow polariscope described in =75=. From +the reading thus obtained the volume of the precipitate and the +degree of correction to be applied are calculated as in the subjoined +example. The flasks should be filled at near the temperature at which +the polarizations are made and the observation room must be kept at +practically a constant temperature of 20° to avoid the complications +which would be produced by changes in the gyrodynat of lactose and +the value of the quartz plates and wedges of the apparatus by marked +variations in temperature. + +_Example._—Weight of milk used in each case 65.52 grams. + + Polarimetric reading from the 100 cubic centimeter flask, 20°.84 + ” ” ” ” 200 ” ” ” 10°.15 + Then 10.15 × 2 = 20.30 + 20.84 - 20.30 = 0.54 + 0.54 × 2 = 1.08 + 20.84 - 1.08 = 19.76 + 19.76 ÷ 4 = 4.94, + +which is the corrected reading showing the percentage of lactose in the +sample used. + +The volume of the precipitate is calculated as follows: + +20.84 ÷ 4 = 5.21, the apparent percentage of lactose present. + +Then 5.21: 4.94 = 100: _x_. + +Whence _x_ = 94.82. From this number it is seen that the true volume of +the milk solution polarized is 94.82 instead of 100 cubic centimeters, +whence the volume occupied by the precipitate is 100 - 94.82 = 5.18 +cubic centimeters. So little time is required to conduct the analysis +by the double dilution method as to render it preferable in all cases +where incontestable data are desired. Where arbitrary corrections are +made the volume allowed for the precipitate may vary from two and a +half cubic centimeters in milks poor in fat, to six for those with a +high cream content. + +For milks of average composition sufficient accuracy is secured by +making an arbitrary correction of five cubic centimeters for the volume +of the precipitate. + + +SEPARATION OF SUGARS BY CHEMICAL AND CHEMICAL-OPTICAL METHODS. + +=245. Conditions of Separation.=—In the foregoing paragraphs the +optical methods for determining certain sugars have been described. +Many cases arise, however, in which these processes are inapplicable or +insufficient. In these instances, the analyst, as a rule, will be able +to solve the problem presented by the purely chemical methods which +have been previously described, or by a combination of the chemical +and optical processes. Not only have the different sugars distinctive +relations to polarized light, but also they are oxidized by varying +quantities of metallic salts and these differences are sufficiently +pronounced to secure in nearly every instance, no matter how complex, +data of a high degree of accuracy. + +The carbohydrates of chief importance, from an agricultural point of +view, are starch and sucrose; while the alternation products of chief +importance, derived therefrom by chemical and biological means, are +dextrin, maltose, dextrose and invert sugar. + +=246. Sucrose, Levulose, and Dextrose.=—The purely chemical methods of +separating these three sugars have been investigated by Wiechmann.[204] +They are based on the data obtained by determining the percentage of +reducing sugars, both before and after the inversion of the sucrose, +and before and after the removal of the levulose. For the destruction +of the levulose, the method of Sieben is employed, and attention is +called to the fact that the complete removal of the levulose by this +process is difficult of accomplishment, and is probably attended with +alterations of the other sugars present. + +=247. Sieben’s Method of Determining Levulose.=—The decomposing action +of hot hydrochloric acid on levulose, and its comparative inaction on +dextrose are the basis of Sieben’s process.[205] The hydrochloric acid +employed should contain about 220 grams of the pure gas per liter, that +is, be of twenty-two per cent strength, corresponding to 1.108 specific +gravity. If the substance acted on be invert sugar, its solution should +be approximately of two and a half per cent strength. To 100 cubic +centimeters of such a solution, sixty of the hydrochloric acid are +added, and the mixture immersed in boiling water for three hours. + +After quickly cooling, the acid is neutralized with sodium hydrate of +thirty-six times normal strength. Ten cubic centimeters of the hydrate +solution will thus neutralize the sixty of hydrochloric acid which have +been used to destroy the levulose. The work of Wiechmann discloses the +fact, easily prevised, that the method used for destroying levulose is +not always effective and that action of the reagent is not exclusively +confined to the levogyrate constituent of the mixture. Nevertheless, +data of reasonable accuracy may be secured by this process, which is +best carried out as described by Wiechmann. In this connection the +possibility of the polymerization of the dextrose molecules, when +heated with hydrochloric acid, must not be overlooked. + +=248. The Analytical Process.=—The total quantity of invert sugar in a +given solution is determined by the methods already given (=136, 141=.) + +After this has been accomplished, the levulose is destroyed as +described above, and the dextrose determined by any approved method +(=136, 140=). In the presence of sucrose, the sum of the reducing +sugars is first determined as in =136, 142=. After the inversion of +the sucrose, the invert sugar is again determined, and the increased +quantity found, calculated to sucrose. The levulose is then destroyed +by hydrochloric acid, and the dextrose determined as described above. +The quantity of sucrose may also be determined by an optical method +(=91, 92, 94=.). + +=249. Calculation of Results.=—If we represent by a the weight of +metallic copper reduced by the invert sugar present in a solution +containing sucrose, and by _b_ that obtained after the inversion of +the sucrose, the quantity of copper corresponding to the sucrose is _b +- a_ = _c_. After the destruction of the levulose, the copper reduced +by the residual dextrose may be represented by _d_. The weight of +copper equivalent to the levulose is, therefore, _b - d_ = _e_. From +the tables already given, the corresponding quantities of the sugars +equivalent to _c, d_, and _e_ are directly taken. Example: + + { 163.8 milligrams invert sugar. + _a_ = 300 milligrams = { 156.5 ” dextrose. + { 185.63 ” levulose. + + _b_ = 500 ” + + _d_ = 275 ” = 142.8 ” dextrose. + + _c_ = 200 ” = 106.3 ” invert sugar. + + _e_ = 225 ” = 133.89 ” levulose. + +The 106.3 milligrams of invert sugar equivalent to _c_, correspond to +101 milligrams of sucrose. The quantity of dextrose equivalent to 275 +milligrams of copper is 142.8. Of this amount 53.15 milligrams are +due to the inverted sucrose, leaving 89.65 milligrams arising from +the invert sugar and dextrose originally present. This quantity is +equivalent to 175 milligrams of copper. + +Of the 300 milligrams of copper obtained in the first instance, 125 +are due to levulose in the original sample, corresponding to 69.73 +milligrams which number, multiplied by two, gives the invert sugar +present. + +The sample examined, therefore, had the following composition: + + Sucrose 101.00 milligrams. + Invert sugar 139.46 ” + Dextrose 19.92 ” + ------ + Sum 260.38 ” + +On the other hand, if the invert sugar be calculated from the quantity +corresponding to the 225 milligrams of copper corresponding to _e_, the +data will be very different from those given above. In this instance of +the levulose found corresponding to 225 milligrams of copper, _viz._, +133.89, 53.15 milligrams are due to the inverted sucrose. Then the +quantity due to the invert sugar at first present is 133.89 - 53.15 = +80.74 milligrams. Since half the weight of invert sugar is levulose, +the total weight of the invert sugar at first present is 161.48, +leaving only 8.91 milligrams due to added dextrose. The difficulties in +these calculations doubtless arise from the imperfect destruction of +the levulose, and from variations in the reducing action of sugars on +copper salts in the presence of such large quantities of sodium chlorid. + +=250. Calculation from Data obtained with Copper Carbonate.=—The +wide variations observed in different methods of calculations in the +preceding paragraph, are due in part to the different degrees of +oxidation exerted on alkaline copper tartrate by the dextrose and +levulose. Better results are obtained by conducting the analytical work +with Ost’s modification of Soldaini’s solution (=128=). + +The relative quantities of levulose and dextrose oxidized by this +solution are almost identical, and the calculations, therefore, result +in nearly the same data, whether made from the numbers obtained with +the residual dextrose or from the levulose destroyed. The method of +applying this method is illustrated in the following calculation. + +_Example._—In a mixture of sucrose, invert sugar, and dextrose, the +quantities of copper obtained by using the copper carbonate solution +were as follows: + + Copper obtained before inversion = _a_ = 150 milligrams. + ” ” after ” = _b_ = 250 ” + ” ” ” destroying lev’e = _d_ = 137.5 ” + ” equivalent to inverted sucrose = _b_ - _a_ = _c_ = 100 ” + ” ” ” levulose = _b_ - _d_ = _e_ = 112.5 ” + + {44.0 milligrams invert sugar. + _a_ = 150 milligrams Cu = {45.3 ” dextrose. + {42.5 ” levulose. + _d_ = 137.5 ” ” = 41.55 ” dextrose. + _c_ = 100 ” ” = 29.5 ” invert sugar = 28.025 + sucrose. + _e_ = 112.5 ” ” = 31.9 ” levulose. + + 14.75 milligrams of dextrose = 48.5 milligrams Cu. + 14.75 ” ” levulose = 51.5 ” ” + + 137.5 - 48.5 = 89.0 milligrams Cu due to dextrose present before + inversion. + 89.0 milligrams Cu = 27 milligrams dextrose before inversion. + 150.0 - 89.0 ” ” = 61.0 ” Cu due to levulose present + before inversion. + 61.0 ” ” = 17.8 ” levulose before inversion. + 17.8 × 2 = 35.6 milligrams invert sugar present before inversion. + 27.0 - 17.8 = 9.2 ” dextrose ” ” ” + +Again: + + 112.5 - 51.5 = 61.0 milligrams Cu due to levulose present + before inversion. + 61.0 milligrams Cu = 17.8 milligrams levulose. + 17.8 ” levulose indicate 35.6 milligrams invert sugar. + Dextrose in invert sugar before inversion = 17.8 milligrams. + Total dextrose before inversion = 27.0 milligrams. + Dextrose above amount required for invert sugar = 27.0 - 17.8 + = 9.2 milligrams. + +The respective quantities of the three sugars in the solution are, +therefore: + + Sucrose = 28.025 milligrams. + Invert sugar = 35.6 ” + Dextrose = 9.2 ” + +The calculations made from the later data (=234=) give almost the same +results. + +=251. Winter’s Process.=—Winter has proposed a method of separating +dextrose and levulose in the presence of sucrose based on the +selective precipitation produced on treating mixtures of these sugars +in solution with ammoniacal lead acetate.[206] + +The reagent is prepared immediately before use by adding ammonia to +a solution of lead acetate until the opalescence which is at first +produced just disappears. The separation is based on the fact that +the compound of sucrose with the reagent is easily soluble in water, +while the salts formed with levulose and dextrose are insoluble. The +separation of the sugars is accomplished as follows: + +The ammoniacal lead acetate is added to the solution of the mixed +sugars until no further precipitate is produced. The precipitated +matters are digested with a large excess of water and finally separated +by filtration. The sucrose is found in the filtrate in the form of a +soluble lead compound, from which it is liberated by treatment with +carbon dioxid. The lead carbonate produced is separated by filtration +and the sucrose is estimated in an aliquot part of the filtrate by +optical or chemical methods. The precipitate containing the lead +compounds of dextrose and levulose is washed free of sucrose, suspended +in water and saturated with carbon dioxid. By this treatment the lead +compound with dextrose is decomposed and, on filtration, the dextrose +will be found in the filtrate, while the lead compound of the levulose +is retained upon the filter with the lead carbonate. After well washing +the precipitate, it is again suspended in water and saturated with +hydrogen sulfid. By this treatment the lead levulosate compound is +broken up and the levulose obtained, on subsequent filtration, in +the filtrate. The dextrose and levulose, after separation as above +described, may be determined in aliquot parts of their respective +filtrates by the usual gravimetric methods. Before determining the +levulose the solution should be heated until all excess of hydrogen +sulfid is expelled. + +This method was used especially by Winter in separating the various +sugars obtained in the juices of sugar cane. It has not been largely +adopted as a laboratory method, and on account of the time and trouble +required for its conduct, is not likely to assume any very great +practical importance. + +=252. Separation of Sugars by Lead Oxid.=—In addition to the +combination with the earthy bases, sugar forms well defined compounds +with lead oxid. One of these compounds is of such a nature as to have +considerable analytical and technical value. Its composition and the +method of preparing it have been pointed out by Kassner.[207] + +Sucrose, under conditions to be described, forms with the lead oxid +a diplumbic saccharate, which separates in spheroidal crystals, and +has the composition corresponding to the formula C₁₂H₁₈O₁₁Pb₂ + 5H₂O. +The precipitation takes place quantitively and should be conducted as +follows: + +The substance containing the sucrose, which may be molasses, sirups +or concentrated juices, is diluted with enough water to make a sirup +which is not too viscous. Lead oxid suspended in water is stirred into +the mass in such proportion as to give about two parts of oxid to one +of the sugar. The stirring is continued for some time until the oxid +is thoroughly distributed throughout the mass and until it becomes +thick by the commencement of the formation of the saccharate. As soon +as the mass is sufficiently thickened to prevent the remaining lead +oxid from settling, the stirring may be discontinued and the mixture +is left for twenty-four hours, at the end of which time the sucrose +has all crystallized in the form of lead saccharate. The crystals of +lead saccharate can be separated by a centrifugal machine or by passing +through a filter press, and are thoroughly washed with cold water, in +which they are almost insoluble. The washed crystals are beaten up with +water into a thick paste and the lead separated as basic carbonate by +carbon dioxid. The sucrose is found in solution in the residual liquor +and is concentrated and crystallized in the usual way. + +Reducing sugars have a stronger affinity for the lead oxid than the +sucrose, and this fact is made use of to effect a nearly complete +separation when they are mixed together. In order to secure this the +lead oxid is added in the first place only in sufficient quantity to +combine with the reducing sugars present, the process being essentially +that described above. The reducing sugars which are precipitated as +lead dextrosates, lead levulosates, etc., are separated in the usual +way by a centrifugal or a filter press, and the resulting liquor, +which contains still nearly all the sucrose, is subjected to a second +precipitation by the addition of lead oxid. The second precipitation +obtained is almost pure diplumbic saccharate. + +In the precipitation of the sugar which is contained in the beet +molasses, where only a trace or very little invert sugar is present, +the sucrose is almost quantitively separated, and by the concentration +of the residual liquor, potash salts are easily obtained. In this case, +after the decomposition of the lead saccharate by carbon dioxid, the +residual sugar solution is found entirely free of lead. Where invert +sugar is present, however, in any considerable proportions, it is found +to exercise a slightly soluble influence on the lead saccharate, and +in this case a trace of lead may pass into solution. For technical +purposes, this is afterwards separated by hydrosulfuric acid or the +introduction of lime sulfid. + +Lead oxid is regenerated from the basic lead carbonate obtained by +heating in retorts to a little above 260°, and the carbon dioxid +evolved can also be used again in the technical process. + +=253. Commercial Glucose and Grape Sugar.=—The commercial products +obtained by the hydrolysis of starch are known in the trade as glucose +or grape sugar. The former term is applied to the thick sirup obtained +by concentrating the products of a partial hydrolysis, while the latter +is applied to the solid semi-crystalline mass, secured by continuing +the hydrolyzing action until the intermediate products are almost +completely changed to dextrose. In this country the starch employed +is obtained almost exclusively from maize, and the hydrolyzing agent +used is sulfuric acid.[208] The products of conversion in glucose are +chiefly dextrins and dextrose with some maltose, and in grape sugar +almost entirely dextrose. When diastase is substituted for an acid, as +the hydrolytic agent, maltose is the chief product, the ferment having +no power of producing dextrose. In the glucose of Japan, known as midzu +ame dextrin and maltose are the chief constituents.[209] + +Commercial glucose is used chiefly by confectioners for manufacturing +table sirups and for adulterating honey and molasses. + +Commercial grape sugar is chiefly employed by brewers as a substitute +for barley and other grains. + +In Europe, the starch which is converted into glucose, is derived +principally from potatoes. The method employed in conversion, whether +an acid or diastatic action, is revealed not only by the nature of +the product, but also by the composition of its ash. In the case of +diastatic conversion the ash of the sample will contain only a trace of +sulfates, no chlorin, and be strongly alkaline, while the product of +conversion with sulfuric acid will give an ash rich in sulfates with a +little lime and be less strongly alkaline. + +The process of manufacture in this country consists in treating the +starch, beaten to a cream with water, with sulfuric acid, usually under +pressure, until the product shows no blue color with iodin. The excess +of acid is removed with marble dust, the sirup separated by filtration, +whitened by bleaching with sulfurous acid or by passing it through +bone-black and evaporated to the proper consistence in a vacuum. The +solid sugar, consisting mostly of dextrose, is made in the same manner, +save that the heating with the acid is continued until the dextrin and +maltose are changed into maltose. The product is either obtained in +its ordinary hydrated form or by a special method of crystallization +secured as bright anhydrous crystals. Solutions of dextrose, when first +made, show birotation, but attain their normal gyrodynatic state on +standing for twenty-four hours in the cold, or immediately on boiling. + +=254. Methods Of Separation.=—The accurate determination of the +quantities of the several optically active bodies formed in commercial +glucose is not possible by any of the methods now known. Approximately +accurate data may be secured by a large number of processes, and these +are based chiefly on the ascertainment of the rotation and reducing +power of the mixed sugars, the subsequent removal of the dextrose and +maltose by fermentation or oxidation and the final polarization of the +residue. The difficulties which attend these processes are alike in +all cases. Fermentation may not entirely remove the reducing sugars or +may act slightly on the dextrin. In like manner the oxidation of these +sugars by metallic salts may not entirely decompose them, may leave an +optically active residue, or may affect the optical activity of the +residual dextrin. The quantitive methods of separating these sugars +by means of phenylhydrazin, lead salts or earthy bases have not been +developed into reliable and applicable laboratory processes. At the +present time the analyst must be contented with processes confessedly +imperfect, but which, with proper precautions, yield data which are +nearly correct. The leading methods depending on fermentation and +oxidation combined with polarimetric observations will be described in +the subjoined paragraphs. + +=255. Fermentation Method.=—This process is based on the assumption +that, under certain conditions, dextrose and maltose may be removed +from a solution and the dextrin be left unchanged. In practice, +approximately accurate results are obtained by this method, although +the assumed conditions are not strictly realized. In the prosecution +of this method the polarimetric reading of the mixed sugars is made, +and the maltose and dextrose removed therefrom by fermentation +with compressed yeast. The residual dextrins are determined by the +polariscope on the assumption that their average gyrodynat is 193. +In the calculation of the quantities of dextrose and maltose their +gyrodynats are fixed at 53 and 138 respectively. The total quantity +of reducing sugar is determined by the usual processes. The relative +reducing powers of dextrose and maltose are represented by 100 and 62 +respectively. The calculations are made by the following formulas:[210] + + _R_ = reducing sugars as dextrose + _d_ = dextrose + _m_ = maltose + _dʹ_ = dextrin + _P_ = total polarization + (calculated as apparent gyrodynat) + _Pʹ_ = rotation after fermentation + (calculated as apparent gyrodynat). + + Whence _R_ = _d_ + 0.62_m_ (1) + _P_ = 53_d_ + 138_m_ + 163_dʹ_ (2) + _Pʹ_ = 193_dʹ_ (3) + +From these three equations the values of _d_, _m_, and _dʹ_ are readily +calculated: + + _Example_: To find _d_ and _m_: + + Subtract (3) from (2) _P_ = 53_d_ + 133_m_ + 193_dʹ_ + _Pʹ_ = 193_dʹ_ + -------------------------------------- + _P_ - _Pʹ_ = 53_d_ + 138_m_ (4) + + Multiply (1) by 53 and subtract from (4) + + _P_ - _Pʹ_ = 53_d_ + 138_m_ + 53_R_ = 53_d_ + 32.86_m_ + ------------------------------------------- + _P_ - _Pʹ_ - 53_R_ = 105.14_m_ (5) + + _P_ - _Pʹ_ - 53_R_ + Whence _m_ = ------------------ (6) + 105.14 + + _d_ = _R_ - 0.62_m_ (7) + + _Pʹ_ + _dʹ_ = ---- (8) + 193 + +Sidersky assigns the values [_a_]_{D} = 138.3 and [_a_]_{D} = 194.8 to +maltose and dextrin respectively in the above formulas.[211] + +_Illustration_: In the examination of a sample 26.048 grams of midzu +ame in 100 cubic centimeters were polarized in a 200 millimeter tube +and the following data were obtained: + +Polarization of sample in angular degrees 69°.06, which is equal to an +apparent gyrodynat of 132.6: + +Total reducing sugar as dextrose 33.33 per cent: + +Polarization in angular degrees after fermentation 30°.84 = [_a_]_{D} = +59.2. + +Substituting these values in the several equations gives the following +numbers: + + (1) 0.3333 = _d_ + 0.62_m_ + (2) 132.6 = 53_d_ + 138_m_ + 193_dʹ_ + (3) 59.2 = 193_dʹ_ + (4) 73.4 = 53_d_ + 138_m_ + (5) 55.74 = 105.14_m_ + (6) _m_ = 0.5301 = 53.01 per cent. + (7) _d_ = 3333 - 3286 = 0.0047 = 00.47 per cent. + (8) _dʹ_ = 59.2 ÷ 193 = 0.3067 = 30.67 ” ” + +_Summary_: Sample of midzu ame: + + Percentage of dextrin 30.67 per cent. + ” ” maltose 53.01 ” ” + ” ” dextrose 00.47 ” ” + ” ” water 14.61 ” ” + ” ” ash 00.31 ” ” + ----- + Sum 99.07 ” ” + Undetermined 0.93 ” ” + +For polarization the lamplight shadow polariscope employed for sugar +may be used, and the degrees of the sugar (ventzke) scale converted +into angular degrees by multiplying by 0.3467. + +The process of fermentation is conducted as described in the paragraph +given further on, relating to the determination of lactose in the +presence of sucrose. + +=256. The Oxidation Method.=—The removal of the reducing sugars may +be accomplished by oxidation instead of fermentation. The process +of analysis is in all respects similar to that described in the +foregoing paragraph, substituting oxidation for fermentation.[212] +For the oxidizing agent mercuric cyanid is preferred, and it is +conveniently prepared by dissolving 120 grams of mercuric cyanid and +an equal quantity of sodium hydroxid in water mixing the solutions +and completing the volume to one liter. If a precipitate be formed +in mixing the solutions it should be removed by filtering through +asbestos. For the polarization, ten grams of the sugars in 100 cubic +centimeters is a convenient quantity. Ten cubic centimeters of +this solution are placed in a flask of water marked at fifty cubic +centimeters, a sufficient quantity of the mercuric cyanid added to +remain in slight excess after the oxidation is finished (from twenty to +twenty-five cubic centimeters) and the mixture heated to the boiling +point for three minutes. The alkali, after cooling, is neutralized with +strong hydrochloric acid and the passing from alkalinity to acidity +will be indicated by a discharge of the brown color which is produced +by heating with the alkaline mercuric cyanid. The heating with the +mercury salt should be conducted in a well ventilated fume chamber. + +The calculation of the results is conducted by means of the formulas +given in the preceding paragraph. In the original paper describing +this method, it was stated that its accuracy depended on the complete +oxidation of the reducing sugar in a manner leaving no optically active +products, and on the inactivity of the reagents used in respect to the +dextrin present. These two conditions are not rigidly fulfilled, as is +shown by Wilson.[213] According to his data maltose leaves an optically +active residue, which gives a somewhat greater right hand rotation than +is compensated for by the diminished rotation of the dextrin. Wilson, +however, confesses that the dextrin used contained reducing sugars, +which would not be the case had it been prepared by the process of +treating it with alkaline mercuric cyanid as above indicated. Upon the +whole, the oxidation of the reducing sugar by a mercury salt gives +results which, while not strictly accurate, are probably as reliable as +those afforded by fermentation. The author has attempted to supplant +both the oxidation and fermentation methods by removing the reducing +sugars with a precipitating reagent, such as phenylhydrazin, but the +methods are not sufficiently developed for publication. + +=257. Removal of Dextrose by Copper Acetate.=—Maercker first called +attention to the fact that Barfoed’s reagent (one part copper acetate +in fifteen parts of water, and 200 cubic centimeters of this solution +mixed with five cubic centimeters of thirty-eight per cent. acetic +acid) reacts readily with dextrose, while it is indifferent to maltose +and dextrins. Sieben’s method of removing dextrose is based on this +fact.[214] It is found that under certain conditions pure maltose +does not reduce either the acidified or neutral solution of copper +acetate, while dextrose or a mixture of dextrose and maltose does so +readily. It is also shown that the fermentation residue under suitable +conditions acts like maltose. Maltose solutions reduce the reagent +after boiling four minutes while at 40°-45° they have no effect even +after standing four days. The amount of copper deposited by dextrose, +under the latter conditions, is found to depend to a certain extent on +the amount of free acetic acid present, and as the solutions of copper +acetate always contain varying quantities of acetic acid which cannot +be removed without decomposition and precipitation of basic salt, the +use of an absolutely neutral solution is impracticable. The reagent +prepared according to Barfoed’s directions is almost saturated, but a +half normal solution is preferable. Sieben proposes two solutions: I, +containing 15.86 grams copper and 0.56 gram acetic anhydrid per liter; +II, containing 15.86 grams copper and three grams acetic anhydrid per +liter. The reduction of the dextrose is secured by placing 100 cubic +centimeters of the solution in a bottle, adding the sugar solution, +stoppering and keeping in a water-bath at 40°-45° two or three days. An +aliquot portion is then drawn off and the residual copper precipitated +by boiling with forty-five cubic centimeters of the alkali solution +of the fehling reagent and forty cubic centimeters of one per cent +dextrose solution, filtered and weighed as usual. The results show that +either solution can be used, and that standing for two days at 45° +is sufficient. One hundred cubic centimeters of the copper solution +are mixed with ten cubic centimeters of the sugar solution containing +from two-tenths to five-tenths gram of dextrose, as this dilution +gives the best results. No reduction is found to have taken place when +solutions containing five-tenths gram maltose or five-tenths gram +fermentation residue are used. The data can not be compiled in the +form of a table similar to Allihn’s, as it is impossible to obtain a +solution of uniform acidity each time, and the solution will have to be +standardized by means of a known pure dextrose solution and the result +obtained with the unknown sugar solution properly diluted compared with +this. This method of Sieben’s has never been practiced to any extent in +analytical separations and can not, therefore, be strongly recommended +without additional experience. + +=258. Removal of Dextrin by Alcohol.=—By reason of its less solubility, +dextrin can be removed from a solution containing also dextrose and +maltose by precipitation with alcohol. It is impracticable, however, to +secure always that degree of alcoholic concentration which will cause +the coagulation of all the dextrins without attacking the concomitant +reducing sugars. In this laboratory it has been found impossible to +prepare a dextrin by alcoholic precipitation, which did not contain +bodies capable of oxidizing alkaline copper solutions. + +The solution containing the dextrin is brought to a sirupy consistence +by evaporation and treated with about ten volumes of ninety per cent +alcohol. After thorough mixing, the precipitated dextrin is collected +on a filter and well washed with alcohol of the strength noted. It is +then dried and weighed. If weaker solutions of dextrin are used, the +alcohol must be of correspondingly greater strength. In the filtrate +the residual maltose and dextrose may be separated and determined by +the chemical and optical methods already described. + + +CARBOHYDRATES IN MILK. + +=259. The Copper Tartrate Method.=—The lactose in milk is readily +estimated by the gravimetric copper method described in paragraph +=143=. Before the application of the process the casein and fat of +the milk should be removed by an appropriate precipitant, and an +aliquot part of the filtrate, diluted to contain about one per cent +of milk sugar, used for the determination. The clarification is very +conveniently secured by copper sulfate or acetic acid, as described +in the next paragraph. A proper correction should be made for the +volume occupied by the precipitate and, for general purposes, with +whole milk of fair quality this volume may be assumed to be five per +cent. One hundred grams of milk will give a precipitate occupying +approximately five cubic centimeters. In the analytical process, to +twenty cubic centimeters of milk, diluted with water to eighty, is +added a ten per cent solution of acetic acid, until a clear whey is +shown after standing a few minutes, when the volume is completed to 100 +cubic centimeters with water, and the whole, after thorough shaking, +thrown on a filter. An aliquot part of the filtrate is neutralized with +sodium carbonate and used for the lactose determination. This solution +contains approximately one per cent of lactose. In a convenient part +of it the lactose is determined and the quantity calculated for the +whole. This quantity represents the total lactose in the twenty +cubic centimeters of milk used. The weight of the milk is found by +multiplying twenty by its specific gravity. From this number the +percentage of lactose is easily found. In this process the milk is +clarified by the removal of its casein and fat. Other albuminoids +remain in solution and while these doubtless disturb the subsequent +determination of lactose, any attempt at their removal would be equally +as disadvantageous. The volume of the precipitate formed by good, whole +milk when the process is conducted as above described, is about one +cubic centimeter, for which a corresponding correction is readily made. + +=260. The Official Method.=—The alkaline copper method of determining +lactose, adopted by the Association of Official Agricultural Chemists, +is essentially the procedure proposed by Soxhlet.[215] + +Dilute twenty-five cubic centimeters of the milk, held in a half +liter flask, with 400 cubic centimeters of water and add ten cubic +centimeters of a solution of copper sulfate of the strength given for +Soxhlet’s modification of Fehling’s solution, page 129; add about seven +and a half cubic centimeters of a solution of potassium hydroxid of +such strength that one volume of it is just sufficient to completely +precipitate the copper as hydroxid from one volume of the solution of +copper sulfate. In place of a solution of potassium hydroxid of this +strength eight and a half cubic centimeters of a half normal solution +of sodium hydroxid may be used. After the addition of the alkali +solution the mixture must still have an acid reaction and contain +copper in solution. Fill the flask to the mark, shake and filter +through a dry filter. + +Place fifty cubic centimeters of the mixed copper reagent in a beaker +and heat to the boiling point. While boiling briskly, add 100 cubic +centimeters of the lactose solution, prepared as directed above, and +boil for six minutes. Filter immediately and determine the amount of +copper reduced by one of the methods already given, pages 149-155. +Obtain the weight of lactose equivalent to the weight of copper found +from the table on page 163. + +=261. The Copper Cyanid Process.=—It has been found by Blyth that the +copper cyanid process of Gerrard gives practically the same results +in the determination of sugar in milk as are obtained by optical +methods.[216] The milk for this purpose is clarified, by precipitating +the casein with acetic acid in the following manner: + +Twenty-five cubic centimeters of milk are diluted with an equal +volume of distilled water, and strong acetic acid added until the +casein begins to separate. The liquid is heated to boiling and, while +hot, centrifugated in any convenient machine. The supernatant liquid +obtained is separated by filtration and the solid matter thrown upon +the filter and well washed with hot water. The filtrate and washings +are cooled and completed to a volume of 100 cubic centimeters. This +liquid is of about the proper dilution for use with the copper cyanid +reagent. The percentage of sugar determined by this reagent agrees well +with that obtained by the optical method, when no other sugars than +lactose are present. If there be a notable difference in the results +of the two methods other sugars must be looked for. The presence +of dextrin may be determined by testing a few drops of the clear +liquor with iodin, which, in the presence of dextrin, gives a reddish +color. Other sugars are determined by obtaining their osazones. For +this purpose the filtrate obtained as above should be concentrated +until the volume is about thirty cubic centimeters. Any solid matter +which separates during the evaporation is removed by filtration. The +osazones are precipitated in the manner described in paragraph =147=. +On cooling, the almost solid crystalline mass obtained is placed on +a filter, washed with a little cold water, the crystals then pressed +between blotting paper and dried at a temperature of 100°. The dry +osazones obtained are dissolved in boiling absolute alcohol, of which +just sufficient is used to obtain complete solution. The alcoholic +solution is set aside for twelve hours and the separation of a +crystalline product after that time shows that dextrose or invert sugar +is present. Milk sugar alone gives no precipitate but only a slight +amorphous deposit. The lactosazone is precipitated by adding a little +water to the hot alcoholic solution and the crystals thus obtained +should be dissolved in boiling absolute alcohol and reprecipitated by +the addition of water at least three times in order to secure them +pure. The osazones are identified by their melting points, paragraph +=172=. The first part of this method does not appear to have any +advantage over the optical process by double dilution (p. 278), and +requires more time. + +=262. Sugars in Evaporated Milks.=—In addition to the lactose normally +present in evaporated milks the analyst will, in most cases, find large +quantities of sucrose. The latter sugar is added as a preservative and +condiment. By reason of the ease with which sucrose is hydrolyzed, +evaporated milk containing it may have also some invert sugar among +its contents. A method of examination is desirable, therefore, which +will secure the determination of lactose, sucrose and invert sugar +in mixtures. The probability of the development of galactose and +dextrose during the evaporation and conservation of the sample, is not +great. The best method of conducting this work is the one developed by +Bigelow and McElroy.[217] The principle on which the method is based +rests on the fact that in certain conditions, easily supplied, the +sucrose and invert sugar present in a sample may be entirely removed +by fermentation and the residual lactose secured in an unchanged +condition. The lactose is finally estimated by one of the methods +already described. + +The details of the process follow: + +On opening a package of evaporated milk, its entire contents are +transferred to a dish and well mixed. Several portions of about +twenty-five grams each are placed in flasks marked at 100 cubic +centimeters. To each of the flasks enough water is added to bring +all the sugars into solution and normal rotation is made certain by +boiling. After cooling, the contents of the flasks are clarified by +mercuric iodid in acetic acid solution. The clarifying reagent is +prepared by dissolving fifty-three grams of potassium iodid, twenty-two +grams of mercuric chlorid, and thirty-two cubic centimeters of +strongest acetic acid in water, mixing the solutions and completing the +volume to one liter. The clarification is aided by the use of alumina +cream (=84=). The flask is filled to the mark, and the contents well +shaken and poured on a filter. After rejecting the first portion of +the filtrate the residue is polarized in the usual manner. Two or more +separate portions of the sample are dissolved in water in flasks of the +size mentioned, heated to 55°, half a cake of compressed yeast added +to each and the temperature kept at 55° for five hours. The residue in +each flask is treated as above described, the mercuric solution being +added before cooling to prevent the fermentative action of the yeast, +and the polarization noted. + +By this treatment the sucrose is completely inverted, while the +lactose is not affected. The percentage of sucrose is calculated by +the formulas given in paragraph =94=, using the factor 142.6. At the +temperature noted the yeast exercises no fermentative, but only a +diastatic action. + +In each case the volume of the precipitated milk solids is determined +by the double dilution method, and the proper correction made (p. 278). +The lactose remaining is determined by chemical or optical methods, +but it is necessary, in all cases where invert sugar is supposed to be +present, to determine the total reducing sugars in the original sample +as lactose. If the quantity thus determined and the amount of sucrose +found as above are sufficient to produce the rotation observed in the +first polarization, it is evident that no invert sugar is present. When +the polarization observed is less than is equivalent to the quantity +of sugar found, invert sugar is present, which tends to diminish the +rotation produced by the other sugars. In this case it is necessary to +remove both the sucrose and invert sugar by a process of fermentation, +which will leave the lactose unchanged. + +This is accomplished by conducting the fermentation in the presence +of potassium fluorid, which prevents the development of the lactic +ferments. For this purpose 350 grams of the evaporated milk are +dissolved in water and the solution boiled to secure the normal +rotation of the lactose. After cooling to 80°, the casein is thrown +down by adding a solution containing about four grams of glacial +phosphoric acid and keeping the temperature at 80° for about fifteen +minutes. After cooling to room temperature, the volume is completed +to one liter with water, well shaken and poured onto a filter. An +aliquot part of the filtrate is nearly neutralized with a noted volume +of potassium hydroxid. Enough water is added to make up, with the +volume of potassium hydroxid used, the total space occupied by the +precipitated solid, corresponding to that part of the filtrate, and if +necessary, refilter. The volume occupied by the precipitated solids is +easily determined by polarization and double dilution. The filtrate, +obtained from the process described above, is placed in portions of +100 cubic centimeters each in 200 cubic centimeter flasks with about +twenty milligrams of potassium fluorid in solution, and half a cake of +compressed yeast. The yeast is broken up and evenly distributed, and +the fermentation is allowed to proceed for ten days at a temperature +of from 25° to 30°. At the end of this time experience has shown that +all of the sucrose and invert sugar has disappeared, but the lactose +remains intact. The flasks are filled to the mark with water and the +lactose determined by chemical or optical methods. By comparing the +data obtained from the estimation of the total reducing sugars before +fermentation or inversion and the estimation of the lactose after +fermentation, the quantity of invert sugar is easily calculated. The +experience of this laboratory shows that invert sugar is rarely present +in evaporated milks, which is an indication that the sucrose added +thereto does not generally suffer hydrolysis. The mean percentage of +added sucrose found in evaporated milks is about forty. + + +SEPARATION AND DETERMINATION OF STARCH. + +=263. Occurrence.=—Many bodies containing starch are presented for +the consideration of the agricultural analyst. First in importance +are the cereals, closely followed by the starchy root crops. Many +spices and other condiments also contain starchy matters. In the +sap of some plants, for instance sorghum, at certain seasons, +considerable quantities of starch occur. In the analysis of cereals +and other feeding stuffs, it has been the usual custom to make no +separate determination of starch, but to put together all soluble +carbohydrates and estimate their percentage by subtracting from 100 +the sum of the percentages of the other constituents of the sample. +This aggregated mass has been known as nitrogen-free extract. Recent +advances in methods of investigation render it advisable to determine +the starch and pentosan carbohydrates separately and to leave among +the undetermined bodies the other unclassed substances, chiefly of a +carbohydrate nature, soluble in boiling dilute acid and alkali. + +=264. Separation of Starch.=—Starch being insoluble in its natural +state, it is impossible to separate it from the other insoluble matters +of plants by any known process. In bringing it into solution it +undergoes certain changes of an unknown nature, but tending to produce +a dextrinoid body. Nevertheless, in order to procure the starch in a +state of purity suited to analytical processes, it becomes necessary +to dissolve the starch from the other insoluble bodies that naturally +accompany it. As has been shown in preceding paragraphs, there are only +two methods of securing the solution of starch which fully meet the +conditions of accurate analysis. These are the methods depending on the +use of diastatic ferments and on the employment of heat and pressure +in the presence of water. These two processes have been described +in considerable detail in paragraphs =179-181=. It is important, in +starch determinations, to remove from the sample the sugar and other +substances soluble in water and also the oils, when present in large +quantities, before subjecting it to the processes for rendering the +starch soluble. + +=265. Desiccation of Amyliferous Bodies.=—The removal of sugars and +oils is best secured in amyliferous substances after they are deprived +of their moisture. As has already been suggested, the desiccation +should be commenced at a low temperature, not above 60°, and continued +at that point until the chief part of the water has escaped. The +operation may be conducted in one of the ways already described +(pp. 12-27). There is great difference of opinion among analysts in +respect of the degree of temperature to which the sample should be +finally subjected, but for the purposes here in view, it will not be +found necessary to go above 105°. Before beginning the operation the +sample should be as finely divided as possible, and at its end the +dried residue should be ground and passed through a sieve of half a +millimeter mesh. + +=266. Indirect Method of Determining: Water in Starch.=—It is claimed +by Block[218] that it is necessary to dry starch at 160° in order to get +complete dehydration. Wet starch as deposited with its maximum content +of water has nine molecules thereof, _viz._, C₆H₁₀O₅ + 9H₂O. Ordinary +commercial starch has about eighteen per cent of water with a formula +of C₆H₁₀O₅ + 2H₂O. + +The percentage of water may be determined by Block’s feculometer or +Block’s dose-fécule. The first apparatus determines the percentage of +anhydrous starch by volume, and the second by weight. + +Block’s assumption that starch can absorb only fifty per cent of its +weight of water is the basis of the determination. + +A noted weight of starch is rubbed up with water until saturated, the +water poured off, the starch weighed, dried on blotting paper until it +gives off no more moisture and again weighed. Half of the lost weight +is water, from which the original per cent of water can be calculated. +This at best seems to be a rough approximation and not suited to +rigorous scientific determination. + +=267. Removal of Oil and Sugar.=—The dried, finely powdered sample, +obtained as described above, is placed in any convenient extractor +(=33-43=) and the oil or fat it contains removed by the usual solvents. +For ordinary purposes, even with cereals, this preliminary extraction +of the oil is not necessary, but it becomes so with oily seeds +containing starch. The sugar is subsequently removed by extraction +with eighty per cent alcohol and the residue is then ready for the +extraction of the starch. In most cases the extraction with alcohol +will be found sufficient. In some bodies, for instance the sweet potato +(batata), the quantity of sugar present is quite large, and generally +some of it is found. If not present in appreciable amount, the alcohol +extraction may also be omitted. The sample having been prepared as +indicated, the starch may be brought into solution by one of the +methods described in paragraphs =179-181=, preference being given to +the aqueous digestion in an autoclave. The dissolved starch is washed +out of the insoluble residue and determined by optical or chemical +methods =186-194=. + +=268. Preparation of Diastase for Starch Solution.=—The methods of +preparing malt extract for use in starch analysis have been described +in paragraph =179=. If a purer form of diastase is desired it may be +prepared by following the directions given by Long and Baker.[219] +Digest 200 grams of ground malt for twenty-four hours with three parts +of twenty per cent alcohol. Separate the extract by filtration and +to the filtrate add about one and a half liters of ninety-three per +cent alcohol and stir vigorously. After the precipitate has subsided +the supernatant alcohol is removed by a syphon, the precipitate is +brought onto a filter and washed with alcohol of a strength gradually +increasing to anhydrous, and finally with anhydrous ether. The diastase +is dried in a vacuum over sulfuric acid and finally reduced to a fine +powder before using. Thus prepared, it varies in appearance from a +white to a slightly brownish powder. Made at different times and from +separate portions of malt, it may show great differences in hydrolytic +power. + +=269. Estimation of Starch in Potatoes by Specific Gravity.=—A roughly +approximate determination of the quantity of starch in potatoes can be +made by determining their specific gravity. Since the specific gravity +of pure starch is 1.65, it follows that the richer a potato is in +starch the higher will be its specific gravity. The specific weight +of substances like potatoes is conveniently determined by suspending +them in water by a fine thread attached to the upper hook of a balance +pan. There may be a variation of the percentage of other constituents +in potatoes as well as of starch, and therefore the data obtained from +the following table can only be correct on the assumption that the +starch is the only variable. In practice, errors amounting to as much +as two per cent may be easily made, and therefore the method is useful +only for agronomic and commercial and not for scientific purposes. The +method is especially useful in the selection of potatoes of high starch +content for planting. The table is constructed on the weight in grams +in pure water of 10000 grams of potatoes and the corresponding per +cents of dry matter and starch are given. It is not always convenient +to use exactly 10000 grams of potatoes for the determination, but the +calculation for any given weight is easy.[220] + +_Example._—Let the weight of a potato in air be 159 grams, and its +weight in water 14.8 grams. + +Then the weight of 10000 grams of potatoes of like nature in water +would be found from the equation 159: 10000 = 14.8: _x_. + +Whence _x_ = 931 nearly. + +In the table the nearest figure to 931 is 930, corresponding to 24.6 +per cent of dry matter and 18.8 per cent of starch. When the number +found is half way between the numbers given in the table the mean of +the data above and below can be taken. In other positions a proper +interpolation can be made if desired but for practical purposes the +data corresponding to the nearest number can be used. + + TABLE FOR CALCULATING STARCH IN + POTATOES FROM SPECIFIC GRAVITY. + + 10000 grams + of potatoes + weigh in Per cent Per cent + water. dry matter. starch. + Grams. + + 750 19.9 14.1 + 760 20.1 14.3 + 770 20.3 14.5 + 780 20.7 14.9 + 790 20.9 15.1 + 800 21.2 15.4 + 810 21.4 15.6 + 820 21.6 15.8 + 830 22.0 16.2 + 840 22.2 16.4 + 850 22.4 16.6 + 860 22.7 16.9 + 870 22.9 17.1 + 880 23.1 17.3 + 890 23.5 17.7 + 900 23.7 17.9 + 910 24.0 18.2 + 920 24.2 18.4 + 930 24.6 18.8 + 940 24.8 19.0 + 950 25.0 19.2 + 960 25.2 19.4 + 970 25.5 19.7 + 980 25.9 20.1 + 990 26.1 20.3 + 1000 26.3 20.5 + 1010 26.5 20.7 + 1020 26.9 21.1 + 1030 27.2 21.4 + 1040 27.4 21.6 + 1050 27.6 21.8 + 1060 28.0 22.2 + 1070 28.3 22.5 + 1080 28.5 22.7 + 1090 28.7 22.9 + 1100 29.1 23.3 + 1110 29.3 23.5 + 1120 29.5 23.7 + 1130 29.8 24.0 + 1140 30.2 24.4 + 1150 30.4 24.6 + 1160 30.6 24.8 + 1170 31.0 25.0 + 1180 31.3 25.5 + 1190 31.5 25.7 + 1200 31.7 25.9 + 1210 32.1 26.3 + 1220 32.3 26.5 + 1230 32.5 26.7 + 1240 33.0 27.2 + 1250 33.2 27.4 + 1260 33.4 27.6 + 1270 33.6 27.8 + 1280 34.1 28.3 + 1290 34.3 28.5 + 1300 34.5 28.7 + 1310 34.9 29.1 + 1320 35.1 29.3 + 1330 35.4 29.6 + 1340 35.8 30.0 + 1350 36.0 30.2 + 1360 36.2 30.4 + 1370 36.6 30.8 + +=270. Constitution of Cellulose.=—The group of bodies known as +cellulose comprises many members of essentially the same chemical +constitution but of varying properties. The centesimal composition of +pure cellulose is shown by the following numbers: + + Carbon, 44.2 per cent + Hydrogen, 6.3 ” ” + Oxygen, 49.5 ” ” + +corresponding to the formula C₆H₁₀H₅. + +According to the view of Cross and Bevan, cellulose conforms in respect +of its ultimate constitutional groups to the general features of the +simple carbohydrates, but differs from them by reason of a special +molecular configuration resulting in a suppression of the activity of +constituent groups in certain respects, and an increase in activity of +others.[221] + +=271. Fiber and Cellulose.=—The carbohydrates of a plant insoluble in +water are not composed exclusively of starch. There are, in addition +to starch, pentosan fibers yielding pentose sugars on hydrolysis and +furfuraldehyd on distillation with a strong acid. The quantitive +methods for estimating the pentosan bodies are given in paragraphs +=150-157=. The method to be preferred is that of Krug (=155=). + +In the estimation of cattlefoods and of plant substances in general the +residue insoluble in dilute boiling acid and alkali is called crude or +indigestible fiber. + +The principle on which the determination depends rests on the +assumption that all the protein, starch and other digestible +carbohydrates will be removed from the sample by successive digestion +at a boiling temperature with acid and alkali solutions of a given +strength. It is evident that the complex body obtained by the treatment +outlined above is not in any sense a definite chemical compound, but it +may be considered as being composed partly of cellulose. + +=272. Official Method of Determining Crude Fiber.=—The method of +estimating crude fiber, adopted by the Association of Official +Agricultural Chemists, is as follows:[222] + +Extract two grams of the substance with ordinary ether, at least +almost completely, or use the residue from the determination of the +ether extract. To this residue, in a half liter flask, add 200 cubic +centimeters of boiling 1.25 per cent sulfuric acid; connect the flask +with an inverted condenser, the tube of which passes only a short +distance beyond the rubber stopper into the flask. Boil at once, and +continue the boiling for thirty minutes. A blast of air conducted into +the flask may serve to reduce the frothing of the liquid. Filter, wash +thoroughly with boiling water until the washings are no longer acid, +rinse the substance back into the same flask with 200 cubic centimeters +of a boiling 1.25 per cent solution of sodium hydroxid, free or nearly +free of sodium carbonate, boil at once and continue the boiling for +thirty minutes in the same manner as directed above for the treatment +with acid. Filter into a gooch, and wash with boiling water until the +washings are neutral, dry at 110°, weigh and incinerate completely. The +loss of weight is crude fiber. + +The filter used for the first filtration may be linen, one of the forms +of glass wool or asbestos filters, or any other form that secures clear +and reasonably rapid filtration. The solutions of sulfuric acid and +sodium hydroxid are to be made up of the specified strength, determined +accurately by titration and not merely from specific gravity. + +The experience of this laboratory has shown that results practically +identical with those got as above, are obtained by conducting the +digestions in hard glass beakers covered with watch glasses. The ease +of manipulation in the modification of the process just mentioned is a +sufficient justification for its use. + +=273. Separation of Cellulose.=—Hoppe-Seyler observed that cellulose, +when melted with the alkalies at a temperature as high as 200°, was not +sensibly attacked.[223] + +Lange has based a process for determining cellulose on this +observation.[224] + +The process, as improved by him, is carried out as follows: + +From five to ten grams of the substance are moistened with water and +placed in a porcelain dish with about three times their weight of +caustic alkali free of nitrates and about twenty cubic centimeters +of water. The porcelain dish should be deep and crucible shaped and +should be placed in an oil-bath, the temperature of which is easily +controlled. The contents of the dish are stirred with the thermometer +bulb until all foaming ceases and the temperature of the mixture is +then kept at from 175° to 180° for an hour. After the melt has cooled +to 80° about seventy-five cubic centimeters of hot water are added to +bring it into solution and it is then allowed to cool. The solution is +acidified with sulfuric and placed in large centrifugal tubes. After +being made slightly alkaline with soda lye, the tubes are subjected +to continued energetic centrifugal action until the cellulose is +separated. The supernatant liquid can be nearly all poured off and the +separated cellulose is broken up, treated with hot water and again +separated by centrifugal action. The cellulose is finally collected +upon the asbestos felt, washed with hot water, alcohol and ether, dried +and weighed. With a little practice it is possible to complete the +separation of cellulose in two and one-half hours. + +=274. Solubility of Cellulose.=—Cellulose resembles starch in its +general insolubility, but, unlike starch, it may be dissolved in some +reagents and afterwards precipitated practically unchanged or in a +state of hydration. One of the simplest solvents of cellulose is zinc +chlorid in concentrated aqueous solution. + +The solution is accomplished with the aid of heat, adding one part by +weight of cotton to six parts of zinc chlorid dissolved in ten parts of +water. + +A homogeneous sirup is obtained by this process, which is used in the +arts for making the carbon filaments of incandescent electric lamps. + +In preparing the thread of cellulose, the solution, obtained as +described above, is allowed to flow, in a fine stream, into alcohol, +whereby a cellulose hydrate is precipitated, which is freed from zinc +hydroxid by digesting in hydrochloric acid. + +Hydrochloric acid may be substituted for water in preparing the reagent +above noted, whereby a solvent is secured which acts upon cellulose +readily in the cold. + +A solution of ammoniacal cupric oxid is one of the best solvents for +cellulose. The solution should contain from ten to fifteen per cent of +ammonia and from two to two and a half of cupric oxid. + +In the preparation of this reagent, ammonium chlorid is added to a +solution of cupric salt and then sodium hydroxid in just sufficient +quantity to precipitate all of the copper as hydroxid. The precipitate +is well washed on a linen filter, squeezed as dry as possible and +dissolved in ammonia of 0.92 specific gravity. The cellulose is readily +precipitated from the solution in cuprammonium by the addition of +alcohol, sodium chlorid, sugar, or other dehydrating agents. Solutions +of cellulose are used in the arts for many purposes.[225] + +=275. Qualitive Reactions for Detecting Cellulose.=—Cellulose may be +identified by its resistance to the action of oxidizing agents, to the +halogens and to alkaline solutions. It is further recognized by the +sirupy or gelatinous solutions it forms with the solvents mentioned +above. The cellulose hydrates precipitated from solutions have in some +instances the property of forming a blue color with iodin. + +A characteristic reaction of cellulose is secured as follows: To a +saturated solution of zinc hydrochlorate, of 2.00 specific gravity, are +added six parts by weight of potassium iodid dissolved in ten parts +of water and this solution is saturated with iodin. Cellulose treated +with this reagent is at once stained a deep blue violet color.[226] For +the characteristics of cellulose occurring in wood the researches of +Lindsey may be consulted.[227] + +=276. More Rarely Occurring Carbohydrates.=—It is not possible here +to give more space to the rarer forms of carbohydrates, to which the +attention of the agricultural analyst may be called. Nearly a hundred +kinds of sugars alone have been detected in the plant world. For +descriptions of the properties of these bodies and the methods of their +detection and determination, the standard works on carbohydrates may be +consulted.[228] + + +AUTHORITIES CITED IN PART THIRD. + +[170] Vines; Physiology of Plants. Nägeli; Beiträge zur näheren +Kenntniss der Stärkegruppe. + +[171] Bulletin 5, Department of Agriculture, Division of Chemistry, pp. +191 et seq.: Bulletin 25, New Hampshire Experiment Station. + +[172] Spencer; Handbook for Sugar Manufacturers, p. 31. + +[173] Vid. op. cit. supra, pp. 102, 108. + +[174] Journal of the American Chemical Society, Vol. 16, p. 677. + +[175] Botanical Gazette, Vol. 12, No. 3. + +[176] Bulletin de l’Association des Chimistes de Sucrerie et de +Distillerie, Tome 13, p. 133. + +[177] Journal of Analytical and Applied Chemistry, Vol. 4, p. 381. + +[178] Spencer’s Handbook for Sugar Manufacturers, pp. 30 et seq. + +[179] Bulletin de l’Association des Chimistes de Sucrerie et de +Distillerie, Tome 13, p. 292. + +[180] Vid. op. cit. supra, Tome 2, p. 369. + +[181] Dosage du Sucre Cristallisable dans la Betterave, pp. 117 et +seq.: Journal of the American Chemical Society, Vol. 16, p. 266. + +[182] Neue Zeitschrift für Rübenzucker-Industrie. Band 3, S. 342; Band +14, S. 286: Zeitschrift des Vereins für die Rübenzucker-Industrie, +1876, S. 692: Dingler’s Polytechnisches Journal, Band 232, S. 461. + +[183] Sidersky: Traité d’ Analyse des Matières Sucrées, p. 304. + +[184] Neue Zeitschrift für Rübenzucker-Industrie, Band 14, S. 286. + +[185] Zeitschrift des Vereins für die Rübenzucker-Industrie, Band 32, +S. 861. + +[186] Spencer’s Handbook for Sugar Manufacturers, p. 42. + +[187] Bulletin No. 4 of the Chemical Society of Washington, pp. 22, et +seq. + +[188] Vid. op. et loc. cit. 7. + +[189] Chemiker-Zeitung, Band 19, S. 1830. + +[190] Vid. op cit. supra, S. 1784. + +[191] Vid. op. cit. supra, S. 1829. + +[192] Zeitschrift des Vereins für die Rübenzucker-Industrie, 1895, S. +844. + +[193] Journal des Fabricants de Sucre, 1895, No. 33. + +[194] Journal of the American Chemical Society, Vol. 2, p. 387: +Agricultural Science, Feb. 1892. + +[195] American Chemical Journal, Vol. 13, p. 24. + +[196] Tucker; Manual of Sugar Analysis, p. 287: Wiechmann; Sugar +Analysis, p. 51. + +[197] Sidersky; vid. op. cit., 14, p. 197. + +[198] Journal of the American Chemical Society, Vol. 18, p. 81: Allen; +Commercial Organic Analysis, Vol. 1, p. 291. + +[199] Handbuch der Physiologisch- und Pathologisch-Chemischen Analyse, +S. 286. + +[200] Kühne und Chittenden; American Chemical Journal, Vol. 6, p. 45. + +[201] Vid. op. cit. supra, p. 289. + +[202] Analyst, Vol. 13, p. 64. + +[203] Journal American Chemical Society, Vol. 18, p. 438. + +[204] School of Mines Quarterly, Vols. 11 and 12. + +In a later method (School of Mines Quarterly, Vol. 13, No. 3) Wiechman +describes the separation of the sugars by one polariscopic and two +gravimetric determinations, one before and one after inversion. The +polariscopic examination is made in a ten per cent solution at a +temperature of 20°. The gyrodynats of sucrose, dextrose and levulose +at the temperature mentioned are fixed at 66.5, 53.5 and -81.9 +respectively. The gravimetric determinations are conducted according +to the methods already described. In the formulas for calculating +the results _a_ represents sucrose, _b_ reducing sugars, _x_ the +dextrose, _y_ the levulose, and _d_ the observed polarization expressed +in degrees angular measure. The gyrodynats of sucrose, dextrose and +levulose divided by 100 are represented by _s_, _d_ and _l_. The +calculations are made from the following formulas: + + (_as_ + _xd_) - _yl_ = _p_. + (_as_ + _xd_) = _p_ + _yl_. + _xd_ = _p_ + _yl_ - _as_. + + _p_ + _yl_ - _as_ + _x_ = -----------------. + _d_ + +In this calculation the gyrodynat of levulose is about ten degrees +lower than that of most authorities. + +[205] Vid. op. cit., 23, Band 24, S. 869. + +[206] Vid. op. cit. supra, 1888, S. 782. + +[207] Neue Zeitschrift für Rübenzucker-Industrie, Band 35, S. 166. + +[208] Journal of the American Chemical Society, Vol. 2, p. 399: +Science, Oct. 1, 1881: Proceedings American Association for the +Advancement of Science, 1881, p. 61: Sugar Cane, Vol. 13, p. 533, pp. +61-66. + +[209] Wiley and McElroy; Agricultural Science, Vol. 6, p. 57. + +[210] Chemical News, Vol. 46, p. 175. + +[211] Vid. op. cit, 14, p. 352. + +[212] Vid. op. et loc. cit., 41. + +[213] Vid. op. cit., 41, Vol. 65, p. 169. + +[214] Zeitschrift des Vereins für die Rübenzucker-Industrie, 1884, S. +854. + +[215] Bulletin 46, Division of Chemistry, U. S. Department of +Agriculture, p. 60. + +[216] The Analyst, Vol. 20, p. 121. + +[217] Journal American Chemical Society, Vol. 15, p. 668. + +[218] Comptes rendus, Tome 118, p. 147. + +[219] Journal of the Chemical Society, Transactions, 1895, p. 735. + +[220] Die agrikultur-chemische Versuchsstation, Halle, a/S., S. 114. + +[221] Cellulose, p. 77. + +[222] Vid. op. cit., 46, p. 63. + +[223] Zeitschrift für physiologische Chemie, Band 13, S. 84. + +[224] Zeitschrift für angewandte Chemie, 1895, S. 561. + +[225] Vid. op. cit., 52, pp. 8 et seq. + +[226] Vid. op. cit. supra, p. 15. + +[227] Composition of Wood, Agricultural Science, Vol. 7, pp. 49, 97 and +161. + +[228] Tollens; Handbuch der Kohlenhydrate: von Lippmann; Chemie der +Zuckerarten. + + + + +PART FOURTH. + +FATS AND OILS. + + +=277. Nomenclature.=—The terms fat and oil are often used +interchangeably and it is difficult in all cases to limit definitely +their application. The consistence of the substance at usual room +temperatures may be regarded as a point of demarcation. The term fat, +in this sense, is applied to glycerids which are solid or semi solid, +and oil to those which are quite or approximately liquid. A further +classification is found in the origin of the glycerids, and this +gives rise to the groups known as animal or vegetable fats and oils. +In this manual, in harmony with the practices mentioned above, the +term fat will be used to designate an animal or vegetable glycerid +which is solid, and the term oil one which is liquid at common room +temperature, _viz._, about 20°. There are few animal oils, and few +vegetable fats when judged by this standard, and it therefore happens +that the term oil is almost synonymous with vegetable glycerid and fat +with a glycerid of animal origin. Nearly related to the fats and oils +is the group of bodies known as resins and waxes. This group of bodies, +however, can be distinguished from the fats and oils by chemical +characteristics. The waxes are ethers formed by the union of fatty +acids and alcohols of the ethane, and perhaps also of the ethylene +series.[229] This chemical difference is not easily expressed and the +terms themselves often add confusion to the meaning, as for instance, +japan wax is composed mostly of fats, and sperm oil is essentially a +wax. + +=278. Composition.=—Fats and oils are composed chiefly of salts +produced by the combination of the complex base glycerol with the fat +acids. Certain glycerids, as the lecithins, contain also phosphorus +in organic combinations, nitrogen, and possibly other inorganic +constituents in organic forms. By the action of alkalies the glycerids +are easily decomposed, the acid combining with the inorganic base and +the glycerol becoming free. The salts thus produced form the soaps +of commerce and the freed base, when collected and purified, is the +glycerol of the trade. + +When waxes are decomposed by alkalies, fatty acids and alcohols of the +ethane series are produced. + +The natural glycerids formed from glycerol, which is a trihydric +(triatomic) alcohol, are found in the neutral state composed of three +molecules of the acid, united with one of the base. If R represent the +radicle of the fat acid the general formula for the chemical process by +which the salt is produced is: + + Glycerol. Acid. Salt. Water. + O.H O.R + C₃H₅O.H + 3R.OH = C₃H₅O.R + 3H₂O. + O.H O.R + +The resulting salts are called triglycerids or neutral glycyl +ethers.[230] In natural animal and vegetable products, only the neutral +salts are found, the mono- and diglycerids resulting from artificial +synthesis. For this reason the prefix tri is not necessarily used in +designating the natural glycerids, stearin, for instance, meaning the +same as tristearin. + +=279. Principal Glycerids.=—The most important glycerids which the +analyst will find are the following: + + Olein, C₃H₅O(O.C₁₈H₃₃O)₃. + Stearin, C₃H₅O(O.C₁₈H₃₅)₃. + Palmitin, C₃H₅O(O.C₁₆H₃₁O)₃. + Linolein, C₃H₅O(O.C₁₈H₃₁O)₃. + Butyrin, C₃H₅O(O.C₄H₇O)₃. + +Olein is the chief constituent of most oils; palmitin is found in palm +oil and many other natural glycerids; stearin is a leading constituent +of the fats of beeves and sheep, and butyrin is a characteristic +constituent of butter, which owes its flavor largely to this glycerid +and its nearly related concomitants. + +=280. Extraction of Oils and Fats.=—Preparatory to a physical and +chemical study of the fats and oils is their separation from the +other organic matters with which they may be associated. In the case +of animal tissues this is usually accomplished by the application +of heat. The operation known as rendering may be conducted in many +different ways. For laboratory purposes, the animal tissues holding the +fat are placed in a convenient dish and a degree of heat applied which +will liquify all the fat particles and free them from their investing +membranes. The temperature employed should be as low as possible to +secure the desired effect, but fats can be subjected for some time to +a heat of a little more than 100°, without danger of decomposition. +The direct heat of a lamp, however, should not be applied, since it +is difficult to avoid too high a temperature at the point of contact +of the flame and dish. The dry heat of an air-bath or rendering in an +autoclave or by steam is preferable. The residual animal matter is +subjected to pressure and the combined liquid fat freed from foreign +matters by filtering through a jacket filter, which is kept at a +temperature above the solidifying point of the contents. + +On a large scale, as in rendering lard, the fat is separated by steam +in closed vats which are strong enough to withstand the steam pressure +employed. For analytical purposes it is best to extract the fat from +animal tissues in the manner described, since the action of solvents +is slow on fat particles enveloped in their containing membranes, and +the fats, when extracted, are liable to be contaminated with extraneous +matters. In dried and ground flesh meal, however, the fat may be +extracted with the usual solvents. For the quantitive determination of +fat in bones or flesh, the sample, as finely divided as possible, is +thoroughly dried, and the fat separated from an aliquot finely powdered +portion by extraction with chloroform, ether, or petroleum. The action +of anhydrous ether on dried and powdered animal matters is apparently +a continuous one. Dormeyer has shown that even after an extraction of +several months additional matter goes into solution.[231] The fat in such +cases can be determined by saponification with alcoholic potash and the +estimation of the free fatty acids produced. + +From vegetable substances, such as seeds, the fat is extracted either +by pressure or by the use of solvents. For quantitive purposes, only +solvents are employed. The dry, finely ground material is exhausted +with anhydrous ether or petroleum spirit, in one of the convenient +forms of apparatus already described (=33->43=). In very oily seeds +great difficulty is experienced in securing a fine state of subdivision +suited to complete extraction. In such cases it is advisable to conduct +the process in two stages. In the first stage the material, in coarse +powder, is exhausted as far as possible and the percentage of oil +determined. The residue is then easily reduced to a fine powder, in an +aliquot part of which the remaining oil is determined in the usual way. + +[Illustration: FIG. 79.—OIL PRESS.] + +In securing oils for physical and chemical examination both pressure +and solution may be employed. The purest oils are secured by pressure +at a low temperature. To obtain anything like a good extraction some +sort of hydraulic pressure must be used. In this laboratory a press +is employed in which the first pressure is secured by a screw and +this is supplemented by hydraulic pressure in which glycerol is the +transmitting liquid. The construction of the press is shown in the +accompanying figure. + +The whole press is warmed to nearly 100°. The hot finely ground oily +material, enclosed in a cloth bag, is placed in the perforated cylinder +and compressed as firmly as possible by turning with the hands the +wheel shown at the top of the figure. The final pressure is secured by +the screw shown at the bottom of the figure whereby a piston is driven +into a cylinder containing glycerol. The degree of pressure obtained is +equal to 300 atmospheres. + +Even with the best laboratory hydraulic pressure not more than +two-thirds of the total oil contents of oleaginous seeds can be secured +and the process is totally inapplicable to securing the oil from +tissues when it exists in quantities of less than ten per cent. To get +practically all of the oil the best method is to extract with carefully +distilled petroleum of low boiling point. + +In the preparation of this reagent the petroleum ether of commerce, +containing bodies boiling at temperatures of from 35° to 80°, is +repeatedly fractioned by distillation until a product is obtained +which boils at from 45° to 60°. The distillation of this material +is conducted in a large flask heated with steam, furnished with a +column containing a number of separatory funnels and connected with an +appropriate condenser. The distillate is secured in a bottle packed +with broken ice, as shown in Fig. 80. A thermometer suspended in the +vapor of the petroleum serves to regulate the process. Too much care +to avoid accidents cannot be exercised in this operation. Not only +must steam be used in heating, but all flame and fire must be rigidly +excluded from the room in which the distillation takes place, and +the doors leading to other rooms where gas jets may be burning must +be kept closed. In the beginning of the process, as much as possible +of the petroleum boiling under 45° must be removed and rejected. The +distillation is then continued until the temperature rises above 60°. +The parts of the distillate saved between these temperatures are +redistilled under similar conditions. Other portions of the petroleum, +boiling at other temperatures, may be secured in the same way. The +products may be in a measure freed of unpleasant odors by redistilling +them from a mixture with lard. When used for quantitive purposes the +petroleum ether must leave no residue when evaporated at 100°. + +[Illustration: FIG. 80.—APPARATUS FOR FRACTIONAL DISTILLATION OF +PETROLEUM ETHER.] + +=281. Freeing Extracted Oils from Petroleum.=—The petroleum ether which +is used for extracting oils tends to give them an unpleasant odor and +flavor and its entire separation is a matter of some difficulty. The +greater part of the solvent may be recovered as described in paragraph +=43=. Heating the extracted oil for several hours in thin layers, will +remove the last traces of the solvent, but affords opportunity for +oxidation, especially in the case of drying oils. An effective means +of driving off the last traces of petroleum is to cause a current of +dry carbon dioxid to pass through the sample contained in a cylinder +and heated to a temperature of from 85° to 90°. The atmosphere of the +inert gas will preserve the oil from oxidation and the sample will, +as a rule, be found free of the petroleum odor after about ten hours +treatment. Ethyl ether or chloroform may be used instead of petroleum, +but these solvents act on other matters than the glycerids, and the +extract is therefore liable to be contaminated with more impurities +than when the petroleum ether is employed. Other solvents for fats +are carbon tetrachlorid, carbon disulfid, and benzene. In general, +petroleum ether should be employed in preference to other solvents, +except in the case of castor oil, which is difficultly soluble in both +petroleum and petroleum ethers. + +=282. Freeing Fats Of Moisture.=—Any excess of water in glycerids will +accumulate at the bottom of the liquid sample and can be removed by +decanting the fat or separating it from the oil by any other convenient +method. The warm oil may be almost entirely freed of any residual +moisture by passing it through a dry filter paper in a jacket funnel +kept at a high temperature. A section showing the construction of such +a funnel with a folded filter paper in place, is shown in Fig. 81. +The final drying, when great exactness is required, is accomplished +in a vacuum, or in an atmosphere of inert gas, or in the cold in an +exsiccator over sulfuric acid. In drying, it is well to expose the +hot oil as little as possible to the action of the air. Wherever +convenient, it should be protected from oxidation by some inert gas or +a vacuum. + +=283. Sampling for Analysis.=—It is a matter of some difficulty to +secure a representative sample of a fat or oil for analytical purposes. +The moisture in a fat is apt to be unevenly distributed, and the +sampling is to be accomplished in a manner to secure the greatest +possible uniformity. When the quantity of material is of considerable +quantity a trier may be used which will remove a cylindrical or partly +cylindrical mass from the whole length or depth. By securing several +subsamples of this kind, and well mixing them, an average sample of the +whole mass may be secured. Where the fat is found in different casks +or packages samples should be drawn from each as described above. The +subsamples are mixed together in weights corresponding to the different +casks from which they are taken and the mass obtained by this mixture +divided into three equal portions. Two of these parts are melted in a +dish at a temperature not exceeding 60°, with constant stirring, and +when fully liquid the third part is added. As a rule, the liquid fat +retains enough heat to melt the added quantity. As soon as the mixed +fats begin to grow pasty the mass is vigorously stirred to secure an +intimate mixture of the water and other foreign bodies.[232] + +[Illustration: FIG. 81.—SECTION SHOWING CONSTRUCTION OF A FUNNEL FOR +HOT FILTRATION.] + +In the case of butter fat the official chemists recommend that +subsamples be drawn from all parts of the package until about 500 grams +are secured. The portions thus drawn are to be perfectly melted in a +closed vessel at as low a temperature as possible, and when melted +the whole is to be shaken violently for some minutes till the mass is +homogeneous, and sufficiently solidified to prevent the separation of +the water and fat. A portion is then poured into the vessel from which +it is to be weighed for analysis, and this should nearly or quite fill +it. This sample should be kept in a cold place till analyzed.[233] + +=284. Estimation of Water.=—In the official method for butter fat, +which may be applied to all kinds, about two grams are dried to +constant weight, at the temperature of boiling water, in a dish with +flat bottom, having a surface of at least twenty square centimeters. + +The use of clean dry sand or asbestos is admissible, and is necessary +if a dish with round bottom be employed. + +In the method recommended by Benedikt, about five grams of the sampled +fat are placed in a small flask or beaker and dried at 100° with +occasional stirring to bring the water to the surface. + +According to the method of Sonnenschein, the sample is placed in a +flask carrying a cork, with an arrangement of glass tubes, whereby a +current of dry air may be aspirated over the fat during the process +of drying. When the flask is properly fitted its weight is taken, the +fat put in and reweighed to get the exact amount. The fat is better +preserved by aspirating carbon dioxid instead of air.[234] The moisture +may also be readily determined by drying on pumice stone, as described +in paragraph =26=. In this case it is well to conduct the desiccation +in vacuum or in an inert atmosphere to prevent oxidation. + + +PHYSICAL PROPERTIES OF FATS. + +=285. Specific Gravity.=—The specific gravity of an oil is readily +determined by a westphal balance (=53=), by a spindle, by a sprengel +tube, or more accurately by a pyknometer. The general principles +governing the conduct of the work have already been given (=48-59=). +The methods described for determining the density of sugar solutions +are essentially the same as those used for oils, but it is to be +remembered that oils and fats are lighter than water and the +graduation of the sinkers for the hydrostatic balance, and the +spindles for direct determination must be for such lighter liquids. +The necessity of determining the density of a fat at a temperature +above its melting point is manifest, and for this reason the use of +the pyknometer at a high temperature (40° to 100°) is to be preferred +to all the other processes, in the case of fats which are solid at +temperatures below 25°. + +[Illustration: FIG. 82.—BALANCE AND WESTPHAL SINKER.] + +When great delicacy of manipulation is desired, combined with +rapid work, an analytical balance and westphal sinker may be used +conjointly.[235] In this case it is well to have two or three sinkers +graduated for 20°, 25°, and 40°, respectively. Nearly all fats, +when melted and cooled to 40°, remain in a liquid state long enough +to determine their density. The sinkers are provided with delicate +thermometers, and the temperature, which at the beginning is a little +above the degree at which the sinker is graduated, is allowed to +fall to just that degree, when the equilibrium is secured in the +usual manner. The sinker is conveniently made to displace just five +grams of distilled water at the temperature of graduation, but it is +evident that a round number is not necessary, but only convenient for +calculation. + +=286. Expression of Specific Gravity.=—Much confusion arises in the +study of data of densities because the temperatures at which the +determinations are made are not expressed. The absolute specific +gravity would be a comparison of the weight of the object at 4°, with +water at the same temperature. It is evident that such determinations +are not always convenient, and for this reason the determinations of +density are usually made at other temperatures. + +In the case of a sinker, which at 35° displaces exactly five grams of +water, the following statements may be made: One cubic centimeter of +water at 35° weighs 0.994098 gram. The volume of a sinker displacing +five grams of water at that temperature is therefore 5.0297 cubic +centimeters. This volume of water at 4° weighs 5.0297 grams. In a given +case the sinker placed in an oil at 35° is found to displace a weight +equal to 4.5725 grams corresponding to a specific gravity of 35°/35° += 0.9145. From the foregoing data the following tabular summary is +constructed: + + Weight of 5.0287 cubic centimeters of oil at 35°, 4.5725 grams. + ” ” 5.0297 ” ” ” water at 35°, 5.0000 ” + ” ” 5.0297 ” ” ” ” ” 4°, 5.0297 ” + + Relative weight of oil at 35°, to water at 35°, 0.9145 grams. + ” ” ” ” ” 35°, ” ” ” 4°, 0.9092 ” + +=287. Coefficient of Expansion of Oils.=—Oils and fats of every +kind have almost the same coefficient of expansion with increasing +temperature. The coefficient of expansion is usually calculated by the +formula + + _D_₀ - _D_₀ʹ + δ = ---------------- + (_tʹ_ - _t_)_D_₀ + +in which δ represents the coefficient of expansion, _D_₀ the density at +the lowest temperature, _D_₀ʹ the density at the highest temperature, +_t_ the lowest, and _tʹ_ the highest temperatures. + +In the investigations made by Crampton it was shown that the formula +would be more accurate, written as follows:[236] + + _D_₀ - _D_₀ʹ + δ = --------------------------- + (_tʹ_ - _t_) × _D_₀ + _D_₀ʹ + --------------- + 2 + + +The absolute densities can be calculated from the formula Δ = δ + +_K_, in which Δ represents the coefficient of absolute expansion, δ +the apparent coefficient of expansions observed in glass vessels, and +_K_ the cubical coefficient of expansion of the glass vessel. The +mean absolute coefficient of expansion for fats and oils, for 1° as +determined by experiment, is almost exactly 0.0008, and the apparent +coefficient of expansion nearly 0.00077.[237] + +=288. Standard of Comparison.=—In expressing specific gravities it is +advisable to refer them always to water at 4°. The temperature at which +the observation is made should also be given. Thus the expression of +the specific gravity of lard, determined at different temperatures, is +made as follows: + + 15°.5 40° + _d_ ------ = 0.91181; _d_ ----- = 0.89679; + 4° 4° + + 100° + and _d_ ---- = 0.85997, + 4° + +indicating the relative weights of the sample under examination at +15°.5, 40°, and 100°, respectively, to water at 4°. + +=289. Densities of Common Fats and Oils.=—It is convenient to have at +hand some of the data representing the densities of common fats and +oils, and the following numbers are from results of determinations made +in this laboratory:[238] + + 15°.5 40° 100° + Temperature. _d_ = -----. _d_ = ---. _d_ = ----. + 4° 4° 4° + Leaf lard 0.91181 0.89679 0.85997 + Lard stearin 0.90965 0.89443 0.85750 + Oleostearin 0.90714 0.89223 0.85572 + Crude cottonseed oil 0.92016 0.90486 0.86739 + Summer ” ” 0.92055 0.90496 0.86681 + Winter ” ” 0.92179 0.90612 0.86774 + Refined ” ” 0.92150 0.90573 0.86714 + Compound lard ” 0.91515 0.90000 0.86289 + Olive oil 0.91505 0.89965 0.86168 + +=290. Melting Point.=—The temperature at which fats become sensibly +liquid is a physical characteristic of some importance. Unfortunately, +the line of demarcation between the solid and liquid states of this +class of bodies is not very clear. Few of them pass _per saltum_ from +one state to the other. In most cases there is a gradual transition, +which, between its initial and final points, may show a difference +of several degrees in temperature. It has been noted, further, that +fats recently melted behave differently from those which have been +solid for several hours. For this reason it is advisable, in preparing +glycerids for the determination of their melting point, to fuse them +the day before the examination is to be made. The temperature at which +a glycerid passes from a liquid to a solid state is usually higher than +that at which it resumes its solid form. If, however, the change of +temperature could be made with extreme slowness, exposing the sample +for many hours at near its critical temperature, these differences +would be much less marked. + +Many methods have been devised for determining the melting point of +fats, and none has been found that is satisfactory in every respect. In +some cases the moment at which fluidity occurs is assumed to be that +one when the small sample loses its opalescence and becomes clear. +In other cases the moment of fluidity is determined by the change of +shape of the sample or by observing the common phenomena presented by +a liquid body. In still other cases, the point at which the sample +becomes fluid is determined by the automatic completion of an electric +circuit, which is indicated by the ringing of a bell. This latter +process has been found very misleading in our experience. Only a few of +the proposed methods seem to demand attention here, and some of those, +depending on the visible liquefaction of a small quantity of the fat or +based on the physical property, possessed by all liquids when removed +from external stress, of assuming a spheroidal state will be described. +Other methods which may demand attention in any particular case may be +found in the works cited.[239] + +=291. Determination in a Capillary Tube.=—A capillary tube is dipped +into the melted fat and when filled one end of the tube is sealed in +the lamp and it is then put aside in a cool place for twenty-four +hours. At the end of this time the tube is tied to the bulb of a +delicate thermometer the length used or filled with fat being of the +same length as the thermometer bulb. The thermometer and attached fat +are placed in water, oil, or other transparent media, and gently warmed +until the capillary column of fat becomes transparent. At this moment +the thermometric reading is made and entered as the melting point of +the fat. In comparative determinations the same length of time should +be observed in heating, otherwise discordant results will be obtained. +As in all other methods, the resulting members are comparative and not +absolute points of fusion, and the data secured by two observers on the +same sample may not agree, if different methods of preparing the fat +and different rates of fusion have been employed. + +[Illustration: FIG. 83.—MELTING POINT TUBES.] + +Several modifications of the method just described are practiced, and +perhaps with advantage in some cases. In one of these a small particle +of the fat is solidified in a bulb blown on a small tube, as indicated +in Fig. 83, tube _a_. The tube, in an upright position, is heated in a +convenient bath until the particle of fat just begins to run assuming +soon the position shown in tube _b_. This temperature is determined +by a thermometer, whose bulb is kept in contact with the part of the +observation tube containing the fat particle. The rise of temperature +is continued until the fat collected at the bottom of the bulb is +entirely transparent. This is called the point of complete fusion.[240] + +Pohl covers the bulb of a thermometer with a thin film of fat, and the +instrument is then fixed in a test tube, in such a way as not to touch +the bottom, and the film of fat warmed by the air-bath until it fuses +and collects in a droplet at the end of the thermometer bulb.[241] + +Carr has modified this process by inserting the thermometer in a round +flask in such a way that the bulb of the thermometer is as nearly +as possible in the center. By this device the heating through the +intervening air is more regular and more readily controlled.[242] + +A particle of fat placed on the surface of clean mercury will melt +when the mercury is raised to the proper temperature. Where larger +quantities of the fat are employed, a small shot or pellet of mercury +may be placed upon the surface and the whole warmed until the metal +sinks. Of the above noted methods, the analyst will find some form +of capillary tube or the use of a film of the fat on the bulb of a +thermometer the most satisfactory.[243] + +Hehner and Angell have modified the sinking point method by increasing +the size of the sinker without a corresponding increase in weight. +This is accomplished by blowing a small pear-shaped float, nearly +one centimeter in diameter and about two long. The stem of the pear +is drawn out and broken off, and while the bulb is still warm, the +open end of the stem is held in mercury, and a small quantity of this +substance, sufficient in amount to cause the float to sink slowly +through a melted fat, is introduced into the bulb of the apparatus +and the stem sealed. The whole bulb should displace about one cubic +centimeter of liquid and weigh, after filling with mercury, about +three and four-tenths grams. In conducting the experiment about thirty +grams of the dry melted fat are placed in a large test tube and cooled +by immersing the tube in water at a temperature of 15°. The tube +containing the solidified fat is placed in a bath of cold water and the +sinker is placed in the center of the surface of the fat. The bath is +slowly heated until the float disappears. The temperature of the bath +is read just as the bulb part of the float disappears. The method is +recommended especially by the authors for butter fat investigations.[244] + +=298. Melting Point Determined by the Spheroidal State.=—The method +described by the author, depending on the assumption of the spheroidal +state of a particle of liquid removed from all external stress, has +been found quite satisfactory in this laboratory, and has been adopted +by the official chemists.[245] In the preparation of the apparatus there +are required: + +(_a_) a piece of ice floating in distilled water that has been recently +boiled, and (_b_) a mixture of alcohol and water of the same specific +gravity as the fat to be examined. This is prepared by boiling +distilled water and ninety-five per cent alcohol for a few minutes to +remove the gases which they may hold in solution. While still hot, the +water is poured into the test tube described below until it is nearly +half full. The test tube is then nearly filled with the hot alcohol, +which is carefully poured down the side of the inclined tube to avoid +too much mixing. If the alcohol is not added until the water has +cooled, the mixture will contain so many air bubbles as to be unfit for +use. These bubbles will gather on the disk of fat as the temperature +rises and finally force it to the top. + +[Illustration: FIG. 84.—APPARATUS FOR THE DETERMINATION OF MELTING +POINT.] + +The apparatus for determining the melting point is shown in Fig. 84, +and consists of (_a_) an accurate thermometer reading easily tenths of +a degree; (_b_) a cathetometer for reading the thermometer (but this +may be done with an eye-glass if held steadily and properly adjusted); +(_c_) a thermometer; (_d_) a tall beaker, thirty-five centimeters +high and ten in diameter; (_e_) a test tube thirty centimeters long +and three and a half in diameter; (_f_) a stand for supporting the +apparatus; (_g_) some method of stirring the water in the beaker (for +example, a blowing bulb of rubber, and a bent glass tube extending to +near the bottom of the beaker). + +The disks of fat are prepared as follows: The melted and filtered fat +is allowed to fall from a dropping tube from a height of about twenty +cubic centimeters on a smooth piece of ice floating in recently boiled +distilled water. The disks thus formed are from one to one and a half +centimeters in diameter and weigh about 200 milligrams. By pressing the +ice under the water the disks are made to float on the surface, whence +they are easily removed with a steel spatula, which should be cooled in +the ice water before using. They should be prepared a day or at least a +few hours before using. + +The test tube containing the alcohol and water is placed in a tall +beaker, containing water and ice, until cold. The disk of fat is then +dropped into the tube from the spatula, and at once sinks until it +reaches a part of the tube where the density of the alcohol-water is +exactly equivalent to its own. Here it remains at rest and free from +the action of any force save that inherent in its own molecules. + +The delicate thermometer is placed in the test tube and lowered until +the bulb is just above the disk. In order to secure an even temperature +in all parts of the alcohol mixture in the vicinity of the disk, the +thermometer is gently moved from time to time in a circularly pendulous +manner. + +The disk having been placed in position, the water in the beaker is +slowly heated, and kept constantly stirred by means of the blowing +apparatus already described. + +When the temperature of the alcohol-water mixture rises to about +6° below the melting point, the disk of fat begins to shrivel, and +gradually rolls up into an irregular mass. + +The thermometer is now lowered until the fat particle is even with the +center of the bulb. The bulb of the thermometer should be small, so as +to indicate only the temperature of the mixture near the fat. A gentle +rotatory movement from time to time should be given to the thermometer +bulb. The rise of temperature should be so regulated that the last +2° of increment require about ten minutes. The mass of fat gradually +approaches the form of a sphere, and when it is sensibly so the reading +of the thermometer is to be made. As soon as the temperature is taken +the test tube is removed from the bath and placed again in the cooler. +A second tube, containing alcohol and water, is at once placed in the +bath. The test tube (ice water having been used as a cooler) is of +low enough temperature to cool the bath sufficiently. After the first +determination, which should be only a trial, the temperature of the +bath should be so regulated as to reach a maximum of about 1°.5 above +the melting point of the fat under examination. + +The edge of the disk should not be allowed to touch the sides of the +tube. This accident rarely happens, but in case it should take place, +and the disk adhere to the sides of the tube, a new trial should be +made. + +Triplicate determinations should be made, and the second and third +results should show a near agreement. + +_Example._—Melting point of sample of butter: + + Degrees. + First trial 33.15 + Second trial 33.05 + Third trial 33.00 + +The fatty acids, being soluble in alcohol, cannot be treated as the +ordinary glycerids. But even those glycerids which are slightly soluble +in alcohol may be subjected to the above treatment without fear of +experiencing any grave disturbance of the fusing points. + +=293. Solidifying Point.=—The temperature at which a fat shows +incipient solidification is usually lower than the point of fusion. +The same difficulties are encountered in determining the temperature +of solidification as are presented in observing the true melting +point. The passage from a transparent liquid to an opaque solid is +gradual, showing all the phases of turbidity from beginning opalescence +to complete opacity. The best the analyst can do is to determine, +as accurately as possible, the temperature at which the more solid +glycerids of the mixture begin to form definite crystals. This point is +affected to a marked degree by the element of time. A fat cooled just +below its melting point will become solid after hours, or days, whereas +it could be quickly cooled far below that temperature and still be +limpid. + +The methods of observation are the same for the glycerids and fatty +acids, and the general process of determination is sufficiently set +forth in the following description of the method as used in this +laboratory.[246] + +[Illustration: FIG. 85.—APPARATUS FOR DETERMINING CRYSTALLIZING POINT.] + +The melted fat or fat acid is placed in a test tube contained in a +large bottle, which serves as a jacket to protect the tube from sudden +or violent changes of temperature. The efficiency of the jacket may +be increased by exhausting the air therefrom, as in the apparatus +for determining the heat of bromination, hereafter described. A very +delicate thermometer, graduated in tenths of a degree, and having a +long bulb, is employed. By means of the reading glass, the reading can +be made in twentieths of a degree. The arrangement of the apparatus +is shown in Fig. 85. The test tube is nearly filled with the melted +matter. The bottom of the jacket should be gently warmed to prevent a +too rapid congelation in the bottom of the test tube containing the +melted fat, and the tube is to be so placed as to leave an air space +between it and the bottom of the bottle. The thermometer is suspended +in such a manner as to have the bulb as nearly as possible in the +center of the melted fat. The thermometer should be protected from air +currents and should be kept perfectly still. In case the congealing +point is lower than room temperature the jacket may be immersed in a +cooling mixture, the temperature of which is only slightly below the +freezing point of the fatty mass. + +When crystals of fat begin to form, the descent of the mercury in the +stem of the thermometer will become very slow and finally reach a +minimum, which should be noted. As the crystallization extends inwards +and approaches the bulb of the thermometer a point will be reached when +the mercury begins to rise. At this time the partially crystallized +mass should be vigorously stirred with the thermometer and again left +at rest in as nearly, the original position as possible. By this +operation the mercury will be made to rise and its maximum position +should be noted as the true crystallizing point of the whole mass. +In comparing different samples, it is important that the elements of +time in which the first crystallization takes place should be kept, as +nearly as possible, the same. A unit of one hour in cooling the mixture +from a temperature just above its point of fusion until the incipient +crystallization is noticed, is a convenient one for glycerids and for +fat acids. + +=294. Determination of Refractive Power.=—The property of refracting +light is possessed by fats in different degrees and these differences +are of great help in analytical work. The examination may be made by +the simple refractometer of Abbe or Bertrand, or by the more elaborate +apparatus of Pulfrich. + +The comparative refractive power of fats can also be observed by +means of the oleorefractometer of Amagat-Jean or the differential +refractometer of Zune.[247] + +For details of the construction of these apparatus, with a description +of the optical principles on which they are based, the papers above +cited may be consulted. In this laboratory the instruments which have +been employed are three in number, _viz._, Abbe’s small refractometer, +Pulfrich’s refractometer using yellow light, and the oleorefractometer +of Amagat-Jean. A brief description of the methods of manipulating +these instruments is all that can be attempted in this manual. + +=295. Refractive Index.=—Refractive index is an expression employed to +characterize the measurement of the degree of deflection caused in a +ray of light in passing from one transparent medium into another. It is +the quotient of the sine of the angle of the incident, divided by the +sine of the angle of the refracted ray. + +In the case of oils which remain liquid at room temperatures, the +determinations can be made without the aid of any device to maintain +liquidity. In the case of fat which becomes solid at ordinary room +temperatures, the determination must either be made in a room +artificially warmed or the apparatus must have some device, as in +the later instruments of Abbe and Pulfrich, and in the apparatus of +Amagat-Jean, whereby the sample under examination can be maintained in +a transparent condition. In each case the accuracy of the apparatus +should be tested by pure water, the refractive index of which at 18° +is 1.333. The refractive index is either read directly on the scale +as in Abbe’s instrument, or calculated from the angles measured as in +Pulfrich’s apparatus. + +[Illustration: FIG. 86.—ABBE’S REFRACTOMETER.] + +=296. Abbe’s Refractometer.=—For practical use the small instrument +invented by Abbe will be found sufficient. The one which has been +in use for many years in this laboratory is shown in Fig. 86. The +illustration represents the apparatus in the position preliminary to +reading the index. In preparing the sample of oil for observation the +instrument is turned on its axis until the prisms between which the +oil is placed assume a horizontal position, as is seen in Fig. 87. The +movable prism is unfastened and laid aside, the fixed prism covered +with a rectangular shaped piece of tissue paper on which one or two +drops of the oil are placed. The movable prism is replaced in such a +manner as to secure a complete separation of the two prisms by the film +of oiled tissue paper. A little practice will enable the analyst to +secure this result. + +After the paper disk holding the fat is secured by replacing the upper +prism, the apparatus is placed in its normal position and the index +moved until the light directed through the apparatus by the mirror +shows the field of vision divided into dark and light portions. The +dispersion apparatus is now turned until the rainbow colors on the part +between the dark and light fields have disappeared. Before doing this, +however, the telescope, the eyepiece of the apparatus, is so adjusted +as to bring the cross lines of the field of vision distinctly into +focus. The index of the apparatus is now moved back and forth until the +line of the two fields of vision falls exactly at the intersection of +the cross lines. The refractive index of the fat under examination is +then read directly upon the scale by means of a small magnifying glass. +To check the accuracy of the first reading, the dispersion apparatus +should be turned through an angle of 180° until the colors have again +disappeared, and, after adjustment, the scale of the instrument again +read. These two readings should nearly coincide, and their mean is the +true reading of the fat under examination. + +[Illustration: FIG. 87.—CHARGING POSITION OF REFRACTOMETER.] + +For butter fats the apparatus should be kept in a warm place, the +temperature of which does not fall below 30°. For reducing the results +obtained to a standard temperature, say 25°, the factor 0.000176 may be +used. As the temperature rises the refractive index falls. + +_Example._—Refractive index of a butter fat determined at 32°.4 = +1.4540, reduced to 25° as follows: 32.4 -25 = 7.4; 0.000176 × 7.4 = +0.0013; then 1.4540 + 0.0013 = 1.4553. + +The instrument used should be set with distilled water at 18°, the +theoretical refractive index of water at that temperature being 1.333. +In the determination above given, the refractive index of pure water +measured 1.3300; hence the above numbers should be corrected for theory +by the addition of 0.0030, making the corrected index of the butter fat +mentioned at the temperature given, 1.4583. + +=297. Pulfrich’s Refractometer.=—For exact scientific measurements, +Pulfrich’s apparatus has given here entire satisfaction. In this +instrument a larger quantity of the oil is required than for the +abbe, and this quantity is held in a cylindrical glass vessel luted +to the top of the prism. The method of accomplishing this and also an +illustration of the refraction of the rays of light are shown in Fig. +88. + +[Illustration: FIG. 88.—PRISM OF PULFRICH’S REFRACTOMETER.] + +The angle _i_ is measured by a divided circle read with the aid of a +small telescope. The index of the prism of highly refractive glass +_N_ is known. The oil is seen at _n_. The light used is the yellow +sodium ray (_D_). From the observed angle the refractive index of n is +calculated from the formula + + ______________ + _n_ = √_N_² - sin²_i_. + + +For convenience the values of _n_ for all usual values of _i_ are +computed once for all and arranged for use in tabular form. The latest +model of Pulfrich’s apparatus, arranged both for liquid and solid +bodies, and also for spectrometric observation is shown in Fig. 89. + +When the sodium light is used it is placed behind the apparatus and +the light is collected and reflected on the refractive prism by the +lens _N_. Through _H_ and _G_ is secured the micrometric reading of the +angle on the scale _D_ by means of the telescopic arrangement _F E_. +For regulating the temperature of the oil and adjacent parts, a stream +of water at any desired temperature is made to circulate through _L_ +and _S_ in the direction indicated by the arrows. The manner in which +this is accomplished is shown in the cross section of that part of the +apparatus as indicated in Fig. 90. + +[Illustration: FIG. 89.—PULFRICH’S NEW REFRACTOMETER.] + +[Illustration: FIG. 90.—HEATING APPARATUS FOR PULFRICH’S REFRACTOMETER.] + +[Illustration: FIG. 91.—SPECTROMETER ATTACHMENT.] + +For further details of the construction and operation of the apparatus +the original description may be consulted.[248] + +In case a spectrometric observation is desired the _H_ ray, for +instance, is produced by the geissler tube _Q_, Fig. 91. The light is +concentrated and thrown upon the refractive prism by the lens _P_, the +lens _N_, Fig. 89, being removed for this purpose. + +Tables, for correcting the dispersion and for calculating the indices +for each angle and fraction thereof, and for corrections peculiar to +the apparatus, accompany each instrument. + +=298. Refractive Indices of some Common Oils.=—The following numbers +show the refractive indices obtained by Long for some of the more +common oils. The light used was the yellow ray of the sodium flame.[249] + + Refractive Calculated + Name. Temperature. index. for 25°. + Olive oil (France) 26°.6 1.4673 1.4677 + ” ” (California) 25°.4 1.4677 1.4678 + Cottonseed oil 24°.8 1.4722 1.4721 + ” ” 26°.3 1.4703 1.4709 + ” ” 25°.3 1.4718 1.4719 + Sesamé oil 24°.8 1.4728 1.4728 + ” ” 26°.8 1.4710 1.4716 + Castor ” 25°.4 1.4771 1.4773 + Lard ” 27°.3 1.4657 1.4666 + Peanut ” 25°.3 1.4696 1.4696 + +In case of the use of Abbe’s apparatus, in which diffused sunlight +is the source of the illumination, the numbers obtained cannot be +compared directly with those just given unless the apparatus be first +so adjusted as to read with distilled water at 18°, 1.333. In this case +the reading of the scale gives the index as determined by the yellow +ray. The numbers obtained with Abbe’s instrument for some common oils +are given below.[250] + +In the determinations the instrument was set with water at 18°, reading +1.3300, and they were corrected by adding 0.0030 in order to compensate +for the error of the apparatus. + + Material. Calculated for 25°. Corrected index. + Lard 1.4620 1.4650 + Cotton oil 1.4674 1.4704 + Olive oil stearin 1.4582 1.4610 + Lard stearin 1.4594 1.4624 + +=299. Oleorefractometer.=—Instead of measuring the angular value of +the refractive power of an oil it may be compared with some standard +on a purely arbitrary scale. Such an apparatus is illustrated by the +oleorefractometer of Amagat-Jean, or by Zeiss’s butyrorefractometer. + +In the first named instrument, Fig. 92, the oil to be examined is +compared directly with another typical oil and the shadow produced by +the difference in refraction is located on a scale read by a telescope +and graduated for two different temperatures.[251] The internal +structure of the apparatus is shown in Fig. 93. + +[Illustration: FIG. 92.—OLEOREFRACTOMETER.] + +[Illustration: FIG. 93.—SECTION SHOWING CONSTRUCTION OF +OLEOREFRACTOMETER.] + +In the center of the apparatus a metal cylinder, _A_, is found carrying +two plate glass pieces, _C B_, so placed as to form an angle of 107°. +This cylinder is placed in a larger one, provided with two circular +glass windows. To these two openings are fixed to the right and left, +the telescopic attachments, _G_, _V, S, E_, and the apparatus _M, H, +Sʹ_, _Eʹ_, for rendering the rays of light parallel. The field of +vision is divided into two portions, light and dark, by a semicircular +stop inserted in the collimator, and contains the double scale shown +in the figure placed at _H_. The field of vision is illuminated by a +gas or oil lamp placed at a convenient distance from the collimator. +The inner metallic cylinder _A_ is surrounded with an outer one, to +which the optical parts are attached at _D Dʹ_ by means of plane glass +plates. This cylinder is in turn contained in the large water cylinder +_P P_, carrying a thermometer in the opening shown at the top on the +left. The manipulation of the apparatus is very simple. The outer +cylinder is filled with water, at a temperature below 22°, the middle +one with the typical oil furnished with the instrument, the cover of +the apparatus carrying the thermometer placed in position and the +cup-shaped funnel inserted in the cylinder _A_, which is at first also +filled with the typical oil. The whole system is next brought slowly +to the temperature of 22° by means of the lamp shown in Fig. 92. The +telescope is adjusted to bring the scale of the field of vision into +focus and the line dividing the light and shadow of the field should +fall exactly on 0°_a_. If this be not the case the 0° is adjusted by +screws provided for that purpose until it is in proper position. The +typical oil is withdrawn from _A_ by the cock _R_, the cylinder washed +with a little of the oil to be examined and then filled therewith. On +again observing the field of vision the line separating the shadow from +the light will be found moved to the right or left, if the oil have +an index different from that of the typical oil. The position of the +dividing line is read on the scale. + +For fats the temperature of the apparatus is brought exactly to 45° +and the scale 0°b is used. In other respects the manipulation for +the fats is exactly that described for oils. In the use of 0°a, in +case the room be warmer than 22°, all the liquids employed should be +cooled below 22° before being placed in the apparatus. It is then only +necessary to wait until the room temperature warms the system to 22°. +In the case of fats it is advisable to heat all the liquids to about +50° and allow them to cool to 45° instead of heating them to that +temperature by means of the lamp. + +One grave objection to this instrument is found in the absence of the +proper scientific spirit controlling its manufacture and sale, as +evidenced by the attempt to preserve the secret of the composition +of the typical oil and the negligence in testing the scale of the +instruments which will be pointed out further along. + +According to Jean[252] the common oils, when purified, give the +following readings at 22°: + + Peanut oil +3.5 to +6.5 + Colza ” +17.5 ” +21.0 + Cotton ” +18.0 ” +18.0 + Linseed ” +47.0 ” +54.5 + Lard ” +5.5 ” +5.5 + Olive ” +1.5 ” 0.0 + Sesamé ” +17.5 ” +19.0 + Oleomargarin -15.0 ” -15.0 + Butter fat -30.0 ” -30.0 + Mutton oil 0.0 ” 0.0 + Fish ” +38.0 ” +38.0 + +In this instrument, therefore, vegetable and fish oils, as a rule, show +a right hand, and animal fats a left hand deviation. + +The oleorefractometer has been extensively used in this laboratory and +the data obtained thereby have been found useful. We have not found, +however, the values fixed by Jean to be constant. The numbers for lard +have varied from -3.0 to -10.0, and other fats have shown almost as +wide a variation from the values assigned by him. + +Jean states that the number for lard, determined by the +oleorefractometer, is -12, and he gives a definite number for each of +the common oils and fats. On trying the pure lards of known origin in +this instrument, I have never yet found one that showed a deviation of +-12 divisions of the scale; but I have no doubt that there are many +such lards in existence. The pure normal lards derived from the fat +of a single animal would naturally show greater variations in their +chemical and physical properties, than a typical lard derived from +the mixed fats of a great many animals. In leaf lard, rendered in the +laboratory, the reading of the oleorefractometer was found to be -10°, +while with the intestinal lard it was -9°. On the other hand, a lard +rendered from the fat from the back of the animal showed a reading of +only -3°, and a typical cottonseed oil a reading of +12°. According +to the statement of Jean, a lard which gives even as low a refractive +number as -9, by his instrument, would be adjudged at least one-quarter +cottonseed oil. + +After a thorough trial of the instrument of Jean, I am convinced that +it is of great diagnostic value, but if used in the arbitrary manner +indicated by the author it would lead to endless error and confusion. +In other words, this instrument is of greater value in analyses than +Abbe’s ordinary refractometer, because it gives a wider expansion in +the limits of the field of vision, and therefore can be more accurately +read, but it is far from affording a certain means of discovering +traces of adulteration with other fats. + +=300. Variations in the Instruments.=—In the use of the +oleorefractometer, attention should be called to the fact that, through +some negligence in manufacture, the instruments do not give, in all +instances, the same reading with the same substance. Allen obtained the +following data with a sample of lard examined in three instruments, +_viz._, 4°.5, 6°, and 11°. Such wide differences in the scales of +the instruments cannot fail to disparage the value of comparative +determinations. + +The variations in samples of known origin, when read on the same +instrument, however, will show the range of error to which the +determinations made with the oleorefractometer are subject. Pearmain +has tabulated a large number of observations of this kind, covering 240 +samples of oils.[253] + +Following are the data relating to the most important oils. + + AT 22°. + + Highest Lowest Mean + reading. reading. reading. + Name of oil. Degrees. Degrees. Degrees. + + Almond 10.5 8.0 9.5 + Peanut 7.0 5.0 6.0 + Castor 42.0 39.0 40.0 + Codliver 46.0 40.0 44.0 + Cottonseed (crude) 17.0 16.0 16.5 + ” (refined) 23.0 17.0 21.5 + Lard oil -1.0 0.0 0.0 + Linseed (crude) 52.0 48.0 50.0 + ” (refined) 54.0 50.0 52.5 + Olive 3.5 1.0 2.0 + Rape 20.0 16.0 17.5 + Sesamé 17.0 13.0 15.5 + Sunflower 35.0 35.0 35.0 + Tallow oil -5.0 -1.0 -3.0 + Oleic acid -33.0 -29.0 -32.0 + + AT 45°. + + Butter -34.0 -25.0 -30.0 + Oleomargarin -18.0 -13.0 -15.0 + Lard -14.0 -8.0 -10.5 + Tallow -18.0 -15.0 -16.0 + Paraffin 58.5 54.0 56.0 + +[Illustration: FIG. 94.—BUTYROREFRACTOMETER.] + +=301. Butyrorefractometer.=—Another instrument graduated on an +arbitrary scale is the butyrorefractometer of Zeiss. This apparatus, +which resembles in some respects the instrument of Abbe, differs +therefrom essentially in dispensing with the revolving prisms of +Amici, whereby the chromatic fringing due to dispersion is corrected, +and on having the scale fixed for one substance, in this instance, +pure butter fat. The form of the instrument is shown in Fig. 94. The +achromatization for the butter fat is secured in the prisms between +which a film of the fat is placed, as in the Abbe instrument. When +a fat, differing from that for which the instrument is graduated is +introduced, the fringes of the dark and light portions of the field +will not only be colored (difference in dispersion), but the line of +separation will also be displaced (difference in refractive power). The +apparatus is therefore used in the differential determination of these +two properties. It must not be forgotten, however, that butter fats +differ so much in these properties among themselves as to make possible +the condemnation of a pure as an adulterated sample. + +=302. Method of Charging the Apparatus.=—The prism casing of the +instrument is opened by turning the pin _F_ to the right and pushing +the half _B_ of the prism casing aside. The prism and its appendages +must be cleaned with the greatest care, the best means for this purpose +being soft clean linen moistened with a little alcohol or ether. + +Melt the sample of butter in a spoon and pour it upon a small paper +filter held between the fingers and apply the first two or three drops +of clear butter fat so obtained to the surface of the prism contained +in prism casing _B_. For this purpose the apparatus should be raised +with the left hand so as to place the prism surface in a horizontal +position. + +Press _B_ against _A_ and replace _F_ by turning it in the opposite +direction into its original position; thereby _B_ is prevented from +falling back and both prism surfaces are kept in close contact. + +=303. Method of Observation.=—While looking into the telescope, give +the mirror _J_ such a position as to render the critical line which +separates the bright left part of the field from the dark right part +distinctly visible. It may also be necessary to move or turn the +instrument about a little. First it will be necessary to ascertain +whether the space between the prism surfaces be uniformly filled with +butter, for, if not, the critical line will not be distinct. + +By allowing a current of water of constant temperature to flow through +the apparatus, some time previous to the taking of the reading, the at +first somewhat hazy critical line approaches in a short time, generally +after a minute, a fixed position and quickly attains its greatest +distinctness. When this point has been reached note the appearance of +the critical line (_i. e._, whether colorless or colored and in the +latter case of what color); also note the position of the critical line +on the centesimal scale, which admits of the tenth divisions being +conveniently estimated, and at the same time read the thermometer. By +making an extended series of successive readings and by employing an +assistant for melting and preparing the small samples of butter, from +twenty-five to thirty refractometric butter tests may, after a little +practice, be made in an hour. + +The readings of the refractive indices of a large number of butter +samples made at 25° are, by means of a table which will be found +below, directly reduced to scale divisions and yield the following +equivalents:[254] + + Natural butter (1.4590-1.4620) : 49.5-54.0 scale divisions. + Margarin (1.4650-1.4700) : 58.6-66.4 ” ” + Mixtures (1.4620-1.4690) : 54.0-64.8 ” ” + +Whenever, in the refractometric examination of butter at a temperature +of 25°, higher values than 54.0 are found for the critical lines these +samples will, according to Wollny, by chemical analysis, always be +found to be adulterated; but in all samples in which the value for +the position of the critical line does not fall below 52.5, chemical +analysis maybe dispensed with and the samples may be pronounced to be +pure butter. + +In calculating the position of the critical line for other temperatures +than 25° allow for 1° variation of temperature a mean value of 0.55 +scale division. The following table, which has been compiled in this +manner, shows the values corresponding to various temperatures, each +value being the upper limit of scale divisions admissible in pure +butter: + + Temp. Sc. div. Temp. Sc. div. Temp. Sc. div. Temp. Sc. div. + 45° 41.5 40° 44.2 35° 47.0 30° 49.8 + 44° 42.0 39° 44.8 34° 47.5 29° 50.3 + 43° 42.6 38° 45.3 33° 48.1 28° 50.8 + 42° 43.1 37° 45.9 32° 48.6 27° 51.4 + 41° 43.7 36° 46.4 31° 49.2 26° 51.9 + 40° 44.2 35° 47.0 30° 49.8 25° 52.5 + +If, therefore, at any temperature between 45° and 25° values be found +for the critical line, which are less than the values corresponding to +the same temperature according to the table, the sample of butter may +safely be pronounced to be natural, _i. e._, unadulterated butter. If +the reading show higher numbers for the critical line the sample should +be reserved for chemical analysis. A special thermometer for use in the +examination of butter will be described in the section devoted to dairy +products. + +=304. Range of Application of the Butyrorefractometer.=—The extended +range of the ocular scale of the refractometer, _n_ = 1.42 to 1.49, +which embraces the refractive indices of the majority of oils and fats, +renders the instrument applicable for testing oils and fats and also +for examining glycerol. + +By reference to the subjoined table the scale divisions may be +transformed into terms of refractive indices. It gives the refractive +indices for yellow light for every ten divisions of the scale. The +differential column Δ gives the change of the refractive indices in +terms of the fourth decimal per scale division. Owing to the accuracy +with which the readings can be taken (0.1 scale division) the error of +the value of _n_ rarely exceeds one unit of the fourth decimal of _n_. + + +TABLE OF REFRACTIVE INDICES. + + Scale div. n_{D}. Δ. Scale div. n_{D}. Δ. + + 0 1.4220 8.0 50 1.4593 6.6 + 10 1.4300 7.7 60 1.4650 6.4 + 20 1.4377 7.5 70 1.4723 6.0 + 30 1.4452 7.2 80 1.4783 5.7 + 40 1.4524 6.9 90 1.4840 5.5 + 50 1.4593 100 1.4895 + +The process of observation is precisely the same as that already +described. In cases, however, where the critical line presents very +broad fringes (turpentine, linseed oil, etc.) it is advisable to repeat +the reading with the aid of a sodium flame. + +=305. Viscosity.=—An important property of an oil, especially when its +lubricating qualities are considered, is the measure of the friction +which the particles exert on other bodies and among themselves, in +other words, its viscosity. In the measure of this property no definite +element can be considered, but the analyst must be content with +comparing the given sample with the properties of some other liquid +regarded as a standard. The usual method of procedure consists in +determining the time required for equal volumes of the two liquids to +pass through an orifice of given dimensions, under identical conditions +of temperature and pressure. In many instances the viscosity of oils is +determined by comparing them with water or rape oil, while, in other +cases, a solution of sugar is employed as the standard of measurement. + +In case rape oil be taken as a standard and its viscosity represented +by 100 the number representing the viscosity of any other oil may be +found by multiplying the number of seconds required for the outflow of +fifty cubic centimeters by 100 and dividing by 535. If the specific +gravity vary from that of rape oil, _viz._, 0.915, at 15°, a correction +must be made by multiplying the result obtained above by the specific +gravity of the sample and dividing the product by 0.915. If _n_ be the +observed time of outflow in seconds and _s_ the specific gravity the +viscosity is expressed as follows:[255] + + _n_ × 100 × _s_ _n_ × 100 × _s_ + _V_ = ---------------- = ----------------. + 535 × 0.195 489.525 + +[Illustration: FIG. 95.—DOOLITTLE’S VISCOSIMETER.] + +It is important that the height of the oil in the cylinders from which +it is delivered be kept constant, and this is secured by supplying +additional quantities, on the principle of the mariotte bottle. + +=306. The Torsion Viscosimeter.=—In this laboratory the torsion +viscosimeter, based on the principle described by Babcock is used. +The instrument employed is the one described by Doolittle.[256] The +construction of the apparatus is illustrated in Fig. 95. + +A steel wire is suspended from a firm support and fastened to a stem +which passes through a graduated horizontal disk, thus permitting the +accurate measurement of the torsion of the wire. The disk is adjusted +so that the index point reads exactly _0_, thus showing that there is +no torsion in the wire. A brass cylinder seven centimeters long by +five in diameter, having a slender stem by which to suspend it, is +immersed in the oil and fastened by a thumbscrew to the lower part of +the stem of the disk. The oil cup is surrounded by a bath of water or +high fire-test oil, according to the temperature at which it is desired +to determine the viscosity. This temperature obtained, while the disk +is resting on its supports, the wire is twisted 360° by rotating the +milled head at the top. The disk being released, the cylinder rotates +in the oil by virtue of the torsion of the wire. + +The action now observed is identical with that of the simple pendulum. + +If there were no resistance to be overcome, the disk would return to +0, and the momentum thus acquired would carry it 360° in the opposite +direction. But the resistance of the oil to the rotation of the +cylinder causes the revolution to fall short of 360°, and the greater +the viscosity of the oil the greater will be the resistance, and also +the retardation. This retardation is found to be a very delicate +measure of the viscosity of the oil. + +This retardation may be read in a number of ways, but the simplest +is to read directly the number of degrees of retardation between the +first and second complete arcs covered by the rotating pendulum. For +example, suppose the wire be twisted 360° and the disk released so that +rotation begins. In order to obtain an absolute reading to start from, +which shall be independent of any slight error in adjustment, ignore +the starting point and make the first reading of the index at the end +of the first swing. The disk is allowed to complete a vibration and +the needle is read again at its nearest approach to the first point +read. The difference in the two readings will measure the retardation +due to the viscosity of the liquid. In order to eliminate errors +duplicate determinations are made, the milled head being rotated in an +opposite direction in the second one. The mean of the two readings will +represent the true retardation. Each instrument is standardized in a +solution of pure cane sugar, as proposed by Babcock, and the viscosity, +in each case, is a number representing the number of grams of sugar in +100 cubic centimeters, which, at 22°, would produce the retardation +noted. + +Each instrument is accompanied by a table which contains the +necessary corrections for it and the number expressing the viscosity, +corresponding to the different degrees of retardation, as read on the +index. The following numbers, representing the viscosity of some oils +as determined by the method of Doolittle, were obtained by Krug.[257] + + Peanut oil 48.50 + Olive ” 53.00 + Cottonseed ” 46.25 + Linseed ” 33.50 + +=307. Microscopic Appearance.=—When fats are allowed to slowly +crystallize from an ethereal solution they may afford crystalline +forms, which, when examined with a magnifying glass, yield valuable +indications of the nature and origin of the substance under +examination.[258] + +The method of securing fat crystals for microscopic examination, which +has been used in this laboratory, is as follows: From two to five +grams of the fat are placed in a test tube and dissolved in from ten +to twenty cubic centimeters of ether. The tube is loosely stoppered +with cotton and allowed to stand, for fifteen hours or longer, in a +moderately warm room where no sudden changes of temperature are likely +to take place. It is advisable to prepare several solutions of the same +substance with varying properties of solvent, for it is not possible +to secure in a given instance those conditions which produce the most +characteristic crystals. The rate and time of the crystallization +should be such that the microscopic examination can take place when +only a small portion of the fat has separated in a crystalline +condition. A drop of the mass containing the crystals is removed by +means of a pipette, placed on a slide, a drop of cotton or olive +oil added, a cover glass gently pressed down on the mixture and the +preparation subjected to microscopic examination. Several slides should +be prepared from the same or different crystallizations. Sometimes the +results of an examination made in this way are very definite, but the +analyst must be warned not to expect definite data in all cases. Often +the microscopic investigations result in the production of negative or +misleading observations, and, at best, this method of procedure must be +regarded only as helpful and confirmatory. + +A modification of the method of preparation described above has been +suggested by Gladding.[259] About five grams of the melted fat are +placed in a small erlenmeyer, dissolved in a mixture of ten cubic +centimeters of absolute alcohol mixed with half that quantity of ether. +The flask is stoppered with a plug of cotton and allowed to stand in a +cool place for about half an hour. By this treatment the more easily +crystallizable portions of the fat separate in a crystalline form, +while the triolein and its nearly related glycerids remain in solution. +The crystalline product is separated by filtration through paper wet +with alcohol and washed once with the solvent mentioned above. After +drying in the air for some time the crystals are removed from the paper +and dissolved in twenty-five cubic centimeters of ether, the cotton +plug inserted, and the erlenmeyer placed, in a standing position, in +a large beaker containing water. The water jacket prevents any sudden +changes of temperature and affords an opportunity for the uniform +evaporation of the ether which should continue for fifteen hours or +longer in a cool place. + +Other solvents, _viz._, alcohol, chloroform, carbon disulfid, carbon +tetrachlorid, petroleum and petroleum ether have been extensively used +in the preparation of fat crystals for microscopic examination, but in +our experience none of these is equal to ether when used as already +described. + +=308. Microscopic Appearance of Crystals of Fats.=—For an extended +study and illustration of the characteristics of fat crystals the +bulletin of the Division of Chemistry, already cited, may be consulted. +In the case of lard, there is a tendency, more or less pronounced, to +form prismatic crystals with rhombic ends. Beef fat on the other hand +shows a tendency to form fan-shaped crystals in which the radii are +often curved. + +Typical crystals of swine and beef fat are shown in the accompanying +figures, 96 and 97.[260] In mixtures of swine and beef fats the typical +crystals are not always developed, but in most cases the fan-shaped +crystals of the beef fat will appear more or less modified when that +fat forms twenty per cent or more of the mixture. When only five or +ten per cent of the beef fat on the one hand or a like amount of +swine fat on the other are present the expectation of developing any +characteristic crystals of the minimum constituent is not likely to be +realized. + +The typical crystals of lard are thought by some experts to be palmitin +and those of beef fat stearin, but no direct evidence has been adduced +in support of these _a priori_ theories. + +In the experience of this laboratory, as described by Crampton,[261] +the differences between the typical crystallization of beef and swine +fats are plainly shown. In mixed fats, on the contrary, confusing +observations are often made. In a mixture of ten per cent of beef +and ninety per cent of swine fats a uniform kind of crystallization +is observed, not distinctly typical, but the characteristics of the +lard crystals predominate. In many cases a positive identification +of the crystals is only made possible by repeated crystallizations. +In the examination of so-called refined lards, which are mixtures of +lard and beef fat, the form of aggregation of the crystals is found +to resemble the fan-shaped typical forms of beef fat. When the single +crystals, however, are examined with a higher magnifying power, they +are not found to be pointed but blunt, and some present the appearance +of plates with oblique terminations, but not so characteristic as +those obtained from pure lard. In other cases in compound lards no +beef fat crystals are observed and these lards may have been made +partly of cotton oil stearin. When a lard crystal presents its edge +to observation it may readily escape identification, or may even be +mistaken for a crystal of beef fat. In order to insure a side view the +cover glass should be pressed down with a slight rotatory movement, +whereby some of the lard crystals at least may be made to present a +side view. + +=309. Observation of Fat Crystals with Polarized Light.=—The +appearance of fat crystals, when observed by means of polarized light +alone or with the adjunct of a selenite plate, is often of value in +distinguishing the nature and origin of the sample.[262] + +Every fat and oil which is amorphous will present the same set of +phenomena when observed with polarized light through a selenite plate, +but when a fat has been melted and allowed to cool slowly the field of +vision will appear mottled and particolored when thus examined. This +method has been largely used in the technical examination of butter +for adulterants, and the microscope is extensively employed by the +chemists of the Bureau of Internal Revenue for this purpose. In the +examination of the crystals of butter fat by polarized light a cross is +usually observed when the nicols are turned at the proper angle, but +the cross, while almost uniformly seen with butter, is not distinctive, +since other fats often show it. These forms of crystals are best +obtained by heating the butter fat to the boiling-point of water for +about a minute and then allowing it to slowly solidify, and stand for +twenty-four hours. + +Pure butter, properly made, is never subjected to fusion, and hence, +when examined through a selenite plate, presents a uniform field of +vision similarly illuminated and tinted throughout. In oleomargarin, +the fats are sometimes, during their preparation, in a fused condition. +The field of vision is therefore filled to a greater or less extent +with crystals more or less perfect in form. Some of these crystals, +being doubly refracting, will impart to a selenite field a mottled +appearance. Such a phenomenon is therefore indicative of a fraudulent +butter or of one which has been at some time subjected to a temperature +at or above its fusing point. + +=310. Spectroscopic Examination of Oils.=—The presence of chlorophyll +or of its alteration products is a characteristic of crude oils of +vegetable origin. In refined oils, even when of a vegetable origin, +all traces of the chlorophyll products may disappear. The absorption +bands given by oils are not all alike and in doubtful cases a suspected +sample should be compared with one of known origin. + +In conducting the examination, the oil in a glass vessel with parallel +sides, is placed in front of the slit of the spectroscope and any +absorption band is located by means of the common divided scale and by +the color of the spectrum on which it falls. Olive and linseed oils +give three sharply defined absorption bands, a very dark one in the +red, a faint one on the orange and a well marked one in the green. + +Sesame, arachis, poppyseed and cottonseed oils also show absorption +bands. Castor and almond oils do not affect the spectrum. + +[Illustration: Fig. 96. LARD CRYSTALS × 65.] + +[Illustration: Fig. 97. REFINED LARD (BEEF FAT) CRYSTALS × 65. + +A. Hoen & Co., Lithocaustic.] + +Rape and flaxseed oils absorb a part of the spectrum but do not affect +the rest of it. The spectroscope is of little practical utility in oil +analysis.[263] + +=311. Critical Temperature of Solution.=—The study of the critical +temperature of solution of oils has been made by Crismer, who finds it +of value in analytical work.[264] If a fatty substance be heated under +pressure, with a solvent, _e. g._, alcohol, it will be noticed that as +the temperature rises the meniscus of separation of the two liquids +tends to become a horizontal plane. If at this point the contents of +the tube be thoroughly mixed by shaking and then be left at rest, a +point will soon be reached at which the two liquids again separate, +and this point is distinctly a function of temperature. Following +is a description of a convenient method of determining the critical +temperature of the solution of fats and oils for experimental purposes. +Tubes are prepared for holding the reagents in such a way that, after +the introduction of the fat and alcohol, they can be easily sealed. The +capacity of these tubes should be about five cubic centimeters. They +should be charged with about one cubic centimeter of the dry filtered +fat and about twice that quantity of ninety-five per cent alcohol. +Care should be exercised to avoid touching the upper sides of the tube +with the reagents. When charged the tubes are sealed in the flame of +a lamp and attached to the bulb of a delicate thermometer in such a +manner as to have the surface of its liquid contents even with the top +of the bulb. The tube is conveniently fastened to the thermometer by a +platinum wire. For duplicate determinations two tubes may be fastened +to the same thermometric bulb. The apparatus thus prepared is placed in +a large vessel filled with strong sulfuric acid. The operator should +be careful to protect himself from the danger which might arise from +an explosion of the sealed tubes during heating. It is advisable in +all cases to observe the reaction through a large pane of clear glass. +The bath of sulfuric acid is heated by any convenient means and an +even temperature throughout the mass is secured by stirring with the +thermometer and its attachments. When the meniscus which separates +the two liquids becomes a horizontal plane the thermometer is removed +and the liquid in the tubes well mixed until it appear homogeneous. +The thermometer is replaced in the bath, which is allowed to cool +slowly, and the phenomena which take place in the sealed tubes are +carefully noted. The critical temperature of solution is that at which +the two liquids begin to separate. This moment is easily noted. It +is, moreover, preceded by a similar phenomenon taking place in the +capillary part of the tube which retains a drop of the mixture on +shaking. In this droplet an opalescence is first noted. In the mass of +the liquid this opalescence, a few seconds afterwards, is observed to +permeate the whole, followed by the formation of zones and finally of +the reappearance of the meniscus of separation between the two liquids. +The temperature at this moment of opalescence preceding the separation +of the liquid is the critical temperature of solution. With alcohol of +0.8195 specific gravity, at 15°.5 (ninety-five per cent), the observed +critical temperatures for some of the more common fats and oils are as +given below: + + Butter fat 100°.0 + Oleomargarin 125°.0 + Peanut oil 123°.0 + Cotton ” 116°.0 + Olive ” 123°.0 + Sesamé ” 121°.0 + Colza ” 132°.5 + Mutton tallow 116°.0 + Beef marrow 125°.0 + Nut oil 100°.5 + +When the alcohol is not pure or if it be of a different density from +that named, the numbers expressing the critical temperature of solution +will vary from those given above. + +=312. Polarization.=—The pure glycerids are generally neutral to +polarized light. In oils the degree of polarization obtained is often +variable, sometimes to the right and sometimes to the left. Olive oil, +as a rule, shows a slight right hand polarization. Peanut, sesamé, and +cottonseed oils vary in polarizing power, but in no case is it very +marked. Castor oil polarizes slightly to the right. + +In determining the polarizing power of an oil it should be obtained in +a perfectly limpid state by filtration and observed through a tube of +convenient length, as a rule, 200 millimeters. The deviation obtained +may be expressed in divisions of the sugar scale of the instrument or +in degrees of angular rotation. + +=313. Turbidity Temperature.=—The turbidity temperature of a fat, when +dissolved in glacial acetic acid, as suggested by Valenta, may prove +of some diagnostic value.[265] The fats are dissolved, with the aid of +heat, in glacial acetic acid and, on slowly cooling, the temperature at +which they become turbid is observed. The following data observed by +Jones are given for comparison.[266] + +The numbers represent the turbidity temperature of the fat when treated +with the glacial acetic acid, and allowed to cool slowly. Butter fat, +from 40° to 70°, mostly from 52° to 65°; oleomargarin, 95° to 106°; +rape oil, 101°; sesamé oil, 77°; linseed oil, 53° to 57°; lard oil, +96°; olive oil, 89°; peanut oil, 61° to 88°. + +It is important in this test that the acetic acid be absolutely +glacial. About three cubic centimeters of the glacial acetic acid, and +three of the fat, should be used. + + +CHEMICAL PROPERTIES. + +=314. Solubility in Alcohol.=—As has already been noted, the glycerids +are freely soluble in ether, chloroform, carbon bisulfid, acetone, +carbon tetrachlorid, and some other less commonly used solvents. Their +solubility in absolute alcohol is variable and the determination of its +degree may often be useful in analytical work. + +The method used by Milliau for determining the degree of solubility +is as follows:[267] The fatty matter is deprived of its free acids by +shaking for half an hour with twice its volume of ninety-five per cent +alcohol. After standing until the liquids are separated, the oil or fat +is drawn off and washed three times with distilled water. The sample +is deprived of water by filtering through a hot jacket filter and a +given weight of the dry sample is well shaken with twice its weight of +absolute alcohol. A weighed portion of the alcoholic solution obtained +is evaporated to remove the alcohol and the weight of the residual +fat determined. From the data obtained the percentage of solubility +is calculated. Olive oils, when treated as described above, show a +solubility of about forty-three parts per thousand of absolute alcohol, +cotton oil sixty-two parts, sesamé forty-one parts, peanut sixty-six +parts, colza twenty parts, and flaxseed seventy parts per thousand. + +=315. Coloration Produced by Oxidants.=—When oils and fats are mixed +with oxidizing reagents, such as sulfuric and nitric acids, the +glycerids are partly decomposed with the production of colors which +have some analytical significance. The most simple method of applying +these tests is by the use of a thick porcelain plate provided with +small cup-shaped depressions for holding the few drops of material +required. Two or three drops of the oil under examination are placed +in each of the cups, a like quantity of the oxidizing reagent added, +and the mixture stirred with a small glass rod. The colors produced +are carefully noted and the mixture is allowed to remain at room +temperature for at least twelve hours in order that the final tint may +be observed. The sulfuric acid used for this reaction should have a +specific gravity of one and seven-tenths and the nitric acid should +have the usual commercial strength of the strongest acid. Pure lard, +when treated with sulfuric acid, as above described, shows but little +change of color while the vegetable oils mostly turn brown or black. In +addition to the reagents mentioned many others, including sulfuric and +nitric acids, sulfuric acid and potassium bichromate, chlorin, ammonia, +hydrogen peroxid, sodium hydroxid and aqua regia are used. Only a few +of these tests seem to have sufficient analytical importance to merit +any detailed description.[268] + +=316. Coloration in Large Masses.=—Instead of applying the color test +in the small way just described, larger quantities of the fat may +be used, either in the natural state or after solution in petroleum +or other solvent. For this purpose about ten cubic centimeters of +the oil are shaken with a few drops of sulfuric acid or sulfuric and +nitric acids. Lard, when thus treated (five drops of sulfuric acid to +ten cubic centimeters of lard) shows practically no coloration. When +treated with an equal volume of sulfuric acid and shaken, the lard on +separating has a brown-red tint.[269] + +Olive oil, with a few drops of sulfuric acid, gives a green color, +while cottonseed, peanut and other vegetable oils, when thus treated +with sulfuric and nitric acids, show brown to black coloration. The +delicacy of the reaction may be increased by first dissolving the fat +or oil in petroleum ether. + +In the use of the coloration test with solvents, a convenient method +is to dissolve about one cubic centimeter of the fat in a test tube in +petroleum ether, add one drop of strong sulfuric acid and shake. + +In the case of lard, the color does not change or becomes yellow +or red. Cottonseed oil, similarly treated, shows a brown or black +color.[270] + +=317. Special Nitric Acid Test.=—A special nitric acid test for +cottonseed oil is made with nitric acid of exactly 1.375 specific +gravity at 15°. This test is especially valuable in detecting +cottonseed in olive oil. The operation is conveniently conducted by +shaking together equal volumes of the oil and acid in a test tube until +an intimate mixture or emulsion is secured. When any considerable +quantity of cottonseed oil is present an immediate brown coloration +is produced, from the intensity of which the relative proportion of +cottonseed oil in the case of a mixture may be roughly approximated. +When only a little cottonseed oil is present in the mixture, the test +tube containing the reagents should be set aside for several hours +before the final observation is made. + +=318. Coloration with Phosphomolybdic Acid.=—Among the color tests, one +which we have found of use is the coloration produced in certain oils, +mostly of a vegetable origin, by phosphomolybdic acid.[271] + +The method of applying the test is extremely simple. A few cubic +centimeters of the oil or melted lard are dissolved in an equal volume +of chloroform, and a third volume of ten per cent phosphomolybdic acid +added. The mouth of the test tube is closed with the thumb, and the +whole is violently shaken. On being left in repose, the phosphomolybdic +acid gathers at the top, and the coloration produced therein is easily +observed. Cottonseed oil and peanut oil both give a beautiful green +when treated in this way, which is turned to a blue on the addition of +ammonia. Linseed oil gives a green color, but forms a kind of emulsion +which obscures the color to some extent. The pure lards rendered in +the laboratory give no coloration whatever to the reagent, but it +retains its beautiful amber color in every case. Mixtures containing +as little as ten per cent cottonseed oil and ninety per cent lard, +show a distinct greenish tint, while twenty per cent cottonseed oil +gives a distinct green. This reaction, therefore, may be considered +of great value, and on account of its easy application it should come +into wide use. But it is probable that different samples of cottonseed +oil, refined to different degrees or in different ways, vary in their +deportment with phosphomolybdic acid as they do with silver nitrate. In +other words, there may be some samples of cottonseed oil which will not +give the green color when treated as above, or so faintly as to have no +diagnostic value in mixtures. + +This reaction shows itself with nearly all vegetable oils but those +which have been chemically treated either for the purpose of bleaching, +or for the removal of the acidity, do not respond to the test at all, +or else in a feeble manner, and that only after standing some time. +Lard, goose fat, tallow, deer fat, butter fat, etc., show no change in +color on being treated with this reagent, either with or without the +addition of alkali. The presence of a small quantity of vegetable oil +betrays itself by the appearance of the above mentioned coloration, +the intensity of which forms an approximate measure of the amount of +vegetable oil present in the sample. In experiments with suspected +lards, which deviated in their iodin absorption numbers from those of +genuine lard, the results were concordant, the color deepening as the +iodin figure rose. The mineral fats (paraffin, vaselin) are without +action on this reagent, and the only animal fat which reduces it is +codliver oil. + +In like manner some samples of lard may be found which exhibit a +deportment with this reagent similar to that shown with vegetable oils, +and tallow and lard oil have been shown to give more distinct reactions +than some of the vegetable oils.[272] + +The phosphomolybdic acid may be prepared by precipitating a solution +of ammonium molybdate with sodium phosphate and dissolving the washed +precipitate in a warm solution of sodium carbonate. The solution is +evaporated to dryness and the dry residue subjected to heat. If a blue +coloration be produced it may be discharged by adding a little nitric +acid and reheating. The residue is dissolved in water, acidified with +nitric and made of such a strength as to contain about ten per cent of +the substance. + +=319. Coloration with Picric Acid.=—If to ten cubic centimeters of oil +a cold saturated solution of picric acid in ether be added and the +latter be allowed to evaporate slowly, the acid remains dissolved in +the oil, to which it communicates a brown color. + +Pure lard, after the evaporation of the ether, appears of a +citron-yellow color; if cottonseed oil be present, however, the mixture +assumes a brown-red color.[273] + +=320. Coloration with Silver Nitrate.=—A modification of Bechi’s +method of reducing silver nitrate, given further on, has been proposed +by Brullé.[274] The reagent employed consists of twenty-five parts +of silver nitrate in 1,000 parts of alcohol of ninety-five per cent +strength. Twelve cubic centimeters of the oil to be examined and five +of the reagent are placed in a test tube, held in a vessel containing +boiling water, and the ebullition continued for about twenty minutes. +At the end of this time an olive oil, even if it be an impure one, +will show a beautiful green tint. With seed oils the results are +quite different. Cotton oil submitted to this treatment becomes +completely black. Peanut oil shows at first a brown-red coloration and +finally a somewhat green tint, losing its transparency. Sesamé oil is +distinguished by a red-brown tint very pronounced and remaining red. +Colza oil takes on a yellowish green coloration, becomes turbid and is +easily distinguished in its reaction from olive oil. In mixtures of +olive oil with the other oils, any notable proportion of the seed oils +can be easily determined by the above reactions. Natural butter treated +with this reagent retains its primitive color. That containing margarin +becomes a brick-red and as little as five per cent of margarin in +butter can be detected by this test. With ten per cent the tint is very +pronounced. + +=321. Coloration with Stannic Bromid.=—This reagent is prepared by +adding dry bromin, drop by drop, to powdered or granulated tin held in +a flask immersed in ice water, until a persistent red color indicates +that the bromin is in excess. In the application of this reagent three +or four drops of it are added successively to a little less than that +quantity of the oil, the mixture well stirred and set aside for a few +minutes. The unsaponifiable matters of castor oil give a green color +when thus treated, sandal wood oil a blood-red color and cedar oil a +purplish color.[275] + +=322. Coloration with Auric Chlorid.=—The use of auric chlorid +for producing colorations in oils and fats was first proposed by +Hirschsohn.[276] One gram of auric chlorid is dissolved in 200 cubic +centimeters of chloroform and about six drops of this reagent added +to five cubic centimeters of the oil to be tested. In the case of +cottonseed oil a beautiful red color is produced. + +I have found that even pure lards give a trace of color sometimes with +this reagent, and therefore the production of a slight red tint cannot +in all cases be regarded as conclusive of the presence of cottonseed +oil.[277] + +In general, it may be said that the color reactions with fats and oils +have a certain qualitive and sorting value, and in any doubtful case +they should not be omitted. Their value can only be established by +comparison under identical conditions with a large number of fats and +oils of known purity. The analyst must not depend too confidingly on +the data found in books, but must patiently work out these reactions +for himself. + +=323. Thermal Reactions.=—The measurement of the heat produced by +mixing glycerids with reagents which decompose them or excite other +speedy chemical reactions, gives valuable analytical data. These +measurements may be made in any convenient form of calorimeter. The +containing vessel for the reagents should be made of platinum or some +other good conducting metal not affected by them. + +The heat produced is measured in the usual way by the increment in +temperature noted in the mass of water surrounding the containing +vessel. The determination of the heat produced in chemical reactions is +a tedious and delicate operation requiring special forms of apparatus +for different substances. The time element in these operations is +a matter of importance, since it is necessary to work in rooms +subject to slight changes of temperature and to leave the apparatus +for some time at rest, in order to bring it and its contents to a +uniform temperature. For these reasons the more elaborate methods of +calorimetric examination are not well suited to ordinary analytical +work, and the reader is referred to standard works on thermal +chemistry for the details of such operations.[278] For our purpose here +a description of two simple thermal processes, easily and quickly +conducted, will be sufficient, while a description of the method of +determining the heat of combustion of foods will be given in another +place. + +=324. Heat of Sulfuric Saponification.=—Maumené was the first to +utilize the production of heat caused by mixing sulfuric acid with a +fat as an analytical process.[279] In conducting the process a sulfuric +acid of constant strength should be employed inasmuch as the rise of +temperature produced by a strong acid is much greater than when a +weaker acid is employed. The process is at best only comparative and it +is evident that the total rise of temperature in any given case depends +on the strength of the acid, the character, and purity of the fat or +oil, the nature of the apparatus and its degree of insulation, the +method of mixing and the initial temperature. For this reason the data +given by different analysts vary greatly.[280] For some of the methods +of conducting the operation the reader may consult the work of Allen, +cited above, or other authorities.[281] + +In this laboratory the process is conducted as follows:[282] The initial +temperature of the reagents should be a constant one. For oils 20° is a +convenient starting point and for fats about 35°, at which temperature +most of them are soft enough to be easily mixed with the reagent. The +acid employed should be the pure monohydrated form, specific gravity at +20°, 1.845. + +The apparatus used is shown in Fig. 98. + +[Illustration: FIG. 98.—APPARATUS FOR DETERMINING RISE OF TEMPERATURE +WITH SULFURIC ACID.] + +The test tube which holds the reagents is twenty-four centimeters in +length and five in diameter. It is provided with a stopper having three +perforations, one for a delicate thermometer, one for a bulb funnel +for delivering the sulfuric acid, and one to guide a stirring rod bent +into a spiral as shown. The thermometer is read with a magnifying +glass. Fifty cubic centimeters of the fat are placed in the test tube +and ten of sulfuric acid in the funnel and the apparatus is exposed +at the temperature desired until all parts of it, together with the +reagents, have reached the same degree. The test tube holding the oil +should be placed in a vacuum-jacket tube, such as will be described in +paragraph =316=. The oil is allowed to run in as rapidly as possible +from the funnel and the stirring rod is moved up and down two or three +times until the oil and acid are well mixed. Care must be exercised +to stir no more than is necessary for good mixing. The mercury is +observed as it ascends in the tube of the thermometer and its maximum +height is noted. With the glass it is easy to read to tenths, when +the thermometer is graduated in fifths of a degree. When oils are +tested which produce a rise of temperature approaching 100°, in the +above circumstances, (cottonseed, linseed and some others) either +smaller quantities should be used or the oil diluted with some inert +substance or dissolved in some inert solvent of high boiling point. +For a study of the variations produced in the rise of temperature when +varying proportions of oil and acid are used, the work of Munroe may be +consulted.[283] + +The thermélaeometer described by Jean is a somewhat complicated piece +of apparatus and does not possess any advantage over the simple form +described above.[284] + +Instead of expressing the data obtained in thermal degrees showing the +rise of temperature, Thompson and Ballentyne refer them to the heat +produced in mixing sulfuric acid and water.[285] + +The observed thermal degree of the oil and acid divided by that of +the water and acid is termed the specific temperature reaction. +For convenience in writing, this quotient is multiplied by 100. +The respective quantities of acid and water are ten and fifty +cubic centimeters. This method of calculation has the advantage of +eliminating to a certain degree the variations which arise in the use +of sulfuric acid of differing specific gravities. In the following +table are given the comparative data obtained for some common oils.[286] + + Acid of 95.4 Acid of 96.8 Acid of 99 + per cent. per cent. per cent. + /-----------------\ /----------------\ /--------------\ + Rise of Specific Rise of Specific Rise of Specific + temp. temp. temp. temp. temp. temp. + with reaction. with reaction with reaction. + Kind of oil. the oil. the oil. the oil. + 0° 0° 0° 0° 0° 0° + + Olive oil 36.5 95 39.4 85 44.8 96 + Rapeseed oil 49.0 127 37.0 89 58.0 124 + Castor oil 34.0 88 + Linseed oil 104.5 270 125.2 269 + +=325. Method of Richmond.=—The rise of temperature produced by +mixing an oil and sulfuric acid is determined by Richmond in a simple +calorimeter, which is constructed by fitting a small deep beaker inside +a larger one with a packing of cotton. The heat capacity of the system +is determined by adding to ten grams of water, in the inner beaker, +at room temperature, twenty-five grams of water of a noted higher +temperature and observing the temperature of the mixture. The cooling +of the system, during the time required for one determination of heat +of sulfuric saponification, does not exceed one per cent of the whole +number of calories produced.[287] Between the limits of ninety-two per +cent and one hundred per cent the rise of temperature observed is +directly proportional to the strength of the acid. + +_Relative Maumené Figure._—The total heat evolved per mean molecule is +called by Richmond the relative maumené figure. It is calculated as +follows: + + Let _x_ = percentage of sulfuric acid in the acid employed; + _h_ = heat capacity of calorimeter in grams of water; + _R_ = observed rise of temperature (twenty-five grams of + oil, five cubic centimeters sulfuric acid); + _K_ = potash absorbed for saponification (19.5 per cent of + potassium hydroxid, standard of comparison); + _M_ = relative maumené figure: + + 21.5 20 + _h_ 19.5 + Then _M_ = _R_ × -----------× --------- × -----. + _x_ - 78.5 20 _K_ + +=326. Heat of Bromination.=—The rise of temperature caused by mixing +fats with sulfuric acid has long been used to discriminate between +different fats and oils. Hehner and Mitchell propose a similar reaction +based upon the rise of temperature produced by mixing bromin with +the sample.[288] The action of bromin on unsaturated fatty bodies is +instantaneous and is attended with a considerable evolution of heat. +Since the action of bromin on many of the oils is very violent it is +necessary to dilute the reagent with chloroform or glacial acetic acid. +Owing to its high boiling point the acetic acid has some advantage +over chloroform for this purpose. The tests are conveniently made in +a vacuum-jacket tube. In such a tube there is no loss of heat by +radiation. The bromin is measured in a pipette having at its upper end +a tube filled with caustic lime held between plugs of asbestos. The +bromin sample to be tested and the diluent employed are kept at the +same temperature before beginning the trial. They are quickly mixed +and the rise of temperature noted. The oil is first dissolved in the +chloroform and the bromin then added. + +A somewhat constant relation is noticed between the rise of temperature +and the iodin number when one gram of oil, ten cubic centimeters of +chloroform and one cubic centimeter of bromin are used. + +If the rise in temperature in degrees be multiplied by 5.5 the product +is approximately the iodin number of the sample. Thus a sample of lard +gave a rise in temperature of 10°.6 and an iodin number of 57.15. The +number obtained by multiplying 10.6 by 5.5 is 58.3. + +In like manner the numbers obtained for some common oils are as follows: + + Material. Rise of temperature Iodin No. Calculated + with bromin. Iodin No. + + Butter fat 6.6 37.1 36.3 + Olive oil 15.0 80.8 82.5 + Maize ” 21.5 122.0 118.2 + Cotton ” 19.4 107.1 106.7 + Castor ” 15.0 83.8 82.5 + Linseed oil 30.4 160.7 167.2 + Codliver ” 28.0 144.0 140.0 + +=327. Modification of the Heat of Bromination Method.=—The method +described above by Hehner and Mitchell presents many grave difficulties +in manipulation, on account of the inconvenience of handling liquid +bromin. The process is made practicable by dissolving both the oil or +fat and the bromin in chloroform, or better in carbon tetrachlorid, in +which condition the bromin solution is easily handled by means of a +special pipette.[289] + +In order to make a number of analyses of the same sample ten grams of +the fat may be dissolved in chloroform or carbon tetrachlorid and the +volume completed with the same solvent to fifty cubic centimeters. +In like manner twenty cubic centimeters of the bromin are dissolved +in one of the solvents named and the volume completed to 100 cubic +centimeters therewith. + +For convenience of manipulation the solutions are thus made of such a +strength that five cubic centimeters of each represent one gram of the +fat and one cubic centimeter of the liquid bromin respectively. + +[Illustration: FIG. 99. APPARATUS FOR DETERMINING HEAT OF BROMINATION.] + +The apparatus used for the work is shown in the accompanying figure. +The pipette for handling the bromin solution is so arranged as to be +filled by the pressure of a rubber bulb, thus avoiding the danger of +sucking the bromin vapor into the mouth. The filling is secured by +keeping the bromin solution in a heavy erlenmeyer with a side tubulure +such as is used for filtering under pressure. The solutions are +mixed in a long tube, held in a larger vessel, from which the air is +exhausted to secure a minimum radiation of heat. A delicate thermometer +graduated in tenths serves to register the rise of temperature. The fat +solution is first placed in the test tube, with care not to pour it +down the sides of the tube but to add it by means of a pipette reaching +nearly to the bottom. The whole apparatus having been allowed to come +to a standard temperature the bromin solution is allowed to run in +quickly from the pipette. No stirring is required as the liquids are +sufficiently mixed by the addition of the bromin solution. The mercury +in the thermometer rapidly rises and is read at its maximum point by +means of a magnifying glass. With a thermometer graduated in tenths, it +is easy to read to twentieths of a degree. + +It is evident that the rise of temperature obtained depends on +similar conditions to those mentioned in connection with sulfuric +saponification. Each system of apparatus must be carefully calibrated +under standard conditions and when this is done the comparative rise +of temperature obtained with various oils and fats will prove of +great analytical use. It is evident that the ratio of this rise of +temperature to the iodin number must be determined for every system of +apparatus and for every method of manipulation employed, and no fixed +factor can be given that will apply in every case. + +With the apparatus above described and with the method of manipulation +given the following data were obtained for the oils mentioned: + + Rise of temperature. + Olive oil 20°.5 + Refined cottonseed oil 25°.7 + Sunflowerseed oil 28°.4 + Calycanthusseed oil 29°.0 + +Bromin and chloroform, when mixed together, give off heat, due to +the chemical reaction resulting from the substitution of bromin for +hydrogen in the chloroform molecule and the formation of hydrobromic +acid. For this reason the data obtained, when chloroform is used as a +solvent, are slightly higher than with carbon tetrachlorid. The use of +the latter reagent is therefore to be preferred. + +=328. Haloid Addition Numbers.=—Many of the glycerids possess the +property of combining directly with the haloids and forming thereby +compounds in which the haloid, by simple addition, has become a part of +the molecule. Olein is a type of this class of unsaturated glycerids. +The process may take place promptly as in the case of bromin or move +slowly as with iodin. The quantity of the haloid absorbed is best +determined in the residual matter and not by an examination of the fat +compound. By reason of the ease with which the amount of free iodin in +solution can be determined, this substance is the one which is commonly +employed in analytical operation on fats. + +In general, the principle of the operation depends on bringing the fat +and haloid together in a proper solution and allowing the addition +to take place by simple contact. The quantity of the haloid in the +original solution being known, the amount which remains in solution +after the absorption is complete, deducted from that originally +present, will give the quantity which has entered into combination with +the glycerid. + +=329. Hübl’s Process.=—In determining the quantity of iodin which +will combine with a fat, the method first proposed by Hübl, or some +modification thereof, is universally employed.[290] In the determination +of the iodin number of a glycerid it is important that it be +accomplished under set conditions and that iodid be always present +in large excess. It is only when data are obtained in the way noted +that they can be regarded as useful for comparison and determination. +Many modifications of Hübl’s process have been proposed, but it is +manifestly impracticable to give even a summary of them here. As +practiced in the chemical laboratory of the Agricultural Department and +by the Association of Official Agricultural Chemists, it is carried out +as follows:[291] + + +(1) PREPARATION OF REAGENTS. + +(_a_). _Iodin Solution._—Dissolve twenty-five grams of pure iodin in +500 cubic centimeters of ninety-five per cent alcohol. Dissolve thirty +grams of mercuric chlorid in 500 cubic centimeters of ninety-five per +cent alcohol. The latter solution, if necessary, is filtered, and then +the two solutions mixed. The mixed solution should be allowed to stand +twelve hours before using. + +(_b_). _Decinormal Sodium Thiosulfate Solution._—Dissolve 24.8 grams +of chemically pure sodium thiosulfate, freshly pulverized as finely as +possible and dried between filter or blotting paper, and dilute with +water to one liter, at the temperature at which the titrations are to +be made. + +(_c_). _Starch Paste._—One gram of starch is boiled in 200 cubic +centimeters of distilled water for ten minutes and cooled to room +temperature. + +(_d_). _Solution of Potassium Iodid._—One hundred and fifty grams of +potassium iodid are dissolved in water and the volume made up to one +liter. + +(_e_). _Solution of Potassium Bichromate._—Dissolve 3.874 grams of +chemically pure potassium bichromate in distilled water and make the +volume up to one liter at the temperature at which the titrations are +to be made. + + +(2). DETERMINATION. + +(_a_). _Standardizing the Sodium Thiosulfate Solution._—Place twenty +cubic centimeters of the potassium bichromate solution, to which have +been added ten cubic centimeters of the solution of potassium iodid, +in a glass stopper flask. Add to this mixture five cubic centimeters +of strong hydrochloric acid. Allow the solution of sodium thiosulfate +to flow slowly into the flask until the yellow color of the liquid +has almost disappeared. Add a few drops of the starch paste, and with +constant shaking continue to add the sodium thiosulfate solution until +the blue color just disappears. The number of cubic centimeters of +thiosulfate solution used multiplied by five is equivalent to one gram +of iodin. + +_Example._—Twenty cubic centimeters of potassium bichromate solution +required 16.2 sodium thiosulfate; then 16.2 × 5 = 81 = number cubic +centimeters of thiosulfate solution equivalent to one gram of iodin. +Then one cubic centimeter thiosulfate solution = 0.0124 gram of iodin: +(Theory for decinormal solution of sodium thiosulfate, one cubic +centimeter = 0.0127 gram of iodin.) + +(_b_). _Weighing the Sample._—About one gram of butter fat is placed +in a glass stopper flask, holding about 300 cubic centimeters, with +the precautions to be mentioned for weighing the fat for determining +volatile acids. + +(_c_). _Absorption of Iodin._—The fat in the flask is dissolved in ten +cubic centimeters of chloroform. After complete solution has taken +place thirty cubic centimeters of the iodin solution (1) (_a_) are +added. The flask is now placed in a dark place and allowed to stand, +with occasional shaking, for three hours. + +(_d_). _Titration of the Unabsorbed Iodin._—One hundred cubic +centimeters of distilled water are added to the contents of the flask, +together with twenty cubic centimeters of the potassium iodid solution. +Any iodin which may be noticed upon the stopper of the flask should +be washed back into the flask with the potassium iodid solution. The +excess of iodin is taken up with the sodium thiosulfate solution, which +is run in gradually, with constant shaking, until the yellow color of +the solution has almost disappeared. A few drops of starch paste are +added, and the titration continued until the blue color has entirely +disappeared. Toward the end of the reaction the flask should be +stoppered and violently shaken, so that any iodin remaining in solution +in the chloroform may be taken up by the potassium iodid solution in +the water. A sufficient quantity of sodium thiosulfate solution should +be added to prevent a reappearance of any blue color in the flask for +five minutes. + +(_e_). _Setting the Value of the Iodin Solution by the Thiosulfate +Solution._—At the time of adding the iodin solution to the fat, two +flasks of the same size as those used for the determination should be +employed for conducting the operation described above, but without the +presence of any fat. In every other respect the performance of the +blank experiments should be just as described. These blank experiments +must be made each time the iodin solution is used. + +_Example of Blank Determinations._—Thirty cubic centimeters of +iodin solution required 46.4 cubic centimeters of sodium thiosulfate +solution: Thirty cubic centimeters of iodin solution required 46.8 +cubic centimeters of sodium thiosulfate solution: Mean, 46.6 cubic +centimeters. + + Weight of fat 1.0479 grams + Quantity of iodin solution used 30.0 cubic centimeters + Thiosulfate equivalent to iodin used 46.6 ” ” + Thiosulfate equivalent to remaining + iodin 14.7 ” ” + Thiosulfate equivalent to iodin absorbed 31.9 ” ” + Percent of iodin absorbed, 31.9 × 0.0124 × 100 ÷ 1.0479 = 37.75. + +=330. Character of Chemical Reaction.=—The exact nature of the chemical +process which takes place in this reaction is not definitely known. +Hübl supposed that the products formed were chloro-iodid-additive +compounds, and he obtained a greasy product from oleic acid, to which +he ascribed the formula C₁₈H₃₄IClO₂. By others it is thought that +chlorin alone may be added to the molecule.[292] + +In general, it may be said that none of the glycerids capable +of absorbing halogens is able to take on a quantity equivalent +to theory.[293] While the saturated fatty acids (stearic series) +theoretically are not able to absorb iodin some of them are found to +do so to a small degree. It is evident, therefore, that it is not +possible to calculate the percentage of unsaturated glycerids in a fat +from their iodin number alone. According to the data worked out by +Schweitzer and Lungwitz both addition and substitution of iodin take +place during the reaction.[294] This fact they determined by titration +with potassium iodate and iodid according to the formula 5HI + HIO₃ = +6I + 6H₂O. The authors confess that whenever free hydriodic acid is +found in the mixture that iodin substitution has taken place and that +for each atom of hydrogen eliminated from the fat molecule two atoms +of iodin disappear, one as the substitute and the other in the form +of hydriodic acid. When carbon bisulfid or tetrachlorid is used as a +solvent no substitution takes place and pure additive compounds are +formed. + +The following process is recommended to secure a pure iodin addition +to a glycerid: About one gram or a little less of the oil or fat is +placed in a glass stopper flask, to which are added about seven-tenths +of a gram of powdered mercuric chlorid and twenty-five cubic +centimeters of a solution of iodin in carbon bisulfid. The stopper is +made tight by smearing it with powdered potassium iodid, tied down, +and the mixture is heated for some time under pressure. By this method +it is found that no hydriodic acid is formed, and hence all the iodin +which disappears is added to the molecule of the glycerid. The additive +numbers obtained for some oils are appended: + + Time of Per cent Per cent + Oil. heating. Temperature. iodin added. hübl number. + + Lard oil 30 minutes. 50°.0 73.0 78.4 + Cottonseed oil 2 hours. 50°.0 103.0 106.5 + Oleic acid 2 ” 65°.5 93.8 + +=331. Solution in Carbon Tetrachlorid.=—Gantter has called attention +to the fact that the amount of iodin absorbed by fat does not depend +alone upon the proportion of iodin present but also upon the amount of +mercuric chlorid in the solution.[295] Increasing amounts of mercuric +chlorid cause uniformly a much greater absorption of the iodin. +For this reason he proposes to discard the use of mercuric chlorid +altogether for the hübl test and to use a solvent which will at the +same time dissolve both the iodin and the fat. For this purpose he uses +carbon tetrachlorid. The solutions are prepared as follows: + +_Iodin Solution._—Ten grams of iodin are dissolved in one liter of +carbon tetrachlorid. + +In the preparation of this solution the iodin must not be thrown +directly into the flask before the addition of the tetrachlorid. Iodin +dissolves very slowly in carbon tetrachlorid and the solution is made +by placing it in a sufficiently large weighing glass and adding a +portion of the carbon tetrachlorid thereto. The solution is facilitated +by stirring with a glass rod until the added tetrachlorid is apparently +charged with the dissolved iodin. The dissolved portion is then poured +into a liter flask, new portions added to the iodin and this process +continued until the iodin is completely dissolved, and then sufficient +additional quantities of the tetrachlorid are added to fill the flask +up to the mark. + +=332. Sodium Thiosulfate Solution.=—Dissolve 19.528 grams of pure +sodium thiosulfate in 1000 cubic centimeters of water. For determining +the strength of the solution by titration, the solution of iodin in +carbon tetrachlorid and a solution of sodium thiosulfate in water are +each placed in a burette. A given volume of the iodin solution is +first run into a flask with a glass stopper and afterward the sodium +thiosulfate added little by little until, after a vigorous shaking, +the liquid has only a little color. Some solution of starch is then +added and shaken until the mixture becomes deep blue. The sodium +thiosulfate solution is added drop by drop, with vigorous shaking after +each addition, until the solution is completely decolorized. If both +solutions have been correctly made with pure materials they will be of +equal strength; that is, ten cubic centimeters of the iodin solution +will be exactly decolorized by ten cubic centimeters of the sodium +thiosulfate solution. + +=333. Method of Conducting the Absorption.=—The quantity of the fat +or oil employed should range from 100 to 200 milligrams, according to +the absorption equivalent. These quantities should be placed in flasks +with glass stoppers in the ordinary way. In the flasks are placed +exactly fifty cubic centimeters of the iodin solution equivalent to +500 milligrams of iodin, and the flask is then stoppered and shaken +until the fat or oil is completely dissolved. In order to avoid the +volatilization of the iodin finally, sufficient water is poured into +the flask to form a layer about one millimeter in thickness over the +solution containing the iodin and fat. The stopper should be carefully +inserted and the flask allowed to stand at rest for fifty hours. + +=334. Estimation of the Iodin Number.=—This is determined in the usual +way by titration of the amount of iodin left in excess after the +absorption as above described. The iodin number is to be expressed +by the number of milligrams of iodin which are absorbed by each 100 +milligrams of fat. + +_Example._—One hundred and one milligrams of flaxseed oil were +dissolved in fifty cubic centimeters of the carbon tetrachlorid +solution of iodin and allowed to stand as above described for fifty +hours. At the end of this time, 42.3 cubic centimeters of the sodium +thiosulfate solution were required to decolorize the excess of iodin +remaining. + +_Statement of Results._—Fifty cubic centimeters of the sodium +thiosulfate equal 500 milligrams of iodin; therefore, 42.3 cubic +centimeters of the thiosulfate solution equal 423 milligrams of iodin. +The difference equals seventy-seven milligrams of iodin absorbed by 101 +milligrams of the flaxseed oil. Therefore, the iodin number equivalent +and the milligrams of iodin absorbed by 100 milligrams of flaxseed oil +equal 76.2. + +It is evident from the above determination that the iodin number of +the oil, when obtained in the manner described, is less than half that +secured by the usual hübl process. Since the solvent employed, however, +is more stable than chloroform when in contact with iodin or bromin, +the proposed variation is one worthy of the careful attention of +analysts. + +McIlhiney has called especial attention to the low numbers given by the +method of Gantter, and from a study of the data obtained concludes that +iodin alone will not saturate glycerids, no matter what the solvents +may be.[296] + +It is clear, therefore, that the process of Gantter cannot give numbers +which are comparable with those obtained by the usual iodin method. Any +comparative value possessed by the data given by the process of Gantter +must be derived by confining it to the numbers secured by the carbon +tetrachlorid process alone. + +=335. Substitution of Iodin Monochlorid for Hübl’s Reagent.=—Ephraim +has shown that iodin monochlorid may be conveniently substituted for +the hübl reagent with the advantage that it can be safely used at once, +while the hübl reagent undergoes somewhat rapid changes when first +prepared. The present disadvantage of the process is found in the fact +that the iodin monochlorid of commerce is not quite pure and each new +lot requires to be titrated for the determination of its purity. + +The reagent is prepared of such a strength as to contain 16.25 grams +of iodin monochlorid per liter. The solvent used is alcohol. The +operation is carried out precisely as in the hübl method, substituting +the alcoholic solution of iodin monochlorid for the iodin reagent +proposed by Hübl.[297] If the iodin monochlorid solution, after acting +on the oil, be titrated without previous addition of potassium iodid a +new value is obtained, the chloriodin number. In titrating, the sodium +thiosulfate is added until the liquid, which is made brown by the +separated iodin, becomes yellow. At this point the solution is diluted, +starch paste added, and the titration completed. + +=336. Preservation of the Hübl Reagent.=—To avoid the trouble due to +changes in the strength of Hübl’s reagent, Mahle adds hydrochloric acid +to it at the time of its preparation.[298] The reagent is prepared as +follows: Twenty-five grams of iodin dissolved in a quarter of a liter +of ninety-five per cent alcohol are mixed with the same quantity of +mercuric chlorid in 200 cubic centimeters of alcohol, the same weight +of hydrochloric acid of 1.19 specific gravity added and the volume of +the mixture completed to half a liter with alcohol. After five days +such a solution gave, on titration, 49.18 instead of 49.31 grams per +liter of iodin. + +It will be observed that this solution is double the usual strength, +but this does not influence the accuracy of the analytical data +obtained. It appears that the hübl number is not, therefore, an iodin +number, but expresses the total quantity of iodin, chlorin and oxygen +absorbed by the fat during the progress of the reaction. + +=337. Bromin Addition Number.=—In the process of Hübl and others an +attempt is made to determine the quantity of a halogen, _e.g._, iodin, +which the oil, fat or resin will absorb under certain conditions. +The numbers obtained, however, represent this absorption only +approximately, because the halogen may disappear through substitution +as well as absorption. Whether or not a halogen is added, _i. e._, +absorbed or substituted, may be determined experimentally. + +The principle on which the determination depends rests on the fact that +a halogen, _e. g._, bromin, forms a molecule of hydrobromic acid for +every atom of bromin substituted, while in a simple absorption of the +halogen no such action takes place. If, therefore, bromin be brought +into contact with a fat, oil or resin, the determination of the +quantity of hydrobromic acid formed will rigidly determine the quantity +of bromin substituted during the reaction. If this quantity be deducted +from the total bromin which has disappeared, the relative quantities of +the halogen added and substituted are at once determined. In the method +of McIlhiney[299] bromin is used instead of iodin because the addition +figures of iodin are in general much too low. + +_The Reagents._—The following solutions are employed: + +1. One-third normal bromin dissolved in carbon tetrachlorid: + +2. One-tenth normal sodium thiosulfate: + +3. One-tenth normal potassium hydroxid. + +_The Manipulation._—From a quarter to one gram of the fat, oil or +resin, is dissolved in ten cubic centimeters of carbon tetrachlorid +in a dry bottle of 500 cubic centimeters capacity, provided with a +well-ground glass stopper. An excess of the bromin solution is added, +the bottle tightly stoppered, well shaken and placed in the dark. At +the end of eighteen hours the bottle is placed in a freezing mixture +and cooled until a partial vacuum is formed. A piece of wide rubber +tubing an inch and a half long is slipped over the lip of the bottle so +as to form a well about the stopper. This well having been filled with +water the stopper is lifted and the water is sucked into the bottle +absorbing all the hydrobromic acid which has been formed. The well +should be kept filled with water, as it is gradually taken in until in +all twenty-five cubic centimeters have been added. The bottle is next +well shaken and from ten to twenty cubic centimeters of a twenty per +cent potassium iodid solution added. + +The excess of bromin liberates a corresponding amount of iodin, which +is determined by the thiosulfate solution in the usual way, after +adding about seventy-five cubic centimeters of water. The total bromin +which has disappeared is then calculated from the data obtained, +the strength of the original bromin solution having been previously +determined. The contents of the bottle are next transferred to a +separatory funnel, the aqueous portion separated, filtered through a +linen filter, a few drops of thiosulfate solution added, if a blue +color persist, and the free hydrobromic acid determined by titration +with potassium hydroxid, using methyl orange as indicator. The end +reaction is best observed by placing the solution in a porcelain +dish, adding the alkali in slight excess, and titrating back with +tenth-normal hydrochloric acid until the pink tint is perceived. +From the number of cubic centimeters of alkali used the amount of +bromin present as hydrobromic acid is calculated, and this expressed +as percentage gives the bromin substitution figure. The bromin +substitution figure multiplied by two and subtracted from the total +absorption gives the addition figure. + +Following are the data for some common substances: + + Total bromin + absorption in Bromin addition Bromin substitution + Substance. eighteen hours. figure. figure. + Rosin 212.70 0.00 106.35 + Raw linseed oil 102.88 102.88 00.00 + Boiled ” ” 103.92 103.92 00.00 + Salad cotton ” 65.54 64.26 0.64 + Sperm ” 56.60 54.52 1.04 + +By the process just described it is possible to detect mixtures of +rosins and rosin oils with animal and vegetable oils. In this respect +it possesses undoubted advantages over the older methods. + +=338. Method Of Hehner.=—The absorption of bromin which takes place +when unsaturated fats are brought into contact with that reagent was +made the basis of an analytical process, proposed by Allen as long +ago as 1880.[300] In the further study of the phenomena of bromin +absorption, as indicated by McIlhiney, Hehner modified the method as +indicated below.[301] From one to three grams of the sample are placed +in a tared wide-mouthed flask and dissolved in a little chloroform. +Bromin is added to the solution, drop by drop, until it is in decided +excess. The flask is placed on a steam-bath and heated until the +greater part of the bromin is evaporated, when some more chloroform is +added and the heating continued until all the free bromin has escaped. +The flask is put in a bath at 125° and dried to constant weight. A +little acrolein and hydrobromic acid escape during the drying and the +residue may be colored, or a heavy bromo oil be obtained. The gain +in weight represents the bromin absorbed. The bromin number may be +converted into the iodin number by multiplying by 1.5875.[302] + +[Illustration: FIG. 100.—OLEIN TUBE.] + +=339. Halogen Absorption and Addition of Fat Acids.=—Instead of +employing the natural glycerids for determining the degree of action +with the halogens the acids may be separated by some of the processes +of saponification hereafter described and used as directed for the +glycerids themselves. It is doubtful if any practical advantage arises +from this variation of the process. If the fat acids be separated, +however, it is possible to get some valuable data from the halogen +absorption of the fractions. Theoretically the stearic series of acids +would suffer no change in contact with halogens while the oleic series +is capable of a maximum absorptive and additive action. On this fact is +based a variation of the iodin process in which an attempt is made to +separate the oleic acid from its congeners and to apply the halogen to +the separated product. + +The method of separation devised by Muter is carried out as +follows:[303] The separatory or olein tube consists of a wide burette +stem, provided with a lateral stopcock, and drawn out below to secure a +clamp delivery tube, and at the top expanded into a bulb closed with a +ground glass stopper, as shown in Fig. 100. Forty cubic centimeters of +liquid are placed in the tube and the surface is marked 0. Above this +the graduation is continued in cubic centimeters to 250, which figure +is just below the bulb at the top. + +The process of analysis is conducted as follows: About three grams of +the oil or fat are placed in a flask, with fifty cubic centimeters of +alcoholic potash lye, containing enough potassium hydroxid to ensure +complete saponification. The flask is closed and heated on a water-bath +until saponification is complete. The pressure flask to be described +hereafter may be conveniently used. After cooling, the excess of alkali +is neutralized with acetic acid in presence of phenolphthalien and then +alcoholic potash added until a faint pink color is produced. In a large +porcelain dish place 200 cubic centimeters of water and thirty of a ten +per cent solution of lead acetate and boil. Pour slowly, with constant +stirring, into the boiling liquid the soap solution prepared as above +described, and allow to cool, meanwhile continuing the stirring. At the +end, the liquid remaining is poured off and the solid residue washed +with hot water by decantation. + +The precipitate of lead salts is finally removed from the dish into a +stoppered bottle, the dish washed with pure ether, the washings added +to the bottle together with enough ether to make the total volume +thereof 120 cubic centimeters. The closed bottle is allowed to stand +for twelve hours with occasional shaking, by which time the lead oleate +will have been completely dissolved. The insoluble lead salts are next +separated by filtration, and the filtrate collected in the olein tube. +The washing is accomplished by ether and, to avoid loss, the funnel +is covered with a glass plate. The ethereal solution of lead oleate +is decomposed by dilute hydrochloric acid, using about forty cubic +centimeters of a mixture containing one part of strong acid to four +of water. The olein tube is closed and shaken until the decomposition +is complete, which will be indicated by the clearing of the ethereal +solution. The tube is allowed to remain at rest until the liquids +separate and the aqueous solution is run out from the pinch-cock at the +lower end. The residue is washed with water by shaking, the water drawn +off as just described, and the process continued until all acidity is +removed. + +Water is then added until the separating plane between the two liquids +is at the zero of the graduation, and enough ether added to make the +ethereal solution of a desired volume, say 200 cubic centimeters. After +well mixing, the ethereal solution or an aliquot part thereof, _e.g._, +fifty cubic centimeters, is removed by the side tubulure and nearly +the whole of the ether removed from the portion by distillation. To +the residue are added fifty cubic centimeters of pure alcohol and the +solution is titrated for oleic acid with decinormal sodium hydroxid +solution. Each cubic centimeter of the hydroxid solution used is +equivalent to 0.0282 gram of oleic acid. The total quantity of oleic +acid contained in the amount of fat used is readily calculated from the +data obtained. + +To determine the iodin absorption of the free acid another measured +quantity of the ethereal solution containing as nearly as possible half +a gram of oleic acid, is withdrawn from the olein tube, and the ether +removed in an atmosphere of pure carbon dioxid. To the residue, without +allowing it to come in contact with the air, fifty cubic centimeters +of Hübl’s reagent are added and the flask put aside in the dark for +twelve hours. At the end of this time thirty-five cubic centimeters of +a ten per cent solution of potassium iodid are added, the contents of +the flask made up to a quarter of a liter with water, fifteen cubic +centimeters of chloroform added, and the excess of iodin titrated +in the way already described. The percentage of iodin absorbed is +calculated as already indicated. + +Lane has proposed a more rapid process for the above determination.[304] +The lead soaps are precipitated in a large erlenmeyer and cooled +rapidly in water, giving the flask meanwhile a circular motion which +causes the soaps to adhere to its walls. Wash with hot water, rinsing +once with alcohol, add 120 cubic centimeters of ether, attach a reflux +condenser, and boil until the lead oleate is dissolved, cool slowly, to +allow any lead stearate which has passed into solution to separate, and +filter into the olein tube. The rest of the operation is conducted as +described above. The percentage of oleic acid and its iodin absorption +in the following glycerids are given in the table below: + + Cottonseed oil. Lard. Peanut oil. + Per cent oleic acid 75.16 64.15 79.84 + Per cent iodin absorbed 141.96 99.48 114.00 + +=340. Saponification.=—In many of the analytical operations which are +conducted on the glycerids it is necessary to decompose them. When this +is accomplished by the action of a base which displaces the glycerol +from its combination with the fat acids, the resulting salts are known +as soaps and the process is named saponification. In general use the +term saponification is applied, not only strictly, as above defined, +but also broadly, including the setting free of the glycerol either by +the action of strong acids or by the application of superheated steam. +In chemical processes the saponification of a glycerid is almost +always accomplished by means of soda or potash lye. This may be in +aqueous or alcoholic solution and the process is accomplished either +hot or cold, in open vessels or under pressure. It is only important +that the alkali and glycerid be brought into intimate contact. The +rate of saponification is a function of the intimacy of contact, the +nature of the solvent and the temperature. For chemical purposes, it +is best that the decomposition of the glycerid be accomplished at a +low temperature and for most samples this is secured by dissolving the +alkali in alcohol. + +In respect of solvents, that one would be most desirable, from +theoretical considerations, which acts on both the glycerids and +alkalies. In the next rank would be those which dissolve one or the +other of the materials and are easily miscible, as, for instance, +carbon tetrachlorid for the glycerid and alcohol for the alkali. +As a rule, the glycerid is not brought into solution before the +saponification process is commenced. Instead of using an alcoholic +solution of sodium or potassium hydroxid the sodium or potassium +alcoholate may be employed, made by dissolving metallic sodium or +potassium in alcohol. It is probable, however, that a little water is +always necessary to complete the process. + +If a fat be dissolved in ether and treated with sodium alcoholate, +a granular deposit of soap is soon formed and the saponification is +completed in twenty-four hours. As much as 150 grams of fat can be +saponified with ten grams of metallic sodium dissolved in 250 cubic +centimeters of absolute alcohol.[305] For practical purposes the +alcoholic solution of the hydroxid is sufficient. + +The chemical changes which fats undergo on saponification are of a +simple kind. When the process is accomplished by means of alkalies, +the alkaline base takes the place of the glycerol as indicated in the +following equation: + + Triolein 884. Potassium hydroxid 168. + C₃H₅(O.C₁₈H₃₃O)₃ + 3KOH = + + Potassium oleate 960. Glycerol 92. + (KO.C₁₈H₃₃O)₃ + C₃H₅(OH)₃. + +The actual changes which take place in ordinary saponification are +not so simple, however, since natural glycerids are mixtures of +several widely differing fats, each of which has its own rate of +decomposition. Palmitin and stearin, for instance, are saponified more +readily than olein and some of the saponifiable constituents of resins +and waxes are extremely resistant to the action of alkalies. The above +equation may be regarded as typical for saponification in aqueous or +alcoholic solutions in open dishes or under pressure. If the alkali +used be prepared by dissolving metallic sodium or potassium in absolute +alcohol (sodium alcoholate or ethoxid) the reaction which takes place +is probably represented by the equation given below: + + C₃H₅(O.C₁₈H₃₃O)₃ + 3C₂H₅.ONa = C₃H₅(ONa)₃ + 3C₁₈H₃₃O.O.C₂H₅, + +in which it is seen that complete saponification cannot occur without +the absorption of some water, by which the sodium glyceroxid is +converted into glycerol and sodium hydroxid, the latter compound +eventually uniting with the ethyl ether of the fat acid.[306] + +Glycerids are decomposed when heated with water under a pressure of +about sixteen atmospheres or when subjected to a current of superheated +steam at 200°. The reaction consists in the addition of the elements +of water, whereby the glyceryl radicle is converted into free glycerol +and the fat acid is set free. The chemical change which ensues is shown +below: + + C₃H₅(O.C₁₈H₃₃O)₃ + 3H₂O = 3C₁₈H₃₄O₂ + C₃H₅(OH)₃. + +The details of saponification with sulfuric acid are of no interest +from an analytical point of view.[307] + +=341. Saponification in an Open Dish.=—The simplest method of +saponifying fats is to treat them with the alkaline reagent in an +open dish. In all cases the process is accelerated by the application +of heat. Vigorous stirring also aids the process by securing a more +intimate mixture of the ingredients. This method of decomposing +glycerids, however, is not applicable in cases where volatile ethers +may be developed. These ethers may escape saponification and thus +prevent the formation of the maximum quantity of soap. While not suited +to exact quantitive work, the method is convenient in the preparation +of fat acids which are to be the basis of subsequent analytical +operations, as, for instance, in the preparation of fat acids for +testing with silver nitrate. Large porcelain dishes are conveniently +used and the heat is applied in any usual way, with care to avoid +scorching the fat. + +=342. Saponification under Pressure.=—The method of saponification +which has given the best satisfaction in my work and which has been +adopted by the Association of Official Agricultural Chemists is +described below.[308] + +_Reagents._—The reagents employed are a solution of pure potash +containing 100 grams of the hydroxid dissolved in fifty-eight grams of +recently boiled distilled water, alcohol of approximately ninety-five +per cent strength redistilled over caustic soda, and sodium hydroxid +solution prepared as follows: + +One hundred grams of sodium hydroxid are dissolved in 100 cubic +centimeters of distilled water. The caustic soda should be as free as +possible from carbonates, and be preserved from contact with the air. + +_Apparatus._—A saponification flask; it has a round bottom and a ring +near the top, by means of which the stopper can be tied down. The flask +is arranged for heating as shown in Fig. 101. A pipette graduated to +deliver forty cubic centimeters is recommended as being more convenient +than a burette for measuring the solutions: A pipette with a long stem +graduated to deliver 5.75 cubic centimeters at 50°. + +_Manipulation._—The fat to be examined should be melted and kept in a +dry warm place at about 60° for two or three hours, until the water +has entirely separated. The clear supernatant fat is poured off and +filtered through a dry filter paper in a jacket funnel containing +boiling water. Should the filtered fat, in a fused state, not be +perfectly clear, it must be filtered a second time. The final drying is +accomplished at 100° in a thin layer in a flat bottom dish, in partial +vacuum or an atmosphere of inert gas. + +The saponification flasks are prepared by thoroughly washing with +water, alcohol, and ether, wiping perfectly dry on the outside, +and heating for one hour at the temperature of boiling water. The +hard flasks used in moist combustions with sulfuric acid for the +determination of nitrogen are well suited for this work. The flasks +should be placed in a tray by the side of the balance and covered with +a silk handkerchief until they are perfectly cool. They must not be +wiped with a silk handkerchief within fifteen or twenty minutes of the +time they are weighed or else the electricity developed will interfere +with weighing. The weight of the flasks having been accurately +determined, they are charged with the melted fat in the following way: + +[Illustration: FIG. 101.—APPARATUS FOR SAPONIFYING UNDER PRESSURE.] + +The pipette with a long stem, marked to deliver 5.75 cubic centimeters, +is warmed to a temperature of about 50°. The fat, having been poured +back and forth once or twice into a dry beaker in order to thoroughly +mix it, is taken up in the pipette, the nozzle of the pipette having +been previously wiped to remove any externally adhering fat, is carried +to near the bottom of the flask and 5.75 cubic centimeters of fat +allowed to flow into the flask. After the flasks have been charged +in this way they should be re-covered with the silk handkerchief and +allowed to stand for fifteen or twenty minutes, when they are again +weighed. + +=343. Methods of Saponification.=—_In the Presence of Alcohol._—Ten +cubic centimeters of ninety-five per cent alcohol are added to the fat +in the flask, and then two cubic centimeters of the sodium hydroxid +solution. A soft cork stopper is inserted and tied down with a piece +of twine. The saponification is completed by placing the flask upon +the water or steam-bath. The flask during the saponification, which +should last one hour, should be gently rotated from time to time, being +careful not to project the soap for any distance up its sides. At the +end of an hour the flask, after having been cooled to near the room +temperature, is opened. + +_Without the Use of Alcohol._—To avoid the danger of loss from the +formation of ethers, and the trouble of removing the alcohol after +saponification, the fat may be saponified with a solution of caustic +potash in a closed flask without using alcohol. The operation is +carried on exactly as indicated above for saponification in the +presence of alcohol, using potassium instead of sodium hydroxid +solution. For the saponification, use two cubic centimeters of the +potassium hydroxid solution which are poured on the fat after it has +solidified in the flask. Great care must be taken that none of the +fat be allowed to rise on the sides of the saponifying flask to a +point where it cannot be reached by the alkali. During the process of +saponification the flask can only be very gently rotated in order to +avoid the difficulty mentioned. This process is not recommended with +any apparatus except a closed flask with round bottom. Potash is used +instead of soda so as to form a softer soap and thus allow a more +perfect saponification. + +The saponification may also be conducted as follows: The alkali and fat +in the melted state are shaken vigorously in the saponification flask +until a complete emulsion is secured. The rest of the operation is then +conducted as above. + +=344. Saponification in the Cold.=—By reason of the danger of loss +from volatile ethers in the hot alcoholic saponification, a method +for successfully conducting the operation in the cold is desirable. +Such a process has been worked out by Henriques.[309] It is based +upon the previous solution of the fat in petroleum ether, in which +condition it is so easily attacked by the alcoholic alkali as to make +the use of heat during the saponification unnecessary. The process is +conveniently conducted in a porcelain dish covered with a watch glass. +Five grams of the fat are dissolved in twenty-five cubic centimeters +of petroleum ether and treated with an equal quantity of four per cent +alcoholic soda lye. The process of saponification begins at once and +is often indicated by the separation of sodium salts. It is best to +allow the action to continue over night and, with certain difficultly +saponifiable bodies, such as wool fat and waxes, for twenty-four hours. +In the case of butter fat an odor of butyric ether may be perceived at +first but it soon disappears. After the saponification is complete, +the excess of alkali is determined by titration in the usual way with +set hydrochloric acid, using phenolphthalien as indicator. For the +determination of volatile acids, the mixture, after saponification is +complete, is evaporated rapidly to dryness, the solid matter being +reduced to powder with a glass rod, after which it is transferred +to a distilling flask and the volatile acids secured by the usual +processes. In comparison with the saponification and reichert-meissl +numbers obtained with hot alcoholic potash, the numbers given by the +cold process are found to be slightly higher with those fats which give +easily volatile ethers. On account of the simplicity of the process +and the absence of danger of loss from ethers, it is to be recommended +instead of the older methods in case a more extended trial of it should +establish the points of excellence claimed above. + +=345. Saponification Value.=—The number of milligrams of potassium +hydroxid required to completely saturate one gram of a fat is known as +the saponification value of the glycerid. The process of determining +this value, as worked out by Koettstorfer and modified in the +laboratory of the Department of Agriculture, is as follows:[310] + +The saponification is accomplished with the aid of potassium hydroxid +and in the flask and manner described in the preceding paragraph. About +two grams of the fat will be found a convenient quantity. Great care +must be exercised in measuring the alkaline solution, the same pipette +being used in each case and the same time for draining being allowed in +every instance. Blanks are always to be conducted with each series of +examinations. As soon as the saponification is complete, the flask is +removed from the bath, allowed to cool and its contents are titrated +with seminormal hydrochloric acid and phenolphthalien as indicator. +The number expressing the saponification value is obtained by +subtracting the number of cubic centimeters of seminormal hydrochloric +acid required to neutralize the alkali after saponification from +that required to neutralize the alkali of the blank determinations, +multiplying the result by 28.06 and dividing the product by the number +of grams of fat employed. + +_Example._—Weight of sample of fat used 1.532 grams: Number of cubic +centimeters half normal hydrochloric acid required to saturate blank, +22.5: Number of cubic centimeters of half normal hydrochloric acid +required to saturate the alkali after saponification 12.0: Difference, +10.5 cubic centimeters: + +Then 10.50 × 28.06 ÷ 1.532 = 192.3. + +This latter number represents the saponification value of the sample. + +=346. Saponification Equivalent.=—Allen defines the saponification +equivalent as the number of grams of fat saponified by one equivalent, +_viz._, 56.1 grams of potassium hydroxid.[311] The saponification +equivalent is readily calculated from the saponification value using +it as a divisor and 56100 as a dividend. Conversely the saponification +value may be obtained by dividing 56100 by the saponification +equivalent. No advantage is gained by the introduction of a new term so +nearly related to saponification value. + +=347. Saponification Value of Pure Glycerids.=—The theoretical +saponification values of pure glycerids are given in the following +table.[312] + + Molecular Saponification + Name. Symbol. weight. value. + Butyrin C₃H₅(O.C₄H₇O)₃ 302 557.3 + Valerin C₃H₅(O.C₅H₉O)₃ 344 489.2 + Caproin C₃H₅(O.C₆H₁₁O)₃ 386 438.3 + Caprin C₃H₅(O.C₁₀H₁₉O)₃ 554 305.0 + Laurin C₃H₅(O.C₁₂H₂₃O)₃ 638 263.8 + Myristin C₃H₅(O.C₁₄H₂₇O)₃ 722 233.1 + Palmitin C₃H₃(O.C₁₆H₃₁O)₃ 806 208.8 + Stearin C₃H₅(O.C₁₈H₃₅O)₃ 890 189.1 + Olein C₃H₅(O.C₁₈H₃₃O)₃ 884 190.4 + Linolein C₃H₅(O.C₁₈H₃₁O)₃ 878 191.7 + Ricinolein C₃H₅(O.C₁₈H₃₃O₂)₃ 932 180.6 + Euricin C₃H₅(O.C₂₂H₁₄O)₃ 1052 160.0 + +From the above table it is seen that in each series of glycerids the +saponification equivalent falls as the molecular weight rises. + +=348. Acetyl Value.=—Hydroxy acids and alcohols, when heated with +glacial acetic acid, undergo a change which consists in substituting +the radicle of acetic acid for the hydrogen atom of the alcoholic +hydroxyl group. This change is illustrated by the equations below:[313] + +_For a Fat Acid_: + + Ricinoleic acid. Acetic anhydrid. + C₁₇H₃₂(OH).COOH + (C₂H₃O)₂O = + + Acetyl-ricinoleic acid. Acetic acid. + C₁₇H₃₂(O.C₂H₃O)COOH + HC₂H₃O₂. + +_For an Alcohol_: + + Cetyl alcohol. Acetic anhydrid. Cetyl acetate. Acetic acid. + C₁₆H₃₃.OH + (C₂H₃O)₂O = C₁₆H₃₃.C₂H₃O + HC₂H₃O₂. + +_Determination._—The method of determining the acetyl value of a fat or +alcohol has been described by Benedikt and Ulzer.[314] The operation is +conducted on the fat acids and not on the glycerids containing them. + +The insoluble fat acids are prepared as directed in paragraph =340=. + +From twenty to fifty grams of the fat acids are boiled with an equal +volume of acetic anhydrid, in a flask with a reflux condenser, for two +hours. The contents of the flask are transferred to a larger vessel +of about one liter capacity, mixed with half a liter of water and +boiled for half an hour. To prevent bumping, some bubbles of carbon +dioxid are drawn through the liquid by means of a tube drawn out to +a fine point and extending nearly to the bottom of the flask. The +liquids are allowed to separate into two layers and the water is +removed with a syphon. The oily matters are treated several times with +boiling water until the acetic acid is all washed out. The acetylated +fat acids are filtered through a dry hot jacket filter and an aliquot +part, from three to five grams, is dissolved in absolute alcohol. +After the addition of phenolphthalien the mixture is titrated as in +the determination of the saponification value. The acid value thus +obtained is designated as the acetyl acid value. A measured quantity +of alcoholic potash, standardized by seminormal hydrochloric acid, +is added, the mixture boiled and the excess of alkali determined by +titration. The quantity of alkali consumed in this process measures the +acetyl value. The sum of the acetyl acid and the acetyl values is the +acetyl saponification value. The acetyl value is therefore equal to +the difference of the saponification and acid values of the acetylated +fat acids. In other words, the acetyl value indicates the number of +milligrams of potassium hydroxid required to neutralize the acetic acid +obtained by the saponification of one gram of the acetylated fat acids. + +_Example._—A portion of the fat acids acetylated as described, weighing +3.379 grams, is exactly neutralized by 17.2 cubic centimeters of +seminormal potassium hydroxid solution, corresponding to 17.2 × 0.02805 += 0.4825 gram of the hydroxid, hence 0.4825 × 1000 ÷ 3.379 = 142.8, the +acetyl acid value of the sample. + +After the addition of 32.8 cubic centimeters more of the seminormal +potash solution, the mixture is boiled to saponify the acetylated +fat acids. The residual potash requires 14.2 cubic centimeters of +seminormal hydrochloric acid. The quantity of potash required for the +acetic acid is therefore 32.8 - 14.3 = 18.5 cubic centimeters or 18.5 × +0.02805 = 0.5189 gram of potassium hydroxid. Then 0.5189 × 1000 ÷ 3.379 += 153.6 = acetyl value of sample. The sum of these two values, _viz._, +142.8 and 153.6 is 296.4, which is the acetyl saponification value of +the sample. As with the iodin numbers, however, it is also found that +acids of the oleic series give an acetyl value when treated as above, +and it has been proposed by Lewkowitsch to determine, in lieu of the +data obtained, the actual quantity of acetic acid absorbed by fats.[315] +This is accomplished by saponifying the acetylated product with +alcoholic potash and determining the free acetic acid by distillation, +in a manner entirely analogous to that used for estimating volatile fat +acids described further on. + +The rôle which the acetyl value plays in analytical determinations is +interesting, but the data it gives are not to be valued too highly. + +=349. Determination of Volatile Fat Acids.=—The fat acids which are +volatile at the temperature of boiling water, consist chiefly of +butyric and its associated acids occurring in the secretions of the +mammary glands. Among vegetable glycerids cocoanut oil is the only +common one which has any notable content of volatile acids. The boiling +points of the above acids, in a pure state, are much higher than the +temperature of boiling water; for instance, butyric acid boils at about +162°. By the expression volatile acids, in analytical practice, is +meant those which are carried over at 100°, or a little above, with +the water vapor, whatever be their boiling point. The great difficulty +of removing the volatile from the non-volatile fat acids has prevented +the formulation of any method whereby a sharp and complete separation +can be accomplished. The analyst, at the present time, must be content +with some approximate process which, under like conditions, will give +comparable results. Instead, therefore, of attempting a definite +determination, he confines his work to securing a partial separation +and in expressing the degree of volatile acidity in terms of a standard +alkali. To this end, a definite weight of the fat is saponified, the +resulting soap decomposed with an excess of fixed acid, and a definite +volume of distillate collected and its acidity determined by titration +with decinormal alkali. The weight of fat operated on is either two and +a half[316] or five grams.[317] + +Numerous minor variations have been proposed in the process, the most +important of which is in the use of phosphoric instead of sulfuric +acid in the distillation. An extended experience with both acids has +shown that no danger is to be apprehended in the use of sulfuric acid +and that on the whole it is to be preferred to phosphoric.[318] + +The process as used in this laboratory and as adopted by the official +agricultural chemists is conducted as follows:[319] + +=350. Removal of the Alcohol.=—The saponification is accomplished in +the manner already described, (=341-344=) and when alcoholic potash is +used proceed as follows: + +The stopper having been laid loosely in the mouth of the flask, the +alcohol is removed by dipping the flask into a steam-bath. The steam +should cover the whole of the flask except the neck. After the alcohol +is nearly removed, frothing may be noticed in the soap, and to avoid +any loss from this cause or any creeping of the soap up the sides of +the flask, it should be removed from the bath and shaken to and fro +until the frothing disappears. The last traces of alcohol vapor may be +removed from the flask by waving it briskly, mouth down, to and fro. + +_Dissolving the Soap._—After the removal of the alcohol the soap +should be dissolved by adding 100 cubic centimeters of recently +boiled distilled water, or eighty cubic centimeters when aqueous +potassium hydroxid has been used for saponification, and warming on the +steam-bath, with occasional shaking, until the solution of the soap is +complete. + +_Setting free the Fat Acids._—When the soap solution has cooled to +about 60° or 70°, the fat acids are separated by adding forty cubic +centimeters of dilute sulfuric acid solution containing twenty-five +grams of acid in one liter, or sixty cubic centimeters when aqueous +potassium hydroxid has been used for saponification. + +_Melting the Fat Acid Emulsion._—The flask is restoppered as in the +first instance and the fat acid emulsion melted by replacing the flask +on the steam-bath. According to the nature of the fat examined, the +time required for the fusion of the fatty acid emulsions may vary from +a few minutes to several hours. + +_The Distillation._—After the fat acids are completely melted, which +can be determined by their forming a transparent, oily layer on the +surface of the water, the flask is cooled to room temperature, and +a few pieces of pumice stone added. The pumice stone is prepared by +throwing it, at a white heat, into distilled water, and keeping it +under water until used. The flask is connected with a glass condenser, +Fig. 102, slowly heated with a naked flame until ebullition begins, and +then the distillation continued by regulating the flame in such a way +as to collect 110 cubic centimeters of the distillate in, as nearly as +possible, thirty minutes. The distillate should be received in a flask +accurately marked at 110 cubic centimeters. + +[Illustration: FIG. 102.—APPARATUS FOR THE DISTILLATION OF VOLATILE +ACIDS.] + +_Titration of the Volatile Acids._—The 110 cubic centimeters of +distillate, after thorough mixing, are filtered through perfectly +dry filter paper, 100 cubic centimeters of the filtered distillate +poured into a beaker holding about a quarter of a liter, half a cubic +centimeter of phenolphthalien solution added and decinormal barium +hydroxid solution run in until a red color is produced. The contents +of the beaker are then returned to the measuring flask to remove any +acid remaining therein, poured again into the beaker, and the titration +continued until the red color produced remains apparently unchanged for +two or three minutes, The number of cubic centimeters of decinormal +barium hydroxid solution required should be increased by one-tenth to +represent the entire distillate. + +The number thus obtained expresses, in cubic centimeters of decinormal +alkali solution, the volatile acidity of the sample. In each case +blank distillations of the reagents used should be conducted under +identical conditions, especially when alcoholic alkali is used for +saponification. It is difficult to secure alcohol which will not yield +a trace of volatile acid in the conditions named. The quantity of +decinormal alkali required to neutralize the blank distillate is to be +deducted from that obtained with the sample of fat. + +=351. Determination of Soluble and Insoluble Fat Acids.=—The volatile +fat acids are more or less soluble in water, while those which are +not distillable in a current of steam are quite insoluble. It is +advisable, therefore, to separate these two classes of fat acids, and +the results thus obtained are perhaps more decidedly quantitive than +are given by the distillation process just described. The methods used +for determining the percentage of insoluble acids are essentially those +of Hehner.[320] Many variations of the process have been proposed, +especially in respect of the soluble acids.[321] + +The process, as conducted in this laboratory and approved by the +Association of Official Agricultural Chemists, is as follows: + +_Preparation of Reagents.—Sodium Hydroxid Solution._—A decinormal +solution of sodium hydroxid is used. Each cubic centimeter contains +0.0040 gram of sodium hydroxid and neutralizes 0.0088 gram of butyric +acid (C₄H₈O₂). + +_Alcoholic Potash Solution._—Dissolve forty grams of good caustic +potash in one liter of ninety-five per cent alcohol redistilled over +caustic potash or soda. The solution must be clear and the potassium +hydroxid free from carbonates. + +_Standard Acid Solution._—Prepare accurately a half normal solution of +hydrochloric acid. + +_Indicator._—Dissolve one gram of phenolphthalien in 100 cubic +centimeters of ninety-five per cent alcohol. + +_Determination.—Soluble Acids._—About five grams of the sample are +placed in the saponification flask already described, fifty cubic +centimeters of the alcoholic potash solution added, the flask stoppered +and placed in the steam-bath until the fat is entirely saponified. The +operation may be facilitated by occasional agitation. The alcoholic +potash is always measured with the same pipette and uniformity further +secured by allowing it to drain the same length of time (thirty +seconds). Two or three blank experiments are conducted at the same time. + +In from five to thirty minutes, according to the nature of the fat, the +liquid will appear perfectly homogeneous and, when this is the case, +the saponification is complete and the flask is removed and cooled. +When sufficiently cool, the stopper is removed and the contents of +the flask rinsed with a little ninety-five per cent alcohol into an +erlenmeyer, of about 200 cubic centimeters capacity, which is placed on +the steam-bath together with the blanks until the alcohol is evaporated. + +The blanks are titrated with half normal hydrochloric acid, using +phenolphthalien as indicator, and one cubic centimeter more of the half +normal hydrochloric acid than is required to neutralize the potash in +the blanks is run into each of the flasks containing the fat acids. The +flask is connected with a reflux condenser and placed on the steam-bath +until the separated fat acids form a clear stratum on the upper surface +of the liquid. The flask and contents are cooled in ice-water. + +The fat acids having quite solidified, the liquid contents of the +flask are poured through a dry filter into a liter flask, taking care +not to break the cake. Between 200 and 300 cubic centimeters of water +are brought into the flask, the cork with the condenser reinserted +and the flask placed on the steam-bath until the cake of acid is +thoroughly melted. During the melting of the cake of fat acids, the +flask should occasionally be agitated with a rotary motion in such a +way that its contents are not made to touch the cork. When the fat +acids have again separated into an oily layer, the flask and its +contents are cooled in ice-water and the liquid filtered through the +same filter into the same liter flask as before. This treatment with +hot water, followed by cooling and filtration of the wash water, is +repeated three times, the washings being added to the first filtrate. +The mixed washings and filtrate are made up to one liter, and 100 +cubic centimeters thereof in duplicate are titrated with decinormal +sodium hydroxid. The number of cubic centimeters of sodium hydroxid +required for each 100 cubic centimeters of the filtrate is multiplied +by ten. The number so obtained represents the volume of decinormal +sodium hydroxid neutralized by the soluble fat acids of the fat, plus +that corresponding to the excess of the standard acid used, _viz._, one +cubic centimeter. The number is therefore to be diminished by five, +corresponding to the excess of one cubic centimeter of half normal +acid. This corrected volume multiplied by 0.0088 gives the weight of +soluble acids as butyric acid in the amount of fat saponified. + +_Insoluble Acids._—The flask containing the cake of insoluble fat acids +from the above determination and the paper through which the soluble +fat acids have been filtered are allowed to drain and dry for twelve +hours, when the cake, together with as much of the fat acids as can be +removed from the filter paper, is transferred to a weighed evaporating +dish. The funnel, with the filter, is then placed in an erlenmeyer and +the paper thoroughly washed with absolute alcohol. The flask is rinsed +with the washings from the filter paper, then with pure alcohol, and +the rinsings transferred to the evaporating dish. The dish is placed +on the steam-bath until the alcohol is evaporated, dried for two hours +at 100°, cooled in a desiccator and weighed. It is again placed in the +air-bath for two hours, cooled as before and weighed. If there be any +considerable decrease in weight, reheat two hours and weigh again. The +final weighing gives the weight of insoluble fat acids in the sample, +from which the percentage is easily calculated. + +The quantity of non-volatile and insoluble acids in common glycerids +is from ninety-five to ninety-seven parts in 100. The glycerids yield +almost the same proportion of fat acids and glycerol when the acids are +insoluble and have high molecular weights. When the acids are soluble +and the molecular weight low the proportion of acids decreases and that +of glycerol increases. + +In the following table will be found the data secured by quantitive +saponification and separation of soluble and insoluble acids found in +the more common glycerids:[322] + + Yield per 100 parts + Molecular weight of of glycerid. + Glycerid. Fat acid. Glycerid. Fat acid. Fat acid. Glycerol. + Stearin Stearic 890 284 95.73 10.34 + Olein Oleic 884 282 95.70 10.41 + Palmitin Palmitic 806 256 95.28 11.42 + Myristin Myristic 722 228 94.47 12.74 + Laurin Lauric 638 200 94.95 14.42 + Caprin Capric 594 172 93.14 15.48 + Caproin Caproic 386 116 90.16 23.83 + Butyrin Butyric 302 88 87.41 30.46 + +The general expression for the saponification of a neutral fat is +C₃H₅O₃.R₃ + 3H₂O = 3R.OH + C₃H₈O₃, in which R represents the acid +radicle. It is evident from this that the yield of more than 100 parts +of fat acids and glycerol given by glycerids is due to the absorption +of water during the reaction. + +=352. Formulas for General Calculations.=—For calculating the +theoretical yields of fat acids and glycerol, the following general +formulas may be used: + + Let _M_ = the molecular weight of the fat acid: + _K_ = saponification value: + _F_ = the quantity of free fat acids in the glycerid: + _N_ = the quantity of neutral fat in the glycerid: + _A_ = the number of milligrams of potassium hydroxid required + to saturate the free acid in one gram of the + sample. + +The free acid is determined by the method given below. + +_M_ grams of a fat acid require 56100 milligrams of potassium hydroxid +for complete neutralization while _F_ grams corresponding to 100 grams +of fat are saturated by 100 × _A_ milligrams of the alkali. + + Then _M_ : 56100 = _F_ : 100_A_. + + _AM_ + Whence _F_ = ------ (1). + 561 + +Likewise since _M_ grams of fat acid require the quantity of potassium +hydroxid mentioned above we have: + + 1 : _K_ = _M_ : 56100, + + 56100 + Whence _M_ = ------ (2). + _K_ + + Substituting this value of _M_ in (1) we have + + _A_ × 56100 100_A_ + _F_ = ------------ = ------- (3). + 561 × _K_ _K_ + +It is evident that it is not necessary to calculate the acid value +(_A_) of the sample and the saponification value (_K_) of the free fat +acids, the ratio _A_/_K_ alone being required. It will be sufficient +therefore to substitute for _A_ and _K_ the number of cubic centimeters +of alkali solutions required for one gram of the fat and one gram of +the fat acids, respectively. If _a_ and _b_ represent these numbers the +formula may be written + + 100_a_ + _F_ = ------- (4); + _b_ + + 100_a_ + and _N_ = 100 - _F_ = 100 - ------ (5). + _b_ + +To simplify the determinations, it may be assumed that the free fat +acids have the same molecular weight as those still in combination with +the glycerol in any given sample. On this assumption, the process may +be carried on by determining the acid value _A_ and the saponification +value _K_ for the total fat acids. The mean molecular weight _M_, the +percentage of free fat acids _F_, and the proportion of neutral fat +_N_, may then be calculated from the formulas (2), (3), (4), and (5). + +Further, let _G_ = the quantity of glycerol and _L_ that of fat acids +obtainable from one gram of neutral fat, that is, ¹/₁₀₀ of _H_ the +percentage of total fat acids. + +The molecular weight of the neutral fat in each case is 3_M_ + 38. +Therefore, 3_M_ + 38 parts of neutral fat yield 3_M_ parts of fat acids +and ninety-two parts of glycerol (C₃H₈O₃ = 92). + + _H_ 3_M_ + Then _L_ = ----- = ---------- (6); + 100 3_M_ + 38 + + 92 + and _G_ = --------- (7). + 3_M_ + 38 + +_N_ per cent of neutral fat yields, therefore, on saponification, the +following theoretical quantities of fat acids _F_, and glycerol _G_ +expressed as parts per hundred. + + 3_M_ + _F_ = _N_ × -------- (8); + 3_M_ + 38 + + 92 + and _G_ = _N_ × --------- (9). + 3_M_ + 38 + +Formula (9) expresses also the total yield of glycerol from any given +sample. For a further discussion of this part of the subject a work of +a more technical character may be consulted.[323] + +=353. Determination of a Free Fat Acid in a Fat.=—The principle of +the method rests upon the comparative accuracy with which a free fat +acid can be titrated with a set alkali solution when phenolphthalien +is used as an indicator. Among the many methods of manipulation which +the analyst has at his command there is probably none more simple and +accurate than that depending on the solution of the sample in alcohol, +ether, chloroform, or carbon tetrachlorid. Any acidity of the solvent +is determined by separate titration and the proper correction made. +Either an aqueous or alcoholic solution of the alkali may be used, +preferably the latter. The alkaline solution may be approximately or +exactly decinormal, but it is easier to make it approximately so and +to determine its real value before each operation by titration against +a standard decinormal solution of acid. About ten grams of the sample +and fifty cubic centimeters of the solvent will be found convenient +quantities. + +_Example._—Ten grams of rancid olive oil dissolved in alcohol ether +require three and eight-tenths cubic centimeters of a solution of +alcoholic potash to saturate the free acid present. When titrated with +decinormal acid the potash solution is found to contain 25.7 milligrams +of potassium hydroxid in each cubic centimeter. The specific gravity +of the oil is 0.917 and the weight used therefore 9.17 grams. Then the +total quantity of potassium hydroxid required for the neutralization of +the acid is 25.7 × 3.8 = 97.7 milligrams. + +The acid value _A_ is therefore: + + 3.8 × 25.7 + _A_ = ----------- = 10.6 + 9.17 + +It is customary to regard free acid as oleic, molecular weight 282. On +this assumption the percentage of free acids in the above case is found +by the formula + + 3.8 × 25.7 × 282 + _A_ (per cent) = ----------------- = 5.35 + 561 × 9.17 + +=354. Identification of Oils and Fats.=—Properly, the methods of +identifying and isolating the different oils and fats should be +looked for in works on food adulteration. There are, however, many +characteristics of these glycerids which can be advantageously +discussed in a work of this kind. Many cases arise in which the analyst +is called upon to determine the nature of a fat and discover whether +it be admixed with other glycerids. It is important often to know in +a given case whether an oil be of animal or vegetable origin. Many of +the methods of analysis already described are found useful in such +discriminations. For instance, a large amount of soluble or volatile +acids in the sample under examination, would indicate the presence +of a fat derived from milk while the form of the crystals in a solid +fat would give a clue to whether it were the product of the ox or the +swine. In the succeeding paragraphs will be briefly outlined some of +the more important additional methods of determining the nature and +origin of fats and oils of which the history is unknown. + +The data obtained by means of the methods which have been described, +both physical and chemical, are all useful in judging the character and +nature of a glycerid of unknown origin. The colorations produced by +oxidizing agents, in the manner already set forth will be found useful, +especially when joined to those obtained with cottonseed and sesame +oils yet to be described. For instance, the red coloration produced by +nitric acid of 1.37 specific gravity is regarded by some authorities +as characteristic of cottonseed oil as well as the reduction by it of +silver nitrate. The coloration tests with silver nitrate (paragraph +=320=) and with phosphomolydic acid (paragraph =318=) are also helpful +in classifying oils in respect of their animal or vegetable origin. +The careful consideration of these tests, together with a study of the +numbers obtained by treating the samples with iodin, and the heat of +bromination and sulfuric saponification, is commended to all who are +interested in classifying oils. In addition to these reactions a few +specific tests are added for more detailed work. + +=355. Consistence.=—It has already been said that oils are mostly of +vegetable origin and the solid fats of animal derivation. In the animal +economy it would be a source of disturbance to have in the tissues a +large body of fat which would remain in a liquid state at the normal +temperature of the body. Nearly all the animal fats are found to have +a higher melting point than the body containing them. An exception is +found in the case of butter fat, but it should be remembered that this +fat is an excretion and not intended for tissue building until it has +undergone subsequent digestion. Fish oils are another notable exception +to the rule, but in this case these oils can hardly be regarded as true +glycerids in the ordinary sense of that term. + +In general, it may be said that a sample of a glycerid, which in its +natural state remains liquid at usual room temperatures, is probably +an oil of vegetable origin. Fish oils have also an odor and taste +which prevent them from being confounded with vegetable oils. In oils +which are manufactured from animal glycerids such as lard oil, the +discrimination is more difficult but peculiarities of taste and color +are generally perceptible. + +=356. Nature of the Fat Acid.=—When it is not possible to discriminate +between samples by the sensible physical properties just described, +much light can be thrown on their origin by the determination of their +other physical properties, such as specific gravity, refractive index, +melting point, etc., in the manner already fully described. Further +light may be furnished by saponification and separation of the fat +acids. The relative quantities of oleic, stearic, palmitic, and other +acids will help to a correct judgment in respect of the nature of the +sample. The vegetable oils and lard oils, for instance, consist chiefly +of olein; lard and tallow contain a large proportion of stearin; palm +oil and butter fat contain considerable portions of palmitin, and +the latter is distinguished moreover by the presence of soluble and +volatile acids combined as butyrin and its associated glycerids. + +Oleic acid can be rather readily separated from stearic and palmitic +by reason of the solubility of its lead salts in ether. One method of +accomplishing this separation has already been described (paragraph +=339=). + +=357. Separation with Lime.=—A quicker, though perhaps not as accurate +a separation of the oleic from the palmitic and stearic acids, is +accomplished by means of lime according to the method developed by +Bondzyuski and Rufi.[324] This process is used chiefly, however, to +separate the free fat acids (palmitic, stearic) from the neutral fat +and the free oleic acid. It probably has no point of superiority over +the lead process. + +=358. Separation of the Glycerids.=—The fact that olein is liquid at +temperatures allowing palmitin and stearin to remain solid, permits of +a rough separation of these two classes of bodies by mechanical means. +The mixed fats are first melted and allowed to cool very slowly. In +these conditions the stearin and palmitin separate from the olein in +a crystalline form and the olein is removed by pressure through bags. +In this way lard is separated into lard oil, consisting chiefly of +olein, and lard stearin, consisting largely of stearin. Beef (caul) fat +is in a similar manner separated into a liquid (oleo-oil) and a solid +(oleo-stearin) portion. It is evident that these separations are only +approximate, but by repeated fractionations a moderately pure olein or +stearin may be obtained. + +=359. Separation as Lead Salts.=—Muter’s process, with a special +piece of apparatus, has already been described (=339=). For general +analytical work the special tube may be omitted. In a mixture of +insoluble free fat acids all are precipitated by lead acetate, and the +resulting soap may be extracted with ether, either with successive +shakings or in a continuous extraction apparatus. In this latter case +a little of the lead stearate or palmitate may pass into solution in +the hot ether and afterwards separate on cooling. When the operation +is conducted on from two to three grams of the dry mixed acids, the +percentage proportions of the soluble and insoluble acids (in ether) +can be determined. The lead salt which passes into solution can be +decomposed and the oleic acid removed, dried and weighed. Dilute +hydrochloric acid is a suitable reagent for decomposing the lead soap. +The difference between the weight of the oleic acid and that of the +mixed acids before conversion into lead soap furnishes the basis for +the calculation. For further details in respect of the fat acids the +reader may consult special analytical works.[325] + +=360. Separation of Arachidic Acid.=—Peanut oil is easily distinguished +from other vegetable glycerids by the presence of arachidic acid. + +The method used in this laboratory for separating arachidic acid is +a modification of the usual methods based on the process as carried +out by Milliau.[326] About twenty grams of the oil are saponified with +alcoholic soda, using twenty cubic centimeters of 36° baumé soda +solution diluted with 100 cubic centimeters of ninety per cent alcohol. +When the saponification is complete, the soda is converted into the +lead soap by treatment with a slight excess of a saturated alcoholic +solution of lead acetate. Good results are also obtained by using +dilute alcohol, _viz._, fifty per cent, instead of ninety per cent, in +preparing the lead acetate solution. + +While still warm the supernatant liquid is decanted, the precipitate +washed by decantation with warm ninety per cent alcohol and triturated +with ether in a mortar four times, decanting the ethereal solution in +each instance. By this treatment all of the lead oleate and hypogaeate +are removed and are found in the ethereal solution, from which they can +be recovered and the acids set free by hydrochloric acid and determined +in the usual way. + +The residue is transferred to a large dish containing two or three +liters of pure water and decomposed by the addition of about fifty +cubic centimeters of strong hydrochloric acid. The lead chlorid formed +is soluble in the large quantity of water present, which should be +warm enough to keep the free acids in a liquid state in which form +they float as a clear oily liquid on the surface. The free acids are +decanted and washed with warm water to remove the last traces of lead +chlorid and hydrochloric acid. The last traces of water are removed +by drying in a thin layer in vacuo. Practically all of the acids, +originally present in the sample except oleic and hypogaeic, are thus +obtained in a free state and their weight is determined. + +The arachidic acid may be separated almost quantitively by dissolving +the mixed acids in forty cubic centimeters of ninety per cent alcohol, +adding a drop of hydrochloric acid, cooling to 16° and allowing to +stand until the arachidic acid has crystallized. The crystals are +purified by washing twice with twenty cubic centimeters of ninety +per cent and three times with the same quantity of seventy per cent +alcohol. The residual impure arachidic acid is dissolved in boiling +absolute alcohol, poured through a filter and washed with pure hot +alcohol. The filtrate is evaporated to dryness and heated to 100° until +a constant weight is obtained. From the above data, the percentages of +oleic, hypogaeic, arachidic and other acids in the sample examined are +calculated. + +In the above process, owing to the pasty state of the lead soaps, the +trituration in a mortar with ether is found troublesome. The extraction +of the lead oleate and hypogaeate is facilitated by throwing the pasty +ethereal mass on a filter and washing it thoroughly with successive +portions of about fifty cubic centimeters of ether. By this variation, +it was found by Krug in this laboratory, that less ether was required +and a more complete removal of the lead oleate effected. The solution +of the lead oleate is completed by about half a dozen washings with +ether as above described. The extraction may also be secured by placing +the lead soaps in a large extracting apparatus and proceeding as +directed in paragraph =40=. The residue is washed from the filter paper +into a large porcelain dish and decomposed as already described with +hydrochloric acid. After the separation is complete, the mixture is +cooled until the acids are solid. The solid acids are then transferred +to a smaller dish, freed of water and dissolved in ether. The ethereal +solution is washed with water to remove any traces of lead salt or of +hydrochloric acid. After the removal of the ether, the arachidic acid +is separated as has already been described. + +The melting point of pure arachidic acid varies from 73° to 75°. + +=361. Detection of Arachis= (=Peanut=) =Oil.=—Kreis has modified +the usual process of Renard for the detection of arachis oil, by +precipitating the solution of the fat acid with an alcoholic instead +of an aqueous solution of lead acetate, in a manner quite similar to +that described above.[327] The fat acids are obtained in the usual +manner, washed with hot water and the acids from twenty grams of the +oil dissolved in 100 cubic centimeters of ninety per cent alcohol. +The solution is cooled in ice-water and the fat acids precipitated by +the addition of fifteen grams of lead acetate dissolved in 150 cubic +centimeters of ninety per cent alcohol. The precipitate, after standing +for two hours, is separated by filtration through cotton wool and is +extracted for six hours with ether. The residue is boiled with 250 +cubic centimeters of five per cent hydrochloric acid until the fat +acids appear as a clear oily layer upon the surface. The acids thus +obtained are washed with hot water to remove lead chlorid, dried by +pressing between blotting paper, dissolved in 100 cubic centimeters of +ninety per cent alcohol, cooled to 15° and allowed to stand for several +hours, after which time any arachidic acid present is separated by +crystallization and identified in the usual manner. + +When it is not important to obtain all of the acid present, the process +may be simplified in the following manner: + +The fat acids obtained from twenty grams of oil are dissolved in 300 +cubic centimeters of ether and treated at the temperature of ice-water +with a quantity of the alcoholic lead acetate solution mentioned above. +Lead oleate remains in solution and the precipitate which forms after +a few hours consists almost wholly of the lead salts of the solid fat +acids. The precipitate is collected, washed with ether and identified +in the usual manner. + +=362. Cottonseed Oil, Bechi’s Test.=—Crude, fresh cottonseed oil, when +not too highly colored, and generally the refined article, may be +distinguished from other oils by the property of reducing silver salts +in certain conditions. The reaction was first noticed by Bechi and has +been the subject of extensive discussions.[328] + +The process as proposed by Bechi has been modified in many ways but +apparently without improving it. It is conducted as follows: One gram +of silver nitrate is dissolved in 200 cubic centimeters of ninety-eight +per cent alcohol and forty cubic centimeters of ether and one drop of +nitric acid added to the mixture. Ten cubic centimeters of the oil +are shaken in a test tube with one cubic centimeter of this reagent, +and then with ten cubic centimeters of a mixture containing 100 +cubic centimeters of amyl alcohol and ten of colza oil. The mixture +is divided into two portions, one of which is put aside for future +comparison and the other plunged into boiling water for fifteen +minutes. A deep brown or black color, due to the reduction of silver, +reveals the presence of cottonseed oil. + +In this laboratory the heating is accomplished in a small porcelain +dish on which is often deposited a brilliant mirror of metallic silver. +The white color of the porcelain also serves as a background for the +observation of the coloration produced. In most instances a green color +has been noticed after the reduction of the silver is practically +complete. Unless cottonseed oil has been boiled or refined in some +unusual way, the test, as applied above, is rarely negative. The +reduction of the silver is doubtless due to some aldehydic principle, +present in extremely minute quantities, and which may be removed by +some methods of technical treatment. The silver nitrate test therefore +is reliable when the reduction takes place, but the absence of a +distinct reaction may not in all cases prove the absence of cottonseed +oil. + +=363. Milliau’s Process.=—Milliau has proposed the application of +the silver salt directly to the free fat acids of the oil instead of +to the oil itself.[329] About fifteen cubic centimeters of the oil +are saponified with alcoholic potash in the usual manner, 150 cubic +centimeters of water added to the dish and the mixture boiled until +the alcohol is evaporated. The fat acids are freed by the addition of +decinormal sulfuric acid and as they rise to the surface in a pasty +condition are removed with a spoon. The free acids are washed with +distilled water. The water is drained off and the free acids dissolved +in fifteen cubic centimeters of ninety-two per cent alcohol and two +cubic centimeters of a three per cent solution of silver nitrate. +The test tube containing the mixture is well shaken and placed in a +water-bath, out of contact with light, and left until about one-third +of the alcohol is evaporated. Ten cubic centimeters of water are +added, the heating continued for a few minutes and the color of the +supernatant fat acids observed. The presence of cottonseed oil is +revealed by the production of a lustrous precipitate which colors the +fat acids black. In some cases the process of Milliau gives better +results than the original method of Bechi, but this is not always the +case. It does away with the use of amyl alcohol and colza oil, but +its manipulation is more difficult. In all doubtful cases the analyst +should apply both methods. + +=364. Detection of Sesame Oil.=—Milliau has pointed out a +characteristic reaction of this oil which may be used with advantage +in cases of doubtful identity.[330] The identification is based on the +fact that the free acids of sesame oil, or some concomitant thereof, +give a rose-red color when brought in contact with a solution of sugar +in hydrochloric acid. + +The analytical process is conducted as follows: About fifteen grams +of the oil are saponified with alcoholic soda and when the reaction +is complete treated with 200 cubic centimeters of hot water and +boiled until the alcohol is removed. The fat acids are set free with +decinormal sulfuric acid and removed with a spoon as they rise to the +surface in a pasty state, in which condition they are washed by shaking +with water in a large test tube. When washed, the acids are placed in +an oven at 105° until the greater part of the water is evaporated and +the acids begin to become fluid. At this point they are treated with +half their volume of hydrochloric acid saturated with finely ground +sugar. On shaking the mixture, a rose color is developed which is +characteristic of the sesame oil. Other oils give either no coloration +or at most a yellow tint. + +=365. The Sulfur Chlorid Reaction.=—Some vegetable oils, when treated +with sulfur chlorid, give a hard product similar to elaidin, while lard +does not. This reaction is therefore helpful in discriminating between +some vegetable and animal glycerids. The process which is described by +Warren has been used with some satisfaction in this laboratory.[331] + +Five grams of the oil or fat are placed in a tared porcelain dish and +treated with two cubic centimeters of carbon bisulfid and the same +quantity of sulfur chlorid. The dish is placed on a steam-bath and its +contents stirred until the reaction is well under way. The heating is +continued until all volatile products are evaporated, the hard mass +being well rubbed up to facilitate the escape of imprisoned vapors. +The powdered or pasty mass is transferred to a filter and washed with +carbon bisulfid to remove all unaltered oil. The washing with carbon +bisulfid is hastened by pressure and about 200 cubic centimeters of the +solvent should be used. After drying, the weight of insoluble matter is +obtained and deducted from the total weight of the sample used. + +The color and tenacity of the hard, insoluble portion are +characteristic. The quantitive part of the operation appears to have +but little value, but applied qualitively in this laboratory it +produces hard, leathery masses with cotton, olive and peanut oils, +and but little change in lard and beef fats. Qualitively applied, +the process is conducted as described above but without making the +weighings. In this instance it is as easy of application as the process +of Bechi and is deserving of greater attention than has been given it +by analysts. + +In the combination which takes place between the sulfur and the fat it +is probable that only addition products are formed, since the quantity +of alkali required for saponification is not diminished by previously +treating the fat with sulfur chlorid.[332] The reactions which take +place are probably well represented by the following equations, in +which oleic acid is treated with sulfur chlorid: + + C₁₈H₃₄O₂ + S = C₁₈H₃₄S.O₂. + C₁₈H₃₄S.O₂ + NaOH = C₁₈H₃₄SO₂Na + H₂O. + +=366. Detection of Cholesterin and Phytosterin in +Glycerids.=—Cholesterin is often found in animal glycerids and a +corresponding body, phytosterin, is sometimes found in oils of a +vegetable origin.[333] When one of these two bodies is present it may +be useful in distinguishing between animal and vegetable glycerids. +They are detected as follows: Fifty grams of the glycerids in each +case are saponified with alcoholic alkali, preferably potash, in order +to have a soft soap. After saponification is complete, the alcohol is +evaporated and the residual soap dissolved in two liters of water. +The mixture is shaken with ether and the ethereal solution evaporated +to a small bulk. The residue, which may contain a small quantity of +unsaponified fat, is again treated with alcoholic potash and subjected +a second time to the action of ether, as indicated above, with the +addition of a few drops of water and of alcohol if the emulsion +separate slowly. The ethereal extract finally secured is allowed to +evaporate slowly and the cholesterin (phytosterin) is obtained in a +crystalline form. The melting point of the cholesterin crystals is 146° +and that of the phytosterin 132°. + +Cholesterin crystallizes in thin rhombic tables while phytosterin +separates in stellar aggregates or in bundles of long needles. + +When dissolved in chloroform the two products show different color +reactions with sulfuric acid, cholesterin giving a cherry and +phytosterin a blue-red tint. In a mixture of animal and vegetable +glycerids the two products are obtained together and the melting point +of the mixture may afford some idea of the relative quantities of each +present. It is evident, however, that no reliable judgment can be +formed from these data of the relative proportions of the two kinds of +glycerids in the original sample. + +=367. Cholesterin and Paraffin in Ether Extracts.=—In ethereal extracts +of some bodies, especially of flowers of the chrysanthemum, paraffin +is found combined with cholesterin. The two bodies may be separated as +follows:[334] + +The ether extract is treated with aqueous then with alcoholic potash +several times; the residue soluble in ether is a solid body melting at +from 70° to 100°. + +If the ethereal solution be cooled in a mixture of snow and salt, a +crystalline deposit is formed. This substance, purified by repeated +precipitations, is obtained colorless in fine crystalline scales +melting at 64°. It is very soluble in ether, benzene and chloroform, +almost insoluble in cold alcohol, and somewhat soluble in hot. + +Its percentage composition is: + + Per cent. + Carbon 85.00 + Hydrogen 14.95 + +It is therefore a paraffin. + +The ethereal solution, freed by the above process from paraffin, leaves +on evaporation a crystalline mass which is cholesterin, retaining +still a small quantity of fat matters. In treating the crystals with +alcoholic potash these fat bodies are saponified and the residue is +taken up with ether. The cholesterin is obtained in fine needles +melting at from 170° to 176°. It presents all the reactions of +cholesterin, especially the characteristic reaction with chloroform and +sulfuric acid. + +=368. Absorption of Oxygen.=—Among oils a distinction is made between +those which oxidize readily and those which are of a more stable +composition. Linseed oil, for instance, in presence of certain metallic +oxids, absorbs oxygen readily and is a type of the drying oils, while +olive oil represents the opposite type. + +The method of determining the quantity of oxygen absorbed is due to +Livache and is carried out as follows:[335] + +Precipitated metallic lead (by zinc) is mixed in a flat dish, with +the oil to be tested, in the proportions of one gram of lead to +three-quarters of a gram of oil, and exposed to the air and light of +the workroom. The dish is weighed from time to time until there is no +longer any increase in weight. + +Instead of lead, finely divided copper has been used by Krug in this +laboratory, but the percentage of absorption of oxygen is not so +high with copper as with lead. Krug found the quantities of oxygen +absorbed, after nine days, by the samples treated with copper and lead +respectively to be the following: + + Copper, per cent Lead, per + oxygen absorbed. cent oxygen + absorbed. + + Olive oil 1.69 2.03 + Cottonseed oil 4.25 5.30 + Peanut oil 2.74 3.87 + Linseed oil 5.55 7.32 + +Livache found that linseed oil absorbed about twice as much oxygen as +indicated by the data just given. + +=369. Elaidin Reactions.=—In discriminating between oils and fats +having a preponderance of olein and others with a smaller proportion of +that glycerid, the conversion of the olein into its isomer elaidin is +of diagnostic value. The following will be found a convenient method of +applying this test:[336] + +About ten cubic centimeters of the oil are placed in a test tube +together with half that quantity of nitric acid and one gram of +mercury. The mixture is shaken until the mercury dissolves when the +mass is allowed to remain at rest for twenty minutes. At the end of +this time it is again shaken and placed aside. In from one to three +hours the reaction is complete. Olive, peanut and lard oils give +very hard elaidins. The depth to which a plunger of given weight and +dimensions sinks into an elaidin mixture at a given temperature, has +been used as a measure of the percentage of olein contained in the +sample of oil, but it is evident that such a determination is only +roughly approximate. Copper may be used instead of mercury for the +generation of the oxids of nitrogen, but it is not so effective. The +vapors of nitric oxids may also be conducted directly into the oil +from a convenient generator. The reaction may also be accomplished +by shaking the oil with nitric acid and adding, a drop at a time, a +solution of potassium nitrite. + + +AUTHORITIES CITED IN PART FOURTH. + +[229] Benedikt and Lewkowitsch; Oils, Fats, Waxes, p. 1. + +[230] Op. cit. supra, p. 46. + +[231] Archiv für Physiologie, 1895, Band 61, S. 341: Chemiker-Zeitung +Repertorium, Band 16, S. 338. + +[232] Vid. op. cit. 1, p. 63. + +[233] Bulletin No. 46, Division of Chemistry, U. S. Department of +Agriculture, p. 25. + +[234] Journal of the Society of Chemical Industry, 1886, p. 508. + +[235] Bulletin No. 13, Division of Chemistry, U. S. Department of +Agriculture, p. 423. + +[236] Vid. op. cit. supra, p. 435. + +[237] Vid. op. cit. supra, p. 437. + +[238] Vid. op. et loc. cit. supra. + +[239] Benedikt and Lewkowitsch; Oils, Fats, and Waxes, pp. 96 et seq.: +Zune; Analyse des Beurres, pp. 26 et seq. + +[240] Journal of the Society of Chemical Industry, 1885, p. 535. + +[241] Vid. op. cit. 1, p. 97. + +[242] Vid. op. cit. 7, p. 443. + +[243] Vid. op. cit. 1, pp. 97 and 98. + +[244] Butter, its Analysis and Adulterations, p. 24. + +[245] Bulletin No. 46, Division of Chemistry, U. S. Department of +Agriculture, p. 34. + +[246] Vid. op. cit. 7, p. 447. + +[247] Analyse des Beurres, pp. 33 et 63: Zeitschrift für +Instrumentenkunde, 1887, Ss. 16, 55, 392, 444: Zeitschrift für +physikalische Chemie, Band 18, S. 294. (Ou. pp. 328-9 and 334 read +Amagat for Armagat.) + +[248] Zeitschrift für physikalische Chemie, Band 18, S. 294. + +[249] American Chemical Journal, Vol. 10, p. 392. + +[250] Vid. op. cit. 7, pp. 473 et seq. + +[251] Jean; Chimie Analytique des Matiéres Grasses, p. 26. + +[252] Vid. op. cit. supra, p. 31. + +[253] The Analyst, Vol. 20, p. 135. + +[254] Schlussbericht über die Butteruntersuchungsfrage, +Milchwirthschaftlicher Verein, Korrespondenzblatt, No. 39, 1891, S. 15. + +[255] Vid. op. cit. 7, p. 75. + +[256] Journal of the American Chemical Society, Vol. 15, p. 173. + +[257] Communicated by Krug to author. + +[258] Vid. op. cit. 7, pp. 449 et seq. + +[259] Vid. op. cit. 28, Vol. 18, p. 189. + +[260] Vid. op. cit. 7, Plates 32 and 35. + +[261] Vid. op. cit. supra, p. 452. + +[262] Vid. op. cit. supra., p. 93. + +[263] Vogel; Practische Spectralanalyse, S. 279: Zune; Analyse des +Beurres, Tome 2, p. 48: Benedikt and Lewkowitsch; Oils, Fats, Waxes, p. +83. + +[264] Bulletin de l’Association Belge des Chimistes, Tome 9, p. 145. + +[265] Journal of the Chemical Society, Abstracts, Vol. 46, p. 1078: +Dingler’s Polytechnisches Journal, Band 252, S. 296. + +[266] The Analyst, July 1894, p. 152. + +[267] Rapport sur les Procédé pour reconnâitre les Falsifications des +Huiles d’Olive, p. 37. + +[268] Vid. op. cit. 7, p. 251. + +[269] Taylor; Annual Report U. S. Department of Agriculture, 1877, p. +622: Milliau; Journal of the American Chemical Society, Vol. 15, p. 153. + +[270] Gantter; Zeitschrift für analytische Chemie, 1893, Band 32, S. +303. + +[271] Welmans; Journal of the Society of Chemical Industry, 1892, p. +548. + +[272] Vid. op. cit. 1, p. 254. + +[273] Pharmaceutische Zeitung, 1891, p. 798: The Analyst, Vol. 17, p. +59. + +[274] Comptes rendus, Tome 112, p. 105. + +[275] Pearmain and Moor; The Analyst, Vol. 20, p. 174. + +[276] Pharmaceutische Zeitschrift für Russland, 1888, S. 721: American +Journal of Pharmacy, 1889, p. 23. + +[277] Vid. op. cit. 7, p. 502. + +[278] Muir; Elements of Thermal Chemistry, p. 25 et seq. + +[279] Comptes rendus, Tome 35 (1852), p. 572. + +[280] Allen; Commercial Organic Analysis, Vol. 2, p. 56. + +[281] Vid. op. cit. 1, p. 235; et op. cit. 23, p. 217. + +[282] Vid. op. cit. 7, p. 44. + +[283] Vid. op. cit. supra, p. 445: Proceedings American Public Health +Association, Vol. 10. + +[284] Vid. op. cit. 23, p. 61. + +[285] Journal of the Society of Chemical Industry, 1891, p. 234. + +[286] Vid. op. cit. 1, p. 240. + +[287] The Analyst, Vol. 22, p. 58. + +[288] Vid. op. cit. supra, Vol. 20, p. 146. + +[289] Journal of the American Chemical Society, Vol. 17, p. 378. + +[290] Dingler’s Polytechnisches Journal, 1884, Ss. 253-281: Journal of +the Society of Chemical Industry, 1884, p. 641. + +[291] Bulletin No. 46, Division of Chemistry, U. S. Department of +Agriculture, p. 32. + +[292] Liebermann; Berichte der deutschen chemischen Gessellschaft, Band +24, S. 4117. + +[293] Vid. op. cit. 1, p. 136. + +[294] Vid. op. cit. 57, 1895, pp. 130 and 1030. + +[295] Zeitschrift für analytische Chemie, Band 32, Ss. 181 et seq. + +[296] Vid. op. cit. 61, Vol. 16, p. 372. + +[297] Zeitschrift für angewandte Chemie, 1895, S. 254. + +[298] Chemiker-Zeitung, Band 19, Ss. 1786 and 1831. + +[299] Vid. op. cit. 61, Vol. 16, p. 277. + +[300] Pharmaceutical Journal, Sept. 25, 1880. + +[301] Vid. op. cit. 59, Vol. 20, p. 50. + +[302] Williams; vid. op. cit. supra, Vol. 20, p. 277. + +[303] Vid. op. cit. 59, 1889, p. 61. + +[304] Vid. op. cit. 61, Vol. 15, p. 110. + +[305] Zeitschrift für physiologische Chemie, Band 14, S. 599; Band 12, +S. 321; Band 16, S. 152. + +[306] Vid. op. cit. 1, p. 60. + +[307] Vid. op. cit. supra, p. 557. + +[308] Vid. op. cit. 7, p. 459; vid. op. cit. 63, p. 27. + +[309] Vid. op. cit. 69, 1895, S. 721. + +[310] Vid. op. cit. 67, Band 18, S. 199: vid. op. cit. 7, pp. 58-461: +vid. op. cit. 63, p. 30. + +[311] Vid. op. cit. 52, p. 40. + +[312] Vid. op. cit. 1, p. 119. + +[313] Vid. op. cit. supra, p. 127. + +[314] Monatshefte für Chemie und verwandte Theile anderer +Wissenschaften, Band 8, S. 40. + +[315] Vid. op. cit. 57, 1890, p. 846. + +[316] Reichert; vid. op. cit. 67, Band 18, S. 68. + +[317] Meissl; vid. op. cit. 62, Band 233, S. 229. + +[318] Vid. op. cit. 1, p. 121. + +[319] Vid. op. cit. 63, p. 28. + +[320] Vid. op. cit. 67, Band 16, S. 145; Band 18, S. 68: vid. op. cit. +7, p. 53: vid. op. cit. 59, 1877, p. 147. + +[321] Vid. op. cit. 57, 1888, pp. 526 and 697: American Chemical +Journal, Vol. 10, p. 326: vid. op. cit. 1, pp. 123-127. + +[322] Vid. op. cit. 1, p. 143. + +[323] Vid. op. ch. 7, p. 143. + +[324] Vid. op. cit. 67, 1890, S. 4. + +[325] Allen; Commercial Organic Analysis, Vol. 2, pp. 224-236. + +[326] Analyse Chimique des Matiéres Grasses, p. 13. + +[327] Chemiker-Zeitung, Band 19, S. 451. + +[328] Annali del Laboratorio Chimico, 1891-92, p. 197: Bulletin No. 13, +Division of Chemistry, U. S. Department of Agriculture, p. 465: Journal +of Analytical and Applied Chemistry, Vol. 1, p. 449; Vol. 2, pp. 119 +and 275; vid. op. cit. 311. + +[329] Rapport presenté a l’Academie Sciences le 20 fevrier, 1883: +Analyse des Matiéres Grasses, p. 17: Bulletin No. 13, Division of +Chemistry, U. S. Department of Agriculture, p. 446. + +[330] Analyse des Matiéres Grasses, p. 15. + +[331] Chemical News, 1888, p. 113: Bulletin No. 13, Division of +Chemistry, U. S. Department of Agriculture, p. 468. + +[332] Vid. op. cit. 69, 1895, S. 535. + +[333] Justus Liebig’s Annalen der Chemie, Band 192, S. 178: vid. op. +cit. 67, Band 26, S. 575: vid. op. cit. 7, p. 514. + +[334] Journal de Pharmacie et de Chimie, 1889, p. 447. + +[335] Moniteur Scientifique, Tome 13, p. 263: vid. op. cit. 69, 1884, +S. 262. + +[336] Vid. op. cit. 7, p. 515. + + + + +PART FIFTH. + +SEPARATION AND ESTIMATION OF BODIES CONTAINING NITROGEN. + + +=370. Nature of Nitrogenous Bodies.=—The nitrogenous bodies, valuable +as foods, belong to the general class of proteids and albuminoids. They +are composed chiefly of carbon, hydrogen, oxygen, sulfur and nitrogen. +Some of them, as lecithin and nuclein, contain phosphorus instead of +sulfur, but these resemble the fats rather than the proteids. + +Nitrogenous organic bodies of the class mentioned above are designated +by the general name proteids. The term albumin is restricted in a +physiological sense to a certain class of proteids. The term albuminoid +is often used synonymously, as above, for proteids, but, more strictly +speaking, it should be reserved for that class of bodies such as +gelatin, mucin, keratin and the like, not really proteids, but, +nevertheless, closely resembling them.[337] In chemical composition the +proteids are characterized by the relative constancy of their nitrogen +content, the mean percentage of this element being about sixteen, but +varying in some instances more than two units from that number. + +=371. Classification of Proteids.=—Many classifications of the +proteids have been given based on physical, chemical and physiological +characteristics. In respect of origin, they are divided into two great +classes, _viz._, vegetable and animal. In respect of their physical and +chemical properties the following classification of the proteids may be +made.[338] + +_Albumins._—These are proteids soluble in water and not precipitated +from their aqueous solutions by sodium chlorid or magnesium sulfate. +They are easily coagulated by heat and are represented by three great +classes, _viz._, egg-, serum-, and lactalbumin. + +Egg albumin occurs in the white of egg; serum albumin is found in the +serum of the blood. Vegetable albumins have been prepared from wheat, +rye, potatoes, and papaws. (_Carica Papaya_). These vegetable albumins +are coagulated by heat at about 70° and are not precipitated by the +salt solutions named above, nor by acetic acid. The myrosin of mustard +seeds also resembles vegetable albumin. + +_Globulins._—These bodies are insoluble in water, soluble in dilute +solutions of neutral salts, but precipitated therefrom by saturation +with sodium chlorid or magnesium sulfate. They are coagulated by heat. +Among others belonging to this group are serum globulin, fibrinogen, +myosin, crystalin, and globin. + +Serum globulin is found in the serum of blood; cell globulin is found +in lymph cells; fibrinogen occurs in the blood plasma; plasmin, in +blood plasma; myosin, in dead muscles; vitellin, in the yolk of eggs; +crystalin, in the lens of the eye; haemoglobin, in the red pigment of +the blood; haemocyanin, in the blood of certain low grade animals. + +Vegetable globulins are found in the cereals, leguminous plants, +papaws and other vegetables, and are divided into two groups, myosins +and paraglobulins. The vegetable myosins coagulate at from 55° to +60° and are precipitated from a saline solution by removing the salt +by dialysis. In this form, however, they lose their true nature as +globulins, becoming insoluble in weak saline solutions. + +The vegetable paraglobulins are coagulated at from 70° to 75°. +Vegetable vitellin, which is not included in this classification, can +be obtained in a crystalline form and of remarkable purity.[339] + +_Albuminates._—This name is given to the compounds of the proteids with +metallic oxids or bases, and also to acid and alkali albumins. They +are insoluble in water or dilute neutral salts, but easily soluble in +strong acids or alkalies. Casein is a type of this group. + +Acid albumin is made from egg albumin by treatment with hydrochloric +acid; alkali albumin is formed in egg albumin by the action of a dilute +alkali; trinitroalbumin is formed from dry albumin by treatment with +nitric acid; casein or caseinogen is the chief proteid in milk. + +The chief vegetable albuminates are legumin and conglutin. Legumin +is a vegetable casein and occurs chiefly in peas, beans and other +leguminous seeds. It is prepared by extracting the meal of the seeds +mentioned with dilute alkali, filtering the extract, precipitating with +acetic acid, washing the precipitate with alcohol, and drying over +sulfuric acid. Treated with sulfuric acid it yields leucin, tyrosin and +glutamic and aspartic acids. Conglutin is prepared in a similar manner +from almonds. + +It is probable that these bodies do not exist as such in the fresh +seeds in question but are produced therein from the other proteids +by the alkali used in extraction. A further description of vegetable +proteids will be found in the special paragraphs devoted to the study +of these bodies in the principal cereals. + +_Proteoses._—This name is applied to proteids which are not coagulated +by heat, but most of them are precipitated by saturated solutions of +neutral salts. They are also precipitated by nitric acid. They are +formed from other proteids by the action of proteolytic ferments. The +albumoses represent this group. + +Protoalbumose is soluble in distilled water and weak saline solutions +and is precipitated by mercuric chlorid and copper sulfate. + +Heteroalbumose is insoluble in distilled water, but soluble in weak +saline solutions, from which it separates when the salts are removed +by dialysis. Deuteroalbumose is soluble in distilled water and saline +solutions and is not precipitated on saturation with sodium chlorid. It +is thrown out by mercuric chlorid but not by copper sulfate. + +Vegetable proteoses are known as phytalbumoses, two of which have been +found in the juice of the papaw mentioned above. They have also been +found in cereals. + +_Peptones._—These bodies are very soluble in water but are not thrown +out by heat, by saturation with neutral salts, nor by nitric acid. They +are completely precipitated by tannin and by strong alcohol. + +The peptones are the only soluble proteids which are not precipitated +by saturation with ammonium sulfate. The principal animal varieties are +hemi- and anti-peptones. These forms of proteids do not appear to exist +as such in vegetable products but are produced in large quantities by +treating other proteids with pepsin or pancreatin. In sprouting plants, +there appears to be a widely diffused ferment capable of converting +the proteids of the cotyledons into peptonoid bodies and thus fitting +them for entering the tissues of the new plant. + +_Insoluble Proteids._—This class includes a miscellaneous collection +of nitrogenous bodies not belonging to any of the definite groups +already mentioned. Fibrin and gluten are types of these insoluble +bodies. Fibrin is formed from the fibrinogen of fresh blood and causes +coagulation. When washed free of red blood corpuscles it is a white +elastic solid. It is insoluble in water and is converted into albumoses +and peptones by trypsin and pepsin. It swells up when treated with +a very weak one-tenth per cent solution of hydrochloric acid and +dissolves to acid albumin when heated therewith. + +Gluten is the most important of the insoluble vegetable proteids and +forms the chief part of the nitrogenous constituents of wheat. It is +readily prepared by washing wheat flour in cold water, as will be +described further on. It is probably a composite body formed by the +process of extraction from at least two proteid bodies existing in +wheat. When dried it forms a horny elastic mass of a yellow-gray color. +Gluten is composed of two bodies, one soluble the other insoluble +in alcohol. The part insoluble in alcohol has been called vegetable +fibrin, and the soluble part is subdivided into two portions, one +unicedin or vegetable unicin, and the other glutin (gliadin) or +vegetable gelatin. Gluten, according to some authorities, does not +properly exist in wheat flour, but is formed therein by the action +of water and certain ferments from free existing proteids. A better +explanation of the composition of gluten is that of Osborne, which will +be given further on. + +=372. Albuminoids.=—In this paragraph the term albuminoids is not +employed as synonymous with proteids but as characteristic of a class +of bodies nearly resembling them, but, nevertheless, differing from +them in many important particulars. Following is an abstract of their +classification as given in Watt’s dictionary.[340] + +_Collagen._—The nitrogenous portions of connective tissues are largely +composed of collagen. By boiling water it is converted into gelatin. +It may be prepared from tendons as follows: The tendinous tissues are +shredded as finely as possible and extracted with cold water to remove +the soluble proteids. Thereafter they are subjected for several days to +the action of lime water, which dissolves the cement holding the fibers +together. The residual insoluble matter is washed with water, weak +acetic acid, and again with water. The residue is chiefly collagen, +mixed, however, with some elastin and nuclein. With dilute acids and +alkalies collagen swells up after the manner of fibrin. The organic +nitrogenous matter of bone consists largely of collagen, which is +sometimes called ossein. + +_Gelatin._—When the white fibers of collagen, obtained as above, are +subjected to the action of boiling water or of steam under pressure +they dissolve and form gelatin. Isinglass is a gelatin made from the +swimming bladder of the sturgeon or other fish. Glue is an impure +gelatin obtained from hides and bones. Pure gelatin may be prepared +from the commercial article by removing all soluble salts therefrom by +treatment with cold water, dissolving in hot water and filtering into +ninety per cent alcohol. The gelatin separates in the form of white +filaments and these are removed and dried. Gelatin is insoluble in +cold but soluble in hot water. It is insoluble in alcohol, ether and +chloroform. Its hot aqueous solutions deflect the plane of polarized +light to the left. Its gyrodynat varies with temperature and degree of +dilution and is also influenced by acids and alkalies. At 30° it is +[α]_{D}³⁰° = -130. + +Gelatin is not precipitated by acetic acid nor lead acetate solution, +in which respect it differs from chondrin. + +If boiled for a day, or in a short time if heated to 140° in a sealed +tube, gelatin loses its power of setting and is split up into two +peptonoid bodies, semi-glutin and hemi-collin. Gelatin is easily +digested but cannot take the place of other proteids in nutrition. + +_Mucin._—This albuminoid, together with globulin, forms the principal +part of connective tissue. It is also present in large quantities in +mucus and is the chief lubricant of mucous membranes. It is extremely +difficult to prepare mucin in a state of purity, and it is not certain +that it has ever been accomplished. It is precipitated but not rendered +subsequently insoluble by sodium chlorid, magnesium sulfate and +alcohol. When boiled with sulfuric acid it yields leucin and tyrosin +and, with caustic soda, pyrocatechin. + +_Met- and Paralbumin._—Metalbumin is a form of mucin and differs from +paralbumin by giving no precipitate when boiled. Both bodies yield +reducing sugars when boiled with dilute sulfuric acid. + +_Nuclein._—The nitrogenous matters which form the nuclei of the +ultimate cells are called nuclein. Nuclein resembles mucin in many +physical properties but contains phosphorus. It is also, like mucin, +resistant to pepsin digestion. The nuclein of eggs and milk probably +contains iron. Nuclein is found also in cells of vegetable origin and +in yeast and mildew. + +_Nucleoproteids._—These are bodies which yield both nuclein and albumin +when boiled with water or treated with dilute acids or alkalies. Many +nucleoproteids have the physical properties of mucus and the sliminess +of the bile and of the synovial liquid is due to them. They are the +chief nitrogenous constituent of all protoplasm. + +_Chondrin._—Chondrin is obtained from cartilage by boiling with water. +The solutions of chondrin set on cooling in the manner of gelatin. They +are precipitated by the same reagents used for throwing out gelatin and +mucin. Chondrin is also levorotatory. By some authorities chondrin is +regarded as a mixture of gelatin and mucin. + +_Elastin._—The elastic fibers of connective tissue are composed of +this material. It can be prepared from the neck muscles by boiling +with ether and alcohol to remove fats and then for a day and a half +with water to extract the collagens. The residue is boiled with strong +acetic acid and thereafter with strong soda until the fibers begin to +smell. It is then treated with weak acetic acid and for a day with +dilute hydrochloric acid. The acid is removed by washing with water +and the residue is elastin. There is no solvent which acts on elastin +without decomposing it. It is digested by both pepsin and trypsin with +the formation of peptones. + +_Keratin._—This nitrogenous substance is found chiefly in hairs, +nails, and horns. It is essentially an alteration proteid product due +to peripheral exposure. It is prepared by digesting the fine ground +material successively with ether, alcohol, water and dilute acids. The +residue is keratin. An imperfect aqueous solution may be secured by +heating for a long time under pressure to 200°. It is also dissolved +by boiling the materials mentioned above with alkalies, and when the +solution thus obtained is treated with water, hydrogen sulfid is +evolved, showing that the sulfur of the molecule is loosely combined. + +Horn swells up when treated with dilute acetic acid and dissolves in +the boiling glacial acid. When treated with hot dilute sulfuric acid it +yields aspartic and volatile fat acids, leucin and tyrosin. Keratin, +when burning, gives off a characteristic odor as is perceived in +burning hair. + +_Other Albuminoids._—Among the albuminoids of less importance may be +mentioned neurokeratin found in the medullary sheath of nerve fibers; +chitin occurring in the tissues of certain invertebrates; conchiolin, +found in the shells of mussels and snails; spongin, occurring in +sponges; fibroin forming silk and spiders webs; and hyalin or hyalogen +found in edible birds’ nests. + +The nitrogenous bases in flesh which are soluble in cold water, +_viz._, kreatin, kreatinin, carnin, sarkin and xanthin are not classed +among the albuminoid bodies, since they have a much higher percentage +of nitrogen than is found in true proteid bodies, and are further +differentiated from them by the absence of sulfur. + +=373. Other Forms of Nitrogen.=—In addition to the proteids and +albuminoids mentioned above, agricultural products may contain nitrogen +in the form of ammonia, amid nitrogen and nitric acid. The quantities +of nitrogen thus combined are not large but often of sufficient +magnitude to demand special study. In general, these bodies belong to +transition products, representing stages in the transfer of nitrogen +from the simple to complex forms of combination, or the reverse. + +For instance, the nitrogen which finally appears in the proteids +of a plant has entered its organism chiefly as nitric acid, and +the nitric acid which is found in a vegetable product is therefore +a representative of the quantity of unabsorbed nitrogen present in +the tissues at the moment when the vital activity of the plant is +arrested. In some instances, it is found that the absorption of +nitrates by vegetable tissues takes place in far larger quantities than +is necessary for their nutrition, and in these cases the excess of +nitrates accumulates, sometimes to a remarkable extent. In a case cited +in the reports of the Kansas Agricultural Experiment Station, where +Indian corn was grown on ground which had been used for a hog pen, the +quantity of potassium nitrate found in the dried stalks was somewhat +remarkable. When one of the stalks was cut in two and tapped lightly +upon a table, crystals of potassium nitrate were easily obtained in the +form of fine powder. On splitting the cornstalk the crystals in the +pith could be seen without the aid of a microscope. On igniting a piece +of the dried stalk it burned rapidly with deflagration. The percentage +of potassium nitrate in the dried material was 18.8. Cattle eating this +fodder were poisoned.[341] + +In preserved meat products large quantities of oxidized nitrogen are +often found, and these come from the use of potassium nitrate as a +preserving and coloring agent. Ammonia is rarely found in vegetable +tissues in greater quantities than mere traces, but may often exist in +weighable amounts in animal products. + +Amid nitrogen is found rather constantly associated with proteid +matters in vegetable products. Asparagin and glutamin are instances +of amid bodies of frequent occurrence. Betain and cholin are found in +cottonseed. + +The occurrence of nitrogen, in the form of alkaloids, is of interest +to agricultural chemists in this country, chiefly from its presence +as nicotin in tobacco and from a toxicological point of view, but +in other localities the production of alkaloids, as for instance in +opium, tea and coffee, is a staple agricultural industry. The methods +of separating and determining these forms of nitrogen will be given +further on. This description can evidently not include an extended +compilation of the methods of separating and determining alkaloidal +bodies, with the exception of those with which the agricultural +analyst will be called upon frequently to deal, _viz._, nicotin and +caffein and nitrogenous bases such as betain and cholin. + + +QUALITIVE TESTS FOR NITROGENOUS BODIES. + +=374. Nitric Acid.=—Any nitric acid or nitrate which an agricultural +product may contain may be leached out by treating the fine-ground +material with cold water. From vegetable matters this extract is +evaporated to a small bulk, filtered, if necessary, and tested for +nitric acid by the usual treatment with ferrous sulfate and sulfuric +acid. In the case of vegetable substances there will not usually +be enough of organic matter to interfere with the delicacy of the +reaction, but in animal extracts this may occur. Colored extracts +should be decolorized with animal char (bone-black) before they are +subjected to examination. It is not well to attempt to remove the +organic matters, but, since they are more insoluble in water than the +nitrates, the solution containing both may be evaporated to dryness +and treated with a quantity of cold water insufficient for complete +solution. The nitrates will be found in the solution obtained in a +larger proportionate quantity than before. + +=375. Amid Nitrogen.=—One or more atoms of the hydrogen in ammonia may +be replaced by acid or basic bodies (alcohol radicles). In the former +cases amids, in the latter amins result. In the ratio of displacement +there are formed primary, secondary, and tertiary bodies determined by +the number of hydrogen atoms replaced. The primary amids are the only +ones of these bodies that are of interest in this connection. + +The amids are easily decomposed, even on heating with water and the +more readily with acids and alkalies, the amido radicle being converted +into ammonia. A type of these reactions is given below. + + CH₃.CO.NH₂ + H₂O = CH₂.CO.OH + H₃N. + +On boiling an amid with hydrochloric acid, the ammonia is procured +as chlorid whence it is easily expelled by heating with an alkali. +In a body free of ammonia, an amid is easily detected by subjecting +the substance containing it to the action of hot hydrochloric acid, +filtering, neutralizing the free acid with sodium hydroxid, adding an +excess thereof and distilling into an acid.[342] In case the quantity +of ammonia produced is very small it may be detected by the nessler +reagent.[343] Amids are soluble in a fresh, well washed preparation +of cupric hydrate suspended in water. The hydrate also passes into +solution forming a liquid of a deep blue color. + +If amids be added to a cold solution of potassium nitrate in sulfuric +acid free nitrogen is evolved. + +=376. Ammoniacal Nitrogen.=—This combination of nitrogen may be +detected by distilling the sample, or an aqueous extract thereof, with +magnesia or barium carbonate. The ammonia is collected in an acid and +detected therein by the usual qualitive reactions. + +=377. Proteid Nitrogen.=—There are a few general qualitive reactions +for proteid nitrogen and some special ones for distinct forms thereof. +Below will be given a few of those reactions which are of most +importance to the agricultural analyst: + +_Conversion into Ammonia._—All proteid matters are converted into +ammonia on boiling with strong sulfuric acid in presence of an oxygen +carrier. Mercury is the substance usually selected to effect the +transfer of the oxygen. Bodies which are found to be free of nitrates, +ammonia and amids, are subjected directly to oxidation with sulfuric +acid, and the ammonia produced thereby is distilled and detected in +the manner already suggested. If nitrogen be present in the form of +ammonia, amids and nitrates, the substance may be heated with an acid, +hydrochloric or acetic, thrown on a filter, washed with hot dilute acid +and the residue tested as above for proteid nitrogen. + +_Biuret Reaction._—When proteid matter is dissolved in sulfuric acid, +the solution, made alkaline with potassium hydroxid and treated with a +few drops of a solution of copper sulfate, gives a violet coloration. +This is commonly known as the biuret reaction, because the substance +C₂H₆N₃O₂, biuret, left on heating urea to 160° gives the coloration +noted in the conditions mentioned. + +It has been found by Bigelow, in this laboratory, that if a solution is +to be examined containing a very small amount of a proteid or similar +body, the copper sulfate solution should not contain more than four +grams of CuSO₄.5H₂O in 100 cubic centimeters of water, and the test +should first be made by adding to the solution one or two drops of +this copper sulfate solution, and then a strong excess of potassium +or sodium hydroxid. The test may be repeated, using from one-half to +two cubic centimeters of the copper sulfate solution, according to the +amount of proteid present. If too much of the copper sulfate solution +be employed its color may conceal that of the reaction. + +Heating to the boiling point sometimes makes the violet color more +distinct. + +If a solid is to be examined it is first suspended in water, and in +this state treated in the same manner as a solution. If solution is not +complete, the mixture should be filtered when the color produced may be +observed in the filtrate. + +Proteoses and peptones give a red to red-violet and other proteids a +violet to violet-blue coloration. + +_Xanthoproteic Reaction._—Strong nitric acid produces a yellow +coloration of proteid matter, which is intensified on warming. On +treating the yellow mixture with ammonia in slight excess the color is +changed to an orange or red tint. + +=378. Qualitive Tests for Albumin.=—Albumin is one of the chief +proteids and exists in both animal and vegetable substances. It is +soluble in cold water and may therefore be separated from many of its +nearly related bodies which are insoluble in that menstruum. In aqueous +solutions its presence may be determined by the general reactions for +proteid matters given above or by the following tests: + +_Precipitation by Heat._—Albumin is coagulated by heat. Vegetable +albumins become solid at about 65° and those of animal origin at a +somewhat higher temperature (75°). Some forms of animal albumin, +however, as for instance that contained in the serum, coagulate at a +lower temperature. + +_Precipitation by Acids._—Dilute acids also precipitate albumins +especially with the aid of heat. Practically all the albumins are +thrown out of solution by application of heat in the presence of dilute +acids. + +_Mercuric Salts._—Acid mercuric nitrate and a mixture of mercuric +chlorid, potassium iodid and acetic acid completely precipitate all +albuminous matters.[344] + +The yellow or red color produced on heating albumin with the mercuric +nitrate is known as Million’s reaction. + +=379. Qualitive Test for Peptones and Albuminates.=—When peptones and +albuminates are dissolved in an excess of glacial acetic acid and the +solution treated with sulfuric acid a violet color is produced and also +a faint fluorescence. + +_Separation of Peptones and Albumoses._—In a solution of peptones and +albumoses the latter may be precipitated by saturating the solution +with finely powdered zinc or ammonium sulfate. + +_Action of Phosphotungstic Acid._—All proteid matters in aqueous, +alkaline or acid solutions, are precipitated by sodium phosphotungstate +in a strongly acid solution. Acetic, phosphoric, or sulfuric acid may +be used for producing the required acidity, preference being given to +the latter. + +_Action of Trichloracetic Acid._—In the precipitation of albumin by +trichloracetic acid, there is formed a compound of the two bodies which +to 100 parts of albumin has 26.8 parts of the trichloracetic acid. + +The different albuminoid bodies obtained by precipitation behave in +a similar manner. There are formed flocculent precipitates insoluble +both in dilute and concentrated acids in the cold and also at a high +temperature, with the exception of the hemialbumose compound.[345] + +Albumin peptone, however, gives with the acid named in concentrated +solution a precipitate easily soluble in an excess of the reagent. In +the analysis of cow’s milk but not of human milk, this acid can be used +for the estimation of the albuminoid substances. With both kinds of +milk it can be used for the estimation of the albumin after the removal +of the casein. + +After precipitation of the albuminoid bodies, the milk sugar can be +estimated by polarizing the filtrate and, volumetrically after removal +of the excess of the acid by evaporation. By means of trichloracetic +acid it is possible to separate albumin peptone from mucus and mucus +peptone. A similar reaction is also produced by dichloracetic acid, but +the reaction with this last agent is less delicate than with the other. +Neither mucus nor albumin is precipitated by chloracetic acid. + +=380. Action of Albumins on Polarized Light.=—Many of the albumins and +albuminates, when in solution, strongly deflect the plane of polarized +light to the left.[346] + +The gyrodynats of some of the albumins and albuminates are given below: + + Serum albumin [α]_{D} = -57°.3 to -64°.6. + Egg albumin [α]_{D} = -35°.5 to -38°.1. + Serum globulin [α]_{D} = -47°.8. + Milk albumin [α]_{D} = -76°.0 to -91°.0. + +Our knowledge of the gyrodynatic numbers of the proteids and allied +bodies is too fragmentary to be of any great help in analytical work. +In practice, the rotatory power of these bodies becomes a disturbing +force in the determination of milk sugar.[347] A further study of this +property of certain proteids may lead to analytical processes for their +detection and determination, but no reliable methods for this can now +be recorded. + +=381. Alkaloidal Nitrogen.=—Only a general statement can be made here +in respect of the detection of alkaloidal nitrogen in vegetable or +animal tissues. Alkaloids are not found in healthy animal tissues and +the description of methods for isolating and detecting ptomaines is +foreign to the purpose of this work. In vegetable tissues the presence +of alkaloids may be established by the following methods of examination. + +The fine-ground tissues are made to pass a sieve of half millimeter +mesh and when suspended in water are acidified with sulfuric. The +mixture is then thoroughly extracted by shaking in a separatory funnel +with petroleum ether, benzene and chloroform, successively. Some +resins, glucosids and a few alkaloidal bodies not important here are +extracted by this treatment. + +The residue is made distinctly alkaline with ammonia and treated +as above with the same solvents. In the solution obtained as last +mentioned nearly all the alkaloidal bodies found in plants are +contained. + +All the alkaloids in a plant may be obtained by digesting the finely +divided material with dilute sulfuric acid. The acid solution thus +obtained is made nearly neutral with ammonia or magnesia, concentrated +to a sirup, and gums, mucilage, etc. thrown out by adding about three +volumes of ninety-five per cent alcohol. The alkaloids are found in the +filtrate. The alcohol is evaporated from the filtrate and the residue +tested for alkaloids by group reagents.[348] Potassium mercuric iodid +and phosphotungstic and molybdic acids are types of these reagents. + +The same group reagents may also be applied to the extracts obtained +with petroleum ether, benzene and chloroform, in all cases, after the +removal of the solvents by evaporation. + + +ESTIMATION OF NITROGENOUS BODIES IN AGRICULTURAL PRODUCTS. + +=382. Total Nitrogen.=—Any one of the methods heretofore described for +the estimation of total nitrogen in soils or fertilizers is applicable +for the same purpose to agricultural products. One among these, +however, is so superior in the matter of convenience and certainty, as +to make it preferable to any other. The moist combustion of the sample +with sulfuric acid with subsequent distillation of the ammonia produced +is the process which is to be recommended.[349] + +The usual precautions for securing a representative sample should be +observed, but no further directions are needed. In all cases hereafter, +where the estimation of nitrogen is enjoined, it is understood that the +moist combustion process is to be used unless otherwise stated. + +=383. Estimation of Ammoniacal Nitrogen.=—If the distillation of +ammonia be accomplished with the aid of magnesia alba or barium +carbonate it may be safely conducted on the finely ground materials +or, in case of animal bodies, in as fine a state of subdivision as +may be conveniently secured. Since the salts of ammonia are easily +soluble in water they may be all obtained in aqueous solution, and the +distillation of this solution with magnesia gives correct results. +Experience has shown that the stronger alkalies, such as sodium and +potassium hydroxids, cannot be safely used in the distillation of +ammonia from mixtures containing organic nitrogenous materials because +of the tendency of these bodies to decomposition, in the circumstances, +yielding a portion of their nitrogen as ammonia. Barium carbonate acts +with less vigor on non-ammoniacal nitrogenous matters than magnesia, +and in some cases, as pointed out further on, may be substituted +therefor with advantage. There is no danger of failing to obtain a part +of the ammonia on distillation with magnesia provided the latter does +not contain more than a trace of carbonate.[350] + +When no easily decomposable organic nitrogenous matters are present, +the distillation may be conducted with the stronger alkalies in the +manner prescribed.[351] All the necessary details of conducting the +distillation are found in the preceding volumes of this work. + +=384. Estimation of Amid Nitrogen.=—In bodies containing no ammonia, +or from which the ammonia has been removed by the method described in +the preceding paragraph, the nitrogen in the amid bodies is converted +into ammonia by boiling for about an hour with five per cent sulfuric +or hydrochloric acid. The ammonia thus produced is estimated in the +usual manner after distillation over magnesia free of carbonate. The +free acid is exactly neutralized with sodium or potassium carbonate +before the addition of the magnesia. The results are given in terms of +asparagin. The reaction which takes place in the decomposition of the +amid body is indicated by the following equation: + + Asparagin. Sulfuric Aspartic Ammonium + acid. acid. sulfate. + 2C₄H₈N₂O₃ + 2H₂O + H₂SO₄ = 2C₄H₇NO₄ + (H₄N)₂SO₄. + +Half of the nitrogen contained in the amid body is thus obtained as +ammonia. + +It is advisable to calculate all the amid nitrogen in agricultural +products as asparagin. + +=385. Sachsse’s Method.=—A method for the determination of amid +bodies by liberation of free nitrogen has been described by Sachsse +and Kormann.[352] It is based on the reaction which takes place when +amid bodies are brought into contact with nitrites in presence of an +acid. The mixture of the reagents by which the gas is set free is +accomplished in the apparatus shown in Fig. 103. The vessel _A_ has a +capacity of about fifty cubic centimeters and carries a stopper with +three perforations for the arrangement shown. + +[Illustration: FIG. 103.—APPARATUS FOR AMID NITROGEN.] + +[Illustration: FIG. 104.—SACHSSE’S EUDIOMETER.] + +About six cubic centimeters of a concentrated aqueous solution of +potassium nitrite are placed in _A_ and the lower parts of the tubes +_a_ and _b_ are filled with water to a little above _e_ in order to +exclude the air therefrom. Dilute sulfuric acid is placed in one of +the funnels and an aqueous solution of the amid in the other. The air +is displaced from the empty part of _A_ by introducing the sulfuric +acid, a little at a time, whereby nitrous acid and nitric oxid are +evolved. This operation is continued until all the air has been driven +out through _c d_, the open end of _d_ being kept in the liquid in the +dish shown in Fig. 104. The eudiometer in which the evolved nitrogen +is measured is shown in Fig. 104, and should have a capacity of about +fifty cubic centimeters, and be graduated to fifths. It is filled +with the solution of ferrous sulfate contained in _B_ by sucking at +_g_, after which the clamp _h_ is replaced, the cock _f_ closed, and +the free end of _d_ placed in the lower end of the eudiometer. The +solution of the amid is run slowly into the generator _A_, Fig. 103, +together with small additional quantities of the sulfuric acid when +the evolution of gas becomes slow. From time to time _h_ is opened +and fresh quantities of the ferrous solution allowed to flow into the +eudiometer. Any trace of the amid remaining in the funnel is washed +into _A_ with pure water, with care to avoid the introduction of air. +When the liquid in _A_ assumes a permanent blue color the decomposition +is complete. The residual gas is driven out of _A_ by filling with +water. The tubes _d_ and _h_, after all the nitric oxid is absorbed, +are removed from the eudiometer which is transferred to a cylinder +containing water and immersed therein until the two liquid surfaces +are at the same level and the volume of the nitrogen observed. After +correction for temperature and pressure, the weight of the nitrogen is +calculated. Twenty-eight parts by weight of nitrogen correspond to 150 +of pure asparagin, 181 of tyrosin and 131 of leucin.[353] This method of +procedure is difficult of manipulation and is apt to give results that +are too high. It cannot be preferred to the more simple and accurate +processes already described. + +=386. Preparation of Asparagin.=—In case the analyst desires to prepare +a quantity of asparagin for comparative purposes it may be easily +accomplished in the following way: A sufficient quantity of pease +or beans is sprouted in a dark place and allowed to grow until the +reserve food of the seed is exhausted. The young sprouts are gathered, +shredded and subjected to strong pressure. The juice thus obtained is +boiled to coagulate the albumin, and thrown on a filter. The filtrate +is evaporated to a thin sirup and set aside to allow the asparagin +to separate in a crystallized form. If the crystals at first formed +are colored they may be dissolved, decolorized with bone-black, and +recrystallized. Instead of the above method the young shoots may be +shredded, extracted with hot water and the extract treated as above. A +larger yield of the asparagin is obtained by the latter process than by +the one mentioned above.[354] + +=387. Detection and Estimation of Asparagin and Glutamin.=—Of all +the amid bodies asparagin is the most important from an agricultural +standpoint, because of its wide distribution in vegetable products.[355] +Asparagin is easily obtained from the aqueous extracts of plants by +crystallization.[356] In addition to its crystalline characteristics +asparagin may be identified by the following tests. Heated with +alkalies, including barium hydroxid, asparagin yields ammonia. Boiled +with dilute acids it forms ammonium salts. A warm aqueous solution +dissolves freshly prepared copper hydroxid with the production of a +deep blue color. Sometimes, on cooling, crystals of the copper compound +formed are separated. Asparagin crystallizes with one molecule of +water. Glutamin gives essentially the reactions characteristic of +asparagin, but crystallizes without water in small white needles. +Asparagin is easily detected with the aid of the microscope by placing +sections of vegetable tissues containing it in alcohol. After some +time microscopic crystals of asparagin are separated. The presence of +large quantities of soluble carbohydrates seriously interferes with the +separation of asparagin in crystalline form. + +For the detection of glutamin the liquid containing it is boiled with +dilute hydrochloric acid, by which ammonia and glutamic acid are +formed. On the addition of lead acetate to the solution the glutamic +acid is thrown out as a lead salt, in which, after its decomposition +with hydrogen sulfid, the characteristic properties of glutamic acid +can be established. + +The above process is chronophagous and also uncertain where the +quantity of glutamin is very small and that of other soluble organic +matters very large. A much better process, both for the detection +of glutamin and asparagin, is the following, based on the property +possessed by mercuric nitrate of precipitating amids. + +The aqueous extract containing the amid bodies is mixed with lead +acetate until all precipitable matters are thrown out and the mixture +poured into a filter. To the filtrate is added a moderately acid +solution of mercuric nitrate. The precipitate produced is collected +on a filter, washed, suspended in water, decomposed with hydrogen +sulfid and again filtered. The amid bodies (glutamin, asparagin, etc.) +are found in the filtrate and can be detected and estimated by the +processes already described. A reaction showing the presence of an amid +body is not a positive proof of the presence of asparagin or glutamin, +since among other amids, allantoin may be present. This substance is +found in the sprouts of young plants and also in certain cereals, as +shown by researches in this laboratory.[357] Allantoin, glutamin, and +asparagin, when obtained in solution by the above process, may be +secured, by careful evaporation and recrystallization, in well defined +crystalline forms. Asparagin gives lustrous, rhombic prisms, easily +soluble in hot water, but insoluble in alcohol and ether. + +Allantoin is regarded as a diureid of glyoxalic acid and has the +composition represented by the formula C₄H₆N₄O₃. It crystallizes in +lustrous prisms having practically the same solubility as asparagin. + +Glutamin is the amid of amidoglutaric acid. It crystallizes in fine +needles. Its structural formula is represented as + + CO.NH₂ + / + C₃H₅(NH₂) + \ + CO₂H. + +=387. Cholin and Betain.=—Cholin is a nitrogenous base found in both +animal and plant tissues. Its name is derived from the circumstance +that it was first discovered in the bile. It is found in the brain, +yolk of eggs, hops, beets, cottonseed and many other bodies. When +united with glycerolphosphoric acid it forms lecithin, a compound of +great physiological importance. From a chemical point of view, cholin +is oxyethyltrimethyl-ammonium hydroxid, + + OH + / + C₂H₄ ; (C₅H₁₅NO₂). + \ + N(CH₃)₃.OH + +It is crystallized with difficulty and is deliquescent. Its most +important compound, from an analytical point of view, is its platinum +salt C₅H₁₄ONCl₂PtCl₄. This salt crystallizes in red-yellow plates and +is insoluble in alcohol. + +Betain, C₅H₁₁NO₂, is the product of the oxidation of cholin. + +In this laboratory the bases are separated from cottonseed and from +each other by the process described below.[358] + +About five pounds of fine-ground cottonseed cake are extracted with +seventy per cent alcohol. The material should not be previously treated +with dilute mineral acids because of the danger of converting a part +of the cholin into betain. The alcohol is removed from the filtered +extract and the residue dissolved in water. The aqueous solution is +treated with lead acetate until no further precipitation takes place, +thrown on a filter, the lead removed from the filtrate with hydrogen +sulfid and the liquid evaporated to a viscous syrup. The sirup is +extracted with alcohol containing one per cent of hydrochloric acid. +The solution thus obtained is placed in a deep beaker and the bases +precipitated by means of an alcoholic solution of mercuric chlorid. The +complete separation of the salts requires at least two weeks. + +The double salts of the bases and mercury thus obtained, after freeing +from the mother liquor, are recrystallized from a solution in water +and from the pure product thus obtained the mercury is removed after +solution in water, by hydrogen sulfid. The filtrate, after separating +the mercury, contains the bases as chlorids (hydrochlorates). The +solution of the chlorids is evaporated slowly in (pene) vacuo to a +thick sirup and set over sulfuric acid to facilitate crystallization. +The hydrochlorates are obtained in this way colorless and in +well-shaped crystalline forms. + +In a quantitive determination, a small amount of the fine meal is +extracted at once with one per cent hydrochloric acid in seventy per +cent alcohol, the salts obtained purified as above and weighed. + +The following process serves to determine the relative proportions of +cholin and betain in a mixture of the two bases. + +A definite weight of the chlorids, prepared as directed above, is +extracted by absolute alcohol. This treatment dissolves all the cholin +chlorid and a little of the betain salt. The alcoholic solution is +evaporated and again extracted with absolute alcohol. This process is +repeated three times and at the end the cholin chlorid is obtained free +of betain. In a sample of cottonseed cake examined in this laboratory +the two bases were found present in the following relative proportions, +_viz._, cholin 17.5 per cent, betain 82.5 per cent. Thus purified the +cholin is finally precipitated by platinum chlorid. For a description +of the special reaction, by means of which cholin and betain are +differentiated, the paper cited above may be consulted. + +These bodies have acquired an economic interest on account of their +occurrence in cottonseed meal, which is so extensively used as a +cattle food. It is evident from the relative proportions in which they +occur that the less nocuous base, betain, is the more abundant. It is +possible, however, that the base originally formed is cholin and that +betain is a secondary product. + +Experience has shown that it is not safe to feed cottonseed meal to +very young animals, while moderate rations thereof may be given to +full-grown animals without much expectation of deleterious results. In +the case of toxic effects it is fair to presume that a meal has been +fed in which the cholin is relatively more abundant than the betain. + +=389. Lecithin.=—Lecithin is a nitrogenous body, allied both to the +fats and proteids and containing glycerol and phosphoric acid. Its +percentage composition is represented with some accuracy by the formula +C₄₂H₈₆NPO₉, or according to Hoppe-Seyler, C₄₄H₉₀NPO₉. It appears to +be a compound of cholin with glycerolphosphoric acid. It is widely +distributed both in animal and vegetable organisms, in the latter +especially in pease and beans. + +From a physiological point of view, lecithin is highly important as the +medium for the passage of phosphorus from the organic to the inorganic +state, and the reverse. This function of lecithin has been thoroughly +investigated in this laboratory by Maxwell.[359] + +In the extraction of lecithin from seeds (pease, beans, etc.) it is not +possible to secure the whole of the substance by treatment with ether +alone.[360] + +The extraction of the lecithin may, however, be entirely accomplished +by successive treatments for periods of about fifteen hours with pure +ether and alcohol. This is better than to mix the solvents, since, in +this case, the ether having the lower boiling point is chiefly active +in the extraction. When the extraction is accomplished by digestion +and not in a continuous extracting apparatus the two solvents may be +mixed together and thus used with advantage. After the evaporation of +the solvents, the lecithin is ignited with mixed sodium and potassium +carbonate whereby the organic phosphorus is secured without loss in an +inorganic form. Where greater care is desired, the method described for +organic phosphorus in soils may be used.[361] The inorganic phosphorus +thus obtained is estimated in the usual way as magnesium pyrophosphate. + +For analytical purposes, the extraction of lecithin from vegetable +substances is conducted in this laboratory as follows:[362] The +fine-ground pea or bean meal is placed in an extraction apparatus +and treated continuously with anhydrous ether for fifteen hours. The +ether in the apparatus is replaced with absolute alcohol and the +extraction continued for six hours longer. The alcoholic extract is +evaporated to dryness and treated with ether. The part of the lecithin +at first insoluble in ether becomes soluble therein after it has been +removed from the vegetable tissues by alcohol. Moreover, any trace of +inorganic phosphorus which may have been removed by the alcohol, is +left undissolved on subsequent treatment with ether. The ether extract +from the alcohol residue is added to that obtained directly, the ether +removed by evaporation, and the total lecithin oxidized and the residue +used for the estimation of phosphorus as already described. + +In determining the lecithin in eggs, the procedure employed for +vegetable tissues is slightly changed.[363] The whole egg, excluding +the shell, is placed in a flask with a reflux condenser and boiled for +six hours with absolute alcohol. The alcohol is then removed from the +flask by evaporation and the residue treated in like manner with ether +for ten hours. The ether is removed and the dry residue rubbed to a +fine powder, placed in an extractor and treated with pure ether for +ten hours. The extract thus secured is oxidized after the removal of +the ether by fusion with mixed alkaline carbonates and the phosphorus +determined in the usual way. + +=390. Factor for Calculating Results.=—The percentage of lecithin is +calculated from the weight of magnesium pyrophosphate obtained by +multiplying it by the factor, 7.2703.[364] This factor is calculated +from the second formula for lecithin given above, in which the +percentage of phosphorus pentoxid, P₂O₅, is 8.789. + +_Example._—In fifty-four grams of egg, exclusive of the shell, is found +an amount of organic phosphorus yielding 0.0848 gram of magnesium +pyrophosphate. Then 0.0848 × 7.2703 = 0.61652 and 0.61652 × 100 ÷ 54 = +1.14. Therefore the percentage of lecithin in the egg is 1.14. + +=391. Estimation of Alkaloidal Nitrogen.=—The alkaloids contain +nitrogen in a form more difficult of oxidation than that contained in +proteid or albuminoid forms. It is doubtful whether any of the nitrogen +in alkaloids becomes available for plant nutrition by any of the usual +processes of fermentation and decay to which nitrogenous bodies are +submitted in the soil. Likewise, it is true that it is not attacked by +the digestive processes in any way preparatory to its assimilation as +food by the animal tissues. Alkaloidal nitrogen is therefore not to be +regarded as a food either for the animal or plant. + +For the general methods of estimating alkaloids the reader is referred +to standard works on plant chemistry and toxicology. The alkaloids of +interest in this manual are those which are found in tobacco, tea, +coffee and a few other products of agricultural importance. The best +methods of isolating and estimating these bodies will be given in the +part of the volume devoted to the special consideration of the articles +mentioned. + + +SEPARATION OF PROTEID BODIES IN VEGETABLE PRODUCTS. + +=392. Preliminary Treatment.=—The chief disturbing components of +vegetable tissues, in respect of their influence on the separation +and estimation of the proteid constituents, are fats and oils and +coloring matters. In many cases these bodies are present in such +small quantities as to be negligible, as, for instance, in rice. In +other cases they exist in such large proportions as to present almost +insuperable difficulties to analytical operations, as is the case with +oily seeds. In all instances, however, it is best to remove these +bodies, even when present in small proportions, provided it can be done +without altering the character of the proteid bodies. This is secured +by extracting the fine-ground vegetable material first with petroleum +ether, and afterwards with strong alcohol and ether. Practically, all +of the fatty bodies and the greater part of the most objectionable +coloring matters are removed by this treatment. The extraction should +in all cases be made at low temperatures, not exceeding 35°, to avoid +the coagulating effect of higher temperatures upon the albuminous +bodies which may be present. + +In this laboratory, fatty seeds, as for instance peanuts, are first +ground into coarse meal, then extracted with petroleum ether, ground +to a fine meal and the fat extraction completed with petroleum ether, +ninety-five per cent alcohol and pure sulfuric ether. The residue of +the last solvent may be removed by aspirating air through the extracted +meal. In some cases, it is advisable to extract with ethyl ether before +as well as after the alcoholic extraction. This treatment removes at +least a part of the water and prevents the dilution of the first part +of alcohol added to such an extent as to make it dissolve some of the +proteid matters. In each case, a portion of the alcoholic extract +should be tested qualitively for proteid matter. If any be found, +stronger alcohol should be used for, at least, the first extraction. A +portion of the meal, prepared as above directed, is extracted with a +ten per cent solution of sodium chlorid, as described further on, and a +measured portion of the filtered extract diluted with water until the +proteid matter in solution begins to be precipitated. By this treatment +the proper strength of the salt solution, to be used for the subsequent +extraction, is determined. To save time in dialyzing, the solution of +salt employed as a solvent should be as dilute as possible. + +The mixture of meal and solvent sometimes filters with difficulty. +In these cases, it is advisable to first pour it into a linen bag +from which the liquid portion can be removed by gentle pressure and +subsequently filtered through paper. As a last resort, the liquid +secured from the linen filter can be saturated with ammonium, zinc or +magnesium sulfate, whereby all the proteid matters are thrown out. +After filtering, the residue is again dissolved in salt solution and +can then be readily filtered through paper. + +The clear filtrate should be tested by fractional precipitation by heat +and the final filtrate by acetic acid, as will be described further on. + +The proteid matter may be further freed from amid compounds by +treatment with copper sulfate.[365] This treatment is not advisable, +however, except for the purpose of determining the total proteid +nitrogen in the sample. The action of the water, heat and cupric +sulfate combined is capable of inducing grave changes in the character +of the residual matter which would seriously interfere with the results +of subsequent studies of the nature of the proteid bodies. + +In many instances, as with cereal grains, the separation of the proteid +bodies is accomplished by no further preliminary treatment than is +necessary to reduce them to the proper degree of fineness. + +=393. Diversity of Character.=—The proteids which occur in vegetable +products are found in all parts of the tissues of the plants, but in +cereals especially in the seeds. In grass crops and in some of the +legumes, such as clover, the nitrogenous matters are chiefly found in +the straw and leaves. The general classification of these bodies has +already been given, but each kind of plant presents marked variations, +not only in the relative proportions of the different classes, but also +in variations in the nature of each class. For this reason the study +of vegetable proteids is, in some respects, a new research for each +kind of plant examined. There are, however, some general principles +which the analyst must follow in his work, and an attempt will be made +here to establish these and to construct thereon a rational method +of conducting the investigation. In the separation and estimation +of complex bodies so nearly related to each other, it is difficult +not only to secure satisfactory results, but also to prevent the +transformation of some forms of proteid matter into others nearly +related thereto by the action of the solvents used for separation and +precipitation. + +=394. Separation of Gluten from Wheat Flour.=—The most important +proteid in wheat is the body known as gluten, a commercial name given +to the nitrogenous matters insoluble in cold water. The gluten thus +obtained does not represent a single chemical compound, but is a +complex consisting of at least two proteid bodies, which together form +an elastic, pasty mass, insoluble in cold water containing a trace +of mineral salts. This mass has the property of holding mechanically +entangled among its particles bubbles of gas, which, expanding under +the action of heat during cooking, give to bread made of glutenous +flours its porous property. + +In respect of proteids, the American wheats, as a rule, are quite +equal to those of foreign origin. This is an important characteristic +when it is remembered that both the milling and food values of a wheat +depend largely on the nitrogenous matter which is present. It must +not be forgotten, however, that merely a high percentage of proteids +is not always a sure indication of the milling value of a wheat. The +percentage of gluten to the other proteid constituents of a wheat is +not always constant, and it is the gluten content of a flour on which +its bread making qualities chiefly depend. The percentage of moist +gluten gives, in a rough way, the property of the glutenous matter of +absorbing and holding water under conditions as nearly constant as +can be obtained. In general, it may be said that the ratio between +the moist gluten and the dry gluten in a given sample is an index for +comparison with other substances in the same sample. Upon the whole, +however, the percentage of dry gluten must be regarded as the safer +index of quality. In respect of the content of glutenous matter, our +domestic wheats are distinctly superior to those of foreign origin. +They are even better than the Canadian wheats in this respect. It may +be fairly inferred, therefore, that while our domestic wheats give a +flour slightly inferior in nutritive properties to that derived from +foreign samples, it is nevertheless better adapted for baking purposes, +and this quality more than compensates for its slight deficiency in +respect of nutrition, a deficiency, which, however, is so minute as to +be hardly worth considering.[366] + +The gluten is separated in this laboratory from the other constituents +of a flour by the following process: + +Ten grams of the fine-ground flour are placed in a porcelain dish, +well wet with nearly an equal weight of water at a temperature of not +to exceed 15°, and the mass worked into a ball with a spatula, taking +care that none of it adheres to the walls of the dish. The ball of +dough is allowed to stand for an hour, at the end of which time it +is held in the hand and kneaded in a stream of cold water until the +starch and soluble matter are removed. The ball of gluten thus obtained +is placed in cold water and allowed to remain for an hour when it is +removed, pressed as dry as possible between the hands, rolled into a +ball, placed in a flat bottom dish and weighed. The weight obtained +is entered as moist gluten. The dish containing the ball of gluten is +dried for twenty hours in a steam-bath, again weighed, and the weight +of material obtained entered as dry gluten. The determination of dry +and moist gluten cannot in any sense be regarded as an isolation and +estimation of a definite chemical compound. For millers’ and bakers’ +purposes, however, the numbers thus obtained have a high practical +value. A typical wheat grown in this country will contain about 26.50 +per cent of moist and 10.25 per cent of dry gluten. + +The gluten of wheat is composed of two proteid bodies, gliadin and +glutenin.[367] Gliadin contains 17.66 per cent, and glutenin 17.49 per +cent of nitrogen. Gliadin forms a sticky mass when mixed with water and +is prevented from passing into solution by the small content of mineral +salts present in the flour. It serves to bind together the other +ingredients of the flour, thus rendering the dough tough and coherent. +Glutenin serves to fix the gliadin and thus to make it firm and solid. +Glutenin alone cannot yield gluten in the absence of gliadin, nor +gliadin without the help of glutenin. Soluble metallic salts are also +necessary to the formation of gluten, and act as suggested above, by +preventing the solution of the gliadin in water, during the process of +washing out the starch. No fermentation takes place in the formation of +gluten from the ingredients named. + +The gluten, which is obtained in an impure state by the process above +described, is, therefore, not to be regarded as existing as such in the +wheat kernel or flour made therefrom, but to arise by a union of its +elements by the action of water. + +=395. Extraction with Water.=—It is quite impossible to get an extract +from fine-ground vegetable matter in pure water because the soluble +salts of the sample pass at once into solution and then a pure water +solvent becomes an extremely dilute saline solution. The aqueous +extract may, however, be subjected to dialysis, whereby the saline +matter is removed and the proteid matter, not precipitated during the +dialytic process, may be regarded as that part of it in the original +sample soluble in pure water. Nevertheless, in many instances, it +is important to obtain an extract with cold water. In oatmeal the +aqueous extract is obtained by Osborne as follows:[368] Five pounds of +fine-ground meal are shaken occasionally with six liters of cold water +for twenty-four hours, the liquid removed by filtration and pressure +and the extraction continued with another equal portion of water in +the manner noted. The two liquid extracts are united and saturated +with commercial ammonium sulfate which precipitates all the dissolved +proteid matter. The filtrate obtained is collected on a filter, washed +with a saturated solution of ammonium sulfate and removed as completely +as possible from the filter paper by means of a spatula. Any residual +precipitate remaining on the paper is washed into the vessel containing +the removed precipitate and the undissolved precipitate well beaten up +in the liquid, which is placed in a dialyzer with a little thymol, to +prevent fermentation, and subjected to dialysis for about two weeks. At +the end of that time, the contents of the dialyzer are practically free +of sulfates. The contents of the dialyzers are then thrown on a filter +and in the filtrate are found those proteids first extracted with +water, precipitated with ammonium sulfate and redissolved from this +precipitated state by pure water. In the case of oatmeal, this proteid +matter is not coagulated by heat, and may be obtained in the dry state +by the evaporation of the filtrate last mentioned at a low temperature +in vacuo. It is evident that the character of the proteid matter thus +obtained will vary with the nature of the substance examined. In the +case of oats, it appears to be a proteose and not an albumin. + +=396. Action of Water on Composition of Proteids.=—When a body, such +as oatmeal, containing many proteids of nearly related character, is +exposed to the action of a large excess of water, the proteid bodies +may undergo important changes whereby their relations to solvents are +changed. After oatmeal has been extracted with water, as described +above, the proteid matter originally soluble in dilute alcohol +undergoes an alteration and assumes different properties. The same +remark is applicable to the proteid body soluble in dilute potash. +Nearly all the proteid matter of oatmeal is soluble in dilute potash, +if this solvent be applied directly, but if the sample be previously +treated with water or a ten per cent salt solution the subsequent +proportion of proteid matter soluble in dilute potash is greatly +diminished.[369] Water applied directly to the oatmeal apparently +dissolves an acid albumin, a globulin or globulins, and a proteose. The +bodies, however, soluble in water, exist only in small quantities in +oatmeal. Experience has shown that in most instances, it is safer to +begin the extraction of a cereal for proteid matter with a dilute salt +solution rather than with water, and to determine the matters soluble +in water alone by subsequent dialysis. + +=397. Extraction with Dilute Salt Solution.=—In general, it is +advisable to begin the work of separating vegetable proteids by +extracting the sample with a dilute brine usually of ten per cent +strength. As conducted by Osborne and Voorhees, on wheat flour, the +manipulation is carried on as follows:[370] + +The fine-ground whole wheat flour, about four kilograms, is shaken with +twice that weight of a ten per cent sodium chlorid solution, strained +through a sieve, to break up lumps, and allowed to settle for sixteen +hours. At the end of this time, about half of the supernatant liquid +is removed by a siphon or by decantation and filtered. Two liters +more of the salt solution are added, the mixture well stirred and the +whole brought onto the filter used above. The filtrate is collected +in successive convenient portions and each portion, as soon as it is +obtained, is saturated with ammonium sulfate. All the proteid matter is +precipitated by this reagent. The precipitate is collected on a filter, +redissolved in a convenient quantity of the salt solution and dialyzed +for fourteen days or until all sulfates and chlorids are removed. The +proteid matter, which is separated on dialysis, in this instance, is a +globulin. + +The proteid matter not precipitated on dialysis is assumed to be that +part of the original substance soluble in water. + +A part of the water soluble proteid matter obtained as above is +coagulated by heat at from 50° to 80°. The part not separated by heat +gives a precipitate on saturation with sodium chlorid. + +In wheat there are found soluble in water two albumins and a +proteose.[371] + +In separating the albumin coagulating at a low boiling point from the +dialyzed solution mentioned above, it is heated to 60° for an hour, +the precipitate collected on a gooch, washed with hot water (60°), and +then successively with ninety-five per cent alcohol, water-free alcohol +and ether. On drying the residual voluminous matter on the filter over +sulfuric acid, it becomes dense and horny, having in an ash free state, +according to Osborne, the following composition: + + Per cent. + Carbon 53.06 + Hydrogen 6.82 + Nitrogen 17.01 + Sulfur 1.30 + Oxygen 21.81 + +=398. Treatment without Precipitation with Ammonium Sulfate.=—Where +abundant means are at hand for dialyzing large volumes of solution, the +preliminary treatment of the solution made with ten per cent sodium +chlorid with ammonium sulfate may be omitted. + +When the precipitated proteids are to be used for the estimation of +the nitrogen therein contained, it has been proposed to substitute +the corresponding zinc salt for the ammonium sulfate.[372] This +reagent has given satisfactory results in this laboratory and while a +larger experience is desirable before commending it as an acceptable +substitute in all cases, yet its obvious advantage, in being free of +nitrogen for the use mentioned, entitles it to careful consideration. + +The manipulation, with the exception of the precipitation with ammonium +sulfate, is the same as that described in the preceding paragraph. The +globulins are completely precipitated when the dialysis is complete and +may be separated from the soluble albumins and proteoses by filtration. + +=399. Separation of the Bodies Soluble in Water.=—_Albumins._—By the +methods of treatment just described, the proteid matters soluble in +ten per cent sodium chlorid solution are separated into two classes, +_viz._, globulins insoluble in pure water and albumins and proteoses +soluble in pure water. The aqueous solution will also contain any amids +or nitrogenous bases soluble in the dilute saline solution and in +water. Osborne and Voorhees have found that the best way of separating +the albumins in the pure aqueous solution is by the application of +heat.[373] By means of a fractional coagulation the albumins are +divided into classes, _viz._, those separating at from 60° to 65° and +those remaining in solution at that temperature but separating up to +85°. The respective quantities of these albumins are determined by +collecting them in a filter and estimating the nitrogen therein by +moist combustion in the usual way. Even a larger number of albumins may +be secured, as in the maize kernel, by such a fractional precipitation +by means of heat. Chittenden and Osborne find in this instance that the +precipitation begins at about 40°.[374] + +_Proteose._—After the separation of the albumins by heat the filtrate +may still contain proteid matter. This matter belongs to the proteose +class. It may be partially secured by concentrating the filtrate, +after the removal of the albumins, to a small bulk when a part of +the proteose body will separate. It may be thrown out entirely by +treating the filtrate above mentioned with fine-ground salt until it is +saturated or by adding salt until the solution contains about twenty +per cent thereof and precipitating the proteose by acetic acid.[375] + +=400. Separation of the Globulins.=—The globulins which are extracted +with ten per cent solution of sodium chlorid and precipitated on +dialysis may be separated by fractional solution into several bodies of +nearly related properties. This solution is conveniently accomplished +by saline solvents of increasing strength. In the case of the maize +globulins, Chittenden and Osborne employ dilute solutions of common +salt for effecting the separation, beginning with a quarter of a per +cent and ending with a two per cent mixture.[376] + +=401. Proteids Soluble in Dilute Alcohol.=—Some of the proteid bodies +which are soluble in dilute salt solution and in water are also soluble +in alcohol. Since these bodies are more easily identified by the +processes already described, attention will be given in this paragraph +solely to those proteid bodies which are insoluble in water or dilute +salt solution and are soluble in dilute alcohol. + +For the extraction of these bodies, the residue, left after extraction +with a ten per cent solution of sodium chlorid or with water, is +mixed with enough strong alcohol to secure by the admixture with the +water present in the sample an alcohol of about seventy-five per cent +strength. The mixture is well shaken and digested for some time, at a +temperature of about 46°, and thrown on a filter which is kept at about +the same temperature. The residue is again mixed with alcohol of the +same strength (seventy-five per cent) using about four liters for two +and a half kilos of the original material. During the second digestion +the temperature is kept at about 60°. The latter operation is repeated +three times and in each case the filtrate obtained is evaporated +separately.[377] This process is especially applicable to the meal from +maize kernels, which contains a high relative percentage of an alcohol +soluble proteid, zein. + +The chief part of the zein is found in the first two extracts, obtained +as described above. On evaporation, the zein separates as a tough, +leathery, yellow-colored mass on the walls of the containing vessel. It +is cut into small pieces and digested for several days in cold, pure +alcohol. This is followed by digestion with a mixture of ether and +pure alcohol, and finally with pure ether. By this treatment a part of +the zein becomes insoluble in seventy-five per cent alcohol. The part +soluble in dilute alcohol is precipitated by pouring it into water. + +Another method of preparing zein is to extract the meal with +seventy-five per cent alcohol after it has been treated with a ten per +cent salt solution. + +In this case the extraction is continued with seventy-five per cent +alcohol in successive portions until no more proteid matter passes +into solution. The several extracts are united and the alcohol removed +by distillation, by which process the zein is separated. It is washed +with distilled water, until the sodium chlorid is removed, dissolved +in warm alcohol of about eighty per cent strength and any insoluble +matter removed by filtration. On evaporating the filtrate nearly +to dryness, the zein is separated and pressed as free of water as +possible, yielding a yellow, elastic substance resembling molasses +candy. This preparation is purified by digestion with pure alcohol +and ether in the manner described. The two zeins which are secured +by the treatment, one soluble and the other insoluble in alcohol, are +practically identical in composition.[378] + +Zein freshly precipitated by pouring its alcoholic solution in water +is wholly insoluble in water, and, on boiling therewith, is changed +into the variety insoluble in dilute alcohol. Boiled with dilute +sulfuric acid, six in 300 cubic centimeters of water, it melts, forming +a gummy mass, which is very slowly attacked by the acid yielding +proteoses and peptones. Heated with stronger sulfuric acid it undergoes +decomposition, yielding leucin, tyrosin, and glutamic acid. + +=402. Solvent Action of Acids and Alkalies.=—In the preceding +paragraphs, a synopsis has been given of the methods of separating +proteid matters in such a manner as to secure them in a pure state in +the same conditions as they exist in the natural substances. A very +large percentage of the proteid matter is still left undissolved after +extraction with the solvents already mentioned. + +Often important information may be gained concerning the nature of the +residual proteid matters by fractional extraction with dilute acids +and alkalies. When the strength of these solutions is such that they +contain about one per cent of the acid or alkali, the whole of the +proteid matter may be dissolved by boiling successively with acid and +alkali for half an hour. The proteid matter passing into solution in +these cases is usually changed in character, assuming the nature of +proteoses or allied bodies, when treated with an acid, and becoming +albuminates when boiled with an alkali. Easily soluble carbohydrate +matter is also removed by this treatment so that the residue obtained +consists largely of cellulose and is known as crude or insoluble fiber. +The removal of all the bodies soluble in dilute boiling acid and alkali +is accomplished by the method described in paragraph =272=. + +For research purposes, the solvent action of dilute alkali is of chief +importance to the analyst, and the extraction of the proteid matter, +after all that is soluble in water, common salt solution and alcohol +has been removed, should commence with a solution of potassium or +sodium hydroxid containing not over two-tenths per cent of the alkali. + +It has been shown by Osborne that the solvent action of very dilute +alkali, in the cold, may be exerted without changing the character of +the dissolved proteid.[379] + +=403. Method of Extraction.=—The solvent employed is usually a +two-tenths per cent solution of potassium hydroxid. It may be added +directly to the substance or may follow extraction with water, +salt solution or alcohol. In the former case, the manipulation +is illustrated by the following description of the treatment of +oatmeal:[380] + +One hundred grams of oatmeal are mixed with half a liter of a +two-tenths per cent potassium hydroxid solution and allowed to stand +for some time at room temperature. The mixture is strained through +a cloth to remove the chaff and the residue is stirred with another +small portion of the solvent, again strained in the same cloth and the +residue squeezed dry. The strained liquids are united and enough more +of the solvent added to make the volume 700 cubic centimeters. After +standing for some time, the insoluble matter settles to the bottom of +the vessel and the supernatant liquid is decanted. More solvent is +added to the residue, well mixed therewith and treated as above. It is +advisable to make a third extraction in the same way. The extracts are +united, passed through a filter, the proteid matter in solution thrown +out by acetic acid, washed with water, alcohol and ether and dried over +sulfuric acid. + +The methods of procedure, when the sample has been previously extracted +with water, salt solution or alcohol, are essentially the same as that +just described and the reader may consult the paper of Osborne for +details.[381] + +=404. Methods of Drying Separated Proteids.=—In the preceding +paragraphs, the analyst has been directed, in most instances, to +dry the proteid matter, after it is secured in as pure a form as +possible, at room temperature, over sulfuric acid. By this treatment +the preparation may be obtained in a form suited to the study of its +physical properties, since its solubility has not been affected by +subjecting it to a high temperature. When it is desired to use the +sample only for chemical analysis it is not necessary to wait on the +slow process above mentioned. In this case the sample may be dried in +an inert atmosphere at the temperature of a steam-bath or even at 110°. +It is better, however, to avoid so high a temperature and to conduct +the desiccation in vacuo at a heat not above that of boiling water. The +sample, before drying, should be reduced to the finest possible state +of comminution, otherwise particles of aqueous vapor may be retained +with great tenacity. + +In many cases it is advisable to dry the sample pretty thoroughly, then +grind to a fine powder and finish the desiccation with the pulverulent +mass. This treatment can be followed when the quantity of the material +is considerably in excess of that required for the analytical +operations. + +=405. Determination of Ash.=—No method of treatment is known by means +of which vegetable proteid matters may be obtained entirely free +of mineral matters. The mineral bases may be naturally present in +the proteid matter as organic and inorganic salts, or they may be +mechanically entangled therewith, having been derived either from +the other tissues of the plant or from the solvents employed. It is +necessary in calculating the analytical data to base the computation on +the ash free substance. The percentage of ash is determined by any of +the standard processes or by heating the sample in a combustion tube, +to very low redness, in a current of oxygen. The total residue obtained +is used in calculating the percentage of ash, and the weights of +material subsequently used for the determination of carbon, hydrogen, +nitrogen and sulfur are corrected for the calculations by deducting the +quantity of mineral matter contained therein. + +By reason of the highly hygroscopic nature of the dry proteid bodies, +they must be kept over a desiccating material and weighed quickly on a +balance, in an atmosphere which is kept free of moisture by the usual +methods. + +=406. Carbon and Hydrogen.=—Carbon and hydrogen are estimated in +proteid matters by combustion with copper oxid. Osborne prefers to burn +the sample in a platinum boat in a current of air or of oxygen free of +moisture and carbon dioxid.[382] It is advisable to use also a layer of +lead chromate in addition to the copper oxid and metallic copper. The +method of conducting the combustion has already been described.[383] The +analyst should have at his disposal a quantity of pure sugar, which +may be used from time to time in testing the accuracy of the work. +In beginning a series of combustions this precaution should never be +omitted. The addition of the lead chromate is to make more certain the +absorption of oxidized sulfur produced during the combustion. + +=407. Estimation of Nitrogen.=—In most cases it is found convenient, +during the progress of separating vegetable proteids, to determine the +quantity of each kind by estimating the nitrogen by moist combustion +and computing the quantity of proteid matter by multiplying the +nitrogen by 6.25. The estimation of the nitrogen is made either on an +aliquot part of the extract or by direct treatment of the residue. + +In the pure extracted proteid matter the nitrogen is most conveniently +determined by moist combustion, but it may also be obtained either by +combustion with soda-lime or with copper oxid, or by other reliable +methods.[384] + +The percentages of nitrogen found in the principal proteid bodies, +together with the factors for computing the weights of the proteid +bodies from the weights of nitrogen found, are given below: + + Name of body. Percentage of nitrogen. Factor. + + Mucin 13.80 to 14.13 7.25 to 7.08 + Chondrin 14.20 to 14.65 7.04 to 6.83 + Albuminates 13.87 7.21 + Oat proteids 15.85 6.31 + Serum globulin 15.63 6.40 + Egg albumin 15.71 to 17.85 6.37 to 5.60 + Maize proteids 16.06 6.22 + Casein 15.41 to 16.29 6.49 to 6.13 + Serum albumin 15.96 6.27 + Syntonin 16.10 6.21 + Keratin 16.20 to 17.70 6.17 to 5.65 + Fibrinogen 16.65 6.01 + Peptones 16.66 to 17.13 6.00 to 5.84 + Elastin 16.75 5.97 + Wheat proteids 16.80 to 18.39 5.95 to 5.44 + Fibrin 16.91 5.91 + Flax seed proteids 17.70 to 18.78 5.65 to 5.33 + +=408. Determination of Sulfur.=—Sulfur is a characteristic constituent +of the proteid bodies, existing in quantities approximating one per +cent of their weight. + +In the estimation of sulfur, it is first converted into sulfuric acid, +which is thrown out by a soluble barium salt and the sulfur finally +weighed as barium sulfate. + +All the sulfur existing in the organic state in a proteid may be +obtained by burning in a current of oxygen and conducting the gaseous +products of combustion through solid sodium or potassium carbonate +at or near a red heat.[385] The organic sulfur may also be converted +into sulfuric acid by fusing the proteid body with a mixture of +sodium hydroxid and potassium nitrate. The fused mass, after cooling, +is dissolved in water, the solution acidified with hydrochloric, +evaporated to dryness to decompose nitrates and remove excess of +hydrochloric acid and dissolved in a large excess of water. After +standing for a day, the solution is filtered and the sulfuric acid +thrown out of the hot filtrate with a slight excess of barium chlorid +solution. The usual precautions in precipitating, filtering and +igniting the barium sulfate are to be observed.[386] + +=409. General Observations.=—In the preceding paragraphs have been +stated the general principles upon which the separation of vegetable +proteid matters depends, and a description has been given of the +several processes by which this separation is accomplished. In +each case, however, special conditions exist which require special +modifications of the general processes, and these can only be +successfully secured by the skill, judgment and patient labor of the +investigator. Many of these cases have been already worked out, and the +valuable data secured by Chittenden, Osborne and others, are accessible +to the analyst in the papers already cited. In the case of the proteids +in the peanut, a similar work has been done in this laboratory by +Bigelow, the data of which have not yet been published. It is only +by a careful study of the work already done as outlined here and as +published in full in the cited papers, that the analyst will be able to +secure trustworthy guidance for future investigations. + +=410. Dialysis.=—One of the most important of the operations connected +with the separation and analysis of proteids is the removal of the +salts whereby their solutions are secured. This is accomplished by +subjecting the solutions of the proteid matters to dialysis. The +solution is placed in bags made of parchment dialysis paper. These +bags are tied about a glass tube, whereby access may be had to their +contents during the progress of the work. Since the volume of the +liquid increases during the process, the bags should not be filled too +full in the beginning. + +[Illustration: FIG. 105. DIALYZING APPARATUS.] + +In this laboratory the dialysis is carried out by Bigelow with the +city water from the Potomac, which is first passed through a battery +of porous porcelain filtering tubes to remove any suspended silt or +micro-organisms. If unfiltered water be used, the germs therein cause +a fermentation in the proteid matter, which seriously interferes with +the value of the data obtained, and which can only be avoided by the +use of an antiseptic, such as an alcoholic solution of thymol. Even +with filtered water, the use of a few drops of the solution mentioned +is often necessary. To avoid the use of too great quantities of the +filtered water, the dialyzers are arranged _en batterie_, as shown in +the figure. The filtered water enters the first vessel and thence +passes through all. The parchment bags are frequently changed from +vessel to vessel, each being brought successively into the first vessel +in contact with the fresh water. In some cases the final steps in the +dialysis may be accomplished in distilled water. + +It is advisable to conduct a fractional preliminary dialysis of the +salt solution containing proteids in such a way as to secure the +globulins precipitated in each interval of twenty-four hours. Each +portion thus secured may be examined with the microscope. Usually a +period of two weeks is required to entirely remove the mineral salts +from solution. If prepared parchment tubes be used for the dialysis, +they should be first tested for leaks, and should not be more than half +filled. By the use of a large number of these tubes a greater surface +is exposed to dialytic action, and the time required to complete the +operation is correspondingly decreased. + + +SEPARATION AND ESTIMATION OF NITROGENOUS BODIES IN ANIMAL PRODUCTS. + +=411. Preparation of the Sample.=—Animal products present many +difficulties in respect of the reduction thereof to a sufficiently +comminuted condition for analytical examination. In the case of bones, +the choppers used for preparing them for feeding to fowls are the most +efficient apparatus for reducing them to fragments. In this condition +they may be ground to a finer state in a sausage machine. The flesh of +animals may be reduced by this machine, with two or three grindings, +to a fairly homogeneous mass. Subsequent grinding in a mortar with +powdered glass or sharp sand may serve to reduce the sample to a finer +pulp, but is not usually necessary and should be avoided when possible. +The sample thus prepared serves for the estimation of water, ash +and fat by methods already described. The sample should be prepared +in quantities of considerable magnitude, the whole of any organ or +separate portion of the body being used when possible. In examining the +whole body the relative weights of blood, bones, viscera, muscle, hide +and other parts should first of all be ascertained and noted. + +=412. Treatment of Muscular Tissues for Nitrogenous Bodies.=—For +the present purpose a brief sketch of the method of separating the +nitrogenous bodies in the muscular tissues of the body is all that +can be attempted. For methods of examining the different organs and +parts of the body in greater detail, standard works on physiological +chemistry may be consulted.[387] + +_Extraction with Cold Water._—A noted quantity of the finely divided +tissues is mixed with several volumes of ice-cold water and well rubbed +occasionally for several hours, the temperature meanwhile being kept +low. The mixture is poured into a linen bag and the liquid portion +removed by gentle pressure. The residue in like manner is treated with +fresh portions of cold water until it gives up no further soluble +matters. An aliquot portion of the extract is concentrated to a small +bulk and serves for the determination of total nitrogen. The methods of +separating and estimating nitrogenous bodies in flesh soluble in water +will be given in considerable detail further on. + +_Extraction with Ammonium Chlorid and Hydrochloric Acid._—The residue, +after exhaustion with cold water, is extracted with a solution of +ammonium chlorid containing 150 grams of the salt in a liter. This +method of extraction is entirely similar to that with water just +described. Globulins and myosin pass into solution by this treatment. +The residual mass is washed as free as possible of the solvent and +is then further extracted with dilute hydrochloric acid containing +four cubic centimeters of the fuming acid in a liter. The treatment +with dilute acid is continued until no further substance passes +into solution. This is determined by neutralizing a portion of the +extract with sodium carbonate, or by the direct addition of potassium +ferrocyanid. In either case absence of a precipitate indicates that no +nitrogenous matters are present in the solution. + +_Extraction with Alkali._—The residue from the acid extraction is +washed with water until the acid is removed and then extracted in a +similar manner with a dilute solution of sodium or potassium hydroxid +containing not to exceed two grams of the caustic to the liter. When +this residue is finally washed with water and a little acetic acid, +it will be found that practically all the purely albuminous bodies +contained in the tissues have been extracted with the exception of any +fibrin, which the blood, present in the tissues at the commencement of +the extraction, may have contained. The extract should be acidified +with acetic as soon as obtained. + +_Extraction with Boiling Water._—The residual matter boiled for some +time with water will part with its collagen, which, when transformed by +the heat into glutin, passes into solution. + +The sarcolemma, membranes, elastic fibers and keratin remain +undissolved. + +=413. Contents of the Several Extracts.=—By the systematic treatment of +muscular tissues in the manner just described, the nitrogenous bodies +they contain are separated into five classes, _viz._: + +_Cold Water Extract._—This contains serum albumin, serum globulin, +muscle albumin, myosin, mucin and peptone. + +_Ammonium Chlorid Extract._—This solution contains the globulins and +also in many cases some myosin and serum globulin. + +_Hydrochloric Acid Extract._—When the extractive matter removed by +hydrochloric acid, thrown out by sodium carbonate and well washed with +water, has a neutral reaction, it consists of syntonin, when acid, of +an albuminate. + +_Alkali Extract._—The acid albumin of the animal tissue is found in the +alkaline solution and may be thrown out by making the solution slightly +acid. + +_Insoluble Residue._—The fifth class contains the insoluble nitrogenous +bodies mentioned above. + +=414. General Observations.=—Only a brief résumé of the methods of +treating animal tissues for nitrogenous bases is given above, since +a more elaborate discussion of these principles and methods would +lead too far away from the main purpose of this manual. For practical +purposes, the most important of these bodies are those soluble in water +and the methods of treating these will be handled at some length. +Unfortunately, the methods of determining the exact qualities of these +bodies are not as satisfactory in case of animal as in vegetable +nitrogenous bodies. The flesh bases, soluble in water, contain a much +larger percentage of nitrogen than is found in true proteid bodies, +and therefore the multiplication of the weight of nitrogen found +therein by 6.25 does not give even a near approximation of the actual +quantities of the nitrogenous bodies present in the sample. + +Some of the flesh bases contain more than twice as much nitrogen as +is found in proteids, and in such cases 3.12, and not 6.25, would be +the more correct factor to use in the computation. When possible, +therefore, these bodies should be precipitated and weighed after +drying, but this is not practicable in many instances. The sole +resource of the chemist in such cases is to determine the nature of the +body as nearly as possible by qualitive reactions, then to determine +the total nitrogen therein and multiply its weight by the corresponding +factor. The principal flesh bases have the following percentages of +nitrogen and the approximate factors for calculating analytical data +are also given: + + Name of base. Formula. Per cent Factor. + nitrogen. + Glutin C₁₃H₂₀N₄O₅ 17.95 5.57 + Carnin C₇H₈N₄O₂ 31.11 3.21 + Kreatin C₄H₁₉N₃O₂ 32.06 3.12 + Kreatinin C₄H₇N₃O₂ 37.17 2.69 + Sarkin C₅H₄N₄O 41.18 2.43 + +=415. Composition of Meat Extracts.=—The meat extracts of commerce +contain all the constituents of meat that are soluble in warm water. +The parts which are soluble in warm water and not in cold are found +in the cold aqueous solution as suspended or sedimentary matters. +Among the nitrogenous bodies present are included albumin, albumose +and peptone among the proteids, carnin, kreatin, kreatinin, sarkin and +xanthin among the non-proteids, and inosinic and uric acids and urea +among other nitrogenous bodies. Among the non-nitrogenous bodies are +found lactic and butyric acids, inosit and glycogen. Among mineral +bodies occurs the phosphates and chlorids of the common bases. In +addition to these bodies, meat extracts may also contain gelatin and +other decomposition products of proteid matter. Since meat extract is +supposed to be prepared by the digestion of the meat free of bones +and put in cold water or in warm water not above 75°, the presence +of gelatin would indicate a different method of preparation, _viz._, +either by boiling water or water heated above the boiling point under +pressure. In a properly prepared extract, the percentage of gelatin is +very small. + +Approximately one-tenth of the whole nitrogen present is in the form +of albumoses and only a trace as peptones. By far the greater part of +the nitrogen exists as flesh bases (kreatin, etc.). The composition of +three meat extracts, numbers one and two solid and number three liquid, +is given in the subjoined table.[388] + + No. 1. No. 2. No. 3. + Per cent. Per cent. Per cent. + + Total nitrogen 9.28 9.14 2.77 + Nitrogen as albumin trace 0.08 trace + ” ” albumose 0.96 1.21 0.70 + ” ” peptone trace trace none + ” ” flesh bases 6.81 5.97 1.56 + ” ” ammonia 0.47 0.41 0.09 + ” in compounds insoluble in + sixty-six per cent alcohol 0.21 0.33 0.25 + ” ” other bodies 0.83 1.14 0.17 + +=417. Analysis of Meat Extracts.=—The analysis of a meat extract +should include the determination of the water, ash and total nitrogen. +After multiplying the nitrogen which exists as proteids by 6.25 and +adding together the percentages of all the ingredients, ash, water, +etc., including ammonia, the sum is to be subtracted from 100 and the +difference entered as non-nitrogenous organic matter. The nature of +this conglomerate has already been explained. + +_Water._—It is advisable to determine the water in a partial vacuum +(=20=) or in an atmosphere of hydrogen (=23-25=). + +The water may also be determined in solid extracts by placing about +five grams of the material in a flat bottom tin foil dish about +fifty-five millimeters in diameter and twenty millimeters deep. The +material is dissolved in enough warm water to fill the dish a little +over one-half and the liquid is then absorbed by adding a weighed +quantity of fibrous asbestos or of dry fragments of pumice stone. +The asbestos is to be preferred because of the fact that it may be +subsequently cut into small bits for the determination of the gelatin. +The dish thus prepared is dried to constant weight in a steam-bath +or vacuum oven. The weight of the dish and of the added absorbent, +together with that of the material employed and of the dried dish and +its contents, give the data for calculating the percentage of water. +The contents of the dish are used as described further on for the +determination of gelatin. In liquid extracts the water is determined in +an entirely analogous manner, using about twenty grams of the material +and omitting the solution in water. + +In solid extracts, the part insoluble in cold water is determined +separately. + +_Ash._—The ash is determined by ignition at the lowest possible +temperature, best in a muffle (=28-32=). The ash should be examined +qualitively. Where a quantitive analysis is desired, larger quantities +of the extract are incinerated and the constituents of the ash +determined in the usual way.[389] + +_Total Nitrogen._—Since nitrates are not present unless added in +the manufacture, the total nitrogen is best determined by moist +combustion.[390] + +_Nitric Nitrogen._—The extract should be tested for nitrates and if +present they are determined in the manner already described.[391] + +_Ammoniacal Nitrogen._—When ammonia is present it is determined by +distillation with magnesia.[392] + +Since boiling with magnesia may cause the distillation of more +ammonia than is present as ammonium salts, the plus being due to the +decomposition of some other nitrogenous compounds, Stutzer replaces the +magnesia with barium carbonate.[393] + +_Proteid Nitrogen Insoluble in Sixty-Two Per Cent Alcohol._—The aqueous +solution is treated with strong alcohol until the mixture contains +about sixty-two per cent of the reagent. The precipitate produced is +separated by filtration, washed with sixty-two per cent alcohol and the +nitrogen therein determined. + +_Albumose Nitrogen._—This is secured by saturating the aqueous solution +with zinc or ammonium sulfate. The separated albumoses are skimmed from +the surface, thrown in a filter, washed with a saturated solution of +zinc sulfate and the nitrogen determined therein by moist combustion. +In the filtrate from the above separation, peptone is detected +qualitively by adding a few drops of dilute solution of copper sulfate +(biuret reaction). + +_Kreatin, Kreatinin and Other Flesh Bases._—The clear, aqueous solution +of the extract is acidified with sulfuric, mixed with a solution of +sodium phosphotungstate and allowed to stand for about six days. The +precipitate is collected, washed with a solution of the precipitant, +and the nitrogen therein determined. The nitrogen found, less that due +to ammonia, represents the total nitrogenous matter precipitated by +the phosphotungstic acid. From this quantity is deducted the nitrogen +in the proteids, precipitated by sixty-two per cent alcohol and by +ammonium or zinc sulfate, and the remainder represents the nitrogen in +flesh bases. + +The nitrogen thrown out by the phosphotungstic acid is deducted from +the total nitrogen, and the remainder represents the nitrogenous bodies +not precipitable by the reagent named. + +This method of separating the nitrogenous matters in meat extracts is +based on the observation that these bodies contain at most only a small +quantity of peptones, so small as to be safely negligible.[394] + +_Quantities used for Analysis._—In conducting the separations above +noted, it will be found convenient to use in each case about five +grams of the solid or twenty of the liquid extract. In the nitrogen +determinations, the weight of the sample should be inversely +proportional to its content of nitrogen. + +=417. Preparation of the Phosphotungstic Reagent.=—The phosphotungstic +reagent is conveniently prepared as follows: + +Dissolve 120 grams of sodium phosphate and 200 of sodium tungstate in +one liter of water and add to the solution 100 cubic centimeters of +strong sulfuric acid. When the reagent is prepared for general purposes +it is customary to acidify with nitric, but in the present instance, +inasmuch as the precipitate is used for the determination of nitrogen, +it is evident that sulfuric should be substituted for nitric acid. +In all cases the analyst must be assured of the strong acidity of +the reagent, and in addition to this the solutions of proteid matter +to which the reagent is added must first be made strongly acid with +sulfuric. + +=418. Zinc Sulfate as Reagent for Separating Albumoses from +Peptones.=—When the albumoses are separated from the peptones, by +precipitation with ammonium sulfate, there may be danger of some of +this reagent adhering to the albumose, and in this way the quantity of +nitrogen obtained on analysis may be increased. To avoid an accident of +this kind Bömer replaces the ammonium by zinc sulfate.[395] + +Since the precipitation of the albumoses by saturated saline solutions +depends on their hydrolytic power, the substitution of another salt +for ammonium sulfate capable of strongly attracting water, may be made +if that salt does not possess any objectionable property. Crystallized +zinc sulfate will dissolve in less than its own weight of cold water +and is therefore well suited for the purpose in view. + +In the case of a meat extract, the precipitation is accomplished as +follows: Fifty cubic centimeters of the extract, freed from all solid +matter by filtration and containing about two grams of the soluble +proteids, are saturated in the cold with finely powdered zinc sulfate. +The separated albumoses collect on the surface and are skimmed off, +poured on a filter and washed with cold saturated zinc sulfate +solution. The filter and its contents are used for the determination of +nitrogen by moist combustion.[396] + +The filtrate from the precipitated albumoses gives no biuret reaction, +and, therefore, as in the use of ammonium sulfate, is free of albumin. + +The biuret reaction is applied to the zinc sulfate filtrate as follows: +The filtrate is greatly diluted with water and freed of zinc by means +of a saturated solution of sodium carbonate. The filtrate free of zinc +is evaporated on the steam-bath, made strongly alkaline with sodium +hydroxid and treated with a few drops of a two per cent copper sulfate +solution, added successively. + +Another advantage possessed by the zinc sulfate is found in the fact +that in the filtrate from the separated albumoses the peptones and +other flesh bases can be thrown out by phosphotungstic acid. Before the +application of the reagent, the filtrate should be made strongly acid +by adding about an equal volume of dilute sulfuric acid (one part of +acid to four of water.) + +The nitrogen in the precipitate thus obtained is determined by moist +combustion in the manner already suggested. + +If the proteid matters contain salts of ammonium it is probable +that a difficultly soluble double sulfate of zinc and ammonium, +(NH₄)₂SO₄.ZnSO₄.6H₂O, will be found in the precipitate. Ammonium salts, +if present, should therefore be removed by distillation with magnesia. +It is better, however, to throw down the ammonia with the first zinc +precipitate, distil this with magnesia and determine the amount of +nitrogen derived from the ammonia compounds. In a second sample, the +total nitrogen is determined by moist combustion and the difference +between the two results gives that due to albumoses. + +=419. Examination for Muscular Tissue.=—Some samples of meat extracts +contain small quantities of finely ground muscular tissue. For +detecting this the extract is treated with cold water and the insoluble +residue examined with a microscope. If muscular tissue be found, about +eight grams of the extract or twenty-five of the fluid preparation, are +treated with cold water, the insoluble matter collected upon a filter, +washed with cold water, and the nitrogen determined in the residue. The +percentage of nitrogen multiplied by 6.25 gives the quantity of muscle +fiber proteids present. The filtrate from the above determination +is acidified with acetic, boiled, any precipitate which is formed +collected and the nitrogen therein determined. The nitrogen obtained +multiplied by 6.25 gives the quantity of coagulable albumin present. +An aliquot portion of the filtrate is used for the determination of +nitrogen and the percentage therein found, deducted from the total +nitrogen of the sample, gives a remainder which may be used as a +representative of the whole of the nitrogen present in the form of +albumin and muscular tissue. + +=420. Estimation of Gelatin.=—The tin foil dish and its contents +used for the determination of water, as above described, are cut +into small pieces, placed in a beaker and extracted four times with +absolute alcohol. After the removal of the alcohol, the residue is +extracted with ice water containing ten per cent of alcohol, in +which a small piece of ice is kept to avoid a rise of temperature. +The beaker should be shaken during the extraction, which should last +for about two minutes. Where large numbers of samples are treated +at once, any convenient form of shaking machine may be employed. At +least two extractions with ice water must be made. The residue is then +collected upon a filter and washed with ice water until the washings +are completely colorless. The residue on the filter is replaced in +the beaker, boiled with water, well washed on the filter with boiling +water, the filtrate and washings concentrated and the nitrogen therein +determined. + +The principle of this determination is based on the fact that gelatin +is almost completely insoluble in ice water while serum peptones and +albumin peptones are almost completely soluble in that reagent. On +the other hand, the flesh bases and the proteids present are almost +completely removed by the preliminary treatment with alcohol and ice +water or are left undissolved by the hot water. The solution in boiling +water, therefore, contains practically nothing but gelatin.[397] + +In a later article, Stutzer modifies the method given above as +follows:[398] + +Of dry and moist extracts from five to seven grams and of liquid +extracts from twenty to twenty-five grams are used for the +determination and placed in tin foil dishes, as described above. In +case of solid extracts, a sufficient quantity of warm water is added to +completely dissolve them, the solution being facilitated by stirring. +In case the solution is too thin it should be concentrated before going +further. It is treated with a sufficient amount of dust-free ignited +sand to completely absorb it, and the dish and its contents are then +dried to a constant weight. The dried contents of the dish are rubbed +up in a mortar, the dish cut into fine bits, and all placed in a +beaker. The solid syrphete[399] is extracted four times with 100 cubic +centimeters of absolute alcohol, the alcohol in each case being poured +through an asbestos filter for the purpose of collecting any matters +suspended therein. In a large flask are placed 100 grams of alcohol, +300 grams of ice and 600 grams of cold water, and the flask is placed +in a large vessel and packed with finely divided ice. Four beakers +marked _b, c, d, e_ are also placed in ice and the beaker containing +the syrphete, left after extraction with absolute alcohol as above +mentioned, is marked _a_ and also placed in pounded ice. The extraction +with cold alcoholic water proceeds as follows: + +In beaker _a_ are poured 100 cubic centimeters of the mixture in the +large flask, its contents are stirred for two minutes and then the +liquid portion poured off into beaker _b_ to which, at the same time, +a piece of ice is added. In beaker _a_ are poured again 100 cubic +centimeters from the large flask, treated as above described, and the +liquid extract poured into beaker _c_. In like manner the extraction +in beaker _a_ is continued until each of the beakers has received +its portion of the extract. By this time the liquid over the sand in +beaker _a_ should be completely colorless. The filtration of the liquid +extract is accomplished as follows: + +In a funnel of about seven centimeters diameter is placed a perforated +porcelain plate about four centimeters in diameter which is covered +with asbestos felt with long fiber. Three filters are prepared in this +way. On the first filter are poured the contents of beaker _b_. After +the liquid has passed through, the sand and other residue in beaker _a_ +are transferred to the filter and the beaker and residue washed with +the alcoholic ice water from the large flask. The filtration should +be accomplished under pressure. On the second filter are poured the +contents of beaker _c_. On the third filter the contents of beakers +_d_ and _e_. The washing with alcoholic ice water from the large flask +is continued in each instance until the filtrate is colorless. At the +same time the asbestos filter, which was used in the first instance for +filtering the absolute alcohol extract, is washed with the alcoholic +ice water mixture from the large flask. At the end the sand remaining +in beaker a together with all the asbestos filters are brought +together into a porcelain dish, boiled two or three times with water, +the aqueous solution filtered and the filtrate concentrated and used +for the estimation of the nitrogen. The quantity of nitrogen found +multiplied by 6.25 represents the proteid matter in the gelatin of the +sample. + +The object of the multiple filters, described above, is to accelerate +the process, and they are required because the gelatin quickly occludes +the filter pores. For this reason the asbestos filters are found +to operate better than those made of paper. It should be mentioned +that the residue of the peptones insoluble in alcohol may contain, +in addition to gelatin, also small quantities of albumoses. From the +quantity of albumose nitrogen found, it is understood that the nitrogen +in the form of coagulable albumin, determined as described in the first +process mentioned above, is to be deducted, since these coagulable +albumins are insoluble in alcohol. + +=421. Estimation of Nitrogen in the Flesh Bases Soluble in +Alcohol.=—About five grams of the dry extract, ten grams of the +extract containing water or twenty-five grams of the liquid extract +are placed in a beaker and enough water added in each case to make +about twenty-five cubic centimeters in all. Usually no water need be +added to the liquid extracts. Very thin peptone solutions should be +evaporated until the content of water is reduced to seventy-five per +cent. The solution, prepared as above indicated, is treated slowly +with constant stirring with 250 cubic centimeters of absolute alcohol, +the stirring continued for some minutes and the vessel set aside for +twelve hours, at the end of which time the precipitate is separated by +filtration and washed repeatedly with strong alcohol. Leucin, tyrosin +and a part of the flesh bases are dissolved by alcohol. The alcohol is +removed by distillation and the residue dissolved in water. Any flocky +residue which remains on solution with water is removed by filtration, +the nitrogen determined therein and the quantity thereof added to the +albumose nitrogen found, as hereafter described. + +The volume of the aqueous solution is completed with water to half a +liter. One hundred cubic centimeters of this solution are used for the +determination of total nitrogen, and another 100 cubic centimeters +for the determination of ammoniacal nitrogen by distillation with +barium carbonate. A part of the ammonia may have escaped during the +preliminary distillation of the alcohol and therefore the amount found +may not represent the whole amount originally present. The use of the +above determination is principally to ascertain the correction to be +made in the amount of total nitrogen found in the first 100 cubic +centimeters of the solution. + +=422. Treatment of the Residue Insoluble in Alcohol.=—The residue +insoluble in alcohol is washed from the filter into the beaker in +which the first solution was made. The aqueous mixture is warmed +on a water-bath until the alcohol adhering to the precipitate is +completely evaporated, when the contents of the beaker are poured upon +a filter free of nitrogen. A small part of the albumose, by reason of +the treatment with alcohol, tends to remain undissolved, and it is +advisable to collect this albumose upon a filter, wash it well with hot +water and estimate the nitrogen therein. The quantity of nitrogen thus +found is to be added to the albumose nitrogen determined as described +later on. + +The total filtrate obtained from the last filtration is made up to +a volume of half a liter, of which fifty cubic centimeters are used +for the determination of total nitrogen, fifty cubic centimeters for +the determination of gelatin, albumose and peptone, and 100 cubic +centimeters for the residual peptones. The albumose, together with the +gelatin and peptones carried down with it, is precipitated with zinc or +ammonium sulfate solution, and its per cent calculated from the amount +of nitrogen found in the precipitate. The true peptone is determined by +subtracting the quantity of nitrogen determined as albumose from the +total nitrogen in solution. + +The rest of the liquid, _viz._, 300 cubic centimeters, is evaporated to +a small volume and tested qualitively for true peptones as follows: + +To separate the albumose and gelatin a concentrated liquor is treated +with an excess of finely divided ammonium sulfate so that a part of +the salt remains undissolved. The separated albumose, gelatin and +undissolved ammonium salts are collected on a filter, the filtrate +mixed with a few drops of dilute copper sulfate solution and a +considerable quantity of concentrated soda or potash lye added. Care +should be taken that the quantity of copper is not too great, otherwise +the peculiar red coloration will be obscured by the blue color of the +copper solution. + +=423. Pancreas Peptone.=—The filtrate obtained as described above, by +treating the portion of the material insoluble in alcohol with warm +water, contains in addition to the albumose and gelatin the whole of +the pancreas peptone which may be present. To separate this peptone, +100 cubic centimeters of the aqueous solution are evaporated in a +porcelain dish until the volume does not exceed ten cubic centimeters. +When cool, at least 100 cubic centimeters of a saturated cooled +solution of ammonium sulfate solution are added, the mixture thoroughly +stirred, the precipitate collected upon a filter and washed with a cold +saturated solution of ammonium sulfate. The contents of the filter +are dissolved in boiling water, the filter thoroughly washed and the +filtrate and washings evaporated in a porcelain dish with the addition +of barium carbonate until, on the addition of new quantities of barium +carbonate, no further trace of ammonia can be discovered. The residue +is extracted with water, the barium sulfate and carbonate present +separated by filtration, well washed and the nitrogen determined in the +evaporated filtrate and washings in the usual way and multiplied by +6.25 to determine the quantity of pancreas peptone. + +=424. Albumose Peptone.=—A part of the albumose peptone which may be +present is determined in conjunction with the other bodies mentioned +above. The chief quantity is found in the solution of the residue +insoluble in alcohol in the following manner: + +Fifty cubic centimeters of the solution of this residue in hot water +are mixed with an equal volume of dilute sulfuric acid, one volume +of acid to three of water, in the cold, and a solution of sodium +phosphotungstate added until it produces no further precipitate. The +precipitate is washed with dilute sulfuric acid and the nitrogen +determined therein. The nitrogen thus found is derived from the +albumose, pancreas peptone and gelatin. The quantity of nitrogen in the +pancreas peptone and gelatin, as above described, is subtracted from +the total quantity found in the phosphotungstic acid precipitated, and +the remainder represents the nitrogen due to the albumose. + +=425. Nitrogen in the Form of Flesh Bases Insoluble in Alcohol.=—This +is determined by subtracting the quantity of nitrogen, determined by +the phosphotungstic acid method already described, from the total +quantity of nitrogen found in the precipitate insoluble in alcohol and +soluble in water. + + +AUTHORITIES CITED IN PART FIFTH. + +[337] Watts’ Dictionary of Chemistry, new edition, Vol. 4, p. 327. + +[338] Vid. op. cit. supra, p. 330. + +[339] Barbieri, Journal für praktische Chemie, neue Folge Band 18, S. +114. + +[340] Vid. op. cit. 1, p. 339. + +[341] Bulletin No. 49, Kansas Experiment Station, May, 1895. + +[342] This work, Vol. 2, p. 208. + +[343] Vid. op. cit. supra, Vol. 1, p. 570. + +[344] Wiley, American Chemical Journal, Vol. 6, No. 5, p. 289. + +[345] Obermayer, Chemiker-Zeitung Repertorium, Oct. 1889, S. 269. + +[346] Hoppe-Seyler, Handbuch der physiologisch- und +pathologisch-chemischen Analyse, S. 269. + +[347] Wiley, American Chemical Journal, Vol. 6, p. 289. + +[348] Dragendorff’s Plant Analysis, p. 55. + +[349] This work, Vol. 2, pp. 192 et seq. + +[350] Chemiker-Zeitung, Band 20, S. 151. + +[351] This work, Vol. 2, p. 207. + +[352] Landwirtschaftlichen Versuchs-Stationen, Band 17, S. 321: +Zeitschrift für analytische Chemie, Band 14, S. 380. + +[353] Vid. op. cit. 12, p. 245. + +[354] Landwirtschaftlichen Versuchs-Stationen, Band 16, S. 61. + +[355] Berichte der deutschen chemischen Gesellschaft, Band 10, Ss. 85, +199; Band 16, S. 312: Chemiker-Zeitung, Band 20, S. 145. + +[356] Zeitschrift für analytische Chemie, Band 22, S. 325. + +[357] Richardson and Crampton, Berichte der deutschen chemischen +Gesellschaft, Band 19, S. 1180. + +[358] Maxwell, American Chemical Journal, Vol. 13, p. 470. + +[359] Vid. op. cit. supra, Vol. 15, p. 185. + +[360] Vid. op. cit. supra, Vol. 13, p. 13: Schulze, Zeitschrift +physiologische Chemie, Band 14, S. 491. + +[361] This work, Vol. I, p. 411. + +[362] Vid. op. cit., 22, Vol. 13, p. 15. + +[363] Vid. op. cit. supra, Vol. 15, p. 188. + +[364] Hoppe-Seyler, Handbuch der physiologisch- und +pathologisch-chemischen Analyse, S. 169. + +[365] This work, Vol. 2, p. 225. + +[366] Bulletin No. 45, Division of Chemistry, U. S. Department of +Agriculture, p. 51. + +[367] Osborne and Voorhees, American Chemical Journal, Vol. 15, p. 470. + +[368] Vid. op. cit. supra, Vol. 13, p. 385. + +[369] Vid. op. cit. supra, p. 412. + +[370] Vid. op. cit. supra, Vol. 15, p. 402. + +[371] Vid. op. cit. supra, p. 404. + +[372] Zeitschrift für analytische Chemie, Band 34, S. 562. + +[373] Vid. op. cit. 34, p. 404. + +[374] Vid. op. cit. 3, p. 455. + +[375] Osborne and Voorhees, vid. op. cit. 34, p. 409. + +[376] Vid. op. cit. supra, Vol. 13, p. 464. + +[377] Chittenden and Osborne, op. cit. supra, Vol. 14, p. 32. + +[378] Vid. op. cit. supra, p. 41. + +[379] Vid. op. cit. supra, p. 639. + +[380] Vid. op. cit. supra, Vol. 13, p. 399. + +[381] Vid. op. cit. supra, pp. 395, 400, 401. + +[382] Vid. op. cit. supra, p. 409. + +[383] This work, Vol. 1, p. 319. + +[384] This work, Vol. 2, pp. 169 et seq. + +[385] Vid. op. cit. 47, p. 420. + +[386] Osborne, vid. op. cit. 44, p. 410. + +[387] Hoppe-Seyler, Handbuch der physiologisch- und +pathologisch-chemischen Analyse. + +[388] König und Bömer, Zeitschrift für analytische Chemie, Band 34, S. +560. + +[389] This work, Vol. 2, pp. 297, 298. + +[390] Vid. op. cit. supra, p. 184. + +[391] Vid. op. cit. supra, p. 206. + +[392] This work, Vol. I, p. 450; Vol. 2, p. 226. + +[393] Zeitschrift für analytische Chemie, Band 34, S. 377. + +[394] König und Bömer, vid. op. cit. supra, S. 560. + +[395] Vid. op. cit. supra, S. 562. + +[396] Vid. op. cit. 53, p. 184. + +[397] Vid. op. cit. 57, S. 374. + +[398] Vid. op. cit. supra, S. 568. + +[399] From συρφετος. + + + + +PART SIXTH. + +DAIRY PRODUCTS. + + +=426. Introductory.=—The importance of dairy products has led to the +publication of a vast amount of literature relating thereto, and it +seems almost a hopeless task to present even a typical abstract of +the various analytical processes which have been proposed and used in +their study. The general principles which have been developed in the +preceding parts of this volume are applicable to the study of dairy +products, and the analyst who is guided by them can intelligently +examine the bodies specially considered in the present part. There +have been developed, however, many valuable processes for the special +examination of dairy products, which are of such a nature that they +could not be properly discussed in the preceding pages. In the present +part an effort will be made to present in a typical form the most +important of these processes and to state the general principles on +which they are based. This subject is naturally subdivided into three +parts, _viz._, milk, butter and cheese. The milk sugar industry is not +of sufficient importance to receive a special classification. + + +MILK. + +=427. Composition of Milk.=—The composition of milk not only varies +with the genus and species of the mammal from which it is derived, but +also depends in a marked degree on idiosyncrasy.[400] + +Milk is a mixture containing water, proteids, fat, carbohydrates, +organic and inorganic acids and mineral salts. There have also been +observed in milk in minute quantities ammonia, urea, hypoxanthin, +chyme, chyle, biliverdin, cholesterin, mucin, lecithin, kreatin, leucin +and tyrosin. In the fermentation which milk undergoes in incipient +decomposition there is sometimes developed from the proteid matter, as +pointed out by Vaughn, a ptomaine, tyrotoxicon, which is a virulent +poison.[401] The presence of these last named bodies is of interest +chiefly to the physiologist and pathologist and can receive no further +attention here. + +From a nutritive point of view, the important components of milk are +the fats, proteids and sugar, but especially in the nourishment of the +young the value of lime and phosphoric acid must be remembered. The +mean composition of the most important milks, as determined by recent +analyses, is given below: + + Water. Sugar. Proteids. Fat. Ash. + Per cent. Per cent. Per cent. Per cent. Per cent. + Cow 86.90 4.80 3.60 4.00 0.70 + Human 88.75 6.00 1.50 3.45 0.30 + Goat 85.70 4.45 4.30 4.75 0.80 + Ass 89.50 6.25 2.00 1.75 0.50 + Mare 90.75 5.70 2.00 1.20 0.35 + Sheep 80.80 4.90 6.55 6.85 0.90 + +The mean composition of milk, as given by Watts and König, is given in +the following tables: + + WATTS. + Mineral + Water. Solids. Proteids. Fats. Sugar. Salts. + Woman 87.65 12.35 3.07 3.91 5.01 0.17 + Ass 90.70 9.30 1.70 1.55 5.80 0.50 + Cow 86.56 13.44 4.08 4.03 4.60 0.73 + Goat 86.76 13.24 4.23 4.48 3.91 0.62 + Sheep 83.31 16.69 5.73 6.05 3.96 0.68 + Mare 82.84 17.16 1.64 6.87 8.65 + + KÖNIG. + Casein and Milk + Water. Fat. albumin. sugar. Ash. + Woman 87.41 3.78 2.29 6.21 0.31 + Mare 90.78 1.21 1.99 5.67 0.35 + Ass 89.64 1.63 2.22 5.99 0.51 + Cow 87.17 3.69 3.55 4.88 0.71 + +The average composition of 120,540 samples of cow milk, as determined +by analysis, extending over a period of eleven years, was found by +Vieth to be as follows:[402] + + Per cent. + Total solids 12.9 + Solids not fat 8.8 + Fat 4.1 + +The quantity of solids and fat in milk is less after longer than after +shorter periods between milkings. + +The quantity of solids and fat in cow milk is less in the spring than +in the autumn. + +The chief organic acid naturally present in milk is citric, which +exists probably in combination with lime. + +The mean content of citric acid in milk is about one-tenth of one per +cent.[403] + +Citric acid is not found in human milk, and probably exists only in the +mammary secretions of herbivores. + +Among the mineral acids of milk, phosphoric is the most important, but +a part of the phosphorus found as phosphoric acid in the ash of milk +may come from pre-existing organic phosphorus (lecithin, nuclein). + +The sulfuric acid, which is found in the ash of milk, is derived from +the sulfur of the proteid matter during ignition. + +Lactic acid is developed from lactose during the souring of milk as the +result of bacterial activity. + +Gases are also found in solutions of milk, notably carbon dioxid, which +gives to freshly drawn milk its brothy appearance. + +The ash of milk has the following composition expressed as grams per +liter of the original milk:[404] + + Grams Probable form Grams + Component. per liter. of combination. per liter. + + { sodium chlorid 0.962 + Chlorin 0.90 { potassium chlorid 0.830 + + { KH₂PO₄ 1.156 + { K₂HPO₄ 0.853 + Phosphoric acid 2.42 { MgHPO₄ 0.336 + { CaHPO₄ 0.671 + { Ca₃(PO₄)₂ 0.806 + + Potassium 1.80 (as shown above) + and as potassium citrate 0.495 + + Sodium 0.49 sodium chlorid 0.962 + + Lime 1.90 (as shown above) + and as calcium citrate 2.133 + + Magnesia 0.20 MgHPO₄ 0.336 + +The percentage composition of the ash of milk, according to Fleischmann +and Schrott, is expressed as follows:[405] + + Per cent. + Potassium oxid, K₂O 25.42 + Sodium oxid, Na₂O 10.94 + Calcium oxid, CaO 21.45 + Magnesium oxid, MgO 2.54 + Iron oxid, Fe₂O₃ 0.11 + Sulfuric acid, SO₃ 4.11 + Phosphoric acid, P₂O₅ 24.11 + Chlorin, Cl 14.60 + ------ + 103.28 + Less Cl as O 3.28 + ------ + 100.00 + +=428. Alterability of Milk.=—The natural souring and coagulation of +milk is attributed by most authorities to bacterial action produced +by infection from the air or containing vessels.[406] Pasteur, however, +shows that fresh milk sterilized at a temperature of 110° may be +exposed to the air without danger of souring.[407] After about three +days, however, a fermentation is set up which is totally different from +that produced by the microzymes naturally present in the milk. This +point has been further investigated by Béchamp, who finds that the +natural souring of milk is accomplished without the evolution of any +gas, while the fermentation produced in sterilized milk by the microbes +of the air, is uniformly attended by a gaseous development.[408] As a +result of his investigations, he concludes that the souring of milk +takes place spontaneously by reason of milk being an organic matter, +in the physiological sense of the term, and that this alteration is +produced solely by the natural microzymes of the milk. + +According to Béchamp, the milk derived from healthy animals is capable +of spontaneous alteration, which consists in the development of lactic +acid and alcohol, and of curd in those milks which contain caseinates +produced by the precipitating action of the acids formed. Oxygen and +the germs which are present in the air, according to him, have nothing +to do with this alteration in the properties of milk. Milk belongs +to that class of organic bodies like blood, which are called organic +from a physiological point of view, on account of containing automatic +forces which produce rapid changes therein when they are withdrawn +from the living organisms. + +After milk has become sour by the spontaneous action of the microzymes +which it contains, there are developed micro-organisms, such as +vibriones and bacteria from a natural evolution from the microzymes. + +Milk which is sterilized at a high temperature, _viz._, that of boiling +water or above, is no longer milk in the true physiological sense of +that term. The globules of the milk undergo changes and the microzymes +a modification of their functions, so that in milk thus altered by +heat, they are able to produce a coagulation without development of +acidity. The microzymes thus modified, however, retain to a large +extent their ability to become active. Human milk differs from cow +milk in containing neither caseinates nor casein, but special proteid +bodies, and also a galactozyme or galactozymase functionally very +different from that which exists in cow milk. The extractive matter +is also a special kind, consisting of milk globules and microzymes +belonging particularly to it and containing three times less phosphate +and mineral salts than cow milk. Boiling the milk of the cow or other +animals does not render it similar to that of woman. There is no +treatment, therefore, of any milk which renders it entirely suited to +the nourishment of infants. The composition of the milk of the cow may +be represented by three groups: + +1. Organic elements in suspension; consisting chiefly of the globules +of the milk, which are mostly composed of the fat, of an epidermoid +membrane containing mineral matter of special soluble albumins and of +microzymes containing also mineral matter. + +2. Dissolved constituents; consisting of caseinates, lactalbuminates, +galactozymase, holding phosphates in combination, lactose, extractive +matter, organic phosphates of lime, acetates, urea and alcohol. + +3. Mineral matters in solution; consisting of sodium and calcium +chlorids, carbon dioxid and oxygen.[409] + +It will be noticed from the above classification that Béchamp fails to +mention citrate of lime. It is scarcely necessary to add to this brief +résumé of the theories of Béchamp that they are entirely at variance +with the opinions held by nearly all his contemporaries. + +=429. Effects of Boiling on Milk.=—On boiling, the albumin in milk is +coagulated and on separating the proteid bodies by saturation with +magnesium sulfate no albumin is found in the filtrate. The total casein +precipitated from boiled is therefore greater than from unboiled milk. +Jager has shown that the casein can be precipitated from boiled milk by +rennet, but with greater difficulty than from unboiled.[410] According +to this author in 3.75 per cent of proteid in milk there are found 3.15 +per cent of casein, 0.35 of albumin and 0.25 of globulin. + +=430. Appearance of the Milk.=—The color, taste, odor and other +sensible characters of the milk are to be observed and noted at the +time the sample is secured. Any variation from the faint yellow color +of the milk is due to some abnormal state. A reddish tint indicates +the admixture of blood, while a blue color is characteristic of the +presence of unusual micro-organisms. Odor and taste will reveal often +the character of the food which the animals have eaten. Any marked +departure of the sample from the properties of normal milk should at +once lead to its condemnation for culinary or dietetic purposes. + +=431. Micro-Organisms of the Milk.=—Milk is a natural culture solution +for the growth of micro-organisms, and they multiply therein with +almost incredible rapidity. Some of these are useful, as, for instance, +those which are active in the ripening of cream, and others are of an +injurious nature, producing fermentations which destroy the sugars or +proteids of the milk and develop acid, alcohol, mucous or ptomaine +products. It is not possible here to even enumerate the kinds of +micro-organisms which abound in milk and the reader is referred to the +standard works on that subject.[411] + +For analytical purposes it is important that the sample be kept as +free as possible of all micro-organisms, good or bad, which may be +accomplished by some of the methods given below. + +=432. Sampling Milk.=—It is not difficult to secure for examination +representative samples of milk, if the proper precautions be taken. +On the other hand, the ease and rapidity with which a milk undergoes +profound changes render necessary a careful control of the methods +of taking samples. The most rapid changes to which a mass of milk is +obnoxious are due to the separation of the fat particles and to the +action of bacteria. Even after standing for a few minutes, it will be +found that the fat globules are not evenly distributed. Before securing +the sample for analysis, it is necessary to well stir or mix the milk. +A mean sample may also be secured from a can of milk by the sampling +tube devised by Scovell, which will be described below. + +In securing samples, a full detailed description of the cow or herd +furnishing them is desirable, together with all other data which seem +to illustrate in any way the general and particular conditions of the +dairy. Samples are to be preserved in clean, well stoppered vessels, +properly numbered and securely sealed. + +[Illustration: FIG. 106.—SCOVELL’S MILK SAMPLING TUBE.] + +=433. Scovell’s Milk Sampler.=—In sampling large quantities of milk +in pails or shipping cans, it is exceedingly inconvenient to mix the +milk by pouring from one vessel to another or by any easy process of +stirring. In order to get representative samples in such conditions, +Scovell has put in use a sampler, by means of which a typical portion +of the milk may be withdrawn from a can without either pouring or +stirring. The construction of the sampler is shown in Fig. 106, +representing it in outline and longitudinal section. The tube _a_, +made of brass, is open at both ends and of any convenient dimensions. +Its lower end slides in a large tube _b_, closed at the bottom and +having three elliptical, lateral openings _c_, which admit the milk as +the tube is slowly depressed in the contents of the can. In getting +the sample, _a_ is raised as shown in profile. When the bottom of +_b_ reaches the bottom of the can _a_ is pushed down as shown in the +section. The milk contained in the sampler is then readily withdrawn. + +=434. Preserving Milk for Analysis.=—Pasteurizing or boiling the sample +is not advisable by reason of the changes produced in the milk by heat. +The milk sample may be preserved by adding to it a little chloroform, +one part in 100 being sufficient. Boric and salicylic acids may also +be used, but not so advantageously as formaldehyd or mercuric chlorid. +Rideal has observed that one part of formaldehyd will preserve 10,000 +parts of milk in a fresh state for seven days. The formaldehyd sold in +the trade contains about one part of formaldehyd in 320 of the mixture. +One-half pint of this commercial article is sufficient for about twenty +gallons of milk, corresponding to about one part of pure formaldehyd +to 45,000 parts of milk. Rideal much prefers formalin (formaldehyd) to +borax or boric acid as a milk preservative. No ill effects due to its +toxic action have been observed, even when it is consumed in a one per +cent solution.[412] + +Samples of milk can be kept in this way from four to six weeks by +adding about one drop of the commercial formaldehyd to each ounce of +sample. The analyst should remember in such cases that the formaldehyd +may not all escape on evaporation, on account of forming some kind of +a compound with the constituents of the milk, as is pointed out by +Bevan.[413] + +Bevan suggests that the formaldehyd may not actually be retained in the +sample, but that the increase in the apparent amount of total solids +is due to the conversion of the lactose into galactose. This point, +however, has not been determined. + +Richmond and Boseley propose to detect formalin by means of +diphenylamin. A solution of diphenylamin is made with water, with the +help of just enough sulfuric acid to secure a proper solvent effect. +The liquid to be tested, which is supposed to contain formaldehyd, or +the distillate therefrom, is added to this solution and boiled. If +formaldehyd be present, a white flocculent precipitate is deposited, +which is colored green if the acid used contain nitrates. For other +methods of detecting formalin and for a partial literature of the +subject the paper mentioned above may be consulted. + +One gram of fine-ground mercuric chlorid dissolved in 2,000 grams of +milk will preserve it, practically unchanged, for several days. One +gram of potassium bichromate dissolved in one liter of milk will also +preserve it for some time. Thymol, hydrochloric acid, carbon disulfid, +ether and other antiseptics may also be employed. No more of the +preserving agent should be used than is required to keep the milk until +the analysis is completed. + +All methods of preservation are rendered more efficient by the +maintenance of a low temperature, whereby the vitality of the bacteria +is greatly reduced. + +=435. Freezing Point of Milk.=—By reason of its content of sugar and +other dissolved solids, the freezing point of milk is depressed below +0°. A good idea of the purity of whole milk is secured by subjecting +it to a kryoscopic test. The apparatus employed for this purpose is +that used in general analytical work in the determination of freezing +points. Pure full milk freezes at about 0°.55 below zero, and any +marked variation from this number shows adulteration or abnormal +composition.[414] A simple apparatus, especially adapted to milk, is +described by Beckmann.[415] The kryoscopic investigation may also be +extended to butter fat dissolved in benzol. + +=436. Electric Conductivity of Milk.=—The electric conductivity of milk +may also be used as an index of its composition. The addition of water +to milk diminishes its conductivity.[416] This method of investigation +has at present but little practical value. + +=437. Viscosity Of Milk.=—The viscosity of milk may be determined by +the methods already described. Any variation from the usual degree +of fluidity is indicated either by the abstraction of some of the +contents of the milk, the addition of some adulterant or the result of +fermentation. + +=438. Acidity and Alkalinity of Milk.=—Fresh milk of normal +constitution has an amphoteric reaction. It will redden blue and blue +red litmus paper. This arises from the presence in the milk of both +neutral and acid phosphates of the alkalies. A saturated alkaline +phosphate, _i. e._, one in which all the acid hydrogen of the acid +has been replaced by the base has an alkaline reaction while the +acid phosphates react acid. When fresh milk is boiled its reaction +becomes strongly alkaline and this arises chiefly from the escape +of the dissolved carbon dioxid. By the action of micro-organisms on +the lactose of milk, the alkaline reaction soon becomes acid, and +delicate test paper will show this decomposition long before it becomes +perceptible to the taste. It is advisable to test the reactions of the +milk as soon as possible after it is drawn from the udder, both before +and after boiling. + +=439. Determination of the Acidity of Milk.=—In the determination of +the acidity of milk it is important that it first be freed of the +carbon dioxid it contains.[417] Van Slyke has found that too high +results are obtained by the direct titration of milk for acidity, and +when the milk is previously diluted the results are also somewhat too +high.[418] Good results are got by diluting the milk with hot water +and boiling for a short time to expel the carbon dioxid. Twenty-five +cubic centimeters of milk are diluted with water to about a quarter of +a liter, as above, two cubic centimeters of a one per cent alcoholic +phenolphthalien added and the titration accomplished by decinormal +alkali. This variation of the methods of procedure, suggested by +Hopkins and Powers, appears to be the best process at present known for +the determination of acidity. The reader is referred to the paper cited +above for references to other methods which have been proposed. + +=440. Opacity Of Milk.=—The white color and opacity of milk are +doubtless due to the presence of the suspended fat particles and to +the colloid casein. On the latter it is probably principally dependent +since the color of milk is not very sensibly changed after it has +passed the extractor, which leaves not to exceed one-tenth of one per +cent of fat in it. Some idea of the quality of the milk, however, may +be obtained by determining its opacity. This is accomplished by the use +of a lactoscope. The one generally employed was devised by Feser and is +shown in Fig. 107. + +The instrument consists of a cylindrical glass vessel of a little more +than 100 cubic centimeters content, in the lower part of which is set a +cone of white glass marked with black lines. Into this part are placed +four cubic centimeters of milk. A small quantity of water is added and +the contents of the vessel shaken. This operation is repeated until the +black lines on the white glass just become visible. The graduations on +the left side show the volume of water which is necessary to bring the +dark lines into view, while those on the right indicate approximately +the percentage of fat present. + +Among the other lactoscopes which have been used may be mentioned +those of Donné, Vogel, Hoppe-Seyler, Trommer, Seidlitz, Reischauer, +Mittelstrass, Hénocque, and Heusner.[419] Since the invention of so many +quick and accurate methods of fat estimation these instruments have +little more than a historical interest. + +[Illustration: FIG. 107.—LACTOSCOPE, LACTOMETER AND CREAMOMETER.] + +=441. Creamometry.=—The volume of cream which a sample of milk affords +under arbitrary conditions of time and temperature is sometimes of +value in judging the quality of milk. A convenient creamometer is +a small cylinder graduated in such a way that the volume of cream +separated in a given time can be easily noted. There are many kinds of +apparatus used for this purpose, a typical one being shown in Fig. 107. + +The usual time of setting is twenty-four hours. A quicker determination +is secured by placing the milk in strong glass graduated tubes and +subjecting these to centrifugal action. The process is not exact and +is now rarely practiced as an analytical method, even for valuing the +butter making properties of milk. + +=442. Specific Gravity.=—The specific gravity of milk is uniformly +referred to a temperature of 15°. Generally no attempt is made to free +the milk of dissolved gases beforehand. This should not be done by +boiling but by placing the sample in a vacuum for some time. Any of the +methods described for determining specific gravity in sugar solutions +may be used for milk (=48-59=). The specific gravity of milk varies in +general from 1.028 to 1.034. Nearly all good cow milk from herds will +show a specific gravity varying from 1.030 to 1.032. In extreme cases +from single cows the limits may exceed those first given above, but +such milk cannot be regarded as normal. + +Increasing quantities of solids not fat in solution, tend to increase +the specific gravity, while an excess of fat tends to diminish it. +There is a general ratio existing between the solids not fat and +the fat in cow milk, which may be expressed as 9: 4. The removal of +cream and the addition of water in such a manner as not to affect the +specific gravity of the sample disturbs this ratio. + +The determination of the specific gravity alone, therefore, cannot be +relied upon as an index of the purity of a milk. + +=443.= =Lactometry.=—A hydrometer especially constructed for use in +determining the density of milk is called a lactometer. In this country +the one most commonly used is known as the lactometer of the New York +Board of Health. It is a hydrometer, delicately constructed, with a +large cylindrical air space and a small stem carrying the thermometric +and lactometric scales. It is shown held in the creamometer in Fig. +107. The milk is brought to a temperature of 60° F. and the reading +of the lactometer scale observed. This is converted into a number +expressing the specific gravity by means of a table of corresponding +values given below. Each mark on the scale of the instrument +corresponds to two degrees and these marks extend from 0° to 120°. +The numbers of this scale can be converted into those corresponding +to the direct reading instrument, described in the next paragraph, by +multiplying them by 0.29. + +The minimum density for whole milk at 60° F. is fixed by this +instrument at 100°, corresponding to a specific gravity of 1.029. The +instrument is also constructed without the thermometric scale. The +mean density of many thousand samples of pure milk, as observed by the +New York authorities, is 1.0319. + +The specific gravity is easily secured, and while not of itself +decisive, should always be determined. The specific gravity of milk +increases for some time after it is drawn and should be made both when +fresh and after the lapse of several hours.[420] + +TABLE SHOWING SPECIFIC GRAVITIES CORRESPONDING TO DEGREES OF THE NEW +YORK BOARD OF HEALTH LACTOMETER. TEMPERATURE 60° F. + + Degree. Sp. gr. Degree. Sp. gr. + 90 1.02619 106 1.03074 + 91 1.02639 107 1.03103 + 92 1.02668 108 1.03132 + 93 1.02697 109 1.03161 + 94 1.02726 110 1.03190 + 95 1.02755 111 1.03219 + 96 1.02784 112 1.03248 + 97 1.02813 113 1.03277 + 98 1.02842 114 1.03306 + 99 1.02871 115 1.03335 + 100 1.02900 116 1.03364 + 101 1.02929 117 1.03393 + 102 1.02958 118 1.03422 + 103 1.02987 119 1.03451 + 104 1.03016 120 1.03480 + 105 1.03045 + +=444.= =Direct Reading Lactometer.=—A more convenient form of +lactometer is one which gives the specific gravity directly on the +scale. The figures given represent those found in the second and third +decimal places of the number expressing the specific gravity. Thus 31 +on the scale indicates a specific gravity of 1.031. This instrument is +also known as the lactometer of Quévenne. For use with milk, the scale +of the instrument does not need to embrace a wider limit than from +25 to 35, and such an instrument is capable of giving more delicate +readings than when the scale extends from 14 to 42, as is usually the +case with the quévenne instrument. + +Langlet has invented a lactoscope with a scale, showing the corrections +to be applied for temperatures other than 15°. A detailed description +of this instrument, as well as the one proposed by Pinchon, is +unnecessary.[421] + +=445. Density of Sour Milk.=—Coagulated milk cannot be used directly +for the determination of the specific gravity, both because of its +consistence and by reason of the fact that the fat is more or less +completely separated. In such a case, the casein may be dissolved by +the addition of a measured quantity of a solvent of a known specific +gravity, the density of the resulting solution determined and that +of the original milk calculated from the observed data. Ammonia is a +suitable solvent for this purpose.[422] + +=446. Density of the Milk Serum.=—The specific gravity of the milk +serum, after the removal of the fat and casein by precipitation and +filtration, may also be determined. For normal cow milk the number is +about 1.027. + +=447. Total Solids.=—The direct gravimetric determination of the total +solids in milk is attended with many difficulties, and has been the +theme of a very extended periodical literature. A mere examination of +the many processes which have been proposed would require several pages. + +The most direct method of procedure is to dry a small quantity of milk +in a flat-bottom dish to constant weight on a steam-bath. The surface +of the dish should be very large, even for one or two grams of milk; +in fact the relation between the quantity of milk and the surface of +the dish should be such that the fluid is just sufficient in amount to +moisten the bottom of the dish with the thinnest possible film. The +dish, during drying, is kept in a horizontal position at least until +its contents will not flow. The water of the sample will be practically +all evaporated in about two hours. The operation may be accelerated by +drying in vacuo. + +The drying may also be accomplished by using a flat-bottom dish +containing some absorbent, such as sand, pumice stone, asbestos or +crysolite. The milk may also be absorbed by a dried paper coil and +dried thereon (=26=). + +It is convenient to determine the water in the sample subsequently +to be used for the gravimetric determination of the fat, and this is +secured by the adoption of the paper coil method, as suggested by the +author, or by the use of a perforated metal tube containing porous +asbestos, as proposed by Babcock.[423] + +The process is conveniently carried out as follows: + +Provide a hollow cylinder of perforated sheet metal sixty millimeters +long and twenty millimeters in diameter, closed five millimeters from +one end by a disk of the same material. The perforations should be +about 0.7 millimeter in diameter and as close together as possible. +Fill loosely with from one and a half to two and a half grams of dry +woolly asbestos and weigh. Introduce a weighed quantity of milk (about +five grams). Dry at 100° for four hours. During the first part of the +drying the door of the oven should be left partly open to allow escape +of moisture. Cool in a desiccator and weigh. Repeat the drying until +the weight remains constant. Place in an extractor and treat with +anhydrous ether for two hours. Evaporate the ether and dry the fat at +100°. The extracted fat is weighed and the number thus obtained may be +checked by drying and weighing the cylinder containing the residue. + +The asbestos best suited for use in this process should be of a woolly +nature, quite absorbent, and, previous to use, be ignited to free it +of moisture and organic matter. A variety of serpentine, crysolite is +sometimes used instead of asbestos. When the content of water alone is +desired, it is accurately determined by drying in vacuo over pumice +stone (page 33). + +The methods above mentioned are typical and will prove a sufficient +guide for conducting the desiccation, either as described or by any +modification of the methods which may be preferred. + +=448. Calculation of Total Solids.=—By reason of the ease and +celerity with which the density of a milk and its content of fat +can be obtained, analysts have found it convenient to calculate the +percentage of total solids instead of determining it directly. This +is accomplished by arbitrary formulas based on the data of numerous +analyses. These formulas give satisfactory results when the samples do +not vary widely from the normal and may be used with advantage in most +cases. + +Among the earliest formulas for the calculation may be mentioned those +of Fleischmann and Morgen,[424] Behrend and Morgen,[425] Claus, Stutzer +and Meyer,[426] Hehner,[427] and Hehner and Richmond.[428] Without doing +more than citing these papers it will be sufficient here to give the +formulas as corrected by the most recent experience. + +In the formula worked out by Babcock the specific gravity of the sample +is represented by _S_, the fat by _F_, and the solids not fat by _t_. +The formula is written as follows:[429] + + 100_S_ - _FS_ + _t_ = ( ----------------- - 1)(250 - 2.5 _F_). + 100 - 1.0753_FS_ + +In this formula it is assumed that the difference between the specific +gravity of the milk serum and that of water is directly proportional +to the per cent of solids in the serum, but this assumption is not +strictly correct. Even in extreme cases, however, the error does not +amount to more than 0.05 per cent. + +Since a given amount of milk sugar increases the density of a milk +more than the same quantity of casein, it is evident that the formula +would not apply to those instances in which the ratio between these two +ingredients is greatly disturbed, as for instance, the whey. + +The formula of Hehner and Richmond, in its latest form, is expressed as +follows: + _G_ + _T_ = 0.2625 ---- + 1.2_F_, + _D_ + +in which _T_ represents the total solids, _G_ the reading of the +quévenne lactometer, _D_ the specific gravity, and _F_ the fat. + +_Example._—Let the reading of the lactometer be 31, corresponding to +_D_ 1.031, and the percentage of fat be three and five-tenths, what is +the percentage of the total solids? + +Substituting these values in the formulas we have + + 31 + _T_ = 0.2625 ----- + 1.2 × 3.5 = 12.09. + 1.031 + +To simplify the calculations, Richmond’s formula may be written + + _G_ 6_F_ + _T_ = ----- + ----- + 0.14. + 4 5 + +Calculated by this shortened formula from the above data _T_ = 12.09, +the same as given in the larger formula. + +Calculating the solids not fat in the hypothetical case given above by +Babcock’s formula, we get _t_ = 8.46, and this plus 3.5 gives 11.96, +which is slightly lower than the number obtained by the richmond +process. + +The babcock formula may be simplified by substituting the number +expressing the reading of the quévenne lactometer for that donating the +specific gravity, in other words, the specific gravity multiplied by +100 and the quotient diminished by 1000. + +The formulas for solids not fat and total solids then become + + _L_ _L_ + _t_ = ---- + 0.2_F_, and _T_ = ----- + 1.2_F_, + 4 4 + +in which _L_ represents the reading of the lactometer. By the addition +of the constant factor 0.14 the results calculated by the formula of +Babcock are the same as those obtained by the method of Richmond. + +In the following table are given the solids not fat in milks as +calculated by Babcock’s formula. To obtain the total solids add the +per cent of fat to solids not fat. To obtain total solids according to +Richmond’s formula increase that number by 0.14. + + TABLE SHOWING PER CENT OF SOLIDS NOT FAT IN MILK CORRESPONDING + TO QUÉVENNE’S LACTOMETER READINGS AND PER CENT OF FAT. + + Per + cent Lactometer reading at 60° F. + of + fat. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. + + 0.0 6.50 6.75 7.00 7.25 7.50 7.75 8.00 8.25 8.50 8.75 9.00 + 0.1 6.52 6.77 7.02 7.27 7.52 7.77 8.02 8.27 8.52 8.77 9.02 + 0.2 6.54 6.79 7.04 7.29 7.54 7.79 8.04 8.29 8.54 8.79 9.04 + 0.3 6.56 6.81 7.06 7.31 7.56 7.81 8.06 8.31 8.56 8.81 9.06 + 0.4 6.58 6.83 7.08 7.33 7.58 7.83 8.08 8.33 8.58 8.83 9.08 + 0.5 6.60 6.85 7.10 7.35 7.60 7.85 8.10 8.35 8.60 8.85 9.10 + 0.6 6.62 6.87 7.12 7.37 7.62 7.87 8.12 8.37 8.62 8.87 9.12 + 0.7 6.64 6.89 7.14 7.39 7.64 7.89 8.14 8.39 8.64 8.89 9.14 + 0.8 6.66 6.91 7.16 7.41 7.66 7.91 8.16 8.41 8.66 8.91 9.16 + 0.9 6.68 6.93 7.18 7.43 7.68 7.93 8.18 8.43 8.68 8.93 9.18 + 1.0 6.70 6.95 7.20 7.45 7.70 7.95 8.20 8.45 8.70 8.95 9.20 + 1.1 6.72 6.97 7.22 7.47 7.72 7.97 8.22 8.47 8.72 8.97 9.22 + 1.2 6.74 6.99 7.24 7.49 7.74 7.99 8.24 8.49 8.74 8.99 9.24 + 1.3 6.76 7.01 7.26 7.51 7.76 8.01 8.26 8.51 8.76 9.01 9.26 + 1.4 6.78 7.03 7.28 7.53 7.78 8.03 8.28 8.53 8.78 9.03 9.28 + 1.5 6.80 7.05 7.30 7.55 7.80 8.05 8.30 8.55 8.80 9.05 9.30 + 1.6 6.82 7.07 7.32 7.57 7.82 8.07 8.32 8.57 8.82 9.07 9.32 + 1.7 6.84 7.09 7.34 7.59 7.84 8.09 8.34 8.59 8.84 9.09 9.34 + 1.8 6.86 7.11 7.36 7.61 7.86 8.11 8.36 8.61 8.86 9.11 9.37 + 1.9 6.88 7.13 7.38 7.63 7.88 8.13 8.38 8.63 8.88 9.14 9.39 + 2.0 6.90 7.15 7.40 7.65 7.90 8.15 8.40 8.66 8.91 9.16 9.41 + 2.1 6.92 7.17 7.42 7.67 7.92 8.17 8.42 8.68 8.93 9.18 9.43 + 2.2 6.94 7.19 7.44 7.69 7.94 8.19 8.44 8.70 8.95 9.20 9.45 + 2.3 6.96 7.21 7.46 7.71 7.96 8.21 8.46 8.72 8.97 9.22 9.47 + 2.4 6.98 7.23 7.48 7.73 7.98 8.23 8.48 8.74 8.99 9.24 9.49 + 2.5 7.00 7.25 7.50 7.75 8.00 8.25 8.50 8.76 9.01 9.26 9.51 + 2.6 7.02 7.27 7.52 7.77 8.02 8.27 8.52 8.78 9.03 9.28 9.53 + 2.7 7.04 7.29 7.54 7.79 8.04 8.29 8.54 8.80 9.05 9.30 9.55 + 2.8 7.06 7.31 7.56 7.81 8.06 8.31 8.57 8.82 9.07 9.32 9.57 + 2.9 7.08 7.33 7.58 7.83 8.08 8.33 8.59 8.84 9.09 9.34 9.59 + 3.0 7.10 7.35 7.60 7.85 8.10 8.36 8.61 8.86 9.11 9.36 9.61 + 3.1 7.12 7.37 7.62 7.87 8.13 8.38 8.63 8.88 9.13 9.38 9.64 + 3.2 7.14 7.39 7.64 7.89 8.15 8.40 8.65 8.90 9.15 9.41 9.66 + 3.3 7.16 7.41 7.66 7.92 8.17 8.42 8.67 8.92 9.18 9.43 9.68 + 3.4 7.18 7.43 7.69 7.94 8.19 8.44 8.69 8.94 9.20 9.45 9.70 + 3.5 7.20 7.45 7.71 7.96 8.21 8.46 8.71 8.96 9.22 9.47 9.72 + 3.6 7.22 7.48 7.73 7.98 8.23 8.48 8.73 8.98 9.24 9.49 9.74 + 3.7 7.24 7.50 7.75 8.00 8.25 8.50 8.75 9.00 9.26 9.51 9.76 + 3.8 7.26 7.52 7.77 8.02 8.27 8.52 8.77 9.02 9.28 9.53 9.78 + 3.9 7.28 7.54 7.79 8.04 8.29 8.54 8.79 9.04 9.30 9.55 9.80 + 4.0 7.30 7.56 7.81 8.06 8.31 8.56 8.81 9.06 9.32 9.57 9.83 + 4.1 7.32 7.58 7.83 8.08 8.33 8.58 8.83 9.08 9.34 9.59 9.85 + 4.2 7.34 7.60 7.85 8.10 8.35 8.60 8.85 9.11 9.36 9.62 9.87 + 4.3 7.36 7.62 7.87 8.12 8.37 8.62 8.88 9.13 9.38 9.64 9.89 + 4.4 7.38 7.64 7.89 8.14 8.39 8.64 8.90 9.15 9.40 9.66 9.91 + 4.5 7.40 7.66 7.91 8.16 8.41 8.66 8.92 9.17 9.42 9.68 9.93 + 4.6 7.43 7.68 7.93 8.18 8.43 8.68 8.94 9.19 9.44 9.70 9.95 + 4.7 7.45 7.70 7.95 8.20 8.45 8.70 8.96 9.21 9.46 9.72 9.97 + 4.8 7.47 7.72 7.97 8.22 8.47 8.72 8.98 9.23 9.48 9.74 9.99 + 4.9 7.49 7.74 7.99 8.24 8.49 8.74 9.00 9.25 9.50 9.76 10.01 + 5.0 7.51 7.76 8.01 8.26 8.51 8.76 9.02 9.27 9.52 9.78 10.03 + 5.1 7.53 7.78 8.03 8.28 8.53 8.79 9.04 9.29 9.54 9.80 10.05 + 5.2 7.55 7.80 8.05 8.30 8.55 8.81 9.06 9.31 9.56 9.82 10.07 + 5.3 7.57 7.82 8.07 8.32 8.57 8.83 9.08 9.33 9.58 9.84 10.09 + 5.4 7.59 7.84 8.09 8.34 8.60 8.85 9.10 9.36 9.61 9.86 10.11 + 5.5 7.61 7.86 8.11 8.36 8.62 8.87 9.12 9.38 9.63 9.88 10.13 + 5.6 7.63 7.88 8.13 8.39 8.64 8.89 9.15 9.40 9.65 9.90 10.15 + 5.7 7.65 7.90 8.15 8.41 8.66 8.91 9.17 9.42 9.67 9.92 10.17 + 5.8 7.67 7.92 8.17 8.43 8.68 8.94 9.19 9.44 9.69 9.94 10.19 + 5.9 7.69 7.94 8.20 8.45 8.70 8.96 9.21 9.46 9.71 9.96 10.22 + 6.0 7.71 7.96 8.22 8.47 8.72 8.98 9.23 9.48 9.73 9.98 10.24 + +=449. Determination of Ash.=—In the determination of the solid residue +obtained by drying milk, it is important to observe the directions +already given (=28-32=). + +In the direct ignition of the sample, a portion of the sulfur and +phosphorus may escape oxidation and be lost as volatile compounds. +This loss may be avoided by the use of proper oxidizing agents or by +conducting the combustion as heretofore described.[430] In the official +method, it is directed to add six cubic centimeters of nitric acid to +twenty of milk, evaporate to dryness and ignite the residue at a low +red heat until free of carbon.[431] It is doubtful if this precaution +be entirely sufficient to save all the sulfur and phosphorus, but +the method is evidently more reliable than the common one of direct +ignition without any oxidizing reagent whatever. + + +ESTIMATION OF FAT. + +=450. Form of Fat in Milk.=—The fat in milk occurs in the form of +globules suspended in the liquid, in other words in the form of an +emulsion. Many authorities have asserted that each globule of fat is +contained in a haptogenic membrane composed presumably of nitrogenous +matter, but there is no convincing evidence of the truth of this +opinion. The weight of experimental evidence is in the opposite +direction. The supposed action of the membrane and the phenomena +produced thereby are more easily explained by the surface tension +existing between the fat globules and the menstruum in which they are +suspended. + +Babcock affirms that the spontaneous coagulation of the fibrin present +in milk tends to draw the fat globules into clusters, and this tendency +can be arrested by adding a little soda or potash lye to the milk as +soon as it is drawn.[432] + +The diameter of the fat globules is extremely variable, extending in +some cases from two to twenty micromillimeters. In cow milk, the usual +diameters are from three to five micromillimeters. + +=451. Number of Fat Globules in Milk.=—The number of fat globules in +milk depends on their size and the percentage of fat. It is evident +that no definite statement of the number can be made. There is a +tendency, on the part of the globules, to diminish in size and increase +in number as the period of lactation is prolonged. To avoid large +numbers, it is convenient to give the number of globules in 0.0001 +cubic millimeter. This number may be found within wide limits depending +on the individual, race, food and other local conditions to which the +animal or herd is subjected. In general, in whole milk this number will +be found between 140 and 250. + +=452. Method of Counting Globules.=—The number of globules in milk is +computed with the aid of the microscope. The most convenient method +is the one devised by Babcock.[433] In carrying out this computation, +capillary tubes, from two to three centimeters long and about one-tenth +millimeter in internal diameter, are provided. The exact diameter of +each tube, in at least three points, is determined by the micrometer +attachment of the microscope, and from these measurements the mean +diameter of the tube is calculated. This known, its cubic content +for any given length is easily computed. Ten cubic centimeters of +the milk are diluted with distilled water to half a liter and one +end of a capillary tube dipped therein. The tube is quickly filled +with diluted milk and each end is closed with a little wax to prevent +evaporation. Several of these tubes being thus prepared, they are +placed in a horizontal position on the stage of the microscope and +covered with glycerol and a cover glass. The tubes are left at rest +for some time until all the fat globules have attached themselves +to the upper surfaces, in which position they are easily counted. +The micrometer is so placed as to lie parallel with the tubes, and +the number of globules, corresponding to each division of its scale, +counted. The mean number of globules corresponding to each division of +the micrometer scale is thus determined. + +To compare the data obtained with each tube they are reduced to a +common basis of the number of globules found in a length of fifty +divisions of the micrometer scale in a tube having a diameter of 100 +divisions, using the formula + + 10000_n_ + _N_ = ---------, + _d_² + +in which _n_ = the number of globules found in the standard length of +tube measured and _d_ = the diameter of the tube. It is not difficult +to actually count all the globules in a length of fifty divisions of +the scale, but the computation may also be made from the mean numbers +found in a few divisions. The usual number of globules found in a +length of 0.1 millimeter in a tube 0.1 millimeter in diameter, varies +from fifty to one hundred. + +_Example._—The length of one division of the micrometer scale is 0.002 +millimeter, and the internal diameter of the tube 0.1 millimeter. The +content of a tube, of a length of 0.002 × 50 = 0.1 millimeter, is +therefore 0.0007854 cubic millimeter. + +The cubic content of a tube 100 scale divisions in diameter and fifty +in length is 0.0031416 cubic millimeter. The number of globules found +in fifty divisions of the tubes used is 40. Then the number which would +be contained in a tube of a diameter of 100 divisions of the micrometer +scale and a length of fifty divisions thereof is + + 10000_n_ 400000 + _N_ = -------- = ------ = 160. + _d_² 2500 + +Since the milk is diluted fifty times, the actual number of globules +corresponding to the volume given is 8000. It is convenient to reduce +the observations to some definite volume, _exempla gratia_, 0.0001 +cubic centimeter. The equation for this in the above instance is +0.0031416: 0.0001 = 8000: _x_, whence _x_ = 223, = number of fat +globules in 0.0001 cubic millimeter. + +In one cubic millimeter of milk there are therefore 2,230,000 fat +globules, and in one cubic centimeter 2,230,000,000 globules. In a +single drop of milk there are from one to two hundred million fat +globules.[434] + +=453. Classification of Methods of Determining Fat in Milk.=—The fat, +being the most valuable of the constituents of milk, is the subject +of a number of analytical processes. An effort will be made here to +classify these various methods and to illustrate each class with one +or more typical processes. In general the methods may be divided into +analytical and commercial, those of the first class being used for +scientific and of the other for trade purposes. For normal milk, some +of the trade methods have proved to be quite as accurate as the more +chronokleptic analytical processes to which, in disputed cases, a final +appeal must be taken. When the analyst is called upon to determine the +fat in a large number of samples of milk some one of the trade methods +may often be adopted with great advantage. + +=454. Dry Extraction Methods.=—Among the oldest and most reliable +methods of determining fat in milk, are included those processes based +on the principle of drying the milk and extracting the fat from the +residue by an appropriate solvent. The solvents generally employed are +ether and petroleum spirit of low boiling point. The methods of drying +are legion. + +In extracting with ether, it must not be forgotten that other bodies +than fat may pass into solution on the one hand and on the other any +substituted glycerid, such as lecithin or nuclein, which may be present +may escape solution, at least in part. Perhaps petroleum spirit, +boiling at from 45° to 60°, is the best solvent for fat, but it is +almost the universal custom in this and other countries to use ether. + +=455. The Official Methods.=—In the methods adopted by the Association +of Official Agricultural Chemists two processes are recommended. + +(1) _The Asbestos Process_: In this process it is directed to extract +the residue from the determination of water by the asbestos method +(=447=) with anhydrous pure ether until the fat is removed, evaporate +the ether, dry the fat at 100° and weigh. The fat may also be +determined by difference, after drying the extracted cylinders at 100°. + +(2) _Paper Coil Method_: This is essentially the method proposed by +Adams as modified by the author.[435] Coils made of thick filter paper +are cut into strips 6.25 by 62.5 centimeters, thoroughly extracted with +ether and alcohol, or the weight of the extract corrected by a constant +obtained for the paper. If this latter method be used, a small amount +of anhydrous sodium carbonate should be added. Paper free of matters +soluble in ether is also to be had for this purpose. From a weighing +bottle about five grams of milk are transferred to the coil by a +pipette, taking care to keep dry the lower end of the coil. The coil, +dry end down, is placed on a piece of glass, and dried at a temperature +of boiling water for one hour, or better, dried in hydrogen at a +temperature of boiling water, transferred to an extraction apparatus +and extracted with absolute ether or petroleum spirit boiling at about +45°. The extracted fat is dried in hydrogen and weighed. Experience +has shown that drying in hydrogen is not necessary. The fat may be +conveniently dried in partial vacuo. + +=456. Variations of Extraction Method.=—The method of preparing the +milk for fat extraction is capable of many variations. Some of the most +important follow: + +(1) _Evaporation on Sand_: The sand should be pure, dry and of uniform +size of grain. It may be held in a dish or tube. The dish may be made +of tin foil, so that it can be introduced with its contents into the +extraction apparatus after the desiccation is complete. For this +purpose, it is cut into fragments of convenient size after its contents +have been poured into the extractor. The scissors used are washed with +the solvent. + +(2) _Evaporation on Kieselguhr_: Dry kieselguhr (infusorial earth, +tripoli) may take the place of the sand as above noted. The +manipulation is the same as with sand. + +(3) _Evaporation on Plaster of Paris_,[436] (_Soxhlet Method_),[437] (4) +_On Pumice Stone_, (5) _On Powdered Glass_, (6) _On Chrysolite_:[438] +The manipulation in these cases is conducted as with sand and no +detailed description is required. + +(7) _Evaporation on Organic Substances_: These variations would fall +under the general heading of drying on paper. The following materials +have been used; _viz._, sponge,[439] lint,[440] and wood pulp.[441] In +these variations the principal precautions to be observed are to secure +the organic material in a dry state and free of any matter soluble in +the solvent used. + +(8) _Dehydration with Anhydrous Copper Sulfate_: In this process the +water of the sample is absorbed by powdered anhydrous copper sulfate, +the residual mass extracted and the butter fat obtained determined by +saponification and titration.[442] In the manipulation about twenty +grams of the anhydrous copper sulfate are placed in a mortar, a +depression made therein in such a manner that ten cubic centimeters of +milk can be poured into it without wetting it through to the mortar. +The water is soon absorbed when the mass is ground with a little dry +sand and transferred to the extractor. + +Petroleum spirit of low boiling point is used as a solvent, successive +portions of about fifteen cubic centimeters each being forced through +the powdered mass under pressure. Two or three treatments with the +petroleum are required. The residual butter fat, after the evaporation +of the petroleum, is saponified with a measured portion, about +twenty-five cubic centimeters, of seminormal alcoholic potash lye. The +residual alkali is determined by titration with seminormal hydrochloric +acid in the usual manner. From the data obtained is calculated the +quantity of alkali employed in the saponification. The weight of butter +fat extracted is then calculated on the assumption that 230 milligrams +of potash are required to saponify one gram of the fat. + +=457. Gypsum Method for Sour Milk.=—In sour milk, extraction of the +dry residue with ether is attended with danger of securing a part of +the free lactic acid in the extract. This may be avoided, at least +in part, by making the milk neutral or slightly alkaline before +desiccation. This method is illustrated by a variation of Soxhlet’s +method of drying on gypsum proposed by Kühn.[443] The curdled milk is +treated with potash lye of forty per cent strength until the reaction +is slightly alkaline. For absorbing the sample before drying, a mixture +is employed consisting of twenty-five grams of plaster of paris, four +of precipitated carbonate of lime and two of acid potassium sulfate. To +this mixture ten grams of the milk, rendered alkaline as above noted, +are added in a desiccating dish, the excess of moisture evaporated at +100°, the residual mass finely ground and extracted with ether for +four hours. A little gypsum may be found in the solution, but in such +small quantities as not to interfere seriously with the accuracy of the +results obtained. + +=458. Estimation of Fat in Altered Milk.=—In altered milk the lactose +has usually undergone a fermentation affording considerable quantities +of lactic acid. If such milks be treated by the extraction method for +fat, the results will always be too high, because of the solubility of +lactic acid in ether. + +Vizern[444] has proposed to avoid this error by first warming the soured +milk for a few minutes to 40°, at which temperature the clabber is +easily divided by vigorous shaking. Of the milk thus prepared, thirty +grams are diluted with two or three volumes of water and poured onto a +smooth and moistened filter. The vessel and filter are washed several +times until the filtrate presents no further acid reaction. The filter +and its contents are next placed in a vessel containing some fine +washed sand. A small quantity of water is added, sufficient to form a +paste. With a stirring rod, the filter is entirely broken up and the +whole mass thoroughly mixed. Dried on the water bath the material is +subjected to extraction in the ordinary way. Several analyses made +on fresh milk and on milk kept for several months show that almost +identical results are obtained. + +In respect of this process there would be danger, on long standing, +of the formation of free acids from butter glycerids, and these acids +would be removed by the process of washing prescribed. In this case the +quantity of fat obtained would be less than in the original sample. + +=459. Comparison of Methods.=—An immense amount of work has been done +by analysts in comparing the various types of extraction methods +outlined above.[445] + +The consensus of opinion is that good results are obtained by all the +methods when properly conducted, and preference is given to the two +methods finally adopted by the Association of Official Chemists. As +solvents, pure ether and petroleum spirit of low boiling point are +preferred. The direct extraction gravimetric processes are important, +since it is to these that all the other quicker and easier methods must +appeal for the proof of their accuracy. + +=460. Wet Extraction Methods.=—It has been found quite impracticable to +extract the fat from milk by shaking it directly with the solvent. An +emulsion is produced whereby the solvent itself becomes incorporated +with the other constituents of the milk, and from which it is not +separated easily even with the aid of whirling. The disturbing element +which prevents the separation of the solvent is doubtless the colloid +casein, since, when this is previously rendered soluble, the separation +of the solvent holding the fat is easily accomplished. + +The principle on which the methods of wet extraction are based is a +simple one; _viz._, to secure a complete or partial solution of the +casein and subsequently to extract the fat with a solvent immiscible +with water. The methods may be divided into three great classes; +_viz._, (1) those in which the solvent is evaporated from the whole of +the extracted fat and the residual matters weighed; (2) processes in +which an aliquot part of the fat solution is employed and the total fat +calculated from the data secured; (3) the density of the fat solution +is determined at a definite temperature and the percentage of fat +corresponding thereto determined from tables or otherwise. Methods (1) +and (2) are practically identical in principle and one or the other may +be applied according to convenience or to local considerations. The +methods may be further subdivided in respect of the reagents used to +secure complete or partial solution of the casein, as, for instance, +alkali or acid.[446] + +=461. Solution in an Acid.=—A good type of these processes is the +method of Schmid.[447] In this process ten cubic centimeters of milk +are placed in a test tube of about five times that content, graduated +to measure small volumes. An equal quantity of hydrochloric acid is +added, the mixture shaken, boiled until it turns dark brown, and cooled +quickly. The fat is extracted by shaking with thirty cubic centimeters +of ether. After standing some time the ethereal solution separates and +its volume is noted. An aliquot part of the solution is removed, the +solvent evaporated, and the weight of fat in the whole determined by +calculation. + +The schmid process has been improved by Stokes,[448] Hill,[449] and +Richmond.[450] The most important of these variations consists in +weighing instead of measuring the milk employed, thus insuring greater +accuracy. Dyer and Roberts affirm that the ether dissolves some of the +caramel products formed on boiling condensed milk with hydrochloric +acid, and that the data obtained in such cases by the process of Schmid +are too high.[451] + +Since lactic acid is also slightly soluble in ether, sour milk should +not be extracted with that solvent. In these cases petroleum spirit, +or a mixture of petroleum and ether, as suggested by Pinette, may be +used.[452] Another variation consists in extracting the fat with several +portions of the solvent and evaporating all the extracts thus obtained +to get the total fat. This method is perhaps the best of those in which +the fat is extracted from the residual liquid after the decomposition +of the casein by an acid, and may be recommended as both reliable and +typical within the limitations mentioned above. + +=462. Solution in an Alkali.=—The casein of milk is not so readily +dissolved in an alkali as in an acid, but the solution is sufficient +to permit the extraction of the fat. Soda and potash lyes and ammonia +are the alkaline bodies usually employed. To promote the separation +of the emulsions, alcohol is added with advantage. The principle of +the process rests on the observed power of an alkali to free the fat +globules sufficiently to allow them to dissolve in ether or some other +solvent. When the solvent has separated from the emulsion at first +formed, the whole or a part of it is used for the determination of fat +in a manner entirely analogous to that employed in the process with the +acid solutions described above. There are many methods based on this +principle, and some of the typical ones will be given below. Experience +has shown that extraction from an alkaline solution is more troublesome +and less perfect than from an acid and these alkaline methods are, +therefore, not so much practiced now as they were formerly. + +=463. Method of Short.=—Instead of measuring the volume of the +separated fat, Short has proposed a method in which the casein is +dissolved in an alkali and the fat at the same time saponified. The +soap thus produced is decomposed by sulfuric acid and the volume of the +separated fat acids noted. This volume represents eighty-seven per cent +of the corresponding volume of fat.[453] + +The solvent employed is a mixture of sodium and potassium hydroxids, +containing in one liter 125 and 150 grams, respectively, of these +alkalies. The sample of milk is mixed with half its volume of the +reagent and placed in boiling water for two hours. By this treatment +the casein is dissolved and the fat saponified. After cooling to about +60°, the soap is decomposed by the addition of equal parts of sulfuric +and acetic acids. The tubes containing the mixture are again placed in +boiling water for an hour and they are then filled with boiling water +to within one inch of the top. The tubes may either be furnished with a +graduation or the column of fat be measured by a scale. + +=464. Method of Thörner.=—The process of Short is conducted by Thörner +as follows:[454] + +Ten cubic centimeters of milk measured at 15° are saponified, in +tubes fitting a centrifugal, by the addition of one and a half cubic +centimeters of an alcoholic potash lye, containing 160 grams of +potassium hydroxid per liter, or one cubic centimeter of an aqueous +fifty per cent soda lye. The saponification is hastened by setting the +tubes in boiling water, where they remain for two minutes. The soap +formed is decomposed with a strong acid, sulfuric preferred, the tubes +placed in the centrifugal and whirled for four minutes, when the fat +acids will be formed in the narrow graduated part of the tube and the +volume occupied thereby is noted after immersion in boiling water. +Thörner’s process is not followed in this country, but is used to a +considerable extent in Germany.[455] + +=465. Liebermann’s Method.=—In this method, fifty cubic centimeters +of milk, at ordinary temperatures, are placed in a glass cylinder +twenty-five centimeters high and about four and a half internal +diameter; five cubic centimeters of potash lye of 1.27 specific gravity +are added, the cylinder closed with a well fitting cork stopper and +thoroughly shaken.[456] After shaking, fifty cubic centimeters of +petroleum spirit, boiling point about 60°, are added. The cylinder is +again stoppered and vigorously shaken until an emulsion is formed. +To this emulsion fifty cubic centimeters of alcohol of ninety-five +per cent strength are added, and the whole again thoroughly shaken. +After four or five minutes the petroleum spirit, containing the fat, +separates. In order to insure an absolute separation of the fat, +however, the shaking may be repeated three or four times for about +one-quarter minute, waiting each time between the shakings until the +spirit separates. + +Of the separated petroleum spirit twenty cubic centimeters are placed +in a small weighed flask. The use of the flask is recommended on +account of the ease with which the petroleum spirit can be evaporated +without danger of loss of fat. Instead of the flask a weighed beaker or +other weighed dish may be employed. + +The petroleum spirit is carefully evaporated on a water-bath and the +residue dried at 110° to 120° for one hour. The weight found multiplied +by five gives the content of fat in 100 cubic centimeters of the +milk. The percentage by weight can then be calculated by taking into +consideration the specific gravity of the milk employed. + +The results obtained by this method agree well with those obtained by +the paper coil method, when petroleum spirit instead of sulfuric ether +is used as the solvent for the fat. Sulfuric ether, however, gives +an apparently higher content of fat because of the solution of other +bodies not fat present in the milk. + +=466. Densimetric Methods.=—Instead of evaporating the separated fat +solution and weighing the residue, its density may be determined and +the percentage of fat dissolved therein obtained by calculation, or +more conveniently from tables. The typical method of this kind is due +to Soxhlet, and until the introduction of modern rapid volumetric +processes, it was used perhaps more extensively than any other +proceeding for the determination of fat in milk.[457] The reagents +employed in the process are ether saturated with water and a potash lye +containing 400 grams of potash in a liter. The principle of the process +is based on the assumption that a milk made alkaline with potash will +give up all its fat when shaken with ether and the quantity of fat in +solution can be determined by ascertaining the specific gravity of the +ethereal solution. + +[Illustration: FIG. 108. AREOMETRIC FAT APPARATUS.] + +The apparatus is arranged as shown in Fig. 108, whereby it is easy to +drive the ethereal fat solution into the measuring vessel by means of +the bellows shown. In the bottle, seen at the right of the engraving +are placed 200 cubic centimeters of milk, ten of the potash lye and +sixty of the aqueous ether. The milk and potash are first added and +well shaken, the ether then added, and the contents of the bottle are +shaken until a homogeneous emulsion is formed. The bottle is then set +aside for the separation of the ethereal solution, which is promoted +by gently jarring it from time to time. When the chief part of the +solution has separated, a sufficient quantity of it is driven over +into the measuring apparatus, by means of the air bulbs, to float the +hydrometer contained in the inner cylinder. After a few moments the +scale of the oleometer is read and the percentage of fat calculated +from the table. All the measurements are made at a temperature of +17°.5. The temperature is preserved constant by filling the outer +cylinder of the apparatus with water. If the room be warmer than 17°.5, +the water added should be at a temperature slightly below that and +_vice versa_. The oleometer carries a thermometer which indicates the +moment when the reading is to be made. + +The scale of the oleometer is graduated arbitrarily from 43 to 66, +corresponding to the specific gravities 0.743 and 0.766, respectively, +or to corresponding fat contents of 2.07 and 5.12 per cent, in the +milk, a range which covers most normal milks. + +In the use of the table the per cents corresponding to parts of an +oleometer division can be easily calculated. + + TABLE FOR CALCULATING PER CENT OF FAT IN MILK BY AREOMETRIC METHOD + OF SOXHLET. + + Reading of Per cent fat Reading of Per cent fat + oleometer. in milk. oleometer. in milk. + + 43 2.07 55 3.49 + 44 2.18 56 3.63 + 45 2.30 57 3.75 + 46 2.40 58 3.90 + 47 2.52 59 4.03 + 48 2.64 60 4.18 + 49 2.76 61 4.32 + 50 2.88 62 4.47 + 51 3.00 63 4.63 + 52 3.12 64 4.79 + 53 3.25 65 4.95 + 54 3.37 66 5.12 + +=467. Application of the Areometric Method.=—Soxhlet’s method, as +outlined above, with many modifications, has been extensively used +in Europe and to a limited degree in this country, and the results +obtained are in general satisfactory, when the sample is a mixed one +from a large number of cows and of average composition. + +The author has shown that the process is not applicable to abnormal +milk and often not to milk derived from one animal alone.[458] + +The chief difficulty is found in securing a separation of the emulsion. +This trouble can usually be readily overcome by whirling. Any +centrifugal machine, which can receive the bottle in which the emulsion +is made, may be employed for that purpose. + +Since the introduction of more modern and convenient methods of fat +determination, the areometric method has fallen into disuse and perhaps +is no longer practiced in this country. It is valuable now chiefly from +the fact that many of the recorded analyses of milk fat were made by +it, and also for its typical character in representing all methods of +analysis of fat in milk based on the density of ethereal solutions. + +=468. The Lactobutyrometer.=—A typical instrument for measuring the +volume of fat in a milk is known as Marchand’s lactobutyrometer. It +is based on the observation that ether will dissolve the fat from +milk when the casein is wholly or partly dissolved by an alkali, and +further, that the fat in an impure form can be separated from its +ethereal solution by the action of alcohol. Experience has shown +that all the fat is not separated from the ethereal solution by this +process, and also that the part separated is a saturated solution in +ether. The method cannot be rigorously placed in the two classes given +above, but being volumetric demands consideration here chiefly because +of its historical interest.[459] + +The instrument employed by Marchand is a tube about thirty centimeters +long and twelve in diameter, closed at one end and marked in three +portions of ten cubic centimeters each. The upper part is divided in +tenths of a cubic centimeter. The superior divisions are subdivided so +that the readings can be made to hundredths of a cubic centimeter. + +The tube is filled with milk to the first mark and two or three drops +of a twenty-five per cent solution of soda lye added thereto. Ether is +poured in to the second mark, the tube closed and vigorously shaken. +Alcohol of about ninety per cent strength is added to the upper mark, +the tube closed, shaken and allowed to stand in a vertical position, +with occasional jolting, until the separation of the liquids is +complete. In order to promote the separation the tube is placed in a +cylinder containing water at 40°. + +When the separation is complete the milk serum is found at the bottom, +the mixture of alcohol and ether in the middle and the fat at the top. +The mixture of ether and alcohol contains 0.126 gram of fat, and each +cubic centimeter of the separated ether fat 0.233 gram of fat. The +total volume of the separated fat, multiplied by 0.233, and the product +increased by 0.126, will give the weight of fat in the ten cubic +centimeters of milk employed. + +_Example._—Milk used, ten cubic centimeters of 1.032 specific gravity += 10.32 grams. The observed volume of the saturated ether fat solution +is two cubic centimeters. Then the weight of fat is 2 × 0.233 + 0.126 = +0.592 gram. The percentage of fat in the sample is 0.592 × 100 ÷ 10.32 += 5.74. + +In the apparatus used in this laboratory the upper division of the +graduation is marked 12.6, because this represents the quantity of fat +which remains in the ether-alcohol mixture for one liter of milk. From +this point the graduation is extended downward to ninety-five, which, +for ten cubic centimeters of milk, represents 0.95 gram. After the fat +has separated, enough ninety-five per cent alcohol is added to bring +the upper surface exactly to the graduation 12.6. The number of grams +per liter of milk is then read directly from the scale. + +In respect of applicability, the observation made regarding Soxhlet’s +areometric method may be repeated. + +In practical work in this country the lactobutyrometer is no longer +used, but many of the recorded determinations of fat in milk have been +made by this method. + +=469. Volumetric Methods.=—For practical purposes, the volumetric +methods of estimating fat in milk have entirely superseded all the +other processes. It has been found that the fat readily separates in a +pure state from the other constituents of milk whenever the casein is +rendered completely soluble; whereas no process has yet been devised +whereby the fat can be easily separated in a pure state from milk +which has not been treated with some reagent capable of effecting a +solution of the casein. The volumetric methods may be divided into +two classes; _viz._, (1) Those in which the fat is separated by the +simple action of gravity, and (2) those in which the natural action +of gravity is supplanted by centrifugal motion. Each of these classes +embraces a large number of variations and some of the typical ones will +be described in the following paragraphs. As solvents for the casein a +large number of reagents has been used, including alkalies and single +and mixed acids. In practice, preference is given to the least complex +and most easily prepared solvents. + +=470. Method Of Patrick.=—A typical illustration of the method of +collecting the fat after solution of the casein, without the aid of +whirling, is found in the process devised by Patrick.[460] + +The solvent employed is a mixture of acetic, sulfuric and hydrochloric +acids, saturated with sodium sulfate, in the respective volumetric +proportions of nine, five and two. The separation is accomplished in a +large test tube drawn out near the top into a constricted neck which is +graduated to measure the volume of the separated fat or to give direct +percentage results. + +The tube should have a content of about twenty-five cubic centimeters +below the upper mark on the neck. In use 10.4 cubic centimeters of +milk and a sufficient quantity of the mixed acids to fill it nearly +to the upper mark are placed in the tube, together with a piece of +pumice stone, and the mixture boiled. On cooling below 100°, the fat +will separate and the volume thereof may be measured in the constricted +portion of the tube. The volume of the fat may be converted into weight +on multiplying by 0.88 at 60°, or more conveniently the percentage +of fat be taken from a table. In practice, the tube is filled with +the milk and acid mixture nearly up to the neck, its contents well +mixed and additional acid mixture added until the liquid is raised +in the tube above the neck. After mixing a second time, the contents +are boiled for five minutes and the fat allowed to collect in the +expanded part of the tube above the neck. When the fat has collected, +the mixture is boiled gently a second time for a few minutes. By this +treatment the fat is mixed with the upper portions of the acid liquid +and clarified. The clearing of the fat may be hastened by sprinkling +over it a little effloresced sodium sulfate. The fat is brought into +the graduated neck by opening a small orifice in the belly of the tube, +which is closed by means of a rubber band. When the temperature has +reached 60°, the space occupied by the fat is noted and the numbers +obtained express the percentage of fat in the sample. + +This process is illustrative of the principle of analysis, but is no +longer used in analytical determinations. + +=471. The Lactocrite.=—One of the earliest methods for fat estimation +in milk, depending on the solution of the casein and the collection +of the fat by means of whirling, is based on the use of a centrifugal +machine known as the lactocrite. This apparatus is modeled very like +the machine usually employed for creamery work,[461] and at one time +was extensively used, but it has now given place to less troublesome +and expensive machines. The acid mixture for freeing the fat of casein +is composed of glacial acetic acid carrying five per cent of sulfuric. +The samples of milk are heated with the acid mixture in test tubes +provided with stoppers and short glass tubes to return the condensation +products. The hot mixture is poured into a small metallic cylindrical +cup holding about three cubic centimeters. This cup fits by means of +an accurately ground shoulder on a metal casing, carrying inside a +heavy glass graduated tube of small internal diameter. The excess of +the milk mixture escapes through a small aperture in the metallic screw +cap of the metal holder. The metal holder is cut away on both sides in +order to expose the graduations on the glass tube. The glass tube is +held water-tight by means of perforated elastic washers. Thus prepared +the tubes are inserted in the radial holes of a revolving steel disk +previously heated to a temperature of 60°. The whirling is accomplished +in a few minutes by imparting to the steel disk a speed of about 6,000 +revolutions per minute. At the end of this operation the fat is found +in a clear column in the small glass tube and the number of the +divisions it occupies in this tube is noted. Each division of the scale +represents one-tenth per cent of fat. + +This apparatus is capable of giving accurate results when all its parts +are in good working order. In this laboratory the chief difficulty +which its use has presented is in keeping the joints in the glass metal +tube tight. + +This description of the apparatus is given to secure an illustration of +the principle involved, a principle which has been worked out in later +times into some of the most rapid and practical processes of estimating +fat in milk. + +=472. Modification of Lindström.=—Many modifications have been proposed +for conducting the determination of fat by means of the lactocrite, +but they do not involve any new principle and are of doubtful merit. +In the modification suggested by Lindström, which has attained quite +an extended practical application, the solvent mixture is composed of +lactic and sulfuric acids and the butyrometer tubes are so changed +as to permit the collection of the fat in the graduated neck after +whirling, by means of adding water. The apparatus is also adjusted +to secure the congelation of the fat column before its volume is +noted.[462] The analyst can read the fat volume at his leisure when it +is in the solid state and is not confused by changes of volume during +the observation. The best acid mixture has been found to be composed of +100 volumes of lactic, an equal amount of acetic and fifteen volumes of +sulfuric acids.[463] + +[Illustration: FIG. 109. BABCOCK’S BUTYROMETER AND ACID MEASURE.] + +=473. The Babcock Method.=—Among the many quick volumetric methods +which have been proposed for the determination of fat in milk, none has +secured so wide an application as that suggested by Babcock.[464] + +The chief point of advantage in the use of this method is found in +effecting the solution of the casein by means of sulfuric acid of about +1.83 specific gravity. By this reagent the casein is dissolved in a few +moments without the aid of any other heat than that generated by mixing +the milk with the reagent. The bottle in which the separation is made +is shown in Fig. 109. The graduations on the neck are based on the use +of eighteen grams of milk. To avoid the trouble of weighing, the milk +is measured from a pipette graduated to deliver eighteen grams of milk +of the usual specific gravity. While it is true that normal milk may +vary somewhat in its density, it has been found that a pipette marked +at 17.6 cubic centimeters delivers a weight which can be safely assumed +to vary but slightly from the one desired. The graduated bottle holds +easily thirty-five cubic centimeters of liquid in its expanded portion +and the volume of milk just noted is mixed with an equal volume of +sulfuric acid, conveniently measured from the lip cylinder shown in the +figure. The complete mixture of the milk and acid is effected by gently +rotating the bottle until its contents are homogeneous. The final color +of the mixture varies from dark brown to black. + +While still hot, the bottles are placed in a centrifugal machine and +whirled for at least five minutes. The most convenient machine, where +it is available, is the one driven by a jet of steam. The revolutions +of the centrifugal should be at least 700 per minute for a twenty inch +and 1,200 for a twelve inch wheel. After five minutes the bottles are +removed and filled to the upper mark or nearly so with hot water, +replaced in the machine and whirled for at least one minute. The fat +will then be found in a clearly defined column in the graduated neck of +the bottle. In reading the scale, the extreme limits between the lowest +point marked by the lower meniscus and the highest point marked by the +edge of the upper meniscus are to be regarded as the termini of the fat +column. + +In testing cream by the babcock process, it may either be diluted +until the column of fat secured is contained in the graduated part of +the neck or specially graduated bottles may be used. + +_Condensed Milk_: In applying the babcock test to condensed milk, it is +necessary to weigh the sample and to use only about eight grams.[465] +This quantity is placed in the bottle and dissolved in ten cubic +centimeters of water and the analysis completed as above. The reading +noted is multiplied by eighteen and divided by the weight of the sample +taken. + +_Skim Milk_: In determining the fat in skim milk and whey, it is +desirable to use a bottle of double the usual capacity, but with the +same graduation on the neck. The percentage of fat noted is divided by +two. + +_Cheese_: Five grams are a convenient quantity of cheese to employ. +To this quantity in the bottle are added fifteen cubic centimeters +of hot water and the flask gently shaken and warmed until the cheese +is softened. The treatment with acid and whirling are the same as +described above. The noted reading is multiplied by eighteen and +divided by five. + +=474. Solution in Amyl Alcohol and Hydrochloric and Sulfuric +Acids.=—Leffmann and Beam have proposed to aid the solution of the +casein in sulfuric acid by the previous addition to the milk of a +mixture of equal volumes of amyl alcohol and hydrochloric acid.[466] +In this process the same graduated flasks may be used as in the +babcock process, or a special flask may be employed. In this case the +graduation of the neck is for fifteen cubic centimeters of milk, and +each one and a half cubic centimeters is divided into eighty-six parts. +The quantity of milk noted is placed in the flask, together with three +cubic centimeters of the mixture of amyl alcohol and hydrochloric acid, +and well shaken. To the mixture, sulfuric acid of 1.83 specific gravity +is added until the belly of the flask is nearly full and the contents +well mixed by shaking. When the casein is dissolved, the addition of +the sulfuric acid is continued until the flask is filled to the upper +mark and again the contents mixed. It is well to close the mouth of +the flask with a stopper while shaking. The bottle is placed in a +centrifugal and whirled for a few moments, when the fat is collected in +the graduated neck and its volume noted. + +The process is also known in this country as the beimling method.[467] +The fat separated in the above process is probably mixed with a little +fusel oil, and therefore it is advisable to use the specially graduated +bottle instead of one marked in absolute volumes.[468] + +The method, when conducted according to the details found in the +papers cited, gives accurate results, but is somewhat more complicated +than the babcock process and is not now used to any great extent in +analytical work. + +=475. Method of Gerber.=—The method proposed by Gerber for estimating +fat in milk is based on the processes of Babcock, Beimling and Beam +already described. The tubes in which the decomposition of the milk and +the measurement of the fat are accomplished are of two kinds, one open +at only one end for milk and the other open at both ends for cheese. +They are closed during the separation by rubber stoppers.[469] + +[Illustration: FIG. 110. GERBER’S BUTYROMETERS.] + +The apparatus have been greatly improved and simplified since the first +description of them was published and have come into extensive use in +Europe and to a limited extent in this country.[470] + +The butyrometer tubes are made of various sizes and shapes, but the +most convenient are those noted above as shown in Fig. 110. + +Before adding the strong sulfuric acid, one cubic centimeter of amyl +alcohol is mixed with the milk in the butyrometer. This admixture +serves to clarify the fat and render the reading more easy. + +The centrifugal is run by hand, and the required speed of rotation is +given it by means of a cord wrapped spirally about its axis, as shown +in Fig. 111. The cord in the new machines is replaced by a leather +strap working on a ratchet. + +[Illustration: FIG. 111. GERBER’S CENTRIFUGAL.] + +The process is more speedy than that of Babcock, and the results have +been shown by a large experience to be reliable and accurate. + +The sulfuric acid employed is of 1.825 to 1.830 specific gravity. There +is no danger of loss by the formation of volatile ethers where the +quantity of amyl alcohol used does not exceed one cubic centimeter. +In a comparison of the respective merits of the methods of Babcock, +Thörner and Gerber, made by Hausamann, the first place is awarded to +the Gerber process.[471] In the figure 110, the butyrometers marked 2, +5 and 8 are for milk, and those numbered 1, 3 and 7 are for cream +and cheese. In conducting the analysis, ten cubic centimeters of the +sulfuric acid are placed in the butyrometer with one cubic centimeter +of the amyl alcohol. When mixed, eleven cubic centimeters of the +milk are added and the contents of the tube well mixed, the tube +stoppered and placed in the centrifugal. The larger tubes, open at both +ends, require double the quantities of the reagents mentioned. The +measurements are made at about 15°. + +Minute directions for conducting the analyses with milk, skim milk, +buttermilk, cream, condensed milk, cheese and butter accompany each +apparatus. + + +PROTEID BODIES IN MILK. + +=476. Kinds of Proteid Bodies in Milk.=—The proteid bodies in milk +are all found in at least partial solution. Some authorities state +that a portion of the casein is present in the form of fine particles +suspended after the manner of the fat globules.[472] The number and +kind of proteid bodies are not known with definiteness. Among those +which are known with certainty are casein, albumin, peptone and fibrin. +The latter body was discovered in milk by Babcock.[473] Lactoglobulin +and lactoprotein are also names given to imperfectly known proteid +bodies in milk. Lactoprotein is not precipitated either by acids or +by heat and is therefore probably a peptone. By far the greater part +of the proteid matters in milk is casein. Casein has been called +caseinogen by Halliburton,[474] and paracasein by Schulze and Röse.[475] +Casein has intimate relations to the mineral matters in milk, and +is probably itself made up of several proteid bodies of slightly +differing properties. In general all that class of proteid matter +contained in milk which is precipitated by rennet or a weak acid, or +spontaneously on the development of lactic acid, is designated by the +term casein, while the albumins and peptones in similar conditions +remain in solution. Casein contains phosphorus, presumably as nuclein. +Fibrin is recognized in milk by the reactions it gives with hydrogen +peroxid or gum guiacum. The decomposition of hydrogen peroxid is not +a certain test for fibrin, inasmuch as pus and many other bodies will +produce the same effect. If the milk decompose hydrogen peroxid, +however, before and not after boiling, an additional proof of the +presence of fibrin is obtained, since boiled fibrin does not act on +the reagent.[476] The gum guiacum test is applied by dipping a strip of +filter paper into the milk and drying. The solution of gum guiacum is +applied to the dried paper and the presence of fibrin is recognized +by the blue color which is produced. The fibrin is probably changed +into some other proteid during the ripening of cream in which the +fibrin is chiefly found. The albumin in milk is coagulated by boiling, +while the casein remains practically unaffected when subjected to that +temperature. + +=477. Estimation of Total Proteid Matter.=—The total proteid matter +in milk is determined by any of the general methods applicable to the +estimation of total nitrogen, but the moist combustion method is by far +the most convenient. From the total nitrogen, that which represents +ammonia or other nonproteid nitrogenous bodies, is to be deducted +and the remainder multiplied by an appropriate factor. Practically +all the nitrogen obtained is derived from the proteid matters and, +as a rule, no correction is necessary. The factors employed for +calculating the weight of proteid matter from the nitrogen obtained +vary from 6.25 to 7.04. It is desirable that additional investigations +be made to determine the magnitude of this factor. It is suggested +that provisionally the factor 6.40 be used. In the method adopted by +the Association of Official Agricultural Chemists it is directed that +about five grams of milk be placed in the oxidizing flask and treated +without previous evaporation exactly as described for the estimation +of total nitrogen in the absence of nitrates. The nitrogen obtained is +multiplied by 6.25 to get the total proteid matter.[477] In order to +prevent the too great dilution of the sulfuric acid, the milk may be +evaporated to dryness or nearly so before oxidation. In this laboratory +it is conveniently done by placing the milk first in the oxidizing +flask, connecting this with the vacuum service and placing the flask in +hot water. The aqueous contents of the milk are quickly given off at +a temperature not exceeding 85°, and the time required is only a few +minutes. + +The milk may also be dried in dishes made of thin glass or tin foil +and, after desiccation, introduced with the fragments of the dishes +into the oxidizing flask. + +The preliminary drying in the oxidizing flask is recommended as the +best. + +Söldner oxidizes the nitrogen in human milk by boiling ten cubic +centimeters thereof for three hours with twenty-five of sulfuric +acid, fifty milligrams of copper oxid and three drops of a four per +cent platinic chlorid solution, and, after distilling the ammonia, +uses the factor 6.39 for calculating the proteid matter. According to +this author human milk is much less rich in nitrogenous constituents +than is generally supposed, containing not more than one and a half +per cent thereof in average samples collected at least a month after +parturition.[478] + +=478. Precipitation of Total Proteids with Copper Sulfate.=—This method +of throwing out the total proteids of milk is due to Ritthausen.[479] +The proteids and fat are precipitated together by the addition of a +measured volume of copper sulfate solution, containing 63.92 grams +of the crystallized salt in one liter. The process, as modified by +Pfeiffer, is conducted as follows:[480] + +Ten grams of milk are diluted with ten times that much water, five +cubic centimeters of the copper sulfate solution added and then soda +lye solution drop by drop until the copper is just precipitated. This +is determined by testing a few drops of the filtrate with soda lye, +which, when the copper is precipitated, will give neither a turbidity +nor a blue color. + +The mixture is poured into a dry tared filter, the precipitate washed +with hot water, dried to constant weight and weighed. The fat is +removed from the dry pulverized mass by extraction with ether and the +residue dried and weighed. + +The quantity of copper oxyhydrate contained in the precipitate is +calculated from the quantity of the copper solution used and amounts +to 0.2026 gram. The casein thus prepared contains not only the copper +compound named, but also some of the sodium sulfate formed on the +addition of the soda lye and other mineral salts present in the milk +and from which it is quite impossible to completely free it. There are +also many other objections to the process, and the product is of such a +nature as to render the data obtained by the method very doubtful. + +This method is chiefly valuable on account of its historical interest. +Not only are the drying and weighing of the precipitate rendered +unnecessary by the modern methods of determining nitrogen, but there +are numerous sources of error which seem to throw doubt on the accuracy +of the results. The copper hydroxid does not lose all its water even on +drying at 125°.[481] The method therefore can only be recommended for +practical purposes when all the tedious processes of drying, extracting +and calculating the quantity of copper oxid are abandoned and the moist +washed precipitate used directly for the determination of nitrogen. + +=479. Proteid Bodies by Ammonium Sulfate.=—All the proteid bodies +except peptones are precipitated from milk on saturation with ammonium +sulfate. This method has little analytical value because of the +presence of nitrogenous salt in the precipitate. Zinc sulfate may be +substituted for the ammonium salt and thus a determination of proteid +matter other than peptone be obtained. This result subtracted from the +total proteid nitrogen gives that due to peptone. + +=480. Total Proteid Matter by Tannic Acid.=—For the determination +of the total proteid matter in milk Sebelien uses the following +process.[482] From three to five grams are diluted with three or four +volumes of water, a few drops of a saline solution added (sodium +phosphate, sodium chlorid, magnesium sulfate, _et similia_), and the +proteid bodies thrown out with an excess of tannic acid solution. +The precipitate is washed with an excess of the precipitant and the +nitrogen therein determined and multiplied by 6.37. + +=481. Separation of Casein from Albumin.=—Sebelien prefers magnesium +sulfate or sodium chlorid to acetic acid as the best reagent for +separating casein from lactalbumin. Of the two saline reagents +mentioned, the former is the better. The milk is first diluted +with a double volume of the saturated saline solution and then the +fine powdered salt added until saturation is secured. The casein +is completely thrown out by this treatment, collected on a filter, +washed with the saturated saline solution, and the nitrogen therein +determined. The difference between the total and casein nitrogen gives +the quantity due to the albumin plus the almost negligible quantity due +to globulin.[483] + +=482. Van Slyke’s Method of Estimating Casein.=—The casein may be +separated from the other albuminoids in milk by the procedure proposed +by Van Slyke.[484] Ten grams of the fresh milk are diluted with ninety +cubic centimeters of water and the temperature raised to 40°. The +casein is thrown down with a ten per cent solution of acetic acid, of +which about one and a half cubic centimeters are required. The mixture +is well stirred and the precipitate allowed to subside. The whey is +decanted onto a filter, and the precipitate washed two or three times +with cold water, brought finally onto the filter and washed once or +twice with cold water. The filter paper and its contents are used +for the determination of nitrogen in the usual way. The casein is +calculated from the nitrogen found by multiplication by the factor +6.25. Milk may be preserved for this method of determination by adding +to it one part of finely powdered mercuric chlorid for each two +thousand parts of the sample. The method is not applicable to curdled +milk. + +=483. Theory of Precipitation.=—Most authorities now agree in supposing +that the state of semisolution in which the casein is held in milk +is secured by the presence of mineral matters in the milk, in some +intimate combination with the casein. Among these bodies lime is of +the most importance. The action of the dilute acid is chiefly on these +mineral bodies, releasing them from combination with the casein, which, +being insoluble in the milk serum, is precipitated. + +=484. Factors for Calculation.=—Most analysts still use the common +proteid factor, 6.25, in calculating the quantity of proteids from the +nitrogen determined by analysis. For casein many different factors have +been proposed. According to Makeris the factor varies from 6.83 to +7.04.[485] Munk gives 6.34 for human and 6.37 for cow milk.[486] Sebelien +adopts the latter factor, and Hammersten nearly the same; _viz._, +6.39. The weight of authority, at the present time, favors a factor +considerably above 6.25 for calculating the casein and, in fact, the +total proteids of milk from the weight of nitrogen obtained. + +=485. Béchamp’s Method of Preparing Pure Casein.=—The casein in about +one liter of milk is precipitated by adding gradually about three +grams of glacial acetic acid diluted with water. The addition of the +acid is arrested at the moment when litmus paper shows a slightly acid +reaction. The precipitate thus produced, containing all the casein, +the milk globules and the microzymes, is separated by filtration, +being washed by decantation before collecting it on the filter. On +the filter it is washed with distilled water and the fat removed by +shaking with ether. The residue is suspended in water, dissolved in the +least possible quantity of ammonium carbonate, any insoluble residue +(microzymes, globules) separated by filtration and the pure casein +thrown out of the filtrate by the addition of acetic acid. The washing +with distilled water, solution in ammonium carbonate, filtration and +reprecipitation are repeated three or four times in order to obtain +the casein entirely free of other substances. Casein thus prepared is +burned to a carbon free ash with difficulty and contains but little +over one-tenth per cent of mineral matter.[487] + +=486. Separation of Casein with Carbon Dioxid.=—The supersaturation of +the lime compounds of casein with carbon dioxid diminishes the solvent +action of the lime and thus helps to throw out the proteid matter. For +this reason Hoppe-Seyler recommends the use of carbon dioxid to promote +the precipitation of the casein.[488] The milk is diluted with about +twenty volumes of water and treated, drop by drop, with very dilute +acetic acid as long as a precipitate is formed. A stream of pure carbon +dioxid is conducted through the mixture for half an hour, and it is +allowed to remain at rest for twelve hours, when the casein will have +all gone down and the supernatant liquid will be clear. Albumins and +peptones are not thrown out by this treatment. + +The method of precipitation is advantageously modified by saturating +the diluted milk with carbon dioxid before adding the acetic acid, less +of the latter being required when used in the order just noted.[489] + +=487. Separation of Albumin.=—In the filtrate from the casein +precipitate the albumin may be separated by heating to 80°. It may also +be precipitated by tannic acid, in which case it may contain a little +globulin. It may also be thrown out by saturation with ammonium or +zinc sulfates. The latter reagent is to be preferred when the nitrogen +is to be determined in the precipitate. The quantities of albumin and +globulin, especially the latter, present in milk are small compared +with its content of casein. + +=488. Separation of Globulin.=—The presence of globulin in milk +is demonstrated by Sebelien in the following manner:[490] The milk +is saturated with finely powdered common salt and the precipitate +produced is separated by filtration. This filtrate in turn is +saturated with magnesium sulfate. The precipitate produced by this +reagent is collected on a filter, dissolved in water and precipitated +by saturation with sodium chlorid. This process is repeated several +times. The final precipitate is proved to be globulin by the following +reactions: When a solution of it is dialyzed the proteid body +separates as a flocculent precipitate, which is easily dissolved in +a weak solution of common salt. The clear solution thus obtained +becomes turbid on adding water, and more so after the addition of a +little acetic acid. A neutral solution of the body is also completely +precipitated by saturation with sodium chlorid. These reactions serve +to identify the body as a globulin and not an albumin. All the globulin +in milk is not obtained by the process, since a part of it is separated +with the casein in the first precipitation. + +=489. Other Precipitants of Milk Proteids.=—Many other reagents besides +those mentioned have been used for precipitating milk proteids, wholly +or in part. Among these may be mentioned the dilute mineral acids, +lactic acid, rennet, mercuric iodid in acetic acid, phosphotungstic +acid, acid mercuric nitrate, lead acetate and many others. + +It has been shown by the author that many of these precipitants do not +remove all the nitrogen but that among others the mercury salts are +effective.[491] When nitrogen is to be subsequently determined the acid +mercuric nitrate cannot be employed. + +=490. Precipitation by Dialysis.=—Since the casein is supposed to be +held in solution by the action of salts it is probable that it may be +precipitated by removing these salts by dialysis. + +=491. Carbohydrates in Milk.=—The methods of determining lactose in +milk, both by the copper reduction and optical processes, have been +fully set forth in foregoing paragraphs (=243, 244, 259, 262=). In +general, the optical method by double dilution is to be preferred +as practically exact and capable of application with the minimum +consumption of time.[492] For normal milks a single polarization is +entirely sufficient, making an arbitrary correction for the volume +occupied by the precipitated proteids and fat. This correction is +conveniently placed at six and a half per cent of the volume of milk +employed. + +The polarimetric estimation of lactose in human milk is likely to give +erroneous results by reason of the existence in the serum of polarizing +bodies not precipitable by the reagents commonly employed for the +removal of proteids.[493] The same statement may be made in respect of +ass and mare milk. The use of acetopicric acid for removing disturbing +bodies, as proposed by Thibonet[494] does not insure results free from +error. With the milks above mentioned, it is safer to rely on the data +obtained by the alkaline copper reagents. + +=492. Dextrinoid Body in Milk.=—In treating the precipitate, produced +in milk by copper sulfate, with alcohol and ether for the purpose +of removing the fat, Ritthausen isolated a dextrin like body quite +different from lactose in its properties.[495] The alcohol ether extract +evaporated to dryness leaves a mass not wholly soluble in ether, and +therefore not composed of fat. This residue extracted with ether, +presents flocky particles, soluble in water and mostly precipitated +therefrom by alcohol. This body has a slight reducing effect on +alkaline copper salts and produces a gray color with bismuth nitrate. +The quantity of this material is so minute as to lead Ritthausen to +observe that it does not sensibly affect the fat determinations when +not separated. It is not clearly demonstrated that it is a dextrinoid +body and the analyst need not fear that the optical determination of +milk sugar will be sensibly affected thereby. + +Raumer and Späth assume that certain discrepancies, observed by them +in the data obtained for lactose by the copper and optical methods, +are due to the presence of this dextrinoid body, but no positive proof +thereof is adduced.[496] + +=493. Amyloid Bodies in Milk.=—Herz has observed in milk a body +having some of the characteristics of starch.[497] Observed by the +microscope, these particles have some of the characteristics of the +starch grains of vegetables, with a diameter of from ten to thirty-five +micromillimeters. They are colored blue by iodin. When boiled with +water, however, these particles differ from starch in not forming +a paste. The particles are most abundant in the turbid layer found +immediately beneath the ether fat solution in the areometric process of +Soxhlet. + +The amyloid particles may be collected from cheese and butter by +boiling with water, when they settle and can be observed on the +sediment after freeing of fat by ether. + +Some of the statements regarding the adulteration of dairy products +with starch may have been made erroneously by reason of the natural +occurrence of these particles. + +As in the case of the dextrin like body mentioned above this starchy +substance, if it really exist, occurs in too minute a quantity to +influence the results of any of the analytical methods heretofore +described. + +In connection with the supposed presence of an amyloid body in milk, +it should be remembered that certain decomposition nitrogenous bodies +give practically the same reactions as are noted above.[498] Among these +may be mentioned chitin, which occurs very extensively in the animal +world. The proof of the existence of dextrinoid and amyloid bodies in +milk rests on evidence which should be thoroughly revised before being +undoubtedly accepted. + + +ANALYSIS OF BUTTER. + +=494. General Principles.=—The general analysis of butter fat is +conducted in accordance with the methods described in the part of this +volume devoted to the examination of fats and oils. The methods of +sampling, drying, filtering, and of determining physical and chemical +properties, are there developed in sufficient detail to guide the +analyst in all operations of a general nature. There remain for +consideration here only the special processes practiced in butter +analysis and which are not applied to fats in general. These processes +naturally relate to the study of those properties of a distinctive +nature, by means of which butter is differentiated from other fats for +which it may be mistaken or with which it may be adulterated. These +special studies, therefore, are directed chiefly to the consideration +of the peculiar physical properties of butter fat, to its content of +volatile acids and to its characteristic forms of crystallization as +observed with the aid of the microscope. For dietetic, economic and +legal reasons, it is highly important that the analyst be able to +distinguish a pure butter from any substitute therefor. + +=495. Appearance of Melted Butter.=—Fresh, pure butter, when slowly +melted, shows after a short time the butter fat completely separated, +of a delicate yellow color and quite transparent. Old samples of +butter do not give a fat layer of equal transparency. Oleomargarin, +or any artificial butter when similarly treated, gives a fat layer +opalescent or opaque. By means of this simple test an easy separation +of pure from adulterated butter may be effected. In mixtures, the +degree of turbidity shown by the separated fats may be regarded as a +rough index of the amount of adulteration. In conducting the work, +the samples of butter, in convenient quantities according to the size +of the containing vessel, are placed in beakers and warmed slowly at +a temperature not exceeding 50°. After a lapse of half an hour the +observations are made. + +If one part of the melted butter be shaken with two volumes of warm +water (40°) and set aside for five minutes the fat is still found as an +emulsion, while oleomargarin, similarly treated, shows the fat mostly +separated. This process has some merit, but must not be too highly +valued.[499] + +=496. Microscopic Examination of Butter.=—The microscope is helpful in +judging the purity of butter and the admixture of foreign fats, if not +in too small quantity to be of any commercial importance, can easily +be detected by this means.[500] The methods of preparing butter fat +in a crystalline state are the same as those described in paragraphs +=307-309=. The crystals of butter fat differ greatly in appearance +with the different methods of preparation. When butter is melted, +filtered, heated to the boiling point of water and slowly cooled, +it forms spheroidal crystalline masses as seen by the microscope, +which present a well defined cross with polarized light. This cross +is not peculiar to butter fat, but is developed therein with greater +distinctiveness than in other fats of animal origin. + +Pure, fresh, unmelted butter, when viewed with polarized light through +a plate of selenite, presents a field of vision of uniform tint, +varying with the relative positions of the nicols. When foreign fats, +previously melted, as in rendering, are mixed with the butter the +crystallization they undergo disturbs this uniformity of tint and the +field of vision appears particolored. Old, rancid or melted butter may +give rise to the same or similar phenomena under like conditions of +examination. The microscope thus becomes a most valuable instrument for +sorting butters and in distinguishing them in a preliminary way from +oleomargarin. + +[Illustration: FIG. 112. THERMOMETER FOR BUTYROREFRACTOMETER.] + +=497. Judgment of Suspected Butter or Lard by Refractive Power.=—In +discriminating between pure and adulterated butters by the aid of the +butyrorefractometer (=301=), the absolute reading of the instrument is +of less importance than the difference which is detected between the +highest permissible numbers, for any degree of temperature, and the +actual reading obtained at that temperature. These differences, within +certain limits, do not perceptibly vary with the temperature, and +heretofore they have been determined with the aid of a table, and in +this respect the observations have been made the more laborious. + +Wollny has rendered these tables unnecessary by constructing a +thermometer in which the mercury column does not indicate degrees of +temperature, but the highest permissible number for butter or lard +at the temperature of observation. The scale of the instrument is so +adjusted as to include temperatures of from 30° to 40°, which renders +it suited to the examination of butter and lard. The oleothermometer is +shown in Fig. 112. + +The side of the scale _B_ is for butter and that marked _S_ for lard. +The use of the instrument is the simplest possible. The sample of fat +is placed in the prisms in the usual manner. When the mercury in the +thermometer is at rest, the scale of the instrument is read. In the +case of a butter, if the reading of the scale give a higher number than +that indicated by the thermometer, the sample is pronounced suspicious +and the degree of suspicion is proportional to the difference of the +two readings. + +=498. Estimation of Water, Fat, Casein, Ash and Salt.=—The methods +proposed by the author for conducting these determinations, with +minor amendments, have been adopted by the Association of Official +Agricultural Chemists.[501] + +_Water._—The sample held in a flat bottom dish is dried to constant +weight at about 100°. The weight of the sample used should be +proportional to the area of the bottom of the dish, which should be +just covered by the film of melted fat. The dish may be previously +partly filled with sand, asbestos or pumice stone. The drying may take +place in the air, in an inert gas or in a vacuum. + +_Fat._—The fat in a sample of butter is readily determined by treating +the contents of the dish after the determination of water with an +appropriate solvent. + +The process is conducted as follows: + +The dry butter from the water determination is dissolved in the dish +with ether or petroleum spirit. The contents of the dish are then +transferred to a weighed gooch with the aid of a wash bottle containing +the solvent, and washed till free of fat. The crucible and contents are +heated at the temperature of boiling water till the weight is constant. +The weight of fat is calculated by difference from the data obtained. + +The fat may also be determined by drying the butter on asbestos or +sand, and subsequently extracting the fat by anhydrous alcohol free +ether. The extract, after evaporation of the ether, is dried to +constant weight at the temperature of boiling water and weighed. + +_Casein or Curd and Ash._—The crucible containing the residue from the +fat determination is covered and heated, gently at first, gradually +raising the temperature to just below redness. The cover may then be +removed and the heat continued till the contents of the crucible are +white. The loss in weight of the crucible and contents represents +casein or curd, and the residue is mineral matter or ash. + +_Salt._—It is the usual custom in the manufacture of butter in this +country to add, as a condiment, a certain proportion of salt. In +Europe, the butter offered for consumption is usually unsalted. A +convenient method of determining the quantity of salt is found in the +removal thereof, from the sample, by repeated washing with hot water +and in determining the salt in the wash water by precipitation with +silver nitrate. The operation is conducted as follows: From five to +ten grams of the sample are placed in a separatory funnel, hot water +added, the stopper inserted and the contents of the funnel well shaken. +After standing until the fat has all collected on top of the water, the +stopcock is opened and the water is allowed to run into an erlenmeyer, +being careful to let none of the fat globules pass. Hot water is again +added to the beaker, and the extraction is repeated several times, +using each time from ten to twenty cubic centimeters of water. The +resulting washings contain all but a mere trace of the sodium chlorid +originally present in the butter. The sodium chlorid is determined in +the filtrate by a set solution of silver nitrate, using a few drops of +a solution of potassium chromate as an indicator. + +It is evident that the quantity of salt may also be determined from the +ash or mineral matter obtained, as above noted, by the same process. +If desirable, which is rarely the case, the gravimetric method of +estimating the silver chlorid may be used. + +=499. Volatile or Soluble Acids.=—The distinguishing feature of butter, +from a chemical point of view, is found in its content of volatile or +soluble fat acids. Among the volatile acids are reckoned those which +are carried over in a current of steam at a temperature only slightly +higher than that of boiling water. As soluble acids are regarded +those which pass without great difficulty into solution in hot water. +These two classes are composed essentially of the same acids. Of these +butyric is the most important, followed by caproic, caprylic and capric +acids. Small quantities or rather traces of acetic, lauric, myristic +and arachidic acids are also sometimes found in butter. Palmitic, +stearic and oleic acids also occur in large quantities. The above named +acids, in combination with glycerol, form the butter fat. + +=500. Relative Proportion of Ingredients.=—The composition of butter +fat is given differently by different authorities.[502] A typical dry +butter fat may be regarded as having the following composition: + + Per cent. + Butyrin 7.00 + Caproin, Caprylin and Caprin 2.30 + Olein 37.70 + Palmitin, stearin, etc. 53.00 + +Pure butter fat consists principally of the above glycerids, some +coloring principles, varying in quantity and composition with the food +of the animal, and a trace of lecithin, cholesterol, phytosterol and a +lipochrome. + +=501. Estimation of Volatile or Soluble Acids.=—The volatile or soluble +acids in butter fat are estimated by the methods already described +(=349, 351=). In practice preference is given to the method of +determining volatile acids, based on the principle that under standard +conditions practically all the acids of this nature are secured in a +certain volume of the distillate. This assumption is not strictly true, +but the method offers a convenient and reliable manner of obtaining +results which, if not absolute, are at least comparative. + +The quantity of acid distilled is determined by titration with tenth +normal alkali and for convenience the data are expressed in terms of +the volume of the alkali consumed. Five grams of normal butter fat +will give a distillate, under the conditions given, requiring about +twenty-eight cubic centimeters of tenth normal alkali for complete +saturation. This is known as the reichert-meissl number. Occasionally +this number may rise to thirty-two or may sink to twenty-five. Cases +have been reported where it fell below the latter number, but such +samples cannot be regarded as normal butter. + +The determination of the reichert-meissl number is the most important +of the chemical processes applied to butter fat analysis. + +=502. Saponification Value and Reichert Number.=—It may often be +convenient to make the same sample of butter fat serve both for the +determination of the saponification value and of the reichert number. +For this purpose it is convenient to use exactly five grams of the +dry filtered fat. The saponification may be accomplished either under +pressure or by attaching a reflux condenser to the flask as suggested +by Bremer.[503] When the saponification, which is accomplished with +alcoholic potash lye containing about 1.25 grams in each ten cubic +centimeters of seventy per cent alcohol, is finished, and the contents +of the flask are cooled, the residual alkali is titrated with a set +sulfuric acid solution, using phenolphthalein as indicator. When the +color has almost disappeared, an additional quantity of the indicator +is added and the titration continued until the liquid is of an amber +tint. A sample of the alkali, treated as above, is titrated at the same +time and from the two sets of data obtained, the saponification number +is calculated as indicated in paragraph (=345=). + +A few drops of the alcoholic lye are added to the contents of the flask +and the alcohol removed by evaporation. The residual soap and potassium +sulfate are dissolved in 100 cubic centimeters of recently boiled +water, some pieces of pumice added, and the volatile acids removed by +distillation in the usual way after adding an excess of sulfuric acid. +It is important to conduct blank distillations in the same form of +apparatus to determine the magnitude of any corrections to be made. +The size of the distilling flask and the form of apparatus to prevent +mechanical projection of sulfuric acid into the distillate should be +the same in all cases. + +=503. Modification of the Reichert-Meissl Method.=—Kreis has proposed +the use of strong sulfuric acid for saponifying the fats, the +saponification and distillation being accomplished in one operation. +A source of error of some inconvenience in this method is due to the +development of sulfurous acid by the reducing action of the organic +matter on the oil of vitriol. Pinette proposes to avoid this difficulty +by adding, before the distillation is begun, sufficient potassium +permanganate to produce a permanent red coloration. By this means the +sulfurous acid is completely oxidized and its transfer to the standard +alkali during distillation entirely prevented. The same result is +accomplished by Micko by the use of potassium bichromate. The details +of the manipulation are as follows:[504] + +About five grams of the fused fat (butter or oleomargarin) are placed +in a flask of approximately 300 cubic centimeters capacity. After +cooling, there are added ten cubic centimeters of sulfuric acid +containing three grams of water to each ninety-seven grams of the +strongest acid. + +The fat and acid are well mixed by a gentle rotatory motion of the +flask and placed in a water bath at a temperature of 35° (_circa_) for +fifteen minutes. At the end of this time the flask is removed from +the bath and 125 cubic centimeters of water added, little by little, +keeping the contents cool. Next are added four cubic centimeters of a +four per cent solution of potassium bichromate. The contents of the +flask are vigorously shaken and, after five minutes, a solution of +ferrous sulfate is added gradually from a burette until the reaction +with a drop of potassium ferrocyanid shows a slight excess of the iron +salt. The volume of the liquor in the flask is then increased to 150 +cubic centimeters by the addition of water and 110 cubic centimeters +distilled. After mixing and filtering through a dry filter, the acid +in 100 cubic centimeters is determined by standard tenth normal barium +hydroxid solution and the number thus obtained increased by one-tenth +representing the total acid obtained. + +=504. Elimination of Sulfurous Acid.=—Prager and Stern[505] propose to +eliminate the sulfurous acid by a stream of air, succeeded by one of +carbon dioxid, and proceed as follows: Five grams of the butter fat are +brought into a liter flask, ten cubic centimeters of strong sulfuric +acid are added and the flask is kept for ten minutes at 30-32° with +constant agitation. When the liquid is cold, air is bubbled through +it until the odor of sulfurous acid has disappeared. One hundred +cubic centimeters of water are added, with precautions against rise +of temperature, and carbon dioxid is bubbled through for ten minutes. +This is then displaced by a stream of air for another ten minutes, the +delivery tube is washed into the flask with fifty cubic centimeters +of water and the distillation is effected. The following results are +quoted: + +Cubic centimeters of tenth normal alkali required by five grams of +butter fat: + + Reichert-Meissl. Prager-Stern. + Sample _a_ 29.86 29.60 + ” _b_ 30.23 29.65 + ” _c_ 28.34 27.76 + ” _d_ 28.20 28.10 + +The authors do not comment on the possibility of loss of acids +other than sulfurous in the stream of air, but they admit that +further investigation is requisite to render the suggestion of Kreis +serviceable. + +=505. Errors Due to Poor Glass.=—The easy solubility of the glass +holding the reagents is the cause of some of the difficulties attending +the determination of the saponification value. The separated silica +tends to carry down, mechanically, a part of the alkali. This is shown +by the fact that after the color has been discharged by titration +with acid and the flask set aside a reappearance of the red color is +noticed, after a time, beginning at the bottom of the flask.[506] In +order to avoid difficulties of this nature, either cold saponification +should be practiced or the digestion vessels used for moist combustion +in sulfuric acid be employed. + +Errors may also be easily introduced by the use of uncalibrated +burettes and from the employment of varying quantities of the +phenolphthalein solution. + +=506. Estimation of the Molecular Weight of Butter and Butter +Substitutes.=—Garelli and Carono have proposed a method for +discriminating between butter and its substitutes by the kryoskopic +determination of molecular weights. + +The molecular weights of stearin, palmitin and olein are 890, 806 +and 884, and of butyrin, caproin and caprylin 303, 386 and 470 +respectively. Pure butter, therefore, has a lower mean molecular weight +than margarin. + +The method and apparatus of Beckmann are used in the determination, +fifteen grams of benzol being employed as a solvent. + +The constant for the molecular depression of the benzol is found to be +53. + +The molecular weight obtained with samples of pure butter varied from +696 to 716, and for oleomargarin from 780 to 883. + +The figures obtained with mixtures of twenty, twenty-five, thirty-three +and fifty per cent of margarin with butter were 761, 720, 728 and 749 +respectively. The method can be relied upon to classify samples as +follows: + +1. Pure butter. + +2. Butter containing margarin. + +3. Suspicious butter.[507] + +=507. Substitutes and Adulterants of Butter.=—In this country, butter +is never adulterated with cocoa or sesame oil, as is sometimes the case +in other lands. The common substitute for butter here is oleomargarin, +and the most common butter adulterant, neutral lard. The methods of +analyses, by means of which these bodies can be identified, have +already been sufficiently described. By the use of certain digestive +ferments and other bodies, butter may be made to hold an excessive +quantity of casein, sugar and water in the form of a somewhat permanent +emulsion.[508] This form of adulteration is revealed at once on melting +the sample. + +=508. Furfurol Reaction with Sesame Oil.=—Olive oil and sometimes +butter are mixed with the cheaper body, sesame oil. The latter is +detected with certainty, from the red coloration it gives when mixed +with furfurol and hydrochloric acid. Instead of furfurol, some body +yielding it when subjected to the action of hydrochloric acid, _viz._, +sucrose or a pentose sugar, may be used. It has been found by Wauters, +however, that an alcoholic solution of two grams of furfuraldehyd in +100 cubic centimeters of alcohol is the best reagent. One-tenth of a +cubic centimeter of this reagent is used for each test.[509] + +The test is made as follows: The quantity of the furfuraldehyd solution +mentioned above is mixed with ten cubic centimeters of hydrochloric +acid, and there are added, without mixing, an equal volume of the +suspected oil. On standing, a red coloration is produced at the zone of +separation of the two liquids. If the oil be sesame, the coloration is +produced instantly. As little as one per cent of sesame in a mixed oil +will show the color in two minutes. The manipulation is also varied by +shaking together the reagents and the melted butter. Turmeric, which +is sometimes used in coloring butter, also gives the rose-red color +when treated with hydrochloric acid, but turmeric supplies its own +furfuraldehyd. It is easy to distinguish therefore the coloration due +to sesame oil, which is developed only when furfuraldehyd is present, +from that due to the turmeric, which is produced without the aid of the +special reagent. + +=509. Butter Colors.=—Where cows are deprived of green food and root +crops, such as carrots, and kept on a poorly balanced ration, the +butter made from their milk may be almost colorless. To remedy this +defect it is quite a common practice to color the product artificially. +Almost the sole coloring matter used in this country is anatto.[510] +Other coloring matters which are occasionally employed are turmeric, +saffron, marigold leaves, yellow wood (_Chlorophora tinctoria_), carrot +juice, chrome yellow (lead chromate) and dinitrocresol. + +The use of small quantities of anatto, turmeric or saffron is +unobjectionable, from a sanitary point of view, but this is not the +case with such a substance as lead chromate. The detection of anatto or +saffron in butter may be accomplished by the method of Cornwall.[511] +About five grams of the warm filtered fat are dissolved in about fifty +cubic centimeters of ordinary ether, in a wide tube, and the solution +is vigorously shaken for from ten to fifteen seconds, with from twelve +to fifteen cubic centimeters of a very dilute solution of caustic +potash or soda in water, only alkaline enough to give a distinct +reaction with turmeric paper, and to remain alkaline after separating +from the ethereal fat solution. The corked tube is set aside, and in +a few hours, at most, the greater part of the aqueous solution, now +colored more or less yellow by the anatto, can be drawn from beneath +the ether with a pipette or by a stopcock below, in a sufficiently +clear state to be evaporated to dryness and tested in the usual way +with a drop of concentrated sulfuric acid. + +Sometimes it is well to further purify the aqueous solution by shaking +it with some fresh ether before evaporating it, and any fat globules +that may float on its surface during evaporation should be removed by +touching them with a slip of filter paper; but the solution should not +be filtered, because the filter paper may retain much of the coloring +matter. + +The dry yellow or slightly orange residue turns blue or violet blue +with sulfuric acid, then quickly green, and finally brownish or +somewhat violet this final change being variable, according to the +purity of the extract. + +Saffron can be extracted in the same way; it differs from anatto very +decidedly, the most important difference being in the absence of the +green coloration. + +Genuine butter, free from foreign coloring matter, imparts at most a +very pale yellow color to the alkaline solution; but it is important +to note that a mere green coloration of the dry residue on addition +of sulfuric acid is not a certain indication of anatto (as some books +state) because the writer has thus obtained from genuine butter, +free from foreign coloring matter, a dirty green coloration, but not +preceded by any blue or violet-blue tint. + +Blank tests should be made with the ether. + +Turmeric is easily identified by the brownish to reddish stratum that +forms between the ethereal fat solution and the alkaline solution +before they are intimately mixed. It may be even better recognized by +carefully bringing a feebly alkaline solution of ammonia in alcohol +beneath the ethereal fat solution with a pipette, and gently agitating +the two, so as to mix them partially. + +Another method of separating artificial coloring matter has been +proposed by Martin.[512] + +A method of determining the relative amount of butter color has been +worked out by Babcock.[513] + + +EXAMINATION OF CHEESE. + +=510. Composition Of Cheese.=—Pure cheese is made from whole milk by +precipitating the casein with rennet. The precipitated casein carries +down also the fat of the milk and a little lactose and whey remain +incorporated with the cheesy mass. The ingredients of cheese are +therefore those of the whole milk less the greater part of the whey, +_id est_, milk sugar, lactalbumin, globulin, soluble mineral matters +and water. In the conversion of the crude precipitate noted above into +the cheese of commerce, it is subjected to a ripening process which is +chiefly conditioned by bacterial action. It is not possible here to +enter into a discussion of methods of isolating and identifying the +bacteria which promote or retard the ripening process.[514] As a rule, +about a month is required for the curing process, before the cheeses +are ready for boxing and shipment. The most important changes during +ripening take place in the proteid matter, which is so altered as to +become more palatable and more digestible as a result of the bacterial +activity. + +The percentage composition of the principal cheeses of commerce are +shown in the following table:[515] + + Water, Casein, Fat, Sugar, Ash, + Per cent. Per cent. Per cent. Per cent. Per cent. + Cheddar 34.38 26.38 32.71 2.95 3.58 + Cheshire 32.59 32.51 26.06 4.53 4.31 + Stilton 30.35 28.85 35.39 1.59 3.83 + Brie 50.35 17.18 25.12 1.94 5.41 + Neufchatel 44.47 14.60 33.70 4.24 2.99 + Roquefort 31.20 27.63 33.16 2.00 6.01 + Edam 36.28 24.06 30.26 4.60 4.90 + Swiss 35.80 24.44 37.40 2.36 + + Full cream, 38.60 25.35 30.25 2.03 4.07 + (mean of 143 analyses) + +It is evident that the composition of the cheese will vary with +the milk from which it is made and the manipulation to which it +is subjected. A good American green cheese made from milk of +the composition noted below will have the composition which is +appended.[516] + + TABLE SHOWING MEAN COMPOSITION OF + MILK AND CHEESE MADE THEREFROM. + Milk. Cheese. + Per cent. water 87.38 36.70 + ” ” fat 3.73 34.18 + ” ” proteids 3.13 23.44 + ” ” sugar, ash etc. 5.76 5.68 + +From the above it is seen that in full milk cheese the ratio of fat to +casein is 1.46: 1, and to solids not fat 1.17: 1. This is a point of +some importance in judging the purity of a cheese. When the full milk +of a mixed herd is used the percentage of fat in a cheese will always +be considerably higher than that of casein. + +=511. Manipulation of the Milk.=—When sweet milk is received at the +cheese factory, a starter of sour milk is added to it in order to +hasten its ripening. When it is thought that the proper degree of +acidity has been secured, it is subjected to a rennet test. In this +test 160 cubic centimeters of the milk are heated to 30° and mixed +with five cubic centimeters of the rennet solution made by diluting +five cubic centimeters of the rennet of commerce with fifty cubic +centimeters of water. The number of seconds required for the milk +to curdle is noted. The observation is facilitated by distributing +throughout the milk a few fine fragments of charcoal. The contents of +the vessel are given a circular motion and, at the moment of setting, +the movement of the black particles is suddenly arrested. If coloring +matter be added to the milk, it should be done before it becomes sour. +The quantity of rennet required is determined by the nature of the +cheese which it is desired to make. For a cheese to be rapidly cured, +enough rennet should be added to produce coagulation in from fifteen +to twenty minutes, and when slow curing is practiced in from thirty +to forty-five minutes. When the mass is solid so that it can be cut +with a knife, the temperature is raised to 37°, and it is tested on a +hot iron until it forms threads an eighth of an inch in length. This +test is made by applying an iron heated nearly to redness to the curd. +When the curd is in proper condition threads from a few millimeters to +two centimeters in length are formed, when the iron is withdrawn. The +longer threads indicate, but only to a limited extent, a higher degree +of acidity.[517] This test is usually made about two and one-half hours +from the time of coagulation. The whey is then drawn off through a +strainer and the curd is placed on racks with linen bottoms in order +that the residual whey may escape, the curd being stirred meanwhile. +In from fifteen to twenty minutes it can be cut into blocks eight or +ten inches square and turned over. This is repeated several times in +order to facilitate the escape of the whey. When the curd assumes a +stringy condition, it is run through a mill and cut into small bits and +is ready for salting, being cooled to 27° before the salt is added. +From two to three pounds of salt are used for each 100 pounds of curd. +The curd is then placed in the molds and pressed into the desired +form. The cheeses thus prepared are placed on shelves in the ripening +room and the rinds greased. They should be turned and rubbed every day +during the ripening, which takes place at a temperature of from 15° to +18°.[518] + +=512. Official Methods of Analysis.=—The methods of cheese analysis +recommended by the Association of Official Agricultural Chemists +are provisional and are not binding on its members. They are as +follows:[519] + +_Preparation of Sample._—Where the cheese can be cut, a narrow wedge +reaching from the edge to the center will more nearly represent the +average composition than any other sample. This should be chopped quite +fine, with care to avoid evaporation of water, and the several portions +for analysis taken from the mixed mass. When the sample is obtained +with a cheese trier, a plug perpendicular to the surface one-third of +the distance from the edge to the center of the cheese more nearly +represents the average composition than any other. The plug should +either reach entirely or half way through the cheese. For inspection +purposes the rind may be rejected, but for investigations where the +absolute quantity of fat in the cheese is required the rind should be +included in the sample. It is well, when admissible, to secure two or +three plugs on different sides of the cheese, and, after splitting them +lengthwise with a sharp knife, use portions of each for the different +determinations. + +_Determination of Water._—From five to ten grams of cheese are placed +in thin slices in a weighed platinum or porcelain dish which contains +a small quantity of freshly ignited asbestos to absorb the fat. The +dish is heated in a water oven for ten hours and weighed; the loss in +weight is considered as water. If preferred, the dish may be placed in +a desiccator over concentrated sulfuric acid and dried to constant +weight. In some cases this may require as much as two months. The acid +should be renewed when the cheese has become nearly dry. + +_Determination of Ether Extract._—Grind from five to ten grams of +cheese in a small mortar with about twice its weight of anhydrous +copper sulfate. The grinding should continue until the cheese is finely +pulverized and evenly distributed throughout the mass, which will have +a uniform light blue color. This mixture is transferred to a glass tube +having a strong filter paper, supported by a piece of muslin, tied over +one end. Put a little anhydrous copper sulfate into the tube next to +the filter before introducing the mixture containing the cheese. On top +of the mixture place a tuft of ignited asbestos, and place the tube in +a continuous extraction apparatus and treat with anhydrous ether for +fifteen hours. Dry the fat obtained at 100° to constant weight. + +_Determination of Nitrogen._—The nitrogen is determined by the kjeldahl +method, using about two grams of cheese, and multiplying the percentage +of nitrogen found by 6.25 for proteid compounds. + +_Determination of Ash._—The dry residue from the water determination +may be used for the ash determination. If the cheese be rich in fat, +the asbestos will be saturated therewith. This may be carefully ignited +and the fat allowed to burn, the asbestos acting as a wick. No extra +heat should be applied during this operation, as there is danger of +spurting. When the flame has died out, the burning may be completed in +a muffle at low redness. When desired, the salt may be determined in +the ash in the manner specified under butter (=498=). + +_Determination of Other Constituents._—The sum of the percentages of +the different constituents, determined as above, subtracted from 100 +will give the amount of organic acids, milk sugar etc., in the cheese. + +=513. Process of Mueller.=—The process of Müller,[520] as modified by +Kruger,[521] is conducted as follows: About ten grams of a good average +sample of cheese are rubbed in a porcelain mortar with a mixture of +three parts of alcohol and one part of ether. After the mixed liquids +have been in contact with the cheese five or ten minutes they are +poured upon a weighed filter of from fifteen to sixteen centimeters +diameter, and this process is repeated from one to three times, after +which the contents of the mortar are brought upon the filter. The +filtrate is received in a weighed flask, the alcohol ether driven off +by evaporation and the residue dried. Since it is difficult to get +all the particles of cheese free from the mortar, it is advisable to +perform the above process in a weighed dish which can afterwards be +washed thoroughly with ether and alcohol and dried and the amount of +matter remaining thereon accounted for. The residue remaining in the +flask after drying is treated several times with pure warm ether, +and the residue also remaining upon the filter mentioned above is +completely extracted with ether. The dried residue obtained in this +way from the filter plus the residue in the flask which received the +filtrate, plus the amount left upon the dish in which the cheese was +originally rubbed up, constitute the total dry matter of the cheese +freed of fat. All the material soluble in ether should be collected +together, dried and weighed as fat. + +By this process the cheesy mass is converted into a fine powder which +can be easily and completely freed from fat by ether, and can be dried +without becoming a gummy or horny mass. + +For the estimation of the nitrogen, about three grams of the well +grated cheese are used and the nitrogen determined by moist combustion +with sulfuric acid.[522] + +For the estimation of ash, about five grams are carbonized, extracted +with water, and the ash determined as described below.[523] + +Char from two to three grams of the substance and burn to whiteness +at the lowest possible red heat. If a white ash cannot be obtained in +this manner, exhaust the charred mass with water, collect the insoluble +residue on a filter, burn, add this ash to the residue from the +evaporation of the aqueous extract and heat the whole to a low redness +till the ash is white. + +=514. Separation of Fat from Cheese.=—It is often desirable to secure +a considerable quantity of the cheese fat for physical and chemical +examination without the necessity of effecting a complete quantitive +separation. In this laboratory this is accomplished by the method of +Henzold.[524] The cheese, in quantities of about 300 grams, is cut +into fragments about the size of a pea and treated with 700 cubic +centimeters of potash lye, which has previously been brought to a +temperature of about 20°. The strength of the lye should be such that +about fifty grams of the caustic potash are contained in each liter of +the solution. + +The treatment is conveniently conducted in a wide neck flask and the +solution of the casein is promoted by vigorous shaking. After from five +to ten minutes, it will be found that the casein is dissolved and the +fat is found swimming upon the surface of the solution in the form of +lumps. The lumps of fat are collected in as large a mass as possible by +a gentle shaking to and fro. Cold water is poured into the flask until +the fat is driven up into the neck, whence it is removed by means of a +spoon. + +The fat obtained in this way is washed a few times with as little cold +water as possible in order to remove the residue of potash lye which +it may contain. Experience shows that the fat by this treatment is +not perceptibly attacked by the potash lye. In a short time, by this +procedure, the fat is practically all separated and is then easily +prepared for chemical analysis by filtering and drying in the manner +already described (=283=). The fat may also be separated, but with less +convenience, by partially drying the sample, reducing it to a finely +divided state and applying any of the usual solvents. The solvent +is removed from the extract by evaporation and the residual fat is +filtered and prepared for examination as usual. + +=515. Filled Cheese.=—The skim milk coming from the separators is +unfortunately too often used for cheese making. The abstracted fat +is replaced with a cheaper one, usually lard. These spurious cheeses +are found in nearly every market and are generally sold as genuine. +The purchasers only discover the fraud when the cheese is consumed. +Many of the States have forbidden by statute the manufacture and sale +of this fraudulent article. Imported cheeses may also be regarded +with suspicion, inasmuch as the method of preparing filled cheese is +well known and extensively practiced abroad. A mere determination of +the percentage of fat in the sample is not an index of the purity of +the cheese. It is necessary to extract the fat by one of the methods +already described and, after drying and filtering, to submit the +suspected fat to a microscopic and chemical examination. A low content +of volatile fat acid and the occurrence of crystalline forms foreign to +butter will furnish the data for a competent judgment. + +When the reichert-meissl number falls below twenty-five the sample +may be regarded with suspicion. The detection of the characteristic +crystals of lard or tallow is reliable corroborating evidence (=308=). + +It is stated by Kühn[525] that the margarin factory of Mohr, at +Bahrenfeld-Altona, has made for many years a perfect emulsion of fat +with skim milk. This product has been much used in the manufacture of +filled cheese which is often found upon the German market. + +=516. Separation of the Nitrogenous Bodies in Cheese.=—The general +methods of separation already described for proteid bodies (=417-425=) +are also applicable to the different nitrogenous bodies present +in cheese, representing the residue of these bodies as originally +occurring in the milk, and also the products which are formed therefrom +during the period of ripening. For practical dietary and analytical +purposes, these bodies may be considered in three groups: + +(_a_) The useless (from a nutrient point of view) nitrogenous bodies, +including ammonia, nitric acid, the phenylamido-propionic acids, +tyrosin, leucin and other amid bodies. + +(_b_) The albumoses and peptones, products of fermentation soluble in +boiling water. + +(_c_) The caseins and albuminates, insoluble in boiling water. + +The group of bodies under (_a_), according to Stutzer, may be separated +from the groups (_b_) and (_c_) by means of phosphotungstic acid. For +this purpose a portion of an intimate mixture of fine sand and cheese +(100 cheese, 400 sand) corresponding to five grams of cheese, is +shaken for fifteen minutes with 150 cubic centimeters of water. After +remaining at rest for another fifteen minutes 100 cubic centimeters of +dilute sulfuric acid (one acid, three water) are added, followed by +treatment with the phosphotungstic acid as long as any precipitate is +produced. The mixture is thrown on a filter and the insoluble matters +washed with dilute sulfuric acid until the filtrate amounts to half +a liter. Of this quantity an aliquot part (200 cubic centimeters) is +used for the determination of nitrogen. From the quantity of nitrogen +found, that representing the ammonia, as determined in a separate +portion, is deducted and the remainder represents the nitrogen present +in the cheese as amids.[526] + +_Albumoses and Peptones._—Albumoses and peptones are determined in +cheese by the following method:[527] A quantity of the sand mixture +already described, corresponding to five grams of the cheese, is +treated with about 100 cubic centimeters of water, heated to boiling, +and the clear liquid above the sand poured into a flask of half a liter +capacity. The extraction is continued with successive portions of water +in like manner until the volume of the extract is nearly half a liter. +When cold, the volume of the extract is completed to half a liter with +water, the liquor filtered, 200 cubic centimeters of the filtrate +treated with an equal volume of dilute sulfuric acid (one to three) and +phosphotungstic acid added until no further precipitate takes place. +The nitrogen is determined in the precipitate after filtration and +washing with dilute sulfuric acid. + +_Casein and Albuminates._—The quantity of casein and albuminates in +cheese is calculated by subtracting from the total nitrogen that +corresponding to ammonia, amids, that in the indigestible residue +and that corresponding to the albumose and peptone. In three samples +of cheese, _viz._, camembert, swiss, and gervais, Stutzer found the +nitrogen, determined as above, distributed as follows:[528] + + Camembert. Swiss. Gervais. + N as ammonia 13.0 3.7 1.6 + N as amids 38.5 9.0 5.2 + N as albumose peptone 30.5 8.6 15.5 + N indigestible 4.0 2.4 8.6 + N as casein, albuminates 14.0 76.3 69.1 + +_Ammoniacal Nitrogen._—The ammoniacal nitrogen is determined by mixing +a quantity of the sand-cheese corresponding to five grams of cheese, +with 200 cubic centimeters of water, adding an excess of barium +carbonate and collecting the ammonia by distillation in the usual way. + +_Digestible Proteids._—The digestible proteids in cheese are determined +by the process of artificial digestion, which will be described in the +part of this volume treating of the nutritive value of foods. + +These data show the remarkable changes which the proteids undergo where +the ripening is carried very far as in the camembert cheese. + +=517. Koumiss.=—Fermented mare milk has long been a favorite beverage +in the East, where it is known as koumiss. In Europe and this country +cow milk is employed in the manufacture of fermented milk, although it +is less rich in lactose than mare milk. The process of manufacture is +simple, provided a suitable starter is at hand. A portion of a previous +brewing is the most convenient one, the fermentation being promoted by +the addition of a little yeast. After the process of fermentation is +finished the koumiss is placed in bottles and preserved in a horizontal +position in a cellar, where the temperature is not allowed to rise +above 12°. + +=518. Determination of Carbon Dioxid.=—The carbon dioxid in koumiss is +conveniently estimated by connecting the bottle by means of a champagne +tap with a system of absorption bulbs.[529] The exit tube from the +koumiss bottle passes first into an erlenmeyer, which serves to break +and retain any bubbles that pass over. The water is next removed by +means of sulfuric acid. The koumiss bottle is placed in a bath of water +which is raised to the boiling point as the evolution of the gas is +accomplished. The arrangement of the apparatus is shown in Fig. 113. At +the end of the operation any residual carbon dioxid in the apparatus +is removed by aspiration after removing the tap and connecting it +with a soda-lime tube to hold the carbon dioxid in the air. A large +balance suited to weighing the koumiss bottle is required for this +determination. The carbon dioxid may also be determined, but less +accurately, by loss of weight in the koumiss bottle after adding weight +of water retained in the apparatus. + +=519. Acidity.=—Although koumiss may contain a trace of acetic acid, +it is best to determine the acid as lactic. The clarification is +most easily accomplished by mixing the koumiss with an equal volume +of ninety-five per cent alcohol, shaking and filtering. The first +filtrate will usually be found clear. If not it is refiltered. In an +aliquot part of the filtrate the acidity is determined by titration +with tenth-normal sodium hydroxid solution, using phenolphthalein as +indicator. The necessary corrections for dilution and volume of the +precipitated casein are to be made. A linen filter may be used when +paper is found too slow. + +[Illustration: FIG. 113. APPARATUS FOR DETERMINING CARBON DIOXID IN +KOUMISS.] + +=520. Alcohol.=—Half a liter of koumiss, to which 100 cubic centimeters +of water have been added, is distilled until the distillate amounts to +500 cubic centimeters. + +If the distillate be turbid 100 cubic centimeters of water are added +and the distillation repeated. The alcohol is determined by the +processes described hereafter. + +=521. Lactose.=—The milk sugar may be determined by any of the methods +described, but most conveniently by double dilution and polarization +(=86=). + +=522. Fat.=—Evaporate twenty grams of the sample to dryness and extract +with pure ether or petroleum spirit in the manner already described +(=455=). + +The analysis is more quickly accomplished by the volumetric method of +Babcock or Gerber (=473-475=). + +=523. Proteids.=—The total proteids are most easily estimated by the +official kjeldahl method.[530] The separation of the proteid bodies is +accomplished by the methods described in paragraphs =475-489=. + +In addition to the methods already described for separating the soluble +and suspended proteid bodies in milk, and which may be used also for +koumiss, the following should also be mentioned as of especial worth: + +_Separation by Filtration through Porous Porcelain._—A purely physical +method, and one which is to be recommended by reason of the absence +of any chemical action upon the different proteid matters, is that +proposed by Lehmann, depending upon the principle that when milk is +forced through porous porcelain, the albumin passes through together +with the milk, sugar and other soluble constituents as a clear +filtrate, while the casein and fat are perfectly retained.[531] + +By this method it is quite certain that the albumin and other perfectly +soluble proteids of milk may be obtained in the purest form. + +_Separation by Precipitation with Alum._—Probably the best chemical +method of separating the two classes of proteid matters is that +proposed by Schlosmann, which is effected by means of precipitating the +casein with a solution of alum.[532] + +The principle of this separation rests upon the fact that a solution +of potash alum, when added to milk diluted with four or five times its +volume of water, will completely separate the casein without affecting +the albumin or globulin. The operation is conducted as follows: + +Ten cubic centimeters of the milk are diluted with from three to five +times that quantity of water and warmed to a temperature of about +40°. One cubic centimeter of a concentrated solution of potash alum +is added, the mixture well stirred and the coagula which are formed +allowed to subside. If the coagulation of the casein does not take +place promptly, a small addition of the alum solution is made, usually +not exceeding half a cubic centimeter, until the precipitation is +complete. The temperature during the process should be kept as nearly +as possible 40°. After a few minutes, the mixture is poured upon a +filter and the filtrate, if not perfectly clear, is poured back until +it is secured free of turbidity. In difficult cases the filtration may +be promoted by the addition of some common salt or calcium phosphate, +the latter acting mechanically in holding back the fine particles of +casein. The precipitate is washed with water at a temperature of 40°, +and afterwards with alcohol, not allowing the alcohol wash water to +flow into the filtrate. When the water has been chiefly removed from +the precipitate by washing with alcohol, the fat of the precipitated +casein is removed with ether and the residue used for the determination +of nitrogen in the usual way. The albumin is removed from the filtrate +by a tannin solution in the manner already described (=480=). If it be +desired to separate the albumin and globulin, the methods described in +paragraph =399= may be used. + +=524. Mercurial Method.=—A volumetric method for determining the total +proteid matter in milk has lately been proposed by Deniges.[533] It is +based upon the observation that in the precipitation of proteid matter +by mercury salts, a definite quantity of mercury in proportion to the +amount of proteid, is carried down therewith. The precipitation is made +with a mercurial salt of known strength and the excess of the mercurial +salt in the filtrate is determined by titration. For the details of the +manipulation, the paper cited above may be consulted. + +=525. Water and Ash.=—From two to five grams of the koumiss are dried +to constant weight in a flat platinum dish over ignited sand, asbestos +or pumice stone, and the dried residue incinerated. + +=526. Composition of Koumiss.=—The composition of koumiss varies with +the character of the milk used and the extent of the fermentation. Some +of the data obtained by analysts are given below:[534] + + COMPOSITION OF KOUMISS. + + Carbon + Kind Water, Sugar, Alcohol, Fat, Proteid, dioxid, Acidity, + of Per Per Per Per Per Per Per + milk. cent. cent. cent cent. cent. cent. cent. + Cow 89.32 4.38 0.76 2.08 2.56 0.83 0.47 + Probably 3.95 1.38 0.88 2.89 0.82 + cow + skim’d + Mare 91.87 0.79 2.89 1.19 1.91 1.04 + +From the above it is seen that koumiss is made either from whole or +skim milk, and that the percentage of alcohol may vary within large +limits, its proportion being inverse to that of the milk sugar. + +Koumiss is a beverage which is very palatable, easily digested and one +which is not appreciated in this country in proportion to its merits, +especially for the use of invalids. + + +AUTHORITIES CITED IN PART SIXTH. + +[400] Wiley; Proceedings of the Society for the Promotion of +Agricultural Science, 1889, p. 84. (Omit “food” before idiosyncrasy.) + +[401] Pharmaceutical Journal and Transactions, Series 3, Vol. 18, p. +479. + +[402] The Analyst, 1892, p. 85. + +[403] Henkel; Wiener Landwirtschaftliche Zeitung, 1888, S. 401: +Bulletin No. 24, Division of Chemistry, U. S. Department of +Agriculture, p. 155. + +[404] Die Landwirtschaftlichen Versuchs-Stationen, Band 35, S. +351: Bulletin No. 24, Division of Chemistry, U. S. Department of +Agriculture, p. 151. + +[405] Baumeister; Milch und Molkerei-Producte, S. 16. + +[406] Bulletins Nos. 9 and 25 of the Office of Experiment Stations, U. +S. Department of Agriculture: Farmers’ Bulletins Nos. 9 and 29, U. S. +Department of Agriculture. + +[407] Annales de Chimie et de Physique, 3e Série, Tome 64, p. 61. + +[408] Bulletin de la Société Chimique de Paris, 3ᵉ Série, Tome 15-16, +p. 248. + +[409] Vid. op. cit. supra, p. 453. + +[410] Central-Blatt für medicinische Wissenschaft, Band 34, S. 145. + +[411] Conn; Farmers’ Bulletins 9 and 25, Office of Experiment Stations, +U. S. Department of Agriculture: Farmers’ Bulletins 9 and 29, +Department of Agriculture: Les Microbes et leur Rôle dans la Laiterie +Freudenreich: Langlois, Le Lait, pp. 95 et seq. + +[412] The Analyst, Vol. 20, p. 157. + +[413] Vid. op. cit. supra, p. 152. + +[414] Forschungs-Berichte über Lebensmittel etc., Band 2, S. 368. + +[415] Vid. op. cit. supra, Band 1, S. 422. + +[416] Vid. op. cit. supra, S. 372. + +[417] Hopkins and Powers; Bulletin No. 47, Division of Chemistry, U. S. +Department of Agriculture, p. 127. + +[418] Bulletin No. 38, Division of Chemistry, U. S. Department of +Agriculture, p. 118. + +[419] Becke; Die Milchprüfungs-Methoden, S. 45: Rouvier; Le Lait, p. 45. + +[420] The Analyst, 1890, Vol. 16, p. 170. + +[421] Rouvier; Le Lait, p. 35. + +[422] Central-Blatt für Nahrungs und Genussmittel Chemie, Band 13, S. +277. + +[423] Bulletin No. 46, Division of Chemistry, U. S. Department of +Agriculture, p. 36. + +[424] Journal für Landwirtschaft, 1882, S. 293; 1885, S. 251. + +[425] Vid. op. cit. supra, 1879, S. 249. + +[426] Forschungen auf dem Gebiete der Viehhaltung, 1879, S. 265. + +[427] The Analyst, Vol. 7, p. 129. + +[428] Vid. op. cit. supra, Vol. 13, p. 26. + +[429] Bulletin No. 47, Division of Chemistry, U. S. Department of +Agriculture, p. 123. + +[430] This work, Vol. 1, page 411. + +[431] Bulletin No. 16, Division of Chemistry, U. S. Department of +Agriculture, p. 36. + +[432] Sixth Annual Report Wisconsin Agricultural Experiment Station, p. +64. + +[433] Fourth Annual Report New York (Geneva) Agricultural Experiment +Station, p. 298. + +[434] Woll; Seventh Annual Report Wisconsin Agricultural Experiment +Station, p. 238. + +[435] The Analyst, 1885, p. 46: Bulletin No. 13, Part 1, Division of +Chemistry, U. S. Department of Agriculture, p. 86. + +[436] Haidlen; Die Milchprüfungs-Methoden, S. 12. + +[437] Dingler’s polytechnisches Journal, Band 232, S. 461. + +[438] Macfarlane; The Analyst, Vol. 18, p. 73. + +[439] Duclaux; Le Lait, p. 176. + +[440] Abraham; The Analyst, Vol. 9, p. 22. + +[441] Gantter; Zeitschrift für analytische Chemie, Band 26, S. 677. + +[442] Morse, Piggot and Burton; American Chemical Journal, Vol. 9, pp. +108 and 222. + +[443] Chemiker-Zeitung Repertorium, 1889, S. 228. + +[444] Journal de Pharmacie et de Chimie, 1890, p. 460. + +[445] Richmond; The Analyst, Vol. 17, p. 48: Bulletins 28, 31, 35, 38, +43, and 46, Division of Chemistry, U. S. Department of Agriculture. + +[446] Bulletin No. 28, Division of Chemistry, U. S. Department of +Agriculture, p. 31. + +[447] Zeitschrift für analytische Chemie, Band 27, S. 464. + +[448] Chemical News, Nov. 1889. + +[449] The Analyst, Vol. 16, p. 67. + +[450] Vid. op. cit. supra, Vol. 18, p. 53. + +[451] Vid. op. cit. supra, Vol. 17, p. 81. + +[452] Chemiker-Zeitung, Band 15, S. 1833. + +[453] Journal of Analytical Chemistry, 1888, Vol. 2, p. 371: Fifth +Annual Report Wisconsin Agricultural Experiment Station. + +[454] Molkerei Zeitung, 1892, No. 1; Chemisches Central-Blatt, 1892, +Band 2, S. 429. + +[455] Chemiker-Zeitung, Band 18, S. 1816; Band 19, S. 348. + +[456] Zeitschrift für analytische Chemie, Band 32, S. 168. + +[457] Zeitschrift des Landwirtschaftlichen Vereins in Bayern, 1880; +Zeitschrift für analytische Chemie, Band 20, S. 452. + +[458] Bulletin No. 13, Division of Chemistry, U. S. Department of +Agriculture, p. 92. + +[459] Instruction sur l’Emploi du Lactobutyrometer, Paris, 1856 et +1878: Becke; Die Milchprüfungs-Methoden, S. 66. + +[460] Bulletin No. 8, Iowa Agricultural Experiment Station, p. 295. + +[461] Dingler’s polytechnisches Journal, Band 261, S. 219. + +[462] Milch Zeitung, Band 21, S. 496. + +[463] Op. cit. supra, Band 22, S. 85. + +[464] Bulletin No. 24, Wisconsin Agricultural Experiment Station. + +[465] Bulletin No. 31, Wisconsin Agricultural Experiment Station. + +[466] The Analyst, Vol. 17, p. 83. + +[467] Bulletin No. 21, Vermont Agricultural Experiment Station. + +[468] Vid. op. cit. 67, Vol. 17, p. 144; Vol. 18, p. 130; Vol. 19, p. +62. + +[469] Chemiker-Zeitung, Band 16, S. 1839. + +[470] Vid. op. cit. supra, Band 19, S. 348; Band 18, S. 1816. + +[471] Vid. op. cit. supra, Band 19, S. 348. + +[472] Comptes rendus, Tome 107, p. 772; Hoppe-Seyler’s Handbuch der +Physiologisch- und Pathologisch-Chemischen Analyse, S. 479. + +[473] Proceedings of the Society for the Promotion of Agricultural +Science, 1888, p. 13. + +[474] Journal of Physiology, Vol. 11, p. 459. + +[475] Die Land wirtschaftlichen Versuchs-Stationen, Band 31, S. 131. + +[476] Sixth Annual Report of the Wisconsin Agricultural Experiment +Station, p. 64. + +[477] Bulletin No. 46, Division of Chemistry, U. S. Department of +Agriculture, p. 36. + +[478] Zeitschrift für Biologie, Band 33, S. 43. + +[479] Journal für praktische Chemie, {2}, Band 15, S. 329. + +[480] Vid. op. cit. 79, Band 33, {Neue Folge, 15}, S. 55. + +[481] Stenberg; Zeitschrift für physiologische Chemie, Band 13, S. 138. + +[482] Vid. op. cit. supra, S. 137. + +[483] Vid. op. cit. supra, S. 160. + +[484] Journal of the American Chemical Society, Vol. 15, p. 644. + +[485] Handbuch der Physiologisch- und Pathologisch-Chemischen Analyse, +S. 285. (Read, Makris instead of Makeris.) + +[486] Zeitschrift für Biologie, Band 23, S. 64. + +[487] Bulletin de la Société Chimique de Paris, 3ᵉ Série, Tome 11, p. +152. + +[488] Vid. op. cit. 86, S. 487. + +[489] Zeitschrift für Nahrungsmittel-Untersuchung, Band 10, S. 104. + +[490] Zeitschrift für physiologische Chemie, Band 9, S. 445. + +[491] American Chemical Journal, Vol. 6, p. 289. + +[492] Journal of the American Chemical Society, Vol. 18, p. 428. + +[493] Journal de Pharmacie et de Chimie, 6e Série, Tome 4, p. 65. + +[494] Contribution à l’Étude des Lactoses, Thèse pour le diplôme +supérieure de Pharmacie, Paris, 1892. (Read Thibault instead of +Thibonet.) + +[495] Journal für praktische Chemie, Neue Folge, Band 15, S. 348. + +[496] Zeitschrift für angewandte Chemie, 1896, S. 72. + +[497] Chemisches Central-Blatt, 1892, Band 2, S. 1028. + +[498] Vid. op. cit. supra, Band 21, S. 753. + +[499] Vid. op. cit. 90, S. 86. + +[500] Bulletin No. 13, Division of Chemistry, U. S. Department of +Agriculture, pp. 29 et seq. + +[501] Vid. op. cit. supra, pp. 73-75: Bulletin No. 46, Division of +Chemistry, U. S. Department of Agriculture, p. 26. + +[502] Benedikt and Lewkowitsch; Oils, Fats and Waxes, p. 490. + +[503] Forsuchungs-Berichte über Lebensmittel, 1895, Band 2, S. 424; +Chemiker-Zeitung Repertorium, 1896, Band 20, S. 15. + +[504] Revue Internationale des Falsifications, Mai, 1893, p. 157. + +[505] Chemiker-Zeitung, 1893, Band 17, S. 468. + +[506] Zeitschrift für angewandte Chemie, 1896, S. 177. + +[507] Vid. op. cit. 90, Aug. 26, 1894, S. 219; Le Stazioni +Sperimentali Agrarie Italiane, 1893, pp. 25-77. + +[508] Farmers’ Bulletin No. 12, U. S. Department of Agriculture. + +[509] Bulletin de l’Association Belge des Chimistes, Tome 9, p. 279. + +[510] Vid. op. cit. 101, p. 26. + +[511] Vid. op. cit. supra, p. 27: Chemical News, Vol. 55, p. 49. + +[512] Vid. op. cit. 111, p. 28. + +[513] Vid. op. et. loc. cit. supra. + +[514] Russell; Dairy Bacteriology. + +[515] Woll; Dairy Calendar, p. 223. + +[516] Van Slyke; Bulletin 82, New Series, New York Agricultural +Experiment Station, p. 654. + +[517] Babcock; Twelfth Annual Report Wisconsin Agricultural Experiment +Station, p. 133. + +[518] Woll; Dairy Calendar, 1895, p. 220. + +[519] Bulletin No. 46, Division of Chemistry, U. S. Department of +Agriculture, p. 37. + +[520] Landwirtschaftliches Jahrbuch, 1872, part 1. + +[521] Molkerei Zeitung, 1893, Nos. 20, 22. + +[522] This work, Vol. 2, p. 204. + +[523] Vid. op. cit. 120, p. 24. + +[524] Milch Zeitung, 1895, Band 24, S. 729: Chemiker-Zeitung +Repertorium, Band 19, S. 372. + +[525] Chemiker-Zeitung, 1895, S. 554. + +[526] Zeitschrift für analytische Chemie, Band 35, S. 497. + +[527] Vid. op. cit. supra, S. 499. + +[528] Vid. op. cit. supra, S. 502. + +[529] Bulletin No. 13, Division of Chemistry, U. S. Department of +Agriculture, pp. 118, 293. + +[530] This work, Vol. 2, p. 204. + +[531] Pflüger’s Archiv, Band 56, S. 558. + +[532] Hoppe-Seyler’s Zeitschrift für physiologische Chemie, Band 22, S. +213. + +[533] Bulletin de la Société Chimique de Paris, Tomes 15-16, p. 1126. + +[534] American Chemical Journal, Vol. 8, p. 200: Bulletin 13, Division +of Chemistry, U. S. Department of Agriculture, p. 120. + + + + +PART SEVENTH. + +MISCELLANEOUS AGRICULTURAL PRODUCTS. + + +=527. Classification.=—In the preceding parts have been set forth the +fundamental principles underlying the conduct of agricultural analysis +and a résumé of the best practice of the art. The analyst, as a rule, +will seldom be required to undertake investigations which are unnoticed +in the preceding pages. Cases will arise, however, in which problems +are presented which can not be solved by the rules already elucidated. +In respect of the great classes of agricultural bodies, it will be +observed that dairy products have already received special mention. +In respect of foods and fodders in general, it is evident that they +are chiefly composed of moisture, ash, carbohydrates, oils and proteid +matters. The methods of identifying, separating and estimating these +constituents have been fully set forth. It is not necessary, therefore, +to study in this part the analytical processes which are applicable +to cereals, cattle foods and other food products, further than is +necessary to present in the most important cases a working résumé of +principles and methods. There remain, however, certain products of +importance which require some special modifications of treatment, +and it is to these that the present part will be chiefly devoted. +Among these are found tobacco, tea and coffee, fruits, fermented and +distilled drinks and certain animal products. It is evident that an +enumeration of all agricultural products, with a description of their +methods of examination, would be impracticable in the available space +and undesirable by reason of the repetition which would be required. +In each case the analyst, in possession of the methods described, will +be able to adapt the means at his disposal to the desired purpose to +better advantage than any rigid directions could possibly secure. + +In respect of the analytical methods of determining the nutritive +value of foods, they may be divided into chemical and physiological. +The chemical methods embrace the thermal and artificial digestion +investigations, and the physiological include those which are carried +out with the help of the animal organisms. In the latter case the +digestive process is checked by the analysis of the foods before +ingestion and of the excreta of all kinds during and after digestion. + +It is evident that a detailed description of this method should be +looked for in works devoted to physiological chemistry. + + +CEREALS AND CEREAL FOODS. + +=528. General Analysis.=—The cereals are prepared for analysis by +grinding until the fragments pass a sieve having circular perforations +half a millimeter in diameter. The moisture, ash, ether extract, +proteids and carbohydrates are determined by some one of the +processes already described in detail. In this country the methods +of the Association of Official Agricultural Chemists are generally +followed.[535] For convenience these methods are summarized below. + +_Moisture._—Dry from two to three grams of the fine-ground sample for +five hours, at the temperature of boiling water, in a current of dry +hydrogen. If the substance be held in a glass vessel, the latter should +not be in contact with the boiling water. + +_Ash._—Char from two to three grams of the sample and burn to whiteness +at the lowest possible red heat. If a white ash can not be obtained in +this manner, exhaust the charred mass with water, collect the insoluble +residue on a filter, burn it, add this ash to the residue from the +evaporation of the aqueous extract and heat the whole to low redness +until the ash is white. + +_Ether Extract._—Pure ether is prepared by washing the commercial +article four or five times with water to free it of the chief part +of the alcohol it contains. The residual water is mostly removed by +treating the liquid with caustic soda or potash. Any residual alcohol +or water is finally removed by the action of metallic sodium. The +ether thus prepared is stoppered, after the evolution of hydrogen has +ceased, and is kept over metallic sodium. Immediately before use it +should be distilled out of contact with moist air. + +The residue from the determination of moisture, as described above, is +extracted in an appropriate apparatus (=39=) with the pure ether for +sixteen hours. The extract is dried to constant weight. The weight may +be checked by drying and weighing the extraction tube and its contents +before and after the operation. + +_Crude Proteids._—Proceed as in the method of determining nitrogen in +the absence of nitrates and multiply the weight of nitrogen obtained +by 6.25. This factor is a general one, but should not be rigidly +applied. In each instance, according to the nature of the cereal, the +appropriate factor, pointed out in paragraph =407= should be used, and +the factor 6.25 be applied only in those cases where a special factor +is not given. The factors for the common cereals are wheat 5.70, rye +5.62, oats 6.06, maize 6.22, barley 5.82 and flaxseed 5.62. + +For separating the proteid matters consult paragraphs =392-410=. In the +case of wheat the methods of Teller may be consulted.[536] + +_Amid Nitrogen._—The albuminoid nitrogen is determined as directed in +paragraph =203= of volume II. The difference between this number and +that representing the total nitrogen gives the nitrogen as amids. + +_Fiber and Carbohydrates._—The methods of analysis are described in +detail in Part Third. + +=529. Bread.=—In general, the same processes are followed in bread +analysis as are used with cereals and flours. In addition to +the regular analytical processes, breads are to be examined for +adulterants, bleaching and coloring matters, and for the purpose of +determining the changes which have taken place in their nutrient +constituents in the processes of fermentation and cooking. + +_Temperature of Baking._—The interior of a loaf during the process +of baking does not attain the high temperature commonly supposed. +This temperature is rarely found to be more than one degree above the +boiling point of water.[537] In biscuits and other thin cakes, which +become practically dry and which by reason of their thinness are the +more readily penetrated by heat, the temperature may go as high as 110°. + +_Soluble Extract._—The quantity of matters both in flour and bread, +soluble in cold water, is determined by extraction in the usual way +and drying the extract. Soluble albuminoids, sugars and mineral salts +are extracted by this process. When possible, the operation should be +conducted both on the bread and the flour from which it is made. + +_Color._—In baker’s parlance is found an apparent contradiction of +terms, since it speaks of bread with “no color” when the loaf is dark +brown, while a white loaf is said to have a high color. An ideal +color for the interior of a loaf is a light cream tint, which is +more desirable than a pure white.[538] The texture, odor and flavor of +the loaf are also to be considered, but these are properties of more +importance to the technical expert than to the analyst. + +_Quantity of Water._—It is not possible to set a rule of limitation in +respect of the quantity of water a bread should hold. For full loaves, +perhaps forty per cent is not too high a maximum, while some authors +put it as low as thirty-four per cent. Some flours are capable of +holding more water than others, and the loaf should have just enough +water to impart to the slice of bread the requisite degree of softness +and the proper texture. Most breads will have a content of water +ranging from thirty to forty per cent. In biscuits and other thin cakes +the moisture is much less in quantity. + +_Acidity._—The acidity of both bread and flour is determined by shaking +ten grams of the sample with 200 cubic centimeters of distilled water +for fifteen minutes, pouring the mass on a filter and titrating an +aliquot part of the filtrate with tenth-normal alkali. The acidity is +reckoned as lactic acid in the case of breads raised by fermentation. + +_Nature of Nitrogenous Compounds._—The methods of investigation are +described in paragraphs =392-410=. + +=530. Determination of Alum in Bread.=—The presence of alum in bread +may be detected by means of logwood. Five grams of fresh logwood chips +are digested with 100 cubic centimeters of amyl alcohol. One cubic +centimeter of this decoction and the same quantity of a saturated +solution of ammonium carbonate are mixed with ten grams of flour and +an equal quantity of water. With pure flour, a slight pink tint is +produced. In the presence of alum the color changes to a lavender or +blue, which is persistent on heating. + +The test may be varied by diluting five cubic centimeters of the +reagents mentioned with ninety cubic centimeters of water and pouring +the mixture over ten grams of the crumbled bread. After standing for +five minutes, any residual liquid is poured off and the residue, washed +once with a little water, is dried in a steam bath, when the blue color +is developed if alum be present.[539] + +=531. Chemical Changes Produced by Baking.=—Changes of a chemical +nature, produced in bread by baking, are found chiefly in modifications +of the starch and proteids. The starch is partly converted into dextrin +and the albumins are coagulated. The changes in digestion coefficient +are determined by the methods which follow. The fermentations which +precede the baking are due to the usual decompositions of the +carbohydrates under the influence of yeast germs. + + +FODDERS, GRASSES AND ENSILAGE. + +=532. General Principles.=—The analyst, in examining the fibrous foods +of cattle, is expected to determine moisture, ash, fiber and other +carbohydrates, ether extract and albuminoid and amid nitrogen. If a +more exhaustive study be required, the sugar and starch are separated +from the other non-nitrogenous matters, the carbohydrate bodies +yielding furfuraldehyd separately determined and the ash subjected to +a quantitive analysis. The processes are conducted in harmony with the +principles and methods of procedure fully set forth in the preceding +pages. + +Green fodders and grasses are easily dried and sampled by comminution +in the shredder described on page 9, and roots by that shown on page +10. The moisture is determined by drying a small sample of the shredded +mass, while the rest of it is dried, first at about 60° and finally +at 100°, or a little above, ground to a fine powder and subjected to +analysis by methods already described. The food values as obtained +by analysis should be compared, when possible, with those secured by +natural and artificial digestion. + +Ensilage is shredded and analyzed in precisely the same way, but in +drying, the content of volatile acids formed during fermentation must +be considered. In other words, the loss on drying ensilage at 100°, or +slightly above, is due not only to the escape of water but also to the +volatilization of the acetic acid, which is one of the final products +of fermentation which the mass undergoes in the silo. + +=533. Organic Acids in Ensilage.=—In the examination of ensilage, the +organic acids which are present may be determined by the processes +described in following paragraphs. The acetic acid, formed chiefly +by fermentation, is conveniently determined by the method given for +tobacco further on. Lactic acid is detected and estimated by expressing +the juice from a sample of ensilage, removing the acetic acid by +distillation, repeated once or twice, and treating the filtered residue +with zinc carbonate in excess, filtering and determining the zinc +lactate in the filtrate. The zinc is determined by the method described +for evaporated apples and the lactic acid calculated from the weight of +zinc found. Crystallized zinc lactate contains 18.18 per cent of water +and 27.27 per cent of zinc oxid.[540] + +=534. Changes due to Fermentation in the Silo.=—Silage differs from +green fodder in having less starch and sugar, more acetic and lactic +acids and alcohol and a higher proportion of amid to albuminoid +nitrogen.[541] There is also a considerable loss of nitrogenous +substances in ensilage, due probably to their conversion into ammonium +acetate, which is lost on drying. + +=535. Alcohol in Ensilage.=—The fermentation which takes place in the +silo is not wholly of an alcoholic nature, as the development of lactic +acid, noted above, clearly indicates. The alcohol which is formed may +escape and but small quantities can be detected in the ripened product. +So small is this quantity of alcohol that it appears to be useless to +try to secure a quantitive estimation of it. Qualitively, it may be +detected by collecting it in a distillate, which is neutralized or made +slightly alkaline with soda or potash lye and redistilled. The greater +part of the alcohol will be found in the first few cubic centimeters, +which are made alkaline with potash lye and as much iodin added as +can be without giving a red tint to the solution. Any alcohol which is +present will soon separate as iodoform. + +=536. Comparative Values of Fodder and Ensilage.=—In judging of the +comparative values of green and dry fodders for feeding purposes, it is +necessary to secure representative samples in the green, quickly dried +and ensilaged condition. It is quite certain that the greater part of +the sugar contained in green fodders is lost both by natural curing and +by placing in a silo. When well cured by the usual processes there is +but little loss of nitrogenous matters, but in the silo this loss is +of considerable magnitude, amounting in some instances to as much as +thirty per cent. + +The ideal way of preparing green fodders in order to preserve the +maximum food value efficiently, is to shred them and dry rapidly by +artificial heat, or in the sunlight, until they are in a condition +which insures freedom from fermentation. In this condition, when placed +in bales, under heavy pressure, the food constituents are preserved in +the highest available form. The immense sugar content of the stalks of +maize and sorghum could be preserved in this way almost indefinitely. + + +FLESH PRODUCTS. + +=537. Names Of Meats.=—The parts of the animal from which the meats are +taken have received distinctive names, which serve to designate the +parts of the carcass offered for sale. These names are not invariable +and naturally are quite different in many markets. In this country +there is some degree of uniformity among butchers in naming the meats +from different parts. The names in scientific use for the parts of +mutton, beef and pork are found in the accompanying illustrations.[542] + +=538. Sampling.=—When possible the whole animal should constitute the +sample. The relative weights of blood, intestinal organs, hide, hoofs, +horns, bones and edible flesh are determined as accurately as possible. +The general method of preparing samples of animal products is given in +paragraph =5=. + +[Illustration: FIG. 114.] + +[Illustration: FIG. 115.] + +[Illustration: FIG. 116.] + +[Illustration: NAMES OF CUTS OF MEAT.] + +The method of sampling employed by Atwater and Woods is essentially +that just noted.[543] The sample, as received at the laboratory, is +weighed, the flesh (edible portion) is then separated from the refuse +(skin, bones etc.) and both portions weighed. There is always a slight +loss in the separation, evidently due to evaporation and to small +fragments of the tissues that adhere to the hands and to the implements +used in preparing the sample. The perfect separation of the flesh from +the other tissues is difficult, but the loss resulting from this is +small. In sampling the material for analysis, it is finely chopped, +either in a tray or in a sausage cutter, and in each case is well mixed. + +=539. Methods of Analysis.=—The general methods for the analyses of +food products are applicable to meats and animal products in general. +In the separation of the nitrogenous constituents the methods described +in paragraphs =411-414= are followed. It is not safe to estimate as +proteids the total nitrogen multiplied by 6.25, since the flesh bases +have much higher percentages of nitrogen than are found in proteid +matters. As indicated in paragraph =280= the complete extraction of +dried meats by ether is difficult of accomplishment. After a few hours +it may be assumed that the total extract will represent the fat, +although additional soluble matters are obtained by continuing the +process. The heat producing power may be calculated from the analytical +data secured. The methods which have been described in the preceding +pages will be found sufficient for guidance in the examination of +animal products, and the analyst will find them, when modified to suit +particular cases, adapted to the isolation and estimation of proximate +food principles. + +The methods of analyses followed by Atwater and Woods are given +below:[544] + +_Water and Water-Free Substance._—The drying is done in ordinary water +ovens at a temperature of nominally 100°, but actually at 96° and 98°. +For each analysis of animal tissues (flesh) one or more samples of from +fifty to one hundred grams of the freshly chopped substance are weighed +on a small plate, heated for from twenty-four to forty-eight hours, +cooled, allowed to stand in the open air for about twenty-four hours, +weighed, ground, sifted through a sieve with circular holes one-half +millimeter in diameter, bottled and set aside for analysis. In case of +fat samples which cannot be worked through so fine a sieve, either a +coarser sieve is used or the substance crushed as finely as practicable +and bottled without sifting. + +For the complete desiccation, about two grams of material are dried for +three hours. It is extremely difficult to get an absolutely constant +weight, though it is found that this is in most cases approximately +attained in four hours. + +_Nitrogen, Protein, Albuminoids etc._—The nitrogen is determined in +the partly dried substance by the method of Kjeldahl. The protein is +calculated by multiplying the percentage of nitrogen by 6.25. The +nitrogenous matters in meats and fish, _i. e._, in the materials which +have practically no carbohydrates, are also estimated by subtracting +the sum of ether extract and ash from the water-free substance, or +the sum of water, ether extract and ash from the fresh substance, the +remainder being taken as proteids, albuminoids etc., by difference. +While this is not an absolutely correct measure of the total +nitrogenous matter, it is doubtless more nearly so than the product of +the nitrogen multiplied by 6.25. + +_Fat (Ether Extract)._—The fat is extracted with ether in the usual +manner. The point at which the extraction is complete is not always +easy to determine. For the most part, the extraction is continued for +such time as experience indicates to be sufficient, and then the flask +is replaced by another and the extraction repeated until the new flask +shows no increase in weight. + +According to experience, the fat of many animal tissues is much more +difficult to extract than that of most vegetable substances. In +general, the greater the percentage of fat in a substance the more +difficult is the removal of the last traces. Dried flesh is frequently +so hard that the fineness of the material to be extracted seems to be a +very important matter. + +_Ash._—Ash is determined by the method recommended by the Association +of Official Agricultural Chemists. + +_Food Value—Potential Energy._—The food materials are not necessarily +burned in the calorimeter, but the fuel value of a pound of each of the +foods, as given in the tables, is obtained by multiplying the number of +hundredths of a pound of protein and of carbohydrates by 18.6 and the +number of hundredths of a pound of fat by 42.2, and taking the sum of +these three products as the number of calories of potential energy in +the materials. + +More reliable results are obtained by using the factors obtained by +Stohmann; _viz._, 5731 calories for proteids, 9500 calories for common +glycerids, 9231 calories for butter fat, 3746 calories for pentose +sugars, 3749 calories for dextrose and levulose and 3953 calories for +sucrose and milk sugar.[545] + +=540. Further Examination of Nitrogenous Bodies.=—It is evident +that both of the methods proposed above for the examination of the +nitrogenous constituents of meats are unreliable. If the total nitrogen +be determined and multiplied by 6.25 the product does not by any means +represent the true quantity of nitrogenous matter since the flesh bases +contain in some instances more than twenty-five per cent of nitrogen. + +If, on the other hand, the water, ash and fat in a meat sample be +determined and the sum of their per cents be subtracted from 100, the +difference represents the nitrogenous bodies plus all undetermined +matters and errors of analysis. The assumption that meats are free +of carbohydrates is not tenable since glycogen is constantly found +therein and in horse flesh in comparatively large amounts. In a +thoroughly scientific analysis of meats, the nitrogenous bodies should +be separated and determined by groups, according to the principles +developed in paragraphs =411-414=. This process requires a great amount +of analytical work and in general it will be sufficient to make a +cold water extract to secure the flesh bases and a hot water extract +to secure the gelatin. The nitrogen is then determined in each of +these portions separately. The nitrogen in the cold water extract is +multiplied by four, in the hot water extract by six and in the residue +by 6.25. The sum of these products represents approximately the total +nitrogenous matter in the sample. + +Aqueous extracts containing nitrogen are easily prepared for moist +combustion by placing them in the digestion flasks, connecting the +latter with the vacuum service and evaporating the contents of the +flask nearly to dryness. The sulfuric acid is then added and the +nitrogen converted into ammonia and determined in the usual manner. + +=541. Fractional Analysis of Meats.=—A better idea of the composition +of a meat is obtained by separating its constituents into several +groups by the action of different solvents. This method has been +elaborated by Knorr.[546] + +The separation of the meats in edible portion and waste and the +determination of moisture and fat are conducted as already described. +The residue from the fat extraction is exhausted with alcohol, and +in the extract are found the nitrogenous bases kreatin, kreatinin, +sarkin and xanthin, and urea, lactic, butyric, acetic and formic acids, +glycogen and inosit. In the residue from the alcohol extraction, the +proteid nitrogen is determined in a separate sample. + +A separate portion of the sample is ground to a fine paste and +repeatedly rubbed up with cold water, which is poured through a tared +filter. When the extraction is complete, the filter and its contents +are dried and the dry residue determined. This residue represents +the nitrogenous constituents of the muscle fibers and their sheaths +together with any other bodies insoluble in cold water. The filtrate +from the cold water extraction is heated to boiling to precipitate +the albuminous matters which are collected, dried and weighed, or the +nitrogen therein determined and the albuminous matters calculated by +multiplying by the usual factor. The filtrate from the coagulated +albuminous bodies is evaporated to dryness and weighed. It consists +essentially of the same materials as the alcoholic extract mentioned +above. The ash and nitrogen in the aqueous extract are also determined. + +The mean content of the edible parts of common meats, expressed as per +cents in groups as mentioned, follow: + + Per cent. + Water 73.11 + Ash 1.18 + Total soluble matter 26.89 + Phosphoric acid 0.49 + + Per cent. + { Proteids insoluble in cold water 13.76 + { Of which coagulable by heat 2.24 + Cold water extract 3.56 + { Ash in water extract 1.09 + { Of which phosphoric acid 0.38 + + Per cent. + Fat 4.93 + Alcohol extract 3.03 + Proteids in residue from alcohol 17.88 + Total nitrogen in sample 3.37 + +=542. Estimation of Starch in Sausages.=—Starchy substances are +sometimes added to sausages for the purpose of increasing their weight. +The presence of starch in a sausage is easily detected by iodin. The +quantity may be determined by the following process:[547] + +The principle of the process is based upon the observation that while +starch is easily soluble in an aqueous solution of the alkalies, it is +insoluble in an alcoholic solution thereof. The chief constituents of +meat, _viz._, fat and proteid matters, on the other hand, are readily +soluble in an alcoholic solution of potash or soda. This renders the +separation of the starch easy. The sample is warmed on a water bath +with a considerable excess of an eight per cent solution of potassium +hydroxid in alcohol whereby the fat and flesh are quickly dissolved. +The starch and other carbohydrate bodies, remain in an undissolved +state. In order to prevent the gelatinizing of the soap which is +formed, the mass is diluted with warm alcohol, the insoluble residue +collected upon a filter and washed with alcohol until the alkaline +reaction disappears. The residue is then treated with aqueous potassium +hydroxid solution, whereby the starch is brought into solution and, +after filtration, is treated with alcohol until it is all precipitated. +The precipitated starch is collected upon a filter, washed with alcohol +and finally with ether, dried and weighed. Starch prepared in this +way contains a considerable quantity of potash, the amount of which +can be determined by incineration. In order to avoid this trouble, +the starch, after separation in the first instance as above mentioned +and solution in aqueous potassium hydroxid, is precipitated on the +addition of enough acetic to render the solution slightly acid. The +precipitated starch, in this instance, is practically free of potash, +since potassium acetate is soluble in alcohol. + +=543. Detection of Horse Flesh.=—Since horse flesh has become an +important article of human food and is often sold as beef and sausage, +a method of distinguishing it is desirable. The comparative anatomist +is able to detect horse flesh when accompanied by its bones, or +in portions sufficiently large for the identification of muscular +characteristics. It is well known that horse flesh contains a much +higher percentage of glycogen than is found in other edible meats. +Niebel has based a method of detecting horse flesh upon this fact, the +glycogen being converted into dextrose and determined in the usual way. +Whenever the percentage of reducing sugars in the dry fat-free flesh +exceeds one per cent, Niebel infers that the sample under examination +is horse flesh.[548] + +The reaction for horse flesh, proposed by Bräutigam and Edelmann, is +preferred by Baumert. In this test about fifty grams of the flesh are +boiled for an hour with 200 cubic centimeters of water, the filtered +bouillon evaporated to about half its volume, treated with dilute +nitric acid and the clear filtrate covered with iodin water. Horse +flesh, by reason of its high glycogen content, produces a burgundy +red zone at the points of contact of the two liquids. In the case of +sausages, if starch have been added, a blue zone is produced, and if +dextrin be present, a red zone, both of which obscure the glycogen +reaction. The starch is easily removed by treating the bouillon with +glacial acetic acid. No method is at present known for separating +dextrin from glycogen. The detection of horse flesh is a matter of +considerable importance to agriculture as well as to the consumers, +especially of sausages. A considerable quantity of horse flesh is +annually sent to the market, little of which presumably is sold under +its own name. As a cheap substitute for beef and pork in sausages, its +use must be regarded as fraudulent, although no objection can be urged +against its sale when offered under its own name.[549] + + +METHODS OF DIGESTION. + +=544. Artificial Digestion.=—The nutrient values of cereals and other +foods are determined both by chemical analysis and by digestion +experiments. The heat forming properties of foods are disclosed by +combustion in a calorimeter, but the quantity of heat produced is not +in every case a guide to the ascertainment of the nutritive value. This +is more certainly shown, especially in the case of proteid bodies, by +the action of the natural digestive ferments. + +It is probable that the digestion, which is secured by the action of +these ferments without the digestive organs, is not always the same as +the natural process, but when the conditions which prevail in natural +digestion are imitated as closely as possible the effects produced can +be considered as approximately those of the alimentary canal in healthy +action. + +Three classes of ferments are active in artificial digestion, _viz._, +amylolytic ferments, serving to hydrolyze starch and sugars and to +convert them into dextrose, maltose and levulose, aliphalytic ferments, +which decompose the glycerids and proteolytic ferments, which act on +the nitrogenous constituents of foods. When these ferments are made +to act on foods under proper conditions of acidity and temperature, +artificial digestion ensues, and by the measurement of the extent +of the action an approximate estimate of their digestibility can be +secured. In artificial digestion, the temperature should be kept near +that of the body, _viz._, at about 40°. + +The soluble ferments which are active in the digestion of foods, as +has been intimated, comprise three great classes. Among the first +class, _viz._, the amylolytic ferments, are included not only those +which convert starch into dextrose, but also those which cause the +hydrolysis of sugars in general. Among these may be mentioned ptyalin, +invertase, trehalase, maltase, lactase, diastase, inulase, pectase +and cyto-hydrolytic ferments which act upon the celluloses and other +fibers. + +Among the aliphalytic ferments, in addition to those which act also +upon proteid matter, may be mentioned a special one, lipase. + +In the third class of ferments are found pepsin, trypsin or pancreatin +and papain. + +For the latest information in regard to the nature of the soluble +ferments and their nomenclature, the work of Bourquelot may be +consulted.[550] + +=545. Amylytic Ferments.=—A very active ferment of this kind is found +in the saliva. Saliva may be easily collected from school boys, who +will be found willing to engage in its production if supplied with +a chewing gum. A gum free of sugar is to be used, or if the chewing +gum of commerce is employed, the saliva should not be collected +until the sugar has disappeared. A dozen boys with vigorous chewing +will soon provide a sufficient quantity of saliva for practical use. +The amylolytic digestion is conducted in the apparatus hereinafter +described for digestion with pepsin and pancreatin. The starch or +sugar in fine powder is mixed with ten parts of water and one part of +saliva and kept at about 37°.5 for a definite time. The product is then +examined for starch, sucrose, maltose, dextrose, dextrin and levulose +by the processes already described. In natural digestion the hydrolysis +of the carbohydrates is not completed in the mouth. The action of +the ferment is somewhat diminished in the stomach, but not perhaps +until half an hour after eating. The dilute hydrochloric acid in the +stomach, which accumulates some time after eating, is not active in +this hydrolysis. On the contrary the amylolytic ferment of the saliva +is somewhat enfeebled by the presence of an acid. The active principle +of the saliva is ptyalin. + +The diastatic hydrolysis of starch has already been described (=179=). +It is best secured at a somewhat higher temperature than that of the +human stomach. + +=546. Aliphalytic Ferments.=—In the hydrolysis of glycerids in the +process of digestion the fat acids and glycerol are set free. Whether +the glycerids be completely hydrolyzed before absorption is not +definitely known. In certain cases where large quantities of oil have +been exhibited for remedial purposes, the fat acids and soaps have been +found in spherical masses in the dejecta[551] and have been mistaken for +gall stones. + +The fat which enters the chyle appears to be mostly unchanged, except +that it is emulsified.[552] The aliphalytic ferment can be prepared +from the fresh pancreas, preferably from animals that have not been +fed for forty hours before killing. It is important to prepare the +ferment entirely free of any trace of acid. The fresh glands are rubbed +to a fine paste with powdered glass and extracted for four days with +pure glycerol, to which one part of one per cent soda solution has +been added. The filtered liquor contains aliphalytic, proteolytic and +amylytic ferments, and is employed for saponification by shaking with +the fat to form an emulsion and keeping the mixture, with occasional +shaking, at a temperature of from 40° to 60°. The free acids can +be titrated or separated from the unsaponified fats by solution in +alcohol.[553] + +Heretofore it has not been possible to separate a pure aliphalytic +ferment from any of the digestive glands. The digestion of +carbohydrates and that of fats are intimately associated, and these +two classes of foods seem to play nearly the same rôle in the animal +economy. + +The aliphalytic ferments, prepared from the fresh pancreas, act also on +the glucosids and other ester-like carbohydrate bodies. Since the fats +may be regarded as ethers, the double action indicates the similarity +of composition in the two classes of bodies.[554] The aliphalytic +ferments exist also in plants and have been isolated from rape seed.[555] + +=547. Proteolytic Ferments.=—The most important process in artificial +digestion is the one relating to the action of the ferments on proteid +matters. The hydrolysis of fats and carbohydrates by natural ferments +takes place best in an alkaline medium, while in the case of proteids +when pepsin is used an acid medium is preferred. Since the acidity +of the stomach is due chiefly to hydrochloric, that acid is employed +in artificial digestion. The hydrolyte used is uniformly the natural +ferment of the gastric secretions, _viz._, pepsin; but this is often +followed by the pancreatic ferment, (pancreatin, trypsin) in an +alkaline medium. During the digestion, the proteids are changed into +peptones, and the measurement of this change determines the degree of +digestion. The total proteid matter is determined in the sample, and +after the digestion is completed, the soluble peptones are removed +by washing and the residual insoluble proteid matter determined by +moist combustion. The difference in the two determinations shows the +quantity of proteid matter digested. The investigations of Kühn on the +digestion of proteids may be profitably consulted.[556] For a summary +of digestion experiments in this country the résumé prepared by Gordon +may be consulted.[557] The method followed in this laboratory is fully +described by Bigelow and Hamilton.[558] + +=548. Ferments Employed.=—Both the pepsins of commerce and those +prepared directly from the stomachs of pigs may be used. The commercial +scale pepsin is found, as a rule, entirely satisfactory, and more +uniform results are secured by its use than from pepsin solutions made +from time to time from pig stomachs. In the preparation of the pepsin +solution one gram of the best scale pepsin is dissolved in one liter +of 0.33 per cent hydrochloric acid. Two grams of the sample of food +products, in fine powder, are suspended in 100 cubic centimeters of the +solution and kept, with frequent shaking, at a temperature of 40° for +twelve hours. The contents of the flask are poured on a wet filter, the +residue on the filter well washed with water not above 40°, the filter +paper and its contents transferred to a kjeldahl flask and the residual +nitrogen determined and multiplied by 6.25 to get the undigested +proteid matter. A large number of digestions can be conducted at once +in a bath shown in Fig. 117.[559] The quantity of water in the bath +should be as large as possible. + +=549. Digestion in Pepsin and Pancreatin.=—The digestion of the +proteids is not as a rule wholly accomplished by the stomach juices, +and, therefore, in order to secure in artificial digestion results +approximating those produced in the living organism, it is necessary +to follow the treatment with pepsin by a similar one with the pancreas +juices. The method employed in this laboratory is essentially that of +Stutzer modified by Wilson.[560] + +[Illustration: FIG. 117. BATH FOR ARTIFICIAL DIGESTION.] + +The residue from the pepsin digestion, after washing, is treated for +six hours at near 40° with 100 cubic centimeters of pancreas solution, +prepared as follows: + +Free the pancreas of a healthy steer of fat, pass it through a sausage +grinder, rub one kilogram in a mortar with fine sand and allow to stand +for a day or longer. Add three liters of lime water, one of glycerol, +of 1.23 specific gravity, and a little chloroform and set aside for +six days. Separate the liquor by pressure in a bag and filter it +through paper. Before using, mix a quarter of a liter of the filtrate +with three-quarters of a liter of water and five grams of dry sodium +carbonate, or its equivalent crystallized, heat from 38° to 40° for +two hours and filter.[561] In order to avoid the trouble of preparing +the pancreas solution pure active pancreatin may be used.[562] One and +a half grams of pure pancreatin and three grams of sodium carbonate +are dissolved in one liter of water and 100 cubic centimeters of this +solution are used for each two grams of the sample. In all cases where +commercial pepsin and pancreatin are used, their activity should be +tested with bodies such as boiled whites of eggs, whose coefficient of +digestibility is well known and those samples be rejected which do not +prove to have the required activity.[563] + +=550. Digestion in Pancreas Extract.=—In order to save the time +required for successive digestions in pepsin and pancreatin Niebling +has proposed to make the digestion in the pancreas extract alone.[564] +This process and also a slight modification of it have been used with +success by Bigelow and McElroy.[565] Two grams of the sample are washed +with ether and placed in a digestion flask with 100 cubic centimeters +of two-tenths per cent hydrochloric acid. The contents of the flask +are boiled for fifteen minutes, cooled, and made slightly alkaline +with sodium carbonate. One hundred cubic centimeters of the unfiltered +pancreas solution, prepared as directed above, are added and the +digestion continued at 40° for six hours. The residue is thrown on a +filter, washed, and the nitrogen determined. The method is simplified +by the substitution of active commercial pancreatin for pancreas +extract. The solution of the ferment is made of the same strength as is +specified above. + +=551. Artificial Digestion of Cheese.=—The artificial digestion of +cheese is conducted by Stutzer as follows:[566] + +The digestive liquor is prepared from the fresh stomachs of pigs by +cutting them into fine pieces and mixing with five liters of water and +100 cubic centimeters of hydrochloric acid for each stomach. To prevent +decomposition, two and a half grams of thymol, previously dissolved in +alcohol, are added to each 600 cubic centimeters of the mixture. The +mixture is allowed to stand for a day with occasional shaking, poured +into a flannel bag and the liquid portion allowed to drain without +pressing. The liquor obtained in this way is filtered, first through +coarse and then through fine paper, and when thus prepared will keep +several months without change. It is advisable to determine the content +of hydrochloric acid in the liquor by titration and this content should +be two-tenths of a per cent. The cheese to be digested is mixed with +sand as previously described, freed of fat by extraction with ether, +and a quantity corresponding to five grams of cheese placed in a +beaker, covered with half a liter of the digestive liquor and kept at +a temperature of 40° for forty-eight hours. At intervals of two hours +the flasks are well shaken and five cubic centimeters of a ten per +cent solution of hydrochloric acid added and this treatment continued +until the quantity of hydrochloric acid amounts to one per cent. After +the digestion is finished, the contents of the beaker are thrown on a +filter, washed with water and the nitrogen determined in the usual way +in the residue. By allowing the pepsin solution to act for two days as +described above, the subsequent digestion with pancreas solution is +superfluous. + +=552. Suggestions Regarding Manipulation.=—The filter papers should +be as quick working as possible to secure the separation of all +undissolved particles. They should be of sufficient size to hold the +whole contents of the digestion flask at once, since if allowed to +become empty and partially dry, filtration is greatly impeded. The +residue should be dried at once if not submitted immediately to moist +combustion. After drying, the determination of the nitrogen can be +made at any convenient time. Beaker flasks, _i. e._, lip erlenmeyers +with a wide mouth are most convenient for holding the materials during +digestion. The flasks are most conveniently held by a crossed rubber +band attached at either end to pins in the wooden slats extending +across the digestive bath. The bath should be suspended by cords from +supports on the ceiling and a gentle rotatory motion imparted to it +resembling the peristaltic action attending natural digestion. + +=553. Natural Digestion.=—The digestion of foods by natural processes +is determined chiefly by the classes of ferments already noted. The +principle underlying digestive experiments with the animal organism +may be stated as follows: A given weight of food of known composition +is fed to a healthy animal under the conditions of careful control and +preparation already mentioned. The solid dejecta of the animal during a +given period are collected and weighed daily, being received directly +from the animal in an appropriate bag, safely secured, as is shown in +the accompanying figure. The dejecta are weighed, dried, ground to a +fine powder, mixed and a representative part analyzed. The difference +between the solid bodies in the dejecta and those given in the food +during the period of experiment represents those nutrients which have +been digested and absorbed during the passage of the food through the +alimentary canal. The urine, containing solid bodies representing the +waste of the animal organism, does not require to be analyzed for the +simple control of digestive activity outlined above. In a complete +determination of this kind the exhalations from the surface of the +body and from the lungs are also determined. In the latter case the +human animal is selected for the experiment; in the former it is more +convenient to employ the lower animals, such as the sheep and cow. + +The arrangement of the stalls and of the apparatus for collecting the +excreta should be such as is both convenient and effective.[567] + +The method of constructing a bag for attachment to a sheep is shown in +Fig. 118. It is made according to the directions given by Gay, of heavy +cloth and in such a way as to fit closely the posterior parts of the +animal.[568] When attached, its appearance is shown in Fig. 119. + +[Illustration: FIG. 118.—BAG FOR COLLECTING FECES.] + +[Illustration: FIG. 119.—FECAL BAG ATTACHMENT.] + +Healthy animals in the prime of life are used, and the feeding +experiments are conducted with as large a number of animals as +possible, in order to eliminate the effects of idiosyncrasy. The food +used is previously prepared in abundant quantity and its composition +determined by the analysis of an average sample. + +The feeding period is divided into two parts. In the first part the +animal is fed for a few days with the selected food until it is certain +that all the excreta are derived from the nutrients used. In the second +part the same food is continued and the excreta collected, weighed, the +moisture determined, and the total weight of the water-free excreta +ascertained. The first part should be of at least seven and the second +of at least five days duration. The urine and dung are analyzed +separately. Males are preferred for the digestion experiments because +of the greater ease of collecting the urine and feces without mixing. +For ordinary purposes the feces only are collected. The methods of +analysis do not differ from those described for the determination of +the usual ingredients of a food. + +_Example._—The following data taken from the results of digestive +experiments, obtained at the Maine Station, will illustrate the method +of comparing the composition of the food with that of the feces and +of determining the degree of digestion which the proteids and other +constituents of the food have undergone. + + COMPOSITION OF MAIZE FODDER AND OF FECES + THEREFROM AFTER FEEDING TO SHEEP. + + BEFORE DRYING. + Water, Ash, Proteid, Fiber, Fat, Undetermined, + per per per per per per + Food. cent. cent. cent. cent. cent. cent. + Sweet maize 83.85 1.13 2.18 4.14 0.62 8.08 + Feces 72.01 ... ... ... ... ... + + DRY. + Ash, Proteid, Fiber, Fat, Undetermined, + per per per per per + Food. cent. cent. cent. cent. cent. + Sweet maize 7.01 13.52 25.63 3.86 49.98 + Feces 14.42 17.52 19.34 2.68 46.04 + + DAILY WEIGHTS. + Green, Dry, + Food. grams. grams. + Sweet maize 2521 407 + Feces 445 125 + + PER CENT DIGESTED. + Food. Ash, Proteid, Fiber, Undetermined, Fat, + Sweet maize 37.0 60.2 76.9 71.8 78.3 + +In the above instance it is seen that the coefficient of digestibility +extended from 37.0 per cent in the case of the mineral components +of the food, to 78.3 per cent in the case of the fats. These data +are taken only from the results obtained from a single sheep and one +article of food. The mean data secured from two animals and three kinds +of maize fodder show the following per cents of digestibility: Ash +39.4, proteid 61.8, fiber 76.7, undetermined matters 72.1, fat 76.4. +The undetermined matters are those usually known as nitrogen free +extract and composed chiefly of pentosans and other carbohydrates.[569] + +=554. Natural Digestibility of Pentosans.=—The digestibility of +pentosan bodies in foods under the influence of natural ferments +has been investigated by Lindsey and Holland.[570] The feeding and +collection of the feces is carried on as described above and the +relative proportions of pentosan bodies in the foods and feces +determined by estimating the furfuraldehyd as prescribed in paragraph +=150=.[571] + + +PRESERVED MEATS. + +=555. Methods of Examination.=—In general the methods of examination +are the same as those applied in the study of fresh meats. The contents +of water, salt and other preservatives, fat and nitrogenous matters are +of most importance. When not already in a fine state, the preserved +meats are run through meat cutters until reduced to a fine pulp. Most +potted meats are already in a state of subdivision well suited to +analytical work. The composition of preserved meats has been thoroughly +studied in this laboratory by Davis.[572] + +=556. Estimation Of Fat.=—Attention has already been called to +the difficulty of extracting the fat from meats by ether or other +solvents.[573] In preserved meats, as well as in fresh, it is preferable +to adopt some method which will permit of the decomposition of the +other organic matters and the separation of the fat in a free state. +The most promising methods are those employed in milk analyses for +the solution of nitrogenous matters. Sulfuric or hydrochloric acid +may be used for this purpose, preference being given to sulfuric. The +separated fats may be taken up with ether or separated by centrifugal +action. A method of this kind for preserved meats, suggested by +Hefelmann, is described below. + +About six grams of the moist preserved meat are placed in a calibrated +test tube and dissolved in twenty-five cubic centimeters of fuming +hydrochloric acid. The tube is placed in a water bath, quickly heated +to boiling and kept at that temperature for half an hour. About twenty +cubic centimeters of cold water are added and the temperature lowered +to 30°, then twenty cubic centimeters of ether and the tube gently +shaken to promote the solution of the fat. When the ether layer has +separated, its volume is read and an aliquot part removed by means of a +pipette, dried and weighed. The separation of the ethereal solution is +greatly promoted by whirling. + +The mean proportions of the ingredients of preserved meats are about as +follows: + + Per cent. + + Water 67.0 + Dry matter 33.0 + +Of which + + Nitrogenous bodies 19.0 + Fats 10.5 + Ash and undetermined 3.5 + +=557. Meat Preservatives.=—Various bodies are used to give taste and +color to preserved meats and to preserve them from fermentation. The +most important of these bodies are common salt, potassium and sodium +nitrates, sulfurous, boric, benzoic and salicylic acids, formaldehyd, +saccharin and hydronaphthol. A thorough study of the methods of +detecting and isolating these bodies has been made in this laboratory +by Davis and the results are yet to be published as a part of Bulletin +13. + + +DETERMINATION OF NUTRITIVE VALUES. + +=558. Nutritive Value of Foods.=—The value of a food as a nutrient +depends on the amount of heat it gives on combustion in the tissues of +the body, _i. e._ oxidation, and in its fitness to nourish the tissues +of the body, to promote growth and repair waste. The foods which +supply heat to the body are organic in their nature and are typically +represented by fats and carbohydrates. The foods which promote growth +and supply waste are not only those which preeminently supply heat, +but also include the inorganic bodies and organic nitrogenous matters +represented typically by the proteids. It is not proper to say that +one class of food is definitely devoted to heat forming and another to +tissue building, inasmuch as the same substance may play an important +rôle in both directions. As heat formers, carbohydrates and proteids +have an almost equal value, as measured by combustion in oxygen, while +fat has a double value for this purpose. The assumption that combustion +in oxygen forms a just criterion for determining the value of a food +must not be taken too literally. There are only a few bodies of the +vast number which burn in oxygen that are capable of assimilation +and oxidation by the animal organism. Only those parts of the food +that become soluble and assimilable under the action of the digestive +ferments, take part in nutrition and the percentage of food materials +digested varies within wide limits but rarely approaches 100. It may +be safely said that less than two-thirds of the total food materials +ingested are dissolved, absorbed, decomposed and assimilated in the +animal system. We have no means of knowing how far the decomposition +(oxidation) extends before assimilation, and therefore no theoretical +means of calculating the quantity of heat which is produced during the +progress of digestion. The vital thermostat is far more delicate than +any mechanical contrivance for regulating temperature and the quantity +of food, in a state of health, converted into heat, is just sufficient +to maintain the temperature of the body at a normal degree. Any excess +of heat produced, as by violent muscular exertion, is dissipated +through the lungs, the perspiration and other secretions of the body. + +Pure cellulose or undigestible fiber, when burned in oxygen, will +give a thermal value approximating that of sugar, but no illustration +is required to show that when taken into the system the bodily heat +afforded by it is insignificant in quantity. + +Thermal values, therefore, have little comparative usefulness +in determining nutritive worth, except when applied to foods of +approximately the same digestive coefficient. + +=559. Comparative Value of Food Constituents.=—It has already been +noted that, judged by combustion in oxygen, carbohydrates and proteids +have about half the thermal value possessed by fats. Commercially, +the values of foods depend in a far greater degree on their flavor +and cooking qualities than upon the amount of nutrition they +contain. Butter fat, which is scarcely more nutritious than tallow, +is worth twice as much in the market, while the prices paid for +vegetables and fruits are not based to any great extent on their food +properties.[574] In cereals, especially in wheat, the quantity of fat is +relatively small, and starch is the preponderating element. In meats, +carbohydrates are practically eliminated and fats and proteids are the +predominating constituents. + +In the markets, fats and proteids command far higher prices than +sugars and starches. The relative commercial food value of a cereal +may be roughly approximated by multiplying the percentages of fat and +protein by two and a half and adding the products to the percentage of +carbohydrates less insoluble fiber. This method was adopted in valuing +the cereals at the World’s Columbian Exposition.[575] + +=560. Nutritive Ratio.=—In solid foods the nutritive ratio is that +existing between the percentage of proteids and that of carbohydrates, +increased by multiplying the fat by two and a half and adding the +product. In a cereal containing twelve per cent of protein, seventy-two +of carbohydrates, exclusive of fiber, and three of fat, the ratio is +12: 72 + 3 × 2.5 = 6.5. Instead of calculating the nutritive ratio +directly from the data obtained by analysis, it may be reckoned from +the per cents of the three substances in the sample multiplied by their +digestive coefficient. Since the relative amounts of proteids, fats and +carbohydrates digested do not greatly differ, the numerical expression +of the nutritive ratio is nearly the same when obtained by each of +these methods of calculation. + +Where the proportion of protein is relatively large the ratio is called +narrow, 1: 4 ... 6. When the proportion of protein is relatively small +the ratio is called broad 1: 8 ... 12. In feeding, the nutritive ratio +is varied in harmony with the purpose in view, a narrow ratio favoring +the development of muscular energy, and a wide one promoting the +deposition of fat and the development of heat. These principles guide +the scientific farmer in mixing rations for his stock, the work horses +receiving a comparatively narrow and the beeves a relatively wide ratio +in their food. + +=561. Calorimetric Analyses of Foods.=—The general principles of +calorimetry have been already noticed. The theoretical and chemical +relations of calorimetry have been fully discussed by Berthelot, +Thomsen, Ostwald and Muir.[576] In the analyses of foods the values +as determined by calculation or combustion are of importance in +determining the nutritive relations. + +Atwater has presented a résumé of the history and importance of +the calorimetric investigations of foods to which the analyst is +referred.[577] + +In the computation of food values the percentages of proteids, +carbohydrates and fats are determined and the required data obtained +by applying the factors 4100, 5500 and 9300 calories for one gram of +carbohydrates, proteids and fats respectively. + +For most purposes the computed values are sufficient, but it is well to +check them from time to time by actual combustions in a calorimeter. + +=562. Combustion in Oxygen.=—The author made a series of combustions +of carbonaceous materials in oxygen at the laboratory of Purdue +University in 1877, the ignition being secured by a platinum wire +rendered incandescent by the electric current. The data obtained were +unsatisfactory on account of the crudeness of the apparatus. The +discovery of the process of burning the samples in oxygen at a high +pressure has made it possible to get expressions of thermal data which +while not yet perfect, possess a working degree of accuracy. The best +form of bomb calorimeter heretofore employed is that of Hempel, as +modified by Atwater and Woods.[578] + +A section of this calorimeter, with all the parts in place, is shown in +Fig. 120. + +In the figure the steel cylinder _A_, about 12.5 centimeters deep and +6.2 in diameter, represents the chamber in which the combustion takes +place. Its walls are about half a centimeter thick and it weighs about +three kilograms. It is closed, when all the parts are ready and the +sample in place, by the collar _C_, which is secured gas tight by means +of a powerful spanner. The cover is provided with a neck _D_ carrying a +screw _E_ and a valve screw _F_. In the neck _D_, where the bottom of +the cylinder screw _E_ rests, is a shoulder fitted with a lead washer. +Through _G_ the oxygen used for combustion is introduced. The upper +edge of the cylinder _A_ is beveled and fits into a groove in the cover +_B_, carrying a soft metal washer. To facilitate the screwing on of the +cover, ball bearings _KK_, made of hard steel, are introduced between +the collar and the cover. The platinum wires _H_ and _I_ support the +platinum crucible holding the combustible bodies which are ignited by +raising the spiral iron wire connecting them to the temperature of +fusion by an electric current. The combustion apparatus when charged is +immersed in a metal cylinder _M_, containing water and resting on small +cylinders of cork. The water is stirred by the apparatus _LL_. The +cylinder _M_ is contained in two large concentric cylinders, _N_, _O_, +made of non-conducting materials and covered with disks of hard rubber. +The space between _O_ and _N_ may be filled with water. The temperature +is measured by the thermometer _P_, graduated to hundredths of a degree +and the reading is best accomplished by means of a cathetometer. + +[Illustration: FIG. 120. HEMPEL AND ATWATER’S CALORIMETER.] + +=563. The Williams Calorimeter.=—The calorimeter bomb has been +improved by Williams by making it of aluminum bronze of a spheroidal +shape. The interior of the bomb is plated with gold. By an ingenious +arrangement of contacts the firing is secured by means of a permanently +insulated electrode fixed in the side of the bomb. The calorimetric +water, as well as that in the insulating vessel, is stirred by means +of an electrical screw so regulated as to produce no appreciable +degree of heat mechanically. The combustion is started by fusing a +fine platinum wire of definite length and thickness by means of an +electric current. The heat value of this fusion is determined and the +calories produced deducted from the total calories of the combustion. +The valve admitting the oxygen is sealed automatically on breaking +connection with the oxygen cylinder. The effluent gases, at the end of +the combustion, may be withdrawn through an alkaline solution and any +nitric acid therein thus be fixed and determined.[579] + +=564. Manipulation and Calculation.=—The material to be burned is +conveniently prepared by pressing it into tablets. The oxygen is +supplied from cylinders, of which two should be used, one at a pressure +of more than twenty atmospheres. By this arrangement a pump is not +required. + +In practical use, a known weight of the substance to be burned is +placed in the platinum capsule, the cover of the bomb screwed on, after +all adjustments have been made, and the apparatus immersed in the water +contained in _M_, which should be about 2° below room temperature. All +the covers are placed in position and the temperature, of the water in +_M_ begins to rise. Readings of the thermometer are taken at intervals +of about one minute for six minutes, at which time the temperature of +the bomb and calorimetric water may be regarded as sensibly the same. +The electric current is turned on, the iron wire at once melts, ignites +the substance and the combustion rapidly takes place. In the case of +bodies which do not burn readily Atwater adds to them some naphthalene, +the thermal value of which is previously determined. The calories due +to the combustion of the added naphthalene are deducted from the total +calories obtained. + +The temperature of the water in _M_ rises rapidly at first, and +readings are made at intervals of one minute for five minutes, and then +again after ten minutes. The first of the initial readings, the one at +the moment of turning on the current, and the last one mentioned above +are the data from which the correction, made necessary by the influence +of the temperature of the room, is calculated by the following +formulas.[580] + +The preliminary readings of the thermometer at one minute intervals +are represented by _t_₁, _t_₂, _t_₃ ... _t_ₙ₁. The last observation +tₙ₁ is taken as the beginning temperature of the combustion and is +represented in the formulas for calculations by Θ₁. The readings after +combustion are also made at intervals of one minute, and are designated +by Θ₂, Θ₃ ... Θₙ. The readings are continued until there is no observed +change between the last two. Generally this is secured by five or six +readings. + +The third period of observations begins with the last reading Θₙ, which +in the next series is represented by _tʹ_₁, _tʹ_₂ ... _tʹ_ₙ₂. + +In order to make the formulas less cumbersome let + + _t_ₙ₁ - _t_₁ + ------------ = _v_, + _n_₁ - 1 + + _tʹ_ₙ₁ - _tʹ_₁ + ------------- = _vʹ_, + _n_₂ - 1 + + _t_₁ + _t_₂ + _t_₃ ... _t_ₙ₁ + --------------------------- = t, + _n_₁ + + _tʹ_₁ + _tʹ_₂ + _tʹ_₃ ... _tʹ_ₙ₂ + and -------------------------------- = _tʹ_. + _n_₂ + +The correction to be made to the difference between Θₙ - Θ₁ for the +influence of the outside temperature is determined by the formula of +Regnault-Pfaundler, which is as follows: + + _v_ - _vʹ_ ⁿ⁻¹ Θₙ + Θ₁ + ∑ Δ_t_ = -------------- ( ∑ Θ_r_ + -------- - _nt_) - (_n_ - 1)_v_, + _tʹ_ - _t_ ₁ 2 + + ⁿ⁻¹ + in which ∑ Θ_r_ + ₁ + +is calculated from the observation of the thermometer Θ₁, Θ₂ etc., +made immediately after the combustion. It is equal to the sum of +observations Θ₁, Θ₂ etc., increased by an arbitrary factor equivalent +to (Θ₂ - Θ₁)/9, which is made necessary by reason of the irregularity +of the temperature increase during the first minute after combustion, +the mean temperature during that minute being somewhat higher than the +mean of the temperatures at the commencement and end of that time. + +The quantity of heat formed by the combustion of the iron wire used for +igniting the sample is to be deducted from the total heat produced. +This correction may be determined once for all, the weight of the +iron wire used being noted and that of any unburned portion being +ascertained after the combustion. + +Ten milligrams of iron, on complete combustion, will give sixteen +calories. + +In the combustion of substances containing nitrogen, or in case the +free nitrogen of the air be not wholly expelled from the apparatus +before the burning, nitric acid is formed which is dissolved by the +water produced. + +The heat produced by the solution of nitric acid in water is 14.3 +calories per gram molecule. The quantity of nitric acid formed is +determined by titration and a corresponding reduction made in the total +calculated calories. + +In the titration of nitric acid it is advisable to make use of an +alkaline solution, of which one liter is equivalent to 4.406 grams of +nitric acid. One cubic centimeter of the reagent is equivalent to a +quantity of nitric acid represented by one calorie. + +Since the materials of which the bomb is composed have a specific heat +different from that of water, it is necessary to compute the water +thermal value of each apparatus. + +The hydrothermal equivalent of the whole apparatus is most simply +determined by immersing it at a given temperature in water of a +different temperature.[581] With small apparatus this method is quite +sufficient, but there are many difficulties attending its application +to large systems weighing several kilograms. In these cases the +hydrothermal equivalent may be calculated from the specific heats of +the various components of the apparatus. + +In calculating these values the specific heats of the various +components of the apparatus are as follows: + + Brass 0.093 + Steel 0.1097 + Platinum 0.0324 + Copper 0.09245 + Lead 0.0315 + Oxygen 0.2389 + Glass 0.190 + Mercury 0.0332 + Hard rubber 0.33125 + +_Example._—It is required to calculate the hydrothermal value of a +calorimeter composed of the following substances: + + Hydrothermal + value. + Steel bomb and cover, 2850 grams × 0.1097 312.65 grams. + Platinum lining, capsule and wires, 120 grams × 0.0324 3.89 ” + Lead washer, 100 grams × 0.0315 3.15 ” + Brass outer cylinder, 500 grams × 0.093 46.50 ” + Mercury in thermometer, 10 grams × 0.0332 0.33 ” + Glass (part of thermometer in water), 10 grams × 0.19 1.90 ” + Brass stirring apparatus (part in water), 100 grams + × 0.093 9.30 ” + ------ + Total water value of system 377.72 ” + +When a bomb of 300 cubic centimeters capacity is filled with oxygen at +a pressure of twenty-four atmospheres it will hold about ten grams of +the gas, equivalent to a water value of 2.40 grams. Hence the water +value of the above system when charged, assuming the bomb to be of the +capacity mentioned, is 380.12 grams. + +If the cylinder holding the water be made of fiber or other +non-conducting substance, its specific heat is best determined by +filling it in a known temperature with water at a definite different +temperature. + +It is advisable to have the water cylinder of such a size as to permit +the use of a quantity of water for the total immersion of the bomb +which will weigh, with the water value of the apparatus, an even number +of grams. In the case above, 2622.28 grams of water placed in the +cylinder will make a water value of 3,000 grams, which is one quite +convenient for calculation. + +=565. Computing the Calories of Combustion.=—In the preceding paragraph +has been given a brief account of the construction of the calorimeter +and of the methods of standardizing it and securing the necessary +corrections in the data directly obtained in its use. An illustration +of the details of computing the calories of combustion taken from the +paper of Stohmann, Kleber and Langbein, will be a sufficient guide for +the analyst in conducting the combustion and in the use of the data +obtained.[582] + +Weight of substance burned, 1.07 grams. + +Water value of system (water + apparatus), 2,500 grams. + +Preliminary thermometric readings, _t_₁ = 26.8; _t_₂ = 27.2; _t_₃ = +27.7; _t_₄ = 28.1; _t_₅ = 28.5; _t_ₙ₁ = 28.9. + +Thermometric reading after combustion, Θ₁ = 28.9; Θ₂ = 202; Θ₃ = 213; +Θ₄ = 214.2; Θₙ = 214.0. + +Final thermometric readings, _tʹ_₁ = 214.0; _tʹ_₂ = 213.8; _tʹ_₃ = +213.6; _tʹ_₄ = 213.5; _tʹ_₅ = 213.3; _tʹ_₆ = 213.1; _tʹ_₇ = 212.9; +_tʹ_₈ = 212.7; _tʹ_₉ = 212.6; _tʹ_₁₀ = 212.4; _tʹ_ₙ₂ = 212.2. + +From the formulas given above the following numerical values are +computed: + + _v_ = 0.42. + _vʹ_ = -0.18. + _t_ = 27.9. + _tʹ_ = 213.1. + _n_ = 5. + + ⁿ⁻¹ Θ₂ - Θ₁ + ∑ Θ_r_ = Θ₁ + Θ₂ + Θ₃ + Θ₄ + ------- = 667. + ₁ 9 + +Substituting these values in the formula of Regnault-Pfaundler, the +value of the correction for the influence of the external air is + + 0.42 - (-0.18) 214 + 29 + ∑ Δt = [--------------- (677 + --------- - (5 × 27.9)) + 213.1 - 27.9 2 + + - (4 × 0.42)] = 0.45, + +which is to be added to the end temperature (Θₙ = 214.0). + +The computation is then made from the following data: + + Corrected end temperature (Θₙ + 0.45) 214.45 = 15°.3699 + Beginning temperature (Θ₁) 28.90 = 12°.8406 + Increase in temperature 185.55 = 2°.5293 + Total calories 2.5293 × 25000 = 6323.3 + Of which there were due to iron burned 9.1 + ” ” ” ” nitric acid dissolved 8.2 + Total calories due to one gram of substance 5893.5 + +The thermometric readings are given in the divisions of the thermometer +which in this case are so adjusted as to have the number 28.90 +correspond to 12°.8406, and each division is nearly equivalent to +0°.014 thermometric degree. + +The number of calories above given is the proper one when the +computation is made to refer to constant volume. By reason of the +consumption of oxygen and the change of temperature, although mutually +compensatory, the pressure may be changed at the end of the operation. +The conversion of the data obtained at constant volume referred to +constant pressure may be made by the following formula, in which [_Q_] +represents the calories from constant volume and _Q_ the desired data +for constant pressure, _O_ the number of oxygen atoms, _H_ the number +of hydrogen atoms in a molecule of the substance, and 0.291 a constant +for a temperature of about 18°, at which the observations should be +made. + + _H_ + _Q_ = [_Q_] + (--- - _O_) 0.291. + 2 + +=566. Calorimetric Equivalents.=—By the term calorie is understood the +quantity of heat required to raise one gram of water, at an initial +temperature of about 18°, one degree. The term ‘Calorie’ denotes the +quantity of heat, in like conditions, required to raise one kilogram of +water one degree. + +For purposes of comparison and for assisting the analyst in adjusting +his apparatus so as to give reliable results, the following data, +giving the calories of some common food materials, are given: + + Substance. Chemical composition. + Proteids. Calories. C. H. N. S. O. + Per Per Per Per Per + cent. cent. cent. cent. cent. + Serum albumin 5917.8 53.93 7.65 15.15 1.18 22.09 + Casein 5867.0 54.02 7.33 15.52 0.75 22.38 + Egg albumin 5735.0 52.95 7.50 15.19 1.51 22.85 + Meat free of + fat and + extracted + with water 5720.0 52.11 6.76 18.14 0.96 22.66 + Peptone 5298.8 50.10 6.45 16.42 1.24 25.79 + Proteids (mean) 5730.8 52.71 7.09 16.02 1.03 23.15 + Glycerids. + Butterfat 9231.3 + Linseed oil 9488.0 + Olive oil 9467.0 + + Carbohydrates. Formula. + Arabinose 3722.0 C₅H₁₀O₅ + Xylose 3746.0 C₅H₁₀O₅ + Dextrose 3742.6 C₆H₁₂O₆ + Levulose 3755.0 C₆H₁₂O₆ + Sucrose 3955.2 C₁₂H₂₂O₁₁ + Lactose 3736.8 C₁₂H₂₂O₁₁ + H₂O + Maltose 3949.3 C₁₂H₂₂O₁₁ + +=567. Distinction between Butter and Oleomargarin.=—Theoretically +the heats of combustion of butter fat and oleomargarin are different +and de Schweinitz and Emery propose to utilize this difference for +analytical purposes.[583] The samples of pure butter fat examined by +them afforded 9320, 9327 and 9362 calories, respectively. The calories +obtained for various samples of oleomargarin varied from 9574 to +9795. On mixing butter fat and oleomargarin, a progressive increase +in calorimetric power is found, corresponding to the percentage of the +latter constituent. Lards examined at the same time gave from 9503 to +9654 calories. + + +FRUITS, MELONS AND VEGETABLES. + +=568. Preparation of Sample.=—Fresh fruits and vegetables are most +easily prepared for analysis by passing them through the pulping +machine described on page 9. Preliminary to the pulping they should +be separated into skins, cores, seeds and edible portions, and the +respective weights of these bodies noted. Each part is separately +reduced to a pulp and, at once, a small quantity of the well mixed +substance placed in a flat bottom dish and dried, first at a low +temperature, and finally at 100°, or somewhat higher, and the +percentage of water contained in the sample determined. The bulk of +the sample, three or four kilograms, is dried on a tray of tinned or +aluminum wire, first at a low and then at a high temperature, until all +or nearly all the moisture is driven off. The dried pulp is then ground +to as fine a powder as possible and subjected to the ordinary processes +of analysis; _viz._, the determination of the moisture, ash, nitrogen, +fiber, fat and carbohydrates. + +In this method of analysis it is customary to determine the +carbohydrates, exclusive of fiber, by subtracting the sum of the per +cents of the other constituents and the nitrogen multiplied by 6.25 +from 100. + +=569. Separation of the Carbohydrates.=—It is often desirable to +determine the relative proportions of the more important carbohydrates +which are found in fruits and vegetables. The pentoses and pentosans +are estimated by the method described in paragraph =150=. The cane +sugar, dextrose and levulose are determined by extracting a portion of +the substance with eighty per cent alcohol and estimating the reducing +sugars in the extract before and after inversion by the processes +described in paragraphs =238-251=. The percentages of sugars deducted +from the percentage of carbohydrates, exclusive of fiber, give the +quantity of gums, pentosans, cellulose and pectose bodies present. + +Pectose exists chiefly in unripe fruits. By the action of the fruit +acids and of a ferment, pectose, in the process of ripening, is +changed into pectin and similar hydrolyzed bodies soluble in water. +The gelatinous properties of boiled fruits and fruit juices are due to +these bodies, boiling accelerating their formation. In very ripe fruits +the pectin is completely transformed into pectic acids. The galactan is +estimated as described in =585=. + +=570. Examination of the Fresh Matter.=—To avoid the changes which take +place in drying fruits and vegetables, it is necessary to examine them +in the fresh state. The samples may be first separated into meat and +waste, as suggested above, or shredded as a whole. The moisture in the +pulp is determined as indicated above, and in a separate portion the +soluble matters are extracted by repeated treatment with cold water. +The seeds, skins, cellulose, pectose and other insoluble bodies are +thus separated from the sugars, pectins, pectic and other acids, and +other soluble matters. The insoluble residue is rapidly dried and the +relative proportions of soluble and insoluble matters determined. The +estimation of these bodies is accomplished in the usual way. + +=571. Examination of Fruit and Vegetable Juices.=—The fruits and +vegetables are pulped, placed in a press and the juices extracted. The +pressure should be as strong as possible and the press described in +paragraph =280= is well suited to this purpose. The specific gravity of +the expressed juice is obtained and the sucrose therein determined by +polarization before and after inversion. The reducing sugars and the +relative proportions of dextrose and levulose are determined in the +usual manner. In grape juice dextrose is the predominant sugar while in +many other fruits left hand or optically inactive sugars predominate. +Soluble gums, dextrin, pectin etc., may be separated from the sugars by +careful precipitation with alcohol, or the total solids, ash, nitrogen, +ether extract and acids be determined and the carbohydrates estimated +by difference. From the carbohydrates the total percentage of sugars is +deducted and the remainder represents the quantity of pectin, gum and +other carbohydrates present. + +=572. Separation of Pectin.=—Pectin exists in considerable quantities +in the juice of ripe fruits (pears) and may be obtained in an +approximately pure state from the juices by first removing proteids +by the careful addition of tannin, throwing out the soluble lime +salts with oxalic acid and precipitating the pectin with alcohol. On +boiling with water, pectin is converted into parapectin, which gives +a precipitate with lead acetate. Boiling with dilute acids converts +pectin into metapectin, which is precipitated by a barium salt. + +Pectic acid may be obtained by boiling an aqueous extract (carrots) +with sodium carbonate and precipitating the pectic with hydrochloric +acid. It is a jelly-like body and dries to a horny mass. + +=573. Determination of Free Acid.=—The free acid, or rather total +acidity of fruits, is determined by the titration of their aqueous +extracts or expressed juices with a set alkali. In common fruits and +vegetables the acidity is calculated to malic C₄H₆O₅, in grapes to +tartaric C₄H₆O₆, and in citrous fruits to citric acid C₆H₈O₇. Many +other acids are found in fruits and vegetables, but those mentioned are +predominant in the classes given. + +=574. Composition of Common Fruits.=—The composition of common +fruits in this country has been extensively investigated at the +California Station and the following data are derived chiefly from its +bulletins.[584] + + Name. Total Rind Seed. Pulp. Juice. Total + weight. skin. sugars Sucrose + in in + juice. juice. + per per per cubic per per + grams. cent. cent. cent. centimeters. cent. cent. + Naval orange 300 28.4 27.7 107 9.92 4.80 + Mediterranean + sweet orange 202 27.0 0.8 24.0 86 9.70 4.35 + St. Michael’s + orange 138 19.2 1.6 25.9 65.4 8.71 3.48 + Malta Blood + orange 177 31.0 24.0 71.0 10.30 5.85 + Eureka lemon 104 32 0.12 24.5 38 2.08 0.57 + Flesh Per cent + Apricot 62.4 93.85 6.15 10.0 90.0 13.31 + Prune 25.6 94.2 5.8 21.2 78.8 20.0 + Plum 60.4 95.2 4.8 24.7 75.3 17.97 + Peach 185 93.8 6.2 22.5 77.5 17.0 + Skin Cores + Apple 183 17.0 7.0 10.26‡ 1.53‡ + + ‡ In whole fresh fruit. + -------------------------------------------------------------------- + In whole fruit. + /-----------------------------\ + Name. + Acid. Nitrogenous Water. Dry Ash. + bodies. organic + matter. + per per per per per + cent. cent. cent. cent. cent. + Naval orange 1.02 1.31 86.56 13.04 0.40 + Mediterranean + sweet orange 1.38 0.96 85.83 13.06 0.41 + St. Michael’s + orange 1.35 1.43 84.10 15.42 0.48 + Malta Blood + orange 1.61 1.05 84.50 15.05 0.45 + Eureka lemon 7.66 0.94 85.99 13.50 0.51 + + Apricot 0.68 1.25 85.16 14.35 0.49 + Prune 0.40 1.01 77.38 22.18 0.44 + Plum 0.48 1.33 77.43 22.04 0.53 + Peach 0.25 82.50 16.95 0.55 + + Apple[585] 0.11 86.43 13.28 0.29 + + +=575. Composition of Ash of Fruits.=—Two or three kilograms of the +dried sample are incinerated at a low temperature and burned to a white +ash in accordance with the directions given in paragraphs =28-32=. + +The composition of the ash is determined by the methods already +described.[586] + +The pure ash of some common whole fruits has the following +composition:[587] + + Name. Per Per Per Per Per Per Per + cent cent cent cent cent cent cent + pure potash. soda. lime. magnesia. ferric mangano- + ash oxid. manganic + in oxid. + fruit. + + Prune 0.47 63.83 2.65 4.66 5.47 2.72 0.39 + Apricot 0.51 59.36 10.26 3.17 3.68 1.68 0.37 + Orange 0.43 48.94 2.50 22.71 5.34 0.97 0.37 + Lemon 0.53 48.26 1.76 29.87 4.40 0.43 0.28 + Apple 1.44 35.68 26.09 4.08 8.75 1.40 + Pear 1.97 54.69 8.52 7.98 5.22 1.04 + Peach 4.90 27.95 0.23 8.81 17.66 0.55 + ------------------------------------------------------------ + Name. Per Per Per Per + cent cent cent cent + phosphorus sulfur silica. chlorin. + pentoxid. trioxid. + + Prune 14.08 2.68 3.07 0.34 + Apricot 13.09 2.63 5.23 0.45 + Orange 12.37 5.25 0.65 0.92 + Lemon 11.09 2.84 0.66 0.39 + Apple 13.59 6.09 4.32 + Pear 15.20 5.69 1.49 + Peach 43.63 0.37 + +=576. Dried Fruits.=—A method of preserving fruits largely practiced +consists in subjecting them, in thin slices or whole, to the action +of hot air until the greater part of the moisture is driven off. The +technique of the process is fully described in recent publications.[588] +It has been shown by Richards that fruit subjected to rapid evaporation +undergoes but little change aside from the loss of water.[589] + +In the analyses of dried fruits the methods already described are used. +The presence of pectin renders the filtration of the aqueous extract +somewhat difficult, and in many cases it is advisable to determine the +sugars present in the extract without previous filtration. + +=577. Zinc in Evaporated Fruits.=—Fruits are commonly evaporated on +trays made of galvanized iron. In these instances a portion of the zinc +is dissolved by the fruit acids, and will be found as zinc malate etc., +in the finished product. The presence of zinc salts is objectionable +for hygienic reasons, and therefore the employment of galvanized trays +should be discontinued. The presence of zinc in evaporated fruits may +be detected by the following process.[590] The sample is placed in +a large platinum dish and heated slowly until dry and in incipient +combustion. The flame is removed and the combustion allowed to proceed, +the lamp being applied from time to time in case the burning ceases. +When the mass is burned out it will be found to consist of ash and +char, which are ground to a fine powder and extracted with hydrochloric +or nitric acid. The residual char is burned to a white ash at a low +temperature, the ash extracted with acid, the soluble portion added to +the first extract and the whole filtered. The iron in the filtrate is +oxidized by boiling with bromin water and the boiling continued until +the excess of bromin is removed. A drop of methyl orange is placed in +the liquid and ammonia added until it is only faintly acid. The iron is +precipitated by adding fifty cubic centimeters of a solution containing +250 grams of ammonium acetate in a liter and raising the temperature to +about 80°. The precipitate is separated by filtration and washed with +water at 80° until free of chlorids. The filtrate is saturated with +hydrogen sulfid, allowed to stand until the zinc sulfid settles and +poured on a close filter. It is often necessary to return the filtrate +several times before it becomes limpid. The collected precipitate is +washed with a saturated solution of hydrogen sulfid containing a little +acetic acid. The precipitate and filter are transferred to a crucible, +dried, ignited and the zinc weighed as oxid. Small quantities of zinc +salts added to fresh apples which were dried and treated as above +described, were recovered by this method without loss. Other methods of +estimating zinc in dried fruits are given in the bulletin cited. + +Evaporated apples contain a mean content of 23.85 per cent of water and +0.931 per cent of ash. + +The mean quantity of zinc oxid found in samples of apples dried in +the United States is ten milligrams for each 100 grams of the fruit, +an amount entirely too small to produce any toxic effects. When zinc +exists in the soil it will be found as a natural constituent in the +crop.[591] + +=578. Composition of Watermelons and Muskmelons.=—In the examination of +melons a separation of the rind, seeds and meat is somewhat difficult +of accomplishment, since the line of demarcation is not distinct. +In watermelons the separation of rind and meat is made at the point +where the red color of the meat disappears. In muskmelons no such +definite point is found and in the examination of these they are taken +as a whole. The total moisture, ash and nitrogen may be determined +in the whole mass or in the separate portions. The sugars are most +conveniently determined in the expressed juices. The mean composition +of the melons given below is that obtained from analyses made in the +Department of Agriculture.[592] + + COMPOSITION OF MELONS. + Total + Total weight, Juice, proteids, Ash, + grams. per cent. per cent. per cent. + ------------------------------------------------------------- + Watermelons 10330 meat 83.99 6.12 0.37 + rind 81.02 + ------------------------------------------------------------- + Muskmelons 3407 80.23 6.45 0.57 + + COMPOSITION OF JUICE. + Sucrose in Reducing sugars Ash in + juice, in juice, juice, + per cent. per cent. per cent. + ------------------------------------------------------------- + Watermelons meat 1.92 meat 4.33 meat 0.31 + rind 0.34 rind 2.47 rind 0.38 + ------------------------------------------------------------- + Muskmelons 1.02 3.04 0.53 + + +TEA AND COFFEE. + +=579. Special Analysis.=—Aside from the examination of teas and coffees +for adulterants, the only special determinations which are required +in analyses are the estimation of the alkaloid (caffein) and of the +tannin contained therein. It is chiefly to the alkaloid that the +stimulating effects of the beverages made from tea and coffee are due. +The determination of the quantity of tannin contained in tea and coffee +is accomplished by the processes described under the chapter devoted to +that glucosid. + +The general analysis, _viz._, the estimation of water, ether extract, +total nitrogen, fiber, carbohydrates and ash, with the exceptions noted +above, is conducted by the methods which have already been given. + +For detailed instructions concerning the detection of adulterants of +tea and coffee the bulletins of the Chemical Division, Department of +Agriculture, may be consulted.[593] + +=580. Estimation of Caffein= (=Thein=).—The method adopted by Spencer, +after a thorough trial of all the usual processes for estimating this +alkaloid, is as follows:[594] To three grams of the finely powdered tea +or coffee, in a 300 cubic centimeter flask, add about a quarter of a +liter of water, slowly heat to the boiling point, using a fragment of +tallow to prevent frothing, and boil gently for half an hour. When +boiling begins, the flask should be nearly filled with hot water +and more added from time to time to compensate for the loss due to +evaporation. After cooling, add a strong solution of basic lead acetate +until no further precipitation is produced, complete the volume to the +mark with water, mix and throw on a filter. Precipitate the lead from +the filtrate by hydrogen sulfid and filter. Boil a measured volume of +this filtrate to expel the excess of hydrogen sulfid, cool and add +sufficient water to compensate for the evaporation. Transfer fifty +cubic centimeters of this solution to a separatory funnel and shake +seven times with chloroform. Collect the chloroform solution in a tared +flask and remove the solvent by gentle distillation. A safety bulb, +such as is used in the kjeldahl nitrogen method, should be employed to +prevent entrainment of caffein with the chloroform vapors. + +The extraction with chloroform is nearly complete after shaking out +four times; a delicate test, however, will usually reveal the presence +of caffein in the watery residue even after five or six extractions, +hence seven extractions are recommended for precautionary reasons. The +residual caffein is dried at 75° for two hours and weighed. + +The principal objection which has been made to Spencer’s method is +that the boiling with water is not continued for a sufficient length +of time. For the water extraction, Allen prescribes at least six hours +cohobation.[595] In this method six grams of the powdered substance +are boiled with half a liter of water for six hours in a flask, with +a condenser, the decoction filtered, the volume of the filtrate +completed to 600 cubic centimeters with the wash water, heated to +boiling, and four cubic centimeters of strong lead acetate solution +added, the mixture boiled for ten minutes, filtered and half a liter +of the filtrate evaporated to fifty cubic centimeters. The excess of +lead is removed with sodium phosphate and the filtrate and washings +concentrated to about forty cubic centimeters. The caffein is removed +by shaking four times with chloroform. Older but less desirable +processes are fully described by Allen.[596] + +In France this method is known as the process of Petit and Legrip, and +it has been worked out in great detail by Grandval and Lajoux and by +Petit and Terbat.[597] + +=581. Estimation of Caffein by Precipitation with Iodin.=—The caffein +in this method is extracted, the extract clarified by lead acetate and +the excess of lead removed as in Spencer’s process described above. The +caffein is determined in the acidified aqueous solution thus prepared, +according to the plan proposed by Gomberg, as follows:[598] + +Definite volumes of the aqueous solution of the caffein are acidulated +with sulfuric and the alkaloid precipitated by an excess of a set +solution of iodin in potassium iodid. After filtering, the excess of +iodin in an aliquot part of the filtrate is determined by titration +with a tenth normal solution of sodium thiosulfate. The filtration of +the iodin liquor is accomplished on glass wool or asbestos. The results +of the analyses are calculated from the composition of the precipitated +caffein periodid; _viz._, C₈H₁₀N₄O₂.HI.I₄. The weight of the alkaloid +is calculated from the amount of iodin required for the precipitation +by the equation 4I: C₈H₁₀N₄O₂ = 508: 194. From this equation it is +shown that one part of iodin is equivalent to 0.3819 part of caffein, +or one cubic centimeter of tenth normal iodin solution is equal to +0.00485 gram of iodin. + +In practice, it is recommended to divide the aqueous extract of the +alkaloid, prepared as directed above, into two portions, one of which +is treated with the iodin reagent without further preparation, and +the other after acidulation with sulfuric. After ten minutes, the +residual iodin is estimated in each of the solutions as indicated +above. The one portion, containing only the acetic acid resulting from +the decomposition of the lead acetate, serves to indicate whether +the aqueous solution of the caffein contains other bodies than that +alkaloid capable of forming a precipitate with the reagent, since the +caffein itself is not precipitated even in presence of strong acetic +acid. + +In the solution acidulated with sulfuric, the caffein, together with +the other bodies capable of combining with iodin, is precipitated. The +residual iodin is determined in each case, and thus the quantity which +is united with the caffein is easily ascertained. The weight of iodin +which has entered into the precipitated caffein periodid multiplied by +0.3819 gives the weight of the caffein in the solution. + +Gomberg’s method has been subjected to a careful comparative study by +Spencer and has been much improved by him in important particulars.[599] + +It is especially necessary to secure the complete expulsion of +the hydrogen sulfid and to observe certain precautions in the +addition of the iodin reagent. The precipitation should be made in a +glass-stoppered flask, shaking thoroughly after the addition of the +iodin and collecting the precipitate on a gooch. As thus modified, the +iodin process gives results comparable with those obtained by Spencer’s +method, and it can also be used to advantage in estimating caffein in +headache tablets in the presence of acetanilid. + +=582. Freeing Caffein of Chlorophyll.=—Any chlorophyll which may pass +into solution and be found in the caffein may be removed by dissolving +the caffein in ten per cent sulfuric acid, filtering, neutralizing +with ammonia and evaporating to dryness. The residue is taken up with +chloroform, the chloroform removed at a low temperature and the pure +caffein thus obtained.[600] + +=583. Proteid Nitrogen.=—The proteid nitrogen in tea and coffee may be +determined in the residue after extraction of the alkaloid by boiling +water as described above. More easily it is secured by determining +the total nitrogen in the sample and deducting therefrom the nitrogen +present as caffein. The remainder, multiplied by 6.25, will give the +quantity of proteid matter. + +=584. Carbohydrates of the Coffee Bean.=—The carbohydrates of the +coffee bean include those common to vegetable substances; _viz._, +cellulose, pentosan bodies (xylan, araban), fiber etc., together +with certain sugars, of which sucrose is pointed out by Ewell as the +chief.[601] In smaller quantities are found a galactose yielding body +(galactan), as pointed out by Maxwell, a dextrinoid and a trace of a +sugar reducing alkaline copper solution. + +The sucrose may be separated from the coffee bean by the following +process:[602] The finely ground flour is extracted with seventy per +cent alcohol, the extract clarified with lead acetate, filtered, the +lead removed from the filtrate with hydrogen sulfid, the excess of the +gas removed by boiling, the filtrate evaporated in a partial vacuum to +a sirup and the sucrose crystallized from a solution of the sirup in +alcohol. + +For a quantitive determination, ten grams of the coffee flour are +extracted with ether and the residue with seventy-five per cent +alcohol. This process, conducted in a continuous extraction apparatus, +should be continued for at least twenty-four hours. The alcohol is +removed by evaporation, the residue dissolved in water, clarified with +basic lead acetate, filtered, the precipitate washed, the lead removed, +again filtered, the filtrate washed and wash water and filtrate +made to a definite volume. In an aliquot part of this solution the +sugars are determined by the alkaline copper method, both before and +after inversion. From the data obtained the percentage of sucrose is +calculated. + +In a coffee examined by Ewell the percentage of sucrose was found to be +6.34. The pentose yielding constituents of the coffee bean amount to +from eight to ten per cent. + +When coffee meal is extracted with a five per cent solution of sodium +carbonate, a gummy substance is obtained, which is precipitable by +alcohol. This gum, after washing with hydrochloric acid containing +alcohol, gives a gray, translucent, hard mass on drying. On hydrolysis +it yielded 75.2 per cent of dextrose, on distillation with hydrochloric +acid, thirteen per cent of furfuraldehyd and, on oxidation with nitric +acid, 18.7 per cent of mucic acid. This gum, therefore, consists +chiefly of a mixture of galactan, xylan and araban. + +=585. Estimation of Galactan.=—From three to five grams of the +substance supposed to contain galactan are placed in a beaker with +sixty cubic centimeters of nitric acid of 1.15 specific gravity. The +mixture is evaporated on a steam bath until it is reduced to one-third +of its original volume, allowed to stand for twenty-four hours, ten +cubic centimeters of water added, well stirred and again allowed +to stand for twenty-four hours, until the mucic acid is separated +in a crystalline form. To remove impurities from the mucic acid it +is separated by filtration, washed with not to exceed twenty cubic +centimeters of water, placed together with the filter in the beaker, +from twenty-five to thirty cubic centimeters of ammonium carbonate +solution, containing one part of dry ammonium carbonate, nineteen +parts of water and one part of ammonium hydroxid, added and heated to +near the boiling point. The mucic acid is dissolved by the ammonium +carbonate solution and any insoluble impurity separated by filtration, +the filtrate being received in a platinum dish, the residue well washed +and the entire filtrate and wash water evaporated to dryness on a steam +bath acidified with dilute nitric, well stirred and allowed to stand +until the mucic acid separates in a crystalline form. The separation +is usually accomplished in half an hour, after which time the crystals +of mucic acid are collected on a tared filter, or gooch, and washed +with not to exceed fifteen cubic centimeters of water followed with +sixty cubic centimeters of alcohol, then with ether, dried at 100° +and weighed. For computing the amount of galactose, one gram of the +mucic acid is equal to 1.333 of galactose and one gram of galactose is +equal to nine-tenths gram of galactan. Before the commencement of the +operation, the material should be freed of fatty matters in the case of +oily seeds and other substances similar thereto.[603] + +=586. Revised Factors for Pentosans.=—The factors given in paragraph +=154= have lately been recalculated by Mann, Kruger and Tollens, and +as a result of their investigations the following factors are now +recommended.[604] The quantity of furfurol is derived from the weight of +furfurolhydrazone obtained by the formula: + + 1. Furfurolhydrazone × 0.516 + 0.0104 = furfurol. + 2. Furfurol × 1.84 = pentosans. + 3. Furfurol × 1.64 = xylan. + 4. Furfurol × 2.02 = araban. + +The pentoses (xylose, arabinose) may be calculated from the pentosans +(xylan, araban) by dividing by 0.88. + +The method of procedure preferred for the estimation of the pentosans +is that described in paragraph =157=. The phloroglucin is dissolved in +hydrochloric acid of 1.06 specific gravity before it is added to the +furfurol distillate. The latest factor for converting the phloroglucid +obtained into furfurol is to divide by 1.82 for small quantities and +1.93 for large quantities. After the furfurol is obtained, the factors +given above are applied. + +=587. Application of Roentgen Rays to Analysis.=—The detection of +mineral matters in vegetable substances by roentgen photography has +been proposed by Ranvez.[605] This process will prove extremely valuable +in detecting the lacing of teas with mineral substances. Practically, +it has been applied by Ranvez in the detection of mineral substances +mixed with saffron with fraudulent intent. + +Barium sulfate is often mixed with saffron for the purpose of +increasing its weight. Pure saffron and adulterated samples are +enclosed in capsules of black paper and exposed on the same sensitive +plate for a definite time to the rays emanating from a crookes tube. +In this case the pure saffron forms only a very faint shadow in the +developed negative, while the parts to which barium sulfate are +attached produce strong shadows. The principle involved is applicable +to a wide range of analytical research. + + +TANNINS AND ALLIED BODIES. + +=588. Occurrence and Composition.=—The tannins and allied bodies, +which are of importance in this connection, are those which occur in +food products and beverages and also those made use of in the leather +industry. The term tannin is applied to a large class of astringent +substances, many of which are glucosids. Tannic acid is the chief +constituent of the tannins, and is found in a state of comparative +purity in nutgalls. The source from which the tannic acid is derived is +indicated by a prefix to the name, _e. g._, gallotannic, from nutgalls, +and caffetannic, from coffee etc. The tannins have lately been the +theme of a critical study by Trimble, and the reader is referred to +his work for an exhaustive study of the subject.[606] Tannin is one of +the most widely diffused compounds, occurring in hundreds of plants. +Commercially, the oaks and hemlocks are the most important plants +containing tannin. The sumach, mangrove, canaigre, palmetto and many +others have also been utilized as commercial sources of tannin. The +tannins as a class are amorphous and odorless. They are slightly acid +and strongly astringent. Their colors vary from dark brown to pure +white. They are soluble in water, alcohol, ether and glycerol and +insoluble in chloroform, benzol, petroleum ether, carbon bisulfid and +the oils. The tannins give blue or green precipitates with iron salts +and most of them brown precipitates with potassium bichromate. They are +all precipitated by gelatin or albumin. Tannins are not only generally +of a glucosidal nature, but are found quite constantly associated with +reducing sugars, or in unstable combination therewith. + +The reducing sugars may be separated from the tannin by precipitating +the latter with lead acetate and determining the glucose in the +filtrate after the removal of the lead. A separate portion of the +tannin is hydrolyzed with sulfuric or hydrochloric acid and the +reducing sugars again determined. Any excess of sugars over the first +determination is due to the hydrolysis of the tannin glucosid. + +=589. Detection and Estimation of Tannins.=—The qualitive reactions +above mentioned serve to detect the presence of a tannin. Of the iron +salts ferric acetate or chlorid is preferred. Ferrous salts do not +give any reaction with dilute tannin solutions. An ammoniacal solution +of potassium ferricyanid forms with tannins a deep red color changing +to brown. In quantitive work the tannins are mostly determined by +precipitation with metallic salts, by treatment with gelatin or hide +powder, or by oxidation with potassium permanganate. Directions for the +estimation of glucosids in general are found in Dragendorff’s book.[607] + +=590. Precipitation with Metallic Salts.=—The methods depending on +precipitation of the tannins with metallic salts are but little used +and only one of them will be mentioned here. A full description of +the others is contained in Trimble’s book.[608] A method for the +determination of caffetannic acid in coffee has been worked out by Krug +and used with some satisfaction.[609] + +In this method two grams of the coffee meal are digested for thirty-six +hours with ten cubic centimeters of water, a little more than twice +that volume of ninety-five per cent alcohol added and the digestion +continued for a day. The contents of the flask are poured on a filter +and the residue washed with alcohol. The filtrate contains tannin, +caffetannic acid and traces of coloring matter and fat. It is heated +to the boiling point and clarified with a solution of lead acetate. +A caffetannate of lead containing forty-nine per cent of the metal +is precipitated. As soon as the precipitate has become flocculent it +is collected on a filter, washed with ninety per cent alcohol until +the soluble lead salts are all removed, then with ether and dried. +The composition of the precipitate is represented by the formula +Pb₃(C₁₅H₁₅O₈)₂. The caffetannic acid is calculated by the equation: +Weight of precipitate: weight of caffetannic acid = 1267: 652. + +=591. The Gelatin Method.=—The precipitation of tannin with gelatin is +the basis of a process for its quantitive estimation which, according +to Trimble, is conducted as follows:[610] Two and a half grams of +gelatin and ten grams of alum are dissolved in water and the volume of +the solution made up to one liter. The solution of gelatin and also +that of the tannin are heated to 70° and the tannin is precipitated +by adding the gelatin reagent slowly, with constant stirring, until +the precipitate coagulates, and, on settling, leaves a clear liquor in +which no further precipitate is produced on adding a few drops more +of the reagent. In case the clearing of the mixture do not take place +readily, the process should be repeated with a more dilute tannin +solution. The precipitate is collected on two counterpoised filter +papers one placed inside the other, dried at 110° and weighed, the +empty filter paper being placed on the pan with the weights. For pure +tannin (gallotannic acid) fifty-four per cent of the weight of the +precipitate are tannin. Ammonium chlorid and common salt have been used +in place of the alum in preparing the reagent, but if the proportion of +alum mentioned above be used, satisfactory results will be obtained in +most cases. + +=592. The Hide Powder Method.=—The principle of this method is based on +the change in specific gravity, _i. e._, total solids, which a tannin +solution will undergo when brought into contact with raw hides in a +state of fine subdivision. The hide powder absorbs the tannin, and the +total solid content of the solution is correspondingly diminished. The +method is conducted according to the official directions as follows:[611] + +_Preparation of the Sample._—The bark, wood, leaves or other materials +holding the tannins, are dried and ground to a fine powder and +thoroughly extracted with water as mentioned below. In each case the +solution or extraction is made as thorough as possible and the volume +of the extract is made up to a definite amount. + +_Quantity of Tanning Material._—Use such an amount of the tanning +material as shall give in 100 cubic centimeters of the filtered +solution about one gram of dry solids. In the case of barks, woods, +leaves and similar materials, transfer to a half liter flask, fill +the flask with water at approximately 80° and let stand over night in +a bath which is kept at 80°, cool, fill to the mark, shake well and +filter. In the case of extracts and sweet liquors, wash the proper +quantity into a half liter flask with water at approximately 80°, +almost filling the flask, cool and fill to the mark. + +_Determination of Moisture._—Dry five grams of the sample in a flat +bottom dish at the temperature of boiling water until the weight +becomes constant. + +_Determination of Total Solids._—Shake the solution, which should +be at a temperature of about 20°, and immediately remove 100 cubic +centimeters with a pipette, evaporate in a weighed dish and dry to +constant weight at the temperature of boiling water. + +_Determination of Soluble Solids._—Filter a portion of the solution +through a folded filter, returning the filtrate to the filter twice +and adding a teaspoonful of kaolin, if necessary. Evaporate 100 cubic +centimeters of the filtrate and dry as above. + +_Determination of Tanning Substances._—Extract twenty grams of hide +powder by shaking for five minutes with 250 cubic centimeters of water, +filter through well washed muslin or linen, repeat the operation three +times and dry as much as possible in a suitable press. Weigh the wet +powder and determine the residual moisture in about one-fourth of the +whole by drying to constant weight at 100°. Shake 200 cubic centimeters +of the unfiltered solution of the tannin with the rest of the moist +hide powder for about five minutes, add five grams of barium sulfate, +shake for one minute and filter through a schleicher and schüll folded +filter, No. 590, fifteen centimeters in diameter, returning the first +twenty-five cubic centimeters of the filtrate. Evaporate 100 cubic +centimeters of the clear filtrate and dry the residue to constant +weight at a temperature of boiling water. The difference between the +soluble solids obtained in the filtered tannin solution and the residue +as obtained above is the amount of tanning material absorbed by the +hide powder. This weight must be corrected for the water retained by +the hide powder. The shaking must be conducted by means of a mechanical +shaker, in order to remove all the tannin substance from the solution. +The simple machine used by druggists, and known as the milkshake, is +recommended. + +_Testing the Hide Powder._—Shake ten grams of the hide powder with 200 +cubic centimeters of water for five minutes, filter through muslin +or linen, squeeze out thoroughly by hand, replace the residue in the +flask and repeat the operation twice with the same quantity of water. +Pass the last filtrate through paper until a perfectly clear liquid is +obtained. Evaporate 100 cubic centimeters of the final filtrate in a +weighed dish, dry at 100° until the weight is constant. If the residue +amount to more than ten milligrams the sample should be rejected. The +hide powder must be kept in a dry place and tested once a month. + +Prepare a solution of pure gallotannic acid by dissolving five grams in +one liter of water. Determine the total solids by evaporating 100 cubic +centimeters of this solution and drying to constant weight. Treat 200 +cubic centimeters of the solution with hide powder exactly as described +above. The hide powder must absorb at least ninety-five per cent of +the total solids present. The gallotannic acid used must be completely +soluble in water, alcohol, acetone and acetic ether and should contain +not more than one per cent of substances not removed by digesting with +excess of yellow mercuric oxid on the steam bath for two hours. + +_Testing the Non-Tannin Filtrate. For Tannin_:—Test a small portion +of the clear non-tannin filtrate with a few drops of a ten per cent +solution of gelatin. A cloudiness indicates the presence of tannin, in +which case the determination must be repeated, using twenty-five grams +of hide powder instead of twenty grams. + +_For Soluble Hide_:—To a small portion of the clear non-tannin +filtrate, add a few drops of the original solution, previously filtered +to remove reds. A cloudiness indicates the presence of soluble hide +due to incomplete washing of the hide powder. In this case, repeat the +determination with perfectly washed hide powder. + +=593. The Permanganate Gelatin Method.=—This process, which is +essentially the method of Löwenthal, as improved by Councler, Schroeder +and Proctor and as used by Spencer for the determination of tannin in +teas, is conducted as described below.[612] The principle of the process +is based on the oxidation of all bodies in solution oxidizable by +potassium permanganate, the subsequent precipitation of the tannin by +a gelatin solution, and the final oxidation, by means of permanganate, +of the remaining organic bodies. The difference between the total +oxidizable matter and that left after the precipitation of the tannin +represents the tannin originally in solution. + +_Reagents Required._—The following reagents are necessary to the proper +conduct of the potassium permanganate process: + +(1). Potassium permanganate solution containing about one and a third +grams of the salt in a liter: + +The potassium permanganate solution is set by titration against the +decinormal oxalic acid solution mentioned below. The end reaction with +the indicator must be of the same tint in all the titrations, _i. e._, +either golden yellow or pink. + +(2). Tenth-normal oxalic acid solution for determining the exact titer +of the permanganate solution: + +(3). Indigo-carmin solution to be used as an indicator and containing +six grams of indigo-carmin and fifty cubic centimeters of sulfuric +acid in a liter. The indigo-carmin must be very pure and quite free of +indigo-blue. + +(4). Gelatin solution, prepared by digesting twenty-five grams of +gelatin at room temperature for one hour in a saturated solution of +sodium chlorid, then heating until solution is complete, cooling and +making the volume up to one liter: + +(5). A salt acid solution, made by adding to 975 cubic centimeters of +a saturated solution of sodium chlorid, enough strong sulfuric acid to +bring the volume of the mixture to one liter: + +(6). Powdered kaolin for promoting filtration. + +_The Process._—Five grams of the finely powdered tea (or other +vegetable substance containing tannin) are boiled with distilled water +in a flask of half a liter capacity for half an hour. The distilled +water should be at room temperature when poured over the powdered tea. +After cooling, the volume of the decoction is completed to half a +liter, and the contents of the flask poured on a filter. To ten cubic +centimeters of the filtered tea infusion are added two and a half times +as much of the indigo-carmin solution and about three-quarters of a +liter of distilled water. + +The permanganate solution is run in from a burette, a little at a time, +with vigorous stirring, until the color changes to a light green, and +then drop by drop until the final color selected for the end of the +reaction, golden yellow or faint pink, is obtained. The number of cubic +centimeters of permanganate required is noted and represented by a in +the formula below. The titration should be made in triplicate and the +mean of the two more nearly agreeing readings taken as the correct one. + +One hundred cubic centimeters of the filtered tea infusion, obtained +as directed above, are mixed with half that quantity of the gelatin +reagent, the first named quantity of the acid salt solution added, +together with ten grams of the powdered kaolin, the mixture well +shaken for several minutes and poured on a filter. Twenty-five cubic +centimeters of the filtrate, corresponding to ten of the original +tea solution are titrated with the permanganate reagent, under the +conditions given above, and the reading of the burette made and +represented by _b_. The quantity of permanganate solution, _viz._, _c_, +required to oxidize the tannin is calculated from the formula _a - b_ = +_c_. The relation between the permanganate, oxalic acid and tannin is +such that 0.04157 gram of gallotannic acid is equivalent to 0.063 gram +of oxalic acid. The relation between the oxalic acid solution and the +permanganate having been previously determined the data for calculating +the quantity of tannin, estimated as gallotannic acid, are at hand. + +=594. The Permanganate Hide Powder Method.=—Instead of throwing out the +tannin with gelatin it may be absorbed by hide powder. The principle of +the process, save this modification, is the same as in the method just +described. As described by Trimble, the analysis is conducted according +to the following directions:[613] + +_Reagents Required._—The reagents required for conducting the +permanganate hide powder process are as follows: + +1. _Permanganate Solution._—Ten grams of pure potassium permanganate +are dissolved in six liters of water. The solution is standardized with +pure tannin. The moisture in the pure tannin is determined by drying at +100° to constant weight and then a quantity of the undried substance, +representing two grams of the dried material, is dissolved in one +liter of water. Ten cubic centimeters of this solution and double +that quantity of the indigo solution to be described below, are mixed +with three-quarters of a liter of water and the permanganate solution +added from a burette with constant stirring until the liquid assumes +a greenish color and then, drop by drop, until a pure yellow color +with a pinkish rim is obtained. Fifty cubic centimeters of the pure +tannin solution are digested, with frequent shaking, with three grams +of hide powder which has been previously well moistened and dried in a +press for eighteen or twenty hours, the contents of the flask thrown +on a filter and ten cubic centimeters of the filtrate titrated with +the permanganate solution as directed above. The difference between +the amount of permanganate solution required for the first and second +titrations represents the amount of pure tannin or oxidizable matter +removed by the hide powder. + +2. _Indigo Solution._—The indicator which is used in the titrations +is prepared by dissolving thirty grams of sodium sulfindigotate in +three liters of dilute sulfuric acid made by adding one volume of the +strong acid to three volumes of water. The solution is shaken for a few +minutes, thrown upon a filter and the insoluble residue washed with +sufficient water to make the volume of the filtrate six liters. + +3. _Hide Powder._—The hide powder used should be white, wooly in +character and sufficiently well extracted with water to afford no other +extract capable of oxidizing the permanganate solution. + +_The Process._—The reagents having been prepared and tested as above, +the solution of the substance containing the tannin, prepared as +described further on, is titrated first with the permanganate solution +in the manner already given. Fifty cubic centimeters of the tannin +solution are then shaken, from, time to time for eighteen hours, +with three grams of hide powder, thrown upon a filter and ten cubic +centimeters of the filtrate titrated with the potassium permanganate as +above described. From the data obtained, the quantity of permanganate +solution corresponding to the tannin removed by the hide powder is +easily calculated. The value of the permanganate solution having been +previously set with a pure tannin, renders easy of calculation the +corresponding amount of pure tannin in the solution under examination. + +=595. Preparation of the Tannin Infusion.=—A sample weighing about a +kilogram should be secured, representing as nearly as possible the +whole of the materials containing tannin in a given lot. The sample +is reduced to a fine powder and passed through a sieve containing +apertures about a millimeter in diameter. The quantity of the sample +used for the extraction depends largely upon its content of tannin. +Five grams of nutgalls, ten grams of sumach or twenty grams of oak +bark represent about the quantities necessary for these classes of +tannin-holding materials. The sample is boiled for half an hour with +half a liter of water, filtered through a linen bag into a liter flask +and washed and pressed with enough water to make the volume of the +filtrate equal to one liter. The proper quantities of this solution are +used for the analytical processes described above. + + +TOBACCO. + +=596. Fermented and Unfermented Tobacco.=—Samples of tobacco may +reach the analyst either in the fermented or unfermented state. As a +basis for comparison, it is advisable in all cases to determine the +constituents of the sample before fermentation sets in. The analysis, +after fermentation is complete, will then show the changes of a +chemical nature which it has undergone during the process of curing and +sweating. Only tobacco which has undergone fermentation is found to be +in a suitable condition for consumption. In addition to the natural +constituents of tobacco, it may contain, in the manufactured state, +flavoring ingredients such as licorice and sugar, coloring matters and +in some instances, it is said, opium or other stimulating drugs. It +is believed, however, that opium is not often found in manufactured +tobacco, and it has never been found in this laboratory in cigarettes, +although all the standard brands have been examined for it.[614] + +In researches made at the Connecticut Station it is shown that +fermentation produces but little change in the relative quantities +of nitric acid, ammonia, fiber and starch in the leaves, while those +of nicotin, albuminoids and amids are diminished. This is not in +harmony with the generally accepted theory that starch is inverted and +fermented during the process.[615] + +The nature of the ferments which are active in producing the changes +which tobacco undergoes in curing, is not definitely understood. Some +of the organic constituents of the tobacco undergo a considerable +change during the process. Any sugar which is found in the freshly +cured leaves disappears wholly or in part. As products of fermentation +may also be found succinic, fumaric, formic, acetic, propionic and +butyric acids. + +=597. Acid and Basic Constituents of Tobacco.=—In unfermented and +fermented tobacco are found certain organic acids, among the most +important of which are citric, malic, oxalic, pectic and tannic. Of +the inorganic acids the chief which are found are nitric, sulfuric +and hydrochloric. Among the bases ammonia and nicotin are the most +important. Ammonia is found in the unfermented tobacco in only small +quantities, but in the fermented product it may sometimes reach as high +as half a per cent. The presence of these two nitrogenous bases in +tobacco renders the estimation of the proteid matter contained therein +somewhat tedious and difficult. + +=598. Composition of Tobacco Ash.=—The mineral constituents of tobacco +are highly important from a commercial point of view. The burning +properties of tobacco depend largely upon the nature of its mineral +constituents. A sample containing a large quantity of chlorids +burns much less freely than one in which the sulfates and nitrates +predominate. For this reason, the use of potash fertilizers containing +large amounts of chlorin is injudicious in tobacco culture, the +carbonates and sulfates of potash being preferred. The leaves of the +tobacco plant contain a much larger percentage of mineral constituents +than the stems, their respective contents of pure ash, that is ash free +from carbon dioxid, carbon and sand, being about seventeen and seven. +The pure ash of the leaves has the following mean composition: Potash +29.1 per cent, soda 3.2 per cent, lime 36.0 per cent, magnesia 7.4 per +cent, iron oxid 2.0 per cent, phosphoric acid 4.7 per cent, sulfuric +acid 6.0 per cent, silica 5.8 per cent, and chlorin 6.7 per cent.[616] + +=599. Composition of Tobacco.=—The mean composition of some of the more +important varieties of water-free tobacco is shown in the following +table:[617] + + Havana, Sumatra, Kentucky, Java, + per cent. per cent. per cent. per cent. + Nicotin 3.98 2.38 4.59 3.30 + Malic acid 12.11 11.11 11.57 6.04 + Citric acid 2.05 2.53 3.40 3.30 + Oxalic acid 1.53 2.97 2.03 3.38 + Acetic acid 0.42 0.29 0.43 0.22 + Tannic acid 1.13 0.98 1.48 0.51 + Nitric acid 1.32 0.60 1.88 0.23 + Pectic acid 11.36 11.88 8.22 10.13 + Cellulose 15.76 10.59 12.48 11.82 + Ammonia 0.49 0.06 0.19 0.23 + Soluble nitrogenous matter 7.74 8.84 13.90 10.39 + Insoluble ” ” 9.75 7.97 8.10 9.53 + Residue and chlorophyll 5.15 8.63 1.99 6.45 + Oil 1.03 1.26 2.28 0.81 + Ash 17.50 17.03 14.36 18.46 + Undetermined 8.68 12.88 13.10 15.20 + +Among the undetermined matters are included those of a gummy or +resinous composition not extracted by ether, the exact nature of +which is not well understood, and the starches, sugars, pentosans and +galactan. + +Tobacco grown in more northern latitudes has less nicotin than the +samples given in the foregoing table. + +The following table shows the composition of tobacco grown in +Connecticut:[618] + + (A)= Unfermented, + (B)= Fermented, + Upper leaves. Short seconds. First wrappers. + (A) (B) (A) (B) (A) (B) + % % % % % % + Water 23.50 23.40 27.40 21.10 27.50 24.90 + Pure ash 14.89 15.27 22.85 25.25 15.84 16.22 + Nicotin 2.50 1.79 0.77 0.50 1.26 1.44 + Nitric acid 1.89 1.97 2.39 2.82 2.59 2.35 + Ammonia 0.67 0.71 0.16 0.16 0.33 0.47 + Proteids 12.19 13.31 6.69 6.81 11.31 11.62 + Fiber 7.90 8.78 7.89 8.95 9.92 10.42 + Starch 3.20 3.36 2.62 3.01 2.89 3.08 + Oil and fat 3.87 3.42 2.95 3.04 2.84 2.92 + Undeterm’d 29.39 27.99 26.28 28.36 25.52 26.88 + +=600. Estimation of Water.=—In the estimation of water in vegetable +substances, as has already been noted, it is usual to dry them in +the air or partial vacuum, or in an inert gas, at a temperature of +100° until a constant weight is reached. By this process, not only +the water, but all substances volatile at the temperature and in the +conditions mentioned are expelled. The quantity of these volatile +substances in vegetable matter, as a rule, is insignificant and hence +the total loss may be estimated as water. In the case of tobacco a +far different condition is presented, inasmuch as the nicotin, which +sometimes amounts to five per cent of the weight of the sample, is also +volatile under the conditions mentioned. It is advisable, therefore, to +dry the sample of tobacco at a temperature not above fifty degrees and +in a vacuum as complete as possible. Tobacco is also extremely rich in +its content of crystallized mineral salts, containing often water of +crystallization, and there is danger of this crystal water being lost +when the sample is dried at 100°. The desiccation is conveniently made +in the apparatus described on page 22. If a high vacuum be employed, +_viz._, about twenty-five inches of mercury, it is better not to allow +the temperature to go above 40° or 45°. A rather rapid current of dry +air should be allowed to pass through the apparatus for the more speedy +removal of the moisture and a dish containing sulfuric acid may also be +placed inside of the drying apparatus. It is possible by proceeding in +this way to secure constant weight in the sample after a few hours. + +=601. Estimation of Nitric Acid.=—The nitric acid in a sample of +tobacco is most easily estimated by the ferrous chlorid process.[619] + +The sample is best prepared by making an alcoholic extract which is +accomplished by exhausting about twenty-five grams of the fine tobacco +powder with 200 cubic centimeters for forty per cent alcohol made +slightly alkaline by soda lye. The mixture is boiled in a flask with +a reflux condenser for about an hour. After cooling, the volume is +completed to a definite quantity, and, after filtering, an aliquot +part is used for the analytical process. It is evident that the nitric +acid cannot be estimated in this case after previous reduction to +ammonia by zinc or iron on account of the presence of ammonia in the +sample itself. If, however, the amount of ammonia be determined in a +separate portion of the sample, the nitric acid may be reduced in the +usual way, by zinc or iron, the total quantity of ammonia determined by +distillation, the quantity originally present in the sample deducted +and the residual ammonia calculated to nitric acid. + +=602. Sulfuric and Hydrochloric Acids.=—These two acids are determined +in the ash of the sample by the usual methods. The sulfuric acid thus +found represents the original sulfuric acid in combination with the +bases in the mineral parts of the plant, together with that produced +by the oxidation of the organic sulfur during combustion. In order to +avoid all loss of sulfur during the combustion, the precautions already +given should be observed. The separation of the sulfur pre-existing as +sulfates from that converted into sulfates during the combustion is +accomplished as previously directed.[620] For ordinary purposes, this +separation is not necessary. + +To avoid loss of chlorin from volatilization during incineration the +temperature should be kept at the lowest possible point until the mass +is charred, the soluble salts extracted from the charred mass and the +incineration completed as usual. + +=603. Oxalic, Citric and Malic Acids.=—The separation and estimation of +organic acids from vegetable tissues is a matter of great difficulty, +especially when they exist as is usually the case, in very minute +proportions. During incineration, the salts of the inorganic acids +are converted into carbonates and the subsequent examination of the +ash gives no indication of the character of the original acids. In +the case of tobacco, the organic acids of chief importance, from an +analytical point of view, are oxalic, citric and malic. These acids may +be extracted and separated by the following process:[621] + +Ten grams of the dry tobacco powder are rubbed up in a mortar with +twelve cubic centimeters of dilute sulfuric acid (one to five) and then +absorbed with coarse pumice stone powder in sufficient quantity to +cause all the liquid to disappear. The mass is placed in an extraction +apparatus of proper size and thoroughly extracted with ether until a +drop of the extract leaves no acid residue on evaporation. Usually +about ten hours are required. The organic acids are thus separated +from the mineral acids. The ether is removed from the extract and +the residue dissolved in hot water, cooled, filtered, if necessary +several times, until the solution is separated from the fat and resin +which have been extracted by the ether. The filtrate is neutralized +with ammonia, slightly acidified with acetic and the oxalic acid +contained therein thrown out by means of a dilute solution of calcium +acetate, which must not be added in excess. The calcium oxalate is +separated by filtration, and determined as lime oxid. To the filtrate +is added drop by drop, with constant stirring, a dilute solution of +lead acetate, prepared by mixing one part of a saturated solution of +lead acetate with four parts of water. When the precipitate formed +has settled, the clear supernatant liquid is tested by adding a drop +of acetic acid and a few drops of the dilute lead acetate. In case a +precipitate be formed, the addition of the lead acetate is continued +until a precipitate is secured which will immediately dissolve in +acetic acid. At this moment the citric acid is almost completely +precipitated. In order to avoid the accumulation of the acetic acid +by reason of the repetition of the process as above described, the +mixture is neutralized each time with dilute ammonia. The precipitated +neutral lead citrate obtained by the above process, is separated by +filtration and, in order to avoid its decomposition when washed with +pure water, it is washed with a very dilute acetic acid solution of +lead acetate. The washing and filtration are accomplished as quickly +as possible, and the final washing is made with alcohol of thirty-six +per cent strength. In the filtrate the residual lead citrate, together +with a little lead malate, are precipitated by the alcohol used as the +wash and this precipitate is also separated by filtration. The filtrate +containing the greater part of the malic acid is evaporated to remove +the alcohol and treated with lead acetate in excess. Afterwards it +is mixed with five times its volume of thirty-six per cent alcohol +containing a half per cent of acetic acid. In these conditions the lead +malate is completely precipitated as neutral salt, and after standing a +few hours, is separated by filtration. The three precipitates, obtained +as above, are dried at 100° and weighed. If the precipitates have +been collected on filter paper they should be removed as completely +as possible, the papers incinerated in the usual way and any reduced +lead converted into nitrate and oxid by treatment with nitric acid and +subsequent ignition. From the quantities of lead oxid obtained, the +weights of the citric and malic acids are computed. The precipitate +which is obtained by the action of alcohol, above noted, is also dried +and ignited and the lead oxid found divided equally between the citric +and malic acids, the respective quantities of which found, are included +in computing their total weights. The weight of the citric acid is +calculated from the formula (C₆H₅O₇)₂Pb₃ + H₂O, and that of the malic +acid from the formula C₄H₄O₅Pb + H₂O. + +=604. Acetic Acid.=—For the determination of the volatile acids of the +fatty series existing in tobacco, the following process, also due to +Schlösing, may be followed:[622] + +The apparatus employed is shown in Fig. 121. Ten grams of the +pulverized tobacco, moistened with water and mixed with a little +powdered tartaric acid, are placed in the tube _A_. The two ends of +the tube, _A_, are stoppered with asbestos or glass wool. Steam, +generated in the flask, _D_, is passed into _B_. After fifteen minutes, +or as soon as it is certain that the contents of _A_ have reached a +temperature of 100°, the dish, _F_, containing mercury, is placed in +the position shown in the figure. The steam, by this arrangement, +is forced into the lower end of _A_, passes into the condenser _E_, +and the condensed water collected in _C_. The operation should be so +conducted as to avoid any condensation of water in _B_. It is advisable +during the progress of the distillation, which should continue for at +least twenty minutes, to neutralize from time to time the acetic acid +collected in _C_ by a set solution of dilute alkali, or, an excess of +the alkaline solution may be placed in _C_ and the part not neutralized +by the acetic acid determined at the end of the distillation by +titration. + +[Illustration: FIG. 121.—APPARATUS FOR ACETIC ACID.] + +=605. Pectic Acid.=—Under this term are included not only the pectic +acid but all the other bodies of a pectose nature contained in tobacco. +These bodies are of considerable interest, although they do not belong +to the most important constituents. In fresh tobacco leaves are found +three pectin bodies. One pectin is soluble in water, another is an +insoluble pectose and the third is the pectose body forming salts +with the alkalies, _i. e._, true pectic acid. In fermented tobacco +pectic acid is found chiefly in combination with lime in the ribs of +the leaves, serving to give them the necessary stiffness. For the +estimation of the pectin bodies (mucilage) the powdered tobacco is +thoroughly extracted with cold water. An aliquot part of the aqueous +extract is mixed with two volumes of strong alcohol and allowed to +stand in a well closed vessel in a cool place for twenty-four hours. +The precipitate is collected on a filter, washed with sixty-six per +cent alcohol, dried and weighed. The dried residue is incinerated and +the amount of ash determined. In general, vegetable mucilages contain +about five per cent of ash. If more than this be found, it is due to +the solution of the salts of the organic acids contained in the sample. +A dried vegetable mucilage, obtained as above, dissolves in water to +a mucilaginous liquid which does not reduce alkaline copper solution +until it has been hydrolyzed by boiling with a dilute mineral acid.[623] + +=606. Tannic Acid.=—This acid is separated and estimated by the +processes given in paragraphs =589-595=. + +=607. Starch and Sugar.=—The unfermented leaves of tobacco contain +considerable quantities of carbohydrates in addition to woody fiber, +pentosans, galactan and cellulose. Among these, starch is the most +important. Sugar exists in small quantities in the fresh leaf, +usually not over one per cent. During fermentation, according to some +authorities, the starch is partially converted into sugar and the +latter substance disappears under the action of the alcoholic ferments. +It has been found at the Connecticut Station, however, that the starch +content of the leaf does not decrease during fermentation. The starch +and sugar may be determined in the fresh leaves by the methods already +given. + +In the manufacture of certain grades of tobacco it is customary to add +a quantity of sugar. The analyst may thus be called upon to determine +in some cases whether the sugar found in a sample is natural or added. +The occurrence of natural sugars in tobacco has been investigated at +the instance of the British Treasury.[624] + +The natural sugars which may be found in sun dried tobaccos usually +disappear entirely during the process of fermentation. It was found by +the Somerset House chemists that the content of sugar in commercial +tobaccos varies from none at all to over fifteen per cent. A remarkable +example of this variation is reported in two samples from this +country, one of which, grown in Kentucky, contained no sugar, and the +other grown in Virginia, 15.2 per cent. + +It was noticed that the saccharin matters in the tobaccos examined +were neutral to polarized light. They are determined by their copper +reducing power. The tobacco sugars are therefore to be classed with the +reducing bodies, not optically active, found in the juices of sorghum +and sugar canes. + +=608. Ammonia.=—As has already been intimated, ammonia exists only +in minute quantities in fresh tobacco leaves, but in considerable +quantities after fermentation. In the estimation of ammonia, twenty +grams of the tobacco powder are digested with 250 cubic centimeters of +water, acidulated with sulfuric and after an hour enough water added +to make the total quantity 400 cubic centimeters. After filtration, an +aliquot part of the filtrate, about 200 cubic centimeters, is treated +with magnesium oxid in excess and the ammonia and nicotin removed by +distillation in a current of steam. The distillate is collected in +dilute sulfuric acid of known strength. The total amount of the two +bases is determined by titration and the quantity of base representing +the nicotin, which has been determined in a separate sample, subtracted +in order to obtain the weight of the ammonia.[625] + +The ammonia in tobacco is determined by Nessler in the following +manner:[626] + +The powdered tobacco is mixed with water and magnesium oxid and after +standing for several hours it is distilled in a current of steam, the +distillate received in dilute sulfuric acid and the process continued +until a drop of the distillate gives no reaction for ammonia with the +nessler reagent. The excess of sulfuric acid in the distillate is +neutralized with pure sodium carbonate and the nicotin precipitated +by a neutral solution of mercuric iodid and potassium iodid. The +precipitate is separated by filtration, the filtrate treated with +sodium sulfid, and the ammonia again obtained by distillation with +an alkali, collected in dilute solution of set sulfuric acid and +determined by titration. The difference of the two determinations +represents the ammonia. + +=609. Nicotin.=—In this laboratory McElroy has made a study of some of +the best approved methods for determining nicotin, and finds the most +simple and reliable to be that proposed by Kissling.[627] The finely +powdered tobacco should be dried at a temperature not exceeding 60°, or +it may be partially dried at that temperature before grinding and the +final drying completed afterwards. Twenty grams of the powdered sample +are intimately mixed by means of a pestle with ten cubic centimeters +of dilute alcoholic solution of soda lye, made by dissolving six grams +of sodium hydroxid in forty cubic centimeters of water and completing +the volume to 100 cubic centimeters with ninety-five per cent alcohol. +The mass is transferred to an extraction paper cylinder, placed in +an extraction apparatus and extracted for three hours with ether. +The ether is nearly all removed by careful distillation, the residue +mixed with fifty cubic centimeters of a very dilute soda lye solution +(4 to 100) and subjected to distillation in a current of steam. The +flask containing the nicotin extract should be connected with the +condensing apparatus by a safety bulb as is usual in the distillation +of substances containing fixed alkali. The distillation should be +conducted rapidly and in such a manner that when 200 cubic centimeters +of the distillate have been collected, not more than fifteen cubic +centimeters of the liquid remain in the distillation flask. In +the distillate, the nicotin is determined by titration with a set +solution of dilute sulfuric acid, using rosolic acid or phenacetolin +as indicator. It is advisable to titrate each fifty cubic centimeters +of the distillate as it is received and the distillation is continued +until the last fifty cubic centimeters give no appreciable quantity +of the alkaloid. In the calculations one molecule of sulfuric acid is +equivalent to two molecules of nicotin according to the equation + + H₂SO₄ = (C₁₀H₁₄N₂)₂. + 98 324 + +_Polarization Method._—Popovici has based a method of detecting the +quantity of nicotin in tobacco on its property of rotating the plane +of polarized light.[628] The gyrodynat of pure nicotin is expressed by +the formula [_a_]_{D} = -161°.6. When ten parts of nicotin are mixed +with ninety parts of water, this value becomes -74°.1. By reason of +this great depression in gyrodynatic value Popovici determined the +relation which exists between the dilute solutions of nicotin and the +number of minutes of angular rotation produced on polarization in a +200 millimeter tube. In a solution in which two grams of nicotin are +contained in fifty cubic centimeters, each minute of angular rotation +is found to correspond to 6.5 milligrams of nicotin. For one gram in +solution in the same volume one minute of angular rotation corresponds +to 5.9 milligrams and for a half gram in solution to 5.7 milligrams. + +The nicotin is prepared for polarization by extracting with ether, as +indicated in the previous paragraph, and the ethereal solution from +twenty grams of tobacco is shaken with a concentrated solution of +sodium phosphotungstate in nitric acid by means of which nicotin and +ammonia are precipitated and rapidly settle. The supernatant liquid +is carefully poured off and the residue made up to a volume of fifty +cubic centimeters with distilled water and the nicotin freed from any +of its compounds by the addition of eight grams of finely powdered +barium hydroxid. In order to promote the decomposition of the nicotin +compounds the mixture should be shaken at intervals for several hours. +The at first blue precipitate changes into blue green and finally into +yellow. It is separated by filtration and the somewhat yellow colored +filtrate placed in an observation tube, polarized, the polarization +calculated to minutes of angular rotation and the number of minutes +thus found multiplied by the nearest factor given above. + +The analyst will find a description of other methods of estimating +nicotin in tobacco in the periodical literature of analytical +chemistry.[629] + +=610. Estimation of Amid Nitrogen.=—For the estimation of amid +nitrogen ten grams of the powdered tobacco are digested with 100 +cubic centimeters of forty per cent alcohol, the extract separated by +filtration, acidified with sulfuric and the albumin, peptone, nicotin +and ammonia precipitated with as little phosphotungstic acid as +possible. The precipitate is separated by filtration and seventy-five +cubic centimeters of the filtrate evaporated in a thin glass or tin +foil capsule after the addition of a little barium chlorid and the +nitrogen determined in the residue. The nitrogen thus obtained is that +which was present in an amid state. The nitrogen present as amids, +ammonia and nicotin subtracted from the total nitrogen leaves that +present as protein. + +=611. Fractional Extraction of Tobacco.=—To determine the character of +the soluble constituents of tobacco it is advisable to subject it to +a fractional extraction with different reagents. The reagents usually +employed in the order mentioned are petroleum ether, ether, absolute +alcohol, water, dilute soda lye and dilute hydrochloric acid. The +extract obtained by petroleum ether contains vegetable wax, chlorophyll +and its alteration products, fat, ethereal oils, and resin bodies. +The extract with ether may be divided into water soluble and alcohol +soluble bodies. Among the first are small quantities of glucosids and +nicotin while in the alcoholic solution resin predominates. + +The alcoholic extract is also divided into water soluble and alcohol +soluble parts. The first contains the nicotin, which is insoluble in +ether, in combination with acids, together with tannic acid and allied +bodies and also the sugar. The part insoluble in water consists chiefly +of resin. + +The aqueous solution contains the vegetable mucilages (pectin) soluble +carbohydrates, soluble proteids and organic acids. + +The dilute soda lye solution contains chiefly proteids. + +The dilute hydrochloric acid solution contains the starch and the +oxalic acid originally combined with lime. The extractions with dilute +soda lye and dilute hydrochloric acid should be made at a boiling +temperature. The residual matter consists of a mixture of carbohydrate +bodies to which the term crude fiber is usually applied. + +=612. Burning Qualities.=—When tobacco is to be used for the +manufacture of cigars, or cigarettes, or for smoking in pipes, its +ability to keep burning is a matter of great importance. The tobacco, +when once ignited, should burn for some time and form, a fluffy ash, +free of fused mineral particles. A tobacco with good burning properties +is one containing nitrates in considerable quantity, not too much sugar +and starch, a porous cellular structure and comparatively free of +chlorin. In determining comparative burning properties the tests may +be applied to the single leaf or the tobacco may be first rolled into +a cigar form and burned in an artificial smoker. + +[Illustration: FIG. 122. APPARATUS FOR SMOKING.] + +In applying the test to the leaf it is important that the ignition +be made with a fuse without flame, which maintains a uniform burning +power. Any good slow burning fuse may be used and it is applied to the +leaf in such a way that a hole may be burned in it, leaving its edges +uniformly ignited. The number of seconds elapsing before the last spark +is extinguished is noted. At the Connecticut Experiment Station a +lighter, proposed by Nessler, is employed. It is prepared by digesting +eighty grams of gum arabic in 120 cubic centimeters, and forty grams of +gum tragacanth in a quarter of a liter of water for two days, mixing +the mucilaginous masses and adding ten grams of potassium nitrate and +about 350 grams of pulverized charcoal. The mixture is rolled, on a +plate sprinkled with charcoal, into sticks a few inches in length and +of the diameter of a cigar and dried at a gentle heat. These fuses +burn slowly and without smoke and are well suited for lighting tobacco +leaves. Several tests, at least six, should be made with each leaf. +Leaves having a uniform burning power should be used as comparators and +the number of seconds they burn be designated by 100. It is important +that all the samples to be tested be exposed for a day or two to the +same atmosphere in order that they may have, as nearly as possible, +the same content of moisture. The burning tests, when possible, should +be made both before and after fermentation. As a rule fermentation +improves the burning quality of second rate leaves, but has little +effect on leaves of the first quality. + +=613. Artificial Smoker.=—For the purpose of comparing the burning +properties of cigars, or of leaves rolled into cigar form, the +artificial smoking apparatus devised by Penfield and modified in this +laboratory is employed.[630] The construction of the apparatus is shown +in the accompanying figure. + +The lighted cigar is set in the tube at the left, so that air entering +the test-tube must pass through the cigar. The test-tube contains +enough water to seal the end of the tube carrying the cigar, and is +connected with the aspirator on the right by the =T= tube, as shown. An +arm of the =T= dips just beneath the surface of the liquid in the cup +in the center. Water flows in a slow stream into the aspirator through +the tube at the extreme right, forcing the air out through the arm +of the =T= until the siphon begins to act. While the water is voided +through the long arm of the siphon, air enters through the cigar, the +liquid rising in the =T=. The action of the apparatus is automatic and +intermittent. When the cigar is about one-third burned, it is removed +without disturbing the ash cone, and the latter examined and compared +with other samples as a standard. The sealing liquid of the long arm of +the =T= may be mercury or water. In case mercury be used, care must be +taken not to immerse the open end of the =T= more than one millimeter +therein. + + +FERMENTED BEVERAGES. + +=614. Description.=—Among fermented beverages are included those +drinks, containing alcohol, prepared by fermenting the sugars or +starches of fruits, cereals or other agricultural products. Wine +and beer, in their various forms, and cider are the chief members +of this class of bodies. Koumiss, although a fermented beverage, is +not included in this classification, having been noticed under dairy +products. The large number of artificial drinks, made by mixing alcohol +with fruit and synthesized essences, is also excluded, although the +methods of analysis which are used may be applied also to them. + +Fermented beverages containing less than two per cent of alcohol are +usually regarded as non-intoxicating drinks. Beers are of several +varieties, and the term includes lager beer, ale, porter and stout. +Distilled liquors are obtained by separating the alcohols and other +volatile matters from the products of fermentation by distillation. +It is not practicable here to attempt a description of the methods of +preparing fermented drinks. Special works on this branch of the subject +are easy of access.[631] + +=615. Important Constituents.=—Alcohol is the most important +constituent of fermented beverages. The solid matters, commonly called +extract, which are obtained on evaporation are composed of dextrins, +sugars, organic acids, nitrogenous bodies and mineral matters affording +ash on combustion. Of these the dextrins and sugars form the chief part +and the proteid bodies nearly ten per cent in the case of beers made of +malt and hops. In beers the bitter principles derived from hops, while +not important by reason of quantity, are of the utmost consequence from +a gustatory and hygienic point of view. The ash of fermented beverages +varies with their nature, or with the character of the water used +in making the mash. In the manufacture of beer, water containing a +considerable proportion of gypsum is often used, and this substance is +sometimes added in the course of manufacture, especially of wine. The +presence of common salt in the ash in any notable quantity is evidence +of the addition of this condiment, either to improve the taste of the +beverage or to increase the thirst of the drinker. In cider the organic +acids, especially malic, are of importance. + +Glycerol is a product of fermentation and of the hydrolysis of the fats +and oils in the substances fermented. + +=616. Specific Gravity.=—In order to secure uniformity of expression, +the specific gravity of fermented beverages is determined at about +15°.6, although that is a temperature much below the average found in +American laboratories. The specific gravity may be determined by an +alcoholometer, pyknometer or hydrostatic balance in harmony with the +directions given in paragraphs =48-54= and =285=. By reason of the +extractive matters held in solution, fermented beverages are usually +heavier than water, even if the content of alcohol be twenty per cent +or more. On the other hand distilled liquors are lighter than water. + +=617. Determination of Alcohol.=—The determination of the percentage of +alcohol present in a solution is based on two general principles. On +the one hand, and this is the base of the methods in common use, the +alcohol is secured mixed only with water and its amount determined by +ascertaining the specific gravity of the mixture. On the other hand the +quantity of alcohol in a mixture may be determined by ascertaining the +temperature of the vapors produced on boiling. This is the principle +involved in the use of the ebullioscope. The latter method is not +employed to any extent in this country. + +_Use of the Alcoholometer._—The alcoholometer usually employed is +known by the name of Gay-Lussac, who first made practical use of it in +the determination of alcohol. It is constructed in such a way as to +read directly the volume of absolute alcohol contained in one hundred +volumes of the liquid at a temperature of 15°.6. The instruments +employed should be carefully calibrated and thoroughly cleaned by +washing with absolute alcohol before use. The stem of the instrument +must be kept free from any greasy substance, and this is secured by +washing it with ether. After this last washing the analyst should be +careful not to touch the stem of the instrument with his fingers. It is +most convenient to make the determination exactly at 15°.6, but when +made at other temperatures the reading of the instrument is corrected +by tables which may be found in works especially devoted to the +analysis of wines.[632] + +In this country the alcoholometer is used to some extent, but the +official method is based upon the determination of the specific gravity +by an instrument constructed in every respect like the alcoholometer, +but giving the specific gravity of the liquor at 15°.6 instead of its +percentage by volume in alcohol. The reading of the instrument having +been determined at a temperature of 15°.6, the corresponding percentage +of alcohol by volume or by weight is taken directly from the table +given further on. + +[Illustration: FIG. 123. METAL DISTILLING APPARATUS.] + +_Methods of Distillation._—The metal apparatus employed in the +laboratory of the Department of Agriculture, for the distillation of +fermented beverages in order to determine the percentage of alcohol +by the method given above, is shown in the accompanying figure. The +apparatus consists of a retort of copper carried on supports in such a +way as to permit an alcohol or bunsen lamp to be placed under it. It +is connected with a block tin condenser and the distillate is received +in a tall graduated cylinder placed under the condenser in such a way +as to prevent the loss of any alcohol in the form of vapor. Exactly +300 cubic centimeters of the wine or fermented beverage are used for +the distillation. Any acid which the wine contains is first saturated +with calcium carbonate before placing in the retort. Exactly 100 +cubic centimeters of distillate are collected and the volume of the +distillate is completed to 300 cubic centimeters by the addition of +recently distilled water.[633] The cylinder containing the distillate is +brought to a temperature of 15°.6, the alcoholometer inserted and its +reading taken with the usual precautions. + +_Official Method._—The alcoholometers employed in the official methods +are calibrated to agree with those used by the officers of the Bureau +of Internal Revenue. They are most conveniently constructed, carrying +the thermometer scale in the same stem with that showing the specific +gravity. It is highly important that the analyst assure himself of +the exact calibration of the instrument before using it. Inasmuch as +the volume of the distillate may not be suited in all cases to the +use of a large alcoholometer, it is customary in this laboratory to +determine the specific gravity by means of the hydrostatic balance, +as described further on. Attention is also called to the fact that, +in the official method, directions are not given to neutralize the +free acid of the fermented beverage before the distillation. Since the +Internal Revenue Bureau is concerned chiefly with the determination of +alcohol in distilled liquors, this omission is of little consequence. +Even in ordinary fermented beverages the percentage of volatile acids, +(acetic etc.,) is so small as to make the error due to the failure to +neutralize it of but little consequence. In order, however, to avoid +every possibility of error, it is recommended that in all instances the +free acids of the sample be neutralized before distillation. In this +laboratory, the distillations are conducted in a glass apparatus shown +in the accompanying figure. The manipulation is as follow:[634] + +[Illustration: FIG. 124. DISTILLING APPARATUS.] + +One hundred cubic centimeters of the liquor are placed in a flask of +from 250 to 300 cubic centimeters capacity, fifty cubic centimeters +of water added, the flask attached to a vertical condenser by means +of a bent bulb tube, 100 cubic centimeters distilled and the specific +gravity of the distillate determined. The distillate is also weighed, +or its weight calculated from the specific gravity. The corresponding +percentage of alcohol by weight is obtained from the appended table, +and this figure multiplied by the weight of the distillate, and the +result divided by the weight of the sample, gives the per cent of +alcohol by weight contained therein. + +The percentage of alcohol by volume of the liquor is the same as that +of the distillate, and is obtained directly from the appended table. + +In distilled liquors about thirty grams are diluted to 150 cubic +centimeters, 100 cubic centimeters distilled and the per cent of +alcohol by weight determined as above. + +The percentage of alcohol by volume in the distillate is obtained from +the appended table. This figure divided by the number expressing the +volume in cubic centimeters of the liquor taken for the determination +(calculated from the specific gravity), and the result multiplied by +100 gives the per cent of alcohol by volume in the original liquor. + +=618. Determining the Specific Gravity of the Distillate.=—The specific +gravity of the distillate may be determined by the pyknometer, +alcoholometer, hydrostatic balance or in any accurate way. The volume +of the distillate is not always large enough to be conveniently used +with an alcoholometer, especially the large ones employed by the Bureau +of Internal Revenue. In the laboratory of the Agricultural Department, +it is customary to determine the density of the distillate by the +hydrostatic balance shown in paragraph =285=. The specific gravity +is in each case determined at 15°.6, referred to water of the same +temperature, or if at a different temperature calculated thereto. + +=619. Table for Use with Hydrostatic Plummet.=—It is more convenient to +determine the density of the alcoholic distillate at room temperature +than to reduce it to the standard for which the plummet is graduated. +In the case of a plummet which displaces exactly five grams, or +multiple thereof, of distilled water at 15°.6, the corrections for +temperatures between 12°.2 and 30° are found in the following table, +prepared by Bigelow.[635] + +If the weight of the alcoholic solution displaced be 4.96075 grams the +apparent specific gravity 0.99215 and the temperature of observation +25°.4, the correction, which is additive, as given in the table is +0.00191 and the true specific gravity is 0.99406 and the percentage of +alcohol by volume 4.08. + +When the plummet does not exactly displace five grams of water at +15°.6, but nearly so, the table may still be used. + +For example, suppose the weight of water displaced be 4.9868 instead of +five grams. The apparent specific gravity of the water by this plummet +is 0.99736 and the difference between this and the true specific +gravity is 0.00264, which is a constant correction to be added to the +specific gravity as determined in each case. + + CORRECTION TABLE FOR SPECIFIC GRAVITY. + + _Below 15°.6 Subtract; Above 15°.6 Add._ + + Temp. Correction. Temp. Correction. Temp. Correction. + + 12.2 0.00047 18.2 0.00043 24.2 0.00163 + 12.4 0.00044 18.4 0.00046 24.4 0.00167 + 12.6 0.00042 18.6 0.00050 24.6 0.00172 + 12.8 0.00039 18.8 0.00053 24.8 0.00176 + 13.0 0.00037 19.0 0.00057 25.0 0.00181 + 13.2 0.00634 19.2 0.00061 25.2 0.00186 + 13.4 0.00032 19.4 0.00065 25.4 0.00191 + 13.6 0.00029 19.6 0.00068 25.6 0.00195 + 13.8 0.00027 19.8 0.00072 25.8 0.00200 + 14.0 0.00024 20.0 0.00076 26.0 0.00205 + 14.2 0.00021 20.2 0.00080 26.2 0.00210 + 14.4 0.00018 20.4 0.00084 26.4 0.00215 + 14.6 0.00015 20.6 0.00087 26.6 0.00220 + 14.8 0.00012 20.8 0.00091 26.8 0.00225 + 15.0 0.00009 21.0 0.00095 27.0 0.00230 + 15.2 0.00006 21.2 0.00099 27.2 0.00235 + 15.4 0.00003 21.4 0.00103 27.4 0.00240 + 15.6 0.00000 21.6 0.00107 27.6 0.00246 + 15.8 0.00003 21.8 0.00111 27.8 0.00251 + 16.0 0.00006 22.0 0.00115 28.0 0.00256 + 16.2 0.00009 22.2 0.00119 28.2 0.00261 + 16.4 0.00012 22.4 0.00123 28.4 0.00267 + 16.6 0.00016 22.6 0.00128 28.6 0.00272 + 16.8 0.00019 22.8 0.00132 28.8 0.00278 + 17.0 0.00022 23.0 0.00136 29.0 0.00283 + 17.2 0.00025 23.2 0.00140 29.2 0.00288 + 17.4 0.00029 23.4 0.00145 29.4 0.00294 + 17.6 0.00032 23.6 0.00149 29.6 0.00299 + 17.8 0.00036 23.8 0.00154 29.8 0.00306 + 18.0 0.00039 24.0 0.00158 30.0 0.00311 + +The table is only accurate when the distillate does not contain over +seven nor less than three per cent of alcohol. If the distillate +contain more than seven per cent of alcohol it is diluted and the +compensating correction made. + +=620. Calculating Results.=—The specific gravity of the alcoholic +distillate having been determined by any approved method and corrected +to a temperature of 15°.6, the corresponding per cents of alcohol by +volume and by weight are found by consulting the following table.[636] +If, for example, the corrected specific gravity be exactly that given +in any figure of the table the corresponding per cents are directly +read. If the specific gravity found fall between two numbers in the +table the corresponding per cents are determined by interpolation. + + TABLE SHOWING PERCENTAGE OF ALCOHOL + BY WEIGHT AND BY VOLUME. + -------------+------------------+--------------- + Specific | Per cent | Per cent + gravity at | alcohol | alcohol + 15°.6/15°.6. | by volume. | by weight. + -------------+------------------+--------------- + 1.00000 | 0.00 | 0.00 + 0.99992 | .05 | .04 + 984 | .10 | .08 + 976 | .15 | .12 + 968 | .20 | .16 + 961 | .25 | .20 + 953 | .30 | .24 + 945 | .35 | .28 + 937 | .40 | .32 + 930 | .45 | .36 + .99923 | 0.50 | 0.40 + 915 | .55 | .44 + 907 | .60 | .48 + 900 | .65 | .52 + 892 | .70 | .56 + 884 | .75 | .60 + 877 | .80 | .64 + 869 | .85 | .67 + 861 | .90 | .71 + 854 | .95 | .75 + .99849 | 1.00 | 0.79 + 842 | .05 | .83 + 834 | .10 | .87 + 827 | .15 | .91 + 819 | .20 | .95 + 812 | .25 | .99 + 805 | .30 | 1.03 + 797 | .35 | .07 + 790 | .40 | .11 + 782 | .45 | .15 + .99775 | 1.50 | 1.19 + 768 | .55 | .23 + 760 | .60 | .27 + 753 | .65 | .31 + 745 | .70 | .35 + 738 | .75 | .39 + 731 | .80 | .43 + 723 | .85 | .47 + 716 | .90 | .51 + 708 | .95 | .55 + .99701 | 2.00 | 1.59 + 694 | .05 | .63 + 687 | .10 | .67 + 679 | .15 | .71 + 672 | .20 | .75 + 665 | .25 | .79 + 658 | .30 | .83 + 651 | .35 | .87 + 643 | .40 | .91 + 636 | .45 | .95 + 0.99629 | 2.50 | 1.99 + 622 | .55 | 2.03 + 615 | .60 | .07 + 607 | .65 | .11 + 600 | .70 | .15 + 593 | .75 | .19 + 586 | .80 | .23 + 579 | .85 | .27 + 571 | .90 | .31 + 564 | .95 | .35 + .99557 | 3.00 | 2.39 + 550 | .05 | .43 + 543 | .10 | .47 + 536 | .15 | .51 + 529 | .20 | .55 + 522 | .25 | .59 + 515 | .30 | .64 + 508 | .35 | .68 + 501 | .40 | .72 + 494 | .45 | .76 + .99487 | 3.50 | 2.80 + 480 | .55 | .84 + 473 | .60 | .88 + 466 | .65 | .92 + 459 | .70 | .96 + 452 | .75 | 3.00 + 445 | .80 | .04 + 438 | .85 | .08 + 431 | .90 | .12 + 424 | .95 | .16 + .99417 | 4.00 | 3.20 + 410 | .05 | .24 + 403 | .10 | .28 + 397 | .15 | .32 + 390 | .20 | .36 + 383 | .25 | .40 + 376 | .30 | .44 + 369 | .35 | .48 + 363 | .40 | .52 + 356 | .45 | .56 + .99349 | 4.50 | 3.60 + 342 | .55 | .64 + 335 | .60 | .68 + 329 | .65 | .72 + 322 | .70 | .76 + 315 | .75 | .80 + 308 | .80 | .84 + 301 | .85 | .88 + 295 | .90 | .92 + 288 | .95 | .96 + 0.99281 | 5.00 | 4.00 + 274 | .05 | .04 + 268 | .10 | .08 + 261 | .15 | .12 + 255 | .20 | .16 + 248 | .25 | .20 + 241 | .30 | .24 + 235 | .35 | .28 + 228 | .40 | .32 + 222 | .45 | .36 + .99215 | 5.50 | 4.40 + 208 | .55 | .44 + 202 | .60 | .48 + 195 | .65 | .52 + 189 | .70 | .56 + 182 | .75 | .60 + 175 | .80 | .64 + 169 | .85 | .68 + 162 | .90 | .72 + 156 | .95 | .76 + .99149 | 6.00 | 4.80 + 143 | .05 | .84 + 136 | .10 | .87 + 130 | .15 | .92 + 123 | .20 | .96 + 117 | .25 | 5.00 + 111 | .30 | .05 + 104 | .35 | .09 + 098 | .40 | .13 + 091 | .45 | .17 + .99085 | 6.50 | 5.21 + 079 | .55 | .25 + 072 | .60 | .29 + 066 | .65 | .33 + 059 | .70 | .37 + 053 | .75 | .41 + 047 | .80 | .45 + 040 | .85 | .49 + 034 | .90 | .53 + 027 | .95 | .57 + .99021 | 7.00 | 5.61 + 015 | .05 | .65 + 009 | .10 | .69 + 002 | .15 | .73 + .98996 | .20 | .77 + 990 | .25 | .81 + 984 | .30 | .86 + 978 | .35 | .90 + 971 | .40 | .94 + 965 | .45 | .98 + 0.98959 | 7.50 | 6.02 + 953 | .55 | .06 + 947 | .60 | .10 + 940 | .65 | .14 + 934 | .70 | .18 + 928 | .75 | .22 + 922 | .80 | .26 + 916 | .85 | .30 + 909 | .90 | .34 + 903 | .95 | .38 + .98897 | 8.00 | 6.42 + 891 | .05 | .46 + 885 | .10 | .50 + 879 | .15 | .54 + 873 | .20 | .58 + 867 | .25 | .62 + 861 | .30 | .67 + 855 | .35 | .71 + 849 | .40 | .75 + 843 | .45 | .79 + .98837 | 8.50 | 6.83 + 831 | .55 | .87 + 825 | .60 | .91 + 819 | .65 | .95 + 813 | .70 | .99 + 807 | .75 | 7.03 + 801 | .80 | .07 + 795 | .85 | .11 + 789 | .90 | .15 + 783 | .95 | .19 + .98777 | 9.00 | 7.23 + 771 | .05 | .27 + 765 | .10 | .31 + 754 | .20 | .39 + 748 | .25 | .43 + 742 | .30 | .48 + 736 | .35 | .52 + 724 | .45 | .60 + .98719 | 9.50 | 7.64 + 713 | .55 | .68 + 707 | .60 | .72 + 701 | .65 | .76 + 695 | .70 | .80 + 689 | .75 | .84 + 683 | .80 | .88 + 678 | .85 | .92 + 672 | .90 | .96 + 666 | .95 | 8.00 + 0.98660 | 10.00 | 8.04 + 654 | .05 | .08 + 649 | .10 | .12 + 643 | .15 | .16 + 637 | .20 | .20 + 632 | .25 | .24 + 626 | .30 | .28 + 620 | .35 | .33 + 614 | .40 | .37 + 609 | .45 | .41 + .98603 | 10.50 | 8.45 + 597 | .55 | .49 + 592 | .60 | .53 + 586 | .65 | .57 + 580 | .70 | .61 + 575 | .75 | .65 + 569 | .80 | .70 + 563 | .85 | .74 + 557 | .90 | .78 + 552 | .95 | .82 + .98546 | 11.00 | 8.86 + 540 | .05 | .90 + 535 | .10 | .94 + 529 | .15 | .98 + 524 | .20 | 9.02 + 518 | .25 | .07 + 513 | .30 | .11 + 507 | .35 | .15 + 502 | .40 | .19 + 496 | .45 | .23 + .98491 | 11.50 | 9.27 + 485 | .55 | .31 + 479 | .60 | .35 + 474 | .65 | .39 + 468 | .70 | .43 + 463 | .75 | .47 + 457 | .80 | .51 + 452 | .85 | .55 + 446 | .90 | .59 + 441 | .95 | .63 + .98435 | 12.00 | 9.67 + 430 | .05 | .71 + 424 | .10 | .75 + 419 | .15 | .79 + 413 | .20 | .83 + 408 | .25 | .87 + 402 | .30 | .92 + 397 | .35 | .96 + 391 | .40 | 10.00 + 386 | .45 | .04 + 0.98381 | 12.50 | 10.08 + 375 | .55 | .12 + 370 | .60 | .16 + 364 | .65 | .20 + 359 | .70 | .24 + 353 | .75 | .28 + 348 | .80 | .33 + 342 | .85 | .37 + 337 | .90 | .41 + 331 | .95 | .45 + .98326 | 13.00 | 10.49 + 321 | .05 | .53 + 315 | .10 | .57 + 310 | .15 | .61 + 305 | .20 | .65 + 299 | .25 | .69 + 294 | .30 | .74 + 289 | .35 | .78 + 283 | .40 | .82 + 278 | .45 | .86 + .98273 | 13.50 | 10.90 + 267 | .55 | .94 + 262 | .60 | .98 + 256 | .65 | 11.02 + 251 | .70 | .06 + 246 | .75 | .14 + 240 | .80 | .15 + 235 | .85 | .19 + 230 | .90 | .23 + 224 | .95 | .27 + .98219 | 14.00 | 11.31 + 214 | .05 | .35 + 209 | .10 | .39 + 203 | .15 | .43 + 198 | .20 | .47 + 193 | .25 | .52 + 188 | .30 | .56 + 182 | .35 | .60 + 177 | .40 | .64 + 172 | .45 | .68 + .98167 | 14.50 | 11.72 + 161 | .55 | .76 + 156 | .60 | .80 + 151 | .65 | .84 + 146 | .70 | .88 + 140 | .75 | .93 + 135 | .80 | .97 + 130 | .85 | 12.01 + 125 | .90 | .05 + 119 | .95 | .09 + 0.98114 | 15.00 | 12.13 + 108 | .05 | .17 + 104 | .10 | .21 + 099 | .15 | .25 + 093 | .20 | .29 + 088 | .25 | .33 + 083 | .30 | .38 + 078 | .35 | .42 + 073 | .40 | .46 + 068 | .45 | .50 + .98063 | 15.50 | 12.54 + 057 | .55 | .58 + 052 | .60 | .62 + 047 | .65 | .66 + 042 | .70 | .70 + 037 | .75 | .75 + 032 | .80 | .79 + 026 | .85 | .83 + 021 | .90 | .87 + 016 | .95 | .91 + .98011 | 16.00 | 12.95 + 005 | .05 | .99 + 001 | .10 | 13.03 + .97996 | .15 | .08 + 991 | .20 | .12 + 986 | .25 | .16 + 980 | .30 | .20 + 975 | .35 | .24 + 970 | .40 | .29 + 965 | .45 | .33 + .97960 | 16.50 | 13.37 + 955 | .55 | .41 + 950 | .60 | .45 + 945 | .65 | .49 + 940 | .70 | .53 + 935 | .75 | .57 + 929 | .80 | .62 + 924 | .85 | .66 + 919 | .90 | .70 + 914 | .95 | .74 + .97909 | 17.00 | 13.78 + 904 | .05 | .82 + 899 | .10 | .86 + 894 | .15 | .90 + 889 | .20 | .94 + 884 | .25 | .98 + 879 | .30 | 14.03 + 874 | .35 | .07 + 869 | .40 | .11 + 864 | .45 | .15 + 0.97859 | 17.50 | 14.19 + 853 | .55 | .23 + 848 | .60 | .27 + 843 | .65 | .31 + 838 | .70 | .35 + 833 | .75 | .40 + 828 | .80 | .44 + 823 | .85 | .48 + 818 | .90 | .52 + 813 | .95 | .56 + .97808 | 18.00 | 14.60 + 803 | .05 | .64 + 798 | .10 | .68 + 793 | .15 | .73 + 788 | .20 | .77 + 783 | .25 | .81 + 778 | .30 | .85 + 773 | .35 | .89 + 768 | .40 | .94 + 763 | .45 | .98 + .97758 | 18.50 | 15.02 + 753 | .55 | .06 + 748 | .60 | .10 + 743 | .65 | .14 + 738 | .70 | .18 + 733 | .75 | .22 + 728 | .80 | .27 + 723 | .85 | .31 + 718 | .90 | .35 + 713 | .95 | .39 + .97708 | 19.00 | 15.43 + 703 | .05 | .47 + 698 | .10 | .51 + 693 | .15 | .55 + 688 | .20 | .59 + 683 | .25 | .63 + 678 | .30 | .68 + 673 | .35 | .72 + 668 | .40 | .76 + 663 | .45 | .80 + .97658 | 19.50 | 15.84 + 653 | .55 | .88 + 648 | .60 | .93 + 643 | .65 | .97 + 638 | .70 | 16.01 + 633 | .75 | .05 + 628 | .80 | .09 + 623 | .85 | .14 + 618 | .90 | .18 + 613 | .95 | .22 + 0.97608 | 20.00 | 16.26 + 603 | .05 | .30 + 598 | .10 | .34 + 593 | .15 | .38 + 588 | .20 | .42 + 583 | .25 | .46 + 578 | .30 | .51 + 573 | .35 | .58 + 568 | .40 | .59 + 563 | .45 | .63 + .97558 | 20.50 | 16.67 + 552 | .55 | .71 + 547 | .60 | .75 + 542 | .65 | .80 + 537 | .70 | .84 + 532 | .75 | .88 + 527 | .80 | .92 + 522 | .85 | .96 + 517 | .90 | 17.01 + 512 | .95 | .05 + .97507 | 21.00 | 17.09 + 502 | .05 | .13 + 497 | .10 | .17 + 492 | .15 | .22 + 487 | .20 | .26 + 482 | .25 | .30 + 477 | .30 | .34 + 472 | .35 | .38 + 467 | .40 | .43 + 462 | .45 | .47 + .97457 | 21.50 | 17.51 + 451 | .55 | .55 + 446 | .60 | .59 + 441 | .65 | .63 + 436 | .70 | .67 + 431 | .75 | .71 + 426 | .80 | .76 + 421 | .85 | .80 + 416 | .90 | .84 + 411 | .95 | .88 + .97406 | 22.00 | 17.92 + 401 | .05 | .96 + 396 | .10 | 18.00 + 391 | .15 | .05 + 386 | .20 | .09 + 381 | .25 | .13 + 375 | .30 | .17 + 370 | .35 | .21 + 365 | .40 | .26 + 360 | .45 | .30 + 0.97355 | 22.50 | 18.34 + 350 | .55 | .38 + 345 | .60 | .42 + 340 | .65 | .47 + 335 | .70 | .51 + 330 | .75 | .55 + 324 | .80 | .59 + 319 | .85 | .63 + 314 | .90 | .68 + 309 | .95 | .72 + .97304 | 23.00 | 18.76 + 299 | .05 | .80 + 294 | .10 | .84 + 289 | .15 | .88 + 283 | .20 | .92 + 278 | .25 | .96 + 273 | .30 | 19.01 + 268 | .35 | .05 + 263 | .40 | .09 + 258 | .45 | .13 + .97253 | 23.50 | 19.17 + 247 | .55 | .21 + 242 | .60 | .25 + 237 | .65 | .30 + 232 | .70 | .34 + 227 | .75 | .38 + 222 | .80 | .42 + 216 | .85 | .46 + 211 | .90 | .51 + 206 | .95 | .55 + .97201 | 24.00 | 19.59 + 196 | .05 | .63 + 191 | .10 | .67 + 185 | .15 | .72 + 180 | .20 | .76 + 175 | .25 | .80 + 170 | .30 | .84 + 165 | .35 | .88 + 159 | .40 | .93 + 154 | .45 | .97 + .97149 | 24.50 | 20.01 + 144 | .55 | .05 + 139 | .60 | .09 + 133 | .65 | .14 + 128 | .70 | .18 + 123 | .75 | .22 + 118 | .80 | .26 + 113 | .85 | .30 + 107 | .90 | .35 + 102 | .95 | .39 + 0.97097 | 25.00 | 20.43 + 092 | .05 | .47 + 086 | .10 | .51 + 081 | .15 | .56 + 076 | .20 | .60 + 071 | .25 | .64 + 065 | .30 | .68 + 060 | .35 | .72 + 055 | .40 | .77 + 049 | .45 | .81 + .97014 | 25.50 | 20.85 + 039 | .55 | .89 + 033 | .60 | .93 + 028 | .65 | .98 + 023 | .70 | 21.02 + 018 | .75 | .06 + 012 | .80 | .10 + 007 | .85 | .14 + 001 | .90 | .19 + .96996 | .95 | .23 + .96991 | 26.00 | 21.27 + 986 | .05 | .31 + 980 | .10 | .35 + 975 | .15 | .40 + 969 | .20 | .44 + 964 | .25 | .48 + 959 | .30 | .52 + 953 | .35 | .56 + 949 | .40 | .61 + 942 | .45 | .65 + .96937 | 26.50 | 21.69 + 932 | .55 | .73 + 926 | .60 | .77 + 921 | .65 | .82 + 915 | .70 | .86 + 910 | .75 | .90 + 905 | .80 | .94 + 899 | .85 | .98 + 894 | .90 | 22.03 + 888 | .95 | .07 + .96883 | 27.00 | 22.11 + 877 | .05 | .15 + 872 | .10 | .20 + 866 | .15 | .24 + 861 | .20 | .28 + 855 | .25 | .33 + 850 | .30 | .37 + 844 | .35 | .41 + 839 | .40 | .45 + 833 | .45 | .50 + 0.96828 | 27.50 | 22.54 + 822 | .55 | .58 + 816 | .60 | .62 + 811 | .65 | .67 + 805 | .70 | .71 + 800 | .75 | .75 + 794 | .80 | .79 + 789 | .85 | .83 + 783 | .90 | .88 + 778 | .95 | .92 + .96772 | 28.00 | 22.96 + 766 | .05 | 23.00 + 761 | .10 | .04 + 755 | .15 | .09 + 749 | .20 | .13 + 744 | .25 | .17 + 738 | .30 | .21 + 732 | .35 | .25 + 726 | .40 | .30 + 721 | .45 | .34 + .96715 | 28.50 | 23.38 + 709 | .55 | .42 + 704 | .60 | .47 + 698 | .65 | .51 + 692 | .70 | .55 + 687 | .75 | .60 + 681 | .80 | .64 + 675 | .85 | .68 + 669 | .90 | .72 + 664 | .95 | .77 + .96658 | 29.00 | 23.81 + 652 | .05 | .85 + 646 | .10 | .89 + 640 | .15 | .94 + 635 | .20 | .98 + 629 | .25 | 24.02 + 623 | .30 | .06 + 617 | .35 | .10 + 611 | .40 | .15 + 605 | .45 | .19 + .96600 | 29.50 | 24.23 + 594 | .55 | .27 + 587 | .60 | .32 + 582 | .65 | .36 + 576 | .70 | .40 + 570 | .75 | .45 + 564 | .80 | .49 + 559 | .85 | .53 + 553 | .90 | .57 + 547 | .95 | .62 + 0.96541 | 30.00 | 24.66 + 535 | .05 | .70 + 529 | .10 | .74 + 523 | .15 | .79 + 517 | .20 | .83 + 511 | .25 | .87 + 505 | .30 | .91 + 499 | .35 | .95 + 493 | .40 | 25.00 + 487 | .45 | .04 + .96481 | 30.50 | 25.08 + 475 | .55 | .12 + 469 | .60 | .17 + 463 | .65 | .21 + 457 | .70 | .25 + 451 | .75 | .30 + 445 | .80 | .34 + 439 | .85 | .38 + 433 | .90 | .42 + 427 | .95 | .47 + .96421 | 31.00 | 25.51 + 415 | .05 | .55 + 409 | .10 | .60 + 403 | .15 | .64 + 396 | .20 | .68 + 390 | .25 | .73 + 384 | .30 | .77 + 378 | .35 | .81 + 372 | .40 | .85 + 366 | .45 | .90 + .96360 | 31.50 | 25.94 + 353 | .55 | .98 + 347 | .60 | 26.03 + 341 | .65 | .07 + 335 | .70 | .11 + 329 | .75 | .16 + 323 | .80 | .20 + 316 | .85 | .24 + 310 | .90 | .28 + 304 | .95 | .33 + .96298 | 32.00 | 26.37 + 292 | .05 | .41 + 285 | .10 | .46 + 279 | .15 | .50 + 273 | .20 | .54 + 267 | .25 | .59 + 260 | .30 | .63 + 254 | .35 | .67 + 248 | .40 | .71 + 241 | .45 | .76 + 0.96235 | 32.50 | 26.80 + 229 | .55 | .84 + 222 | .60 | .89 + 216 | .65 | .93 + 210 | .70 | .97 + 204 | .75 | 27.02 + 197 | .80 | .06 + 191 | .85 | .10 + 185 | .90 | .14 + 178 | .95 | .19 + .96172 | 33.00 | 27.23 + 166 | .05 | .27 + 159 | .10 | .32 + 153 | .15 | .36 + 146 | .20 | .40 + 140 | .25 | .45 + 133 | .30 | .49 + 127 | .35 | .53 + 120 | .40 | .57 + 114 | .45 | .62 + .96108 | 33.50 | 27.66 + 101 | .55 | .70 + 095 | .60 | .75 + 088 | .65 | .79 + 082 | .70 | .83 + 075 | .75 | .88 + 069 | .80 | .92 + 062 | .85 | .97 + 056 | .90 | 28.00 + 049 | .95 | .05 + .96043 | 34.00 | 28.09 + 036 | .05 | .13 + 030 | .10 | .18 + 023 | .15 | .22 + 016 | .20 | .26 + 010 | .25 | .31 + 003 | .30 | .35 + .95996 | .35 | .39 + 990 | .40 | .43 + 983 | .45 | .48 + .95977 | 34.50 | 28.52 + 970 | .55 | .56 + 963 | .60 | .61 + 957 | .65 | .65 + 950 | .70 | .70 + 943 | .75 | .74 + 937 | .80 | .78 + 930 | .85 | .83 + 923 | .90 | .87 + 917 | .95 | .92 + 0.95910 | 35.00 | 28.96 + 903 | .05 | 29.00 + 896 | .10 | .05 + 889 | .15 | .09 + 883 | .20 | .13 + 876 | .25 | .18 + 869 | .30 | .22 + 862 | .35 | .26 + 855 | .40 | .30 + 848 | .45 | .35 + .95842 | 35.50 | 29.39 + 835 | .55 | .43 + 828 | .60 | .48 + 821 | .65 | .52 + 814 | .70 | .57 + 807 | .75 | .61 + 800 | .80 | .65 + 794 | .85 | .70 + 787 | .90 | .74 + 780 | .95 | .79 + .95773 | 36.00 | 29.83 + 766 | .05 | .87 + 759 | .10 | .92 + 752 | .15 | .96 + 745 | .20 | 30.00 + 738 | .25 | .05 + 731 | .30 | .09 + 724 | .35 | .13 + 717 | .40 | .17 + 710 | .45 | .22 + .95703 | 36.50 | 30.26 + 695 | .55 | .30 + 688 | .60 | .35 + 681 | .65 | .39 + 674 | .70 | .44 + 667 | .75 | .48 + 660 | .80 | .52 + 653 | .85 | .57 + 646 | .90 | .61 + 639 | .95 | .66 + .95632 | 37.00 | 30.70 + 625 | .05 | .74 + 618 | .10 | .79 + 610 | .15 | .83 + 603 | .20 | .88 + 596 | .25 | .92 + 589 | .30 | .96 + 581 | .35 | 31.01 + 574 | .40 | .05 + 567 | .45 | .10 + 0.95560 | 37.50 | 31.14 + 552 | .55 | .18 + 545 | .60 | .23 + 538 | .65 | .27 + 531 | .70 | .32 + 523 | .75 | .36 + 516 | .80 | .40 + 509 | .85 | .45 + 502 | .90 | .49 + 494 | .95 | .54 + .95487 | 38.00 | 31.58 + 480 | .05 | .63 + 472 | .10 | .67 + 465 | .15 | .72 + 457 | .20 | .76 + 450 | .25 | .81 + 442 | .30 | .85 + 435 | .35 | .90 + 427 | .40 | .94 + 420 | .45 | .99 + .95413 | 38.50 | 32.03 + 405 | .55 | .07 + 398 | .60 | .12 + 390 | .65 | .16 + 383 | .70 | .20 + 375 | .75 | .25 + 368 | .80 | .29 + 360 | .85 | .33 + 353 | .90 | .37 + 345 | .95 | .42 + .95338 | 39.00 | 32.46 + 330 | .05 | .50 + 323 | .10 | .55 + 315 | .15 | .59 + 307 | .20 | .64 + 300 | .25 | .68 + 292 | .30 | .72 + 284 | .35 | .77 + 277 | .40 | .81 + 269 | .45 | .86 + .95262 | 39.50 | 32.90 + 254 | .55 | .95 + 246 | .60 | .99 + 239 | .65 | 33.04 + 231 | .70 | .08 + 223 | .75 | .13 + 216 | .80 | .17 + 208 | .85 | .22 + 200 | .90 | .27 + 193 | .95 | .31 + 0.95185 | 40.00 | 33.35 + 177 | .05 | .39 + 169 | .10 | .44 + 161 | .15 | .48 + 154 | .20 | .53 + 146 | .25 | .57 + 138 | .30 | .61 + 130 | .35 | .66 + 122 | .40 | .70 + 114 | .45 | .75 + .95107 | 40.50 | 33.79 + 099 | .55 | .84 + 091 | .60 | .88 + 083 | .65 | .93 + 075 | .70 | .97 + 067 | .75 | 34.02 + 059 | .80 | .06 + 052 | .85 | .11 + 044 | .90 | .15 + 036 | .95 | .20 + .95028 | 41.00 | 34.24 + 020 | .05 | .28 + 012 | .10 | .33 + 004 | .15 | .37 + .94996 | .20 | .42 + 988 | .25 | .46 + 980 | .30 | .50 + 972 | .35 | .55 + 964 | .40 | .59 + 956 | .45 | .64 + .94948 | 41.50 | 34.68 + 940 | .55 | .73 + 932 | .60 | .77 + 924 | .65 | .82 + 916 | .70 | .86 + 908 | .75 | .91 + 900 | .80 | .95 + 892 | .85 | 35.00 + 884 | .90 | .04 + 876 | .95 | .09 + .94868 | 42.00 | 35.13 + 860 | .05 | .18 + 852 | .10 | .22 + 843 | .15 | .27 + 835 | .20 | .31 + 827 | .25 | .36 + 820 | .30 | .40 + 811 | .35 | .45 + 802 | .40 | .49 + 794 | .45 | .54 + 0.94786 | 42.50 | 35.58 + 778 | .55 | .63 + 770 | .60 | .67 + 761 | .65 | .72 + 753 | .70 | .76 + 745 | .75 | .81 + 737 | .80 | .85 + 729 | .85 | .90 + 720 | .90 | .94 + 712 | .95 | .99 + .94704 | 43.00 | 36.03 + 696 | .05 | .08 + 687 | .10 | .12 + 679 | .15 | .17 + 670 | .20 | .21 + 662 | .25 | .23 + 654 | .30 | .30 + 645 | .35 | .35 + 637 | .40 | .39 + 628 | .45 | .44 + .94620 | 43.50 | 36.48 + 612 | .55 | .53 + 603 | .60 | .57 + 595 | .65 | .62 + 586 | .70 | .66 + 578 | .75 | .71 + 570 | .80 | .75 + 561 | .85 | .80 + 553 | .90 | .84 + 544 | .95 | .89 + .94536 | 44.00 | 36.93 + 527 | .05 | .98 + 519 | .10 | 37.02 + 510 | .15 | .07 + 502 | .20 | .11 + 493 | .25 | .16 + 484 | .30 | .21 + 476 | .35 | .25 + 467 | .40 | .30 + 459 | .45 | .34 + .94450 | 44.50 | 37.39 + 441 | .55 | .44 + 433 | .60 | .48 + 424 | .65 | .53 + 416 | .70 | .57 + 407 | .75 | .62 + 398 | .80 | .66 + 390 | .85 | .71 + 381 | .90 | .76 + 373 | .95 | .80 + 0.94364 | 45.00 | 37.84 + 355 | .05 | .89 + 346 | .10 | .93 + 338 | .15 | .98 + 329 | .20 | 38.02 + 320 | .25 | .07 + 311 | .30 | .12 + 302 | .35 | .16 + 294 | .40 | .21 + 285 | .45 | .25 + .94276 | 45.50 | 38.30 + 267 | .55 | .35 + 258 | .60 | .39 + 250 | .65 | .44 + 241 | .70 | .48 + 232 | .75 | .53 + 223 | .80 | .57 + 214 | .85 | .62 + 206 | .90 | .66 + 197 | .95 | .71 + .94188 | 46.00 | 38.75 + 179 | .05 | .80 + 170 | .10 | .84 + 161 | .15 | .89 + 152 | .20 | .93 + 143 | .25 | .98 + 134 | .30 | 39.03 + 125 | .35 | .07 + 116 | .40 | .12 + 107 | .45 | .16 + .94098 | 46.50 | 39.21 + 089 | .55 | .26 + 080 | .60 | .30 + 071 | .65 | .35 + 062 | .70 | .39 + 053 | .75 | .44 + 044 | .80 | .49 + 035 | .85 | .53 + 026 | .90 | .58 + 017 | .95 | .62 + .94008 | 47.00 | 39.67 + .93999 | .05 | .72 + 990 | .10 | .76 + 980 | .15 | .81 + 971 | .20 | .85 + 962 | .25 | .90 + 953 | .30 | .95 + 944 | .35 | .99 + 934 | .40 | 40.04 + 925 | .45 | .08 + 0.93916 | 47.50 | 40.13 + 906 | .55 | .18 + 898 | .60 | .22 + 888 | .65 | .27 + 879 | .70 | .32 + 870 | .75 | .37 + 861 | .80 | .41 + 852 | .85 | .46 + 842 | .90 | .51 + 833 | .95 | .55 + .93824 | 48.00 | 40.60 + 815 | .05 | .65 + 808 | .10 | .69 + 796 | .15 | .74 + 786 | .20 | .78 + 777 | .25 | .83 + 768 | .30 | .88 + 758 | .35 | .92 + 740 | .40 | .97 + 739 | .45 | 41.01 + .93730 | 48.50 | 41.06 + 721 | .55 | .11 + 711 | .60 | .15 + 702 | .65 | .20 + 692 | .70 | .24 + 683 | .75 | .29 + 673 | .80 | .34 + 664 | .85 | .38 + 655 | .90 | .43 + 645 | .95 | .47 + .93636 | 49.00 | 41.52 + 626 | .05 | .57 + 617 | .10 | .61 + 607 | .15 | .66 + 598 | .20 | .71 + 588 | .25 | .76 + 578 | .30 | .80 + 569 | .35 | .85 + 559 | .40 | .90 + 550 | .45 | .94 + .93540 | 49.50 | 41.99 + 530 | .55 | 42.04 + 521 | .60 | .08 + 511 | .65 | .13 + 502 | .70 | .18 + 492 | .75 | .23 + 482 | .80 | .27 + 473 | .85 | .32 + 463 | .90 | .37 + 454 | .95 | .41 + -------------+------------------+------------------ + +=621. Determination of Percentage of Alcohol by Means Of Vapor +Temperature.=—The temperature of a mixture of alcohol and water vapors +is less than that of water alone and the depression is inversely +proportional to the quantity of alcohol present. This principle is +utilized in the construction of the ebullioscope or ebulliometer. +In this apparatus the temperature of pure boiling water vapor is +determined by a preliminary experiment. This point must be frequently +revised in order to correct it for variations in barometric pressure. +The water is withdrawn from the boiler of the apparatus, the same +volume of a wine or beer placed therein, and the vapor temperature +again determined. By comparing the boiling point of the wine, with a +scale calibrated for different percentages of alcohol, the quantity +of spirit present is determined. When water vapor is at 100° a _vin +ordinaire_ having eight per cent of alcohol gives a vapor at 93°.8. The +presence of extractive matters in the sample, which tend to raise its +boiling point, is neglected in the calculation of results. + +=622. Improved Ebullioscope.=—The principle mentioned in the above +paragraph may be applied with a considerable degree of accuracy, by +using the improved ebullioscope described below.[637] + +The apparatus consists of a glass flask F, shaped somewhat like an +erlenmeyer, closed at the top with a rubber stopper carrying a central +aperture for the insertion of the delicate thermometer A B, and a +lateral smaller aperture for connecting the interior of the flask with +the condenser D. The return of the condensed vapors from D is effected +through the tube entering the flask F in such a manner as to deliver +the condensed liquid beneath the surface of the liquid in F as shown +in the figure. The flask F contains pieces of scrap platinum or pumice +stone to prevent bumping and secure an even ebullition. The flask F +rests upon a disk of asbestos, perforated in such a way as to have the +opening fully covered by the bottom of the flask. To protect F against +the influence of air currents it is enclosed in the glass cylinder E +resting on the asbestos disk below and closed with a detachable soft +rubber cover at the top. The temperature between the cylinder E and +the flask F is measured by the thermometer C and the flame of the +lamp G should be so adjusted as to bring the temperature between the +flask F and the cylinder E to about 90° at the time of reading the +thermometer B. The bulb of the thermometer B may be protected by a thin +glass tube carrying distilled water, so adjusted as to prevent the +escape of the watery vapor into F. The thermometer B is such as is used +for determining molecular weights by the cryoscopic method. It has a +cistern at A which holds any excess of mercury not needed in _adjusting +the thermometer_ for any required temperature. + +[Illustration: FIG. 125. IMPROVED EBULLIOSCOPE.] + +A second apparatus, exactly similar to the one described, is +conveniently used for measuring the changes in _barometric_ pressure +during the process of the analysis. The temperature of the vapor of +boiling water having been first determined, the beer or wine is placed +in F, and the temperature of the vapor of the boiling liquid determined +after the temperature of the air layer between E and F reaches about +90°, measured on the thermometer C. By using alcoholic mixtures of +known strength the depression for each changing per cent of alcohol +is determined for each system of apparatus employed, and this having +once been done, the percentage of alcohol in any unknown liquid is +at once determined by inspecting the thermometer, the bulb of which +is immersed in the vapor from the boiling liquid. In the apparatus +figured, a depression of 0°.8 is equivalent to one per cent of alcohol +by volume. Full directions for the manipulation of the apparatus may be +found in the paper cited above. + +=623. Total Fixed Matters.=—The residue left on evaporating a fermented +beverage to dryness is commonly known as extractive matter, or +simply extract. It is composed chiefly of unfermented carbohydrates, +organic acids, nitrogenous bodies, glycerol and mineral substances. +Hydrochloric and sulfuric acids may also be found therein. If any +non-volatile preservatives have been used in the sample, such as +borax, salicylates and the like, these will also be found in the +solid residue. The bodies which escape are water, alcohols, ethers +and essential oils. The character of the residue left by wines and +beers is evidently different. In each case it should contain typical +components which aid in judging of the purity of the sample. For +instance, in beers the substitution for malt of carbohydrate bodies +comparatively free of proteids, produces a beer containing a deficiency +of nitrogenous bodies. Pure malt beer will rarely have less than +one-half of a per cent of proteids, while beer made largely of glucose, +rice or hominy grits, will have a much smaller quantity. First will be +described below the methods of determining the fixed residue left on +evaporation, and thereafter the processes for ascertaining its leading +components. + +=624. Methods of the Official Chemists.=—Two methods are in use by +the official chemists for determining the fixed solids in fermented +beverages.[638] They are as follows: + +_Direct Method._—Fifty cubic centimeters of the sample are weighed, +placed in a platinum dish about eighty millimeters in diameter and +capable of holding about seventy-five cubic centimeters and evaporated +on the steam bath to a sirupy consistence. The residue is heated for +two and a half hours in a drying oven at the temperature of boiling +water and weighed. + +_In Sweet Wines._—Ten cubic centimeters of the liquor are weighed and +diluted to 100 with water. Fifty cubic centimeters of this solution are +evaporated as described above. + +_Optional Method._—Fifty cubic centimeters of the sample are placed in +a platinum or porcelain dish and evaporated on the steam bath until +the volume is reduced to one-third. The dealcoholized liquid is washed +into a fifty cubic centimeter flask, cooled and made up to the original +volume. It is mixed thoroughly and the specific gravity ascertained +with a pyknometer, hydrostatic balance or an accurately standardized +hydrometer. The percentage of total solids is obtained from the +appended table. The column on the left of the specific gravity gives +the percentage of extract in a wine, as calculated by Hager, and that +on the right the percentage of extract in a beer or wort, as calculated +by Schultze. According to Baumert, however, Schultze’s table gives +results which approximate more closely the data obtained by direct +estimation than does Hager’s. + + TABLES OF HAGER AND SCHULTZE FOR + THE DETERMINATION OF EXTRACT + BY THE INDIRECT METHOD. + ======+==================+=========== + Hager.| Specific gravity.| Schultze. + ------+------------------+----------- + 0.84 | 1.0038 | 1.00 + 0.86 | 1.0039 | 1.02 + 0.88 | 1.0040 | 1.05 + 0.90 | 1.0041 | 1.08 + 0.92 | 1.0042 | 1.10 + 0.94 | 1.0043 | 1.13 + 0.96 | 1.0044 | 1.15 + 0.98 | 1.0045 | 1.18 + 1.00 | 1.0046 | 1.21 + 1.02 | 1.0047 | 1.23 + 1.04 | 1.0048 | 1.26 + 1.06 | 1.0049 | 1.29 + 1.08 | 1.0050 | 1.31 + 1.10 | 1.0051 | 1.34 + 1.12 | 1.0052 | 1.36 + 1.15 | 1.0053 | 1.39 + 1.17 | 1.0054 | 1.41 + 1.19 | 1.0055 | 1.44 + 1.22 | 1.0056 | 1.46 + 1.25 | 1.0057 | 1.49 + 1.27 | 1.0058 | 1.51 + 1.30 | 1.0059 | 1.54 + 1.32 | 1.0060 | 1.56 + 1.34 | 1.0061 | 1.59 + 1.37 | 1.0062 | 1.62 + 1.39 | 1.0063 | 1.64 + 1.42 | 1.0064 | 1.67 + 1.44 | 1.0065 | 1.69 + 1.46 | 1.0066 | 1.72 + 1.48 | 1.0067 | 1.74 + 1.50 | 1.0068 | 1.77 + 1.52 | 1.0069 | 1.79 + 1.55 | 1.0070 | 1.82 + 1.57 | 1.0071 | 1.84 + 1.59 | 1.0072 | 1.87 + 1.61 | 1.0073 | 1.90 + 1.64 | 1.0074 | 1.92 + 1.66 | 1.0075 | 1.95 + 1.68 | 1.0076 | 1.97 + 1.70 | 1.0077 | 2.00 + 1.72 | 1.0078 | 2.02 + 1.75 | 1.0079 | 2.05 + 1.77 | 1.0080 | 2.07 + 1.79 | 1.0081 | 2.10 + 1.82 | 1.0082 | 2.12 + 1.84 | 1.0083 | 2.15 + 1.86 | 1.0084 | 2.17 + 1.88 | 1.0085 | 2.20 + 1.90 | 1.0086 | 2.23 + 1.92 | 1.0087 | 2.25 + 1.94 | 1.0088 | 2.28 + 1.96 | 1.0089 | 2.30 + 1.98 | 1.0090 | 2.33 + 2.00 | 1.0091 | 2.35 + 2.03 | 1.0092 | 2.38 + 2.05 | 1.0093 | 2.41 + 2.07 | 1.0094 | 2.43 + 2.09 | 1.0095 | 2.46 + 2.11 | 1.0096 | 2.48 + 2.14 | 1.0097 | 2.51 + 2.16 | 1.0098 | 2.53 + 2.18 | 1.0099 | 2.56 + 2.21 | 1.0100 | 2.58 + 2.23 | 1.0101 | 2.61 + 2.25 | 1.0102 | 2.64 + 2.27 | 1.0103 | 2.66 + 2.30 | 1.0104 | 2.69 + 2.32 | 1.0105 | 2.71 + 2.34 | 1.0106 | 2.74 + 2.36 | 1.0107 | 2.76 + 2.38 | 1.0108 | 2.79 + 2.40 | 1.0109 | 2.82 + 2.42 | 1.0110 | 2.84 + 2.44 | 1.0111 | 2.87 + 2.46 | 1.0112 | 2.89 + 2.48 | 1.0113 | 2.92 + 2.50 | 1.0114 | 2.94 + 2.52 | 1.0115 | 2.97 + 2.54 | 1.0116 | 2.99 + 2.57 | 1.0117 | 3.02 + 2.59 | 1.0118 | 3.05 + 2.61 | 1.0119 | 3.07 + 2.64 | 1.0120 | 3.10 + 2.66 | 1.0121 | 3.12 + 2.68 | 1.0122 | 3.15 + 2.70 | 1.0123 | 3.17 + 2.72 | 1.0124 | 3.20 + 2.75 | 1.0125 | 3.23 + 2.77 | 1.0126 | 3.25 + 2.79 | 1.0127 | 3.28 + 2.82 | 1.0128 | 3.30 + 2.84 | 1.0129 | 3.33 + 2.86 | 1.0130 | 3.35 + 2.88 | 1.0131 | 3.38 + 2.90 | 1.0132 | 3.41 + 2.92 | 1.0133 | 3.43 + 2.94 | 1.0134 | 3.46 + 2.96 | 1.0135 | 3.48 + 2.98 | 1.0136 | 3.51 + 3.00 | 1.0137 | 3.54 + ======+==================+=========== + +If it be desired to use this table for the examination of liquors +containing a higher percentage of extract, Schultze’s table (intended +originally for wort) may be consulted. + +Gautier regards the fixed solids as the residue obtained on +evaporating, in a flat platinum dish, ten cubic centimeters of wine at +100° for four hours and a half.[639] + +The official French method is as follows: Twenty cubic centimeters of +wine are placed in a flat bottom, platinum dish of such a diameter +that the depth of the liquid therein does not exceed one millimeter. +The dish should be immersed as totally as possible in the steam. The +heating is continued for six hours. + +The following method is used at the municipal laboratory of Paris: + +Twenty-five cubic centimeters of wine are placed in a flat bottom, +platinum dish seventy millimeters in diameter and twenty-five deep. The +dish is placed on a water bath in such a manner that it just touches +the surface of the water which is kept at a constant level. The heating +is continued for seven hours.[640] + +=625. Determination in a Vacuum.=—To avoid the changes and +decomposition produced by heating, the fixed solids may also be +determined by drying the sample in a vacuum over sulfuric acid. In +this laboratory, it has been found that the process may be greatly +facilitated by connecting the desiccating apparatus with the vacuum +service of the working desks in which a vacuum corresponding to a +mercurial column of 600 millimeters is obtained. The desiccator is +provided with a valve whereby a minute current of dry air is allowed +to flow through it. This current is not large enough to lessen the +vacuum but is sufficient to greatly accelerate the rapidity of the +evaporation. The evaporation is hastened also, in a marked degree, by +absorbing the liquid with a piece of filter paper previously dried in a +vacuum. When it is desired to examine the residue, however, it must be +obtained in a flat dish exposing a large surface to evaporation. + +=626. Estimation of Water.=—It is evident that the percentage of water +in a fermented beverage is easily calculated when the percentage of +alcohol by weight and that of the dry residue are known. In a given +case, if the number of grams of alcohol in 100 of the sample be five +and that of fixed solids four and a half, the quantity of water therein +is 100 - (5.0 + 4.5) = 90.5 grams. In this case the volatile essences +are counted as water, but these, at most, are so small in quantity as +to be practically unweighable. Nevertheless, it must be admitted that +direct drying, in many cases, may give erroneous results, especially +when the sample contains an abundance of ethers and of glycerol. The +loss which takes place on evaporation may be diminished by adding to +the sample, before evaporation, a known weight of potassium sulfate +in crystals, which serves to increase the surface of evaporation, to +hasten the process and to obtain a quantity of residue in excess of +that secured by direct evaporation in an open dish. + +=627. Total Acidity.=—The acidity found in fermented beverages is +due both to the natural acids of the materials from which they are +made, and to those caused by fermentation. The typical acids also +indicate the nature of the original materials, as malic in cider and +tartaric in wine. The acids of beers are due almost exclusively to +fermentation, and acetic is probably the dominant one. In determining +total acidity, it is not always convenient to ascertain beforehand what +acid predominates, nor to accurately distribute the acid among its +various components. In the analytical work it is advisable, therefore, +to estimate the total acid of cider as malic, of wines as tartaric and +of beers as acetic. The process of titration is conducted as follows: + +Expel any carbon dioxid that is present by continued shaking. Transfer +ten cubic centimeters to a beaker and, in the case of white wines, add +about ten drops of a neutral litmus solution. Add decinormal sodium +hydroxid solution until the red color changes to violet. Then add the +reagent, a few drops at a time, until a drop of the liquid, placed on +delicate red litmus paper, shows an alkaline reaction. + +One cubic centimeter of decinormal sodium hydroxid solution = 0.0075 +gram tartaric, 0.0067 of malic and 0.006 gram of acetic acid. + +=628. Determination of Volatile Acids.=—Fifty cubic centimeters of the +sample, to which a little tannin has been added to prevent foaming, +are distilled in a current of steam. The flask is heated until the +liquid boils, when the lamp under it is turned down and the steam +passed through until 200 cubic centimeters have been collected in the +receiver. The distillate is titrated with decinormal sodium hydroxid +solution and the result expressed as acetic acid. + +One cubic centimeter of decinormal sodium hydroxid solution = 0.0060 +gram acetic acid. + +The acidity due to volatile acids may be determined by ascertaining the +total acidity as above described, evaporating 100 cubic centimeters to +one-third of their volume, restoring the original volume with water and +again titrating. The difference between the first and second titrations +represents the volatile acidity. + +A method of determining volatile acidity in wines, without the +application of heat, has been proposed by de la Source.[641] The +sample, five cubic centimeters, freed of carbon dioxid by shaking, +is placed in a flat dish about eight centimeters in diameter. In a +separate portion of the sample, the total acidity is determined in the +presence of phenolphthalien by a set solution of barium hydroxid, one +cubic centimeter of which is equal to four milligrams of sulfuric acid. +The sample in the flat dish is placed in a desiccator, which contains +both sulfuric acid and solid potassium hydroxid, and left for two days, +by which time it is practically dry. The residue is dissolved in two +cubic centimeters of warm water and the dish is kept in the desiccator +for an additional two days. By this time the volatile acids, even +acetic, will have disappeared and the residual acidity is determined +after solution in water. + +The method is also applicable when wines have been treated with an +alkali. In this case two samples of five cubic centimeters each are +acidified with two cubic centimeters of a solution of tartaric acid +containing twenty-five grams per liter. This treatment sets free the +volatile acids, and their quantity is determined as before. + +=629. Titration with Phenolphthalien.=—The total acidity is also easily +determined by titration with a set alkali, using phenolphthalien as +indicator. Colored liquors must be treated with animal black before the +analysis. The sample is shaken to expel carbon dioxid and five cubic +centimeters added to 100 of water containing phenolphthalien. The set +alkali (tenth normal soda) is added until the red color is discharged. +Even wines having a considerable degree of color may be titrated in +this way.[642] The acidity, expressed as tartaric, may be stated as due +to sulfuric by dividing by 1.53. + +=630. Determination of Tartaric Acid.=—The determination of potassium +bitartrate is necessary when an estimation of the free tartaric acid is +desired.[643] + +Fifty cubic centimeters of wine are placed in a porcelain dish and +evaporated to a sirupy consistence, a little quartz sand being added +to render subsequent extraction easier. After cooling, seventy cubic +centimeters of ninety-six per cent alcohol are added with constant +stirring. After standing for twelve hours, at as low a temperature as +practicable, the solution is filtered and the precipitate washed with +alcohol until the filtrate is no longer acid. The alcoholic filtrate +is preserved for the estimation of the tartaric acid. The filter and +precipitate are returned to the porcelain dish and repeatedly treated +with hot water, each extraction being filtered into a flask or beaker +until the washings are neutral. The combined aqueous filtrates and +washings are titrated with decinormal sodium hydroxid solution. + +One cubic centimeter of decinormal sodium hydroxid solution = 0.0188 +gram potassium bitartrate. + +The alcoholic filtrate is made up to a definite volume with water and +divided into two equal portions. One portion is exactly neutralized +with decinormal sodium hydroxid solution, the other portion added, +the alcohol evaporated, the residue washed into a porcelain dish and +treated as above. + +One cubic centimeter decinormal sodium hydroxid solution = 0.0075 gram +tartaric acid. + +As, however, only half of the free tartaric acid is determined by this +method: + +One cubic centimeter decinormal sodium hydroxid = 0.0150 gram of +tartaric acid. + +=631. Modified Berthelot-Fleury Method.=—Ten cubic centimeters of +wine are neutralized with potassium hydroxid solution and mixed in a +graduated cylinder with forty cubic centimeters of the same sample. +To one-fifth of the volume, corresponding to ten cubic centimeters of +wine, fifty cubic centimeters of a mixture of equal parts of alcohol +and ether are added and allowed to stand twenty-four hours. The +precipitated potassium bitartrate is separated by filtration, dissolved +in water and titrated. The excess of potassium bitartrate over the +amount of that constituent present in the wine corresponds to the free +tartaric acid.[644] + +=632. Determination of Tartaric, Malic and Succinic Acids.=—Two hundred +cubic centimeters of wine are evaporated to one-half, cooled and lead +subacetate solution added until the reaction is alkaline.[645] The +precipitate is separated by filtration and washed with cold water until +the filtrate shows only a slight reaction for lead. The precipitate +is washed from the filter into a beaker, by means of hot water, and +treated hot with hydrogen sulfid until all the lead is converted into +sulfid. It is then filtered hot and the lead sulfid washed with hot +water until the washings are no longer acid. The filtrate and washings +are evaporated to fifty cubic centimeters and accurately neutralized +with potassium hydroxid. An excess of a saturated solution of calcium +acetate is added and the liquid allowed to stand from four to six +hours with frequent stirring. It is then filtered and the precipitate +washed until the filtrate amounts to exactly 100 cubic centimeters. +The precipitate of calcium tartrate is converted into calcium oxid by +igniting in a platinum crucible. After cooling, from ten to fifteen +cubic centimeters of normal hydrochloric acid are added, the solution +washed into a beaker and accurately titrated with normal potassium +hydroxid solution. Every cubic centimeter of normal acid saturated +by the calcium oxid is equivalent to 0.0750 gram tartaric acid. To +the amount so obtained, 0.0286 gram must be added, representing the +tartaric acid held in solution in the filtrate as calcium tartrate. The +sum represents the total tartaric acid in the wine. + +The filtrate from the calcium tartrate is evaporated to about +twenty-five cubic centimeters, cooled and mixed with three times +its volume of ninety-six per cent alcohol. After standing several +hours, the precipitate is collected on a weighed filter, dried at +100° and weighed. It represents the calcium salts of malic, succinic +and sulfuric acids and of the tartaric acid which remained in +solution. This precipitate is dissolved in a minimum quantity of +hydrochloric acid, filtered and the filter washed with hot water. +Potassium carbonate solution is added to the hot filtrate, and the +precipitated calcium carbonate separated by filtration and washed. +The filtrate contains the potassium salts of the above named acids. +It is neutralized with acetic acid, evaporated to a small volume +and precipitated hot with barium chlorid. The precipitate of barium +succinate and sulfate is separated by filtration, washed with hot +water and treated on the filter with dilute hydrochloric acid. The +barium sulfate remaining is washed, dried, ignited and weighed. In +the filtrate, which contains the barium succinate, the barium is +precipitated hot with sulfuric acid, washed, dried, ignited and +weighed. Two hundred and twenty-three parts of barium sulfate equal 118 +parts of succinic acid. The succinic and sulfuric acids, as well as the +tartaric acid remaining in solution, which is equal to 0.0286 gram, +are to be calculated as calcium salts and the result deducted from the +total weight of the calcium precipitate. The remainder is the calcium +malate, of which 172 parts equal 134 parts malic acid. + +According to Macagno, succinic acid may be estimated in wines by the +following process:[646] One liter of the wine is digested with lead +hydroxid, evaporated on the water bath and the residue extracted with +strong alcohol. The residual salts of lead are boiled with a ten +per cent solution of ammonium nitrate, which dissolves the salts of +succinic acid. The solution is filtered, the lead removed by hydrogen +sulfid, boiled, neutralized with ammonia and treated with ferric +chlorid as long as a precipitate is formed. The ferric succinate is +separated by filtration, washed and ignited. The succinic acid is +calculated from the weight of ferric oxid obtained. + +Malic acid in wines and ciders is determined by the method of Berthelot +in the following manner:[647] The sample is evaporated until reduced +to a tenth of its volume. To the residue an equal volume of ninety per +cent alcohol is added and the mixture set aside for some time. The +tartaric acid and tartrates separate, together with the greater part of +the salts of lime which may be present. + +The supernatant liquid is decanted and a small quantity of lime +added to it until in slight excess of that required to neutralize +the acidity. Calcium malate is separated mixed with lime. The solid +matters are separated by filtration, dissolved in a ten per cent +solution of nitric acid, from which the lime bimalate will separate in +a crystalline form. The weight of calcium bimalate multiplied by 0.59 +gives that of the malic acid. + +=633. Polarizing Bodies in Fermented Beverages.=—The study of the +nature of the carbohydrates, which constitute an important part of the +solid matters dissolved in fermented beverages, is of the greatest +importance. These bodies consist of grape sugars, sucrose, tartaric +acid and the unfermented hydrolytic products derived from starch. A +natural grape sugar (chiefly dextrose) is found in wines. Sucrose +is also a very important constituent of sweet wines. The hydrolytic +products of starch are found in beers, either as a residue from the +fermentation of malt or from the rice, glucose, hominy grits etc., +added in brewing. The character and quantities of these residues can +be determined by the methods already given in the parts of this volume +relating to sugars and starches. For convenience, however, and for +special application to the investigation of fermented beverages a +résumé of the methods adopted by the official chemists follows:[648] + +=634. Determination of Reducing Sugars.=—The reducing sugars are +estimated as dextrose, and may be determined by any of the methods +given for the estimation thereof (=113-140=). + +=635. Polarization.=—All results are to be stated as the polarization +of the undiluted sample. The triple field shadow saccharimeter is +recommended, and the results are expressed in the terms of the sugar +scale of this instrument. If any other instrument be used, or if it be +desirable to convert to angular rotation, the following factors may be +employed: + + 1° Schmidt and Haensch = 0°.3468 angular rotation D. + 1° angular rotation D = 2°.8835 Schmidt and Haensch. + 1° Schmidt and Haensch = 2°.6048 Wild (sugar scale). + 1° Wild (sugar scale) = 0°.3840 Schmidt and Haensch. + 1° Wild (sugar scale) = 0°.1331 angular rotation D. + 1° angular rotation D = 0°.7511 Wild (sugar scale). + 1° Laurent (sugar scale) = 0°.2167 angular rotation D. + 1° angular rotation D = 4°.6154 Laurent (sugar scale). + +In the above table D represents the angular rotation produced with +yellow monochromatic light. + +(_a_) _In White Wines or Beers._—Sixty cubic centimeters of wine are +decolorized with three cubic centimeters of lead subacetate solution +and filtered. Thirty cubic centimeters of the filtrate are treated with +one and five-tenths cubic centimeters of a saturated solution of sodium +carbonate, filtered and polarized. This gives a solution of nearly +ten to eleven, which must be considered in the calculation, and the +polariscope reading must accordingly be increased one-tenth. + +(_b_) _In Red Wines._—Sixty cubic centimeters of wine are decolorized +with six cubic centimeters of lead subacetate solution and filtered. To +thirty cubic centimeters of the filtrate, three cubic centimeters of +a saturated solution of sodium carbonate are added, filtered and the +filtrate polarized. The dilution in this case is nearly five to six, +and the polariscope reading must accordingly be increased one-fifth. + +(_c_) _In Sweet Wines._ (1) _Before Inversion._—One hundred cubic +centimeters are decolorized with two cubic centimeters of lead +subacetate solution and filtered after the addition of eight cubic +centimeters of water. One-half cubic centimeter of a saturated solution +of sodium carbonate and four and five-tenths cubic centimeters of water +are added to fifty-five cubic centimeters of the filtrate, the liquids +mixed, filtered and polarized. The polariscope reading is multiplied by +1.2. + +(2) _After Inversion._—Thirty-three cubic centimeters of the filtrate +from the lead subacetate in (1) are placed in a flask with three cubic +centimeters of strong hydrochloric acid. After mixing well, the flask +is placed in water and heated until a thermometer, placed in the flask +with the bulb as near the center of the liquid as possible, marks 68°, +consuming about fifteen minutes in the heating. It is then removed, +cooled quickly to room temperature, filtered and polarized, the +temperature being noted. The polariscope reading is multiplied by 1.2. +Because of the action of lead subacetate on invert sugar (=87=) it is +advisable to decolorize the samples with other reagents (=87-89=). + +(3) _After Fermentation._—Fifty cubic centimeters of wine, which have +been dealcoholized by evaporation and made up to the original volume +with water, are mixed, in a small flask, with well washed beer yeast +and kept at 30° until fermentation has ceased, which requires from +two to three days. The liquid is washed into a 100 cubic centimeter +flask, a few drops of a solution of acid mercuric nitrate and then lead +subacetate solution, followed by sodium carbonate, added. The flask +is filled to the mark with water, shaken, the solution filtered and +polarized and the reading multiplied by two. + +=636. Application of Analytical Methods.=—(1) _There is no +rotation._—This may be due to the absence of any rotatory body, to the +simultaneous presence of the dextrorotatory nonfermentable constituents +of commercial dextrose and levorotatory sugar, or to the simultaneous +presence of dextrorotatory cane sugar and levorotatory invert sugar. + +(_a_) _The Wine is Inverted._—A levorotation shows that the sample +contains cane sugar. + +(_b_) _The Wine is Fermented._—A dextrorotation shows that both +levorotatory sugar and the unfermentable constituents of commercial +dextrose are present. + +If no change take place in either (_a_) or (_b_) in the rotation, +it proves the absence of unfermented cane sugar, the unfermentable +constituents of commercial dextrose and of levorotatory sugar. + +(2) _There is right rotation._—This may be caused by unfermented cane +sugar, the unfermentable constituents of commercial dextrose or both. + +(_a_) The sugar is inverted: + +(_a_₁) _It rotates to the left after inversion._—Unfermented cane sugar +is present. + +(_a_₂) _It rotates more than 2°.3 to the right._—The unfermentable +constituents of commercial dextrose are present. + +(_a_₃) _It rotates less than 2°.3 and more than 0°.9 to the right._—It +is in this case treated as follows: + +Two hundred and ten cubic centimeters of the sample are evaporated +to a thin sirup with a few drops of a twenty per cent solution of +potassium acetate. To the residue 200 cubic centimeters of ninety per +cent alcohol are added with constant stirring. The alcoholic solution +is filtered into a flask and the alcohol removed by distillation +until about five cubic centimeters remain. The residue is mixed with +washed bone-black, filtered into a graduated cylinder and washed until +the filtrate amounts to thirty cubic centimeters. When the filtrate +shows a dextrorotation of more than 1°.5, it indicates the presence of +unfermentable constituents of commercial dextrose. + +(3) _There is left rotation._—The sample contains unfermented +levorotatory sugar, derived either from the must or mash or from +the inversion of added cane sugar. It may, however, also contain +unfermented cane sugar and the unfermentable constituents of commercial +dextrose. + +(_a_) The wine sugars are fermented according to directions in =262=. + +(_a_₁) _It polarizes 3° after fermentation._—It contains only +levorotatory sugar. + +(_a_₂) _It rotates to the right._— It contains both levorotatory sugar +and the unfermentable constituents of commercial dextrose. + +(_b_₁) The sucrose is inverted according to (_c_), in (2). + +(_b_₂) It is more strongly levorotatory after inversion. In contains +both levorotatory sugar and unfermented cane sugar. + +=637. Estimation of Sucrose, Dextrose, Invert Sugar, Maltose and +Dextrin.=—The total and relative quantities of these carbohydrates are +determined by the processes already described (=237-262=). + +=638. Determination of Glycerol.=—(_a_) _In Dry Wines and Beers._—One +hundred cubic centimeters of wine are evaporated in a porcelain dish +to about ten cubic centimeters, a little quartz sand and milk of lime +added and the evaporation carried almost to dryness. The residue is +mixed with fifty cubic centimeters of ninety per cent alcohol, using +a glass pestle or spatula to break up any solid particles, heated to +boiling on the water bath, allowed to settle and the liquid filtered +into a small flask. The residue is repeatedly extracted in a similar +manner, with small portions of boiling alcohol, until the filtrate in +the flask amounts to about 150 cubic centimeters. A little quartz sand +is added, the flask connected with a condenser and the alcohol slowly +distilled until about ten cubic centimeters remain. The evaporation +is continued on the water bath until the residue becomes sirupy. It +is cooled and dissolved in ten cubic centimeters of absolute alcohol. +The solution may be facilitated by gentle heating on the steam bath. +Fifteen cubic centimeters of anhydrous ether are added, the flask +stoppered and allowed to stand until the precipitate has collected on +the sides and bottom of the flask. The clear liquid is decanted into +a tared weighing bottle, the precipitate repeatedly washed with a few +cubic centimeters of a mixture of one part of absolute alcohol and +one and five-tenths parts anhydrous ether and the washings added to +the solution. The ether-alcohol is evaporated on the steam bath, the +residue dried one hour in a water oven, weighed, the amount of ash +determined and its weight deducted from that of the weighed residue to +get the quantity of glycerol. + +(_b_) _In Sweet Wines._—One hundred cubic centimeters of wine are +evaporated on the steam bath to a sirupy consistence, a little quartz +sand being added to render subsequent extraction easier. The residue +is repeatedly treated with absolute alcohol until the united extracts +amount to from 100 to 150 cubic centimeters. The solution is collected +in a flask and for every part of alcohol one and five-tenths parts of +ether are added, the liquid well shaken and allowed to stand until it +becomes clear. The supernatant liquor is decanted into a beaker and the +precipitate washed with a few cubic centimeters of a mixture of one +part alcohol and one and five-tenths parts ether. The united liquids +are distilled, the evaporation being finished on the water bath, the +residue is dissolved in water, transferred to a porcelain dish and +treated as under (_a_). + +=639. Determination of Coloring Matters in Wines.=—The methods of +detecting the more commonly occurring coloring matters in wines as +practiced by the official chemists are given below. + +(_a_) _Cazeneuve Reaction._—Add two-tenths gram of precipitated +mercuric oxid to ten cubic centimeters of wine, shake for one minute +and filter. + +Pure wines give filtrates which are colorless or light yellow, while +the presence of a more or less red coloration indicates that an anilin +color has been added to the wine. + +(_b_) _Method of Sostegni and Carpentieri._—Evaporate the alcohol from +200 cubic centimeters of wine. Add from two to four cubic centimeters +of a ten per cent solution of hyrochloric acid, immerse therein some +threads of fat-free wool and boil for five minutes. Remove the threads, +wash them with cold water acidified with hydrochloric, then with hot +water acidified with hydrochloric, then with pure water and dissolve +the color in a boiling mixture of fifty cubic centimeters of water and +two cubic centimeters of concentrated ammonia. Replace the threads by +new ones, acidify with hydrochloric and boil again for five minutes. +In the presence of anilin colors to the amount of two milligrams per +liter, the threads are dyed as follows: + + Safranin light rose-red. + Vinolin rose-red to violet. + Bordeaux-red rose-red to violet. + Ponceau-red rose-red. + Tropæolin oo straw yellow. + Tropæolin ooo light orange. + +(_c_) _Detection of Fuchsin and Orseille._—To twenty cubic centimeters +of wine add ten cubic centimeters of lead acetate solution, heat +slightly and mix by shaking. Filter into a test-tube, add two cubic +centimeters of amyl alcohol and shake. If the amyl alcohol be +colored red, separate it and divide it into two portions. To one add +hydrochloric acid, to the other ammonia. When the color is due to +fuchsin, the amyl alcohol will in both cases be decolorized; when due +to orseille, the ammonia will change the color of the amyl alcohol to +purple-violet. + +=640. Determination of Ash.=—The residue from the direct extract +determination is incinerated at as low a heat as possible. Repeated +moistening, drying and heating to low redness is advisable to get rid +of all organic substances. When a quantitive analysis of the ash is +desired, large quantities of the sample are evaporated to dryness and +the residue incinerated with the usual precautions. + +=641. Determination Of Potash.=—(_a_) _Kayser’s Method._—Dissolve +seven-tenths gram pure sodium hydroxid and two grams of tartaric +acid in 100 cubic centimeters of wine, add 150 cubic centimeters of +ninety-two to ninety-four per cent alcohol and allow the liquid to +stand twenty-four hours. The precipitated potassium bitartrate is +collected on a small filter and washed with fifty per cent alcohol +until the filtrate amounts to 260 cubic centimeters. The precipitate +and filter are transferred to the beaker in which the precipitation was +made, the precipitate dissolved in hot water, the volume made up to 200 +cubic centimeters and fifty cubic centimeters thereof titrated with +decinormal sodium hydroxid solution, adding 0.004 gram to the final +result, representing the potash which remains in solution as bitartrate. + +(_b_) _Platinum Chlorid Method._—Evaporate 100 cubic centimeters of the +wine to dryness, incinerate the residue and determine the potash as in +ash analysis.[649] + +=642. Determination of Sulfurous Acid.=—One hundred cubic centimeters +of wine are distilled in a current of carbon dioxid, after the addition +of phosphoric acid, until about fifty cubic centimeters have passed +over. The distillate is collected in accurately set iodin solution. +When the distillation is finished, the excess of iodin is determined +with set sodium thiosulfate solution and the sulfurous acid calculated +from the iodin used. + +=643. Detection of Salicylic Acid.=—(_a_) _Spica’s Method._—Acidify 100 +cubic centimeters of the liquor with sulfuric and extract with sulfuric +ether. Evaporate the extract to dryness, warm the residue carefully +with one drop of concentrated nitric acid and add two or three drops +of ammonia. The presence of salicylic acid in the liquor is indicated +by the formation of a yellow color due to ammonium picrate and may be +confirmed by dyeing therein a thread of fat-free wool. + +(_b_) _Bigelow’s Method._—Place 100 cubic centimeters of the wine +in a separatory funnel, add five cubic centimeters of sulfuric acid +(1-3) and extract with a sufficient quantity of a mixture of eight or +nine parts of ether to one part of petroleum ether. Throw away the +aqueous part of the extract, wash the ether once with water, then shake +thoroughly with about fifty cubic centimeters of water, to which from +six to eight drops of a one-half per cent solution of ferric chlorid +have been added. Discard the aqueous solution, which contains the +greater part of the tannin in combination with iron, wash again with +water, transfer the ethereal solution to a porcelain dish and allow +to evaporate spontaneously. Heat the dish on the steam bath, take up +the residue with one or two cubic centimeters of water, filter into +a test-tube and add one to two drops of one-half per cent solution +of ferric chlorid. The presence of salicylic acid is indicated by +the appearance of a violet-red coloration. In the case of red wines, +a second extraction of the residue with ether mixture is sometimes +necessary. This method cannot be used in the examination of beers and +ales. + +(_c_) _Girard’s Method._—Extract a portion of the acidified liquor +with ether as in the preceding methods, evaporate the extract to +dryness and exhaust the residue with petroleum ether. The residue from +the petroleum ether extract is dissolved in water and treated with a +few drops of a very dilute solution of ferric chlorid. The presence +of salicylic acid is indicated by the appearance of a violet-red +coloration. + +=644. Detection of Gum and Dextrin.=—Four cubic centimeters of the +sample are mixed with ten cubic centimeters of ninety-six per cent +alcohol. When gum arabic or dextrin is present, a lumpy, thick and +stringy precipitate is produced, whereas pure wine becomes at first +opalescent and then gives a flocculent precipitate. + +=645. Determination of Nitrogen.=—The best method of determining +nitrogen in fermented beverages is the common one of moist combustion +with sulfuric acid. The sample is placed in the kjeldahl digestion +flask, which is attached to the vacuum service and placed in a +steam bath until its contents are dry or nearly so. The process is +then conducted in harmony with the well known methods. Where large +quantities of the sample are to be employed, as in drinks containing +but little nitrogen, the preliminary evaporation may be accomplished in +an open dish, the contents of which are transferred to the digestion +flask before any solid matter is deposited. The same procedure may be +followed when the sample foams too much on heating. + +=646. Substitutes for Hops.=—It is often claimed that cheap and +deleterious bitters are used in brewing in order to save hops. While +it is doubtless true that foreign bitters are sometimes employed, the +experience of this laboratory goes to show that such an adulteration +is not very prevalent in this country.[650] Possibly strychnin, +picrotoxin, quassin, gentian and other bitter principles have sometimes +been found in beer, but their use is no longer common. It is difficult +to decide in every case whether or not foreign bitters have been added. +A common process is to treat the sample with lead acetate, filter, +remove the lead from the filtrate and detect any remaining bitters by +the taste. All the hop bitters are removed by the above process. Any +remaining bitter taste is due to other substances. For the methods of +detecting the special bitter principles in hops and other substances, +the work of Dragendorff may be consulted.[651] + +=647. Bouquet of Fermented and Distilled Liquors.=—The bouquet of +fermented and distilled liquors is due to the presence of volatile +matters which may have three different origins. In the first place the +materials from which these beverages are made contain essential oils +and other odoriferous principles.[652] In the grape, for instance, the +essential oils are found particularly in the skins. These essential +principles may be secured by distilling the skins of grapes in a +current of steam. This method of separation, however, cannot be +regarded as strictly quantitive. + +In the second place, the yeasts which produce the alcoholic +fermentation are also capable of producing odoriferous products. +These minute vegetations, resembling in their biological relations +the mushrooms, grow in the soil and reach their maturity at about +the time of the harvest of the grapes. Their spores are transmitted +through the air, reach the expressed grape juice and produce the vinous +fermentation. The particular odor due to any given yeast persists +through many generations of culture showing that the body which +produces the odor is the direct result of the vegetable activity of +the yeast. A beer yeast, after many generations of culture, will still +give a product which smells like beer, and in like manner a wine yeast +will produce one which has the odor of wine. The quantity of odorant +matter produced by this vegetable action is so minute as to escape +detection in a quantitive or qualitive way by chemical means. These +subtle perfumes arise moreover not only from the breaking up of the +sugar molecule, but are also the direct results of molecular synthesis +accomplished under the influence of the yeast itself. + +In the third place, the fermented and distilled liquors contain +odoriferous principles due to the chemical reactions which take place +by the breaking up of the sugar and other molecules during the process +of fermentation. The alcohols and acids produced have distinct odors by +which they are often recognized. This is particularly true of ethylic, +propylic, butylic, amylic and oenanthylic alcohols and acetic acid. +These alcohols themselves also undergo oxidation, passing first into +the state of aldehyds which, together with ethers, produce the peculiar +aroma which is found in various fruits. The etherification noted above +is of course preceded by the formation of acids corresponding to the +various aldehyds present. The formation of these ethers takes place +very slowly during aging, and it therefore requires three or four +years for the proper ripening of wines or distilled liquors. By means +of artificial heat, electricity and aeration, the oxidizing processes +above noted may be hastened, but it is doubtful whether the products +arising from this artificial treatment are as perfect as those which +are formed in the natural processes. + + +AUTHORITIES CITED IN PART SEVENTH. + +[535] Bulletin 46, Chemical Division U. S. Department of Agriculture, +pp. 24-25. + +[536] Bulletin 42, Arkansas Agricultural Experiment Station, pp. 81 et +seq. + +[537] Balland; Recherches sur les Blés, les Farines et le Pain, p. 229. + +[538] Jago; Flour and Bread, p. 457. + +[539] Jago; op. cit. supra, p. 465. + +[540] Richardson; Journal of the Chemical Society, Transactions, 1885, +pp. 84 et seq. + +[541] Auct. et op. cit. supra, pp. 80 et seq. + +[542] Bulletin 28, Office of Experiment Stations, U. S. Department of +Agriculture, pp. 9, 10. + +[543] Bulletin 29, Office of Experiment Stations U. S. Department of +Agriculture, p. 8. + +[544] Op. et. loc. cit. supra. + +[545] Experiment Station Record, Vol. 6, pp. 590 et seq. + +[546] Annual Report, U. S. Department of Agriculture, 1884, p. 365. + +[547] Forschungs-Berichte über Lebensmittel, Band 3, S. 142. + +[548] Zeitschrift für angewandte Chemie, 1895, S. 620. + +[549] Op. et loc. cit. supra. + +[550] Les Ferments Solubles; Diastases—Enzymes. + +[551] Wiley; Medical News, July, 1888. + +[552] Virchow’s Archiv., Band 123, S. 230: Journal of the Chemical +Society, Abstracts, 1892, p. 755. + +[553] Ladenberg; Handwörterbuch der Chemie, Band 4, S. 122. + +[554] Chemisches Centralblatt, 1892, Band 2, S. 579. + +[555] Op. cit. supra, 1890, Band 2, S. 628. + +[556] Die Landwirtschaftlichen Versuchs-Stationen, Band 44, S. 188; +Experiment Station Record, Vol. 6, p. 12. + +[557] Experiment Station Record, Vol. 6, pp. 5 et seq. (Read Jordan +instead of Gordon.) + +[558] Journal of the American Chemical Society, Vol. 16, pp. 590 et seq. + +[559] From photograph made in this laboratory by Bigelow. + +[560] Journal of the Society of Chemical Industry, Vol. 10, p. 118. + +[561] Die Landwirtschaftlichen Versuchs-Stationen, Band 36, S. 321: +Bulletin 13, Chemical Division, U. S. Department of Agriculture, p. +1028. + +[562] Wilson; Vid. op. et loc. cit. 26. + +[563] U. S. Dispensatory, p. 1088. + +[564] Landwirtschaftliche Jahrbücher, 1890, Band 19, S. 149. + +[565] Bulletin 13, Chemical Division, U.S. Department of Agriculture, +p. 1028. + +[566] Zeitschrift für analytische Chemie, Band 35, S. 498. + +[567] Annual Report of the Maine Agricultural Experiment Station, 1891, +p. 25: Gay; Annales Agronomiques, 1885, p. 145, et 1896, pp. 145 et seq. + +[568] Annales Agronomiques, Tome 21, pp. 149, 150. + +[569] Vid. op. et loc. cit. primo sub 33. + +[570] Twelfth Annual Report of the Massachusetts Agricultural +Experiment Station, 1894, p. 175. + +[571] See also paragraph =586= this volume. + +[572] Manuscript prepared for publication as a part of Bulletin 13, +Chemical Division, U. S. Department of Agriculture. + +[573] Vid. this volume, paragraph =280=. + +[574] Vid. op. cit. 31, p. 1020. + +[575] Bulletin 45, Chemical Division, U.S. Department of Agriculture, +p. 12. + +[576] Berthelot; Essai de Chimie Mécanique: Thomsen; Thermo Chemische +Untersuchungen: Ostwald; Algemeine Chemie: Muir; Elements of Thermal +Chemistry. + +[577] Bulletin 21, Office of Experiment Stations, U. S. Department +of Agriculture, pp. 113 et seq.: Seventh Annual Report Connecticut +(Storr’s) Agricultural Experiment Station, pp. 133 et seq. + +[578] Vid. op. et loc. cit. 43. + +[579] From personal inspection by author in Williams’ laboratory, 161 +Tremont St., Boston, Mass. + +[580] Journal für praktische Chemie, Band 147 {Neue Folge Band 39}, +Ss. 517 et seq. + +[581] Berthelot; Annales de Chemie et de Physique, 6e Série, Tome 10, +p. 439. + +[582] Vid. op. cit. 46, Ss. 522-523. The data in paragraph =566= are +taken from Stohmann, Zeitschrift für Biologie, Band 31, S. 364 and +Experiment Station Record, Vol. 6, p. 590. + +[583] Journal of the American Chemical Society, Vol. 18, p. 174. + +[584] Bulletins 93, 97, 101 and 102, California Agricultural Experiment +Station. + +[585] Annual Report, U. S. Department of Agriculture, 1886, p. 354. + +[586] This work, Vol. 2, p. 318. + +[587] Vid. California Bulletins cited under 50: Wolff; Aschen Analyse, +S. 126. + +[588] Bulletin 100, Cornell Agricultural Experiment Station: Bulletin +48, Chemical Division, U. S. Department of Agriculture. + +[589] Vid. op. cit. 51, p. 353. + +[590] Vid. op. cit. ultimo sub 54. + +[591] Bulletin No. 42, Arkansas Agricultural Experiment Station, p. 78. + +[592] Annual Report, U. S. Department of Agriculture, 1884, p. 347. + +[593] Spencer; Bulletin 13, U. S. Department of Agriculture, pp. 875 et +seq. + +[594] Journal of Analytical and Applied Chemistry, Vol. 4, p. 390; +Bulletin 13, Chemical Division, U. S. Department of Agriculture, p. 889. + +[595] Pharmaceutical Journal, Vol. 52, p. 213. + +[596] Commercial Organic Analysis, Vol. 3, part 2, p. 484. + +[597] Journal de Pharmacie et de Chimie, 6ᵉ Série, Tome 3, p. 529. + +[598] Journal of the American Chemical Society, Vol. 18, p. 338. + +[599] Manuscript communication to author. + +[600] Vid. op. cit. 63, p. 533. + +[601] American Chemical Journal, Vol. 14, p. 473. + +[602] Op. et loc. cit. supra. + +[603] Lindsey; Report made to Thirteenth Annual Convention of the +Association of Official Agricultural Chemists, Nov. 6th, 1896: Tollens; +Handbuch der Kohlenhydrate, Band 2, S. 52. + +[604] Zeitschrift für angewandte Chemie, 1896, p. 195, + +[605] Comptes rendus hebdomadaires de Seances de l’Academie des +Sciences, Tome 122, p. 841. + +[606] The Tannins, two volumes. + +[607] Dragendorff; Plant Analysis. + +[608] The Tannins, Vol. 1, p. 33. + +[609] Bulletin 13, Chemical Division, U. S. Department of Agriculture, +p. 908. + +[610] Vid. op. cit. 74, p. 38. + +[611] Bulletin 46, Chemical Division, U. S. Department of Agriculture, +p. 77 as revised at 13th annual meeting of the Association of Official +Agricultural Chemists. + +[612] Vid. op. cit. 75, p. 890: Zeitschrift für analytische Chemie, +Band 25, S. 121: Journal of the Society of Chemical Industry, Vol. 3, +p. 82: Trimble; The Tannins, Vol. 1, p. 44. + +[613] Vid. op. cit. 74, p. 48. + +[614] McElroy; Analyses made in this laboratory. + +[615] Annual Report Connecticut Agricultural Experiment Station (New +Haven) 1892, p. 30. + +[616] Kissling; Tabakkunde, S. 40. + +[617] Vid. op. cit. supra, S. 58. + +[618] Vid. op. cit. 81, p. 29. + +[619] This work, Vol 1, pp. 500 et seq. + +[620] This work, Vol. 1, p. 420. + +[621] Vid. op. cit. 82, S. 62. + +[622] Vid. op. cit. supra, S. 64. + +[623] Dragendorff; Plant Analysis, p. 65. + +[624] Sugar, 1896, March 15th, p. 11. + +[625] Vid. op. cit. 82, S. 65. + +[626] Der Tabak, S. 144. + +[627] Vid. op. cit. 82, S. 65: Zeitschrift für analytische Chemie, Band +21, S. 76: Band 22, S. 199: Band 32, S. 277: Band 34, Ss. 413-731. + +[628] Zeitschrift für physiologische Chemie Band 13, S. 445: Band 14, +S. 182. + +[629] Zeitschrift für analytische Chemie, Band 34, S. 413, Band 35, Ss. +309, 731. + +[630] Annual Report Connecticut Agricultural Experiment Station (New +Haven), 1892, p. 17. + +[631] Buell; The Cider-makers’ Manual: Southby; Systematic Text-Book +of Practical Brewing: Moritz and Morris; Text-Book of the Science of +Brewing. + +[632] Gautier; Sophistication et Analyse des Vins, p. 49. + +[633] Auct. et. op. cit. supra, p. 44. + +[634] Bulletin 46, Chemical Division, U. S. Department of Agriculture, +p. 63. + +[635] Manuscript not yet published. + +[636] Vid. op. cit. 100, pp. 95 et seq. + +[637] Wiley; Journal of the American Chemical Society, Vol. 18, p. 1063. + +[638] Vid. op. cit. 100, p. 70. + +[639] Vid. op. cit. 98, p. 65. + +[640] Vid. op. cit. 98, p. 67. + +[641] The Analyst, Vol. 21, p. 158. + +[642] Vid. op. cit. 98, p. 98. + +[643] Vid. op. cit. 100, p. 74. + +[644] Vid. op. cit. 100, p. 75. + +[645] Vid. op. cit. 100, p. 75. + +[646] Bulletin de la Société Chimique de Paris, Série {2}, Tome 24, p. +288; Berichte der deutschen chemischen Gesellschaft, Band 8, S. 257. + +[647] Vid. op. cit. 98, p. 120. + +[648] Bulletin 46, Chemical Division U. S. Department of Agriculture, +pp. 72, et. seq. + +[649] This work, Vol. 2, pp. 267 and 326. + +[650] Bulletin 13, Chemical Division, U. S. Department of Agriculture, +p. 296. + +[651] Plant Analysis, pp. 38, et seq. + +[652] Repertoire de Pharmacie, Série 3e, Tome 7, p. 436. + + + + +INDEX. + + + Page. + A + Abbe, refractometer, 329 + Acetic acid, estimation in tobacco, 602 + Acetyl value, 384, 385 + Acidity, estimation in fermented beverages, 27 + of milk, determination, 473 + Acids, determination in fruits and vegetables, 579 + Agricultural products, classification of miscellaneous, 541 + description, 1 + Air-bath, drying, 16 + Albumin, definition, 410 + gyrodynals of hydrates, 276 + precipitants in milk, 276 + qualitive tests, 420 + separation in milk, 509 + Albuminates, definition and properties, 411 + estimation in cheese, 531 + qualitive tests, 421 + Albuminoids, 413 + definition, 410 + Albumins, action, on polarized light, 422 + gyrodynats, 422 + properties, 410 + separation, 439 + Albumose peptone, 461 + Albumoses, estimation, in cheese, 531 + separation, from peptones, 455 + Alcohol, calculating, in fermented beverages, 616 + digestion, 245 + estimation, by vapor temperature, 622 + in ensilage, 546 + fermented beverages, 612 + koumiss, 534 + sugar analysis, 186 + reagent for precipitating dextrin, 292 + table showing percentage, 617-621 + Alcoholic digestion, sugar beets, 248-250 + Alcoholometer, 612 + Aliphalytic ferments, 556 + Alkali, action on reducing sugars, 131, 132 + Alkaline copper solutions, comparison, 127-129 + Alkaloidal nitrogen, estimation, 432 + qualitive tests, 422, 423 + Alkaloids, occurrence, 417 + Allantoin, 428 + Allein and Gaud, modification of Pavy’s process, 145 + Allen, modification of Pavy’s process, 144 + potassium cyanid process, 146 + Allihn, gravimetric dextrose method, 155-158 + Alum, occurrence, in bread, 544 + reagent for casein, 535 + Alumina cream, clarification, 100 + Aluminum dishes, drying, 33 + Amagat-Jean, refractometer, 334-338 + Amid nitrogen, estimation, 424 + in cereals, 543 + tobacco, 607 + occurrence, 417 + qualitive test, 418 + separation, in cheese, 530 + Ammonia, estimation, in tobacco, 605 + Ammoniacal copper solution, 143 + nitrogen, estimation, 423, 424 + in cheese, 531 + qualitive test, 419 + Ammonium sulfate, reagent for milk proteids, 507 + precipitating proteids, 433 + Amyl alcohol, use, in milk fat analysis, 501 + Amyliferous bodies, desiccation, 299 + Amyloid bodies in milk, 512 + Amylolytic ferments, 556 + Anatto, 522 + Animal products, sampling, 448 + substances, preparation, 4, 5 + Anoptose, 234 + Antipeptones, 412 + Aqueous diffusion, sugar beet analysis, 251, 252 + Araban, occurrence, 586 + Arabinose, molecular weight, 177 + Arachidic acid, separation, 398, 399 + Areometric method, application in milk fat analysis, 494, 495 + Areometry, 70 + Artificial digestion, 555 + manipulation, 561 + smoker, 609 + Ash, composition, in milk, 466, 467 + of fruit, 580 + estimation, 36 + in butter, 516 + cereals, 542 + fermented beverages, 637 + koumiss, 536 + meats, 550 + milk, 482 + proteids, 444 + German method, 39 + Asparagin, 417 + estimation, 426, 427 + preparation, 426 + Aspartic acid, 412 + Atwater and Woods, calorimeter, 569 + methods of meat analysis, 549 + preparation of fish, 12 + Auric chlorid, color test with fats and oils, 356 + Authorities cited in Part + Fifth, 462, 463 + First, 56, 57 + Fourth, 406-409 + Second, 222 + Seventh, 641-644 + Sixth, 536-540 + Third, 306-308 + + B + Babcock, formula, for calculating total solids, 479, 480 + method of counting fat globules, 483, 484 + milk fat analysis, 499, 500 + Bacteria, reactions, on sugar, 196 + Bagasse, analysis, 239, 240 + Barfoed, reagent, for removing dextrose, 291, 292 + Barium saccharate, 187 + Barley starch, 221 + Basic lead acetate, clarification, 101 + Baumé and brix degrees, comparison, 73 + Bean starch, 220 + Bechi’s test for cottonseed oil, 400, 401 + Beet rasp, 10, 251 + Beimling, method of milk fat analysis, 502 + Betain, 417 + separation from cholin, 429 + Bigelow and McElroy, estimation of sugar in evaporated milks, 296-298 + method of dialysis, 447 + table for correcting hydrostatic plummet, 615 + Biliverdin, occurrence in milk, 464 + Birotation, 118 + mathematical theory, 177, 178 + Biuret reaction, 419 + Block, feculometer, 300 + Bone-black, decolorization, 104 + Bordeaux-red, determination, in wines, 637 + Bouquet of fermented and distilled liquors, 640, 641 + Bread, acidity, 544 + amount of water, 544 + baking, temperature, 543 + chemical changes in baking, 545 + color, 544 + methods of analysis, 543-545 + nitrogenous compounds, 544 + soluble extract, 543 + Brix and baumé degrees, comparison, 73 + Bromin addition number, 371-373 + Brullé, color test for fats and oils, 355 + Butter, adulterants, 521 + appearance of melted, 513 + with polarized light, 514 + calorimetric distinction, from oleomargarin, 576 + colors, 522 + detection, 523 + fat analysis, classification of methods, 484 + estimation, 482-504 + methods of analysis, 512-523 + microscopic examination, 513 + molecular weight, 520 + refractive index, 514 + relative proportion of ingredients, 517 + substitutes, molecular weight, 520, 521 + Butyrin, 310 + Butyrorefractometer, 339-341 + range of application, 342 + + C + Caffein, estimation, 583 + Caffetannic acid, 590 + Calcium saccharates, 188 + Caldwell, hydrogen drying oven, 26, 27 + Calories, computation, 574-578 + definition, 576 + Calorimeter, description, 569 + formulas for calculation, 572 + hydrothermal value, 573 + manipulation, 571 + Calorimetric equivalents, 576 + Calorimetry, 568-576 + Canada balsam, mounting starches, 219 + Cane cutting machines, 236, 237 + pulp, determination of sugar, 238 + drying and extraction, 238 + sugar, gyrodynat, 117, 118 + Carbohydrates, 58 + estimation in cereals, 543 + kind, 58 + milk, 511 + molecular weights, 175 + nomenclature, 59 + occurrence, 58 + in coffee, 585 + of rare, 306 + separation, 279 + in fruits and vegetables, 577 + Carbon, estimation in proteids, 444 + dioxid, determination, in sugar analysis, 186 + estimation, in koumiss, 532 + reagent for casein, 509 + tetrachlorid, reagent in iodin addition, 368 + Carnin, 416 + composition, 451 + Carr, vacuum drying oven, 22, 23 + Casein, estimation, in butter, 516 + cheese, 531 + with mercurial salts, 535 + factors for calculating, 508 + method of estimating, 508 + precipitants in milk, 276 + precipitation, by alum, 535 + preparation, 509 + separation, by filtering through porous porcelain, 534 + from albumin, 507 + solution, in acid, 489 + theory of precipitation, 508 + Caseinogen, 504 + Cassava starch, 222 + Cattle foods, 545 + Cellulose, constitution, 303 + qualitive reactions, 306 + separation, 304 + solubility, 305 + Cereals, general principles of analysis, 542 + Chalmot and Tollens, method of estimating pentosans, 182 + Chandler and Ricketts, polariscope, 266 + Cheese, artificial digestion, 561 + composition, 524 + constituents, 530 + filled, 529 + methods of analysis, 526-533 + manufacture, 525 + proteids, separation, 530, 531 + Chitin, 416 + character of reaction, 512 + Chlorophyll, separation, from caffein, 585 + Cholesterin, detection, 403, 404 + occurrence, in milk, 464 + Cholin, 417 + separation, from cottonseed, 428, 429 + Chondrin, 415 + Chrome yellow, 522 + Chrysolite, use, in drying, 486 + Chyle, occurrence, in milk, 464 + Chyme, occurrence, in milk, 464 + Citric acid, estimation, in tobacco, 601 + occurrence, in milk, 466 + Clerget, method of inversion, 105-107 + Cobaltous nitrate, reagent for nitrate, 189 + Collagen, 413 + Coloring matters, determination, in wines, 636, 637 + Combustion products, 36, 37 + Conchiolin, 416 + Conglutin, 411 + Constant monochromatic flame, 85 + Control observation tube, 95, 96 + Copper carbonate process, 138-140 + use, in estimating sucrose, dextrose and levulose, 282, 283 + cyanid, reagent for estimating lactose, 294, 295 + oxid, weighing, in sugar analysis, 262 + reagent in determining oxygen absorption of oils, 405 + salts, reduction, by sugar, 123 + solution, action on dextrose, 125 + sulfate, reagent for milk proteids, 506 + separating proteid from amid nitrogen, 433 + titration of residual, 148, 149 + Cottonseed oil, detection, 400 + Courtonne, ash muffle, 40 + Crampton, preparation of fat crystals, 347 + Creamometry, 474 + Creydt, formula, 110 + Crismer, critical temperature, 349, 350 + Critical temperature, fats and oils, 349 + Crude proteids, estimation, in cereals, 543 + Crystallin, 411 + Crystallization, temperature, 327 + Cuprous oxid, specific gravity, 137 + Curd, estimation, in butter, 516 + + D + Dairy products, importance, 464 + Davis, meat preservatives, 566 + Density, determination, 63 + of sour milk, 477 + Deuteroalbumose, 412 + Dextrin, detection, in fermented beverages, 639 + occurrence, in glucose, 264 + precipitation, by alcohol, 292 + separation, from dextrose and maltose, 287-293 + Dextrinoid bodies in milk, 511 + Dextrosazone, 193 + Dextrose, action of alkaline copper solution, 125 + estimation, in presence of sucrose, 274, 275 + and levulose, 280-285 + group, qualitive test, 190 + gyrodynat, 118 + molecular weight, 176 + removal, by copper acetate, 291 + separation from maltose and dextrin, 287-293 + table for calculating, from copper, 260 + Dialysis, 447 + application, for precipitating milk proteids, 511 + Diastase, action, on starch, 198 + preparation, 300 + Diffusion, instantaneous, 243 + Digestion, alcoholic, 245 + Distillation, methods, 612, 613 + Doolittle, viscosimeter, 343, 344 + Double dilution, milk analysis, 278 + polarization, 102 + Dreef grinding machine, 11 + Dry substance, estimation, for factory control, 263 + Drying samples, general principles, 34, 35 + + E + Earth bases, reagents for precipitating sugar, 187 + Ebullioscope, 622, 623 + Edson, preserving sugar juices, 235 + Elaidin, 406 + Elastin, 415 + Electric drying oven, 19 + Ensilage, alcohol, 546 + changes, due to fermentation, 546 + comparative value, 547 + organic acids, 546 + Ether extract, estimation, in cereals, 542 + solvent, 41 + Evaporated fruits, 580 + milk, estimation of sugar, 296 + Ewell, method of estimating coffee carbohydrates, 585 + permanganate method, 136 + Excreta, collection, 562, 563 + Extract, composition, in fermented beverages, 624 + estimation, by indirect method, 625 + in fermented beverages, 624 + vacuum, 626 + Extraction apparatus, 43-51 + by digestion, 42 + percolation, 43 + compact apparatus, 48-51 + methods, 41, 42 + with alcohol, 245 + + F + Fat acids, determinations of nature, 396 + formulas for calculating yield, 392, 393 + temperature of crystallization, 327 + crystals, appearance, with polarized light, 347 + microscopic appearance, 346, 347 + estimation, in altered milk, 487, 488 + butter, 515 + koumiss, 534 + meats, 550 + preserved meats, 563 + extraction, methods, adapted to milk, 486 + form of globules in milk, 482 + globules, method of counting, 483 + number, in milk, 482 + in milk, classification of methods of analysis, 484 + comparison of methods of analysis, 488 + wet extraction methods, 488 + Fats and oils, coloration, produced by oxidants, 352 + consistence, 396 + drying, for analysis, 316 + estimation of water, 317 + extraction, 41 + melting point, 320-323 + microscopic appearance, 345 + physical properties, 317-345 + polarization, 350 + preparation, for microscope, 345, 346 + refractive index, 328 + sampling, 315 + solubility, in alcohol, 351 + specific gravity, 317-319 + table of densities, 320 + temperature of crystallization, 327 + thermal reactions, 356-363 + turbidity temperature, 351 + composition, 309, 310 + freeing, of moisture, 315 + nomenclature, 309 + Feculometer, Block, 300 + Fehling solutions, comparison, 127 + composition, 126 + historical, 124 + Fermentation, method of separating sugars, 288, 289 + use, in sugar analysis, 185 + Fermented beverages, constituents, 611 + description, 610 + distillation, 612-614 + polarization, 632 + specific gravity, 611 + Ferments, aliphalytic, 556 + amylolytic, 556 + proteolytic, 557 + Fiber, estimation, 303, 304 + in canes, 241 + cereals, 543 + occurrence, 303 + Fibrin, 413, 504 + Fibrinogen, 411 + Fibroin, 416 + Field of vision, appearance, 81 + Filled cheese, 529 + Fischer, carbohydrates, 59 + Fish, preparation, 12 + Flesh bases, treatment of residue, insoluble in alcohol, 460 + Foods, constituents, comparative values, 567 + fuel value, 551 + nutritive values, 566 + potential energy, 551 + Free fat acid, determination, 394 + Fruits, composition, 579 + evaporated, 580 + sampling, 577 + Fuchsin, detection, in wines, 637 + Furfurol, determination, 180 + precipitation, with pyrogalol, 183 + qualitive tests, for sugars, 194 + reactions, 194, 195 + + G + Galactan, method of estimating, 586 + occurrence, 586 + Galactosazone, 193 + Galactose, products of oxidation, with nitric acid, 191 + Gelatin, 414 + estimation, 456-459 + reagent for tannins, 590 + Gerber, butyrometer, 502 + method of milk fat analysis, 502-503 + Gerrard, potassium cyanid process, 146 + Ginger starch, 220 + Gird, gravimeter, 233 + Glacial acetic acid, reagent for fats and oils, 351 + Gladding, method of preparing fats for the microscope, 346 + Glass, errors due to poor, 520 + Gliadin, 436 + Globin, 411 + Globulin, separation in milk, 510 + Globulins, properties, 411 + separation, 440 + Glucosazone, 171 + Glucose, commercial, 286 + process of manufacture, 287 + Glutamic acid, 412 + Glutamin, 417 + estimation, 426, 427 + Gluten, 413 + composition, 426 + separation, from wheat flour, 434, 435 + Glutenin, 436 + Glutin, 413 + composition, 451 + Glycerids, principal, 310 + saponification value, 383, 384 + separation, 397 + Glycerol, estimation, in fermented beverages, 635, 636 + formulas for calculating yield, 392, 393 + Gomberg, method of estimating caffein, 584, 585 + Grape sugar, birotation, 287 + commercial, 264, 286 + Gravimeter, 233 + Green samples, grinding, 9 + Grinding apparatus, 6-11 + Gum, detection, in fermented beverages, 639 + Gypsum, use, in drying sour milk, 487 + Gyrodynat, definition, 116 + + H + Haemocyanin, 411 + Haemoglobin, 411 + Halle drying apparatus, 29 + Haloid absorption by fat acids, 374-376 + addition numbers, 364 + Heat of bromination, improved method of determining, 361-363 + Hehner and Mitchell, method of determining heat of bromination, 361 + Richmond, formula for calculating total solids, 479, 480 + bromin addition number, 373 + Hemi-peptones, 412 + Hempel, calorimeter, 569 + Heteroalbumose, 412 + Hibbard, estimation of starch, 207 + Hide powder, reagent for tannins, 590 + testing, 592 + Honey, composition, 264 + Hoppe-Seyler, cellulose, separation, 304 + Hops, bitter principles, 640 + substitutes, 640 + Horse flesh, detection, 554 + Hübl’s process, 364-367 + reagent, preservation, 371 + Hyalin, 416 + Hyalogen, 416 + Hydrochloric acid, estimation, in tobacco, 600 + Hydrogen, drying, 24 + estimation, in proteids, 444 + Hydrometer, balling, 71 + baumé, 71 + brix, 71 + Hydrometers, 71 + Hydrometry, correction for temperature, 72 + Hydrostatic balance, 68 + plummet, 615 + correction table, 615 + Hypogaeic acid, separation, 399 + Hypoxanthin, occurrence, in milk, 464 + + I + Impurities, error due, 74 + Incineration, purpose and conduct, 37, 38 + Insoluble fat acids, determination, 391, 392 + Inversion, application of the process, 114 + calculation of results, 108, 109 + determination of sucrose, 105 + influence of strength of solution, 108 + time of heating, 108 + Invert sugar, estimation of minute quantities, 257 + gyrodynat, 119 + influence of temperature on gyrodynat, 265 + occurrence, 264 + official method, 161-162 + optical neutrality, 265 + separation and estimation, 264 + table for calculating, from copper, 260 + estimating, 159, 258 + Invertase, determination of activity, 111, 112 + use, in inversion, 110, 111 + Invertose, molecular weight, 177 + Iodin addition, 364-367 + character of chemical reaction, 367 + monochlorid, substitution for hübl reagent, 370 + number, estimation, 369-370 + reaction with starch, 196 + reagent for caffein, 584 + + J + Juices, analysis of fruit and vegetable, 578 + + K + Keratin, 416 + Kieselguhr, use in drying, 486 + Knorr, extraction apparatus, 44 + fractional analysis of meats, 552 + Koettstorfer, saponification value, 382, 383 + Koumiss, acidity, 532 + composition, 532, 536 + Kreatin, 416 + composition, 431 + determination, 454 + occurrence in milk, 464 + Kreatinin, 416 + composition, 451 + determination, 454 + Krug, method of determining oxygen absorption of oils, 405, 406 + estimating pentosans, 179, 183 + separation of oleic and hypogaeic acids, 399 + viscosity of oils, 345 + + L + Lactobutyrometer, 495, 496 + Lactocrite, use in milk fat analysis, 498 + Lactoglobulin, 504 + Lactometer, direct reading, 476 + New York Board of Health, 476 + Lactometry, 475, 476 + Lactoprotein, 504 + Lactosazone, precipitation, 295 + Lactoscopes, 473, 474 + Lactose, estimation, 293 + in Koumiss, 534 + milk, 275 + gyrodynat, 119 + molecular weight, 177 + official method of estimation, 294 + Laurent lamp, 83, 84 + polariscope, 83 + construction, 86-88 + manipulation, 88 + Lead acetate, preserving agent, 235 + oxid, separation of sugars, 284, 285 + reagent for determining oxygen absorption of oils, 405 + salts, reagents for separating fat acids, 397 + solutions, errors, 102 + subacetate, action on levulose, 103 + Lecithin, extraction from seeds, 430, 431 + factors for calculating, 431 + occurrence and properties, 430 + in milk, 464 + Leffmann and Beam, method of milk fat analysis, 501 + Legumin, 411 + Leucin, 412 + occurrence in milk, 464 + Levulosazone, 193 + Levulose, estimation, 168 + in presence of sucrose and dextrose, 280-285 + general formula for calculation, 274 + gyrodynat, 119 + optical determination, 267 + preparation, 167 + principles of calculation, 270-273 + table for calculating from copper, 260 + estimation, 169-171 + Liebermann, method of milk fat analysis, 471, 492 + Liebig ente, 28 + Light, kind used for polarization, 82 + Lindet, method of inversion, 109, 110 + Lindsey and Holland, digestibility of pentosans, 564 + Lindström, modification of lactocrite, 499 + Lineolin, 310 + Lint, use, in drying, 486 + Livache, method of determining oxygen absorption, 405 + Long and Baker, diastase preparation, 300 + table of refractive indices, 334 + + Mc + McElroy and Bigelow, estimation of sugar in + evaporated milks, 296-298 + estimation of nicotin, 605 + McIlhiney, bromin addition number, 372 + + M + Maercker, apparatus for hydrolysis of starch, 204 + method of sugar analysis, 153-155 + Magnesium sulfate, reagent for precipitating proteids, 433 + Maize starch, 221 + Malic acid, estimation in fermented beverages, 630, 631 + tobacco, 601 + Malt extract, 301 + Maltosazone, 193 + Maltose, estimation, 165 + gyrodynat, 119, 206 + molecular weight, 177 + occurrence, in glucose, 264 + separation from dextrin and dextrose, 287-293 + table for calculating from copper, 261 + determination, 165-167 + Maple sugar, 228 + conditions of manufacture, 228 + Maranta starch, 219 + Massecuites, analysis, 254 + determination of ash, 256 + reducing sugars, 256 + water, 255 + Maumené, heat of sulfuric saponification, 357, 358 + Maxwell, method of extracting lecithin, 430, 431 + Meat extracts, analysis, 452-454 + composition, 451, 452 + Meats, estimation of proteids, 550 + fractional analysis, 552 + methods of analysis, 549-554 + sampling, 547 + scientific names,547 + Meissl, table for invert sugar, 158 + Melons, sampling, 577 + Melting point, determination by spheroidal state, 323-326 + methods of determining, 321-326 + of fats and oils, 320-323 + Mercuric compounds, clarification, 104 + cyanid, reagent for destroying reducing sugars, 290, 291 + salts, reagent for casein, 535 + reduction by sugar, 121 + Metabolism, vegetable and animal, 2 + Metalbumin, 415 + Methyl blue, qualitive test for invert sugar, 192 + Micro-organisms, occurrence in milk, 469 + Midzu ame, Japan glucose, 286 + composition, 264 + Milk, acidity, 475 + alkalinity, 472 + alterabitity, 467, 468 + appearance, 469 + carbohydrates, 293, 511 + composition, 464, 465, 468 + determination of total solids, 477 + effects of boiling, 469 + electric conductivity, 472 + error due to volume of precipitate in polarization, 277 + fat analysis, volumetric methods, 496-504 + extraction, asbestos process, 485 + paper coil method, 485 + variation of methods, 486 + freezing point, 472 + mean composition, 465 + opacity, 473 + polarization, 277 + preservatives, 471, 472 + proteids, 504 + estimation, 505 + precipitants, 510 + sampling, 469, 470 + serum, density, 477 + specific gravity, 474 + sterilized, 468 + sugar, estimation, 163, 164 + table for estimation, 163 + viscosity, 472 + Millian, method for determining solubility, 351 + modification of Bechi’s test, 401 + process of separating arachidic acid, 398 + Million’s reagent, 421 + Mills, grinding, 7-11 + Mitscherlich, determination of ash, 83 + reducing sugars, 256 + water, 255 + specific gravity, 254, 255 + Monochromatic flame, constant, 85 + Mother beets, determination of sugar, 250, 251 + Mucic acid, test for lactose, 190 + Mucin, 414 + Munroe, thermal reactions of oils, 359 + Muscular tissues, occurrence in meat extracts, 456 + separation of nitrogenous bodies, 448-450 + Muskmelons, composition, 581, 582 + Muter, method of determining haloid addition, 374-376 + process of separating fat acids, 397 + table, for identifying starches, 214-217 + Mycsin, 411 + Myrosin, 411 + + N + Natural digestion, 562 + Neuclein, 415 + Neucleoproteids, 415 + Neurokeratin, 416 + Nickel prism, 77 + theory, 77-80 + Nicotin, estimation in tobacco, 605-607 + gyrodynat, 606 + polarization, 606 + Nitric acid, color test, fats and oils, 353 + estimation in tobacco, 600 + qualitive test, 418 + Nitrogen, estimation in fermented beverages, 639 + flesh bases, 459 + proteids, 445 + of total, 423 + percentage in proteids, 445 + Nitrogenous bases, occurrence in animal tissues, 450, 451 + bodies, composition, 410 + estimation in meats, 551 + occurrence in animal products, 448-462 + qualitive tests, 418-421 + separation in cheese, 530 + Nutritive ratio, 568 + values, 566 + + O + Oat starch, 221 + Observation tube, continuous, 253 + Oil press, 312 + removal from starchy bodies, 300 + Oils and fats, extraction, 310, 314 + identification, 395, 406 + physical properties, 317, 345 + coefficient of expansion, 319 + composition, 309, 310 + heat of bromination, 360 + nomenclature, 309 + spectroscopic examination, 348 + Oleic acid separation from palmitic, 397 + Olein, 310 + Oleomargarin, calometric distinction from butter, 576 + Oleorefractometer, 334 + Oleothermometer, 514 + Organic acids, occurrence in ensilage, 546 + Orseille, detection in wines, 637 + Osazones, melting points, 193 + Ost, copper carbonate method, 258, 259 + solution, 257 + Oven, electric, 19 + hydrogen drying, 25, 27 + steam coil, 20, 21 + water jacket, drying, 14 + Oxalic acid, estimation in tobacco, 601 + Oxygen, absorption by oils, 405 + combustion, 569 + + P + Palm sugar, 228 + Palmitic acid, separation from oleic, 397 + Palmitin, 310 + Pancreas extract, digestion, 560 + peptone, 461 + Pancreatin digestion, 558 + preparation, 560 + Paraffin, occurrence in plants, 404, 405 + Paralbumin, 415 + Patrick, volumetric method of milk fat analysis, 497 + Pavy’s process, 143 + Pea starch, 220 + Peanut oil, detection, 400 + Pectic acid, estimation in tobacco, 603 + Pectin, occurrence, 577, 578 + separation, 578 + Pectose, occurrence, 577 + Pellet, continuous observation tube, 253 + method of cold diffusion, 243, 244 + Pentosans, digestibility, 564 + estimation, 178 + revised factors for calculating, 587 + Pentose sugars, estimation, 177 + Pepsin digestion, 558 + preparation, 558 + Peptones, 412 + qualitive tests, 420, 421 + separation from albumoses, 455 + in cheese, 531 + Permanganate gelatin, method for tannins, 593, 594 + hide powder method for tannins, 595 + process, 132 + modified by Ewell, 136 + Peska’s process, 144 + Petroleum ether, preparation, 312 + removal from extracted oils, 314 + solvent, 41 + Phenylhydrazin, action on sugars, 172, 174 + compounds with sugar, 192 + reagent for precipitating furfurol, 180 + sugar, 171 + Phloroglucin, modification of furfurol method, 588 + reagent for precipitating furfurol, 184 + Phosphomolybdic acid, color test, fats and oils, 353 + Phosphorus, loss of organic in combustion, 37 + Phosphotungstic acid, preparation of reagent, 454 + Phytalbumoses, 412 + Phytosterin, detection, 403, 404 + Picric acid, color test, with fats and oils, 355 + Plasmin, 411 + Plaster of paris, use in drying, 486 + Polarimeter, 83 + Polarimètre, 83 + Polarimetry, general principles, 92, 93 + Polariscope, adjustment, 93, 94 + of quartz plates, 96, 97 + definition, 80 + for estimating levulose, 267, 270 + kinds, 80, 81 + rotation instruments, 82 + Polaristrobometer, 83 + Polarization, analytical use of data in + fermented beverages, 634, 635 + for factory control, 263 + of fermented beverages, 632, 633 + Polarized light, 75, 76 + application to butter analysis, 514 + relation to sugar analysis, 74 + Politis, method of sugar analysis, 148 + Ponceau-red, detection in wines, 637 + Potash, estimation in wines, 637 + Potassium cyanid, use in sugar analysis, 146 + hydroxid, solvent for proteids, 443 + nitrate, occurrence in maize stalks, 417 + preserving agent, 417 + permanganate, reagent for tannins, 593 + Potato starch, 220 + Potatoes, estimation of starch, 301, 302 + Powdered glass, use in drying, 486 + Preserved meats, 563 + Proteid bodies, separation, 432, 448 + nitrogen, estimation in tea and coffee, 585 + qualitive test, 419, 420 + Proteids, action of acids, 442 + classification, 410 + diversity of character, 434 + estimation in cereals, 543 + koumiss, 534 + meats, 550 + milk, 505 + of digestible in cheese, 531 + general principles of separation, 446 + insoluble, 413 + kinds in milk, 504 + methods of drying, 443, 444 + precipitation, 439 + separation, soluble in water, 439, 440 + soluble in dilute alcohol, 440 + salt solution, 438 + water, 436 + solution in alkalies, 442 + Proteolytic ferments, 557 + Proteoses, definition and properties, 412 + separation, 440 + Protoalbumose, 412 + Pulfrich, refractometer, 331, 333 + Pumice stone, use in drying, 33, 486 + Purity, apparent, 263 + Pyknometer, formulas for calculating volume, 67 + use, 63, 64 + at high temperature, 65, 66 + Pyrogalol, reagent for furfurol determination, 183 + + Q + Quartz plates, 96, 97 + applicability, 98 + corrections, 97, 98 + Quévenne, lactometer, 466 + + R + Raffinose, estimation, 115 + in presence of sucrose, 266 + gyrodynat, 119 + molecular weight, 177 + Raoul, method of determining molecular weights, 175 + Reducing sugars, estimation, 234 + in fermented beverages, 632 + factors for computation, 141, 142 + relation to quantity of copper suboxid, 141 + Refractive index of fats and oils, 328 + indices, 333, 334 + Refractometers, 329, 333 + variation, 338 + Regnault-Pfaundler, calorimetric formula, 572 + Reichert number, 518, 519 + Resorcin, qualitive test for levulose, 191 + Richmond, thermal reaction of oils, 359, 360 + Ritthausen, method of precipitating milk proteids, 506 + Roentgen rays, application to analyses, 588 + Rye starch, 221 + + S + Saccharic acid, test for dextrose group, 190 + Sachsse’s method of determining amid bodies, 424, 425 + solution, 122 + Saffron, 522 + Safranin, detection in wines, 637 + Sago starch, 220 + Salicylic acid, detection in fermented beverages, 638, 639 + Salt, estimation in butter, 516 + Samples, collecting, 5 + grinding, 6 + preparation, 3, 4 + preserving, 5 + Sand, use in drying, 486 + Saponification, 376, 384 + chemical reactions, 377, 378 + equivalent, 383 + in the cold, 381, 382 + methods of conducting, 378, 382 + under pressure, 379, 380 + value, 382, 383 + of butter, 518 + with alcohol, 381 + without alcohol, 381 + Sarkin, 416 + composition, 451 + Sausages, occurrence of starch, 553 + Scheibler, double polarization, 102 + extraction tube, 248 + Schmidt, method of milk fat analysis, 489 + deSchweinitz and Emery, calorimetric distinction between + butter and oleomargarin, 576 + Schweitzer and Lungwitz, iodin addition, 367, 368 + Scovell, milk sampler, 470 + Selenite plate, microscopic examination of starches, 219 + use in examination of fat crystals, 343 + Sesame oil, detection, 402 + furfurol reaction, 521 + Shadow polariscope, 90 + Short, method of milk fat analysis, 490 + Shredding apparatus, 9, 10, 236 + Sidersky, modification of Soldaini’s process, 147 + Sieben, method of determining levulose, 280 + Silver nitrate, color test with fats and oils, 355 + Sirups, analysis, 254 + determination of ash, 256 + reducing sugars, 256 + water, 255 + specific gravity, 254 + Sodium chlorid, reagent for extracting proteids, 433 + thiosulfate solution, preparation, 369 + Soldaini copper carbonate process, 139, 140 + gravimetric method, 258 + Soleil-Ventzke polariscope, 88, 89 + Solidifying point of fats and oils, 326, 327 + Soluble acids, estimation in butter, 517 + fat acids, determination, 389, 390 + Solvent, recovery, 53, 55 + in open dish, 55 + Solvents, 52, 53 + object, 40 + Sour milk, density, 477 + Soxhlet, areometric method of milk fat estimation, 492, 494 + extraction apparatus, 47 + Specific gravity, areometric method, 70 + determination in distillate, 615 + example, 68 + method of expressing, 319 + standard of comparison, 320 + heats of materials in calorimeter, 573 + rotatory power, 115, 116 + causes of variation, 117 + Spectroscopy, oils and fats, 348 + Spencer, air-drying oven, 17 + method of estimating caffein, 583 + observation tube, 253 + Spheroidal state, melting point, 323, 326 + Sponge, use in drying, 486 + Spongin, 416 + Stannic bromate, color test with fats and oils, 356 + Starch, colorimetric estimation, 210 + composition, 196 + disturbing bodies in estimation, 209 + estimation in potatoes, 301, 302 + sausages, 553 + of ash, 203 + nitrogen, 203 + water, 202, 299 + with barium hydroxid, 208 + factor for calculating from dextrose, 205 + fixation of iodin, 211 + gyrodynat of soluble, 206 + hydrolysis at high temperatures, 199 + in an autoclave, 199, 200 + with acids, 203, 204 + occurrence, 298 + in tobacco, 604 + particles, separation, 197 + polarization, 205 + principles of determination, 201 + properties, 196 + rapid estimation, 207 + separation, 399 + solution at high pressure, 206 + in nitric acid, 206 + Starches, classification, 218 + description of typical, 219 + identification, 211 + microscopic examination, 219 + occurrence in the juices of plants, 228 + Steam coil oven, 20, 21 + Stearin, 310 + Stone, method of estimating pentosans, 181 + Strontium saccharates, 187 + Stutzer, artificial digestion of cheese, 561 + method of estimating gelatin, 457 + Succinic acid, estimation in fermented beverages, 630, 631 + Sucrose, cobaltous nitrate test, 189 + estimation in coffee, 586 + presence of dextrose, 274 + levulose and dextrose, 280, 285 + raffinose, 266 + molecular weight, 176 + occurrence, 264 + pipette, 231, 232 + qualitive optical test, 188 + separation and estimation, 264 + Sugar analysis, chemical methods, 120 + classification of methods, 61, 62 + general remarks, 104 + gravimetric copper methods, 149, 170 + Halle method, 153, 155 + laboratory gravimetric method, 150-153 + permanganate process, 132-135 + volumetric laboratory method, 129, 130 + methods, 121 + Sugar beets, analysis, 242 + apparatus for grinding, 10 + extraction with alcohol, 245, 247 + content in maple sap, 228 + direct determination in canes, 235 + estimation in cane and beet pulp, 238 + sap, 228-230 + sugar beets, 242 + extraction from plants, 230 + flask, diffusion and alcohol digestion, 245 + Sugar flasks, 98, 99 + instantaneous diffusion, 243 + juices, preservation, 235 + mills, 230 + preparation of pure, 60 + removal from starchy bodies, 300 + solutions, preparation for polarization, 99-104 + specific gravity, 62 + Sugars, determination in dried material, 239 + without weighing, 253 + estimation in fermented beverages, 632, 635 + hexose, 59 + miscellaneous qualitive tests, 193 + occurrence in tobacco, 604 + optical properties, 74, 75 + pentose, 59 + qualitive tests, 188 + separation by lead oxid, 284, 285 + state of existence in plants, 227 + Sulfur chlorid, reagent for oils, 402, 403 + determination in proteids, 446 + loss of organic in combustion, 37 + Sulfuric acid, color test for fats and oils, 352 + estimation in tobacco, 600 + saponification, 357, 358 + Sulfurous acid, elimination, 519 + estimation in fermented beverage, 638 + + T + Tannic acid, estimation in tobacco, 604 + reagent for milk proteids, 507 + + Tannin, composition, 588 + detection, 589, 593 + estimation 589, 596 + by hide powder method, 590, 592 + infusion, preparation, 596 + occurrence, 588 + permanganate gelatin method, 593, 594 + precipitation with metallic salts, 589 + Tartaric acid, estimation in wine, 229, 230 + Tea and coffee, 582 + Thein, 583 + Thermal reactions, fats and oils, 356, 363 + Thermostat for steam bath, 15 + Thörner, method of milk fat analysis, 491 + Tobacco, acid and basic constituents, 597 + burning qualities, 608 + composition, 598, 599 + of ash, 598 + fermentation, 596, 597 + fractional extraction, 608 + Tollens and Günther, method of estimating pentosans, 180 + Torsion Viscosimeter, 342 + Total solids, calculation, 478 + formulas for calculating, 479, 480 + Trinitroalbumin, 411 + Triple shadow polariscope, 91, 92 + Tropæolin, detection in wines, 637 + Turbidity temperature, fats and oils, 351 + Turmeric, 522 + Tyrosin, 412 + occurrence in milk, 464 + Tyrotoxicon, occurrence in milk, 464 + + U + Ulsch, drying oven, 31 + Unicedin, 413 + Urea, occurrence, in milk, 464 + + V + Vacuum, drying, 18 + Van Slyke, method of estimating casein, 508 + Vegetable substances, preparation, 3, 4 + Vegetables, sampling, 577 + Vinolin, detection in wines, 637 + Viscosimetry, 342, 345 + Viscosity of fats and oils, 342 + Viscous liquids, drying, 32, 33 + Vitellin, 411 + Vogel, table for identifying starches, 212, 213 + Volatile acids, estimation in butter, 517 + fermented beverages, 627, 628 + bodies, drying, 13 + fat acids, determination, 386, 388 + distillation, 387, 388 + titration, 388 + Volume of precipitate, calculation, 279 + + W + Water, action on composition of proteids, 437 + estimation in butter, 515 + cereals, 542 + fermented beverages, 626 + fruits and vegetables, 578 + koumiss, 536 + meats, 549 + tobacco, 599 + Watermelons, composition, 581, 582 + Wax, occurrence in tobacco, 608 + Waxes, composition, 309 + Westphal balance, 69 + Wheat starch, 221 + Wiechmann, formula for calculating sugars, 307 + method of estimating levulose, sucrose and dextrose, 280, 281 + Williams, calorimeter, 570 + Winter, estimation of levulose and dextrose + in presence of sucrose, 283, 284 + Wood pulp, use in drying, 486 + Wrampelmayer, drying oven, 30 + + X + Xanthin, 416 + Xanthoproteic reaction, 420 + Xylan, occurrence, 586 + + Y + Yeast, inversion, 113 + + Z + Zein, 441 + Zeiss, butyrorefractometer, 339, 341 + Zinc, occurrence, in evaporated fruits, 380, 581 + sulfate, reagent for precipitating proteids, 433 + separating albumoses from peptones, 455 + + + + +CORRECTIONS FOR VOL. I. + + +Page 5, 11th line, insert “ten” before “thousand.” + +Page 21, read “Magdeburg” instead of “Madgeburg” in both instances. + +Page 61, for per cent. of oxygen in ulmin read “28.7” instead of “8.7.” + +Page 62, for per cent. of carbon in apocrenic acid read “54.4” instead +of “34.4.” + +Page 112, 2d line from bottom, read “14” instead of “13.” + +Page 140, the sentence beginning “The burette is lowered etc.” is +repeated. _Dele_ one of them. + +Page 141, 6th line, insert “or air dried” after “moisture.” + +Page 141, in example read 10.25, 10.22, 13.07 and 76.21 for 9.52, 9.22, +12.07, and 60.35 respectively. + +Page 158, 3d line of =172=, insert “and estimating soluble matters +therein” after “flow.” ____ Page 159, omit “√ ” in first formula. + +Page 293, 12th line, read “U” for “V.” + +Page 309, last line, read “sixth” for “sixteenth.” + +Page 312, 1st line, read “atmosphere” instead of “room.” + +Page 315, 6th and 7th lines, read =299=, and =300=, for =294=, and +=295=, respectively. + +Page 323, 3d line from bottom, read =301= for =299=. + +Page 333, 5th line of =316=, insert after “is” “to eliminate carbon +dioxid and.” + +Page 354, 3d line from end of (6) insert after “solution” “acidified +with acetic and;” same line, transfer “r” from “ther,” to “pecipitate.” + +Page 357, 6th line from end of (4) insert after “difference” “and the +phosphoric acid estimated as in =380=, deducted therefrom.” + +Page 367, 4th line, add after “taken,” “The phosphoric acid may be +determined as described in =372=, or following paragraphs.” + +Page 410, line 21, _dele_ “dilute” and insert “one per cent nitric.” + +Page 410, line 25, after “capsule” insert “adding water once or twice.” + +Page 449, 4th line read “hydrobromic” for “hydrochloric.” + +Page 457, reference “30” read “Band 38” instead of “Band 37.” + +Page 468, 8th line, _dele_ “or gypsum” and read “200” instead of “50.” + +Page 471, last line, read “not” for “very.” + +Page 472, 4th line, insert “un” before “successful.” + +Page 496, 5th line, insert “the” before “soil.” + +Page 515, next to last and last lines, read “stannous” instead of +“zinc.” + +Page 516, second line, read “stannous” instead of “zinc.” + +Page 557, 9th line of =500= read “red-yellow” instead of “blue.” + + +CORRECTIONS FOR VOL. II. + +Page 14, line 21, read “61.74 per cent.” for “16.74 per cent.” + +Page 23, 8th line from bottom, read “0.0025.” for “0.0035.” + +Page 54, 9th line, read “white” instead of “yellow.” + +Page 54, 13th line, read “phosphate” instead of “phosphomolybdate.” + +Page 57, 8th line from bottom, read “saturated” instead of “citrate.” + +Page 73, read “Kosmann” in 10th line of paragraph =72= for “Kormann.” + +Page 158, reference number 72, read “1889” instead of “1888.” + + +CORRECTIONS FOR VOL. III. + +Page 11, name of Figure 4 read “Dreef” instead of “Dree.” + +Page 40, fifth and seventh lines, read “Courtonne” instead of +“Courtoune.” + +Page 59, eighth line from bottom, insert “original” before “optical.” + +Page 60, second line, read “_d_” instead of “_l_” before “fructose.” + +Page 68, legend of Figure 29, read “areometers” instead of +“aereometers.” + +Page 146, sixth line from bottom, insert “cyanid” after “potassium.” + +Page 159, instead of headings for table as given, substitute those on +page 160. + +Page 177, in formula for lactose, read “H₂₂” instead of “H₃₂”; in +formula for arabinose, read “H₁₀” instead of “H₁₉.” + +Page 180, seventh line from bottom, read “Günther” instead of “Gunther.” + +Page 191, read paragraph =169=. Seventh line read “resorcin” instead of +“resorsin.” + +Page 268, legend of Figure 77, read “Desiccating” instead of +“Dessicating.” + +Page 288, in formula (2) read “53d” instead of “54d.” + +Pages 328, 329 and 334, read “Amagat” instead of “Armagat.” + +Page 348, fourth line from bottom, omit accent in “sesame.” + +Page 365, (b) first line, read 24.8 instead of 24.6. + +Page 424, 4th line from bottom, read “nitrites” instead of “nitrates.” + +Page 425, 4th and 5th lines, read “nitrite” instead of “nitrate.” + +Page 445, in table of factors for computing proteids under Maize +Proteids, read “16.06 and 6.22” instead of “15.64 and 6.39” +respectively. + +Page 451, sixth line from bottom, read “occur” instead of “occurs.” + +Page 464, twelfth line from bottom, _dele_ “food.” + +Page 499, ninth line from bottom, read “Babcock.” + +Page 543, sixteenth line, read “6.06 and 6.22” for “6.31 and 6.39” +respectively. + +Pages 555-556, read “amylolytic” for “amylytic.” + +Page 555, fifth and seventeenth lines from bottom, insert after “into +dextrin, maltose.” + +Page 572, second equation, read “_tʹ_ₙ₂” instead of “_tʹ_ₙ₁.” + +Page 573, 3rd line from bottom, read “stirring” for “storing.” + +Page 574, 6th line from bottom, read “Θ₄” for “O_4.” + +Page 575, 16th line from bottom, insert “one gram of” before +“substance.” + +Page 576 instead of 567, fourth line of paragraph 566, read “Calorie” +instead of “calorie.” + +Page 644, _dele_ “Band 12, Ss. 64 und 199” in reference 94. + + + +*** END OF THE PROJECT GUTENBERG EBOOK 75389 *** |