METHODS OP ORGANIC ANALYSIS THE MACMILLAN COMPANY NEW YORK BOSTON CHICAGO DALLAS SAN FRANCISCO MACMILLAN & CO., LIMITED LONDON BOMBAY CALCUTTA MELBOURNE THE MACMILLAN CO. OF CANADA, LTD. TORONTO METHODS OF ORGANIC ANALYSIS BY HENRY C. SHERMAN, PH.D. PROFESSOR OF FOOD CHEMISTRY IN COLUMBIA UNIVERSITY AUTHOR OF " CHEMISTRY OF FOOD AND NUTRITION " SECOND EDITION REWRITTEN AND ENLARGED gork THE MACMILLAN COMPANY 1912 All rights reserved ! e,r' COPYRIGHT, 1905, BY HENRY C. SHERMAN. COPYRIGHT, 1912, BY THE MACMILLAN COMPANY. Set up and electrotyped. Published June, 1912. Norfaooi J. 8. Gushing Co. Berwick & Smith Co. Norwood, Mass., U.S.A. PREFACE TO SECOND EDITION IN rewriting this work after six years of constant use in the classroom and laboratory, the author has endeavored to keep in mind the needs both of students and of practising chemists. Methods which are commonly used as exercises for beginners (such, for example, as the determination of alcohol by the distillation method) are fully described with detailed explana- tory and precautionary notes, while those methods which are apt to be used only by advanced students or professional chemists are given more concisely. At the end of each chapter will be found, first a list of reference books arranged alphabetically by authors, and then a chronological list of journal articles, bulletins, etc., particularly of the last few years. The abbreviations are those used by the American Chemical Society in the publication of Chemical Abstracts. The scope of the work has been somewhat extended, the new matter including a chapter on solid and liquid fuels, and sections on industrial alcohol, drying oils, crude petroleum, the new international methods of glycerin analysis, and quan- titative methods for the testing of enzymes. The discussions of aldehydes, sugars, proteins, and food preservatives are also much fuller than in the first edition. These additions, and the rewriting of the original text to embody recent advances and certain changes of arrangement which have been found advantageous from the standpoint of teaching, make the present edition practically a new work. The text and references are designed to cover, along with the directions for laboratory work, so much at least of the technology of the various topics considered as is involved in a proper appreciation of the purposes of the analyses and the significance of the analytical results. 258727 VI PREFACE TO SECOND EDITION The writer takes pleasure in acknowledging his indebtedness to Dr. C. A. Browne, Chemist-in-charge of the New York Sugar Trade Laboratory, and Mr. T. T. Gray, Chief Chemist of the Tide-Water Oil Company, for criticism of the sections relating to their specialties, and to his colleague, Dr. J. M. Nelson, for many helpful suggestions. H. C. S. JANUARY, 1912. PREFACE TO FIRST EDITION THE purpose of this work is to give a connected introductory training in organic analysis, especially as applied to plant and animal substances and their manufactured products. No attempt is made to touch upon all important branches of this subject, but representative topics are treated in considerable detail with reference both to analytical methods and to the interpre- tation of results. The greater part of the book is devoted to quantitative methods for food materials and related substances. Standard works of reference and the publications of the Association of Official Agricultural Chemists have been freely used. The nomenclature adopted in these publications has been followed as closely as possible. As a rule, footnotes show the original sources of statements or methods included in the text, while general or additional references are given at the end of each chapter. The references have been carefully selected and are believed to be sufficient to put the reader in touch with the most important literature. The descriptions of methods were written primarily for the use of third-year students in the School of Chemistry, Colum- bia University, and therefore presuppose a knowledge of inor- ganic quantitative analysis, elementary organic chemistry, and general physics. The writer takes pleasure in acknowledging his indebtedness to Professor Edmund H. Miller for helpful advice and sugges- tions throughout the work, and to Mr. Roland H. Williams for assistance in testing methods and in the revision of parts of the manuscript. H. C. S. NEW YORK, July 1, 1905. vii CONTENTS \ PACK INTRODUCTION xv CHAPTER I ALCOHOLS 1 Ethyl Alcohol 2 Detection and Identification of Ethyl Alcohol ... 5 Determination of Ethyl Alcohol 6 Determination and Identification of Small Amounts of Alcohol 23 Detection and Determination of Methyl Alcohol ... 24 Determination of Amyl Alcohols or Fusel Oil ... 28 Official Requirements of Purity 29 References 31 CHAPTER II ALDEHYDES ............ 34 Formaldehyde , . . 36 Detection and Identification ....... 38 Quantitative Determination 40 Benzaldehyde 46 Vanillin 48 References 48 CHAPTER III CARBOHYDRATES GENERAL METHODS 50 Occurrence and Relations ,50 Solubilities ... . . " .54 Reactions with Acids ......... 56 Reactions with Hydrazines 61 Reduction of Copper Solutions . . 69 Rotation of Polarized Light * . . . . . . . .78 References . . 85 ix CONTENTS CHAPTER IV PAGB SPECIAL METHODS OF SUGAR ANALYSIS . . . . .87 Analysis of Raw Sugar ......... 87 Polariscopic Examination ....... 87 Determination of Reducing Sugars . . . . . .96 Determination of Moisture and Ash . . . . .97 Determination~of Sucrose in Beets and Cane . . . .98 Density and Purity of Sugar Solutions 100 Identification and Analysis of " Unknown " Sugars . . . 101 References ' ... 103 CHAPTER V STARCH AND AMYLASE 106 Determination of Starch . . . . . / . . 106 Diastatic Power of Amylases . . . . . ..'.113 References . . . . . . . . . . . 121 CHAPTER VI VINEGAR AND ACETATE * ;. . 123 Vinegar . . . . . . . . . .- . .123 Determination of Source . . . . . . . 124 Methods of Analysis . . . . . . . . . 127 Acetic Acid and Acetates . . 129 References . 131 CHAPTER VII FATTY ACIDS .133 Acids of Series C;H 2n O 2 . .133 Acids of Series C n H 2n _ 2 O 2 . . 135 Acids of Series C w H 2n _ 4 O 2 ",..'. 136. Acids of Series C n H 2n _ 6 O 2 ' . . 137 Acids of Series C n H 2n _ 8 O 2 . ' . .137 Saturated Hydroxy Acids .137 Hydroxy Acids of Series C n Il 2n _ 2 O 3 . . . . . .138 References . .139 CHAPTER VIII OILS, FATS, AND WAXES GENERAL METHODS .... 140 Classification 141 Properties of Fats and Fatty Oils ...... 142 CONTENTS XI PAGE Analytical Methods 143 Saponification Number 144 Acid and Ester Numbers 147 Hehner Number 148 Reichert-Meissl Number . . 148 Iodine Numbers . . . . . . . . . 148 Maumene Number Specific Temperature Reaction . . 157 Acetyl Number 160 Specific Gravity 162 Index of Refraction . . . . . . . . .164 Alcohols of Fats and Waxes Unsaponifiable Matter . . . 169 References 172 CHAPTER IX EDIBLE OILS AND FATS ......... 174 Salad Oils .174 Analytical Properties of Olive Oil 175 Detection of Adulterants 177 Butter . 185 Determination of Water, Fat, Curd, Ash . . . . 186 Examination of Butter Fat 188 References . . 199 CHAPTER X DRYING OILS 203 Oils Used as Drying Oils 203 Analytical Properties of Linseed Oil ....... 205 Adulterants and Methods of Detection ...... 206 Oils Altered by Age or Oxidation 210 " Unknown " Oils and Mixtures . 213 References 214 CHAPTER XI PETROLEUM AND LUBRICATING OILS 218 Examination of. Crude Petroleum 218 Examination of Lubricating Oils . . . . . . . 222 Determination of Constituents 223 Viscosity 226 Acidity 231 Cold Test and Chilling Point or Cloud Test . . . . 232 Flashing and Burning Points . 233 Additional Determinations ....... 234 Examination of Lubricating Greases 235 References . . . 236 Xll CONTENTS CHAPTER XII PAGE FUELS 239 Determination of Calorific Power . . . . . . 239 Chemical Composition and Calorific Power of Organic Compounds 245 Fuel Oils and Gasoline 247 Woods and Similar Fuels . . ... . . .251 Coal 254 Ultimate Composition and Calorific Power .... 254 Proximate Analysis . . . . 256 Relation of Proximate Composition to Calorific Power . . 257 References 261 CHAPTER XIII SOAP AND GLYCERIN 266 Analysis of Commercial Soap 266 Scheme of Analysis 266 Details of Determinations . . . . . 267 Glycerol ....'....,*... 275 Analysis of Crude Glycerin . . . . .... 276 References , 286 CHAPTER XIV NITROGEN, SULPHUR, AND PHOSPHORUS . . . . . 288 Determination of Nitrogen ........ 288 Determination of Sulphur . . . . . . . 295 Determination of Phosphorus ........ 303 References ........... 306 CHAPTER XV PROTEINS AND PROTEASES . . . . ... . . 308 Analytical Reactions of Proteins . . ... . . 313 Color Reactions ......... 313 Protein Precipitants . . . . . . 316 Separation of Proteins from Simpler Nitrogen Compounds and from each other . . . . . 322 Proteases or Proteolytic Enzymes . . . . . 323 References . . . . . * 329 CONTENTS xiii CHAPTER XVI PAGE GRAIN PRODUCTS . . ... . . . . 334 Preparation of Samples . . .... . . . 334 Methods of Analysis . . . . 335 Interpretation of Results . . . . ... . . 343 References 346 CHAPTER XVII MILK . . . ... . . . ..'.. .349 'Sampling and Preservation of Samples . . . . 352 Preliminary or Partial Examination . . . , . . 353 Determination of Fat, Proteins, Milk Sugar, and Ash . . . 356 Interpretation of Results . '. . . 362 Examination of Milk Serum for Added Water .... 364 Chemical Preservatives . . ... . . . . 365 References . . . . . . . . 366 CHAPTER XVIII FOOD PRESERVATIVES . . r . . ;. 369 Formaldehyde ........... 369 Hydrogen Peroxide 372 Boric Acid and Borates 373 Fluorides . . . . . . 375 Fluoborates and Fluosilicates . 376 Sulphurous Acid . . * . ' . . . . ... 377 Salicylic Acid . . . . ... . . . . 378 Benzoic Acid and Benzoates . . . . . . . . 385 Saccharin. . . . 388 Beta-Naphthol . . . . v . >. . . . . . . 389 Abrastol 389 Sucrol or Dulcin ........ \ 390 References . . . ... . . . . . 391 INTRODUCTION ULTIMATE organic analysis is the determination of the ele- ments composing any organic substance. Proximate Organic analysis is the determination of the compounds present in a mixture or of the radicles present in a compound. Both ultimate and proximate analysis are often required in the examination of organic materials. In the case of a complex mixture, however, proximate analysis is frequently directed to the determination of the principal groups of related com- pounds rather than of each individual compound present. Thus in the analysis of a food it may sometimes suffice to determine the percentage of moisture and of each of the groups of substances represented by the terms proteins, fats, carbo- hydrates, and ash constituents. In the analysis of ordinary animal and vegetable substances it is usually difficult to make a clear distinction between organic and inorganic constituents, because in many cases the inorganic compounds found in the ash are formed during combustion, the bases having existed in combination with organic acids or with proteins, while the acid radicles may also have existed in organic combination or may have been formed by the oxida- tion of the carbon, sulphur, and phosphorus of the organic matter. The chapters which follow are devoted chiefly to methods of proximate analysis. The determination of carbon and hydrogen by means of the combustion furnace is doubtless already familiar to most users of this book and is therefore omitted, but methods for determining nitrogen, sulphur, and phosphorus will be found in Chapter XIV. XVI INTRODUCTION The sequence of topics is in the main that usually followed in textbooks of organic chemistry, but exceptions are made in some cases in order to bring together subjects which are closely related in practical interest. METHODS OF OKGANIC ANALYSIS METHODS OF ORGANIC ANALYSIS CHAPTER I Alcohols ALCOHOLS are neutral hydroxyl derivatives capable of react- ing with acids to form esters, and the most characteristic alcohol reactions are those involving the replacement of the hydroxyl by an acid radicle. For detailed discussions of the analytical behavior of the hydroxyl radicle and characteristic reactions of the different groups of alcohols, the reader is re- ferred to the works of Meyer, 1 and of Meyer and Tingle, 1 while Mulliken's tables 1 may be followed in the systematic identifica- tion of individual pure preparations. Of the analytical methods involving reactions of the hydroxyl group, the more important are those in which the acetyl or benzoyl ester is formed. The preparation of the dinitrobenzo- ate serves for the identification of ethyl alcohol, and glycerol may be determined quantitatively by acetylation. Often, how- ever, esterification is either not quantitative or is less con- venient or less delicate than other methods of determination. Thus the monatomic alcohols containing less than four carbon atoms mix freely with water and are not readily separated from it, but may be determined in aqueous solution either by physi- cal methods or by the behavior of the alcohols on oxidation. The present chapter will deal with ethyl alcohol and the de- tection and determination of a few of its more important homo- logues. Glycerol will be considered in connection with soap in a later chapter. For more extended discussions of the analyti- cal chemistry of the alcohols the works cited at the end of the chapter may be consulted. 1 The titles of these and other works of reference will be found at the end of the chapter. B 1 METHODS OF ORGANIC ANALYSIS ETHYL ALCOHOL Pure ethyl alcohol is a colorless, mobile liquid of characteris- tic penetrating odor and " hot " pungent taste, boiling at about 78.4 and showing at 15.56 (60 F.), compared with water at the same temperature, a specific gravity of 0.79387 (Bureau of Standards). It mixes with water in all proportions and is only with great difficulty obtained in the anhydrous or " absolute " state. According to Allen the presence of as small proportion as 0.5 per cent of water in alcohol is indicated by the pink color assumed by the liquid on introducing a crystal of potas- sium permanganate. The so-called " absolute alcohol " used in analytical operations ordinarily contains from 0.2 to 1 per cent of water. Alcohol is of great importance in organic analysis not only as a constituent to be determined but also as a solvent in ana- lytical operations. It dissolves many organic substances which are not soluble in water, but inorganic compounds insoluble in water are usually also insoluble in alcohol. As a rule chlorides, bromides, iodides, and acetates are soluble in fairly strong alcohol, while carbonates, borates, sulphates, phosphates, oxa- lates, and tartrates are only very sparingly soluble. Three strengths of alcohol are recognized in the U. S. Pharmacopoeia of 1905 : "Alcohol" containing about 92.3 per cent by weight or about 94.9 per cent by volume of actual ethyl alcohol and about 7.7 per cent by weight of water ; specific gravity about 0.816 at 60 F. "Absolute Alcohol" containing not more than 1 per cent by weight of water; specific gravity not higher than 0.798 at 60 F. " Diluted Alcohol " made by mixing equal volumes of " alcohol " and water and containing about 41.5 per cent by weight or 48.9 per cent by volume of actual ethyl alcohol ; specific gravity about 0.936 at 60 F. On mixing alcohol with water a considerable evolution of heat takes place and the volume of the mixture after cooling ALCOHOLS 6 is less than the sum of the volumes of the alcohol and water mixed. This contraction is not uniformly proportional to the amount of alcohol in the mixture. Hence in mixtures of water and alcohol the relation between the percentages of alcohol " by volume " and " by weight " varies somewhat with the strength of the solutions. These variations together with the differences in the density of the supposedly absolute alcohol used as stand- ard by the various observers account for the small discrepancies found on comparing the commonly used tables showing the re- lation between density and percentage. In the United States the tables principally used are (1) those of the U. S. Bureau of Standards recalculated from the determina- tions made by Mendelejeff and (2) those based on Squibb's de- terminations, which have been very generally used in this country and incorporated in the methods of the Association of Official Agricultural Chemists and the U. S. Pharmacopoeia. The official chemists have under consideration the question of substituting the tables of the Bureau of Standards for those now official. The two systems differ chiefly in the conditions taken as standard, the final results when properly calculated being very nearly the same, especially for solutions containing less than 25 per cent of alcohol. The methods and data given in this chapter provide for the use of either system, Table I (beyond) being from the Bureau of Standards, and Table II condensed from the tables of the Official Agricultural Chemists and of the U. S. Pharmacopoeia. The table of Morley (J". Am. Ohem. Soc., 26, 1185-1193), also based on the observations of Mendelejeff, gives the specific gravity of alcohol for each integral percentage by weight and for each degree of the hydrogen thermometer from 15 to 22 C. For revenue purposes, both in the United States and Great Britain, the strength of alcoholic liquors is expressed in terms of " proof spirit," but the term has different meanings in the two countries. American proof spirit is defined by Section 3249 of the Re- vised Statutes of the United States as follows: " Proof spirit shall be held to be that alcoholic liquor which contains one half 4 METHODS OF ORGANIC ANALYSIS its volume of alcohol of a specific gravity of seven thousand nine hundred and thirty-nine ten-thousandths at 60 F." It is therefore practically 50 per cent by volume or about 42.5 per cent by weight. British proof spirit is denned by Parliament as having such a density that at 57 F. thirteen volumes shall weigh the same as twelve volumes of water at the same temperature. This cor- responds to about 49.2 per cent by weight. "Rectified spirit" of the British Pharmacopoeia has 84 per cent by weight and British "methylated spirit" consists of nine parts of rectified spirit to one part of commercial " wood spirit " or " wood naphtha," the latter containing, according to the observations of Thorpe and Holmes, from 72 to 80 per cent of methyl alcohol by volume. Denatured Alcohol A statute of the United States of June 7, 1906, provides that domestic alcohol may be withdrawn from bond for 'use in the arts and industries, and for fuel, light, and power, without the payment of an internal revenue tax, on condition that it shall have been denatured by the admixture of some material which destroys its character as a beverage and renders it unfit for liquid medicinal purposes. The regular formula for denaturing alcohol is as follows : To 100 parts by volume of ethyl alcohol (not less than 90 per cent strength) add 10 parts of approved methyl (wood) alcohol and 0.5 part of approved benzine. Such alcohol is classed as completely denatured and becomes a regular article of commerce. The wood alcohol used in denaturing must have a specific gravity not above 0.830 at 60 F. (corresponding, if impurities be neglected, to about 88 per cent strength, or to 91 on Tralles scale) and meet several other requirements which include maxi- mum and minimum limits for acetone and bromine-absorbing constituents and a maximum limit for esters. The Bureau of Internal Revenue of the Treasury Department establishes the detailed regulations governing both the regular method of denaturing and the special formulas permitted in the ALCOHOLS 5 several classes of industries in which tax-free alcohol may be used, but for which the alcohol denatured by the regular formula would not be suitable. These formulae and specifications are given in Regulation No. 30 of the U. S. Internal Revenue, together with the supplementary regulations and the series of "Treasury Decisions." DETECTION AND IDENTIFICATION OF ETHYL ALCOHOL Lieberis Iodoform Test The " iodoform test," while not distinctive, is often useful. It may be carried out as follows : To 10 cc. of the clear liquid to be tested, add 5 or 6 drops of 10 per cent solution of sodium or potassium hydroxide, heat to about 50 C., and add drop by drop with constant shaking a saturated solution of iodine in aqueous potassium iodide until the liquid becomes just per- manently yellowish brown, then carefully decolorize by adding more of the hydroxide solution, avoiding excess. If alcohol were present, iodoform gradually separates out as a yellow or yellowish white crystalline deposit. Even when very little iodoform is produced its odor can usually be recognized. While the test is quite delicate, the appearance of a precipitate of iodoform does not prove the presence of ethyl alcohol, since it may result from various other compounds, especially acetone, aldehydes, and the propyl and butyl alcohols. If the original liquid contained carbohydrate or organic acid, it should be neutralized, distilled, and the first portion of the distillate used for the test. Ethyl Dinitrobenzoate Test As a specific test for ethyl alcohol Mulliken 1 recommends the preparation of Ethyl 3, 5-Dinitrobenzoate as follows : Heat together gently in a three-inch test tube held over a small flame, 0.15 gm. of 3, 5-dinitrobenzoic acid and 0.20 gm. of phosphorus pentachloride. When signs of chemical action are seen, remove the heat for a few seconds. Then heat again, 1 Identification of Pure Organic Compounds, Vol. I, p. 168. D METHODS OF ORGANIC ANALYSIS boiling the liquefied mixture very gently for one minute. Pour out on a very small watchglass and allow to solidify. As soon as solidification occurs, remove the liquid phosphorus oxy- chloride, with which the crystalline mass is impregnated, by rubbing the latter between two small pieces of porous tile. Place the powder in a dry five- or six-inch test tube. Allow four drops of the alcohol (which must contain not more than about 10 per cent of water) to fall upon it, and then stopper the tube tightly without delay. Immerse the lower part of the test tube in water having a temperature of 75-85. Shake gently, and continue the heating for ten minutes. To purify the ester produced in the reaction, crush with a stirring rod any hard lumps which may form when the mixture cools and boil gently with 15 cc. of methyl alcohol (2 : 1) until all is dissolved, or for a minute or two. Filter boiling hot if the solution is not clear. Cool, shake and filter. Wash with 3 cc. cold methyl alcohol (2:1). Recrystallize from 9 cc. of boiling methyl alcohol (2:1). Wash with 2 cc. of the same solvent. Spread out the product on a piece of tile. Allow to become air-dry, and determine the melting point. Ethyl 3, 5-Dinitrobenzoate, the product of this test, crystal- lizes in white needles melting at 92-93 (uncorr.). The corresponding derivatives of methyl, propyl, butyl, and isobutyl alcohols melt at 107.5 (uncorr.), 73-73.5 (uncorr.), 64 (uncorr.), and 83-83.5 (uncorr.), respectively. Other Methods The methods recommended by Bacon as being especially adapted to the detection and determination of such small amounts of alcohol as may be present as the result of incipient spoilage in food material are described later in this chapter. DETERMINATION OF ETHYL ALCOHOL As already stated, the complete separation of ethyl alcohol from water is very difficult. The quantitative determination of the alcohol is therefore carried out in water solution and is usually accomplished by one of the following methods: from ALCOHOLS 7 (1) the density, (2) the index of refraction, (3) the boiling point of the solution, or (4) by quantitative oxidation of the alcohol to acetic acid by heating in acid solution with potassium dichromate. The first and second methods are seriously influenced by the presence of any substance other than the alcohol and water and are ordinarily applied only after distillation ; the third method is less influenced by dissolved solids, and for approximate work may be applied directly to ordinary alcoholic liquors, but is not so accurate as the specific gravity method; the fourth method is applicable only in the absence of any other substance capable of reacting with the acid-dichromate mixture, and is not readily adaptable to the determination of more than small amounts. Generally the most accurate and satisfactory method is to separate the alcohol from substances other than water by dis- tillation and find the amount of alcohol in the distillate by a careful determination of its specific gravity. Preparations containing chloroform, ether, or essential oils may be treated as follows : 1 Dilute 25 cc. of the sample with water to about 100 cc. in a separatory funnel, add sodium chloride to saturation and then 50 to 80 cc. of light petroleum distillate (boiling below 60). Shake vigorously for five min- utes, allow to stand for half an hour, draw off the lower layer into another separatory funnel, wash again in the same way with a small amount of petroleum ether, and then draw off into a distillation flask. Unite the petroleum ether layers, wash with two successive portions each 25 cc. of water saturated with salt, adding these washings to the main solution. The alcohol remains dissolved in the aqueous salt solution, which (after neutralizing if necessary) may be distilled as described below. Carbon dioxide if present in large amount should be removed as well as possible in advance by shaking the liquor in a large flask at room temperature and avoiding the froth when taking the portions for analysis. . l Thorpe and Holmes : J. Chem. 8oc., 1903, 83, 314. 8 METHODS OF ORGANIC ANALYSIS Unless the appearance or odor of the sample suggests the presence of carbon dioxide or of chloroform, ether, or essential oil, it is usually permissible to proceed with the determination as described below without subjecting the sample to any of the above preliminary treatment. Specific Gravity Method In the case of a fermented liquor or other sample containing less than 25 per cent of alcohol (Note 1), dry and weigh a 100 cc. graduated flask, fill to the mark with the sample and weigh (Note 2); transfer to a distilling flask of about 300 cc. ca- pacity, rinsing the measuring flask with about 50 cc. of water, bringing the total volume in the distilling flask to about 150 cc. Drop into the solution a small piece of delicate litmus paper, or better a few milligrams of solid phenolphthalein, and neutralize with a dilute solution of sodium or potassium hy- droxide in order that no volatile acid may be distilled with the alcohol (Note 3). Connect the distilling flask with a well- cooled condenser and distill, collecting the distillate in a 100 cc. flask, preferably the one used in measuring the sample. The distillate should not be unnecessarily exposed to the air but should be conducted well into the receiving flask. If the con- denser is vertical or nearly so, it is only necessary to support the flask in such a position that the condenser tube projects into the neck of the flask, otherwise an adapter may be used (Note 4). The apparatus may be arranged as shown in Fig. 1. When the distillate amounts to nearly 100 cc. remove the receiver ; and if the alcohol is to be reported in percentage by volume, the temperature should be brought to that at which the sample was measured out for analysis and water then added to bring the distillate exactly to 100 cc., but if the results are to be reported by weight, this adjustment is not important. See that the flask is clean and dry outside, weigh and subtract the weight of the empty flask as found at the beginning of the experiment. Mix the distillate well by shaking, and by means of a pyknometer (Note 5) determine carefully its specific ALCOHOLS 9 gravity to the fifth decimal place, with special attention to the accurate control of the temperature, which should be governed according to the table which is to be used in finding the per- centage of alcohol from the specific gravity (Note 6). Find in Table 1 the percentage of alcohol by weight, or in Table 2 FIG. 1. Arrangement of distillation apparatus for alcohol determination. the percentages by weight and by volume, corresponding to this specific gravity. The percentage by weight of alcohol in the distillate, multi- plied by the weight of the latter, shows the actual weight of alcohol distilled over, and this divided by the weight of sample taken gives the percentage by weight of alcohol in the liquor. The percentage by volume is of course the same for the origi- nal sample as for the distillate, if both are measured in the same flask at the same temperature. Note 1. If the sample contains more than 25 per cent of alcohol, a proportionately smaller amount than 100 cc. should be taken for the alcohol determination. Of cordials, 50 cc., and of distilled liquors or commercial alcohol, 25 cc., will usu- ally be a suitable amount. If results are to be expressed in 10 METHODS OF ORGANIC ANALYSIS percentage by weight, the weight as well as the volume of the sample taken must of course be known. Note 2. If the specific gravity of the sample, is known, or if the results of the analysis are to be expressed only in per- centage by volume or grams per 100 cc., the portion desired for the determination may be measured by means of a pipette. If, however, the same 100 cc. flask is used for measuring the sample and for the distillate and each is weighed as well as measured, the accuracy of the result whether expressed in per- centage by weight or by volume will be independent of the accuracy of calibration of this measuring flask. Note 3. Most alcoholic beverages, unless they have under- gone acetic fermentation, contain too little volatile acid to affect materially the result of the alcohol determination. It is, however, not safe to neglect the neutralization unless volatile acids are known to be absent. Note 4. A suitable arrangement for this distillation is shown in Fig. 1. The bulb in the connecting tube serves to prevent the mechanical carrying over of spray, and permits the distillation to be carried on at a fairly rapid rate, provided a sufficient stream of cold water flows through the condenser to thoroughly chill the distillate. If a distilling flask having side tube on the neck is used, the distillation should be con- ducted more slowly, in order to avoid danger from spray, and if possible a flask should be selected which has the exit tube high on the neck. If it is desired to expel the alcohol quickly, the solution may be saturated with salt before distilling. Note 5. On account of the importance of exact control of temperature, it is desirable to use either a thin-walled Sprengel- Ostwald pyknometer, which may be hung in water of the de- sired temperature for adjustment, or a pyknometer bottle of the type carrying a thermometer in the ground glass stopper and having a capillary side tube for overflow and adjustment. In either case the pyknometer is calibrated by weighing first empty and dry, then filled with recently boiled distilled water of known temperature. When using the pyknometer, care must be exercised to avoid warming it by contact with the hand. ALCOHOLS 11 \ FIG. 2. The Ostwald pyknometer. The Ostwald pyknometer (Fig. 2) has one arm (^4.) of uni- form bore, while the other arm carries a pipette body (B) and ends in a capillary tip ((7). It is ^ filled by attaching a small rubber tube at and drawing the liquid through A until the entire pyknome- ter is full, when it is suspended in water at the desired temperature, and when this is reached the adjust- ment is made by touching the point <7 with filter paper until just enough liquid is removed to bring the menis- cus in A to the graduation mark M ; then cap the tips (if caps are provided), remove and care- fully wipe the pyk- nometer, suspend it from the hook over the balance pan, and weigh. The specific gravity bottle of the form shown in Fig. 3, while not so good in principle as the Ostwald pyknometer, is more widely used in analytical laboratories. The thermometer of such a bottle should (unless it bears a "con- jf trol stamp ") be tested, by comparison with a JL thermometer known to be accurate, at the >> temperature at which it is to be used. This pyknometer should always be filled either at or slightly below the temperature at which the weighing is to be made. Fill to about the middle of the ground portion of the neck so that when the stopper is in- serted the liquid will overflow not only around the stopper, but also through the side tube. The stopper should be inserted snugly but without unnecessary force. As FIG. 3. Specific grav- ity bottle with ther- mometer in stopper. 12 METHODS OF ORGANIC ANALYSIS soon as the stopper is in place, wipe off the tip of the side tube, place the pyknometer in water (or air) maintained at the desired temperature, and notice whether any further overflow occurs. When the reading of the thermometer in the pyknom- eter and the position of the surface of the liquid at the tip of the side tube remain constant, the contents are evidently at the same temperature with the surrounding water or air. The glass cap is now placed over the tip, the pyknometer carefully wiped with a clean, dry cloth, weighed, and the weight of the empty pyknometer deducted. Note 6. The pyknometer, having been weighed with water as just described, is either dried or repeatedly rinsed with small portions of the distillate and then filled with the latter at a little below the standard temperature for the alcohol table which is to be used, then allowed to warm slowly up to the exact tem- perature desired and at exactly this temperature the volume is carefully adjusted, the pyknometer capped, wiped, and weighed as described in Note 5. To use Table 1, correct the weighings of water and of distillate by adding 0.00106 gram to each gram of water or distillate which the pyknometer apparently contains. (This corrects for the buoyant effect of the air when weighings are made with brass weights in air of average humidity at room temperature.) To simplify calculations adopt either 20 or 25 as the tempera- ture for adjusting the pyknometer both with water and with the distillate. Divide the corrected weight of distillate by the corrected weight of water, thus obtaining the specific gravity at f -j} or |f. Divide this by the density of water at 20 (0.998234) or 25 (0.997077) as the case may be to obtain the density of the distillate at -^ or %-, from which by interpola- tion in Table 1 find the percentage by weight of alcohol in the distillate to the second decimal place. If for any reason the temperature of adjustment has been be- tween 20 and 25, the density of water at the known temperature of adjustment can be found from Table 3 below and the cor- rected weight of water calculated thus to 4 can be used in find- ing the density of distillate for the temperature of adjustment. ALCOHOLS 13 TABLE 1. DENSITY OF MIXTURES OF ETHYL ALCOHOL AND WATER (BUREAU OF STANDARDS) Per cent alcohol by weight D^i D D2jf- Per cent alcohol t>y weight &f & B* 0.99913 0.99824 0.99708 50 0.917S7 0.91386 0.90983 1 .99725 .99636 .99521 51 .91566 .91164 .90758 2 .99543 .99453 .99338 52 .91344 .90940 .90533 3 .99366 .99274 .99159 53 .91120 .90715 .90307 4 .99197 .99102 .98984 54 .90895 .90488 .90079 5 .99033 .98936 .98815 55 .90670 .90262 .89851 6 .98877 .98776 - .98651 56 .90443 .90034 .89622 1 .98726 .98620 .98491 57 .90215 .89805 .89392 8 .98581 .98470 .98336 58 .89987 .89576 .89162 9 .98442 .98325 .98185 59 .89758 .89346 .88931 10 .98307 .98185 .98038 60 .89528 .89115 .88700 11 .98176 .98047 .97893 61 .89297 .88883 .88467 12 .98049 .97913 .97752 62 .89066 .88651 .88234 13 .97925 .97781 .97612 63 .88834 .88418 .88000 14 .97803 .97651 .97474 64 .88601 .88185 .87766 15 .97683 .97522 .97336 65 .88368 .87950 .87530 16 .97563 .97393 .97199 66 .88134 .87716 .87295 17 .97444 .97264 .97061 67 .87899 .87480 .87058 18 .97324 .97134 .96922 68 .87664 .87244 .86821 19 .97203 .97003 .96782 69 .87428 .87008 .86583 20 .97080 .96870 .96640 70 .87192 .86770 .86344 21 .96956 .96736 .96497 71 .86954 .86532 .86105 22 .96829 .96599 .96352 72 .86716 .86292 .85864 23 .96699 .96459 .96203 73 .86477 .86052 .85622 24 .96566 .96317 .96052 74 .86237 .85812 .85380 25 .96430 .96171 .95897 75 .85997 .85570 .85137 26 .96289 .96021 .95739 . 76 .85755 .85328 .84893 2T .96145 .95868 .95577 77 .85513 .85084 .84648 28 .95997 .95711 .95412 78 .85270 .84840 .84403 29 .95845 .95550 .95244 79 .85026 .84595 .84157 30 .95688 .95385 .95071 80 .84781 .84349 .83909 31 .95526 .95215 .94894 81 .84534 .84101 .88660 82 k .95360 .95042 .94713 82 .84286 .83852 .83410 88 .95191 .94865 .94529 83 .84037 .83602 .83159 34 .95017 .94684 .94342 84 .83786 .83350 .82906 35 .94839 .94499 .94152 85 .83534 .83097 .82652 36 .94657 .94311 .93957 86 .83279 .82842 .82396 37 .94471 .94119 .93760 87 .83022 .82583 .82137 38 .94282 .93924 .93560 88 .82762 .82323 .81876 39 .94089 .93725 .93356 89 .82500 .82060 .81613 40 .93893 .93524 .93151 90 .82235 .81795 .81348 41 .93694 .93320 .92943 91 .81966 .81527 .81080 42 .93491 .93113 .92732 92 .81694 .81255 .80809 43 .93286 .92904 .92519 93 .81418 .80979 .80534 44 ,93078 .92693 .92305 94 .81138 .80700 .80256 45 .92868 .92480 .92088 95 .80854 .80417 .79974 46 .92655 .92264 .91870 96 .80564 .80129 .79689 47 .92441 .92047 .91650 97 .80271 .79838 .79400 48 .92225 .91828 .91429 98 .79972 .79541 .79106 49 .92006 .91608 .91207 99 .79668 .79240 .78809 50 .91787 .91386 .90983 100 .79358 .78933 .78507 = Density at 15 C. referred to water at 4 C. 14 METHODS OF ORGANIC ANALYSIS Suppose this is found to be 0.98123 at -^-; by comparing the figures in Table 1 for ^- and %- at about this density it will be seen that the decrease of density in alcohol of this strength is about 0.000294 for each 1 C. between 20 and 25. Hence by adding 3 x 0.000294 to 0.98123 (the density at -\ 3 -) we obtain 0.98211 as the density at -^j -, which is found from Table 1 to correspond to 9.81 per cent of alcohol by weight. The interpo- lation for temperature is apt to introduce a slight error, for which reason as well as to simplify calculation it is preferable to adjust the pyknometer always at 20 or 25 if practicable. To use Table 2 find the weight of distillate which the pyk- nometer holds at 15.56 and divide by the weight of water which it holds at the same temperature. In this case the weighings are not corrected for displaced air (since this correc- tion was apparently not made in determining the data from which the table was constructed), but it is necessary to exer- cise great care in the control of the temperature, since 15.56 is much below the usual working temperature of the laboratory. The water content of the pyknometer may be observed at a higher temperature and calculated to 15.56 from the data in Table 3 ; but since with rising temperature alcohol expands at an uneven rate the adjustment of the pyknometer with the dis- tillate must be at or near the standard temperature, though if only a few degrees from standard the temperature may be taken, the weight divided by the weight of water at 15.56 to find the " apparent specific gravity," then correct this by the following empirical formula given by Allen, 1 in which D is the specific gravity at 15.56, D f the "apparent specific gravity," and d the difference (in degrees centigrade) between 15.56 and the temperature at which D f was determined : D = D' d (o. 00014 + The correction is added to or subtracted from D r according as the pyknometer was filled and adjusted at a temperature above or below the standard. 1 Commercial Organic Analysis, Vol. I (3d Ed.) p. 93. ALCOHOLS 15 TABLE 2. PERCENTAGE OF ALCOHOL BY WEIGHT AND BY VOLUME [Recalculated from the determinations of Gilpin, Drinkwater, and Squibb, by EDGAR RICHARDS.] Specific gravity at|8'F. Per cent alcohol by volume Per cent alcohol by weight Specific gravity at eg F. Per cent alcohol by volume Per cent alcohol by weight Specific gravity atf8F. Per cent alcohol by volume Per cent alcohol by weight . 1.00000 0.00 0.00 0.99281 5.00 4.00 0.98660 10.00 8.04 0.99984 .10 .08 268 .10 .08 649 .10 .12 968 .20 .16 255 .20 .16 637 .20 .20 953 .30 .24 241 .30 .24 626 .30 .29 937 .40 .32 228 .40 .32 614 .40 .37 .99923 0.50 0.40 .99215 5.50 4.40 .98603 10.50 8.45 907 .60 .48 202 .60 .48 592 .60 .53 892 .70 .56 '189 .70 .56 580 .70 .61 877 .80 .64 175 .80 .64 569 .80 .70 861 .90 .71 162 .90 .72 557 .90 .78 .99849 1.00 0.79 .99149 6.00 4.80 .98546 11.00 8.86 834 .10 .87 136 .10 .88 535 .10 .94 819 .20 .95 123 .20 .96 524 .20 9.02 805 .30 1.03 111 .30 5.05 513 .30 .11 790 .40 .11 098 .40 .13 502 .40 .19 .99775 1.50 1.19 .99085 6.50 5.21 .98491 11.50 9.27 760 .60 .27 072 .60 .29 479 .60 .35 745 .70 .35 059 .70 .37 468 .70 .43 731 .80 .43 047 .80 .45 457 .80 .51 716 .90 .51 034 .90 .53 446 .90 .59 .99701 2.00 1.59 .99021 7.00 5.61 .98435 12.00 9.67 687 .10 .67 009 .10 .69 424 .10 .75 672 .20 .75 .98996 .20 .77 413 .20 .83 658 .30 .83 984 .30 .86 402 .30 .92 643 .40 .91 971 .40 .94 391 .40 10.00 .99629 2.50 1.99 .98959 7.50 6.02 .98381 12.50 10.08 615 .60- 2.07 947 .60 .10 370 .60 .16 600 .70 .15 934 .70 .18 359 .70 .24 586 .80 .23 922 .80 .26 348 .80 .33 571 .90 .31 909 .90 .34 337 .90 .41 .99557 3.00 2.39 .98897 8.00 6.42 .98326. 13.00 10.49 543 .10 .47 885 .10 .50 315 .10 .57 529 .20 .55 873 .20 .58 305 .20 .65 515 .30 .64 861 .30 .67 294 .30 .74 501 .40 .72 849 .40 .75 283 .40 .82 .99487 3.50 2.80 .98837 8.50 6.83 .98273 13.50 10.90 473 .60 .88 825 .60 .91 262 .60 .98 459 .70 .96 813 .70 .99 251 .70 11.06 445 .80 3.04 801 .80 7.07 240 .80 .15 431 .90 .12 789 .90 .15 230 .90 .23 .99417 4.00 3.20 .98777 9.00 7.23 .98219 14.00 11.31 403 .10 .28 765 .10 .31 209 .10 .39 390 .20 .36 754 20 .39 198 .20 .47 376 .30 .44 742 .30 .48 188 .30 .56 363 .40 .52 730 .40 .56 177 .40 .64 .99349 4.50 3.60 .98719 9.50 7.64 .98167 14.50 11.72 335 .60 .68 707 .60 .72 156 .60 .80 322 .70 .76 695 .70 .80 146 .70 .88 308 .80 .84 683 .80 .88 135 .80 .97 295 .90 .92 672 .90 .96 125 .90 12.05 16 METHODS OF ORGANIC ANALYSIS PERCENTAGE OF ALCOHOL BY WEIGHT AND BY VOLUME. Continued. [Recalculated from the determinations of Gilpin, Drinkwater, and Squibb, by EDGAR RICHARDS.] , ^ *3 te ^ "S _>> -3 "o c . o 0> ~ i , | '!> - u lj 8 3a 2 f It II *" "3 1| +> <3/ It s a 03 s ** "3 !| tt "3 2 O te o SS a ? C Jj o wo o S c til w >> 1 K > ft s Bb 04 h S Z- fe- 1" P, &* IH ^ 3 OQ PH HH OQ Pi 02 PH 0.98114 15.00 12.13 0.97608 20.00 16.26 0.97097 25.00 20.43 104 .10 .21 598 .10 .34 086 .10 .51 093 .20 .29 588 .20 .42 076 .20 .60 083 .30 .38 578 .30 .51 065 .30 X .68 073 .40 .46 568 .40 .59 055 .40 .77 .98063 15.50 12.54 .97558 20.50 16.67 .97044 25.50 20.85 052 .60 .62 547 .60 .75 033 .60 .93 042 .70 .70 537 .70 .84 023 .70 21.02 032 .80 .79 527 .80 .92 012 .80 .10 021 .90 .87 517 .90 17.01 001 .90 .19 .98011 16.00 12.95 .97507 21.00 17.09 .96991 26.00 21.27 001 .10 13.03 497 .10 .17 980 .10 .35 .97991 .20 .12 487 .20 .26 969 .20 .44 980 .30 .20 477 .30 .34 959 .30 .52 970 .40 .29 467 .40 .43 949 .40 .61 .97960 16.50 13.37 .97457 21.50 17.51 .96937 26.50 21.69 950 .60 .45 446 .60 .59 926 .60 .77 940 .70 .53 436 .70 .67 915 .70 .86 929 .80 .62 426 .80 .76 905 .80 .94 919 .90 .70 416 .90 .84 894 .90 22.03 .97909 17.00 13.78 .97406 22.00 17.92 .96883 27.00 22.11 899 .10 .86 396 .10 18.00 872 .10 .20 889 .20 .94 386 .20 .09 861 .20 .28 879 .30 14.03 375 .30 .17 850 .30 .37 869 .40 .11 365 .40 .26 839 .40 .45 .97859 17.50 14.19 .97355 22.50 18.34 .96828 27.50 22.54 848 .60 .27 345 .60 .42 816 .60 .62 838 .70 .35 335 .70 .51 805 .70 .71 828 .80 .44 324 .80 .59 794 .80 .79 818 .90 .52 314 .90 .68 783 .90 .88 .97808 18.00 14.60 .97304 23.00 18.76 .96772 28.00 22.96 798 .10 .68 294 .10 .84 761 .10 23.04 788 .20 .77 283 .20 .92 749 .20 .13 778 .30 .85 273 .30 19.01 738 .30 .21 768 .40 .94 263 .40 .09 726 .40 .30 .97758 18.50 15.02 .97253 23.50 19.17 .96715 28.50 23.38 748 .60 .10 242 .60 .25 704 .60 .47 738 .70 .18 232 .70 .34 692 .70 .55 728 .80 .27 222 .80 .42 681 .80 .64 718 .90 .38 211 .90 .51 669 .90 .72 .97708 19.00 15.43 .97201 24.00 19.59 .96658 29.00 23.81 698 .10 .51 191 .10 .67 646 .10 .89 688 .20 .59 180 .20 .76 635 .20 .98 678 .30 .68 170 .30 .84 623 .30 24.06 668 .40 .76 159 .40 .93 611 .40 .15 .97658 19.50 15.84 .97149 24.50 20.01 .96600 29.50 24.23 648 .60 .93 139 .60 .09 587 .60 .32 638 .70 16.01 128 .70 .18 576 .70 .40 628 .80 .09 118 .80 .2(5 564 .80 .49 618 .90 .18 107 .90 .35 553 .90 .57 ALCOHOLS 17 In the writer's laboratory this formula has given fairly satis- factory results when applied to temperatures differing but little from the standard. The actual weighing of the pyknometer must always be made at a temperature above the " dew point," but a pyknometer which provides for expansion of its contents without loss may be filled and adjusted at the standard tem- perature and then allowed to stand until it reaches room temperature before weighing. TABLES. DENSITY OF PURE WATER FREE FROM AIR l Temperature Density Temperature Density 4C. 1.0000000 21 C. 0.9980233 15 0.9991266 22 0.9978019 15.56 0.9990415 23 0.9975702 16 0.9989705 24 0.9973286 17 0.9988029 25 0.9970770 18 0.9986244 26 0.9968158 19 0.9984347 27 0.9965451 20 0.9982343 28 0.9962652 Refractometer Method In solutions containing only alcohol and water, such as the distillates obtained in the specific gravity method above de- scribed, the percentage of alcohol can be found from the index of refraction as well as from the specific gravity. The recently introduced " immersion refractometer " is the most convenient form of apparatus for this purpose. Figure 4 shows the appara- tus complete in position for an observation, while Fig. 5 shows the positions of the principal parts. The liquid to be examined is placed in a small beaker sur- rounded by water of the required temperature, usually 17.5 or 20, and the refractometer is suspended from the wire frame 1 According to Chappius (Bureau international des Poids et Mesures, Travaux et Me"moires XIII, 1907). The data given above are taken from the table pub- lished by the Bureau of Standards, which shows density for each tenth of a degree from to 41 C., referred to water at 4 C. as unity, c 18 METHODS OF ORGANIC ANALYSIS in such a position that the prism is immersed in the liquid to be observed. By means of a mirror the light from a Avindow is reflected through the glass bottom of the water bath and upward through the refractometer. On looking downward through the ocular Oc and the telescope of the refractometer FIG. 4. General view of immersion refractometer. (Courtesy of Eimer and Amend.) one observes the border line of total reflection, the upper part of the field of vision being light and the lower part shaded. A scale, marked in degrees of arbitrary but known and con- stant value, extends from top to bottom of the field of vision, and the position of the border of the shadow upon this scale indicates the index of refraction. The index of refraction cor- ALCOHOLS 19 responding to each degree of the immersion refractometer scale is shown in Table 4. In using this refractometer, after everything is in position it should be allowed to stand ten minutes before taking obser- vations in order to insure uniformity of temperature. The FIG. 5. Sectional view of immersion refractometer. (Courtesy of Eimer and Amend.) adjustment of the instrument should first be tested by taking reading on distilled water. The index at R should stand at 5 and the ocular Oo should be focused so that the edge of the shadow is clearly marked. If when the micrometer screw Z stands at zero the line lies between two of the scale degrees, its 20 METHODS OF ORGANIC ANALYSIS TABLE 4. INDEX OF REFRACTION FOR EACH DEGREE ON SCALE OF THE IMMERSION REFRACTOMETER Scale reading Index of refraction Scale reading Index of refraction Scale reading Index of refraction -5 1.32539 32 1.33972 69 1.35352 -4 1.32578 33 1.34010 70 1.35388 -3 1.32618 34 1.34048 71 1.35425 -2 1.32657 35 1.34086 72 1.35461 -1 1.32696 36 1.34124 73 1.35497 1.32736 37 1.34162 74 1.35533 1 1.32775 38 1.34199 75 1.35569 2 1.32814 39 1.34237 76 1.35606 3 1.32854 40 1.34275 77 1.35642 4 1.32893 41 1.34313 78 1.35678 5 1.32932 42 1.34350 79 1.35714 6 1.32971 43 1.34388 80 1.35750 7 1.33010 44 1.34426 81 1.35786 8 1.33049 45 1.34463 82 1.35822 9 1.33087 46 1.34500 83 1.35858 10 1.33126 47 1.34537 84 1.35894 11 1.33165 48 1.34575 85 1.35930 12 1.33204 49 1.34612 86 1.35966 13 1.33242 50 1.34650 87 1.36002 14 1.33281 51 1.34687 88 1.36038 15 1.33320 52 1.34724 89 1.36074 16 1.33358 53 1.34761 90 1.36109 17 1.33397 54 1.34798 91 1.36145 18 1.33435 55 1.34836 92 1.36181 19 1.33474 56 1.34873 93 1.36217 20 1.33513 57 1.34910 94 1.36252 21 1.33551 58 1.34947 95 1.36287 22 1.33590 59 1.34984 96 1.36323 23 1.33628 60 1.35021 97 1.36359 24 1.33667 61 1.35058 98 1.36:394 25 1.33705 62 1.35095 99 1.36429 26 1.33743 63 1.35132 100 1.36464 27 1.33781 64 1.35169 101 1.36500 28 1.33820 65 1.35205 102 1.36535 29 1.33858 66 1.35242 103 1.36570 30 1.33896 67 1.35279 104 1.36605 31 1.33934 68 1.35316 105 1.36640 ALCOHOLS 21 position may be estimated in tenths of a degree by the eye or the micrometer screw Z may be turned until the line of the shadow comes exactly to a scale division. . The latter then in- dicates the whole degrees and the tenths are read from the scale on the micrometer screw. This method is usually more accurate than estimating the tenths by the eye alone, and has the advantage that one may quickly turn the micrometer screw back to zero and repeat the operation as often as desired, finally averaging the readings. With water the reading, depending upon the temperature, should be as follows : Temperature C. Scale reading 17.5 15.0 20 14.5 21 14.25 22 14.0 23 13.75 24 13.5 25 13.25 26 13.0 27 12.7 28 12.4 29 12.1 30 11.8 Having thus tested the adjustment of the instrument by readings in water, the readings in the alcoholic solution are taken in the same way. The percentages by weight of alcohol corresponding to the scale readings at 17.5 as found by Acker- niann and Steinmann l are as follows : TABLE 5. PERCENTAGES BY WEIGHT OF ALCOHOL CORRESPONDING TO SCALE READINGS OF ZEISS IMMERSION REFRACTOMETER AT 17.5 C. (ACKERMANN AND STEINMANN) Scale reading Alcohol per cent Scale reading Alcohol per cent 15.0 0.00 19.5 2.80 15.5 0.32 20.0 3.10 16.0 0.64 20.5 3.38 16.5 0.95 21.0 3.67 17.0 1.25 21.5 3.96 17.5 1.57 22.0 4.22 18.0 1.87 22.5 4.49 18.5 2.19 23.0 4.76 19.0 2.49 23.5 5.02 For tables extending to higher percentages of alcohol (some- times expressed as percentage by volume or grams per 100 cc.), 1 Ztschr. f. der gesamte Brauwesen, 28 (1905). 22 METHODS OF ORGANIC ANALYSIS see papers by Wagner, Wagner and Schultze, arid Doroshevski and Dvorzhanchik among references given at the end of this chapter. The table given by the last-named authors is particu- larly noteworthy, since it gives the actual index of refraction for alternate percentages of alcohol from to 100 and for the temperatures 17.5, 20, 22, 24 C. For a fuller description of the immersion refractometer see Leach's Food Inspection and Analysis (latest edition) or the circulars furnished by the manufacturer of the instrument. Boiling Point Method In mixtures of alcohol and water containing no appreciable amount of other volatile substances and only small quantities of dissolved solids, the difference between the boiling point of the mixture and that of pure water under the same conditions gives a measure of the percentage of alcohol present. For the rapid determination of alcohol on this principle, several forms of ebullioscope have been devised. The liquid to be tested is boiled under a reflux condenser while a thermometer bulb is fixed just above the surface of the liquid so as to be entirely surrounded by the vapor. The more common technical forms, such as those of Pohl and Kappeller, have scales reading per- centage of alcohol instead of thermometer scales. Water is first boiled in the apparatus and the scale adjusted so that the mercury stands at zero. If then the water be removed and the sample introduced and brought to boiling under the same baro- metric conditions, the point reached by the mercury column shows the amount of alcohol present. Wiley 1 uses a delicate differential thermometer with an apparatus similar to that employed for the determination of molecular weights by the boiling point method. Up to five per cent of alcohol, the depression of the boiling point is said to be so regular that the results are entirely satisfactory for practical work. In the ebullioscopes bearing scales graduated in terms of alcohol, the variations in the boiling point curve at 1 J. Am. Chem. Soc., 1896, 18, 1063. ALCOHOLS 23 the higher percentages are, of course, allowed for. The boiling point method is very rapid and gives results sufficiently accu- rate for many purposes. Reference may be made to Vaubel x for a general discussion of methods based on the determination of the boiling point and to Freyer 2 for experimental results on the influence of dissolved solids in the ebullioscopic determina- tion of alcohol. Oxidation Method Under suitable conditions ethyl alcohol can be quantitatively oxidized to acetic acid by means 'of potassium dichromate in the presence of sulphuric acid. The amount of alcohol can then be ascertained either by determining the amount of dichro- mate reduced 3 or by distilling and titrating the acetic acid formed. 4 The conditions of oxidation must be carefully regu- lated, and as a rule the method is used only for the determina- tion of very small amounts of alcohol, the specific gravity method being preferable for the examination of any but very dilute solutions. The alcohol must of course be separated by distillation from any other oxidizable matter before the oxida- tion method can be applied. A comparison of the results ob- tained by oxidation with those shown by the specific gravity method may be useful in demonstrating the presence of homol- ogous alcohols. DETERMINATION AND IDENTIFICATION OF SMALL AMOUNTS OF ALCOHOL This subject has been studied by Bacon 5 with special refer- ence to its application in demonstrating alcoholic fermentation in food products. Bacon recommends that the alcohol be con- centrated by distillation after addition of salt (which as noted above results in the alcohol being removed with a smaller 1 Quantitative Bestimmung organischer Verbindungen. Berlin, 1902. 2 Z. angew. Chem., 1896, 654. 3 Hehner: Analyst, 1887, 12, 25. Benedict and Norris : J. Am. Chem. Soc., 1898, 20, 293. * r>upre : J. Chem. Soc., 1867, 20, 496. 5 U. S. Dept. Agriculture, Bureau of Chemistry, Circular No. 74. 24 METHODS OF ORGANIC ANALYSIS amount of water) after which the distillate is examined as to density and index of refraction as well as by chemical tests to 'demonstrate the presence of alcohol. In a typical experiment given by Bacon, 1000 cc. of a 0.1 per cent solution of alcohol were three fourths saturated with salt, 150 cc. distilled off, this again three fourths saturated with salt and 25 cc. distilled off. This distillate showed at 17.5 a refractometer reading of 21.3 corresponding to 3.84 per cent of alcohol in the dis- tillate or 0.096 per cent in the original sample, a recovery of 96 per cent of the amount present. Bacon considers that if the refractometer reading and the specific gravity indicate the same percentage of alcohol it is almost a certainty that it is ethyl alcohol which is present, and that the substance under examination contains the percentage of eth}d alcohol equivalent to these constants. As a further demonstration Bacon recommends that the solution containing the alcohol be treated with a slight excess of paranitrobenzoyl chloride and an equivalent amount of sodium hydroxide and the mixture shaken until the odor of the acid chloride disap- pears, when the crystalline ester (readily identified by its melt- ing point of 57 C. 1 ) may be collected and weighed. The yield is stated to be 70 to 90 per cent when working with small quantities of alcohol. If the paranitrobenzoyl chloride is not available, Bacon suggests benzoyl chloride, which when used in the same way yields the benzoic acid ethyl ester (ethyl benzoate), which may be weighed (yield said to be nearly quantitative) and identified by its odor and its boiling point, 212 C., using for the determination of the latter the method of Smith and Menzies 2 ; and that in addition the iodoform reaction be applied since the only benzoic ester having an odor similar to ethyl benzoate is the methyl ester, and methyl alcohol does not give the iodoform reaction. DETECTION AND DETERMINATION OF METHYL ALCOHOL The great difference in price between denatured alcohol and alcohol which has been subject to internal revenue tax some- 1 Ber., 1905, 38, 620. 2 J. Am. Chem. Soc., 1910, 32, 897. ALCOHOLS 25 times results in the substitution of denatured alcohol in cases where only ethyl alcohol should be used. This is detected by demonstrating the presence of methyl alcohol, but since dena- tured alcohol contains only one part of methyl to ten parts of ethyl alcohol, it is evident that methods to be useful for this purpose must be applicable to the detection of relatively small amounts of methyl alcohol in the presence of relatively large amounts of ethyl alcohol. Many methods have been proposed for this purpose. Those which probably have been most generally used are based upon the oxidation of methyl alcohol to formaldehyde and the detec- tion of the latter by one of the methods described in the next chapter. In the methods of Mulliken and Scudder 1 and of the U. S. Pharmacopoeia (Edition of 1905, p. 34) the oxidation of methyl alcohol to formaldehyde is accomplished by means of a copper spiral which is heated until covered with oxide and then plunged into the liquid, the copper being reduced and the alcohol partially oxidized. Yorisek 2 prefers to oxidize by means of chromic acid; and Bacon (loc. cit.) recommends that 5 to 8 grams of chromic acid be added to 100 cc. of the aqueous methyl alcohol in a 200 cc. distilling flask and the first 10 cc. of the distillate be tested for formaldehyde. The results of such oxidation methods must be interpreted with caution, since several observers have reported formaldehyde among the prod- ucts of oxidation of ethyl alcohol. The Association of Official Agricultural Chemists have pro- visionally adopted the methods of Trillat and of Riche and Bardy. Trillat's method 3 is based upon the observation that the products of oxidation of ethyl and methyl alcohol combine with dimethyl aniline to form bases which differ in their color reactions. The method of Riche and Bardy 4 depends upon the formation of methyl aniline violet. 1 Am. Chem. J., 1900, 24, 444. 2 J. Soc. Chem. Ind., 1909, 28, 823. 3 Compt. rend., 1898, 127, 232. Ann. chim. anal, 1899, 4, 42. J. pharm. chim., 1899, 9, 372. Analyst, 24, 13, 211, 212. Leach's Food Inspec- tion and Analysis, 2d Ed., p. 750. U. S. Dept. Agr., Bur. Chem., Bui. 107, p. 99. 4 Compt. rend., 1875, 80, 1076. Allen's Commercial Organic Analysis, 4th Ed., Vol. I, p. 98. Leach's Eood Inspection and Analysis, 2d Ed., p. 751. U. S. Dept. Agr., Bur. Chem., Bui. 107, p. 99. 26 METHODS OF ORGANIC ANALYSIS The Method of Leach and Lythgoe^- depends upon the fact that although ethyl and methyl alcohol in solutions have very similar densities, they differ considerably in their indices of refraction. This difference in properties is utilized both in detecting the presence of methyl alcohol and in estimating its amount as follows : Submit the alcoholic distillate obtained in the determination of alcohol to refraction with the immersion refractometer at exactly 20 C. and note the reading. If on reference to the table the refraction shows the percentage of alcohol agreeing with that obtained from the specific gravity in the regular manner, it may safely be assumed that no methyl alcohol is present. If, however, there is an appreciable amount of methyl alcohol, the low refractometer reading will at once in- dicate the fact. Addition of methyl to ethyl alcohol decreases the refraction in direct proportion to the amount present ; hence the quan- titative calculation is readily made by interpolation in the table, using the figures for pure ethyl and methyl alcohol of the same alcoholic strength as the sample. Example : Suppose the distillate from a vanilla extract made up to the original volume of the measured portion taken for the alcohol determination has a specific gravity of 0.97350, corresponding to 18.38 per cent alcohol by weight, and has a refraction of 35.8 on the immersion refractometer at 20. By interpolation in the refractometer table the readings of ethyl and methyl alcohol corresponding to 18.38 per cent alcohol are 47.2 and 25.4, respectively, the difference being 21.8 ; 47.2- 35.8 = 11.4 ; (11. 4 -21.8)100 = 52.3, showing that 52.3 per cent of the alcohol present is methyl. The Method of Thorpe and Holmes 2 is probably the best for the quantitative analysis of mixtures of ethyl and methyl alcohols in cases in which the immersion refractometer is not available. It depends upon the oxidation of the mixture of 1 J. Am. Chem. Soc., 1905, 27, 964. U. S. Dept. Agr., Bur. Chem., Cir. 29 and Bui. 107. 2 J. Chem. Soc., 1904, 85, 1. ALCOHOLS 27 TABLE 6. SCALE READINGS ON ZEISS IMMERSION REFRACTOMETER AT 20 C., CORRESPONDING TO EACH PER CENT BY WEIGHT OF ETHYL AND METHYL ALCOHOLS (LEACH AND LYTHGOE) 4J f <=- ** III PHa,0 Scale readings Per cent alcohol by weight Scale readings lit ,?.(>> H C3 ,O Scale readings Per cent alcohol bv weight Scale readings Methyl alcohol Ethyl alcohol Methyl alcohol Ethyl alcohol Methyl alcohol Ethyl alcohol Methyl alcohol Ethyl alcohol 14.5 14.5 26 30.3 61.9 51 39.7 91.1 76 29.0 101.0 1 14.8 16.0 27 30.9 63.7 52 39.6 91.8 77 28.3 100.9 2 15.4 17.6 28 31.6 65.5 53 39.6 92.4 78 27.6 100.9 3 16.0 19.1 29 32.2 67.2 54 39.5 93.0 79 26.8 100.8 4: 16.6 20.7 30 32.8 69.0 55 39.4 93.6 80 26.0 100.7 5 17.2 22.3 31 33.5 70.4 56 39.2 94.1 81 25.1 100.6 6 17.8 24.1 32 34.1 71.7 57 39.0 94.7 82 24.3 100.5 7 18.4 25.9 33 34.7 73.1 58 38.6 95.2 83 23.6 100.4 8 19.0 27.8 34 35.2 74.4 59 38.3 95.7 84 22.8 100.3 9 19.6 29.6 35 35.8 75.8 60 37.9 96.2 85 21.8 100.1 10 20.2 31.4 36 36.3 76.9 61 37.5 96.7 86 20.8 99.8 11 20.8 33.2 37 36.8 78.0 62 37.0 97.1 87 19.7 99.5 12 21.4 35.0 38 37.3 79.1 63 36.5 97.5 88 18.6 99.2 13 22.0 36.9 39 37.7 80.2 64 36.0 98.0 89 17.3 98.9 14 22.6 38.7 40 38.1 81.3 65 35.5 98.3 90 16.1 98.6 15 23.2 40.5 41 38.4 82.3 66 35.0 98.7 91 14.9 98.3 16 23.9 42.5 42 38.8 83.3 67 34.5 99.1 92 13.7 97.8 17 24.5 44.5 43 39.2 84.2 68 34.0 99.4 93 12.4 97.2 18 25.2 46.5 44 39.3 85.2 69 33.5 99.7 94 11.0 96.4 19 25.8 48.5 45 39.4 86.2 70 33.0 100.0 95 9.6 95.7 20 26.5 50.5 46 39.5 87.0 71 32.3 100.2 96 8.2 94.9 21 27.1 52.4 47 39.6 87.8 72 31.7 100.4 97 6.7 94.0 22 27.8 54.3 48 39.7 88.7 73 31.1 100.6 98 5.1 93.0 23 28.4 56.3 49 39.8 89.5 74 30.4 100.8 99 3.5 92.0 24 29.1 58.2 50 39.8 90.3 75 29.7 101.0 100 2.0 91.0 25 29-.7 60.1 ethyl and methyl alcohols under such conditions that the former is converted into acetic acid while the latter is com- pletely oxidized to carbon dioxide and water. The total amount of alcohols (estimated as ethyl alcohol) having been 28 METHODS OF ORGANIC ANALYSIS determined by distillation and specific gravity, a part of the distillate is mixed with water in such proportions that 50 cc. of the mixture shall contain not more than 1 gram of methyl alcohol nor more than 4 grams of ethyl and methyl alcohols together. Fifty cubic centimeters of this mixture are intro- duced into a 300-cc. flask having a tight stopper and fitted with a funnel and side tube, 20 grams of potassium dichromate and 80 cc. of dilute sulphuric acid (1 : 4) added, and the mix- ture allowed to remain for 18 hours. A further quantity of 10 grams of potassium dichromate and 50 cc. of sulphuric acid mixed with an equal volume of water are now added, and the Contents of the flask heated to the boiling point for about 10 minutes, the evolved carbon dioxide being swept out of the apparatus by a current of air and collected in weighed soda- lime tubes. Under these conditions each gram of ethyl alco- hol was found to yield about 0.01 gram of carbon dioxide. The remaining carbon dioxide found is calculated as being derived from the complete oxidation of methyl aclohol. DETERMINATION OF AMYL ALCOHOLS OR FUSEL OIL The amyl alcohols are the principal constituents of fusel oil, and most of the methods proposed for the determination of fusel oil in distilled liquors are essentially attempts to estimate the amyl alcohols. In order to separate the amyl alcohols from the relatively large amounts of ethyl alcohol ordinarily present, advantage is taken of the fact that the former are much more soluble in chloroform or carbon tetrachloride than is the latter, so that on shaking a small amount of chloroform or carbon tetrachloride with a distillate containing ethyl and amyl alcohols practically all of the amyl alcohols and only a little of the ethyl alcohol is extracted by the chloroform or the carbon tetrachloride. The amount of amyl alcohols or of fusel oil is usually esti- mated either: 1. By extracting under fixed conditions with an accurately known volume of chloroform and estimating the fusel oil from ALCOHOLS 29 the increase in volume of the chloroform layer (Roese's method). 2. By extracting with carbon tetrachloride, oxidizing the alcohols of the extracted fusel oil to the corresponding acids by means of potassium dichromate, and distilling and titrating the acids thus formed (Allen-Marquardt method). Both of these methods have been adopted provisionally by the Association of Official Agricultural Chemists and are given in full on pp. 97-98, Bui. 107 (Revised), Bureau of Chemistry, U. S. Dept. Agriculture. OFFICIAL REQUIREMENTS AS TO PURITY Ethyl Alcohol The U. S. Pharmacopoeia in addition to the requirements above given and a test which is supposed to show methyl alcohol if more than two per cent is present, prescribes the fol- lowing tests of purity for alcohol : It should not affect the color of blue or red litmus paper previously moistened with water. If 50 cc. of alcohol be evaporated in a clean vessel, no color or weighable residue should remain. If 10 cc. of alcohol be mixed with 5 cc. of water and 1 cc. of glycerin, and the mixture allowed to evaporate spontaneously from a piece of clean, odorless blotting paper, no foreign odor should become perceptible when the last traces of the alcohol leave the paper (absence of fusel oil constitu- ents) . If 25 cc. be allowed to evaporate spontaneously in a porcelain evaporat- ing dish, carefully protected from dust, until the surface of the dish is barely moist, no red or brown color should be produced upon the addition of a few drops of colorless, concentrated sulphuric acid (absence of amyl alcohol, or non-volatile carbonizable, organic impurities, etc.). If 10 cc. of alcohol be mixed in a test tube with 5 cc. of potassium hydroxide test solution, the liquid should not at once assume a yellow color (absence of aldehyde or oak tannin). If 20 cc. of alcohol be shaken in a clean glass-stoppered vial with 1 cc. of silver nitrate test solution, the mixture should not become more than faintly opalescent, nor acquire more than a faint brownish tint when exposed for six hours to diffused daylight (limit of organic impurities, amyl alcohol, aldehyde, etc.). 30 METHODS OF ORGANIC ANALYSIS Methyl Alcohol The following requirements have been established by the Bureau of Internal Revenue for methyl alcohol to be used in denaturing grain alcohol : The methyl alcohol submitted must be partially purified wood alcohol obtained by the destructive distillation of wood. It must conform to the following analytical requirements : 1. Color. This shall not be darker than that produced by a freshly pre- pared solution of 2 cc. of tenth-normal iodine diluted to 1000 cc. with distilled water. 2. Specific Gravity. It must have a specific gravity of not more than 0.830 at 60 F. (15.56 C.), corresponding to 91 of Tralles' scale. 3. Boiling Point. One hundred cubic centimeters slowly heated in a flask under conditions as described below must give a distillate of not less than 90 cc. at a temperature not exceeding 75 C. at the normal pressure of the barometer (760 mm.) . One hundred cc. of wood spirit are run into a short- necked copper flask of about 180-200 cc. capacity and the flask placed on an asbestos plate having a circular opening of 30 mm. diameter. In the neck of this flask is fitted a fractionating tube 12 mm. wide and 170 mm. long, with a bulb just 1 centimeter below the side tube which is connected with a Lie- big's condenser having a water jacket not less than 400 mm. long. In the upper opening of the fractionating tube is placed a standardized thermome- ter, so adjusted that its mercury bulb comes in the center of the bulb. The distillation is conducted in such a manner that 5 cc. pass over in one minute. The distillate is run into a graduated cylinder, and when the temperature of 75 C. has been reached at the normal barometric pressure of 760 mm. at least 90 cc. shall have been collected. Should the barometer vary from 760 mm. during the distillation, 1 C. shall be allowed for every variation of 30 mm. For example, at 770 mm. 90 cc. should have distilled at 75.3 C., and at 750 mm. 90 cc. should have distilled at 74.7 C. 4. Miscibility with Water. It must give a clear or only slightly opales- cent solution when mixed with twice its volume of water. 5. Acetone Content. It must contain not more than 25 nor less than 15 grams per 100 cc. of acetone and other substances estimated as acetone when tested by the following method (Messinger). One cubic centimeter of a mixture of 10 cc. wood alcohol with 90 cc. of water is treated with 10 cc. of double normal soda solution. Then 50 cc. of tenth-normal iodine solution are added while shaking, and the mixture made acid with dilute sulphuric acid three minutes after the addition of the iodine. The excess of iodine is titrated back with tenth-normal sodium thiosulphate solution, using a few drops of starch solution for an indicator. ALCOHOLS 31 From 15.5 to 25.8 cc. of tenth-normal iodine solution should be used by the spirit. The solution should be kept at a temperature between 15 and 20 C. Calculation : x = grams of acetone in 100 cc. of spirit. y = number of cubic centimeters of tenth-normal iodine solution required. JV = volume of spirit taken for titration. N 6. Esters. It should contain not more than 5 grams of esters per 100 cc. of spirit, calculated as methyl acetate and determined as follows : Five cubic centimeters of wood spirit are run into a flask and 10 cc. normal sodium hydroxide free from carbonates are added, and the flask connected with a return condenser and boiled for two hours. Instead of digesting at boiling temperature the flask may be allowed to stand overnight at room temperature and then heated on a steam bath for thirty minutes with an ordinary tube condenser. The liquid after digestion is cooled and titrated with normal sulphuric acid, using phenolphthalein as an indicator. Methyl acetate, in grams 1 _ .074 x cc. of normal soda required x 100 per 100 cc. of spirit j cc. of spirit taken 7. Bromine Absorption. It must contain a sufficient quantity of impurities derived from the wood so that not more than 25 cc. nor less than 15 cc. shall be required to decolorize a standard solution containing 0.5 gram of bromine, as follows : The standard bromine solution is made by dissolving 12.406 grams of potassium bromide and 3.481 grams of potassium bromate (which is of tested purity and has been dried for two hours at 100 C.) in a liter of water. Fifty cubic centimeters of the standard solution containing 0.5 gram of bromine are placed in a glass-stoppered flask having a capacity of about 200 cc. This is acidified by the addition of 10 cc. of diluted sulphuric acid (1:4), and the whole shaken and allowed to stand a few minutes. The wood alcohol is then allowed to flow slowly into the mixture, drop by drop, from a burette until the color is entirely discharged. The temperature of the mixture should be 20 C. In addition to the above requirements the methyl alcohol must be of such a character as to render the ethyl alcohol with which it is mixed unfit for use as a beverage. REFERENCES ALLEN : Commercial Organic Analysis, Vol. 1. LEACH : Food Inspection and Analysis. LUNGE : Chemisch-technische Untersuchungsmethoden, Bd. III. 32 METHODS OF ORGANIC ANALYSIS MEYER : Analyse und Konstitutionsermittehmg organischer Verbindungen. MEYER-TINGLE : Determination of Radicles in Carbon Compounds. MULLIKEN : Identification of Pure Organic Compounds, Vol. I. SCHMIDT : Ausfiihrliches Lehrbuch der pharmaceutischen Chemie, Bd. II, Abth. I. U. S. Dept. Agriculture, Bureau of Chemistry, Bui. 107 (Revised). Official and Provisional Methods of Analysis. U. S. Dept. Commerce and Labor, Bureau of Standards, Circular No. 19, Standard Density and Volumetric Tables. U. S. Pharmacopoeia. VAUBEL : Die physikalischen und chemischen Methoden der quantitativen Bestimmung organischer Verbindungen. YOUNG : Fractional Distillation. II 1905. LEACH and LYTHGOE : The Detection and Determination of Ethyl and Methyl Alcohols in Mixtures by the Immersion Ref ractom- eter. /. Am. Chem. Soc., 27, 964. SCHIDROWITZ and KAYE : The Determination of Higher Alcohols in Spirits. Analyst, 30, 190. WINKLER: (Preparation of Ethyl Alcohol) . Ber., 38, 3612 ; Analyst, 31, 76. 1906. SCUDDER and RIGGS : The Detection of Methyl Alcohol. J. Am. Chem. Soc., 28, 1202. TOLMAN and TRESCOTT : Methods for the Determination of Esters, Aldehydes, and Furfural in Alcoholic Liquors. J. Am. Chem. Soc., 28, 1619. 1907. FLEISCHER and FRANK : (Rapid Estimation of Alcohol and Ether in their Mixtures). Chem. Ztg., 31, 665. SCHIDROWITZ : The Estimation of Higher Alcohols (" Fusel Oil ") in Distilled Liquors. J. Am. Chem. Soc., 29, 561. U.S. Internal Revenue Regulations, No. 30 Revised. Concerning Denatured Alcohol, Central Denaturing Bonded Warehouses, and Industrial Distilleries. WAGNER and SCHULTZE : (Estimation of Ethyl Alcohol with the Zeiss Immersion Ref rac tome ter). Z. anal. Chem., 46, 508. 1908. ANDREWS: The Refractive Indices of Alcohol-Water Mixtures. /. Am. Chem. Soc., 30, 353. CRAMPTON and TOLMAN: A Study of the Changes taking Place in Whiskey stored in Wood. J. Am. Chem. Soc., 30, 98. DOROSHEVSKII and DVORZHANCHIK : Index of Refraction of Mix- tures of Alcohol and Water. J. Russ. Phys.-Chem. Soc., 40, 101 ; Chem. Abs., 2, 2181. ALCOHOLS 33 DUDLEY: Notes on the Roese Method for the Determination of Fusel Oil, and a Comparison of the Results by the Allen-Mar - quardt Method. J. Am. Chem. Soc., 30, 1271. HINKEL : The Detection of Small Quantities of Methyl Alcohol in the Presence of Ethyl Alcohol. Analyst, 33, 417. 1909. PLUCKER : Preparation of Pure Ethyl Alcohol. Z. Nahr. Genussm., 17, 454. TOLMAN and HILLYER : Methods of Analysis of Distilled Spirits. U. S. Dept. Agriculture, Bur. Chem., Bui. 122, p. 206. VORICEK : Detection of Methyl Alcohol in Ethyl Alcohol. /. Soc. Chem. Ind., 28, 823. 1910. WILEY : Manufacture of Denatured Alcohol. U. S. Dept. Agricul- ture, Bur. Chem., Bui. 130. 1911. BACON : Detection and Determination of Small Quantities of Ethyl and Methyl Alcohol and of Formic Acid. U. S. Dept. Agri- culture, Bur. Chem., Cir. No. 74. GORE : An Electrically Controlled Constant Temperature Water Bath for the Immersion Refractometer. J. Ind. Eng. Chem., 3, 506. CHAPTER II Aldehydes THE most important methods for the detection and deter- mination of aldehydes are based upon reactions of oxidation, of condensation, and of direct addition. In this chapter the analytical application of these reactions will be illustrated by methods for formaldehyde, benzaldehyde, and vanillin. The readiness with which aldehydes undergo oxidation gives them the property of reducing ammoniacal silver solution, which is the basis of one of the most delicate qualitative tests for this group of compounds. The test maybe carried out as follows: 1 Mix, in a test tube previously cleaned with hot sodium hy- droxide solution, 1 cc. of ammoniacal silver nitrate solution (containing one part of silver nitrate in ten parts of ammonium hydroxide of 0.923 sp. gr.) and 1 cc. of ten per cent sodium hydroxide solution. Shake the mixture in the tube and then allow two or three drops of the solution to be tested to flow slowly down the moistened glass surface into the reagent. Shake and allow to stand cold for five minutes. Aldehydes (and a few other compounds including some of the polyatomic alcohols) cause the production of a dark brown or black pre- cipitate or mirror of metallic silver. This reaction is given by all of the ordinary aldehydes of the fatty series, including the aldose carbohydrates, but not by all aromatic aldehydes. The ammoniacal silver solution and the sodium hydroxide must not be mixed in advance and must always be kept cool, as a dangerously explosive precipitate is apt to form on warming 1 Noyes and Mulliken : Identification and Class Reactions of Organic Sub- stances. Mulliken : Identification of Pure Organic Compounds, Vol. I., p. 22. 34 ALDEHYDES 35 or on long standing. The use of a mixture of sodium hydroxide and ammoniacal silver nitrate (Tollens' aldehyde reagent) makes the test more delicate than when the ammoniacal silver solution is used alone. Alkaline solutions of other metals are reduced by many alde- hydes, especially on boiling, and many quantitative methods for individual aldehydes are based upon the determination of the amount of metal reduced. Condensation reactions, especially with phenylhydrazine, hydroxylamine, and phenols, are often used for the detection and sometimes for the determination of aldehydes. A general dis- cussion of such methods will be found in the works of Vaube and of Meyer. Several special methods will be described in this and the two following chapters. Of the addition reactions of aldehydes, that with bisulphite is of especially wide application. On shaking a liquid aldehyde or a concentrated solution of aldehyde in water or ether, with an equal volume of strong sodium bisulphite solution, addition takes place with the formation of the saturated compound RCH(OH)SO 3 Na which usually separates as a white crystalline precipitate. Ketones containing the CH 3 CO group also give the reaction. A negative result is not conclusive, as the addi- tion product may be too soluble to appear as a precipitate. According to Ripper, 1 the bisulphite reaction can be utilized for the determination of any aldehyde soluble in water or which can be brought into solution by a small amount of alcohol. A one half per cent solution of the aldehyde is mixed with twice its volume of a solution of potassium bisulphite of known strength (about 12 grams per liter), and after 15 minutes the excess of bisulphite is determined by titration with iodine. Ripper .applied this method with satisfactory results to solu- tions of formaldehyde, acetaldehyde, benzaldehyde, and vanillin. A similar addition reaction gives rise to the well-known and delicate " fuchsin test " for aldehydes. This test, as developed by Mulliken, 2 is as follows: 1 Monatsh. Chem., 1900, 21, 1079. 2 Identification of Pure Organic Compounds, Vol. I, p. 15. 36 METHODS OF ORGANIC ANALYSIS To prepare the fuchsin aldehyde reagent, dissolve 0.2 gram of rosanilin, or, if the free base cannot be obtained, of the hydro- chloride or acetate, in 10 cc. of a freshly prepared, cold, saturated aqueous solution of sulphur dioxide. Allow the solution to stand until all signs of pink disappear and it becomes colorless or pale yellow. This will require several hours. Then dilute with water to 200 cc. and preserve for use in a tightly stoppered bottle. To 5 cc. of this reagent add 0.05 gram, or one drop, of the substance to be tested (if pure, or a few drops if in solution). If the substance is a liquid, or dissolves in the reagent, allow to stand two minutes and observe the color. If the substance does not dissolve, shake gently for two minutes and then observe the color. / The appearance of a distinct pink, red, purple, or blue coloration indicates the presence of an aldehyde/ The test to be of value must be applied under carefully regulated conditions. The reagent is reddened by alkalies or alkaline salts of weak acids, by heating or by long exposure to air at ordinary tem- perature. In general, the test as here described distinguishes aldehydes other than carbohydrates from the latter and from ke- tones. A few acetals show the reaction through being partially hydrolyzed to aldehydes under the conditions of the test. Ace- tone and some other soluble ketones prepared by destructive distillation gradually redden the reagent if added to it in large quantity or if allowed to remain in contact for a number of minutes ; but this is thought to be due chiefly, if not wholly, to the presence of traces of aldehydes or acetals (Mulliken). This reaction serves for the detection of minute quantities of aldehydes present as impurities in commercial alcohol, and for the colorimetric estimation of aldehydes in distilled liquors. 1 FORMALDEHYDE Formaldehyde gas, produced by the partial oxidation of methyl alcohol, is freely soluble in water and is most com- 1 Medicos : Forschungsber. uber Lebensmittel, 1895, 1, 299. Bui. 107, Bur. Chem., U. S. Dept. Agriculture. Tolman and Trescott : J. Am. Chem. Soc., 1906, 28, 1619. ALDEHYDES 37 monly handled as a 35 to 40 per cent aqueous solution. Such solutions are often designated formalin, formol, or formal. More dilute solutions are sometimes sold as food preservatives under fanciful or misleading names. In dilute aqueous solution, formaldehyde exists in the " monomolecular " state, as CH 2 O. Such solutions do not change if kept at ordinary temperature in closed vessels. When an aqueous solution is concentrated either by sponta- neous evaporation or by heating, a white flocculent deposit appears. If the solution is then separated from the deposit, it is found to contain condensed or polymerized formaldehyde. 1 The material which deposits from a concentrated aqueous solution of formaldehyde has, after drying, the composition (CH 2 O) 6 H 2 O to (CH 2 O) 8 - H 2 O. 2 It is amorphous, soluble in warm water, and has an odor resembling that of formal- dehyde. The paraformaldehyde of commerce consists essen- tially of this material. Metaformaldehyde (oxymethylene, " trioxymethylene "), (CHgO),,., may also be formed by evaporation of formaldehyde solutions. By prolonged digestion at ordinary temperature or by heating for a short time at 130-150 with a large excess of water, metaformaldehyde passes into solution and into the " mono-molecular " form. Polymeric modifications of formal- dehyde in aqueous solution resemble closely the original sub- stance in its behavior toward reagents, so that, as measured by the ordinary methods, a solution does not lose strength by the partial polymerization of the formaldehyde so long as all re- mains in solution. Commercial solutions of formaldehyde commonly contain methyl alcohol and may contain small amounts of any of the impurities of commercial wood spirit. Solutions of the usual strength, from 35 to 40 per cent., should have specific gravities of about 1.08 to 1.11 at 15, lower figures ordina- 1 Tollens and Mayer : Ser., 1888, 21, 1571, 3503. Kraut, Eschweiler, and Grossmann: Ann. Chem., 1890, 258, 103. 2 Losekann : Chem. Ztg., 1890, 14, 1408. Delephine: Compt. rend., 1897, 124, 1525. Beilstein: Organische Chemie, Erganzbd., I., 467. 38 METHODS OF ORGANIC ANALYSIS rily indicating the presence of excessive amounts of methyl alcohol. 1 The methods given in this chapter for the detection and de- termination of formaldehyde refer especially to the examina- tion of commercial solutions containing only such impurities as ordinarily occur in crude preparations of formaldehyde, or substances which might be used with formaldehyde in preserva- tive mixtures. The examination of food products for formalde- hyde will be discussed in connection with other food preserva- tives in a subsequent chapter. If a solution to be examined contains dissolved solids which interfere with the direct application of the tests as described, it can be acidified with a small excess of phosphoric or sulphuric acid, distilled, and the test applied to the distillate. The latter, however, will never contain all of the formaldehyde, since some is always polymerized and left as paraformaldehyde in the distilling flask. DETECTION AND IDENTIFICATION Resorcin Test* Mix one drop of a 1 per cent aqueous solution of resorcin with 1 cc. of a dilute aqueous solution (preferably about 0.2 per cent) of the aldehyde. Allow the mixture to flow gently down the side of an inclined test-tube containing 3-5 cc. of pure con- centrated sulphuric acid (or incline the test tube containing the mixture and pour in the acid). Impart a gentle rotary motion to the liquids by cautiously swaying the lower end of the tube through a circle about a decimeter in diameter, in such a man- ner as not to cause the disappearance of the two layers. If formaldehyde is present, a red ring slightly tinged with violet will soon appear. Above this ring a light flocculent precip- 1 On the determination of methyl alcohol in formaldehyde solutions see Duyk : Ann. chim. anal., 1901, 6, 407 ; J. Chem. Soc., 1902, 82, ii, 110. Stri- trar : Z. anal. Chem., 1904, 43, 401. Gnehm and Kaufler: Z. angew. Chem., 1904, 17, 673; 1905, 18, 93. Bamberger: Ibid., 1904, 17, 1246. 2 Mulliken and Scuddrr : Am. Chem. J., 1900, 24, 451. Mulliken : Identi- fication of Pure Organic Compounds, Vol. I, p. 24. ALDEHYDES 39 itate, at first nearly white on its upper surface and red- violet beneath, but soon changing to flocks that are red throughout, will be seen suspended in the aqueous upper layer. This reaction is very satisfactory for solutions containing one part of formaldehyde in 100 to 5000 parts of solution and can be detected to a dilution of 1 : 100,000. A similar reaction is obtained if phenol is used instead of resorcin. Gallic Acid Test 1 Mix 0.2 cc. of a saturated solution of gallic acid in pure ethyl alcohol, with 1 to 2 cc. of the solution to be tested, and introduce a layer of concentrated sulphuric acid, as in the resorcin test. In the presence of formaldehyde, a green zone appears at the line of contact of the two liquids. This gradu- ally changes to a pure blue ring, which, in the case of pure aqueous solutions of formaldehyde, can be detected without difficulty at a dilution of 1 : 500,000. If the solution tested contains as much as one part of formaldehyde in 20,000, a yel- lowish color appears immediately at the line of contact of the two liquids. This quickly turns green, and the blue color de- velops both above and below the green zone. If other sub- stances which give color reactions are also present, the upper layer will vary in color, but the green and lower blue ring will still appear beneath (Mulliken and Scudder). On swaying the tube, or allowing it to stand for some time, the blue color spreads throughout the zone and a pure blue ring is usually obtained. The color is quite permanent and apparently quite characteristic, no other substance having been noted as giving the blue ring. Acetaldehyde tested in the same way gives a reddish brown coloration. Hydrochloric Acid and Casein Test* Mix 5 cc. of the solution to be tested with 5 cc. of pure milk in a porcelain casserole, add 10 cc. of concentrated hydrochloric iBarbier and Jandrier : Ann. chim. anal, 1, 325; Abs. Analyst, 1896, 21, 295. Mulliken and Scudder : Am. Chem. J., 1900, 24, 444. 2 Leach : Ann. Kept. Mass. State Board of Health, 1897, 558 ; 1899, 699. 40 METHODS OF ORGANIC ANALYSIS acid containing 0.002 gram of ferric chloride, and heat slowly over a free flame nearly to boiling, meanwhile giving the cas- serole a rotary motion to break up the curd. -A violet colora- tion indicates formaldehyde. According to Leach, various aldehydes give color reactions under this treatment, but form- aldehyde alone shows the unmistakable violet coloration. This test is especially useful for the detection of formaldehyde in milk, and will be more fully discussed in that connection. Meihylene-di-ft-naphthol Test Since formaldehyde is frequently sold under other names, its identification by some method independent of the above color reactions may be a matter of importance. In such cases the following test given by Mulliken l will be useful. Place in a test tube 3 drops of a 30 to 40 per cent, or 10 drops of a 10 per cent, solution of the formaldehyde, 3 cc. of dilute alcohol (1 : 2), 0.04 to 0.06 gram /3-naphthol, and 3 to 5 drops of concentrated hydrochloric acid. Boil gently until the liquid becomes filled with an abundant precipitate of small white needles. Filter while hot. Wash with 1 cc. of dilute alcohol (1:2). Boil the precipitate with 4 cc. of dilute alcohol (1 : 1). (It is not necessary that all should dissolve.) Cool and filter off the precipitate. Wash with 1 cc. of dilute alcohol (1:1). Dry on porous tile in a warm place and determine the melting point. Methylene-di-/3-naphthol, the product, forms white needles, which, when the temperature in the neighborhood of the melt- ing point is raised at the rate of 1 in 15 seconds, begin to turn brown at 180. It melts with decomposition to a red-brown liquid at 189-192 (uncorr.). DETERMINATION BY OXIDATION lodimetric Method 2 This method depends upon the oxidation of formaldehyde to formic acid by means of iodine in alkaline solution. Two atoms 1 Identification of Pure Organic Compounds, Vol. I, p. 24. 2 Romijn: Z. anal. Chem., 1897, 36, 18. Williams: J. Am. Chem. Soc., 1905, 27, 596. ALDEHYDES 41 of iodine oxidize one molecule of formaldehyde, and the excess of iodine is liberated by acidulation and determined by titration with sodium thiosulphate. CH 2 + 1 2 + H 2 = CH 2 2 + 2 HI. Reagents. Standard solutions of iodine and sodium thiosul- phate, preferably about tenth-normal. Approximately normal solutions of sodium hydroxide and hydrochloric acid. Determination. Dilute a weighed portion of the sample with a known quantity of water so as to obtain a solution containing 0.5 to 1 per cent of actual formaldehyde. Mix 10 cc. of this solution with 25 cc. normal sodium hydroxide and add from a burette 50 to 75 cc. of tenth-normal iodine solution or enough to assure an excess of iodine as shown by the permanent yellow- color of the solution. Shake or stir thoroughly and after ten minutes add 35 cc. normal hydrochloric acid and 'titrate with sodium thiosulphate in the usual way, using starch solution as indicator. At the same time determine the strength of the iodine in terms of thiosulphate solution and from the amount of iodine consumed in oxidizing the formaldehyde calculate the weight of the latter in the 10 cc. taken for the determination. Notes. Under the conditions given the oxidation of form- aldehyde is rapid and complete but the method is applicable only in the absence of all other substances capable of consuming iodine under these conditions. Other aldehydes, acetone, and alcohol cause high results, the latter probably through absorb- ing iodine with the formation of iodoform. In the absence of interfering compounds, the method is very satisfactory, even for solutions containing only 0.1 per cent of formaldehyde. Variations in the excess of iodine added have no appreciable influence upon the results. Hydrogen Peroxide Method 1 In this method, formaldehyde is oxidized to formic acid by means of hydrogen peroxide in the presence of a known amount of alkali. 1 Blank and Finkenbeiner : J5er, 1898, 31, 2979. Haywood and Smith: J. Am. Chem. Soc., 1905, 27, 1183. Bui. 107, Bar. Chem., U. S. Dept. Agriculture. 42 METHODS OF ORGANIC ANALYSIS The excess of alkali, over that required to combine with the formic acid produced, is determined by titration. The method is here described as used by the Association of Official Agricultural Chemists. Reagents. Normal solutions of sodium hydroxide and sul- phuric acid. Neutral 1 3 per cent solution of hydrogen peroxide. Solution of purified litmus as indicator. Determination. Measure 50 cc. of normal sodium hydroxide into a 500-cc. Erlenmeyer flask, add 50 cc. hydrogen peroxide solution, then 3 grams of the formaldehyde solution. Place a funnel in the neck of the flask and stand it on a steam bath for 5 minutes, shaking occasionally during this time. Remove, wash funnel with water, cool to room temperature, and titrate excess of alkali with normal acid, using litmus as indicator. Each molecule of sodium hydroxide which has been consumed (deducting the amount required to neutralize any free acid which the peroxide solution or the original solution of form- aldehyde may have contained) represents one molecule of formaldehyde oxidized to formic acid. Notes. The use of exactly 3 grams of formaldehyde is not essential, but the exact weight must of course be known. It is convenient to use a weighing bottle containing a small pipette, measure out about 3 cc. and obtain the weight by difference. Acetaldehyde is partially oxidized under the same conditions. Its presence, therefore, causes high results, but not so high as by the iodimetric method. The results are not influenced by the presence of paraldehyde, acetone, or ethyl or methyl alcohol. Commercial formalin containing only traces of acetone or acetaldehyde should show the same percentage of formaldehyde by the peroxide as by the iodimetric method. DETERMINATION BY CONDENSATION REACTIONS Several of the condensation reactions of formaldehyde have been utilized for its quantitative determination. One of the 1 If all available peroxide is acid, the acidity must be determined by titration, using litmus as indicator, and allowed for in calculating the amount of alkali consumed in the formaldehyde determination. ALDEHYDES 43 oldest and best-known methods is based upon the fact that formaldehyde and ammonia when mixed in not too dilute solution condense to form hexamethylene tetramine : 6 CH 2 O + 4 NH 4 OH = N 4 (CH 2 ) 6 + 10 H 2 O. If a known amount of ammonia is used, the determination of the excess shows the amount of formaldehyde originally present. Legler's Ammonia Method**- Weigh about 1.5 grams of the solution containing 30 to 40 per cent formaldehyde, or an equivalent amount of a more dilute solution, into a 250-cc. glass-stoppered flask or bottle. Add 100 cc. of fifth-normal ammonia solution ; stopper tightly at once ; mix and allow to stand overnight at room tempera- ture. Standing for two or three days does no harm, provided the stopper fits so tightly as to prevent any loss of ammonia. Finally, add a very small amount of rosolic acid as indicator ahd titrate the excess of ammonia with standard sulphuric acid. Calculate the quantity of formaldehyde originally present from the amount of ammonia consumed in condensing with it ac- cording to the equation given above. Notes and Precautions. In order to prevent loss of ammonia during the determination, the flask or bottle must be tightly closed, the stopper being coated with vaseline if necessary. For the same reason the excess of ammonia should be titrated quickly after opening the flask. Normal or half-normal ammonia is commonly recommended for this method, but the fifth-normal solution is less likely to lose strength and has been found by Williams to give as complete reactions as the stronger solutions. In titrating the excess of ammonia the end reaction is usually unsatisfactory, especially when the solution is highly colored by the indicator. Two drops of a freshly prepared 0.1 per cent solution of rosolic acid have been found sufficient. The results are not affected by the presence of acetone, iLegler: Ber., 1883, 16, 1333. Smith : J. Am. Chem. Soc., 1903, 25, 1028. Williams : loc. cit. 44 METHODS OF ORGANIC ANALYSIS methyl or ethyl alcohol, paraldehyde, or benzaldehyde. Acetal- dehyde reacts with ammonia and thus causes high results if present in the formaldehyde solution. This method was formerly much used in analysis of com- mercial formalin, but on account of the tendency toward low results it is now generally displaced by the hydrogen peroxide method. DETERMINATION BY ADDITION REACTIONS The general addition reaction of aldehydes with bisulphites has been used quantitatively by Ripper, as already noted. For the determination of formaldehyde, however, the reaction with potassium cyanide has been found especially useful. Potassium Cyanide Method^- On mixing aqueous solutions of formaldehyde and potassium cyanide an addition product is formed, which, according to Romijn, is probably the potassium compound of oxyacetonitril : CH 2 O + KCN = CH 2 OK . CN. The addition product reduces silver nitrate in alkaline solu- tion, but has no effect in the presence of an excess of nitric acid. If, therefore, the formaldehyde to be tested be mixed with a known solution of potassium cyanide, the latter being in excess, and the mixture added to a standard solution of silver nitrate acidulated with nitric acid, only the excess of potassium cyanide reacts with the silver nitrate. The amount of formal- dehyde originally present is shown by the quantity of potassium cyanide consumed in the formation of the addition product. The details of the method as here given are nearly identical with those originally recommended by Romijn. Reagents. Tenth-normal solutions of silver nitrate and ammonium thiocyanate. A solution of potassium cyanide 6.2 grams per liter.. Saturated solution of ferric ammonium sulphate. Nitric acid 1.32 sp. gr. (50 per cent). 1 Romijn: Z. anal. Chem., 1897, 36, 18. Smith: loc. cit. Williams: Joe. cit. ALDEHYDES 45 Determination. (1) Measure 15 cc. tenth-normal silver nitrate into a 100-cc. flask, add 6 to 8 drops of the nitric acid and 10 cc. of the cyanide solution ; shake, dilute to the mark, mix thoroughly, and filter through a dry paper. Titrate 50 cc. of the nitrate with tenth-normal ammonium thiocyanate, using 5 cc. of the ferric solution as indicator. The strength of the silver and of the thiocyanate solutions being known, this titra- tion shows the strength of the cyanide. (2) Dilute the sample until it contains about 1 per cent of formaldehyde, mix 10 cc. of this dilute solution with 35 cc. of the cyanide solution, and rinse the mixture into another portion of 15 cc. tenth-normal silver nitrate, acidulated with 6 to 8 drops of the nitric acid and contained in a 100-cc. flask ; shake and determine the excess of silver by means of thiocyanate in the same way as before. Twice the difference between the two titrations (since only half the liquid was used in each case) represents the amount of cyanide consumed by the formaldehyde. If the thiocyanate solution is exactly tenth-normal, twice the difference (in cubic centimeters) between the two titrations, multiplied by 0.0030Q2, gives the weight of formaldehyde (in grams) in the portion taken for the determination. Notes. This method is applicable to very dilute solutions of formaldehyde, larger volumes being used in place of the 10 cc. called for in the above directions. Smith obtained accurate results upon a solution containing 0.01 per cent. Ethyl and methyl alcohols, acetone, benzaldehyde, and paral- dehyde do not interfere. Acetaldehyde causes high results if allowed to stand for some time in contact with the cyanide solution, but if the formaldehyde solution is added to the cyanide and, after mixing, poured at once into the silver nitrate solution, the presence of acetaldehyde does not influence the results. With commercially pure solutions of formaldehyde in water Romijn and Smith obtained concordant results by the iodimetric and cyanide methods. Williams obtained concord- ant results by the ammonia and the cyanide methods, which were slightly lower than those obtained by the oxidation methods. 46 METHODS OF OKGANIC ANALYSIS BENZALDEHYDE Many methods have been advanced for the determination of benzaldehyde. Among these the methods based on reactions with phenylhydrazine and its derivatives, and with neutral sulphite, are worthy of special notice in that they are fairly accurate and serve to illustrate the analytical application of fairly general aldehyde reactions. PHENYLHYDRAZINE METHOD By simple condensation benzaldehyde and phenylhydrazine yield an insoluble derivative which may be collected, washed, dried, and weighed. C 6 H 5 CHO + C 6 H 5 NH.NH 2 =C 6 H 5 CH : N . NH . C 6 H 5 + H 2 O. This method, as developed by Denis and Dunbar 1 and adopted by the Association of Official Agricultural Chemists, 2 is as follows : Determination of Benzaldehyde in Almond Extract (Denis and Dunbar) Reagent. Add 1.5 cc. of glacial acetic acid to 20 cc. of water and mix with 1 cc. of phenylhydrazine. Manipulation. Measure out two portions of 10 cc. each of the extract in 300 cc. Erlenmeyer flasks and add 10 cc. of the reagent to one flask and 15 cc. to the other. Allow to stand in a dark place overnight, add 200 cc. of water, and filter on a weighed Gooch crucible having a thin felt of asbestos. Wash first with cold water, finally with 10 cc. of 10 per cent alcohol, and dry for three hours in a vacuum oven at 70 C., or to con- stant weight over sulphuric acid. If the duplicate results do not agree, repeat the determination, using a larger quantity of the reagent. NEUTRAL SULPHITE METHOD Benzaldehyde, in common with many other aldehydes, reacts with neutral sodium sulphite in such a way that there results 1 J. Ind. Eng. Chem., 1907, 1, 256. 2 U. S. Dept. Agr., Bur. Chem., Bui. 137, pp. 74, 121. ALDEHYDES 47 the aldehyde-bisulphite addition product and sodium hydroxide, thus: C 6 H 6 CHO + Na 2 SO 3 + H 2 O == C 6 H 5 CH(OH)SO 3 Na + NaOH. The sodium hydroxide formed is titrated and furnishes a measure of the amount of benzaldehyde which was present. The details of this method as adopted in the U.S. Pharma- copoeia are as follows : Assay of Benzaldehgde ( U. S. Pharmacopoeia) Introduce into a 150-cc. flask 10 cc. purified kerosene, note the exact weight, add 12 drops of benzaldehyde, and again note the weight; add 20 cc. of water, 6 drops of phenolphthalein solution, and neutralize exactly by the addition of tenth-normal sodium hydroxide, shaking thoroughly. Then add from a burette, gradually, a solution of sodium sulphite (1 in 5), alternating with half-normal hydrochloric acid from a second burette, until 10 cc. of the sodium sulphite solution have been added, and enough half-normal hydrochloric acid to maintain the neutrality of the mixture ; after adding a few drops of phenolphthalein solution and shaking the flask frequently, allow it to stand two hours to insure a permanent condition of neutrality, and then note the volume of half -normal acid used. Carry out a blank test identical with the foregoing except that the benzaldehyde is omitted and note the amount of acid consumed. From the difference in volume of the half-normal acid used in the two cases, calculate the amount of benzaldehyde which reacted with sulphite according to the equation given above. For discussion of the application of the neutral sulphite method to other aldehydes see the papers of Burgess 1 and Sadtler 2 . When this method is applied to aldehydes which have an ethylene linkage, there may occur a further reaction with addition of bisulphite at this point as well as at the carbonyl group and a correspondingly increased liberation of sodium hydroxide. 1 Analyst, 29, 78. 2 J. Am. Chem. Soc., 27, 1321. 48 METHODS OF ORGANIC ANALYSIS VANILLIN In the case of vanillin, the sulphite method appears to be inapplicable, and in this laboratory the bisulphite method has given results somewhat too low. 1 Better results have been obtained by the alkalimetric method of Welmans, 2 and by the Hanus 3 method of condensation with p. bromphenylhydrazine : C 6 H 3 (OH)(OCH 3 )CHO + C 6 H 4 (Br)NH NH 2 = C 6 H 3 (OH)(OCH 3 ) CH: NH . NHC 6 H 4 Br + H a O. Twenty-five cubic centimeters of a water solution containing 0.5 to 1 per cent of vanillin are treated with 75 cc. of a hot water solution containing 0.5 to 0.75 gram of p. bromphenyl- hydrazine. At the conclusion of the precipitation the mixed liquid should be at about 50 ; the precipitate is allowed to stand 5 hours, filtered on a Gooch crucible, washed with hot water till washings show no precipitation nor distinct color- ation with silver nitrate, dried at 95-100 to constant weight, and weighed. With pure vanillin solutions this method gave nearly theoretical results. 1 REFERENCES ALLEN : Commercial Organic Analysis. LUNGE : Chemisch-technische Untersuchungsmethoden. MEYER : Analyse und Konstitutioiisermittelung organise her Verbindungen. MULLIKEN : Identification of Pure Organic Compounds, Vol. I. VAUBEL : Quantitative Bestimmung organischer Verbindungen. II 1904. BURGESS : Estimation of Aldehydes and Ketones in Essential Oils. Analyst, 29, 78. 1905. HANUS : Ueber eine quantitative Bestimmung des Vanillins. Z. Nahr. Genussm., 10, 585. HAYWOOD and SMITH : A Study of the Hydrogen Peroxide Method of Determining Formaldehyde. J. Am. Chem. Soc., 27, 1183. 1 Determinations by B. G. Feinberg, not yet published. *Pharm. Ztg., 1898, 34, 634 ; Vaubel, II, 88. 8 Z. Nahr. Genussm., 3, 531 ; 10, 585. ALDEHYDES 49 SADTLER : A Fuller Study of the Neutral Sulphite Method for Deter- mining Some Aldehydes and Ketones in Essential Oils. /. Am. Chem. Soc., 27, 1321. WILLIAMS : A Study of Methods for the Determination of Formalde- hyde. J. Am. Chem. Soc., 27, 596. WINTON and BAILEY : The Determination of Vanillin, Coumarin, and Acetanilid in Vanilla Extract. J. Am. Chem. Soc., 27, 719. 1906. CHASE : A Method for the Determination of Citral in Lemon Oils and Extracts. J. Am. Chem. Soc., 28, 1472. 1907. DOBY: (Comparison of Methods for Determination of Formalde- hyde). Z. angew. Chem., 20, 353. 1908. WOODMAN and LYFORD : The Colorimetric Estimation of Benzalde- hyde in Almond Extracts. J. Am. Chem. Soc., 30, 1607. 1909. DENIS and DUNBAR: Determination of Benzaldehyde in Almond Flavoring Extract. J. Ind. Eng. Chem., 1, 256. CHAPTER III Carbohydrates General Methods THE carbohydrates include the simple sugars (monosaccha- rides) and the substances which can be converted into simple sugars by hydrolysis. The monosaccharides are aldehyde alco- hols or ketone alcohols, each molecule containing a carbonyl group, either as such or in tautomeric form, and several hydroxyl groups, one of the latter being adjacent to the carbonyl group. The purpose of this chapter is to outline the more important general methods and analytical properties of the following car- bohydrates: Monosaccharides: Hexoses Dextrose (d. glucose), Levulose (d. fructose), Galactose, Mannose; Pentoses Xylose, Arabin- ose. Disaccharides : Sucrose, Lactose, Maltose. Trisaccharide: Raffinose. Polysaccharides : Starch, Dextrin, Glycogen, Galactan, Cellu- lose, Pentosans. OCCURRENCE AND RELATIONS Monosaccharides (glucoses, glycoses, monoses) have the com- position (CHgO).,. 1 and are called tetroses, pentoses, hexoses, etc., according to the number of carbon atoms in the molecule. Only pentoses and hexoses are of sufficient practical importance to call for consideration in connection with ordinary methods of analysis. The pentoses do not occur free in nature but 1 This statement docs not apply to the methyl derivatives now frequently classified as monosaccharides. 50 CARBOHYDRATES GENERAL METHODS 51 are met by the analyst as products of the hydrolysis of the pentosans. The hexoses include all of the monosaccharides of present commercial importance and all whose biological relations have been thoroughly studied. Dextrose (d. glucose, grape sugar, starch sugar, diabetic sugar, ordinary glucose) is widely distributed in nature, occur- ring especially in fruits and plant juices, often mixed with other sugars. It is a normal constituent of blood and is the form of carbohydrate ordinarily found in the urine in diabetes or glycosuria. With the exception of the pentosans and galactan, all of the di-, tri-, and polysaccharides mentioned above yield dextrose on hydrolysis. Levulose (d. fructose, fruit sugar) occurs with dextrose in plant juices and especially in fruits and honey. It is also a product of the hydrolysis of sucrose and of raffinose. Galactose does not occur free ; but as a product of hydrolysis of lactose, raffinose, and the galactans it is of considerable analytical importance. Mannose also is not found free, but has been detected among the products of hydrolysis of the insoluble carbohydrate matter of a number of thick-walled vegetables tissues, nut shells, etc., and of several Japanese vegetables. The disaccharides considered here are all hexo-bioses (C 12 H 22 O n ). Sucrose (saccharose, cane sugar) is widely distributed in the vegetable kingdom, being found in considerable quantity, gen- erally mixed with dextrose and levulose, in the fruits and juices of many plants. The most important sources of sucrose are the sugar beet, the sugar and sorghum canes, and the sugar maple. A molecule of sucrose yields on hydrolysis one mole- cule each of dextrose and levulose. The hydrolysis of sucrose is often called " inversion " and the resulting mixture of equal parts dextrose and levulose is known as "invert sugar." Lactose (lactobiose, milk sugar) occurs in the milk of most mammals, constituting usually from 4 to 7 per cent of the fresh secretion. Lactose crystallizes with one molecule of water which it retains on drying at room temperature over 52 METHODS OF ORGANIC ANALYSIS sulphuric acid or on heating in the air at 100, but loses at about 130. A molecule of lactose yields on hydrolysis one molecule each of dextrose and galactose. Maltose (malt sugar) is formed from starch by the action of diastatic enzymes and is therefore an important constituent of germinating cereals, malt, malt extract, and beer wort. It is also formed as an intermediate product when starch is hydro- lyzed to dextrose by boiling with dilute mineral acids, as in the manufacture of commercial glucose. Maltose crystallizes with one molecule of water, which it loses on heating in the air at 100. Each molecule of maltose yields two molecules of dex- trose on hydrolysis. The only trisaccharide of practical importance is raffinose (C 18 H 32 O 16 ), also called meletriose and formerly melitose or gossypose. It occurs in cotton seed and in small quantity in the germs of various other seeds including wheat and barley. Sugar beets, especially if unhealthy or injured, sometimes contain raffinose in sufficient quantity to affect the refining process. Raffinose crystallizes with five molecules of water in needles or slender prisms and has a marked influence upon the crystallization of the cane sugar present. 1 Raffinose loses its water of crystallization at 100. On hydrolysis it yields one molecule each of dextrose, levulose, and galactose. Partial hydrolysis results in the formation of levulose and the disaccharide, melibiose. Starch (C 6 H 10 O 5 ) Z is the most important of the polysac- charides, being the principal form of carbohydrate in grains and most other edible seeds, as well as in potatoes and other tubers. It is the main product of the assimilation process and the principal reserve carbohydrate of most green plants. Commercially it is of great importance as a constituent of foods, as the source of dextrin, maltose, and commercial glucose, and as the principal raw material of many of the fermentation industries. Starch constitutes over one half of the solid matter of all ordinary cereals and about three fourths of the total solids in potatoes. Starch granules of different plants vary in size 1 Stone and Baird: J. Am. Chem. Soc., 1897, 19, 116. CARBOHYDRATES GENERAL METHODS 53 and structure so that in most cases the source of a starch which has not been altered by heat, ferments, or chemical reagents can be determined by microscopical examination. All starches yield dextrose only, as the final product of complete hydrolysis. Dextrins, (C 6 H 10 O 5 ) X or (C 6 H 10 O 5 ) X H 2 O, are, formed from starch by the action of enzymes, acids, or heat. Small amounts of dextrin are found in normal, and larger amounts in germi- nating, cereals. Malt diastase acting upon starch in fairly concentrated solution yields usually about one part of dextrin to four of maltose. During acid hydrolysis, dextrin is formed as an intermediate product between soluble starch and maltose. Commercial dextrin, the principal constituent of " British gum," is obtained by heating starch, either alone or with a small amount of dilute acid. Glycogen, (C 6 H 10 O 5 ) X or perhaps (C 6 H 10 O 5 ) X . H 2 O, is the principal carbohydrate of the animal organism, being found in small quantity in the muscles and more abundantly in the liver of all well-nourished animals. It is a white amorphous powder intermediate in properties between starch and dextrin and is sometimes called animal starch. The determination of glycogen is often important in physiological investigations and is sometimes useful in distinguishing horseflesh from beef, the latter containing usually less than 0.7 per cent of glycogen, the former often two or three times this amount. On com- plete hydrolysis glycogen yields only dextrose. Galactans, amorphous polysaccharides yielding galactose on hydrolysis, occur in small quantity in many plants and in relative abundance in the seeds of the legumes where they largely replace starch as reserve carbohydrate. Since the galactans are readily hydrolyzed by hot dilute acids and are digested by some of the diastatic enzymes, it is probable that galactan has been reported as starch in many analyses. Cellulose occurs in the cell walls of all vegetable tissues. The term is sometimes applied to the whole of the fiber which is unattacked by boiling dilute acids and alkalies, but should be restricted to that constituent of the fiber which is of a true carbohydrate nature. " Normal " cellulose, such as is derived 54 METHODS OF ORGANIC ANALYSIS from cotton and flax fibers, yields dextrose on hydrolysis. A few celluloses have been found to yield mannose or a pentose (probably xylose) in addition to dextrose (Tollens). Pentosans, anhydrides of arabinose and xylose, are the prin- cipal constituents of the vegetable gums, araban occurring espe- cially in the soluble gums such as cherry gum and gum arable, xylan in the so-called wood gum of fibrous tissues such as wood, straw, vegetables, and the outer portion of the cereal grains. The wheat grain, for example, contains 3 to 5 per cent of pentosan, which in the milling process is largely left in the bran. The so-called patent flour obtained from the interior of the grain contains hardly any pentosan, while the breakfast cereals and the so-called entire wheat and graham flours have usually about as much as the original grain. SOLUBILITIES IN WATER Of the carbohydrates mentioned above, all except the poly- saccharides are crystallizable compounds dissolving in water to form clear solutions. Milk sugar dissolves in six parts of water at ordinary temperature; all of the other members are more freely soluble. Among the polysaccharides, dextrin, glycogen, and some of the galactans and pentosans are soluble ; starch, cellulose, some of the galactans, and most of the pento- sans of ordinary food materials are insoluble in cold water. Glycogen gives a strongly opalescent solution, which is not cleared by repeated filtration but loses its opalescence on the addition of a little potassium hydroxide or acetic acid. On heating with water, starch grains swell and finally gelatinize with the formation of " starch paste." Different starches vary considerably in the temperature at which they gelatinize and in the physical properties of the paste produced. Thin starch pastes can be filtered through paper, but almost always leave some gelatinous residue upon the filter. Pastes containing only a few hundredths of one per cent of starch become clear on boiling and can be filtered without loss. Water-soluble starch CARBOHYDRATES GENERAL METHODS 55 can be prepared 1 by chemical treatment and is sometimes found in natural products, for example in immature grains. Cellulose and the ordinary pentosans of foods and fibers are insoluble in water and not gelatinized by boiling. IN ALCOHOL AND ETHER Levulose is soluble in 5 parts of cold absolute alcohol and is somewhat soluble in mixtures of ether and strong alcohol. The other monosaccharides are sparingly soluble in cold alcohol, in- soluble in ether, and practically insoluble in the alcohol-ether mixture. Dextrose is much more readily soluble in hot than in cold alcohol ; 100 parts of 90 per cent alcohol dissolve about 2 parts dextrose at 18, about 22 parts at boiling temperature. The di-, tri-, and polysaccharides are insoluble in ether. Di- and trisaccharides are less soluble in alcohol than is dextrose. Lactose is practically insoluble in alcohol, even when the latter is diluted to 60 per cent. Sucrose is much more readily soluble in diluted than in con- centrated alcohol. According to Scheibler : 100 parts 90 per cent alcohol dissolve 0.9 parts at 14 ; 2.3 parts at 40 100 parts 80 per cent alcohol dissolve 6.6 parts at 14 ; 13.3 parts at 40 100 parts 70 per cent alcohol dissolve 18.8 parts at 14 ; 31.4 parts at 40 100 parts 60 per cent alcohol dissolve 33.9 parts at 14 ; 49.9 parts at 40 100 parts 50 per cent alcohol dissolve 47.1 parts at 14 ; 63.4 parts at 40 Sucrose dissolves in about 80 parts of boiling absolute alcohol. All of the polysaccharides are insoluble in alcohol. Those which are soluble in water can be precipitated from their aqueous solutions by the addition of strong alcohol. IN ACIDS AND ALKALIES Among the carbohydrates which are insoluble in water, separations can sometimes be made by the use of acid or alka- line solutions. Cellulose is soluble in concentrated sulphuric acid, but in- 1 Lintner : J. prakt. Chem., 1886, [2] 34, 381. Wroblewski : Z. physiol. Chem., 1898, 24, 173. Vaubel : II., 500. 56 METHODS OF ORGANIC ANALYSIS soluble in any ordinary aqueous solution of acid or alkali. It dissolves in Schweitzer's reagent (aqueous ammonia saturated with cupric hydroxide) to a viscous solution, from which it is precipitated by neutralization with acid. Starch is insoluble in Schweitzer's reagent or in solutions of ammonia. Treated with dilute aqueous solutions of sodium or potassium hydroxide, starch swells, gelatinizes, and becomes soluble. It can be completely precipitated from such a solution by neutralizing with acetic acid and adding strong alcohol. Starch is not affected by dilute solutions of alkalies in strong alcohol. By diluted solutions of strong acids starch is first dissolved, then hydrolyzed. Some weak organic acids dissolve starch with little if any hydrolysis. Boiling water containing 1 per cent of salicylic acid dissolves starch to an opalescent so- lution which niters much more readily than a corresponding starch paste made with water alone. The pentosans of foods and fibers so-called wood gums con- sisting mainly of xylan are insoluble in dilute ammonia or in Schweitzer's reagent, largely soluble in dilute aqueous solutions of sodium or potassium hydroxide and in cold dilute acids. From such solutions the pentosan is precipitated by alcohol. Boiling dilute mineral acids dissolve and hydrolyze pentosans almost as readily as starch. Hence pentosans have frequently been reported as starch when the latter has been estimated by direct hydrolysis with acid and determination of the resulting glucose. REACTIONS WITH ACIDS All carbohydrates on treatment with moderately strong hydrochloric or sulphuric acid yield furfural, which is probably the cause of the color reaction with a-naphthol described below. Hexacarbohydrates form only small amounts of furfural and relatively large amounts of levulinic acid. Pentacarbohy- drates yield large amounts of furfural and no levulinic acid. In all cases, however, there is more or less formation of " humus " and other by-products. The following is widely used as a general qualitative test for carbohydrates. CARBOHYDRATES GENERAL METHODS 57 MOLISCH'S -NAPHTHOL REACTION l Mix 2 to 3 cc. of the very dilute solution to be tested with 2 or 3 drops of a 15 per cent solution of a-naphthol in alcohol or chloroform, incline the tube, and pour in carefully 2 to 3 cc. of pure concentrated sulphuric acid. In the presence of carbo- hydrate, a violet zone appears quickly and spreads by diffusion. If the solution tested contains more than a few milligrams of carbohydrate, it quickly blackens and on dilution with water gives a dull violet precipitate. A similar test with thymol gives a crimson or carmine-red solution which soon becomes turbid. FURFURAL TEST FOR PENTOSES AND PENTOSANS Place the substance to be tested in an Erlenmeyer flask, add hydrochloric acid (1.06 sp. gr.), and boil. Lay over the mouth of the flask a small filter paper moistened with a solution of anilin acetate. 2 If the vapor escaping from the flask contains more than traces of furfural, a bright red coloration appears. A test with sucrose or pure starch will show the amount of color to be expected from hexacarbohydrates. If the reaction obtained from the unknown substance is much stronger than from sucrose or starch, the presence of pentoses or pentosans is indicated. A few other substances such as glycuronic acid and oxycellulose have been found to yield considerable amounts of furfural, but these rarely occur in sufficient quantities to require consideration. DETERMINATION OF PENTOSES AND PENTOSANS Under carefully regulated conditions the yield of furfural from either xylose or arabinose, or from the corresponding anhydride, is nearly constant. If the furfural is distilled and collected, its amount can be estimated by adding anilin acetate 1 Molisch : Monatsh. Chem., 1886, 7, 198. Udranszky : Z. physiol. Chem., 1888, 12, 355, 377. Tollens : Handbuch der Kohlenhydrate, II, 101. Mulli- ken : Identification of Pure Organic Compounds, I, 26. 2 Prepared by mixing equal volumes of anilin and 50 per cent acetic acid. 58 METHODS OF ORGANIC ANALYSIS and comparing the red color with that shown by furfural solu- tions of known strength. 1 This method is delicate, but gives only approximate results. Unless the quantity is very small, it should be determined gravimetrically. Furfural forms sparingly soluble condensation products with a number of bases and phenols. Phenylhydrazine and phloro- glucin have been principally used as precipitants. 2 The Association of Official Agricultural Chemists has adopted provisionally the phloroglucin method. 3 LEVTJLINIC ACID REACTION OF HEXACARBOHYDRATES According to Wehmer and Tollens, 4 all hexacarbohydrates yield levulinic acid in sufficient quantity for identification when treated as follows : Heat 5 to 20 grams of the substance with 100 cc. of hydro- chloric acid (1.10 sp. gr.) for 18 hours on a boiling water bath; filter the solution, shake with ether to extract the levulinic acid, and convert the latter into the zinc or silver salt for identifica- tion. As this method is purely qualitative and is not often used in ordinary analytical work, reference must be made to the original paper for details of manipulation. OXIDATION BY NITRIC ACID By heating with moderately strong nitric acid, xylose and arabinose are oxidized, each yielding a trioxyglutaric acid, COOH(CHOH) 3 COOH, while aldoses of the hexose group yield the corresponding acids, COOH(CHOH) 4 COOH, sac- 1 This colorimetric method is also used for the estimation of furfural in dis- tilled liquors. 2 Barbituric acid has recently been recommended as preferable to phenyl- hydrazine and phloroglucin as a precipitant for furfural, linger : Dissertation, Munich, 1904. Jager and Unger: Ber., 1902, 35, 4440. 8 Bull. 107, Bur. Chem., U. S. Dept. Agriculture. For a review and discus- sion of the methods of determining the pentosans and the practical applications of the results see Krober, Rimbach, and Tollens, Z. angew. Chem., 1902, 15, 477, 508. Also Fraps : Am. Chem. J., 1901, 25, 501, 4 Ann. Chem., 1887, 243, 314. CARBOHYDRATES GENERAL METHODS 59 charic, mannosaccharic, and raucic acids being obtained respec- tively from dextrose, maiinose, and galactose. All of these oxidation products are freely soluble except mucic acid, which is practically insoluble in water or dilute nitric acid. Since mucic acid is produced in fairly constant proportion, its insolubility affords a means for the approximate determination of galactose or any substance which yields galac- tose on hydrolysis. Mucic Acid Method for G-alactose, Lactose, Raffinose, and Gralactans. 1 Weigh 1 to 3 grams of substance according to the amount of mucic acid expected ; remove fat if necessary by washing with ether ; transfer to a beaker about 5.5 cm. in diameter and T cm. deep ; add 60 cc. of nitric acid of 1.15 sp. gr. and evaporate the solution to exactly one third its volume on a water bath at a temperature of 94 to 96. After standing 24 hours, add 10 cc. of water to the precipitate and allow it to stand another 24 hours. The mucic acid has now crystallized, and, unless contaminated with insoluble residue from the sample, it can be transferred to a weighed filter, washed with 30 cc. of water, then with alcohol and ether, dried at 100, and weighed. In case other insoluble substances are present, return the filter and mucic acid, after washing with water, to the beaker ; warm 15 minutes with a mixture of 1 part strong ammonia, 1 part ammonium carbonate, and 19 parts of water : filter and wash ; evaporate filtrate to dryness over a water bath ; add 5 cc. nitric acid of 1.15 sp. gr. ; stir thoroughly and allow to stand for 30 minutes. Collect the mucic acid on a weighed filter, wash with 10 to 15 cc. of water, then with 60 cc. of alcohol and a number of times with ether ; dry at 100 and weigh. When these directions are strictly followed, galactose yields about three fourths its weight of mucic acid. The weight of 1 Tollens and Rischbiet : Ber., 1885, 18, 2616. Creydt : Ibid., 1886, 19, 3116. Bull. 107, Bur. Chem., U. S. Dept. Agriculture. 60 METHODS OF ORGANIC ANALYSIS 'mucic acid obtained from lactose, raffinose, or galactan is calcu- lated as three fourths the weight of galactose which could be obtained by hydrolysis. The yield of mucic acid is, however, considerably influenced by the details of manipulation. In carrying out the method comparative determinations with pure milk sugar (both alone and mixed in known proportions with the sample under examination) should always be made, as sub- stances may be present which prevent the crystallization of the mucic acid. 1 HYDROLYSIS BY DILUTE ACIDS Monosaccharides, as the name implies, cannot be hydrolyzed to simpler sugars. As a rule, they are unaffected by dilute acids except on prolonged heating, when they are gradually attacked, yielding in part decomposition products like those produced by stronger acids, and undergoing a partial " rever- sion " with the formation of di- or polysaccharides. Dextrose, for example, when heated too long with dilute hydrochloric or sulphuric acid, is changed partially to " isomaltose " and to dex- trin-like anhydrides, such as " gallisin," the unfermentable con- stituent of crude commercial glucose. Levulose decomposes much more readily than dextrose on heating with dilute acids. Disaccharides differ considerably in the readiness with which they are hydrolyzed .by acids. Sucrose is very easily hydro- lyzed, a 20 per cent solution being completely changed to invert sugar by mixing with one tenth its volume of concen- trated hydrochloric acid (making about 3 per cent of actual acid in the mixture) and warming to 68 at such a rate as to require 15 minutes' heating. 2 Maltose is less easily hydrolyzed than sucrose, a 2 per cent solution in 2 to 3 per cent hydrochloric acid requiring 30 to 40 iHerzfeld: Z. Vereins Rubenzucker Ind., 1890, 40, 265; Abs. Chem. Ztg., 1890, 14, Rep., 108. Stone and Baird : J. Am. Chem. Soc., 1897, 19, 119. 2 Clerget's method, described by Wiley: Agricultural Analysis, Vol. III., pp. 105-107. According to Borntrager and to Samelson (Z. angew. Chem., 1892, 334 ; 1893, 690 ; 1894. 267, 351), the sucrose in such a mixture is completely hydrolyzed by standing overnight at room temperature. CARBOHYDRATES GENERAL METHODS 61 minutes' boiling, or 2 to 3 hours' heating on a water bath, for complete hydrolysis to dextrose. 1 Lactose is also less readily hydrolyzed than sucrose, the difference being especially marked in the case of weak acid or low temperature. Raffinose is hydrolyzed by dilute mineral acids, slowly in the cold, much more rapidly on boiling, requiring in either case more vigorous treatment than does sucrose. Dextrin, glycogen, and starch are hydrolyzed to dextrose under the same conditions as maltose, but require somewhat longer heating. Pentosans and galactans (at least the more common forms) are hydrolyzed by acids almost as readily as starch. Normal cellulose is not hydrolyzed by boiling dilute acids. Hemicellulose is a term commonly applied to the carbo- hydrate matter in the cell walls of plants, more resistant to enzymes than starch, but dissolved and hydrolyzed on boiling with dilute mineral acids. The hemicellulose of many of the common food plants consists largely of pentosans. REACTIONS WITH HYDRAZINES All of the monosaccharides, and maltose and lactose among the disaccharides, have the carbonyl group (as such or in the tautomeric form), and therefore on treatment with hydrazines yield hydrazones or osazones. The hydrazines chiefly used are phenylhydrazine, para-bromphenylhydrazine, methylphe- nylhydrazine (C 6 H 5 N(CH 3 ) NH 2 ), diphenylhydrazine, and naphthylphenylhydrazine. Phenylhydrazine has been most commonly employed, and its behavior with the different sugars has been most fully investi- gated. The analytical application of the phenylhydrazine re- action is discussed below. The other hydrazines are used largely for confirmatory tests and also for the purpose of distinguishing between sugars whose behavior with phenylhydrazine is not sufficiently 1 It is sometimes stated that maltose cannot be made to yield more than about 98 per cent of the theoretical amount of dextrose, the acid always begin- ning to attack the latter before the hydrolysis of the former is quite complete. See notes on the determination of starch. 62 METHODS OF ORGANIC ANALYSIS different to permit of satisfactory identification by that reagent alone. Mannose is distinguished from the other sugars by forming with phenylhydrazine an insoluble hydrazone, and this reaction has been applied quantitatively by Bourquelot and Herissey. 1 The phenylhydrazones of the other sugars are soluble, but when heated with an excess of phenylhydrazine react to form osazones. 2 To form the osazone, 2 dissolve one part of the sugar, two of pure phenylhydrazine hydrochloride, and three of crystallized sodium acetate in 20 parts of water in a test tube ; filter if not clear ; cork' loosely to avoid evaporation and place the tube in boiling water. Under these conditions the maximum yield of osazone is usu- ally obtained by warming the solution in the water bath for one to two hours and then allowing it to cool. The osazone thus obtained is ordinarily a yellow iridescent precipitate, more or less distinctly crystalline according to the purity and concentra- tion of the solution. To purify it, filter on a small paper, wash with a little cold water, dissolve in the smallest possible amount of boiling 50 per cent alcohol, and filter hot. The osazone which separates from the alcohol solution may, if desired, be further recrystallized from alcohol or from pyridine. Dextrose and levulose yield the same osazone, glucosazone, which crystallizes in needles melting at 204-205 when heated at such a rate that the melting point is reached in three to four minutes. G-alactosazone crystallizes in needles which melt at 193 ; maltosazone, in independent needles or tables melting at 206 ; lactosazone, in masses of microscopic prisms melting at 200. Xylosazone and arabinosazone melt at about 160. A conclusive method of ascertaining whether an osazone is that of a pentose, a hexose, or a disaccharide is to determine the percentage of nitrogen. In the case of a mixture, the hex- *J. Pharm. Chim., 1899, [6], 10, 206. See also Pellet : Bui. Assoc. Chim. Sucr. Distill, 1900-01, 18, 758 ; Abs. Z. Nahr. Genussm., 1902, 5, 74. 2 Fischer: Ber , 1884 17, 579 ; 1887, 20, 821 ; 1888, 21, 1805, 2631; 1889, 22, 87 ; 1890, 23, 2117. CARBOHYDRATES GENERAL METHODS 63 osazone can be freed from osazones of the other two groups by washing with hot water. Maltosazone is soluble in about 75 parts, xylosazone in about 50 parts, and lactosazone in 80 to 90 parts, of boiling water, while the pure hexosazones are nearly insoluble. Since the melting points of osazones are not very sharply defined and may be appreciably below the figures just given if the osazones are not entirely pure or are not heated at a suf- ficiently rapid rate, an entirely conclusive identification of a sugar by means of phenylhydrazine alone is usually not to be expected, but by similar reactions with some of the derivatives of phenylhydrazine above mentioned a characteristic hydrazone or osazone may be obtained. Thus Neuberg 1 identified arabinose by its diphenylhydra- zone and distinguished between glucose and fructose by the difference in their behavior toward methylphenylhydrazine ; Tollens and Maurenbrecker 2 also separate arabinose from xylose by the preparation of the diphenylhydrazone. Kahl 3 found that parabrombenzylhydrazide condenses to form an insoluble hydrazone with glucose, galactose, mannose, or arabinose ; not with levulose, maltose, or lactose ; and to only a slight extent with xylose. Kendall and Sherman 4 worked out a method by which this reaction can be made to serve for the identification of any one of the four reacting sugars, but the usefulness of this method is restricted by the fact that the preparation of the reagent is troublesome and time-consuming. Neuberg 6 made use of the differences in optical activity of the osazones as a means of identification. Dissolving 0.2 gram osazone in 4 cc. pyridine plus 6 cc. absolute alcohol and examin- ing the solution in a 100-mm. tube in the circular-scale polari- scope (see discussion of polariscope methods beyond), he obtained the following results: 1 Ber., 33, 2243 ; 35, 959. 3 Dissertation, Freiburg, 1904. 2 Ber., 38, 600. * J. Am. Chem. Soc., 1908, 30, 1451. 6 Ber., 1899, 32, 3384 64 METHODS OF ORGANIC ANALYSIS Compound Melting point Eotation Arabinosephenylosazone 160 _|_ 1 10' Arabinose-p-brornphenylosazone . . 196 00 -f 8' Xylosephenylosazone 158 15' Xylose-p-bromphenyloStizonc 9Q8 4-0 204-205 - 1 30' 222 31' Galactosephenyloso/zone 196-197 + 48' IVlaltosephenylosEizone . 206 -f 1 30' Lactosephenylosazone ' ... ... 200 4- Glucuronic acid-p-bromphenylhydrazine compound 216 - 7 25' Only rarely are the rotatory powers of the osazones of assist- ance in analytical work, since the differences are small and difficult to observe. By the application of the different hydrazines as reagents,, and the study of the products obtained with reference to solu- bility, crystalline form, melting point, etc., it is possible to identify the individual sugars even in such mixtures as are ob- tained by the hydrolysis of plant tissues or in complex artificial mixtures of carbohydrates which do not occur together in nature. For a full account of such methods see the works of Abderhalden, Browne, Lippmann, Oppenheimer, and Tollens. If, however, the problem is to identify a pure sugar or a simple mixture of sugars, it will usually be more convenient to apply the osazone reaction in the manner described below and depend upon other methods to complete the identification as described later. ANALYTICAL APPLICATION OF THE OSAZONE REACTION Maqueiine 1 found that the reducing sugars when treated in a uniform way with phenylhydrazine showed considerable differ- ences both in the yield of osazone and in the time required for the appearance of the osazone precipitate. Mulliken 2 has 1 Compt. rend., 112, 799. 2 Identification of Pure Organic Compounds, Vol. I. CARBOHYDRATES GENERAL METHODS 65 studied these differences in rapidity of osazone formation and makes use of them to an important extent in his scheme, for the identification of pure sugars. According to Mulliken, 0.1 gram sugar, 0.2 gram pure phenylhydrazine hydrochloride, 0.3 gram sodium acetate, and 2 cc. water are mixed in a small test tube, corked loosely to prevent evaporation, and heated in' boil- ing water. If the tube is occasionally shaken without removing it from the boiling water, the osazone precipitate usually sepa- rates out quite suddenly so that duplicate experiments usually give results that agree within half a minute. Under these, con- ditions the time required for the appearance of the osazone in the hot solution is given by Mulliken as follows : Fructose, two minutes ; sorbinose, three and one half minutes ; glucose, four to five minutes ; xylose, seven minutes ; rhamnose, nine min- utes ; arabinose, ten minutes ; galactose, fifteen to nineteen minutes. Sucrose, after about thirty minutes' heating, ifs suffi- ciently hydrolyzed to yield a slight precipitate of glucosazone. Maltose and lactose give no precipitate in the hot solution even when the heating is continued for two hours. Sherman and Williams 1 followed closely the conditions adopted by Mulliken except that, for greater convenience of manipulation, twice the quantities were used throughout. Having confirmed the results above given for glucose, fructose, sucrose, maltose, and lactose at the standard dilution, they de- termined the times required for the osazone precipitation with smaller amounts of glucose or fructose in pure solution and also when different amounts of other sugars were present at the same time. Every test was carried out as has been described!^ using 0.4 gram phenylhydrazine hydrochloride, 0.6 gram sodium acetate, and 4 cc. water, 2 so that the only variable factor was the amount of sugar or sugars present. The tabular statements which follow show the time of heating required for the appearance of an osazone precipitate in each case. 1 J. Am. Chem. Soc., 1906, 28, 629. 2 If the solution at this point is not clear, it is filtered through a dry paper before heating. F 66 METHODS OF ORGANIC ANALYSIS PURE SOLUTIONS OF GLUCOSE, FRUCTOSE, INVERT SUGAR, OR SUCROSE Weight of sugar taken Gram Glucose Minutes Fructose Minutes Invert sugar Minutes Sucrose Minutes 0.2 4-5 l ; l_ll 1HI 31 0.1 5 lf-2 2 35 - 0.05 6| 2* 3 78 0.01 17 5 6-6$ No ppt. 0.005 34 10 14 0.0025 65 17 With less than 0.005 gram glucose or 0.0025 gram fructose, the amount of osazone separating in the hot solution was small and the time of its appearance doubtful. INFLUENCE OF MALTOSE ON GLUCOSE Weight of glucose Gram 0.01 0.02 Weight of maltose 0.2 grain No ppt. 26-28 min. 0.1 gram 40 min. 0.05 gram 30 rnin. 0.01 gram 22 min. Weight of glucose Gram 0.01 0.02 INFLUENCE OF LACTOSE ON GLUCOSE Weight of lactose 0.2 gram No ppt. 0.1 gram 50 min. 0.05 gram 32 min. 0.01 gram 25 min. 45-48 min. In absence of maltose 17 min. 12-13 min. In absence of lactose 17 min. 12-13 min. It is evident that both maltose and lactose interfere seriously with the formation and precipitation of glucosazone and that the influence of lactose is greater than that of maltose. Thus a mixture of 0.01 gram glucose and 0.1 gram lactose required ten minutes' longer heating than a parallel mixture with 0.1 gram maltose, and when the quantities of glucose, lactose, and maltose are doubled (the amounts of reagents and the volume of the solution remaining the same) the lactose mixture required twenty minutes' longer heating than the maltose mixture. INFLUENCE OF SUCROSE ON GLUCOSE Weight of glucose Gram 0.005 0.01 AVeight of sucrose In absence of sucrose 33-39 min. 17 min. 0.2 gram 15-17 min. 14-16 min. 0.1 gram 15-17 min. 16 min. 0.05 gram 22 min. 17 min. 0.01 gram 30 min. 17 min. CARBOHYDRATES GENERAL METHODS 67 Weight of fructose Gram 0.01 Weight of fructose Gram 0.01 INFLUENCE OF MALTOSE ON FRUCTOSE Weight of maltose 0.2 gram 7-8 min. 0.1 gram min. 0.05 gram 0.01 gram 5| min. INFLUENCE OF LACTOSE ON FRUCTOSE Weight of lactose 0.2 gram MO min. 0.1 gram 7| min. 0.05 gram 6| min. 0.01 gram 6 min. In absence of maltose 5 min. In absence of lactose 5 min. Comparing these results with the corresponding figures for glucose, it will be seen that the interference of maltose and lac- tose is less marked with fructose than with glucose. In both cases, however, the appearance of the osazone precipitate is re- tarded distinctly by maltose and to a greater extent by lactose. Weight of fructose Gram 0.005 INFLUENCE OF SUCROSE ON FRUCTOSE Weight of sucrose 0.2 gram 8 min. 0.1 gram 8| min. 0.05 gram 9i min. 0.01 gram 9 min. In absence of sucrose 9i min. Here the effect of the sucrose was not so noticeable. Interpretation of time relations in applying the osazone test to sugar mixtures or to samples in which the concentration of the reacting sugar is not known or is different from that of the conventional method, should be based on a careful study of the above data. The delicacy of the test in the presence of dextrin (which is known to retard the formation of the osazone), and of sub- stances other than carbohydrates, has not been studied in de- tail, except in the case of certain constituents of urine. 1 Hence in applying the osazone test to an unknown solution, it is well to make at the same time two check experiments, one with a mixture of carbohydrates corresponding to that which the un- known solution is believed to contain, the other with a portion 1 The influence of other substances likely to be present, upon the osazone test for dextrose in urine has been studied by Hirschl : Z. physiol. Chem., 1890, 14, 377. Jaff: Ibid., 22, 532. Neuberg: Ibid., 1900, 20, 274. Neumann- Wender : Pharmac. Post., 26, 673, 614 (Vaubel ; I. c., II, 68 METHODS OF ORGANIC ANALYSIS of the unknown solution to which has been added a very small amount of dextrose. The yield of osazone has been studied especially by Maquenne, 1 Laves, 2 Fischer, 3 Lintrier and Krober, 4 Raimann, 5 and Davis and Ling. 6 In each of these cases the osazone was formed by heating for. one or two hours, in a more dilute solution than that above given, with a considerable excess of a slightly acid solution of phenylhydrazine acetate. The precipitate of osa- zone can then be filtered, washed with water, and weighed. According to Laves the following corrections should be applied for the amount of glucosazone left in solution or dissolved by washing : 100 parts boiling water dissolve 0.01 part osazone 100 parts water at 20 dissolve 0.0042 part osazone 100 parts 2 per cent acetic acid at 20 dissolve . . . 0.007 part osazone 100 parts 3 per cent acetic acid at 20 dissolve . . . 0.0145 part osazone 100 parts 4 per cent acetic acid at 20 dissolve . . . 0.022 part osazone 100 parts 5 per cent acetic acid at 20 dissolve . . . 0.031 part osazone 100 parts 10 per cent alcohol slightly acidulated dis- solve at 20 0.0075 part osazone Corresponding corrections have not been worked out for other osazones, as the attempts to apply the osazone method quantitatively have been mainly with the object of determining dextrose or dextrose and levulose. In comparative tests carried out in exactly the same manner, the weight of osazone obtained is proportional to the amount of sugar originally present, but slight differences of manipulation affect the yield to such an extent that the osazone precipitation cannot yet be regarded as a satisfactory quantitative method. Williams 7 has recently studied the conditions affecting the yield of glucosazone from pure glucose solutions. It was found best to allow one hour of heating for the formation of the 1 Compt. rend., 1891, 112, 799 ; Z. anal. Chem., 1894, 33, 226. 2 Archiv. der Pharm., 1893, 231, 366. Vaubel's Bestimmung organischer Verbindungen, II, 307, 311, 312. 3 Ber., 1895, 28, 1437. 4 Z. /. d. ges. Brauwesen, 1895, 18, 153; Z. anal. Chem., 1896, 35, 95; Analyst, 1897, 20, 167. 5 Z. anal. Chem., 1901, 40, 390. 6 J. Chem. Soc., 1904, 85, 24. 7 Data not yet published. CARBOHYDRATES GENERAL METHODS 69 osazone, since longer heating, although increasing the yield, gave a less pure product. A solution of phenylhydrazine acetate made by dissolving the free base in acetic acid gave as good results as the reagent made by mixing phenylhydrazine hydrochloride with sodium acetate and was found more conven- ient. The amounts of phenylhydrazine and acetic acid greatly influenced the yield of osazone. In a series of experiments in which 0.2 gram pure dextrose in 20 cc. water was heated with varying amounts of reagents for one hour, then washed with 100 cc. water, dried and weighed, it was found that maximum yields, about 60 per cent of the theoretical, were obtained by using 2 grams of phenylhydrazine in 8 cc. of 50 per cent acetic acid, 2.4 grams phenylhydrazine in 9.2 cc. of 50 per cent acetic acid, or 3.2 grams of phenylhydrazine in 6.4 cc. of 50 per cent acetic acid. Thus while a large excess of phenylhydrazine was always essential to high yields, the amount required for a maxi- mum yield depended upon the ratio between the phenylhydra- zine and the acetic acid, as well as upon that between the reagents and the sugar. REDUCTION OF COPPER SOLUTIONS The same sugars which react with phenylhydrazine have the power of reducing certain metallic salts, especially salts of copper, silver, and mercury in alkaline solution. This "re- ducing power," due to the susceptibility of the carbonyl sugars to oxidation, can be utilized for the detection and determina- tion of these sugars. When for example an alkaline cupric solution, such as- Feh- ling's, is boiled with one of these " reducing " sugars, the copper is reduced to the cuprous state and the sugar is at- tacked in two ways : (1) oxidation and (2) decomposition by the alkali. Tartronic acid is often cited as a typical oxida- tion product formed by the action of boiling alkaline copper solution upon dextrose, but in practice less than half as much copper is reduced as would correspond to a complete oxidation of glucose to tartronic acid, because the boiling alkali so largely decomposes the glucose into other than oxidation products. 70 METHODS OF ORGANIC ANALYSIS In order that the amount of copper reduced may indicate quantitatively the amount of reducing sugar, it is necessary to control the decomposing action of the alkali. This is usu- ally accomplished by prescribing the amount and final dilu- tion of the alkaline solution and either (1) adjusting the sugar solution so as to find the amount which in measured volume will exactly reduce the copper as described below for Fehling's volumetric method, or (2) treating a fixed amount of the reagent with a prescribed volume of the reducing sugar solu- tion of a strength insufficient to reduce all the copper and collecting and weighing the copper reduced as in Defren's gravimetric method described later. FEHLING'S VOLUMETRIC METHOD Reagents. (1) Copper solution : Dissolve 34.64 grams of pure crystallized copper sulphate in water, and dilute to ex- actly 500 c.c. (2) Alkaline tartrate solution : Dissolve 175 grams of pure sodium potassium tartrate and 50 grams of pure sodium hy- droxide in water, and dilute to 500 c.c. This reagent does not keep well unless carefully protected from the air (Note 1). Determination. Measure accurately into a small flask or casserole or a deep porcelain dish, 5 cc. of each of the above solutions, making 10 cc. of the "mixed Fehling reagent." Add 40 cc. of water, mix, and boil. To the boiling liquid, add from a burette a solution which contains not over 1 per cent of the reducing sugar to be determined, boiling two minutes after each addition of sugar until the blue color is entirely discharged, showing that all of the copper has been reduced. This test indicates approximately the amount of reducing sugar in the sample. Now adjust the strength of the sugar solution, if necessary, so that about 20 cc. will be re- quired to reduce 10 cc. of the mixed Fehling reagent. Repeat the test, adding the calculated amount of sugar solution at once to the boiling copper solution ; regulate the heating so that the mixture will again begin to boil about one minute CARBOHYDRATES GENERAL METHODS 71 after the addition of the sugar ; note the exact time that actual boiling commences and continue to boil for just two minutes, then remove the flame and at once test the liquid for unre- duced copper (Note 2). Repeat the test, using more or less of the sugar solution depending upon the presence or absence of an excess of copper in the preceding experiment until two amounts of sugar solu- tion are found which differ by only 0.1 or 0.2 cc., one giving complete reduction and the other leaving a small amount of copper in the cupric state. The mean of these two amounts is taken as the volume of solution required for complete reduc- tion of the copper reagent. Note 1. All of the reagents used must be the purest ob- tainable, and the two constituents of the "mixed Fehling reagent " must be kept separate instead of being made up in one solution as was formerly done. If the solutions are not fresh, make a blank test in a casserole, boiling as above with- out the addition of any sugar, allow to stand for a few minutes, then decant off the liquid and notice whether any cuprous oxide has been precipitated. Finally wipe out the casserole with a small piece of filter paper and examine the latter, which may show traces of cuprous oxide not visible in the presence of the blue solution. If the liquid shows any change of color in this blank test, or if the slightest trace of cuprous oxide is found, the alkaline tartrate solution must be rejected and another blank test made with a freshly prepared solution. Note 2. So long as the solution shows a distinct blue color it is unnecessary to apply other tests for cupric copper. When the test is made in a flask, the blue color is best seen by holding level with the eye and looking horizontally through the meniscus, but on account of the presence of the cuprous oxide the disappearance of the blue color is not alone a safe criterion. When the blue color is no longer apparent, test for cupric copper by one of the following methods. It is well for each analyst to try all three methods and select the one which he finds most satisfactory for the conditions of his work. 72 METHODS OF ORGANIC ANALYSIS Whichever test is used it is important to work quickly lest reduced copper be oxidized by contact with the air. Ferrocyanide Test. Quickly filter a portion of the liquid through two or three thickness of paper, repeating the filtration if necessary, observe carefully that the filtrate is free from any trace of cuprous oxide, then acidulate with acetic acid and add a few drops of a dilute solution of potassium f errocyanide when if copper is present a red-brown coloration or precipitate will appear. Watts and Tempany suggest as a modification of this test that the liquid instead of filtering be poured upon a small pad consisting of several layers of filter paper, and the bottom paper (which should be wet with the solution but free from cuprous oxide) be removed, acidulated with acetic acid, and then tested with ferrocyanide. Starch-iodide Test. 1 As soon as the sugar solution and the copper reagent have been boiled together for the required two minutes, add a drop or two of the solution (which need not be filtered from cuprous oxide) to a considerable excess of cold acidulated starch-iodide solution, when, if cupric copper is present, a blue or purple coloration is obtained. The starch-iodide solution is prepared as follows: Boil 0.02 gram starch with 15 to 20 cc. of water, cool, add 4 to 5 grams of potassium iodide and dilute to 50 cc. A fresh solution must be prepared each day. When the test is to be made, pour about 1 cc. of this starch-iodide solution into a test tube, add two or three drops of acetic acid and then immediately a drop or two of the solution to be tested. Thiocyanate Test. 2 This requires a solution of ferrous thio- cyanate which may be prepared as follows : Dissolve 1 gram ferrous sulphate and 1.5 gram ammonium thiocyanate in 10 cc. water at about 45 to 50 C., cool immediately, add 2| cc. con- centrated hydrochloric acid and a trace of zinc dust to decolor- ize the solution. 1 Harrison: Pharm. J., 1903, 170 (Button's Volumetric Analysis, 9th Ed., p. 312.) 2 Ling and Rendle : Analyst, 1905, 30, 183. Ling and Jones : IUd., 1908, 33, 160. CARBOHYDRATES GENERAL METHODS 73 Place a drop of the freshly prepared ferrous thiocyanate solution on a porcelain plate and add a drop of the solution to be tested (which need not be filtered), when, if cupric copper is present, the well-known red color of ferric thiocyanate will be produced. Calculation and Verification of Results. In calculating the results it is commonly assumed that 10 cc. of the mixed Fehl- ing reagent require for reduction under the above conditions: 0.0500 gram of anhydrous dextrose, levulose, or invert sugar. 0.0678 gram of dry crystallized lactose (C 12 H 22 O n - H 2 O). 0.0807 gram of anhydrous maltose. That these factors are not absolute, but are dependent upon exact uniformity of conditions has already been explained. To secure the greatest accuracy, therefore, the result obtained should be verified by a check experiment, carried out under the exact conditions of the analysis, with a known solution of pure sugar of the kind actually determined. It is sometimes convenient to express the reducing power of some other sugar or of a mixture in terms of the reducing power of dextrose, the latter being taken as 100. Thus, if 0.0678 gram lactose or 0.0807 gram maltose has the same reducing power as 0.05 gram dextrose, then on the basis of dextrose = 100, the reducing power of lactose is X 100 05 = 74, and that of maltose is X 100 = 62. .0807 According to Soxhlet, levulose and galactose have distinctly less reducing power than dextrose. It is quite commonly as- sumed, however, that these three monosaccharides have the same reducing power, but that invert sugar often fails to show its full effect because of the decomposing action of the acid used for inversion. Xylose and arabinose reduce Fehling's solution somewhat more strongly than does dextrose. 74 METHODS OF ORGANIC ANALYSIS DEFREN'S GRAVIMETRIC METHOD 1 This method is based on that of O'Sullivan 2 and provides a uniform procedure for the determination of dextrose, maltose, or lactose. Reagents. (1) Dissolve 34.64 grams of copper sulphate in water, add 0.5 cc. strong sulphuric acid, and dilute to 500 cc. (2) Dissolve 178 grams of sodium potassium tartrate and 50 grains of sodium hydroxide in water and dilute to 500 cc. Determination. Mix 15 cc. of each of the above reagents in an Erlenmeyer flask having a capacity of 250 to 300 cc., dilute with 50 cc. of freshly boiled distilled water, and place the flask in a boiling water bath for five minutes ; then add 25 cc., ac- curately measured from a burette or pipette, of a solution con- taining approximately 0.5 per cent of the sugar to be deter- mined and allow the mixture to stand in the boiling water bath for fifteen minutes. Remove the flask from the bath and filter at once (using moderate suction) through asbestos prepared as described below (Note 1); wash the cuprous oxide with boiling distilled water until the filtrate is no longer alkaline. The cu- prous oxide can now be (1) washed with alcohol and then with ether, dried in a boiling water oven for 20 minutes, and weighed (Note 2) ; (2) ignited and weighed as cupric oxide, as recom- mended by Defren ; or (3) dissolved in nitric acid and the copper determined by electrolysis or by any other reliable method, in which case it will not be necessary to use asbestos especially pre- pared by boiling with acid and alkali. From the weight of copper or cuprous oxide determined, calculate the equivalent amount of cupric oxide and find the corresponding weight of reducing sugar from Defren's table (Note 3). Note 1. The filtrate from the cuprous oxide must always be distinctly blue, showing that a sufficient excess of Fehling solution was used, otherwise the determination must be repeated, using a more dilute solution of the reducing sugar. If the copper reduced is to be weighed as cuprous or cupric oxide on the asbestos filter, the latter must be especially prepared in *J. Amer. Chem. Soc., 1396, 18, 749. 2 J. Chem. Soc., 1876, 30, 130. CARBOHYDRATES GENERAL METHODS 75 order that it shall lose no weight when treated with the hot alkaline Fehling solution. Asbestos of good quality is boiled with nitric acid (1.05 to 1.10 sp. gr.), washed with water, then boiled with 25 per cent sodium hydroxide, washed, and the treatment with acid and alkali repeated. The prepared asbes- tos is used to make a tight felt about 1 centimeter thick in a Gooch crucible. When the crucible has been prepared for use and weighed, it should be tested by running through it a " blank " of hot alkaline Fehling solution and washing with water as in a regular determination. The loss of weight should not exceed one half milligram. After each determination the precipitate is dissolved in nitric acid, and the crucible washed, ignited, and reweighed. If a loss of over one milligram is found, the determination should be rejected and the filter treated alternately with acid and alkali until it ceases to lose in weight. Note 2. The method of weighing the cuprous oxide as such is convenient, and when working with fairly pure sugar mixtures is accurate, but impure material, such as crude raw sugar, molasses, or malt extract, is liable to leave some organic matter with the cuprous oxide on the filter so that for the most accurate results with such materials, one should ignite to cupric oxide or dissolve and determine copper. Note 3. Defren determined the amount of copper reduced by fifteen to twenty known solutions each of dextrose, maltose, and lactose, with the following results: Dextrose= (0.4400 + 0.000037 W) W. Maltose =(0.7215 + 0.000061 W)W. Lactose =(0.6270 + 0.000053 W)W. In which Wis the weight of cupric oxide obtained, the values of W varying from 30 to 320 milligrams. From these formulae it is apparent that the reducing power of each of the sugars increases slightly with the concentration ; hence, to find the amount of reducing sugar corresponding to any given weight of copper, one must use, not a simple factor, but a formula or (more conveniently) a table. 76 METHODS OF ORGANIC ANALYSIS Table 7 is condensed from that given in Defren's paper (Z. Dextrin s 160 to 210 190 to 210 In order to avoid confusing the determination of specific ro- tatory power with the conventional polariscope examination of *' raw sugar " described in the next chapter, it is well for the student at this point to determine the specific rotatory power of a sample of sugar of known purity. REFERENCES I ABDERHALDEN : Handbuch der Biochemisches Arbeitsmethoden. ALLEN : Commercial Organic Analysis. BROWNE : Handbook of Sugar Analysis. HOPPE-SEYLER : Physiologisch und Pathologisch-Chemischen Analyse. LANDOLT : Optical Rotation of Organic Substances. LIPPMANN : Chemie der Zuckerarten. MAQUENNE : Les Sucres. MEYER and JACOBSON : Organische Chemie. MULLIKEN : Identification of Pure Organic Compounds. OPPENHEIMER : Handbuch der Biochemie. ROLFE : The Polariscope in the Chemical Laboratory. TOLLENS : Chemie der Kohlen hydrate. VAUBEL : Bestimmung Organischer Verbindungen. WILEY : Agricultural Analysis, Vol. III. II 1905. LING and RENDLE : The Volumetric Determination of Reducing Sugars. Analyst, 30, 182. 1906. BROWNE: The Analysis of Sugar Mixtures. J. Am. Chem. Soc., 28, 439. MUNSON and WALKER : Unification of Reducing Sugar Methods. /. Am. Chem. Soc., 28, 663. See also Walker, same, 29, 541 ; 34, 202. 86 METHODS OF ORGANIC ANALYSIS 1907. BANG : (Determination of Reducing Sugars by means of Hydroxyl- amine). Biochem. Z., 2, 271 ; Chem. Abs., 1, 590. BATES : A Quartz Compensating Polariscope with Adjustable Sen- sibility. U. S. Bur. Standards, Bui. 4. Also Z. Ver. Zuckerind., 58, 105, 821 ; Chem. Abs., 2, 1219, 3165. BENEDICT: The Detection and Estimation of Reducing Sugars. J. Biol. Chem., 3, 101. BROWNE and HALLIGAN : Report on Sugar. U. S. Dept. Agr., Bur. Chem., Bui. 105, p. 116. HINKEL and SHERMAN : Experiments on Barfoed's Acid Cupric Acetate Solution as a Means of Distinguishing Glucose from Maltose, Lactose, and Sucrose. J. Am. Chem. Soc., 29, 1744. HUDSON: Action of Acids and Bases on the Mutarotation of Glu- cose. /. Am. Chem. Soc., 29, 1571. MATHEWS arid McGuiGAN: The Oxidation of Sugars by Cupric Acetic Acid Mixtures. Proc. Soc. Exp. Biol. Med., 1907 ; Chem. Abs., 1, 1780. NEF : Behavior of Sugars toward Fehling Solution as well as toward other Oxidizing Agents. Ann. Chem., 357, 214; Chem. Abs., 2, 799. 1908. BUNZEL : The Rate of Oxidation of Sugars in an Acid Medium. Am. J. Physiol., 21, 23. BROWNE : Determination of Reducing Sugars from Weight of Cuprous Oxide. Intern. Sugar J., 10, 537; Chem. Abs., 3, 385. KENDALL and SHERMAN : The Detection and Identification of Cer- tain Reducing Sugars by Condensation with p. Brombenzylhy- drazide. J. Am. Chem. Soc., 30, 1451. LING and JONES : (Accuracy of Determination of Reducing Sugars) . Analyst, 33, 160. MEISENHEIMER : Behavior of Glucose, Fructose, and Galactose toward dilute Sodium Hydroxide. Ber., 41, 1009. 1910. HERSTEIN: (Historical Sketch of Fehling's Solution). J. Am. Chem. Soc., 32, 779. HUDSON: Mutarotation. J. Am. Chem. Soc., 32, 889. NEF : Behavior of Sugars toward Caustic Alkalies. Ann. Chem., 376, 1. SCHLIEPHACKE : Mutarotation of Maltose. Ann. Chem., 397, 164. 1911. BANG: Preparation of Copper Solutions for Sugar Titration. Biochem. Z., 32, 443. BENEDICT : A Method for the Estimation of Reducing Sugars. /. Biol. Chem., 9, 57. FISCHER : (Determination of Rotatory Power on Small Amounts of Material). Ber., 44, 129. 1912. KENDALL : A New Method for the Determination of the Reducing Sugars. J. Am. Chem. Soc., 34, 317. CHAPTER IV Special Methods of Sugar Analysis ANALYSIS OF RAW SUGAR POLAKISCOPIC EXAMINATION A SPECIAL room of even temperature which can be darkened when desired, or a dark screen which can be placed around the instrument on the laboratory table, should be provided for the polariscopic examination. For illumination of the white-light saccharimeter, a triple flame or an Argand burner with incan- descent mantle is used. In taking readings the polariscope must be brought only near enough to the burner to secure a good illumination of the field, never within less than eight inches. As soon as the reading is taken the polariscope should be turned away or the flame lowered, in order to avoid any possible warming of the instrument. Determination of the Zero Point. The trough of the sac- charimeter being empty, set the scale within a few degrees of zero and focus the eyepiece so that the field of vision is clear and the perpendicular line or band dividing it is perfectly dis- tinct. Now rotate the milled head so as to move the zero point of the scale toward that of the vernier. When the neutral point (the true zero point for the instrument as it stands) is reached, the appearance of the entire field is uniform. Approach the zero point first from one side then from the other, taking the reading each time as soon as the entire field appears uni- form, until successive readings do not differ by more than 0.2 on the Ventzke sugar scale. The average of six to ten such readings is taken as the zero point in the subsequent work of 87 88 METHODS OF ORGANIC ANALYSIS the day, unless the instrument should be jarred or moved, in which case the zero point should be redetermined. Test with Pure Sugar. Weigh 26 l grams of pure sucrose, dissolve in water in an accurately calibrated 100-cc. flask, fill to the mark at 20 to 22, and mix thoroughly by shaking, hold- ing the flask in such a way as not to warm the solution. The latter should have a density very nearly 1.10 and should rotate the plane of polarized light 34. 68 to the right, giving a reading of 100.0 on the Ventzke scale. Rinse the 200-mm. tube several times with the solution, then fill (by pouring through a dry filter, rejecting the first portions of the filtrate) until the curved surface of the liquid projects above the open end of the tube ; see that all air bubbles have risen to the surface, and then slide on the cover-glass horizontally in such a manner that the excess of liquid is carried over the side, leaving the cover glass exactly closing the tube, with no air bubbles beneath it and none of the liquid upon its upper surface. The cover glass being in position, the tube is closed by screwing on the cap. The latter should be only tight enough to prevent leakage, as any considerable pressure on the glass plate may cause it to become optically active. Look through the tube lengthwise to be sure that the cover glasses are clean and dry and the contents of the tube perfectly clear. Place the tube in the trough of the saccharimeter or polari- scope and take readings as in setting the zero point. Unless the ends of the polariscope tube are ground absolutely parallel, the position of the tube in the instrument may influence slightly the length of column of sugar solution in the line of vision. 1 The value of the Ventzke scale was originally fixed by means of pure sucrose solutions of 1.100 sp. gr. at 17.5 and it was found that 100 cc. of such a solution contains 26.048 grams of sucrose (weighed in air with brass weights). For many years, however, it has been the custom of instrument makers to cali- brate the Ventzke scale by means of solutions containing 26.048 grams of sucrose in 100 Mohr's cubic centimeters. A solution of very nearly the same strength is obtained by dissolving 26 grams and completing the volume at 20 to 100 metric cubic centimeters (the volume occupied by 100 grains of water at 4 weighed in vacuo). The latter proportions have been adopted by the Interna- tional Commission for Unifying Methods of Sugar Analysis. SPECIAL METHODS OF SUGAR ANALYSIS 89 In order to be sure that the length whose rotation is measured is the true length of the polariscope tube, take two or four readings, then turn the tube two thirds over in the trough of the instrument, and after taking two or four readings in this position turn the tube again two thirds over in the same direc- tion for the final two or four readings. After a little practice it will probably be found sufficient to take one reading approach- ing the end point from the right and one from the left in each of three positions of the tube, or six readings in all. The aver- age reading (corrected for zero point) should not differ from 100.0 by more than 0.2 on the Yentzke scale, nor from 34 41' on the Laurent polariscope, by more than 5'. Polarization of the Raw Sugar. Mix the sample, weigh out 26 grams, and dissolve in 60 to 80 cc. of water in a 100 cc. flask. When the sugar is entirely dissolved, add from 1 to 5 cc. (according to the nature of the sample ; only very dark sugars should require more than 2 cc.) of a solution of basic lead acetate of about 1.25 sp. gr. 1 A decided excess of basic acetate should be carefully avoided. This may be done by add- ing the lead solution a few drops at a time, shaking after each addition, and stopping as soon as another drop of the solution produces no further precipitate. After the lead acetate has been added and mixed, add twice its volume of "alumina cream." 2 This assists in the clarifica- tion, precipitates the excess of lead, and facilitates filtration. A moderate excess of alumina cream does no harm. With high grade sugars the use of alumina cream alone may be sufficient for clarification. Make up to volume with distilled 1 This may be prepared by dissolving the solid basic salt or by boiling an ex- cess of litharge with a strong solution of neutral lead acetate. 2 Prepared as follows : Shake powdered commercial alum with water at ordinary temperature until a saturated solution is obtained. Set aside a little of the solution, and to the residue add ammonia, little by little, stirring between additions, until the mixture is alkaline to litmus paper. Then drop in additions of the portion left aside, until the mixture is just acid to litmus paper. By this procedure a cream of aluminium hydroxide is obtained suspended in a solution of ammonium sulphate. This sulphate is advantageous when added after the basic acetate, since it precipitates whatever excess of lead may be present. 90 METHODS OF ORGANIC ANALYSIS water, 1 shake well, and then pour the whole solution, or as much as practicable, on a dry filter. Reject the first 20 or 30 cc. of filtrate and then polarize the remainder as already de- scribed for the pure sugar solution. The average reading of the Ventzke scale, corrected for the deviation of the zero point, is reported as "polarization." Notes and Precautions. Care must be taken to avoid the following errors due to manipulation : (1) change in moisture content of sample during weighing, (2) change in volume of solution due to fluctuations of temperature, (3) imperfect mix- ing of solution after diluting to volume in the graduated flask, (4) evaporation during filtration, (5) too great compression of cover glasses in closing the polariscope tube. Clarification is very important. All proteins are levorota- tory and must be entirely removed. The solution must be free from turbidity, but not necessarily free from color. Only so much of the clarifying agents should be used as is necessary to free the solution from optically active impurities and from turbidity. Any excess increases the error due to the presence of precipitate when the solution is diluted to volume. The volume occupied by the precipitate varies between 0.05 and 1.0 cc. for ordinary raw sugars. It can be determined by Scheiber's method of double dilution, 2 in which a duplicate determination is made in a flask of twice the volume, or by determining the weight and density of the precipitate as rec- ommended by Sachs. 3 The latter method is preferred by Wiechmann 4 and Home, 5 each of whom determined the volume of the precipitate for a number of raw sugars from different localities. Home has found (loc. cit.) that the error can be almost entirely avoided by clarifying with anhydrous basic acetate after the sugar solution has been diluted to volume, and this method of clarification, after extended investigation 1 If frothing interferes at this point, add two or three drops of ether. 2 Z. Vereins. Biibenzucker Industrie, 1875, 25, 1054. 3 /bid., 1880, 50, 229. See also the paper by Home. 4 Ibid., 1903, [n. f.] 40, 498 ; Abs. J. Chem. Soc., 1903, 84, ii, 699. 6 J. Am. Chem. Soc., 1904, 26, 186. SPECIAL METHODS OF SUGAR ANALYSIS 91 and discussion, has now been adopted by the International Commission on a parity with the usual method. According to Browne, the amount of dry acetate used should not exceed 0.5 gram. Regulations of the International Commission The following resolutions, 1 adopted by the International Commission for the Unification of Sugar Analysis, in 1900, have since been generally accepted. 1. In general, all sugar tests shall be made at 20 C. 2 2. The graduation of the saccharimeter shall be made at 20 C. Twenty-six grams of pure sugar, dissolved in water, and the volume made up to 100 metric cubic centimeters, or during the period of transition 26.048 grams of pure sugar in 100 Mohr cubic centimeters, all weighings to be made in air with brass weights, the completion of the volume and the polariza- tions to be made at 20 on an instrument graduated at 20, should give an indication of 100 on the scale of the saccharim- eter. For countries where temperatures are usually higher than 20, it is permissible that saccharimeters be graduated at 30, or any other suitable temperature, under the conditions specified above, providing that the analysis of the sugar be made at the same temperature that is, that the volume be completed and the polarization made at the temperature specified. 3. Preparation of pure sugar: Purest commercial sugar is to be further purified in the following manner: A hot satu- rated aqueous solution is prepared and the sugar precipitated with absolute ethyl alcohol ; the sugar is carefully spun in a 1 U. S. Dept. Agriculture, Bur. Chem., Bull. 73, p. 58. J. Am. Chem. Soc., 1901, 23, 59. 2 In polarizing pure sucrose with quartz wedge saccharimeter there is a falling off in polarization of about 0.03 Ventzke for each degree C. that the tempera- ture rises above 20. Crude sugars, however, may contain enough levulose so that their polariscope readings are increased by a rise of temperature. Browne has clearly shown that temperature errors cannot be compensated by corrections based on pure sucrose, but must be avoided by actually polarizing at or near 20 C. 92 METHODS OF ORGANIC ANALYSIS small centrifugal machine and washed in the latter with abso- lute alcohol. The sugar thus obtained is redissolved in water, the saturated solution again precipitated with alcohol and washed as above. The product of the second crop of crystals is dried between blotting paper and preserved in glass vessels for use. The moisture still contained in the sugar is deter- mined and taken into account when weighing the sugar which is to be used. The committee further decided that central stations shall be designated in each country which are to be charged with the preparation and distribution of chemically pure sugar. Wherever this arrangement is not feasible, quartz plates, the values of which have been determined by means of chemically pure sugar, shall serve for the control of the saccharimeters. The committee further decided that the above control of quartz plates by means of chemically pure sugar should, as a rule, apply only to the central stations which are to test the correctness of saccharimeters ; for those who execute commer- cial analyses, the repeated control of the instruments is to be accomplished, now as before, by quartz plates. 4. In effecting the polarization of substances containing sugar, half-shaded instruments, or triple field, only are to be employed. 5. During the observation the apparatus must be in a fixed position and so far removed from the source of light that the polarizing nicol is not warmed. 6. Sources of light may be gas, triple burner with metallic cylinder, lens, and reflector ; gas lamp with Auer (Welsbach) burner ; electric lamp ; petroleum duplex lamp ; sodium light. Several readings are to be made and the mean thereof taken, but any one reading must not be neglected. 7. In making a polarization the whole normal weight for 100 cubic centimeters is to be used, or a multiple thereof for any corresponding volume. 8. As clarifying and decolorizing reagents there may be used : (a) subacetate of lead, (3 parts by weight of acetate of lead, one part by weight of oxide of lead, 10 parts by weight SPECIAL METHODS OF SUGAR ANALYSIS 93 of water) ; (5) alumina cream ; ( t> O O r-l r-l CO T I O CO IO "<;fl "* t- r-l tQ r-l COCO^f OCO OOC^OOO t CM iO CO * CO CO iO CO -I -5 ^-/ 0< s"8 SH ^ ll a pa 1 1? a jj x .1 si ^ s co 3 O S Je d eS OPQ^I P^O Petroleum, ozoke Wood of several CM oo o :t Paraffin llosin ( OILS, FATS, AND WAXES 171 ce .3 I I I fl ,-H I A ';=! li fi "5 rO C8 S.S- 1| Sp=^ Soga cc co fe s-i- g~ I s ^ B 3Stf ; T3 ? - 1 S ^I- eo :: ^9 o *g "o o II a% 51 ^ fcc ^ e D B gja ^ga o _ fco (N COOCD CO^CM-HHOi OiCO CO GNI Oi Oi ir to T-H tO ^ Tf^ TJI CO "^ CO CO ^^ "^ "^ ^ "^ CO Oi oT oToToT orororoTor ccToT it : 5 2 78 ^H O CO O? Ci Oi 77 7 iH -* to i CO 'M- v^l 172 METHODS OF ORGANIC ANALYSIS dissolves readily in ether, chloroform, carbon bisulphide, fatty oils, turpentine, or hot strong alcohol. Phytosterol and sitosterol are alcohols, probably isomeric with cholesterol and resembling it closely in physical properties, which occur in vegetable fats, the latter being found especially in the oils of the cereal grains. The identification of one of these alcohols may, therefore, aid in determining the origin of an oil or fat or in the detection of vegetable fats present as adul- terants in the more expensive fats of animal origin, as will be explained in the next chapter. REFERENCES ALDER- WRIGHT and MITCHELL : Animal and Vegetable Fixed Oils, Fats, Butters, and Waxes. ALLEN : Commercial Organic Analysis, Vol. II. BENEDIKT and ULZER : Analyse der Fette und Wachsarten. GILL : Short Handbook of Oil Analysis. HEFTER : Technologic der Fette und Oele. HOPKINS : Oil-Chemists' Handbook. LEACH : Food Inspection and Analysis. LEWKOWITSCH : Chemical Technology and Analysis of Oils, Fats, and Waxes. Laboratory Companion to Fat and Oil Industries. LUNGE : Chemisch-technische Untersuchungsmethoden. UBBELOHDE : Handbuch der Chemie und Technologic der Oele und Fette. WRIGHT : Analysis of Oils and Allied Substances. II 1902. HUNT : A Comparison of Methods used to determine Iodine Values of Oils. J. Soc. Chem. Ind., 21, 454. TOLMAN and MUNSON: Refractive Indices of Salad Oils. /. Am. Chem. Soc., 24, 754. 1903. LYTHGOE : Zeiss Butyro-refracto meter Readings of Edible Oils and Fats. Technology Quarterly, 16, 222. TOLMAN and MUNSON : Iodine Absorption of Oils and Fats. J. Am. Chem. Soc., 25, 244. 1905. LYTHGOE : Refraction Indices of Oils. J. Am. Chem. Soc., 27, 887. 1906. DUNLAP : Preparation of Aldehyde-free Ethyl Alcohol for use in Oil and Fat Analysis. J. Am. Chem. Soc., 28, 395. HALLER : Alcoholysis of Fatty Substances. Compt. rend., 143, 657, 803. OILS, FATS, AND WAXES 173 RADCLIFF : Analytical Constants of Carnaiiba Wax. J. Soc. Chem. Ind., 25, 158. SCHNEIDER and BLUMENFELD : Characteristics of Certain Animal Fats. Chem. Ztg., 30, 53. THOMPSON and DUNLOP: (Revision of Iodine Numbers). Analyst, 31, 281. 1907. BERG : Examination of Beeswax. Chem. Ztg., 31, 337. DONS : Index of Refraction of Fats and Fatty Acids. Z. Nahr. Genussm., 13, 257. Louis and SAUVAGE : New Characteristic Constant of Oils. Compt. rend., 145, 183. MEYER ; Determination of Unsaponifiable Matter. Chem. Ztg., 31, 423. RAKUSIN : Optical Properties of Animal Fats. Chem. Ztg., 30, 1247. RICHMOND : Temperature Corrections of the Zeiss Butyro-refractom- eter. Analyst, 32, 44. RICHTER : Maumene Test and Iodine Number of Certain Oils. Z. angew. Chem., 37, 1605. SCHICHT and HALPERN : Estimation of Unsaponifiable Matters in Fats. Chem. Ztg., 31, 279. TWITCHELL : A Reagent in the Chemistry of Fats. J. Am. Chem. Soc., 29, 566. 1908. BERG : Analytical Chemistry of Beeswax. Chem. Ztg., 32, 777. FAHRION : Progress in Fat Analysis in 1907. Z. angew. Chem., 1908, 1125. HALLA : Preparation of Alcoholic Potassium Hydroxide. Chem. Ztg. 32, 890. INGLE : Notes on Wijs Solution. /. Soc. Chem. Ind., 27, 314. 1909. BARTLETT and SHERMAN : Effect of Excess of Reagent and Time of Reaction in the Determination, of Iodine Numbers of Fatty Oils. School of Mines Quarterly, 31, 55. BOMER : Mixed Glycerides of Palmitic and Stearic Acids. Z. Nahr. Genussm., 17, 353. BOYNTON and SHERMAN : A Comparison of the Calculated and De- termined Values for the Specific Temperature Reactions of Oil Mixtures with Sulphuric Acid. School of Mines Quarterly, 31, 64. FREUDLICH : Analytical Methods in Stearin Manufacture. Chem. Rev. Fette- Harz.-Ind., 15, 224, 246, 277; Chem. Abs., 3, 253. 1911. COMMITTEE REPORT: (Methods for Moisture, Volatile Matter, Sus- pended Impurities, Free Fatty Acids, Unsaponifiable Matter, Metallic Soaps, and Titer Test in Fats and Fatty Oils). J. Lid. Eng. Chem., 3, 50. FAHRION: Fat Analysis and Fat Chemistry in 1910. Z. angew. Chem., 24, 241. CHAPTER IX Edible Oils and Fats SALAD OILS MOST salad oils ai;e sold as olive oil. The principal substitutes and adulterants are cottonseed, arachis (peanut), sesame, maize, poppyseed, and lard oils. Both quantitative and qualitative methods must be used in any thorough examination of an oil for adulterants. As a rule an oil should be pronounced adul- terated only when quantitative determinations yield results which could not be obtained from a pure oil of normal character. Qualitative tests are usually required to show which of several possible adulterants is present. As yet certain color reactions are indispensable for this purpose, but in using these tests and interpreting the results, it must be remembered that they de- pend upon the presence of constituents which may be removed or destroyed by improved methods of refining, and that olive oil which has been altered by long exposure to air in loosely stoppered or partially filled vessels, or oil containing a small amount of some accidental impurity, may give a reaction which cannot be distinguished from that of the adulterant sought. The results of even the best of the color reactions must there- fore be interpreted with great caution and must usually be regarded as of much less significance than the quantitative numbers. On the other hand, a good grade of arachis oil, or a carefully prepared mixture of lard oil with one of the seed oils, can be added to olive oil in large proportion without affecting the ordinary constants to such an extent as to pass the limits which can fairly be regarded as normal. It is necessary, therefore, not only to compare each number found with the established 174 EDIBLE OILS AND FATS 175 limits for pure oil, but also to view the quantitative results in their relations to each other and to the indications of the quali- tative tests. While the system to be followed and the number of tests re- quired will naturally vary in different laboratories, the follow- ing may be recommended. Determine accurately the specific gravity or the index of refraction, the iodine number, and sa- ponification number. If an abundance of the sample is at hand, determine the specific temperature reaction and the acidity. Apply the nitric acid test, Halphen's test, one or more of the color reactions for sesame oil, and examine for arachidic acid by Renard's method. Finally, if the importance of the sample justifies the time acquired, separate and examine the unsaponi- fiable matter and the mixed fatty acids and determine the vis- cosity of the soap solution by Abraham's method described below. ANALYTICAL PROPERTIES OF OLIVE OIL The numbers included in the table of "constants" already given are intended to cover the range of normal variations in oils found in the American markets. Miintz, Durand, and Milliau 1 examined samples from Africa, Spain, Portugal, Greece, Turkey, and the Levant without finding any significant variation in the specific gravity, iodine number, or temperature reaction. According to Milliau, Bertainchand, and Malet, 2 however, Tunis oils have high specific gravities, the average of 49 samples being 0.9183. Tolman and Munson have recently examined a large number of olive oils many of which were of known origin. The following are taken from their results: 3 The average iodine number of the oils from California is therefore higher than that of the French and Italian oils and, as might be expected, the higher iodine number is accompanied 1 Bulletin du Ministere de PAgriculture, 1895. Quoted from Bui. 77, Bur. Chem., U. S. Dept. Agriculture. 2 Bulletin de PAgriculture et Commerce de Tunis. Quoted from Bull. 77, loc. cit. 3 Bui. 77, Bur. Chem., U. S. Dept. Agriculture. 176 METHODS OF ORGANIC ANALYSIS TABLE 16. ANALYTICAL PROPERTIES OF OLIVE OILS Description Iodine number g l * n <** c S.2 8l| ^^s l-< Specific temperature reaction Free acid as oleic per cent California oils of known origin 1 ' (42 samples). | Avg< 89.8 78.5 85.3 0.9180 0.9162 0.9170 1.4718 1.4703 1.4713 109.7 94.5 101.8 8.21 1 0.20 1.20 Italian oils of known origin Max. Min. 86.1 79.2 0.9180 0.9155 1.4713 1.4705 104.7 95.6 2.79 0.57 (17 samples). Avg. 81.6 0.9163 1.4709 99.1 1.11 Italian oils (commercial) not found adulterated (57 samples). 2 Max. Min. Avg. 84.5 77.5 80.9 0.9179 0.9150 0.9161 1.4712 1.4701 1.4706 108.4 88.4 97.8 5.30 0.72 2.42 French oils (commercial) not found adulterated (60 samples). Max. Min. Avg. 85.0 79.0 81.3 0.9183 3 0.9150 0.9166 1.4713 1.4699 1.4708 114.4 90.4 100.1 3.63 0.45 1.59 by a higher specific gravity, refractive index, and temperature reaction. Although individual samples will show slight varia- tions, the same general relation is found on comparing oils which differ in iodine numbers though obtained from the same locality. Thus taking at random, from among the California oils of known origin examined by Tolman and Munson, 10 sam- ples with high and 10 with low iodine numbers the following average figures were found : Iodine number Specific gravity 15.5 15.5 Index of refraction 15.5 Specific temperature reaction First group . .... Second group . , 88.44 83.12 0.9172 0.9166 1.4716 1.4711 105.7 98.6 1 Two samples with larger amounts of free acids were found, but were ex- cluded from average as being unfit for use as salad oils. 2 Omitting one sample containing 15.25 per cent of free acid and having a specific gravity of 0.9134. 3 Omitting one sample having an abnormally high specific gravity (0.9196) which may have been due to exposure. EDIBLE OILS AND FATS 177 The normal relations of the constants to each other and the changes which may occur as the result of age or exposure 1 must always be taken into consideration when interpreting the results of an analysis. For records of individual samples of olive oil showing iodine numbers of 90.5 to 94.7 see Allen's Com- mercial Organic Analysis, Fourth Edition, II, 113. The standard adopted by the United States Department of Agriculture is as follows : Olive oil is the oil obtained from the sound, mature fruit of the cultivated olive tree (Olea europcea L.) and subjected to the usual refining processes ; is free from rancidity ; has a refractive index (25 C.) not less than 1.4660 and not exceed- ing 1.4680 ; and an iodine number not less than 79 and not exceeding 90. DETECTION OF COTTONSEED OIL The presence of cottonseed oil in olive oil raises the specific gravity, iodine number, and temperature reaction and lowers the viscosity of the soap solution. A mixture of cottonseed and lard oils may, however, be added to olive oil in large quan- tity without greatly affecting any but the last of these " con- stants." The qualitative tests for cottonseed oil are therefore of considerable importance. Halpherts Reaction* Dissolve 1 part of sulphur in 100 parts of carbon bisulphide and mix the solution with an equal volume of amyl alcohol. Mix equal volumes, 2 to 3 cc. each, of the reagent and the oil to be tested and heat the test tube containing the mixture gently at first until violent boiling ceases, then in a bath of boiling saturated solution of common salt. Heat for 2 hours unless a color develops sooner. If cottonseed oil is present, the solution turns orange or red. This is probably the most sensitive and characteristic test for 1 See section on this subject beyond. 2 Halphen : Ann. chim. anal., 1898, 3, 9 ; Analyst., 1898, 23, 131 ; Bui. 107. Bur. Chem., U. S. Dept. Agriculture. N 178 METHODS OF ORGANIC ANALYSIS cottonseed oil and the least liable to give unsatisfactory results in the hands of an inexperienced person. The presence of 1 or 2 per cent of unchanged cottonseed oil in olive oil is detected without difficulty. A reaction is often obtained l with lard or lard oil or even with butter fat 2 from animals which have been fed upon cottonseed meal. Copac (or kapok) oil, which is closely related to cottonseed oil, gives the same reaction. When heated at 250 for 10 to 20 minutes, cottonseed oil loses the prop- erty of giving this reaction. No pure olive oil has yet been known to give a similar coloration. Hence a positive result is con- sidered conclusive, but a negative result is not. The substance to which the reaction is due cannot be removed by treatment with animal charcoal 3 and is supposed to be an unsaturated acid which combines with sulphur giving a red compound. 4 Nitric Acid Test Cottonseed oil shaken at room temperature with an equal volume of nitric acid, of 1.37 to 1.38 specific gravity, gives a brown coloration, sometimes only on standing overnight. Other seed oils give similar reactions. Normal olive oil under the same treatment shows no change of color. The test is not so delicate as that of Halphen, but is applicable to cottonseed oil which has been heated until it no longer colors the Halphen reagent. * It may also be of value in determining whether a weak test with the latter reagent is due to a small amount of unheated cottonseed oil or to a larger amount which has been heated sufficiently to weaken the Halphen reaction (Tolman and Munson). According to Lewkowitsch this re- action cannot be relied upon to detect less than 10 to 20 per cent of American cottonseed oil in olive oil. Tolman and Munson consider the test much more delicate. A positive re- iSoltsein: Z. offentl Chem., 1901, 7, 140. 2 Wauters: Bull. Assoc. Belg. Chem., 13, 404; J. Soc. Chem. Ind., 1900, 19, 172. 8 Utz : Chem. Rev. Fett.-Harz-Ind., 1902, 9, 125 ; Gill's Oil Analysis, p. 73. * Raikow : Chem. Ztg., 1900, 24, 562, 583 ; 1902, 26, 10. See also Halphen : Bull. Soc. Chim., 1905, [3], 33, 108. EDIBLE OILS AND FATS 179 suit should always be confirmed by finding a high iodine number or by proving the presence of some other oil having a very low iodine number ; for pure olive oil if much altered as the result of age and exposure will sometimes give a reaction which cannot be distinguished from that of cottonseed oil. DETECTION OF ARACHIS (PEANUT) OIL Arachis oil has usually a higher specific gravity and iodine number and practically always a higher temperature reaction than olive oil. The specific temperature reaction of arachis oil with concentrated sulphuric acid is usually 40 to 60 units higher than the iodine number of the same sample, whereas olive oil usually shows a difference of less than 20 and very rarely of more than 25 units between the iodine and the specific Maumene numbers. The presence of peanut oil in olive oil greatly diminishes the viscosity of the soap solution obtained on saponification. Any of these changes, however, might be due to other adulterants. Arachis oil is shown conclusively by isolating and identifying arachidic acid. This can be done with approximately quantitative results by Tolman's modifica- tion of Renard's method. Renard-Tolman Test for Arachidic Acid 1 Weigh 20 grams of oil in an Erlenmeyer flask. Saponify with alcoholic potash, neutralize exactly with dilute acetic acid, using phenolphthalein as indicator, and wash the solution into a 500-cc. flask containing a boiling mixture of 100 cc. of water and 120 cc. of a 20 per cent lead acetate solution. Boil one minute and then cool by immersing the flask in water, occa- sionally giving it a whirling motion to cause the precipitated lead soaps to stick to the sides of the flask. After thorough cooling, pour off the water containing the excess of lead acetate and wash the soap with cold water and then with 90 per cent alcohol. After pouring off the alcohol as completely as 1 Renard : Compt. rend., 1871, 73, 1330. Tolman : U. S. Dept. Agriculture, Bur. Chem., Bui. 81, p. 64. See also Archbutt: J. Soc. Chem. 2nd., 1898, 17, 1124. 180 METHODS OF ORGANIC ANALYSIS possible, add 200 cc. of ether, cork the flask and allow to stand until the soap is disintegrated, then connect with a reflux con- denser, heat gently to boiling, and boil for 5 minutes on a safety water bath or an electric heater. Cool to 15 and allow to stand overnight. Filter, wash the residue thoroughly with ether, and then transfer it from the filter to the flask by means of a stream of hot water acidified with hydrochloric acid. Add an excess of dilute hydrochloric acid and 200 cc. of hot water and heat until the fatty acids separate as a clear oily layer. Nearly fill the flask with hot water and allow to stand at room temperature until the layer of fatty acids has completely separated and solid- ified. Remove and drain the cake of fatty acids, wash again with hot water, then dissolve in 100 cc. of boiling alcohol, 90 per cent by volume. Cool the solution to 15, shaking fre- quently, and allow it to stand as long as any acid continues to crystallize out, or overnight, at a temperature not above 20. Filter, wash the crystals twice with 10 cc. of 90 per cent alcohol, noting the total volume of filtrate and washings, and then with alcohol, 70 per cent by volume (in which the crystals are practically insoluble). Dissolve the crystals by means of hot absolute alcohol in a weighed dish, evaporate, dry, and weigh. To this weight add 0.0045 gram for each 10 cc. of 90 per cent alcohol in the filtrate and washings if the temperature of filtration was 20; or 0.0025 gram for each 10 cc. if the tem- perature was 15. The melting point of arachidic acid obtained in this way is 71 to 73. According to Tolman and Munson 1 the determina- tion of the melting point must not be neglected since cotton- seed and lard oils have been found to give crystals resembling arachidic acid in appearance, but having a lower melting point. Tolman finds that from 5 to 10 per cent of the oil can be de- tected by this method. On the usual assumption that the oil yields 5 per cent of the acid, 2 each centigram found as described 1 U. S. Dept. Agriculture, Bur. Chem., Bui. 77, p. 35. 2 This is probably more nearly a maximum than an average yield. Tolman and Munson (loc. cit.) obtained from 3.41 to 4.24 per cent. EDIBLE OILS AND FATS 181 above (using 20 grams of oil) indicates 1 per cent of arachis (peanut) oil in the sample. Archbutt (Allen's Commercial Organic Analysis, 4th Ed., II, 99-100) recommends the following method of Bellier 1 as giving satisfactory qualitative results. Solutions. (1) Alcoholic potassium hydroxide, made by dissolving 8.5 grams pure potassium hydroxide in 70 per cent alcohol and making up to 100 cc. (2) Acetic acid of such strength that 1.5 cc. will exactly neutralize 5 cc. of the potash solution (about 28-29 per cent of actual acetic acid). Test. Weigh 1 gram of sample into a dry test tube, add 5 cc. of the potash solution, and boil gently, avoiding evapora- tion, over a free flame until saponification is complete, probably a little over 2 minutes; then add 1.5 cc. acetic acid, or just sufficient to neutralize the alkali, mix well, cool rapidly in water at 17 to 19, and let stand at this temperature for at least 30 minutes, shaking occasionally ; then add 50 cc. 70 per cent alcohol containing 1 per cent by volume of hydrochloric acid of 1.16 sp. gr., shake well, and again place in the cold water for 1 hour. In the absence of arachis oil the liquid should remain clear or become merely opalescent, while if the sample contained more than 10 per cent of arachis oil a flocculent, crystalline precipitate is obtained. DETECTION OF SESAME OIL Sesame oil affects the usually determined constants in the same way as cottonseed oil and to practically the same extent. The color reactions are usually considered quite characteristic. Baudouin's Test Dissolve 0.1 gram of sugar in 10 cc. of hydrochloric acid of 1.18 to 1.20 specific gravity and add 20 cc. of the oil. Shake thoroughly in a test tube for one minute and allow to stand. The water solution separates quickly and shows a distinct red 1 Ann. chim. anal., 1899, 4, 4. 182 METHODS OF ORGANIC ANALYSIS or "crimson color if the sample contains 1 per cent or more of sesame oil. The active reagent is probably furfural formed by the action of the acid upon the sugar. Villivecchia's modification consists in shaking 10 cc. of the oil with 10 cc. of hydrochloric acid (1.20 sp. gr.) to which has been added 0.1 cc. of a 2 per cent solution of furfural in 95 per- cent alcohol. Olive oils of known purity have usually been found to give only a slight pink color, but sometimes the reddening of the water solution is so pronounced as to cause confusion with that produced by a small amount of sesame oil. Check experiments therefore should always be made. If much sesame oil is present, the red color should be perceptible in the oily layer as well as in the water solution. Tochers Test Dissolve 1 gram of pyrogallol in 15 cc. of concentrated hydro- chloric acid. Shake this solution with 15 cc. of oil in a sep- aratory funnel and allow to stand for 1 or 2 minutes. Draw off the aqueous solution and boil for 5 minutes. The presence of sesame oil is indicated if the solution after boiling appears red by transmitted and blue by reflected light. This test has not been so generally used nor so thoroughly studied as the preceding. The Association of Official Agricul- tural Chemists authorize the use of either Baudouin's, Villivec- chia's, or Tocher's test for the detection of sesame oil in edible oils and fats. DETECTION OF MAIZE, POPPYSEED, AND LARD OILS Each of these oils has a characteristic odor or taste, the odor of lard oil being intensified by heating. These properties, however, cannot be relied upon, as the substances to which they are due can be almost entirely eliminated in the refining process. The effect of maize or poppy oil in raising the iodine number would be very noticeable, but might be neutralized by the addition of a somewhat greater quantity of lard oil. The difference between the iodine number and the specific tern- EDIBLE OILS AND FATS 183 perature reaction would be appreciably greater in such a mix- ture than in pure olive oil (compare detection of arachis oil). Maize and poppyseed oils react with nitric acid, giving brown colors similar to that produced by cottonseed oil. Lard oil often gives the same reaction and might be indicated by the melting point of the fatty acids (those of lard oil having a relatively high melting point, 33 to 38 according to Tolman and Munson) or by the character of the unsaponifiable matter phytosteryl acetate test. 1 (See references at the end of this chapter.) All of these oils (as well as most others) yield soap solutions of much lower viscosity than those obtained from pure olive oils. 2 The determination and significance of this property has been studied in some detail 3 and the method has been found capable of giving valuable results, especially if the con- ditions worked out by Abraham are carefully observed. For a full discussion of these conditions the original paper must be consulted. The essential features of the process are as follows : Abraham 's Modification of Blasdales Viscosity Test The saponification number having been determined, weigh 3 grams of oil in an accurately graduated 100-cc. flask, add 2 cc. of alcohol and an amount of standard potash solution sufficient to saponify the oil and leave an excess of 0.625 gram of potas- sium hydroxide. Close the flask with a stopper having a Kroonig valve and saponify on a water bath. After saponifi- cation expel the alcohol by warming and allowing air freed from carbon dioxide to pass through the flask, while a partial vacuum is maintained by means of a suction pump. In this way the alcohol is entirely removed in 5 to 10 minutes. Evap- oration should not be carried to complete dryness. Without allowing the flask to cool, add 50 cc. of hot water, rotate gently 1 Bonier : Z. Nahr.-Genussm., 1901, 4, 1091. 2 Blasdale : J. Am. Chem. Soc., 1895, 17, 937. 3 Abraham : Ibid., 1903, 25, 968. Sherman and Abraham : Ibid., 1903. 25, 977. 184 METHODS OF ORGANIC ANALYSIS until a homogeneous solution of the soap is obtained, cool to 20, fill to the mark with distilled water, and mix well by shak- ing or by repeatedly pouring the solution from one flask to another. Determine the viscosity of the solution in an Ostwald viscosimeter immersed in water kept at 20. Care must be taken to avoid the introduction of air bubbles into the vis- cosimeter and to maintain the exact temperature. Repeat the readings until five or more concordant results are obtained. The viscosimeter is standardized by means of distilled water, and it is advisable to select for this work an instrument in which the time of flow of water is about 100 seconds. Successive readings of a soap solution should then agree within 2 seconds. The viscosity is calculated as follows : v l = 1001 where v 1 = the viscosity number. j = the time of flow (in seconds). s 1 specific gravity of the solution. t = time of flow of distilled water. The viscosity numbers obtained by this method were : Olive oil of known purity (9 samples) 168.0-185.7 Olive oil of doubtful purity (4 samples) 145.8-165.8 Lard oil (5 samples) 122.9-135.0 Arachis (peanut) oil (1 sample) 126.6 Cottonseed oil (3 samples) 126.6-127.9 Rapeseed oil (3 samples) 124.7-125.7 Sesame oil (1 sample) 139.2 Maize oil (1 sample) 126.6 Poppyseed oil (1 sample) 123.9 Mixtures of olive and lard oils gave viscosity numbers agree- ing closely with those obtained by calculation, but cottonseed or arachis oil when added to olive oil lowered the viscosity to a much greater extent than would have been predicted, indicating that the viscosity number is a more useful means of detecting adulteration than appears from a comparison of EDIBLE OILS AND FATS 185 the results obtained on testing the olive oil and its adulterants separately. The usefulness of the method for testing isolated samples is limited by the fact that comparable results can be obtained only under strictly uniform conditions, the viscosity of the soap solution being greatly influenced by slight variations in strength, alkalinity, or temperature, while the terms in which the results are expressed will naturally vary with the form of viscosimeter used ; but in cases of sufficient importance to justify the time required to arrange the apparatus and make comparative deter- minations, the " viscosity number " will be found an important factor in the examination of olive oil for adulterants. BUTTER Butter is officially defined 1 as "the product made by gathering in any manner the fat of fresh or ripened cream into a mass which also contains a small portion of the other milk constituents, with or without salt." Standard butter contains not less than 82.5 per cent of butter fat, having a Reichert-Meissl number not less than 24 and a specific gravity not less than 0.905 at 40/40. By acts of Congress approved August 2, 1886, and May 9, 1902, butter may also contain additional coloring matter. Butter may fail to meet the requirements of the official stand- ard either because of a deficiency in the percentage of fat or be- cause of the presence of foreign fat or butter fat of abnormal character, though the sample may contain nothing which can be regarded as unwholesome. Butter analysis therefore includes (1) the examination of the whole butter, (2) the examination of the butter fat. The methods of examining butter fat will be given fully be- low, but for convenience the determination of water, fat, curd, and ash will be described first. The point at which tests for preservatives can conveniently be made will be indicated, but the detection of foreign colors will not be considered, as these are not regarded as adulterants. 1 Circular No 19, Office of the Secretary, U. S. Dept. Agriculture. 186 METHODS OF ORGANIC ANALYSIS DETERMINATION OF WATER, FAT, CURD, AND ASH Especial care must be taken in sampling butter, since the water, salt, and curd are often unevenly distributed and an attempt to mix by stirring is apt to result in squeezing out drops of brine. If a large quantity is to be sampled, a butter trier should be used, and the portions thus drawn united until a working sample of about 500 grams is obtained. Melt the sample at the lowest possible temperature in a wide- mouthed glass-stoppered bottle, shake violently to insure a homo- geneous mixture, and continue, the shaking while cooling the sample until it is thoroughly solidified. Great care is necessary here to prevent a separation of water and fat. Thoroughly clean and dry a lipped dish or beaker having a flat bottom of at least 20 square centimeters. Weigh the dish, introduce 1.5 to 2 grams of butter, and re weigh quickly to avoid evaporation. Dry to constant weight in a boiling water oven. The loss is water. Treat the dry residue with petroleum ether or benzine, transfer it to a weighed Gooch crucible having a felt of ignited asbestos, and wash with the solvent until all fat is re- moved. Dry the crucible and residue to constant weight in a water oven or an air bath not above 110. The material dis- solved by the petroleum ether or benzine is fat. Burn the curd at a temperature below a red heat and weigh the crucible con- taining the ash. Notes. The method given is essentially that of the Associ- ation of Official Agricultural Chemists. 1 If preferred the but- ter can be dried on clean dry sand or asbestos. When the latter is not used, it is important that the butter form only a very thin layer on the bottom of the dish or beaker ; otherwise the water sinks into the melted fat and is only very slowly expelled at the temperature of the boiling water oven. For convenience in transferring and washing the residue with petroleum ether the latter should be used in a small wash bottle having a ground glass stopper. Instead of drying in a dish and transferring the residue to a crucible, the butter may. be weighed directly in a 1 Bui. 107, Bur. Chem., U. S. Dept. Agriculture. EDIBLE OILS AND FATS 187 crucible two thirds filled with fibrous asbestos, dried to constant weight, and then extracted. 1 A device for facilitating this drying by passing a current of air through the heated crucible has been described by Bird. 2 References to a number of other quick methods for moisture in butter will be found at the end of the chapter. Great care must be taken to burn the curd at the lowest tem- perature possible in order to avoid loss of chlorine (see Chapter XVII). Any small amount of milk sugar which the butter might contain would be counted as curd in this analysis. The percentage of salt can be found by determining chlorine in the ash, or by repeatedly washing the butter with hot water in a separatory funnel and titrating the combined washings with a standard solution of silver nitrate. Good butter usually con- tains 10 to 14 per cent water, 84 to 87 per cent fat, 0.5 to 1.5 per cent curd, 2.0 to 4.0 per cent ash if salted ; if unsalted, 0.25 to 0.5 per cent. The water content of butter has often been limited by legal or trade standards to 16 per cent. This is considerably above the present average for creamery butter, as shown by an investigation made by the United States Depart- ment of Agriculture in 1902. 3 Of 800 samples from 400 cream- eries in 18 states, the average water content was 11.78 per cent; 85 per cent of the samples contained between 10 and 14 per cent; the extreme limits were 7.20 and 17.62 per cent. The amount of salt added to butter varies greatly with the demands of differ- ent markets, but over 5 per cent would be excessive unless the butter were intended for export to a tropical country. An ex- cessive amount of curd indicates careless manufacture or fraud- ulent increase of weight and is likely to injure the keeping qualities of the butter. 1 Richards and Woodman: Air, Water, and Food (2d Ed.), 201. 2 J. Am. Chem. Soc., 1905, 27, 818. 3 Circular No. 39, Bureau of Animal Industry ; Analyst, 1903, 28, 184. 188 METHODS OF ORGANIC ANALYSIS . EXAMINATION OF BUTTER FAT Preparation Melt 100 grams or more of the butter and allow it to stand at 45 to 55 until the water and salt settle to the bottom. 1 Pour off the melted fat by decantation and filter it through a dry paper in a funnel heated by a water jacket or supported in a drying oven kept at about 60. The filtered fat, which must be free from turbidity, is received in a wide-mouthed bottle and kept stoppered in a cool place until analyzed. Reichert-Meissl or Reichert-Wollny Number* This number is a comparative measure of the proportion of volatile acids, and is the most important basis for deciding the purity of butter fat. Often the presence or absence of foreign fat in butter, or the proportion of butter fat in oleomargarine, is inferred from this number alone. In the interest of uni- formity of results official chemists have sought to describe the method in such detail as to eliminate variations due to manipulation. Reagents. 1. Caustic soda solution, made by dissolving sodium hydroxide (nearly free from carbonate) in an equal weight of distilled water. 2. Alcohol, 92 to 95 per cent, containing no appreciable amount of volatile acid, either free or combined. 3. Dilute sulphuric acid, made by mixing pure concentrated sulphuric acid with four times its volume of water. 4. An accurately standardized approximately tenth-normal solution of barium (or sodium) hydroxide. 5. A 1 per cent solution of phenolphthalein in alcohol. Determination. Thoroughly clean and dry a flask of 250 to 1 This water solution can be tested for preservatives, of which boric acid and borax are most likely to be found in butter. For methods see Chapter XVIII. 2 Reichert: Z. anal. Chem., 1879, 18, 69. Meissl : Dingier^ s poly tech. Journ., 1879, 233, 229. Wollny : Milch Ztg., 1887, 16, 609 ; Analyst, 1887, 12, 203, 235 ; 1888, 13, 8, 38. These papers are reprinted in Ephraim's Origi- nal Arbeiten iiber Analyse der Nahrungsinittel. . EDIBLE OILS AND FATS 189 800 cc. capacity Weigh the flask, thoroughly mix the melted fat, introduce 5.6 to 5.8 cc. measured at about 50, allow the flask and fat to cool for 15 to 20 minutes, and reweigh (or, if convenient, weigh exactly 5 grams of fat into the flask). Add 10 cc. of the alcohol and 2 cc. of the caustic soda solution, attach the flask to a reflux condenser, and boil on a water bath or electric heater for at least half an hour to insure complete saponification. 1 Evaporate the alcohol by heating the flask in a steam bath, shaking occasionally to avoid danger of loss from frothing and to facilitate the removal of the alcohol. Add 132 cc. of recently boiled distilled water, warm at 60 to 70 until the soap is com- pletely dissolved, add 8 cc. of the dilute sulphuric acid and a few pieces of pumice stone, re-stopper the flask or connect it with a condenser, and warm without boiling until the fatty acids separate as a clear layer. Distill 2 through a glass condenser, collecting the distillate in a flask accurately graduated at 110 cc. The distillation should be so regulated that 110 cc. will be col- lected in from 28 to 32 minutes. Mix the distillate, filter through dry paper, and titrate 100 cc. of the filtrate, using 0.5 cc. of the phenolphthalein solution as indicator, until the red color remains apparently unchanged for 2 minutes. Increase the burette reading by one tenth (on account of the 10 cc. of distillate not titrated), and calculate the number of cubic centi- meters of tenth-normal alkali which would have been required if exactly 5 grams of fat were taken for the determination. This is the Reichert-Meissl number. Notes. The number thus found does not represent the total volatile acids present. The yield is fairly uniform if the given conditions of dilution and distillation are maintained. Wollny submitted this method to an exhaustive examination and pointed out the following sources of error : (1) absorption of 1 The saponification can also be accomplished by heating in a closed flask, using either aqueous or alcoholic alkali, or by means of the glycerol-soda solution proposed by Leffmann and Beam : Analyst, 1891, 16, 153. 2 In ordinary work a form of apparatus similar to that shown in Fig. 1, Chapter I, may be used. 190 METHODS OF ORGANIC ANALYSIS carbon dioxide during saponification, (2) formation of esters during saponification, (3) formation of esters during distillation, (4) coherence of fatty acids during distillation, resulting in holding back some of the volatile acid, (5) variations in the proportion of volatile acid carried over, due to differences in size and shape of distillation apparatus. In order to avoid dis- crepancies from these and other causes he published an elabo- rately detailed system of manipulation and precautions. It has been shown that Wollny greatly overestimated the probable er- rors of the method as previously carried out and that some of his precautions are unnecessary; but as in the main they tend toward greater uniformity of results, they have been adopted with slight modifications by the Association of Official Agricul- tural Chemists, whose methods l are usually accepted as stand- ard in the United States and should be followed exactly in any determination which is likely to be made the basis of legal action. In Great Britain, a joint committee representing the Govern- ment Laboratory and the Society of Public Analysts has adopted the method essentially as described above with the following specifications for the apparatus to be employed : 2 Flask used for saponification and distillation: capacity, 300 cc. ; length of neck, 7 to 8cm.; width of neck, 2 cm. The flask is connected with the condenser by means of a bent glass tube 7 mm. wide, so placed that the bend is 15 cm. above the top of the cork. At a distance of 5 cm. above the cork is a bulb 5 cm. in diameter. The flask is supported on a circular piece of asbestos 12 cm. in diameter, having a hole 5 cm. in diameter in the center, so that the bottom of the flask is heated by a free flame during the dis- tillation. The British committee further prescribed that blank determinations be made and the volume of alkali found necessary to neutralize the distillate (which volume should not exceed 0.3 cc.) be deducted in calculating the results of each determination. The number so obtained is called the Reichert- Wollny number. 1 Bui. 107, Bur. Chem., U. S. Dept. Agriculture. 2 Analyst, 1900, 25, 309. EDIBLE OILS AND FATS 191 The Reichert-Meissl or Reichert-Wollny number of butter fat is usually between 24 and 34 ; that of coconut fat, between 6 and 8 ; of other edible fats and oils, usually less than 1. Specific Gravity The specific gravity of butter fat has often been determined either at 100 or at 37.8 (100 F.). The standard recently established for the United States prescribes a minimum specific gravity at 40, water at the same temperature being taken as unity. Either a specific gravity flask or an Ostwald pyknom- eter can be used conveniently for the determination, the pyknometer being filled and adjusted while surrounded by water kept at the required temperature, then removed from the water bath, wiped dry on the outside, allowed to cool to the tempera- ture of the balance, and weighed. Saponification Number This is determined as described in Chapter VIII. Since the normal saponification numbers of butter fat are only about 15 per cent in excess of those of the fats commonly used as adulter- ants, the determination in order to be of much value must be very accurately made. Insoluble Fatty Acids Hehner Number Reagents. 1. The alcoholic potash solution used in the de- termination of the saponification number. 2. Alcohol, about 95 per cent by volume, which leaves no appreciable residue upon evaporation. Determination. Saponify 4 grams of butter fat with 50 cc. of the alcoholic potash solution, evaporate to a paste to expel alcohol, dissolve the soap in about 400 cc. of hot water in a weighed beaker, add hydrochloric acid in excess of the amount required to neutralize the potash used, and heat nearly to boil- ing with occasional stirring until the fatty acids have collected in a clear layer on the surface. Cool thoroughly, pour the so- lution through a filter, and wash the cake with cold water with- 192 METHODS OF ORGANIC ANALYSIS out removing it from the beaker. Stir up the fatty acids in the beaker with another portion of hot water (200 to 300 cc.), cool thoroughly, filter, and wash again. Repeat this treatment three times. After a final thorough washing with cold water, put the beaker containing the fatty acids beneath the funnel and dissolve any fatty acids which the filter may contain by washing with hot 95 per cent alcohol, allowing the washings to run into the beaker. Evaporate off the alcohol and dry the beaker containing the fatty acids to constant weight in a boiling water oven. Notes. This is the modification of Hehner's method adopted by the Association of Official Agricultural Chemists l and by the chemists of the Government Laboratory, London. 2 The original method, 3 which is still largely used, involves washing the melted fatty acids with hot water on a paper filter. The results thus obtained are usually 1 to 2 per cent lower than those by the official method. In the hot filtration method there is danger of washing some of the melted fatty acid through the paper. Iodine Number The determination of the iodine number has been fully de- scribed in Chapter VIII. As butter fat absorbs only 26 to 38 per cent of its weight of iodine, a gram of sample can be used for each determination. According to Patrick,* butter fat shows iodine numbers about 1 unit higher by the Hanus than by the Hiibl method. Melting Point Wiley's Method 5 Apparatus and Reagents. 1. An accurate thermometer read- ing to 0.1 degree. 1 Bui. 107, Bur. Chem., U. S. Dept. Agriculture. 2 Thorpe : J. Chem. Soc., 1904, 85, 248. 3 Hehner and Angell : Butter, its Composition and Adulterations. London, 1874. Hehner : Z. anal. Chem., 1877, 16, 145. Ephraim, loc. cit. 4 U. S. Dept. Agriculture, Bur. Chem., Bui. 81, p. 49. 5 Wiley's Agricultural Analysis, Vol. Ill ; Bui. 107, Bur. Chem., U. S. Dept. Agriculture. EDIBLE OILS AND FATS 193 2. A tall beaker nearly filled with water and arranged to be heated gradually with constant stirring from bottom to top. 3. A wide test tube suspended in the water in the beaker and nearly filled with water and alcohol as follows : Half fill the tube with hot recently boiled distilled water, then pour a nearly equal volume of hot recently boiled alcohol into the tube, carefully floating the alcohol on the water with as little mixing of the liquids as possible. Determination. Allow a drop of the melted butter fat to fall upon a smooth piece of ice floating in recently boiled dis- tilled water. A thin disk of fat about 1 cm. in diameter should be obtained. Remove the disk from the ice by forcing the latter below the water when the fat will come to the surface whence it is removed by means of a steel spatula or knife blade and dropped into the tube containing the water and alcohol. The disk sinks to the point where the density of the alcohol- water mixture is equal to its own. It must not touch the side of the tube. Suspend the thermometer so that the bulb hangs in the tube exactly level with the disk of fat. Gradually heat the beaker, keeping the water well stirred. After the disk begins to shrivel, indicating that the temperature is within a few degrees of the melting point, the heat must be applied very carefully. The temperature at which the fat becomes a sphere is taken as the melting point. Repeat the determina- tion twice, heating the bath at such a rate that 8 to 10 minutes are required to raise the temperature through the last 2 degrees. The second and third determinations should agree within 0.2. Notes. The special -advantage of this method is that it avoids the discrepancies caused by the adherence of the melting fat to solid surfaces, which in most other methods makes it difficult to determine the exact point of fusion. It is important to secure a very thin disk of fat for the determination, to avoid all adherence of air bubbles, and to secure uniform heating of the thermometer bulb and the disk by occasionally swaying the former around the latter as the temperature approaches the 194 METHODS OF ORGANIC ANALYSIS melting point. By using hot recently boiled water and alcohol in preparing the test tube for the determination the danger of air bubbles is avoided. Additional Determinations The index of refraction and Crismer's test 1 based upon the critical temperature of solution in alcohol are considerably used in the examination of butter fat. They are especially useful where rapid " sorting tests " are required, as in food inspection laboratories, where only the suspected samples are submitted to complete examination. The phytosteryl acetate test 2 is occasionally employed as a means of detecting the presence of vegetable fat, but requires too much time and skill for routine use and is liable to give misleading results in the hands of an inexperienced person. The chief vegetable fats in use as butter substitutes are cot- tonseed oil, the methods for which have already been described, and coconut oil, whose detection is discussed below. (See also the references at the end of this chapter.) Acidity of butter fat is sometimes determined and interpreted as a measure of the rancidity, although the odor and taste which cause a butter to be regarded as rancid are more largely due to aldehydes and other decomposition products than to free fatty acids. On the assumption that acidity can serve as a measure of rancidity, the term degree of rancidity is sometimes used as synonymous with degree of acidity, i.e. to show the number of cubic centimeters of normal alkali required to neutralize the free acid in 100 grams of fat. One " degree " is, therefore, equivalent to an acid number of 0.56 or to 0.28 per cent of free oleic acid. iCrismer: Bull. Assoc. beige. Chim., 1895, 9, 71; 1896, 10,312; Abs. Analyst., 1895, 20, 209; 1897, 22, 71. See also Weiss: Pharm. Ztg., 41, 268; Chem. CentrbL, 1896, I, 1212. Asboth : Chem. Ztg., 1896, 20, 685. Browne: J. Am. Chem. Soc., 1899, 21, 990. 2 Bomer : Z. Nahr.-Genussm., 1901, 4, 865, 1070 ; 1902, 5, 1018. Juckenack and Pasternack : Ibid., 1904, 7, 193. Gill and Tufts : J. Am. Chem. Soc., 25, 251, 254, 498. Tolman : J. Am. Chem. Soc., 27, 589. Lewkowitsch: Oils, Fats, and Waxes, 4th Ed., p. 473. EDIBLE OILS AND FATS COMPOSITION OF BUTTER FAT 195. Browne analyzed the mixture of fatty acids from a sample having a rather low iodine number (29.28) with the following results : 1 Acids Percentage of acid in fat Corresponding per- centage of tri- glyceride Oleic 32.50 33.95 Dioxystetiric 1.00 1 04 Ste&ric . . 1.83 1 91 Palmitic 3861 40 51 9.89 10.44 Laurie 2.57 2.73 Capric .... 032 034 C a, pry lie . . . 049 053 Caproic 2 09 2 3 9 Butyric 5.45 6.23 Total 94.75 100 00 This calculation neglects the unsaponifiable matter, which according to Browne amounts to only about 0.1 per cent. The composition of butter fat is, however, quite variable, as will be seen from the range in analytical properties. VARIATIONS AND RELATIONS OF ANALYTICAL PROPERTIES The Reichert-Meissl or Reichert-Wollny number is much the most important of the data obtained in the examination of butter fat. The proportion of volatile acids tends to decrease as the period of lactation advances. The estimated normal range for the other important properties has been given in the table at the end of Chapter VIII. Any of these properties may be influenced by the feeding or health of the animal and occasionally vary much beyond the usually accepted " normal " limits, as is shown by the following data collected by Browne: 2 1 J. Am. Chem. Soc., 1899, 21, 823. 2 J. Am. Chem. Soc., 1899, 21, 632. 196 METHODS OF ORGANIC ANALYSIS General limits Extreme limits Reichert-Meissl number Saponification number . . Iodine number 20-33 220-236 26-38 11.2[Morse]-41 [Nilson] 216 [Samelson]-245[Fischer] 19.5 [Moore] -49.57 f Farnsteiner "I \_ and Karsch J The results of analyses of 357 authentic samples of butter fat collected from various parts of Great Britain and examined at the Government Laboratory, London, have been arranged by Thorpe l according to the Reichert-Wollny numbers and averaged by groups to show the relations between the principal physical and chemical properties of pure butter fat. The following table shows the averages for each group of samples. The first line, for example', gives the average Reichert-Wollny number for all samples in which this number lay between 22.00 and 22.99, with the average of the same samples for each of the other determinations. The second line shows the averages for all samples having Reichert-Wollny numbers from 23.00 to 23.99, etc. TABLE 17. RELATION OF PHYSICAL AND CHEMICAL PROPERTIES OF BUTTER FAT (THORPE) Number of samples Reichert- Wollny number Specific gravity 37.8 37. b Butyro- refractometer reading at 45 Saponification number Insoluble fatty acids per cent Mean molecular weight of insoluble acids 7 22.5 0.9101 42.0 219.9 90.1 266.9 17 23.5 0.9104 41.5 221.6 89.7 265.5 15 24.5 0.9108 41.5 223.5 89.4 265.0 27 25.5 0.9110 41.3 223.6 89.3 264.2 37 26.5 0.9113 41.0 225.6 88.9 261.9 51 27.5 0.9114 40.6 227.0 88.7 261.7 78 28.8 0.9118 40.1 228.6 88.4 260.9 56 29.5 . 0.9120 40.1 230.2 88.3 259.6 41 30.5 0.9123 39.9 231.7 87.9 260.1 18 31.3 0.9125 39.7 232.5 87.9 258.0 10 32.6 0.9130 39.4 232.8 87.7 257.8 1 J. Chem. 8oc., 1904, 85, 248. EDIBLE OILS AND FATS 197 In order to show to what extent increase of volatile acids takes place at the expense of oleic acid, the iodine numbers of 50 of the above samples were determined. Arranging these in groups of 20 and 30, respectively, according to the Reichert- Wollny values, the following average figures were obtained : Reichert- Wollny number Iodine number Oleic acid per cent Insoluble acid per cent Mean molecular weight of insoluble acids First group 04 9 40.0 44.4 89.6 264.6 Second jjroup . . 30.8 32.4 36.0 88.1 259.8 DETECTION OF OLEOMARGARINE Butter substitutes or "artificial butters," unless sold under special names indicating their origin, are collectively termed " oleomargarine " in America or " margarine " in England. The oleomargarine made in America l consists chiefly of refined 1 The materials used in the manufacture of oleomargarine in the United States during the fiscal year ending June 30, 1899 (Senate Document No. 168, 57th Congress, 1st Session), were as follows : TABLE 18. MATERIALS USED IN MANUFACTURE OF OLEOMARGARINE, 1899 Material Quantity Percentage of the whole Value per pound Total value Neutral lard Pounds 31,297,251 34.27 Cents 8 $2,503 780.08 Oleo oil . .... 24 491 769 26.82 9 2 144 917 69 Cottonseed oil . ..... " Butter oil " 4,357,514 4,342,904 4.77 4.76 6 6 522,025.08 260,520.00 Sesame oil 486,310 .53 10 4,863 10 Coloring matter .... 148,970 .16 20 29 296 00 Sugar . 110 164 .12 4 4 406 50 Glycerin 8 963 01 10 896 30 Stearin 5,890 .007 8 459 60 Glucose 2,550 003 3 76 50 Milk . . ... 14 200 576 15 55 1 142 005 76 Salt . ... 6 773 670 7 42 1 67 726 70 Butter 1 568 319 1 72 20 313 663 80 Cream 3 527 410 3 86 5 176 370 50 Total 91,322,260 100 $6,171,007.61 198 METHODS OF ORGANIC ANALYSIS lard, " oleo oil " (the soft part of beef fat), and cottonseed oil, often mixed with a small amount of butter and almost always churned with milk or cream. Palm oil and sesame oil are known to be used to some extent and other semi-drying and non-drying oils are probably utilized in some factories. Hence, in comparison with any of the common constituents of oleomargarine, butter fat is characterized by its high proportion of soluble volatile acids, together with low percentages of oleic and stearic acids. The presence of oleomargarine in butter fat, therefore, lowers the Reichert-Meissl and saponification numbers and the specific gravity, while it raises the percentage of in- soluble acids and either the melting point or the iodine number or both. Several European governments require that sesame oil be added to oleomargarine in order to faciliate its detection when mixed with, or substituted for, butter-. Similarly, the addition of butter to oleomargarine is sometimes forbidden or restricted in order to prevent the production of mixtures too closely resembling genuine butter. The essential features of the oleo- margarine laws of the principal countries are given in Lewko- witsch's Oils, Fats, and Waxes. DETECTION OF COCONUT FAT Since coconut fat consists largely of glycerides of saturated acids of low molecular weight, it could be added in considerable quantity to butter fat of average composition without causing the latter to vary beyond the normal limits in any of the im- portant analytical properties. A comparison of the Reichert- Meissl and saponification numbers, however, would lead to the detection of this adulteration since the former number is higher and the latter lower in butter than in coconut fat. In pure "Butter oil" is commonly stated to be a special brand of cottonseed oil ; but the high federal tax laid upon artificially colored oleomargarine by the law of May 9, 1902, practically prohibits the use of coloring matters employed before that date and has led to the introduction of "butter oils" containing palm oil which is naturally highly colored (Crampton and Simons : J. Am. Chem. Soc , 1905,27,270). EDIBLE OILS AND FATS 199 butter fat the value of the factor [Saponification number (200 + Reichert-Meissl number)] varies from 3.4 to 4.1; in pure coconut fat it varies from 47 to 50. 7. * Another method of showing the presence of coconut oil is to determine the volume of tenth-normal alkali required to neu- tralize the insoluble volatile acids from 5 grams of fat. Under the conditions described by Polenske 2 the results thus obtained are approximately quantitative, the percentage of insoluble volatile acids (mainly lauric acid) being much higher in coco- nut fat than in butter. REFERENCES I (See book references at end of preceding chapter.) II 1887-89. WILEY: Butter and Lard, Parts 1 and 4, Bui. 13, Bur. Chem., U. S. Dept. Agriculture. 1897. SADTLER : Arachis (Peanut) Oil. Am. J. Pharm., 69, 490 ; Analyst, 22, 284. 1898. ARCHBUTT : Estimation of Arachidic Acid. J. Soc. Chem. Ind., 17, 1124. HOPKINS: Maize Oil. J. Am. Chem. Soc., 20, 948. TORTELLI and RUGGERI : Detection of Cottonseed Oil. Z. angew. Chem., 1898, 464. 1898-99. BOMER: Phytosterol Test. Z. Nahr. Genussm., 1, 21, 81, 532; 2, 46, 705. 1899. BELLIER: Color Reactions for Sesame Oil. Ann. de Chim. Anal., 4, 217; Analyst, 25, 50. BROWNE : Butter Fat. J. Am. Chem Soc., 21, 632, 823, 975. COCHRAN : Butter and Butter Adulterants. J. Franklin Inst., 147, 85. 1900. BELLIER : Detection of Arachis Oil. Bull. Soc. Chim., [3], 23, 358. ESTCOURT : Butters from Various Countries Compared. Analyst, 25, 113. OILAR : Halphen Test. Am. Chem. J., 24, 355. WILLIAMS : Maize Oil. Analyst, 25, 146. 1 Juckenack and Posternack : Z. Nahr. Genussm., 1904, 7, 193. 2 Polenske: Ibid., 1904,7, 273. See also, Lewkowitsch's Oils, Fats, and Waxes. Muntz and Coudon : Ann. d. Inst. Agron., 1904; Analyst, 1905, 30, 155. Hesse : Milchwirthschaftl. Centrbl, 1905, 1, 13 ; Chem. Centrbl., 1905, I, 566. 200 METHODS OF ORGANIC ANALYSIS 1900-01. VULTE and GIBSON : Maize Oil. /. Am. Chem. Soc., 22, 453 ; 23, 1. 1901. HOLM, KRARUP, and PETERSON: Refraction, Iodine Number, and Volatile Acid Content of Butter. Z. Nahr. Genussm. 4, 746. PFRRIN : Separation of Arachidic Acid. Monat. scientif., [4], 15, 320; Z. Nahr. Genussm., 4, 986. SIEGFELD : Judgment of Butter by Reichert-Meissl Number. Z. Nahr. Genussm., 4, 433. WEEMS and GRETTENBERG : Analytical Characters of Different Cot- tonseed Oils. Proc. Iowa Acad. Sci., 1901 ; Z. Nahr. Genuxsm., 1902, 5, 465. 1901-02. BOMER: Phytosterol Test. Z.Nahr. Genussm,, 4, 865, 1070; 5, 1018. 1902. BEHREND and WOLFS : Butter Fats from Individual Cows. Z. Nahr. Genussm., 5, 689. FULMER : Halphen's Test. J. Am. Chem. Soc., 24, 1148. LAXA : Change of Butter Fat by Microorganisms. Arch. Hyg., 41, 119. RITTER : Phytosterol. Z. physiol. Chem., 34, 430, 461 ; 35, 550. 1902. TOLMAN and MUNSON : Olive Oil and its Substitutes. Bui. 77, Bur. Chem., U. S. Dept. Agriculture. 1903. CRAMPTON: Composition of Process or Renovated Butter. J. Am. Chem. Soc., 25, 358. GILL and TUFTS : (Cholesterol, Phytosterol, Sytosterol.) J. Am. Chem. Soc., 25, 251, 254, 498. KREIS : Detection of Sesame Oil. Chem. Ztg., 27, 1030. SWAYING : Influence of Feeding Cottonseed and Sesame Meal on the Properties of Butter Fat. Z. Nahr. Genussm., 6, 97. TOLMAN and MUNSON : (Analysis of Salad Oils). J. Am. Chem. Soc., 25, 954. Wus : (Iodine Numbers of Edible Oils). Z. Nahr. Genussm., 6, 692. 1904. GROSSMANN arid MEINHARD : Dutch Butter. Z. Nahr. Genussm., 3, 237. KRAUS: (Conditions Affecting the Keeping Qualities of Butter). Arb. kaiserl. Gesundheitsamte, 22, 235 ; Z. Nahr. Genussm., 9, 286. 1905. ARNOLD: (Analysis of Food Fats). Z. Nahr. Genussm., 10, 201. CRAMPTON and SIMONS : Detection of Palm Oil when used as a Color- ing Material in Oils and Fats. J. Am. Chem. Soc., 27, 270. FISCHER and PEYAN: (Detection of Cottonseed Oil). Z. Nahr. Genussm., 9, 81. LYTHGOE : Refractive Index of Codliver Oil. J. Am. Chem. Soc., 27, 887. PARRY: Codliver Oil Standards. Chemist and Druggist, 46, 491; Analyst, 30, 208. POLENSKE: (Analysis of Lard and Butter). Arb. kaiserl. Gesund- heitsamte, 22, 557, 576; Analyst, 31, 46. EDIBLE OILS AND FATS 201 1905. TOLMAN: Phytosteryl Acetate Test for Examination of Lard from Cottonseed -meal-fed Hogs. /. Am. Chem. Soc., 27, 589. WESSON and LANE : Quantitative Analysis of Lard. /. Soc. Chem. Ind., 24, 714. 1906. ASCHMANN and AREND : Determination of Water in Butter and Other Fats. Chem. Ztg., 30, 953. FARNSTEINER : (Examination of Lard). Z. Nahr. Genussm., 11, 1 ; Analyst, 31, 72. HARRIS : Estimation of Coconut Oil in Butter Fat. Analyst, 31, 353. PATRICK: Rapid Determination of Water in Butter. J. Am. Chem. Soc., 28, 1611. EJDEAL and HARRISON: On the Polenske Method for Coconut Oil in Butter. Analyst, 31, 254. TOLMAN: American Codliver Oils. J. Am. Chem. Soc., 28, 388. WALKER: Coconut Oil. Philippine J. Sci., 1, 117; Analyst, 31, 165. 1907. AMBERGER: Influence of Food on Composition of Butter Fat. Z. Nahr. Genussm., 13, 614. ARCHBUTT : Tunisian and Algerian Olive Oils. J. Soc. Chem. Ind., 26, 453. BELLIER : Analysis of Butter (New Method). Ann. de Chim. Anal., 11, 412 ; Analyst, 32, 22. HANUS: (Detection of Coconut Oil). Z. Nahr. Genussm., 13, 18. HENSEVAL and HUVART : Contribution to the Study of Fish Liver 011. Chem. Rev. Fette- Harz-Ind., 14, 191; Chem. Abs., 1, 2751. HINKS : Detection of Coconut Oil in Butter. Analyst, 32, 160. HODGSON : Detection of Coconut Oil in Butter. Chem. News, 95, 121. JEAN: Examination of Butter. Rev. gen. Chim., 10, 253; Chem. Abs., 1, 2617. KUHN and BENGEN : Cause of Halphen Reaction. Z. Nahr. Genussm., 12, 145. KUHN and HALEPAAP: Critical Study of Welman's Reaction. Z. Nahr. Genussm., 12, 449. MCPHERSON and RUTH : Corn Oil Its Possibilities as an Adulter- ant in Lard and its Detection. J. Am. Chem. Soc., 29, 921. PATRICK: Rapid Determination of Water in Butter. J. Am. Chem. Soc., 29, 1126. SIEGFELD : (Detailed Study of Polenske Number) . Chem. Ztg., 32, 511. : Influence of Feed on Butter Fat. Z. Nahr. Genussm., 13, 513: SMITH : Application of Arachidic Acid Test to Solid Fats. J. Am. Chem. Soc., 29, 1756. SOLTSEIN : Detection of Tallow and Lard in Presence of Each Other. Chem. Rev. Fette-Harz-lnd., 13, 240; Chem. Abs., 1, 109. WINDAUS : Separation of Cholesterol and Phytosterol. Chem. Ztg., 30, 1011. 202 METHODS OF ORGANIC ANALYSIS 1908. CORNELISON : Detection of Synthetic Color in Butter. J. Am. Chem. Soc., 30, 1478. EMERY: Detection of Beef Fat in Lard. U. S. Dept. Agr., Bur. Animal Ind., Cir. 132 ; Chem. Abs., 2, 2461. FARRINGTON : Determination of Water in Butter. Wis. Agl. Expt. Station, Bui. 154 ; Chem. Abs., 2, 870. FULMER and MANCHESTER : Effect of Heat on Cottonseed Oil Con- stants. /. Am. Chem. Soc., 30, 1477. FRITSCHE: (Polenske Number of Dutch Butter). Z. Nahr. Genussm., 15, 193. HANUS and STEHL : The Ethyl Ester Number, A New Method for Coconut Fat. Z. Nahr. Genussm., 15, 576. KREIS : Influence of Rancidity on Baudouin Reaction. Chem. Abs., 2, 2165, 2166. WAGNER and CLEMENT : Cottonseed Oil. Z. Nahr. Genussm., 16, 145. 1909. KOHNIG and SCHLUCKEBIER : Influence of Fat in Feed upon Fat of Pigs with Special Regard to Phytosterol. Z. Nahr. Genussm., 15, 641. TATLOCK and THOMSON : The Value of the Polenske Method. J. Soc. Chem. Ind., 28, 69. MILLIAU : (Reactions of Kapok and Baobab Oils). Matieres grasses, 2, 1545 ; Chem. Abs., 4, 969. 1910. BULL and SAETHER : Can One Determine the Nature of the Vege- table Oil on Sardines? Chem. Ztg., 34, 733. GIBBS and AGCAOILI: Lard from Wild and Domestic Philippine Hogs and the Changes Effected by Feeding Copra Cake. Philippine. J. Sci., 5A, 33; Chem. Abs., 4, 2748. HARE : Some Effects of Feeds upon the Properties of Lards. /. Ind. Eng. Chem., 2, 264. LINDSAY ET AL: (Effect of Feed on Butter Fat). Mass. Agl. Expt. Sta. Report, 1908, 66-110; Chem. Abs., 4, 1774. 1911. ARNOLD: Determination of Coconut Oil in Edible Fats. Z. Nahr. Genussm., 21, 587. MARCILLE: Olive Oils from Tunis. Ann. falsif., 3, 372; Chem. Abs., 5, 732. REVIS and BOLTON : Methods of Estimating Coconut Oil and Butter in Butter and Margarine. Analyst, 36, 333. RICHARDSON : Coconut Oil of High Iodine Value. /. Ind. Eng. Chem., 3, 574. STEENBOCK : Modification of Wiley's Method for Melting Point of Fats. /. Ind. Eng. Chem., 2, 480. TORTELLI and FORTINI : (Detection of Rape Oil in Salad Oils). Ann. falsif., 4, 139; Chem. Abs., 5, 1947. CHAPTER X Drying Oils SEVERAL oils of both vegetable and animal origin (among the latter more particularly the fish oils) contain glycerides of highly unsaturated fatty acids which on exposure in thin layers to air readily absorb oxygen and become converted into solids. This property gives rise to the term " drying " oils. A few of the more prominent drying oils may be listed as follows : VEGETABLE DRYING OILS Linseed oil, pressed from the seeds of the flax plant, pro- duced in large quantities in North and South America, Russia, and India, is the most important of the drying oils and is ex- tensively used for direct application as a protective coating to wood and metal, and in the manufacture of paints and varnishes, oilcloths, linoleum, printing inks, rubber substitutes, etc. Lewkowitsch estimates that there were produced in 1907, 1,200,000 tons of linseed in Argentina ; 646,275 tons in the United States; 450,000 tons in Russia; and 419,900 tons in India. Linseed yields in general about 35 per cent of its weight of oil. The seed are usually crushed between rollers, then heated to about 160 F. and pressed while warm. The oil so obtained is yellowish brown or brownish yellow and somewhat turbid from traces of moisture and mucilaginous material, which impurities, however, gradually settle out when the oil is stored. Oil which has been thus purified by long standing is sometimes called "tanked oil." A more rapid method of refining is to 203 204 METHODS OF ORGANIC ANALYSIS treat the oil with 1 to 2 per cent of sulphuric acid. This pro- duces a charred mass which carries down with it the greater part of the impurities. Boiled linseed oils are made by heating linseed oil with a "drier," formerly to temperatures of 210 to 260 C., now, according to Lewkowitsch, to a temperature of about 150 C. The extent to which the analytical characters are changed depends upon the details of the process and the extent to which the oil is exposed to air. Tung oil, also called Chinese or Japanese wood oil, is pressed from the nuts of the tung tree. According to Ennis the use of this oil in the United States is steadily increasing and experi- ments are being made with the cultivation of the tree in Cali- fornia. At present the oil comes chiefly from China, where the natives roast and crush the nuts and press from them about 40 per cent of oil, which is only about three fourths of the amount present. According to Lewkowitsch the oil is also produced in Madagascar under the name of " Bakoly oil." Tung oil has a characteristic persistent odor which is not easily removed by refining. It is used largely for oiling wood and waterproofing paper. Lewkowitsch states that while many patents have been taken out for the substitution of tung oil for linseed oil in manufactures, little progress has yet been made in this direction. Walnut oil and poppyseed oil, while they have not such pronounced drying properties as linseed and tung oils, have the advantage of yielding almost colorless films which are not likely to crack. These oils are therefore especially adapted to use in white or delicately colored paints for artists. Maize oil has not sufficient drying property to be useful as a paint oil but " dries " well enough to permit of its use in putty. It is also used like linseed oil in making rubber substitute. It has probably been used to some extent as an adulterant of lin- seed oil. Soy (soja, soya) bean oil is used to some extent in admixture with linseed oil. DRYING OILS 205 FISH OILS In recent years the fish oil industry has been much altered through the introduction of steam trawlers and the prompt rendering of the oil, which results in a product largely free from the dark color and rank odor formerly regarded as characteristic of fish oils. Menhaden oil is obtained from the bodies of the menhaden, a fish somewhat larger than a herring, which abounds in the Atlantic especially off the coast of New Jersey. Toch 1 states that menhaden is the best fish oil for use in paint and that the winter-bleached variety is to be preferred. This should be fairly pale in color, with an iodine number of 150 or over, and with little or no fishy odor. Such an oil, Toch finds, may be mixed with linseed oil even up to 75 per cent of the mixture with good results when used for exterior painting where its odor is not noticeable. In fact paint made from such a fish oil is said to be more resistant to heat than a linseed oil paint and therefore preferable for smoke stacks, boiler fronts, etc. It is also said to be more resistant to the salt air of the seacoast. Menhaden oil has also been used in place of linseed oil in the manufacture of enamel leather and of printing inks. Japanese sardine oil, sardine oil, and herring oil have some drying property and are used to some extent as partial sub- stitutes for, or adulterants of, linseed oil. LINSEED OIL ANALYTICAL PROPERTIES OF LINSEED OIL Commercial linseed oil is usually designated by the region of its origin. It varies considerably, the variation being due mainly to the presence of foreign seeds in the linseed at the time of pressing. Hempseed is practically always present, sometimes in very small proportions but often to the extent of 5 per cent, or more, of the weight of seed. The drying properties of lin- 1 J. Ind. Eng. Chem., 3, "627. 206 METHODS OF ORGANIC ANALYSIS seed oil are better the purer the seeds from which it is pressed. The iodine number and the drying power of the oil decrease as the proportion of hempseed increases. Hence a linseed oil con- taining much hemp oil wo.uld be shown to be of inferior quality by its low iodine number, but could not be pronounced adulterated so long as this did not fall below 170. The maximum iodine number of linseed oil is difficult to fix, since the results obtained by the method of Wijs often exceed the Hubl numbers, but since no other common oil has a higher iodine number than linseed, the maximum limit is of little practical importance in the detec- tion of adulterations. The usual range of the more important analytical constants of linseed oil has been given in the table at the end of Chap- ter VIII. The interpretation of " constants," their relations to each other, and their use in the detection of adulterations having been discussed in some detail in connection with the exam- ination of salad oils, it will be sufficient in this case to mention briefly the principal adulterants with means for the detection of each, and describe the hexabromide test which distinguishes linseed from practically all other oils. ADULTERANTS AND METHODS OF DETECTION Mineral Oil Mineral oil would greatly lower the iodine number, tempera- ture reaction, and saponification number. Whenever a low saponification number is found, the unsaponifiable matter should be separated and examined. Any mineral oil which is not volatile at 100 can be separated quantitatively, dried, and weighed. Volatile mineral oil can be distilled by means of a current of steam, separated from water in the distillate, and measured or weighed. In case turpentine were present, as in some so-called " boiled " oils, it would be distilled with steam and would separate from water in the distillate in the same way. The optical rotatory power of turpentine affords an easy means of distinguishing it from benzine or other volatile mineral oil. DRYING OILS 207 Rosin and Rosin Oil Rosin dissolved in linseed oil raises the specific gravity and index of refraction while the saponification and iodine numbers are appreciably decreased only when large amounts of rosin are added. Presence of rosin greatly increases the acid number, which in pure linseed oil is usually less than 7. Rosin acids can be separated and determined by Twitchell's method as described under soap analysis beyond. Rosin oil in linseed oil would raise the specific gravity and greatly lower the saponification and iodine numbers. Rosin oil is a mixture of substances many of which are unsaponifiable, so that its presence in linseed oil would increase the amount of unsaponifiable matter. Either rosin or rosin oil can be detected by the Liebermann- Storch color reaction or by determining the bromine substitution number. Liebermann-StorcTi Reaction. Shake 2 cc. of the oil with 5 cc. of acetic anhydride, warming gently. Allow to cool, draw off the anhydride, and test by adding one drop of sulphuric acid (1:1). A violet color (not permanent) is produced in the presence of rosin or rosin oil. Cholesterol, which might be found in linseed oil if fish oil were present as an adulterant, gives a similar color reaction. Bromine Substitution Number (Mcllhiney). Fatty oils take up bromine by direct addition, little or no substitution taking place. With rosin and rosin oil much the greater part of the bromine is taken up by substitution, a molecule of hydrobromic acid being formed for each molecule of bromine which disappears. The hydrobromic acid thus affords a means of measuring the amount of substitution. It is determined by adding an excess of potassium iodate and titrating the liberated iodine. The same apparatus can be used as in the determination of the iodine number and the manipulation is similar. From 0.2 to 0.3 gram of the drying oil is dissolved in 10 cc. of carbon tetrachloride and 20 cc. of a one third normal solution of bromine in carbon tetrachloride is added. After two minutes potassium iodide is 208 METHODS OF ORGANIC ANALYSIS added and the excess of halogen titrated by means of thiosul- phate as in the determination of the iodine number. This shows the total amount of bromine which has disappeared. As soon as this titration is finished, add 5 cc. of a 2 per cent solution of potassium iodate and titrate the iodine set free from the iodate by the action of the free halogen acids, according to the reaction : 6 HI + KIO 3 = 3 I 2 + KI + 3 H 2 O. The bromine thus found is equal in amount to that which has combined with the sample by substitution. For further details of manipulation the reader must be referred to the original papers. 1 According to Mcllhiney the bromine substitution number of raw or boiled linseed oil is always less than 7, while rosin oil gives numbers from 40 to 100 and rosin from 65 to 80. Maize Oil The presence of maize oil in linseed oil lowers the specific gravity, index of refraction, iodine number, and temperature reaction. The amount which can be added without carrying these numbers below the normal limits of variation will depend upon the quality of the linseed oil in the mixture. Since the maize oil used as an adulterant of linseed would probably not be highly refined, the characteristic odor and taste would aid in its detection. Cottonseed Oil The " constants " of linseed oil would be lowered by cotton- seed in the same way as by maize oil and to a somewhat greater extent. If the cottonseed oil had not been heated, its presence would be detected by the Halphen test as described under salad oils. Fish Oils Menhaden and other fish oils are often used as adulterants, and are difficult to detect with certainty since their " constants " are frequently within the limits found for pure linseed oil. Their presence is often indicated by the odor, but this cannot be relied upon, as the difference in odor between refined men- 1 Mcllhiney: J. Am. Chem. Soc., 1894, 16, 275; 1899, 21, 1084 ; 1902, 24, 1109. See also Tolman : Ibid., 1904, 26, 826. DRYING OILS 209 haden and low grade linseed oil is not so pronounced as might be supposed. Lewkowitsch recommends the determination of the melting point of the phytosteryl acetate obtained from the oil. The crystals of phytosteryl acetate from pure linseed oil melt at 128-129 (Bomer and Winter), while in the presence of cholesterol from fish oil much lower melting points are obtained. The method of Eisenschiml and Copthorne, as adopted by the Association of Official Agricultural Chemists, for the detection of fish oil in the presence of vegetable oils is as follows : Dissolve in a test tube about 6 grams of the oil in 12 cc. of a mixture of equal parts of chloroform and glacial acetic acid. Add bromine drop by drop until a slight excess is indicated by the color, keeping the solution at about 20 C. Allow to stand 15 minutes or more and then place the test tube in boiling water. If only vegetable oils are present, the solution will be- come perfectly clear, while fish oils will remain cloudy or con- tain a precipitate due to the presence of insoluble bromides. This method is based on the fact that the bromides of the vegetable oils, although they may be precipitated abundantly in the cold, will be completely soluble in the mixture of chloro- form and glacial acetic acid when heated as described in this test. Boiled linseed oil containing metallic driers cannot be tested by this method unless the metals are first removed. This method of Eisenschiml and Copthorne is evidently an outgrowth of the " hexabromide " test of Hehner and Mitchell, the description of which follows. " HEXABROMIDE " TEST Hehner and Mitchell a showed that linseed and fish oils differ from other oils in yielding considerable quantities of insoluble bromides when treated with bromine in ether solution. They applied the test directly to the oil as follows : Dissolve 1 to 2 grams of oil in 40 cc. of ether acidulated with glacial acetic acid, cool the solution to 5, and add bromine, drop by drop, until the solution is permanently colored brown. 1 Analyst, 1898, 23, 310. Also Mitchell in Allen's Commercial Organic Analysis, 4th Ed., Vol. II, p. 28. 210 METHODS OF ORGANIC ANALYSIS After standing for at least 3 hours, preferably in an ice box, filter through a Gooch crucible, leaving most of the precipitate in the vessel in which it was formed, wash 4 times with ice-cold ether, dry the precipitate at 100, and weigh. Linseed oil yields 23 to 38 per cent (usually about 25 per cent) of the insoluble bromide, the amount increasing with the iodine number of the oil. Some of the fish oils yield equal or greater amounts; but tung, poppy, and walnut oils, and such seed oils as maize and cottonseed, yield almost none according to the figures compiled by Lewkowitsch never over 2 per cent. Mitchell regards the products precipitated in these tests as bromides of mixed glycerides containing one radicle of linolenic acid (or an isomeric acid), and states that the insoluble bromide from linseed oil contains about 56 per cent of bromine and melts at 143.5 to 144, whereas the corresponding bromides from marine animal oils decompose before melting so that even small amounts of such oils in linseed oil can be detected by this test. Lewkowitsch recommends that the test be applied to the mixed fatty acids rather than to the oil itself. In the separa- tion of the acids care must be taken to avoid oxidation by ex- posure to the air. The mixture of fatty acids from linseed oil yields 30 to 42 per cent of hexabromide, melting to a clear liquid at 175 to 180, whereas the corresponding products from fish, liver, and blubber oils do not melt at this temperature but be- come darker and are completely blackened at about 200. Lewkowitsch states that this test is capable of showing the presence of 10 per cent of fish oil in linseed oil. For further discussion of this test see the papers just cited and also Proctor: J. Soc. Chem. Ind., 25, 798 (1906). OILS ALTERED BY AGE OR OXIDATION It has been assumed in discussing the analytical " constants " that the oils under examination are fresh or have been kept under such conditions as to prevent any material alteration. Age alone probably has no appreciable effect upon the analyt- ical properties of commercially pure fatty oils, but such oils when kept for a long time in contact with the air, for example, DRYING OILS 211 in partially filled or loosely stoppered vessels, take up atmos- pheric oxygen and gradually become considerably altered in those properties which are commonly regarded as "constants." This atmospheric oxidation naturally takes place much more rapidly with drying than with non-drying or semi-drying oils, and in open vessels than in those in which the oil is exposed to only a limited amount of air. It is probable that oils which have been thus altered are more frequently encountered in analytical work than has been supposed. The influence of such oxidation upon the more important analytical properties is to increase the specific gravity, index of refraction, and temperature reaction with sulphuric acid, and to decrease the iodine number, the specific refractive power, 1 and, in the case of olive oil, the viscosity of the soap solution. The acidity of the oil may increase at the same time, but this change does not always occur. The following results were obtained upon oils intentionally exposed to the air, arid while large as compared with the changes which should occur under ordinary conditions of laboratory storage, they do not represent the maximum change which may result from atmospheric exposure. TABLE 19. EFFECTS OF EXPOSURE OF OILS TO AIR Oil Iodine number Sp. Gr. 15.5 15.5 Index of refraction at 15.5 Specific refractive power Specific tempera- ture reaction Olive oil before exposure . . . 83.8 77 3 0.9165 9240 1.4712 1 4722 0.5141 5100 100 127 2 Lard oil before exposure Same after exposure 73.3 56 2 0.917 943 1.4697 1.4724 0.5122 05010 106 141 Cottonseed oil before exposure Same after exposure 105.2 90.2 0.923 0.939 1.4737 1.4779 0.5132 0.5090 171 217 2 Linseed oil before exposure . . 177.1 136 9 0.934 0969 1.4835 1 4886 0.5177 05042 1 Calculated by Landolt's formula JV-1 D (Ber., 1882, 15, 1031), in which is the index of refraction and D is the specific gravity. 2 These numbers were determined earlier than the other data and presumably represent a lesser degree of change. 212 METHODS OF ORGANIC ANALYSIS Many other oils have been tested with similar results. It is evident that oils thus altered are very likely to be misjudged, especially if only one or two quantitative determinations are made. Thus if only the specific gravity and temperature re- action of the olive oil had been determined, the results would have been interpreted as indicating the presence of some seed oil. The iodine number of the linseed oil taken alone would indicate extensive adulteration with some oil of lower drying power. The results emphasize the importance of determining the iodine number and either the specific gravity or the index of refraction in all cases, and show the advantage of determin- ing the temperature reaction, not as a substitute for the iodine number but for comparison with it. For a fuller discussion of this subject with the results obtained upon a number of other oils the reader is referred to two papers in the Journal of the American Chemical Society (July, 1903, and May, 1905). As the result of this work it appears that the increase in specific gravity and the decrease in iodine number are almost exactly proportional to each other in non-drying and semi- drying oils, so that in examining an altered oil belonging to either of these classes the original iodine number can be esti- mated by adding 0.8 to the number found on the exposed sample for each increase of 0.001 in the specific gravity. When the original specific gravity is not known, the calculation must be based upon the average specific gravity for oil of the species under examination. The error of this assumption can hardly be sufficient to affect the interpretation of the results. The iodine numbers of exposed samples of linseed and fish oils cannot be corrected accurately by the rule given for semi-drying and non-drying oils, the number thus obtained being always too low. It has also been found that when linseed oil is thus changed by atmospheric oxidation the amount of insoluble bromide which it will yield in the hexabromide test is greatly reduced. Commercial "blown" oils show greatly increased specific gravities and decreased iodine numbers ; the changes appear to bear much the same relation to each other as in the oils which have been altered by exposure. DRYING OILS 213 "UNKNOWN" OILS AND MIXTURES In the examination of an unknown oil the appearance, odor, and taste should be compared with those of typical oils of known purity. In testing transfer a drop of the oil by means of a glass rod to the back of the tongue and note both the first impression and the after taste. The odor may be observed not only cold, but also after heating a portion in a porcelain dish to 140-150. Also after cooling sufficiently pour a few drops of the oil into the palm of one hand, rub with the other and smell again. These preliminary superficial observations and the determination of the iodine and saponification numbers and either the specific gravity or the index of refraction should locate the sample as one of a small group of oils, after which any special tests available for the detection of individual mem- bers of the group can be applied. The tests described in this and the preceding chapter taken in connection with the quanti- tative determinations mentioned enable the analyst, in the majority of cases, to identify the oil or, if a mixture, the prin- cipal constituent. In case of doubt one should not fail to consult the larger works, especially Lewkowitsch's Oils, Fats, and Waxes. If a saponification number indicates that only fatty oil is present, but the relation of the specific gravity to the iodine number does not correspond to that ordinarily found in any pure oil, the determination of the specific temperature reaction and the acidity will usually show whether the discrepancy is to be attributed to oxidation or adulteration. The relative commercial value will of course determine what oils can profitably be used as adulterants. Prices vary greatly in different markets, as well as with the degree to which the oils are refined, and are also likely to fluctuate from year to year so that no fixed order of commercial value can be given. The list of oils in order of commercial value given by Gill and by Lewkowitsch show considerable variation, which doubt- less is due largely to the differences between American and English markets. In each of the lists the highest-priced oils are given first. 214 METHODS OF ORGANIC ANALYSIS Gill. Almond, castor, sesame, neatsf oot, rape, olive, sperm, whale, peanut (aracbis), linseed, tallow, lard, fish, cottonseed, mineral, rosin oil. Lewkowitsch. Almond, sperm, olive, neatsfoot, lard, cod liver, arctic sperm, arachis, poppy, sesame, seal, rape, castor, cottonseed, maize, linseed, whale, fish, mineral, rosin oil. In the examination of mixtures containing other than fatty oils, it may be necessary to separate the mixed fatty acids and examine this mixture in order to identify the fatty oils origin- ally present. The " constants " of the mixed fatty acids of various oils, as well as of many oils and fats not mentioned in this work, are conveniently tabulated in Lewkowitsch's Lab- oratory Companion to the Fat and Oil Industries. REFERENCES ALLEN : Commercial Organic Analysis. CHURCH : Chemistry of Paints and Painting. ENNIS : Linseed Oil and other Seed Oils. FAHRION : Die Chemie der trocknenden Oele. GILL : Handbook of Oil Analysis. HOLLEY and LADD : Analysis of Mixed Paints, Color Pigments, and Varnishes. LEWKOWITSCH : Chemical Technology and Analysis of the Oils, Fats, and Waxes. SABIN : Technology of Paint and Yarnish. SCOTT : White Paints and Painting Materials. TOCH : Materials for Permanent Painting. : Technology of Mixed Paints. II 1891. BALLANTYNE: (Oxidized Oils). /. Soc. Chem.Ind., 10, 29. 1892. THOMPSON and BALLANTYNE : (Same). J. Soc. Chem. 2nd., 11, 506. 1898. FAHRION: (Same). Z. angew. Chem., 1898, 781. HEHNER and MITCHELL : (" Hexabromide" Test). Analyst, 23, 310. 1899. GILL and LAMB : American Linseed Oil. /. Am. Chem. Soc., 21, 29. MC!LHINEY: (Bromine Substitution Number). /. Am. Chem. Soc., 21, 1084. 1901. MclLHiNEY : Report on Linseed Oil and its Adulterants to the New York State Commissioner of Agriculture. (Reprinted in Sabin's Technology of Paint and Varnish, Chapter V.) DRYING OILS 215 1902. LEWKOWITSCH : (Oxidized Oils). Analyst, &7, 139. MC!LHINEY: Further notes on the Bromine Absorption of Oils. /. Am. Chem. Soc., 24, 1109. 1903. DUNLAP and SCHENK : Oxidation of Linseed Oil. J. Am. Chem. Soc., 25, 826. SHERMAN and FALK : Influence of Atmospheric Oxidation upon the Composition and Analytical Constants of Fatty Oils. J. Am. Chem. Soc., 25, 711. SJOLLEMA : Linseed Oil. Z. Ndhr. Genussm., 6, 631. UTZ : Poppyseed Oil. Chem Ztg., 27, 1176. 1904. LEWKOWITSCH: Linseed Oil. Analyst, 29, 2. MCCANDLESS : Examination of Turpentine. J. Am. Chem. Soc., 26, 981. 1904-07. Reports and Discussions on Preservative Coatings for Iron and Steel. Proc. Am. Soc. Testing Materials, 4, 137 ; 5, 79 ; 6, 63 ; 7, 140. 1905. LANGMUIR : Determination of Rosin in Shellac. J. Soc. Chem. Ind., 24, 12. McGiLL : Examination of Turpentine. Bui. 79, Canadian Inland Revenue Laboratory. RABY : Rotatory Power of Turpentine. Ann. de Chim. Anal., 10, 146 ; Analyst, 30, 210. SHERMAN and FALK: Influence of Atmospheric Oxidation upon the Analytical Constants of Fatty Oils. J. Am. Chem. Soc., 27, 605. THOMPSON: Proper Methods in Conducting Painting Tests. Proc. Am. Soc. Testing Materials, 5, 417. VALENTA : Examination of Turpentine. Chem. Ztg., 29, 807 ; Analyst, 30, 342. 1906. BEADLE and STEVENS: Analysis of Rosin Size. Chem. News, 93, 155 ; Chem. Eng., 4, 263. BOHME : Detection of Adulterants in Turpentine. Chem. Ztg., 30, 631. GENTHER : Drying Process of Linseed Oil. Z. angew. Chem., 1906 ; Chem. Abs., 1, 912. GILL: Determination of Rosin in Varnishes. J. Am. Chem. Soc., 23, 1723. HOLLEY: Turpentine and its Substitutes. 17th Ann. Rpt. North Dakota Agl. Expt. Station. LEVY : American Colophony. Ber., 39, 3043. PROCTOR and BENNETT: Examination of Marine Oils. J. Soc. Chem. Ind., 25, 798. 1907. CHEESMAN: Priming Coats for Metal Surfaces Linseed Oil vs. Paint. Proc. Am. Soc. Testing Materials, 7, 479. CLOVER : Philippine Wood Oils. Phil. J. Sci., 1, 191. 216 METHODS OF ORGANIC ANALYSIS 1907. Committee Report on Shellac Analysis. J. Am. Chem. Soc., 29, 1221. ENDEMANN : Testing Shellac for Purity. J. Frank. Inst., 164, 285. HUGHES: Deleterious Ingredients in Paints (with Discussion). Proc. Am. Soc. Testing Materials, 7, 486. KRESS : Analytical Properties of some Pine Wood Oils. School of Mines Quarterly, 29, 46. LADD: Paint Legislation (with Discussion). Proc. Am. Soc. Testing Materials, 7, 523. McGiLL : Examination of Turpentine. J. Soc. Chem. Ind., 26, 847. PERRY: Physical Properties of Paint Films. Proc. Am. Soc. Testing Materials, 7, 511. PHOKIN : (Oxidation and Polymerization Processes in Drying Oils). J. Russ. Phys. Chem. Soc., 39, 307, 308 ; Chem. Abs., 1, 1752, 1775. RYAN and MARSHALL : Influence of Oxygen and Nitrogen, and San- light and Darkness on Olive Oil. Am. J. Pharm., 79, 308 ; Chem. Abs., 1, 2275. SMITH : Physical Testing of Oil Varnishes (with Discussion). Proc. Am. Soc. Testing Materials, 7, 499. UTZ : Specific Gravity of Linseed Oil. Chem. Rev. Fett-Harz-Ind., 14,137; Chem. Abs., 1, 2181. 1908. COSTE: Examination of Turpentine and its Substitutes. Analyst, 33, 219. FREY : Rapid Determination of Naphtha in Turpentine. J. Am. Chem. Soc., 30, 420. GILL: Oxidation of Olive Oil. J. Am. Chem. Soc., 30, 874. LORENTZ : Unsaponifiable Matter of Linseed Oil. Chem. Ztg., 32, 819. MCILHINEY: Analysis of Oil Varnishes. Eng. News, 60, 31 ; Chem. Abs., 2, 2630. MC!LHINEY: Method of Analyzing Shellac. J. Am. Chem. Soc., 30, 867. MARCUSSON : Determination of Benzine in Turpentine. Chem. Rev. Fett-Harz-Ind., 17, 6; Chem. Abs., 4, 1541. RICHARDSON and BOWEN: Analysis of Turpentine Oils. J. Soc. Chem. Ind., 27, 613. SCHULTZE: (Nature of Rosin Oil). Ann. Chem., 359, 129; Chem. Abs., 2, 1715. 1909. Committee Report on Preservative Coatings for Structural Materials. Proc. Am. Soc. Testing Materials, 9, 139. GEER : Analysis of Turpentine by Fractional Distillation. Cir. 152, Forest Service, U. S. Dept. Agriculture. MARCUSSON : Turpentine and Its Substitutes. Chem. Ztg., 33, 966, 978, 985; Chem. Abs., 4, 1236, 1542. DRYING OILS 217 PAUL : Turpentine and Its Adulterants. J. Ind. Eng. Chem., 1, 27. WHITE: Paints for Concrete Their Need and Requirements (with Discussion). Proc. Am. Soc. Testing Materials, 9, 526. 1910. AGRESTINI: Changes in Olive Oil kept for Over Two Centuries. Staz. sper. agrar. ital., 43, 283 ; Chem. Abs., 4, 3147. EIBNER and HUE : Determination of Benzine in Turpentine. Chem. Ztg., 34, 643, 657. EISENSCHIML and COPTHORNE : Detection of Fish Oils in Vegetable Oil. J. Ind. Eng. Chem., 2, 43. HEPBURN : Natural Changes Occurring in Fats and Oils. J. Frank. Inst., 168, 365, 421 ; 169, 23. KREIKENBAUM : Analytical Constants of Chinese Wood Oil. J. Ind. Eng. Chem., 2, 205. MORRELL : Testing Turpentine. J. Soc. Chem. Ind., 29, 241. SINGH : Analytical Constants of Shellac. J. Soc. Chem. Ind., 29, 1435. YAUBEL : Analysis of Shellac. Chem. Ztg., 34, 991, 1008; Chem. Abs., 5, 1196. WALKER : Some Technical Methods of Testing Miscellaneous Sup- plies. U. S. Dept. Agriculture, Bur. Chem., Bui. 109, Revised. 1911. CHERCHEFFSKY: Methods of Testing Turpentine. Matieres grasses, 3, 1925; Chem. Abs., 5, 1677. COSTE and NASH : Turpentine Substitutes. Analyst, 36, 207. FORREST : Characteristics of Creosote and Tar Oils Available for Wood Preservation. J. Soc. Chem. Ind., 30, 193. INGLE: Linseed and Other Oils Relations of "Constants" and Effect of Heat. /. Soc. Chem. Ind., 30, 344. JENSEN : Examination of Linseed Oil. Pharm. J., 86, 839 ; Chem. Abs., 5, 2976. LANGMUIR and WHITE : The Analysis of Shellac. J. Soc. Chem. Ind., 30, 786. TOCH : Fish Oil as a Paint Vehicle. /. Ind. Eng. Chem., 3, 627. VEITCH: Commercial Turpentines of the United States. J. Ind. Eng. Chem., 3, 521. VEITCH and DONK : Wood Turpentine; Its Production, Refining, Properties, and Uses. U. S. Dept. Agriculture, Bur. Chem., Bui. 144. CHAPTER XI Petroleum and Lubricating Oils THE petroleums from different localities vary considerably both in the hydrocarbons of which they are chiefly composed and in the amounts of nitrogen, sulphur, and oxygen com- pounds present. The Appalachian field, which includes the oil regions of Pennsylvania, New York, West Virginia, Ken- tucky, and southeastern Ohio, yields petroleum consisting chiefly of paraffin hydrocarbons of which all the members of the series from CH 4 to C 16 H 34 , as well as C 25 H 52 , C 27 H 56 , and C 30 H 62 , have been shown to be present together with small amounts of the olefines and traces of the hydrocarbons of the benzene and napthene series. This is generally considered the best grade of petroleum produced in large quantities. It contains very little sulphur (between 0.05 and 0.1 per cent) practically no asphaltic matter and in refining gives good yields of gasoline, illuminating oils, and paraffin wax. Oils from the middle western, Texas, and California fields now produced in much larger quantities than Pennsylvania petroleum, contain in general a somewhat lower proportion of the paraffin hydrocarbons, and larger percentages of the less desirable or undesirable constituents. For descriptions of the composition of petroleum oils refer- ence may be made to the works cited at the end of the chapter and particularly to a series of papers by Mabery in the "Pro- ceedings of the American Academy of Arts and Sciences," Vol. 32, and subsequently. EXAMINATION OF CRUDE PETROLEUM An examination of crude petroleum may consist of a few simple tests' or an elaborate investigation both by analytical 218 PETROLEUM AND LUBRICATING OILS 219 methods and a laboratory imitation of actual refining processes, according to the importance of the case and the judgment and experience of the chemist. We shall consider here only a few factors of the superficial examination and fractional distillation. A sample of petroleum as received at the laboratory may contain water, either naturally or because of having been put into a wet bottle. It should be allowed to stand so that any water may settle out as completely as possible before making any tests. If any water or sediment appear on standing, de- cant the oil and then pass it through a dry filter. In some cases it will be desirable to make a quantitative determination of the moisture and of the sediment. Note the color and general appearance of the oil in a stand- ard 4-oz. sample bottle. With experience the odor is also of much assistance in indicating the general character of the sample. The density of the sample is usually taken with a delicate hydrometer reading either in specific gravity or degrees Baume. In the oil trade the latter is more common, and the relation be- tween specific gravity and Baume density becomes a matter of importance. In the past the different manufacturers of Baume hydrom- eters have given many different values to the Baume scale. 1 Recently the U. S. Bureau of Standards has defined the value of the Baume scale (for liquids lighter than water) in terms of specific gravity as follows : Degrees Baume = 130. o c. .!_ oU r . Specific gravity ^-^ The same value was adopted by the Manufacturing Chemists' Association of the United States in 1903. Hydrometers pur- porting to read degrees Baume should not be used unless they have been graduated in strict conformity with this scale. In the absence of a sufficiently accurate and delicate hydrom- eter the specific gravity should be determined by means of a 1 Chandler : The Baum^ Hydrometers. Memoirs of the National Academy of Sciences, Vol. Ill (1881). 220 METHODS OF ORGANIC ANALYSIS pyknometer or a good Westphal balance. The results obtained may then be converted to Baume (if desired) by means of the above equation. In routine work it is customary to use a Baume hydrometer with Fahrenheit thermometer attached and add or subtract 0.1 from the Baume reading for each degree Fahrenheit below or above 60. 1 When accuracy is desired, the density should be observed at the standard temperature so as to avoid the necessity for tempera- ture corrections. As a rough preliminary indication of the purposes for which the crude petro- f leum may be useful, it is often submitted to an Engler distillation test, in which 100 cc. of the oil are placed in a distilling flask of the size and shape specified by Engler (Fig. 13) and sub- mitted to distillation, heat- ing first over a wire gauze and then with a free flame with care to avoid drafts. The top of the thermometer bulb should be on a level with the bottom of the opening in the neck of the flask. Raise the temperature carefully (especially when in the neighborhood of 100, as here drops of water may condense on the thermometer bulb and fall back upon the hot oil, producing small explosions which may endanger the experiment 2 ) and at such a rate as to dis- till over as nearly as possible 2* cc. per minute. When the ther- 1 Better results are obtained by the use of the correction table given in Tagliabue's Manual for Inspectors of Coal Oil. 2 Water should be removed in advance by settling as described above. FIG. 13. Diagram of Engler distillation flask. PETROLEUM AND LUBRICATING OILS 221 mometer registers 150 C., remove the flame and allow the temperature to fall 20, and then heat again to 150, cool again, and repeat as long as any distillate is obtained below 150. Then raise the temperature and collect separately the second distillate, which is usually that obtained between 150 and 200, the distillation being conducted in the same way as before. Similarly collect and measure the distillates between 200 and 250, between 250 and 300 ; and finally all above 300 until nothing but coke is left in the flask. In order that the heavier portions may not undergo loss by hardening in the cold condenser tube, Low suggests that the water be drained from the condenser after the boiling point passes 200. Usually the three fractions collected between 150 and 300 are added together as an indication of the "illuminating oil," while that below 150 is taken as an indication of the "naphtha," and that above 300 the "lubricating oil," obtain- able from the sample. Grray's method for oils of the Pennsylvania type consists in distilling from 2 to 4 liters of the crude oil by means of outside heat and a current of steam, collecting the distillate in fractions each of which is 1 per cent (by volume) of the sample. The Baume density of each fraction is observed as the distillation proceeds. All fractions reading over 57 Baume (lighter than 0.7487 specific gravity) are added together as "naphtha"', those between 57 and 43 B. (0.7487 to 0.8092 specific gravity) as " illuminating oils " ; and all the distillate from this point until 18 per cent of the original oil remains in the still as "heavy distillate" the residue being "steam refined cylinder stock" unless the petroleum was of the asphaltic type. Interpretation. It is to be remembered that the competent petroleum expert uses these fractional distillations only as one factor in the detailed examination upon which to base a final opinion of the industrial possibilities of a crude petroleum. Much depends upon the character of the different fractions and their adaptability to further refining. Petroleums are sometimes spoken of as having a "paraffin 222 METHODS OF ORGANIC ANALYSIS base " or an " asphalt base," according as the heavier hydrocar- bons are solid paraffins or asphaltic substances. A pronounced example of either type may be recognized by the examination of the heavier fractions or residue obtained in a fractional dis- tillation, but since the introduction of the middle western oils and those from northern Texas, there is no sharp dividing line between the paraffin and asphaltic base crude oils, many oils yielding both asphaltic material and paraffin wax. When the refining process is a destructive distillation, the asphaltic mate- rial may be broken up by " cracking " into fuel oil, lubricating oil, etc., and the paraffin wax recovered ; but if the process is one of steam distillation, the residue being utilized for cylinder stock, the asphaltic matter is detrimental to the quality of the cylinder stock obtained. The question sometimes arises whether a given sample is a genuine crude oil or a mixture of fractions of lesser commercial or refining value. In such cases it is usual to distill, collect- ing the distillate in fractions each one tenth (by volume) of the sample taken, and determine the density of each. A regu- lar gradation of densities in the fractions is an indication of genuineness of the oil. EXAMINATION OF LUBRICATING OILS A thorough examination of lubricating oil involves : (1) the determination of the nature of the oil and, if it is a mixture, the proportion of each constituent ; (2) tests to determine the efficiency and safety of the oil as a lubricant with special refer- ence to the conditions of temperature, pressure, etc., to which it will be subjected in use. Among the more important prop- erties of lubricating oils which can be measured in the labora- tory are specific gravity, viscosity, acidity, or alkalinity, the temperature at which the oil solidifies, the flashing and burn- ing points, asphaltic matter, and freedom from grit or other objectionable impurities. The usefulness of other determina- tions will depend upon the purposes for which the oil is intended. PETROLEUM AND LUBRICATING OILS 223 DETERMINATION OF CONSTITUENTS Pure fatty and mineral oils are largely used as lubricants, both singly and mixed with each other in all proportions. Other substances are, however, often added to increase the vis- cosity of the oil, among the most common being " gelatin oils " containing aluminium oleate or other soaps. A better but more expensive means of increasing viscosity is to use castor oil or a " blown " oil. In beginning the examination of a lubricating oil, note care- fully any color, odor, turbidity, or fluorescence which may aid in identifying the oil or detecting foreign substances. The presence of soap is easily shown by burning a weighed portion of the oil, as refined fatty and mineral oils should not yield over 0.05 per cent of ash. Rosin oil can be detected by the Liebermann-Storch reaction. Qualitative test for saponifiable oil is conveniently made by the Lux-Ruhemann method as follows : l Put 3 to 4 cc. of the oil to be tested in a dry test tube, add a small piece of sodium hydroxide, or (better) metallic sodium, and heat in a paraffin bath for 15 minutes at 230 C. in the case of pale-colored, or 250 C. in the case of dark-colored or cylinder oils. On re- moving the tube from the bath and allowing it to cool, the presence of saponifiable oil is indicated by the partial or com- plete gelatinization of the contents of the tube or by the appear- ance of a soapy froth on the surface. After making these preliminary observations the saponifica- tion number should be determined unless the sample is a pure mineral oil. In saponifying mixtures consisting largely of heavy mineral oil there is difficulty in securing sufficient con- tact between the sample and the alcoholic potash solution even though petroleum ether or gasoline be added. In such cases a Soxhlet extractor can be placed between the flask and the re- flux condenser. 2 The intermittent syphoning of the condensed solvent from the extractor into the saponification flask mixes 1 J. Soc. Chem. Ind., 12, 470. Archbutt and Deeley : Lubrication and Lubri- cants, p. 209. 2 Conradson: J. Am. Chem, Soc., 1904, 26, 672. 224 METHODS OF ORGANIC ANALYSIS the contents and facilitates saponification. In order to dimin- ish the volume of solvent required and the interval between stirrings, the body of the extractor is partially filled with glass beads. Having found the saponification number (Chapter VIII), if the sample appears to be a mixture, the proportions of saponi- fiable and unsaponifiable matter are found either by separating and weighing the latter, or by estimating the former from the amount of fatty acids recovered from the soap solution after saponification. DETERMINATION OF UNSAPONIFIABLE OILS Weigh 1 to 5 grams of oil (depending upon the saponification number and the method to be followed), saponify by heating with alcoholic potash on a water bath, 1 evaporate off the alco- hol, and separate the unsaponifiable matter by one of the follow- ing methods. Method of Immiscible Solvents. To the residue from the evaporation of alcohol add 75 cc. of water, stir thoroughly, transfer to a separatory funnel, add about an equal volume of petroleum ether or washed ethyl ether, close the funnel, shake vigorously, and allow to stand over night or until the aqueous and ethereal solutions separate completely. Draw off the aqueous layer into another separatory funnel ; wash it again with ether and the ethereal layer again with water ; repeat if necessary. Finally unite the ether solutions in a weighed flask,, distill off the ether, and dry the unsaponifiable oil to constant weight in a boiling water oven. If desired, the fatty acids can be recovered from the aqueous soap solution by adding an excess of mineral acid and shaking with ether or by separating the fatty acids as in soap analysis. The principal objection to the separation by immiscible sol- vents is that emulsions frequently form in the separating funnel which remain even on standing for a day or more. The addi- tion of 1 to 2 cc. of alcohol often helps to break the emulsion, 1 See also the method involving cold saponification given by Fahrion : Z. angew. Chem., 1898, 267. PETROLEUM AND LUBRICATING OILS 225 but if more alcohol is added it tends to carry soap into the ether layer. The separation of the solvents is also facilitated by chilling the funnel and twirling it gently, or, if the apparatus is available, by whirling in a centrifuge. Petroleum ether dis- solves less soap than ethyl ether and gives less troublesome emulsions, but does not always extract the unsaponifiable mat- ter completely. Extraction of the Dry Soap. To avoid the difficulties just noted the following modification of the method recommended by A. C. Wright 1 may be used: Saponify 2 to 4 grams of oil, using 2 grams of caustic potash ; after evaporating off the alcohol, add 3 grams of sodium bicarbonate and 10 cc. of pure methyl alcohol, stir well and evaporate, add 5 cc. more of methyl alcohol and 10 grams of precipitated chalk, mix well, dry on a water bath and then for a few minutes at 110. Transfer the thoroughly dried mixture quickly to a Soxhlet extractor and extract the unsaponifiable matter with petroleum ether. Dry the extract to constant weight in a boiling water oven and weigh. The mixture of calcium carbonate and soap from which the unsaponifiable matter has been extracted can be treated with hydrochloric acid to dissolve the carbonate and liberate the fatty acids, which can then be separated and examined further. ESTIMATION AND IDENTIFICATION OF FATTY OILS From the weight of fatty acid recovered as described above, the percentage of fatty oil can be calculated on the assumption that the oil yields 95 per cent of insoluble fatty acids. The result thus found serves as a check upon the determination of unsaponifiable oil. If only fatty and mineral oils are present and the percentage of the former is small, it can be estimated with sufficient ac- curacy for most purposes from the saponification number, since the fatty oils which are likely to be present in mixed lubricants do not vary greatly in their saponification numbers. See table 1 Analysis of Oils and Allied Substances, p. 111. 226 METHODS OF ORGANIC ANALYSIS at end of Chapter VIII. If the fatty oil is identified, the aver- age number for that species of oil should be used in estimating the percentage. If the lubricant consists entirely of fatty oil with a known small amount of inert unsaponifiable matter, the usual methods for the identification of fatty oils can be employed. Otherwise the identification is based upon the examination of the separated fatty acids. VISCOSITY Apparatus and Methods The viscosity of an oil can be determined either by measur- ing the resistance which it offers to the movement of a sub- merged solid, or by observing the rate at which it flows through an aperture under given conditions of temperature and pressure. Torsion viscosimeters, such as that of Doolittle, depend upon the first principle ; but those depending upon the measurement of the rate of flow are much more generally used. Viscosime- ters of this kind are made in a great variety of forms, for de- scriptions of which the reference books at the end of the chapter can be consulted. Among the viscosimeters most commonly used are those of Engler, Redwood, and Saybolt. The Engler viscosimeter is probably more widely used the world over than any other form. It has long been regarded as the standard instrument in Germany, and has now been adopted in the United States government specifications, and by the American Society for Testing Materials. It consists of a cylindrical reservoir with concave bottom, in the center of which is a capillary outlet. The reservoir is of brass, sometimes gold-lined, is surrounded by a water or oil jacket, and provided with a cover through which passes a ther- mometer for taking the temperature of the oil and a plug which closes the outlet. In testing an oil, 240 cc. are poured into the reservoir, and should fill it to the points of the studs which serve to indicate the correct leveling of the apparatus. The reservoir is then covered, the temperature regulated, and finally the plug is withdrawn, and the time required for the outflow of PETROLEUM AND LUBRICATING OILS 227 200 cc. is carefully noted. The time in seconds required by the oil divided by that required by water at 20 C. is taken as the Engler viscosity number. Unless the oil is too viscous, or for some other reason a higher temperature is desired, it should be tested at 20 C. In any case the temperature should be stated in reporting results. The instrument must be very carefully cleaned and dried before and after using, as any trace of oil around the outlet will interfere with the flow of water, and vice versa. Serious errors may also be caused by dust, grit, or scratches about the outlet. The time of flow of 200 cc. water from the Engler viscosimeter should be from 51 to 53 seconds. The " normal apparatus " is made according to strict specifications, and is carefully standardized by the German officials. Figure 14 shows a sec- tion of the apparatus in which A represents the oil cylinder, B the outer bath or jacket, C the flask marked at 200 and 240 cc. which serves both to measure the oil for the test and to receive it as it flows from the capil- lary, b the plug, t the thermometer, D the tripod support, and d the ring burner, which is used only when making tests at temperatures above that of the room. The correct dimensions of the apparatus are also indicated on this cut. Redwood's viscosimeter, which has been largely used in Great Britain, consists of a cylinder about 4.7 cm. in diameter and 8.7 cm. high, having in the center of the bottom a cup-shaped FIG. 14. Diagram of Engler viscosimeter. From Lewkowitsch's Oils, Fats, and Waxes. (Macmillan and Co.) 228 METHODS OF ORGANIC ANALYSIS agate jet which can be closed by means of a spherical plug. Inside the cylinder is a small fixed bracket of thick bent wire with an upturned point to indicate the height to which the oil should extend at the beginning of the test. The apparatus is jacketed and provided with a closed side tube and a revolving stirrer so that determinations can be made at high temperatures if desired. The apparatus is intended to deliver 50 cc. of water at 15.5 in 25.5 seconds, but as the rate of flow is in- fluenced by many conditions, it must be determined by each observer for his own apparatus and method of working. To use the apparatus at room temperature place it on a level support, insert the plug, and fill with the liquid to be tested until the surface comes exactly to the upturned point already mentioned. Place beneath the outlet a narrow-necked flask graduated at 50 cc., open the jet by lifting the ball valve, and observe the time required for 50 cc. to flow into the receiving flask. Whatever method is adopted for expressing the results, the report should always show the actual time of flow for the oil and for water and the temperature at which the test was made. The same precautions should be taken with this as with the Engler apparatus. For a description of Saybolt's viscosimeter consult Gill's Oil Analysis. Significance of Results Since the object of lubricating with oil is to separate the moving surfaces by a fluid layer, it is important that the oil have sufficient viscosity or " body " to stay in place and keep the moving surfaces apart under the maximum pressure to which they will be subjected. The greater the pressure the more viscous the oil should be, but any viscosity beyond that which is necessary to keep the oil in place means an increase of fluid friction and consequent loss of power. The viscosity of the oil is, therefore, the most direct indication of its suita- bility as a lubricant under given conditions. For several rea- sons, however, the viscosity alone is not a conclusive measure of the lubricating power. The adhesion to solid surfaces which PETROLEUM AND LUBRICATING OILS 229 prevents the displacement of the oil by pressure is not always directly proportional to the true viscosity or internal friction. Oils vary greatly in the rate of change of viscosity with in- creasing temperature and pressure. The viscosity as measured by the rate of flow depends not only upon the internal friction of the oil, but also to some extent upon its adhesion to the sides of the outlet and upon the specific gravity. Hence, it is not to be assumed that any two oils having the same viscosity can be used interchangeably as lubricants. In order to duplicate an oil which has been found satisfactory in use, the kind of oil, the specific gravity, and the viscosity at least, should be specified. Viscosity is especially important in dealing with mineral oils because of the ease with which they can be varied in this re- spect, while any particular kind of fatty 'oil varies only within comparatively narrow limits. For a full theoretical discussion of viscosity and lubrication, the work of Archbutt and Deeley should be consulted. The reader must also be referred to this and other books and articles given below, for discussion of the many practical considerations affecting the selection of lubricating oils. Mixed oils do not always show the viscosities which would be expected from the proportions and viscosities of the con- stituents of the mixture. In other words, viscosity is not an additive property in oil mixtures. In mixtures of oils whose viscosities are similar, the discrepancy between the estimated and observed viscosity of the mixture may not be apparent, but where oils of widely different viscosities are mixed, the discrepancy may be considerable. Sherman, Gray, and Hammerschlag 1 in a series of experiments with nine sets of mixtures found that the observed viscosity of the mixture was lower than the calculated value whether the mixture was that of two mineral oils, a mineral and a fatty oil, or a mineral oil and sperm oil; and in general, the greater the difference in viscosities between the oils mixed, the greater was the difference between the calculated and observed viscosity numbers of the mixtures. In a typical case a series was pre- ij; Ind.Eng, Chem., 1, 13. 230 METHODS OF ORGANIC ANALYSIS pared, consisting of mixtures in tenths by weight of a high viscosity lubricating oil (" H ") and a low viscosity lubricating oil ("L"), both made from Pennsylvania petroleum. The pure oils and mixtures tested as follows: ENGLER VISCOSITIES AT 20 C. Calculated Found Difference Hisrh viscosity oil ( ; 'H") 25.56 90 per cent " H," 10 per cent " L " . . . . 23.41 20.04 3.37 80 per cent " H," 20 per cent " L " .... 21.27 16.25 5.02 70 per cent " H," 30 per cent " L " .... 19.12 13.37 5.75 60 per cent " H," 40 per cent " L " .... 16.97 10.90 6.07 50 per cent " H," 50 per cent " L " .... 14.83 9.04 5.79 40 per cent " H," 60 per cent " L " .... 12.68 7.69 4.99 30 per cent " H," 70 per cent " L " .... 10.54 6.38 4.16 20 per cent " H," 80 per cent " L " .... 8.39 5.52 2.87 10 per cent " H," 90 per cent " L " .... 6.24 4.67 1.57 Low viscosity oil C" L ") 4.10 In every case the viscosity of the mixture was less than the value obtained by calculation from the percentages and viscosi- ties of the constituents. The difference between the calculated .,3 i 20$ 3 o# 405* sojj 6056 70:* Percentage of lower viscosity oil. FIG. 15. Viscosities of oil mixtures. 9036 PETROLEUM AND LUBRICATING OILS 231 and determined values increased with the increasing propor- tions of light oil in the mixture up to 40 per cent of light and 60 per cent of heavy oil ; with further increments of the light oil, the difference gradually decreased. The determined vis- cosities are plotted in Fig. 15 (the viscosities as ordinates, the percentages of " L " as abscissae), and will be seen to form a very regular curve, dropping away from the calculated values somewhat more abruptly when the light oil is added to the heavy than when the heavy oil is added to light. Subsequently Kessler and Mathiason 1 obtained similar results. ACIDITY Weigh accurately 5 to 10 grams of oil in a 250-cc. Erlen- meyer flask, add 50 cc. of neutralized 85 per cent alcohol con- taining phenolphthalein as indicator, and titrate with standard sodium or potassium hydroxide, shaking vigorously after each addition until a permanent pink color is obtained. It is often necessary to allow the flask to stand after shaking until the oil separates from the alcohol solution before observing the color of the latter. 2 To test for free mineral acid, shake 10-15 grams of oil with 100 cc. of warm water in a separatory funnel, allow to separate, draw off the water, filter through wet paper, cool, and add methyl orange. If mineral acid is found, shake the oil remain- ing in the funnel repeatedly with small portions of hot water until all mineral acid is extracted, filter as before, add the filtrate to the first portion containing methyl orange, and titrate very carefully with standard alkali. Concentrate the neutralized solution, test qualitatively to determine the nature of the min- eral acid, and calculate the percentage. If the identification of the mineral acid is prevented by the presence of salts, calculate the mineral acidity as due to sulphuric acid. The acidity due 1 J. Ind. Eng. Chem., 3, 66. 2 For determining acidity in very dark colored fats the use as indicator of 10 cc. of a 2 per cent solution of " Alkali Blue II OLA " (Meister, Lucius and Brunig) in 99 per cent alcohol has been recommended by Freundlich : Oesterr. Chem. Ztg., 1901, 4, 441 ; Z. Nahr. Genussm., 1902, 5, 460. 232 METHODS OF ORGANIC ANALYSIS to organic acids, or the total acidity if only this is determined, is usually calculated as percentage of oleic acid. As much as 15 per cent of free oleic acid is sometimes allowed in lubricating oils. The best grades of lard oil do not contain over 1.5 per cent. Free mineral acids should be absent. COLD TEST AND CHILLING POINT OR CLOUD TEST The "cold test" indicates the temperature at which the sample just ceases (or just begins) to flow ; the "chilling point" that at which the oil begins to become turbid or to show flocks or scales of solid. In either case the temperature required will be influenced by details of manipulation, so that an arbitrary method must be followed to obtain strictly comparable results. The directions below follow the procedure recommended by Gray. Gold Test. Pour about 25 cc. of oil into an ordinary bottle of about 100 cc. capacity and insert a stopper carrying a ther- mometer the bulb of which reaches just below the surface of the oil, cool the sample slowly to 50 F. and then place in a freezing mixture of ice and salt. As the temperature falls every few degrees remove and tilt the bottle until the tempera- ture is found where the oil just ceases to flow. This is called the "cold test" or "setting point." Cylinder and black oils which have not been treated either by acid or by filtration through fuller's earth may show abnormally low and irregular cold tests. With oils having cold tests higher than 45 F., it is custom- ary to reverse the process, freezing the sample first and allowing it then to warm slowly until it just flows. Chilling Point. Usually it is only necessary to know whether the oil remains clear for a given number of minutes at a given temperature. Use the same bottle, amount of sample, and ther- mometer as for the cold test. Expose the liquid to cold, stirring with the thermometer, and hold at the required temperature for the specified time (usually ten minutes). If the oil remains transparent and free from flocks or scales, it meets the require- ment as to chilling test. PETROLEUM AND LUBRICATING OILS 233 If it is required to find the chilling point, the procedure is similar ; but the liquid after remaining clear as described is ex- posed to a temperature 3 lower, allowed to stand with constant watching arid occasional stirring with the thermometer until the oil is as cold as the bath, repeat this cooling until opacity or flocks or scales begin to show. The reading of the thermometer when this occurs shows the "chilling point" or "cloud test." For further information on the cold test or setting point see the works of Archbutt and Deeley, Gill, Holde, Lewkowitsch, and Stillman. FLASHING AND BURNING POINTS For the most accurate results closed testers such as are used in examining illuminating oils should be employed. Among these the Perisky-Martens apparatus is perhaps the best for this purpose. An illustrated description of this apparatus will be found in Lewkowitsch's Oils, Fats, and Waxes, 4th Ed. Vol. III. pp. 58-60. For routine work in this country the " Cleveland Cup" tester is often used. This is a shallow, cylindrical, jacketed cup, open to the air and heated by a Bunseii flame. 1 The test cup is filled to about 0.5 cm. from the top and the thermometer is suspended in such a position that the bulb is entirely immersed in the oil at the center of the dish without touching the bottom. Heat by means of a Bunsen burner, and as the flashing point is approached, test at each second or third degree by slowly pass- ing the test flame across the dish horizontally about 0.5 cm. above the level of the oil and directly in front of the thermom- eter. Record the temperature at which the first flash is seen as the flashing point. Continue heating and testing in the same way until the liquid takes fire ; note this temperature as the burning point. Remove the thermometer and blow out the flame or smother it by sliding a watch glass over the dish. Notes. The flashing and burning points must be determined in a place free from drafts. The heating should be so regulated 1 In the absence of either of these forms of apparatus a porcelain dish, 4 cm. deep and 4 cm. in diameter, set deep in a sand bath, is sometimes used. 234 METHODS OF ORGANIC ANALYSIS that on approaching the flashing or burning point the rate of rise of temperature of the oil is not greater than 6 C. per min- ute. Tests may then be made every half minute using a test flame not over 5 mm. long. The test flame may be obtained from a narrow glass jet (similar to that used on a wash bottle) connected with the ordinary gas tubing, the flow of gas being regulated to give a flame of the size desired. Any variation of the conditions, either in size and form of dish, the rate of heating and testing, or the manner of applying the test flame may cause an appreciable discrepancy in the result. In oil mixtures the flashing and burning points are not addi- tive properties and cannot be predicted by simple interpolation. Sherman, Gray, and Hammerschlag 1 found that in mixtures of mineral oils of different flashing and burning points, and of mineral oil with a fatty oil, or sperm oil, the flashing and burn- ing points found were invariably lower than the figures which would be found by simple interpolation from the known prop- erties and proportions of the oils in the mixture. It was also observed that when a high-test and a low-test oil were mixed in different proportions the discrepancy was greater in mixtures containing a high proportion of the high-test oil ; or, differently stated, the flashing and burning points were lowered by the presence of 25 per cent of the low-test oil to a greater extent than they were raised by the presence of 25 per cent of the high-test oil. Kessler and Mathiason 2 have also shown that the flashing and burning points are not additive properties in oil mixtures. ADDITIONAL DETERMINATIONS Additional tests and determinations are frequently required to show the suitability of the lubricant for the particular use intended. Friction tests on oil testing machines especially designed for the work are sometimes of great importance. A full discussion of such mechanical methods of testing will be found in Archbutt and Deeley's Lubrication and Lubricants. 1 J. Ind. Eng. Chem., 1, 13. 2 J. Ind. Eng. Chem., 3, 66. PETROLEUM AND LUBRICATING OILS 235 Loss by evaporation and tendency to "gum" are tested by heating a small amount of oil on a watch glass for several hours at the highest temperature to which it is likely to be subjected in use. The oil must not become sticky and the loss of weight should usually be less than 1 per cent. Suspended matter which may be invisible in a dark oil is detected by diluting the sample with gasoline or petroleum ether. Antifluorescents, added to destroy the fluorescence or u bloom " of mineral oils, can often be detected by boiling 1 cc. of the oil with 3 cc. of a 10 per cent solution of potassium hydroxide in alcohol. A red color indicates nitronaphthalene or nitrobenzene, which are the principal antifluorescents used. According to Holde, 1 asphaltic matter can be approximately determined as follows: Dissolve 5 grams in 125 cc. of ether at 15, add, drop by drop with constant shaking, 62.5 cc. of 96 per cent alcohol ; after standing 5 hours at 15, filter, wash with a mixture of alcohol and ether (1 : 2 by volume) until nothing more than traces of pitchlike substance is removed. Dissolve the residue in ben- zol, evaporate, dry one half hour at 105, and weigh. For other tests and determinations and fuller discussions of most of those here given the reader is. referred to the works given for reference below. EXAMINATION OF LUBRICATING GREASES Lubricating greases are usually mixtures of soaps with fats, hydrocarbons, rosin, or tar, containing water and sometimes large amounts of mineral matter. On melting, the grease often separates into a soap solution and an oily layer. The soaps used in making such lubricants may contain salts of sodium, potassium, calcium, or heavy metals with either fatty or resin acids. Some greases consisting essentially of fats and hydro- carbons melt at temperatures to which they are subjected in use and may therefore be examined in the melted state by the methods used for lubricating oils. For most greases, however, it is necessary to adapt the analytical method to the nature of l Mitt. Kgl. Techn. Versuchsanstalt, Berlin, 1902, 20, 253; Z. Nahr. Genussm., 1903, 6, 855. 236 METHODS OF OEGANIC ANALYSIS the mixture to be examined in each case, since the composition of these greases is too variable to allow the use of any fixed system of examination. It may often be necessary to resort to a combination of the methods used in the analysis of soaps, fats, and lubricating oils. For detailed information on the composi- tion and testing of lubricating greases, see the reference books cited below and a review by Conradson: J. Am. Ohem. >SW., 1904, 26, 705-712. REFERENCES ALDER- WRIGHT and MITCHELL : Oils, Fats, Butters, and Waxes. ALLEN : Commercial Organic Analysis. ARCHBUTT and DEELEY : Lubrication and Lubricants. DAVIS : Friction and Lubrication. GILL : Short Handbook of Oil Analysis. HOLDE : Untersuchung der Schmiermittel. LEWKOWITSCH : Oils, Fats, and Waxes. LUNGE : Chemisch-technische Untersuchungsmethoden. POST : Chemisch-technisehe Analyse. RAKUSIN : Die Untersuchung des Erdoles und seine Producte. REDWOOD : Petroleum and its Products. STILLMAN : Engineering Chemistry. UBBELOHDE : Handbuch der Chemie und Technologic der Oele und Fette. II 1904. EGER : Testing of Mineral Lubricating Oils. Z. angew Chem., 1904, 1577. 1905. RICHARDSON and HANSON : Valuation of Lubricants with Special Reference to Cylinder Oils. J. Soc. Chem. Ind., 24,. 315. 1906. GILL : Apparatus for Testing Liability of Oils to produce Spontane- ous Combustion. J. Soc. Chem. Ind., 26, 185. Ross and LEATHER : Valuation of Oils for Gas-making. Analyst, 31, 284. TOLMAN : Cooperative Work on the Cloud and Cold Test for 1906. U. S. Dept. Agriculture, Bur. Chem., Bui. 105, p. 29. VALENTA : Determination of Coal Tar Oils in Mineral and Rosin Oils. Chem. Ztg., 30, 266. 1907. ACHESON : A New Lubricant. Defloculated Graphite. Eng. News, 53, 127. PETROLEUM AND LUBRICATING OILS 237 CHARITSCHKOW: Influence of Water on Flash-Point and Viscosity. Chem. Rev. Fett- Harz-Ind., 14, 225 : Chem. Abs., 2, 176. KISSLING: Constants in Mineral Oil Analysis. Chem. Ztg., 31 r 328. Physikalisch-technische Reichsanstalt. Calibration of Engler Visco- simeters. Z. angew. Chem., 20, 832 ; Z. offenil. Chem., 13, 204. SCHLICHT and HALPHEN: Determination of Unsaponifiable Matter. Chem. Ztg., 31, 279. SCHREIBER : Determination of Saponification Number in Lubricat- ing Oils. /. Am. Chem. Soc., 29, 74. STOLZENBURG : Technical Examination of Lubricating Oils. Chem. Rev. Fett- Harz-Ind., 14, 239, 274 ; Chem. Abs., 2, 456. UBBELOHDE: Improvements in Engler Viscosimeter. Chem. Ztg., 31, 38. 1908. HOLDE : (Physical Behavior of Lubricants). Z. angew. Chem., 1908, 31, 2138. HOLDE and EICKMANN : Resinous Products in Mineral Oils. Pe- troleum, 1908, 3, 1077; Chem. Abs., 2, 1341. KISSLING : New Constants in Mineral Lubricating Oil Analysis. Chem. Ztg., 32, 938. LECOCQ : Determination of Asphalt in Mineral Oils. Bull. Soc. Chim. Belg., 22, 81 ; Chem. Abs., 2, 1880. MABERY and MATHEWS : Viscocity and Lubrication. J. Am. Chem. Soc., 30, 992. Review on Lubrication. Engineering, 85, 86; Chem. Abs., 2, 1342. U. S. Senate Document No. 469, 60th Congress, 1st Session. Plans for International Standards for Testing Mineral Oil Products. 1909. DAY and GILPIN : Changes in Crude Petroleum effected by Diffusion through Clay. /. Ind. Eng. Chem., 1, 449. GILLETT : Analyses and Friction Tests of Lubricating Greases. J. Ind. Eng. Chem., 1, 351. HYDE : Definition of Gasoline. J. Ind. Eng. Chem., 1, 377. LADD : Experiments with Burning Oils. N. Dak. Agl. Expt. Sta. r 18th Ann. Report, pp. 34-43 ; Chem. Abs., 3, 243. MAGRUDER : Rapid Method for the Determination of Sulphur in Crude Petroleum. Chem. Abs., 3, 115. SADTLER : (Methods of avoiding Emulsions in Extraction of tTnsa- ponifiable Oil). J. Ind. Eng. Chem., 1, 479. SHERMAN, GRAY, and HAMMERSCHLAG : Comparison of Calculated and Determined Viscosity Numbers and Flashing and Burning Points in Oil Mixtures. /. Ind. Eng. Chem., 1, 13. STORMER : Viscosimeter. /. Ind. Eng. Chem., 1, 317. UBBELOHDE : Viscosity of Illuminating Oils and an Apparatus for its Determination. Petroleum, 1909, 4, 861 ; Chem. Abs., 3, 2376. 238 METHODS OF ORGANIC ANALYSIS 1910. CONRADSON : Laboratory Tests of Lubricants Interpretation of Analyses. /. Ind. Eng. Chem., 2, 171. Goss : Oils and Lubricants. Modern Power, 1, No. 4; Chem. Abs., 5, 379. KISSLING : Examination of Crude Petroleum and of its Products. Petroleum, 1910, 5, 505; Chem. Abs., 4, 1365. KISSLING : Determination of Asphalt in Cylinder Oils. Chem. Rev. Fett- Harz-Ind., 17, 35; Chem. Abs., 4, 1367. MABERY: Lubrication and Lubricants. J. Ind. Eng. Chem., 2, 115. MEISSNER: Influence of Errors in the Dimensions of Engler's Yis- cosimeter. Chem. Rev. Fett- Harz-Ind., 17, 202; Chem. Abs., 4, 3148. ROBERTS and FRASER : Estimation of Water in Petroleum. /. Soc. Chem. Ind., 29, 197. WATERS : Action of Sunlight and Air upon some Lubricating Oils. J. Ind. Eng. Chem., 2, 451. 1911. DAY: The Production of Petroleum in 1910. Published by U.S. Geological Survey. GROSCHUFF : Solubility of Water in Benzene, Petroleum, and Par- affin Oils. Z. Electrochem., 17, 348 ; Chem. Abs., 5, 2550. KESSLER and MATHIASON: On the Interpolation Method of Oil Analysis. J. Ind. Eng. Chem., 3, 66. LOEBELL: Determination of Asphaltum Insoluble in a Mixture of Alcohol and Ether in Mineral Lubricating Oils. Petroleum, 6, 774; Chem. Abs., 5, 3149. WATERS : The Effect of Added Fatty and Other Oils upon the Car- bonization of Mineral Lubricating Oils. J. Ind. Eng. Chem., 3, 812. CHAPTER XII Fuels THE purpose of this chapter is to outline the direct determi- nation of the calorific value of solid and liquid fuels and then to consider the analytical determinations of most importance for the judgment of each of the chief types of fuel and especi- ally the relation of the chemical composition to the calorific power. DETERMINATION OF CALORIFIC POWER The heat of combustion or calorific power of a solid or liquid fuel is best determined by burning in oxygen in a bomb calorimeter according to the general method of Berthelot. The chief modifications of the Berthelot bomb in use in this country are those of At water, Emerson, and Mahler. The Atwater apparatus has been fully described by Atwater and Snell in the Journal of the American Chemical Society for July, 1903. The original description of the Emerson calori- meter will be found in the Journal of Industrial and Engineering Chemistry for January, 1909, and the Mahler bomb is well de- scribed by Gill in his Gras and Fuel Analysis for Engineers. Since these descriptions as well as others referred to at the end of this chapter are readily accessible and full directions are usu- ally furnished with the apparatus by the manufacturer, it is not so important here to discuss the exact details of manipulation, which depend to some extent upon the form of bomb calorimeter used, as to outline certain principles and precautions of general application. The heat of combustion is determined by burning in the bomb in an atmosphere of oxygen a small amount of the sub- 239 240 METHODS OF ORGANIC ANALYSIS stance, usually enough to yield about 5 or 6 large calories, and measuring the rise of temperature of the known amount of water in which the bomb is immersed. In order to be able to determine the quantity of heat from the rise in temperature it is necessary to know not only the amount of water surround- ing the bomb but also the heat capacity of the apparatus. There are four general methods of estimating this heat capacity : (1) By calculation from the weights and specific heats of the materials of which the apparatus is composed. (2) By some application of the " method of mixtures," such as placing the bomb in a known amount of water and after the temperature has become uniform adding a known amount of water of a different (known) temperature and noting the change of temperature of the system. (3) By burning in the bomb under the conditions of an ordinary determination a weighed amount of substance whose heat of combustion is accurately known and noting the rise of temperature produced by this known amount of heat. (4) By generating a known amount of heat in the bomb electrically and measuring the resulting rise of temperature. The first method is not sufficiently accurate for final calibra- tion of a bomb but is sometimes useful for a preliminary calcu- lation of approximate heat capacity. The second method is accurate only when carried out under conditions and with precautions so troublesome as to be prac- tically prohibitive. The third method is more accurate than the first or second, and is the one now generally used, but it involves the assump- tion that the heat of combustion of the "standard substance" is accurately known. The fourth method l is the most accurate but is hardly neces- sary except as an ultimate basis in the preparation of standard substances. The Bureau of Standards recommends that calorimeters generally be calibrated by the third method using standard 1 Jaeger and Steinmehr: Ann. d. Phys., 21, 23-63 (1906) and Bulletin of the Bureau of Standards, Reprint No. 135. FUELS 241 combustion samples, the heats of combustion of which have been carefully determined in calorimeters calibrated by elec- trical means. The heat capacity of the apparatus is usually expressed as its "water equivalent" or " hydrothermal equivalent." Thus a " water equivalent " or " hydrothermal equivalent " of 408 would mean that the heat capacity of the apparatus was equal to that of 408 grams of water. The specific heat of water changes somewhat with the tem- perature so that a slight error is introduced if a calorie be taken in one case as the heat capacity of water per degree centigrade at zero and in another case at room temperature. The Bureau of Standards adopts the heat capacity of water at 15 C. as unity so that the calorie is defined as the heat capacity of one gram of water per degree centigrade at a temperature of 15 C. and the B. T. U. as the heat capacity of one pound of water per degree Fahrenheit at a temperature of 60 F. For- tunately it is not necessary in ordinary determinations of heat of combustion to change the basis of calculation with the tem- perature of working between 15 and 25 C., because the Bureau of Standards has found that the total heat capacity of an ordinary combustion calorimeter does not change apprecia- bly with temperature between 15 and 25 C. This is due to the fact that the metal of the bomb and accessories has a posi- tive temperature coefficient, while the water has a negative temperature coefficient throughout this range of temperature and the two nearly neutralize each other. Standard Materials. Among the substances which have been used in the standardization of combustion calorimeters are cane sugar, benzoic acid, naphthalene, glycocoll, hippuric acid, and camphor. The Bureau of Standards recommends the first three and supplies standard samples of these for this pur- pose. Sucrose is not volatile nor strongly hygroscopic, but is rather difficult to ignite, sometimes fails to burn completely, and has a heat of combustion only about half as high as that of good coal. 242 METHODS OF ORGANIC ANALYSIS Benzole acid is only slightly volatile, not very hygroscopic, burns readily, and has a heat of combustion about four-fifths that of ordinary coal. Naphthalene is more volatile than benzoic acid, but is not hygroscopic, burns very readily, and has a heat of combustion a little higher than that of coal. The Bureau of Standards reports that the loss by sublimation from naphthalene pressed into pellets for combustion will hardly exceed 0.1 to 0.2 per cent in an hour, so that any error in standardization due to volatility of the naphthalene should be less than 0.1 per cent. Although sugar has been most used in the past, the Bureau of Standards finds benzoic acid the most satisfactory substance for accurate standardization of bomb calorimeters. Preparation of sample or charge. Since the amount of mate- terial used for a determination is usually not over a gram, the sample must be finely ground and thoroughly mixed. A por- tion of the mixed powdered sample is then pressed into a pellet or small briquet, which -is weighed in the combustion crucible and then placed in the bomb. Pressing the charge into a pel- let avoids the danger of portions being blown out of the cruci- ble either when the oxygen is admitted to the bomb or during the rapid combustion which takes place when the substance is fired. In standardizing with napthalene the loss by volatiliza- tion is also much smaller from a pellet than from the same weight of loose material. Liquids, and anthracite coals which cannot be pressed into pellets, are conveniently burned in weighed hard " gelatin " capsules of determined calorific power. The objections sometimes offered to this method are probably due to the use of capsules of unsuitable char- acter. The writer uses Parke-Davis capsules of "five-grain" size which weigh about one tenth gram each, have a heat of combustion of about 4480 calories per gram, are neither sticky nor hygroscopic, and burn readily, thus aiding the complete combustion of high-ash anthracite coals. Some prefer to burn anthracites in a loose condition and, if difficulty is experienced in obtaining complete combustion, to place beneath the anthra- cite a weighed amount of bituminous coal of known calorific FUELS 243 power. Blakeley and Chance hold that the failure of high-ash coals to burn completely when placed directly in the combus- tion crucible is largely due to the cooling of that portion of the coal which is in contact with the metal floor of the crucible and may be avoided by placing a disk of asbestos beneath the coal. Pasty solids and non-volatile liquids may be weighed and burned directly in the combustion crucibles. In the case of fatty oils it was found advantageous to add a little loose as- bestos (previously ignited) to regulate the burning and pre- vent possible loss by spattering. For a volatile liquid select a capsule having a body with a smooth edge and a snug-fitting cap, transfer the liquid from the sample bottle to the weighed capsule by means of a pipette or medicine dropper, close the capsule, stand it in the combus- tion crucible and weigh, then allow to stand about 5 minutes and weigh again to make sure that none of the sample is being lost by evaporation. Should it be found that the capsule per- mits loss by evaporation, it must be rejected and another pre- pared and tested in the same way. Both in standardizing the bomb and in the testing of fuels the amount of the charge should depend upon its calorific power so that there may not be great differences in the amount of heat to be measured in the different determinations. When the charge has been prepared in the combustion crucible, it is placed in position, the fuse wire adjusted, the bomb closed and charged with oxygen. The oxygen used for combustion must be the purest obtainable commercially, especially as regards freedom from combustible gases such as hydrogen, carbon monoxide, and hydrocarbons. Each cylinder of oxygen should be tested by determinations upon standard combustion samples such as are used in deter- mining the heat capacity of the apparatus. The bomb is charged with oxygen to a pressure of about 25 atmospheres, and, after being properly closed to prevent any es- cape of gas, is immersed in a weighed quantity of water, the terminals of the firing circuit adjusted, the covers placed in position, and the stirring and thermometric observations begun. 244 METHODS OF ORGANIC ANALYSIS The details of these operations vary with the different forms of apparatus and are explained for each in the descriptions already cited. At the end of the " fore-period " in which the rate of change due to the temperature of the surroundings has been determined, the charge is ignited by closing the switch of the firing circuit so as to pass through the fuse wire a current sufficient to heat it to redness if platinum, or cause it to burn if iron, but of low potential so as to avoid danger of arcing with evolution of heat within the bomb. Temperature measurements should be made with delicate dif- ferential thermometers of the Beckmann type which should be carefully calibrated, preferably by the Bureau of Standards or the Reichsanstalt. Accuracy requires that the proper correc- tions be determined and applied for errors of the scale, for the emergent stem, and for the amount of mercury removed from the bulb. The first, called " caliber correction," should be ap- plied to each reading ; the second and third together are often called " thermometer correction," and this correction, which will depend upon the room temperature and the setting of the thermometer, is applied to the apparent rise of temperature ob- served during the combustion, to obtain the rise in true degrees. The radiation correction is calculated from the readings taken before, during, and after the combustion, preferably by means of the Regnault-Pfaundler formula. At the end of the after-period following a combustion the bomb is opened and a careful examination made for any evi- dences of incomplete combustion. Any portions of the fuse- wire which may remain unburned are weighed and deducted from the original weight. The bomb is rinsed out and the nitric acid which has been formed during the combustion is determined by titration. The heat of formation and solution of the nitric acid formed (230 calories per gram), as well as the heat of combustion of the iron fuse wire burned (1600 calories per gram), and of the gelatin capsule if used, must be allowed for in the calculation of the results. The full explanation of the calculation of results given by Atwater and Snell should be FUELS 245 consulted. Fuels containing sulphur yield sulphuric acid on combustion in the bomb, so that a slight error is introduced if the total acidity of the bomb rinsings be calculated as nitric acid. To correct for this the usual subtraction for total acidity calculated as nitric acid may be made, then the sulphuric acid in the rinsings may be found by precipitation and the correction increased by 13 calories per gram for each per cent of sulphur in the fuel. 1 This is based on the assumption that as fuel is ordinarily used tjie sulphur burns to sulphur dioxide. The calorific powers as found in the bomb calorimeter repre- sent somewhat more heat than is actually obtained from the fuel in ordinary use, not only because the combustion is complete, but especially because the water vapor produced in the combus- tion condenses in the bomb and the latent heat of this vapor is not lost as is usually the case when the fuel is burned in use. Occasionally an attempt is made to distinguish between " heat of combustion " and " calorific power," confining the latter term to the values obtained on subtracting from the heat of com- bustion the estimated heat of vaporization of the water. More often the heat of combustion is called the " calorific power (high value) " or simply " calorific power," and the term " calorific power (low value)" is used to designate the value obtained on deducting the latent heat of water vapor. In this chapter the "high values" are always given without the use of that term, or in other words the term calorific power is used as synonymous with heat of combustion. CHEMICAL COMPOSITION AND CALORIFIC POWER OF ORGANIC COMPOUNDS According to the usually accepted determinations, the heat of combustion of carbon is 8080, and of hydrogen 34,500, calories per gram. The heat of combustion of a compound consisting of carbon and hydrogen only is the sum of the heats of combus- tion of the carbon and hydrogen it contains minus the heat of formation of the compound, which is for hydrocarbons a rela- 1 Lord : U. S. Geological Survey, Professional Paper No. 48. 246 METHODS OF ORGANIC ANALYSIS lively small factor. In compounds containing oxygen the heat of formation is larger and the heat of combvistion is proportion- ately less than the heat which would be obtained by burning the quantities of carbon and hydrogen present, for the obvious rea- son that these elements are already partially " oxidized " by the oxygen present in the molecule. From the heats of combustion of carbon and hydrogen one may readily calculate that a given weight of oxygen will cause a greater evolution of heat in burn- ing hydrogen to water than in burning carbon to carbon di- oxide. Hence in the case of a compound of carbon, hydrogen, and oxygen, the estimated calorific power will be lower if we assume that the oxygen in the molecule is to be considered as combined with hydrogen than if we assume that it is to be con- sidered as combined with carbon. Usually estimates of calorific power from ultimate analysis have been based on " Welter's rule," which assumes that the oxygen present is combined with hydrogen, or, that we shall offset the heat of formation if we deduct the quantity of heat which would be produced if the oxygen present combined with the hydrogen. On this assumption the heat of combustion of such a compound would be represented by the formula: X = 8080 C + 34,500 (H - -J O). Walker, in his Introduction to Physical Chemistry, pointed out that the values thus obtained are considerably too low in the cases of sugar and of butyric acid, and stated that " a better result may usually be obtained by subtracting the oxygen, not with the corresponding quantity of hydrogen, but with the corresponding quantity of carbon, and then estimating the heat of combustion of the elements in the residue." Putting this suggestion (which for convenience we may call " Walker's rule " as contrasted with " Welter's rule ") in the form of an equation, we have, X = 8080 (C - O) + 34,500 H. If, now, these two formulae be applied to the various classes of pure organic compounds, and the results compared with the experimentally determined heats of combustion as compiled by FUELS 247 Berthelot, or Vaubel, it will be found that in some cases the higher, in other cases the lower, of the calculated results ap- proximates the true value, while for still other compounds the true value lies between them and at a distance from either. The relative values of these formulae as applied to different classes of fuels is considered below. In some cases fuel values are estimated from proximate, rather than ultimate, chemical composition. Thus, since the calorific power of pure alcohol and the relation of specific gravity to percentage strength of alcohol-water mixtures are both known, the specific gravity of any commercial alcohol which is essentially a mixture of pure alcohol and water will show the amount of alcohol present, and hence the calorific power of the liquid. In food analysis the fuel value is commonly estimated from the percentages of proteins, fats, and carbohydrates as has been fully explained in another volume. 1 FUEL OILS AND GASOLINE With appliances sufficiently well adapted to its properties, any petroleum oil can be burned with an evolution of heat much greater than that of any coal. Since American petroleums are essentially mixtures of hydro- carbons and chiefly of the methane series, it was to be expected that the greater the specific gravity of the sample the greater would be the mean molecular weight and percentage of carbon and the less would be the percentage of hydrogen and the heat of combustion per gram. In general a specific gravity of 0.7 -0.75 indicates about 11,500-11,300 calories per gram ; 0.75-0.8 indicates about 11,300-11,100 calories per gram ; 0.8 -0.85 indicates about 11,100-10,900 calories per gram ; 0.85-0.9 indicates about 10,900-10,700 calories per gram ; 0.9 -0.933 indicates about 10,700-10,500 calories per gram. More commonly in English-speaking countries the density of petroleum oils is stated in terms of the Baume scale and the 1 Sherman : Chemistry of Food and Nutrition, Chapter IV. 248 METHODS OF ORGANIC ANALYSIS calorific power in British thermal units. The relations of the values are as follows : calories per gram x 1.8 = B. T. U. per pound ; * specific gravity From the densities and calorific powers of 86 samples of American petroleum oils, 70 of which were examined by the writer and his associates at Columbia University, and 16 by Allen and Strong of the U. S. Geological Survey, is constructed the following table 1 for estimating the calorific power from the Baume density in commercially pure petroleum oils. TABLE 20. APPROXIMATE CALORIFIC POWERS, IN BRITISH THERMAL UNITS PER POUND, OF PETROLEUM OILS OF 20 to 67 BAUME Density degrees Baume Calorific power B. T. U. per pound Density degrees Baume Calorific power B. T. U. per pound Density degrees Baum6 Calorific power B. T. U. per pound 20 18930 36 19735 52 20220 21 18990 37 19770 53 20245 22 19050 38 19805 54 20270 23 19110 39 19840 55 20290 24 19170 40 19875 56 20310 . 25 19225 41 19910 57 20330 26 19280 42 19940 58 20350 27 19335 43 19970 59 20370 28 19390 44 20000 60 20390 29 19445 45 20030 61 20410 30 19495 46 20060 62 20430 31 19545 47 20090 63 20450 32 19590 48 20120 64 20470 33 34 19630 19665 49 , 50 20145 20170 65 66 20490 20510 35 19700 51 20195 67 20530 The values given in the table were found by plotting the data of all the samples, drawing a smooth curve through the approxi- mate mean results, and taking from this curve the calorific power (in the nearest multiple of 5 units) corresponding to each degree of density on the Baume* scale. On taking the calorific 1 Revision of the work of Sherman and Kropff (J. Am. Chem. Soc., 30, 1C26). FUELS 249 powers from the table for each individual sample and compar- ing with the value determined in the bomb calorimeter, it was found that of 86 samples, ranging from gasoline to the heaviest crude oils, in only 1 case in 15 did the estimated value differ from that experimentally determined by as much as 1 per cent; in only 1 in 43 was the difference as much as 2 per cent ; in no case was the difference as much as 3 per cent. It is evident, therefore, that in commercially pure American petroleum oils the calorific power may be estimated from the density by means of the above table with a sufficient degree of accuracy for many practical purposes. Since in gasoline engines, and in some of the machines using other fuel oils, the combustion is preceded by vaporization of the fuel, it is evident that volatility is an important factor in determining the adaptability of the gasoline or fuel oil to the engine in which it is to be used. In general, the mechanical engineer judges the volatility from the density, but since commercial gasoline or other fuel oil is a mixture of numerous lighter and heavier hydrocarbons, "a definite constant density is not a guarantee that the compo- sition may not change sufficiently to affect the action of the fuel in an engine" (Lucke and Woodward). In order to determine the character of a sample in this re- spect, it may be submitted to distillation in an ordinary distill- ing flask, collecting the distillate in convenient fractions of the volume of the sample taken, and noting the temperature of dis- tillation of each fraction by means of a thermometer so placed that the top of the mercury bulb is on a level with the bottom of the outlet in the neck of the distilling flask, so as to show the temperature of the vapors as they pass from the flask into, the condenser. The " motor gasoline " used by Lucke and Woodward in their comparison of alcohol and gasoline as fuel for internal combus- tion engines in 190607 was examined in this manner, 150 cc. being distilled, and the distillate collected in fractions of 10 cc. each, with the following results: l 1 U. S. Dept. Agriculture, Office of Experiment Stations, Bui. 191, p. 23. 250 METHODS OF ORGANIC ANALYSIS Number of fraction Temperature of distillation C. Number of fraction Temperature of distillation C. 1 46-60 9 100-104 2 64-75 10 104-108 3 75-80 11 108-112 4 80 12 112-120 5 80-86 13 120-126 6 86-92 14 126-140 7 8 92-97 97-100 15 (5 cc.) 140-155 Residue at 155 C. = 5 cc. or 3.3 pet. A sample purchased in New York City in 1908, and believed to be a representative specimen of satisfactory automobile gaso- line, was examined by the writer with the following results: (1) DISTILLATION OF 300 cc. IN FRACTIONS ACCORDING TO VOLUME Number of fraction Temperature of distillation C. Volume cc. Specific Gravity at 15 C. 1 40-68 30 0.669 2 68-74 30 0.678 3 74-81 30 0.692 4 81-86 30 0.704 5 86-91 30 0.712 6 91-96 30 0.720 7 96-102 30 0.727 8 102-110 30 0.734 9 110-121 30 0.741 Residue at 121 C. 30 0.756 (2) DISTILLATION OF 300 cc. IN FRACTIONS ACCORDING TO TEMPERATURE Number of fraction Temperature of distillation C. Volume Specific gravity at 15 C. cc. % 1 40-70 40.5 13.5 0.670 2 70-80 47.5 15.8 0.690 3 80-90 60. 20. 0.706 4 90-100 47.5 15.8 0.722 5 100-110 42.5 14.2 0.733 6 110-120 29.5 9.9 0.741 7 Residue at 120 C. 0.755 FUELS 251 These data may be useful for purposes of comparison when examining commercial gasolines with reference to their utility as fuel for internal combustion engines. Three samples of "fuel oil" purchased by gas manufacturers in New York City in 1905 and 1906, showed, when divided into fifths by fractional distillation, the following results : TABLE 21. COMPARISON OF FRACTIONS OF COMMERCIAL "FUEL OILS' Temperature of Distillation C. Density at 15.5 C. (60 F.) Specific Gravity Degrees Baume Sample A. 1st fraction 165-257 257-290 290-318 318-340 138-243 243-288 288-327 327-353 160-260 260-286 286-305 305-330 0.832 0.870 0.880 0.886 0.900 0.770 0.851 0.871 0.883 0.906 0.795 0.843 0.853 0.861 0.881 38.3 30.9 29.1 28.0 25.5 51.8 34.5 30.7 28.5 24.5 46.1 36.1 34.1 32.6 28.9 2d fraction 3d fraction 4th fraction Residue Sample B. 1st fraction 2d fraction 3d fraction 4th fraction Sample C. 3d fraction 4th fraction .... Residue WOODS AND SIMILAR FUELS Wood, peat, spent tan, bagasse, and other similar materials used as fuel differ greatly in calorific power because of wide fluctua- tions in moisture and ash content and considerable differences in the nature of their organic constituents; for instance, some woods approximate cellulose, while others contain large amounts 252 METHODS OF ORGANIC ANALYSIS of resinous material of much higher calorific power. All the fuels of this general group are, however, characterized by high oxygen content and low heat of combustion as compared with good coal, and so in attempts to estimate the calorific power from the ultimate analysis the differences between the results obtained by the use of Welter's rule or Walker's suggestion as explained above are relatively larger for this group of fuels than for coals. In order to ascertain which method of calculation is preferable and whether either yields accurate results, Sherman and Amend analyzed eight fuels of this type and compared the results obtained by each method of calculation with those actually determined by combustion in oxj^gen. The results of ultimate chemical analysis, reduced to the basis of dry matter were as follows: TABLE 22. ULTIMATE COMPOSITION OF DRY MATTER OF WOOD, ETC. Sample Carbon Hydrogen % Oxygen Nitrogen Sulphur Ash Chestnut wood chips . . . 50.28 5.58 43.21 0.10 0.03 0.80 Chestnut wood chips, leached 50.09 5.65 43.33 0.10 0.02 0.81 Hemlock tan ...... 53.74 5.66 39.05 0.24 0.04 1.27 Hemlock tan, leached . . . 54.97 5.73 37.69 0.26 0.02 1.33 Oak tan, leached 49.51 5.53 39.24 0.40 0.05 5.27 Bagasse 49.04 5.96 42.58 . 0.31 0.07 2.04 " Oil cake " . ... 48. 9 6.47 38.02 4.24 0.25 2.82 Peat ... . . 57.30 4.66 19.26 1.13 0.77 16.88 From these analyses the calorific power or heat of combustion was estimated (1) according to Dulong's formula X= 8080 C + 34,500 (H - JO) + 2250 S, based on Welter's rule of calculating the oxygen with the hydro- gen, (2) according to Walker's suggestion of calculating the oxygen with the carbon. Using here the same calorific values for the elements as in Dulong's formula, we have the formula: X = 8080 (C - | O) + 34,500 H + 2250 S. FUELS 253 which must of course be applied to the percentages in the water- free substance. The results (in calories) obtained by these two formulae along with those determined directly by means of the Atwater- Mahler bomb calorimeter were as follows : TABLE 23. ESTIMATED AND DETERMINED CALORIFIC POWERS Welter's rule Walker's rule Direct determination Chestnut wood chips 4125 4659 4632 Chestnut wood chips, leached . . 4129 4612 4684 5113 4661 5106 4794 5277 5217 Oak tan leached . .... 4217 4720 4798 Basrasse . . . 4185 4731 4625 4594 5078 4953 Peat 5422 5671 5606 Averagre 4510 4992 4950 It will be seen that in the case of these woody fuels high, in oxygen the calorific powers estimated by means of Dulong's formula based on Welter's rule are all much too low, the esti- mated values in terms of the actual ranging from 88.4 to 96.7 per cent and averaging 90.4 per cent, while the values estimated by a corresponding formula based on Walker's rule show a fair approximation to the values actually determined by the bomb calorimeter, the estimated values in terms of the determined ranging from 100.1 to 102.5 per cent and averaging 100.8 per cent. The two methods of calculation were also applied to seven analyses of lignites taken from Bulletin 290 of the U. S. Geo- logical Survey. In one of the seven cases the calorific power actually determined was lower than that found by Welter's rule ; in one it was materially higher than that found by Walker's rule ; in each of the other five cases and also in the average of all seven the results by Welter's rule were much too 254 METHODS OF ORGANIC ANALYSIS low and those calculated according to the suggestion of Walker were approximately correct. The results indicate : (1) that too much reliance should not be placed upon estimates of calorific power from ultimate chemical composition, especially in fuels high in oxygen ; (2) that Dulong's formula or any simular formula based on " Welter's rule " of calculating the oxygen with the hydrogen is likely to give results much below the truth; (3) that the higher results obtained by calculating the oxygen of the water- free sample as combining with the carbon according to the suggestion of Walker are much more nearly correct, and in most cases show a fair approximation to the values determined directly. COAL Ultimate Composition and Calorific Power The data above given having shown that the calorific power of wood and similar fuels is related to the ultimate composition much more nearly according to Walker's than according to Welter's rule, a similar comparison of these two methods of calculating calorific power was made for coal. 1 For this pur- pose the analyses and calorific powers of 67 coals examined by the U. S. Geological Survey and described in Professional Paper No. 48 were used. These data were determined in connection with the studies made by the Survey in its coal- testing plant at the St. Louis Exposition in 1904 and cover coals mined in 17 states and differing widely in composition and character. A comparison of the calorific powers as calculated by Dulong's formula based on Welter's rule with those found by actual determination in a bomb calorimeter showed that the values calculated by this formula were below those found by the calorimeter in seven eighths of the cases and averaged 98.9 per cent of the determined values. In more than half of the cases the calculated differed from the determined values by 1 Sherman, Bartlett, and Weatherless ; results not yet published. FUELS 255 over 1 per cent ; in 12 of the 67 cases or about 1 in 6, by more than 2 per cent ; in 4 cases or about 1 in 17 by more than 3 per cent, the greatest difference being 3.8 per cent. Thus the error involved in the use of the Dulong formula based on Welter's rule was quite variable. The values thus calculated averaged 1.1 per cent too low, but it is evident that the calculated results of individual samples cannot be made accurate by raising them all by a corresponding percentage ; for the extent of the discrepancy in each calculated value de- pends primarily upon the oxygen present in the sample, so that to increase all the calculated values by a fixed percentage would give calorific powers too high where the oxygen content was low and too low where the oxygen content was high. Better results were obtained by calculating the calorific powers in accordance with Walker's suggestion that the oxygen be figured as combining with the carbon rather than the hydrogen. Calculating the calorific powers for the same 67 coals accord- ing to the formula, X = 8080 (C - f O) + 34,500 H + 2250 S which must of course be applied to the percentages in the water-free coal, it was found that the calculated averaged 100.37 per cent of the determined values, and were about twice as often above the latter as below. In about two thirds of the cases the calculated and determined values agreed within 1 per cent, and in about one third of the cases differed by 1 per cent or more; 6 of the 67 cases, or about 1 in 11, differed by more than 2 per cent; none differed by as much as 3 per cent. Comparing the results obtained by the two methods of calculation it will be seen that the method of calculating the combined oxygen with the carbon instead of with the hydrogen gives a much better average result, a smaller proportion of cases in which the error exceeds a given margin (whether this be one or two per cent) and a smaller maximum error. Of the formulae at present available for calculating the calo- rific power of coal from its ultimate analysis the one last given 256 METHODS OF ORGANIC ANALYSIS should therefore be used, but data thus calculated are much less reliable than those obtained by properly conducted determina- tions with an accurately standarized bomb calorimeter. Proximate Analysis of Coal The proximate analysis of coal consists in determining the moisture and ash, and separating the organic matter by an arbitrary heat treatment into volatile matter and fixed carbon. In order that results of different analysts may be comparable, a uniform method has been agreed upon as follows : * The sample should be air-dried and ground to pass a 100-mesh sieve. Moisture. Dry 1 gram of the fine-ground, air-dry sample in an open platinum or porcelain crucible in an oven at 104 to 107 C. for 1 hour ; cool in a desiccator and weigh covered. The loss of weight is considered as moisture. Volatile matter. Heat one gram of the air-dry sample (or the portion which has been used for the determination of moisture) in a platinum crucible weighing 20-30 grams with a well-fitting cover over the full flame of a good Bunsen burner for 7 minutes, in a place free from drafts. The flame should be 25 cm. high and the crucible supported on a platinum triangle so that the bottom of the crucible is 6 to 8 cm. above the top of the burner and the entire crucible is surrounded by the hottest part of the flame. The upper surface of the cover should burn clean; any carbon on the under surface of the cover is weighed with the residue in the crucible. The loss in weight by this heating, corrected for moisture if neces- sary, is called the volatile combustible, or simply the volatile matter. Ash. Burn one gram of sample, or the residue from one of the above determinations, in a platinum crucible (open and in- clined) first over a very low flame, then at a higher temperature, till free from carbon, and weigh the residue as ash. 1 Report of Committee of American Chemical Society : J. Am. Chem. Soc.. 21, 1119. FUELS 257 Fixed carbon is found by subtracting the sum of the percent- ages of moisture, volatile matter, and ash from 100. The determination of sulphur, while not apart of the proximate analysis, is so often required in connection with it that the usual method may be outlined here. This is the modified Eschka method. One gram of the finely pulverized coal is mixed with I'gram of light magnesium oxide and 0.5 gram of dry sodium carbonate in a Meissen porcelain crucible (or plati- num dish of about 75 cc. capacity) and heated with an alcohol lamp (or with a gas flame nearly free from sulphur, the dish being set in a smooth hole in an asbestos pad so that its contents are protected as much as possible from the products of combus- tion). The heat should be applied gently at first, especially with soft coals, and the mixture stirred frequently with a stout platinum wire. Gradually increase the heat until the bottom of the dish reaches low redness, and maintain at about this temperature with frequent stirring until all carbon is burned; then cool and transfer to a beaker with about 50 cc. water, add 15 cc. bromine water (saturated) and boil for at least 5 minutes to complete the oxidation of the sulphur compounds to sulphates; allow to settle, decant the solution through a filter, and boil the residue a second and a third time with about 30 cc. portions of water and then \vash thoroughly with hot water. Acidify the filtrate with hydrochloric acid so as to have about 1 cc. of the latter in excess, boil to expel bromine, and precipitate the sul- phates by means of barium chloride, observing the usual pre- cautions in obtaining, washing, igniting, and weighing the barium sulphate. From the weight of the latter calculate the percentage of sulphur in the coal. Relation of Proximate Composition to Calorific Power The proximate analysis shows the amount, and something of the character, of the organic matter in a coal. The perform- ance of such an analysis requires relatively little time and no special apparatus; the results thus readily obtained are un- doubtedly often of value in helping to determine the adapta- 258 METHODS OF ORGANIC ANALYSIS bility of a coal to some particular purpose, and if the origin or general character of the coal is sufficiently well known, the proxi- mate analysis may give a fair indication of its probable calo- rific power. Formulae for estimating the calorific power of coal from its proximate analysis have been proposed, that of Goutal being most often quoted ; but on the other hand, it has been found by experiments in the laboratories of the United States Geological Survey, that the volatile matter driven off by heat, as in the usual proximate analysis, consists to a consider- able degree of inert gases, and that the proportion of these in the volatile matter " varies in different coal deposits, and makes it impossible to determine the heating value of the coal from the proximate analysis alone." 1 That there should be considerable differences in the calorific power of the volatile matter of different coals is obvious when one considers that the material volatilized from some bituminous coals is rich in hydrocarbons, while that from woody lignites contains a relatively large amount of water vapor. In order to obtain definite data (1) on the average relation of calorific power to proximate composition, and (2) on the extent of the variations from the average relation to expected in individual samples or different types of coal, Sherman and Regester compiled and computed the results of analyses made in the laboratories of the United States Geological Survey, the Ohio Geological Survey, the West Virginia Geological Survey, and Columbia University covering in all 500 samples of coal from different parts of the United States, and believed to rep- resent the principal American types of coal. In all cases the proximate composition had been determined by the method outlined above, and the calorific power by combustion in oxy- gen in a bomb calorimeter of standard type, such as the Atwater, Mahler, or Williams instrument. When the data of the 500 .samples were calculated to the basis of dry, ash-free material, and grouped according to percentage of volatile mat- ter, it was found that the coals containing from 2 to 10 per 1 U. S. Geological Survey, Bui. 339, p. 9. FUELS 259 cent of volatile in the combustible matter averaged about 14,900 B. T. U. per pound of combustible ; beyond this the calorific power increased with the volatile matter until the lat- ter reached about 17 per cent, after which it declined gradu- ally with increasing proportion of volatile in the combustible matter, up to about 40 per cent, beyond which the average calorific power decreased more rapidly. The average calorific powers for different percentages of volatile matter on the basis of dry, ash-free coal were then plotted, and the values estimated from this curve were com- pared with those found by the bomb calorimeter for each of the 500 coals. The first hundred coals showed from 1.74 to 8.50 per cent of volatile matter in the dry, ash-free substance and a mean dif- ference between the calculated and the determined values of 0.98 per cent ; in the second hundred coals with 8.62 to 22.17 per cent volatile, the mean difference was 1.14 per cent; in the third hundred with 22.53 to 38.42 per cent volatile, the mean difference was 1.32 per cent; the next 160 coals showed 38.44 to 47.93 per cent volatile and a mean difference of 1.96 per cent ; while in the last 40 coals which contained 48.22 to 59.78 per cent volatile in the dry, ash-free substance the mean difference was 6.30 per cent. It will be seen from the latter figure that among the coals having over 48 per cent of volatile matter in the dry, ash-free substance the probable variations are so great that a state- ment of the average relation of proximate analysis to calorific power cannot be made the basis of any general rule for the estimation of the latter value from the former. For coals in this region of proximate composition such relations if used at all should be worked out for each particular type or vein of coal. On the other hand for coals containing up to 48 per cent of volatile matter in the dry, ash-free substance the proximate analysis gives some indication of the probable calorific power, the average relation being approximately as shown in the follow- ing table. 260 METHODS OF ORGANIC ANALYSIS TABLE 24. APPROXIMATE AVERAGE RELATION OF VOLATILE MATTER AND CALORIFIC POWER IN THE DRY, ASH-FREE SUBSTANCE OF AMERICAN COALS Volatile matter in dry, ash-free substance per cent British Thermal Units per pound of dry, ash-free substance Volatile matter in dry, ash-free substance per cent British Thermal Units per pound of dry, ash-free substance Volatile matter in dry, ash-free substance per cent British Thermal Units per pound of dry, ash -free substance 14544 * 17 15900 33 15500 2 14900 ' 18 15850 34 15500 3 14900 19 15800 35 15500 4 14900 20 15800 36 15500 5 14900 21 15700 37 15300 6 14900 22 15700 38 15100 7 14900 23 15700 39 15100 8 14900 24 15700 40 15100 9 14900 25 15700 41 14600 10 14900 26 15700 42 14600 11 15050 27 15700 43 14600 12 15200 28 15700 44 14600 13 15350 29 15700 45 14500 14 15500 30 15600 46 14400 15 15650 31 15500 47 14300 16 15800 32 15500 48 14200 This table probably gives as good an idea as is now possible of the average relation of proximate composition to calorific power in American coals, but these average relations must not be given undue weight, because individual samples of coal may vary greatly from the average. An idea of the variations to be ex- pected in individual cases may be obtained from the fact that among the 460 cases considered by Sherman and Regester which fall within the range of this table the calorific powers actually determined by combustion in oxygen differed from those calcu- lated from the above table by 1.00 per cent or more in 233 cases or almost exactly one half of the total ; by 2.00 per cent or more in 110 cases (about 1 in 4) ; by 3.00 per cent or more in 53 cases (about 1 in 8) ; by 4.00 per cent or more in 27 cases 1 Corresponding to 8080 calories per gram, the usually accepted value for carbon. FUELS 261 (about 1 in 17) ; and by 5.00 per cent or more in 17 cases or about 1 case in 27. If this comparison were extended to the coals containing a larger amount of volatile matter, the proportion of cases show- ing serious discrepancies would of course be much greater. It is evident that in general the proximate analysis of a coal is of less value as an indication of its calorific power than is the ulti- mate analysis. The results of either ultimate or proximate analysis become more significant when interpreted in the light of the foregoing data; but in cases in which an accurate knowledge of the calo- rific power of coal is required, one should accept only the results of direct determinations by a skilled observer using an accu- rately standardized bomb calorimeter. REFERENCES I BERTHELOT : Thermochemie. GILL : Gas and Fuel Analysis for Engineers. HEMPEL : Gasanalytische Methoden. JUPTNER : Heat Energy and Fuels. LOUGUININE : Bestimmung der Verbrennungswarme. LUNGE : Chemisch-technische Untersuchungsmethoden. Ohio Geological Survey, Fourth Series, Bui. 9, Coal. POOLE : Calorific Power of Fuels. SCHEURER-KESTNER : Pouvoir Calorifique des Combustibles. SOREL : Carburetting and Combustion in Alcohol Engines (Transl. by Woodward and Preston). THOMSEN : Thermochemistry (Transl. by Burke). U. S. Geological Survey, Professional Paper 48, and Bulletins 261, 290, 323, 329, 339, 341, 362, 378, 382, 392, 428. VAUBEL: Quantitative Bestimmung organischer Verbindungen, Vol. I. West Virginia Geological Survey, Vol. II, Coal Report, and Vol. II A, Supplementary Coal Report. II 1877-78. FRASER: Classification of Coals. Trans. Am. Inst. Mining Engineers, 6, 430. 1889. STOHMANN : (Heats of Combustion of Various Organic Substances). /. prakt. Chem., 1889 et seq. 262 METHODS OF ORGANIC ANALYSIS 1895. NOYES, MCTAGGART, and GRAVER : The Determination of the Heating Effects of Coals. J. Am. Chem. Soc., 17, 843. 1897. KENT : Calorific Power of American Coals. Trans. Am. Inst. Mining Engineers, Nov., 1897. 1899. Committee Report on Coal Analysis. J. Am. Chem. Soc., 21, 1116. 1902. STODDART: Determination of Sulphur in Coal. J. Am. Chem. Soc., 24, 852. 1903. ATWATER and SNELL: Description of a Bomb Calorimeter and Method of its Use. J. Am. Chem. Soc., 25, 659. 1904. SOMMERMEIER : Sulphur in Coal, Effect on Calorific Power. /. Am. Chem. Soc., 26, 555. 1905. CAMPBELL : The Classification of Coals. Trans. Am. Inst. Mining Engineers, Sept., 1905. HENDERSON : Heat of Combustion of Atoms and Molecules. /. Phys. Chem., 9, 40. RICHARDS, HENDERSON, and FORBES : The Elimination of Thermo- metric Lag and Accidental Loss of Heat in Calorimetry. Proc. Am. Acad. Arts and Sci., 41, 1. TERRY, ARNOLD, and FISHER : Molasses as Fuel. School of Mines Quarterly, 26, 283. THOMSEN: Heats of Combustion and Formation of Volatile Organic Compounds. Z. physik. Chem., 52, 343 ; J. Chem. Soc., 88, li, 571-574. 1906. ABBOTT : Some Characteristics of Coal as Affecting Performance with Steam Boilers. J. Western Soc. Eng., 2, 529; Chem Abs., 1, 98. PARR : The Classification of Coals. J. Am. Chem. Soc., 28, 1425. : Composition and Character of Illinois Coals. Bui. 3, Illinois State Geological Survey, 1906, 86 pp.; Chem. Abs., 1, 838. SOMMERMEIER : (Moisture and Volatile Combustible Matter, in Coals and Lignites). /. Am. Chem. Soc., 28, 1002, 1630. 1907. BEMENT : Analytical Data on American Coals. J. Soc. Chem. Ind., 26, 670. BENEDICT and FLETCHER : Calorimetric Bomb Investigation. /. Am. Chem. Soc., 29, 739. ENNIS : Efficiency of Fuel under Steam Boilers. Eng. Mag., 33, 413; Chem. Abs., 1, 2027. FRANKFORTER: Lignites of the Northwest. J. Am. Chem. Soc., 29, 1488. FRIES : Investigations in the Use of the Bomb Calorimeter. U. S. Dept. Agriculture, Bureau of Animal Industry, Bui. 94. GROUT : The Composition of Coals. Econ. GeoL, 2, 225 ; Chem. Abs., 1, 1839. FUELS 263 1907. HOLMES and RANDALL: Testing of Coals used by the United States' Government. Proc. Am. Soc. Testing Materials, 7, 537. Junker Gas Calorimeter, new Automatic. Journal fur Gasbeleuchtung, 50, 520; Chem. Abs., 1, 2632. RICHARDS, HENDERSON, and FREVERT : Heat of Combustion by Adiabatic Method. Z. physik. Chem., 59, 532; Chem. Abs., 1, 2971. SY : Alcohol as a Fuel. J. Frank Inst., 163, 57. VOORHEES: Methods of Testing Coal. Proc. Am. Soc. Testing Materials, 7, 560. WOODWELL: Purchase of Coal under Specifications. Proc. Am. Soc. Testing Materials, 7, 543. 1908. JAKOB : (Calorimetry). Z. chem. Apparatenkunde, 2, 281, 313, 337, 369, 499, 533, 565, 597 ; Chem Abs., 2, 1803. LORD : Coals of the United States. Chem. Eng., Sept. 1908 ; Chem. ^^.,2,3278. LYON and CARPENTER: Peats of Indiana. /. Am. Chem. Soc., 30, 1307. NYSTROM : Peat and Lignite ; their Manufacture and Uses in Europe. Canada Dept. Mines, Ottawa, 1908, 247 pp. PARR and HAMILTON : Weathering of Coal. Econ. GeoL, 2, 693 ; Chem. Abs., 2, 650. PARR and WHEELER : Deterioration of Coal Samples. University of Illinois, Engineering Experiment Station, Bui. 17, p. 26. PORTER and OVITZ : The Nature of the Volatile Matter of Coal as Evolved under Different Conditions. J. Am. Chem. Soc., 30, 1486. REDWOOD : Supply and Use of Mineral Oil. Engineering, 86, 118 ; Chem. Abs., 3, 372. WOODWELL : Commercial Results in the Purchase of Coal on Specifi- cations. Proc. Am. Soc. Testing Materials, 8, 582. 1909. BAILEY : Accuracy in Sampling Coal. /. Ind. Eng. Chem., 1, 161. : Calorimeter Standardization. J. Ind. Eng. Chem., 1, 328. BRINSMAID : Amount of Inert Volatile Matter in the Mineral Con- stituents of Coal. J. Ind. Eng. Chem., 1, 65. Cox : Coal Calorimetry. Philippine J. Sci. (A), 4, 171; Chem. Abs., 3, 3006. EMERSON : A New Bomb Calorimeter. /. Ind. Eng. Chem., 1, 17. FISCHER and WREDE : (Use of Platinum Resistance Thermometer in Determining Heat of Combustion). Z. physik. Chem., 69, 218 ; Chem. Abs., 4, 537. FRIES : Methods and Standards in Bomb Calorimetry. U. S. Dept. Agriculture, Bureau of Animal Industry, Bui. 124. LANGBEIN : Modified Bomb Calorimeter. Chem. Ztg., 33, 1055. 264 METHODS OF ORGANIC ANALYSIS 1909. LORD : Coal Analysis. /. Ind. Eng. Chem., 1, 307. PARR et al : Determination of Sulphur in Coal. J. Ind Eng. Chem., 1, 689. PARR and WHEELER : The Ash of Coal and its Relation to Actual or Unit Coal Value. J. Ind. Eng. Chem., 1, 636. SHIMER : The Determination of the Volatile Combustible Matter in Coke and Anthracite. J. Ind. Eng. Chem., 1, 518. 1910. BEMENT : Influence of Oxygen on the Value of Coal. Science, 30, 922. BENEDICT and HIGGINS : An Adiabatic Calorimeter for Use with the Calorimetric Bomb. J. Am. Chem. Soc., 32, 461. BURGESS and WHEELER: Volatile Constituents of Coal. J. Chem. Soc., 97, 1917. FIELDNER and DAVIS : Some Variations in the Official Method for the Determination of Volatile Matter in Coal. J. Ind. Eng. Chem., 2, 304. HUNTLEY : Accuracy Obtainable in Fuel Calorimetry. J. Soc. Chem. Ind., 29, 917. MYERS: Tan Bark as a Boiler Fuel. School of Mines Quarterly, 31, 116. PARR : A New Gas Calorimeter. J. Ind. Eng. Chem., 2, 337. PARR and BARKER : Occluded Gases in Coal. Univ. of III. Bui., 6, 32. PARR and WHEELER : Unit Coal and the Composition of Coal Ash. Univ. of III. Bui, 6, 43 ; Chem. Abs., 4, 2199. PORTER and OVITZ : Losses in the Storage of Coal. /. Ind. Eng. Chem., 2, 77. - : Volatile Matter in Coal. U. S. Bur. Mines, Bui. 1. RICHARDS and JESSE : Heats of Combustion of the Octanes and Xylenes. J f Am. Chem. Soc., 32, 268. WALSH: Standard Gas Coal. Progressive Age, 28, 328; Chem. Abs., 4, 2724. WELD: Accuracy in Sampling Coal. J. Ind. Eng. Chem., 2, 426. (See also p. 543.) WHITE : (Temperature Measurements in Calorimeter Work). Physi- cal Review, 31, 562. 1911. ALLEN : Specifications for the Purchase of Fuel Oil for the Govern- ment with Directions for Sampling Oil and Natural Gas. J. Ind. Eng. Chem., 3, 730. ANONYMOUS : Estimation of Moisture in Fuel Oil. Chem. Tech. Ztg., 1911 (6), 29, 47; Chem. Abs., 5, 2425. BLAKELEY and CHANCE : Accurate Technical Estimation of the Calorific Power of Anthracite Coal. J. Ind. Eng. Chem., 3, 557. BURGESS and WHEELER: The Volatile Constituents of Coal. /. Chem. Soc., 99, 649. COSTE and JAMES : New Gas Calorimeter. J. Soc. Chem. Ind., 30, 258, FUELS 265 1911. DOANE : The Purchase of Coal on the Efficiency Basis. Eng. Rec., 61, 502; Chem. Abs., 5, 584. HOLMES : The Sampling of Coal in the Mine. Technical Paper 1, U. S. Bur. Mines. KENT and ALLEN : Formula for Purchase of Coal on Heat Unit Basis. Eng. News, 65, 109. PARKER : The Production of Coal in 1910. Published by U. S. Geol. Survey. (This includes a classified list (pp. 226-241) of the papers dealing with coal, coke, lignite, and peat contained in publications of the U. S. Geological Survey.) PARR : The Determination of Volatile Matter in Coal. /. Ind. Eng. Chem., 3, 900. POPE : Purchase of Coal by the Government under Specifications. U. S. Geol. Survey, Bur. Mines, Bui. 11. U. S. Bureau of Standards. Circular No. 11, The Standardization of Bomb Calorimeters. WHITE : Recent Progress in Calorimetry. Met. Chem. Eng., 9, 202, 296, 449. CHAPTER XIII Soap and Glycerin ANALYSIS OF COMMERCIAL SOAP THE determinations required in the examination of a com- mercial soap depend largely upon the purpose for which it is intended. The scheme outlined below is adaptable to almost all cases, as it can be easily extended to include additional deter- minations or shortened by omitting such steps as are unneces- sary when only a partial analysis is required. OUTLINE SCHEME OF ANALYSIS 1 FIRST PORTION. Dry 2 to 5 grams of soap (I) 2 and introduce into Soxhlet extractor, either in a thimble or supported by a firm plug of cotton, and extract with petroleum ether. Solution. Evap- orate ether and weigh as unsaponi- Jied fat and unsa- ponifidble matter. (II.) Residue. Allow petroleum ether to evaporate, trans- fer the soap to a beaker, dissolve in hot water, and filter. Solution. Decompose with a con- siderable excess of standard sul- phuric (or hydrochloric) acid, sep- arate aqueous solution from fatty layer. (III.) Residue. Dry and weigh as in- soluble matter. Ignite and weigh as insoluble mineral matter. (X.) Solution. Add methyl orange and titrate for total alkali. Test for chlorides (or sulphates), sugar, and glycerin. (IV.) Fatty Layer. Dry and weigh as fatty and resin acids. (V.) 1 Allen : Commercial Organic Analysis, Vol. II. 2 Numbers in parenthesis refer to sections which follow. 266 SOAP AND GLYCERIN 267 SECOND PORTION. Exhaust 2 to 5 grams of the fresh sample with care- fully neutralized alcohol. (VI.) Solution. Add pheuolphthalein and titrate for free caustic alkali or free fatty acid. (VII.) Residue. Dry and weigh. (VIII.) Exhaust thoroughly with boiling water. Solution. Divide into aliquot parts. Residue. May be examined instead of residue from first portion or used for additional tests or determina- tions. (X.) Add methyl orange and ti- trate for alkali carbonate (and b or ate, silicate, or aluminate if present). (IX.) Test for (and if necessary de- termine) sulphate, borate, silicate, and aluminate. (IX.) A cake of soap exposed to the air dries rapidly at the surface, forming a horny layer which to some extent prevents the evap- oration of water from the interior. In sampling such a soap it is important to remember this variation in water content of different parts of the same cake. If the sample is to represent the individual cake as it existed at the time of commencing the analysis, a number of sections through the entire cake should be taken either by slicing or by means of a cork borer or a cheese or butter sampler. Often the purpose of the analysis is to show the composition of the sample as originally sold, in which case the dried surface should be rejected and the sample taken from the center of the cake. The sample for analysis should be reduced quickly to fine shavings and kept in a tightly stoppered bottle. DETAILS OF DETERMINATIONS INDICATED IN THE SCHEME I. Determination of Water If the soap is very hard and dry, it may be reduced to fine shavings and dried on a watchglass, heating first for some time at 40 to 60 and then finishing at 105 to 110. Very few soaps can be completely freed from moisture in this way, but some others may be sufficiently dried for the ether extraction, while the moisture determination is made on a separate sample 268 METHODS OF ORGANIC ANALYSIS as follows : Dissolve about two grams of soap in the minimum quantity of hot strong alcohol and evaporate on clean dry sand. Finish the drying at 110 with frequent stirring, a small rod having been weighed with the dish. For approximate deter- mination of moisture Allen recommends Smith's method: Heat 5 to 10 grams of finely divided soap in a large porcelain crucible on a sand bath over a small Bunseu flame. Stir continually .with a small glass rod (weighed with the crucible) having a roughened end to facilitate breaking any lumps of soap which may be formed. Continue heating until there is no more evi- dence of water being expelled, then test by removing the burner and placing a cold glass at once over the crucible. If no moist- ure condenses on the glass, the soap is considered dry. With practice the drying can be finished in 20 to 30 minutes. Any burning of the soap is readily detected by the odor. The results are said to be reliable to 0.25 per cent. II. Petroleum Ether Extract Transfer the thoroughly dried soap to a paper " fat extraction thimble," plug tightly with fat-free cotton and treat in a .Soxh- let extractor, 1 heated on a safety water bath or electric heater, with petroleum ether of nearly constant boiling point. Regu- late the heating so that the extractor fills to the siphoning point in ten or fifteen minutes and continue the extraction for four to six hours. Disconnect the extraction apparatus (observing that no free flame is near) as the solvent flows through the siphon to the flask; remove the thimble, reconnect the appara- tus, and recover the solvent by placing a wide, short test-tube in the space previously occupied by the thimble, or by allowing the solvent to collect in this space and removing it before it reaches the top of the siphon. Having expelled nearly all of the petroleum ether, heat the flask containing the extract in a boiling water oven to constant weight. The petroleum ether extract of a commercially pure soap 1 The parts of the Soxhlet extractor may be connected by ground glass joints, by mercury seal, or by corks covered with tinfoil. SOAP AND GLYCERIN 269 may contain unsaponified fat (or free fatty acids, which are now largely used in soap-making) as well as the " unsaponifiable matter " of the original soap grease. Hydrocarbons, phenols, and other substances soluble in petroleum ether may also be found in mixed and " medicated " soaps. Allen l gives direc- tions for the systematic examination of this extract, including the quantitative determination of phenol if present. III. Liberation of Fatty and Resin Acids Decompose the water solution of the soap by adding a consid- erable excess (allow 5 to 7 cc. of normal acid for each gram of dry soap) of normal or half-normal sulphuric, nitric, or hydro- chloric acid. Add the entire amount of acid at once, boil, stir thoroughly for some minutes, and keep the solution hot until the fatty acids collect at the surface, leaving the water solution nearly clear, then complete the separation as in the determina- tion of insoluble acids in butter. It is convenient to liberate the fatty acids in a tared beaker in which they can afterward be dried and weighed (V). If the fatty acids are liquid at ordinary temperature or form a cake too soft to be handled conveniently, a known weight of dry bleached beeswax or stearic acid may be added to the hot solution. The fatty acids become incorporated with the wax and on cooling a firm cake is obtained. IV. Solution separated from Fatty Acids This solution contains, in the form of sulphate (or chloride if hydrochloric acid be used to decompose the soap), all the alkali originally present as soap, as carbonate (silicate or borate), or as hydroxide. On titrating this solution with alkali, using methyl orange as indicator, the amount of acid found to have been neutralized gives a measure of the total alkali of the soap. 2 1 Commercial Organic Analysis, Vol. II. 2 A quick determination of total alkali can also be made by burning a weighed portion of the soap to a white ash and determining the alkalinity of this ash, using methyl orange as indicator. 270 METHODS OF ORGANIC ANALYSIS Unless potash is known to be present, this total alkali is usually calculated as sodium oxide. The solution also contains any chlo- rides or other soluble salts, soluble fatty acids, glycerol, sugar, etc., which the soap may have contained. After titration the solution can be diluted to a known volume and separate por- tions taken for qualitative tests and quantitative determinations. When sulphuric acid has been used to liberate the fatty acids, chlorides can be determined in a portion of the neutralized solu- tion. If it is desired to determine both chloride and sulphate in this solution, the soap can be decomposed by means of stand- ard nitric acid. It is usually more convenient to test for sul- phates in the residue from the alcohol extraction as described below. Soluble fatty acids will be found in this solution if the liberated acids of coconut or palm-nut oil soaps are washed with hot water, as is often recommended. When the fatty acids are separated cold and washed with cold water only, the amount dissolved can usually be neglected without appreciable error. Sugar, if present, is detected in a part of this solution. After further treatment with acid to insure complete hydrolysis, the invert sugar is determined either volumetrically or gravimetri- cally (Chapter III). Sugar may also be determined by means of the polariscope, using a separate portion of the sample and precipitating the fatty acids as insoluble barium soaps. 1 In the absence of sugar and other interfering substances, glycerol can be determined by treating a portion of the neu- tralized solution directly with sulphuric acid and standard dichromate as described beyond. Since the results thus found are often too high, because of the presence of organic impurities, Lewkowitsch recommends the following method: Decompose the water solution of the soap with sulphuric acid, separate the fatty acids, neutralize the nitrate with barium carbonate, evapo- rate to a sirup, and extract with a mixture of 3 parts 95 per cent alcohol and 1 part ether. The glycerol thus obtained can be determined by the acetin method after complete removal of alcohol. iFreyer: Oesterr. Chem. Ztg., 1900, 3, 25; Analyst, 1900, 25, 127. SOAP AND GLYCERIN 271 V. Mixed Fatty and Resin Acids The mixture of acids liberated as already described (III) is dried to constant weight in a boiling water oven in a weighed flat-bottomed dish or beaker as in the determination of the insoluble acids in butter fat. The weight having been found, test a portion for resin acid by the Liebermann-Storch reaction as described under drying oils (Chapter X). In the absence of resin acids it may be possible to show the nature of the fat from which the soap was made, by examining the mixed fatty acids according to the methods used in identifying fats and oils as described in Chapters VIII to XI, consulting special works such as those of Lewkowitsch or Benedikt-Ulzer for the "con- stants " which cannot be inferred from the properties of the cor- responding fats. The separation of fatty and resin acids is best accomplished by TwitcheWs method based upon the difference of behavior of these acids when exposed in alcoholic solution to the action of hydrochloric acid. By this treatment fatty acids are converted to ethyl esters, while resin acids remain practically unchanged. The method is carried out by Lewkowitsch l as follows: Weigh 2 to 3 grams of the mixed acids in a flask, dissolve in 10 times their volume of absolute alcohol, immerse the flask in cold water, and pass a current of dry hydrochloric acid gas through the solution for an hour; then dilute the con- tents of the flask (which will have separated into two layers) with 5 times its volume of water and boil until the aqueous solution has become clear, the esters, with resin acids in solu- tion, floating on top. Transfer the contents of the flask to a separating funnel by means of 50 cc. of petroleum ether (boil- ing below 80); shake, allow to separate, draw off the acid solution, and wash the petroleum ether layer once with water. After the latter has separated completely and been removed, add a solution of 0.5 gram of potassium hydroxide and 5 cc. of alcohol in 50 cc. of water; shake, and allow to separate. The ethyl esters remain dissolved in the petroleum ether, while the resin acids are extracted by the dilute alkaline solution 1 Oils, Fats, and Waxes (4th Ed.), p. 501. 272 METHODS OF ORGANIC ANALYSIS forming soaps. Draw off the soap solution, wash the petro- leum ether solution again with dilute alkali, unite the alkaline solutions; liberate the resin acids by means of hydrochloric acid, collect, dry, and weigh them as in the determination of liberated fatty acids. The resin acids can be titrated after washing free from hydrochloric acid, instead of being separated and weighed. The volumetric method is more rapid than the gravimetric, but necessitates the assumption of a combining weight (346) for the resin acids, which is liable to considerable inaccuracy. According to Lewkowitsch, the results by the volumetric method are likely to be too high ; those by the gravimetric method too low. VI. Extraction with Alcohol Dry soap can be extracted with 95 per cent alcohol ; for wet soap stronger alcohol should be used, so that after taking up the moisture of the sample it will still be too strong to dis- solve an appreciable amount of carbonate. The alcohol to be used must first be very carefully neutralized, using phenol- phthalein as indicator. In this neutralization there is danger of adding an excess of alkali unless it is remembered that the full pink color of the indicator will not appear in alcohol of this strength. If difficulty is experienced in detecting the neutral point, a small amount of the alcohol can be removed and mixed with an equal volume of boiling water to bring out the color of the indicator. While the extraction of the soap with alcohol is often carried out in open vessels, filtering and washing in the ordinary way, it is usually more satisfactory to use the Soxhlet extractor. The soap can be put in a paper thimble as in the petroleum ether extraction, or between plugs of cotton in a glass tube with perforated bottom. In the latter case the progress of the extraction can be watched without disconnecting the apparatus. When the extraction is complete, the residue should be in powder form. If distinct pieces remain, these may contain soap which has been protected from the action SOAP AND GLYCERIN 273 of the alcohol by the formation of a layer of insoluble salts. In this case remove and crush the residue, replace, and extract again. VII. Free Caustic Alkali or Fatty Acid To the alcoholic extract add a few drops of neutralized phe- nolphthalein solution. If the solution reacts alkaline, titrate with tenth-normal acid for caustic alkali; if acid, titrate with tenth-normal alkali for free acid. For a further discussion of this extract, including a rapid method for the partial analysis of soaps, see Allen. VIII. Residue Insoluble in Alcohol It is advisable to dry and weigh this residue so that the per- centage of impurities not actually determined can be found by difference. A microscopic examination may also be of use in determining the subsequent treatment. Starch and gelatin if present could be separated from carbonate, borate, and sulphate by dissolving the latter salts in cold water ; but silicate would probably be incompletely dissolved, and it is therefore better as a rule to extract with hot water and to use separate portions of the soap, if necessary, for the determination of starch and gelatin. In such a case extract the soap with alcohol and in the residue determine starch as described in Chapter V, or determine nitrogen by the Kjeldahl method and calculate the corresponding amount of gelatin, taking the nitrogen content of the latter as 17.9 per cent. 1 IX. Carbonate, Silicate, Borate, and Aluminate Add to the water extract from the residue insoluble in alcohol an excess of standard acid and. boil to insure decom- position of the silicate. If it is important to distinguish quan- titatively between carbonate and the other alkaline salts present, the carbonic acid given off during this boiling can be collected and weighed. Add methyl orange as indicator and 1 Richards and Gies : Am. J. PhysioL, 1902, 7, 129. 274 METHODS OF ORGANIC ANALYSIS titrate with standard alkali to determine the total amount of alkali which was present as carbonate, silicate, borate, and aluminate. To a portion of the solution add hydrochloric acid in excess, evaporate to small volume, and test for boric acid by means of turmeric paper ; when dry, heat at 110, take up with dilute hydrochloric acid, filter out, and determine silica, if present. Other portions of the solution or the filtrate from silica can be used for the detection and determination of sulphates, alu- rninates, etc. X. Insoluble Matter This residue should be dried to constant weight at 100, a portion examined microscopically and the remainder ignited and weighed. If over 1 per cent of insoluble mineral matter is found, it should be analyzed. Among the substances which may be found in this residue are oatmeal, bran, sawdust, clay, chalk, steatite, infusorial earth, pumice, sand, mineral pigments, etc. CALCULATION AND INTERPRETATION OF RESULTS In the case of hard soap, the results of the partial analysis usually required may be reported as follows : Water ; unsaponified fat and unsaponifiable matter ; fatty and resin anhydrides (97 per cent of the weight of free acids); sodium oxide combined as soap ; sodium hydroxide ; sodium carbonate ; insoluble organic matter ; insoluble mineral matter. It is well to report also the total alkali in terms of sodium oxide. The purpose for which a soap is intended must be known be- fore an opinion as to its quality can safely be formed. In most cases the percentage of alkali combined as soap is the best measure of the amount of actual soap in the material, but for special purposes the presence or absence of other constituents is often of greater importance. Toilet soaps should contain as little free alkali (either caustic or carbonate) as possible. Alder-Wright divided toilet soaps into three classes according to the proportion of free alkali to SOAP AND GLYCERIN 275 alkali combined as soap. The first class included those soaps which contained less than 2.5 per cent as much free as com- bined alkali ; the second, those in which the percentage was 2.5 to 7.5 ; the third, those containing over 7.5 per cent as much free as combined alkali. In judging the quality of toilet soaps it is also important to consider the proportions and nature of all foreign matter, the amount of water, the hardness of the soap, and in some cases the origin must be sought by an exami- nation of the fatty acids. The more expensive " transparent " toilet soaps may contain alcohol or glycerin ; in cheaper grades a similar appearance is obtained by the addition of sugar. Household soaps are made from cheaper and softer fats than those used for toilet soap. Alkali in the form of carbonate, silicate, or borate is not objectionable unless present in exces- sive amount. No appreciable amount of sugar or glycerol is likely to be present. Scouring soaps often contain large amounts of pulverized quartz, infusorial earth, etc., and are sometimes strongly alkaline with sodium carbonate or hydrox- ide. For discussion of the adaptability of different types of soaps to specific uses see references at the end of the chapter. GLYCEROL Glycerol is a colorless, odorless, viscous liquid of sweet taste and neutral reaction, miscible in all proportions with water and with alcohol. It also dissolves in mixtures of alcohol and ether, but is only very sparingly soluble in pure ether 1 and is practically insoluble in chloroform, carbon disulphide, and benzene. The specific gravity of pure glycerol at 15 referred to water at the same temperature is variously stated at from 1.265 to 1.2677. Anhydrous glycerol boils at about 290, but evaporates rapidly at lower temperatures (160 or over), and the evaporation is greatly accelerated by the presence of a small amount of water. When kindled, glycerol burns with a blue flame and leaves no carbonaceous residue. 1 According to Lewkowitsch, one part of glycerol of 1.23 sp. gr. dissolves in about 500 parts of ether. 276 METHODS OF ORGANIC ANALYSIS These properties, together with the fact that it yields acrolein when heated with acid potassium sulphate, are usually sufficient for the identification of glycerol when in a fairly pure and con- centrated state. Glycerol is a good solvent for many substances, both organic and inorganic, and its presence often increases their solubility in aqueous and alcoholic solutions. This fact and the difficulty of distilling without loss make it troublesome to separate glycerol as a pure aqueous solution as is done in the determination of alcohol. The percentage of glycerol in commercial glycerin is usually determined either by acetylating the glycerol and finding the amount of acetin by saponification (acetin method), or by quantitative oxidation of the glycerol by means of standard potassium dichromate (dichromate method). The acetin method requires that the glycerol be concentrated, and that other acetylizable substances if present shall be corrected for ; the dichromate method requires the removal of chlorides and all organic substances which would be oxidized by the dichro- mate treatment. ANALYSIS OF CRUDE GLYCERIN In recent years the increase in price of crude glycerin has resulted in the manufacture of glycerin from lower-grade fats than before, with the result that the product often contains impurities which behave so much like glycerol as to introduce serious discrepancies in the analytical determination of glycerol in crude glycerin. This led to the appointment of committees, both in this country and in Europe, to study methods of glyc- erin analysis. Representatives of these committees met as an international committee, which, after investigation, decided that the acetin method should be the basis on which glycerin should be bought and sold, but that the dichromate method, be- ing more convenient for factory control, might continue to be used for some technical purposes in a properly standardized form. The methods recommended by the international com- mittee for sampling and analysis of glycerin are as follows : 1 *J. Ind. Eng. Chem., 3, 679-686. SOAP AND GLYCEKIN 277 Sampling The most satisfactory method available for sampling crude glycerin liable to contain suspended matter, or which is liable to deposit salt on settling, is to have the glycerin sampled by a sampler mutually approved by the buyer and seller as soon as possible after the glycerin is filled into drums, but in any case before any separation of salt has taken place. In such cases he shall sample with a sectional sampler (see appendix to original report), then seal the drums, brand them with a number for identification, and keep a record of the brand number. The presence of any visible salt or other suspended matter is to be noted by the sampler, and a report of the same made in his cer- tificate, together with the temperature of the glycerin. Each drum must be sampled. Glycerin which has deposited salt or other solid matter cannot be accurately sampled from the drums, but an approximate sample can be obtained by means of a sec- tional sampler which will allow a complete vertical section of the glycerin to be taken, including any deposit. Analysis 1. Determination of Free Caustic Alkali. Put 20 grams of the sample into a 100-cc. flask, dilute with approximately 50 cc. of freshly boiled distilled water, add an excess of neutral barium chloride solution, 1 cc. of phenolphthalein solution, make up to the mark, and mix. Allow the precipitate to settle, draw off 50 cc. of the clear liquid, and titrate with normal acid. Calcu- late the percentage of caustic alkali as Na 2 O. 2. Determination of Ash and Total Alkalinity. Weigh 2-5 grams of the sample in a platinum dish, burn off the glycerin over a luminous Argand burner or other source of heat at a low temperature, to avoid volatilization and the formation of sul- phides. When the mass is thoroughly charred, stir with hot water, filter, wash, and ignite the residue in the platinum dish. Return the filtrate and washings to the dish, evaporate the water, ignite carefully, avoiding fusion, and weigh the ash. Dis- solve the ash in water and titrate total alkalinity, using as indi- 278 METHODS OF ORGANIC ANALYSIS cator methyl orange in a cold solution, or litmus, if the solution is boiled. 3. Determination of Alkali present as Carbonate. Take 10 grams of the sample, dilute with 50 cc. water, add sufficient normal acid to neutralize the total alkali found at (2), boil under a reflux condenser for 1520 minutes, wash down the condenser tube with water free from carbon dioxide, and then titrate the free acid in the solution with normal sodium hydrox- ide, using phenolphthalein as indicator. Calculate the percent- age of Na 2 O, deduct the Na 2 O found in (1). The difference is the alkali present as carbonate expressed in terms of Na 2 O. 4. Alkali combined with Organic Acids. The sum of the percentages of Na 2 O found at (1) and (3) deducted from the percentage found at (2) is a measure of the Na 2 O or other alkali combined with organic acids. 5. Determination of Acidity. Take 10 grams of the sample, dilute with 50 cc. distilled water free from carbon dioxide, ti- trate with normal sodium hydroxide, using phenolphthalein as indicator, and express the result in terms of Na 2 O required to neutralize 100 grams. 6. Determination of Total Residue at 160 C. For this de- termination the crude glycerin should be slightly alkaline with sodium carbonate, not exceeding 0.2 per cent Na 2 O in order to prevent loss of organic acids. To avoid the formation of poly- glycerols,' this alkalinity must not be exceeded. Ten grams of the sample are put in a 100-cc. flask, diluted with water, and the calculated quantity of normal hydrochloric acid or sodium carbonate added to give the required degree of alkalinity. The flask is filled to 100 cc., the contents mixed, and 10 cc. measured into a weighed flat-bottomed glass dish 2.5 inches in diameter and 0.5 inches deep. In the case of crude glycerins abnormally high in organic residue a smaller amount should be taken so that the organic residue shall not materially exceed 30-40 milligrams. The dish is placed on a water bath (or on top of the oven kept at 160) until most of the water has evaporated, then placed in an oven at 160, leaving the door, of the oven open so as to have SOAP AND GLYCERIN 279 a temperature of 130-140, until the glycerin, or most of it, has evaporated. When only a slight vapor is seen to come off, the dish is removed, allowed to cool, and 0.5 to 1 cc. of water added and by a rotary motion the residue brought wholly or nearly into solution. The dish is then allowed to stand on top of the oven until the water has evaporated and the residue is sufficiently dry to prevent spurting, when it is placed in the oven at 160 C. The dish is then kept in the oven carefully maintained at 160 C. for one hour, when it is removed, cooled, the residue treated with water, the water evaporated, and the residue subjected to a second baking of one hour, after which the dish is allowed to cool in a desiccator over sulphuric acid and weighed. The treatment with water, etc., is repeated until a constant loss of 1 to 1.5 milligram per hour is obtained. In the case of acid glycerin, a correction must be made for the alkali added, 1 cc. normal alkali representing an addition of 0.03 gram to the residue. In the case of alkaline glycerins a correction should be made for the acid added, by deducting the increase in weight due to the conversion of sodium hydroxide and sodium carbonate to sodium chloride. The corrected weight multiplied by 100 gives the percentage of total residue at 160 C. This residue is used for determination of the non-volatile acetylizable impurities as described under the acetin method below. 7. Organic Residues. Subtract the ash from the total residue at 160 C. Report as organic residue at 160 C. (it should be noted that alkaline salts of fatty acids are converted into car- bonates on ignition and that the carbon dioxide thus derived is not included in the organic residue). G-lycerol by Acetin Method This process is the one agreed upon at a conference of dele- gates from the American, British, French, and German Commit- tees, and has been confirmed by each of the above committees as giving results nearer to the truth than the dichromate method on crude glycerins in general. It is the process to be 280 METHODS OF ORGANIC ANALYSIS used (if applicable) whenever only one method is employed. On pure glycerins the results are identical with those obtained by the dichromate process. For the application of this method the crude glycerin should not contain over 60 per cent water. Reagents. (yl) Best acetic anhydride. This should be carefully selected. A good sample must not require more than 0.1 cc. normal sodium hydroxide for saponification of the im- purities in a blank test on 7.5 cc. Only a slight color should develop during digestion of the blank. The anhydride may be tested for strength by the following method: Into a weighed stoppered vessel, containing 10 to 20 cc. of water, run about 2 cc. of the anhydride, replace the stop- per, and weigh. Let stand, with occasional shaking, for several hours, to permit the hydrolysis of all the anhydride; then dilute to about 200 cc., add phenolphthalein, and titrate with normal sodium hydroxide. This gives the total acidity due to free acetic acid and acid formed from the anhydride. It is worthy of note that in the presence of much free anhydride a compound is formed with phenolphthalein, soluble in alkali and acetic acid, but insoluble in neutral solutions. If a turbidity is noticed toward the end of the neutralization, it is an indication that the anhydride is incompletely hydrolized, and inasmuch as the indicator is withdrawn from the solution, results may be incorrect. Into a stoppered weighing bottle containing a known weight of recently distilled aniline (from 10 to 20 cc.) measure about 2 cc. of the sample, stopper, mix, cool, and weigh. Wash the contents into about 200 cc. of cold water and titrate the acidity as before. This yields the acidity due to the original preformed acetic acid plus one half the acid due to anhydride (the other half having formed acetanilide) ; subtract the second result from the first (both calculated to 100 grams) and double the result, obtaining the cubic centimeters of normal sodium hydroxide per 100 grams of the sample. Each cubic centimeter equals 0.0510 gram an- hydride. (5) Pure fused sodium acetate. The purchased salt is again completely fused in a platinum, silica, or nickel dish, SOAP AND GLYCERIN 281 avoiding charring, powdered quickly and kept in a stoppered bottle or desiccator. It is important that the sodium acetate be anhydrous. ((7) A solution of sodium hydroxide for neutralizing, of about normal strength, free from carbonate. This can be readily made by dissolving pure sodium hydroxide in its own weight of water (preferably free from carbon dioxide) and allowing to settle until clear or filtering through an asbestos or paper filter. The clear solution is diluted with water free from carbon dioxide for the strength required. (D) Normal sodium hydroxide free from carbonate. Pre- pared as above and carefully standardized. Some sodium hy- droxide solutions show a marked diminution in strength after being boiled; such solutions should be rejected. (.Z7) Normal acid carefully standardized. (^) Phenolphthalein solution, a one half per cent solution in alcohol, neutralized. Method. In a narrow-mouthed flask (preferably round bottom), capacity about 120 cc., which has been thoroughly cleaned and dried, weigh accurately and as rapidly as possible 1.25 to 1.5 grams of the glycerin. A Grethan or Lunge pipette will be found convenient. Add about 3 grams of the anhy- drous sodium acetate, then 7.5 cc. of the acetic anhydride, and connect the flask with an upright Liebig condenser. For con- venience the inner tube of this condenser should not be over 50 cm. long and 9 to 10 mm. inside diameter. The flask is con- nected to the condenser by either a ground glass joint (prefer- ably) or a rubber stopper. If a rubber stopper is used, it should have a preliminary treatment with hot acetic anhydride vapor. Heat the contents and keep just boiling for one hour, taking precautions to prevent the salts drying on the sides of the flask. Allow the flask to cool somewhat, and through the condenser tube add 50 cc. of distilled water, free from carbon dioxide, at a temperature of about 80 C., taking care that the flask is not loosened from the condenser. The object of cool- ing is to avoid any sudden rush of vapors from the flask on adding water and to avoid breaking the flask. Time is saved 282 METHODS OF ORGANIC ANALYSIS by adding the water before the contents of the flask solidifies, but the contents may be allowed to solidify and the test pro- ceeded with the next day without detriment, bearing in mind that the anhydride in excess is much more effectively hydro- lized in hot than in cold water. The contents of the flask may be warmed to, but must not exceed, 80 C., until the solution is complete except a few dark flocks representing organic impur- ities in the crude glycerin. By giving the flask a rotary motion, solution is more quickly effected. Cool the flask and contents without loosening from the con- denser. When quite cold wash down the inside of the con- denser tube, detach the flask, wash off the stopper or ground glass connection into the flask, and filter the contents through an acid-washed filter into a Jena glass flask of about one liter capacity. Wash thoroughly with cold distilled water, free from carbon dioxide. Add 2 cc. of phenolphthalein solution (-F), then run in caustic soda solution ( (7) or (D) until a faint pinkish yellow color appears throughout the solution. This neutralization must be done most- carefully ; the alkali should be run down the sides of the flask, the contents of which are kept rapidly swirling with occasional agitation or change of motion until the solution is nearly neutralized, as indicated by the slower disappearance of the color developed locally by the alkali running into the mixture. When this point is reached, the sides of the flask are washed down with carbon-dioxide-free water, and the alkali subsequently added drop by drop, mixing after each drop until the desired tint is obtained. Now run in from a burette 50 cc. or a calculated excess of normal sodium hydroxide {D) and note carefully the exact amount. Boil gently for fifteen minutes, the flask being fitted with a glass tube acting as a partial condenser. Cool as quickly as possible and titrate the excess of sodium hydroxide with normal acid (J7) until the pinkish yellow er chosen end point color just remains. 1 1 A precipitate at this point is an indication of the presence of iron or alu- minium and high results will be obtained unless a correction is made as described below. SOAP AND GLYCERIN 283 A further addition of the indicator at this point will cause an increase of the pink color; this must be neglected and the first end point taken. From the amount of normal sodium hydroxide consumed, calculate the percentage of glycerol (including acetylizable impurities) after making correction for the blank test described below. 1 cc. normal sodium hydroxide corresponds to 0.03069 gram glycerol. The coefficient of expansion for normal solutions is 0.00033 per cubic centimeter for each degree C. A correction should be made on this account, if necessary. Blank Test. As the acetic anhydride and sodium acetate may contain impurities which affect the result, it is necessary to make a blank test, using the same quantities of acetic anhydride, sodium acetate, and water as in the analysis. It is not necessary to filter the solution of the melt in this case, but sufficient time must be allowed for the hydrolysis of the anhydride before proceeding with the neutralization. After neutralization it is not necessary to add more than 10 cc. of normal alkali (-0), as this represents the excess usually present after the saponification of the average soap lye crude glycerin. In determining the acid equivalent of the normal sodium hydroxide, however, the entire amount taken in the analysis, 50 cc., should be titrated after dilution with 300 cc. of water free from carbon dioxide and without boiling. Determination of the Crlycerol Value of the Acetylizable Im- purities. The total residue at 160 C. is dissolved in one or two cubic centimeters of water, washed into the acetylizing flask, and evaporated to dryness. Then add anhydrous sodium acetate and acetic anhydride in the usual amounts and proceed as described in the regular analysis. After correcting for the blank calculate the result to glycerol. Calculation of the Actual Crlycerol Contents. (1) Determine the apparent percentage of glycerol in the sample by the acetin process as described. The result will include acetylizable impurities, if any are present. 284 METHODS OF ORGANIC ANALYSIS (2) Determine the total residue at 160 C. (3) Determine the acetin value of this residue in terms of glycerol. (4) Deduct the result found at (3) from the percentage obtained at (1) and report this corrected figure as glycerol. If volatile acetylizable impurities are present, these are included in this figure. Trimethyleneglycol is more volatile than glycerol and can therefore be concentrated by fractional distillation. An ap- proximation to the quantity can be obtained from the differ- ence between the results by the acetin and by the dichromate method on such distillates. The difference multiplied by 1.736 will give the glycol. Crlycerol by Dichromate Method Reagents. {A) Pure potassium dichromate powdered and dried in a'ir free from dust or organic vapors at 110 to 120 C. This is taken as the standard. (.#) Dilute dichromate solution. 7.4564 grams of the above dichromate are dissolved in distilled water and the solu- tion made up to one liter at 15.5 C. ((7) Ferrous ammonium sulphate. It is never safe to as- sume this salt to be constant in composition and it must be standardized against the dichromate as follows : Dissolve 3.7282 grams of dichromate (A) in 50 cc. of water. Add 50 cc. of 50 per cent sulphuric acid (by volume) and to the cold undiluted solution add from a weighing bottle a moderate excess of the ferrous ammonium sulphate and titrate back with the dilute dichromate (-5). Calculate the value of the ferrous salt in terms of dichromate. (-Z>) Silver carbonate. This is prepared as required for each test from 140 cc. of 0.5 per cent silver sulphate solution by precipitation with about 4.9 cc. normal sodium carbonate solution (a little less than the calculated quantity of sodium carbonate should be used, as an excess prevents rapid settling). Settle, decant, and wash once by decantation. (JS) Subacetate of lead. Boil a 10 per cent solution of pure . SOAP AND GLYCERIN 285 lead acetate with an excess of litharge for one hour, keeping the volume constant, and filter while hot. Disregard any precipi- tate which subsequently forms. Preserve out of contact with carbon dioxide. (jP) Potassium ferricyanide. A very dilute freshly pre- pared solution containing about 0.1 per cent. Method. Weigh 20 grams of the glycerin, dilute to 250 cc., and take 25 cc. Add the silver carbonate, allow to stand with occasional agitation for about ten minutes, and add a slight excess (about 5 cc. in most cases) of the basic lead acetate (^E) ; allow to stand a few minutes, dilute with distilled water to 100 cc. and then add 0.15 cc. to compensate for the volume of the precipitate ; mix thoroughly, filter through an air-dry filter into a suitable narrow-mouthed vessel, rejecting the first 10 cc., and return the filtrate, if not clear and bright. Test a portion of the filtrate with a little basic lead acetate, which should produce no further percipitate (in the great majority of cases 5 cc. are ample, but occasionally a crude glycerin will be found requiring more, and in this case another portion of 25 cc. of the dilute glycerin should be taken and purified with 6 cc. of the basic acetate). Care must be taken to avoid a marked excess of basic acetate. Measure off 25 cc. of the clear filtrate into a flask or beaker, previously cleaned with potassium dichromate arid sulphuric acid. Add 12 drops of sulphuric acid (1 to 4) to precipitate the small excess of lead as sulphate. Add 3.7282 grams of the powdered potassium dichromate (^1). Rinse down the dichromate with 25 cc. of water and let stand with occasional shaking until all the dichromate is dissolved (no reduction will take place in the cold). Now add 50 cc. of 50 per cent sulphuric acid (by volume) and immerse the vessel in boiling water for two hours and keep protected from dust and organic vapors, such as alcohol, until the titration is completed. Add from a weighing bottle a slight excess of the ferrous ammonium sulphate ((7), making spot tests on a porcelain plate with the potassium ferricyanide (-F). Titrate back with the dilute dichromate. From the 286 METHODS OF ORGANIC ANALYSIS amount of dichromate reduced, calculate the percentage of glycerol. One gram glycerol = 7.4564 grains dichromate One gram dichromate = 0.13411 gram glycerol. The percentage of glycerol obtained above includes any oxidizable impurities present after purification. A correction for the non-volatile impurities may be made by running a di- chromate test on the residue at 160 C. Notes. (1) It is important that the concentration of acid in the oxidation mixture and the time of oxidation should be strictly adhered to. (2) Before the dichromate is added to the glycerin solution it is essential that the slight excess of lead be precipitated with sulphuric acid as stipulated. (3) For crude glycerins practically free from chlorides the quantity of silver carbonate may be reduced to one-fifth and the basic lead acetate to 0.5 cc. (4) It is sometimes advisable to add a little potassium sul- phate to insure a clear filtrate. An appendix to the committee report from which the above methods are taken describes and illustrates a new form of sampling tube for taking samples of crude glycerin from drums. REFERENCES I ALDER-WRIGHT and MITCHELL : Animal and Vegetable Fixed Oils, Fats, Butters, and Waxes. ALLEN : Commercial Organic Analysis, Vol. II. BENEDIKT-ULZER : Analyse der Fette und Wachsarten. LAMBORN: Modern Soaps, Candles, and Glycerin. LEWKOWITSCH : Chemical Technology and Analysis of the Oils, Fats, and Waxes. II 1900. DEVINE: A Method of Determining Free Alkali in Soaps. J. Am. Chem. Soc., 22, 693. HENRIQUES and MAYER : (Determination of Total, Free, and Car- bonated Alkali in Soaps). Z. angew. Chem., 1900, 785. 1902. DOANE : The Disinfectant Properties of Washing Powders. Bui. 79, Maryland Agricultural Experiment Station. SOAP AND GLYCERIN 287 FRIEDRICH: (Soap Analysis) 4 Bericht des Vereins gegen Verfal- schung der Lebensmittel, etc., Chemnitz, 1902, 132 ; Z. Ndhr. Genussm., 1903, 6, 851. 1903. HELLER: (Significance of Soaps in Disinfectants). Arch. Hygiene, 47, 213. 1904. HEERMANN: Determination of Caustic and Carbonated Alkali in Soaps. Chem. Ztg., 28, 531. MARTIN : Determination of Glycerol in Soap. Moniteur Scientifique, [4], 17, 797. VAN SLYKE and URNER : The Composition of Commercial Soaps in Relation to Spraying. Bui. 257, New York State Agricultural Experiment Station. 1905. DEVINE : Determination of Rosin in Soaps. Chem. Eng., 1, 207. MATTHEWS : The Effects of Alkaline Scouring Agents on the Strength of Woolen Yarns. /. Soc. Chem. Ind., 24, 659. RODET: Experiments on the Antiseptic Value of Common Soaps. Revue d' Hygiene, 27, 301. 1907. JACKSON : Detergents and Bleaching Agents used in Laundry Work. J. Soc. Arts, 55, 1101, 1122 ; Chem. Abs., 2, 327. 1908. BORNEMANN : Fat, Soap, and Candle Industry in 1907. Chem. Ztg., 32, 741, 755. FENDLER and FRANK: Determination of Fatty Acid Content of Soaps. Z. angew. Chem., 22, 252. STEINER : (Recent Development of the Soap Industry). Chem. Ztg.j 32, 445, 458. 1909. DOMINIKIEWICZ : New Method for Determination of Fatty Acids in Soap. Chem. Ztg., 33, 728. SY : Mercury Seals in Fat Extraction Apparatus and a New Form of Flask. J. Ind. Eng. Chem., 1, 314. 1910. COMEY and BACKUS : The Coefficient of Expansion of Glycerin. /. Ind. Eng. Chem., 2, 11. 1911. Committee Report on Glycerin Analysis. J. Ind. Eng. Chem., 3, 679. HAMILTON : Soaps from Different Glycerides ; their Germicidal and Insecticidal Values Alone and Associated with Active Agents. J. Ind. Eng. Chem., 3, 582. CHAPTER XIV Nitrogen, Sulphur, and Phosphorus THE DETERMINATION OF NITROGEN THE well-known copper oxide method of Duinas, sometimes called the absolute method, has/the advantage of being applicable to all classes of nitrogen compounds. In the great majority of cases, however, it is equally accurate and much more conven- ient to use one of the methods based upon the conversion of nitrogen to ammonia and the determination of the latter by titration. Before the introduction of the Kjeldahl process, the soda- lime method of Will and Varrentrap was commonly used. In this method the finely ground substance is mixed with a large excess of soda lime and heated in a combustion tube, the am- monia given off being absorbed in standard acid. In order to insure the reduction of nitro-compounds or nitrates, this method was modified by the introduction of stannous sulphide (Gold- berg) ; of sodium formate and sodium thiosulphate (Arnold) ; or of sodium thiosulphate and a mixture of equal parts sulphur and powdered sugar or charcoal (Ruffle). The soda-lime method has now been very generally superseded by the various modifications of the Kjeldahl process. THE KJELDAHL METHOD In this process the substance is decomposed by heating with strong sulphuric acid, usually with the addition of some reagent which assists the decomposition either by raising the boiling point or by acting as a carrier of oxygen. When decomposition is complete, the nitrogen remains as ammonium sulphate in the sulphuric acid, the carbon and hydrogen of the substance hav- 288 NITROGEN, SULPHUR, AND PHOSPHORUS 289 ing been oxidized and the products of oxidation boiled out of the solution. The oxidation takes place partly at the expense of the sulphuric acid, so that a considerable evolution of sulphur dioxide occurs, especially in the earlier stages of the process. After the completion of the digestion, the ammonia is liberated by means of fixed alkali and determined by distilling into standard acid. In the va^gus modifications of the process, different reagents or combinations of reagents are used to hasten the decomposition of the organic matter by the sulphuric acid. Kjeldahl originally directed * that the substance be heated with sulphuric acid, with or without the addition of phosphoric anhydride, until a clear solution is obtained ; then potassium permanganate added in small portions to the hot solution until it remains permanently colored, the permanganate being added very cautiously on account of the danger of a loss of nitrogen if the reaction becomes too vigorous. Willfarth 2 introduced the use of mercuric oxide to facilitate the action of sulphuric acid, and stated that if the solution be boiled until colorless, neither phosphoric anhydride nor potas- sium permanganate need be used. In his earlier experiments Willfarth used copper instead of mercury. Arnold 3 used both mercury and copper in addition to phosphoric anhydride. Gunning * used for the digestion a simple mixture of sulphuric acid with one third to one half its weight of potassium sulphate. Arnold and Wedemeyer 5 modified the Gunning process by the use of mercury and copper in addition to the potassium sulphate. By this modification the time required to decompose the organic matter was greatly reduced and good results were obtained with a number of alkaloids and other compounds which had not readily yielded the whole of their nitrogen when treated by the methods previously used. Independently Dyer 6 ob- tained equally good results on a wide range of organic com- 1 Z. anal. Chem., 1883, 22, 366. 2 Chem. ZentrbL, 1885, [3], 16, 17, 113. 3 Archw. der Pharm., [3], 24, 785 ; Z. anal. Chem., 26, 249. *Z. anal, Chem., 1889, 28, 188. 5 Z. anal. Chem., 1892, 31, 525. 6 J. Chem. Soc., 1895, 67, 811. u 290 METHODS OF ORGANIC ANALYSIS pounds by the use of mercury and potassium sulphate without copper. The Official Agricultural Chemists authorize l the Kjeldahl- Willfarth and the Gunning methods for the analysis of foods, spices other than peppers, and fertilizers not containing nitrates. For peppers, to secure complete ammonification of the alkaloidal nitrogen, the Arnold-Wedemeyer modification of the Gunning method was provisionally adopted in 1902. In the experience of this laboratory, the Dyer modification has been found more rapid and slightly more accurate than either the Kjeldahl-Willfarth or Gunning method and fully as efficient in the case of alkaloids as is the Arnold-Wedemeyer method, while it has the advantage over the latter of requiring one less reagent and of yielding a colorless solution. Applicability of the Kjeldahl Method. The Gunning- Arnold- Dyer modification, which for the reasons just mentioned is rec- ommended for general use, is applicable to all classes of animal and vegetable substances, including such difficultly decomposable bases as betaine and pyridine and chinoline alkaloids, and to cyanides, ferrocyanides, and ferricyanides. It has also been tested 2 with good results on many other compounds, including acetanilid, sulphanilic acid, orthobenzoic sulphinid, aminoben- zoic acid, benzamid, diaminophenol, naphthylamine, diphenyl- amine, diphenylthiourea, nitroso-dimethylaniline, indigotin, pyridine, and oxyphenyl methylpyrimidine. Jodlbauer's modification, 3 devised for the determination of nitrogen in nitrates, was found by Dyer to be applicable to nitro- compounds, to azo-, hydrazo-, and amidoazo-benzene, to carbazol, and, with the addition of 1 or 2 grams of sugar, to hydroxyl- amine and oximes. Dyer did not obtain the whole of the nitro- gen of hydrazine derivatives, but Dafert 4 and Milbauer 6 have published modifications which are said to give accurate results with this class of compounds. 1 Bulletin 107, Revised, Bureau of Chemistry, U. S. Dept. Agriculture. 2 In some cases without the use of mercury. See in addition to the papers al- ready cited, that of Gibson in J. Am. Chem. Soc., 1904, 26, 105. 8 Chem. Zentrbl., 1886, 3, 17, 433. *Landw. Versuchs-Sta., 1887, 34, 311. 5 Z. anal. Chem., 1903, 42, 725. NITROGEN, SULPHUR, AND PHOSPHORUS 291 GUNNING-ARNOLD-DYER MODIFICATION Reagents. Pure concentrated sulphuric acid. Mercury. Pure potassium sulphate. Potassium sulphide solution, 40 grams per liter. Saturated solution of sodium hydroxide (commercial) . Granulated zinc or pumice stone. Paraffin. Solution of methyl orange, congo red, cochineal, lacmoid, or any other indicator suitable for titration in the presence of ammonium salts. Standard solutions of acid and al- kali, preferably one fifth or one tenth normal. Determination. Weigh 0.5 to 5.0 grams sample, or so much as will probably yield from 50 to 75 milligrams of ammonia, and trans- fer to a pear-shaped Kjeldahl flask of 550 to 750 cc. capacity. Add 20 to 25 cc. concentrated sulphuric acid and about 0.7 gram of mer- cury. Place the flask in an in- clined position (Fig. 16) and heat gently until the first vigorous frothing ceases, then raise the heat gradually until the liquid boils ; remove the flame for a few minutes, add 10 to 12 grams of potassium sulphate, and boil. If the liquid -, n T .,. ., .,, FIG. 16. Kjeldahl digestion flask IS kept actually boiling, it Will in position on ordinary ring stand. usually be clear and colorless within 30 minutes after the addition of the sulphate. Con- tinue the boiling for at least 30 minutes after the solution becomes colorless or in any case for one hour from the time the potassium sulphate is added. If the sample contains al- kaloids, the boiling should be continued for at least three hours in all and not less than two hours after the solution is colorless. When the digestion is finished, allow the flask to cool for 10 to 15 minutes or to 40-60 (if allowed to become thoroughly cold *_* 292 METHODS OF ORGANIC ANALYSIS the solution solidifies) ; then dilute carefully with 150 to 200 cc. of water ; allow to cool, add 25 cc. potassium sulphide solu- tion, mix well, and then add 75 to 100 cc. (or enough to make the reaction strongly alkaline - 1 ) of a cold saturated solution of sodium hydroxide, pouring it carefully down the side of the flask, so that it does not mix immediately with the acid solution. Add a few pieces of granulated zinc or pumice stone to prevent bump- ing, and a piece of paraffin the size of a pea to diminish frothing ; connect the flask, preferably by means of a Hopkins distilling head (Fig. 17), with a condenser, the deliv- ery tube of which dips into 50 cc. of tenth normal sulphuric or hydrochloric acid or its equivalent in the receiver ; mix the contents of the flask by shaking, and distill until about one half of the liquid has passed into the re- ceiver. Titrate the excess of acid in the receiver by means of standard alkali, using one of the indicators mentioned above. The reagents used should be tested by making a blank determination with pure sugar or cellulose, carrying out all opera- tions in exactly the same way as in a regular analysis. Notes. If in transferring the substance to the flask any par- ticles or drops should lodge in the neck, they can be washed down by the sulphuric acid subsequently added. Dry samples can usually be weighed on a watch glass and brushed into the flask through a funnel having a wide stem which has been cut down to a length of about 1 cm., or transferred by means of a narrow strip of glazed paper. It is often more convenient to weigh the sample on, or transfer to, a small piece of pure filter paper, fold the latter loosely over the weighed portion, and in- troduce the whole into the flask in such a way that the sample can be easily shaken free from the paper. The amount of cel- lulose thus introduced with the sample does not materially pro- 1 Corallin (rosolic acid) may be used as indicator to show that the contents of the flask are alkaline before beginning the distillation. - FIG. 17. The Hop- kins distilling head in position. NITROGEN, SULPHUR, AND PHOSPHORUS 293 long the time required for digestion, while its reducing effect may aid in the ammonificatioii of any firmly bound nitrogen present. Time should be allowed for thorough wetting of the sample by the sulphuric acid before heat is applied. During digestion a small funnel or balloon stopper may be placed loosely in the mouth of the flask to retard the evaporation of acid and guard against mechanical loss. The flask may rest upon a wire gauze, an iron plate, or a piece of asbestos having a circular hole about 4 cm. in diameter which permits the free flame of the Bunsen burner to play upon the flask below the surface of the boiling liquid. The digestion should be con- ducted in a well-ventilated hood where the air is free from any considerable amount of ammonia. In order to hasten the de- composition of organic matter, Dakin 1 and Milbauer (1. ., 1896, 29, 298. PROTEINS AND PROTEASES 315 The Xanthoproteic Reaction l Proteins are colored yellow by nitric acid of 1.2 specific gravity or stronger. The color is intensified by heating and changes to orange or red on treatment with an excess of am- monia. According to Halliburton 2 this reaction depends upon the presence of an oxybenzene nucleus. Salkowski has proposed (Z. physiol. Chem., 12, 219 ; Vaubel, I, 381) to make use of this reaction for the approximate colon- metric estimation of peptones in solution. The Millon Reaction 3 On heating with Millon's reagent 4 (a solution of mercuric nitrate containing nitrous acid) protein matter (including gelatin) gives a brick-red coloration which according to Nasse is also due to the presence of any oxybenzene nucleus in the protein molecule. For general description see Mann, p. 7, and for detailed discussions the papers of Vaubel 5 and Nasse. 6 The Tryptophan Reaction 9 ? When protein is treated first with glacial acetic acid and then with concentrated sulphuric a violet color usually appears. Hopkins and Cole 8 found that the reaction occurred only when the acetic acid contained traces of glyoxylic acid and that a better reaction is obtained by mixing the protein with a little 1 Fourcroy and Vauquelin : Ann. Chim., 56, 37. Fiirth : Einwirkung von Sal- petersaure auf Eiweisstoffe, Habilitationsschrift, Straussburg, 1899. Salkowski : Z. physiol. Chem., 12, 215 (1887). Rohde, Ibid., 44, 161 (1905). 2 Schafer's Textbook of Physiology, I, 47. 3 Millon: Compt. rend., 28, 40 (1849). 4 To prepare the reagent dissolve mercury in twice its weight of nitric acid, 1.42 sp. gr., and dilute the solution obtained with three times its volume of water. According to Nasse a better method is to use a solution of mercuric acetate containing a few milligrams of sodium or potassium nitrite, the solution having been recently acidulated with acetic acid. 5 Z. angew. Chem., 1900, 1125. 6 Arch. ges. Physiol., (Pfluger), 1901, 83, 361. 7 Adamkiewicz : Arch. ges. Physiol., 9, 156 (1874) and Ber., 8, 161 (1875). 8 Proc. Royal Soc., 1901, 68, 21 ; J. Physiol., 27, 418 ; 29, 451. 316 METHODS OF ORGANIC ANALYSIS glyoxylic acid solution l and afterward adding concentrated sulphuric acid. The color then appears at the line of contact of the liquids. This reaction, which is due to the presence of the tryptophan group in the protein molecule, is not given by gelatin. For other color reactions due to tryptophan, see Cole : J. Physiol., 1903, 30, 311. Several other more or less characteristic color reactions of proteins may be found by consulting the references given at the end of the chapter. PROTEIN PRECIPITANTS Heat coagulation and salting out processes have already been referred to in the general description of the proteins and have been made use of in classification. Zinc sulphate is most used for salting-out in analytical work where the amount of protein precipitated is to be found by determining nitrogen in the precipitate. Heavy metals have been considerably used as precipitants. Among these may be mentioned iron as chloride or acetate, copper as sulphate, acetate, or hydroxide, lead as neutral or basic acetate, mercury as chloride or as mercury-potassium-iodide, uranium as acetate. Halogens form insoluble or sparingly soluble compounds with proteins, and bromine especially has been used as precipitant in analytical work. Among the acids which are good precipitants for both proteins and alkaloids are phosphotungstic and phosphomolybdic acids, tannic acid, picric and picrolonic acids, trichlor-acetic acid. Zinc sulphate? In general on saturating a solution with zinc sulphate all proteins present except peptones are precipitated. This precipitation may therefore be used to separate peptones 1 Prepared as follows : Place a saturated solution of oxalic acid in a tall cylinder, add lumps of sodium amalgam (about 60 grams per liter of solution), allow to stand as long as hydrogen is evolved, then filter and dilute the solution with twice its volume of water. 2 Bonier: Z. anal. Chem., 1895, 34, 562. Baumann and Bonier : Nahr.Zen- ussm., 1898, 1, 106. Zunz : Z. physiol. Chem., 1899, 27, 217. Van Slyke and Hart : Am. Chem. J., 1903, 29, 150. PROTEINS AND PROTEASES 317 from other proteins. It is especially employed in the analysis of mixtures of proteins in connection with artificial digestion experiments. Here the proteins other than proteoses and pep- tones can usually be removed in other ways (for instance by neutralizing and heating the solution), the proteoses separated by saturating the solution with zinc sulphate, and the peptones determined in the filtrate. Ferric acetate is recommended by Allen 1 as an efficient pre- cipitant. A neutral solution of the reagent is added in excess and the liquid rapidly boiled, when all protein will be precipi- tated and can be determined from the amount of nitrogen found in the precipitate by the Kjeldahl method. Copper 2 is probably more generally used than any of the other heavy metals for the analytical precipitation of proteins, espe- cially for the separation of protein from non-protein nitrogen in vegetable products. Moist cupric hydroxide (often called Stutzer's reagent) is most commonly used. This method has been adopted by the Association of Official Agricultural Chemists in the following form : Separation of Proteins from Amids, etc. Preparation of Reagent. Dissolve 100 grams of pure cupric sulphate in 5 liters of water, add 25 cc. of glycerol, and then a dilute solution of sodium hydroxide until the liquid is alkaline; filter; rub the precipitate up with water containing 5 cc. of glycerol per liter, and wash by decantation or filtration until the washings are no longer alkaline. Rub the precipitate up again in a mortar with water containing 10 per cent of glycerol, thus preparing a uniform gelatinous mass that can be measured out with a pipette. Determine the quantity of cupric hy- droxide per cubic centimeter of this mixture. .Determination. Place 0.7 gram of the substance in a beaker, add 100 cc. of water, heat to boiling, or, in the case of sub- 1 Commercial Organic Analysis, IV (2), 38 (1898); and Vaubel: Bestimmung organischer Verbindungen, I, 227 (1902). 2 Stutzer, J. Landw., 1880, 28, 103 ; 1881, 29, 473 ; Z. anal Chem., 1895, 34, 568. Mallet, U. S. Dept. Agriculture, Bur. Chem., Bui. 54. Fraps and Bizzell, J. Am. Chem. Soc., 22, 709. 318 METHODS OF ORGANIC ANALYSIS stances rich in starch, heat on the water bath ten minutes; add a quantity of cupric hydroxide mixture containing about 0.5 gram of the hydroxide, stir thoroughly, filter when cold, wash with cold water, and, without removing the precipitate from the filter, determine nitrogen by the Kjeldahl method, adding sufficient potassium sulphide solution to completely precipitate all copper and mercury. The filter papers used must be practi- cally free from nitrogen. If the substance examined consists of seed of any kind, or residues of seeds, such as oil cake or anything else rich in alkaline phosphates, add a few cubic centimeters of a concentrated solution of alum just before adding the cupric hydroxide, and mix well by stirring. This serves to decom- pose the alkaline phosphates. If this be not done, cupric phos- phate and free alkali may be formed, and the protein copper precipitate may be partially dissolved in the alkaline liquid. This method has been considerably criticized, and to some extent replaced by precipitation with other reagents, especially phosphotungstic acid, but Fraps and Bizzell consider it still the best method available for vegetable materials. According to Schjerning's work it is likely to precipitate a considerable part of any purin body which may be present. Lead l is an effective precipitant of proteins as shown by its successful use as a " clarifier " in preparing liquids containing sugar for polariscopic examination, for all proteins are optically active, and if not completely removed from the solution, would vitiate the polariscopic determination of sugar. Mercuric chloride 2 precipitates the proteins, including pep- tones, but is also liable to give precipitates with alkaloids and even with ammonium salts. Uranium acetate precipitates proteins, and according to Schjerning's experiments 3 does not precipitate the alkaloids or simpler nitrogen compounds likely to be met in analytical work, with the exception of piperazine. 1 Hofmeister: Z. physiol. Chem., 1878, 2, 288. 2 Kuhne : Z. Biol., 1885, 22, 423. Neumeister : Z. Biol., 1890, 26, 234. Sieg- fried: Z. physiol. Chem., 1902,35, 164. 8 Summarized by Vaubel : Bestimmung organischer Verbindungen, I, 224- 232. PROTEINS AND PROTEASES 319 According to Schultz and Barbieri as quoted by Allen, the greatest source of error in the quantitative separation of pro- teins from simpler nitrogen compounds by means of reagents such as the above lies in the precipitation of more or less of the other nitrogen compounds along with the proteins. Hence they recommend that parallel precipitations be made with several different reagents. If then all the nitrates be proven free from protein by testing with acetic acid and potassium ferrocyanide, and the nitrogen* be determined in each precipitate, the lowest result found is presumably the most nearly correct. Bromine precipitation is sometimes employed for separation of proteins from simpler nitrogen compounds, such as the " ex- tractives " of meat. The following example is from the meth- ods of the Association of Official Agricultural Chemists: Separation of Proteoses, Peptones, and Gelatin from Extractives This is a combination of Bomer's 1 method with that of Allen and Searle, 2 as modified by Wiley. 3 Evaporate the filtrate from 'the globulins to small volume, add 2 or 3 drops of (1 : 3) sulphuric acid, and saturate with pow- dered zinc sulphate. The excess of zinc sulphate added should not be large, as otherwise serious " bumping " is likely to ensue. About 80 grams of the salt are required for each 50 cc. of liquid. Allow the coagulated proteins to settle, filter, and wash with a saturated solution of zinc sulphate. Acidulate the filtrate from the zinc sulphate precipitate with 2 or 3 drops of strong hydrochloric acid, dilute with an equal volume of water, add about 2 cc. of liquid bromine, and shake the contents of the flask vigorously. (This can be most con- veniently done in a Kjeldahl flask.) If the bromine be all taken up, add more until about 0.5 cc. of liquid bromine is left undis- solved and the supernatant liquid thoroughly saturated. Allow the mixture to stand over night, decant the supernatant liquid through a filter paper, and wash with water, so directing the jet that the globule of bromine is stirred up and saturates the wash 1 Z. anal. Chem., 1895, 5, 562. 2 Analyst, 1897, 22, 258-263. 3 U. S. Dept. Agriculture, Bur. Chem., Bui. 54. 320 METHODS OF ORGANIC ANALYSIS water. Return the filter paper and precipitate to the flask, add the zinc sulphate precipitate and filter paper containing it, and determine the nitrogen. The percentage of nitrogen so found, multiplied by 6.25, gives the percentage of proteoses, peptones, and gelatin, including gelatin peptone. Alkaloids, if present, are likely to be more or less completely precipitated by bromine along with the protein matter. Phosphotungstic acid is an efficient precipitant of the proteins other than peptones. At ordinary temperatures simpler nitro- gen compounds are also thrown down, but according to Mallet (Bui. 54, Div. Chem.) a satisfactory separation of ordinary proteins from the usual nitrogenous extractives, such as occur in meat extract, may be effected by precipitating at 90 and washing with water at the same temperature. Fraps and Biz- zell (J. Am. Chem. Soc., 22, 709) question the completeness of precipitation of proteins at temperatures above 60. Alkaloids if present would be likely to be precipitated about as com- pletely as the proteins. Stutzer (Z. anal. Chem., 1895, 34, 568; 1896, 35, 493) and also Bondzynski (Landw. Jahrb. der Schweiz, 1894) used phos- photungstic acid as precipitant to separate proteins, including peptones, from amino-compounds. Van Slyke and Hart (Bui. 215, New York Agricultural Experiment Station, Geneva) con- sider that precipitation with this reagent in the cold gives a satisfactory separation of the proteins of cheese from mono- amino compounds, but is liable to precipitate di-amino-com- pounds if these are present, arginin giving a precipitate with phosphotungstic acid which is soluble when hot, but separates out on cooling, while the lysin, histidin, and putrescine precip- itates failed to redissolve completely even on boiling. In using phosphotungstic acid for the determination of pro- tein nitrogen in meat extract according to the suggestion of Mallet, the solution is first acidulated with acetic acid and boiled, filtered if necessary, and then treated with a slight ex- cess of a solution of phosphotungstic acid in 2.5 per cent hydro- chloric acid, heated to 90, filtered hot, and the precipitate washed with water at about the same temperature. The re- PROTEINS AND PROTEASES 321 suits thus obtained are usually slightly higher and somewhat more uniform than those obtained by precipitation with bromine. Tannin l precipitates proteins, including peptones, from solu- tions acidulated with acetic acid, the precipitation being favored by the presence of sodium chloride. According to Allen and also Halliburton, this precipitation, even in the case of pep- tones, is complete. Van Slyke and Hart, however, found that tannin precipitated much less of the nitrogen compounds formed by peptic digestion of cheese than was precipitated by phosphotungstic acid ; and after investigation they attributed this discrepancy mainly to incomplete precipitation of peptones by tannin. They concluded that phosphotungstic acid should be used to separate peptones from amino bodies when the amount of the latter is relatively small or when they consist largely of mono-amino-compounds ; while tannin should be pre- ferred as a precipitant in those cases in which the amino-acids are present in relatively large amount as compared with pep- tones or when considerable amounts of di-amino-compounds are present. Bigelow and Cook (J". Am. Chem. Soc., 1906, 28, 1485) as the result of a detailed study recommend, for the separation of proteoses and peptones from the amino bodies, the use of liberal amounts of tannin and salt at a temperature of approximately 12 C. Their method is essentially as follows : Dissolve 1 gram of the sample if a powder, 2 grams if a paste, or 10 grams if a concentrated solution, in a little cold water. in a 100-cc. flask, keeping the volume within 20 cc. [Or measure into the flask 20 cc. of the sample if a dilute solution.] Add 50 cc. of a solution containing 15 grams of sodium chlo- ride, shake well, cool to approximately 12, add 30 cc. of a 24 per cent tannin solution at the same temperature, dilute with cold water to 100 cc., shake thoroughly and allow to stand over night in an ice box at about 12 ; then filter at the same temperature and determine nitrogen both in an aliquot part of 1 Allen : Commercial Organic Analysis, Vol. IY (2d. ed.), p. 19. Schjerning : Z. anal. Chem., 1900, 39, 545. 322 METHODS OF ORGANIC ANALYSIS the filtrate and in a similar portion of the nitrate from a blank in which the reagents alone have been used. The corrected nitrogen of the nitrate represents that which was present as ammonia and amino compounds except that creatin if present is partly precipitated by the tannin-salt reagent. The amount of creatin thus thrown down may be found by estimating the amounts in the original solution and in the nitrate colorimetri- cally by the method of Folin, the excess of tannin, being re- moved from the filtrate by means of barium. Picric acid precipitates proteins very completely from solu- tions acidulated with acetic acid, but as the reagent contains nitrogen its use in quantitative analytical work is limited. Gelatin is precipitated when the picric acid is in excess, and milk or cream may be tested for gelatin by removing the milk proteins with acid mercuric nitrate and adding to the clear filtrate an excess of a saturated solution of picric acid, when gelatin, if present, yields a yellow precipitate. Trichlor acetic acid appears to be a convenient reagent for the separation of proteins under certain conditions, but has not been much used in analytical work. According to Halliburton * the protein solution is mixed with an equal volume of a 10 per cent solution of trichloracetic acid, boiled and filtered hot, when the filtrate will contain the proteoses and peptones while the other proteins are precipitated. In the cold the proteoses are partially precipitated (Martin). SEPARATION OF PROTEINS FROM SIMPLER NITROGEN COM- POUNDS AND FROM EACH OTHER The separation of proteins from simpler nitrogen compounds is usually based upon the precipitation of the former by some one of the above reagents which will not precipitate the other nitrogen compounds believed to be present in the substance under examination. The choice of precipitant will therefore depend upon the nature of the substance from which the pro- 1 Schafer's Physiology, I. 40-41, where the following references are given: Obermayer, Med. Jalirb., Wien, 1888, 375; Starling, J. PhysioL, 14, 131; Martin, same, 15, 375 ; Halliburton, same, 17, 169, and J. Path, and Bact., 3, 295. See also Mendel and Blood, J. Biol. Chem., 8, 186, 189. PROTEINS AND PROTEASES 323 tein is to be separated. Cupric hydroxide used as described above has been most commonly employed for vegetable mate- rials, while precipitation with phosphotungstic acid, bromine or tannin and salt as described in the foregoing paragraphs has been more commonly used for animal substances. The methods of separating proteins from each other are based almost entirely upon differences in solubilities and coagulation or other precipitation reactions such as have been given in characterizing the groups already mentioned. As the reactions of the peptones approach those of some of the compounds of known structure, the separation of proteins from simpler com- pounds and from each other can best be studied as parts of the same problem. The methods available for these separations are so dependent upon the particular combination of com- pounds to be separated that any attempt to give detailed direc- tions would be of less value than references to the original publications, a list of which will be found at the end of this chapter. PROTEASES OR PROTEOLYTIC ENZYMES For the determination of proteolytic powers of substances containing proteases (pepsin, trypsin, papain, etc.) many differ- ent methods have been used. Some of these may be grouped as follows : 1. The enzyme acts upon an insoluble protein and the rate at which the latter is digested into soluble products is observed (methods of Bidder and Schmidt, Griinhagen, Grutzner, Mett, Palladin, U. S. Pharmacopoeia). 2. The enzyme acts upon a solution or suspension of protein and the time required to carry the digestion to a definite stage, or the amount of protein remaining undigested at the end of a definite time, is determined (methods of Allen, Einhorn, Fuld and Levison, Gross, Jacoby-Solms, Robertson, Rose, Thomas and Weber, U. S. Pharmacopoeia, Witte). 3. The enzyme acts upon protein or polypeptid, and the cleavage products are determined by chemical or physical meth- ods (methods of Abderhalden, Allen, Hedin, Kober, Koelker, Levene and Rouiller, Schiff, Schiitz, Sorenson, Volhard). 324 METHODS OF ORGANIC ANALYSIS 4. The enzyme is allowed to act on a protein solution, and the progress of the digestion is measured by increase of electri- cal conductivity or decrease of turbidity or viscosity of the solu- tion (methods of Bayliss, Hata, Liebermann, Spriggs). Since the newer methods, such as the copper method of Kober and the optical method of Abderhalden and Koelker, are still being developed in detail, it seems best for the purposes of this work to describe fully only a few of the older methods which are now in more general use, after which will be given a chrono- logical list of references which will guide the reader to the literature of the more recent methods. U. S. PHARMACOPOEIA METHOD FOR PEPTIC ACTIVITY Mix 9 cc. of hydrochloric acid of 10 per cent strength by weight (1.049 sp. gr. at 25 C.) with 291 cc. of distilled water and dissolve 0.100 gram of the pepsin to be tested in 150 cc. of the acid liquid. Immerse a hen's egg, which should be fresh, during 15 minutes in boiling water ; remove the pellicle and all of the yolk ; rub the white coagulated albumin through a clean No. 40 sieve. Reject the first portion that passes through the sieve, and place 10 grams of the succeeding portion in a wide- mouthed bottle of 100 cc. capacity. Add 20 cc. of the acid liquid and, with the aid of a glass rod tipped with cork or black rubber tubing, completely disintegrate the albumin ; then rinse the rod with 15 cc. more of the acid liquid and add 5 cc. of the solution of pepsin. Cork the bottle securely, invert it three times, and place it in a water bath that has previously been regulated to maintain a temperature of 52 C. Keep at this temperature for 2^ hours, shaking every 10 minutes by invert- ing the bottle once. Then remove it from the water bath, add 50 cc. of cold distilled water, transfer the mixture to a narrow graduated cylinder, and allow it to stand for half an hour. The deposit of undissolved albumin should not then measure more than 1 cc. The relative proteolytic power of pepsin stronger or weaker than that just described may be determined by ascertaining through repeated trials the quantity of the above pepsin solu- PROTEINS AND PROTEASES 325 tion required to digest, under the prescribed conditions, 10 grams of boiled and disintegrated egg albumin. Divide 15,000 by this quantity expressed in cubic centimeters to ascertain how many parts of egg albumin one part of pepsin will digest. U. S. PHARMACOPOEIA TEST FOR TRYPTIC ACTIVITY OF PANCREATIN If 0.28 gram of pancreatin and 1.5 grams of sodium bicar- bonate be added to 100 cc. of tepid water contained in a flask, and if 400 cc. of fresh cows' milk, which has been previously heated to 38 C., be then added, and the temperature of the mixture maintained at this point for 30 minutes, the milk should be so completely peptonized that, if a small portion of it be transferred to a test tube and mixed with some nitric acid, no coagulation should occur. If it be desired to compare the powers of two preparations, they may be tested side by side as above described and portions of each withdrawn and acidulated at frequent intervals. The rapidity with which the end point is reached will then give an indication of the comparative proteolytic power. METT METHOD This method consists in allowing a solution of the proteolytic enzyme to act upon the ends of a column of coagulated egg albumin contained in a narrow glass tube and observing the rate at which the column is shortened by the digesting of the coagulum. While this cannot be considered a strictly quanti- tative method, it has obvious advantages as a means of demon- strating the presence or absence of proteolytic enzyme or any pronounced difference in proteolytic power between substances which it is desired to compare. For this method obtain the white of a fresh egg, cut it thor- oughly with scissors or stir it with an equal volume of water, and filter or strain through muslin or cheesecloth. The albumin thus prepared should be homogeneous, nearly clear, and entirely free from air bubbles. Fill some clean, dry capillary glass tubes of 1 to 2 mm. diameter with the prepared albumin. 326 METHODS OF ORGANIC ANALYSIS This may be done either by drawing up the fluid into the tube as into a pipette or by lowering the tube into a column of the fluid. Great care must be taken to avoid air bubbles in filling the tubes. In order to coagulate uniformly the contents of a capillary tube, hold it (after filling with albumin) in the same manner as a pipette and with the finger over the upper end touch the lower end to the surface of the boiling water in a water bath until coagulation begins, then lay the tube hori- zontally in the boiling water ; after 15 minutes immersion in boiling water, allow the tube of coagulated albumin to cool slowly, preferably under water, then remove and if the tube is not to be used at once, seal the ends by means of sealing wax or paraffin. For use cut the tube of coagulated albumin into sections about 2 cm. in length (being careful to scratch the tube at right angles to its axis and to break it with all possible care to secure clean-cut square ends). Reject the end sections and any which contain air bubbles or in which the albumin has shrunken and does not completely fill the capillary. Immerse a perfect section of " Mett tube " thus prepared and selected in a measured volume of the liquid to be tested, with " blanks " alongside in which all of the conditions are the same except that the enzyme is omitted, and note the length of column digested out of the tube after standing 10 hours at 38 C. or for such time and at such temperature as may best meet the requirements of the particular case. When the test is applied to a liquid (e.g. to gastric juice) an equal volume of the same liquid, boiled to destroy the enzyme, may be used for the blank test. In testing a solid, it should be dissolved in an acid, alkaline, or neutral medium according to the nature of the enzyme sup- posed to be present. For pepsin or trypsin the acidity or alka- linity prescribed respectively in the above methods from the U. S. Pharmacopoeia may be used, and comparisons may be made with a sample of pepsin or trypsin whose power is known to approximate the requirement of the Pharmacopoeia test. See also the methods of expressing proteolytic power given by Rose and others in the journal articles cited below. PROTEINS AND PROTEASES 327 ALLEN METHOD MODIFIED Dissolve 1 gram of dry powdered egg albumin in 20 cc. of lukewarm water in a 100-cc. flask, heat in a boiling water bath to coagulate the albumin (Note 1), and cool to 40 C., add 0.1 gram of the sample of pepsin to be tested and 25 cc. of tenth- normal hydrochloric acid (Note 2) ; warm to 40 C., and main- tain at this temperature for 3 hours (Note 3) ; then add a volume of tenth-normal sodium carbonate solution exactly equivalent to the acid previously added (Note 4), mix thor- oughly by shaking, and place the flask in a water bath at 90 C. for 10 minutes to destroy the enzyme and coagulate any meta- protein (syntonin) which may have been dissolved by the acid and liberated when the latter was neutralized by the alkali. Cool, dilute with water to 100 cc., and filter through a dry paper, protecting the solution from unnecessary exposure to air so that it shall not change by evaporation. This filtrate (A) contains the proteoses, peptones, peptids and ammo-acids which have been formed by digestive hydrolysis of the protein plus what existed in the pepsin added. Measure 25 cc. of filtrate A into a Kjeldahl flask and determine total nitrogen. To 50 cc. of filtrate A (representing one half of the solution), add powdered zinc sulphate in the cold with thorough stirring until the solution is saturated, allow to stand with occasional stirring for half an hour, filter through pure filter paper, and wash the precipitate with a saturated solution of zinc sulphate. Trans- fer the precipitate with the paper to a Kjeldahl flask and deter- mine nitrogen as a measure of the proteoses ; acidulate the filtrate (B) with hydrochloric acid and precipitate with bro- mine as described above under determination of proteins by bromine precipitation. The nitrogen of the bromine precipi- tate serves as a measure of the amount of peptones ; the filtrate from the bromine precipitate (filtrate C) contains the ammo- acids and any peptids not precipitated by bromine. The amount of nitrogen in these forms may be found by difference. From the total nitrogen of proteoses, peptones, peptids, and amino-acids as found from an aliquot portion of filtrate A 328 METHODS OF ORGANIC ANALYSIS above, subtract the nitrogen of proteoses (zinc sulphate pre- cipitate) and of peptones (bromine precipitate) ; the remainder is the nitrogen of peptids and amino-acids. A blank* test should be conducted, using a boiled solution of the pepsin, but in all other respects exactly as above described. Such a blank test will supply the combined corrections for the nitrogen of the pepsin and for the cleavage products which may possibly be produced by the action of the hydrochloric acid on the coagulated albumin. For data illustrating the application of this method see Allen's Commercial Organic Analysis, Volume IV (2d ed.), pp. 357- 358. Note 1. The coagulated albumin should be in the form of finely divided flocks so as to be readily accessible to the enzyme. So far as the action of the enzyme on the albumin is concerned, it would seem better to leave the latter uncoagulated ; but when this is done there may be difficulty in filtration at the end of the digestion period. Note 2. The addition of acid at this point is intended to give the reaction most favorable for the pepsin activity. If the enzyme under test works best in a neutral solution, the acid may be omitted ; or in the case of trypsin, sodium carbonate may be added at this point to give the favorable degree of alkalinity. Note 3. While three hours is a suitable time for digestion in the case of commercial pepsin such as Allen was engaged in testing, it is obvious that a longer or a shorter period of diges- tion maybe adopted when working with substances of much lower or much higher proteolytic power. Note 4. Compare Note 2. If no acid was added at the be- ginning of the digestion, no alkali should be added here. If al- kali was added at the beginning of the digestion, an equivalent amount of acid must be added at the end. The object here is to restore the neutrality of the solution so that the subsequent heating and filtration will remove any dissolved but undigested protein along with the undissolved coagulated albumin. PKOTEINS AND PROTEASES 329 OTHER METHODS As indicated above, many other methods, some of them very promising, have been proposed for the measurement of proteo- lytic power. In research work especially, the various methods available should be carefully studied with a view to the selection of the one best adapted to the particular problem in hand. The references given at the end of this chapter will put the reader in touch with the literature of the subject. REFERENCES ABPERHALDEN: Biochemisches Handlexicon. : Handbuch der Biochemischen Arbeitsmethoden. : Lehrbuch der Physiologische Chemie. ALLEN : Commercial Organic Analysis. COHNHEIM : Chemie der Eiweisskorper. FISCHER : Untersuchungen iiber Aminosauren, Polypeptide, und Proteine. FRANKEL : Descriptive Biochemie. HAMMARSTEN : Physiological Chemistry (translated by Mandel). HAWK : Practical Physiological Chemistry. KONIG : Chemie der Menschliche Nahrungs- und Genussmittel. MANN : Chemistry of the Proteids. NEUBERG : Der Harn und Korperflussigkeiten. OPPENHEIMER : Die Fermente und ihre Wirkung. : Handbuch der Biochemie. OSBORNE : Proteins of the Wheat Kernel. : The Vegetable Proteins. PLIMMER : The Chemical Constitution of the Proteins. SCHAEFER : Text-book of Physiology. SHERMAN : Chemistry of Food and Nutrition. II On Proteins (See also the references given in the text) 1880-86. STUTZER: Untersuchungen ueber die quantitative Bestimmung des Proteinstickstoffes und die Trennung der Proteinstoffe von anderen in Pflanzen vorkommenden StickstofrVerbindungen. Journ.f. Landwirthschaft, 28, 103; 29, 473; 34, 151. 330 METHODS OF ORGANIC ANALYSIS SCHULZE und BARBIERI : Zur Bestimmung des Eiweisstoffe und der nichteiweissartigen Stickstoffverbiudungen in der Pflanzen. Landw. Vers. Stat., 26, 218. 1896. TELLER: The Quantitative Separation of Wheat Proteids. Bui. 42, Ark. Agl. Expt, Sta., p. 81. 1897. ALLEN and SEARLE : Improved Method of determining Proteid and Gelatinoid Substances. Analyst, 22, 258. 1898. BAUMANN UND BOMER : Ueber die Fallung der Albumosen durch Zinksulfat. Z. Nahr.-Genussm., 1, 106. 1899. MALLET : Analytical Methods for distinguishing between the Nitro- gen of Proteids and that of the Simpler Amids or Amino-acids. Bui. 54, Div. Chem., U. S. Dept. Agriculture ; Chem. News, 80, 117, 168, 179. VIVIAN : A Comparison of Reagents for Milk Proteids. 16th Ann. llpt. Wis. Agricl. Expt. Sta., p. 179. . 1900. BARNSTEIN : Ueber eine Modifikation des von Ritthausen vorge- schlagenen Verfahrens der Eiweissbestimmung. Landw. Vers. Stat., 54, 327; Z. Nahr.-Genussm., 1901, 4, 688. 1894-1901. SCHJERNING : [A series of papers on the quantitative separation and precipitation of proteins] . Z. anal. Chem., 33, 263 ; 34, 135; 35, 285; 36, 643; 37, 73, 413; 39, 545, 633. 1900. TRAPS and BIZZELL: Metlwds of determining Protein Nitrogen in Vegetable Matter. J. Am. Chem. Soc., 22, 709. 1901. HART : Ueber die Quantitative Bestimmung der Spaltungsprodukte von Eiweisskorpern. Z. physiol. Chem., 33, 347. 1903. TEBB : The Precipitation of Proteins by Alcohol and Certain Other Reagents. J. Physiol., 30, 25. VAN SLYKE and HART : Methods for the Estimation of the Proteo- lytic Compounds, contained in Cheese and Milk. Bui. 215, New York Agl. Expt. Sta. ; Am. Chem. J., 29, 150. BIGELOW : Meat and Meat Products, U. S. Dept. Agriculture, Bur. Chem., Bui. 65, pp. 10, 17; Bui. 13, Part 10, p. 1396; Bui. 81, p. 104. 1904. GRINDLEY : A Study of the Nitrogenous Constituents of Meats, U. S. Dept. Agriculture, Bur. Chem., Bui. 81, p. 110; J. Am. Chem. Soc., 26, 1086. HASLAM : Separation of Proteins. J. Physiol., 32, 267. SNYDER : The Determination of Gliadin in Wheat Flour by means of the Polariscope. J. Am. Chem. Soc., 26, 263. 1905. CHAMBERLAIN: Determination of Gliadin and Glutenin in Flour. U. S. Dept. Agriculture, Bur. Chem., Bui. 81, p. 118; Circular 20, p. 14. GRINDLEY and EMMETT : The Chemistry of Flesh. J. Am. Chem. Soc., 27, 658. PROTEINS AND PROTEASES 331 1906. BIGELOW and COOK : The Separation of Proteoses and Peptones from the Simpler Amino Bodies. J. Am. Chem. Soc., 28, 1485. CHAMBERLAIN : Investigations on Properties of Wheat Proteins. .7. Am. Chem. Soc., 28, 1657. MATHEWSON : The Optical Rotation of Gliadin in Certain Organic Solvents. J. Am. Chem. Soc., 28, 1482. 1907. AGREE : A Formaldehyde Color Test for Proteins. Am. Chem. J., 37, 604. COOK and TRESCOTT : A Modification of the Tannin-Salt Method. J. Am. Chem. Soc., 29, 605. 1908. BARDACH : A New Protein Reaction. Z. physiol. Chem., 54, 355. LIEBERMANN : (Formaldehyde Color Reaction for Protein). Z. Nahr.-Genussm., 16, 231. SEAMAN and GIES: An Examination of Bardach's New Protein Test. Proc. Soc. Exp. Biol. Med., 5, 125; Chem. Abs., 2, 2829. 1910. OSBORNE: Die Pflanzenproteine. Ergebnisse der Physiologic, 10, 47-215. WEYL : (Precipitation of proteins and some amino-acids by acetone). Z. physiol. Chem., 65, 246. Also Ber., 43, 508. 1911. MICRO : (Examination of Meat Juices). Z. Nahr.-Genussm., 20, 537. OSBORNE and GUEST: (Analysis of the Products of Hydrolysis of Proteins). /. Biol. Chem., 9, 333, 425. VAN SLYKE: The Analysis of Proteins by Determination of the Chemical Groups Characteristic of the Diiferent Amino-acids. /. Biol. Chem., 10, 15. On Proteases 1881. ROBERTS : Estimation of the Amylolytic and Proteolytic Activity of Pancreatic Extracts. Proc. Royal Soc., 32, 145. 1897. ALLEN: (Valuation of Pepsin). Pharm. J., 1897, 561; Z. anal. Chem., 42, 466. 1901. KRUGER : Quantitative Observations on Pepsin -Action. Z. Biol., 1901, 41, 467; J. Chem. Soc., 1902, 82, ii, 33. SAMOJLOFF : Mett's Method of estimating Peptic Activity. Arch. ges. Physiol. (Pfluger}, 85, 86; J. Chem. Soc., 1901, 80, ii, 401. THOMAS and WEBER : Quantitative Determination of Proteolytic Power. Centrbl. f. Stoffwechselversuche und Verdauungskrank- heiten, 901, 2, 365; Z. Nahr.-Genussm., 1902, 5, 723. 1902. PECKELHARING : On Pepsin. Z. physiol. Chem., 35, 8. SPRIGGS : New Method of Determining Pepsin Activity. Z. physiol. Chem., 35, 465. 1903. WINOGRADOW: Quantitative Experiments with Peptic Digestion. Z. Nahr.-Genussm., 6, 589. 332 METHODS OF ORGANIC ANALYSIS 1905. COBB : Contribution to our Knowledge of the Action of Pepsin, with Special Reference to its Quantitative Estimation. Am. J. Physiol., 13, 448. LOEHLEIN : Volhard's Titrimetric Method for Estimation of Pepsin and Trypsin. Beitr. chem. Physiol Path., 7, 120. 1906. LEVENE and ROUILLER : Estimation of Tryptophan in Protein Cleavage Products. J. Biol. Chem., 2, 481. ROBERTSON : (Hydrolysis of Casein by Trypsin). J. Biol. Chem., 2 t 318. 1907. ABDERHALDEN and KOELKER : Employment of Optically Active Poly- peptids as Proof of the Activity of Proteolytic Enzymes. Z. physiol. Chem.., 51, 294. KUTTNER : The Volhard Method for Estimation of Pepsin. Z. physiol. Chem., 52, 63. 1908. ARRHENIUS : Law of Schiitz and Reaction Velocities. Medd. Vetens- kapsakademiems Nobelinst., 1, No. 9 ; Chem. Abs., 2, 2589. EINHORN: Simplification of Jacoby-Solm Ricin Method of Pepsin Estimation. Berlin, klin. Woch., 45, 1567. FULD and LEVISON : Determination of Pepsin by Means of Edestin Test. Biochem. Z., 6, 473. GOODMAN: Ricin Method of Jacoby-Solms for the Quantitative Estimation of Pepsin. Am. J. Med. Sci., 136, 734. GROSS : Activity of Trypsin and a Simple Method for its Estimation. Arch. exp. Path. Pharm., 58, 157; Chem. Abs., 2, 1570. MEYER: Is the Schiitz Law for Peptic Digestion Invalid? Berlin. klin. Woch., 45, 1485. SORENSEN : (Estimation of Proteolytic Power by Titration of Amino- acids). Biochem. Z., 7, 45; Chem. Abs., 2, 1288. WITTE : (Modification of Jacoby-Solms Method). Berlin, klin. Woch., 1908, p. 643. 1909. BERG : Comparative Study of Digestibility of Different Proteins in Pepsin-Hydrochloric-Acid Solutions. Am. J. Physiol., 23, 420. DEZANI : Contribution to the Study of Pepsin. Atti della R. Accad. della Scienze di Torino, 45, 225. LIEBERMANN: New Method for Clinical Determination of Pepsin. Med. Klin., 1909, 1784. 1910. BLOOD : The Erepsin of the Cabbage (Brassica oleracea.) J. Biol. Chem., 8, 215. FRANK : Digestibility of White of Egg as Influenced by the Tempera- ture at which it is Coagulated. J. Biol. Chem., 9, 463. HAT A : Estimation of Pepsin by the Clearing of Turbid Solutions of Egg Albumin. Biochem. Z., 23, 179. KOELKER: The Study of Enzymes by Means of Synthetical Poly- peptids. J. Biol. Chem., 8, 145. PROTEINS AND PROTEASES 333 MENDEL and BLOOD : Some Peculiarities of the Proteolytic Activity of Papain. J. Biol. Chem., 8, 177. PALLADIN: (Trypsin Activity and its Determination). Arch. ges. PhysioL (Pftiiger), 134, 337; Chem. Abs., 5, 1615. ROSE : Modified Method for Clinical Estimation of Pepsin. Arch. Intern. Med., 5, 459; Chem. Abs., 4, 1980. 1911. ABDERHALDEN and STEINBECK : Study of the Action of Pepsin. Z. physiol. Chem., 68, 293. AMBERG and JONES : On the Application of the Optical Method to a Study of the Enzymatic Decomposition of the Nucleic Acids. J. Biol. Chem., 10, 81. GRABER : Some Observations upon the Assay of Digestive Ferments. /. Ind. Eng. Chem., 3, 919. KOBER : A Method for the Study of Proteolytic Ferments. J. Biol. Chem., 10, 9. RAMSAY: Method of Determining the Tryptic Value of Pancreatin. J. Ind. Eng. Chem., 3, 822. CHAPTER XVI Grain Products IN the routine analysis of vegetable foods and feeding-stuffs it is customary to determine moisture, fat, protein, fiber, and ash and to estimate the remaining substances as " carbohydrates by difference" or as "nitrogen-free extract." Often the separate determination of fiber is omitted, and this, as well as the sugars, starches, pentosans, etc., is included in the "carbohydrates by difference." The present chapter will include the methods for such deter- minations, which are of fairly general application in food anal- ysis, and also some special methods for the examination of grain products in particular. PREPARATION OF SAMPLES Samples for analysis should, if moist, be weighed into large flat-bottom dishes, dried until brittle (though not necessarily quite to constant weight) at a temperature not above that of the water oven, then allowed to stand exposed to the air until they neither gain or lose in weight, and the weight of the air-dry material recorded in percentage of the weight of the fresh sub- stance. The air-dry material is then ground in a coffee or drug mill l until all will pass through a sieve of one-half-milli- meter mesh, or, if this is not feasible, through a sieve having round holes one millimeter in diameter. Fineness of grinding is important, not only to secure suffi- ciently accurate portions for the separate determinations, but also to permit of efficient extraction in the determinations described below. 1 In laboratories where many such samples are to be ground a special power mill is sometimes provided. 334 GRAIN PRODUCTS 335 DETERMINATION OF MOISTURE AND FAT Dry 2 grams for 5 hours, or to constant weight, at the tem- perature of boiling water, if possible in a current of dry hydro- gen or in vacuo. Consider the loss of weight as moisture. Extract the dried sample in a Soxhlet or continuous extractor, with anhydrous alcohol-free ether for sixteen hours. Dry the extract to constant weight in a boiling water oven. The ether extraction should be carried out at a distance from any free flame, the flask being heated by a safety water bath or, more conveniently, by an electric heater. Notes. The ether extract of vegetable substances often contains in addition to fat more or less of coloring matters and resinous substances, these being more readily soluble in ether containing fatty oils than in ether alone. In the cereal grains and especially in the milling products from which the outer layers of the grains have been separated the amount of such impurities is usually negligible. If the extract is made to percolate a layer of animal charcoal, prac- tically pure fat is obtained. 1 The reason for drying in a current of hydrogen rather than in air is that the oils of the cereal grains belong to the "semi- drying " group and therefore absorb oxygen when exposed to air, especially at high temperature. This will of course increase the weight of the fat and make the apparent percentage of moisture too low. The partially oxidized oils are also apt to be incompletely extracted by ether. For a full discus- sion of the determination of water in foods and physiological preparations, see Benedict and Manning: Am. J. Physiol., 1905, 13, 309. Fat may also fail of complete extraction, even when un- changed, by being occluded or mechanically inclosed in car- bohydrate or protein material which is impervious to the ether. Although ether extracts may be evaporated at the tempera- ture of boiling water without loss of fat, such loss has been 1 Patterson: Am. Chem. J., 12, 261. 336 METHODS OF ORGANIC ANALYSIS found to occur in drying moist samples even in a current of hydrogen. That the loss in such cases is, in part at least, due to an actual volatilization of material may be shown by passing the current of hydrogen in which the sample is dried into strong sulphuric acid. This loss is probably due to the action of the escaping steam and may be practically avoided by drying at a lower temperature, preferable in a partial vacuum. All three of the causes just mentioned tend toward a defi- ciency of fat in the analysis of cooked foods prepared from cereal products. Thus in a number of experiments on bread making l the fat found by analysis of the dried bread was less than half of that contained in the materials used, and the iodine figure of the ether extract of the bread was only 60.4 as against 101.4 in that of the original flour, showing that a very consider- able oxidation had taken place even in that portion of the fat which was still soluble in ether. Berntrop's method for the determination of fat in breadstuffs is as follows : 2 Mix 150 grams of fresh bread with 500 cc. of water, add 100 cc. of concentrated hydrochloric acid, and boil for two hours connected with a reflux condenser. 3 Cool the resulting brown liquid to room temperature, filter, with suction through a moistened' fat-free paper, and wash with cold water. Dry the paper and residue for an hour at 100 to 110, remove the residue as completely as possible from the filter paper, and grind it with sand in a mortar. Cut up and add the filter paper, and transfer the dry mixture to a paper extrac- tion thimble and treat with ether or petroleum ether in an extractor. Dormeyer's method^ designed originally for the determi- nation of fat in animal tissues, has been adapted to vege- table foods by Beger. 5 From 3 to 5 grams of substance are mixed with 480 cc. of water, 20 cc. of 25 per cent hy- 1 Bui. 67, Office of Experiment Stations, U. S. Dept. Agriculture. 2 Z. angew. Chem., 1902, 121. 8 In treating meal or flour, heat for an hour in a water bath and then boil for an hour with the reflux condenser attached. 4 Arch. ges. Physiol. (Pfluger), 1895, 61, 341 ; 1896, 65, 90. 5 Chem. Ztg., 1902, 26, 112. GRAIN PRODUCTS 337 drochloric acid, and 1 gram of fat-free pepsin. The mixture is kept at 37 to 40 for twenty-four hours, filtered with suction through a paper supported on a porcelain plate and covered with asbestos, and both the filtrate and the residue extracted with ether. DETERMINATION OF CRUDE FIBER 1 Extract 2 grams of the substance with ordinary ether, or use the residue from the determination of the ether extract. To this residue, in a 500-cc. flask, add 200 cc. of boiling 1.25 per cent sulphuric acid; connect the flask with an inverted con- denser, the tube of which passes only a short distance beyond the rubber stopper into the flask. Boil at once, and continue the boiling for 30 minutes. A blast of air conducted into the flask may serve to reduce the frothing of the liquid. Filter, wash with boiling water till the washings are no longer acid; rinse the substance back into the same flask with 200 cc. of a boiling 1.25 per cent solution of sodium hydroxide, practically free from sodium carbonate ; boil at once, and continue the boiling for 30 minutes in the same manner as directed above for the treatment with acid. Filter on a Gooch crucible, and wash with boiling water till the washings are neutral; dry at 110; weigh; 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 sulphuric acid and sodium hydroxide are to be made up of the specified strength, determined accurately by titration and not merely from specific gravity. DETERMINATION OF ASH Char about 2 grams and burn to whiteness at the lowest pos- sible red heat, preferably in a flat-bottomed platinum dish in a muffle. 1 Bui. 107, Revised, Bur. Chem., U. S. Dept. Agriculture. See also Thatcher: J. Am. Chem. Soc., 1902, 24, 1210; Browne: Ibid., 1904,25, 315. z 338 METHODS OF ORGANIC ANALYSIS If considerable quantities of phosphates are present, these may fuse over some of the carbon and render its combus- tion very slow. In such cases, extract the charred mass with a little hot acetic acid, set aside the solution till the char is burned, then evaporate it to dry ness in the same dish and heat the residue to dull redness till the ash is white or nearly so. Samples containing added salt should be ex- tracted with water before charring, and the determination finished as just described. DETERMINATION OF PROTEIN Determine total nitrogen by one of the modifications of the Kjeldahl method as described in Chapter XIV. On the assumption that proteins in general contain approxi- mately 16 per cent of nitrogen, it has been customary to multi- ply the percentage of nitrogen by 6.25 as an estimate of the percentage of protein. The extended investigations by Osborne and his associates, 1 and by Ritthausen, 2 have shown that nearly all the cereal pro- teins contain over 16 per cent of nitrogen, so that the results obtained by multiplying the nitrogen by 6.25 are too high. The factors now regarded as most nearly correct are : for wheat, rye, and barley, 5.7 3 ; for maize, oats, rice, and buckwheat, 6.00. The old factor 6.25 is, however, still fre- quently used for the sake of uniformity or for comparison with earlier work. In reporting results, therefore, the factor used should always be given. SEPARATION OF WHEAT PROTEINS Nearly all the nitrogenous material in wheat, and especially in wheat flour, is in the form of proteins. The proteins of wheat have been studied with -great thorough- 1 Reports Conn. Agl. Expt. Station, 1890 et seq. Much of the work has also appeared in J. Am. Chem. Soc., Am. Chem. J., Am. J. Physiol., or J. Biol. Chem. 2 Summarized in Landw. Vers. Stat., 1896, 47, 391. 3 The factor 5.68 has recently been proposed for wheat flour. GRAIN PRODUCTS 339 ness by Osborne, who finds five forms and estimates the amount of each in average wheat as follows: f Albumin (leucosin) 0.3-0.4 per cent Soluble in water 4 _ ^ , , . [Proteose (about) 0.3 per cent Soluble in ten per cent NaCl Globulin (edestin) . . 0.6-0.7 per cent Soluble in 70 per cent alcohol Gliadin ( about ) 4.25 per cent Insoluble in neutral solvents Glutenin 4.0-4.5 per cent In fine flour the relative amount of gliadin is higher than in the whole grain, from one half to three fifths of the total nitrogen of fine flour being usually in the form of alcohol-soluble protein. The gliadin and glutenin together make up the gluten, which to the bread maker is of greater importance than the other pro- teins of the flour. In addition to the percentage of gluten in the flour the proportion of gliadin in the gluten is important to its baking qualities. Hence the protein separations of direct importance in establish- ing the commercial value of the flour are (1) to separate the water- and salt-soluble proteins from the gluten, (2) to determine the alcohol-soluble protein or gliadin. Knowing the amounts of these and the total protein present, the amount of glutenin may be found by difference. On the basis of Osborne's studies, Chamberlain has developed the following methods for the routine determination of alcohol- soluble and salt-soluble proteins. Determination of alcohol-soluble protein {"crude gliadin" ). Treat 5 grams of the sample with 250 cc. of alcohol, 70 per cent by volume, for 24 hours, shaking every half hour during the first 8 hours; filter through a dry paper, determine nitrogen in 100 cc. of the filtrate, and multiply the result by 5.7. In making this determination of nitrogen the 100 cc. of solu- tion may be transferred to a Kjeldahl flask, 3 cc. of sulphuric acid added, and the liquid boiled down to a small volume, after which the remainder of the acid is added and the determination completed as usual. This determination will usually give a result very slightly higher than the true amount of gliadin, since any amino-acids or amids present are likely to be dissolved by the alcohol. 340 METHODS OF ORGANIC ANALYSIS Determination of salt-soluble protein. Treat 12 grams of the sample with 300 cc. of 5 per cent potassium sulphate as described under the determination of alcohol-soluble protein. Determine nitrogen in 100 cc. of the filtrate and multiply by 5.7 to estimate the salt-soluble proteins. SEPARATION OF CARBOHYDRATES IN CEREAL PRODUCTS Determination of Reducing Sugars, Sucrose, Dextrin, Starch, Pentosans, and Cellulose The following scheme 1 provides for each of the substances or groups mentioned and avoids the danger (inherent in any plan of making a number of independent determinations) of includ- ing the same substance as a constituent of more than one group. Free the sample from fat by washing with ether. Extract with boiling alcohol. Solution A. Evaporate the alcohol, dilute with water, and determine the reducing power of portions of the solution before and after hydrolysis. Calculate the reducing sugar and sucrose. Residue A. Extract with cold water. Solution B. Hydrolyze a portion and determine the result- ing dextrose, calculate dextrin and soluble starch. In another portion, precipitate soluble starch by barium hydroxide, 2 filter, and determine dextrin in the filtrate. Residue B. Boil with water and treat with malt extract or saliva, filter, and wash thoroughly. Solution O. Hydrolyze, determine resulting dextrose, and calculate starch. Residue O. Boil with 2 per cent hydrochloric or sulphuric acid until the maximum reducing power of the solution is reached. 3 Solution D. Determine reducing power in the same manner as for dextrose. Calculate as xylose, the reducing power of 1 Based on the following papers : Stone : J. Am. Chem. Soc., 1897, 19, 183. Sherman : Ibid., 1897, 19, 291. Browne and Beistle : Ibid., 1901, 23, 229. 2 Asboth: Chem. Ztg., 1889, 13, 591. 8 For sulphuric acid this was found to be 4 to 6 hours. Stone prefers hydro- chloric acid and states that the reaction is nearly complete in 1 hour. GRAIN PRODUCTS 341 which is 1.03 times that of dextrose. 1 From the pentose thus found calculate the percentage of pentosan. In the case of wheat it has been found 2 that the material (" hemicellulose ") dissolved and hydrolyzed at this point is entirely pentosan. The same is probably true of the other cereals. The pentosan thus dissolved and hydrolyzed does not include necessarily the entire furfural-yielding substance of the cereal. Residue D. Boil for 30 minutes with 1 per cent sodium hydroxide, filter and wash, press out most of the water, and ex- pose the moist fiber to chlorine gas for one hour. Wash with water, boil with a solution containing 2 per cent sodium sulphite and 0.2 per cent sodium hydroxide; filter, wash with warm water until the washings are neutral and colorless, then wash with strong alcohol, dry, and weigh. Deduct the ash which the residue contains and calculate the organic matter as cellulose. , 3 Determination of Maltose, Dextrin, and Starch in Malted Cereal The absence of any considerable amount of dextrose or invert sugar must be shown by stirring some of the sample with about ten times its weight of water and testing the fil- tered extract by means of phenylhydrazine or Barfoed's solu- tion as described in Chapter III. If 110 monosaccharide is present, the percentages of maltose, dextrin, and starch can be estimated as follows : Mix 5 grams of sample with 125 cc. of cold water 4 in a 250-cc. flask ; allow to stand at room temperature for one hour, shaking frequently ; fill to the mark, shake, and filter through dry paper. Determine reducing power of one or more 25-cc. portions of 1 Stone : Am. Chem. J., 1891, 13, 82. Since the reducing power of arabinose does not differ greatly from that of xylose, this calculation would still be -nearly correct in case both pentoses were present. 2 J. Am. Chem. Soc., 1897, 19, 294. 3 This is the method of Cross and Bevan. For a comparison of this with other methods see J. Am. Chem. Soc., 1897, 19, 304. 4 If the sample contains an active enzyme, some of the carbohydrate may be changed during this treatment with water. To prevent this a very dilute alkali solution, containing 0.02 per cent potassium hydroxide or an equivalent amount of sodium or ammonium hydroxide, may be used. Ling and Rendle ; J. Inst. Brewing, 1904, 10, 238 ; Abs. J. Chem. Soc., 1904, 86, ii, 507. 342 METHODS OF ORGANIC ANALYSIS this filtrate by either Defren's or Allihn's method and calculate the amount of maltose. 1 Measure 50 cc. of the same filtrate into a 100-cc. flask, add 5 cc. of hydrochloric acid of 1.125 sp. gr., and hydrolyze as in the determination of starch. Determine the re- sulting dextrose, deduct the amount due to maltose, and estimate the remainder as due to dextrin. Soluble starch if present would be counted as dextrin in this analysis. Treat another portion of the original sample as described under the determination of starch, but without extracting the soluble carbohydrates. From the dextrose found, subtract that yielded by maltose and dextrin, and estimate the remainder as derived from starch. The results require a slight correction on account of the pres- ence of the insoluble residue when the solution is diluted to vol- ume in the graduated flask. Although some details of the method are open to criticism, it gives results sufficiently exact for the purpose for which it is mainly used, which is to show whether the starch of the cereal has been largely changed to soluble products. The same plan may be used in the examination of cereal foods prepared by parching or in other ways, provided only one reduc- ing sugar is present in appreciable quantity. The amount of soluble carbohydrate in such preparations is usually too small for satisfactory determination by means of the polariscope. ACIDITY Acidity in flour is objectionable both as an indication of deterioration and because it acts upon the gliadin, injuring the physical properties which are especially important in bread making. To determine acidity, shake 10 grams of the dry sample with 100 cc. of cold water, filter, and titrate an aliquot part with tenth-normal sodium or potassium hydroxide, using phenolphthalein as indicator. In fine flour the acidity calcu- lated as lactic acid should not exceed 0.10 per cent. 1 If Allihn's method is used, assume the reducing power of maltose to be 0.61 that of dextrose. GRAIN PRODUCTS 343 INTERPRETATION OF RESULTS Official Definitions and Standards 1 Grain is the fully matured, clean, sound, air-dry seed of wheat, maize, rice, oats, rye, buckwheat, barley, sorghum, millet, or spelt. Meal is the sound product made by grinding grain. Flour is the fine, sound product made by bolting wheat meal and contains not more than 13. 5 per cent of moisture, not less than 1.25 per cent of nitrogen, not more than 1.0 per cent of ash, and not more than 0.50 per cent of fiber. Grraham flour is unbolted wheat meal. Grluten flour is the product made from flour by the removal of starch and contains not less than 5.6 per cent of nitrogen and not more than 10 per cent of moisture. Maize meal, corn meal, or Indian corn meal is meal made from sound maize grain and contains not more than 14 per cent of moisture, not less than 1.12 per cent of nitrogen, and not more than 1.6 per cent of ash. Rice is the hulled and polished grain of Oryza sativa. Oatmeal is meal made from hulled oats and contains not more than 8 per cent of moisture, nor more than 1.5 per cent of crude fiber, not less than 2.24 per cent of nitrogen, and not more than 2.2 per cent of ash. Rye flour is the fine sound product made by bolting ryemeal and contains not more than 13.5 per cent of moisture, not less than 1.36 per cent of nitrogen, and not more than 1.25 per cent of ash. Buckwheat flour is bolted buckwheat meal and contains not more than 12 per cent of moisture, not less than 1.28 per cent of nitrogen, and not more than 1.75 per cent of ash. Where percentages are given in these standards they are, of course, intended to represent normal limits rather than averages or extreme limits. 1 Circular No. 19, Office of the Secretary, U. S. Dept. Agriculture. 344 METHODS OF ORGANIC ANALYSIS Composition of Entire Grains Wiley 1 estimates the approximate composition of average typical American grains as follows : TABLE 25. PERCENTAGE COMPOSITION OF ENTIRE GRAINS (WILEY) Barley Buck- wheat Maize Oats Rice unhulled Eye Wheat Moisture 10.85 12.00 10.75 10.00 10.50 10.50 10.60 Protein (Nitrogen x 6.25) Fat (Ether extract) . . Crude fiber 11.00 2.25 3.85 10.75 2.00 10.75 10.00 4.25 1.75 12.00 4.50 12.00 7.50 1.60 9.00 12.25 1.50 2.10 12.25 1.75 2.40 Ash 2.50 1.75 1.50 3.50 4.00 1.90 1.75 Carbohydrates (diff.) . . 69.45 62.75 71.75 58.00 67.40 71.75 71.25 Composition of Mitt Products An extended study of the mill products of wheat, made by Teller at the Arkansas Experiment Station, 1894 to 1898, 2 in- cluded a milling experiment in which the principal products of a long process (7 break) roller mill were analyzed with the following results : TABLE 26. PERCENTAGE COMPOSITION OF MILL PRODUCTS OF WHEAT (TELLER) Patent Flour Straight Flour Low Grade Flour Ship Stuff Bran Whole Wheat Pure Germ Moisture 13.75 1390 1322 12 5 1285 1390 680 Ash . 33 47 90 3 12 5 80 2 15 4.65 Crude fiber 17 26 74 3 55 6 14 2 17 1.60 Fat . . . 1 05 125 1 70 4 80 5 2 15 1438 Protein (Nitrogen x 5.7) Carbohydrates (diff.) . . Total Nitrogen .... Protein Nitrogen . . . Amid Nitrogen .... 9.69 75.01 1.70 1.65 .05 10.37 73.75 1.82 1.72 .10 12.88 70.56 2.26 2.20 .06 16.36 59.02 2.87 2.68 .19 15.56 54.45 2.73 2.51 .22 12.31 63.32 2.16 1.98 .18 36.00 36.55 6.34 3 !Bul. 45, Bur. Chem., U. S. Dept. Agriculture. 2 Buls. 42 and 53, Ark. Expt. Station (Fayetteville, Ark.). 3 The germ is richer in amid nitrogen than other parts of the wheat kernel. GRAIN PRODUCTS 345 Under the system of milling now practiced in the Northwest a number of " streams " of flour are produced which are after- ward united in different proportions to form the market grades of flour. Snyder has recently published 1 the following analyses of the different " streams " as obtained in milling No. 1 North- ern wheat by typical modern machinery. TABLE 27. SXYDER'S ANALYSES OF DIFFERENT MILL PRODUCTS FROM ONE SAMPLE OF WHEAT Name of sample, or " stream " Water. Per cent Ash. Per cent Gliadin. Number 2 Acidity. Per cent Protein (N x 6.25) Per cent Carbo- hydrates and fat. Per cent 90 per cent Patent . . . 10.89 .48 60.75 .09 13.38 75.25 Clear grade . 10.53 .85 54.63 .13 14.19 74 43 First break 11.68 .61 60.83 .09 13.56 i tr.:rO 74 1 i Second break .... 11.10 .52 59.17 .09 15.00 I J . -I *J 73.38 Third break .... 10.97 .49 59.09 .10 16.50 72.04 Fourth break .... 11.14 .71 58.31 .11 18.44 69.71 First germ 10.90 .48 ' 54.17 ' .08 12.00 76 69 I U.U*rf Second germ 10.37 .59 56.44 .10 12.63 76.41 First middlings .... 10.37 .42 57.43 .08 12.63 76.57 Second middlings . . . 10.69 .42 63.85 .08 13.31 75.58 Third middlings . . . 10.29 .37 66.67 .08 12.56 76.78 Fourth middlings . . . 11.08 .38 64.29 .07 12.25 76.29 Fifth middlings . . . 10.21 .42 62.44 .09 12.81 76.55 Sixth middlings . . . 10.15 .37 57.97 .09 12.94 76.53 Seventh middlings . . . 10.30 .47 59.16 .10 13.31 79.92 First tailings .... 9.01 .77 50.00 .12 13.50 76.72 Second tailings .... 9.54 .65 57.94 .10 13.38 76.43 Second tailings cut . . 9.32 .83 54.88 .11 13.44 76.42 Shorts duster .... 9.36 1.61 39.51 .18 15.19 73.84 Shorts middlings . . . 9.79 4.03 17.47 Wheat 13.07 1.82 14.25 It will be seen that a sample of wheat containing 13.07 per cent moisture and 2.28 per cent nitrogen gave streams of flour containing from 9.01 to 11.68 per cent moisture, and from 1.92 to 2.95 per cent of nitrogen. The "gliadin number," or 1 Bui. 85, Minn. Agl. Expt. Station, St. Anthony Park, Minn., 1904. 2 Alcohol-soluble nitrogen in percentage of the total nitrogen. 346 METHODS OF ORGANIC ANALYSIS percentage of the total nitrogen existing in the form of alcohol- soluble proteins, varied from 39.51 to 66.67. It is interesting to note that some of the streams of flour thus obtained from average wheat in the ordinary milling process contain con- siderably more nitrogen than is sometimes found in so-called gluten and diabetic flours obtained in the market. For additional analyses and results of experiments upon the digestibility and nutritive value of cereal products see Bui. 13, Part 9, Bureau of Chemistry, and Buls. 28, 67, 85, 101, 126, and 143, Office of Experiment Stations, U. S. Department of Agriculture. REFERENCES ALLEN : Commercial Organic Analysis. ATWATER and BRYANT : The Chemical Composition of American Food Materials. Bui. 28, Revised, Office of Experiment Stations, U. S. Dept. Agriculture. JAGO : Science and Art of Breadmaking, Chemistry and Analyses of Wheat. : Technology of Breadmaking. KONIG : Chemie der Menschliche-Nahrungs- und Genussrnittel. LEACH : Food Inspection and Analysis. MAURIZIO : Getreide, Mehl und Brot. OSBORNE : Proteins of the Wheat Kernel. SNYDER : Studies in Bread and Breadmaking, Buls. 67, 101, 126, Office of Experiment Stations, U. S. Dept. Agriculture. VOGL : Die wichtigsten vegetabilischen Nahrungs- und Genussmittel. WILEY : Foods and their Adulteration. WINTON : The Microscopy of Vegetable Foods. II 1894. OSBORNE and VOORHEES: Proteins of Wheat. /. Am. CJiem. Soc., 16, 524. 1895. WILEY: Analyses of Cereals collected at the World's Columbian Exposition. U. S. Dept. Agriculture, Bur. Chem., Bui. 45. 1897. OSBORNE : The Amount and Properties of the Proteins of the Maize Kernel. J. Am. Chem. Soc., 19, 525. SHERMAN: The Insoluble Carbohydrates of Wheat. J. Am. Chem. Soc., 19, 291. 1898. WILEY et al.: Cereals and Cereal Products. U. S. Dept. Agiiculture, Bur. Chem., Bui. 13, Part IX. GRAIN PRODUCTS 347 1899. KRAEMER : An Examination of Commercial Flours. J. Am. Chem. Soc., 21, 650. 1900. GUESS: The Gluten Constituents of Wheat and Flour and their Relation to Bread-making Qualities. J. Am. Chem. Soc., 22, 263. 1904. SNYDER: Wheat and Flour Investigations. Bui. 85, Minn. Agl. Expt. Station. 1905. COCHRAN: Estimation of Fat in Infants' and Invalids' Foods. J. Am. Chem. Soc., 27, 906. HARPER and PETER : Protein Content of the Wheat Kernel. Bui. 113, Kentucky Agricultural Experiment Station. HOPKINS, SMITH, and EAST : Breeding Corn. Bui. 100, Illinois Agricultural Experiment Station. SNYDER : Testing Wheat Flour for Commercial Purposes. J. Am. Chem. Soc., 27, 1068. 1906. BREMER : (Grading of Flour according to its Catalase Content). Z. Nahr.-Genussm., 11, 569. CHAMBERLAIN: Investigation on the Properties of Wheat Proteins. /. Am. Chem. Soc., 28, 1657. NORTON: Crude Gluten. /. Am. Chem. Soc., 28, 8. WINTON: Diabetic Foods. Ann. Kept. Conn. Expt. Sta., 1906, p. 153. 1907. ALWAY and GORTNER : The Detection of Bleached Flours. J. Am. Chem. Soc., 29, 1503. AVERY: A Contribution to the Chemistry of Bleached Flour. J. Am. Chem. Soc., 29, 571. HARCOURT : Breakfast Foods ; their Chemical Composition, Digesti- bility, and Cost. /. Soc. Chem. Ind., 26, 240, and Ontario Dept. Agriculture, Bui. 162. SHAW : Polariscopic Method for Determination of Gliadin. J. Am. Chem. Soc., 29, 1747. THATCHER: A Comparison of Various Methods of estimating the Baking Qualities of Flour. J. Am. Chem. Soc., 29, 910. WOOD : Test for Strength of Wheat Flour. Nature, 75, 391 ; Chem. Abs., 1, 1150. 1908. BAKER and HULTON: Considerations affecting the Strength of Wheat Flours. J. Soc. Chem. Ind., 27, 368. MATHEWSON: On the Analytical Estimation of Gliadin. /. Am. Chem. Soc., 30, 74. 1909. HERTY: Rapid Determination of Oil in Cottonseed Products. J. Ind. Eng. Chem., 1, 76. HOLDFLEISS and W T ESSLING : Laboratory Experiments on the De- termination of the Milling and Breadmaking Qualities of Wheat. J. Soc. Chem. Ind., 28, 808. 348 METHODS OF ORGANIC ANALYSIS 1910. POLENSKE : The Determination of Fat in Foods. Arb. kais. Gesund- heitsamte, 33, 563; Chem. Abs., 5, 325. WILLARD : Changes in Weight of Stored Flour. Kansas State Board of Health, 7, 9; Chem. Abs., 5, 1636. WINTON : Diabetic Foods. British Food /., 12, 23 ; Chem. Abs., 4, 1632. 1911. GREAVES : Some Factors influencing the Quantitative Determination of Gliadin. /. Biol. Chem., 9, 271. HOAGLAND : The Determination of Gliadin or Alcohol-soluble Pro- tein in Wheat Flour. J.-Ind. Eng. Chem., 3, 838. CHAPTER XVII Milk Cows' milk is concisely described as essentially an aqueous solution of milk sugar, albumin, and certain salts, holding in suspension globules of fat and in a state of semisolution casein together with mineral matter (Richmond). Small amounts of other compounds are also present, but need not be considered here. Standard milk (whole milk) is defined 1 as the lacteal secre- tion obtained by the complete milking of one or more healthy cows properly kept and fed, excluding that obtained within fifteen days before and five days after calving, and contains not less than 8.5 per cent of solids not fat, and not less than 3.25 per cent of milk fat. These limits are considerably below the average and consider- ably above the lowest authentic figures which have been found. Average milk may be assumed to contain 12.9 to 13 per cent of total solids, made up of Fat Protein Milk Sugar 2 Ash In round numbers 3 Estimated average 4.0 4.0 3.3 3.35 5.0 4.88 0.7 0.72 The protein content of average milk is, therefore, about one- fourth of the total solids. In general the same relation holds in milk which is richer than the average. Each increase of 1 1 Circular No. 19, Office of the Secretary, U. S. Dept. Agriculture. 2 The figures for milk sugar include the small amount of undetermined non- nitrogenous matter. 3 These are the figures used in most publications of the U. S. Dept. Agri- culture. 349 350 METHODS OF ORGANIC ANALYSIS per cent in total solids thus involves on the average an increase of 0.25 per cent of proteins, the remaining 0.75 per cent being practically all fat. This increase in proteins and fat is usually accompanied by a slight increase in ash and decrease in milk sugar. The following average percentages illustrate these relations in rich milk : Total Solids Fat Protein Milk Ash Sugar Average for 5 years ; mixed even- ing milk of 400 to 500 cows . . 14.62 5.39 3.66 4.82 0.75 Average of 13 unusually rich samples from individual cows 18.03 7.76 4.68 4.76 0.83 The composition of milk of less than average richness cannot be so definitely stated. In some cases there is a deficiency of fat and protein with no decrease in milk sugar, while in other cases the reverse is true. Usually if an unadulterated milk is poor in fat, it will be found proportionately poor in protein; while if the fat is normal, the protein is usually also normal and the low percentage of solids -not fat is due to a deficiency in milk sugar. Several hundred American analyses, made before 1890, com- piled and averaged in ten groups arranged according to per- centage of total solids, gave the following results : 1 TABLE 28. COOKE'S COMPILATION OF AMERICAN ANALYSIS OF MILK Group No. Total solids. Per cent Fat. Per cent Proteins. Per cent Sugar and ash. Per cent Group No. Total solids. Per cent Fat. Per cent Proteins. Per cent Sugar and ash. Per cent 1 11.35 3.20 2.99 5.16 6 13.71 4.46 3.48 5.77 2 11.77 3.36 3.03 5.38 7 14.25 4.87 3.65 5.73 3 12.21 3.60 3.10 5.51 8 14.77 5.20 3.87 5.70 4 12.75 3.82 3.29 5.64 9 15.17 5.47 4.07 5.63 5 13.17 4.09 3.40 5.68 10 15.83 5.88 4.26 5.69 1 Cooke : Vermont Agricultural Experiment Station Report for 1890, p. 97. MILK 351 As a rule the percentage of milk sugar and ash is most nearly constant, that of fat is most variable, while the protein varies with the fat, but to a much smaller extent. The variations which may be regarded as usual, and the extreme variations Avhich the writer has found authentically recorded, are as fol- lows : Fat. Per cent Solids not fat. Per cent Proteins. Per cent Milk sugar. Per cent Ash. Per cent Usual variations . . 3-6 8.5-9.5 3-4 4.6-5 0.7-0.78 Extreme variations J . 1.04-14.67 4.90-13.76 2.86-9.98 2.33-5.28 0.66-1.44 The extreme variations are of no practical value as a means of determining the limits within which milk shall be considered unadulterated, partly because it is possible to practice "adulter- ation through the cow" (i.e. by selection, feeding, and manner of milking to obtain " genuine " milk much below the normal quality), but mainly because the milk which reaches market is practically always the mixed product of several cows so that individual variations have comparatively little effect. There are many causes of variation in the composition of cows' milk. Only the most important can be given here. Other conditions being normal, the percentages of fat and proteins are higher in autumn and winter than in spring and summer; they also increase as the amount of milk decreases toward the end of each period of lactation. Milk drawn in the evening is generally 0.3 to 0.4 per cent richer in fat than that obtained in the morning, and at any one milking the last portions drawn are much richer than the first. The influence of a change of food upon the percentage composition of milk is usually only temporary. In general the peculiarities of breed 2 1 Including only results obtained from apparently healthy cows, believed to have been milked regularly under normal conditions. 2 For comparison of the milk of different breeds see Richmond's Dairy Chemistry, pp. 122-126, Report of the New York State Expt. Station for 1891 (abstracted in the Expt. Station Record, 4, 263) , and Report of the Wisconsin Expt. Station for 1901, p. 85. 352 METHODS OF ORGANIC ANALYSIS and the qualities of individual animals are the most important factors in determining the richness -of milk. Aside from all these conditions the milk of individual cows is subject to con- siderable fluctuation, especially in fat content. Thus the analyses of 60 monthly samples of the mixed milk of about 500 cows showed a variation of 0.89 in the percentage of fat, the greatest deviation from the average being 0.50 per cent. About one half of the determinations of fat in the milk of in- dividual cows of the herd during the same period were more than 0.50 per cent and about one fifth were more than 1.0 per cent above or below the average. Milk representing the mixed product of several farms, such as is now commonly sold in large cities, should, therefore, be much more uniform in composition than that of a single cow or a small herd. 1 SAMPLING AND PRESERVATION OF SAMPLES If the lot of milk to be sampled is small it can be mixed by pouring from one vessel to another from two to ten times, according to the extent to which the cream has separated, and the portion for analysis dipped, or withdrawn by means of a pipette, from near the center. When the sample is too large to be handled in this way it should be transferred if necessary to a cylindrical can and sampled by means of a Scovell tube. 2 In order to obtain a proper sample of a large lot of milk delivered in cans of the same diameter, it is only necessary to sample each can with the Scovell tube and mix the portions thus obtained. The sample should be placed at once in a clean, dry, sterile bottle, tightly stoppered, and analyzed as soon as possible. Before withdrawing each portion for analysis the sample must be thoroughly mixed by pouring not by shaking. 1 Fuller discussions of the variations in the milk of individual cows and mixed milk of herds will be found in some of the reference books given at the end of the chapter, in the papers on the composition of milk published annually by Richmond in the Analyst, and in Hittcher's Gesammtbericht liber die Unter- suchung der Milch, Berlin, 1899. 2 Wiley's Agricultural Analysis, Vol. III., p. 470; Leach's Food Inspection and Analysis, 2d ed., p. 131. These sampling tubes are sold by dealers in dairy apparatus. MILK 353 If the analysis cannot be made at once or if the sample is to be kept for some time after the analysis, it must either be stored at a temperature near the freezing point or preserved by the addition of an antiseptic. Formaldehyde 1 added, while the milk is still fresh, in the proportion of 1 : 1000 will preserve the sample for months with- out apparent change. This amount of form- aldehyde has a scarcely perceptible influence upon the analytical results. If preservation for only a few days is required, a smaller amount of formaldehyde should be used, 1: 2000 to 1: 10,000, according to the freshness of the milk. \ PRELIMINARY OR PARTIAL EXAMINATION DETERMINATION OF SPECIFIC GRAVITY The specific gravity of milk is usually be- tween 1.029 and 1.034. Since cream is con- siderably lighter than milk, the specific gravity would be lowered by the addition of water or of cream, but cases in which genuine milk shows a low specific gravity as a result of high fat content are very rare. As already ex- plained, high percentages of fat are normally accompanied by high percentages of proteins, so that in most cases the specific gravity is higher in rich than in poor milk. With practice the samples which are exceptions to this rule can usually be detected by noticing the apparent FIG. is. Lactometer ., ., ,, . ' , of Quevenne or viscosity and opacity of the milk as it runs from Soxhiet type, with the surface of the lower bulb of the lactometer. thermometer in the stem. The specific gravity taken in connection with this appearance is much used as a preliminary test by milk in- spectors and is recommended by Richmond as the best means of rapidly testing each lot of milk received by a large dairy. 1 Other preservatives are sometimes useful. See Richmond's Dairy Chem- istry, p. 144; and Grelat: Chem. Abs., 1, 1588. 2A 354 METHODS OF ORGANIC ANALYSIS The Quevenne, Veith, and Soxhlet lactometers are hydrom- eters of sufficient range for use with milk and so graduated as to read the "excess gravity " over water taken as 1000. Thus a milk of 1.0315 specific gravity gives a lactometer reading of 31.5. These instruments are often made to include a Fahren- heit thermometer, the scale of the latter being on the same stem with the lactometer scale (Fig. 18). The lactometer read- ing should be taken between 50 and 65 F. and corrected for temperature by adding or subtracting 0.1 for each degree F. above or below 60. The New York Board of Health lactometer has a scale reading zero in pure water and 100 at 1.029 specific gravity. VOLUMETRIC DETERMINATION OF FAT Babcock, in 1890, introduced the first satisfactory rapid method for the determination of fat in milk. On mixing milk with approximately an equal volume of strong sulphuric acid, the casein is dissolved while the fat remains unchanged and can be separated by centrifugal force. The test is performed in a bottle with a neck so graduated that the percentage of fat can be read off directly upon removing the bottle from the centrifuge. Determination. Measure 17.6 cc. of milk at 14 to 18 (about 55 to 65 F.) and introduce into the test bottle. Add 17.5 cc. of sulphuric acid of 1.82 sp. gr. (commercial concen- trated acid is usually the right strength), allowing the acid to flow down the side of the bottle so as not to mix with the milk. When acid has been added to all of the bottles and everything is ready to start the whirling, mix the milk and acid quickly and thoroughly by shaking, and continue the shaking until the curd is in solution and the liquid has reached a permanent and uniform color. Then place an even number of the bottles in opposite pockets in the machine and whirl at the rate of 800 to 1200 revolutions per minute, according to the diameter of the wheel which carries the bottle. After whirling five minutes, fill with hot water to the shoulder of the bottle and whirl two minutes, then fill with hot water to near the top of the gradua- tion in the neck and whirl again for two minutes. The per- MILK 355 centage of fat is now shown by the height of the column in the graduated neck of the bottle. Notes. The capacity of the graduated neck of the bottle from to 10 is 2 cc. It is assumed that the 17.6 cc. of milk taken for the determination will weigh 10 times as much as 2 cc. of warm butter fat. It is important that the final read- ings be taken while the fat is still warm. On account of the unavoidable contraction of the fat while taking these readings it is customary to read from the bottom of the lower to the top of the upper meniscus. The result is usually within 0.2 per cent of that found by the gravimetric method. The column of fat should be of a clear yellow color throughout. If the acid used is too weak, flocks of undissolved casein are apt to be found in the lower part of the fat column ; if too strong, the acid may char the fat. For a full discussion of the details of the test, with directions for applying it to other dairy products, see the work of Farrington and Woll. 1 The most important modifications of the Babcock method are fully described in Richmond's Dairy Chemistry, pp. 1.74-192. CALCULATION OP SOLIDS FROM SPECIFIC GRAVITY AND FAT Many formulae have been proposed by which to calculate the percentage of solids in milk from the percentage of fat and the specific gravity. The results thus obtained are sufficiently ac- curate for many technical purposes and often for routine in- spection work which is not to be made the basis of legal action. Such formulae may be found in many of the works referred to at the end of this chapter. They are necessarily based on the assumption that each per cent of fat causes a definite decrease, and each per cent of solids not fat a definite increase, in the specific gravity. Since the solution densities of proteins, milk sugar, and milk ash differ considerably, 2 any change in the relative 1 Testing Milk and Its Products. Madison, Wisconsin. 2 Allen : Commercial Organic Analysis, Vol. IV. (2d ed.), p. 166. 356 METHODS OF ORGANIC ANALYSIS proportions of these constituents must alter the solution density of the solids not fat and thus diminish the accuracy of this method. One of the simplest of these formulae is that of Richmond : Total solids = Lactometer reading ^ fet Q u> 4 With samples which do not differ greatly from the average composition the results thus calculated are usually accurate within 0.25 per cent. DETERMINATION OF FAT, PROTEINS, MILK SUGAR, AND ASH TOTAL SOLIDS AND ASH Into an accurately weighed flat-bottomed platinum dish intro- duce two to five grams (depending upon the size of the dish ; see below) of the thoroughly mixed milk and weigh quickly to the nearest milligram. If this weighing cannot be accomplished within one minute, the dish should be covered with a weighed watch glass to retard evaporation. Place the open dish on a water bath or on top of the boiling water oven until nearly all of the water is expelled ; dry to constant weight in a boiling water oven or an air bath kept constantly at 97 to 100. The residue is somewhat hydroscopic and must be weighed quickly upon removal from the desiccator in order to obtain the correct amount of total solids. Ignite the dry solids in a muffle at 550 to 600, or, if this is not feasible, regulate a Bunsen burner to give a very small col- orless flame and apply this carefully with constant attention so that no part of the dish is heated abcTve the lowest possible red- ness. The ash should be white or very light gray. After obtaining the weight of the ash it may be used in testing for preservatives, as described below. Notes on Total Solids. When the same portion is not to be used for the determination of ash a platinum dish is not essential. Lead foil bottle caps are then very convenient, as they are eas- ily numbered by scratching, quickly heated and cooled, and so MILK 357 cheap that each dish can be rejected after being used once. In order that a larger surface may be exposed, the dish may contain dry sand or other porous material and a small stirring rod weighed with the dish and used to stir the residue while drying. Unless absorbed upon porous material, no more than 0.5 gram of milk for each square centimeter of the area of the bottom of the dish should be taken for the determination. The methods used in the Government Laboratory, London, for the determination of solids in sour or fermented milk and the estimation of the solids lost in fermentation have recently been described by Thorpe. 1 Notes on Ash. Normally about two-thirds of the ash of cows' milk is insoluble in hot water. The presence of a larger proportion of soluble ash may be due to the use of mineral preservatives or to the addition of salts to restore the density and ash content to milk which has been watered. Special tests for some of the mineral preservatives are given beyond. In important cases it may be necessary to analyze the ash to show whether it is of normal character. Richmond 2 gives the following as the average composition of milk ash : calcium oxide, 20.27 per cent; magnesium oxide, 2.80 per cent; potassium oxide, 28.71 per cent; sodium oxide, 6.67 per cent; phosphoric anhydride, 29.33 per cent ; chlorine, 14.00 per cent ; carbonic anhydride, 0.97 per cent ; sulphuric an- hydride, trace ; ferric oxide, etc., 0.40 per cent. According to most other writers 3 milk ash contains sulphates, but only traces of carbonates. A sample of ash from the mixed milk of about 500 cows examined by Thompson and the writer showed no appreciable amount of carbonates and only traces of sul- phates. When the ash was prepared at known temperatures, there was no volatilization of chlorides below 650, but even at 450 to 500 there was considerable loss of chlorine, due doubtless to the formation of acid products in the combus- tion of the organic constituents of the milk. 1 J. Chem. Soc., 1905, 87, 206. 2 Dairy Chemistry, p. 32. 8 See tabulated analyses in Stohmann's Milch- und Molkereiproducte, p. 89. 358 METHODS OF ORGANIC ANALYSIS FAT GRAVIMETRIC DETERMINATION Adams' Paper Coil Method In this method the milk is dried on porous paper, the fat ex- tracted by means of ether into a weighed flask, the ether evapo- rated, and the fat weighed. Apparatus. (1) Strips of thick absorbent fat-free paper about 55 cm. long and 6.25 cm. wide, 1 each rolled into a loose coil and fastened by means of a piece of wire or fat-free thread. If difficulty is found in making a loose coil, two pieces of fat-free string may be laid lengthwise upon the paper strip before rolling it up. This, however, should not be necessary. (2) A Soxhlet apparatus for ether extraction, the form hav- ing ground glass connections being recommended. (3) A safety water bath or, preferably, an electric heater which can be easily regulated. Determination. Mix the milk thoroughly and absorb a known amount, about 5 grams, on the paper coil. The milk can be meas- ured by means of a 5-cc. pipette and delivered directly upon the coil, but as milk is more viscous than water, an ordinary 5- cc. pipette will deliver less than 5 cc. of milk, so that this method can be made accurate only by determining experimentally the amount of milk which the pipette actually delivers. A better method is to pour about 5 cc. into a very small beaker, weigh quickly to centigrams and at once absorb the milk by standing the coil in the beaker. The absorption can be hastened by in- clining the beaker and rotating the coil. The last drops in the beaker must be carefully absorbed. Stand the coil upon the dry end and reweigh the beaker quickly to centigrams. If carried out rapidly, this method is considerably more accurate than measuring with a pipette. Dry the coil thoroughly in a boiling water oven, place in a Soxhlet extractor, and extract with an- hydrous ether, using the electric heater or safety water bath and keeping the apparatus as far as possible from free flames. If 1 Strips of paper especially prepared for this purpose are made by Schleicher and Schlill. If these are not available, the paper strips must be very carefully extracted before use. See Richmond's Dairy Chemistry, pp. 91-93. MILK 359 not more than 5 grams of milk is used and the extractor siphons at intervals of 10 to 15 minutes, the extraction need not be con- tinued longer than three hours. At the end of extraction dis- connect the apparatus, remove the coil, replace the extractor, recover nearly all the ether by allowing it to collect in the space formerly occupied by the coil, return the ether to its bottle, and heat the flask containing the fat in a boiling water oven until the weight is practically constant. Notes. As the milk is absorbed by the paper the greater part of the fat is left on or near the surface, so that it is very rapidly extracted by the ether. The coil must be thoroughly dried before extracting with ether. The drying can be hastened by pressing in the dry end of coil so that the inner layers of the wet end are made to project in the form of a cone. Such a coil will usually be dry after standing two to three hours in the boil- ing-water oven. On removing from the oven press back the projecting end of the coil and place it, milk end down, in the extractor ; connect with the flask, pour in ether until it siphons into the flask, then enough more ether to cover about half the coil. This is sufficient to avoid any danger of the flask going dry during the extraction, if the heat is so regulated that no perceptible amount of ether escapes the reflux condenser. To dry the extract, leave it in the boiling water oven for three hours, allow to cool for one half hour, weigh, and then repeat, heating about one hour each time, until two successive weighings show a loss of less than one milligram. In laboratories where many determinations are made it is customary to dry the extract for a fixed length of time (usually five hours), which has been found by experience to be sufficient. Babcock Asbestos Method l Provide a hollow cylinder of perforated sheet metal, 60 mm. long and 20 mm. in diameter, closed 5 mm. from one end by a disk of the same material. The perforations should be about 0.7 mm. in diameter and about 0.7 mm. apart. Fill loosely with from 1.5 to 2.5 grams of freshly ignited, woolly asbestos, 1 Bui. 107, Bur. Chem., U. S. Dept. Agriculture. 360 METHODS OF ORGANIC ANALYSIS free from fine and brittle material, cool in a desiccator, and weigh. Introduce a weighed quantity of milk (between 3 and 5 grams) and dry at 100 to constant weight. This weight shows the percentage of total solids. Place the cylinder in an extractor and complete the determination of fat as described above. This method avoids the possibility of having any ether- soluble matter in the porous substance used to absorb the milk. It is especially recommended for the determination of fat in cream which cannot be absorbed upon the paper coil without previous dilution. PROTEINS Formerly milk proteins were precipitated, or the milk evapo- rated to dryness, and the residue after washing with ether and dilute alcohol was dried, weighed, burned, and the ash deducted. On account of the difficulty of completely removing the sugar and fat, the results thus obtained were usually too high ; so that in the older statements of the composition of milk (some of which are still often quoted) the proteins were usually over- estimated. Protein in milk is now calculated from the nitrogen content, multiplying the latter by the usual factor 6.25 or sometimes by a special factor, 6.33 or 6.3T, based on analyses of milk proteins showing less than 16 per cent of nitrogen. To determine the total nitrogen in milk pour 5 to 10 grams of the sample into a small beaker, weigh quickly to centigrams, pour the rnilk carefully into a Kjeldahl flask, re weigh the beaker, and introduce 20 to 25 cc. of concentrated sulphuric acid into the flask in such a way as to wash down any milk which may have remained in the neck. Add 0.7 gram of mer- cury, heat gently over a very small flame until most of the water is expelled and no more frothing or spirting occurs, then increase the size of the flame and complete the determination as described in Chapter XIV. Casein can be precipitated by acidulating the milk or by means of magnesium sulphate. Determination of nitrogen in the washed precipitate shows the amount of casein in the milk. Albumin can be precipitated by boiling the filtrate, and deter- MILK 361 mined in the same manner. Detailed directions for these determinations will be found in Bulletin 107, Bureau of Chem- istry, U. S. Department of Agriculture. MILK SUGAR OR LACTOSE In most cases the direct determination of lactose is unneces- sary, as the difference between the percentage of total solids and the sum of the percentages of fat, proteins, and ash should not differ from the true percentage of lactose by more than 0.1 to 0.2 per cent. When direct determination is desired, either the polariscopic method or one of the methods based upon the reduction of copper can be used. In the former case the pro- teins are precipitated and the solution clarified by means of mercuric nitrate or iodide; in the latter, by cupric hydroxide or acetic acid, alum, and aluminium hydroxide. Optical Determination 1 Place 65.8 grams of milk in each of two flasks, one graduated at 100, the other at 200 cc., to each add 4 cc. of mercuric nitrate solution, 2 fill to the mark, shake, filter through dry paper, and polarize in a 200-mm. tube in the Schmidt and Haensch polar- iscope. In each case the reading is too high on account of the volume occupied by the precipitate which contains the proteins and fat of the milk. This volume is twice as great in proportion in the 100-cc. as in the 200-cc. flask. The corrected reading and the volume occupied by the precipitate can, therefore, be calculated by the method of double dilution as in the following example : 3 Weight of milk taken, 65.8 grams, or twice the "lactose normal " weight 4 for the Ventzke scale. 1 Wiley and Ewell : J. Am. Chem. Soc., 1896, 18, 428. 2 To prepare this solution dissolve mercury in twice its weight of nitric acid, 1.42 specific gravity, and add to the solution an equal volume of water ; or pre- pare a solution of equal strength by dissolving solid mercuric nitrate in water acidulated with nitric acid. 3 Compare Wiley's Agricultural Analysis, Vol. Ill, pp. 102, 278. 4 Calculated from the sucrose normal weight and the approximate specific rotatory powers of sucrose and lactose. These data and directions for the manipulation of the polariscope have been given in Chapters III and IV. 362 METHODS OF ORGANIC ANALYSIS Average reading from 100-cc. flask, 10.45. Average reading from 200-cc. flask, 5.075. Then 10.45- (5.075x2) = 0.30 (half the error in the higher reading). 10.45-(0.30 x2) = 9.85 (corrected reading for 100-cc. flask). 9.85 -j- 2 = 4.925, corrected percentage of lactose. The volume of the precipitate is calculated as follows : 10.45 -r- 2 = 5.225, apparent percentage of lactose (100-cc. flask). Then 5. 225: 4. 925:: 100: a;. x = 94.26, the volume of solution in the 100-cc. flask. Hence the volume of the precipitate is 5.74 cc. Determination by Fehling Solution* Dilute 25 cc. of milk with 400 cc. of water in a 500-cc. flask, add 10 cc. of the copper sulphate solution used in the Fehling method, mix and add 4.4 cc. of normal sodium or potassium hydroxide (or an equivalent amount of a weaker standard solu- tion), fill to the mark, mix, and filter through dry paper. The filtrate must contain copper in order to insure the absence of any trace of free alkali. In this clear filtrate lactose can be determined by means of Fehling solution either by Defren's method as described in Chapter III., or by Soxhlet's method, Bui. 107, I. c. The milk is so greatly diluted in clarifying the solution that the volume of the precipitated proteins and fat can be neglected. If lactose is to be determined volumetrically, the proteins can be precipitated and the solution clarified as described in Richards and Woodman's Air, Water, and Food. INTERPRETATION OF RESULTS The principal adulterations affecting the percentages of nutrients in milk are the addition of water (sometimes con- taining dissolved solids) and the removal of cream. These 1 Bui. 107, loc. cit. MILK 363 adulterations are sometimes difficult to detect with certainty because genuine cows' milk varies considerably both, in fat and in other solids. Since the percentage of fat is more variable than that of solids not fat, skimming is more difficult to detect than watering. If as much as one fourth of the fat were removed, the skimming would usually be indicated by the dis- turbance of the normal relation between the percentage of fat and that of proteins or of solids not fat ; but the analysis can- not be said to prove the removal of cream unless it shows a lower percentage of fat than is ever found in genuine normal milk. Starting with average milk containing 4 per cent fat and 9 per cent solids not fat, one tenth of the fat could be removed by skimming and the resulting product containing 3.6 per cent fat could not be distinguished by analysis from genuine milk ; while if the fat were reduced to 3.6 per cent by watering, the solids not fat would be reduced to 8.1 per cent, which is sufficiently below the normal to be detected without difficulty. Occasionally genuine milk contains even less than 8.0 per cent of solids not fat (the deficiency in most of these cases falling mainly upon the milk sugar), so that the limit of 8.5 for solids not fat might indicate watering where none had been practiced. Such errors are avoided by taking account of the proteins and ash. Milk should contain not less than 8.5 per cent of solids not fat, 3.0 per cent of proteins, 0.7 per cent of total ash, 0.5 per cent of ash insoluble in hot water. These four determinations, especially if supplemented by the refractometer examination of the serum as described below, will usually suffice to show whether the milk is gen- uine or has been watered with or without the addition of soluble solids. In most cases it is not necessary to show conclusively whether milk has been skimmed or watered, but only whether it meets the requirements of a legal or trade standard. The principal standards in force in the United States in 1910 are given in the accompanying table, from the Twenty-seventh Annual Report, Bureau of Animal Industry, U. S. Department of Agriculture. 364 METHODS OF ORGANIC ANALYSIS TABLE 29. UNITED STATES AND STATE STANDARDS FOR MILK, 1910 State. Total solids. , Per cent 1 Solids not fat. Per cent Fat. Per cent State. Total solids. Per cent Solids not fat. Per cent 1 & i United States l . . . 8.5 3.25 New Hampshire . . 13 9.5 3.5 California .... 8.5 3 New Jersey . . . 11.5 3 Colorado 8.5 3.25 New Mexico Connecticut .... 11.75 8.5 3.25 New York .... 11.5 3 Dist. Columbia . . . 9 3.5 North Carolina . . 8.5 3.25 Delaware 2 .... North Dakota . . 12 3 Florida 2 . . . Ohio 12 3 12 8.5 3 25 12 5 3 Hawaii 11.5 3.50 Oregon . . 12.2 9 3 2 Idaho 11 8 3 Pennsylvania 2 . . Illinois . . . 8.5 3 Porto Rico 12 3 8 5 3 25 Iowa 12.5 3 South Dakota 13 8.5 3 25 Kentucky .... 12 8.5 3.25 Tennessee .... 8.5 3.25 Louisiana .... 13 95 3.5 Texas 8.5 3.25 11.75 8.5 3.25 Utah 12 9 32 Maryland .... 12.5 3.5 Vermont .... 12.5 9.25 4 Massachusetts . . . 12.15 3.35 May and June . . 12 Michigan . 12 5 3 8.5 3 5 Minnesota .... 13 3.5 Washington . . . 12 8.75 3.25 Missouri .... 8 5 3 25 8 5 3 Montana 12 9 3 "Wyoming 12 2 4 Nebraska .... 3 May and June . . 11.5 1 Standards of Purity of Food Products. Office of the Secretary, Circular 19. 2 Municipal control ; no State standard. United States Dept. Agriculture, EXAMINATION OF MILK SERUM FOR ADDED WATER In general the serum or whey obtained from milk under fixed conditions is believed to be much more uniform in prop- erties than the milk itself, in which case watering will be more certainly detected by an examination of the whey than of the whole milk. Woodman's method, adopted by Leach, for preparing the serum is as follows : To 100 cc. of milk at room temperature in a beaker, add 2 cc. of 25 per cent acetic acid, cover and heat in a water bath at 70 C. for 20 minutes ; then place the beaker MILK 365 in ice water for 10 minutes, after which filter through a dry paper. This should result in a clear filtrate (the serum or whey) which may be tested either for specific gravity or with the im- mersion refractometer. A specific gravity below 1.027 at 15 C., or a reading of the refractometer below 39 at 20 C., is a strong indication that the sample is watered. The great advantage in rapidity and convenience of such a method over the determination of solids not fat commends it to inspection laboratories where many samples must be rapidly examined for adulteration, and in several such laboratories the refractometer reading of the serum is now taken as the chief criterion of watering. It should be noted, however, that the usual legal criterion is a minimum percentage of solids not fat. According to data determined by Tice 1 and by Leach 2 it would appear that milk very poor in solids but free from added water may fall far below the usual legal minimum of 8.5 per cent solids not fat while yielding serum readings of 39 to 42 on the immersion refractometer scale. Hence if milk is de- clared watered only when both the percentage of solids not fat and the refractometer reading of the serum are below minimum limits, there will be much less danger of prosecutions for watering in cases of milk not watered but naturally poor in solids. CHEMICAL PRESERVATIVES The chemical preservatives most likely to be used in milk are formaldehyde, hydrogen peroxide, boric acid or borax, and fluo- rides/ Benzoates and salicylates may perhaps be used in rare instances. Methods for the detection and determination of these and other preservatives will be found in the next chapter. Carbonate or bicarbonate is sometimes added to milk, not as a preservative properly so called, but as an adulterant to hide the fact that the milk has undergone acid fermentation, and so to give it a fraudulent appearance of freshness. When milk contains the equivalent of 0.05 per cent of 1 Report of the New Jersey State Board of Health, 1909, pp. 191-194. 2 Leach : Food Inspection and Analysis, 2d ed., pp. 166-169. 366 METHODS OF ORGANIC ANALYSIS sodium carbonate, the ash obtained by direct ignition of the solids shows effervescence on addition of hydrochloric acid. Such effervescence is rarely if ever seen in the ash of pure milk, but since Richmond has found small amounts of carbonic acid in the ash of milk believed to have been pure, the presence of carbonate or bicarbonate should be confirmed by applying Schmidt's test, in which 10 cc. of milk are mixed with an equal volume of alcohol and a few drops of a 1 per cent solution of rosolic acid. The color is brownish yellow in pure milk but rose-red in milk containing carbonate or bicarbonate. A com- parative test with pure milk should always be made. The re- action is nearly as delicate as the test for effervescence in the ash. REFERENCES ALLEN : Commercial Organic Analysis. CHAPIN: Theory and Practice of Infant Feeding. CONN : Bacteria in Milk and its Products. FARRINGTON and WOLL : Testing Milk and its Products. FLEISCHMANN : Lehrbuch der Milchwirthschaft. GROTENFELT : The Principles of Modern Dairy Practice. KONIG : Chemie der menschliche Nahrungs- und Genussmittel. LEACH : Food Inspection and Analysis. RICHMOND : Dairy Chemistry. ROSENAU : Milk in its Relation to Public Health. ROTHSCHILD : Bibliographia Lactaria. RUSSELL : Dairy Bacteriology. SOMMERFELD : Haudbuch der Milchkunde. STOHMAXN : Milch- und Molkereiproducte. SWITHINBANK and NEWMAN : Bacteriology of Milk. U. S. Dept. Agriculture, Farmers' Bulletins 42 (Facts about Milk), 74 (Milk as Food). VAN SLYKE : Modern Methods of Testing Milk and Milk Products. WINSLOW : Production arid Handling of Clean Milk. Wisconsin Agricultural Experiment Station, Bulletins and Reports. II 1899. RICHMOND: The Composition of Milk and Milk Products. Analyst, 24, 197. WOODMAN : On the Determination of Added Water in Milk. J. Am. Chem. Soc., 21, 503. MILK 367 1900. RICHMOND: The Composition of Milk and Milk Products. Analyst, 25, 225. WHITAKER : The Milk Supply of Boston and Other New England Cities. U. S. Dept. Agriculture, Bureau of Animal Industry, Bui. 26. 1901. RICHMOND : The Composition of Milk. Analyst, 26, 310. 1903. RICHMOND: The Composition of Milk. Analyst, 28, 289. SHERMAN : On the Composition of Cow's Milk. J. Am. Chem. Soc., 25, 132. 1904. LEACH and LYTHGOE : The Detection of Watered Milk. /. Am. Chem. Soc., 26, 1195. 1905. RICHMOND: The Composition and Analysis of Milk. Analyst, 30, 325. 1906. FREAR : American Milk and Milk Standards. Proc. Assn. State and National Dairy and Food Departments, 1906, p. 172. LEACH: Report on Dairy Products (Refractometer Test for Water- ing). U. S. Dept. Agriculture, Bur. Chem., Bui. 105, p. 37. MULLER : Methylene Blue as a Test for the Freshness of Milk. Arch. Hyg., 56, 108; Analyst, 31, 299. RICHMOND : Estimation of Fat in Homogenized Milk. Analyst, 31, 218, 219-224. RICHMOND and MILLER : Methods of Analysis of Milk Used by the Government Laboratory. Analyst, 31, 317. ROUSSEAU: Investigations on Sterilization of Milk by Means of Hydrogen Peroxide. Bull. soc. pharmacol., 13, 606 ; Chem. Abs., 1, 1592. SELIGMANN: (Detection of Heated Milk). Z. angew. Chem.,1906, 1540. SHERMAN : Seasonal Variations in the Composition of Cow's Milk. /. Am. Chem. Soc., 28, 1719. 1907. ACKERMANN : Refractometric Detection of Added Water in Milk. Z. Nahr.-Genussm., 13, 186. ANDERSON : The Detection of Cane Sugar in Milk and Cream. Analyst, 32, 87. BAIER and NEUMANN: Refractometer Examination of Milk. Z. Nahr.-Genussm., 13, 369. DUBOIS: Analysis of Milk Chocolate. J. Am. Chem. Soc., 29, 556. 1907. HENKEL : Acidity of Cows' Milk. Milchwirtsch. ZentrU., 3, 340 ; Chem. Abs., 1, 2480. HOWARD: Analysis of Ice Cream. J. Am. Chem. Soc., 29, 1622. Low : The Test for Formaldehyde in Milk by Leach's Modification of the Hydrochloric Acid and Ferric Chloride Test. J. Am. Chem. Soc., 29, 786. 1908. BAIER and NEUMANN : Detection of Calcium Sucrate in Milk and Cream. Z. Nahr.-Genussm., 16, 51. 368 METHODS OF ORGANIC ANALYSIS BURR, BERBERICH and LAUTERWALD : Investigations of Milk Serum Milchwirtsch. Zentrbl., 4, 145, 210, 262; Chem. Abs., 2, 2961. FRERICHS : Detection of Calcium Sucrate in Milk and Cream. Z. Nahr.-Genussm., 16, 682. HART : Centrifugal Method for Casein in Milk. Wisconsin Expt. Sta., Bui. 156 ; Chem. Abs., 2, 675. MAI and ROTHENFUSSER : Detection of Added Water in Milk by Means of the Refractometer. Z. Nahr.-Genussm., 16, 7. 1909. FENDLER and KUHN : Determination of Dirt in Milk. Z. Nahr.- Genussm., 17, 513. LYTHGOE and NURENBERG : A Comparison of Methods for the Prep- aration of Milk Serum. J. Ind. Eng. Chem., 1, 38. ROBERTSON : A Rapid Method of Determining the Percentage of Casein in Milk. /. Ind. Eng. Chem., 1, 723. ROTHENFUSSEH : (Nitrate Test as Evidence of Added Water in Milk). Z. Nahr.-Genussm., 18, 353. VAN SLYKE and BOSWORTH : Volumetric Method for the Determina- tion of Casein in Milk. J. Ind. Eng. Chem., 1, 768. 1910. AUZINGER : (Test for Abnormal Milk). Milchwirtsch. Zentrbl., 5, 293, 352, 393, 430; Chem. Abs., 4, 619. ECKLES : Seasonal Variations in Percentages of Fat in Cows' Milk. Milchwirtsch. Zentrbl., 5, 488; Chem. Abs., 4, 620. . HART : A Volumetric Method for the Estimation of Casein in Milk. J. Biol. Chem., 6, 445. LYTHGOE and MARSH : The Relation Between Fat and Calcium in Cream. J. Ind. Eng. Chem., 2, 327. POETSCHKE : The Determination of Sodium Chloride in Milk. J. .Ind. Eng. Chem., 2, 210. ROTHENFUSSER : Detection of Cane Sugar and Calcium Sucrate in Milk and Cream. Z. Nahr.-Genussm., 19, 465. TILLMANS: (Determination and Significance of Nitrates in Milk). Z. Nahr.-Genussm., 20, 676. 1911. BACKE: Analysis of Sweetened Condensed Milk. Analyst, 36, 138. BULL: A Comparison Between the Refraction and the Specific Gravity of Milk Serum for the Detection of Added Water. J. Ind. Eng. Chem., 3, 44. CHAPTER XVIII Food Preservatives 1 FORMALDEHYDE 2 Detection OF the many methods available for the detection of formalde- hyde in milk and other foods only three of the best known and most delicate will be given here. For other methods see Bui. 107, Revised, Bureau of Chemistry, U. S. Department of Agri- culture. Sulphuric Acid Test.* Dilute 2 to 3 cc. of milk with an equal volume of water in a test tube, add carefully, so as not to mix the layers, from 3 to 5 cc. of concentrated commercial sul- phuric acid or pure acid to which a trace of ferric salt has been added. If formaldehyde is present, a violet ring forms at the junction of the two liquids. The charring of the milk by the sulphuric acid makes it difficult to define the delicacy of the test. One part of formaldehyde in 100,000 of milk can be detected if the milk is fresh and the test is applied soon after adding the preservative. Hydrochloric Acid Test.* Mix 10 cc. of milk and 10 cc. of concentrated hydrochloric acid containing about 2 mg. of ferric chloride and heat slowly nearly to boiling, rotating the mixture occasionally to insure solution of the curd. In the presence of 1 Trade names and analyses of many proprietary preservatives will be found in the Year-Book of the U. S. Department of Agriculture for 1900, Chapin's Theory and Practice of Infant Feeding, and the Zeitschrift fur die Untersuchung der Nahrungs- und Genussmittel. 2 See also the methods for detection and determination of f onnaldehyde given in Chapter II. 3 Hehner : Analyst, 1896, 21, 95. 4 Leach: Ann. Rpts. Mass. State Board of Health, 1897, 558; 1899, 699; Food Inspection and Analysis, p. 140. See also Chapter II. 2B 369 370 METHODS OF ORGANIC ANALYSIS formaldehyde a violet color develops, otherwise the solution slowly turns brown. The test is best performed in a porcelain casserole, and in case of doubt the violet color is made much more distinct by adding 50 to 75 cc. of water after having heated just below boiling for about a minute. The liquid must be observed carefully at the moment of dilution as the color brought out in this way fades very rapidly. This test is delicate to 1 : 250,000, but formaldehyde added to milk in such small quantities soon disappears. When added to the extent of 1: 50,000 to 1: 100,- 000 the presence of formaldehyde in the milk will be shown by this test for from 1 to 5 days. 1 In testing sour or stale milk the brown color noted above will often obscure the reaction given by a small amount of formaldehyde until the solution is diluted with water, but at this point the violet color can be seen even though the milk may have been much charred by the acid. Grallic Acid Test. This test has been described in Chapter II. To apply it to milk or other liquid food acidulate 30 cc. with 2 cc. of normal sulphuric acid and distill. To the first 5 cc. of distillate add 0.2 to 0.3 cc. of a saturated solution of gallic acid in pure alcohol, incline the test tube, and pour in slowly 3 to 5 cc. of concentrated sulphuric acid. The presence of formaldehyde is shown by the characteristic blue ring described in detail in Chapter II. In the writer's experience this test is at least twice as delicate as either the sulphuric or the hydrochloric acid test. The latter would be sufficiently delicate for all practical pur- poses if milk samples could always be tested while fresh, but when small amounts of formaldehyde have been added one or two days previously, the gallic acid test may show the preservative where either of the other tests would fail. In a laboratory ex- periment, 2 a sample of milk which originally contained 1: 50,000 formaldehyde ceased to give any reaction by the hydrochloric acid and ferric chloride test after five days, but the distillate subsequently obtained from 30 cc. of this sample gave an un- mistakable formaldehyde reaction when tested with gallic acid. iEivas: University of Pennsylvania Medical Bulletin, 1904, 17, 175. Williams and Sherman: J. Am. Chem. Soc., 1905, 27, 1497. 2 Williams and Sherman: J. Am. Chem. Soc. t 27, 1499. FOOD PRESERVATIVES 371 This gallic acid reaction may also be used very satisfactorily as a means of confirming any doubtful results obtained by either of the preceding tests, as there is little danger of inter- ference due to charring or to the appearance of other colors. Determination Very small amounts of formaldehyde in milk can be deter- mined by the following method, which is essentially that of Smith, 1 except that a larger quantity of sample is used. To 300 cc. of milk in a round-bottomed flask of about one liter capacity add 3 cc. of (1 : 3) sulphuric acid and some glass beads to prevent bumping, heat gradually to boiling, preferably by means of a small rose-top burner, and distill until the dis- tillate measures 60 cc. Transfer this to a 100-cc. flask, add 10 cc. of standard potassium cyanide solution, approximately tenth-normal, and mix ; add a mixture of 15 cc. of tenth-nor- mal silver nitrate and 6 to 8 drops of 50 per cent nitric acid, fill to the mark, shake, and filter through dry paper. Deter- mine the excess of silver by the Volhard method and calculate the results as explained in Chapter II. under the description of the cyanide method as used for commercial solutions of formaldehyde. The precautions and the directions for stand- ardizing there given should also be noted. By this method from 32 to 39 per cent of the formaldehyde in the milk is recovered and determined in the distillate. Assuming that the amount recovered represents 35 per cent, of the quantity in the milk, the latter can be estimated with a probable error of about one tenth. Using this as a means of studying the disappearance of formaldehyde in milk, it was found in a typical experiment in which the proportion added was 1: 40,000, that nearly three fourths of the preservative had disappeared after two days at room temperature. After four days no formaldehyde was shown by this method, but the violet color on dilution with water after heating with hydrochloric acid containing ferric chloride was unmistakable. The latter reaction could still be obtained after the mixture had stood for 1 J. Am. Chem. Soc., 1903, 25, 1036. 372 METHODS OF ORGANIC ANALYSIS two weeks. When formaldehyde is added to milk in large proportion, 1 : 1000 to 1 : 10,000, as in the preservation of samples for analysis or reference, the rate of disappearance is much slower. HYDROGEN PEROXIDE Detection Hydrogen peroxide in uncooked milk is easily detected by adding, to 10 to 15 cc. of the milk, 2 to 3 drops of a 2 per cent aqueous solution of paraphenylene diamine hydrochloride. In the presence of hydrogen peroxide a blue color appears either imme- diately upon shaking or after a few minutes, depending upon the amount present. The reaction depends upon the action of an oxidizing enzyme in the milk, and the condition of the milk, there- fore, affects the delicacy of the test. According to Arnold and Mentzel 1 1 part in 40,000 can be detected. Under ordinary con- ditions the delicacy is probably somewhat less than this. In comparative tests made immediately after adding the same amounts of peroxide to sweet milks 1 to 2 days old and to very sour curdled milks 3 to 4 days old, the former were found to give the reaction much more strongly than the latter. In practice, however, the preservative would be added while the milk was sweet and would probably disappear entirely before the occurrence of curdling. Milk which has been boiled can be tested after adding an equal volume of fresh milk known to be free from peroxide. Determination Chick, 2 in an investigation of the germicidal properties and rate of disappearance of hydrogen peroxide in milk, used a method based upon the titration of the iodine liberated by the peroxide on adding potassium iodide and sulphuric acid. 2 KI + H 2 2 + H 2 S0 4 = K 2 S0 4 + I 2 + H 2 O. Mettler 3 has used the method with satisfactory results in the following modified form: To 40 cc. of water, 0.5 gram of 1 Z. Nahr.-Genussm., 1903, 6, 306. 2 CentralUatt fur Bacteriologie und Parasitenkunde, II. Abth., 1901, 1, T05. 3 Thesis for the degree of Bachelor of Science, Columbia University, 1905. FOOD PRESERVATIVES 373 potassium iodide, and 10 cc. of 12 per cent sulphuric acid in a glass-stoppered flask, add 10 cc. of the milk, stopper tight, and allow to stand in a cool, dark place for two and one half hours. In order to guard against any possible loss of iodine during this time, use a flask with flaring mouth as described in connection with the determination of the iodine number (Chapter VIII), and fill the gutter around the stopper with a solution of potas- sium iodide. Finally titrate the iodine which has been set free in the milk, using a fiftieth-normal solution of sodium thiosul- phate. In this titration it is not necessary to use starch as in- dicator, since the disappearance of the yellow color produced by the action of the iodine upon the proteins affords a satisfactory end point. Test analyses gave results about 3 per cent too low, doubtless because of the absorption of iodine by the milk fat. This source of error can be avoided by curdling the milk with acid, filtering, and adding the iodide to a measured amount of filtrate; but this is considered inadvisable in -view of the fact that the peroxide may be undergoing decomposition during the filtration. BORIC ACID AND BORATES In routine milk analysis 1 the ash obtained in the usual way is treated with two drops of dilute hydrochloric acid and about a cubic centimeter of water. A strip of turmeric paper is then placed in the dish, allowed to soak for a minute, removed, and allowed to dry in the air. A deep red color changing to green or blue when treated with dilute alkali shows the presence of boric acid. According to Leach this reaction is delicate to 1 part in 8000. The well-known flame test with methyl alcohol is less delicate, but can be used in confirmation. The methods adopted by the Association of Official Agricul- tural Chemists are as follows: 2 Qualitative Detection Render decidedly alkaline with lime water about 25 grams of the sample and evaporate to dryness on a water bath. Ignite the residue -to destroy 1 Leach : Food Inspection and Analysis, 2d ed., p. 184. 2 U. S. Dept. Agriculture, Bur. Chem., Bui. 107, Revised. 374 METHODS OF ORGANIC ANALYSIS organic matter. Digest with about 15 cc. of water, add hydrochloric acid, drop by drop, until all is dissolved, and add 1 cc. in excess. Moisten a piece of delicate turmeric paper with the solution ; if borax or boric acid is present, the paper on drying will acquire a peculiar red color, which is changed by ammo- nium hydroxide to a dark blue-green, but is restored by acid. A preliminary test may be made by immersing a strip of turmeric paper in about 100 cc. of liquid foods, to which about 7 cc. of concentrated hydro- chloric acid has been added. Solid and pasty foods may be heated with enough water to make them thoroughly fluid, hydrochloric acid added in about the proportion of 1 to 13, and tested in the same manner. Quantitative Estimation Render 100 grams of the sample decidedly alkaline with sodium hydroxide and evaporate to dryness in a platinum dish. Ignite the residue thoroughly, heat with about 20 cc. of water, and add hydrochloric acid drop by drop r until all is dissolved. Transfer to a 100-cc. flask, the volume not being allowed to exceed 50 to 60 cc. Add 0.5 gram of calcium chloride and a few drops of phenolphthalein, then a ten per cent solution of caustic soda until a permanent slightly pink color is produced, and finally add 25 cc. of limewater. Make the volume up to 100 cc. Mix and filter through a dry filter. To 50 cc. of the filtrate add normal sulphuric acid until the pink color disappears, then methyl orange, and continue the addition of the acid until the yellow is just changed to pink. Boil to expel carbon dioxide. Add fifth-normal caustic soda until the liquid assumes the yellow tinge, excess of soda being avoided. Cool the solution, add a little phenolphthalein and an equal volume of glycerin. Titrate with standardized sodium hydroxide until a permanent pink color is produced. One cubic centimeter of fifth-normal soda solution is equal to 0.0124 gram of crystallized boric acid. Low 1 proposes the following modification of the turmeric test. Ten grams of the sample (hashed meat for instance) are mixed with 5 cc. of half-normal solution of sodium carbonate, dried, and heated until volatile matter is completely driven off. The charred mass is powdered and treated with 10 cc. of water and 1 cc. of strong hydrochloric acid. After testing a small portion of the filtrate as usual, the remainder is placed in a shallow dish with a piece of turmeric paper and allowed to evaporate at 40-50 in a desiccator, if necessary in a vacuum, when the usual color should be developed. 1 7. Am. Chem. Soc. t 28, 807. FOOD PRESERVATIVES 375 This test is said to be much more delicate than drying on a steam bath. For full details and discussion of delicacy see the original paper. In this same paper, Low gives an improved quantitative method for the determination of boric acid in food, and data on the occurrence of boric acid in common salt and in laboratory apparatus and reagents. FLUORIDES If food containing a small amount of fluoride is burned to ash in the usual way, the fluorine is likely to be almost entirely lost. In the presence of a considerable excess of alkali this loss of fluorine does not occur. To detect or determine fluorides, add 1 gram of sodium car- bonate to 100 cc. of milk, 1 evaporate, and burn to ash. If only qualitative results are required, examine for fluorides by the well-known etching test on glass. For a quantitative deter- mination of the fluorine, leach the mixture of ash and sodium carbonate thoroughly with hot water, nearly neutralize with sulphuric acid, leaving the solution slightly alkaline, and then apply Rose's method as modified by Treadwell and Koch, Z. anal. Chem., 1904, 43, 469. The details of the methods adopted by the Association of Official Agricultural Chemists are as follows : 2 Modified Method of Blarez Thoroughly mix the sample and heat 150 cc. to boiling (in the case of solid foods the filtrate prepared as directed under salicylic acid may be employed). Add to the boiling liquor 5 cc. of a 10 per cent solution of potassium sulphate and 10 cc. of a 10 per cent solution of barium acetate. Collect the precipitate in a compact mass (a centrifuge may be used advan- tageously) and wash upon a small filter. Transfer to a platinum crucible and ash. Prepare a glass plate (preferably of the thin variety commonly used for lantern slide covers) as follows : First thoroughly clean, polish, and coat on one side by carefully dipping the plate while hot in a mixture of equal parts of Carnaiiba wax and paraffin. Near the middle of the plate make a distinc- 1 Or equivalent amount of other food. 2 U. S. Dept. Agriculture, Bur. Chem., Bui. 107, Revised. 376 METHODS OF ORGANIC ANALYSIS tive mark through the wax with a sharp instrument, such as a pointed piece of wood or ivory, which will remove the wax and expose the glass without scratching the latter. Add a few drops of concentrated sulphuric acid to the residue in the cru. cible and cover with the waxed plate, having the mark nearly over the cen- ter and making sure that the crucible is firmly embedded in the wax. Place in close contact with the top or unwaxed surface of the plate a cooling device, consisting of a glass tube considerably larger in diameter than the crucible, the bottom of the tube being covered tightly with a thin sheet of pure rub- ber. A constant stream of cold water is passed through the tube. Heat the crucible for an hour at as high a temperature as practicable without melting the wax (an electric stove gives the most satisfactory form of heat). Remove the glass plate and indicate the location of the distinguishing mark on the unwaxed surface of the plate by means of gummed strips of paper, then melt off the wax by heat or a jet of steam, and thoroughly clean the glass with a soft cloth. If fluorine be present, a distinct etching will be apparent on the glass where it was exposed. Second Method If it is desired, the preceding method may be varied by mixing a small amount of precipitated silica with the precipitated calcium fluoride and applying the method given below for the detection of fluosilicates. This method is of value in the presence of foods whose ash contains a considerable amount of silica, which unites with fluorine and forms fluosili- cates. The sulphuric acid then liberates hydrofluosilicic acid, which would escape detection by the Blarez modified method. FLUOBORATES AND FLUOSILICATES (Methods of the Association of Official Agricultural Chem- ists. U. S. Dept. Agriculture, Bur. Chem., Bui. 107, Revised.) Make about 200 grams of the sample alkaline with lime water, evaporate to dryness, and incinerate. Extract the crude ash first obtained with water, to which sufficient acetic acid has been added to decompose carbonates, filter, burn the insoluble portion, extract with dilute acetic acid, and again filter. The insoluble portion now contains calcium silicate and fluoride, while the filtrate will contain all the boric acid present. First Method l Incinerate the filter containing the insoluble portion, mix with a little precipitated silica, and place, with the addition of 1 or 2 cc. of concentrated sulphuric acid, in a short test tube, which is attached to a small U-tube con- 1 Niviere and Hubert, Moniteur scientifique, 1895 [4], 9, 324. FOOD PRESERVATIVES 377 taining a few drops of water. Place the test tube in a beaker of water and keep it hot on the steam bath for from 30 to 40 minutes. If any fluoride be present, the silicon fluoride generated will be decomposed by the water in the U-tube and will form a gelatinous deposit on the walls of the tube. Now test the filtrate as directed under boric acid. If both hydrofluoric and boric acids be present, it is probable that they are combined as boro- fluoride. If, however, silicon fluoride is detected and not boric acid, the operation is repeated without the introduction of the silica, in which case the formation of the silicon skeleton is conclusive evidence of the presence of fluosilicate. Second Method Incinerate the filter containing the insoluble portion in a platinum cru- cible, mix with a little precipitated silica, and add 1 cc. of concentrated sul- phuric acid. Cover the crucible with a watch glass, to the under side of which a drop of water is suspended, and heat an hour at the temperature of 70 to 80 C. 1 The silicon fluoride which is formed is decomposed by the water, leaving a gelatinous deposit of silica and etching a ring at the pe- riphery of the drop of water. Test the filtrate for boric acid as described above. SULPHUROUS ACID The methods adopted by the Association of Official Agri- cultural Chemists are as follows : Qualitative Detection 2 To about 25 grams of the sample (with the addition of water, if necessary), placed in a 200-cc. Erlenmeyer flask, add some sulphur -free zinc and several cubic centimeters of hydrochloric acid. In the presence of sulphites hydro- gen sulphide will be generated and may be tested for with lead paper. Traces of metallic sulphides are occasionally present in vegetables, and the above test will indicate sulphites. Hence positive results obtained by this method should be verified by the distillation method. It is always advisable to make the quantitative determination of sulphites, owing to the danger that the test may be due to traces of sulphides. A trace is not to be considered sufficient indication of the presence of sulphur dioxide either as a bleaching agent or as a preservative. Quantitative Distillation Method 8 Distill from 20 to 100 grams of the sample (adding recently boiled water, if necessary) in a current of carbon dioxide, after the addition of about 5 cc. 1 The watch glass may be kept cool by means of a piece of ice. 2 U. S. Dept. Agriculture, Bur. Chein., Bui. 107, Revised. 3 Ibid., Bui. 137, p. 116. 378 METHODS OF ORGANIC ANALYSIS of a 20 per cent solution of glacial phosphoric acid, until 150 cc. have passed over. Collect the distillate in about 100 cc. of nearly saturated bromine water. Allow the end of the condenser to dip below the surface of the liquid in the receiver. The method and apparatus may be simplified without mate- rial loss in accuracy by omitting the current of carbon dioxide, adding 10 cc. of phosphoric acid instead of 5 cc., and dropping into the distilling flask a piece of sodium bicarbonate weighing not more than a gram, immediately before attaching the condenser. The carbon dioxide liberated is not sufficient to expel the air entirely from the apparatus, but will prevent oxidation to a large extent. When the distillation is finished,, boil off the excess of bro- mine, dilute the solution to about 250 cc., add 5 cc. of hydrochloric acid (1 part of the concentrated acid to 3 of water), heat to boiling, and precipi- tate the sulphuric acid with a 10 per cent solution of barium chloride. Boil for a few minutes longer, allow to stand overnight in a warm place, filter on a weighed Gooch crucible, wash with hot water, ignite at a dull red heat, and weigh as barium sulphate. Horne 1 suggests that in distilling sulphurous acid it be passed through cadmium chloride solution to remove hydrogen sulphide before it reaches the bromine or iodine solution. For other methods of detecting and determining sulphurous acid and sulphites, as well as for discussion and interpretation, see references at the end of this chapter. SALICYLIC ACID The method and precautionary notes of the Association of Official Agricultural Chemists are as follows: 2 A small amount of salicylic acid occurs naturally in many fruits, and not more than 50 grams should be used for its qualitative detection in the ex- amination of foods. A reaction obtained with this amount is due to added salicylic acid. The method described below is intended for the quantitative determination of salicylic acid. If only a qualitative determination be de- sired, many of the details may be omitted. If the material be a solid or semisolid, macerate the sample in a mortar with water made slightly alkaline, and strain through a cotton bag or sepa- rate by means of a centrifuge. If preferred, macerate from 200 to 300 grams with about 400 cc. of water, and use aliquots of the filtrate for the deter- mination of preservatives. In quantitative work place the macerated mass in a graduated flask, make up to a definite volume with water, and shake from time to time until solu- 1 U. S. Dept. Agriculture, Bur. Chem., Bui. 105, p. 125. 2 Ibid., Bui. 107, Revised. FOOD PRESERVATIVES 379 tion is complete. Then strain as directed above and use an aliquot of the nitrate for extraction. Extract in a separatory funnel 100 cc. of the sample or of the aqueous solution prepared from the sample as described above with a sufficient amount of sulphuric ether 1 to prevent emulsion after the addition of 2 or 3 cc. of dilute (1-3) sulphuric acid. Separate the clear aqueous solution, and if any emulsion is present, give the separatory funnel a quick, vigorous shake, and allow to settle again. If the emulsion is not broken up in this way, it may be accomplished by means of a centrifuge, or by adding 10 or 15 cc. of low boiling point gasoline or petroleum ether, and shaking again. Separate the clear, aqueous portion obtained from the emulsion and add it to the first aqueous portion separated. Then pour the ether into another separatory funnel, care being taken that none of the aqueous portion is left with the ether. Return the aqueous portion to the separatory funnel and again extract with ether, following the same procedure as before. Repeat this operation twice again, four separate extractions with ether being made in all. In case of special difficulty in breaking up the emulsion in any of the 1 If the nature of the substance is such that extraction with organic solvents is not practicable, as in the case of the presence of a large amount of fat, the salicylic acid may first be separated by distillation. In such cases acidify the macerated material with phosphoric acid and transfer to a distilling flask with a very short neck and wide mouth. An Erlenmeyer flask with inside diameter of mouth of 1| inches is a good shape. The tube connecting the flask with con- denser should be very short, with an inside diameter of not less than f of an inch. Conduct steam through a small tube passing through the stopper and dipping deeply into the material in the flask. The distillation of the salicylic acid is facilitated by submerging the distilling flask almost to the stopper in an oil bath and distilling with the temperature of the oil at from 120 to 130 C. , or by adding about 20 grams of sodium chloride to the contents of the flask for each 100 cc. of the substance, to raise the boiling point. Care must be taken not to let the contents of the flask get too low, as the heat will decompose the organic matter. Collect at least 600 cc. of the distillate and continue the distillation until the last 200 cc. gives no color on the addition of a drop of ferric solution. The dis- tilling apparatus should in all cases be tested with known amounts of salicylic acid in order to determine the amount of distillate necessary to carry over a definite weight of salicylic acid. It is sometimes practicable to determine the salicylic acid directly in the dis- tillate by the colorimetric method with ferric chloride given above. If the min- eral acid used in the distillation be carried over mechanically, however, the accuracy of the method is greatly impaired. Salicylic acid may be recovered from the distillate after making alkaline and evaporating, if desired, by extrac- tion with ether and estimating colorimetrically as directed above. 380 METHODS OF ORGANIC ANALYSIS extractions, a small amount of ether may be allowed to remain with the aqueous portion rather than the reverse, as it is removed in successive ex- tractions. Wash the combined ether extracts by shaking in a separatory funnel with one tenth their volume of water (using, however, not less than 20 cc. of water at each washing). Care must be taken at each washing to separate the aqueous portion completely from the ether, but none of the ether should be allowed to run into the wash water. Distill slowly the greater part of the ether, transfer the remainder to a porcelain dish, and allow to evaporate spontaneously. Thoroughly dry in a vacuum desiccator 1 over sulphuric acid, extract the dry residue with ten portions of 10 or 15 cc. each of carbon bisulphide or low boiling point petro- leum ether, rubbing the contents of the dish with a glass rod or other suit- able instrument and transferring the successive portions of solvent to a second porcelain dish. The extracted residue should finally be tested with a drop of ferric-alum solution, and if any reaction for salicylic acid be given it should be taken up in water, reextracted with ether, and the operation repeated. The gasoline extract is finally allowed to evaporate spontane- ously. Dissolve the residue in a small amount of hot water and dilute to a defi- nite volume. Dilute aliquots of the solution and match, in Nessler tubes or with a colorimeter, the color obtained by adding a few drops of ferric chlo- ride or ferric alum solution with that of a standard solution of salicylic acid containing about 1 mg. of salicylic acid in 50 cc. A 0.5 per cent solution of ferric chloride should be used, or a 2 per cent solution of ferric alum. 2 In either case, and especially with ferric chlorid, an excess of reagent should be avoided, although an excess of 0.5 cc. of 2 per cent ferric alum solution may be added to 50 cc. of the solution of salicylic acid without impairing the results. Salicylic acid may often be separated from fat extracted with the ether by washing the ether solution with dilute ammonium hydroxide. Then evaporate the aqueous liquid almost to dryness and test with ferric solution. 1 In examining a substance whose ether extract does not give a color or pre- cipitate with ferric solution, the drying of the residue and its extraction with gasoline may be omitted. The residue may then be transferred by means of warm water directly from the distilling flask to the graduated flask, in which it is made up to a definite volume. Substances interfering with the ferric reaction may often be removed by precipitation with ferric chloride or lime. 2 This solution should be boiled until a precipitate appears, allowed to 'settle, and filtered. The acidity of the solution is slightly increased in this manner, but so precipitated it keeps clear for a considerable time, and the turbidity caused by its dilution with water is much less and does not appear for a much longer time than if the unboiled solution is employed. This turbidity is espe- cially objectionable in the quantitative estimation of salicylic acid, as it interferes with the exact matching of the color. FOOD PRESERVATIVES 381 In the case of foods which yield to the gasoline solution of the ether resi- due a color that obscures the ferric chloride reaction (for example, tomatoes), the ether solution may be evaporated, the residue dried in a desiccator or in a current of dry air, sublimed, and collected on a watch glass cooled with ice. Then dissolve the sublimate in hot water and test with ferric alum. The same difficulty may often be avoided, and in fact the extraction with gasoline of the dry residue from the ether extraction may sometimes be ob- viated, by precipitating before extraction with ferric chloride or calcium chloride, making alkaline, and filtering. By this means tannin is entirely separated from the product, and other substances whose color masks the sali- cylic acid reaction are often removed. Delicacy of the Ferric Chloride Test. Using fresh 1 per cent ferric chloride as reagent the test is delicate in our hands to a dilution of about 1 : 400,000 when applied to 10 c6. of solution, about 1 : 750,000 to 1 : 1,000,000 if 25 cc. of solution be tested. The violet color obtained with such small amounts of salicylic acid must be observed quickly, as it fades rapidly, passing through a rose-red color. A faint rose color may also be obtained on addition of ferric chloride to solutions containing salicylic acid in amounts too small to show violet reaction. Interpretation. The formation of a violet color with ferric chloride is a reaction by no means confined to salicylic acid. Mulliken's tables 1 include many colorless compounds which give more or less distinctly violet reactions with ferric chloride, and some of these also resemble salicylic acid in solubilities and even in volatility. It is not safe to assume in testing foods that a constituent volatile with steam, soluble in ether, capable of sublimation and crystallization, and giving a violet reaction with ferric chloride, is necessarily salicylic acid. Brand 2 found that an extract of caramel malt yielded a sub- stance not salicylic acid which showed all of these properties. This substance he called " maltol." The same or similar interfering substances have been found in dark beers 3 and in solid foods consisting partly of baked cereal products. 4 1 Identification of Pure Organic Compounds. 2 Z. ges. Brauw., 15, 303 ; and Ber., 27, 806. 8 Abraham : J. de Pharm. de Liege, 1898, 5, 173 ; Z. Nahr.-Genussm., 1, 157. 4 Backer Ann. de falsifications, Nov., 1909. Sherman: J. Ind. Eng. Chem., 2, 24. Backe: Compt. rend., 150, 540 ; 151, 78. 382 METHODS OF ORGANIC ANALYSIS Among the tests for salicylic acid, other than the ferric chloride reaction, are the formation of the methyl ester or the nitro-compound, the reactions with bromine water and with Millons's reagent, and the Jorissen test. The adoption by Mulliken of the methyl-ester and nitration tests for the identification of salicylic acid is sufficient evidence of their value for cases in which enough salicylic acid is involved to make them available; but these tests and also the test with bromine water seem not to be sufficiently delicate for the detec- tion of very small amounts. The Millon and Jorissen tests, however, are very delicate, and should be commonly used to confirm the findings of the official ferric chloride test. Test with Millori s Reagent l To 10 to 20 cc. of the final aqueous solution to be tested, add 2 drops of Millon's reagent (prepared as described in Chapter XV), mix by shaking, and immerse in boiling water for 45 minutes unless a sufficient color develops in a shorter time. In the presence of salicylic acid a red or pink color is obtained. By heating, if necessary, for as long as 45 minutes, this test is made so delicate that with practice and with blank tests for comparison no difficulty was found in detecting the presence of 1 part salicylic acid in 2,000,000 of water when 20 cc. were tested; when only 10 cc. were tested, the pinkish tint was barely perceptible at this dilution. Longer heating and varia- tions in the amount of reagent added were tried without appre- ciably altering the result. The limit of delicacy of the test with Millon's reagent as here used seems, therefore, to be reached by heating in boiling water for 45 minutes and to lie at a dilution of about 1:2,000,000. The Millon reaction also has the advantage over the ferric chloride test that the color produced even with very small amounts of salicylic acid shows no evidence of fading on stand- ing overnight; but on account of the large number of sub- 1 Sherman and Gross : J. Ind. Eng. Chem., 3, 492. FOOD PRESERVATIVES 383 stances which respond to the Millon reagent 1 it seems unlikely that this reaction will prove as useful as that of Jorissen. Jorissen Test 2 This test in its original form is as follows : To the solution to be tested add 4 or 5 drops of a 10 per cent solution of potassium (or sodium) nitrite, 4 or 5 drops of acetic acid, 1 drop of a 10 per cent solution of copper sulphate, and heat to boiling. In the presence of salicylic acid the solution turns reddish and with more than a very minute amount be- comes blood-red. Jorissen found that phenol behaved in the same way, but benzoic acid did not. Abraham found that maltol does not give this reaction and recommended it as the most reliable test for salicylic acid. By diminishing the amount of copper used in the above directions and prolonging the heating, this test can be made much more delicate than at first reported and considerably more delicate than the ferric chloride ' reaction. The longer heating is necessary to fully develop the characteristic color, at least when only very small amounts of salicylic acid are present, and the reduction in the amount of copper diminishes the slight green color due to the reagent which otherwise may interfere with the more delicate tests. The modified Jorissen test as now used for very small amounts of salicylic acid is as follows : 3 Bring the solution to be tested into a test tube, add 4-5 drops of 10 per cent sodium or potassium nitrite, 4-5 drops of 50 per cent acetic acid, and 1 drop of one per cent copper sulphate. Shake after addition of each reagent and finally place in a boiling water bath in such a position that the test liquid is completely immersed in the boiling water and allow to stand for 45 minutes, then 1 Vaubel: Z. angew. Chem., 1900, 1125. Nasse: Pfluger's. Archiv f. d. ges. Physiol., 83, 361 (1901). Mann: Physiological Histology, pp. 321-323, and Chemistry of the Proteids, p. 7. 2 Jorissen : Bulletins de I" 1 Academic Royal des Sciences, etc., Belgique, 3d series, 3, 259. Sherman: J. Ind. Eng. Chem., 2, 24. Sherman and Gross: Ibid., 3, 492. 3 Sherman and Gross : J. Ind. Eng. Chem., 3, 492. 384 METHODS OF ORGANIC ANALYSIS remove, allow to cool, and examine against a white background, viewing the tube both vertically and horizontally and compar- ing with a blank test in which the same amounts of reagents have been added to pure water. In this way, the presence of as little as 0.005 to 0.01 milli- gram of salicylic acid in pure water solution can be detected. Faint but perceptible reactions were obtained with 5 to 8 cc. of a solution of 1 : 1,000,000 and with 18 to 25 cc. of solutions of 1 : 3,000,000 to 1 : 3,500,000. No advantage has been found in a brine bath over a water bath, in longer heating than 45 minutes, nor in varying the amounts of nitrite and acetic acid used. When larger amounts of salicylic acid are present, a drop of stronger copper sulphate solution may be used, up to a 10 per cent solution as originally recommended. Except with very small amounts of salicylic acid the red color of the Jorissen reaction develops quickly on heating, and the long immersion in the water bath then becomes unnecessary if only qualitative results are required. A feature which will be of great importance in colorimetric estimations of small amounts of salicylic acid is that while the violet color of the ferric chloride test fades rapidly, the red color of the Jorissen test is quite stable. Even the faint colors obtained by long heating, where only very minute amounts of salicylic acid are involved, have shown no deterioration when, allowed to stand overnight. It may also be noted that the ferric chloride and Jorissen. tests may be applied to the same portion of solution. After making the ferric chloride test the solution is cautiously diluted with water until the violet color just disappears and then very carefully submitted to the modified Jorissen test, when if salicylic acid is present a pink color will appear. Maltol, isomaltol, orcin, arbutin, resorcin, phlprizin, and methyl-ethyl-aceto-acetate (all of which are among the sub- stances giving blue, violet, or violet-red colors with ferric chloride) do not respond to the Jorissen reaction. Phenol gives about the same color as salicylic acid in both the Millon and the Jorissen tests, but the limits of delicacy are FOOD PRESERVATIVES 385 quite different. Phenol can be detected by the Millon reaction to about 1 : 2,000,000. In the Jorissen test, phenol 1 : 100,000 gives practically the same color as salicylic acid 1 : 1,000,000. Saligenin gives, in the Jorissen reaction, a red color at 1:10,000; a yellowish tint at 1:100,000; no . reaction at 1 : 1,000,000. The limit of delicacy for the ferric chloride re- action with saligenin lies between 1 : 10,000 and 1 : 20,000. 2-oxy-isophthalic acid gives the Jorissen reaction up to a dilution of 1 : 100,000 but is easily distinguished from salicylic acid in the color which it gives with ferric chloride. BENZOIC ACID AND BENZOATES The methods of the Association of Official Agricultural Chemists l are as follows : QUALITATIVE DETECTION Separate benzole acid as directed for salicylic acid. If benzoic acid be present in considerable quantity, it will crystallize from the evaporated ether in shining leaflets with characteristic odor on heating. Dissolve the residue in hot water, divide into two portions (a) and (6), and test by the following methods : (1) First Method Make portion (a) alkaline with ammonium hydroxide, expel the excess of ammonia by evaporation, take up the residue with water, and add a few drops of a neutral 0.5 per cent solution of ferric chloride. The presence of benzoic acid will be indicated by the formation of a brownish colored pre- cipitate of ferric benzoate. (2) Second Method (Mahler's Method Modified) Add to the water solution (portion 5), prepared as described above, from 1 to 3 cc. of third-normal sodium hydroxide and evaporate to dryness. To the residue, add 5 to 10 drops of concentrated sulphuric acid and a small crystal of potassium nitrate. Heat for 10 minutes in glycerol bath at 120 to 130 C., or for 20 minutes in a boiling water bath. This causes the formation of meta-di-nitro-benzoic acid. In no case must the temperature exceed 130 C. After cooling, add 1 cc. of water, and make decidedly ammoniacal ; boil the solution, to break up any ammonium nitrite which may have been formed. Cool and add a drop of fresh colorless ammonium sulphide, without allowing the layers to mix. A red-brown ring indicates benzoic acid. This is due to 1 U. S. Dept. Agriculture, Bur. Chem.,Bul. 107, Revised, p. 181; and Bui. 137, pp. 110-112, 113, 117-118. 2c 386 METHODS OF ORGANIC ANALYSIS the formation of ammonium rneta-di-amido-benzoic acid. On mixing, the color diffuses through the whole liquid; on heating it finally changes to greenish yellow, owing to the decomposition of the amido acid. This fur- nishes a means of distinguishing benzoic acid from salicylic or cinnamic acids. Both the latter form amido compounds, which are not destroyed by heating. The presence of phenolphthalein interferes with this test. QUANTITATIVE ESTIMATION General Method of Preparation Grind in a sausage machine, if solid or semisolid, thoroughly mix the sample, and transfer a convenient quantity (about 150 grams) to a 500-cc. graduated flask. Add enough pulverized sodium chloride to saturate the water in the sample, render alkaline with sodium hydroxide or milk of lime, and dilute to the mark with a saturated salt solution. Allow to stand for at least two hours with frequent shaking, and filter. If the sample contains large amounts of matter precipitable by salt solution, it is advisable to follow a method similar to that given under " Salt or dried fish." When alcohol is present, follow the method given under " Cider and similar products containing al- cohol." Where large amounts of fats are present, it is well to make an alka- line extraction of the filtrate before proceeding as directed under Extraction and Titration. The following will illustrate the manner of applying the method to various classes of food products : Special Methods of Preparation Ketchup. To 150 grams of the sample add 15 grams of pulverized sodium chloride and transfer the mixture to a 500-cc. graduated flask, using about 150 cc. of a saturated solution of sodium chloride for rinsing. Make slightly alkaline to litmus paper with strong sodium hydroxide and complete the dilution to 500 cc. with saturated salt solution. Allow to stand at least two hours with frequent shaking and then filter through a large folded filter. If any difficulty is experienced, the mixture may be centrif uged or squeezed through a muslin bag before filtering. Jellies, Jams, Preserves and Marmalades. Dissolve 150 grams of the sample in about 150 cc. of saturated salt solution and add 15 grams of pulverized sodium chloride. Render alkaline to litmus paper with milk of lime. Transfer to a 500-cc. graduated flask and dilute to the mark with saturated salt solution. Allow to stand at least two hours with frequent shaking, centrifuge if necessary, and filter through a large folded filter. Cider and Similar Products containing Alcohol. Render 250 cc. of the sample alkaline to litmus paper with sodium hydroxide and evaporate on the steam bath to about 100 cc. Transfer the sample to a 250-cc. flask, add 30 grams of pulverized sodium chloride, and shake until dissolved. Dilute to the FOOD PRESERVATIVES 387 original volume, 250 cc., with saturated salt solution, allow to stand at least two hours with frequent shaking, and filter through a folded filter. Salt or Dried Fish. Transfer 50 grams of the ground sample to a 500-cc. flask with water. Make slightly alkaline to litmus paper with strong sodium hydroxide and dilute to the mark with water. Allow to stand at least two hours with frequent shaking and then filter through a folded filter. Pipette accurately as large a portion of the filtrate as possible (at least 300 cc.) into a second 500-cc. flask. Add 30 grams of pulverized sodium chloride for each 100 cc. of solution. Shake until the salt has dissolved and dilute to the mark with saturated salt solution. Mix thoroughly and filter off the precipitated protein matter on a folded filter. Extraction and Titration Pipette a convenient portion of the filtrate (100 to 200 cc.), obtained as above, into a separatory funnel. Neutralize the solution to litmus paper with hydrochloric acid (1 : 3) and add an excess of 5 cc. of the same acid. In the case of salt fish a precipitation of protein matter usually occurs on acidifying, but the precipitate does not interfere with the extraction. Ex- tract carefully with chloroform, using successive portions of 70, 50, 40, and 30 cc. To avoid emulsion shake each time cautiously (vigorous shaking is not necessary). The chloroform layer usually separates readily at the bottom of the funnel after standing a few minutes. If any emulsion forms, it can be broken up by stirring the chloroform layer with a glass rod. If this is unsuccessful, the emulsified portion may be drawn off into a second funnel and given one or two sharp shakes from one end of the funnel to the other. If this also fails, the emulsion should be centrif uged for a few moments. As this is a progressive extraction great care must be taken to draw off as much of the clear chloroform solution as possible after each extraction, but under no circumstances must any of the emulsion be drawn off with the chloroform layer. If care is taken not to draw off any of the emulsion, it is unnecessary to wash the chloroform extract. Transfer the combined chloroform extract to a porcelain dish, rinsing the container several times with a few cubic centimeters of chloroform, and evaporate to dryness at room temperature in a current of dry air. (See note.) Dry the residue overnight (or until no odor of acetic acid can be detected in case the product is a ketchup) in a sulphuric acid desiccator. Dissolve the residue of benzoic acid in neutral alcohol (30 to 50 cc.), add about one fourth this volume of water, a drop or two of phenolphthalein solution, and titrate with twentieth-normal sodium hydroxide ; 1 cc. of twentieth-normal sodium hydroxide = 0.0072 gram anhydrous sodium benzoate. Note. If a blast is convenient, it is preferable to evaporate the whole ex- tract at room temperature. For this purpose the following simple apparatus may be used : A wide-mouth salt bottle is fitted with a cork ; a glass tube extends through the center of the cork to the bottom of the bottle, and its 388 METHODS OF ORGANIC ANALYSIS upper end is attached to the blast by a rubber tube. As many other glass tubes as convenient are passed through the cork around the central tube. These terminate just inside the cork, and outside the cork are bent outward and downward at an angle of about 45 C. The bottle is filled with calcium chlorid and by this means a current of dry air can be delivered to the dish containing the extract. In the absence of a blast an electric fan may be used for evaporating the extract. If it is impracticable to evaporate the chloroform spontaneously or by means of a blast it maybe transferred from the separatory funnel to a300-cc. Erlenmeyer flask, rinsing the separatory funnel three times with 5 or 10 cc. of chloroform. Distill very carefully to about one-fifth the original volume, keeping the temperature down so that the chloroform comes over in drops, not in a steady stream. Then transfer the extract to a porcelain evaporating dish, rinsing the flask three times with 5 or 10 cc. portions of chloroform and evaporate to dryness spontaneously. SACCHAKIN The usual method of testing for saccharin is to' extract it by means of ether, then convert it into sodium salicylate by heat- ing with sodium hydroxide, and finally apply the test for salicylate. The sweet taste of the saccharin serves as a pre- liminary test for its presence in the ether extract. The recent ruling under the Federal Food and Drugs Act against the presence of saccharin in foods adds greatly to the significance of its presence or absence. The analyst therefore should not only follow the details of the official method with care in order to avoid confusing saccharin with " false saccharin" or salicylic acid, but should also consult the original papers on both saccharin and salicylic acid which are given in the refer- ences at the end of this chapter. ' The qualitative method and precautionary notes of the Asso- ciation of Official Agricultural Chemists are as follows : Extract with ether (after maceration and exhaustion with water, if nec- essary), as described under salicylic acid. Allow the ether extract to evap- orate spontaneously and note the taste of the residue. The presence of saccharin to the amount of 20 mg. per liter is indicated by a sweet taste. This may be confirmed by heating with sodium hydroxide, as described below, and detecting the salicylic acid formed thereby. Results by this method indicating the presence of a faint trace of saccharin in wines which did not contain it have been frequently obtained, owing to the presence in wine of so-called " false saccharin." FOOD PRESERVATIVES 389 Acidify 50 cc. of a liquid food (or the aqueous extract of 50 grams of a solid or semisolid, prepared as directed in the official method for salicylic acid as given above) and extract with ether. Test the extracted matter in the usual way for salicylic acid, ^return the gasoline extract to the dish con- taining the residue, dilute the whole to about 10 cc. volume, and add 2 cc. of sulphuric acid (1 : 3.) Bring the solution to the boiling point and add a 5 per cent solution of potassium permanganate, drop by drop, to slight excess; partly cool the solution, dissolve in it a piece of sodium hydroxide, and filter the mixture into a silver dish (silver crucible lids are well adapted to the purpose) ; evaporate to dryness and heat for 20 minutes at 210 to 215 C. Dissolve the residue in water, acidify and extract with ether, evaporate the ether, and test the residue with two drops of a 2 per cent solution of ferric alum. By this method all the so-called false saccharin and the salicylic acid naturally present (also added salicylic acid when not present in too large amount) are destroyed, while 5 mg. of saccharin per liter is detected with certainty. BETA-NAPHTHOL * Extract 200 cc. of the sample (or of its aqueous extract prepared as on page 378) with 10 cc. of chloroform in a separatory funnel, add a few drops of alcoholic potash to the chloroform extract in a test tube, and place in a boiling water bath for two minutes. The presence of beta-naphthol is indicated by the formation of a deep blue color, which changes through green to yellow. ABRASTOL 1 (Calcium o-mono-sulphonate of ^3-naphthol) (a) Sinibaldi's Method' 2 Make 50 cc. of the sample alkaline with a few drops of ammonium hy- droxide and extract with 10 cc. of amyl alcohol (ethyl alcohol is added if an emulsion is formed). Decant the amyl alcohol, filter if turbid, and evap- orate to dryness. Add to the residue 2 cc. of a mixture of equal parts of strong nitric acid and water, heat on the water bath until half of the water is evaporated, and transfer to a test tube with the addition of 1 cc. of water. Add about 0.2 cc. of ferrous sulphate and an excess of ammonium hydroxide, drop by drop, with constant shaking. If the resultant precipitate is of a reddish color, dissolve it in a few drops of sulphuric acid, and add ferrous sulphate and ammonium hydroxide as before. As soon as a dark-colored or greenish precipitate has been obtained, introduce 5 cc. of alcohol, dissolve the precipitate in sulphuric acid, and shake the fluid well and filter. In 1 The methods given are those adopted by the Association of Official Agri- cultural Chemists. U. S. Dept. Agriculture, Bur. Chein., Bui. 107, Revised. 2 Moniteur scientifique, 1893 (4), 7, 842. 390 METHODS OF ORGANIC ANALYSIS the absence of abrastol this method gives a colorless or light yellow liquid, while a red color is produced in the presence of 0.01 gram of abrastol. (&) Sangle'-Ferriere's Method 1 Boil 200 cc. of the sample with 8 cc. of concentrated hydrochloric acid for one hour in a flask with a reflux condenser attached. Abrastol is thus converted into beta-naphthol and is detected as directed above. SUCROL OR DULCIN 2 (para-phenetol carbamid) (a) Morpurgo's Method 3 Evaporate about 100 cc. of the sample (or of the aqueous extract pre- pared as directed on page 378) to a sirupy consistency after the addition of about 5 grams of lead carbonate, and extract the residue several times with alcohol of about 90 per cent; evaporate the alcohol extract to dryness; extract the residue with ether, and allow the ether to evaporate sponta- neously in a porcelain dish. Add 2 or 3 drops each of phenol and concen- trated sulphuric acid and heat for about 5 minutes on the water bath ; cool ; transfer to a test tube and pour ammonium hydroxide or sodium hydroxide over the surface with the least possible mixing. The presence of dulcin is indicated by formation of a blue zone at the plane of contact. (&) Jorissen's Method 4 Suspend the residue from the ether extract obtained as directed above in about 5 cc. of water ; add from 2 to 4 cc. of an approximately 10 per cent solution of mercuric nitrate, and heat from 5 to 10 minutes on the water bath. In the presence of sucrol a violet-blue color is formed, which is changed to a deep violet by the addition of lead peroxide. In the foregoing selection of methods for the detection of food preservatives, preference has been given to those which have been adopted by the Association of Official Agricultural Chemists, whose methods are usually accepted as standard in questions relating to adulteration of food. Other methods, however, should not be neglected. It is believed that the fol- lowing references will put the reader in touch with the most important literature. 1 Comp. rend., 1893, 117, 796. 2 Method of the Association of Official Agricultural Chemists (Joe. eft.). 3 Z. anal. Chem., 1896, 35, 104. 4 Ibid., p. 628. FOOD PRESERVATIVES 391 REFERENCES 1 ALLEN : Commercial Organic Analysis. BLYTH : Foods, their Composition and Analysis. LEACH : Food Inspection and Analysis. LEFFMANN and BEAM : Select Methods in Food Analysis. U. S. Dept. Agriculture, Bur. Chem., Bui. 107, Revised. Methods of the Association of Official Agricultural Chemists. II 1899. BEYTHIEN and HEMPEL : (Determination of Boric Acid and Borax in Meat Products). Z. Nahr.-Genussm., 2, 842. 1901. PELLET : Nature of the Substance giving the Ferric Chloride Re- action ; Presence of Salicylic Acid in Pure Wines. Ann. chim. anal., 6, 328 ; J. Chem. Soc., 80, ii, 701. PORTES and DESMOULIERES : Normal Occurrence of Salicylic Acid in Strawberries. Ann. chim. anal., 1901, 6, 401 ; Z. Nahr.- Genussm., 1902, 5, 468. 1902. ARNOLD and MENTZEL : Detection of Formaldehyde. Z. Nahr.- Genussm., 5, 353. WINDISCH : On the Question of the Occurrence of Salicylic Acid in Natural Wines. Z. Nahr.-Genussm., 5, 653. 1903. ARNOLD and MENTZEL : Detection of Hydrogen Peroxide in Milk. Z. Nahr.-Genussm., 6, 305. GRUNHUT : (Review of Methods for Determination of Boric Acid in Foods). Z. anal. Chem., 42, 119. TRAPHAGEN and BURKE: Occurrence of Salicylic Acid in Fruits. J. Am. Chem. Soc., 25, 242. WINDISCH : Natural Occurrence of Salicylic Acid in Strawberries and Raspberries. Z. Nahr.-Genussm., 6, 447. 1904. ALLEN and TANKARD : The Determination of Boric Acid in Cider, Fruits, etc. Analyst, 29, 301. BEYTHIEN : (Detection of Sulphurous Acid in Foods.) Z. Nahr.- Genussm., 8, 36. DESMOULIERES : Action of Ferric Chloride on Salicylic Acid, Methyl Salicylate, Hydrosalicylic Acid, and Some Other Phenol Deriva- tives. Ann. chim. anal., 8, 85; Z. Nahr.-Genussm., 7, 316. FARNSTEINER: Organically Combined Sulphurous Acid in Foods. Z. Nahr.-Genussm., 7, 449. 1 These references are to literature on the occurrence, detection, and deter- mination of preservatives and do not include articles dealing primarily with their uses and effects. 392 METHODS OF ORGANIC ANALYSIS KEEP: The Behavior of Sulphurous Acid in Foods. Z. Nahr.- Genussm., 8, 53. 1905. LEFFMANN : Detection of Abrastol. Chem. Ztg., 29, 1086. MASON : The Occurrence of Benzoic Acid Naturally in Cranberries. /. Am. Chem. Soc., 27, 613. WILLIAMS and SHERMAN : The Detection, Determination, and Rate of Disappearance of Formaldehyde in Milk. J. Am. Chem. Soc., 27, 1497. 1906. AGREE : On the Detection of Formaldehyde in Milk. /. Biol. Chem., 2, 145. CRIBB and ARNAUD : Detection of Boric Acid. Analyst, 31, 147. DUBOIS : Estimation of Salicylic Acid. J. Am. Chem. Soc., 28, 1616. FRABOT : (Detection of Fluorides in Eggs). Ann. chim. anal., 11, 330; Analyst, 31, 362. HOLLEY : The Amount of Sodium Sulphite Recoverable from Food Products as a Basis for the Estimation of the Amount Originally Present. /. Am. Chem. Soc., 28, 993. HORNE : Determination of Sulphites in Sugar Products. U. S. Dept. Agriculture, Bur. Chem., Bui. 105. KASTLE : A Test for Saccharin. Public Health and Marine Hospital Service, Hygienic Laboratory, Bui. 26 ; Review of Amer. Chem. Research, 1906, 331. Low: Boric acid; Its Detection and Determination in Large and Small Amounts. J. Am. Chem. Soc., 28, 807. MENTZEL : Determination of Sulphurous Acid in Meat. Z. Nahr.- Genussm., 11, 320. PERRIER : Occurrence of Formaldehyde in Foods. Compt. rend., 143, 600; Chem. Abs., 1, 205. SHREWSBURY: The Estimation of Preservatives in Milk. Analyst, 32, 5. TRILLAT: Production of Formaldehyde in the Caramelization of Sugar. Bull. Soc. Chim., 35, 685; Analyst, 31, 410. WOODMAN and TALBOT : The Etching Test for Small Amounts of Fluorides. J. Am. Chem. Soc., 28, 1437. 1907. ALEXANDER: Determination of Sulphurous Acid in Gelatine. J. Am. Chem. Soc., 29, 783. AZZARELLO: (On the Presence of Boric Acid in Genuine Wine). Gazz. chim. ital., 36, ii, 575; Chem. Abs., 1, 1040. DUBOIS: Determination of Salicylic Acid. J. Am. Chem. Soc., 29, 293. RAMSEY: The Formation of Formaldehyde in Solutions of Cane Sugar and its Bearing on Hehner's Test for Formaldehyde in Saccharine Mixtures. J. Proc. Royal Soc. N. S. W., 41, 172; Chem. Abs., 3, 126. FOOD PRESERVATIVES 393 RICHARDSON : Nitrates in Food. J. Am. Chem. Soc., 29, 1757. SALOMONS : Test for Abrastol. Giorn. farm, chim., 55, 481 ; Chem. Abs., 1, 1447. SCOVILLE : Berizoic vs. Cinnamic Acid in Food Analysis. Am. J. Pharm., 79, 549; Chem. Abs., 2, 674. VITALI : Detection of Salicylic Acid in Wine. J. Soc. Chem. Ind., 26, 269. WINTON and BAILEY: The Formation of Volatile Sulphur Com- pounds in Meat and their Influence on the Detection of Added Sulphites. J. Am. Chem. Soc., 29, 1499. WOODMAN and TALBOT : Fluorides in Malt Liquors. /. Am. Chem. Soc., 29, 1362. 1908. CARLES : (Fluorides in Wines believed to be Pure). Ann. chim. anal., 13,102; Chem. Abs., 2, 1994. FEDER: Detection of Hydrogen Peroxide in Milk. Z. Nahr.-Ge- nussm., 15, 234. GENERSICH : Detection and Estimation of Boric Acid, Salicylic Acid, and Benzoic Acid in Foods. Z. Nahr.-Genussm., 16, 210. GUDEMAN: Determination of Sulphurous Acid in Foods. J. 2nd. Eng. Chem., 1, 81. HOPFGARTNER : Reaction between Iron and Salicylic Acid. Mo- natsh. Chem., 29, 689; Chem. Abs., 3, 309. LAVALLE : Detection of Boric Acid in Foods by Means of Turmeric Paper. Chem. Ztg., 32, 816. LA WALL and BRADSHAW : The Quantitative Estimation of Benzoic Acid in Ketchup. Am. J. Pharm., 80, 171 ; Chem. Abs., 2, 1845. MANNICH and PREISS : Sensitive Test for Detection of Boric Acid in Foods. Chem. Ztg., 32, 314. PADJK : Determination of Sulphurous Acid in Gelatine. Ann. chim. anal, 13, 299; Chem. Abs., 2, 3250. ROTHENFUSSER : The Detection of Hydrogen Peroxide, Formalde- hyde, and Persulphates. Z. Nahr.-Genussm., 16, 589. SHREWSBURY and KNAPP : Rapid Method for Detection and Estima- tion of Formaldehyde in Milk. Analyst, 34, 12. STEIGER : Estimation of Small Amounts of Fluorine. J. Am. Chem. Soc., 30, 219. WEST : The Determination of Benzoic Acid in Tomato Ketchup and Other Food Products. J. Ind. Eng. Chem., 1, 190. WILKINSON and PETERS : Influence of Formaldehyde on the Detec- tion of Hydrogen Peroxide in Milk. Z. Nahr.-Gcnussm., 16, 515. WILLARD : Sulphurous Acid in Green Corn (and other vegetables) . Bull. Kansas State Board of Health, 4, 216 ; Chem. Abs., 3, 87. WOODMAN and BURWELL : Detection of Formic Acid in Food. Tech. Quart., 21, 1. 394 METHODS OF ORGANIC ANALYSIS ZERBAN and NAQUIN: Determination of Sulphurous Acid in Mo- lasses. U. S. Dept. Agriculture, Bur. Chem., Bui. 116. 1909. BACKE : (Source of Error in Salicylic Acid Test). Ann. falsifications, Nov., 1909. CARLINFANTI and TUFFI : Use and Detection of Fluorides in Tomato Conserves. Arch. farm, sper., 8, 377; Chem. Zentr., 1909, II, 1765. FISCHER and GRUENERT: Detection of Benzoic Acid in Meat and Fats. Z. Nahr.-Genussm., 17, 721. French Official Methods. Verh. kais. Gesundheitsamt., 32, 1292; Chem. Abs., 3, 554. GENTH : Test for Saccharin in Foods and Beverages. Am. J. Pharm., 81, 536 ; Chem. Abs., 4, 352. HILLYER : Method for determining Sodium Benzoate in Ketchups or Other Food Materials. J. Ind. Eng. Chem., 1, 538. JORGENSON: Detection of Saccharin in Beer. Ann. falsifications, 2, 58. LANGE : Sulphurous Acid in Gelatine. Arb. kais. Gesundheitsamte, 32, 144; Chem. Abs., 3, 2989. ROBIN: Test for Benzoic Acid in Fatty Substances. Ann. chim. anal., 13, 431 ; Chem. Abs., 3, 1938. SCHWARZ and WEBER : Quantitative Determination of Formic Acid in Fruit Sirups. Z. Nahr.-Genussm., 17, 194. TESTONI : Estimation of Saccharin in Foods. Z. Nahr.-Genussm., 18, 577. U. S. Dept. Agriculture, Bur. Chem., Bui. 116, 12. WAUTERS : Detection of Saccharin. /. Soc. Chem. Ind., 28, 733. 1910. BACKE : (Substances mistaken for Salicylic Acid in Ferric Chloride Test). Campt. rend., 150, 540; 151, 78. BERTAINCHAND and GAUVRY : Presence of Boron in Tunisian Wines. Ann. chim. anal. ,15, 179; Chem. Abs., 4, 2179. BERTRAND and AGULHON : Determination of Boric Acid. Bui. soc. chim., 7, 90, 125; Chem. Abs., 4, 1439. CASSEL : Estimation of Salicylic Acid by.the Distillation of its Dilute Aqueous Solutions. Chem. News, 101, 289 ; Chem. Abs., 4, 2426. COLLINS: The Transfer of Boric Acid from Cattle Food to Cows' Milk. Durham County Council Bui, 3, 21 ; Chem. Abs., 4, 1324. COMANDUCCI : Detection of Saccharin. Boll. chim. farm., 49, 791 ; Chem. Zentrbl., 1910, II, 1951. DOWZARD : Detection and Determination of Sulphurous Acid in Lime Juice. Am. J. Pharm., 81, 561 ; Chem. Abs., 4, 1348. DUGAST : Presence of Boron in the Wines of Algeria. Compt. rend., 150, 839; Chem. Abs., 4, 2974. FISCHER and GREUNERT : Detection of Benzoic Acid in Meat and Fats. Z. Nahr.-Genussm., 20, 580. FOOD PRESERVATIVES 395 GREIBEL : Benzole Acid in Cranberries. Z. Nahr.-Genussm., 19, 241. HUBERT : Disappearance of Sulphurous Acid (when added to wine). Ann. chim. anal, 14, 453; Chem. Abs., 4, 1080. KICKTON and BEHNCKE : Occurrence of Fluorine in Wines. Z. Nahr.-Genussm., 20, 193. KUHN and RUHLE : Determination of Sulphurous Acid in Meats. Z. Nahr.-Genussm., 20, 10; Chem. Abs., 4, 2851. PAWLOWSKI : Detection of Saccharin in Beer. Z. ges. Brauw., 32, 281; Chem. Abs., 4, 948. PELLET : Normal Presence of Salicylic Acid in Wines. Rev. soc. hyg. alim., 5, 806 ; Chem. Abs., 4, 1647. PERKIER : Presence of Formaldehyde in Certain Foods. Rev. soc. liyg. alim., 5, 804; Chem. Abs., 4, 1635. BOSSET : Detection of Fluoride in Foods. Ann. chim. anal., 14, 365 ; Chem. Abs., 4, 623. SHERMAN : A Source of Error in the Examination of Foods for Salicylic Acid. J. Ind. Eng. Chem., 2, 24. VON DER HEIDE and JAKOB : The Detection of Benzoic, Cinnamic, and Salicylic Acids in Wine. Z. Nahr.-Genussm., 19, 137. VON FELLENBERG : Determination of Salicylic Acid in Preserves. Z. Nahr.-Genussm., 20, 63. YODER and TAGGART : Occurrence of Formaldehyde in Sugar Cane Juice and Sugar-House Products. J. Ind. Eng. Chem., 2, 260. 1911. FINCKE: Determination of Formic Acid in Foods. Z. Nahr.- Genussm., 21, 1. FOLIN and FLANDERS : Determination of Benzoic Acid. J. Am. Chem. Soc., 33, 1622. FRANZEN and EGGER : Quantitative Determination of Formic Acid. J. prakt. Chem., 83, 323. LYTHGOE and MARSH : The Detection of Benzoic Acid in Coffee Extract. /. Ind. Eng. Chem., 3, 842. LOOCK : Preservatives in Fruit Juices with Special Reference to the Detection of Formic Acid. Z. offentl. Chem., 16, 350; Chem. Abs., 5. 537. POLENSKE : Detection of Benzoic Acid in Food. Arb. kais. Gesundh., 38, 149; Chem. Abs., 5, 3860. POLENSKE and KOPKE : Determination of Saltpeter in Meat. Arb. kais. Gesundh., 36, 291 ; Chem. Abs., 5, 1802. PRICE and INGERSOLL : Effects of Nitrates and Nitrites on the Turmeric Test for Boric Acid. U. S. Dept. Agr., Bur. Chem., Bui. 137, 115. SHERMAN and GROSS : The Detection of Salicylic Acid. /. Ind. Eng. Chem., 3, 492. VIERHOUT : Quantitive Estimation of Salicylic Acid in Fruit Juices. Z. Nahr.-Genussm., 21, 664. SUBJECT INDEX Abbe refractometer, 165. Abderhalden and Koelker's optical method for proteolytic enzymes, (ref.) 332. Abraham's viscosity method, 183. Abrastol, 389, 390, (ref.) 392-393. Absolute alcohol, 2, 32, 33. Acetanilid, determination in vanilla extract, (ref.) 49. Acetate, 129, (ref.) 131-132. Acetic acid, 129, (ref.) 131-132; de- termination in calcium acetate, 129- 131 ; in vinegar, 128. Acetin method for glycerol, 279. Acetone determination, 30. Acetylizable impurities in glycerin, 283. Acetyl number, 144, 160. Acid, arachidic, 134, 179, 180; behenic, 134; benzoic, 385, 386; boric, 373, 374; butyric, 133, 195; capric, 133, 195 ; caproic, 133, 195 ; caprylic, 133 ; 195 ; carnaubic, 134, 139 ; cerotic, 135; clupanodonic, 137; dihydroxystearic, 137, 195; erucic 135 ; hypogseic, 135 ; isolinolenic, 137 ; lanoceric, 138 ; lauric, 134, 195; lignoceric, 134; linoleic, 136; linolenic, 137, 139; linusic, 137; melissic, 135; myristic, 134, 195; oleic, 135, 139, 195, 197; palmitic, 134, 195; phycetoleic, 135; ricino- leic, 138; sativic, 137; stearic, 134, 195; sulphurous, 377. (See also Acids.) Acid-albumin, 311. Acidity, degrees of, 147. Acidity of butter fat, 194 ; of glycerin, 278; of lubricating oil, 231; of milk, (ref.) 367. Acid method for phosphorus, 304. Acid number, 147. Acids, fatty, 133, 138, 139, 167, 196; fatty and resin, 271 ; in butter fat, 139, 195. (See also Acid.) Acree's formaldehyde color reaction for proteins, (ref.) 331. . Added water, detection of, in milk, 363, 364, (ref.) 366-368. Addition reactions of aldehydes, 35; of formaldehyde, 44. Adiabatic calorimeter, (ref.) 264. Adulterants in oils, 174-185, 199-202, 206-210, 213-217. Albuminates, 311. Albuminoids, 308, 311. Albumins, 308, 310. Alcohol, 2, 31-33; absolute, 2, 32, 33; aldehyde-free, 172; cetyl, 169; denatured, 4, 32, 33; density of solutions, 13, 15, 16 ; detection of, 5, 23, 32, 33 ; determination of, 6-24, 31-33; fusel oil in, 28, 32, 33; methyl alcohol in, 24, 32, 33 ; myri- cyl, 169; octodecyl, 169; refer- ences, 31-33 ; standards of purity, 29 ; TL S. P., 2, 29. Alcohol-ether mixtures, analysis of, (ref.) 32. Alcohols, 1, 31-33, 169. (See also Alcohol.) Alcohol-soluble proteins, 308, 310. Alcoholysis of fatty substances, (ref.) 172. Aldehydes, 34, 47, 48, 49 ; detection of, 34; determination of in alcoholic liquors, (ref.) 32. (See also Ben- zaldehyde, Formaldehyde, Vanillin.) Alkali-albumin, 311. Alkali method for phosphorus, 303 ; for sulphur, 298. Alkalinity of vinegar ash, 123, 125, 126, 128. Allein and Gaud reagent, 78. Allen method for peptic activity, 327; for protein nitrogen, (ref.) 330. Allihn method for dextrose, 107. Allihn's table for dextrose, 108. Allspice, starch content of, 112. Almond extract, determination of ben- zaldehyde in, 46, 49. Almond oil, 171. Almond shells,, starch content of, 112. Alumina cream, 89. Aluminate in soap, 273. Ammoniacal silver nitrate test for aldehydes, 34. Amyl alcohol, 28, 32, 33. Amylases, 113-120, (ref.) 121-122. Angular rotation, 83. Apple vinegar, 123. 397 398 SUBJECT INDEX Arabinosazone, 62, 64. Arabinose, 50, 54, 63, 85. (See also Pentosans, Pentoses.) Arachidic acid, 134, 179, 180, (ref.) 199-201. Arachis oil, 171, 179, 184, (ref.) 199-201. Arnold-Wedemeyer method for nitro- gen, 289. Ash : in butter, 186 ; in coal, 256, 264 ; in grain products, 337 ; in milk, 356, 357 ; in sugar, 97 ; in vinegar, 125. Asphaltic matter in oils, 222, 235, 238. Atwater calorimeter, 239. Azo-compounds, determination of nitro- gen in, (ref.) 306. Balance, Westphal, 162. Balling hydrometer ("spindle"), 100. Bang method for reducing sugars, (ref.) 86. Baobab oils, (ref.) 202. Bardach's protein reaction, (ref.) 331. Barfoed's method for reducing sugars, 76. Barlow's method for sulphur, 296. Bates polariscope, (ref.) 86. Baudouin test, 181 ; influence of ran- cidity on, (ref.) 202. Baume hydrometer, 219. Baume scale, 219. Beef tallow, 170. Beeswax, 170, (ref.) 173. Beets, determination of sugar in, 98. Behenic acid, 134. Bellier method for arachidic acid, 181. Benedict method for reducing sugars, (ref.) 86. Benedict reagent, 78, (ref.) 86. Benzaldehyde, 46, 47, 49. Benzene, water in, (ref.) 238. Benzoate, detection and determination, 385, 386, (ref.) 393-395. Benzoic acid, detection and determina- tion of, 385, 386, (ref.) 391-395; occurrence of, in cranberries, (ref.) 392, 395 ; standardization of calo- rimeter by means of, 242; titration of, 387 ; vs. cinnamic acid in food analysis, (ref.) 393. Berthelot bomb calorimeter, 239. Berthelot method for sulphur, 301. Beta-naphthol, 389. Bicarbonate, detection of, in milk, 365. Bisulphite reaction of aldehydes, 35. Biuret reaction, 313. Blarez method for fluorides, 375. Blasdale's viscosity test, 183. Boiling point, determination of, 24. Boiling point method for alcohol, 22. Bomb calorimeter, 239. Borates, in food, 373, (ref.) 392-395; in soap, 273. Boric acid, detection and determination, 373-375, (ref.) 391-395; natural oc- currence in foods, 375, (ref.) 391- 395. Boron in wines, (ref.) 394. Bran, starch content of, 112. British thermal unit, 241. Brix hydrometers ("spindles"), 100. Bromine substitution number, 207. Bromine water test for salicylic acid, 382. Brugelmann method for sulphur, 296. Buckwheat hulls, starch content of, 112. Burning oils, (ref.) 236-237. (See also Illuminating oils.) Burning point, 233. Butter, 185 ; acid content of, (ref.) 200 ; analysis of, 186 ; detection of coco- nut fat, 198, (ref.) 202 ; Dutch, (ref.) 200, 202 ; iodine number, (ref.) 200 ; keeping qualities of, (ref.) 200 ; renovated, (ref.) 200; standard, 185; substitutes, 197-202 ; synthetic color in, (ref.) 202 ; volatile acid content, 195-197, (ref.) 200; water content, 186-187, (ref.) 201-202. Butter fat, analysis of, 1-88-194; com- position of, 195-197; references, 199-202; relation of physical and chemical characters, 196 ; separation of acids in, (ref.) 139 ; variations in properties, 195. Butyric acid, 133. Butyrin, 146. Butyro-refractometer, 165. Cacao butter, 170. Calcium acetate, 12*9. Calorific power, 245 ; determination of, 239 ; estimated and determined in wood, etc., 253; of coal, 257-265; of fatty oils, 171 ; of fuel oils and gasoline, 247 ; of organic compounds, 245; of petroleum oils, 248; rela- tion to chemical composition, 257, 260. Calorimeter, adiabatic, (ref.) 264; At- water, 239; Berthelot, 239; Emer- son, 239; Junker, 263; Mahler, 239; radiation correction, 244; ref- erences, 261-265. Calorimetry, 239-265. Cane, determination of sucrose in, 98. Cane sugar, see Sucrose. SUBJECT INDEX 399 Cane sugar factories, chemical control of, (ref.) 104. Capric acid, 133, 195. Caproic acid, 133, 195. Caprylic acid, 133, 195. Carbohydrates (general) , 50-57 ; be- havior on oxidation, 58 ; detection of, by Molisch reaction, 57 ; hy- drolysis of, 60, 94, 106, 109, 110; reactions with acids, 56 ; reactions with hydrazines, 61 ; rotation of polarized light, 78-85 ; separation by pure yeasts, (ref.) 104 ; separation in grain products, 340 ; specific ro- tatory powers, 82-85. Carbonate, in glycerin, 278 ; in milk, 365 ; in soap, 273. Carius method, (ref.) 307. Carnaliba wax, 170. Carnaiibic acid, 134, (ref.) 139. Casein determination, (ref.) 368. Casein test for formaldehyde, 39. Castor oil, 141, 142, 171. Cellulose, 53, 55, 61. Cereals, determination of starch in, 106, 110, 112, (ref.) 121 ; loss of phosphorus in ashing, (ref.) 307. Cereals, see Grain products. Cerotic acid, 135. Cetyl alcohol, 169. Chilling point, 232. Chinese wood oil, 204, (ref.) 217. Cholesterol, 169, (ref.) 200; separa- tion from phytosterol, (ref.) 201. Cider, composition of, (ref.) 132 ; de- termination of benzoic acid in, 386. Cider vinegar, 123-126, (ref.) 132. Cinnamic acid, detection of, .(ref.) 393, 395. Citral, determination of, (ref.) 49. Clarification in sugar analysis, 79, 89, 90, 92, (ref.) 105. Classon's method for sulphur, 296. Clerget method for sucrose, 94. Cleveland cup tester, 233. Cloud test, 232, (ref.) 236. Cloves, starch content of, 112. Clupanodonic acid, 137. Coagulated proteins, 312. Coal, 254-265 ; accuracy in sampling, (ref.) 263; American, (ref.) 262; calorific power, 254-265 ; classifica- tion, (ref.) 261, 262; composition in relation to calorific power, 254-260, (ref.) 262-265 ; deterioration of sam- ples, (ref.) 263; gases occluded in, (ref.) 264 ; influence of oxygen in, 254, (ref.) 264 ; losses in storage, (ref.) 264 ; production of, in 1910, (ref.) 265 ; proximate analysis, 256 ; purchase on specifications, (ref.) 263-265 ; sam- pling, (ref.) 263, 265; standard, for gasmaking, (ref.) 264 ; sulphur deter- mination, 257 ; volatile matter, 256, (ref.) 263-265 ; weathering of, (ref.) 263. Coals of Illinois, (ref.) 262. Coals of the United States, (ref.) 263. Coal tar oils, determination in other oils, (ref.) 236. Cocoa products, determination of starch in, (ref.) 121. Cocoa shells, starch content of, 112. Coconut fat, detection in butter, 198 ; ethyl ester number, (ref.) 202. Coconut oil, 141, 170; detection of, 198, (ref.) 201-202; of high iodine value, (ref.) 202. (See also Coco- nut fat.) Codliveroil, 171, (ref.) 200-201.. Coefficient of purity, see Quotient of purity. Cold test, 232, (ref.) 236. Collagens, 311. Colophony, 170, (ref.) 215: Color reactions of proteins, 313-316. Colza oil, 171. Combustion, heat of, 168, 239, 245, (ref.) 261-265. Compensator, 81. Compressed oxygen method for sulphur, 301. Condensation reactions of aldehydes, 42, 46, 48. Condensed milk, analysis of sweetened, (ref.) 368. "Constants," analytical, of oils, fats, and waxes, 143, 170, 171, 176, 196. Copak oil, 178. Copper as protein precipitant, 317. Corn stover, starch content of, 112. Cottonseed meal, influence of feeding, on fat, (ref.) 200. Cottonseed oil, 171, 177, 178, 184, 208, (ref.) 199-202. Cottonseed stearin, 170. Coumarin, determination in vanilla ex- tract, (ref.) 49. Cream, 267, 268. Creosote, characteristics of, (ref.) 217. Crismer's test, 194. Crude fiber, 337. Crude petroleum, 218, (ref.) 237. (See also Petroleum.) Cyanide method for formaldehyde, 44. 400 SUBJECT INDEX Cylinder oils, (ref .) 236. Cylinder stock, 221. Defren's method for reducing sugars, 74. Degrees of acidity (fats), 147. Denatured alcohol, 4. Density : of alcohol solutions, 13, 15, 16 ; of sugar solutions, 100 ; of water, 17. Detergents, see Soap. Dextrin, 53, 61, 85. Dextrose, 51 ; detection, 62-68, 77 ; determination, 70, 74, 107 ; reducing power, 69, 73, 75 ; rotating power, 84, 85, (ref.) 105. Diabetic foods, (ref.) 348. Diabetic sugar, 51. (See also Dex- trose.) Diastase, see Amylases. Diastase method for determination of starch, 110. Diastatic power, 113-120, (ref.) 121- 122. Dichromate method for alcohol, 23 ; for glycerol, 284. Dichromate solution as light filter, 93. Dinitrobenzoate test for alcohol, 5. Dioxystearic acid, 137, 195. Diphenyl hydrazine as reagent for sugars, 63. Dirt in milk, (ref.) 368. Disaccharides, 50, 51. Distillation method for alcohol, 8 ; for gasoline, 249 ; for petroleum, 220. Distilled vinegar, 124. Drying oils, 203. Dry lead clarification of sugar solutions, 90, (ref.) 104. Dulcin, 390. Dulong's formula, 252, 254. Dyer method for nitrogen, 291. Ebullioscope, 22. Edestin, 339. Edible oils and fats, 174. Elaidin test, 136. Elastins, 311. Emerson calorimeter, 239, (ref.) 263. Engler distillation test, 220. Engler flask, 220. . Engler viscosimeter, 226, (ref.) 237, 238. Enzymes, amylolytic, 113-120, (ref.) 121-122 ; proteolytic, 323-328, (ref.) 329, 331-333. Erucic acid, 135. Eschka method for sulphur, 257. Essential oils, aldehydes and ketones in, (ref.) 48, 49. Ester number, 147. Esters, glyceryl, 143 ; in alcoholic liquors, (ref.) 32 ; in wood alcohol, 31 ; saponification data of, 146. Ether-alcohol mixture, analysis of, (ref.) 32. Ether extract, see Fat. Ethyl ester number, (ref.) 202. Evaporation test for oils, 235. Factories, cane sugar, chemical control, (ref.) 104. Fat, analysis, 143-217 ; determination, 268, 335, 354, 358. (See also Butter, Olive Oil, etc.) Fats, 140-217; acetyl number, 160; alcohols of, 169 ; analytical methods, 143 ; analytical properties, 170, 171 ; characteristics of animal, 169, (ref.) 172, 173; classification, 141; "con- stants" of, 143, 170-171; edible, 174 ; heat of combustion, 168, 171 ; index of refraction, 164, 170, 171, 176, 196, 211; iodine number, 148-157, 170, 171, 176, 197, 211; Maumene number, 157, 171, 176, 211; melting point, 144, 167, 170; solubilities, 142 ; specific gravity, 162, 170, 171. Fatty acids, 133-139, 167. (See also Butter, Fats, etc.) Fatty and resin acids in soap, 269. Fatty oils in lubricants, 225. Fehling's method for reducing sugars, 70. Fehling's solution, 69, 70. Ferric acetate as protein precipitant, 317. Ferric chloride test for salicylic acid, 381. Fish liver oil, (ref.) 201. Fish oil, 137, 139, 205, 208, 217. Fixed carbon in coal, 257. Flashing point, 233, (ref.) 237. Flour ; analysis of, 334-340 ; composition of, 344-346; starch content of, 112. Fluoborates, 376. Fluorides, 375, (ref.) 392-395. Fluorine, see Fluorides. Fluosilicates, 376. Folin's method for sulphur, (ref.) 307. Food preservatives, 369-395. Foods, see under names of individual articles of food. Formaldehyde, 36 ; detection of, 38-40, 369-371, (ref.) 391-395; deter- mination, 40-45, 48, 49, 371, (ref.) 392, 393; influence of, on detection of hydrogen peroxide in milk, (ref.) 393 ; occurrence in foods, (ref.) 392-395. SUBJECT INDEX 401 Formic acid, detection and determina- tion in foods, (ref.) 394-395. Friction tests on lubricants, 234, (ref.) 237. Fructose, see Levulose. Fruit juices, (ref.) 395. Fruit sugar, see Levulose. Fuchsin test for aldehydes, 35. Fuels, 239-265. (See also Coal, etc.) Furfural, 32, 57. Fusel oil, 28, 32, 33. Galactans, 53, 59, 61. Galactosazone, 62, 64. Galactose, 51, 59, 62, 64, 84, 85. Gallic acid test for formaldehyde, 39. Gas-making, valuation of oils for, (ref.) 236. Gasoline, 237, 247, 250. Gelatin, 311. Gerard reagent, 78. Gliadin, 339, (ref.) 346-348. Globulins, 308, 310. Glucosazone, 62, 64-69. Glucose, see Dextrose. Glucose vinegar, 124. Glutelins, 308. Glutenin, 339. Glycerides, 142-143. (See also Fats.) Glycerin, 276-287. (See also Glycerol.) Glycerol, 1, 140, 275, 283; determina- tion by acetin method, 279 ; by dichromate method, 284 ; in soap, 270, 287 ; significance in vinegar, (ref.) 132. Glyceryl esters, 143. Glycogen, 53, 54, 61, 85. Glycoproteins, 309, 312. Grain products, 334-348 ; analysis of, 334-342 ; composition, 344-346 ; defi- nitions and standards, 343 ; diges- tibility and nutritive value, 346; references, 346-348. Grain vinegar, 124. Grape sugar, see Dextrose. Grape vinegar, 123. Graphite, deflocculated, (ref.) 236. Gray's method for crude petroleum, 221. Greases, lubricating, 235. Gunning method for nitrogen, 289, 291. Half-shade polariscope, 80, 81. Halogens, as protein precipitants, 316, 319 ; determination in organic sub- stances, (ref.) 306. Halphen's reaction, 177, (ref.) 199-201. Hanus method for iodine number, 153, 156. 2D Heat capacity of calorimeter, 240. Heat coagulation of proteins, 316. Heat of combustion, 239-245 ; adiabatic method, (ref.) 263; of fats and fatty oils, 168, 171; of fuels, 245- 265 ; of other organic substances, (ref.) 261. Heavy distillate, 221. Heavy metals as protein precipitants, 316. Hehner number, 144, 148, 191. Hemicellulose, 61. Hemoglobins, 309. Hemp oil, 171. Herzfeld's method for reducing sugars, 96. Hexabromide test, 209, 214. Histones, 308. Homogenized milk, (ref.) 367. Honey, (ref.) 105. Hopkins distilling head, 292. Hubl number, 148, 156, 170, 171. Hydrazine reactions of sugars, 61. Hydrazones, determination of nitro- gen in, (ref.) 306. Hydrochloric-acid-casein test for for- maldehyde, 39. Hydrogen peroxide, detection and de- termination, 372, (ref.) 391-393; method for formaldehyde, 41. Hydrolysis of carbohydrates, 60. Hydrometer, Baume, 219. Hydrothermal equivalent of calorimeter, 241. Hydroxy acids, 137, 138, 161. Hypogseic acid, 135. Ice cream, analysis of, (ref.) 367. Illuminating oils, 218, 221, (ref.) 236- 238. Immersion refractometer, 17-21, 32, 33. Immiscible solvents, 224. Imported sugars and molasses, testing of, (ref.) 104. Index of refraction, 17-22, 164-166. (See also under Fats.) International methods, for oil testing, (ref.) 237; for sugar analysis, 91. Invertase method for sucrose, (ref.) 105. Invert sugar, 51, 85. lodimetric method for formaldehyde, 40. Iodine number, 148-157. (See also Fats.) lodoform test for alcohol, 5. Isolinolenic acid, 137. 402 SUBJECT INDEX Japan wax, 170. Jodlbauer method for nitrogen, 290. Jorissen method for sucrol or dulcin, 390. Jorissen reaction for salicylic acid, 382. Junker gas calorimeter, (ref.) 263. Kapok oil, 178, 202. Kendall method for reducing sugars, (ref.) 86. Keratins, 311. Kjeldahl method for nitrogen, 288- 295, 306-307. Kjeldahl-Wilfarth method, 289. Koettstorfer number, 144. Lactobiose, see Lactose. Lactometer, 353. Lactosazone, 62, 64. Lactose, 51, 59, 61, 64; determination, 59, 70, 74, 361; hydrolysis of, 52, 104 ; rotating power, 84, 85 ; separa- tion from maltose, (ref.) 103 ; solu- bility, 54, 55. Lanoceric acid, 138. Lard, 170, (ref.) 199-202; effect of feed upon properties, (ref.) 202. Lard oil, 141, 171, 182, 184. Laurent polariscope, 80. Laurie acid, 134, 195. "Lead number" of maple products, (ref.) 104. Lecithoproteins, 309. Legler's method for formaldehyde, 43. Lemon oils and extracts, determina- tion of citral, (ref.) 49. Leucosin, 339. Levulinic acid reaction, 58. Levulose, 51, 84, 85, 103, 105. Lieben's test for alcohol, 5. Liebermann-Storch reaction, 207. Liebig's method for phosphorus, 303 ; for sulphur, 296, 297. Light filter, 93. Lignites, 253, 262. Lignoceric acid, 134. Linoleic acid, 136. Linolein, 146. Linolenic acid, 137, 139. Linolenin, 146. Linseed meal, starch content of, 112. Linseed oil, 141, 171, 203, 205, (ref.) 215-216 ; vs. paint as priming coat, (ref.) 215. Lintner's method for diastatic power, 115, 121. Lintner's scale, 115, 116. Linusic acid, 137. Low's test for boric acid, 374. Lubricants, 218, 222-235, (ref.) 236-238. Lubricating greases, see Lubricants. Lubricating oils, see Lubricants. Lux-Ruhemann test for saponifiable oil, 223. Mace, starch content of, 112. Magnesium nitrate method for phos- phorus, 305. Mahler calorimeter, 239. Maize oil, 171, 182, 184, 199, 200, 204, 208. Malic acid in vinegar, 125. Malt extracts, 114, 115. Maltosazone, 62, 64. Maltose, 52, 60, 70, 74, 84-86, 104-105. Malt sugar, see Maltose. Malt vinegar, 123. Mannose, 51, 85. Maple sugar, (ref.) 104. Maple sirup, (ref.) 104. Margarine, 197, 202. Marine oils, (ref.) 215. (See also Fish oils.) Maumene number, 144, 157, 159. (See also Fats.) Meat, determination of starch in, 112. Melissic acid, 135. Melting points, 6, 40, 64, 134-135, 144, 167, 170, 180, 192, 193, 202. Menhaderi oil, 171, 205. Metaformaldehyde, 37. Metaproteins, 309, 311. Methyl alcohol, 24-26, 30-33. Methylene blue test for freshness of milk, (ref.) 367. Methylene-di-/3-Naphthol test for for- maldehyde, 40. Methyl ester test for salicylic acid, 382. Methylphenylhydrazine as reagent for sugars, 63. Mett's method for proteolytic enzymes, 325, 331-332. Milk, 349-368; analysis of, 352-368; composition of, 349-352, 366-368; con- densed, (ref.) 368; detection of added water, 363, 364, 366-368; of cane sugar and calcium sucrate, (ref.) 367-368: of heated, 367; of pre- servatives in, 365 (see also Food preservatives) ; determination of dirt, (ref.) 368 ; homogenized, (ref.) 367 ; preservation of, 352, 366, 367 ; sampling, 352; serum, 363, 366- 368; standards, 349, 364, 367; watered, 363, 366-368. Milk chocolate, (ref.) 104, 105, 367. SUBJECT INDEX 403 Milk sugar, see Lactose. Milk supply, (ref.) 366-368. Millon reaction, 315, 382. Mineral oil, 171, 206, 238, 263. (See also Lubricants, Petroleum.) Mixed oils, viscosity of, 229. Moisture determination, in butter, 186 ; in grain products, 335 ; in milk, 356 ; in sugar, 97. Molasses, (ref.) 103-105; as fuel, (ref.) 262. Molisch reaction for carbohydrates, 57. Monosaccharides, 50. Morley's alcohol table, (ref.) 3. Morpurgo's method for sucrol or dulcin, 390. Mucic acid method, 59. Multirotation, 79, 86. Mulliken's test for alcohol, 5. Munson and Walker method for re- ducing sugars, (ref.) 85. Mustard oil, 171. Mutarotation, 79, 86. Muter's method for fatty acids, 136, 138. Mutton tallow, 170. Myricyl alcohol, 169. Myristic acid, 134, 195. Naphtha, 221. Naphthalene, 242. Neatsfoot oil, 171. Neumann's method for phosphorus, 304. New method for diastatic power, 117- 120. New scale for diastatic power, 117-118. New York Board of Health lactometer, 354. New York Sugar Trade Laboratory, (ref.) 105. Nitrates, in food, (ref.) 393; in milk, (ref.) 368; nitrogen in presence of, 294. Nitric acid test for oils, 178. Nitrocompounds, 294. Nitrogen determination, 288-295, (ref.) 306-307; error due to methane, (ref.) 307. Nitrogen-free extract, 334. Nitrogenous extractives, 312. Nucleoproteins, 309, 312. Nutmeg, starch content of, 112. Oatmeal, definition and standard, 343 ; starch content of, 112. (For methods of analysis, see Grain products.) Octodecyl alcohol, 169. Oil, almond, 171; arachis, 171, 179, 184, (ref.) 199-202; baobab, (ref.) 199-202 ; castor, 142, 171 ; coconut, 170, 198, (ref.) 199-202; codliver, 171, (ref.) 199-202; copac, 178; corn, 171, 182, (ref.) 199-202; cot- tonseed, 171, 177, 208, (ref.) 199- 202 ; fish, 17, 208 ; fish-liver, 171, (ref.) 199-202; groups, 141; hemp, 171; Japanese sardine, 205 ; kapok, 178, (ref.) 202; lard, 171, 182, 184; lin- seed, 171, 203, (ref.) 214-217; maize, 171, 182, 184, 204, 208, (ref.) 199- 202; menhaden, 171, 205, 208; mineral, 171, 206, 218-238; mix- tures, 159, 213, 229 ; mustard, 171 ; oleo, 171, 197; olive, 171, 174- 185, (ref.) 199-202; palm, (ref.) 199-202 ; peanut, see Arachis ; poppy- seed, 171, 182, 184, 204; rapeseed, 171, 184, (ref.) 199-202; rosin, 171, 207; salad, see Olive; seal, 171; sesame, 171, 181, 184, (ref.) 199- 202; soy bean, 204; sperm, 171; sunflower, 171; tung, 171, 204; walnut, 204. (See also Oils.) Oil analysis, 140-185, 199-238. Oil coatings, preservative, for iron and steel, (ref.) 215. Oils, acetyl number, 160 ; adulterants, 213 (see also under individual oils) ; altered by age or oxidation, 210 ; analysis (see Oil analysis) ; analytical properties, table, 170-171 ; blown, 168 ; bromine absorption, (ref.) 215 ; classification, 141 ; commercial value, 214 ; committee report on method of analysis, (ref.) 173 ; dry- ing, 141, 203-217; edible, 174-202; fatty, 140-185, 199-217, 223-226; fish, 139, 205; identification of, 213; index of refraction, 164, 170- 171 ; iodine number, 148-157, 170- 172; lubricating, 222-238; Mau- mene number, 157, 171; "new con- stant," (ref.) 173 ; non-drying, 141 ; olive, from different countries, 175- 176; oxidized, 210, (ref.) 214-215; prices, 214; refractometer readings, 164, 170-171, 172, 176, 211; salad, 172, 174, (see also Oil, olive) ; semi- drying, 141 ; specific gravity, 162, 171; "unknown," 213; unsaponi- fiable matter, 169, 173; vegetable, drying, 203; viscosity, 168, 226- 231, (ref.) 236-238. Oleic acid, 135, 195, 197 ; manufacture and examination of commercial, (ref.) 139. 404 SUBJECT INDEX Olein, 146. Oleomargarine, 197. Oleo oil, 170, 197. Olive oil, 141, 171, 174-185, (ref.) 199- 202, 216. Optical activity, of carbohydrates, 78- 85 ; of osazones, 63-64 ; of sugars, 78-85 ; of vinegars, 125. Optical method for proteolytic enzymes, (ref.) 332, 333. Organic compounds, composition and calorific power, 245. Osazones, 62-69, 306. Osborne's method for sulphur, 299. Ost reagent, 78. Ostwald pyknometer, 11. Oxidation, of alcohols, 23 ; of carbohy- drates, 58; of oils, 211. Oxygen calorimeter, 168. Oxygen method for sulphur, 301. Oxymethylene, 37. Paint, 203-205, 216, 217. Palmitic acid, 134, 173, 195. Palmitin, 146, 173. Palm oil, 170, 200. Parabrombenzylhydrazide as reagent for sugars, 63. Paraffin, 170. "Paraffin base" petroleum, 221. Paraffin oils, water in, (ref.) 238. Paraformaldehyde, 37. Paraphenetol carbamid, 390. Pavy's reagent, 78. Peanut oil, 171, 179. Peat, (ref.) 263. Pensky-Martens oil tester, 233. Pentosans, 54-58, 61. Pentoses, 50, 57, 58. Pepper, starch content of, 112. Pepsin, 324, (ref.) 331-333. Peptids, 310. Peptones, 310, 311. Petroleum, 218-238, 247-251. Petroleum oils, calorific powers, 248. Petroleum production, (ref.) 238. Phenylhydrazine method for benzalde- hyde, 46. Phosphoproteins, 309, 312. Phosphorus determination, 303-307. Phosphotungstic acid as protein pre- cipitant, 320. Phycetoleic acid, 135. Phytosterol, 162, 172, 183, 194, (ref.) 200-201. Phytosterylacetate test, 183, 194, (ref.) 200-201. Picric acid as protein precipitant, 322. Pine wood oils, (ref.) 216. Platinum resistance thermometer, (ref.) 263. Polariscope, 80-94. Polariscopic methods, 79-105. Polarization, relation to sugar content, 94. Polarized light, 78. Polenske method, 199, (ref.) 202. Polysaccharides, 50, 52. Poppyseed oil, 171, 182, 184, 204, 215. Potassium cyanide method for formalde- hyde, 44. Potatoes, determination of starch in, 106, 112, (ref.) 121. Preservative coatings for structural materials, (ref.) 216. Preservatives for food, see Food pre- servatives. Proof spirit, 3, 4. Protamines, 309. Proteans, 309. Proteases, 323, 329, 331-333. Proteins, 308-323, 329-331 ; coagulated, 310 ; conjugated, 309 ; derived, 309 ; in grain products, 338 ; in milk, 369 ; simple, 308. Proteolytic enzymes, 323-329, 331- 333. Proteolytic power, 324-329, 331-333. Proteoses, 310, 311. Pulfrich refractometer, 165. Purity of sugar solutions, 100. Pyknometer, 11, 164. Quartz compensator, 81. Quartz wedges, 81. Quevenne lactometer, 353, 354. Quotient of purity, 100. Radiation correction, 244. Rafinose, 50, 52, 59, 61, 85, 104. Rancidity of butter fat, 194. Rapeseed oil, 141, 171, 184, 202. Raw sugar, 87, 104. Rectified spirit, 4. Reducing sugars, 69, 85, 86, 96. Redwood viscosimeter, 227. Refraction, index of, 17-19, 32-33, 144, 164, 166, 170-171, 172, 176, 211. Refractometer, 17-19, 32-33, 165, 364. Refractometric methods, 17-19, 32-33, 165, 364, 367-368. Regnault-Pfaundler cooling correction, 244. Regulations of international commission on sugar analysis, 91. SUBJECT INDEX 405 Reichert-Meissl (Reichert-Wollny) num- ber, 144, 148, 188, 196, 200-202. * Renard-Tolman method for arachidic acid, 179. Resin acids in soap, 271, 287. Resin and resin oil in linseed oil, 207. Resinous products in mineral oils, (ref.) 237. Resorcin test for formaldehyde, 38. Ricinoleic acid, 138. Ricinolein, 146. Rose method for fluorides, 375. Rosin, 170, 207; in shellac, (ref.) 215; in soaps, 271, (ref.) 287 ; in varnishes, (ref.) 215 ; size, (ref.) 215. Rosin oil, 171, 207. Rotation and rotating power, 78-86. Rotation dispersion, 83. Rubber, determination of sulphur in, (ref.) 307. Saccharimeter, 81, 82; observations, unification of, 104. Saccharin, detection, 388, (ref.) 391- 395; estimation in foods, 394. Saccharose, see Sucrose. Salad oil, 174-185, (ref.) 199-202. Salicylic acid, 378-385, (ref.) 391-395; action of ferric chloride on, (ref.) 391 ; bromine water test, 382 ; detection, 378-385, (ref.) 393, 395 ; determina- tion, 378-384 ; (ref.) 391-394 ; ferric chloride test, delicacy, interpreta- tion, 381 ; in pure wines, (ref.) 391, 395; in strawberries, (ref.) 391 ; in fruits, (ref.) 391 ; Jorissen test, 382-383 ; methyl ester test, 382 ; Millon's reagent test, 383 ; reaction with iron, (ref.) 393 ; substances mistaken for, in ferric chloride test, 381, (ref.) 394, 395. Saliva method for determination of starch, 110. Saltpeter, determination in meat, (ref.) 395. Sangle Ferrieres method for abrastol, 390. Saponifiable oil, test for, in lubricating oil, 223. Saponification, data on pure esters, 146 ; equivalent, 146 ; number, 144 ; num- ber of butter fat, 191, 196; num- ber (table), 170-171. Sardine oil, Japanese, 205. Sativic acid, 137. Sauer's method for sulphur, 296. Sausages, determination of starch in, 112. Seal oil, 171. Sesame, meal, influence of feeding, on fat, (ref.) 200; oil, 171, 181, 184, (ref.) 199-202. Shellac analysis, (ref.) 216, 217. Sidersky reagent, 78. Silicate in soap, 273. Sinibaldi's method for abrastol, 389. Sitosterol, 172. Skeletins, 311. Smith and Menzies' method for deter- mination of boiling point, 24. Soap, 266-275,- (ref.) 286-287; alumi- nate in, 273; borate in, 273; car- bonate in, 273 ; chlorides, 271 ; com- mercial, analysis, 266 ; determina- tion of water, 267 ; extraction with alcohol, 272 ; fatty acid content, (ref.) 287; fatty and resin acids, 269, 271; free alkali or acid, 273; glycerol in, 270, (ref.) 287 ; industry, recent development, (ref.) 287 ; insoluble matter, 274 ; petroleum ether extract, 268 ; residue insoluble in alcohol, 273. Soaps, antiseptic value, (ref.) 287; commercial, composition, (ref.) 287 ; free alkali, (ref.) 286 ; from different glycerides, (ref.) 287 ; germicidal and insecticidal values, (ref.) 287 ; rosin in, 287 ; silicate, 273 ; soluble fatty acids, 270; significance in disinfectants, (ref.) 287; total, free, and carbonated alkali, (ref.) 286, 287 ; sugar in, 270. Soldani reagent, 78. Solidifying points of fats, 167. Solids, of milk, calculation of, 355 ; of milk, determination of, 356 ; of vinegar, 124. Solution densities of sugars, (ref.) 105. Sorensen's method for proteolytic power, (ref.) 332. Soxhlet lactometer, 353-354. Soy bean oil, 204. Specific gravity, 11, 112, 133, 134, 162, 170-171, 176, 185, 191, 196, 353, 365. Specific refractive power, 211. Specific rotating (rotatory) power, 82, 83. Specific temperature reaction, 157 ; of olive oils (table), 176. Spermaceti, 142, 170. Sperm oil, 142, 171. Spices, starch content of, 112. Spirit vinegar, 124. Spontaneous combustion, test for oils, (ref.) 236. 406 SUBJECT INDEX Sprigg's method for peptic activity, (ref.) 331. Standard materials for calorimeter, 241. Standards, alcohol, 2, 4, 29, 30 ; butter, 185 ; grain products, 343 ; milk, 364; olive oil, 177; vinegar, 123. Starch, 52, 61; soluble, 54, 55, 56; content of foods and spices, 112; determination of, 106-113, (ref.) 121 ; determination in cocoa prod- ucts, (ref.) 121 ; determination in potatoes, (ref.) 121 ; hydrolysis of, by acid, 106 ; hydrolysis of, by enzymes, 110, 113-120, (ref.) 121- 122; rotating power, 85; sugar, 51. Steam-refined cylinder stock, 221. Stearic acid, 134, 195 ; glycerides of, (ref.) 173. Stearin, cottonseed, 170 ; manufacture, methods, (ref.) 173 ; saponification number, 146. Straw, starch content of, 112. Stutzer method for protein nitrogen, (ref.) 329 ; reagent, 317. Sucrol, 390. Sucrose, 51 ; Clerget method for, 94 ; combustion, 241; determination by use of invertase, (ref.) 105 ; determi- nation in beets and cane, 98 ; deter- mination in condensed milk, (ref.) 103 ; determination in milk, (ref.) 367, 368 ; determination in molasses, (ref.) 105 ; determination in presence of commercial glucose (ref.) 103 ; hydrolysis, 60; hydrolysis, (ref.) 104; rotating power, 84, 85 ; solubility in alcohol, 55. Sugar, see also Sucrose ; analysis, 87 ; beet, optically active non-sugars of, (ref.) 105; beets, analysis of, 98, (ref.) 104, 105 ; beets, see also Beets ; cane, determination of sucrose in, 98 ; in soap, 270 ; manufacture con- trol, (ref.) 104, 105; mixtures, analy- sis of, 101, 102 ; mixtures, analysis of, (ref.), 85, 86; periodicals devoted to, 103 ; solutions, density, and pu- rity of, 100; vinegar, 124, 126, 127. Sugars, 50-105 ; behavior on oxidation, 58; toward caustic alkalies, (ref.), 86; toward Fehling's solution, 69, (ref.) 86 ; toward hydrazines, 61 ; estimation by means of refractom- eter, 101, (ref.) 104, 105; identifi- cation, 101 ; oxidation of, (ref.) 86; rotatory powers of, 84, 85; " unknown," 101. (See also Car- bohydrates.) Sulphite method for benzaldehyde, 46 ; sodium, recoverable from food, (ref.) 392. (See also Sulphites.) Sulphites, determination in foods, (ref.) 393; in gelatin, (ref.) 392, 393, 394; in green corn, (ref.) 393; in lime juice, (ref.) 394 ; in meat, (ref.) 392, 395; in molasses, (ref.) 394; in sugar products, (ref.) 392. (See also Sulphurous acid.) Sulphur, 295-303 ; compounds, volatile in meat and influence on detection of added sulphites, (ref.) 393; de- termination, 295-303, (ref.) 306-307 ; determination in petroleum, 237; in coal, 257, 26-2, 264. Sulphurous acid, 377 ; behavior in foods, (ref.) 392 ; detection in foods, (ref.) 391 ; detection, 377 ; disappearance when added to wine, (ref.) 395 ; organically combined in foods, (ref.) 391 ; quantitative estimation, 377. Sunflower oil, 171. Suspended matter in oils, 235. Suzzi, notes on Maumene number, 160. Tallow, beef, 170; detection of lard, (ref.) 201 ; group, 141 ; mutton, 170. Tan bark as boiler fuel, (ref.) 264. Tannin, as protein precipitant, 321. Tar oils, (ref.) 217. Temperature coefficients in polarization of raw sugars, (ref.) 104. Thomas and Weber's method for pro- teolytic power, (ref.) 331. Titer test, 144, 167. Tocher's test, 182. Tollen's aldehyde reagent, 34, 35. Total solids, determination in milk, 356. Treasury Dept. methods of sugar analy- sis, (ref.) 104. Triazo nitrogen, determination of, (ref.) 307. Trichloracetic acid, as protein precipi- tant, 322. Triglycerides, in butter fat, 195 ; mixed, 143; simple, 143. Trioxy methylene, 37. Trisaccharide, 50, 52. Tryptic activity, 325. Tryptophan reaction, 315. Tung oil, 171, 204. Turmeric test for boric acid or borates, 374; Low's modification, 374. Turpentine, (ref.) 215-216 ; adulterants, (ref.) 215, 217; benzene in, (ref.) 216, 217; commercial, of United States, (ref.) 217 ; naphtha in, (ref.) SUBJECT INDEX 407 216; oils, analysis, (ref.) 216; sub- stitutes, (ref.) 215-217; wood, pro- duction, etc., (ref.) 217. Twitchell's Method for fatty acids, 271. Unification, of methods for diastatic power, 120 ; of reducing sugar methods, (ref.) 85 ; of saccharimeter observations, (ref.) 104. Unsaponifiable matter of fats and waxes, 169, (ref.) 173, 237. Unsaponifiable oils, determination of, in lubricants, 224. Vanilla extract, analysis of, (ref.) 49. Vanillin, determination, 48, 49. Van Slyke's method for analysis of proteins, (ref.) 331. Vegetable drying oils, 203. Ventzke scale, 82, 88. Vinegar, 123-132, (ref.) 131, 132; analyses of typical, 126 ; determina- tion of source, 124 ; glycerol con- tent of, (ref.) 132 ; identification of, 124; methods of analysis, 127; standards, 123, 132. Viscosimeter, 226. Viscosity, 168, 226, (ref.) 236-238; of fats, 144 ; of illuminating oils, (ref.) 237 ; numbers, 184 ; test, Abraham's modification, 183 ; test, Blasdale's, 183. Volatile matter, average relation to calorific power (table), 260; in coke and anthracite, (ref.) 264 ; of coal, 256. Volhard's method for proteolytic power, (ref.) 332. Walker's rule, 246. Walnut, oil, 204 ; shells, starch content of, 112. Washing powders, disinfectant prop- erties, (ref.) 286. Water determination, see Moisture. Water, equivalent of calorimeter, 241 ; density of, 17 ; in butter, 186. Watering of milk, detection of, 363, 364. Wax, bees, 170 ; carnatiba, 170, 173 ; Japan, 170. Waxes, alcohols of, 169 ; analytical properties (table), 170-171 ; classifi- cation, 141 ; general methods, 140 ; Unsaponifiable matter of, 169. Welman's reaction, (ref.) 201. Welter's rule, 246. Westphal balance, 162. Whale oil, 142, 171. Wheat, see Grain products. Whisky, (ref.) 32, 33. Wijs method and solution, 152, 156, (ref.) 173. Wiley's method for melting point, 192, 202. Wilfarth method for nitrogen, 389. Wine vinegar, 123. Wohlgemuth's method for diastatic power, 116, (ref.) 122; scale, 117, (ref.) 122. Wood, calorific power, 253 ; oil, Chinese, 204; oils, Philippine, (ref.), 215; ultimate composition, 252 ; and similar fuels, 251. Wood alcohol, requirements for use in denaturing, 30. Wright's factors for specific gravity calculations, 164. Xanthoproteic reaction, 315. Xylosazone, 62, 63, 64. Xylose, 63, 85. Yeasts, pure, separation of carbo- hydrates by, 104. Zeiss refractometer, 165. Zero-point of polariscope, 80, 81, 87. Zinc sulphate, as protein precipitant, 316. T HE following pages contain advertisements of Macmillan books by the same author or on kindred subjects. Chemistry of Food and Nutrition BY HENRY C. SHERMAN, PH.D. Professor in Columbia University Cloth, i2mo, viii+ 355 pages, $1.50 net The purpose of this volume is to present the principles of the chemistry of food and nutrition with special reference to the food requirements of man and the considerations which should underlie our judgment of the nutritive values of food. The food is here considered chiefly in its nutritive relations. It is hoped that the more detailed description of individual foods and the chemical and legal control of the food industry may be treated in a companion volume later. 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