part 1

.

[521] Molkerei Zeitung, 1893, Nos. 20, 22.

[522] This work, Vol. 2, p. 204.

[523] Vid. op. cit. 120, p. 24.

[524] Milch Zeitung, 1895, Band 24, S. 729: Chemiker-Zeitung Repertorium, Band 19, S. 372.

[525] Chemiker-Zeitung, 1895, S. 554.

[526] Zeitschrift für analytische Chemie, Band 35, S. 497.

[527] Vid. op. cit. supra, S. 499.

[528] Vid. op. cit. supra, S. 502.

[529] Bulletin No. 13, Division of Chemistry, U. S. Department of Agriculture, pp. 118, 293.

[530] This work, Vol. 2, p. 204.

[531] Pflüger’s Archiv, Band 56, S. 558.

[532] Hoppe-Seyler’s Zeitschrift für physiologische Chemie, Band 22, S. 213.

[533] Bulletin de la Société Chimique de Paris, Tomes 15-16, p. 1126.

[534] American Chemical Journal, Vol. 8, p. 200: Bulletin 13, Division of Chemistry, U. S. Department of Agriculture, p. 120.

PART SEVENTH.

MISCELLANEOUS AGRICULTURAL PRODUCTS.

=527. Classification.=—In the preceding parts have been set forth the fundamental principles underlying the conduct of agricultural analysis and a résumé of the best practice of the art. The analyst, as a rule, will seldom be required to undertake investigations which are unnoticed in the preceding pages. Cases will arise, however, in which problems are presented which can not be solved by the rules already elucidated. In respect of the great classes of agricultural bodies, it will be observed that dairy products have already received special mention. In respect of foods and fodders in general, it is evident that they are chiefly composed of moisture, ash, carbohydrates, oils and proteid matters. The methods of identifying, separating and estimating these constituents have been fully set forth. It is not necessary, therefore, to study in this part the analytical processes which are applicable to cereals, cattle foods and other food products, further than is necessary to present in the most important cases a working résumé of principles and methods. There remain, however, certain products of importance which require some special modifications of treatment, and it is to these that the present part will be chiefly devoted. Among these are found tobacco, tea and coffee, fruits, fermented and distilled drinks and certain animal products. It is evident that an enumeration of all agricultural products, with a description of their methods of examination, would be impracticable in the available space and undesirable by reason of the repetition which would be required. In each case the analyst, in possession of the methods described, will be able to adapt the means at his disposal to the desired purpose to better advantage than any rigid directions could possibly secure.

In respect of the analytical methods of determining the nutritive value of foods, they may be divided into chemical and physiological. The chemical methods embrace the thermal and artificial digestion investigations, and the physiological include those which are carried out with the help of the animal organisms. In the latter case the digestive process is checked by the analysis of the foods before ingestion and of the excreta of all kinds during and after digestion.

It is evident that a detailed description of this method should be looked for in works devoted to physiological chemistry.

CEREALS AND CEREAL FOODS.

=528. General Analysis.=—The cereals are prepared for analysis by grinding until the fragments pass a sieve having circular perforations half a millimeter in diameter. The moisture, ash, ether extract, proteids and carbohydrates are determined by some one of the processes already described in detail. In this country the methods of the Association of Official Agricultural Chemists are generally followed.[535] For convenience these methods are summarized below.

_Moisture._—Dry from two to three grams of the fine-ground sample for five hours, at the temperature of boiling water, in a current of dry hydrogen. If the substance be held in a glass vessel, the latter should not be in contact with the boiling water.

_Ash._—Char from two to three grams of the sample and burn to whiteness at the lowest possible red heat. If a white ash can not be obtained in this manner, exhaust the charred mass with water, collect the insoluble residue on a filter, burn it, add this ash to the residue from the evaporation of the aqueous extract and heat the whole to low redness until the ash is white.

_Ether Extract._—Pure ether is prepared by washing the commercial article four or five times with water to free it of the chief part of the alcohol it contains. The residual water is mostly removed by treating the liquid with caustic soda or potash. Any residual alcohol or water is finally removed by the action of metallic sodium. The ether thus prepared is stoppered, after the evolution of hydrogen has ceased, and is kept over metallic sodium. Immediately before use it should be distilled out of contact with moist air.

The residue from the determination of moisture, as described above, is extracted in an appropriate apparatus (=39=) with the pure ether for sixteen hours. The extract is dried to constant weight. The weight may be checked by drying and weighing the extraction tube and its contents before and after the operation.

_Crude Proteids._—Proceed as in the method of determining nitrogen in the absence of nitrates and multiply the weight of nitrogen obtained by 6.25. This factor is a general one, but should not be rigidly applied. In each instance, according to the nature of the cereal, the appropriate factor, pointed out in paragraph =407= should be used, and the factor 6.25 be applied only in those cases where a special factor is not given. The factors for the common cereals are wheat 5.70, rye 5.62, oats 6.06, maize 6.22, barley 5.82 and flaxseed 5.62.

For separating the proteid matters consult paragraphs =392-410=. In the case of wheat the methods of Teller may be consulted.[536]

_Amid Nitrogen._—The albuminoid nitrogen is determined as directed in paragraph =203= of volume II. The difference between this number and that representing the total nitrogen gives the nitrogen as amids.

_Fiber and Carbohydrates._—The methods of analysis are described in detail in Part Third.

=529. Bread.=—In general, the same processes are followed in bread analysis as are used with cereals and flours. In addition to the regular analytical processes, breads are to be examined for adulterants, bleaching and coloring matters, and for the purpose of determining the changes which have taken place in their nutrient constituents in the processes of fermentation and cooking.

_Temperature of Baking._—The interior of a loaf during the process of baking does not attain the high temperature commonly supposed. This temperature is rarely found to be more than one degree above the boiling point of water.[537] In biscuits and other thin cakes, which become practically dry and which by reason of their thinness are the more readily penetrated by heat, the temperature may go as high as 110°.

_Soluble Extract._—The quantity of matters both in flour and bread, soluble in cold water, is determined by extraction in the usual way and drying the extract. Soluble albuminoids, sugars and mineral salts are extracted by this process. When possible, the operation should be conducted both on the bread and the flour from which it is made.

_Color._—In baker’s parlance is found an apparent contradiction of terms, since it speaks of bread with “no color” when the loaf is dark brown, while a white loaf is said to have a high color. An ideal color for the interior of a loaf is a light cream tint, which is more desirable than a pure white.[538] The texture, odor and flavor of the loaf are also to be considered, but these are properties of more importance to the technical expert than to the analyst.

_Quantity of Water._—It is not possible to set a rule of limitation in respect of the quantity of water a bread should hold. For full loaves, perhaps forty per cent is not too high a maximum, while some authors put it as low as thirty-four per cent. Some flours are capable of holding more water than others, and the loaf should have just enough water to impart to the slice of bread the requisite degree of softness and the proper texture. Most breads will have a content of water ranging from thirty to forty per cent. In biscuits and other thin cakes the moisture is much less in quantity.

_Acidity._—The acidity of both bread and flour is determined by shaking ten grams of the sample with 200 cubic centimeters of distilled water for fifteen minutes, pouring the mass on a filter and titrating an aliquot part of the filtrate with tenth-normal alkali. The acidity is reckoned as lactic acid in the case of breads raised by fermentation.

_Nature of Nitrogenous Compounds._—The methods of investigation are described in paragraphs =392-410=.

=530. Determination of Alum in Bread.=—The presence of alum in bread may be detected by means of logwood. Five grams of fresh logwood chips are digested with 100 cubic centimeters of amyl alcohol. One cubic centimeter of this decoction and the same quantity of a saturated solution of ammonium carbonate are mixed with ten grams of flour and an equal quantity of water. With pure flour, a slight pink tint is produced. In the presence of alum the color changes to a lavender or blue, which is persistent on heating.

The test may be varied by diluting five cubic centimeters of the reagents mentioned with ninety cubic centimeters of water and pouring the mixture over ten grams of the crumbled bread. After standing for five minutes, any residual liquid is poured off and the residue, washed once with a little water, is dried in a steam bath, when the blue color is developed if alum be present.[539]

=531. Chemical Changes Produced by Baking.=—Changes of a chemical nature, produced in bread by baking, are found chiefly in modifications of the starch and proteids. The starch is partly converted into dextrin and the albumins are coagulated. The changes in digestion coefficient are determined by the methods which follow. The fermentations which precede the baking are due to the usual decompositions of the carbohydrates under the influence of yeast germs.

FODDERS, GRASSES AND ENSILAGE.

=532. General Principles.=—The analyst, in examining the fibrous foods of cattle, is expected to determine moisture, ash, fiber and other carbohydrates, ether extract and albuminoid and amid nitrogen. If a more exhaustive study be required, the sugar and starch are separated from the other non-nitrogenous matters, the carbohydrate bodies yielding furfuraldehyd separately determined and the ash subjected to a quantitive analysis. The processes are conducted in harmony with the principles and methods of procedure fully set forth in the preceding pages.

Green fodders and grasses are easily dried and sampled by comminution in the shredder described on page 9, and roots by that shown on page 10. The moisture is determined by drying a small sample of the shredded mass, while the rest of it is dried, first at about 60° and finally at 100°, or a little above, ground to a fine powder and subjected to analysis by methods already described. The food values as obtained by analysis should be compared, when possible, with those secured by natural and artificial digestion.

Ensilage is shredded and analyzed in precisely the same way, but in drying, the content of volatile acids formed during fermentation must be considered. In other words, the loss on drying ensilage at 100°, or slightly above, is due not only to the escape of water but also to the volatilization of the acetic acid, which is one of the final products of fermentation which the mass undergoes in the silo.

=533. Organic Acids in Ensilage.=—In the examination of ensilage, the organic acids which are present may be determined by the processes described in following paragraphs. The acetic acid, formed chiefly by fermentation, is conveniently determined by the method given for tobacco further on. Lactic acid is detected and estimated by expressing the juice from a sample of ensilage, removing the acetic acid by distillation, repeated once or twice, and treating the filtered residue with zinc carbonate in excess, filtering and determining the zinc lactate in the filtrate. The zinc is determined by the method described for evaporated apples and the lactic acid calculated from the weight of zinc found. Crystallized zinc lactate contains 18.18 per cent of water and 27.27 per cent of zinc oxid.[540]

=534. Changes due to Fermentation in the Silo.=—Silage differs from green fodder in having less starch and sugar, more acetic and lactic acids and alcohol and a higher proportion of amid to albuminoid nitrogen.[541] There is also a considerable loss of nitrogenous substances in ensilage, due probably to their conversion into ammonium acetate, which is lost on drying.

=535. Alcohol in Ensilage.=—The fermentation which takes place in the silo is not wholly of an alcoholic nature, as the development of lactic acid, noted above, clearly indicates. The alcohol which is formed may escape and but small quantities can be detected in the ripened product. So small is this quantity of alcohol that it appears to be useless to try to secure a quantitive estimation of it. Qualitively, it may be detected by collecting it in a distillate, which is neutralized or made slightly alkaline with soda or potash lye and redistilled. The greater part of the alcohol will be found in the first few cubic centimeters, which are made alkaline with potash lye and as much iodin added as can be without giving a red tint to the solution. Any alcohol which is present will soon separate as iodoform.

=536. Comparative Values of Fodder and Ensilage.=—In judging of the comparative values of green and dry fodders for feeding purposes, it is necessary to secure representative samples in the green, quickly dried and ensilaged condition. It is quite certain that the greater part of the sugar contained in green fodders is lost both by natural curing and by placing in a silo. When well cured by the usual processes there is but little loss of nitrogenous matters, but in the silo this loss is of considerable magnitude, amounting in some instances to as much as thirty per cent.

The ideal way of preparing green fodders in order to preserve the maximum food value efficiently, is to shred them and dry rapidly by artificial heat, or in the sunlight, until they are in a condition which insures freedom from fermentation. In this condition, when placed in bales, under heavy pressure, the food constituents are preserved in the highest available form. The immense sugar content of the stalks of maize and sorghum could be preserved in this way almost indefinitely.

FLESH PRODUCTS.

=537. Names Of Meats.=—The parts of the animal from which the meats are taken have received distinctive names, which serve to designate the parts of the carcass offered for sale. These names are not invariable and naturally are quite different in many markets. In this country there is some degree of uniformity among butchers in naming the meats from different parts. The names in scientific use for the parts of mutton, beef and pork are found in the accompanying illustrations.[542]

=538. Sampling.=—When possible the whole animal should constitute the sample. The relative weights of blood, intestinal organs, hide, hoofs, horns, bones and edible flesh are determined as accurately as possible. The general method of preparing samples of animal products is given in paragraph =5=.

[Illustration: FIG. 114.]

[Illustration: FIG. 115.]

[Illustration: FIG. 116.]

[Illustration: NAMES OF CUTS OF MEAT.]

The method of sampling employed by Atwater and Woods is essentially that just noted.[543] The sample, as received at the laboratory, is weighed, the flesh (edible portion) is then separated from the refuse (skin, bones etc.) and both portions weighed. There is always a slight loss in the separation, evidently due to evaporation and to small fragments of the tissues that adhere to the hands and to the implements used in preparing the sample. The perfect separation of the flesh from the other tissues is difficult, but the loss resulting from this is small. In sampling the material for analysis, it is finely chopped, either in a tray or in a sausage cutter, and in each case is well mixed.

=539. Methods of Analysis.=—The general methods for the analyses of food products are applicable to meats and animal products in general. In the separation of the nitrogenous constituents the methods described in paragraphs =411-414= are followed. It is not safe to estimate as proteids the total nitrogen multiplied by 6.25, since the flesh bases have much higher percentages of nitrogen than are found in proteid matters. As indicated in paragraph =280= the complete extraction of dried meats by ether is difficult of accomplishment. After a few hours it may be assumed that the total extract will represent the fat, although additional soluble matters are obtained by continuing the process. The heat producing power may be calculated from the analytical data secured. The methods which have been described in the preceding pages will be found sufficient for guidance in the examination of animal products, and the analyst will find them, when modified to suit particular cases, adapted to the isolation and estimation of proximate food principles.

The methods of analyses followed by Atwater and Woods are given below:[544]

_Water and Water-Free Substance._—The drying is done in ordinary water ovens at a temperature of nominally 100°, but actually at 96° and 98°. For each analysis of animal tissues (flesh) one or more samples of from fifty to one hundred grams of the freshly chopped substance are weighed on a small plate, heated for from twenty-four to forty-eight hours, cooled, allowed to stand in the open air for about twenty-four hours, weighed, ground, sifted through a sieve with circular holes one-half millimeter in diameter, bottled and set aside for analysis. In case of fat samples which cannot be worked through so fine a sieve, either a coarser sieve is used or the substance crushed as finely as practicable and bottled without sifting.

For the complete desiccation, about two grams of material are dried for three hours. It is extremely difficult to get an absolutely constant weight, though it is found that this is in most cases approximately attained in four hours.

_Nitrogen, Protein, Albuminoids etc._—The nitrogen is determined in the partly dried substance by the method of Kjeldahl. The protein is calculated by multiplying the percentage of nitrogen by 6.25. The nitrogenous matters in meats and fish, _i. e._, in the materials which have practically no carbohydrates, are also estimated by subtracting the sum of ether extract and ash from the water-free substance, or the sum of water, ether extract and ash from the fresh substance, the remainder being taken as proteids, albuminoids etc., by difference. While this is not an absolutely correct measure of the total nitrogenous matter, it is doubtless more nearly so than the product of the nitrogen multiplied by 6.25.

_Fat (Ether Extract)._—The fat is extracted with ether in the usual manner. The point at which the extraction is complete is not always easy to determine. For the most part, the extraction is continued for such time as experience indicates to be sufficient, and then the flask is replaced by another and the extraction repeated until the new flask shows no increase in weight.

According to experience, the fat of many animal tissues is much more difficult to extract than that of most vegetable substances. In general, the greater the percentage of fat in a substance the more difficult is the removal of the last traces. Dried flesh is frequently so hard that the fineness of the material to be extracted seems to be a very important matter.

_Ash._—Ash is determined by the method recommended by the Association of Official Agricultural Chemists.

_Food Value—Potential Energy._—The food materials are not necessarily burned in the calorimeter, but the fuel value of a pound of each of the foods, as given in the tables, is obtained by multiplying the number of hundredths of a pound of protein and of carbohydrates by 18.6 and the number of hundredths of a pound of fat by 42.2, and taking the sum of these three products as the number of calories of potential energy in the materials.

More reliable results are obtained by using the factors obtained by Stohmann; _viz._, 5731 calories for proteids, 9500 calories for common glycerids, 9231 calories for butter fat, 3746 calories for pentose sugars, 3749 calories for dextrose and levulose and 3953 calories for sucrose and milk sugar.[545]

=540. Further Examination of Nitrogenous Bodies.=—It is evident that both of the methods proposed above for the examination of the nitrogenous constituents of meats are unreliable. If the total nitrogen be determined and multiplied by 6.25 the product does not by any means represent the true quantity of nitrogenous matter since the flesh bases contain in some instances more than twenty-five per cent of nitrogen.

If, on the other hand, the water, ash and fat in a meat sample be determined and the sum of their per cents be subtracted from 100, the difference represents the nitrogenous bodies plus all undetermined matters and errors of analysis. The assumption that meats are free of carbohydrates is not tenable since glycogen is constantly found therein and in horse flesh in comparatively large amounts. In a thoroughly scientific analysis of meats, the nitrogenous bodies should be separated and determined by groups, according to the principles developed in paragraphs =411-414=. This process requires a great amount of analytical work and in general it will be sufficient to make a cold water extract to secure the flesh bases and a hot water extract to secure the gelatin. The nitrogen is then determined in each of these portions separately. The nitrogen in the cold water extract is multiplied by four, in the hot water extract by six and in the residue by 6.25. The sum of these products represents approximately the total nitrogenous matter in the sample.

Aqueous extracts containing nitrogen are easily prepared for moist combustion by placing them in the digestion flasks, connecting the latter with the vacuum service and evaporating the contents of the flask nearly to dryness. The sulfuric acid is then added and the nitrogen converted into ammonia and determined in the usual manner.

=541. Fractional Analysis of Meats.=—A better idea of the composition of a meat is obtained by separating its constituents into several groups by the action of different solvents. This method has been elaborated by Knorr.[546]

The separation of the meats in edible portion and waste and the determination of moisture and fat are conducted as already described. The residue from the fat extraction is exhausted with alcohol, and in the extract are found the nitrogenous bases kreatin, kreatinin, sarkin and xanthin, and urea, lactic, butyric, acetic and formic acids, glycogen and inosit. In the residue from the alcohol extraction, the proteid nitrogen is determined in a separate sample.

A separate portion of the sample is ground to a fine paste and repeatedly rubbed up with cold water, which is poured through a tared filter. When the extraction is complete, the filter and its contents are dried and the dry residue determined. This residue represents the nitrogenous constituents of the muscle fibers and their sheaths together with any other bodies insoluble in cold water. The filtrate from the cold water extraction is heated to boiling to precipitate the albuminous matters which are collected, dried and weighed, or the nitrogen therein determined and the albuminous matters calculated by multiplying by the usual factor. The filtrate from the coagulated albuminous bodies is evaporated to dryness and weighed. It consists essentially of the same materials as the alcoholic extract mentioned above. The ash and nitrogen in the aqueous extract are also determined.

The mean content of the edible parts of common meats, expressed as per cents in groups as mentioned, follow:

Per cent. Water 73.11 Ash 1.18 Total soluble matter 26.89 Phosphoric acid 0.49

Per cent. { Proteids insoluble in cold water 13.76 { Of which coagulable by heat 2.24 Cold water extract 3.56 { Ash in water extract 1.09 { Of which phosphoric acid 0.38

Per cent. Fat 4.93 Alcohol extract 3.03 Proteids in residue from alcohol 17.88 Total nitrogen in sample 3.37

=542. Estimation of Starch in Sausages.=—Starchy substances are sometimes added to sausages for the purpose of increasing their weight. The presence of starch in a sausage is easily detected by iodin. The quantity may be determined by the following process:[547]

The principle of the process is based upon the observation that while starch is easily soluble in an aqueous solution of the alkalies, it is insoluble in an alcoholic solution thereof. The chief constituents of meat, _viz._, fat and proteid matters, on the other hand, are readily soluble in an alcoholic solution of potash or soda. This renders the separation of the starch easy. The sample is warmed on a water bath with a considerable excess of an eight per cent solution of potassium hydroxid in alcohol whereby the fat and flesh are quickly dissolved. The starch and other carbohydrate bodies, remain in an undissolved state. In order to prevent the gelatinizing of the soap which is formed, the mass is diluted with warm alcohol, the insoluble residue collected upon a filter and washed with alcohol until the alkaline reaction disappears. The residue is then treated with aqueous potassium hydroxid solution, whereby the starch is brought into solution and, after filtration, is treated with alcohol until it is all precipitated. The precipitated starch is collected upon a filter, washed with alcohol and finally with ether, dried and weighed. Starch prepared in this way contains a considerable quantity of potash, the amount of which can be determined by incineration. In order to avoid this trouble, the starch, after separation in the first instance as above mentioned and solution in aqueous potassium hydroxid, is precipitated on the addition of enough acetic to render the solution slightly acid. The precipitated starch, in this instance, is practically free of potash, since potassium acetate is soluble in alcohol.

=543. Detection of Horse Flesh.=—Since horse flesh has become an important article of human food and is often sold as beef and sausage, a method of distinguishing it is desirable. The comparative anatomist is able to detect horse flesh when accompanied by its bones, or in portions sufficiently large for the identification of muscular characteristics. It is well known that horse flesh contains a much higher percentage of glycogen than is found in other edible meats. Niebel has based a method of detecting horse flesh upon this fact, the glycogen being converted into dextrose and determined in the usual way. Whenever the percentage of reducing sugars in the dry fat-free flesh exceeds one per cent, Niebel infers that the sample under examination is horse flesh.[548]

The reaction for horse flesh, proposed by Bräutigam and Edelmann, is preferred by Baumert. In this test about fifty grams of the flesh are boiled for an hour with 200 cubic centimeters of water, the filtered bouillon evaporated to about half its volume, treated with dilute nitric acid and the clear filtrate covered with iodin water. Horse flesh, by reason of its high glycogen content, produces a burgundy red zone at the points of contact of the two liquids. In the case of sausages, if starch have been added, a blue zone is produced, and if dextrin be present, a red zone, both of which obscure the glycogen reaction. The starch is easily removed by treating the bouillon with glacial acetic acid. No method is at present known for separating dextrin from glycogen. The detection of horse flesh is a matter of considerable importance to agriculture as well as to the consumers, especially of sausages. A considerable quantity of horse flesh is annually sent to the market, little of which presumably is sold under its own name. As a cheap substitute for beef and pork in sausages, its use must be regarded as fraudulent, although no objection can be urged against its sale when offered under its own name.[549]

METHODS OF DIGESTION.

=544. Artificial Digestion.=—The nutrient values of cereals and other foods are determined both by chemical analysis and by digestion experiments. The heat forming properties of foods are disclosed by combustion in a calorimeter, but the quantity of heat produced is not in every case a guide to the ascertainment of the nutritive value. This is more certainly shown, especially in the case of proteid bodies, by the action of the natural digestive ferments.

It is probable that the digestion, which is secured by the action of these ferments without the digestive organs, is not always the same as the natural process, but when the conditions which prevail in natural digestion are imitated as closely as possible the effects produced can be considered as approximately those of the alimentary canal in healthy action.

Three classes of ferments are active in artificial digestion, _viz._, amylolytic ferments, serving to hydrolyze starch and sugars and to convert them into dextrose, maltose and levulose, aliphalytic ferments, which decompose the glycerids and proteolytic ferments, which act on the nitrogenous constituents of foods. When these ferments are made to act on foods under proper conditions of acidity and temperature, artificial digestion ensues, and by the measurement of the extent of the action an approximate estimate of their digestibility can be secured. In artificial digestion, the temperature should be kept near that of the body, _viz._, at about 40°.

The soluble ferments which are active in the digestion of foods, as has been intimated, comprise three great classes. Among the first class, _viz._, the amylolytic ferments, are included not only those which convert starch into dextrose, but also those which cause the hydrolysis of sugars in general. Among these may be mentioned ptyalin, invertase, trehalase, maltase, lactase, diastase, inulase, pectase and cyto-hydrolytic ferments which act upon the celluloses and other fibers.

Among the aliphalytic ferments, in addition to those which act also upon proteid matter, may be mentioned a special one, lipase.

In the third class of ferments are found pepsin, trypsin or pancreatin and papain.

For the latest information in regard to the nature of the soluble ferments and their nomenclature, the work of Bourquelot may be consulted.[550]

=545. Amylytic Ferments.=—A very active ferment of this kind is found in the saliva. Saliva may be easily collected from school boys, who will be found willing to engage in its production if supplied with a chewing gum. A gum free of sugar is to be used, or if the chewing gum of commerce is employed, the saliva should not be collected until the sugar has disappeared. A dozen boys with vigorous chewing will soon provide a sufficient quantity of saliva for practical use. The amylolytic digestion is conducted in the apparatus hereinafter described for digestion with pepsin and pancreatin. The starch or sugar in fine powder is mixed with ten parts of water and one part of saliva and kept at about 37°.5 for a definite time. The product is then examined for starch, sucrose, maltose, dextrose, dextrin and levulose by the processes already described. In natural digestion the hydrolysis of the carbohydrates is not completed in the mouth. The action of the ferment is somewhat diminished in the stomach, but not perhaps until half an hour after eating. The dilute hydrochloric acid in the stomach, which accumulates some time after eating, is not active in this hydrolysis. On the contrary the amylolytic ferment of the saliva is somewhat enfeebled by the presence of an acid. The active principle of the saliva is ptyalin.

The diastatic hydrolysis of starch has already been described (=179=). It is best secured at a somewhat higher temperature than that of the human stomach.

=546. Aliphalytic Ferments.=—In the hydrolysis of glycerids in the process of digestion the fat acids and glycerol are set free. Whether the glycerids be completely hydrolyzed before absorption is not definitely known. In certain cases where large quantities of oil have been exhibited for remedial purposes, the fat acids and soaps have been found in spherical masses in the dejecta[551] and have been mistaken for gall stones.

The fat which enters the chyle appears to be mostly unchanged, except that it is emulsified.[552] The aliphalytic ferment can be prepared from the fresh pancreas, preferably from animals that have not been fed for forty hours before killing. It is important to prepare the ferment entirely free of any trace of acid. The fresh glands are rubbed to a fine paste with powdered glass and extracted for four days with pure glycerol, to which one part of one per cent soda solution has been added. The filtered liquor contains aliphalytic, proteolytic and amylytic ferments, and is employed for saponification by shaking with the fat to form an emulsion and keeping the mixture, with occasional shaking, at a temperature of from 40° to 60°. The free acids can be titrated or separated from the unsaponified fats by solution in alcohol.[553]

Heretofore it has not been possible to separate a pure aliphalytic ferment from any of the digestive glands. The digestion of carbohydrates and that of fats are intimately associated, and these two classes of foods seem to play nearly the same rôle in the animal economy.

The aliphalytic ferments, prepared from the fresh pancreas, act also on the glucosids and other ester-like carbohydrate bodies. Since the fats may be regarded as ethers, the double action indicates the similarity of composition in the two classes of bodies.[554] The aliphalytic ferments exist also in plants and have been isolated from rape seed.[555]

=547. Proteolytic Ferments.=—The most important process in artificial digestion is the one relating to the action of the ferments on proteid matters. The hydrolysis of fats and carbohydrates by natural ferments takes place best in an alkaline medium, while in the case of proteids when pepsin is used an acid medium is preferred. Since the acidity of the stomach is due chiefly to hydrochloric, that acid is employed in artificial digestion. The hydrolyte used is uniformly the natural ferment of the gastric secretions, _viz._, pepsin; but this is often followed by the pancreatic ferment, (pancreatin, trypsin) in an alkaline medium. During the digestion, the proteids are changed into peptones, and the measurement of this change determines the degree of digestion. The total proteid matter is determined in the sample, and after the digestion is completed, the soluble peptones are removed by washing and the residual insoluble proteid matter determined by moist combustion. The difference in the two determinations shows the quantity of proteid matter digested. The investigations of Kühn on the digestion of proteids may be profitably consulted.[556] For a summary of digestion experiments in this country the résumé prepared by Gordon may be consulted.[557] The method followed in this laboratory is fully described by Bigelow and Hamilton.[558]

=548. Ferments Employed.=—Both the pepsins of commerce and those prepared directly from the stomachs of pigs may be used. The commercial scale pepsin is found, as a rule, entirely satisfactory, and more uniform results are secured by its use than from pepsin solutions made from time to time from pig stomachs. In the preparation of the pepsin solution one gram of the best scale pepsin is dissolved in one liter of 0.33 per cent hydrochloric acid. Two grams of the sample of food products, in fine powder, are suspended in 100 cubic centimeters of the solution and kept, with frequent shaking, at a temperature of 40° for twelve hours. The contents of the flask are poured on a wet filter, the residue on the filter well washed with water not above 40°, the filter paper and its contents transferred to a kjeldahl flask and the residual nitrogen determined and multiplied by 6.25 to get the undigested proteid matter. A large number of digestions can be conducted at once in a bath shown in Fig. 117.[559] The quantity of water in the bath should be as large as possible.

=549. Digestion in Pepsin and Pancreatin.=—The digestion of the proteids is not as a rule wholly accomplished by the stomach juices, and, therefore, in order to secure in artificial digestion results approximating those produced in the living organism, it is necessary to follow the treatment with pepsin by a similar one with the pancreas juices. The method employed in this laboratory is essentially that of Stutzer modified by Wilson.[560]

[Illustration: FIG. 117. BATH FOR ARTIFICIAL DIGESTION.]

The residue from the pepsin digestion, after washing, is treated for six hours at near 40° with 100 cubic centimeters of pancreas solution, prepared as follows:

Free the pancreas of a healthy steer of fat, pass it through a sausage grinder, rub one kilogram in a mortar with fine sand and allow to stand for a day or longer. Add three liters of lime water, one of glycerol, of 1.23 specific gravity, and a little chloroform and set aside for six days. Separate the liquor by pressure in a bag and filter it through paper. Before using, mix a quarter of a liter of the filtrate with three-quarters of a liter of water and five grams of dry sodium carbonate, or its equivalent crystallized, heat from 38° to 40° for two hours and filter.[561] In order to avoid the trouble of preparing the pancreas solution pure active pancreatin may be used.[562] One and a half grams of pure pancreatin and three grams of sodium carbonate are dissolved in one liter of water and 100 cubic centimeters of this solution are used for each two grams of the sample. In all cases where commercial pepsin and pancreatin are used, their activity should be tested with bodies such as boiled whites of eggs, whose coefficient of digestibility is well known and those samples be rejected which do not prove to have the required activity.[563]

=550. Digestion in Pancreas Extract.=—In order to save the time required for successive digestions in pepsin and pancreatin Niebling has proposed to make the digestion in the pancreas extract alone.[564] This process and also a slight modification of it have been used with success by Bigelow and McElroy.[565] Two grams of the sample are washed with ether and placed in a digestion flask with 100 cubic centimeters of two-tenths per cent hydrochloric acid. The contents of the flask are boiled for fifteen minutes, cooled, and made slightly alkaline with sodium carbonate. One hundred cubic centimeters of the unfiltered pancreas solution, prepared as directed above, are added and the digestion continued at 40° for six hours. The residue is thrown on a filter, washed, and the nitrogen determined. The method is simplified by the substitution of active commercial pancreatin for pancreas extract. The solution of the ferment is made of the same strength as is specified above.

=551. Artificial Digestion of Cheese.=—The artificial digestion of cheese is conducted by Stutzer as follows:[566]

The digestive liquor is prepared from the fresh stomachs of pigs by cutting them into fine pieces and mixing with five liters of water and 100 cubic centimeters of hydrochloric acid for each stomach. To prevent decomposition, two and a half grams of thymol, previously dissolved in alcohol, are added to each 600 cubic centimeters of the mixture. The mixture is allowed to stand for a day with occasional shaking, poured into a flannel bag and the liquid portion allowed to drain without pressing. The liquor obtained in this way is filtered, first through coarse and then through fine paper, and when thus prepared will keep several months without change. It is advisable to determine the content of hydrochloric acid in the liquor by titration and this content should be two-tenths of a per cent. The cheese to be digested is mixed with sand as previously described, freed of fat by extraction with ether, and a quantity corresponding to five grams of cheese placed in a beaker, covered with half a liter of the digestive liquor and kept at a temperature of 40° for forty-eight hours. At intervals of two hours the flasks are well shaken and five cubic centimeters of a ten per cent solution of hydrochloric acid added and this treatment continued until the quantity of hydrochloric acid amounts to one per cent. After the digestion is finished, the contents of the beaker are thrown on a filter, washed with water and the nitrogen determined in the usual way in the residue. By allowing the pepsin solution to act for two days as described above, the subsequent digestion with pancreas solution is superfluous.

=552. Suggestions Regarding Manipulation.=—The filter papers should be as quick working as possible to secure the separation of all undissolved particles. They should be of sufficient size to hold the whole contents of the digestion flask at once, since if allowed to become empty and partially dry, filtration is greatly impeded. The residue should be dried at once if not submitted immediately to moist combustion. After drying, the determination of the nitrogen can be made at any convenient time. Beaker flasks, _i. e._, lip erlenmeyers with a wide mouth are most convenient for holding the materials during digestion. The flasks are most conveniently held by a crossed rubber band attached at either end to pins in the wooden slats extending across the digestive bath. The bath should be suspended by cords from supports on the ceiling and a gentle rotatory motion imparted to it resembling the peristaltic action attending natural digestion.

=553. Natural Digestion.=—The digestion of foods by natural processes is determined chiefly by the classes of ferments already noted. The principle underlying digestive experiments with the animal organism may be stated as follows: A given weight of food of known composition is fed to a healthy animal under the conditions of careful control and preparation already mentioned. The solid dejecta of the animal during a given period are collected and weighed daily, being received directly from the animal in an appropriate bag, safely secured, as is shown in the accompanying figure. The dejecta are weighed, dried, ground to a fine powder, mixed and a representative part analyzed. The difference between the solid bodies in the dejecta and those given in the food during the period of experiment represents those nutrients which have been digested and absorbed during the passage of the food through the alimentary canal. The urine, containing solid bodies representing the waste of the animal organism, does not require to be analyzed for the simple control of digestive activity outlined above. In a complete determination of this kind the exhalations from the surface of the body and from the lungs are also determined. In the latter case the human animal is selected for the experiment; in the former it is more convenient to employ the lower animals, such as the sheep and cow.

The arrangement of the stalls and of the apparatus for collecting the excreta should be such as is both convenient and effective.[567]

The method of constructing a bag for attachment to a sheep is shown in Fig. 118. It is made according to the directions given by Gay, of heavy cloth and in such a way as to fit closely the posterior parts of the animal.[568] When attached, its appearance is shown in Fig. 119.

[Illustration: FIG. 118.—BAG FOR COLLECTING FECES.]

[Illustration: FIG. 119.—FECAL BAG ATTACHMENT.]

Healthy animals in the prime of life are used, and the feeding experiments are conducted with as large a number of animals as possible, in order to eliminate the effects of idiosyncrasy. The food used is previously prepared in abundant quantity and its composition determined by the analysis of an average sample.

The feeding period is divided into two parts. In the first part the animal is fed for a few days with the selected food until it is certain that all the excreta are derived from the nutrients used. In the second part the same food is continued and the excreta collected, weighed, the moisture determined, and the total weight of the water-free excreta ascertained. The first part should be of at least seven and the second of at least five days duration. The urine and dung are analyzed separately. Males are preferred for the digestion experiments because of the greater ease of collecting the urine and feces without mixing. For ordinary purposes the feces only are collected. The methods of analysis do not differ from those described for the determination of the usual ingredients of a food.

_Example._—The following data taken from the results of digestive experiments, obtained at the Maine Station, will illustrate the method of comparing the composition of the food with that of the feces and of determining the degree of digestion which the proteids and other constituents of the food have undergone.

COMPOSITION OF MAIZE FODDER AND OF FECES THEREFROM AFTER FEEDING TO SHEEP.

BEFORE DRYING. Water, Ash, Proteid, Fiber, Fat, Undetermined, per per per per per per Food. cent. cent. cent. cent. cent. cent. Sweet maize 83.85 1.13 2.18 4.14 0.62 8.08 Feces 72.01 ... ... ... ... ...

DRY. Ash, Proteid, Fiber, Fat, Undetermined, per per per per per Food. cent. cent. cent. cent. cent. Sweet maize 7.01 13.52 25.63 3.86 49.98 Feces 14.42 17.52 19.34 2.68 46.04

DAILY WEIGHTS. Green, Dry, Food. grams. grams. Sweet maize 2521 407 Feces 445 125

PER CENT DIGESTED. Food. Ash, Proteid, Fiber, Undetermined, Fat, Sweet maize 37.0 60.2 76.9 71.8 78.3

In the above instance it is seen that the coefficient of digestibility extended from 37.0 per cent in the case of the mineral components of the food, to 78.3 per cent in the case of the fats. These data are taken only from the results obtained from a single sheep and one article of food. The mean data secured from two animals and three kinds of maize fodder show the following per cents of digestibility: Ash 39.4, proteid 61.8, fiber 76.7, undetermined matters 72.1, fat 76.4. The undetermined matters are those usually known as nitrogen free extract and composed chiefly of pentosans and other carbohydrates.[569]

=554. Natural Digestibility of Pentosans.=—The digestibility of pentosan bodies in foods under the influence of natural ferments has been investigated by Lindsey and Holland.[570] The feeding and collection of the feces is carried on as described above and the relative proportions of pentosan bodies in the foods and feces determined by estimating the furfuraldehyd as prescribed in paragraph =150=.[571]

PRESERVED MEATS.

=555. Methods of Examination.=—In general the methods of examination are the same as those applied in the study of fresh meats. The contents of water, salt and other preservatives, fat and nitrogenous matters are of most importance. When not already in a fine state, the preserved meats are run through meat cutters until reduced to a fine pulp. Most potted meats are already in a state of subdivision well suited to analytical work. The composition of preserved meats has been thoroughly studied in this laboratory by Davis.[572]

=556. Estimation Of Fat.=—Attention has already been called to the difficulty of extracting the fat from meats by ether or other solvents.[573] In preserved meats, as well as in fresh, it is preferable to adopt some method which will permit of the decomposition of the other organic matters and the separation of the fat in a free state. The most promising methods are those employed in milk analyses for the solution of nitrogenous matters. Sulfuric or hydrochloric acid may be used for this purpose, preference being given to sulfuric. The separated fats may be taken up with ether or separated by centrifugal action. A method of this kind for preserved meats, suggested by Hefelmann, is described below.

About six grams of the moist preserved meat are placed in a calibrated test tube and dissolved in twenty-five cubic centimeters of fuming hydrochloric acid. The tube is placed in a water bath, quickly heated to boiling and kept at that temperature for half an hour. About twenty cubic centimeters of cold water are added and the temperature lowered to 30°, then twenty cubic centimeters of ether and the tube gently shaken to promote the solution of the fat. When the ether layer has separated, its volume is read and an aliquot part removed by means of a pipette, dried and weighed. The separation of the ethereal solution is greatly promoted by whirling.

The mean proportions of the ingredients of preserved meats are about as follows:

Per cent.

Water 67.0 Dry matter 33.0

Of which

Nitrogenous bodies 19.0 Fats 10.5 Ash and undetermined 3.5

=557. Meat Preservatives.=—Various bodies are used to give taste and color to preserved meats and to preserve them from fermentation. The most important of these bodies are common salt, potassium and sodium nitrates, sulfurous, boric, benzoic and salicylic acids, formaldehyd, saccharin and hydronaphthol. A thorough study of the methods of detecting and isolating these bodies has been made in this laboratory by Davis and the results are yet to be published as a part of Bulletin 13.

DETERMINATION OF NUTRITIVE VALUES.

=558. Nutritive Value of Foods.=—The value of a food as a nutrient depends on the amount of heat it gives on combustion in the tissues of the body, _i. e._ oxidation, and in its fitness to nourish the tissues of the body, to promote growth and repair waste. The foods which supply heat to the body are organic in their nature and are typically represented by fats and carbohydrates. The foods which promote growth and supply waste are not only those which preeminently supply heat, but also include the inorganic bodies and organic nitrogenous matters represented typically by the proteids. It is not proper to say that one class of food is definitely devoted to heat forming and another to tissue building, inasmuch as the same substance may play an important rôle in both directions. As heat formers, carbohydrates and proteids have an almost equal value, as measured by combustion in oxygen, while fat has a double value for this purpose. The assumption that combustion in oxygen forms a just criterion for determining the value of a food must not be taken too literally. There are only a few bodies of the vast number which burn in oxygen that are capable of assimilation and oxidation by the animal organism. Only those parts of the food that become soluble and assimilable under the action of the digestive ferments, take part in nutrition and the percentage of food materials digested varies within wide limits but rarely approaches 100. It may be safely said that less than two-thirds of the total food materials ingested are dissolved, absorbed, decomposed and assimilated in the animal system. We have no means of knowing how far the decomposition (oxidation) extends before assimilation, and therefore no theoretical means of calculating the quantity of heat which is produced during the progress of digestion. The vital thermostat is far more delicate than any mechanical contrivance for regulating temperature and the quantity of food, in a state of health, converted into heat, is just sufficient to maintain the temperature of the body at a normal degree. Any excess of heat produced, as by violent muscular exertion, is dissipated through the lungs, the perspiration and other secretions of the body.

Pure cellulose or undigestible fiber, when burned in oxygen, will give a thermal value approximating that of sugar, but no illustration is required to show that when taken into the system the bodily heat afforded by it is insignificant in quantity.

Thermal values, therefore, have little comparative usefulness in determining nutritive worth, except when applied to foods of approximately the same digestive coefficient.

=559. Comparative Value of Food Constituents.=—It has already been noted that, judged by combustion in oxygen, carbohydrates and proteids have about half the thermal value possessed by fats. Commercially, the values of foods depend in a far greater degree on their flavor and cooking qualities than upon the amount of nutrition they contain. Butter fat, which is scarcely more nutritious than tallow, is worth twice as much in the market, while the prices paid for vegetables and fruits are not based to any great extent on their food properties.[574] In cereals, especially in wheat, the quantity of fat is relatively small, and starch is the preponderating element. In meats, carbohydrates are practically eliminated and fats and proteids are the predominating constituents.

In the markets, fats and proteids command far higher prices than sugars and starches. The relative commercial food value of a cereal may be roughly approximated by multiplying the percentages of fat and protein by two and a half and adding the products to the percentage of carbohydrates less insoluble fiber. This method was adopted in valuing the cereals at the World’s Columbian Exposition.[575]

=560. Nutritive Ratio.=—In solid foods the nutritive ratio is that existing between the percentage of proteids and that of carbohydrates, increased by multiplying the fat by two and a half and adding the product. In a cereal containing twelve per cent of protein, seventy-two of carbohydrates, exclusive of fiber, and three of fat, the ratio is 12: 72 + 3 × 2.5 = 6.5. Instead of calculating the nutritive ratio directly from the data obtained by analysis, it may be reckoned from the per cents of the three substances in the sample multiplied by their digestive coefficient. Since the relative amounts of proteids, fats and carbohydrates digested do not greatly differ, the numerical expression of the nutritive ratio is nearly the same when obtained by each of these methods of calculation.

Where the proportion of protein is relatively large the ratio is called narrow, 1: 4 ... 6. When the proportion of protein is relatively small the ratio is called broad 1: 8 ... 12. In feeding, the nutritive ratio is varied in harmony with the purpose in view, a narrow ratio favoring the development of muscular energy, and a wide one promoting the deposition of fat and the development of heat. These principles guide the scientific farmer in mixing rations for his stock, the work horses receiving a comparatively narrow and the beeves a relatively wide ratio in their food.

=561. Calorimetric Analyses of Foods.=—The general principles of calorimetry have been already noticed. The theoretical and chemical relations of calorimetry have been fully discussed by Berthelot, Thomsen, Ostwald and Muir.[576] In the analyses of foods the values as determined by calculation or combustion are of importance in determining the nutritive relations.

Atwater has presented a résumé of the history and importance of the calorimetric investigations of foods to which the analyst is referred.[577]

In the computation of food values the percentages of proteids, carbohydrates and fats are determined and the required data obtained by applying the factors 4100, 5500 and 9300 calories for one gram of carbohydrates, proteids and fats respectively.

For most purposes the computed values are sufficient, but it is well to check them from time to time by actual combustions in a calorimeter.

=562. Combustion in Oxygen.=—The author made a series of combustions of carbonaceous materials in oxygen at the laboratory of Purdue University in 1877, the ignition being secured by a platinum wire rendered incandescent by the electric current. The data obtained were unsatisfactory on account of the crudeness of the apparatus. The discovery of the process of burning the samples in oxygen at a high pressure has made it possible to get expressions of thermal data which while not yet perfect, possess a working degree of accuracy. The best form of bomb calorimeter heretofore employed is that of Hempel, as modified by Atwater and Woods.[578]

A section of this calorimeter, with all the parts in place, is shown in Fig. 120.

In the figure the steel cylinder _A_, about 12.5 centimeters deep and 6.2 in diameter, represents the chamber in which the combustion takes place. Its walls are about half a centimeter thick and it weighs about three kilograms. It is closed, when all the parts are ready and the sample in place, by the collar _C_, which is secured gas tight by means of a powerful spanner. The cover is provided with a neck _D_ carrying a screw _E_ and a valve screw _F_. In the neck _D_, where the bottom of the cylinder screw _E_ rests, is a shoulder fitted with a lead washer. Through _G_ the oxygen used for combustion is introduced. The upper edge of the cylinder _A_ is beveled and fits into a groove in the cover _B_, carrying a soft metal washer. To facilitate the screwing on of the cover, ball bearings _KK_, made of hard steel, are introduced between the collar and the cover. The platinum wires _H_ and _I_ support the platinum crucible holding the combustible bodies which are ignited by raising the spiral iron wire connecting them to the temperature of fusion by an electric current. The combustion apparatus when charged is immersed in a metal cylinder _M_, containing water and resting on small cylinders of cork. The water is stirred by the apparatus _LL_. The cylinder _M_ is contained in two large concentric cylinders, _N_, _O_, made of non-conducting materials and covered with disks of hard rubber. The space between _O_ and _N_ may be filled with water. The temperature is measured by the thermometer _P_, graduated to hundredths of a degree and the reading is best accomplished by means of a cathetometer.

[Illustration: FIG. 120. HEMPEL AND ATWATER’S CALORIMETER.]

=563. The Williams Calorimeter.=—The calorimeter bomb has been improved by Williams by making it of aluminum bronze of a spheroidal shape. The interior of the bomb is plated with gold. By an ingenious arrangement of contacts the firing is secured by means of a permanently insulated electrode fixed in the side of the bomb. The calorimetric water, as well as that in the insulating vessel, is stirred by means of an electrical screw so regulated as to produce no appreciable degree of heat mechanically. The combustion is started by fusing a fine platinum wire of definite length and thickness by means of an electric current. The heat value of this fusion is determined and the calories produced deducted from the total calories of the combustion. The valve admitting the oxygen is sealed automatically on breaking connection with the oxygen cylinder. The effluent gases, at the end of the combustion, may be withdrawn through an alkaline solution and any nitric acid therein thus be fixed and determined.[579]

=564. Manipulation and Calculation.=—The material to be burned is conveniently prepared by pressing it into tablets. The oxygen is supplied from cylinders, of which two should be used, one at a pressure of more than twenty atmospheres. By this arrangement a pump is not required.

In practical use, a known weight of the substance to be burned is placed in the platinum capsule, the cover of the bomb screwed on, after all adjustments have been made, and the apparatus immersed in the water contained in _M_, which should be about 2° below room temperature. All the covers are placed in position and the temperature, of the water in _M_ begins to rise. Readings of the thermometer are taken at intervals of about one minute for six minutes, at which time the temperature of the bomb and calorimetric water may be regarded as sensibly the same. The electric current is turned on, the iron wire at once melts, ignites the substance and the combustion rapidly takes place. In the case of bodies which do not burn readily Atwater adds to them some naphthalene, the thermal value of which is previously determined. The calories due to the combustion of the added naphthalene are deducted from the total calories obtained.

The temperature of the water in _M_ rises rapidly at first, and readings are made at intervals of one minute for five minutes, and then again after ten minutes. The first of the initial readings, the one at the moment of turning on the current, and the last one mentioned above are the data from which the correction, made necessary by the influence of the temperature of the room, is calculated by the following formulas.[580]

The preliminary readings of the thermometer at one minute intervals are represented by _t_₁, _t_₂, _t_₃ ... _t_ₙ₁. The last observation tₙ₁ is taken as the beginning temperature of the combustion and is represented in the formulas for calculations by Θ₁. The readings after combustion are also made at intervals of one minute, and are designated by Θ₂, Θ₃ ... Θₙ. The readings are continued until there is no observed change between the last two. Generally this is secured by five or six readings.

The third period of observations begins with the last reading Θₙ, which in the next series is represented by _tʹ_₁, _tʹ_₂ ... _tʹ_ₙ₂.

In order to make the formulas less cumbersome let

_t_ₙ₁ - _t_₁ ------------ = _v_, _n_₁ - 1

_tʹ_ₙ₁ - _tʹ_₁ ------------- = _vʹ_, _n_₂ - 1

_t_₁ + _t_₂ + _t_₃ ... _t_ₙ₁ --------------------------- = t, _n_₁

_tʹ_₁ + _tʹ_₂ + _tʹ_₃ ... _tʹ_ₙ₂ and -------------------------------- = _tʹ_. _n_₂

The correction to be made to the difference between Θₙ - Θ₁ for the influence of the outside temperature is determined by the formula of Regnault-Pfaundler, which is as follows:

_v_ - _vʹ_ ⁿ⁻¹ Θₙ + Θ₁ ∑ Δ_t_ = -------------- ( ∑ Θ_r_ + -------- - _nt_) - (_n_ - 1)_v_, _tʹ_ - _t_ ₁ 2

ⁿ⁻¹ in which ∑ Θ_r_ ₁

is calculated from the observation of the thermometer Θ₁, Θ₂ etc., made immediately after the combustion. It is equal to the sum of observations Θ₁, Θ₂ etc., increased by an arbitrary factor equivalent to (Θ₂ - Θ₁)/9, which is made necessary by reason of the irregularity of the temperature increase during the first minute after combustion, the mean temperature during that minute being somewhat higher than the mean of the temperatures at the commencement and end of that time.

The quantity of heat formed by the combustion of the iron wire used for igniting the sample is to be deducted from the total heat produced. This correction may be determined once for all, the weight of the iron wire used being noted and that of any unburned portion being ascertained after the combustion.

Ten milligrams of iron, on complete combustion, will give sixteen calories.

In the combustion of substances containing nitrogen, or in case the free nitrogen of the air be not wholly expelled from the apparatus before the burning, nitric acid is formed which is dissolved by the water produced.

The heat produced by the solution of nitric acid in water is 14.3 calories per gram molecule. The quantity of nitric acid formed is determined by titration and a corresponding reduction made in the total calculated calories.

In the titration of nitric acid it is advisable to make use of an alkaline solution, of which one liter is equivalent to 4.406 grams of nitric acid. One cubic centimeter of the reagent is equivalent to a quantity of nitric acid represented by one calorie.

Since the materials of which the bomb is composed have a specific heat different from that of water, it is necessary to compute the water thermal value of each apparatus.

The hydrothermal equivalent of the whole apparatus is most simply determined by immersing it at a given temperature in water of a different temperature.[581] With small apparatus this method is quite sufficient, but there are many difficulties attending its application to large systems weighing several kilograms. In these cases the hydrothermal equivalent may be calculated from the specific heats of the various components of the apparatus.

In calculating these values the specific heats of the various components of the apparatus are as follows:

Brass 0.093 Steel 0.1097 Platinum 0.0324 Copper 0.09245 Lead 0.0315 Oxygen 0.2389 Glass 0.190 Mercury 0.0332 Hard rubber 0.33125

_Example._—It is required to calculate the hydrothermal value of a calorimeter composed of the following substances:

Hydrothermal value. Steel bomb and cover, 2850 grams × 0.1097 312.65 grams. Platinum lining, capsule and wires, 120 grams × 0.0324 3.89 ” Lead washer, 100 grams × 0.0315 3.15 ” Brass outer cylinder, 500 grams × 0.093 46.50 ” Mercury in thermometer, 10 grams × 0.0332 0.33 ” Glass (part of thermometer in water), 10 grams × 0.19 1.90 ” Brass stirring apparatus (part in water), 100 grams × 0.093 9.30 ” ------ Total water value of system 377.72 ”

When a bomb of 300 cubic centimeters capacity is filled with oxygen at a pressure of twenty-four atmospheres it will hold about ten grams of the gas, equivalent to a water value of 2.40 grams. Hence the water value of the above system when charged, assuming the bomb to be of the capacity mentioned, is 380.12 grams.

If the cylinder holding the water be made of fiber or other non-conducting substance, its specific heat is best determined by filling it in a known temperature with water at a definite different temperature.

It is advisable to have the water cylinder of such a size as to permit the use of a quantity of water for the total immersion of the bomb which will weigh, with the water value of the apparatus, an even number of grams. In the case above, 2622.28 grams of water placed in the cylinder will make a water value of 3,000 grams, which is one quite convenient for calculation.

=565. Computing the Calories of Combustion.=—In the preceding paragraph has been given a brief account of the construction of the calorimeter and of the methods of standardizing it and securing the necessary corrections in the data directly obtained in its use. An illustration of the details of computing the calories of combustion taken from the paper of Stohmann, Kleber and Langbein, will be a sufficient guide for the analyst in conducting the combustion and in the use of the data obtained.[582]

Weight of substance burned, 1.07 grams.

Water value of system (water + apparatus), 2,500 grams.

Preliminary thermometric readings, _t_₁ = 26.8; _t_₂ = 27.2; _t_₃ = 27.7; _t_₄ = 28.1; _t_₅ = 28.5; _t_ₙ₁ = 28.9.

Thermometric reading after combustion, Θ₁ = 28.9; Θ₂ = 202; Θ₃ = 213; Θ₄ = 214.2; Θₙ = 214.0.

Final thermometric readings, _tʹ_₁ = 214.0; _tʹ_₂ = 213.8; _tʹ_₃ = 213.6; _tʹ_₄ = 213.5; _tʹ_₅ = 213.3; _tʹ_₆ = 213.1; _tʹ_₇ = 212.9; _tʹ_₈ = 212.7; _tʹ_₉ = 212.6; _tʹ_₁₀ = 212.4; _tʹ_ₙ₂ = 212.2.

From the formulas given above the following numerical values are computed:

_v_ = 0.42. _vʹ_ = -0.18. _t_ = 27.9. _tʹ_ = 213.1. _n_ = 5.

ⁿ⁻¹ Θ₂ - Θ₁ ∑ Θ_r_ = Θ₁ + Θ₂ + Θ₃ + Θ₄ + ------- = 667. ₁ 9

Substituting these values in the formula of Regnault-Pfaundler, the value of the correction for the influence of the external air is

0.42 - (-0.18) 214 + 29 ∑ Δt = [--------------- (677 + --------- - (5 × 27.9)) 213.1 - 27.9 2

- (4 × 0.42)] = 0.45,

which is to be added to the end temperature (Θₙ = 214.0).

The computation is then made from the following data:

Corrected end temperature (Θₙ + 0.45) 214.45 = 15°.3699 Beginning temperature (Θ₁) 28.90 = 12°.8406 Increase in temperature 185.55 = 2°.5293 Total calories 2.5293 × 25000 = 6323.3 Of which there were due to iron burned 9.1 ” ” ” ” nitric acid dissolved 8.2 Total calories due to one gram of substance 5893.5

The thermometric readings are given in the divisions of the thermometer which in this case are so adjusted as to have the number 28.90 correspond to 12°.8406, and each division is nearly equivalent to 0°.014 thermometric degree.

The number of calories above given is the proper one when the computation is made to refer to constant volume. By reason of the consumption of oxygen and the change of temperature, although mutually compensatory, the pressure may be changed at the end of the operation. The conversion of the data obtained at constant volume referred to constant pressure may be made by the following formula, in which [_Q_] represents the calories from constant volume and _Q_ the desired data for constant pressure, _O_ the number of oxygen atoms, _H_ the number of hydrogen atoms in a molecule of the substance, and 0.291 a constant for a temperature of about 18°, at which the observations should be made.

_H_ _Q_ = [_Q_] + (--- - _O_) 0.291. 2

=566. Calorimetric Equivalents.=—By the term calorie is understood the quantity of heat required to raise one gram of water, at an initial temperature of about 18°, one degree. The term ‘Calorie’ denotes the quantity of heat, in like conditions, required to raise one kilogram of water one degree.

For purposes of comparison and for assisting the analyst in adjusting his apparatus so as to give reliable results, the following data, giving the calories of some common food materials, are given:

Substance. Chemical composition. Proteids. Calories. C. H. N. S. O. Per Per Per Per Per cent. cent. cent. cent. cent. Serum albumin 5917.8 53.93 7.65 15.15 1.18 22.09 Casein 5867.0 54.02 7.33 15.52 0.75 22.38 Egg albumin 5735.0 52.95 7.50 15.19 1.51 22.85 Meat free of fat and extracted with water 5720.0 52.11 6.76 18.14 0.96 22.66 Peptone 5298.8 50.10 6.45 16.42 1.24 25.79 Proteids (mean) 5730.8 52.71 7.09 16.02 1.03 23.15 Glycerids. Butterfat 9231.3 Linseed oil 9488.0 Olive oil 9467.0

Carbohydrates. Formula. Arabinose 3722.0 C₅H₁₀O₅ Xylose 3746.0 C₅H₁₀O₅ Dextrose 3742.6 C₆H₁₂O₆ Levulose 3755.0 C₆H₁₂O₆ Sucrose 3955.2 C₁₂H₂₂O₁₁ Lactose 3736.8 C₁₂H₂₂O₁₁ + H₂O Maltose 3949.3 C₁₂H₂₂O₁₁

=567. Distinction between Butter and Oleomargarin.=—Theoretically the heats of combustion of butter fat and oleomargarin are different and de Schweinitz and Emery propose to utilize this difference for analytical purposes.[583] The samples of pure butter fat examined by them afforded 9320, 9327 and 9362 calories, respectively. The calories obtained for various samples of oleomargarin varied from 9574 to 9795. On mixing butter fat and oleomargarin, a progressive increase in calorimetric power is found, corresponding to the percentage of the latter constituent. Lards examined at the same time gave from 9503 to 9654 calories.

FRUITS, MELONS AND VEGETABLES.

=568. Preparation of Sample.=—Fresh fruits and vegetables are most easily prepared for analysis by passing them through the pulping machine described on page 9. Preliminary to the pulping they should be separated into skins, cores, seeds and edible portions, and the respective weights of these bodies noted. Each part is separately reduced to a pulp and, at once, a small quantity of the well mixed substance placed in a flat bottom dish and dried, first at a low temperature, and finally at 100°, or somewhat higher, and the percentage of water contained in the sample determined. The bulk of the sample, three or four kilograms, is dried on a tray of tinned or aluminum wire, first at a low and then at a high temperature, until all or nearly all the moisture is driven off. The dried pulp is then ground to as fine a powder as possible and subjected to the ordinary processes of analysis; _viz._, the determination of the moisture, ash, nitrogen, fiber, fat and carbohydrates.

In this method of analysis it is customary to determine the carbohydrates, exclusive of fiber, by subtracting the sum of the per cents of the other constituents and the nitrogen multiplied by 6.25 from 100.

=569. Separation of the Carbohydrates.=—It is often desirable to determine the relative proportions of the more important carbohydrates which are found in fruits and vegetables. The pentoses and pentosans are estimated by the method described in paragraph =150=. The cane sugar, dextrose and levulose are determined by extracting a portion of the substance with eighty per cent alcohol and estimating the reducing sugars in the extract before and after inversion by the processes described in paragraphs =238-251=. The percentages of sugars deducted from the percentage of carbohydrates, exclusive of fiber, give the quantity of gums, pentosans, cellulose and pectose bodies present.

Pectose exists chiefly in unripe fruits. By the action of the fruit acids and of a ferment, pectose, in the process of ripening, is changed into pectin and similar hydrolyzed bodies soluble in water. The gelatinous properties of boiled fruits and fruit juices are due to these bodies, boiling accelerating their formation. In very ripe fruits the pectin is completely transformed into pectic acids. The galactan is estimated as described in =585=.

=570. Examination of the Fresh Matter.=—To avoid the changes which take place in drying fruits and vegetables, it is necessary to examine them in the fresh state. The samples may be first separated into meat and waste, as suggested above, or shredded as a whole. The moisture in the pulp is determined as indicated above, and in a separate portion the soluble matters are extracted by repeated treatment with cold water. The seeds, skins, cellulose, pectose and other insoluble bodies are thus separated from the sugars, pectins, pectic and other acids, and other soluble matters. The insoluble residue is rapidly dried and the relative proportions of soluble and insoluble matters determined. The estimation of these bodies is accomplished in the usual way.

=571. Examination of Fruit and Vegetable Juices.=—The fruits and vegetables are pulped, placed in a press and the juices extracted. The pressure should be as strong as possible and the press described in paragraph =280= is well suited to this purpose. The specific gravity of the expressed juice is obtained and the sucrose therein determined by polarization before and after inversion. The reducing sugars and the relative proportions of dextrose and levulose are determined in the usual manner. In grape juice dextrose is the predominant sugar while in many other fruits left hand or optically inactive sugars predominate. Soluble gums, dextrin, pectin etc., may be separated from the sugars by careful precipitation with alcohol, or the total solids, ash, nitrogen, ether extract and acids be determined and the carbohydrates estimated by difference. From the carbohydrates the total percentage of sugars is deducted and the remainder represents the quantity of pectin, gum and other carbohydrates present.

=572. Separation of Pectin.=—Pectin exists in considerable quantities in the juice of ripe fruits (pears) and may be obtained in an approximately pure state from the juices by first removing proteids by the careful addition of tannin, throwing out the soluble lime salts with oxalic acid and precipitating the pectin with alcohol. On boiling with water, pectin is converted into parapectin, which gives a precipitate with lead acetate. Boiling with dilute acids converts pectin into metapectin, which is precipitated by a barium salt.

Pectic acid may be obtained by boiling an aqueous extract (carrots) with sodium carbonate and precipitating the pectic with hydrochloric acid. It is a jelly-like body and dries to a horny mass.

=573. Determination of Free Acid.=—The free acid, or rather total acidity of fruits, is determined by the titration of their aqueous extracts or expressed juices with a set alkali. In common fruits and vegetables the acidity is calculated to malic C₄H₆O₅, in grapes to tartaric C₄H₆O₆, and in citrous fruits to citric acid C₆H₈O₇. Many other acids are found in fruits and vegetables, but those mentioned are predominant in the classes given.

=574. Composition of Common Fruits.=—The composition of common fruits in this country has been extensively investigated at the California Station and the following data are derived chiefly from its bulletins.[584]

Name. Total Rind Seed. Pulp. Juice. Total weight. skin. sugars Sucrose in in juice. juice. per per per cubic per per grams. cent. cent. cent. centimeters. cent. cent. Naval orange 300 28.4 27.7 107 9.92 4.80 Mediterranean sweet orange 202 27.0 0.8 24.0 86 9.70 4.35 St. Michael’s orange 138 19.2 1.6 25.9 65.4 8.71 3.48 Malta Blood orange 177 31.0 24.0 71.0 10.30 5.85 Eureka lemon 104 32 0.12 24.5 38 2.08 0.57 Flesh Per cent Apricot 62.4 93.85 6.15 10.0 90.0 13.31 Prune 25.6 94.2 5.8 21.2 78.8 20.0 Plum 60.4 95.2 4.8 24.7 75.3 17.97 Peach 185 93.8 6.2 22.5 77.5 17.0 Skin Cores Apple 183 17.0 7.0 10.26‡ 1.53‡

‡ In whole fresh fruit. -------------------------------------------------------------------- In whole fruit. /-----------------------------\ Name. Acid. Nitrogenous Water. Dry Ash. bodies. organic matter. per per per per per cent. cent. cent. cent. cent. Naval orange 1.02 1.31 86.56 13.04 0.40 Mediterranean sweet orange 1.38 0.96 85.83 13.06 0.41 St. Michael’s orange 1.35 1.43 84.10 15.42 0.48 Malta Blood orange 1.61 1.05 84.50 15.05 0.45 Eureka lemon 7.66 0.94 85.99 13.50 0.51

Apricot 0.68 1.25 85.16 14.35 0.49 Prune 0.40 1.01 77.38 22.18 0.44 Plum 0.48 1.33 77.43 22.04 0.53 Peach 0.25 82.50 16.95 0.55

Apple[585] 0.11 86.43 13.28 0.29

=575. Composition of Ash of Fruits.=—Two or three kilograms of the dried sample are incinerated at a low temperature and burned to a white ash in accordance with the directions given in paragraphs =28-32=.

The composition of the ash is determined by the methods already described.[586]

The pure ash of some common whole fruits has the following composition:[587]

Name. Per Per Per Per Per Per Per cent cent cent cent cent cent cent pure potash. soda. lime. magnesia. ferric mangano- ash oxid. manganic in oxid. fruit.

Prune 0.47 63.83 2.65 4.66 5.47 2.72 0.39 Apricot 0.51 59.36 10.26 3.17 3.68 1.68 0.37 Orange 0.43 48.94 2.50 22.71 5.34 0.97 0.37 Lemon 0.53 48.26 1.76 29.87 4.40 0.43 0.28 Apple 1.44 35.68 26.09 4.08 8.75 1.40 Pear 1.97 54.69 8.52 7.98 5.22 1.04 Peach 4.90 27.95 0.23 8.81 17.66 0.55 ------------------------------------------------------------ Name. Per Per Per Per cent cent cent cent phosphorus sulfur silica. chlorin. pentoxid. trioxid.

Prune 14.08 2.68 3.07 0.34 Apricot 13.09 2.63 5.23 0.45 Orange 12.37 5.25 0.65 0.92 Lemon 11.09 2.84 0.66 0.39 Apple 13.59 6.09 4.32 Pear 15.20 5.69 1.49 Peach 43.63 0.37

=576. Dried Fruits.=—A method of preserving fruits largely practiced consists in subjecting them, in thin slices or whole, to the action of hot air until the greater part of the moisture is driven off. The technique of the process is fully described in recent publications.[588] It has been shown by Richards that fruit subjected to rapid evaporation undergoes but little change aside from the loss of water.[589]

In the analyses of dried fruits the methods already described are used. The presence of pectin renders the filtration of the aqueous extract somewhat difficult, and in many cases it is advisable to determine the sugars present in the extract without previous filtration.

=577. Zinc in Evaporated Fruits.=—Fruits are commonly evaporated on trays made of galvanized iron. In these instances a portion of the zinc is dissolved by the fruit acids, and will be found as zinc malate etc., in the finished product. The presence of zinc salts is objectionable for hygienic reasons, and therefore the employment of galvanized trays should be discontinued. The presence of zinc in evaporated fruits may be detected by the following process.[590] The sample is placed in a large platinum dish and heated slowly until dry and in incipient combustion. The flame is removed and the combustion allowed to proceed, the lamp being applied from time to time in case the burning ceases. When the mass is burned out it will be found to consist of ash and char, which are ground to a fine powder and extracted with hydrochloric or nitric acid. The residual char is burned to a white ash at a low temperature, the ash extracted with acid, the soluble portion added to the first extract and the whole filtered. The iron in the filtrate is oxidized by boiling with bromin water and the boiling continued until the excess of bromin is removed. A drop of methyl orange is placed in the liquid and ammonia added until it is only faintly acid. The iron is precipitated by adding fifty cubic centimeters of a solution containing 250 grams of ammonium acetate in a liter and raising the temperature to about 80°. The precipitate is separated by filtration and washed with water at 80° until free of chlorids. The filtrate is saturated with hydrogen sulfid, allowed to stand until the zinc sulfid settles and poured on a close filter. It is often necessary to return the filtrate several times before it becomes limpid. The collected precipitate is washed with a saturated solution of hydrogen sulfid containing a little acetic acid. The precipitate and filter are transferred to a crucible, dried, ignited and the zinc weighed as oxid. Small quantities of zinc salts added to fresh apples which were dried and treated as above described, were recovered by this method without loss. Other methods of estimating zinc in dried fruits are given in the bulletin cited.

Evaporated apples contain a mean content of 23.85 per cent of water and 0.931 per cent of ash.

The mean quantity of zinc oxid found in samples of apples dried in the United States is ten milligrams for each 100 grams of the fruit, an amount entirely too small to produce any toxic effects. When zinc exists in the soil it will be found as a natural constituent in the crop.[591]

=578. Composition of Watermelons and Muskmelons.=—In the examination of melons a separation of the rind, seeds and meat is somewhat difficult of accomplishment, since the line of demarcation is not distinct. In watermelons the separation of rind and meat is made at the point where the red color of the meat disappears. In muskmelons no such definite point is found and in the examination of these they are taken as a whole. The total moisture, ash and nitrogen may be determined in the whole mass or in the separate portions. The sugars are most conveniently determined in the expressed juices. The mean composition of the melons given below is that obtained from analyses made in the Department of Agriculture.[592]

COMPOSITION OF MELONS. Total Total weight, Juice, proteids, Ash, grams. per cent. per cent. per cent. ------------------------------------------------------------- Watermelons 10330 meat 83.99 6.12 0.37 rind 81.02 ------------------------------------------------------------- Muskmelons 3407 80.23 6.45 0.57

COMPOSITION OF JUICE. Sucrose in Reducing sugars Ash in juice, in juice, juice, per cent. per cent. per cent. ------------------------------------------------------------- Watermelons meat 1.92 meat 4.33 meat 0.31 rind 0.34 rind 2.47 rind 0.38 ------------------------------------------------------------- Muskmelons 1.02 3.04 0.53

TEA AND COFFEE.

=579. Special Analysis.=—Aside from the examination of teas and coffees for adulterants, the only special determinations which are required in analyses are the estimation of the alkaloid (caffein) and of the tannin contained therein. It is chiefly to the alkaloid that the stimulating effects of the beverages made from tea and coffee are due. The determination of the quantity of tannin contained in tea and coffee is accomplished by the processes described under the chapter devoted to that glucosid.

The general analysis, _viz._, the estimation of water, ether extract, total nitrogen, fiber, carbohydrates and ash, with the exceptions noted above, is conducted by the methods which have already been given.

For detailed instructions concerning the detection of adulterants of tea and coffee the bulletins of the Chemical Division, Department of Agriculture, may be consulted.[593]

=580. Estimation of Caffein= (=Thein=).—The method adopted by Spencer, after a thorough trial of all the usual processes for estimating this alkaloid, is as follows:[594] To three grams of the finely powdered tea or coffee, in a 300 cubic centimeter flask, add about a quarter of a liter of water, slowly heat to the boiling point, using a fragment of tallow to prevent frothing, and boil gently for half an hour. When boiling begins, the flask should be nearly filled with hot water and more added from time to time to compensate for the loss due to evaporation. After cooling, add a strong solution of basic lead acetate until no further precipitation is produced, complete the volume to the mark with water, mix and throw on a filter. Precipitate the lead from the filtrate by hydrogen sulfid and filter. Boil a measured volume of this filtrate to expel the excess of hydrogen sulfid, cool and add sufficient water to compensate for the evaporation. Transfer fifty cubic centimeters of this solution to a separatory funnel and shake seven times with chloroform. Collect the chloroform solution in a tared flask and remove the solvent by gentle distillation. A safety bulb, such as is used in the kjeldahl nitrogen method, should be employed to prevent entrainment of caffein with the chloroform vapors.

The extraction with chloroform is nearly complete after shaking out four times; a delicate test, however, will usually reveal the presence of caffein in the watery residue even after five or six extractions, hence seven extractions are recommended for precautionary reasons. The residual caffein is dried at 75° for two hours and weighed.

The principal objection which has been made to Spencer’s method is that the boiling with water is not continued for a sufficient length of time. For the water extraction, Allen prescribes at least six hours cohobation.[595] In this method six grams of the powdered substance are boiled with half a liter of water for six hours in a flask, with a condenser, the decoction filtered, the volume of the filtrate completed to 600 cubic centimeters with the wash water, heated to boiling, and four cubic centimeters of strong lead acetate solution added, the mixture boiled for ten minutes, filtered and half a liter of the filtrate evaporated to fifty cubic centimeters. The excess of lead is removed with sodium phosphate and the filtrate and washings concentrated to about forty cubic centimeters. The caffein is removed by shaking four times with chloroform. Older but less desirable processes are fully described by Allen.[596]

In France this method is known as the process of Petit and Legrip, and it has been worked out in great detail by Grandval and Lajoux and by Petit and Terbat.[597]

=581. Estimation of Caffein by Precipitation with Iodin.=—The caffein in this method is extracted, the extract clarified by lead acetate and the excess of lead removed as in Spencer’s process described above. The caffein is determined in the acidified aqueous solution thus prepared, according to the plan proposed by Gomberg, as follows:[598]

Definite volumes of the aqueous solution of the caffein are acidulated with sulfuric and the alkaloid precipitated by an excess of a set solution of iodin in potassium iodid. After filtering, the excess of iodin in an aliquot part of the filtrate is determined by titration with a tenth normal solution of sodium thiosulfate. The filtration of the iodin liquor is accomplished on glass wool or asbestos. The results of the analyses are calculated from the composition of the precipitated caffein periodid; _viz._, C₈H₁₀N₄O₂.HI.I₄. The weight of the alkaloid is calculated from the amount of iodin required for the precipitation by the equation 4I: C₈H₁₀N₄O₂ = 508: 194. From this equation it is shown that one part of iodin is equivalent to 0.3819 part of caffein, or one cubic centimeter of tenth normal iodin solution is equal to 0.00485 gram of iodin.

In practice, it is recommended to divide the aqueous extract of the alkaloid, prepared as directed above, into two portions, one of which is treated with the iodin reagent without further preparation, and the other after acidulation with sulfuric. After ten minutes, the residual iodin is estimated in each of the solutions as indicated above. The one portion, containing only the acetic acid resulting from the decomposition of the lead acetate, serves to indicate whether the aqueous solution of the caffein contains other bodies than that alkaloid capable of forming a precipitate with the reagent, since the caffein itself is not precipitated even in presence of strong acetic acid.

In the solution acidulated with sulfuric, the caffein, together with the other bodies capable of combining with iodin, is precipitated. The residual iodin is determined in each case, and thus the quantity which is united with the caffein is easily ascertained. The weight of iodin which has entered into the precipitated caffein periodid multiplied by 0.3819 gives the weight of the caffein in the solution.

Gomberg’s method has been subjected to a careful comparative study by Spencer and has been much improved by him in important particulars.[599]

It is especially necessary to secure the complete expulsion of the hydrogen sulfid and to observe certain precautions in the addition of the iodin reagent. The precipitation should be made in a glass-stoppered flask, shaking thoroughly after the addition of the iodin and collecting the precipitate on a gooch. As thus modified, the iodin process gives results comparable with those obtained by Spencer’s method, and it can also be used to advantage in estimating caffein in headache tablets in the presence of acetanilid.

=582. Freeing Caffein of Chlorophyll.=—Any chlorophyll which may pass into solution and be found in the caffein may be removed by dissolving the caffein in ten per cent sulfuric acid, filtering, neutralizing with ammonia and evaporating to dryness. The residue is taken up with chloroform, the chloroform removed at a low temperature and the pure caffein thus obtained.[600]

=583. Proteid Nitrogen.=—The proteid nitrogen in tea and coffee may be determined in the residue after extraction of the alkaloid by boiling water as described above. More easily it is secured by determining the total nitrogen in the sample and deducting therefrom the nitrogen present as caffein. The remainder, multiplied by 6.25, will give the quantity of proteid matter.

=584. Carbohydrates of the Coffee Bean.=—The carbohydrates of the coffee bean include those common to vegetable substances; _viz._, cellulose, pentosan bodies (xylan, araban), fiber etc., together with certain sugars, of which sucrose is pointed out by Ewell as the chief.[601] In smaller quantities are found a galactose yielding body (galactan), as pointed out by Maxwell, a dextrinoid and a trace of a sugar reducing alkaline copper solution.

The sucrose may be separated from the coffee bean by the following process:[602] The finely ground flour is extracted with seventy per cent alcohol, the extract clarified with lead acetate, filtered, the lead removed from the filtrate with hydrogen sulfid, the excess of the gas removed by boiling, the filtrate evaporated in a partial vacuum to a sirup and the sucrose crystallized from a solution of the sirup in alcohol.

For a quantitive determination, ten grams of the coffee flour are extracted with ether and the residue with seventy-five per cent alcohol. This process, conducted in a continuous extraction apparatus, should be continued for at least twenty-four hours. The alcohol is removed by evaporation, the residue dissolved in water, clarified with basic lead acetate, filtered, the precipitate washed, the lead removed, again filtered, the filtrate washed and wash water and filtrate made to a definite volume. In an aliquot part of this solution the sugars are determined by the alkaline copper method, both before and after inversion. From the data obtained the percentage of sucrose is calculated.

In a coffee examined by Ewell the percentage of sucrose was found to be 6.34. The pentose yielding constituents of the coffee bean amount to from eight to ten per cent.

When coffee meal is extracted with a five per cent solution of sodium carbonate, a gummy substance is obtained, which is precipitable by alcohol. This gum, after washing with hydrochloric acid containing alcohol, gives a gray, translucent, hard mass on drying. On hydrolysis it yielded 75.2 per cent of dextrose, on distillation with hydrochloric acid, thirteen per cent of furfuraldehyd and, on oxidation with nitric acid, 18.7 per cent of mucic acid. This gum, therefore, consists chiefly of a mixture of galactan, xylan and araban.

=585. Estimation of Galactan.=—From three to five grams of the substance supposed to contain galactan are placed in a beaker with sixty cubic centimeters of nitric acid of 1.15 specific gravity. The mixture is evaporated on a steam bath until it is reduced to one-third of its original volume, allowed to stand for twenty-four hours, ten cubic centimeters of water added, well stirred and again allowed to stand for twenty-four hours, until the mucic acid is separated in a crystalline form. To remove impurities from the mucic acid it is separated by filtration, washed with not to exceed twenty cubic centimeters of water, placed together with the filter in the beaker, from twenty-five to thirty cubic centimeters of ammonium carbonate solution, containing one part of dry ammonium carbonate, nineteen parts of water and one part of ammonium hydroxid, added and heated to near the boiling point. The mucic acid is dissolved by the ammonium carbonate solution and any insoluble impurity separated by filtration, the filtrate being received in a platinum dish, the residue well washed and the entire filtrate and wash water evaporated to dryness on a steam bath acidified with dilute nitric, well stirred and allowed to stand until the mucic acid separates in a crystalline form. The separation is usually accomplished in half an hour, after which time the crystals of mucic acid are collected on a tared filter, or gooch, and washed with not to exceed fifteen cubic centimeters of water followed with sixty cubic centimeters of alcohol, then with ether, dried at 100° and weighed. For computing the amount of galactose, one gram of the mucic acid is equal to 1.333 of galactose and one gram of galactose is equal to nine-tenths gram of galactan. Before the commencement of the operation, the material should be freed of fatty matters in the case of oily seeds and other substances similar thereto.[603]

=586. Revised Factors for Pentosans.=—The factors given in paragraph =154= have lately been recalculated by Mann, Kruger and Tollens, and as a result of their investigations the following factors are now recommended.[604] The quantity of furfurol is derived from the weight of furfurolhydrazone obtained by the formula:

1. Furfurolhydrazone × 0.516 + 0.0104 = furfurol. 2. Furfurol × 1.84 = pentosans. 3. Furfurol × 1.64 = xylan. 4. Furfurol × 2.02 = araban.

The pentoses (xylose, arabinose) may be calculated from the pentosans (xylan, araban) by dividing by 0.88.

The method of procedure preferred for the estimation of the pentosans is that described in paragraph =157=. The phloroglucin is dissolved in hydrochloric acid of 1.06 specific gravity before it is added to the furfurol distillate. The latest factor for converting the phloroglucid obtained into furfurol is to divide by 1.82 for small quantities and 1.93 for large quantities. After the furfurol is obtained, the factors given above are applied.

=587. Application of Roentgen Rays to Analysis.=—The detection of mineral matters in vegetable substances by roentgen photography has been proposed by Ranvez.[605] This process will prove extremely valuable in detecting the lacing of teas with mineral substances. Practically, it has been applied by Ranvez in the detection of mineral substances mixed with saffron with fraudulent intent.

Barium sulfate is often mixed with saffron for the purpose of increasing its weight. Pure saffron and adulterated samples are enclosed in capsules of black paper and exposed on the same sensitive plate for a definite time to the rays emanating from a crookes tube. In this case the pure saffron forms only a very faint shadow in the developed negative, while the parts to which barium sulfate are attached produce strong shadows. The principle involved is applicable to a wide range of analytical research.

TANNINS AND ALLIED BODIES.

=588. Occurrence and Composition.=—The tannins and allied bodies, which are of importance in this connection, are those which occur in food products and beverages and also those made use of in the leather industry. The term tannin is applied to a large class of astringent substances, many of which are glucosids. Tannic acid is the chief constituent of the tannins, and is found in a state of comparative purity in nutgalls. The source from which the tannic acid is derived is indicated by a prefix to the name, _e. g._, gallotannic, from nutgalls, and caffetannic, from coffee etc. The tannins have lately been the theme of a critical study by Trimble, and the reader is referred to his work for an exhaustive study of the subject.[606] Tannin is one of the most widely diffused compounds, occurring in hundreds of plants. Commercially, the oaks and hemlocks are the most important plants containing tannin. The sumach, mangrove, canaigre, palmetto and many others have also been utilized as commercial sources of tannin. The tannins as a class are amorphous and odorless. They are slightly acid and strongly astringent. Their colors vary from dark brown to pure white. They are soluble in water, alcohol, ether and glycerol and insoluble in chloroform, benzol, petroleum ether, carbon bisulfid and the oils. The tannins give blue or green precipitates with iron salts and most of them brown precipitates with potassium bichromate. They are all precipitated by gelatin or albumin. Tannins are not only generally of a glucosidal nature, but are found quite constantly associated with reducing sugars, or in unstable combination therewith.

The reducing sugars may be separated from the tannin by precipitating the latter with lead acetate and determining the glucose in the filtrate after the removal of the lead. A separate portion of the tannin is hydrolyzed with sulfuric or hydrochloric acid and the reducing sugars again determined. Any excess of sugars over the first determination is due to the hydrolysis of the tannin glucosid.

=589. Detection and Estimation of Tannins.=—The qualitive reactions above mentioned serve to detect the presence of a tannin. Of the iron salts ferric acetate or chlorid is preferred. Ferrous salts do not give any reaction with dilute tannin solutions. An ammoniacal solution of potassium ferricyanid forms with tannins a deep red color changing to brown. In quantitive work the tannins are mostly determined by precipitation with metallic salts, by treatment with gelatin or hide powder, or by oxidation with potassium permanganate. Directions for the estimation of glucosids in general are found in Dragendorff’s book.[607]

=590. Precipitation with Metallic Salts.=—The methods depending on precipitation of the tannins with metallic salts are but little used and only one of them will be mentioned here. A full description of the others is contained in Trimble’s book.[608] A method for the determination of caffetannic acid in coffee has been worked out by Krug and used with some satisfaction.[609]

In this method two grams of the coffee meal are digested for thirty-six hours with ten cubic centimeters of water, a little more than twice that volume of ninety-five per cent alcohol added and the digestion continued for a day. The contents of the flask are poured on a filter and the residue washed with alcohol. The filtrate contains tannin, caffetannic acid and traces of coloring matter and fat. It is heated to the boiling point and clarified with a solution of lead acetate. A caffetannate of lead containing forty-nine per cent of the metal is precipitated. As soon as the precipitate has become flocculent it is collected on a filter, washed with ninety per cent alcohol until the soluble lead salts are all removed, then with ether and dried. The composition of the precipitate is represented by the formula Pb₃(C₁₅H₁₅O₈)₂. The caffetannic acid is calculated by the equation: Weight of precipitate: weight of caffetannic acid = 1267: 652.

=591. The Gelatin Method.=—The precipitation of tannin with gelatin is the basis of a process for its quantitive estimation which, according to Trimble, is conducted as follows:[610] Two and a half grams of gelatin and ten grams of alum are dissolved in water and the volume of the solution made up to one liter. The solution of gelatin and also that of the tannin are heated to 70° and the tannin is precipitated by adding the gelatin reagent slowly, with constant stirring, until the precipitate coagulates, and, on settling, leaves a clear liquor in which no further precipitate is produced on adding a few drops more of the reagent. In case the clearing of the mixture do not take place readily, the process should be repeated with a more dilute tannin solution. The precipitate is collected on two counterpoised filter papers one placed inside the other, dried at 110° and weighed, the empty filter paper being placed on the pan with the weights. For pure tannin (gallotannic acid) fifty-four per cent of the weight of the precipitate are tannin. Ammonium chlorid and common salt have been used in place of the alum in preparing the reagent, but if the proportion of alum mentioned above be used, satisfactory results will be obtained in most cases.

=592. The Hide Powder Method.=—The principle of this method is based on the change in specific gravity, _i. e._, total solids, which a tannin solution will undergo when brought into contact with raw hides in a state of fine subdivision. The hide powder absorbs the tannin, and the total solid content of the solution is correspondingly diminished. The method is conducted according to the official directions as follows:[611]

_Preparation of the Sample._—The bark, wood, leaves or other materials holding the tannins, are dried and ground to a fine powder and thoroughly extracted with water as mentioned below. In each case the solution or extraction is made as thorough as possible and the volume of the extract is made up to a definite amount.

_Quantity of Tanning Material._—Use such an amount of the tanning material as shall give in 100 cubic centimeters of the filtered solution about one gram of dry solids. In the case of barks, woods, leaves and similar materials, transfer to a half liter flask, fill the flask with water at approximately 80° and let stand over night in a bath which is kept at 80°, cool, fill to the mark, shake well and filter. In the case of extracts and sweet liquors, wash the proper quantity into a half liter flask with water at approximately 80°, almost filling the flask, cool and fill to the mark.

_Determination of Moisture._—Dry five grams of the sample in a flat bottom dish at the temperature of boiling water until the weight becomes constant.

_Determination of Total Solids._—Shake the solution, which should be at a temperature of about 20°, and immediately remove 100 cubic centimeters with a pipette, evaporate in a weighed dish and dry to constant weight at the temperature of boiling water.

_Determination of Soluble Solids._—Filter a portion of the solution through a folded filter, returning the filtrate to the filter twice and adding a teaspoonful of kaolin, if necessary. Evaporate 100 cubic centimeters of the filtrate and dry as above.

_Determination of Tanning Substances._—Extract twenty grams of hide powder by shaking for five minutes with 250 cubic centimeters of water, filter through well washed muslin or linen, repeat the operation three times and dry as much as possible in a suitable press. Weigh the wet powder and determine the residual moisture in about one-fourth of the whole by drying to constant weight at 100°. Shake 200 cubic centimeters of the unfiltered solution of the tannin with the rest of the moist hide powder for about five minutes, add five grams of barium sulfate, shake for one minute and filter through a schleicher and schüll folded filter, No. 590, fifteen centimeters in diameter, returning the first twenty-five cubic centimeters of the filtrate. Evaporate 100 cubic centimeters of the clear filtrate and dry the residue to constant weight at a temperature of boiling water. The difference between the soluble solids obtained in the filtered tannin solution and the residue as obtained above is the amount of tanning material absorbed by the hide powder. This weight must be corrected for the water retained by the hide powder. The shaking must be conducted by means of a mechanical shaker, in order to remove all the tannin substance from the solution. The simple machine used by druggists, and known as the milkshake, is recommended.

_Testing the Hide Powder._—Shake ten grams of the hide powder with 200 cubic centimeters of water for five minutes, filter through muslin or linen, squeeze out thoroughly by hand, replace the residue in the flask and repeat the operation twice with the same quantity of water. Pass the last filtrate through paper until a perfectly clear liquid is obtained. Evaporate 100 cubic centimeters of the final filtrate in a weighed dish, dry at 100° until the weight is constant. If the residue amount to more than ten milligrams the sample should be rejected. The hide powder must be kept in a dry place and tested once a month.

Prepare a solution of pure gallotannic acid by dissolving five grams in one liter of water. Determine the total solids by evaporating 100 cubic centimeters of this solution and drying to constant weight. Treat 200 cubic centimeters of the solution with hide powder exactly as described above. The hide powder must absorb at least ninety-five per cent of the total solids present. The gallotannic acid used must be completely soluble in water, alcohol, acetone and acetic ether and should contain not more than one per cent of substances not removed by digesting with excess of yellow mercuric oxid on the steam bath for two hours.

_Testing the Non-Tannin Filtrate. For Tannin_:—Test a small portion of the clear non-tannin filtrate with a few drops of a ten per cent solution of gelatin. A cloudiness indicates the presence of tannin, in which case the determination must be repeated, using twenty-five grams of hide powder instead of twenty grams.

_For Soluble Hide_:—To a small portion of the clear non-tannin filtrate, add a few drops of the original solution, previously filtered to remove reds. A cloudiness indicates the presence of soluble hide due to incomplete washing of the hide powder. In this case, repeat the determination with perfectly washed hide powder.

=593. The Permanganate Gelatin Method.=—This process, which is essentially the method of Löwenthal, as improved by Councler, Schroeder and Proctor and as used by Spencer for the determination of tannin in teas, is conducted as described below.[612] The principle of the process is based on the oxidation of all bodies in solution oxidizable by potassium permanganate, the subsequent precipitation of the tannin by a gelatin solution, and the final oxidation, by means of permanganate, of the remaining organic bodies. The difference between the total oxidizable matter and that left after the precipitation of the tannin represents the tannin originally in solution.

_Reagents Required._—The following reagents are necessary to the proper conduct of the potassium permanganate process:

(1). Potassium permanganate solution containing about one and a third grams of the salt in a liter:

The potassium permanganate solution is set by titration against the decinormal oxalic acid solution mentioned below. The end reaction with the indicator must be of the same tint in all the titrations, _i. e._, either golden yellow or pink.

(2). Tenth-normal oxalic acid solution for determining the exact titer of the permanganate solution:

(3). Indigo-carmin solution to be used as an indicator and containing six grams of indigo-carmin and fifty cubic centimeters of sulfuric acid in a liter. The indigo-carmin must be very pure and quite free of indigo-blue.

(4). Gelatin solution, prepared by digesting twenty-five grams of gelatin at room temperature for one hour in a saturated solution of sodium chlorid, then heating until solution is complete, cooling and making the volume up to one liter:

(5). A salt acid solution, made by adding to 975 cubic centimeters of a saturated solution of sodium chlorid, enough strong sulfuric acid to bring the volume of the mixture to one liter:

(6). Powdered kaolin for promoting filtration.

_The Process._—Five grams of the finely powdered tea (or other vegetable substance containing tannin) are boiled with distilled water in a flask of half a liter capacity for half an hour. The distilled water should be at room temperature when poured over the powdered tea. After cooling, the volume of the decoction is completed to half a liter, and the contents of the flask poured on a filter. To ten cubic centimeters of the filtered tea infusion are added two and a half times as much of the indigo-carmin solution and about three-quarters of a liter of distilled water.

The permanganate solution is run in from a burette, a little at a time, with vigorous stirring, until the color changes to a light green, and then drop by drop until the final color selected for the end of the reaction, golden yellow or faint pink, is obtained. The number of cubic centimeters of permanganate required is noted and represented by a in the formula below. The titration should be made in triplicate and the mean of the two more nearly agreeing readings taken as the correct one.

One hundred cubic centimeters of the filtered tea infusion, obtained as directed above, are mixed with half that quantity of the gelatin reagent, the first named quantity of the acid salt solution added, together with ten grams of the powdered kaolin, the mixture well shaken for several minutes and poured on a filter. Twenty-five cubic centimeters of the filtrate, corresponding to ten of the original tea solution are titrated with the permanganate reagent, under the conditions given above, and the reading of the burette made and represented by _b_. The quantity of permanganate solution, _viz._, _c_, required to oxidize the tannin is calculated from the formula _a - b_ = _c_. The relation between the permanganate, oxalic acid and tannin is such that 0.04157 gram of gallotannic acid is equivalent to 0.063 gram of oxalic acid. The relation between the oxalic acid solution and the permanganate having been previously determined the data for calculating the quantity of tannin, estimated as gallotannic acid, are at hand.

=594. The Permanganate Hide Powder Method.=—Instead of throwing out the tannin with gelatin it may be absorbed by hide powder. The principle of the process, save this modification, is the same as in the method just described. As described by Trimble, the analysis is conducted according to the following directions:[613]

_Reagents Required._—The reagents required for conducting the permanganate hide powder process are as follows:

1. _Permanganate Solution._—Ten grams of pure potassium permanganate are dissolved in six liters of water. The solution is standardized with pure tannin. The moisture in the pure tannin is determined by drying at 100° to constant weight and then a quantity of the undried substance, representing two grams of the dried material, is dissolved in one liter of water. Ten cubic centimeters of this solution and double that quantity of the indigo solution to be described below, are mixed with three-quarters of a liter of water and the permanganate solution added from a burette with constant stirring until the liquid assumes a greenish color and then, drop by drop, until a pure yellow color with a pinkish rim is obtained. Fifty cubic centimeters of the pure tannin solution are digested, with frequent shaking, with three grams of hide powder which has been previously well moistened and dried in a press for eighteen or twenty hours, the contents of the flask thrown on a filter and ten cubic centimeters of the filtrate titrated with the permanganate solution as directed above. The difference between the amount of permanganate solution required for the first and second titrations represents the amount of pure tannin or oxidizable matter removed by the hide powder.

2. _Indigo Solution._—The indicator which is used in the titrations is prepared by dissolving thirty grams of sodium sulfindigotate in three liters of dilute sulfuric acid made by adding one volume of the strong acid to three volumes of water. The solution is shaken for a few minutes, thrown upon a filter and the insoluble residue washed with sufficient water to make the volume of the filtrate six liters.

3. _Hide Powder._—The hide powder used should be white, wooly in character and sufficiently well extracted with water to afford no other extract capable of oxidizing the permanganate solution.

_The Process._—The reagents having been prepared and tested as above, the solution of the substance containing the tannin, prepared as described further on, is titrated first with the permanganate solution in the manner already given. Fifty cubic centimeters of the tannin solution are then shaken, from, time to time for eighteen hours, with three grams of hide powder, thrown upon a filter and ten cubic centimeters of the filtrate titrated with the potassium permanganate as above described. From the data obtained, the quantity of permanganate solution corresponding to the tannin removed by the hide powder is easily calculated. The value of the permanganate solution having been previously set with a pure tannin, renders easy of calculation the corresponding amount of pure tannin in the solution under examination.

=595. Preparation of the Tannin Infusion.=—A sample weighing about a kilogram should be secured, representing as nearly as possible the whole of the materials containing tannin in a given lot. The sample is reduced to a fine powder and passed through a sieve containing apertures about a millimeter in diameter. The quantity of the sample used for the extraction depends largely upon its content of tannin. Five grams of nutgalls, ten grams of sumach or twenty grams of oak bark represent about the quantities necessary for these classes of tannin-holding materials. The sample is boiled for half an hour with half a liter of water, filtered through a linen bag into a liter flask and washed and pressed with enough water to make the volume of the filtrate equal to one liter. The proper quantities of this solution are used for the analytical processes described above.

TOBACCO.

=596. Fermented and Unfermented Tobacco.=—Samples of tobacco may reach the analyst either in the fermented or unfermented state. As a basis for comparison, it is advisable in all cases to determine the constituents of the sample before fermentation sets in. The analysis, after fermentation is complete, will then show the changes of a chemical nature which it has undergone during the process of curing and sweating. Only tobacco which has undergone fermentation is found to be in a suitable condition for consumption. In addition to the natural constituents of tobacco, it may contain, in the manufactured state, flavoring ingredients such as licorice and sugar, coloring matters and in some instances, it is said, opium or other stimulating drugs. It is believed, however, that opium is not often found in manufactured tobacco, and it has never been found in this laboratory in cigarettes, although all the standard brands have been examined for it.[614]

In researches made at the Connecticut Station it is shown that fermentation produces but little change in the relative quantities of nitric acid, ammonia, fiber and starch in the leaves, while those of nicotin, albuminoids and amids are diminished. This is not in harmony with the generally accepted theory that starch is inverted and fermented during the process.[615]

The nature of the ferments which are active in producing the changes which tobacco undergoes in curing, is not definitely understood. Some of the organic constituents of the tobacco undergo a considerable change during the process. Any sugar which is found in the freshly cured leaves disappears wholly or in part. As products of fermentation may also be found succinic, fumaric, formic, acetic, propionic and butyric acids.

=597. Acid and Basic Constituents of Tobacco.=—In unfermented and fermented tobacco are found certain organic acids, among the most important of which are citric, malic, oxalic, pectic and tannic. Of the inorganic acids the chief which are found are nitric, sulfuric and hydrochloric. Among the bases ammonia and nicotin are the most important. Ammonia is found in the unfermented tobacco in only small quantities, but in the fermented product it may sometimes reach as high as half a per cent. The presence of these two nitrogenous bases in tobacco renders the estimation of the proteid matter contained therein somewhat tedious and difficult.

=598. Composition of Tobacco Ash.=—The mineral constituents of tobacco are highly important from a commercial point of view. The burning properties of tobacco depend largely upon the nature of its mineral constituents. A sample containing a large quantity of chlorids burns much less freely than one in which the sulfates and nitrates predominate. For this reason, the use of potash fertilizers containing large amounts of chlorin is injudicious in tobacco culture, the carbonates and sulfates of potash being preferred. The leaves of the tobacco plant contain a much larger percentage of mineral constituents than the stems, their respective contents of pure ash, that is ash free from carbon dioxid, carbon and sand, being about seventeen and seven. The pure ash of the leaves has the following mean composition: Potash 29.1 per cent, soda 3.2 per cent, lime 36.0 per cent, magnesia 7.4 per cent, iron oxid 2.0 per cent, phosphoric acid 4.7 per cent, sulfuric acid 6.0 per cent, silica 5.8 per cent, and chlorin 6.7 per cent.[616]

=599. Composition of Tobacco.=—The mean composition of some of the more important varieties of water-free tobacco is shown in the following table:[617]

Havana, Sumatra, Kentucky, Java, per cent. per cent. per cent. per cent. Nicotin 3.98 2.38 4.59 3.30 Malic acid 12.11 11.11 11.57 6.04 Citric acid 2.05 2.53 3.40 3.30 Oxalic acid 1.53 2.97 2.03 3.38 Acetic acid 0.42 0.29 0.43 0.22 Tannic acid 1.13 0.98 1.48 0.51 Nitric acid 1.32 0.60 1.88 0.23 Pectic acid 11.36 11.88 8.22 10.13 Cellulose 15.76 10.59 12.48 11.82 Ammonia 0.49 0.06 0.19 0.23 Soluble nitrogenous matter 7.74 8.84 13.90 10.39 Insoluble ” ” 9.75 7.97 8.10 9.53 Residue and chlorophyll 5.15 8.63 1.99 6.45 Oil 1.03 1.26 2.28 0.81 Ash 17.50 17.03 14.36 18.46 Undetermined 8.68 12.88 13.10 15.20

Among the undetermined matters are included those of a gummy or resinous composition not extracted by ether, the exact nature of which is not well understood, and the starches, sugars, pentosans and galactan.

Tobacco grown in more northern latitudes has less nicotin than the samples given in the foregoing table.

The following table shows the composition of tobacco grown in Connecticut:[618]

(A)= Unfermented, (B)= Fermented, Upper leaves. Short seconds. First wrappers. (A) (B) (A) (B) (A) (B) % % % % % % Water 23.50 23.40 27.40 21.10 27.50 24.90 Pure ash 14.89 15.27 22.85 25.25 15.84 16.22 Nicotin 2.50 1.79 0.77 0.50 1.26 1.44 Nitric acid 1.89 1.97 2.39 2.82 2.59 2.35 Ammonia 0.67 0.71 0.16 0.16 0.33 0.47 Proteids 12.19 13.31 6.69 6.81 11.31 11.62 Fiber 7.90 8.78 7.89 8.95 9.92 10.42 Starch 3.20 3.36 2.62 3.01 2.89 3.08 Oil and fat 3.87 3.42 2.95 3.04 2.84 2.92 Undeterm’d 29.39 27.99 26.28 28.36 25.52 26.88

=600. Estimation of Water.=—In the estimation of water in vegetable substances, as has already been noted, it is usual to dry them in the air or partial vacuum, or in an inert gas, at a temperature of 100° until a constant weight is reached. By this process, not only the water, but all substances volatile at the temperature and in the conditions mentioned are expelled. The quantity of these volatile substances in vegetable matter, as a rule, is insignificant and hence the total loss may be estimated as water. In the case of tobacco a far different condition is presented, inasmuch as the nicotin, which sometimes amounts to five per cent of the weight of the sample, is also volatile under the conditions mentioned. It is advisable, therefore, to dry the sample of tobacco at a temperature not above fifty degrees and in a vacuum as complete as possible. Tobacco is also extremely rich in its content of crystallized mineral salts, containing often water of crystallization, and there is danger of this crystal water being lost when the sample is dried at 100°. The desiccation is conveniently made in the apparatus described on page 22. If a high vacuum be employed, _viz._, about twenty-five inches of mercury, it is better not to allow the temperature to go above 40° or 45°. A rather rapid current of dry air should be allowed to pass through the apparatus for the more speedy removal of the moisture and a dish containing sulfuric acid may also be placed inside of the drying apparatus. It is possible by proceeding in this way to secure constant weight in the sample after a few hours.

=601. Estimation of Nitric Acid.=—The nitric acid in a sample of tobacco is most easily estimated by the ferrous chlorid process.[619]

The sample is best prepared by making an alcoholic extract which is accomplished by exhausting about twenty-five grams of the fine tobacco powder with 200 cubic centimeters for forty per cent alcohol made slightly alkaline by soda lye. The mixture is boiled in a flask with a reflux condenser for about an hour. After cooling, the volume is completed to a definite quantity, and, after filtering, an aliquot part is used for the analytical process. It is evident that the nitric acid cannot be estimated in this case after previous reduction to ammonia by zinc or iron on account of the presence of ammonia in the sample itself. If, however, the amount of ammonia be determined in a separate portion of the sample, the nitric acid may be reduced in the usual way, by zinc or iron, the total quantity of ammonia determined by distillation, the quantity originally present in the sample deducted and the residual ammonia calculated to nitric acid.

=602. Sulfuric and Hydrochloric Acids.=—These two acids are determined in the ash of the sample by the usual methods. The sulfuric acid thus found represents the original sulfuric acid in combination with the bases in the mineral parts of the plant, together with that produced by the oxidation of the organic sulfur during combustion. In order to avoid all loss of sulfur during the combustion, the precautions already given should be observed. The separation of the sulfur pre-existing as sulfates from that converted into sulfates during the combustion is accomplished as previously directed.[620] For ordinary purposes, this separation is not necessary.

To avoid loss of chlorin from volatilization during incineration the temperature should be kept at the lowest possible point until the mass is charred, the soluble salts extracted from the charred mass and the incineration completed as usual.

=603. Oxalic, Citric and Malic Acids.=—The separation and estimation of organic acids from vegetable tissues is a matter of great difficulty, especially when they exist as is usually the case, in very minute proportions. During incineration, the salts of the inorganic acids are converted into carbonates and the subsequent examination of the ash gives no indication of the character of the original acids. In the case of tobacco, the organic acids of chief importance, from an analytical point of view, are oxalic, citric and malic. These acids may be extracted and separated by the following process:[621]

Ten grams of the dry tobacco powder are rubbed up in a mortar with twelve cubic centimeters of dilute sulfuric acid (one to five) and then absorbed with coarse pumice stone powder in sufficient quantity to cause all the liquid to disappear. The mass is placed in an extraction apparatus of proper size and thoroughly extracted with ether until a drop of the extract leaves no acid residue on evaporation. Usually about ten hours are required. The organic acids are thus separated from the mineral acids. The ether is removed from the extract and the residue dissolved in hot water, cooled, filtered, if necessary several times, until the solution is separated from the fat and resin which have been extracted by the ether. The filtrate is neutralized with ammonia, slightly acidified with acetic and the oxalic acid contained therein thrown out by means of a dilute solution of calcium acetate, which must not be added in excess. The calcium oxalate is separated by filtration, and determined as lime oxid. To the filtrate is added drop by drop, with constant stirring, a dilute solution of lead acetate, prepared by mixing one part of a saturated solution of lead acetate with four parts of water. When the precipitate formed has settled, the clear supernatant liquid is tested by adding a drop of acetic acid and a few drops of the dilute lead acetate. In case a precipitate be formed, the addition of the lead acetate is continued until a precipitate is secured which will immediately dissolve in acetic acid. At this moment the citric acid is almost completely precipitated. In order to avoid the accumulation of the acetic acid by reason of the repetition of the process as above described, the mixture is neutralized each time with dilute ammonia. The precipitated neutral lead citrate obtained by the above process, is separated by filtration and, in order to avoid its decomposition when washed with pure water, it is washed with a very dilute acetic acid solution of lead acetate. The washing and filtration are accomplished as quickly as possible, and the final washing is made with alcohol of thirty-six per cent strength. In the filtrate the residual lead citrate, together with a little lead malate, are precipitated by the alcohol used as the wash and this precipitate is also separated by filtration. The filtrate containing the greater part of the malic acid is evaporated to remove the alcohol and treated with lead acetate in excess. Afterwards it is mixed with five times its volume of thirty-six per cent alcohol containing a half per cent of acetic acid. In these conditions the lead malate is completely precipitated as neutral salt, and after standing a few hours, is separated by filtration. The three precipitates, obtained as above, are dried at 100° and weighed. If the precipitates have been collected on filter paper they should be removed as completely as possible, the papers incinerated in the usual way and any reduced lead converted into nitrate and oxid by treatment with nitric acid and subsequent ignition. From the quantities of lead oxid obtained, the weights of the citric and malic acids are computed. The precipitate which is obtained by the action of alcohol, above noted, is also dried and ignited and the lead oxid found divided equally between the citric and malic acids, the respective quantities of which found, are included in computing their total weights. The weight of the citric acid is calculated from the formula (C₆H₅O₇)₂Pb₃ + H₂O, and that of the malic acid from the formula C₄H₄O₅Pb + H₂O.

=604. Acetic Acid.=—For the determination of the volatile acids of the fatty series existing in tobacco, the following process, also due to Schlösing, may be followed:[622]

The apparatus employed is shown in Fig. 121. Ten grams of the pulverized tobacco, moistened with water and mixed with a little powdered tartaric acid, are placed in the tube _A_. The two ends of the tube, _A_, are stoppered with asbestos or glass wool. Steam, generated in the flask, _D_, is passed into _B_. After fifteen minutes, or as soon as it is certain that the contents of _A_ have reached a temperature of 100°, the dish, _F_, containing mercury, is placed in the position shown in the figure. The steam, by this arrangement, is forced into the lower end of _A_, passes into the condenser _E_, and the condensed water collected in _C_. The operation should be so conducted as to avoid any condensation of water in _B_. It is advisable during the progress of the distillation, which should continue for at least twenty minutes, to neutralize from time to time the acetic acid collected in _C_ by a set solution of dilute alkali, or, an excess of the alkaline solution may be placed in _C_ and the part not neutralized by the acetic acid determined at the end of the distillation by titration.

[Illustration: FIG. 121.—APPARATUS FOR ACETIC ACID.]

=605. Pectic Acid.=—Under this term are included not only the pectic acid but all the other bodies of a pectose nature contained in tobacco. These bodies are of considerable interest, although they do not belong to the most important constituents. In fresh tobacco leaves are found three pectin bodies. One pectin is soluble in water, another is an insoluble pectose and the third is the pectose body forming salts with the alkalies, _i. e._, true pectic acid. In fermented tobacco pectic acid is found chiefly in combination with lime in the ribs of the leaves, serving to give them the necessary stiffness. For the estimation of the pectin bodies (mucilage) the powdered tobacco is thoroughly extracted with cold water. An aliquot part of the aqueous extract is mixed with two volumes of strong alcohol and allowed to stand in a well closed vessel in a cool place for twenty-four hours. The precipitate is collected on a filter, washed with sixty-six per cent alcohol, dried and weighed. The dried residue is incinerated and the amount of ash determined. In general, vegetable mucilages contain about five per cent of ash. If more than this be found, it is due to the solution of the salts of the organic acids contained in the sample. A dried vegetable mucilage, obtained as above, dissolves in water to a mucilaginous liquid which does not reduce alkaline copper solution until it has been hydrolyzed by boiling with a dilute mineral acid.[623]

=606. Tannic Acid.=—This acid is separated and estimated by the processes given in paragraphs =589-595=.

=607. Starch and Sugar.=—The unfermented leaves of tobacco contain considerable quantities of carbohydrates in addition to woody fiber, pentosans, galactan and cellulose. Among these, starch is the most important. Sugar exists in small quantities in the fresh leaf, usually not over one per cent. During fermentation, according to some authorities, the starch is partially converted into sugar and the latter substance disappears under the action of the alcoholic ferments. It has been found at the Connecticut Station, however, that the starch content of the leaf does not decrease during fermentation. The starch and sugar may be determined in the fresh leaves by the methods already given.

In the manufacture of certain grades of tobacco it is customary to add a quantity of sugar. The analyst may thus be called upon to determine in some cases whether the sugar found in a sample is natural or added. The occurrence of natural sugars in tobacco has been investigated at the instance of the British Treasury.[624]

The natural sugars which may be found in sun dried tobaccos usually disappear entirely during the process of fermentation. It was found by the Somerset House chemists that the content of sugar in commercial tobaccos varies from none at all to over fifteen per cent. A remarkable example of this variation is reported in two samples from this country, one of which, grown in Kentucky, contained no sugar, and the other grown in Virginia, 15.2 per cent.

It was noticed that the saccharin matters in the tobaccos examined were neutral to polarized light. They are determined by their copper reducing power. The tobacco sugars are therefore to be classed with the reducing bodies, not optically active, found in the juices of sorghum and sugar canes.

=608. Ammonia.=—As has already been intimated, ammonia exists only in minute quantities in fresh tobacco leaves, but in considerable quantities after fermentation. In the estimation of ammonia, twenty grams of the tobacco powder are digested with 250 cubic centimeters of water, acidulated with sulfuric and after an hour enough water added to make the total quantity 400 cubic centimeters. After filtration, an aliquot part of the filtrate, about 200 cubic centimeters, is treated with magnesium oxid in excess and the ammonia and nicotin removed by distillation in a current of steam. The distillate is collected in dilute sulfuric acid of known strength. The total amount of the two bases is determined by titration and the quantity of base representing the nicotin, which has been determined in a separate sample, subtracted in order to obtain the weight of the ammonia.[625]

The ammonia in tobacco is determined by Nessler in the following manner:[626]

The powdered tobacco is mixed with water and magnesium oxid and after standing for several hours it is distilled in a current of steam, the distillate received in dilute sulfuric acid and the process continued until a drop of the distillate gives no reaction for ammonia with the nessler reagent. The excess of sulfuric acid in the distillate is neutralized with pure sodium carbonate and the nicotin precipitated by a neutral solution of mercuric iodid and potassium iodid. The precipitate is separated by filtration, the filtrate treated with sodium sulfid, and the ammonia again obtained by distillation with an alkali, collected in dilute solution of set sulfuric acid and determined by titration. The difference of the two determinations represents the ammonia.

=609. Nicotin.=—In this laboratory McElroy has made a study of some of the best approved methods for determining nicotin, and finds the most simple and reliable to be that proposed by Kissling.[627] The finely powdered tobacco should be dried at a temperature not exceeding 60°, or it may be partially dried at that temperature before grinding and the final drying completed afterwards. Twenty grams of the powdered sample are intimately mixed by means of a pestle with ten cubic centimeters of dilute alcoholic solution of soda lye, made by dissolving six grams of sodium hydroxid in forty cubic centimeters of water and completing the volume to 100 cubic centimeters with ninety-five per cent alcohol. The mass is transferred to an extraction paper cylinder, placed in an extraction apparatus and extracted for three hours with ether. The ether is nearly all removed by careful distillation, the residue mixed with fifty cubic centimeters of a very dilute soda lye solution (4 to 100) and subjected to distillation in a current of steam. The flask containing the nicotin extract should be connected with the condensing apparatus by a safety bulb as is usual in the distillation of substances containing fixed alkali. The distillation should be conducted rapidly and in such a manner that when 200 cubic centimeters of the distillate have been collected, not more than fifteen cubic centimeters of the liquid remain in the distillation flask. In the distillate, the nicotin is determined by titration with a set solution of dilute sulfuric acid, using rosolic acid or phenacetolin as indicator. It is advisable to titrate each fifty cubic centimeters of the distillate as it is received and the distillation is continued until the last fifty cubic centimeters give no appreciable quantity of the alkaloid. In the calculations one molecule of sulfuric acid is equivalent to two molecules of nicotin according to the equation

H₂SO₄ = (C₁₀H₁₄N₂)₂. 98 324

_Polarization Method._—Popovici has based a method of detecting the quantity of nicotin in tobacco on its property of rotating the plane of polarized light.[628] The gyrodynat of pure nicotin is expressed by the formula [_a_]_{D} = -161°.6. When ten parts of nicotin are mixed with ninety parts of water, this value becomes -74°.1. By reason of this great depression in gyrodynatic value Popovici determined the relation which exists between the dilute solutions of nicotin and the number of minutes of angular rotation produced on polarization in a 200 millimeter tube. In a solution in which two grams of nicotin are contained in fifty cubic centimeters, each minute of angular rotation is found to correspond to 6.5 milligrams of nicotin. For one gram in solution in the same volume one minute of angular rotation corresponds to 5.9 milligrams and for a half gram in solution to 5.7 milligrams.

The nicotin is prepared for polarization by extracting with ether, as indicated in the previous paragraph, and the ethereal solution from twenty grams of tobacco is shaken with a concentrated solution of sodium phosphotungstate in nitric acid by means of which nicotin and ammonia are precipitated and rapidly settle. The supernatant liquid is carefully poured off and the residue made up to a volume of fifty cubic centimeters with distilled water and the nicotin freed from any of its compounds by the addition of eight grams of finely powdered barium hydroxid. In order to promote the decomposition of the nicotin compounds the mixture should be shaken at intervals for several hours. The at first blue precipitate changes into blue green and finally into yellow. It is separated by filtration and the somewhat yellow colored filtrate placed in an observation tube, polarized, the polarization calculated to minutes of angular rotation and the number of minutes thus found multiplied by the nearest factor given above.

The analyst will find a description of other methods of estimating nicotin in tobacco in the periodical literature of analytical chemistry.[629]

=610. Estimation of Amid Nitrogen.=—For the estimation of amid nitrogen ten grams of the powdered tobacco are digested with 100 cubic centimeters of forty per cent alcohol, the extract separated by filtration, acidified with sulfuric and the albumin, peptone, nicotin and ammonia precipitated with as little phosphotungstic acid as possible. The precipitate is separated by filtration and seventy-five cubic centimeters of the filtrate evaporated in a thin glass or tin foil capsule after the addition of a little barium chlorid and the nitrogen determined in the residue. The nitrogen thus obtained is that which was present in an amid state. The nitrogen present as amids, ammonia and nicotin subtracted from the total nitrogen leaves that present as protein.

=611. Fractional Extraction of Tobacco.=—To determine the character of the soluble constituents of tobacco it is advisable to subject it to a fractional extraction with different reagents. The reagents usually employed in the order mentioned are petroleum ether, ether, absolute alcohol, water, dilute soda lye and dilute hydrochloric acid. The extract obtained by petroleum ether contains vegetable wax, chlorophyll and its alteration products, fat, ethereal oils, and resin bodies. The extract with ether may be divided into water soluble and alcohol soluble bodies. Among the first are small quantities of glucosids and nicotin while in the alcoholic solution resin predominates.

The alcoholic extract is also divided into water soluble and alcohol soluble parts. The first contains the nicotin, which is insoluble in ether, in combination with acids, together with tannic acid and allied bodies and also the sugar. The part insoluble in water consists chiefly of resin.

The aqueous solution contains the vegetable mucilages (pectin) soluble carbohydrates, soluble proteids and organic acids.

The dilute soda lye solution contains chiefly proteids.

The dilute hydrochloric acid solution contains the starch and the oxalic acid originally combined with lime. The extractions with dilute soda lye and dilute hydrochloric acid should be made at a boiling temperature. The residual matter consists of a mixture of carbohydrate bodies to which the term crude fiber is usually applied.

=612. Burning Qualities.=—When tobacco is to be used for the manufacture of cigars, or cigarettes, or for smoking in pipes, its ability to keep burning is a matter of great importance. The tobacco, when once ignited, should burn for some time and form, a fluffy ash, free of fused mineral particles. A tobacco with good burning properties is one containing nitrates in considerable quantity, not too much sugar and starch, a porous cellular structure and comparatively free of chlorin. In determining comparative burning properties the tests may be applied to the single leaf or the tobacco may be first rolled into a cigar form and burned in an artificial smoker.

[Illustration: FIG. 122. APPARATUS FOR SMOKING.]

In applying the test to the leaf it is important that the ignition be made with a fuse without flame, which maintains a uniform burning power. Any good slow burning fuse may be used and it is applied to the leaf in such a way that a hole may be burned in it, leaving its edges uniformly ignited. The number of seconds elapsing before the last spark is extinguished is noted. At the Connecticut Experiment Station a lighter, proposed by Nessler, is employed. It is prepared by digesting eighty grams of gum arabic in 120 cubic centimeters, and forty grams of gum tragacanth in a quarter of a liter of water for two days, mixing the mucilaginous masses and adding ten grams of potassium nitrate and about 350 grams of pulverized charcoal. The mixture is rolled, on a plate sprinkled with charcoal, into sticks a few inches in length and of the diameter of a cigar and dried at a gentle heat. These fuses burn slowly and without smoke and are well suited for lighting tobacco leaves. Several tests, at least six, should be made with each leaf. Leaves having a uniform burning power should be used as comparators and the number of seconds they burn be designated by 100. It is important that all the samples to be tested be exposed for a day or two to the same atmosphere in order that they may have, as nearly as possible, the same content of moisture. The burning tests, when possible, should be made both before and after fermentation. As a rule fermentation improves the burning quality of second rate leaves, but has little effect on leaves of the first quality.

=613. Artificial Smoker.=—For the purpose of comparing the burning properties of cigars, or of leaves rolled into cigar form, the artificial smoking apparatus devised by Penfield and modified in this laboratory is employed.[630] The construction of the apparatus is shown in the accompanying figure.

The lighted cigar is set in the tube at the left, so that air entering the test-tube must pass through the cigar. The test-tube contains enough water to seal the end of the tube carrying the cigar, and is connected with the aspirator on the right by the =T= tube, as shown. An arm of the =T= dips just beneath the surface of the liquid in the cup in the center. Water flows in a slow stream into the aspirator through the tube at the extreme right, forcing the air out through the arm of the =T= until the siphon begins to act. While the water is voided through the long arm of the siphon, air enters through the cigar, the liquid rising in the =T=. The action of the apparatus is automatic and intermittent. When the cigar is about one-third burned, it is removed without disturbing the ash cone, and the latter examined and compared with other samples as a standard. The sealing liquid of the long arm of the =T= may be mercury or water. In case mercury be used, care must be taken not to immerse the open end of the =T= more than one millimeter therein.

FERMENTED BEVERAGES.

=614. Description.=—Among fermented beverages are included those drinks, containing alcohol, prepared by fermenting the sugars or starches of fruits, cereals or other agricultural products. Wine and beer, in their various forms, and cider are the chief members of this class of bodies. Koumiss, although a fermented beverage, is not included in this classification, having been noticed under dairy products. The large number of artificial drinks, made by mixing alcohol with fruit and synthesized essences, is also excluded, although the methods of analysis which are used may be applied also to them.

Fermented beverages containing less than two per cent of alcohol are usually regarded as non-intoxicating drinks. Beers are of several varieties, and the term includes lager beer, ale, porter and stout. Distilled liquors are obtained by separating the alcohols and other volatile matters from the products of fermentation by distillation. It is not practicable here to attempt a description of the methods of preparing fermented drinks. Special works on this branch of the subject are easy of access.[631]

=615. Important Constituents.=—Alcohol is the most important constituent of fermented beverages. The solid matters, commonly called extract, which are obtained on evaporation are composed of dextrins, sugars, organic acids, nitrogenous bodies and mineral matters affording ash on combustion. Of these the dextrins and sugars form the chief part and the proteid bodies nearly ten per cent in the case of beers made of malt and hops. In beers the bitter principles derived from hops, while not important by reason of quantity, are of the utmost consequence from a gustatory and hygienic point of view. The ash of fermented beverages varies with their nature, or with the character of the water used in making the mash. In the manufacture of beer, water containing a considerable proportion of gypsum is often used, and this substance is sometimes added in the course of manufacture, especially of wine. The presence of common salt in the ash in any notable quantity is evidence of the addition of this condiment, either to improve the taste of the beverage or to increase the thirst of the drinker. In cider the organic acids, especially malic, are of importance.

Glycerol is a product of fermentation and of the hydrolysis of the fats and oils in the substances fermented.

=616. Specific Gravity.=—In order to secure uniformity of expression, the specific gravity of fermented beverages is determined at about 15°.6, although that is a temperature much below the average found in American laboratories. The specific gravity may be determined by an alcoholometer, pyknometer or hydrostatic balance in harmony with the directions given in paragraphs =48-54= and =285=. By reason of the extractive matters held in solution, fermented beverages are usually heavier than water, even if the content of alcohol be twenty per cent or more. On the other hand distilled liquors are lighter than water.

=617. Determination of Alcohol.=—The determination of the percentage of alcohol present in a solution is based on two general principles. On the one hand, and this is the base of the methods in common use, the alcohol is secured mixed only with water and its amount determined by ascertaining the specific gravity of the mixture. On the other hand the quantity of alcohol in a mixture may be determined by ascertaining the temperature of the vapors produced on boiling. This is the principle involved in the use of the ebullioscope. The latter method is not employed to any extent in this country.

_Use of the Alcoholometer._—The alcoholometer usually employed is known by the name of Gay-Lussac, who first made practical use of it in the determination of alcohol. It is constructed in such a way as to read directly the volume of absolute alcohol contained in one hundred volumes of the liquid at a temperature of 15°.6. The instruments employed should be carefully calibrated and thoroughly cleaned by washing with absolute alcohol before use. The stem of the instrument must be kept free from any greasy substance, and this is secured by washing it with ether. After this last washing the analyst should be careful not to touch the stem of the instrument with his fingers. It is most convenient to make the determination exactly at 15°.6, but when made at other temperatures the reading of the instrument is corrected by tables which may be found in works especially devoted to the analysis of wines.[632]

In this country the alcoholometer is used to some extent, but the official method is based upon the determination of the specific gravity by an instrument constructed in every respect like the alcoholometer, but giving the specific gravity of the liquor at 15°.6 instead of its percentage by volume in alcohol. The reading of the instrument having been determined at a temperature of 15°.6, the corresponding percentage of alcohol by volume or by weight is taken directly from the table given further on.

[Illustration: FIG. 123. METAL DISTILLING APPARATUS.]

_Methods of Distillation._—The metal apparatus employed in the laboratory of the Department of Agriculture, for the distillation of fermented beverages in order to determine the percentage of alcohol by the method given above, is shown in the accompanying figure. The apparatus consists of a retort of copper carried on supports in such a way as to permit an alcohol or bunsen lamp to be placed under it. It is connected with a block tin condenser and the distillate is received in a tall graduated cylinder placed under the condenser in such a way as to prevent the loss of any alcohol in the form of vapor. Exactly 300 cubic centimeters of the wine or fermented beverage are used for the distillation. Any acid which the wine contains is first saturated with calcium carbonate before placing in the retort. Exactly 100 cubic centimeters of distillate are collected and the volume of the distillate is completed to 300 cubic centimeters by the addition of recently distilled water.[633] The cylinder containing the distillate is brought to a temperature of 15°.6, the alcoholometer inserted and its reading taken with the usual precautions.

_Official Method._—The alcoholometers employed in the official methods are calibrated to agree with those used by the officers of the Bureau of Internal Revenue. They are most conveniently constructed, carrying the thermometer scale in the same stem with that showing the specific gravity. It is highly important that the analyst assure himself of the exact calibration of the instrument before using it. Inasmuch as the volume of the distillate may not be suited in all cases to the use of a large alcoholometer, it is customary in this laboratory to determine the specific gravity by means of the hydrostatic balance, as described further on. Attention is also called to the fact that, in the official method, directions are not given to neutralize the free acid of the fermented beverage before the distillation. Since the Internal Revenue Bureau is concerned chiefly with the determination of alcohol in distilled liquors, this omission is of little consequence. Even in ordinary fermented beverages the percentage of volatile acids, (acetic etc.,) is so small as to make the error due to the failure to neutralize it of but little consequence. In order, however, to avoid every possibility of error, it is recommended that in all instances the free acids of the sample be neutralized before distillation. In this laboratory, the distillations are conducted in a glass apparatus shown in the accompanying figure. The manipulation is as follow:[634]

[Illustration: FIG. 124. DISTILLING APPARATUS.]

One hundred cubic centimeters of the liquor are placed in a flask of from 250 to 300 cubic centimeters capacity, fifty cubic centimeters of water added, the flask attached to a vertical condenser by means of a bent bulb tube, 100 cubic centimeters distilled and the specific gravity of the distillate determined. The distillate is also weighed, or its weight calculated from the specific gravity. The corresponding percentage of alcohol by weight is obtained from the appended table, and this figure multiplied by the weight of the distillate, and the result divided by the weight of the sample, gives the per cent of alcohol by weight contained therein.

The percentage of alcohol by volume of the liquor is the same as that of the distillate, and is obtained directly from the appended table.

In distilled liquors about thirty grams are diluted to 150 cubic centimeters, 100 cubic centimeters distilled and the per cent of alcohol by weight determined as above.

The percentage of alcohol by volume in the distillate is obtained from the appended table. This figure divided by the number expressing the volume in cubic centimeters of the liquor taken for the determination (calculated from the specific gravity), and the result multiplied by 100 gives the per cent of alcohol by volume in the original liquor.

=618. Determining the Specific Gravity of the Distillate.=—The specific gravity of the distillate may be determined by the pyknometer, alcoholometer, hydrostatic balance or in any accurate way. The volume of the distillate is not always large enough to be conveniently used with an alcoholometer, especially the large ones employed by the Bureau of Internal Revenue. In the laboratory of the Agricultural Department, it is customary to determine the density of the distillate by the hydrostatic balance shown in paragraph =285=. The specific gravity is in each case determined at 15°.6, referred to water of the same temperature, or if at a different temperature calculated thereto.

=619. Table for Use with Hydrostatic Plummet.=—It is more convenient to determine the density of the alcoholic distillate at room temperature than to reduce it to the standard for which the plummet is graduated. In the case of a plummet which displaces exactly five grams, or multiple thereof, of distilled water at 15°.6, the corrections for temperatures between 12°.2 and 30° are found in the following table, prepared by Bigelow.[635]

If the weight of the alcoholic solution displaced be 4.96075 grams the apparent specific gravity 0.99215 and the temperature of observation 25°.4, the correction, which is additive, as given in the table is 0.00191 and the true specific gravity is 0.99406 and the percentage of alcohol by volume 4.08.

When the plummet does not exactly displace five grams of water at 15°.6, but nearly so, the table may still be used.

For example, suppose the weight of water displaced be 4.9868 instead of five grams. The apparent specific gravity of the water by this plummet is 0.99736 and the difference between this and the true specific gravity is 0.00264, which is a constant correction to be added to the specific gravity as determined in each case.

CORRECTION TABLE FOR SPECIFIC GRAVITY.

_Below 15°.6 Subtract; Above 15°.6 Add._

Temp. Correction. Temp. Correction. Temp. Correction.

12.2 0.00047 18.2 0.00043 24.2 0.00163 12.4 0.00044 18.4 0.00046 24.4 0.00167 12.6 0.00042 18.6 0.00050 24.6 0.00172 12.8 0.00039 18.8 0.00053 24.8 0.00176 13.0 0.00037 19.0 0.00057 25.0 0.00181 13.2 0.00634 19.2 0.00061 25.2 0.00186 13.4 0.00032 19.4 0.00065 25.4 0.00191 13.6 0.00029 19.6 0.00068 25.6 0.00195 13.8 0.00027 19.8 0.00072 25.8 0.00200 14.0 0.00024 20.0 0.00076 26.0 0.00205 14.2 0.00021 20.2 0.00080 26.2 0.00210 14.4 0.00018 20.4 0.00084 26.4 0.00215 14.6 0.00015 20.6 0.00087 26.6 0.00220 14.8 0.00012 20.8 0.00091 26.8 0.00225 15.0 0.00009 21.0 0.00095 27.0 0.00230 15.2 0.00006 21.2 0.00099 27.2 0.00235 15.4 0.00003 21.4 0.00103 27.4 0.00240 15.6 0.00000 21.6 0.00107 27.6 0.00246 15.8 0.00003 21.8 0.00111 27.8 0.00251 16.0 0.00006 22.0 0.00115 28.0 0.00256 16.2 0.00009 22.2 0.00119 28.2 0.00261 16.4 0.00012 22.4 0.00123 28.4 0.00267 16.6 0.00016 22.6 0.00128 28.6 0.00272 16.8 0.00019 22.8 0.00132 28.8 0.00278 17.0 0.00022 23.0 0.00136 29.0 0.00283 17.2 0.00025 23.2 0.00140 29.2 0.00288 17.4 0.00029 23.4 0.00145 29.4 0.00294 17.6 0.00032 23.6 0.00149 29.6 0.00299 17.8 0.00036 23.8 0.00154 29.8 0.00306 18.0 0.00039 24.0 0.00158 30.0 0.00311

The table is only accurate when the distillate does not contain over seven nor less than three per cent of alcohol. If the distillate contain more than seven per cent of alcohol it is diluted and the compensating correction made.

=620. Calculating Results.=—The specific gravity of the alcoholic distillate having been determined by any approved method and corrected to a temperature of 15°.6, the corresponding per cents of alcohol by volume and by weight are found by consulting the following table.[636] If, for example, the corrected specific gravity be exactly that given in any figure of the table the corresponding per cents are directly read. If the specific gravity found fall between two numbers in the table the corresponding per cents are determined by interpolation.

TABLE SHOWING PERCENTAGE OF ALCOHOL BY WEIGHT AND BY VOLUME. -------------+------------------+--------------- Specific | Per cent | Per cent gravity at | alcohol | alcohol 15°.6/15°.6. | by volume. | by weight. -------------+------------------+--------------- 1.00000 | 0.00 | 0.00 0.99992 | .05 | .04 984 | .10 | .08 976 | .15 | .12 968 | .20 | .16 961 | .25 | .20 953 | .30 | .24 945 | .35 | .28 937 | .40 | .32 930 | .45 | .36 .99923 | 0.50 | 0.40 915 | .55 | .44 907 | .60 | .48 900 | .65 | .52 892 | .70 | .56 884 | .75 | .60 877 | .80 | .64 869 | .85 | .67 861 | .90 | .71 854 | .95 | .75 .99849 | 1.00 | 0.79 842 | .05 | .83 834 | .10 | .87 827 | .15 | .91 819 | .20 | .95 812 | .25 | .99 805 | .30 | 1.03 797 | .35 | .07 790 | .40 | .11 782 | .45 | .15 .99775 | 1.50 | 1.19 768 | .55 | .23 760 | .60 | .27 753 | .65 | .31 745 | .70 | .35 738 | .75 | .39 731 | .80 | .43 723 | .85 | .47 716 | .90 | .51 708 | .95 | .55 .99701 | 2.00 | 1.59 694 | .05 | .63 687 | .10 | .67 679 | .15 | .71 672 | .20 | .75 665 | .25 | .79 658 | .30 | .83 651 | .35 | .87 643 | .40 | .91 636 | .45 | .95 0.99629 | 2.50 | 1.99 622 | .55 | 2.03 615 | .60 | .07 607 | .65 | .11 600 | .70 | .15 593 | .75 | .19 586 | .80 | .23 579 | .85 | .27 571 | .90 | .31 564 | .95 | .35 .99557 | 3.00 | 2.39 550 | .05 | .43 543 | .10 | .47 536 | .15 | .51 529 | .20 | .55 522 | .25 | .59 515 | .30 | .64 508 | .35 | .68 501 | .40 | .72 494 | .45 | .76 .99487 | 3.50 | 2.80 480 | .55 | .84 473 | .60 | .88 466 | .65 | .92 459 | .70 | .96 452 | .75 | 3.00 445 | .80 | .04 438 | .85 | .08 431 | .90 | .12 424 | .95 | .16 .99417 | 4.00 | 3.20 410 | .05 | .24 403 | .10 | .28 397 | .15 | .32 390 | .20 | .36 383 | .25 | .40 376 | .30 | .44 369 | .35 | .48 363 | .40 | .52 356 | .45 | .56 .99349 | 4.50 | 3.60 342 | .55 | .64 335 | .60 | .68 329 | .65 | .72 322 | .70 | .76 315 | .75 | .80 308 | .80 | .84 301 | .85 | .88 295 | .90 | .92 288 | .95 | .96 0.99281 | 5.00 | 4.00 274 | .05 | .04 268 | .10 | .08 261 | .15 | .12 255 | .20 | .16 248 | .25 | .20 241 | .30 | .24 235 | .35 | .28 228 | .40 | .32 222 | .45 | .36 .99215 | 5.50 | 4.40 208 | .55 | .44 202 | .60 | .48 195 | .65 | .52 189 | .70 | .56 182 | .75 | .60 175 | .80 | .64 169 | .85 | .68 162 | .90 | .72 156 | .95 | .76 .99149 | 6.00 | 4.80 143 | .05 | .84 136 | .10 | .87 130 | .15 | .92 123 | .20 | .96 117 | .25 | 5.00 111 | .30 | .05 104 | .35 | .09 098 | .40 | .13 091 | .45 | .17 .99085 | 6.50 | 5.21 079 | .55 | .25 072 | .60 | .29 066 | .65 | .33 059 | .70 | .37 053 | .75 | .41 047 | .80 | .45 040 | .85 | .49 034 | .90 | .53 027 | .95 | .57 .99021 | 7.00 | 5.61 015 | .05 | .65 009 | .10 | .69 002 | .15 | .73 .98996 | .20 | .77 990 | .25 | .81 984 | .30 | .86 978 | .35 | .90 971 | .40 | .94 965 | .45 | .98 0.98959 | 7.50 | 6.02 953 | .55 | .06 947 | .60 | .10 940 | .65 | .14 934 | .70 | .18 928 | .75 | .22 922 | .80 | .26 916 | .85 | .30 909 | .90 | .34 903 | .95 | .38 .98897 | 8.00 | 6.42 891 | .05 | .46 885 | .10 | .50 879 | .15 | .54 873 | .20 | .58 867 | .25 | .62 861 | .30 | .67 855 | .35 | .71 849 | .40 | .75 843 | .45 | .79 .98837 | 8.50 | 6.83 831 | .55 | .87 825 | .60 | .91 819 | .65 | .95 813 | .70 | .99 807 | .75 | 7.03 801 | .80 | .07 795 | .85 | .11 789 | .90 | .15 783 | .95 | .19 .98777 | 9.00 | 7.23 771 | .05 | .27 765 | .10 | .31 754 | .20 | .39 748 | .25 | .43 742 | .30 | .48 736 | .35 | .52 724 | .45 | .60 .98719 | 9.50 | 7.64 713 | .55 | .68 707 | .60 | .72 701 | .65 | .76 695 | .70 | .80 689 | .75 | .84 683 | .80 | .88 678 | .85 | .92 672 | .90 | .96 666 | .95 | 8.00 0.98660 | 10.00 | 8.04 654 | .05 | .08 649 | .10 | .12 643 | .15 | .16 637 | .20 | .20 632 | .25 | .24 626 | .30 | .28 620 | .35 | .33 614 | .40 | .37 609 | .45 | .41 .98603 | 10.50 | 8.45 597 | .55 | .49 592 | .60 | .53 586 | .65 | .57 580 | .70 | .61 575 | .75 | .65 569 | .80 | .70 563 | .85 | .74 557 | .90 | .78 552 | .95 | .82 .98546 | 11.00 | 8.86 540 | .05 | .90 535 | .10 | .94 529 | .15 | .98 524 | .20 | 9.02 518 | .25 | .07 513 | .30 | .11 507 | .35 | .15 502 | .40 | .19 496 | .45 | .23 .98491 | 11.50 | 9.27 485 | .55 | .31 479 | .60 | .35 474 | .65 | .39 468 | .70 | .43 463 | .75 | .47 457 | .80 | .51 452 | .85 | .55 446 | .90 | .59 441 | .95 | .63 .98435 | 12.00 | 9.67 430 | .05 | .71 424 | .10 | .75 419 | .15 | .79 413 | .20 | .83 408 | .25 | .87 402 | .30 | .92 397 | .35 | .96 391 | .40 | 10.00 386 | .45 | .04 0.98381 | 12.50 | 10.08 375 | .55 | .12 370 | .60 | .16 364 | .65 | .20 359 | .70 | .24 353 | .75 | .28 348 | .80 | .33 342 | .85 | .37 337 | .90 | .41 331 | .95 | .45 .98326 | 13.00 | 10.49 321 | .05 | .53 315 | .10 | .57 310 | .15 | .61 305 | .20 | .65 299 | .25 | .69 294 | .30 | .74 289 | .35 | .78 283 | .40 | .82 278 | .45 | .86 .98273 | 13.50 | 10.90 267 | .55 | .94 262 | .60 | .98 256 | .65 | 11.02 251 | .70 | .06 246 | .75 | .14 240 | .80 | .15 235 | .85 | .19 230 | .90 | .23 224 | .95 | .27 .98219 | 14.00 | 11.31 214 | .05 | .35 209 | .10 | .39 203 | .15 | .43 198 | .20 | .47 193 | .25 | .52 188 | .30 | .56 182 | .35 | .60 177 | .40 | .64 172 | .45 | .68 .98167 | 14.50 | 11.72 161 | .55 | .76 156 | .60 | .80 151 | .65 | .84 146 | .70 | .88 140 | .75 | .93 135 | .80 | .97 130 | .85 | 12.01 125 | .90 | .05 119 | .95 | .09 0.98114 | 15.00 | 12.13 108 | .05 | .17 104 | .10 | .21 099 | .15 | .25 093 | .20 | .29 088 | .25 | .33 083 | .30 | .38 078 | .35 | .42 073 | .40 | .46 068 | .45 | .50 .98063 | 15.50 | 12.54 057 | .55 | .58 052 | .60 | .62 047 | .65 | .66 042 | .70 | .70 037 | .75 | .75 032 | .80 | .79 026 | .85 | .83 021 | .90 | .87 016 | .95 | .91 .98011 | 16.00 | 12.95 005 | .05 | .99 001 | .10 | 13.03 .97996 | .15 | .08 991 | .20 | .12 986 | .25 | .16 980 | .30 | .20 975 | .35 | .24 970 | .40 | .29 965 | .45 | .33 .97960 | 16.50 | 13.37 955 | .55 | .41 950 | .60 | .45 945 | .65 | .49 940 | .70 | .53 935 | .75 | .57 929 | .80 | .62 924 | .85 | .66 919 | .90 | .70 914 | .95 | .74 .97909 | 17.00 | 13.78 904 | .05 | .82 899 | .10 | .86 894 | .15 | .90 889 | .20 | .94 884 | .25 | .98 879 | .30 | 14.03 874 | .35 | .07 869 | .40 | .11 864 | .45 | .15 0.97859 | 17.50 | 14.19 853 | .55 | .23 848 | .60 | .27 843 | .65 | .31 838 | .70 | .35 833 | .75 | .40 828 | .80 | .44 823 | .85 | .48 818 | .90 | .52 813 | .95 | .56 .97808 | 18.00 | 14.60 803 | .05 | .64 798 | .10 | .68 793 | .15 | .73 788 | .20 | .77 783 | .25 | .81 778 | .30 | .85 773 | .35 | .89 768 | .40 | .94 763 | .45 | .98 .97758 | 18.50 | 15.02 753 | .55 | .06 748 | .60 | .10 743 | .65 | .14 738 | .70 | .18 733 | .75 | .22 728 | .80 | .27 723 | .85 | .31 718 | .90 | .35 713 | .95 | .39 .97708 | 19.00 | 15.43 703 | .05 | .47 698 | .10 | .51 693 | .15 | .55 688 | .20 | .59 683 | .25 | .63 678 | .30 | .68 673 | .35 | .72 668 | .40 | .76 663 | .45 | .80 .97658 | 19.50 | 15.84 653 | .55 | .88 648 | .60 | .93 643 | .65 | .97 638 | .70 | 16.01 633 | .75 | .05 628 | .80 | .09 623 | .85 | .14 618 | .90 | .18 613 | .95 | .22 0.97608 | 20.00 | 16.26 603 | .05 | .30 598 | .10 | .34 593 | .15 | .38 588 | .20 | .42 583 | .25 | .46 578 | .30 | .51 573 | .35 | .58 568 | .40 | .59 563 | .45 | .63 .97558 | 20.50 | 16.67 552 | .55 | .71 547 | .60 | .75 542 | .65 | .80 537 | .70 | .84 532 | .75 | .88 527 | .80 | .92 522 | .85 | .96 517 | .90 | 17.01 512 | .95 | .05 .97507 | 21.00 | 17.09 502 | .05 | .13 497 | .10 | .17 492 | .15 | .22 487 | .20 | .26 482 | .25 | .30 477 | .30 | .34 472 | .35 | .38 467 | .40 | .43 462 | .45 | .47 .97457 | 21.50 | 17.51 451 | .55 | .55 446 | .60 | .59 441 | .65 | .63 436 | .70 | .67 431 | .75 | .71 426 | .80 | .76 421 | .85 | .80 416 | .90 | .84 411 | .95 | .88 .97406 | 22.00 | 17.92 401 | .05 | .96 396 | .10 | 18.00 391 | .15 | .05 386 | .20 | .09 381 | .25 | .13 375 | .30 | .17 370 | .35 | .21 365 | .40 | .26 360 | .45 | .30 0.97355 | 22.50 | 18.34 350 | .55 | .38 345 | .60 | .42 340 | .65 | .47 335 | .70 | .51 330 | .75 | .55 324 | .80 | .59 319 | .85 | .63 314 | .90 | .68 309 | .95 | .72 .97304 | 23.00 | 18.76 299 | .05 | .80 294 | .10 | .84 289 | .15 | .88 283 | .20 | .92 278 | .25 | .96 273 | .30 | 19.01 268 | .35 | .05 263 | .40 | .09 258 | .45 | .13 .97253 | 23.50 | 19.17 247 | .55 | .21 242 | .60 | .25 237 | .65 | .30 232 | .70 | .34 227 | .75 | .38 222 | .80 | .42 216 | .85 | .46 211 | .90 | .51 206 | .95 | .55 .97201 | 24.00 | 19.59 196 | .05 | .63 191 | .10 | .67 185 | .15 | .72 180 | .20 | .76 175 | .25 | .80 170 | .30 | .84 165 | .35 | .88 159 | .40 | .93 154 | .45 | .97 .97149 | 24.50 | 20.01 144 | .55 | .05 139 | .60 | .09 133 | .65 | .14 128 | .70 | .18 123 | .75 | .22 118 | .80 | .26 113 | .85 | .30 107 | .90 | .35 102 | .95 | .39 0.97097 | 25.00 | 20.43 092 | .05 | .47 086 | .10 | .51 081 | .15 | .56 076 | .20 | .60 071 | .25 | .64 065 | .30 | .68 060 | .35 | .72 055 | .40 | .77 049 | .45 | .81 .97014 | 25.50 | 20.85 039 | .55 | .89 033 | .60 | .93 028 | .65 | .98 023 | .70 | 21.02 018 | .75 | .06 012 | .80 | .10 007 | .85 | .14 001 | .90 | .19 .96996 | .95 | .23 .96991 | 26.00 | 21.27 986 | .05 | .31 980 | .10 | .35 975 | .15 | .40 969 | .20 | .44 964 | .25 | .48 959 | .30 | .52 953 | .35 | .56 949 | .40 | .61 942 | .45 | .65 .96937 | 26.50 | 21.69 932 | .55 | .73 926 | .60 | .77 921 | .65 | .82 915 | .70 | .86 910 | .75 | .90 905 | .80 | .94 899 | .85 | .98 894 | .90 | 22.03 888 | .95 | .07 .96883 | 27.00 | 22.11 877 | .05 | .15 872 | .10 | .20 866 | .15 | .24 861 | .20 | .28 855 | .25 | .33 850 | .30 | .37 844 | .35 | .41 839 | .40 | .45 833 | .45 | .50 0.96828 | 27.50 | 22.54 822 | .55 | .58 816 | .60 | .62 811 | .65 | .67 805 | .70 | .71 800 | .75 | .75 794 | .80 | .79 789 | .85 | .83 783 | .90 | .88 778 | .95 | .92 .96772 | 28.00 | 22.96 766 | .05 | 23.00 761 | .10 | .04 755 | .15 | .09 749 | .20 | .13 744 | .25 | .17 738 | .30 | .21 732 | .35 | .25 726 | .40 | .30 721 | .45 | .34 .96715 | 28.50 | 23.38 709 | .55 | .42 704 | .60 | .47 698 | .65 | .51 692 | .70 | .55 687 | .75 | .60 681 | .80 | .64 675 | .85 | .68 669 | .90 | .72 664 | .95 | .77 .96658 | 29.00 | 23.81 652 | .05 | .85 646 | .10 | .89 640 | .15 | .94 635 | .20 | .98 629 | .25 | 24.02 623 | .30 | .06 617 | .35 | .10 611 | .40 | .15 605 | .45 | .19 .96600 | 29.50 | 24.23 594 | .55 | .27 587 | .60 | .32 582 | .65 | .36 576 | .70 | .40 570 | .75 | .45 564 | .80 | .49 559 | .85 | .53 553 | .90 | .57 547 | .95 | .62 0.96541 | 30.00 | 24.66 535 | .05 | .70 529 | .10 | .74 523 | .15 | .79 517 | .20 | .83 511 | .25 | .87 505 | .30 | .91 499 | .35 | .95 493 | .40 | 25.00 487 | .45 | .04 .96481 | 30.50 | 25.08 475 | .55 | .12 469 | .60 | .17 463 | .65 | .21 457 | .70 | .25 451 | .75 | .30 445 | .80 | .34 439 | .85 | .38 433 | .90 | .42 427 | .95 | .47 .96421 | 31.00 | 25.51 415 | .05 | .55 409 | .10 | .60 403 | .15 | .64 396 | .20 | .68 390 | .25 | .73 384 | .30 | .77 378 | .35 | .81 372 | .40 | .85 366 | .45 | .90 .96360 | 31.50 | 25.94 353 | .55 | .98 347 | .60 | 26.03 341 | .65 | .07 335 | .70 | .11 329 | .75 | .16 323 | .80 | .20 316 | .85 | .24 310 | .90 | .28 304 | .95 | .33 .96298 | 32.00 | 26.37 292 | .05 | .41 285 | .10 | .46 279 | .15 | .50 273 | .20 | .54 267 | .25 | .59 260 | .30 | .63 254 | .35 | .67 248 | .40 | .71 241 | .45 | .76 0.96235 | 32.50 | 26.80 229 | .55 | .84 222 | .60 | .89 216 | .65 | .93 210 | .70 | .97 204 | .75 | 27.02 197 | .80 | .06 191 | .85 | .10 185 | .90 | .14 178 | .95 | .19 .96172 | 33.00 | 27.23 166 | .05 | .27 159 | .10 | .32 153 | .15 | .36 146 | .20 | .40 140 | .25 | .45 133 | .30 | .49 127 | .35 | .53 120 | .40 | .57 114 | .45 | .62 .96108 | 33.50 | 27.66 101 | .55 | .70 095 | .60 | .75 088 | .65 | .79 082 | .70 | .83 075 | .75 | .88 069 | .80 | .92 062 | .85 | .97 056 | .90 | 28.00 049 | .95 | .05 .96043 | 34.00 | 28.09 036 | .05 | .13 030 | .10 | .18 023 | .15 | .22 016 | .20 | .26 010 | .25 | .31 003 | .30 | .35 .95996 | .35 | .39 990 | .40 | .43 983 | .45 | .48 .95977 | 34.50 | 28.52 970 | .55 | .56 963 | .60 | .61 957 | .65 | .65 950 | .70 | .70 943 | .75 | .74 937 | .80 | .78 930 | .85 | .83 923 | .90 | .87 917 | .95 | .92 0.95910 | 35.00 | 28.96 903 | .05 | 29.00 896 | .10 | .05 889 | .15 | .09 883 | .20 | .13 876 | .25 | .18 869 | .30 | .22 862 | .35 | .26 855 | .40 | .30 848 | .45 | .35 .95842 | 35.50 | 29.39 835 | .55 | .43 828 | .60 | .48 821 | .65 | .52 814 | .70 | .57 807 | .75 | .61 800 | .80 | .65 794 | .85 | .70 787 | .90 | .74 780 | .95 | .79 .95773 | 36.00 | 29.83 766 | .05 | .87 759 | .10 | .92 752 | .15 | .96 745 | .20 | 30.00 738 | .25 | .05 731 | .30 | .09 724 | .35 | .13 717 | .40 | .17 710 | .45 | .22 .95703 | 36.50 | 30.26 695 | .55 | .30 688 | .60 | .35 681 | .65 | .39 674 | .70 | .44 667 | .75 | .48 660 | .80 | .52 653 | .85 | .57 646 | .90 | .61 639 | .95 | .66 .95632 | 37.00 | 30.70 625 | .05 | .74 618 | .10 | .79 610 | .15 | .83 603 | .20 | .88 596 | .25 | .92 589 | .30 | .96 581 | .35 | 31.01 574 | .40 | .05 567 | .45 | .10 0.95560 | 37.50 | 31.14 552 | .55 | .18 545 | .60 | .23 538 | .65 | .27 531 | .70 | .32 523 | .75 | .36 516 | .80 | .40 509 | .85 | .45 502 | .90 | .49 494 | .95 | .54 .95487 | 38.00 | 31.58 480 | .05 | .63 472 | .10 | .67 465 | .15 | .72 457 | .20 | .76 450 | .25 | .81 442 | .30 | .85 435 | .35 | .90 427 | .40 | .94 420 | .45 | .99 .95413 | 38.50 | 32.03 405 | .55 | .07 398 | .60 | .12 390 | .65 | .16 383 | .70 | .20 375 | .75 | .25 368 | .80 | .29 360 | .85 | .33 353 | .90 | .37 345 | .95 | .42 .95338 | 39.00 | 32.46 330 | .05 | .50 323 | .10 | .55 315 | .15 | .59 307 | .20 | .64 300 | .25 | .68 292 | .30 | .72 284 | .35 | .77 277 | .40 | .81 269 | .45 | .86 .95262 | 39.50 | 32.90 254 | .55 | .95 246 | .60 | .99 239 | .65 | 33.04 231 | .70 | .08 223 | .75 | .13 216 | .80 | .17 208 | .85 | .22 200 | .90 | .27 193 | .95 | .31 0.95185 | 40.00 | 33.35 177 | .05 | .39 169 | .10 | .44 161 | .15 | .48 154 | .20 | .53 146 | .25 | .57 138 | .30 | .61 130 | .35 | .66 122 | .40 | .70 114 | .45 | .75 .95107 | 40.50 | 33.79 099 | .55 | .84 091 | .60 | .88 083 | .65 | .93 075 | .70 | .97 067 | .75 | 34.02 059 | .80 | .06 052 | .85 | .11 044 | .90 | .15 036 | .95 | .20 .95028 | 41.00 | 34.24 020 | .05 | .28 012 | .10 | .33 004 | .15 | .37 .94996 | .20 | .42 988 | .25 | .46 980 | .30 | .50 972 | .35 | .55 964 | .40 | .59 956 | .45 | .64 .94948 | 41.50 | 34.68 940 | .55 | .73 932 | .60 | .77 924 | .65 | .82 916 | .70 | .86 908 | .75 | .91 900 | .80 | .95 892 | .85 | 35.00 884 | .90 | .04 876 | .95 | .09 .94868 | 42.00 | 35.13 860 | .05 | .18 852 | .10 | .22 843 | .15 | .27 835 | .20 | .31 827 | .25 | .36 820 | .30 | .40 811 | .35 | .45 802 | .40 | .49 794 | .45 | .54 0.94786 | 42.50 | 35.58 778 | .55 | .63 770 | .60 | .67 761 | .65 | .72 753 | .70 | .76 745 | .75 | .81 737 | .80 | .85 729 | .85 | .90 720 | .90 | .94 712 | .95 | .99 .94704 | 43.00 | 36.03 696 | .05 | .08 687 | .10 | .12 679 | .15 | .17 670 | .20 | .21 662 | .25 | .23 654 | .30 | .30 645 | .35 | .35 637 | .40 | .39 628 | .45 | .44 .94620 | 43.50 | 36.48 612 | .55 | .53 603 | .60 | .57 595 | .65 | .62 586 | .70 | .66 578 | .75 | .71 570 | .80 | .75 561 | .85 | .80 553 | .90 | .84 544 | .95 | .89 .94536 | 44.00 | 36.93 527 | .05 | .98 519 | .10 | 37.02 510 | .15 | .07 502 | .20 | .11 493 | .25 | .16 484 | .30 | .21 476 | .35 | .25 467 | .40 | .30 459 | .45 | .34 .94450 | 44.50 | 37.39 441 | .55 | .44 433 | .60 | .48 424 | .65 | .53 416 | .70 | .57 407 | .75 | .62 398 | .80 | .66 390 | .85 | .71 381 | .90 | .76 373 | .95 | .80 0.94364 | 45.00 | 37.84 355 | .05 | .89 346 | .10 | .93 338 | .15 | .98 329 | .20 | 38.02 320 | .25 | .07 311 | .30 | .12 302 | .35 | .16 294 | .40 | .21 285 | .45 | .25 .94276 | 45.50 | 38.30 267 | .55 | .35 258 | .60 | .39 250 | .65 | .44 241 | .70 | .48 232 | .75 | .53 223 | .80 | .57 214 | .85 | .62 206 | .90 | .66 197 | .95 | .71 .94188 | 46.00 | 38.75 179 | .05 | .80 170 | .10 | .84 161 | .15 | .89 152 | .20 | .93 143 | .25 | .98 134 | .30 | 39.03 125 | .35 | .07 116 | .40 | .12 107 | .45 | .16 .94098 | 46.50 | 39.21 089 | .55 | .26 080 | .60 | .30 071 | .65 | .35 062 | .70 | .39 053 | .75 | .44 044 | .80 | .49 035 | .85 | .53 026 | .90 | .58 017 | .95 | .62 .94008 | 47.00 | 39.67 .93999 | .05 | .72 990 | .10 | .76 980 | .15 | .81 971 | .20 | .85 962 | .25 | .90 953 | .30 | .95 944 | .35 | .99 934 | .40 | 40.04 925 | .45 | .08 0.93916 | 47.50 | 40.13 906 | .55 | .18 898 | .60 | .22 888 | .65 | .27 879 | .70 | .32 870 | .75 | .37 861 | .80 | .41 852 | .85 | .46 842 | .90 | .51 833 | .95 | .55 .93824 | 48.00 | 40.60 815 | .05 | .65 808 | .10 | .69 796 | .15 | .74 786 | .20 | .78 777 | .25 | .83 768 | .30 | .88 758 | .35 | .92 740 | .40 | .97 739 | .45 | 41.01 .93730 | 48.50 | 41.06 721 | .55 | .11 711 | .60 | .15 702 | .65 | .20 692 | .70 | .24 683 | .75 | .29 673 | .80 | .34 664 | .85 | .38 655 | .90 | .43 645 | .95 | .47 .93636 | 49.00 | 41.52 626 | .05 | .57 617 | .10 | .61 607 | .15 | .66 598 | .20 | .71 588 | .25 | .76 578 | .30 | .80 569 | .35 | .85 559 | .40 | .90 550 | .45 | .94 .93540 | 49.50 | 41.99 530 | .55 | 42.04 521 | .60 | .08 511 | .65 | .13 502 | .70 | .18 492 | .75 | .23 482 | .80 | .27 473 | .85 | .32 463 | .90 | .37 454 | .95 | .41 -------------+------------------+------------------

=621. Determination of Percentage of Alcohol by Means Of Vapor Temperature.=—The temperature of a mixture of alcohol and water vapors is less than that of water alone and the depression is inversely proportional to the quantity of alcohol present. This principle is utilized in the construction of the ebullioscope or ebulliometer. In this apparatus the temperature of pure boiling water vapor is determined by a preliminary experiment. This point must be frequently revised in order to correct it for variations in barometric pressure. The water is withdrawn from the boiler of the apparatus, the same volume of a wine or beer placed therein, and the vapor temperature again determined. By comparing the boiling point of the wine, with a scale calibrated for different percentages of alcohol, the quantity of spirit present is determined. When water vapor is at 100° a _vin ordinaire_ having eight per cent of alcohol gives a vapor at 93°.8. The presence of extractive matters in the sample, which tend to raise its boiling point, is neglected in the calculation of results.

=622. Improved Ebullioscope.=—The principle mentioned in the above paragraph may be applied with a considerable degree of accuracy, by using the improved ebullioscope described below.[637]

The apparatus consists of a glass flask F, shaped somewhat like an erlenmeyer, closed at the top with a rubber stopper carrying a central aperture for the insertion of the delicate thermometer A B, and a lateral smaller aperture for connecting the interior of the flask with the condenser D. The return of the condensed vapors from D is effected through the tube entering the flask F in such a manner as to deliver the condensed liquid beneath the surface of the liquid in F as shown in the figure. The flask F contains pieces of scrap platinum or pumice stone to prevent bumping and secure an even ebullition. The flask F rests upon a disk of asbestos, perforated in such a way as to have the opening fully covered by the bottom of the flask. To protect F against the influence of air currents it is enclosed in the glass cylinder E resting on the asbestos disk below and closed with a detachable soft rubber cover at the top. The temperature between the cylinder E and the flask F is measured by the thermometer C and the flame of the lamp G should be so adjusted as to bring the temperature between the flask F and the cylinder E to about 90° at the time of reading the thermometer B. The bulb of the thermometer B may be protected by a thin glass tube carrying distilled water, so adjusted as to prevent the escape of the watery vapor into F. The thermometer B is such as is used for determining molecular weights by the cryoscopic method. It has a cistern at A which holds any excess of mercury not needed in _adjusting the thermometer_ for any required temperature.

[Illustration: FIG. 125. IMPROVED EBULLIOSCOPE.]

A second apparatus, exactly similar to the one described, is conveniently used for measuring the changes in _barometric_ pressure during the process of the analysis. The temperature of the vapor of boiling water having been first determined, the beer or wine is placed in F, and the temperature of the vapor of the boiling liquid determined after the temperature of the air layer between E and F reaches about 90°, measured on the thermometer C. By using alcoholic mixtures of known strength the depression for each changing per cent of alcohol is determined for each system of apparatus employed, and this having once been done, the percentage of alcohol in any unknown liquid is at once determined by inspecting the thermometer, the bulb of which is immersed in the vapor from the boiling liquid. In the apparatus figured, a depression of 0°.8 is equivalent to one per cent of alcohol by volume. Full directions for the manipulation of the apparatus may be found in the paper cited above.

=623. Total Fixed Matters.=—The residue left on evaporating a fermented beverage to dryness is commonly known as extractive matter, or simply extract. It is composed chiefly of unfermented carbohydrates, organic acids, nitrogenous bodies, glycerol and mineral substances. Hydrochloric and sulfuric acids may also be found therein. If any non-volatile preservatives have been used in the sample, such as borax, salicylates and the like, these will also be found in the solid residue. The bodies which escape are water, alcohols, ethers and essential oils. The character of the residue left by wines and beers is evidently different. In each case it should contain typical components which aid in judging of the purity of the sample. For instance, in beers the substitution for malt of carbohydrate bodies comparatively free of proteids, produces a beer containing a deficiency of nitrogenous bodies. Pure malt beer will rarely have less than one-half of a per cent of proteids, while beer made largely of glucose, rice or hominy grits, will have a much smaller quantity. First will be described below the methods of determining the fixed residue left on evaporation, and thereafter the processes for ascertaining its leading components.

=624. Methods of the Official Chemists.=—Two methods are in use by the official chemists for determining the fixed solids in fermented beverages.[638] They are as follows:

_Direct Method._—Fifty cubic centimeters of the sample are weighed, placed in a platinum dish about eighty millimeters in diameter and capable of holding about seventy-five cubic centimeters and evaporated on the steam bath to a sirupy consistence. The residue is heated for two and a half hours in a drying oven at the temperature of boiling water and weighed.

_In Sweet Wines._—Ten cubic centimeters of the liquor are weighed and diluted to 100 with water. Fifty cubic centimeters of this solution are evaporated as described above.

_Optional Method._—Fifty cubic centimeters of the sample are placed in a platinum or porcelain dish and evaporated on the steam bath until the volume is reduced to one-third. The dealcoholized liquid is washed into a fifty cubic centimeter flask, cooled and made up to the original volume. It is mixed thoroughly and the specific gravity ascertained with a pyknometer, hydrostatic balance or an accurately standardized hydrometer. The percentage of total solids is obtained from the appended table. The column on the left of the specific gravity gives the percentage of extract in a wine, as calculated by Hager, and that on the right the percentage of extract in a beer or wort, as calculated by Schultze. According to Baumert, however, Schultze’s table gives results which approximate more closely the data obtained by direct estimation than does Hager’s.

TABLES OF HAGER AND SCHULTZE FOR THE DETERMINATION OF EXTRACT BY THE INDIRECT METHOD. ======+==================+=========== Hager.| Specific gravity.| Schultze. ------+------------------+----------- 0.84 | 1.0038 | 1.00 0.86 | 1.0039 | 1.02 0.88 | 1.0040 | 1.05 0.90 | 1.0041 | 1.08 0.92 | 1.0042 | 1.10 0.94 | 1.0043 | 1.13 0.96 | 1.0044 | 1.15 0.98 | 1.0045 | 1.18 1.00 | 1.0046 | 1.21 1.02 | 1.0047 | 1.23 1.04 | 1.0048 | 1.26 1.06 | 1.0049 | 1.29 1.08 | 1.0050 | 1.31 1.10 | 1.0051 | 1.34 1.12 | 1.0052 | 1.36 1.15 | 1.0053 | 1.39 1.17 | 1.0054 | 1.41 1.19 | 1.0055 | 1.44 1.22 | 1.0056 | 1.46 1.25 | 1.0057 | 1.49 1.27 | 1.0058 | 1.51 1.30 | 1.0059 | 1.54 1.32 | 1.0060 | 1.56 1.34 | 1.0061 | 1.59 1.37 | 1.0062 | 1.62 1.39 | 1.0063 | 1.64 1.42 | 1.0064 | 1.67 1.44 | 1.0065 | 1.69 1.46 | 1.0066 | 1.72 1.48 | 1.0067 | 1.74 1.50 | 1.0068 | 1.77 1.52 | 1.0069 | 1.79 1.55 | 1.0070 | 1.82 1.57 | 1.0071 | 1.84 1.59 | 1.0072 | 1.87 1.61 | 1.0073 | 1.90 1.64 | 1.0074 | 1.92 1.66 | 1.0075 | 1.95 1.68 | 1.0076 | 1.97 1.70 | 1.0077 | 2.00 1.72 | 1.0078 | 2.02 1.75 | 1.0079 | 2.05 1.77 | 1.0080 | 2.07 1.79 | 1.0081 | 2.10 1.82 | 1.0082 | 2.12 1.84 | 1.0083 | 2.15 1.86 | 1.0084 | 2.17 1.88 | 1.0085 | 2.20 1.90 | 1.0086 | 2.23 1.92 | 1.0087 | 2.25 1.94 | 1.0088 | 2.28 1.96 | 1.0089 | 2.30 1.98 | 1.0090 | 2.33 2.00 | 1.0091 | 2.35 2.03 | 1.0092 | 2.38 2.05 | 1.0093 | 2.41 2.07 | 1.0094 | 2.43 2.09 | 1.0095 | 2.46 2.11 | 1.0096 | 2.48 2.14 | 1.0097 | 2.51 2.16 | 1.0098 | 2.53 2.18 | 1.0099 | 2.56 2.21 | 1.0100 | 2.58 2.23 | 1.0101 | 2.61 2.25 | 1.0102 | 2.64 2.27 | 1.0103 | 2.66 2.30 | 1.0104 | 2.69 2.32 | 1.0105 | 2.71 2.34 | 1.0106 | 2.74 2.36 | 1.0107 | 2.76 2.38 | 1.0108 | 2.79 2.40 | 1.0109 | 2.82 2.42 | 1.0110 | 2.84 2.44 | 1.0111 | 2.87 2.46 | 1.0112 | 2.89 2.48 | 1.0113 | 2.92 2.50 | 1.0114 | 2.94 2.52 | 1.0115 | 2.97 2.54 | 1.0116 | 2.99 2.57 | 1.0117 | 3.02 2.59 | 1.0118 | 3.05 2.61 | 1.0119 | 3.07 2.64 | 1.0120 | 3.10 2.66 | 1.0121 | 3.12 2.68 | 1.0122 | 3.15 2.70 | 1.0123 | 3.17 2.72 | 1.0124 | 3.20 2.75 | 1.0125 | 3.23 2.77 | 1.0126 | 3.25 2.79 | 1.0127 | 3.28 2.82 | 1.0128 | 3.30 2.84 | 1.0129 | 3.33 2.86 | 1.0130 | 3.35 2.88 | 1.0131 | 3.38 2.90 | 1.0132 | 3.41 2.92 | 1.0133 | 3.43 2.94 | 1.0134 | 3.46 2.96 | 1.0135 | 3.48 2.98 | 1.0136 | 3.51 3.00 | 1.0137 | 3.54 ======+==================+===========

If it be desired to use this table for the examination of liquors containing a higher percentage of extract, Schultze’s table (intended originally for wort) may be consulted.

Gautier regards the fixed solids as the residue obtained on evaporating, in a flat platinum dish, ten cubic centimeters of wine at 100° for four hours and a half.[639]

The official French method is as follows: Twenty cubic centimeters of wine are placed in a flat bottom, platinum dish of such a diameter that the depth of the liquid therein does not exceed one millimeter. The dish should be immersed as totally as possible in the steam. The heating is continued for six hours.

The following method is used at the municipal laboratory of Paris:

Twenty-five cubic centimeters of wine are placed in a flat bottom, platinum dish seventy millimeters in diameter and twenty-five deep. The dish is placed on a water bath in such a manner that it just touches the surface of the water which is kept at a constant level. The heating is continued for seven hours.[640]

=625. Determination in a Vacuum.=—To avoid the changes and decomposition produced by heating, the fixed solids may also be determined by drying the sample in a vacuum over sulfuric acid. In this laboratory, it has been found that the process may be greatly facilitated by connecting the desiccating apparatus with the vacuum service of the working desks in which a vacuum corresponding to a mercurial column of 600 millimeters is obtained. The desiccator is provided with a valve whereby a minute current of dry air is allowed to flow through it. This current is not large enough to lessen the vacuum but is sufficient to greatly accelerate the rapidity of the evaporation. The evaporation is hastened also, in a marked degree, by absorbing the liquid with a piece of filter paper previously dried in a vacuum. When it is desired to examine the residue, however, it must be obtained in a flat dish exposing a large surface to evaporation.

=626. Estimation of Water.=—It is evident that the percentage of water in a fermented beverage is easily calculated when the percentage of alcohol by weight and that of the dry residue are known. In a given case, if the number of grams of alcohol in 100 of the sample be five and that of fixed solids four and a half, the quantity of water therein is 100 - (5.0 + 4.5) = 90.5 grams. In this case the volatile essences are counted as water, but these, at most, are so small in quantity as to be practically unweighable. Nevertheless, it must be admitted that direct drying, in many cases, may give erroneous results, especially when the sample contains an abundance of ethers and of glycerol. The loss which takes place on evaporation may be diminished by adding to the sample, before evaporation, a known weight of potassium sulfate in crystals, which serves to increase the surface of evaporation, to hasten the process and to obtain a quantity of residue in excess of that secured by direct evaporation in an open dish.

=627. Total Acidity.=—The acidity found in fermented beverages is due both to the natural acids of the materials from which they are made, and to those caused by fermentation. The typical acids also indicate the nature of the original materials, as malic in cider and tartaric in wine. The acids of beers are due almost exclusively to fermentation, and acetic is probably the dominant one. In determining total acidity, it is not always convenient to ascertain beforehand what acid predominates, nor to accurately distribute the acid among its various components. In the analytical work it is advisable, therefore, to estimate the total acid of cider as malic, of wines as tartaric and of beers as acetic. The process of titration is conducted as follows:

Expel any carbon dioxid that is present by continued shaking. Transfer ten cubic centimeters to a beaker and, in the case of white wines, add about ten drops of a neutral litmus solution. Add decinormal sodium hydroxid solution until the red color changes to violet. Then add the reagent, a few drops at a time, until a drop of the liquid, placed on delicate red litmus paper, shows an alkaline reaction.

One cubic centimeter of decinormal sodium hydroxid solution = 0.0075 gram tartaric, 0.0067 of malic and 0.006 gram of acetic acid.

=628. Determination of Volatile Acids.=—Fifty cubic centimeters of the sample, to which a little tannin has been added to prevent foaming, are distilled in a current of steam. The flask is heated until the liquid boils, when the lamp under it is turned down and the steam passed through until 200 cubic centimeters have been collected in the receiver. The distillate is titrated with decinormal sodium hydroxid solution and the result expressed as acetic acid.

One cubic centimeter of decinormal sodium hydroxid solution = 0.0060 gram acetic acid.

The acidity due to volatile acids may be determined by ascertaining the total acidity as above described, evaporating 100 cubic centimeters to one-third of their volume, restoring the original volume with water and again titrating. The difference between the first and second titrations represents the volatile acidity.

A method of determining volatile acidity in wines, without the application of heat, has been proposed by de la Source.[641] The sample, five cubic centimeters, freed of carbon dioxid by shaking, is placed in a flat dish about eight centimeters in diameter. In a separate portion of the sample, the total acidity is determined in the presence of phenolphthalien by a set solution of barium hydroxid, one cubic centimeter of which is equal to four milligrams of sulfuric acid. The sample in the flat dish is placed in a desiccator, which contains both sulfuric acid and solid potassium hydroxid, and left for two days, by which time it is practically dry. The residue is dissolved in two cubic centimeters of warm water and the dish is kept in the desiccator for an additional two days. By this time the volatile acids, even acetic, will have disappeared and the residual acidity is determined after solution in water.

The method is also applicable when wines have been treated with an alkali. In this case two samples of five cubic centimeters each are acidified with two cubic centimeters of a solution of tartaric acid containing twenty-five grams per liter. This treatment sets free the volatile acids, and their quantity is determined as before.

=629. Titration with Phenolphthalien.=—The total acidity is also easily determined by titration with a set alkali, using phenolphthalien as indicator. Colored liquors must be treated with animal black before the analysis. The sample is shaken to expel carbon dioxid and five cubic centimeters added to 100 of water containing phenolphthalien. The set alkali (tenth normal soda) is added until the red color is discharged. Even wines having a considerable degree of color may be titrated in this way.[642] The acidity, expressed as tartaric, may be stated as due to sulfuric by dividing by 1.53.

=630. Determination of Tartaric Acid.=—The determination of potassium bitartrate is necessary when an estimation of the free tartaric acid is desired.[643]

Fifty cubic centimeters of wine are placed in a porcelain dish and evaporated to a sirupy consistence, a little quartz sand being added to render subsequent extraction easier. After cooling, seventy cubic centimeters of ninety-six per cent alcohol are added with constant stirring. After standing for twelve hours, at as low a temperature as practicable, the solution is filtered and the precipitate washed with alcohol until the filtrate is no longer acid. The alcoholic filtrate is preserved for the estimation of the tartaric acid. The filter and precipitate are returned to the porcelain dish and repeatedly treated with hot water, each extraction being filtered into a flask or beaker until the washings are neutral. The combined aqueous filtrates and washings are titrated with decinormal sodium hydroxid solution.

One cubic centimeter of decinormal sodium hydroxid solution = 0.0188 gram potassium bitartrate.

The alcoholic filtrate is made up to a definite volume with water and divided into two equal portions. One portion is exactly neutralized with decinormal sodium hydroxid solution, the other portion added, the alcohol evaporated, the residue washed into a porcelain dish and treated as above.

One cubic centimeter decinormal sodium hydroxid solution = 0.0075 gram tartaric acid.

As, however, only half of the free tartaric acid is determined by this method:

One cubic centimeter decinormal sodium hydroxid = 0.0150 gram of tartaric acid.

=631. Modified Berthelot-Fleury Method.=—Ten cubic centimeters of wine are neutralized with potassium hydroxid solution and mixed in a graduated cylinder with forty cubic centimeters of the same sample. To one-fifth of the volume, corresponding to ten cubic centimeters of wine, fifty cubic centimeters of a mixture of equal parts of alcohol and ether are added and allowed to stand twenty-four hours. The precipitated potassium bitartrate is separated by filtration, dissolved in water and titrated. The excess of potassium bitartrate over the amount of that constituent present in the wine corresponds to the free tartaric acid.[644]

=632. Determination of Tartaric, Malic and Succinic Acids.=—Two hundred cubic centimeters of wine are evaporated to one-half, cooled and lead subacetate solution added until the reaction is alkaline.[645] The precipitate is separated by filtration and washed with cold water until the filtrate shows only a slight reaction for lead. The precipitate is washed from the filter into a beaker, by means of hot water, and treated hot with hydrogen sulfid until all the lead is converted into sulfid. It is then filtered hot and the lead sulfid washed with hot water until the washings are no longer acid. The filtrate and washings are evaporated to fifty cubic centimeters and accurately neutralized with potassium hydroxid. An excess of a saturated solution of calcium acetate is added and the liquid allowed to stand from four to six hours with frequent stirring. It is then filtered and the precipitate washed until the filtrate amounts to exactly 100 cubic centimeters. The precipitate of calcium tartrate is converted into calcium oxid by igniting in a platinum crucible. After cooling, from ten to fifteen cubic centimeters of normal hydrochloric acid are added, the solution washed into a beaker and accurately titrated with normal potassium hydroxid solution. Every cubic centimeter of normal acid saturated by the calcium oxid is equivalent to 0.0750 gram tartaric acid. To the amount so obtained, 0.0286 gram must be added, representing the tartaric acid held in solution in the filtrate as calcium tartrate. The sum represents the total tartaric acid in the wine.

The filtrate from the calcium tartrate is evaporated to about twenty-five cubic centimeters, cooled and mixed with three times its volume of ninety-six per cent alcohol. After standing several hours, the precipitate is collected on a weighed filter, dried at 100° and weighed. It represents the calcium salts of malic, succinic and sulfuric acids and of the tartaric acid which remained in solution. This precipitate is dissolved in a minimum quantity of hydrochloric acid, filtered and the filter washed with hot water. Potassium carbonate solution is added to the hot filtrate, and the precipitated calcium carbonate separated by filtration and washed. The filtrate contains the potassium salts of the above named acids. It is neutralized with acetic acid, evaporated to a small volume and precipitated hot with barium chlorid. The precipitate of barium succinate and sulfate is separated by filtration, washed with hot water and treated on the filter with dilute hydrochloric acid. The barium sulfate remaining is washed, dried, ignited and weighed. In the filtrate, which contains the barium succinate, the barium is precipitated hot with sulfuric acid, washed, dried, ignited and weighed. Two hundred and twenty-three parts of barium sulfate equal 118 parts of succinic acid. The succinic and sulfuric acids, as well as the tartaric acid remaining in solution, which is equal to 0.0286 gram, are to be calculated as calcium salts and the result deducted from the total weight of the calcium precipitate. The remainder is the calcium malate, of which 172 parts equal 134 parts malic acid.

According to Macagno, succinic acid may be estimated in wines by the following process:[646] One liter of the wine is digested with lead hydroxid, evaporated on the water bath and the residue extracted with strong alcohol. The residual salts of lead are boiled with a ten per cent solution of ammonium nitrate, which dissolves the salts of succinic acid. The solution is filtered, the lead removed by hydrogen sulfid, boiled, neutralized with ammonia and treated with ferric chlorid as long as a precipitate is formed. The ferric succinate is separated by filtration, washed and ignited. The succinic acid is calculated from the weight of ferric oxid obtained.

Malic acid in wines and ciders is determined by the method of Berthelot in the following manner:[647] The sample is evaporated until reduced to a tenth of its volume. To the residue an equal volume of ninety per cent alcohol is added and the mixture set aside for some time. The tartaric acid and tartrates separate, together with the greater part of the salts of lime which may be present.

The supernatant liquid is decanted and a small quantity of lime added to it until in slight excess of that required to neutralize the acidity. Calcium malate is separated mixed with lime. The solid matters are separated by filtration, dissolved in a ten per cent solution of nitric acid, from which the lime bimalate will separate in a crystalline form. The weight of calcium bimalate multiplied by 0.59 gives that of the malic acid.

=633. Polarizing Bodies in Fermented Beverages.=—The study of the nature of the carbohydrates, which constitute an important part of the solid matters dissolved in fermented beverages, is of the greatest importance. These bodies consist of grape sugars, sucrose, tartaric acid and the unfermented hydrolytic products derived from starch. A natural grape sugar (chiefly dextrose) is found in wines. Sucrose is also a very important constituent of sweet wines. The hydrolytic products of starch are found in beers, either as a residue from the fermentation of malt or from the rice, glucose, hominy grits etc., added in brewing. The character and quantities of these residues can be determined by the methods already given in the parts of this volume relating to sugars and starches. For convenience, however, and for special application to the investigation of fermented beverages a résumé of the methods adopted by the official chemists follows:[648]

=634. Determination of Reducing Sugars.=—The reducing sugars are estimated as dextrose, and may be determined by any of the methods given for the estimation thereof (=113-140=).

=635. Polarization.=—All results are to be stated as the polarization of the undiluted sample. The triple field shadow saccharimeter is recommended, and the results are expressed in the terms of the sugar scale of this instrument. If any other instrument be used, or if it be desirable to convert to angular rotation, the following factors may be employed:

1° Schmidt and Haensch = 0°.3468 angular rotation D. 1° angular rotation D = 2°.8835 Schmidt and Haensch. 1° Schmidt and Haensch = 2°.6048 Wild (sugar scale). 1° Wild (sugar scale) = 0°.3840 Schmidt and Haensch. 1° Wild (sugar scale) = 0°.1331 angular rotation D. 1° angular rotation D = 0°.7511 Wild (sugar scale). 1° Laurent (sugar scale) = 0°.2167 angular rotation D. 1° angular rotation D = 4°.6154 Laurent (sugar scale).

In the above table D represents the angular rotation produced with yellow monochromatic light.

(_a_) _In White Wines or Beers._—Sixty cubic centimeters of wine are decolorized with three cubic centimeters of lead subacetate solution and filtered. Thirty cubic centimeters of the filtrate are treated with one and five-tenths cubic centimeters of a saturated solution of sodium carbonate, filtered and polarized. This gives a solution of nearly ten to eleven, which must be considered in the calculation, and the polariscope reading must accordingly be increased one-tenth.

(_b_) _In Red Wines._—Sixty cubic centimeters of wine are decolorized with six cubic centimeters of lead subacetate solution and filtered. To thirty cubic centimeters of the filtrate, three cubic centimeters of a saturated solution of sodium carbonate are added, filtered and the filtrate polarized. The dilution in this case is nearly five to six, and the polariscope reading must accordingly be increased one-fifth.

(_c_) _In Sweet Wines._ (1) _Before Inversion._—One hundred cubic centimeters are decolorized with two cubic centimeters of lead subacetate solution and filtered after the addition of eight cubic centimeters of water. One-half cubic centimeter of a saturated solution of sodium carbonate and four and five-tenths cubic centimeters of water are added to fifty-five cubic centimeters of the filtrate, the liquids mixed, filtered and polarized. The polariscope reading is multiplied by 1.2.

(2) _After Inversion._—Thirty-three cubic centimeters of the filtrate from the lead subacetate in (1) are placed in a flask with three cubic centimeters of strong hydrochloric acid. After mixing well, the flask is placed in water and heated until a thermometer, placed in the flask with the bulb as near the center of the liquid as possible, marks 68°, consuming about fifteen minutes in the heating. It is then removed, cooled quickly to room temperature, filtered and polarized, the temperature being noted. The polariscope reading is multiplied by 1.2. Because of the action of lead subacetate on invert sugar (=87=) it is advisable to decolorize the samples with other reagents (=87-89=).

(3) _After Fermentation._—Fifty cubic centimeters of wine, which have been dealcoholized by evaporation and made up to the original volume with water, are mixed, in a small flask, with well washed beer yeast and kept at 30° until fermentation has ceased, which requires from two to three days. The liquid is washed into a 100 cubic centimeter flask, a few drops of a solution of acid mercuric nitrate and then lead subacetate solution, followed by sodium carbonate, added. The flask is filled to the mark with water, shaken, the solution filtered and polarized and the reading multiplied by two.

=636. Application of Analytical Methods.=—(1) _There is no rotation._—This may be due to the absence of any rotatory body, to the simultaneous presence of the dextrorotatory nonfermentable constituents of commercial dextrose and levorotatory sugar, or to the simultaneous presence of dextrorotatory cane sugar and levorotatory invert sugar.

(_a_) _The Wine is Inverted._—A levorotation shows that the sample contains cane sugar.

(_b_) _The Wine is Fermented._—A dextrorotation shows that both levorotatory sugar and the unfermentable constituents of commercial dextrose are present.

If no change take place in either (_a_) or (_b_) in the rotation, it proves the absence of unfermented cane sugar, the unfermentable constituents of commercial dextrose and of levorotatory sugar.

(2) _There is right rotation._—This may be caused by unfermented cane sugar, the unfermentable constituents of commercial dextrose or both.

(_a_) The sugar is inverted:

(_a_₁) _It rotates to the left after inversion._—Unfermented cane sugar is present.

(_a_₂) _It rotates more than 2°.3 to the right._—The unfermentable constituents of commercial dextrose are present.

(_a_₃) _It rotates less than 2°.3 and more than 0°.9 to the right._—It is in this case treated as follows:

Two hundred and ten cubic centimeters of the sample are evaporated to a thin sirup with a few drops of a twenty per cent solution of potassium acetate. To the residue 200 cubic centimeters of ninety per cent alcohol are added with constant stirring. The alcoholic solution is filtered into a flask and the alcohol removed by distillation until about five cubic centimeters remain. The residue is mixed with washed bone-black, filtered into a graduated cylinder and washed until the filtrate amounts to thirty cubic centimeters. When the filtrate shows a dextrorotation of more than 1°.5, it indicates the presence of unfermentable constituents of commercial dextrose.

(3) _There is left rotation._—The sample contains unfermented levorotatory sugar, derived either from the must or mash or from the inversion of added cane sugar. It may, however, also contain unfermented cane sugar and the unfermentable constituents of commercial dextrose.

(_a_) The wine sugars are fermented according to directions in =262=.

(_a_₁) _It polarizes 3° after fermentation._—It contains only levorotatory sugar.

(_a_₂) _It rotates to the right._— It contains both levorotatory sugar and the unfermentable constituents of commercial dextrose.

(_b_₁) The sucrose is inverted according to (_c_), in (2).

(_b_₂) It is more strongly levorotatory after inversion. In contains both levorotatory sugar and unfermented cane sugar.

=637. Estimation of Sucrose, Dextrose, Invert Sugar, Maltose and Dextrin.=—The total and relative quantities of these carbohydrates are determined by the processes already described (=237-262=).

=638. Determination of Glycerol.=—(_a_) _In Dry Wines and Beers._—One hundred cubic centimeters of wine are evaporated in a porcelain dish to about ten cubic centimeters, a little quartz sand and milk of lime added and the evaporation carried almost to dryness. The residue is mixed with fifty cubic centimeters of ninety per cent alcohol, using a glass pestle or spatula to break up any solid particles, heated to boiling on the water bath, allowed to settle and the liquid filtered into a small flask. The residue is repeatedly extracted in a similar manner, with small portions of boiling alcohol, until the filtrate in the flask amounts to about 150 cubic centimeters. A little quartz sand is added, the flask connected with a condenser and the alcohol slowly distilled until about ten cubic centimeters remain. The evaporation is continued on the water bath until the residue becomes sirupy. It is cooled and dissolved in ten cubic centimeters of absolute alcohol. The solution may be facilitated by gentle heating on the steam bath. Fifteen cubic centimeters of anhydrous ether are added, the flask stoppered and allowed to stand until the precipitate has collected on the sides and bottom of the flask. The clear liquid is decanted into a tared weighing bottle, the precipitate repeatedly washed with a few cubic centimeters of a mixture of one part of absolute alcohol and one and five-tenths parts anhydrous ether and the washings added to the solution. The ether-alcohol is evaporated on the steam bath, the residue dried one hour in a water oven, weighed, the amount of ash determined and its weight deducted from that of the weighed residue to get the quantity of glycerol.

(_b_) _In Sweet Wines._—One hundred cubic centimeters of wine are evaporated on the steam bath to a sirupy consistence, a little quartz sand being added to render subsequent extraction easier. The residue is repeatedly treated with absolute alcohol until the united extracts amount to from 100 to 150 cubic centimeters. The solution is collected in a flask and for every part of alcohol one and five-tenths parts of ether are added, the liquid well shaken and allowed to stand until it becomes clear. The supernatant liquor is decanted into a beaker and the precipitate washed with a few cubic centimeters of a mixture of one part alcohol and one and five-tenths parts ether. The united liquids are distilled, the evaporation being finished on the water bath, the residue is dissolved in water, transferred to a porcelain dish and treated as under (_a_).

=639. Determination of Coloring Matters in Wines.=—The methods of detecting the more commonly occurring coloring matters in wines as practiced by the official chemists are given below.

(_a_) _Cazeneuve Reaction._—Add two-tenths gram of precipitated mercuric oxid to ten cubic centimeters of wine, shake for one minute and filter.

Pure wines give filtrates which are colorless or light yellow, while the presence of a more or less red coloration indicates that an anilin color has been added to the wine.

(_b_) _Method of Sostegni and Carpentieri._—Evaporate the alcohol from 200 cubic centimeters of wine. Add from two to four cubic centimeters of a ten per cent solution of hyrochloric acid, immerse therein some threads of fat-free wool and boil for five minutes. Remove the threads, wash them with cold water acidified with hydrochloric, then with hot water acidified with hydrochloric, then with pure water and dissolve the color in a boiling mixture of fifty cubic centimeters of water and two cubic centimeters of concentrated ammonia. Replace the threads by new ones, acidify with hydrochloric and boil again for five minutes. In the presence of anilin colors to the amount of two milligrams per liter, the threads are dyed as follows:

Safranin light rose-red. Vinolin rose-red to violet. Bordeaux-red rose-red to violet. Ponceau-red rose-red. Tropæolin oo straw yellow. Tropæolin ooo light orange.

(_c_) _Detection of Fuchsin and Orseille._—To twenty cubic centimeters of wine add ten cubic centimeters of lead acetate solution, heat slightly and mix by shaking. Filter into a test-tube, add two cubic centimeters of amyl alcohol and shake. If the amyl alcohol be colored red, separate it and divide it into two portions. To one add hydrochloric acid, to the other ammonia. When the color is due to fuchsin, the amyl alcohol will in both cases be decolorized; when due to orseille, the ammonia will change the color of the amyl alcohol to purple-violet.

=640. Determination of Ash.=—The residue from the direct extract determination is incinerated at as low a heat as possible. Repeated moistening, drying and heating to low redness is advisable to get rid of all organic substances. When a quantitive analysis of the ash is desired, large quantities of the sample are evaporated to dryness and the residue incinerated with the usual precautions.

=641. Determination Of Potash.=—(_a_) _Kayser’s Method._—Dissolve seven-tenths gram pure sodium hydroxid and two grams of tartaric acid in 100 cubic centimeters of wine, add 150 cubic centimeters of ninety-two to ninety-four per cent alcohol and allow the liquid to stand twenty-four hours. The precipitated potassium bitartrate is collected on a small filter and washed with fifty per cent alcohol until the filtrate amounts to 260 cubic centimeters. The precipitate and filter are transferred to the beaker in which the precipitation was made, the precipitate dissolved in hot water, the volume made up to 200 cubic centimeters and fifty cubic centimeters thereof titrated with decinormal sodium hydroxid solution, adding 0.004 gram to the final result, representing the potash which remains in solution as bitartrate.

(_b_) _Platinum Chlorid Method._—Evaporate 100 cubic centimeters of the wine to dryness, incinerate the residue and determine the potash as in ash analysis.[649]

=642. Determination of Sulfurous Acid.=—One hundred cubic centimeters of wine are distilled in a current of carbon dioxid, after the addition of phosphoric acid, until about fifty cubic centimeters have passed over. The distillate is collected in accurately set iodin solution. When the distillation is finished, the excess of iodin is determined with set sodium thiosulfate solution and the sulfurous acid calculated from the iodin used.

=643. Detection of Salicylic Acid.=—(_a_) _Spica’s Method._—Acidify 100 cubic centimeters of the liquor with sulfuric and extract with sulfuric ether. Evaporate the extract to dryness, warm the residue carefully with one drop of concentrated nitric acid and add two or three drops of ammonia. The presence of salicylic acid in the liquor is indicated by the formation of a yellow color due to ammonium picrate and may be confirmed by dyeing therein a thread of fat-free wool.

(_b_) _Bigelow’s Method._—Place 100 cubic centimeters of the wine in a separatory funnel, add five cubic centimeters of sulfuric acid (1-3) and extract with a sufficient quantity of a mixture of eight or nine parts of ether to one part of petroleum ether. Throw away the aqueous part of the extract, wash the ether once with water, then shake thoroughly with about fifty cubic centimeters of water, to which from six to eight drops of a one-half per cent solution of ferric chlorid have been added. Discard the aqueous solution, which contains the greater part of the tannin in combination with iron, wash again with water, transfer the ethereal solution to a porcelain dish and allow to evaporate spontaneously. Heat the dish on the steam bath, take up the residue with one or two cubic centimeters of water, filter into a test-tube and add one to two drops of one-half per cent solution of ferric chlorid. The presence of salicylic acid is indicated by the appearance of a violet-red coloration. In the case of red wines, a second extraction of the residue with ether mixture is sometimes necessary. This method cannot be used in the examination of beers and ales.

(_c_) _Girard’s Method._—Extract a portion of the acidified liquor with ether as in the preceding methods, evaporate the extract to dryness and exhaust the residue with petroleum ether. The residue from the petroleum ether extract is dissolved in water and treated with a few drops of a very dilute solution of ferric chlorid. The presence of salicylic acid is indicated by the appearance of a violet-red coloration.

=644. Detection of Gum and Dextrin.=—Four cubic centimeters of the sample are mixed with ten cubic centimeters of ninety-six per cent alcohol. When gum arabic or dextrin is present, a lumpy, thick and stringy precipitate is produced, whereas pure wine becomes at first opalescent and then gives a flocculent precipitate.

=645. Determination of Nitrogen.=—The best method of determining nitrogen in fermented beverages is the common one of moist combustion with sulfuric acid. The sample is placed in the kjeldahl digestion flask, which is attached to the vacuum service and placed in a steam bath until its contents are dry or nearly so. The process is then conducted in harmony with the well known methods. Where large quantities of the sample are to be employed, as in drinks containing but little nitrogen, the preliminary evaporation may be accomplished in an open dish, the contents of which are transferred to the digestion flask before any solid matter is deposited. The same procedure may be followed when the sample foams too much on heating.

=646. Substitutes for Hops.=—It is often claimed that cheap and deleterious bitters are used in brewing in order to save hops. While it is doubtless true that foreign bitters are sometimes employed, the experience of this laboratory goes to show that such an adulteration is not very prevalent in this country.[650] Possibly strychnin, picrotoxin, quassin, gentian and other bitter principles have sometimes been found in beer, but their use is no longer common. It is difficult to decide in every case whether or not foreign bitters have been added. A common process is to treat the sample with lead acetate, filter, remove the lead from the filtrate and detect any remaining bitters by the taste. All the hop bitters are removed by the above process. Any remaining bitter taste is due to other substances. For the methods of detecting the special bitter principles in hops and other substances, the work of Dragendorff may be consulted.[651]

=647. Bouquet of Fermented and Distilled Liquors.=—The bouquet of fermented and distilled liquors is due to the presence of volatile matters which may have three different origins. In the first place the materials from which these beverages are made contain essential oils and other odoriferous principles.[652] In the grape, for instance, the essential oils are found particularly in the skins. These essential principles may be secured by distilling the skins of grapes in a current of steam. This method of separation, however, cannot be regarded as strictly quantitive.

In the second place, the yeasts which produce the alcoholic fermentation are also capable of producing odoriferous products. These minute vegetations, resembling in their biological relations the mushrooms, grow in the soil and reach their maturity at about the time of the harvest of the grapes. Their spores are transmitted through the air, reach the expressed grape juice and produce the vinous fermentation. The particular odor due to any given yeast persists through many generations of culture showing that the body which produces the odor is the direct result of the vegetable activity of the yeast. A beer yeast, after many generations of culture, will still give a product which smells like beer, and in like manner a wine yeast will produce one which has the odor of wine. The quantity of odorant matter produced by this vegetable action is so minute as to escape detection in a quantitive or qualitive way by chemical means. These subtle perfumes arise moreover not only from the breaking up of the sugar molecule, but are also the direct results of molecular synthesis accomplished under the influence of the yeast itself.

In the third place, the fermented and distilled liquors contain odoriferous principles due to the chemical reactions which take place by the breaking up of the sugar and other molecules during the process of fermentation. The alcohols and acids produced have distinct odors by which they are often recognized. This is particularly true of ethylic, propylic, butylic, amylic and oenanthylic alcohols and acetic acid. These alcohols themselves also undergo oxidation, passing first into the state of aldehyds which, together with ethers, produce the peculiar aroma which is found in various fruits. The etherification noted above is of course preceded by the formation of acids corresponding to the various aldehyds present. The formation of these ethers takes place very slowly during aging, and it therefore requires three or four years for the proper ripening of wines or distilled liquors. By means of artificial heat, electricity and aeration, the oxidizing processes above noted may be hastened, but it is doubtful whether the products arising from this artificial treatment are as perfect as those which are formed in the natural processes.

AUTHORITIES CITED IN PART SEVENTH.

[535] Bulletin 46, Chemical Division U. S. Department of Agriculture, pp. 24-25.

[536] Bulletin 42, Arkansas Agricultural Experiment Station, pp. 81 et seq.

[537] Balland; Recherches sur les Blés, les Farines et le Pain, p. 229.

[538] Jago; Flour and Bread, p. 457.

[539] Jago; op. cit. supra, p. 465.

[540] Richardson; Journal of the Chemical Society, Transactions, 1885, pp. 84 et seq.

[541] Auct. et op. cit. supra, pp. 80 et seq.

[542] Bulletin 28, Office of Experiment Stations, U. S. Department of Agriculture, pp. 9, 10.

[543] Bulletin 29, Office of Experiment Stations U. S. Department of Agriculture, p. 8.

[544] Op. et. loc. cit. supra.

[545] Experiment Station Record, Vol. 6, pp. 590 et seq.

[546] Annual Report, U. S. Department of Agriculture, 1884, p. 365.

[547] Forschungs-Berichte über Lebensmittel, Band 3, S. 142.

[548] Zeitschrift für angewandte Chemie, 1895, S. 620.

[549] Op. et loc. cit. supra.

[550] Les Ferments Solubles; Diastases—Enzymes.

[551] Wiley; Medical News, July, 1888.

[552] Virchow’s Archiv., Band 123, S. 230: Journal of the Chemical Society, Abstracts, 1892, p. 755.

[553] Ladenberg; Handwörterbuch der Chemie, Band 4, S. 122.

[554] Chemisches Centralblatt, 1892, Band 2, S. 579.

[555] Op. cit. supra, 1890, Band 2, S. 628.

[556] Die Landwirtschaftlichen Versuchs-Stationen, Band 44, S. 188; Experiment Station Record, Vol. 6, p. 12.

[557] Experiment Station Record, Vol. 6, pp. 5 et seq. (Read Jordan instead of Gordon.)

[558] Journal of the American Chemical Society, Vol. 16, pp. 590 et seq.

[559] From photograph made in this laboratory by Bigelow.

[560] Journal of the Society of Chemical Industry, Vol. 10, p. 118.

[561] Die Landwirtschaftlichen Versuchs-Stationen, Band 36, S. 321: Bulletin 13, Chemical Division, U. S. Department of Agriculture, p. 1028.

[562] Wilson; Vid. op. et loc. cit. 26.

[563] U. S. Dispensatory, p. 1088.

[564] Landwirtschaftliche Jahrbücher, 1890, Band 19, S. 149.

[565] Bulletin 13, Chemical Division, U.S. Department of Agriculture, p. 1028.

[566] Zeitschrift für analytische Chemie, Band 35, S. 498.

[567] Annual Report of the Maine Agricultural Experiment Station, 1891, p. 25: Gay; Annales Agronomiques, 1885, p. 145, et 1896, pp. 145 et seq.

[568] Annales Agronomiques, Tome 21, pp. 149, 150.

[569] Vid. op. et loc. cit. primo sub 33.

[570] Twelfth Annual Report of the Massachusetts Agricultural Experiment Station, 1894, p. 175.

[571] See also paragraph =586= this volume.

[572] Manuscript prepared for publication as a part of Bulletin 13, Chemical Division, U. S. Department of Agriculture.

[573] Vid. this volume, paragraph =280=.

[574] Vid. op. cit. 31, p. 1020.

[575] Bulletin 45, Chemical Division, U.S. Department of Agriculture, p. 12.

[576] Berthelot; Essai de Chimie Mécanique: Thomsen; Thermo Chemische Untersuchungen: Ostwald; Algemeine Chemie: Muir; Elements of Thermal Chemistry.

[577] Bulletin 21, Office of Experiment Stations, U. S. Department of Agriculture, pp. 113 et seq.: Seventh Annual Report Connecticut (Storr’s) Agricultural Experiment Station, pp. 133 et seq.

[578] Vid. op. et loc. cit. 43.

[579] From personal inspection by author in Williams’ laboratory, 161 Tremont St., Boston, Mass.

[580] Journal für praktische Chemie, Band 147 {Neue Folge Band 39}, Ss. 517 et seq.

[581] Berthelot; Annales de Chemie et de Physique, 6e Série, Tome 10, p. 439.

[582] Vid. op. cit. 46, Ss. 522-523. The data in paragraph =566= are taken from Stohmann, Zeitschrift für Biologie, Band 31, S. 364 and Experiment Station Record, Vol. 6, p. 590.

[583] Journal of the American Chemical Society, Vol. 18, p. 174.

[584] Bulletins 93, 97, 101 and 102, California Agricultural Experiment Station.

[585] Annual Report, U. S. Department of Agriculture, 1886, p. 354.

[586] This work, Vol. 2, p. 318.

[587] Vid. California Bulletins cited under 50: Wolff; Aschen Analyse, S. 126.

[588] Bulletin 100, Cornell Agricultural Experiment Station: Bulletin 48, Chemical Division, U. S. Department of Agriculture.

[589] Vid. op. cit. 51, p. 353.

[590] Vid. op. cit. ultimo sub 54.

[591] Bulletin No. 42, Arkansas Agricultural Experiment Station, p. 78.

[592] Annual Report, U. S. Department of Agriculture, 1884, p. 347.

[593] Spencer; Bulletin 13, U. S. Department of Agriculture, pp. 875 et seq.

[594] Journal of Analytical and Applied Chemistry, Vol. 4, p. 390; Bulletin 13, Chemical Division, U. S. Department of Agriculture, p. 889.

[595] Pharmaceutical Journal, Vol. 52, p. 213.

[596] Commercial Organic Analysis, Vol. 3,