Part 54

=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.