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]

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,

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.

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.
• 98324

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.