Part 82

Extraction with Ammonium Chlorid and Hydrochloric Acid.—The residue, after exhaustion with cold water, is extracted with a solution of ammonium chlorid containing 150 grams of the salt in a liter. This method of extraction is entirely similar to that with water just described. Globulins and myosin pass into solution by this treatment. The residual mass is washed as free as possible of the solvent and is then further extracted with dilute hydrochloric acid containing four cubic centimeters of the fuming acid in a liter. The treatment with dilute acid is continued until no further substance passes into solution. This is determined by neutralizing a portion of the extract with sodium carbonate, or by the direct addition of potassium ferrocyanid. In either case absence of a precipitate indicates that no nitrogenous matters are present in the solution.

Extraction with Alkali.—The residue from the acid extraction is washed with water until the acid is removed and then extracted in a similar manner with a dilute solution of sodium or potassium hydroxid containing not to exceed two grams of the caustic to the liter. When this residue is finally washed with water and a little acetic acid, it will be found that practically all the purely albuminous bodies contained in the tissues have been extracted with the exception of any fibrin, which the blood, present in the tissues at the commencement of the extraction, may have contained. The extract should be acidified with acetic as soon as obtained.

Extraction with Boiling Water.—The residual matter boiled for some time with water will part with its collagen, which, when transformed by the heat into glutin, passes into solution.

The sarcolemma, membranes, elastic fibers and keratin remain undissolved.

=413. Contents of the Several Extracts.=—By the systematic treatment of muscular tissues in the manner just described, the nitrogenous bodies they contain are separated into five classes, viz.:

Cold Water Extract.—This contains serum albumin, serum globulin, muscle albumin, myosin, mucin and peptone.

Ammonium Chlorid Extract.—This solution contains the globulins and also in many cases some myosin and serum globulin.

Hydrochloric Acid Extract.—When the extractive matter removed by hydrochloric acid, thrown out by sodium carbonate and well washed with water, has a neutral reaction, it consists of syntonin, when acid, of an albuminate.

Alkali Extract.—The acid albumin of the animal tissue is found in the alkaline solution and may be thrown out by making the solution slightly acid.

Insoluble Residue.—The fifth class contains the insoluble nitrogenous bodies mentioned above.

=414. General Observations.=—Only a brief résumé of the methods of treating animal tissues for nitrogenous bases is given above, since a more elaborate discussion of these principles and methods would lead too far away from the main purpose of this manual. For practical purposes, the most important of these bodies are those soluble in water and the methods of treating these will be handled at some length. Unfortunately, the methods of determining the exact qualities of these bodies are not as satisfactory in case of animal as in vegetable nitrogenous bodies. The flesh bases, soluble in water, contain a much larger percentage of nitrogen than is found in true proteid bodies, and therefore the multiplication of the weight of nitrogen found therein by 6.25 does not give even a near approximation of the actual quantities of the nitrogenous bodies present in the sample.

Some of the flesh bases contain more than twice as much nitrogen as is found in proteids, and in such cases 3.12, and not 6.25, would be the more correct factor to use in the computation. When possible, therefore, these bodies should be precipitated and weighed after drying, but this is not practicable in many instances. The sole resource of the chemist in such cases is to determine the nature of the body as nearly as possible by qualitive reactions, then to determine the total nitrogen therein and multiply its weight by the corresponding factor. The principal flesh bases have the following percentages of nitrogen and the approximate factors for calculating analytical data are also given:

Name of base. Formula. Per cent Factor. nitrogen. Glutin C₁₃H₂₀N₄O₅ 17.95 5.57 Carnin C₇H₈N₄O₂ 31.11 3.21 Kreatin C₄H₁₉N₃O₂ 32.06 3.12 Kreatinin C₄H₇N₃O₂ 37.17 2.69 Sarkin C₅H₄N₄O 41.18 2.43

=415. Composition of Meat Extracts.=—The meat extracts of commerce contain all the constituents of meat that are soluble in warm water. The parts which are soluble in warm water and not in cold are found in the cold aqueous solution as suspended or sedimentary matters. Among the nitrogenous bodies present are included albumin, albumose and peptone among the proteids, carnin, kreatin, kreatinin, sarkin and xanthin among the non-proteids, and inosinic and uric acids and urea among other nitrogenous bodies. Among the non-nitrogenous bodies are found lactic and butyric acids, inosit and glycogen. Among mineral bodies occurs the phosphates and chlorids of the common bases. In addition to these bodies, meat extracts may also contain gelatin and other decomposition products of proteid matter. Since meat extract is supposed to be prepared by the digestion of the meat free of bones and put in cold water or in warm water not above 75°, the presence of gelatin would indicate a different method of preparation, viz., either by boiling water or water heated above the boiling point under pressure. In a properly prepared extract, the percentage of gelatin is very small.

Approximately one-tenth of the whole nitrogen present is in the form of albumoses and only a trace as peptones. By far the greater part of the nitrogen exists as flesh bases (kreatin, etc.). The composition of three meat extracts, numbers one and two solid and number three liquid, is given in the subjoined table.

No. 1. No. 2. No. 3. Per cent. Per cent. Per cent.

Total nitrogen 9.28 9.14 2.77 Nitrogen as albumin trace 0.08 trace ” ” albumose 0.96 1.21 0.70 ” ” peptone trace trace none ” ” flesh bases 6.81 5.97 1.56 ” ” ammonia 0.47 0.41 0.09 ” in compounds insoluble in sixty-six per cent alcohol 0.21 0.33 0.25 ” ” other bodies 0.83 1.14 0.17

=417. Analysis of Meat Extracts.=—The analysis of a meat extract should include the determination of the water, ash and total nitrogen. After multiplying the nitrogen which exists as proteids by 6.25 and adding together the percentages of all the ingredients, ash, water, etc., including ammonia, the sum is to be subtracted from 100 and the difference entered as non-nitrogenous organic matter. The nature of this conglomerate has already been explained.

Water.—It is advisable to determine the water in a partial vacuum (=20=) or in an atmosphere of hydrogen (=23-25=).

The water may also be determined in solid extracts by placing about five grams of the material in a flat bottom tin foil dish about fifty-five millimeters in diameter and twenty millimeters deep. The material is dissolved in enough warm water to fill the dish a little over one-half and the liquid is then absorbed by adding a weighed quantity of fibrous asbestos or of dry fragments of pumice stone. The asbestos is to be preferred because of the fact that it may be subsequently cut into small bits for the determination of the gelatin. The dish thus prepared is dried to constant weight in a steam-bath or vacuum oven. The weight of the dish and of the added absorbent, together with that of the material employed and of the dried dish and its contents, give the data for calculating the percentage of water. The contents of the dish are used as described further on for the determination of gelatin. In liquid extracts the water is determined in an entirely analogous manner, using about twenty grams of the material and omitting the solution in water.

In solid extracts, the part insoluble in cold water is determined separately.

Ash.—The ash is determined by ignition at the lowest possible temperature, best in a muffle (=28-32=). The ash should be examined qualitively. Where a quantitive analysis is desired, larger quantities of the extract are incinerated and the constituents of the ash determined in the usual way.

Total Nitrogen.—Since nitrates are not present unless added in the manufacture, the total nitrogen is best determined by moist combustion.

Nitric Nitrogen.—The extract should be tested for nitrates and if present they are determined in the manner already described.

Ammoniacal Nitrogen.—When ammonia is present it is determined by distillation with magnesia.

Since boiling with magnesia may cause the distillation of more ammonia than is present as ammonium salts, the plus being due to the decomposition of some other nitrogenous compounds, Stutzer replaces the magnesia with barium carbonate.

Proteid Nitrogen Insoluble in Sixty-Two Per Cent Alcohol.—The aqueous solution is treated with strong alcohol until the mixture contains about sixty-two per cent of the reagent. The precipitate produced is separated by filtration, washed with sixty-two per cent alcohol and the nitrogen therein determined.

Albumose Nitrogen.—This is secured by saturating the aqueous solution with zinc or ammonium sulfate. The separated albumoses are skimmed from the surface, thrown in a filter, washed with a saturated solution of zinc sulfate and the nitrogen determined therein by moist combustion. In the filtrate from the above separation, peptone is detected qualitively by adding a few drops of dilute solution of copper sulfate (biuret reaction).