Part 41

=406. Carbon and Hydrogen.=—Carbon and hydrogen are estimated in proteid matters by combustion with copper oxid. Osborne prefers to burn the sample in a platinum boat in a current of air or of oxygen free of moisture and carbon dioxid.[382] It is advisable to use also a layer of lead chromate in addition to the copper oxid and metallic copper. The method of conducting the combustion has already been described.[383] The analyst should have at his disposal a quantity of pure sugar, which may be used from time to time in testing the accuracy of the work. In beginning a series of combustions this precaution should never be omitted. The addition of the lead chromate is to make more certain the absorption of oxidized sulfur produced during the combustion.

=407. Estimation of Nitrogen.=—In most cases it is found convenient, during the progress of separating vegetable proteids, to determine the quantity of each kind by estimating the nitrogen by moist combustion and computing the quantity of proteid matter by multiplying the nitrogen by 6.25. The estimation of the nitrogen is made either on an aliquot part of the extract or by direct treatment of the residue.

In the pure extracted proteid matter the nitrogen is most conveniently determined by moist combustion, but it may also be obtained either by combustion with soda-lime or with copper oxid, or by other reliable methods.[384]

The percentages of nitrogen found in the principal proteid bodies, together with the factors for computing the weights of the proteid bodies from the weights of nitrogen found, are given below:

Name of body. Percentage of nitrogen. Factor.

Mucin 13.80 to 14.13 7.25 to 7.08 Chondrin 14.20 to 14.65 7.04 to 6.83 Albuminates 13.87 7.21 Oat proteids 15.85 6.31 Serum globulin 15.63 6.40 Egg albumin 15.71 to 17.85 6.37 to 5.60 Maize proteids 16.06 6.22 Casein 15.41 to 16.29 6.49 to 6.13 Serum albumin 15.96 6.27 Syntonin 16.10 6.21 Keratin 16.20 to 17.70 6.17 to 5.65 Fibrinogen 16.65 6.01 Peptones 16.66 to 17.13 6.00 to 5.84 Elastin 16.75 5.97 Wheat proteids 16.80 to 18.39 5.95 to 5.44 Fibrin 16.91 5.91 Flax seed proteids 17.70 to 18.78 5.65 to 5.33

=408. Determination of Sulfur.=—Sulfur is a characteristic constituent of the proteid bodies, existing in quantities approximating one per cent of their weight.

In the estimation of sulfur, it is first converted into sulfuric acid, which is thrown out by a soluble barium salt and the sulfur finally weighed as barium sulfate.

All the sulfur existing in the organic state in a proteid may be obtained by burning in a current of oxygen and conducting the gaseous products of combustion through solid sodium or potassium carbonate at or near a red heat.[385] The organic sulfur may also be converted into sulfuric acid by fusing the proteid body with a mixture of sodium hydroxid and potassium nitrate. The fused mass, after cooling, is dissolved in water, the solution acidified with hydrochloric, evaporated to dryness to decompose nitrates and remove excess of hydrochloric acid and dissolved in a large excess of water. After standing for a day, the solution is filtered and the sulfuric acid thrown out of the hot filtrate with a slight excess of barium chlorid solution. The usual precautions in precipitating, filtering and igniting the barium sulfate are to be observed.[386]

=409. General Observations.=—In the preceding paragraphs have been stated the general principles upon which the separation of vegetable proteid matters depends, and a description has been given of the several processes by which this separation is accomplished. In each case, however, special conditions exist which require special modifications of the general processes, and these can only be successfully secured by the skill, judgment and patient labor of the investigator. Many of these cases have been already worked out, and the valuable data secured by Chittenden, Osborne and others, are accessible to the analyst in the papers already cited. In the case of the proteids in the peanut, a similar work has been done in this laboratory by Bigelow, the data of which have not yet been published. It is only by a careful study of the work already done as outlined here and as published in full in the cited papers, that the analyst will be able to secure trustworthy guidance for future investigations.

=410. Dialysis.=—One of the most important of the operations connected with the separation and analysis of proteids is the removal of the salts whereby their solutions are secured. This is accomplished by subjecting the solutions of the proteid matters to dialysis. The solution is placed in bags made of parchment dialysis paper. These bags are tied about a glass tube, whereby access may be had to their contents during the progress of the work. Since the volume of the liquid increases during the process, the bags should not be filled too full in the beginning.

In this laboratory the dialysis is carried out by Bigelow with the city water from the Potomac, which is first passed through a battery of porous porcelain filtering tubes to remove any suspended silt or micro-organisms. If unfiltered water be used, the germs therein cause a fermentation in the proteid matter, which seriously interferes with the value of the data obtained, and which can only be avoided by the use of an antiseptic, such as an alcoholic solution of thymol. Even with filtered water, the use of a few drops of the solution mentioned is often necessary. To avoid the use of too great quantities of the filtered water, the dialyzers are arranged _en batterie_, as shown in the figure. The filtered water enters the first vessel and thence passes through all. The parchment bags are frequently changed from vessel to vessel, each being brought successively into the first vessel in contact with the fresh water. In some cases the final steps in the dialysis may be accomplished in distilled water.

It is advisable to conduct a fractional preliminary dialysis of the salt solution containing proteids in such a way as to secure the globulins precipitated in each interval of twenty-four hours. Each portion thus secured may be examined with the microscope. Usually a period of two weeks is required to entirely remove the mineral salts from solution. If prepared parchment tubes be used for the dialysis, they should be first tested for leaks, and should not be more than half filled. By the use of a large number of these tubes a greater surface is exposed to dialytic action, and the time required to complete the operation is correspondingly decreased.

SEPARATION AND ESTIMATION OF NITROGENOUS BODIES IN ANIMAL PRODUCTS.

=411. Preparation of the Sample.=—Animal products present many difficulties in respect of the reduction thereof to a sufficiently comminuted condition for analytical examination. In the case of bones, the choppers used for preparing them for feeding to fowls are the most efficient apparatus for reducing them to fragments. In this condition they may be ground to a finer state in a sausage machine. The flesh of animals may be reduced by this machine, with two or three grindings, to a fairly homogeneous mass. Subsequent grinding in a mortar with powdered glass or sharp sand may serve to reduce the sample to a finer pulp, but is not usually necessary and should be avoided when possible. The sample thus prepared serves for the estimation of water, ash and fat by methods already described. The sample should be prepared in quantities of considerable magnitude, the whole of any organ or separate portion of the body being used when possible. In examining the whole body the relative weights of blood, bones, viscera, muscle, hide and other parts should first of all be ascertained and noted.

=412. Treatment of Muscular Tissues for Nitrogenous Bodies.=—For the present purpose a brief sketch of the method of separating the nitrogenous bodies in the muscular tissues of the body is all that can be attempted. For methods of examining the different organs and parts of the body in greater detail, standard works on physiological chemistry may be consulted.[387]

_Extraction with Cold Water._—A noted quantity of the finely divided tissues is mixed with several volumes of ice-cold water and well rubbed occasionally for several hours, the temperature meanwhile being kept low. The mixture is poured into a linen bag and the liquid portion removed by gentle pressure. The residue in like manner is treated with fresh portions of cold water until it gives up no further soluble matters. An aliquot portion of the extract is concentrated to a small bulk and serves for the determination of total nitrogen. The methods of separating and estimating nitrogenous bodies in flesh soluble in water will be given in considerable detail further on.

_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.[388]

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.[389]

_Total Nitrogen._—Since nitrates are not present unless added in the manufacture, the total nitrogen is best determined by moist combustion.[390]

_Nitric Nitrogen._—The extract should be tested for nitrates and if present they are determined in the manner already described.[391]

_Ammoniacal Nitrogen._—When ammonia is present it is determined by distillation with magnesia.[392]

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.[393]

_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).

_Kreatin, Kreatinin and Other Flesh Bases._—The clear, aqueous solution of the extract is acidified with sulfuric, mixed with a solution of sodium phosphotungstate and allowed to stand for about six days. The precipitate is collected, washed with a solution of the precipitant, and the nitrogen therein determined. The nitrogen found, less that due to ammonia, represents the total nitrogenous matter precipitated by the phosphotungstic acid. From this quantity is deducted the nitrogen in the proteids, precipitated by sixty-two per cent alcohol and by ammonium or zinc sulfate, and the remainder represents the nitrogen in flesh bases.

The nitrogen thrown out by the phosphotungstic acid is deducted from the total nitrogen, and the remainder represents the nitrogenous bodies not precipitable by the reagent named.

This method of separating the nitrogenous matters in meat extracts is based on the observation that these bodies contain at most only a small quantity of peptones, so small as to be safely negligible.[394]

_Quantities used for Analysis._—In conducting the separations above noted, it will be found convenient to use in each case about five grams of the solid or twenty of the liquid extract. In the nitrogen determinations, the weight of the sample should be inversely proportional to its content of nitrogen.

=417. Preparation of the Phosphotungstic Reagent.=—The phosphotungstic reagent is conveniently prepared as follows:

Dissolve 120 grams of sodium phosphate and 200 of sodium tungstate in one liter of water and add to the solution 100 cubic centimeters of strong sulfuric acid. When the reagent is prepared for general purposes it is customary to acidify with nitric, but in the present instance, inasmuch as the precipitate is used for the determination of nitrogen, it is evident that sulfuric should be substituted for nitric acid. In all cases the analyst must be assured of the strong acidity of the reagent, and in addition to this the solutions of proteid matter to which the reagent is added must first be made strongly acid with sulfuric.

=418. Zinc Sulfate as Reagent for Separating Albumoses from Peptones.=—When the albumoses are separated from the peptones, by precipitation with ammonium sulfate, there may be danger of some of this reagent adhering to the albumose, and in this way the quantity of nitrogen obtained on analysis may be increased. To avoid an accident of this kind Bömer replaces the ammonium by zinc sulfate.[395]

Since the precipitation of the albumoses by saturated saline solutions depends on their hydrolytic power, the substitution of another salt for ammonium sulfate capable of strongly attracting water, may be made if that salt does not possess any objectionable property. Crystallized zinc sulfate will dissolve in less than its own weight of cold water and is therefore well suited for the purpose in view.

In the case of a meat extract, the precipitation is accomplished as follows: Fifty cubic centimeters of the extract, freed from all solid matter by filtration and containing about two grams of the soluble proteids, are saturated in the cold with finely powdered zinc sulfate. The separated albumoses collect on the surface and are skimmed off, poured on a filter and washed with cold saturated zinc sulfate solution. The filter and its contents are used for the determination of nitrogen by moist combustion.[396]

The filtrate from the precipitated albumoses gives no biuret reaction, and, therefore, as in the use of ammonium sulfate, is free of albumin.

The biuret reaction is applied to the zinc sulfate filtrate as follows: The filtrate is greatly diluted with water and freed of zinc by means of a saturated solution of sodium carbonate. The filtrate free of zinc is evaporated on the steam-bath, made strongly alkaline with sodium hydroxid and treated with a few drops of a two per cent copper sulfate solution, added successively.

Another advantage possessed by the zinc sulfate is found in the fact that in the filtrate from the separated albumoses the peptones and other flesh bases can be thrown out by phosphotungstic acid. Before the application of the reagent, the filtrate should be made strongly acid by adding about an equal volume of dilute sulfuric acid (one part of acid to four of water.)

The nitrogen in the precipitate thus obtained is determined by moist combustion in the manner already suggested.