399. Separation of the Bodies Soluble in Water.—Albumins.—By the methods of treatment just described, the proteid matters soluble in ten per cent sodium chlorid solution are separated into two classes, viz., globulins insoluble in pure water and albumins and proteoses soluble in pure water. The aqueous solution will also contain any amids or nitrogenous bases soluble in the dilute saline solution and in water. Osborne and Voorhees have found that the best way of separating the albumins in the pure aqueous solution is by the application of heat.[373] By means of a fractional coagulation the albumins are divided into classes, viz., those separating at from 60° to 65° and those remaining in solution at that temperature but separating up to 85°. The respective quantities of these albumins are determined by collecting them in a filter and estimating the nitrogen therein by moist combustion in the usual way. Even a larger number of albumins may be secured, as in the maize kernel, by such a fractional precipitation by means of heat. Chittenden and Osborne find in this instance that the precipitation begins at about 40°.[374]

Proteose.—After the separation of the albumins by heat the filtrate may still contain proteid matter. This matter belongs to the proteose class. It may be partially secured by concentrating the filtrate, after the removal of the albumins, to a small bulk when a part of the proteose body will separate. It may be thrown out entirely by treating the filtrate above mentioned with fine-ground salt until it is saturated or by adding salt until the solution contains about twenty per cent thereof and precipitating the proteose by acetic acid.[375]

400. Separation of the Globulins.—The globulins which are extracted with ten per cent solution of sodium chlorid and precipitated on dialysis may be separated by fractional solution into several bodies of nearly related properties. This solution is conveniently accomplished by saline solvents of increasing strength. In the case of the maize globulins, Chittenden and Osborne employ dilute solutions of common salt for effecting the separation, beginning with a quarter of a per cent and ending with a two per cent mixture.[376]

401. Proteids Soluble in Dilute Alcohol.—Some of the proteid bodies which are soluble in dilute salt solution and in water are also soluble in alcohol. Since these bodies are more easily identified by the processes already described, attention will be given in this paragraph solely to those proteid bodies which are insoluble in water or dilute salt solution and are soluble in dilute alcohol.

For the extraction of these bodies, the residue, left after extraction with a ten per cent solution of sodium chlorid or with water, is mixed with enough strong alcohol to secure by the admixture with the water present in the sample an alcohol of about seventy-five per cent strength. The mixture is well shaken and digested for some time, at a temperature of about 46°, and thrown on a filter which is kept at about the same temperature. The residue is again mixed with alcohol of the same strength (seventy-five per cent) using about four liters for two and a half kilos of the original material. During the second digestion the temperature is kept at about 60°. The latter operation is repeated three times and in each case the filtrate obtained is evaporated separately.[377] This process is especially applicable to the meal from maize kernels, which contains a high relative percentage of an alcohol soluble proteid, zein.

The chief part of the zein is found in the first two extracts, obtained as described above. On evaporation, the zein separates as a tough, leathery, yellow-colored mass on the walls of the containing vessel. It is cut into small pieces and digested for several days in cold, pure alcohol. This is followed by digestion with a mixture of ether and pure alcohol, and finally with pure ether. By this treatment a part of the zein becomes insoluble in seventy-five per cent alcohol. The part soluble in dilute alcohol is precipitated by pouring it into water.

Another method of preparing zein is to extract the meal with seventy-five per cent alcohol after it has been treated with a ten per cent salt solution.

In this case the extraction is continued with seventy-five per cent alcohol in successive portions until no more proteid matter passes into solution. The several extracts are united and the alcohol removed by distillation, by which process the zein is separated. It is washed with distilled water, until the sodium chlorid is removed, dissolved in warm alcohol of about eighty per cent strength and any insoluble matter removed by filtration. On evaporating the filtrate nearly to dryness, the zein is separated and pressed as free of water as possible, yielding a yellow, elastic substance resembling molasses candy. This preparation is purified by digestion with pure alcohol and ether in the manner described. The two zeins which are secured by the treatment, one soluble and the other insoluble in alcohol, are practically identical in composition.[378]

Zein freshly precipitated by pouring its alcoholic solution in water is wholly insoluble in water, and, on boiling therewith, is changed into the variety insoluble in dilute alcohol. Boiled with dilute sulfuric acid, six in 300 cubic centimeters of water, it melts, forming a gummy mass, which is very slowly attacked by the acid yielding proteoses and peptones. Heated with stronger sulfuric acid it undergoes decomposition, yielding leucin, tyrosin, and glutamic acid.

402. Solvent Action of Acids and Alkalies.—In the preceding paragraphs, a synopsis has been given of the methods of separating proteid matters in such a manner as to secure them in a pure state in the same conditions as they exist in the natural substances. A very large percentage of the proteid matter is still left undissolved after extraction with the solvents already mentioned.

Often important information may be gained concerning the nature of the residual proteid matters by fractional extraction with dilute acids and alkalies. When the strength of these solutions is such that they contain about one per cent of the acid or alkali, the whole of the proteid matter may be dissolved by boiling successively with acid and alkali for half an hour. The proteid matter passing into solution in these cases is usually changed in character, assuming the nature of proteoses or allied bodies, when treated with an acid, and becoming albuminates when boiled with an alkali. Easily soluble carbohydrate matter is also removed by this treatment so that the residue obtained consists largely of cellulose and is known as crude or insoluble fiber. The removal of all the bodies soluble in dilute boiling acid and alkali is accomplished by the method described in paragraph 272.

For research purposes, the solvent action of dilute alkali is of chief importance to the analyst, and the extraction of the proteid matter, after all that is soluble in water, common salt solution and alcohol has been removed, should commence with a solution of potassium or sodium hydroxid containing not over two-tenths per cent of the alkali.

It has been shown by Osborne that the solvent action of very dilute alkali, in the cold, may be exerted without changing the character of the dissolved proteid.[379]

403. Method of Extraction.—The solvent employed is usually a two-tenths per cent solution of potassium hydroxid. It may be added directly to the substance or may follow extraction with water, salt solution or alcohol. In the former case, the manipulation is illustrated by the following description of the treatment of oatmeal:[380]

One hundred grams of oatmeal are mixed with half a liter of a two-tenths per cent potassium hydroxid solution and allowed to stand for some time at room temperature. The mixture is strained through a cloth to remove the chaff and the residue is stirred with another small portion of the solvent, again strained in the same cloth and the residue squeezed dry. The strained liquids are united and enough more of the solvent added to make the volume 700 cubic centimeters. After standing for some time, the insoluble matter settles to the bottom of the vessel and the supernatant liquid is decanted. More solvent is added to the residue, well mixed therewith and treated as above. It is advisable to make a third extraction in the same way. The extracts are united, passed through a filter, the proteid matter in solution thrown out by acetic acid, washed with water, alcohol and ether and dried over sulfuric acid.

The methods of procedure, when the sample has been previously extracted with water, salt solution or alcohol, are essentially the same as that just described and the reader may consult the paper of Osborne for details.[381]

404. Methods of Drying Separated Proteids.—In the preceding paragraphs, the analyst has been directed, in most instances, to dry the proteid matter, after it is secured in as pure a form as possible, at room temperature, over sulfuric acid. By this treatment the preparation may be obtained in a form suited to the study of its physical properties, since its solubility has not been affected by subjecting it to a high temperature. When it is desired to use the sample only for chemical analysis it is not necessary to wait on the slow process above mentioned. In this case the sample may be dried in an inert atmosphere at the temperature of a steam-bath or even at 110°. It is better, however, to avoid so high a temperature and to conduct the desiccation in vacuo at a heat not above that of boiling water. The sample, before drying, should be reduced to the finest possible state of comminution, otherwise particles of aqueous vapor may be retained with great tenacity.

In many cases it is advisable to dry the sample pretty thoroughly, then grind to a fine powder and finish the desiccation with the pulverulent mass. This treatment can be followed when the quantity of the material is considerably in excess of that required for the analytical operations.

405. Determination of Ash.—No method of treatment is known by means of which vegetable proteid matters may be obtained entirely free of mineral matters. The mineral bases may be naturally present in the proteid matter as organic and inorganic salts, or they may be mechanically entangled therewith, having been derived either from the other tissues of the plant or from the solvents employed. It is necessary in calculating the analytical data to base the computation on the ash free substance. The percentage of ash is determined by any of the standard processes or by heating the sample in a combustion tube, to very low redness, in a current of oxygen. The total residue obtained is used in calculating the percentage of ash, and the weights of material subsequently used for the determination of carbon, hydrogen, nitrogen and sulfur are corrected for the calculations by deducting the quantity of mineral matter contained therein.

By reason of the highly hygroscopic nature of the dry proteid bodies, they must be kept over a desiccating material and weighed quickly on a balance, in an atmosphere which is kept free of moisture by the usual methods.

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:

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:

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]

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.

If the proteid matters contain salts of ammonium it is probable that a difficultly soluble double sulfate of zinc and ammonium, (NH₄)₂SO₄.ZnSO₄.6H₂O, will be found in the precipitate. Ammonium salts, if present, should therefore be removed by distillation with magnesia. It is better, however, to throw down the ammonia with the first zinc precipitate, distil this with magnesia and determine the amount of nitrogen derived from the ammonia compounds. In a second sample, the total nitrogen is determined by moist combustion and the difference between the two results gives that due to albumoses.

419. Examination for Muscular Tissue.—Some samples of meat extracts contain small quantities of finely ground muscular tissue. For detecting this the extract is treated with cold water and the insoluble residue examined with a microscope. If muscular tissue be found, about eight grams of the extract or twenty-five of the fluid preparation, are treated with cold water, the insoluble matter collected upon a filter, washed with cold water, and the nitrogen determined in the residue. The percentage of nitrogen multiplied by 6.25 gives the quantity of muscle fiber proteids present. The filtrate from the above determination is acidified with acetic, boiled, any precipitate which is formed collected and the nitrogen therein determined. The nitrogen obtained multiplied by 6.25 gives the quantity of coagulable albumin present. An aliquot portion of the filtrate is used for the determination of nitrogen and the percentage therein found, deducted from the total nitrogen of the sample, gives a remainder which may be used as a representative of the whole of the nitrogen present in the form of albumin and muscular tissue.

420. Estimation of Gelatin.—The tin foil dish and its contents used for the determination of water, as above described, are cut into small pieces, placed in a beaker and extracted four times with absolute alcohol. After the removal of the alcohol, the residue is extracted with ice water containing ten per cent of alcohol, in which a small piece of ice is kept to avoid a rise of temperature. The beaker should be shaken during the extraction, which should last for about two minutes. Where large numbers of samples are treated at once, any convenient form of shaking machine may be employed. At least two extractions with ice water must be made. The residue is then collected upon a filter and washed with ice water until the washings are completely colorless. The residue on the filter is replaced in the beaker, boiled with water, well washed on the filter with boiling water, the filtrate and washings concentrated and the nitrogen therein determined.

The principle of this determination is based on the fact that gelatin is almost completely insoluble in ice water while serum peptones and albumin peptones are almost completely soluble in that reagent. On the other hand, the flesh bases and the proteids present are almost completely removed by the preliminary treatment with alcohol and ice water or are left undissolved by the hot water. The solution in boiling water, therefore, contains practically nothing but gelatin.[397]

In a later article, Stutzer modifies the method given above as follows:[398]

Of dry and moist extracts from five to seven grams and of liquid extracts from twenty to twenty-five grams are used for the determination and placed in tin foil dishes, as described above. In case of solid extracts, a sufficient quantity of warm water is added to completely dissolve them, the solution being facilitated by stirring. In case the solution is too thin it should be concentrated before going further. It is treated with a sufficient amount of dust-free ignited sand to completely absorb it, and the dish and its contents are then dried to a constant weight. The dried contents of the dish are rubbed up in a mortar, the dish cut into fine bits, and all placed in a beaker. The solid syrphete[399] is extracted four times with 100 cubic centimeters of absolute alcohol, the alcohol in each case being poured through an asbestos filter for the purpose of collecting any matters suspended therein. In a large flask are placed 100 grams of alcohol, 300 grams of ice and 600 grams of cold water, and the flask is placed in a large vessel and packed with finely divided ice. Four beakers marked b, c, d, e are also placed in ice and the beaker containing the syrphete, left after extraction with absolute alcohol as above mentioned, is marked a and also placed in pounded ice. The extraction with cold alcoholic water proceeds as follows:

In beaker a are poured 100 cubic centimeters of the mixture in the large flask, its contents are stirred for two minutes and then the liquid portion poured off into beaker b to which, at the same time, a piece of ice is added. In beaker a are poured again 100 cubic centimeters from the large flask, treated as above described, and the liquid extract poured into beaker c. In like manner the extraction in beaker a is continued until each of the beakers has received its portion of the extract. By this time the liquid over the sand in beaker a should be completely colorless. The filtration of the liquid extract is accomplished as follows:

In a funnel of about seven centimeters diameter is placed a perforated porcelain plate about four centimeters in diameter which is covered with asbestos felt with long fiber. Three filters are prepared in this way. On the first filter are poured the contents of beaker b. After the liquid has passed through, the sand and other residue in beaker a are transferred to the filter and the beaker and residue washed with the alcoholic ice water from the large flask. The filtration should be accomplished under pressure. On the second filter are poured the contents of beaker c. On the third filter the contents of beakers d and e. The washing with alcoholic ice water from the large flask is continued in each instance until the filtrate is colorless. At the same time the asbestos filter, which was used in the first instance for filtering the absolute alcohol extract, is washed with the alcoholic ice water mixture from the large flask. At the end the sand remaining in beaker a together with all the asbestos filters are brought together into a porcelain dish, boiled two or three times with water, the aqueous solution filtered and the filtrate concentrated and used for the estimation of the nitrogen. The quantity of nitrogen found multiplied by 6.25 represents the proteid matter in the gelatin of the sample.

The object of the multiple filters, described above, is to accelerate the process, and they are required because the gelatin quickly occludes the filter pores. For this reason the asbestos filters are found to operate better than those made of paper. It should be mentioned that the residue of the peptones insoluble in alcohol may contain, in addition to gelatin, also small quantities of albumoses. From the quantity of albumose nitrogen found, it is understood that the nitrogen in the form of coagulable albumin, determined as described in the first process mentioned above, is to be deducted, since these coagulable albumins are insoluble in alcohol.

421. Estimation of Nitrogen in the Flesh Bases Soluble in Alcohol.—About five grams of the dry extract, ten grams of the extract containing water or twenty-five grams of the liquid extract are placed in a beaker and enough water added in each case to make about twenty-five cubic centimeters in all. Usually no water need be added to the liquid extracts. Very thin peptone solutions should be evaporated until the content of water is reduced to seventy-five per cent. The solution, prepared as above indicated, is treated slowly with constant stirring with 250 cubic centimeters of absolute alcohol, the stirring continued for some minutes and the vessel set aside for twelve hours, at the end of which time the precipitate is separated by filtration and washed repeatedly with strong alcohol. Leucin, tyrosin and a part of the flesh bases are dissolved by alcohol. The alcohol is removed by distillation and the residue dissolved in water. Any flocky residue which remains on solution with water is removed by filtration, the nitrogen determined therein and the quantity thereof added to the albumose nitrogen found, as hereafter described.

The volume of the aqueous solution is completed with water to half a liter. One hundred cubic centimeters of this solution are used for the determination of total nitrogen, and another 100 cubic centimeters for the determination of ammoniacal nitrogen by distillation with barium carbonate. A part of the ammonia may have escaped during the preliminary distillation of the alcohol and therefore the amount found may not represent the whole amount originally present. The use of the above determination is principally to ascertain the correction to be made in the amount of total nitrogen found in the first 100 cubic centimeters of the solution.

422. Treatment of the Residue Insoluble in Alcohol.—The residue insoluble in alcohol is washed from the filter into the beaker in which the first solution was made. The aqueous mixture is warmed on a water-bath until the alcohol adhering to the precipitate is completely evaporated, when the contents of the beaker are poured upon a filter free of nitrogen. A small part of the albumose, by reason of the treatment with alcohol, tends to remain undissolved, and it is advisable to collect this albumose upon a filter, wash it well with hot water and estimate the nitrogen therein. The quantity of nitrogen thus found is to be added to the albumose nitrogen determined as described later on.

The total filtrate obtained from the last filtration is made up to a volume of half a liter, of which fifty cubic centimeters are used for the determination of total nitrogen, fifty cubic centimeters for the determination of gelatin, albumose and peptone, and 100 cubic centimeters for the residual peptones. The albumose, together with the gelatin and peptones carried down with it, is precipitated with zinc or ammonium sulfate solution, and its per cent calculated from the amount of nitrogen found in the precipitate. The true peptone is determined by subtracting the quantity of nitrogen determined as albumose from the total nitrogen in solution.

The rest of the liquid, viz., 300 cubic centimeters, is evaporated to a small volume and tested qualitively for true peptones as follows:

To separate the albumose and gelatin a concentrated liquor is treated with an excess of finely divided ammonium sulfate so that a part of the salt remains undissolved. The separated albumose, gelatin and undissolved ammonium salts are collected on a filter, the filtrate mixed with a few drops of dilute copper sulfate solution and a considerable quantity of concentrated soda or potash lye added. Care should be taken that the quantity of copper is not too great, otherwise the peculiar red coloration will be obscured by the blue color of the copper solution.

423. Pancreas Peptone.—The filtrate obtained as described above, by treating the portion of the material insoluble in alcohol with warm water, contains in addition to the albumose and gelatin the whole of the pancreas peptone which may be present. To separate this peptone, 100 cubic centimeters of the aqueous solution are evaporated in a porcelain dish until the volume does not exceed ten cubic centimeters. When cool, at least 100 cubic centimeters of a saturated cooled solution of ammonium sulfate solution are added, the mixture thoroughly stirred, the precipitate collected upon a filter and washed with a cold saturated solution of ammonium sulfate. The contents of the filter are dissolved in boiling water, the filter thoroughly washed and the filtrate and washings evaporated in a porcelain dish with the addition of barium carbonate until, on the addition of new quantities of barium carbonate, no further trace of ammonia can be discovered. The residue is extracted with water, the barium sulfate and carbonate present separated by filtration, well washed and the nitrogen determined in the evaporated filtrate and washings in the usual way and multiplied by 6.25 to determine the quantity of pancreas peptone.

424. Albumose Peptone.—A part of the albumose peptone which may be present is determined in conjunction with the other bodies mentioned above. The chief quantity is found in the solution of the residue insoluble in alcohol in the following manner:

Fifty cubic centimeters of the solution of this residue in hot water are mixed with an equal volume of dilute sulfuric acid, one volume of acid to three of water, in the cold, and a solution of sodium phosphotungstate added until it produces no further precipitate. The precipitate is washed with dilute sulfuric acid and the nitrogen determined therein. The nitrogen thus found is derived from the albumose, pancreas peptone and gelatin. The quantity of nitrogen in the pancreas peptone and gelatin, as above described, is subtracted from the total quantity found in the phosphotungstic acid precipitated, and the remainder represents the nitrogen due to the albumose.

425. Nitrogen in the Form of Flesh Bases Insoluble in Alcohol.—This is determined by subtracting the quantity of nitrogen, determined by the phosphotungstic acid method already described, from the total quantity of nitrogen found in the precipitate insoluble in alcohol and soluble in water.

AUTHORITIES CITED IN PART FIFTH.