Chapter XVII: Preface: To Volume Third (17)

For the detection of glutamin the liquid containing it is boiled with dilute hydrochloric acid, by which ammonia and glutamic acid are formed. On the addition of lead acetate to the solution the glutamic acid is thrown out as a lead salt, in which, after its decomposition with hydrogen sulfid, the characteristic properties of glutamic acid can be established.

The above process is chronophagous and also uncertain where the quantity of glutamin is very small and that of other soluble organic matters very large. A much better process, both for the detection of glutamin and asparagin, is the following, based on the property possessed by mercuric nitrate of precipitating amids.

The aqueous extract containing the amid bodies is mixed with lead acetate until all precipitable matters are thrown out and the mixture poured into a filter. To the filtrate is added a moderately acid solution of mercuric nitrate. The precipitate produced is collected on a filter, washed, suspended in water, decomposed with hydrogen sulfid and again filtered. The amid bodies (glutamin, asparagin, etc.) are found in the filtrate and can be detected and estimated by the processes already described. A reaction showing the presence of an amid body is not a positive proof of the presence of asparagin or glutamin, since among other amids, allantoin may be present. This substance is found in the sprouts of young plants and also in certain cereals, as shown by researches in this laboratory.[357] Allantoin, glutamin, and asparagin, when obtained in solution by the above process, may be secured, by careful evaporation and recrystallization, in well defined crystalline forms. Asparagin gives lustrous, rhombic prisms, easily soluble in hot water, but insoluble in alcohol and ether.

Allantoin is regarded as a diureid of glyoxalic acid and has the composition represented by the formula C₄H₆N₄O₃. It crystallizes in lustrous prisms having practically the same solubility as asparagin.

Glutamin is the amid of amidoglutaric acid. It crystallizes in fine needles. Its structural formula is represented as

CO.NH₂
/
C₃H₅(NH₂)
\
CO₂H.

=387. Cholin and Betain.=—Cholin is a nitrogenous base found in both animal and plant tissues. Its name is derived from the circumstance that it was first discovered in the bile. It is found in the brain, yolk of eggs, hops, beets, cottonseed and many other bodies. When united with glycerolphosphoric acid it forms lecithin, a compound of great physiological importance. From a chemical point of view, cholin is oxyethyltrimethyl-ammonium hydroxid,

OH
/
C₂H₄ ; (C₅H₁₅NO₂).
\
N(CH₃)₃.OH

It is crystallized with difficulty and is deliquescent. Its most important compound, from an analytical point of view, is its platinum salt C₅H₁₄ONCl₂PtCl₄. This salt crystallizes in red-yellow plates and is insoluble in alcohol.

Betain, C₅H₁₁NO₂, is the product of the oxidation of cholin.

In this laboratory the bases are separated from cottonseed and from each other by the process described below.[358]

About five pounds of fine-ground cottonseed cake are extracted with seventy per cent alcohol. The material should not be previously treated with dilute mineral acids because of the danger of converting a part of the cholin into betain. The alcohol is removed from the filtered extract and the residue dissolved in water. The aqueous solution is treated with lead acetate until no further precipitation takes place, thrown on a filter, the lead removed from the filtrate with hydrogen sulfid and the liquid evaporated to a viscous syrup. The sirup is extracted with alcohol containing one per cent of hydrochloric acid. The solution thus obtained is placed in a deep beaker and the bases precipitated by means of an alcoholic solution of mercuric chlorid. The complete separation of the salts requires at least two weeks.

The double salts of the bases and mercury thus obtained, after freeing from the mother liquor, are recrystallized from a solution in water and from the pure product thus obtained the mercury is removed after solution in water, by hydrogen sulfid. The filtrate, after separating the mercury, contains the bases as chlorids (hydrochlorates). The solution of the chlorids is evaporated slowly in (pene) vacuo to a thick sirup and set over sulfuric acid to facilitate crystallization. The hydrochlorates are obtained in this way colorless and in well-shaped crystalline forms.

In a quantitive determination, a small amount of the fine meal is extracted at once with one per cent hydrochloric acid in seventy per cent alcohol, the salts obtained purified as above and weighed.

The following process serves to determine the relative proportions of cholin and betain in a mixture of the two bases.

A definite weight of the chlorids, prepared as directed above, is extracted by absolute alcohol. This treatment dissolves all the cholin chlorid and a little of the betain salt. The alcoholic solution is evaporated and again extracted with absolute alcohol. This process is repeated three times and at the end the cholin chlorid is obtained free of betain. In a sample of cottonseed cake examined in this laboratory the two bases were found present in the following relative proportions, _viz._, cholin 17.5 per cent, betain 82.5 per cent. Thus purified the cholin is finally precipitated by platinum chlorid. For a description of the special reaction, by means of which cholin and betain are differentiated, the paper cited above may be consulted.

These bodies have acquired an economic interest on account of their occurrence in cottonseed meal, which is so extensively used as a cattle food. It is evident from the relative proportions in which they occur that the less nocuous base, betain, is the more abundant. It is possible, however, that the base originally formed is cholin and that betain is a secondary product.

Experience has shown that it is not safe to feed cottonseed meal to very young animals, while moderate rations thereof may be given to full-grown animals without much expectation of deleterious results. In the case of toxic effects it is fair to presume that a meal has been fed in which the cholin is relatively more abundant than the betain.

=389. Lecithin.=—Lecithin is a nitrogenous body, allied both to the fats and proteids and containing glycerol and phosphoric acid. Its percentage composition is represented with some accuracy by the formula C₄₂H₈₆NPO₉, or according to Hoppe-Seyler, C₄₄H₉₀NPO₉. It appears to be a compound of cholin with glycerolphosphoric acid. It is widely distributed both in animal and vegetable organisms, in the latter especially in pease and beans.

From a physiological point of view, lecithin is highly important as the medium for the passage of phosphorus from the organic to the inorganic state, and the reverse. This function of lecithin has been thoroughly investigated in this laboratory by Maxwell.[359]

In the extraction of lecithin from seeds (pease, beans, etc.) it is not possible to secure the whole of the substance by treatment with ether alone.[360]

The extraction of the lecithin may, however, be entirely accomplished by successive treatments for periods of about fifteen hours with pure ether and alcohol. This is better than to mix the solvents, since, in this case, the ether having the lower boiling point is chiefly active in the extraction. When the extraction is accomplished by digestion and not in a continuous extracting apparatus the two solvents may be mixed together and thus used with advantage. After the evaporation of the solvents, the lecithin is ignited with mixed sodium and potassium carbonate whereby the organic phosphorus is secured without loss in an inorganic form. Where greater care is desired, the method described for organic phosphorus in soils may be used.[361] The inorganic phosphorus thus obtained is estimated in the usual way as magnesium pyrophosphate.

For analytical purposes, the extraction of lecithin from vegetable substances is conducted in this laboratory as follows:[362] The fine-ground pea or bean meal is placed in an extraction apparatus and treated continuously with anhydrous ether for fifteen hours. The ether in the apparatus is replaced with absolute alcohol and the extraction continued for six hours longer. The alcoholic extract is evaporated to dryness and treated with ether. The part of the lecithin at first insoluble in ether becomes soluble therein after it has been removed from the vegetable tissues by alcohol. Moreover, any trace of inorganic phosphorus which may have been removed by the alcohol, is left undissolved on subsequent treatment with ether. The ether extract from the alcohol residue is added to that obtained directly, the ether removed by evaporation, and the total lecithin oxidized and the residue used for the estimation of phosphorus as already described.

In determining the lecithin in eggs, the procedure employed for vegetable tissues is slightly changed.[363] The whole egg, excluding the shell, is placed in a flask with a reflux condenser and boiled for six hours with absolute alcohol. The alcohol is then removed from the flask by evaporation and the residue treated in like manner with ether for ten hours. The ether is removed and the dry residue rubbed to a fine powder, placed in an extractor and treated with pure ether for ten hours. The extract thus secured is oxidized after the removal of the ether by fusion with mixed alkaline carbonates and the phosphorus determined in the usual way.

=390. Factor for Calculating Results.=—The percentage of lecithin is calculated from the weight of magnesium pyrophosphate obtained by multiplying it by the factor, 7.2703.[364] This factor is calculated from the second formula for lecithin given above, in which the percentage of phosphorus pentoxid, P₂O₅, is 8.789.

_Example._—In fifty-four grams of egg, exclusive of the shell, is found an amount of organic phosphorus yielding 0.0848 gram of magnesium pyrophosphate. Then 0.0848 × 7.2703 = 0.61652 and 0.61652 × 100 ÷ 54 = 1.14. Therefore the percentage of lecithin in the egg is 1.14.

=391. Estimation of Alkaloidal Nitrogen.=—The alkaloids contain nitrogen in a form more difficult of oxidation than that contained in proteid or albuminoid forms. It is doubtful whether any of the nitrogen in alkaloids becomes available for plant nutrition by any of the usual processes of fermentation and decay to which nitrogenous bodies are submitted in the soil. Likewise, it is true that it is not attacked by the digestive processes in any way preparatory to its assimilation as food by the animal tissues. Alkaloidal nitrogen is therefore not to be regarded as a food either for the animal or plant.

For the general methods of estimating alkaloids the reader is referred to standard works on plant chemistry and toxicology. The alkaloids of interest in this manual are those which are found in tobacco, tea, coffee and a few other products of agricultural importance. The best methods of isolating and estimating these bodies will be given in the part of the volume devoted to the special consideration of the articles mentioned.

SEPARATION OF PROTEID BODIES IN VEGETABLE PRODUCTS.

=392. Preliminary Treatment.=—The chief disturbing components of vegetable tissues, in respect of their influence on the separation and estimation of the proteid constituents, are fats and oils and coloring matters. In many cases these bodies are present in such small quantities as to be negligible, as, for instance, in rice. In other cases they exist in such large proportions as to present almost insuperable difficulties to analytical operations, as is the case with oily seeds. In all instances, however, it is best to remove these bodies, even when present in small proportions, provided it can be done without altering the character of the proteid bodies. This is secured by extracting the fine-ground vegetable material first with petroleum ether, and afterwards with strong alcohol and ether. Practically, all of the fatty bodies and the greater part of the most objectionable coloring matters are removed by this treatment. The extraction should in all cases be made at low temperatures, not exceeding 35°, to avoid the coagulating effect of higher temperatures upon the albuminous bodies which may be present.

In this laboratory, fatty seeds, as for instance peanuts, are first ground into coarse meal, then extracted with petroleum ether, ground to a fine meal and the fat extraction completed with petroleum ether, ninety-five per cent alcohol and pure sulfuric ether. The residue of the last solvent may be removed by aspirating air through the extracted meal. In some cases, it is advisable to extract with ethyl ether before as well as after the alcoholic extraction. This treatment removes at least a part of the water and prevents the dilution of the first part of alcohol added to such an extent as to make it dissolve some of the proteid matters. In each case, a portion of the alcoholic extract should be tested qualitively for proteid matter. If any be found, stronger alcohol should be used for, at least, the first extraction. A portion of the meal, prepared as above directed, is extracted with a ten per cent solution of sodium chlorid, as described further on, and a measured portion of the filtered extract diluted with water until the proteid matter in solution begins to be precipitated. By this treatment the proper strength of the salt solution, to be used for the subsequent extraction, is determined. To save time in dialyzing, the solution of salt employed as a solvent should be as dilute as possible.

The mixture of meal and solvent sometimes filters with difficulty. In these cases, it is advisable to first pour it into a linen bag from which the liquid portion can be removed by gentle pressure and subsequently filtered through paper. As a last resort, the liquid secured from the linen filter can be saturated with ammonium, zinc or magnesium sulfate, whereby all the proteid matters are thrown out. After filtering, the residue is again dissolved in salt solution and can then be readily filtered through paper.

The clear filtrate should be tested by fractional precipitation by heat and the final filtrate by acetic acid, as will be described further on.

The proteid matter may be further freed from amid compounds by treatment with copper sulfate.[365] This treatment is not advisable, however, except for the purpose of determining the total proteid nitrogen in the sample. The action of the water, heat and cupric sulfate combined is capable of inducing grave changes in the character of the residual matter which would seriously interfere with the results of subsequent studies of the nature of the proteid bodies.

In many instances, as with cereal grains, the separation of the proteid bodies is accomplished by no further preliminary treatment than is necessary to reduce them to the proper degree of fineness.

=393. Diversity of Character.=—The proteids which occur in vegetable products are found in all parts of the tissues of the plants, but in cereals especially in the seeds. In grass crops and in some of the legumes, such as clover, the nitrogenous matters are chiefly found in the straw and leaves. The general classification of these bodies has already been given, but each kind of plant presents marked variations, not only in the relative proportions of the different classes, but also in variations in the nature of each class. For this reason the study of vegetable proteids is, in some respects, a new research for each kind of plant examined. There are, however, some general principles which the analyst must follow in his work, and an attempt will be made here to establish these and to construct thereon a rational method of conducting the investigation. In the separation and estimation of complex bodies so nearly related to each other, it is difficult not only to secure satisfactory results, but also to prevent the transformation of some forms of proteid matter into others nearly related thereto by the action of the solvents used for separation and precipitation.

=394. Separation of Gluten from Wheat Flour.=—The most important proteid in wheat is the body known as gluten, a commercial name given to the nitrogenous matters insoluble in cold water. The gluten thus obtained does not represent a single chemical compound, but is a complex consisting of at least two proteid bodies, which together form an elastic, pasty mass, insoluble in cold water containing a trace of mineral salts. This mass has the property of holding mechanically entangled among its particles bubbles of gas, which, expanding under the action of heat during cooking, give to bread made of glutenous flours its porous property.

In respect of proteids, the American wheats, as a rule, are quite equal to those of foreign origin. This is an important characteristic when it is remembered that both the milling and food values of a wheat depend largely on the nitrogenous matter which is present. It must not be forgotten, however, that merely a high percentage of proteids is not always a sure indication of the milling value of a wheat. The percentage of gluten to the other proteid constituents of a wheat is not always constant, and it is the gluten content of a flour on which its bread making qualities chiefly depend. The percentage of moist gluten gives, in a rough way, the property of the glutenous matter of absorbing and holding water under conditions as nearly constant as can be obtained. In general, it may be said that the ratio between the moist gluten and the dry gluten in a given sample is an index for comparison with other substances in the same sample. Upon the whole, however, the percentage of dry gluten must be regarded as the safer index of quality. In respect of the content of glutenous matter, our domestic wheats are distinctly superior to those of foreign origin. They are even better than the Canadian wheats in this respect. It may be fairly inferred, therefore, that while our domestic wheats give a flour slightly inferior in nutritive properties to that derived from foreign samples, it is nevertheless better adapted for baking purposes, and this quality more than compensates for its slight deficiency in respect of nutrition, a deficiency, which, however, is so minute as to be hardly worth considering.[366]

The gluten is separated in this laboratory from the other constituents of a flour by the following process:

Ten grams of the fine-ground flour are placed in a porcelain dish, well wet with nearly an equal weight of water at a temperature of not to exceed 15°, and the mass worked into a ball with a spatula, taking care that none of it adheres to the walls of the dish. The ball of dough is allowed to stand for an hour, at the end of which time it is held in the hand and kneaded in a stream of cold water until the starch and soluble matter are removed. The ball of gluten thus obtained is placed in cold water and allowed to remain for an hour when it is removed, pressed as dry as possible between the hands, rolled into a ball, placed in a flat bottom dish and weighed. The weight obtained is entered as moist gluten. The dish containing the ball of gluten is dried for twenty hours in a steam-bath, again weighed, and the weight of material obtained entered as dry gluten. The determination of dry and moist gluten cannot in any sense be regarded as an isolation and estimation of a definite chemical compound. For millers’ and bakers’ purposes, however, the numbers thus obtained have a high practical value. A typical wheat grown in this country will contain about 26.50 per cent of moist and 10.25 per cent of dry gluten.

The gluten of wheat is composed of two proteid bodies, gliadin and glutenin.[367] Gliadin contains 17.66 per cent, and glutenin 17.49 per cent of nitrogen. Gliadin forms a sticky mass when mixed with water and is prevented from passing into solution by the small content of mineral salts present in the flour. It serves to bind together the other ingredients of the flour, thus rendering the dough tough and coherent. Glutenin serves to fix the gliadin and thus to make it firm and solid. Glutenin alone cannot yield gluten in the absence of gliadin, nor gliadin without the help of glutenin. Soluble metallic salts are also necessary to the formation of gluten, and act as suggested above, by preventing the solution of the gliadin in water, during the process of washing out the starch. No fermentation takes place in the formation of gluten from the ingredients named.

The gluten, which is obtained in an impure state by the process above described, is, therefore, not to be regarded as existing as such in the wheat kernel or flour made therefrom, but to arise by a union of its elements by the action of water.

=395. Extraction with Water.=—It is quite impossible to get an extract from fine-ground vegetable matter in pure water because the soluble salts of the sample pass at once into solution and then a pure water solvent becomes an extremely dilute saline solution. The aqueous extract may, however, be subjected to dialysis, whereby the saline matter is removed and the proteid matter, not precipitated during the dialytic process, may be regarded as that part of it in the original sample soluble in pure water. Nevertheless, in many instances, it is important to obtain an extract with cold water. In oatmeal the aqueous extract is obtained by Osborne as follows:[368] Five pounds of fine-ground meal are shaken occasionally with six liters of cold water for twenty-four hours, the liquid removed by filtration and pressure and the extraction continued with another equal portion of water in the manner noted. The two liquid extracts are united and saturated with commercial ammonium sulfate which precipitates all the dissolved proteid matter. The filtrate obtained is collected on a filter, washed with a saturated solution of ammonium sulfate and removed as completely as possible from the filter paper by means of a spatula. Any residual precipitate remaining on the paper is washed into the vessel containing the removed precipitate and the undissolved precipitate well beaten up in the liquid, which is placed in a dialyzer with a little thymol, to prevent fermentation, and subjected to dialysis for about two weeks. At the end of that time, the contents of the dialyzer are practically free of sulfates. The contents of the dialyzers are then thrown on a filter and in the filtrate are found those proteids first extracted with water, precipitated with ammonium sulfate and redissolved from this precipitated state by pure water. In the case of oatmeal, this proteid matter is not coagulated by heat, and may be obtained in the dry state by the evaporation of the filtrate last mentioned at a low temperature in vacuo. It is evident that the character of the proteid matter thus obtained will vary with the nature of the substance examined. In the case of oats, it appears to be a proteose and not an albumin.

=396. Action of Water on Composition of Proteids.=—When a body, such as oatmeal, containing many proteids of nearly related character, is exposed to the action of a large excess of water, the proteid bodies may undergo important changes whereby their relations to solvents are changed. After oatmeal has been extracted with water, as described above, the proteid matter originally soluble in dilute alcohol undergoes an alteration and assumes different properties. The same remark is applicable to the proteid body soluble in dilute potash. Nearly all the proteid matter of oatmeal is soluble in dilute potash, if this solvent be applied directly, but if the sample be previously treated with water or a ten per cent salt solution the subsequent proportion of proteid matter soluble in dilute potash is greatly diminished.[369] Water applied directly to the oatmeal apparently dissolves an acid albumin, a globulin or globulins, and a proteose. The bodies, however, soluble in water, exist only in small quantities in oatmeal. Experience has shown that in most instances, it is safer to begin the extraction of a cereal for proteid matter with a dilute salt solution rather than with water, and to determine the matters soluble in water alone by subsequent dialysis.

=397. Extraction with Dilute Salt Solution.=—In general, it is advisable to begin the work of separating vegetable proteids by extracting the sample with a dilute brine usually of ten per cent strength. As conducted by Osborne and Voorhees, on wheat flour, the manipulation is carried on as follows:[370]

The fine-ground whole wheat flour, about four kilograms, is shaken with twice that weight of a ten per cent sodium chlorid solution, strained through a sieve, to break up lumps, and allowed to settle for sixteen hours. At the end of this time, about half of the supernatant liquid is removed by a siphon or by decantation and filtered. Two liters more of the salt solution are added, the mixture well stirred and the whole brought onto the filter used above. The filtrate is collected in successive convenient portions and each portion, as soon as it is obtained, is saturated with ammonium sulfate. All the proteid matter is precipitated by this reagent. The precipitate is collected on a filter, redissolved in a convenient quantity of the salt solution and dialyzed for fourteen days or until all sulfates and chlorids are removed. The proteid matter, which is separated on dialysis, in this instance, is a globulin.

The proteid matter not precipitated on dialysis is assumed to be that part of the original substance soluble in water.

A part of the water soluble proteid matter obtained as above is coagulated by heat at from 50° to 80°. The part not separated by heat gives a precipitate on saturation with sodium chlorid.

In wheat there are found soluble in water two albumins and a proteose.[371]

In separating the albumin coagulating at a low boiling point from the dialyzed solution mentioned above, it is heated to 60° for an hour, the precipitate collected on a gooch, washed with hot water (60°), and then successively with ninety-five per cent alcohol, water-free alcohol and ether. On drying the residual voluminous matter on the filter over sulfuric acid, it becomes dense and horny, having in an ash free state, according to Osborne, the following composition:

Per cent.
Carbon 53.06
Hydrogen 6.82
Nitrogen 17.01
Sulfur 1.30
Oxygen 21.81

=398. Treatment without Precipitation with Ammonium Sulfate.=—Where abundant means are at hand for dialyzing large volumes of solution, the preliminary treatment of the solution made with ten per cent sodium chlorid with ammonium sulfate may be omitted.

When the precipitated proteids are to be used for the estimation of the nitrogen therein contained, it has been proposed to substitute the corresponding zinc salt for the ammonium sulfate.[372] This reagent has given satisfactory results in this laboratory and while a larger experience is desirable before commending it as an acceptable substitute in all cases, yet its obvious advantage, in being free of nitrogen for the use mentioned, entitles it to careful consideration.

The manipulation, with the exception of the precipitation with ammonium sulfate, is the same as that described in the preceding paragraph. The globulins are completely precipitated when the dialysis is complete and may be separated from the soluble albumins and proteoses by filtration.

=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:

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=).