Part 79

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.

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.

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.

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. 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: 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. 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. 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. 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.