Part 81

=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.

=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:

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.

=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. 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. 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.

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. 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.

=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.

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.