Part 78

The gyrodynats of some of the albumins and albuminates are given below:

Serum albumin [α]{D} = -57°.3 to -64°.6. Egg albumin [α]{D} = -35°.5 to -38°.1. Serum globulin [α]{D} = -47°.8. Milk albumin [α]{D} = -76°.0 to -91°.0.

Our knowledge of the gyrodynatic numbers of the proteids and allied bodies is too fragmentary to be of any great help in analytical work. In practice, the rotatory power of these bodies becomes a disturbing force in the determination of milk sugar. A further study of this property of certain proteids may lead to analytical processes for their detection and determination, but no reliable methods for this can now be recorded.

=381. Alkaloidal Nitrogen.=—Only a general statement can be made here in respect of the detection of alkaloidal nitrogen in vegetable or animal tissues. Alkaloids are not found in healthy animal tissues and the description of methods for isolating and detecting ptomaines is foreign to the purpose of this work. In vegetable tissues the presence of alkaloids may be established by the following methods of examination.

The fine-ground tissues are made to pass a sieve of half millimeter mesh and when suspended in water are acidified with sulfuric. The mixture is then thoroughly extracted by shaking in a separatory funnel with petroleum ether, benzene and chloroform, successively. Some resins, glucosids and a few alkaloidal bodies not important here are extracted by this treatment.

The residue is made distinctly alkaline with ammonia and treated as above with the same solvents. In the solution obtained as last mentioned nearly all the alkaloidal bodies found in plants are contained.

All the alkaloids in a plant may be obtained by digesting the finely divided material with dilute sulfuric acid. The acid solution thus obtained is made nearly neutral with ammonia or magnesia, concentrated to a sirup, and gums, mucilage, etc. thrown out by adding about three volumes of ninety-five per cent alcohol. The alkaloids are found in the filtrate. The alcohol is evaporated from the filtrate and the residue tested for alkaloids by group reagents. Potassium mercuric iodid and phosphotungstic and molybdic acids are types of these reagents.

The same group reagents may also be applied to the extracts obtained with petroleum ether, benzene and chloroform, in all cases, after the removal of the solvents by evaporation.

ESTIMATION OF NITROGENOUS BODIES IN AGRICULTURAL PRODUCTS.

=382. Total Nitrogen.=—Any one of the methods heretofore described for the estimation of total nitrogen in soils or fertilizers is applicable for the same purpose to agricultural products. One among these, however, is so superior in the matter of convenience and certainty, as to make it preferable to any other. The moist combustion of the sample with sulfuric acid with subsequent distillation of the ammonia produced is the process which is to be recommended.

The usual precautions for securing a representative sample should be observed, but no further directions are needed. In all cases hereafter, where the estimation of nitrogen is enjoined, it is understood that the moist combustion process is to be used unless otherwise stated.

=383. Estimation of Ammoniacal Nitrogen.=—If the distillation of ammonia be accomplished with the aid of magnesia alba or barium carbonate it may be safely conducted on the finely ground materials or, in case of animal bodies, in as fine a state of subdivision as may be conveniently secured. Since the salts of ammonia are easily soluble in water they may be all obtained in aqueous solution, and the distillation of this solution with magnesia gives correct results. Experience has shown that the stronger alkalies, such as sodium and potassium hydroxids, cannot be safely used in the distillation of ammonia from mixtures containing organic nitrogenous materials because of the tendency of these bodies to decomposition, in the circumstances, yielding a portion of their nitrogen as ammonia. Barium carbonate acts with less vigor on non-ammoniacal nitrogenous matters than magnesia, and in some cases, as pointed out further on, may be substituted therefor with advantage. There is no danger of failing to obtain a part of the ammonia on distillation with magnesia provided the latter does not contain more than a trace of carbonate.

When no easily decomposable organic nitrogenous matters are present, the distillation may be conducted with the stronger alkalies in the manner prescribed. All the necessary details of conducting the distillation are found in the preceding volumes of this work.

=384. Estimation of Amid Nitrogen.=—In bodies containing no ammonia, or from which the ammonia has been removed by the method described in the preceding paragraph, the nitrogen in the amid bodies is converted into ammonia by boiling for about an hour with five per cent sulfuric or hydrochloric acid. The ammonia thus produced is estimated in the usual manner after distillation over magnesia free of carbonate. The free acid is exactly neutralized with sodium or potassium carbonate before the addition of the magnesia. The results are given in terms of asparagin. The reaction which takes place in the decomposition of the amid body is indicated by the following equation:

Asparagin. Sulfuric Aspartic Ammonium acid. acid. sulfate. 2C₄H₈N₂O₃ + 2H₂O + H₂SO₄ = 2C₄H₇NO₄ + (H₄N)₂SO₄.

Half of the nitrogen contained in the amid body is thus obtained as ammonia.

It is advisable to calculate all the amid nitrogen in agricultural products as asparagin.

=385. Sachsse’s Method.=—A method for the determination of amid bodies by liberation of free nitrogen has been described by Sachsse and Kormann. It is based on the reaction which takes place when amid bodies are brought into contact with nitrites in presence of an acid. The mixture of the reagents by which the gas is set free is accomplished in the apparatus shown in Fig. 103. The vessel A has a capacity of about fifty cubic centimeters and carries a stopper with three perforations for the arrangement shown.

About six cubic centimeters of a concentrated aqueous solution of potassium nitrite are placed in A and the lower parts of the tubes a and b are filled with water to a little above e in order to exclude the air therefrom. Dilute sulfuric acid is placed in one of the funnels and an aqueous solution of the amid in the other. The air is displaced from the empty part of A by introducing the sulfuric acid, a little at a time, whereby nitrous acid and nitric oxid are evolved. This operation is continued until all the air has been driven out through c d, the open end of d being kept in the liquid in the dish shown in Fig. 104. The eudiometer in which the evolved nitrogen is measured is shown in Fig. 104, and should have a capacity of about fifty cubic centimeters, and be graduated to fifths. It is filled with the solution of ferrous sulfate contained in B by sucking at g, after which the clamp h is replaced, the cock f closed, and the free end of d placed in the lower end of the eudiometer. The solution of the amid is run slowly into the generator A, Fig. 103, together with small additional quantities of the sulfuric acid when the evolution of gas becomes slow. From time to time h is opened and fresh quantities of the ferrous solution allowed to flow into the eudiometer. Any trace of the amid remaining in the funnel is washed into A with pure water, with care to avoid the introduction of air. When the liquid in A assumes a permanent blue color the decomposition is complete. The residual gas is driven out of A by filling with water. The tubes d and h, after all the nitric oxid is absorbed, are removed from the eudiometer which is transferred to a cylinder containing water and immersed therein until the two liquid surfaces are at the same level and the volume of the nitrogen observed. After correction for temperature and pressure, the weight of the nitrogen is calculated. Twenty-eight parts by weight of nitrogen correspond to 150 of pure asparagin, 181 of tyrosin and 131 of leucin. This method of procedure is difficult of manipulation and is apt to give results that are too high. It cannot be preferred to the more simple and accurate processes already described.

=386. Preparation of Asparagin.=—In case the analyst desires to prepare a quantity of asparagin for comparative purposes it may be easily accomplished in the following way: A sufficient quantity of pease or beans is sprouted in a dark place and allowed to grow until the reserve food of the seed is exhausted. The young sprouts are gathered, shredded and subjected to strong pressure. The juice thus obtained is boiled to coagulate the albumin, and thrown on a filter. The filtrate is evaporated to a thin sirup and set aside to allow the asparagin to separate in a crystallized form. If the crystals at first formed are colored they may be dissolved, decolorized with bone-black, and recrystallized. Instead of the above method the young shoots may be shredded, extracted with hot water and the extract treated as above. A larger yield of the asparagin is obtained by the latter process than by the one mentioned above.

=387. Detection and Estimation of Asparagin and Glutamin.=—Of all the amid bodies asparagin is the most important from an agricultural standpoint, because of its wide distribution in vegetable products. Asparagin is easily obtained from the aqueous extracts of plants by crystallization. In addition to its crystalline characteristics asparagin may be identified by the following tests. Heated with alkalies, including barium hydroxid, asparagin yields ammonia. Boiled with dilute acids it forms ammonium salts. A warm aqueous solution dissolves freshly prepared copper hydroxid with the production of a deep blue color. Sometimes, on cooling, crystals of the copper compound formed are separated. Asparagin crystallizes with one molecule of water. Glutamin gives essentially the reactions characteristic of asparagin, but crystallizes without water in small white needles. Asparagin is easily detected with the aid of the microscope by placing sections of vegetable tissues containing it in alcohol. After some time microscopic crystals of asparagin are separated. The presence of large quantities of soluble carbohydrates seriously interferes with the separation of asparagin in crystalline form.

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. 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,