385. Sachsse’s Method.—A method for the determination of amid bodies by liberation of free nitrogen has been described by Sachsse and Kormann.[352] 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.
Fig. 103.—Apparatus
for Amid Nitrogen.
Fig. 104.—Sachsse’s Eudiometer.
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.[353] 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.[354]
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.[355] Asparagin is easily obtained from the aqueous extracts of plants by crystallization.[356] 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.