ISOLATION AND ESTIMATION OF DI-AMINO ACIDS 57 



sulphate, which is thoroughly extracted in the usual manner with 

 boiling water, is evaporated down to remove the hydrogen sulphide 

 and again made up to I litre ; a Kjeldahl nitrogen determination in 

 20 c.c. now gives the amount of nitrogen in the substances precipi- 

 tated by silver and baryta. 



IV. Estimation and Isolation of Histidine. 



(a) The solution is freed from sulphuric acid by neutralising 

 to litmus with baryta and adding barium nitrate as long as a preci- 

 pitate is formed ; the barium sulphate is filtered off and washed. 



The solution is concentrated to 300 c.c., acidified with nitric acid, 

 if necessary, and treated with silver nitrate, as before, till a test drop 

 gives a yellow-brown colour with baryta ; when this occurs it is exactly 

 neutralised to litmus with baryta and 5 c.c. of a cold saturated solu- 

 tion of baryta are added. If 10 c.c. of the filtered solution when tested 

 with a drop of baryta give a precipitate which indicates that the silver 

 salt of histidine is not completely thrown down, 2 c.c. of saturated 

 baryta solution are added to the main bulk, and this test is repeated 

 until a test portion remains clear. The precipitate of the silver salt of 

 histidine is then filtered off. 



Instead of adding excess of baryta, Kossel and Pringle direct that 

 a suspension of barium carbonate be added to the neutral solution, the 

 solution warmed on the water-bath and then raised to the boiling- 

 point. After cooling, the histidine silver salt is filtered off and washed 

 with baryta till free from nitric acid. The filtrate and washings are 

 treated as in V. for arginine. 



The precipitate of the silver salt of histidine is suspended and 

 heated in water to which sulphuric acid is added until the reaction is 

 acid and decomposed with hydrogen sulphide. Excess of hydrogen 

 sulphide is removed by boiling and the silver sulphide is filtered off 

 and washed. The solution and washings are concentrated and made 

 up to 250 c.c. A nitrogen estimation in 20-25 c.c. by Kjeldahl's 

 method gives the amount of histidine. 



The histidine is isolated from the remainder of the solution as 

 hydrochloride or as picrolonate : 



I. As Hydrochloride. The solution is made alkaline with baryta, 

 the barium sulphate formed is filtered off, excess of baryta is removed 

 by carbon dioxide, and the whole is evaporated to dryness. The resi- 

 due is extracted with boiling water and to the solution, filtered from 

 barium carbonate, hydrochloric acid is added. Histidine dichloride, 



