ISOLATION OF GLYCINE AND ALANINE 51 



v. Separation of Glycine and Alanine. 



There are two methods of separating these compounds : 



(a) By reconverting the mixture into esters, separating the glycine 

 as ester hydrochloride, and distilling the alanine ester, which 

 is then decomposed by boiling with water and the alanine 

 obtained by crystallisation. 



(&} By precipitating the glycine as picrate [Levene, 1906 ; Levene 

 and Van Slyke, 1912]. 



Glycine picrate, which was first described by Levene, was shown 

 by Levene and Van Slyke to have the composition of 2 molecules of 

 glycine and I molecule of picric acid. The pure compound on heat- 

 ing softens at 200 and melts at 202 ; it is soluble to the extent of 

 '35 gram (= '14 gram glycine) in 20 c.c. of water. In preparing it, 

 it is not advisable to use a large excess of picric acid, as this increases 

 the solubility of the picrate though the solubility is lessened in the 

 presence of alanine. 



The separation of glycine and alanine is effected by dissolving the 

 mixture in 3-4 parts of hot water and then adding an amount of 

 picric acid exceeding that required to combine with the glycine (1-5 

 grams per I gram glycine) but not exceeding the amount required to 

 combine with the whole of the amino nitrogen in the mixture if this 

 be calculated as glycine. The solution is cooled to o ; glycine picrate 

 crystallises out completely in about one hour. It is filtered off and 

 washed with a small quantity of water, followed by 95 per cent, 

 alcohol. Its purity is controlled by a melting-point determination and 

 amino nitrogen content. The filtrate is acidified with a known excess 

 of normal sulphuric acid and freed from picric acid by shaking out with 

 the ether. The sulphuric acid is removed by adding an equivalent of 

 standard baryta solution and the filtrate from the barium sulphate is 

 evaporated to dryness. The residue consists of alanine, generally of 

 over 90 per cent, purity, the impurity being glycine. Pure alanine 

 results on recrystallisation and a separation of the remainder can be 

 effected by repeating the process. 



Glycine is identified by the melting-point and analysis of its ester 

 hydrochloride and picrate ; its amount in the protein is given by the 

 yield. 



Alanine is identified by elementary analysis and its rotation in 

 hydrochloric acid solution. The amount in the protein is also given 

 by the yield. 



4* 



