ISOLATION OF VALINE, LEUCINE AND ISOLEUCINE 45 



tallisation of the amino acids obtained from the esters after removal 

 of the proline : 



1. Valine + leucines. 



2. Valine + alanine. 



3. Alanine. This is purified by recrystallisation from dilute alcohol. 



4. Alanine + glycine. 



If the esters be separated by distillation into three fractions, the 

 residues from fraction I. will contain chiefly glycine and alanine ; from 

 fraction II. valine, leucine and isoleucine; from fraction III. leucine 

 and isoleucine. 



i. Separation of Valine from Leucine and Isoleucine. 



These three compounds are the most difficult to separate from each 

 other. Their separation has really only been accomplished by chance. 



In those cases where the isolation of the individual substance has 

 succeeded it has been effected by the fractional crystallisation of the 

 amino acids themselves and of their copper salts and by the different 

 solubility of the compounds in methyl alcohol. Valine is soluble in 

 methyl alcohol, isoleucine is insoluble in the cold solvent, but soluble 

 in the hot. On cooling the solution, however, the presence of valine 

 prevents its separation. The copper salt of leucine is very insoluble, 

 but the mixed copper salts are relatively soluble. Leucine and iso- 

 leucine were first separated by F. Ehrlich [1904] by the different 

 solubility of their copper salts in methyl alcohol ; that of leucine is 

 insoluble. The separation is most tedious and not at all satisfactory. 



In order to give us more information about the constituents of this 

 fraction, Levene and Van Slyke [1909, I, 2] worked out a method 

 which depends upon the precipitation of leucine and isoleucine as their 

 lead salts from ammoniacal solution and the subsequent separation of 

 these two amino acids by means of the different solubility of their 

 copper salts in methyl alcohol. The details are as follows : 



The mixture is carefully analysed and the amount of leucine + 

 isoleucine is calculated from the carbon content : 



Leucine and isoleucine contain 54-92 per cent. C. j difference = 3 . 68 per cent> 

 Valine contains . . . 51-24 ,, ,, J 



Per cent, carbon - 51*24 = cent . of leucine isomers in the mixture . 

 3-68 



The mixture is pulverised and suspended in 7 parts of water and 

 the water is raised to the boiling-point : for each gram of substance 

 I -5 c.c. of concentrated ammonia solution is then added. The flask 



