48 THE CHEMICAL CONSTITUTION OF THE PROTEINS 



The dry and finely pulverised copper salts are then shaken in a shaking 

 machine with 94 per cent, methyl alcohol. The insoluble copper salt 

 of leucine is filtered off and washed with solvent. The soluble copper 

 salt of isoleucine may be contaminated with some of the copper salt 

 of leucine. It is therefore decomposed with hydrogen sulphide, re- 

 converted into copper salt and again extracted with methyl alcohol. 

 Both the leucine and isoleucine are obtained in the usual way from 

 the copper salt and recrystallised from water. 



They are identified by elementary analysis, rotation and estimation 

 of copper in their copper salts. 



iv. Separation of Valine and Alanine. 



Levene and Van Slyke [1913] described a method for separat- 

 ing these two amino acids. It depends (i) upon the greater insolu- 

 bility of alanine phosphotungstate than valine phosphotungstate in a 

 solution containing 10 per cent, of sulphuric acid and 20 per cent, of 

 phosphotungstic acid ; (2) the greater insolubility of valine in 80 per 

 cent, acetone. Alanine combines with 14 parts of phosphotungstic 

 acid. The solubility of alanine phosphotungstate in the above solution 

 is 0*1 5 gram per 100 c.c. ; that of valine phosphotungstate is i '2 gram. 

 A mixture of the two compounds can be separated by crystallisation 

 from this solution. Glycine behaves like alanine; its phosphotung- 

 state has the solubility of O'2 gram per 100 c.c. If leucine be present 

 in the mixture it must be first separated as lead salt (above). 



In carrying out the separation the mixture should preferably con- 

 tain not more than 50 per cent, of valine, and in precipitating the 

 alanine the volume of liquid should be as small as possible. There 

 are two alternatives : (a) if the alanine phosphotungstate is not re- 

 crystallised, the volume should be 100 c.c. for every gram of valine 

 present; (&) if the alanine phosphotungstate be recrystallised, the 

 volume need only be 30-40 c.c. per gram of valine; one recrys- 

 tallisation from the same volume yields pure alanine phosphotungstate. 



The most satisfactory separation is conducted by dissolving the 

 mixture of valine and alanine in a hot sulphuric acid solution contain- 

 ing 10 grams per 100 c.c. in the proportion of 30-40 c.c. per gram 

 of valine present. This amount is given by an elementary analysis 

 of the mixture. Phosphotungstic acid l in the ratio of 14 : i according 



1 The phosphotungstic acid is purified by Winterstein's method by dissolving in a 

 small volume of water and extracting with ether. An oily solution heavier than water 



