ISOLATION OF VALINE, LEUCINE AND ISOLEUCINE 47 



iii. Leucine and Isoleucine. 



The purity of the lead salt, as obtained above, is tested by analysis. 

 This is performed by dissolving about 0*3 gram in 5 c.c. of normal 

 nitric acid in a 100 c.c. beaker and precipitating the lead with 5 c.c. of 

 normal sulphuric acid followed by 50 c.c. of absolute alcohol. The 

 lead sulphate, which settles rapidly in a granular form, is collected 

 after about fifteen minutes in a Gooch crucible and washed with 95 

 per cent, alcohol acidified with sulphuric acid. The crucible is placed 

 in another crucible, heated gently until the alcohol is driven off, and 

 then with the full heat of the Bunsen burner for ten minutes. 



If the lead content be too high, due to contamination with the lead 

 salt of valine, the mixture is purified by dissolving it, after thorough 

 pulverisation, in 5 parts of hot water + | part of glacial acetic acid 

 and reprecipitating by adding 0*5 c.c. of concentrated ammonia solu- 

 tion for every gram of salt. The precipitate is collected and treated 

 as above under the conditions where the amount of valine present is 

 small. 



The last portions of the mixed leucines are recovered by repeating 

 the entire process. 



The mixed lead salts are dissolved in 15-20 parts of hot water + 

 J part of glacial acetic acid, and hydrogen sulphide is passed into the 

 solution. The filtrate from the lead sulphide is evaporated in vacuo to 

 dryness, and the residue is washed with a mixture of equal parts of 

 alcohol and ether to remove acetic acid. 



Since these isomers have been shown not to be racemised by heating 

 with acids, the composition of the mixture can be ascertained by de- 

 termining the rotation in 20 per cent, hydrochloric acid : 



d-isoleucine has MD = + 37'4 



Meucine . . + I5 -6 difference 



hence percentage of d-isoleucine = 100 x 



a ~ 



21*8 



,, /-leucine = 100 x ^7 4 ~ c t 



21-8 



From the weight of the mixed amino acids and these data the 

 amount of each isomer can be calculated. 



The mixed isomers are converted into their copper salts by boiling 

 with excess of copper oxide, which is thoroughly boiled out with water 

 to remove the last traces of the copper salt of leucine which is pale 

 blue in colour and very insoluble. 



The solution of the copper salts is evaporated to dryness in vacuo, 



