ISOLATION OF VALINE AND ALANINE 49 



to the maximum amount of alanine in the mixture and I gram excess 

 for every 5 c.c. of solution is dissolved in the hot solution. A greater 

 excess does not interfere with the separation, but it requires removal 

 subsequently. The solution is kept at o, or in ice water in an ice 

 box for at least twenty-four hours. 1 The alanine phosphotungstate 

 separates in large transparent crystals forming a solid layer on the 

 bottom and sides of the vessel. The supernatant solution is decanted 

 and the crystals are redissolved in the same volume of 10 per cent, 

 sulphuric acid as that originally used, I gram of phosphotungstic acid 

 per 4 or 5 c.c. of liquid are added, and the solution kept at o for 

 twenty-four hours. The alanine phosphotungstate is filtered off by 

 suction and washed with 10 per cent, sulphuric acid containing 20 per 

 cent, of phosphotungstic acid. 



The alanine in the phosphotungstate is determined and recovered 

 by dissolving the salt in hot water in which it gives a somewhat 

 turbid solution and making up to a known volume containing 50-100 

 mgm. of alanine per 10 c.c. The nitrogen in this solution is esti- 

 mated by Van Slyke's method (p. 89) using 10 c.c., or 2 c.c. if 

 the micro-apparatus be used. It can also be estimated by Kjeldahl's 

 method, but this is not so convenient on account of the violent bump- 

 ing caused by the separation of tungstic acid. This estimation gives 

 a more accurate value for the alanine than its isolation, as loss occurs 

 during the removal of the phosphotungstic acid ; the actual amount 

 isolated is from 90-95 per cent, of that found by the nitrogen esti- 

 mation. A correction for the solubility of alanine phosphotungstate 

 '15 gram in 100 c.c. solution can be applied. 



The remainder of the solution is washed into a beaker, heated to 

 boiling, and treated with excess of pure 20 per cent, lead acetate solu- 

 tion ; 2 excess of lead acetate is ascertained by removing a drop and 

 testing with dilute sulphuric acid. Lead sulphate and phosphotung- 

 state are filtered off and thoroughly washed. The filtrate and wash- 

 ings are concentrated to about 50 c.c. per gram of alanine, an equal 

 volume of 95 per cent, alcohol added, and the mixture heated on the 

 water-bath for about one hour. This removes the remainder of the 



results. It is washed several times with water and the ether removed on the water-bath. 

 The product is not hygroscopic and forms a colourless solution. It should leave no residue 

 after precipitation with pure lead acetate and evaporation of the filtrate. 



1 Working at room temperature, about 75 per cent, of the alanine is precipitated if one 

 half of the volumes given are used. 



2 This should leave no residue after removal of the lead with hydrogen sulphide and 

 evaporating to dryness. 



PT. I. 4 



