RESULTS OF ANALYSIS 67 



3. Separation of Glutamic acid Hydrochloride. The quantity of 

 glutamic acid precipitated as hydrochloride never represents the total 

 quantity present in the protein. The amount precipitated depends 

 very largely on conditions. The precipitate usually contains am- 

 monium chloride ; this is removed by boiling with baryta, and if the 

 baryta be removed with carbon dioxide the barium carbonate may 

 contain barium glutamate, which is very insoluble, and consequently 

 there is loss of this constituent. The remainder of the glutamic acid 

 is recovered with the esters. 



4. Esterification. The loss in this process is not great, especially 

 if it be repeated. Loss is chiefly due to hydrolysis of the esters when 

 the water is removed by evaporation in vacuo. Phelps and Tillotson's 

 method seems to be preferable. 



5. Separation of Glycine Ester Hydrochloride. The glycine is never 

 completely isolated as ester hydrochloride ; the remainder is obtained 

 as ester. 



6. Extraction of Esters. Loss always occurs in this part of the pro- 

 cess, but is covered when the process is repeated. The loss is largely 

 mechanical and cannot be avoided. A small quantity of organic 

 matter is retained by the sodium sulphate used for drying the esters. 



7. Distillation of Esters. (a) In distilling off the ether, especially 

 if its volume be large, a considerable quantity of esters distils at the 

 same time. Two receivers should be used and the esters extracted 

 from the distillate. 



(#) Decomposition of the esters occurs during the distillation, and 

 a more or less large residue represents the loss. The products can 

 be recovered, if necessary. 



Abderhalden and Weil have found that in the process up to this 

 stage 30 per cent, of glutamic acid, 40-45 per cent, of aspartic acid, 

 37-5 per cent, of glycine, 30 per cent, of alanine, and 20 per cent, 

 of leucine are lost ; some of the glutamic acid is lost by conversion 

 into pyrrolidone carboxylic acid. In the case of a mixture, of the 

 glycine 50 per cent, was recovered, of the alanine 57 per cent, of the 

 leucine 66 per cent., of the glutamic acid 58 per cent, and of the 

 aspartic acid 40 per cent. The esters were liberated by treatment 

 with caustic soda and sodium carbonate, alcoholic soda or ammonia. 

 The best yields were obtained with alcoholic soda. The loss of higher 

 boiling esters is greatly diminished if the distillation of fraction IV. be 

 omitted. Osborne and Jones found that the separation of the esters 

 in this fraction is not more troublesome than when they are distilled. 



5* 



