i8 THE CHEMICAL CONSTITUTION OF THE PROTEINS 



(2) Esterification and Isolation of Glycine as Ester Hydro- 

 chloride. 



The filtrate from the glutamic acid hydrochloride, to which the 

 mother liquors from the recrystallisations are added, is again concen- 

 trated in vacuo at a low temperature to a thick syrup ; this is dissolved 

 in absolute alcohol (3 litres to I kilo protein), and the amino acids are 

 esterified by saturating the alcohol with dry gaseous hydrochloric acid 

 at the ordinary temperature and then warming on the water-bath for 

 half an hour. In the process of esterification a large amount of water 

 is formed, which prevents its completion ; the alcohol is therefore 

 evaporated off in vacuo at a temperature below 50 C., and the re- 

 sulting syrup again dissolved in absolute alcohol and saturated with 

 dry gaseous hydrochloric acid. In some cases it may be necessary to 

 repeat this operation once more. 



The esterification, according to Osborne and Jones, is more advan- 

 tageously effected by the method of Phelps and Tillotson. The con- 

 centrated solution of amino acid hydrochlorides is dissolved in alcoholic 

 hydrochloric acid and zinc chloride is added as a catalyst. The solution 

 is maintained at a temperature of 100 C. and the vapours of absolute 

 alcohol containing some hydrochloric acid are passed into the solution. 

 The water arising during the process is removed by the alcohol vapours 

 as fast as it is formed, and complete esterification results in a shorter 

 time. 



At this stage, glycine, if it occurs in the protein, e.g., in gelatin, in 

 any considerable amount, is separated as glycine ester hydrochloride 

 by concentrating in vacuo at 40 C. to two-thirds and seeding the solu- 

 tion with a crystal of this compound and allowing to stand for twenty- 

 four hours at o C. The precipitate is filtered off while the liquid is kept 

 cold and is washed with ice-cold alcohol ; the mother liquor, on further 

 concentration in vacuo and saturation again with hydrochloric acid, 

 may give another crop of glycine ester hydrochloride, which is treated in 

 same way. The glycine ester hydrochloride is dried in vacuo over lime 

 and sulphuric acid, and is purified by recrystallisation from absolute 

 alcohol, charcoal being used to decolorise the solution. Almost the 

 whole of the glycine may be isolated in this way. 



The filtrate containing the esters of the hydrochlorides of the other 

 amino acids and the filtrate from the recrystallised glycine ester hydro- 

 chloride are combined and concentrated to a syrup in vacuo at 40 C. ; 

 the process of esterification is best repeated again, and any further quan- 

 tities of glycine ester hydrochloride separated. 



