ESTERIFICATION 37 



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

 lime and sulphuric acid, and is purified by recrystallisation from ab- 

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

 the whole of the glycine may be isolated in this way [Fischer, 1902]. 

 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, 

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

 quantities of glycine ester hydrochloride separated. 



(4) Extraction of the Esters of the Amino Acids. 



The esters are liberated from their hydrochlorides in the solution 

 which has been concentrated in vacuo at 40 to a syrup by one of 

 the following methods : 



(a) About one-third to one-half the volume of water is added to 

 dissolve the syrup, and, if I kilo, of protein has been used, the solution 

 is divided into two or four portions for convenience and to ensure the 

 subsequent thorough cooling ; to each portion two or three volumes of 

 ether are added, and the mixture is thoroughly cooled in a freezing 

 mixture of ice and salt; strong caustic soda (33 per cent.) is now 

 added till the free hydrochloric acid is neutralised ; this can be tested 

 by adding a small quantity of a saturated solution of potassium 

 carbonate. The feebly basic esters of aspartic and glutamic acids, 

 which are very sensitive to free alkali, are thus liberated and are dis- 

 solved by the ether, which is quickly poured off and replenished by a 

 fresh quantity. Caustic soda and solid potassium carbonate added in 

 small portions at a time set free the other esters from their hydro- 

 chlorides ; these are dissolved by the ether, which is frequently renewed 

 throughout the process and thoroughly mixed with the mass of salt and 

 potassium carbonate ; sufficient caustic soda must be added to com- 

 bine with the whole of the hydrochloric acid, and as much potassium 

 carbonate as is necessary to form finally a pasty mass in order that 

 the esters, which are very easily soluble in water, are salted out and 

 dissolved by the ether. A large amount of ether is required for this 

 extraction, which is continued until the ether separates in a colour- 

 less state, and an essential condition is that, throughout the process 

 of extraction the various portions be kept thoroughly cold by shaking 

 in the freezing mixture. 



The several ethereal extracts are each dried by shaking for about 

 five minutes with potassium carbonate ; they are then combined to- 



