ESTERIFICATION 39 



(&) In order to separate the amino acids as completely as possible, 

 Fischer introduced another method of liberating the esters from their 

 hydrochlorides, i.e., treatment with sodium ethylate. The hydrochloric 

 acid is removed as completely as possible by evaporation in vacua and 

 the mixture of ester hydrochlorides is dissolved in five times its 

 quantity of absolute alcohol. The amount of chlorine is estimated in 

 a small portion of this, and to the remainder the calculated quantity 

 of sodium dissolved in absolute alcohol and freshly prepared, so as to 

 make a 3 per cent, solution, is added. The sodium chloride formed is 

 filtered off. Its separation is greatly facilitated by the addition of 

 ether and cooling to o. The alcohol is removed by evaporation in 

 vacuo. A small quantity of the lower boiling esters of the amino acids 

 passes into the distillate with the alcohol, but is recovered by acidify- 

 ing with hydrochloric acid and evaporating when the amino acid 

 hydrochlorides are obtained. A dark-brown oil again results, which 

 is fractionally distilled in vacuo. 



Although this method prevents loss by the action of alkali, the 

 yield of the higher boiling fractions is not so great on account of the 

 more complex nature of the mixture of esters. The residue which 

 does not distil contains the tyrosine, the di-amino acids, and other 

 substances. 



(c] Instead of employing caustic soda and potassium carbonate for 

 the liberation and salting out of the esters, Levene[i9O5] proposed the 

 use of barium oxide, for which he claims the following advantages : 



(i) On account of its small solubility in water, a large excess of 

 alkali which causes saponification of the esters is avoided. 



(ii) In neutralising the free acid the rise in temperature is less than 

 with caustic soda. 



(iii) When the second esterification is required it is more easily 

 removed. 



The procedure described by Levene and Van Slyke [1909, 2] is 

 as follows : 



The concentrated solution of the ester hydrochlorides is poured 

 into a porcelain or enamelled vessel of capacity of I litre for every 125- 

 150 grams protein, and the flask is washed out with ice-cold baryta 

 solution. The vessel is placed in a freezing mixture. When the con- 

 tents are cold excess of crystallised baryta is added, and the mixture 

 is thoroughly stirred with a wooden or porcelain spatula. The peculiar 

 sticky mass in a few minutes becomes liquid and the solution becomes 

 alkaline in reaction. Several volumes of ether are then poured over 



