io THE CHEMICAL CONSTITUTION OF THE PROTEINS 



chloride being filtered off as it separates out ; the residue is extracted 

 with alcohol and the above process of esterification is repeated. 



In order to avoid this loss and to obtain the amino acids as com- 

 pletely as possible, another method was introduced to liberate the 

 esters from their hydrochlorides, i.e., by treatment with sodium ethylate. 

 The hydrochloric acid is therefore removed as completely as possible 

 by evaporation in vacuo 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, so as 

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

 is filtered off, and 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 acidifying 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 diamino acids 

 and other substances. 



The fractional distillation of the brown oil, which is obtained by 

 either of these methods, is carried out firstly at a pressure of io to 12 

 mm. produced by a water pump, and then at a pressure of 0*5 mm. 

 produced by a Geryck vacuum pump, as described by Fischer and 

 Harries. 1 



The temperatures at which the various fractions are collected are 

 those of the vapours of the esters at io mm. pressure, and those of the 

 water bath at 100 C. and of an oil bath, which replaces the water bath 

 for the higher temperatures up to 160 C., at 0*5 mm. pressure. 



The lower boiling fractions are again distilled in vacuo to obtain 

 a further fractionation, but each fraction, even then, does not generally 

 contain a single ester of an amino acid ; in the case of the higher 

 boiling fractions a second fractionation is not essential, since the esters 

 contained in them can be separated by their varying solubility in water, 

 ether and petroleum ether. The following table shows the fractions 

 which are collected, and the amino acid esters which they may contain : 



1 In this process liquid air is used for condensing the vapours in order to preserve the 

 high vacuum ; carbonic acid has been used by other investigators, and Levene and van 

 Slyke have recently employed sulphuric acid, cooled by a freezing mixture, as an absorbent 

 for this purpose. 



