20 THE CHEMICAL CONSTITUTION OF THE PROTEINS 



A considerable amount of the lower boiling esters nevertheless 

 distils with the ether, especially those of alanine and glycine. Leucine 

 ester has also been found in the distillate. They are recovered by 

 shaking out the ether with dilute hydrochloric acid and evaporating 

 the acid aqueous solution to dryness, when they remain as amino acid 

 hydrochlorides. The glycine is separated as ester hydrochloride and 

 the alanine, and leucine if present, are separated as esters by fractional 

 distillation in vacua (see p. 22). They may also be obtained by evaporat- 

 ing their solution with water, removing the hydrochloric acid with lead 

 oxide and silver carbonate, the excess of these by hydrogen sulphide 

 and fractional crystallisation. Leucine, if present, separates out first, 

 and subsequently the alanine. A mixture of small quantities of glycine 

 and alanine generally remains which is too small for further separation ; 

 it may be treated with picric acid (see p. 30). 



By this method of extracting the esters from their hydrochlorides, 

 neither that of tyrosine, which remains behind combined with alkali, 

 nor those of the diamino acids, which are soluble with difficulty in 

 ether, are obtained. This is advantageous for the subsequent process 

 of separation, but the method has the disadvantage that the whole 

 quantity of esters is not taken up by the ether. Extraction with 

 chloroform after ether increases the amount of ester ; it separates the 

 pasty mass of potassium carbonate into particles which can be washed 

 with the solvent ; this extract contains the ester of tyrosine. 



The main loss seems to be caused by the destruction of the esters 

 by the alkali. In order to avoid this loss, the mass of carbonate is 

 treated with excess of hydrochloric acid and evaporated down, the 

 potassium chloride being filtered off as it separates out ; the residue 

 is extracted with alcohol and the above process of esterification is re- 

 peated. It is simpler to suspend the mass of carbonate in absolute 

 alcohol and to saturate the solution with gaseous hydrochloric acid. 

 The salts remain insoluble and are filtered off and the alcoholic solution 

 is treated as has been described. 



(ft) 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 evapora- 

 tion 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 cal- 

 culated quantity of sodium dissolved in absolute alcohol and freshly pre- 



