22 THE CHEMICAL CONSTITUTION OF THE PROTEINS 



undissolved. The solution is freed from that which has dissolved by 

 the equivalent quantity of sulphuric acid, acidified with hydrochloric 

 acid and esterified in the usual manner. 



(d) Pribram finds that the esters may be liberated very con- 

 veniently by the action of ammonia. His preliminary experiment 

 with glycirie gave a yield of 69 per cent, and an equally good yield 

 of esters was obtained from gelatin. Abderhalden also finds this 

 method very serviceable. 



The concentrated solution of ester hydrochlorides is mixed with 

 ether and surrounded with a freezing mixture. Ammonia, dried by 

 passing through three towers of caustic soda and lime, is then passed 

 into the solution. The ammonium chloride which is formed is filtered 

 off and washed with absolute alcohol. The ether is distilled off and 

 the esters fractionally distilled in vacua. 



(e) Zelinsky, Annenkoff and Kulikoff have quite recently sug- 

 gested a still simpler procedure for obtaining the esters. The con- 

 centrated solution of the ester hydrochlorides is mixed with lead oxide 

 (200 grammes to 100 grammes protein) and the mass is directly dis- 

 tilled in vacua. 



(4) Fractional Distillation of the Esters in Vacuo 



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

 either of these methods, is carried out firstly at a pressure of 10-12 

 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. In order to preserve the high vacuum in this process liquid 

 air is used for condensing the alcohol vapour arising from the decom- 

 position of the esters ; carbonic acid has been used by other investigators, 

 and Levene and Van Slyke have employed sulphuric acid, cooled by 

 a freezing mixture, as an absorbent for this purpose. A small quantity 

 of ester also passes over with the alcohol ; this cannot be recovered if 

 Levene and Van Slyke's modification be used. The accompanying 

 figure (p. 23) shows the arrangement for the distillation at low pressure. 

 At 10-12 mm. pressure the receiver should be cooled in a freezing 

 mixture. 



The temperatures at which the various fractions are collected are 

 those of the vapours of the esters at 10 mm. pressure, and those of 

 the water-bath at iooC. and of an oil-bath, which replaces the water- 

 bath for the higher temperatures up to i6oC., at 0*5 mm. pressure. 



