40 THE CHEMICAL CONSTITUTION OF THE PROTEINS 



the solution. The ether is then poured off and a fresh quantity added. 

 More baryta is added and the stirring continued. The ether is re- 

 plenished and more baryta added several times. During the process 

 the mixture becomes cloudy and pasty, but finally a light dry mass of 

 baryta is left. 



The ethereal solutions are treated as described in the (a) method. 



For the second esterification process the residue is stirred up several 

 times with water, filtered through asbestos, and washed with water till 

 no more organic matter is extracted. Most of the baryta remains un- 

 dissolved. 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 [1910] found that the esters may be liberated very 

 conveniently by the action of ammonia. His preliminary experiment 

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

 esters was obtained from gelatin. Abderhalden has also found 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 [1911] suggested a still simpler procedure 

 for obtaining the esters. The concentrated solution of the ester hydrochlorides is mixed 

 with lead oxide (200 grams to 100 grams protein) and the mass is directly distilled in 

 vacuo. Abderhalden and Weil [1912, 3] do not recommend this method on account of the 

 great loss which occurs during extraction. 



(/) Foreman (see p. 36) dissolves the syrup of ester hydrochlorides in 1000-1500 c.c. of 

 dry chloroform and filters from ammonium chloride. 300-350 grams of anhydrous baryta are 

 added in small quantities at a time and the mixture well shaken after each addition. The 

 liquid is tested with silver nitrate and nitric acid and if a precipitate forms it is allowed to 

 stand until this no longer occurs. The excess of baryta is filtered off and washed with 

 dry chloroform, the chloroform is evaporated off in vacuo and the residue dissolved in ether ; 

 only a gum-like substance remains undissolved. The ether is removed by distillation in 

 vacuo and the residue of ester distilled in vacuo. The baryta was found to contain some 

 amino acids ; it was decomposed with sulphuric acid and the solution treated with lead 

 hydroxide in the same way as the original solution. 



(5) 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. 



