CASEIN 39 



centage calculated on the supposition that casein contains two 

 atoms each of phosphorus and sulphur. Previous estimates, 

 derived from analyses of casein prepared by Hammarsten's 

 method, indicated a phosphorus content of about 0.85 per 

 cent. 



Casein "nach Hammarsten" is obtainable upon the market in 

 a high degree of purity. For the investigations described in this 

 work I have always employed the C. P. casein "nach Hammar- 

 sten" which is manufactured in Germany for Messrs. Eimer and 

 Amend of New York. This substance is free from appreciable 

 fat and would appear to be contaminated only by a small amount 

 of an acid, water-soluble substance. This I remove by further 

 purification in the following manner (30) (35) : 



Half a pound of casein is triturated with about 12 litres of dis- 

 tilled water, the water being added in six successive portions. 

 On each addition of water the casein is well stirred up in a 

 porcelain mortar and allowed to settle, the supernatant water 

 is then poured off and fresh water is added. It is next washed 

 in a similar manner in 5 kilos of absolute alcohol, and then in 

 5 kilos of ether " distilled over sodium." The mortar, containing 

 the casein drained as free from superfluous ether as possible,* is 

 then placed in an incubator over sulphuric acid at 40-50 C., 

 the flame is turned out under the incubator, and it is allowed to 

 cool for about 24 hours. The casein is now found, if these op- 

 erations have been conducted carefully, to be in the form of a 

 dry, pure white powder, still containing, however, a considerable 

 quantity of ether. The presence of a color in the powder indi- 

 cates contamination, either with moisture or with some other 

 impurity. 



The casein is now spread out, within the incubator, in a layer 

 not over 1 cm. deep, the flame under the incubator is lighted and 

 it is allowed to stand for 24 hours at 40-50 C. The casein is 

 then found to be free from appreciable water or ether. 



The casein which is thus prepared gives every indication of being 

 a pure product and a definite chemical individual. It is insoluble 

 in distilled water, save in traces which adhere to the casein par- 



* At this point it is necessary to avoid exposing the mortar to the moist air 

 of the room for a moment longer than is necessary, otherwise the evaporating 

 ether causes the condensation of sufficient moisture to spoil the product unless 

 it is again treated with alcohol and ether. 



