314 Report of the Department of Chemistry ok the 

 PART I. CASEIN AND SOME OF ITS COMPOUNDS. 



METHOD OF PREPARING ASH-FREE CASEIN. 



Casein that is to be used in studying its relation to mineral bases 

 must be free from all such bases. The preparation of really ash-free 

 casein is much more difficult than has been commonly assumed. 

 The so-called chemically-pure casein furnished by chemical-supply 

 houses usually contains 0.6 per ct. of ash. The preparations used 

 in various investigations in which the ash content has been reported 

 rarely contain less than 0.2 per ct. of ash and not infrequently as 

 much as . 6 per ct. 



The principal basic element in casein preparations, as usually 

 made, is calcium. The calcium in casein preparations is usually due 

 to the presence of a compound of calcium and casein, containing 

 0.22 per ct. Ca (equal to 0.31 per ct. CaO), as we shall show later. 

 This salt is insoluble in water but easily soluble in a 5 per ct. solution 

 of sodium chloride, while base-free casein is insoluble in both water 

 and the brine solution. 



When casein is carefully precipitated by dilute acids from milk 

 or from lime-water solutions of casein, the precipitate is apt to 

 contain more or less of the above-mentioned calcium caseinate 

 as well as base-free casein. The precipitation of this calcium salt 

 occurs most readily when the usual precautions in precipitating 

 casein from milk are most rigidly observed, that is, when excess of 

 acid is avoided. We have examined casein preparations obtained 

 from chemical-supply houses and have found that some of them are 

 soluble in a 5 per ct. solution of sodium chloride to the extent of 

 50 per ct., or more, of their weight. 



After trying different methods of preparing casein so as to contain 

 a minimum amount of calcium, we have obtained the most satis- 

 factory results by the method described below. We have been able 

 to prepare casein containing only 0.06 per ct. of ash, consisting 

 largely of calcium phosphate, derived from the trace of calcium not 

 removed and the phosphorus of the casein molecule. The amount 

 of calcium present in 5 grams of such material was too small to 

 determine quantitatively. 



Our method of preparation is to dilute separator skim-milk with 

 seven or eight times its volume of distilled water and carefully add 

 dilute acetic acid (6 cc. of glacial acetic acid diluted to 1 liter) until 

 the casein separates completely, after which the clear solution is 

 removed by siphon as soon as the precipitate settles. Distilled 

 water is then added, the mixture stirred vigorously and the precipi- 

 tate allowed to settle, after which the wash-water is siphoned off. 

 More water is then added and the casein is dissolved by adding, 

 for each liter of milk used, 1 liter of dilute ammonium hydroxide 

 (6 cc. of strong reagent diluted to 1 liter). When the solution is 

 complete, the whole is filtered through a thick layer of absorbent 



