CAROTIN, THE PRINCIPAL YELLOW PIGMENT OF MILK FAT 34! 



emulsions, and more vigorously with subsequent washings. When 

 the wash water no longer reacted alkaline toward phenolphthalein, the 

 ether was either dried over fused CaCl 2 or, after standing several 

 hours, decanted from the precipitated moisture. The ether was then 

 evaporated, leaving a salve-like residue of various tints of yellow to 

 orange to red, depending upon the amount of fat used and the depth 

 of its original color. 



Although the methods of study eventually adopted did not make 

 the procedure necessary, it was found possible to completely free 

 the pigment from its chief impurity, i. e., cholesterol, by means of 

 the- digitonin method of Windaus x for the quantitative estimation 

 of cholesterol. A hot one per cent solution of digitonin in ninety 

 per cent alcohol, when added to an alcoholic solution of the un- 

 saponifiable residues from butter fat, completely precipitated the 

 cholesterol as a colorless compound, leaving the pigment in solution. 

 This solution was still contaminated, however, with traces of fat and 

 lecithin decomposition products. 



GENERAL PROPERTIES OF THE BUTTER FAT PIGMENT. 



The unsaponifiable residues from butter fat, either crude or freed' 

 from cholesterol by digitonin, were readily soluble in hot alcohol and 

 in ether, chloroform, petroleum ether, etc. with a golden yellow color; 

 and in carbon bisulphide with a color which varied, according to the 

 concentration, from a red orange to a blood red color. The freshly 

 prepared crude, and also, the cholesterol-free residues usually gave 

 an instantaneous but transient purple color on adding a drop of con- 

 centrated sulphuric acid, a light green color quickly changing to a 

 greenish blue color on adding a drop of concentrated nitric acid, and 

 a dark blue color with the combined acids. These color reactions 

 were usually shown a little clearer by the cholesterol free residues. 

 Small amounts of impurities often interfered with the color reactions ; 

 in fact they were often rendered negative by some slight decompo- 

 sition of the pigment which was not apparent in the intensity of the 

 color. This was -due to the fact that the crude samples of butter fat 

 pigment were very unstable, quickly bleaching in the air, especially 

 with the aid of heat in the presence of a little water. It was necessary 

 therefore to take great care to have the ether solutions of the pigment 

 as free as possible from water before evaporation, or to transfer the 

 pigment to some solvent such as petroleum ether, which does not 

 absorb water so readily. 



1. Zeit. f. Physiol. Chem. 65, p. 110 (1909). 



