CAROTIN, THE PRINCIPAL YELLOW PIGMENT OF MILK FAT 43! 



perfectly clear bright yellow filtrate. It was saturated with (NH 4 ),>. 

 SO 4 in substance, throwing down a small amount of deep yellow 

 precipitate. The precipitate was filtered off on a large (n-inch) 

 Biichner funnel, using suction, so that the layer of yellow protein 

 would be as free as possible from occluded (NH. 4 ) 2 SO 4 . The golden- 

 yellow precipitate was sucked as dry as possible on the funnel and the 

 sticky mass covering the paper in very thin layer dissolved in warm 

 water, in which it was readily soluble, and the clear yellow solution 

 set aside. 



Portion B was diluted with an equal volume of distilled water, 

 a little sodium chloride added, and the solution raised to a temperature 

 of 75 C. in a water bath. Acetic acid was now added carefully until 

 a heavy definite coagulation took place. The coagulated proteins car- 

 ried down some of the pigment but by far the greatest part was in the 

 clear yellow filtrate. This filtrate was saturated with (NH 4 ) 2 SO 4 

 in substance and the precipitated pigmented protein filtered off in the 

 same way as in the case of portion A. After being made comparatively 

 dry by suction, the deep yellow residue was readily soluble in a small 

 amount of cold distilled water. 



The two similar solutions from A and B were now combined and 

 filtered on a small Biichner funnel through several layers of fine paper 

 to free it from dirt and other foreign matter introduced by the (NH t ) 2 

 SO 4 . The golden-brownish-yellow filtrate of about 250 c.c. volume 

 had a faint cloudiness when viewed by transmitted light and contained 

 some (NH 4 ) 2 SO 4 . That the pigment of this solution was carotin was 

 shown by the fact that when an equal volume of alcohol was added to 

 15 c.c. of the solution and the mixture was shaken with petroleum 

 ether, the petroleum ether rose to the top as a golden-yellow solution, 

 leaving the lower cloudy alcoholic layer colorless. The pigment in 

 the petroleum ether layer gave a red-orange carbon bisulphide solution, 

 and in this solvent showed the usual carotin absorption bands. 



The aqueous solution was now dialysed in a parchment bag 

 against running water for eight days. At the end of this time the 

 solution was still giving a precipitate with barium chloride indicating 

 that the solution was not free from (NH 4 ) 2 SO 4 . No protein crystal- 

 lization had occurred, but decomposition had begun, for the solution 

 was cloudy, and showed a very fine coagulation. This coagulum was 

 filtered off. It had a dirty brown color and when almost dry was quite 

 sticky. It was not soluble in water, but both in the dry state and in 

 suspension in water it gave up a golden-yellow color to ether, leaving- 

 the precipitate dirty white in color. The extracted pigment showed 

 the three absorption bands of carotin in carbon bisulphide solution.. 



