METHODS OF ISOLATION OF CAROTINOIDS 209 



the pigment arc concerned. The writer has long been impressed with 

 the possibility of throwing light upon the formation of milk fat 

 through a study of this interesting complex in the blood of cattle, 

 inasmuch as it is unquestionably the source from which the milk 

 fat derives its natural pigment. The failure of the fat solvents to 

 remove the pigment from this protein complex by direct extraction 

 indicates that the pigment becomes a part of the milk fat through 

 a process much more deep-seated than a mere solvent action. It 

 seems very probable, therefore, that this caroto-albumin plays some 

 important part in the process of fat synthesis in the mammary gland 

 of the cow. 



Isolation o/ Xanthophylls 



Green leaves. It was clearly shown in Chapter II that a group of 

 xanthophyll pigments accompany chlorophyll and carotin in green 

 leaves. It is not known, however, whether the crystalline xanthophyll 

 which can be isolated from the green leaves of plants is a mixture 

 of the xanthophyll isomers or consists of the major xanthophyll 

 constituent, the xanthophyll a of Tswett. The evidence on both sides 

 of the question was presented in Chapter II. 



Willstatter and Mieg (1907) were the first to isolate crystalline 

 xanthophyll in quantity. Their method was as follows. Air-dried, 

 powdered nettle leaves were extracted with cold 95 per cent alcohol, 

 which extracted the chlorophylls and xanthophylis, but very little 

 of the carotin. The extract was treated in the cold with KOH, con- 

 verting the chlorophylls into chlorophyllins, which precipitated in 

 part directly from the alcoholic solution and partly on addition of 

 much ether. The alkaline alcoholic-ether solution was shaken with 

 successive portions of fresh water until all the green color was re- 

 moved from the ether layer. The combined ether solutions obtained 

 in this way from 100 kgs. of dried nettle leaves were concentrated to 

 a volume of 6 liters and after more washing with alcoholic potash and 

 water and drying with anhydrous sodium sulfate, were mixed with 

 two volumes of petroleum ether. This precipitated the xanthophyll 

 and a considerable amount of colorless, high molecular weight alcohol, 

 the xanthophyll coming down as a reddish-yellow precipitate. 



To remove the impurities from the precipitated xanthophyll Will- 

 statter and Mieg boiled the precipitate with 1200 cc. of acetone, which 

 left a part of the colorless substance undissolved. The warm acetone 

 solution was treated with about two volumes of methyl alcohol. In 



