258 CAROTINOIDS AND RELATED PIGMENTS 



carotin is not possible in fruits in which these two carotinoids are 

 present together. 



For animal tissues and fluids containing only a single carotinoid an 

 extraction with ether or petroleum ether either directly, if sufficiently 

 dry, or after treatment with alcohol, if much water is present or if the 

 pigment is bound to protein, should yield a solution which may be used 

 at once for quantitative estimation, colorimetrically. A preliminary 

 concentration of the extract, previous to comparing with the standard, 

 may be advisable. For blood work the writer concentrates the extracts 

 to the original volumes of blood taken (usually 10 cc.) so that the 

 colorimeter readings can be calculated directly to the percentage of 

 carotinoid in the blood. In the case of animal fats, like butter fat or 

 adipose tissue fat, the approximate concentration of carotinoid present 

 (assuming that only one is present I can be determined at once by com- 

 paring the rendered, melted fat with the standard in the colorimeter. 

 If the fat is highly colored the necessity of keeping the fat melted can 

 be avoided by diluting with an equal volume of ether, inasmuch as no 

 great difficulties in the calculation of the results are thereby introduced. 



For animal tissues and fluids containing both types of carotinoids 

 in sufficient quantities so that the assumption of only a single type in- 

 volves too great an error, it is not likely that a saponification of the 

 extracts can be avoided because of the presence of more or less fat in 

 nearly all animal tissues containing the chromolipoids. In carrying 

 out this saponification and subsequent recovery of the pigments in the 

 unsaponifiable matter, care should be taken to avoid the production of 

 aldehyde resins pigments which might be caused by the use of impure 

 alcohol. One to two cc. of 10 to 20 per cent alcoholic potash for each 

 gram of material extracted for the pigment analysis would insure a 

 large excess of alkali for the saponification and would keep the volume 

 of fluids within the realm of easily conducted analyses. Following the 

 saponification and extraction of the fat-free pigments from the soap, 

 the combined pigments must be washed free from alkali and then con- 

 centrated to the lowest possible volume, preferably in vacuum, then 

 diluted with petroleum ether of low boiling point and the pigments sub- 

 mitted to fractionation by the phase test between the petroleum ether 

 and 80-90 per cent alcohol, preferably methyl alcohol. The separated 

 carotin and xanthophylls can then be compared with the standard in the 

 colorimeter, after diluting or concentrating to a suitable volume. In 

 the case of xanthophyll pigments, it is well to first transfer to ether, as 

 in the Willstatter technic for plant tissues. 



