QUANTITATIVE ESTIMATION OF CAROTINOIDS 253 



methyl alcohol and twice with 50 cc. of 92 per cent methyl alcohol. 

 The last extract is generally colorless; if not, another extraction is 

 made with 92 per cent alcohol. 



The methyl alcohol extracts contain the xanthophylls. They are 

 also free from carotin, according to Willstatter and Stoll. The com- 

 bined methyl alcohol extracts are now mixed with 130 cc. of ether and 

 the pigments transferred to the ether by a slow addition of water. 

 The ether solution of the xanthophylls thus obtained and the petro- 

 leum ether solution of carotin are freed from methyl alcohol by wash- 

 ing twice with water. The solutions are then filtered through dry 

 filters into 100 cc. graduated flasks, the solutions cleared up by the 

 addition of a few drops of absolute alcohol and the flasks filled to 

 the mark with ether and petroleum ether respectively. 



Colorimetric comparison with standard solutions. The carotin and 

 xanthophyll fractions, representing 20 grams of fresh leaves, are now 

 ready for comparison with standard solutions in a colorimeter. For 

 this purpose one can use either pure carotin or xanthophyll solutions 

 in petroleum ether and ether, respectively, or their color equivalents, 

 namely, 0.25 per cent alazirin in chloroform, or a 0.2 per cent aqueous 

 solution of K,Cr 2 7 . The pure pigment standards are not satisfactory 

 because of their instability, the xanthophyll standard, especially, fad- 

 ing quite rapidly. The dichromate solution is especially well suited 

 for a substitute because a standard solution once made will keep in- 

 definitely. It is necessary, however, to know its color value in terms 

 of the pure carotinoid pigment solutions. Using 5 x 10~ 5 molar so- 

 lutions of carotin and xanthophyll, respectively, equivalent to 0.0268 

 per cent carotin solution and 0.0284 per cent xanthophyll solution, Will- 

 statter and Stoll found the following relations to exist between the 

 standard 0.2 per cent K 2 Cr 2 7 solution and the carotinoids. 



100 mm. carotin solution equals 101 mm. K^CrzOi solution 

 5Q u n 4^ it (i 



25 " " " " 19 " " " 



100 " xanthophyll " " 72 " 



rn Q7 " " " 



o~ " "11 " " " 



In Willstatter and Stoll experiments the standard carotinoid solu- 

 tions only were apparently used. The standard solutions were always 

 set at a depth of 100 mm. and the height of the unknown adjusted 

 until the colors matched. Readings were then taken with the cups 

 reversed in the colorimeter and the results averaged. A Wolff colori- 

 meter was used by these investigators but a Dubosque or Kober 



