252 CAROTINOIDS AND RELATED PIGMENTS 



portions, or until the filtrate is colorless. According to Willstlitter 

 and Stoll the grinding and preliminary extraction require 15 to 30 

 minutes. The preliminary extracts are discarded. 



Extraction of the pigments. The dry mixture of leafy material 

 and sand on the filter is carefully loosened with a spatula and macer- 

 ated for a few minutes with a small amount of pure acetone, and the 

 acetone quickly sucked away. This is repeated until the acetone 

 comes through colorless, at which time the powder on the filter will 

 also be colorless. The total volume of acetone extract will vary be- 

 tween 400 and 600 cc., depending on the kind of leaves used. 



Removal of the chlorophyll. The green acetone extract is divided 

 into parts of 100-200 cc., to each of which 200-250 cc. of ether are 

 added and the acetone washed out with distilled water. The ether 

 fractions are now combined, dried over anhydrous sodium sulfate and 

 filtered through a dry filter into a 200 cc. graduated flask which is 

 filled to the mark with dry ether. 



One hundred cc. of this ether are saponified with 2 cc. of concentrated 

 methyl alcohol solution of KOH by shaking carefully by hand and 

 then in a shaking machine for 30 minutes. After standing a little 

 while the ether is usually a pure yellow color, but if it still shows 

 a red fluorescence the shaking is continued, if necessary with the ad- 

 dition of more alkali. After complete saponification of the chloro- 

 phyll the ether solution is decanted from the alkali-chlorophyllines 

 into a small separatory funnel and the chlorophyll salts washed gently 

 with ether. In order, however, to completely free the precipitate of 

 occluded xanthophylls 30 cc. more ether are added to the alkaline 

 material, the mixture shaken, and, after adding water, allowed to 

 stand until the emulsion has broken. If necessary this is repeated 

 with fresh ether. 



The ethereal solutions thus obtained are washed with water to which 

 a little methyl alcohol solution of KOH is added in order to sepa- 

 rate traces of chlorophylline and small amounts of brown acid organic 

 substances. The ether is finally washed twice with pure water and 

 evaporated to a volume of a few cubic centimeters in a vacuum dis- 

 tillation flask at room temperature. 



Separation of the carotinoids. The concentrated ether solution of 

 carotinoids in the vacuum distillation flask is washed into a separatory 

 funnel with 80 cc. of petroleum ether, the flask being washed out 

 finally with a little ether. This solution is now mixed successively 

 with 100 cc. of 85 per cent methyl alcohol, 100 cc. of 90 per cent 



