METHODS OF ISOLATION OF CAROTINOIDS 203 



is stated that when the petroleum ether had evaporated the carotin 

 was found as glistening crystals embedded in a dark, waxy mass. 



The removal of the waxy impurities and the final purification are 

 carried out as follows. The mixture of wax and pigment is carefully 

 shaken with three liters of the lowest boiling petroleum ether (pre- 

 sumably that boiling under 50 C.) which removes the bulk of the 

 wax. The solution is filtered, leaving the carotin on the filter but 

 still somewhat contaminated with phytosterol and other colorless sub- 

 stances. According to Willstatter and Mieg the carotin lost in the 

 filtrate can be recovered through precipitation by alcohol, but the 

 details of this recovery are not given. The final purification is car- 

 ried out as in the case of the isolation of carotin from carrots, namely, 

 by dissolving in a small amount of carbon disulfidc, in which a part 

 of the colorless substances do not readily dissolve, and then by adding 

 absolute alcohol cautiously to secure the fractional precipitation of 

 the other impurities from the carotin. The colorless substances come 

 down first and can be quickly filtered off. Then the carotin precipi- 

 tates as sparkling crystals. At this point the yield was a little over 3 

 grams of crystals in Willstatter and Mieg's work. This is mentioned 

 because it gives a good idea of the scale- on which it is apparently 

 necessary to operate in order to secure even small quantities of rela- 

 tively pure pigment. With the facilities available in most laboratories 

 10 kgs. of dried, highly pigmented leaves would be somewhat burden- 

 some to carry through rapidly enough to avoid loss of pigment by 

 oxidation. The yield of relatively pure pigment by this method would 

 not be over 0.5 grams at the most, using highly pigmented spinach 

 leaves as the source of material. 



The final purification of carotin is carried out in the usual manner, 

 namely, by repeated precipitations from carbon disulfide by absolute 

 alcohol and a final crystallization from the lowest boiling petroleum 

 ether. The final yield of perfectly pure pigment would naturally be 

 somewhat less than the figures mentioned above. 



The most tedious features of this process are the removal of the 

 small amount of extracted chlorophyll from the dried leaves, and the 

 final purification of the crude carotin. It is not likely that the frac- 

 tional precipitations and recrystallizations can well be avoided. It 

 seems feasible, however, to substitute a more direct method for remov- 

 ing the chlorophyll. Tswctt (19001)) has shown that if a petroleum 

 ether solution of chlorophyll and carotin is shaken with an excess of 

 dry, finely divided CaCO,, inulin or sucrose, the chlorophyll is com- 



