METHODS OF ISOLATION OF CAROTINOIDS 211 



acetone being removed each time by adding 4 successive liters of 

 water with gentle rotation of the liquid and drawing off the lower layer 

 each time. 



The petroleum ether solution now remaining contains the xantho- 

 phylls, the chlorophylls and the carotin. The xanthophylls, with some 

 chlorophyll, are removed by shaking with three successive additions 

 of 2 liters of 80 per cent methyl alcohol. After each addition and 

 shaking the methyl alcohol layer is removed. If the last extract is 

 still considerably yellow additional extractions are made until the 

 alcohol layer is practically colorless. The xanthophyll in the com- 

 bined methyl alcohol extracts is next freed from chlorophyll by trans- 

 ferring to ether in the following manner: 4 to 5 liters of ether, 

 a quantity of water and 30-50 cc. of concentrated methyl alcoholic 

 potash are added, and the mixture shaken. The liquids are allowed 

 to separate, the lower layer is drawn off and discarded and the ether 

 washed with water until no more green color is extracted. The ether 

 is now dried with anhydrous Na 2 S0 4 , evaporated in vacuum to a 

 volume of 30 cc., 200 to 300 cc. of methyl alcohol added, and the 

 ether removed completely by further concentration in vacuum. 

 Xanthophyll precipitates out on cooling the hot, concentrated methyl 

 alcohol, the addition of a little water helping the precipitation. The 

 yield of crude xanthophyll by this method is stated by Jorgensen and 

 Stiles to be 0.8 grams from 2 kgs. of dried nettle leaves which is 

 over three times as much as Willstatter and Mieg secured by their 

 method. The method just described has the additional advantage that 

 the xanthophyll-free petroleum ether can be used for the isolation 

 of carotin. 5 



The foregoing methods are best suited for the isolation of crystal- 

 line xanthophyll in quantity. A solution of mixed xanthophylls for 

 macroscopic examination can be secured by the following simple pro- 

 cedure. About 25 grams of dried powdered leaves or fresh leaves 



'The method recommended is to wash the petroleum ether, now consisting of about 

 3.5 liters, four times with two liter portions of water to ivnmvo tin- last traces of 

 aeetone and methyl alcohol. As the last traces of these solvents are removed the 

 chlorophyll present in the petroleum ether precipitates as a fine suspension. A little 

 anhydrous Na 2 SO 4 is added to take up the water and then 150 Drains of CaCO 3 , and 

 the solution finally filtered through a layer of CaCO 3 on a Biiclnn T funnel. This treat- 

 ment takes out the chlorophyll suspension. The filtrate is evaporated in vacuum at 

 40 C. and the oily residue treated with 300 cc. of nu per cent alcohol. The carotin 

 hegins to crystallize out immediately and is complete on standing in the cold. Purifica- 

 tion is effected by shaking up the crystalline mass with 200-300 cc. of petroleum ether 

 and filtering quickly and repeating the washing with a mixture of two parts ol 

 petroleum ether and one part of absolute alcohol. The yield of <>.:_>:, -rams from two 

 kgs. of dried nettle leaves is much greater than Willstatter and Mieg secured. 



