PROPERTIES AND METHODS OF IDENTIFICATION 241 



methods which have been proposed for the microchemical crystalliza- 

 tion of the earotinoids only one can be depended upon to assure this 

 result, namely the alkali method of Molisch. He has shown that it is 

 possible by this method to secure the microcrystallization of all types 

 of known carotinoids occurring; in plants, with the possible excep- 

 tion of fueoxanthin. While van Wisselingh found that the brown 

 al.ua 1 give excellent crystals it is not clear whether these are fueo- 

 xanthin or the carotin and xanthophyll which accompany it in these 

 plants. 



The method is carried out as follows. Several small pieces of plant 

 tissue or sections of the same (leaf, petal, slice of fruit or other bulky 

 tissue) are placed in 100 to 200 cc. of alcoholic potash containing 40 

 per cent alcohol by volume and 20" per cent KOH by weight. The 

 tissue and solvent are placed in darkness protected from the air and 

 allowed to stand until the substances associated with the carotinoids 

 have dissolved or become saponified and the carotinoids present have 

 crystallized out. The time of crystallization will vary with the ob- 

 ject from minutes to months but can be greatly speeded up for the 

 latter cases by warming the preparation for a few hours at 70-80 C., 

 on several successive days. Van Wisselingh has shown that crystals 

 can be obtained in a few days by this modification which may require 

 over a year at room temperature. A piece of the tissue being studied 

 is withdrawn from time to time for examination as to the progress of 

 the reaction. It is first washed thoroughly with water and finally 

 allowed to rest in distilled water for several hours before preparing 

 the section for microscopic examination. 



When carotin and xanthophylls are present the crystals which will 

 form divide themselves into two general classes according to their 

 color, one group, probably due to xanthophylls, being orange-yellow 

 to orange and the other, probably due to carotin, being orange-red 

 to red. It is not safe, however, to depend upon the color of the crys- 

 tals for determining their character, because rhodoxanthin, if pres- 

 ent, would undoubtedly crystallize in the red group. In fact, van 

 Wisselingh encountered red xanthophyll-like crystals in his investi- 

 gation. 



Lycopin docs not form microcrystals in the Molisch method. A 

 slight modification, however, permits their formation, namely, heat- 

 ing the tissue to 140 C. in glycerol alone or in glyccrol containing 

 10 per cent KOH. Lycopin forms reddish violet microscopic crystals 

 under these conditions. 



