30 CAROTINOIDS AND RELATED PIGMENTS 



served for a product of rather indefinite composition which results 

 from the action of acid on chlorophyll b (Tswett, 1907, 1908b) . 



In a later study of methods of isolation of phylloxanthine, however, 

 Fremy (1865) undoubtedly obtained much more valid proof of the 

 existence of yellow pigments associated with chlorophyll although he 

 regarded the pigment which he isolated as the same yellow phylloxan- 

 thine isolated by the ether-HCl method. He found that a careful 

 addition of Mg(OH) 2 , or A1(OH) 3 to alcoholic chlorophyll solutions 

 carried down the green pigment only, leaving the yellow pigment in 

 solution. Ca(OH) 2 and Ba(OH) 2 gave similar results, but the best 

 procedure with the last named reagent was to add an excess, which 

 threw down all the pigments, from which the phylloxanthine (caroti- 

 noids) could be extracted with alcohol. Especially interesting was 

 Fremy's observation that when his chlorophyll was saponified with 

 strong bases, alcohol took up the yellow "phylloxanthin" from the 

 residue, and yellow plate-formed or reddish colored prismatic crystals, 

 soluble in alcohol and ether, could be obtained from this solution. 

 The red crystals were described as being very much like crystals of 

 'potassium dichromate, and having a strong coloring power. It would 

 appear as though Fremy succeeded in obtaining for the first time 

 crystals of carotin, and possibly xanthophyll also, from green plants. 



Fremy's observations precipitated a lively interest in the subject of 

 yellow pigments in the chloroplastids which resulted in a number of 

 investigations during the succeeding years, some quite independent of 

 the others. These investigations seem to fall quite naturally into 

 several groups. The first of these was a series of studies confirming 

 the presence of yellow substances accompanying chlorophyll through 

 the development of suitable methods for their separation. 



Separation of Yellow Pigments from Chlorophyll 



Stokes (1864a) is to be credited with first discovering a method for 

 separating the actual yellow pigments accompanying chlorophyll and 

 for recognizing the existence of distinct green and yellow constituents 

 in the plastids. This investigator states, "I find the chlorophyll of 

 land-plants to be a mixture of four substances, two green and two 

 yellow, all possessing distinctive properties"; and later referring to 

 phylloxanthine he states, "When prepared by removing the green 

 bodies by A1(OH) 3 and a little water, it (phylloxanthin) is mainly 

 one of the yellow bodies, but when prepared by HC1 and ether, it is a 



