= 9 = 



and roots , in yellow autumn leaves after the natural fading of the 

 chlorophylls^^, and in yellow etiolated leaves grown without chloro- 

 phyll in the dark. But in these yellow organs without chlorophyll there 

 is no photosynthesis in the presence of sunlight. 



In spite of the effectiveness of the separatory methods developed 

 for the examination of the chloroplast pigments, no single separatory 

 procedure has been found to serve for the separation of all these pig- 

 ments obtained from a single plant. It has been necessary to combine 

 several methods or modifications of i;he methods to effect complete 

 separations o 



ANALYTICAL METHODS FOR ISOLATION OF CHLOROPLAST PIGMENTS 



Separation of Pigments by Partition between Immiscible Solvents 



An effective method for the partial separation of the chloroplast 

 pigments depends upon their distribution or partition between two immis= 

 cible solvents'' . As early as 18614., Stokes shook an alcoholic extract 

 of fresh leaves Tirith carbon disulphide, which was immiscible with the 

 aqueous alcohol, and observed that some of the green pigment was removed 

 preferentially in the carbon disulfide layer. This pigment, chlorophyll 

 a, was spectrally different from the green pigment that remained in the 

 alcohol, chlorophyll b. Upon continued extraction of the aqueous alco- 

 hol with fresh batches of carbon disulfide, all the green was removed, 

 and a yellow alcoholic solution remained. This yellow solution contained 

 the alcohol-soluble xanthophylls, or oxy carotenoids, which could be 

 fractionated further by continued batchwise extraction with carbon di- 

 sulfide. 



