- 29 - 



the orange zones could easily be mistaken for an orange xanthophyll, 

 siphonaxanthin, of the siphonalean green algae. Upon continued washing 

 with petroleum ether plus propanol, the orange zones gradually became 

 light yellow. 



Many variations of the solvent and of the wash liquid were tested,, 

 As a rule unsaturated hydrocarbon solvents, such as benzene, were better 

 solvents for the pigments than petroleum ether, but as wash liquids for 

 formation of the chromatogram, these solvents provided less effective 

 separations than petroleum ether plus n-propanol. In these hydrocarbon 

 solvents, some of the pigments, such as chlorophyll c^ appeared to be 

 altered, and isomers of the chlorophylls were not readily separated. 

 Substitution of other alcohols for the n-propanol in the wash petroleuM 

 ether yielded less effective separations. Methanol frequently sepa- 

 rated from the petroleijm ether as a distinct phase as the solution 

 filtered through the colxjmno Ethanol provided satisfactory separa- 

 tions only at the beginning of the washing. Iso-propanol did not effect 

 as complete separation of the chlorophyll a from the lutein plus 

 zeaxanthin as did n-propanol. A complex mixture of various monohy- 

 droxy and dihydroxy aliphatic alcohols plus several ketones and amines, 

 when substituted for the n-propanol, produced equivalent separation of 

 the pigment zones. Substitution of 14. per cent acetone for the ri-pro- 

 panol, produced a variation of the pigment sequence with the chloro- 

 phyll a zone above the lutein plus zeaxanthin. 



Various sorbents were also tested for the chromatographic sepa- 

 ration of the pigments. Starch, regenerated starch, cellulose and 

 Celite did not yield as distinct zones with as little alteration of 



the pigments as did powdered sugar. Separations in paper were also 



T 20 



less effective than those in columns of sugar^>*-"» 



