A reversed-phase thin-layer chromatographic (TLC) procedure using layers of Kieselguhr G impregnated with triolein, castor oil or paraffin oil is described. Eighteen identified and eight unknown derivatives of chlorophylls a, b and c can be resolved from the parent compounds. These include allomers, isomers, pheophytins, pheophorbides and chlorophyllides. In addition, at least six derivatives of bacteriochlorophyll a and a variety of algal and bacterial carotenoids can also be fractionated.
Recoveries are not quantitative, ranging from 65 to 95 %, but are highly reproducible (standard deviation 3 % or less). Hydrated extracts can be chromatographed directly, thereby eliminating the need for time-consuming and potentially destructive drying procedures. When the pigments are applied to the layers under a nitrogen atmosphere, no degradation is observed. Pigment zones are sharp and uniform and double tailing effects do not occur. The procedure is rapid, simple and relatively insensitive to many variables that have caused difficulties in adsorption TLC, such as relative humidity and the complexity of the pigment mixture.