Quantitative Semiroutine Method For Determining Algal And Sedimentary Chlorophyll Derivatives

An integrated, quantitative method is described for the determination of chlorophylls and chlorophyll degradation products in freshwater phytoplankton and sediments. Prior to extraction, algal samples were concentrated by filtration through glass fiber filters while sediment samples were lyophilized. All materials were extracted in a mixture of acetone, methanol, and water (80:15:5) by ultrasonication in a glass extraction chamber refrigerated at −10 C. Extracts were filtered through solvent-resistent membrane filters, rather than centrifuged, and then fractionated without prior drying by reverse-phase thin-layer chromatography on oil-impregnated layers of kieselguhr G. Using three chromatographic systems consisting of different developing solvents and oil phases (triolein, parrafin oil, and castor oil), rapid, artefact-free separations of chlorophylls a, b, and c and 18 of their derivatives were obtained. Individual pigments were estimated by direct, in situ thin-layer scanning with a filter fluorometer. The sensitivity and reproducibility of the procedure are ca. 2 ng and 11%, respectively. In routine operation, the method is fast and relatively simple, a complete analysis being accomplished in 1.5 hr.