Analysis of Chlorophyll a in Sediments by High-Pressure Liquid Chromatography

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Abstract
We have developed a method that uses high-pressure liquid chromatography (HPLC) with fluorometric detector for the determination of concentrations of chlorophylls a and b and their pheophytins in sediments. A reversed-phase column allowed direct injection of extracts prepared in the conventional manner (90% acetone or other solvent mixtures containing water). Concentrations of chlorophyll a in acetone estimated by a spectrophotometric method (using the acidification ratio) agreed with those estimated by HPLC. Chlorophyll a concentrations in sediments estimated by fluorometry were sometimes quite different from those estimated by HPLC or spectrophotometry. Estimates of concentrations of chlorophyll b and pheophytins a and b can be made by the HPLC method even in the presence of degradation products other than pheophytins that would interfere with the determination by spectrophotometry or fluorometry. The method is sensitive, rapid (14 min per sample), probably compatible with a variety of solvent mixtures, and can be automated for processing large numbers of samples.Key words: chlorophyll a, chlorophyll b, pheophytins, high-pressure liquid chromatography, sediments, algae, epipelic, biomass, primary productivity, suspended particulate matter