IDENTIFICATION AND QUANTIFICATION OF CHLO ROPHYLL AND CAROTENOID-PIGMENTS IN MARINE-SEDIMENTS - A PROTOCOL FOR HPLC ANALYSIS

A HPLC protocol is described which permits analysis of the pigment com position of coastal and intertidal sediments. The HPLC technique has b een extensively applied to phytoplankton pigments for fifteen years. P apers dealing with living benthic microphytic populations have been mo re rare, due to complexity existing in the sediment surface, where pig ments (chlorophylls and carotenoids) from several taxonomic groups of microalgae are mixed with degraded pigments from senescent phytoplankt on cells and macrophytic material. The present paper describes in deta il a protocol adapted from Kraay et al. (1992), which allows a good di scrimination of photosynthetic pigments of microphytobenthos communiti es. The following procedure is recommended. Sediment samples are colle cted using hand-held cores, during emersion of sites in intertidal zon es or by scuba-diving in submerged zones. Cores are sectioned in slice s of 5 mm, weighed and lyophilized. Subsamples of 0.3-3 g are extracte d in 95 % methanol buffered with ammonium acetate, during 10-20 min in the dark, at 5 degrees C; the extract is then filtered with the Sween ex system with GF/F Whatman filters. Fluorescence excitation is perfor med at 430 nm and emission is detected beyond 580 nm. Absorbance is de tected simultaneously at 436 and 450 nm. A binary solvent system is em ployed to separate the pigments. Standards for chlorophyll a, chloroph yll b and beta-carotene were obtained from Sigma; chlorophyllide a was prepared from thallus of the Phaeophyta Halopteris; pheophytins a and b and a major pheophorbide a were prepared by acidification (HCl 0.4 N, final molarity 3 10(-3) M); chlorophylls c(1) + c(2) and several ca rotenoids were also obtained from Halopteris. The remaining carotenoid s were identified by comparison with published chromatograms. Calibrat ion of standards was done spectrophotometrically in acetone or other s olvents, subject to the existence of published extinction coefficients . Pigments were dried and redissolved in methanol in order to calculat e the HPLC factor. A table of the coefficients used is given. Five chr omatograms of three different intertidal stations from the Tagus estua ry are presented as examples; 29 pigments were identified. Chromatogra ms obtained in fluorescence and absorbance at 436 nm and 450 nm from t he same sample are shown to permit comparison between performances of the different types of detection. All chlorophylls, active or degraded , are distinguished in fluorescence. Some of them appear clearly in ab sorbance at 436 nm, while 450 nm is the best wavelength for identifyin g carotenoids. Pigment composition of sediment samples is related to m icroscopic species identification and cell abundance. Chromatograms ob tained with absorbance at 450 nm from a mud sample in a salt-marsh sho wed high levels of violaxanthin, lutein (due to the abundance of plant detritus), zeaxanthin (corresponding to a population of 20 % cyanobac teria), and neoxanthin, diadinoxanthin and chlorophyll b (matching the abundance of euglenophytes: 10 %). A sample from a sandy station pres ented high levels of chlorophylls c(1) and c(2), neofucoxanthin and di atoxanthin; cell countings confirmed a major abundance of diatoms (99 %) with a dominance of Cylindrotheca closterium. A mud sample from a l ow-tide station exhibited a great variety of pheophorbides, which is r elated to the high grazing pressure at this site. Although no method e xists that is perfect for all types of pigments, the one described sho wed a good capability for analysing the taxonomic composition of micro phytobenthos communities.