Data/hora: 01/06/2024 16:34:24

Provedor de dados: | 5
País: | France
Título: | Identification et quantification des pigments chlorophylliens et caroténoïdes des sédiments marins : un protocole d'analyse par HPLC
Autores: | Brotas, V; Plantecuny, Mr.
Data: | 1996
Ano: | 1996
Palavras-chave: | Plants pigments; Sediments; Protocol; HPLC.
Resumo: | A HPLC protocol is described which permits analysis of the pigment composition of coastal and intertidal sediments. The HPLC technique has been extensively applied to phytoplankton pigments for fifteen years. Papers dealing with living benthic microphytic populations have been more rare, due to complexity existing in the sediment surface, where pigments (chlorophylls and carotenoids) from several taxonomic groups of microalgae are mixed with degraded pigments from senescent phytoplankton cells and macrophytic material. The present paper describes in detail a protocol adapted from Kraay et al. (1992), which allows a good discrimination of photosynthetic pigments of microphytobenthos communities. The following procedure is recommended. Sediment samples are collected using hand-held cores, during emersion of sites in intertidal zones or by scuba-diving in submerged zones. Cores are sectioned in slices of 5 mm, weighed and lyophilized. Subsamples of 0.3-3 g are extracted 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 Sweenex system with GF/F Whatman filters. Fluorescence excitation is performed at 430 nm and emission is detected beyond 580 nm. Absorbance is detected simultaneously at 436 and 450 nm. A binary solvent system is employed to separate the pigments. Standards for chlorophyll a, chlorophyll 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 carotenoids were also obtained from Halopteris. The remaining carotenoids were identified by comparison with published chromatograms. Calibration of standards was done spectrophotometrically in acetone or other solvents, subject to the existence of published extinction coefficients. Pigments were dried and redissolved in methanol in order to calculate the HPLC factor. A table of the coefficients used is given. Five chromatograms of three different intertidal stations from the Tagus estuary are presented as examples; 29 pigments were identified. Chromatograms obtained in fluorescence and absorbance at 436 nm and 450 nm from the 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 absorbance at 436 nm, while 450 nm is the best wavelength for identifying carotenoids. Pigment composition of sediment samples is related to microscopic species identification and cell abundance. Chromatograms obtained with absorbance at 450 nm from a mud sample in a salt-marsh showed high levels of violaxanthin, lutein (due to the abundance of plant detritus), zeaxanthin (corresponding to a population of 20 % cyanobacteria), and neoxanthin, diadinoxanthin and chlorophyll b (matching the abundance of euglenophytes: 10 %). A sample from a sandy station presented high levels of chlorophylls c(1) and c(2), neofucoxanthin and diatoxanthin; cell countings confirmed a major abundance of diatoms (99 %) with a dominance of Cylindrotheca closterium. A mud sample from a low-tide station exhibited a great variety of pheophorbides, which is related to the high grazing pressure at this site. Although no method exists that is perfect for all types of pigments, the one described showed a good capability for analysing the taxonomic composition of microphytobenthos communities.
Tipo: | Text
Idioma: | Francês
Identificador: | http://archimer.ifremer.fr/doc/00096/20730/18367.pdf
Editor: | Gauthier-Villars
Relação: | http://archimer.ifremer.fr/doc/00096/20730/
Formato: | application/pdf
Fonte: | Oceanologica Acta (0399-1784) (Gauthier-Villars), 1996 , Vol. 19 , N. 6 , P. 623-634