SPECTROFLUOROMETRIC ASSAY OF CHLOROPHYLLS AND PHEOPIGMENTS USING THE LEAST-SQUARES APPROXIMATION TECHNIQUE

A spectrofluorometric method is described that provides a relatively r apid analysis of chlorophylls and phaeopigments (chl a, chl b, chl c, phe a, phe b, phe c) including 8-desethyl-8-vinyl derivatives of chl a and chl b (pchl a, pchl b) and their phaeopigments (pphe a, pphe b). Fluorescence of pigments in 90% acetone is measured at 24 fixed excita tion and emission wavelengths by means of a programming system. Standa rdization is performed from pure solutions of the n different compound s (n = 10). Pigment concentrations in crude acetone extracts of marine phytoplankton or algal cultures are determined by resolving a system of 24 linear equations and n+1 unknowns using the least squares approx imation technique in a matrix form. The method appears very well adapt ed to routine analysis of chlorophylls in oligotrophic waters where fr ee-living prochlorophytes are generally abundant; its use, however, is not limited to such areas. The discrimination between chl a and 8-des ethyl-8-vinyl chl a is clearly achieved. The discrimination between ch l b and 8-desethyl-8-vinyl chl b is not so efficient, although pchl b appears better assessed in oligotrophic waters than chl b. Some shortc omings could be related to the presence of identified or unidentified representative pigments and insufficient information on the fluorescen ce characteristics of the extracts. However, the pigment analysis coul d be improved potentially by including new representative but spectral ly different pigment standards and/or fluorescence measurements at add itional fixed wavelengths.