Spectrofluorometric assay of chlorophylls and phaeopigments using the least squares approximation technique

A spectrofluorometric method is described that provides a relatively rapid 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 excitation and emission wavelenghts by means of a programming system. Standardization is performed from pure solutions of the n different compounds (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 approximation technique in a matrix form. The method appears very well adapted to routine analysis of chlorophylls in oligotrophic waters where free-living prochlorophytes are generally abundant; its use, however, is not limited to such areas. The discrimination between chl a and 8-desethyl-8-vinyl chl a is clearly achieved. The discrimination between chl 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 shortcomings could be related to the presence of identified or unidentified representative pigments and insufficient information on the fluorescence characteristics of the extracts. However, the pigment analysis could be improved potentially by including new representative but spectrally different pigment standards and/or fluorescence measurements at additional fixed wavelenghts.