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ANALYTICAL CHEMISTRY, VOL. 50, NO. 3, MARCH 1978
Selective Excitation Fluorometry for the Determination of Chlorophylls and Pheophytins Kevin G. Boto” and John S. Bunt Australian Institute of Marine Science, Cape Ferguson, P.M.B. No. 3, Townsville M.S.O., 0.48 70, Australia
A modified fluorometric method for the determination of chlorophylls a, b, and c and their corresponding pheophytins is presented. The use of selected monochromatic excitation and emission wavelengths enables each component to be determined with greater selectivity than with previous spectrophotometrlc or fluorometric methods. The factors limiting the accuracy of determination of each pigment in the usual multicomponent mixtures encountered are discussed.
Table I. Chlorophyll a, b, and c Spectral Properties in 90% Acetone Ratios Chloro~ m , , nm AII AII A111 phyll I 11 111 AI1 AI11 AI11 a 432 664 1.11 b 459 641 2.61 c 444 580 631 1.22 0.63 the use of relatively broad band filters for emission.
Estimates of chlorophyll concentrations in natural waters are frequently required in studies related to primary production. Probably the most widely used analytical technique depends upon spectrophotometric measurements of extinctions of 90% acetone extracts a t three different wavelengths. Solution of appropriate simultaneous equations (trichromatic) provides estimates of chlorophylls a, b, and c ( I ) . Pheophytin a can also be estimated by this method after acidification of t h e extract solution. Various improvements t o the trichromatic equations have been published by a number of authors; for example, new data for t h e extinction coefficients of t h e chlorophylls, including c1 and c2, based on highly purified samples of each pigment ( 2 ) . Fluorometric methods, most of which give crude estimates of chlorophyll a only, have also been used (3). Loftus and Carpenter ( 4 ) have refined the fluorometric method for the analysis of chlorophylls using the Turner Fluorometer, employing a series of emission filters to improve t h e selectivity between t h e emission spectra of each chlorophyll. Their method involves t h e measurement of the emission through three separate filter combinations and again requires the solution of simultaneous equations to yield the concentrations of each pigment. Acidification of the extract, followed by a further three measurements through the filter combinations yields, in theory, the concentration of the corresponding pheophytins. However, only pheophytin a can be estimated with any degree of certainty, and then only if chlorophyll b is not present as a significant component of the mixture. Improved accuracy of chlorophyll b and c determinations compared with spectrophotometric methods is claimed. T h e most obvious advantage of a fluorometric technique is t h e greatly increased sensitivity compared with spectrophotometry. An increase in sensitivity of two orders of magnitude is easily achieved and, provided that no loss of accuracy is incurred, fluorometric techniques should provide a much faster method than conventional spectrophotometry for performing chlorophyll analyses in natural water samples. I n addition, fluorometry eliminates any possible interference by the absorption of nonchlorophyllous pigments in the 600+ region of t h e spectrum. This paper describes a new approach t o the usual fluorometric method. Variable monochromatic excitation is used to greatly improve selectivity between chlorophylls a, b, and c and their corresponding pheophytins. Thus, appropriate selection of excitation and emission wavelengths reduces the overlap between the emission spectra of each pigment t o a greater extent than is possible with broad band excitation and 0003-2700/78/0350-0392$01 OO/O
EXPERIMENTAL Chlorophylls a and b were extracted from local land plants (e.g., Melaleuca alba) methanol or acetone. The crude pigment extract was purified by thin-layer chromatography (3)using Merck Silica Gel 0.2-mm plates with dioxane (30%) in hexane as the solvent. Chlorophyll c was similarly isolated from locally available Sargassum sp. All solvents were of analytical reagent grade. The purified pigments were stored at -20 “C until used although samples were not kept for more than 24 h. The purity of each pigment was checked by UV-visible spectrophotometry with an Aminco DW-2 spectrophotometer. No significant decomposition products or xanthophyll impurities were detected although some chlorophyll a samples contained a small amount (
