Determinations of total carotenoids and chlorophylls a and b of leaf extracts in different solvents

Various equations for the determinations of total chlorophyll and individual amounts of chlorophylls a and b in extracts from plant tissues exist (see Holden, 1976) and some of them (e.g. Arnon, 1949) are widely used. Additional modifications to the equations have also been developed so as to permit an estimate of total carotenoids to be made from the spectrum of the same mixture in diethyl ether (Ziegler & Egle, 1965; Gaudillire, 1974). During the course of studies that involved the use of various solvents we noted large discrepancies (>40%) between estimations made using the different published equations for particular solvents, all of which were known to contain the same amount of pigments. Taking advantage of a t.1.c. method (Lichtenthaler 8t Pfister, 1978) that permits a distinct separation of the two chlorophylls and also the major carotenoids using light petroleum (b.p. 40-6O0C)/dioxane/propan-2-ol (7 :3 : 1, by vol.) as developing solvent, fresh samples of chlorophyll a and b uncontaminated with each other were readily available for re-evaluation of the published specific absorption coefficients. Those values published by Smith & Benitez (1955), using diethyl ether, were found to be still the most acceptable, and relative specific absorption coefficients to these values were established (Table 1) for various other solvents. The red peak maxima of the chlorophylls were shifted to longer wavelengths with increasing polarity of the solvents; in our case diethyl ether, acetone, 80% (v/v) acetone, 96% (v/v) ethanol and methanol. The red absorption peaks of the chlorophylls were also broadened in the same sequence and the values for the specific absorption coefficients decreased. At the same time suitable values for total carotenoids at 470nm were also determined. On the basis of these coefficients the following equations were derived to determine the individual levels of both chlorophyll a (C,) and chlorophyll b (C,) and the total amounts of carotenoids (Cx+c) and chlorophylls (C,+ C,) [in pg.(ml of plant extract)-'] the measured absorbance values (A) at different wavelengths: