Photometric method for the quantification of chlorophylls and their derivatives in complex mixtures: Fitting with Gauss-peak spectra

Accurate quantification of pigments in mixtures is essential in all cases i n which separation of pigments by chromatography is impracticable for one r eason or another. An example is the analysis of in vivo formation of heavy metal-substituted chlorophylls in heavy metal-stressed plants. We describe here a novel, accurate UV/VIS spectrophotometric method for the quantificat ion of individual chlorophyll derivatives in complex mixtures, which has th e potential for universal applicability for mixtures difficult to separate. The method is based on. the description of each pigment spectrum by a seri es of Gaussian peaks. A sample spectrum is then fitted by a linear combinat ion. of these "Gauss-peak spectra" including an automatic correction. of wa velength inaccuracy and baseline instability of the spectrometer as well as a correction of the widening of absorbance peaks in more concentrated pigm ent solutions. The automatic correction of peak shifts can also partially c orrect shifts caused by processes like allomerization, In this paper, we pr esent the Gauss-peak spectra for Mg-chlorophyll a, b, c, pheophytin a, b, c , Cu-chlorophyll a, b, c, and Zn-chlorophyll a in acetone; Mg-chlorophyll a , b, pheophytin a, b, Cu-chlorophyll a, b, allomerized Cu-chlorophyll a, b, and Zn-chlorophyll a, b in cyclohexane; Mg-chlorophyll a, b, pheophytin a, b, and Cu-chlorophyll a, b in. diethyl ether. (C) 2000 Academic Press.