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 in which separation of pigments by chromatography is impracticable for one reason or another. An example is the analysis of in vivo formation of heavy metal-substituted chlorophylls in heavy metalstressed plants. We describe here a novel, accurate UV/ VIS spectrophotometric method for the quantification of individual chlorophyll derivatives in complex mixtures, which has the potential for universal applicability for mixtures difficult to separate. The method is based on the description of each pigment spectrum by a series of Gaussian peaks. A sample spectrum is then fitted by a linear combination of these "Gauss-peak spectra" including an automatic correction of wavelength inaccuracy and baseline instability of the spectrometer as well as a correction of the widening of absorbance peaks in more concentrated pigment solutions. The automatic correction of peak shifts can also partially correct shifts caused by processes like allomerization. In this paper, we present the Gauss-peak spectra for Mg-chlorophyll a, b, c, pheophytin a, b, c, Cu-chlorophyll a, b, c, and Znchlorophyll 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.