SPECTROFLUOROMETRIC DETERMINATION OF CHLOROPHYLL(IDE) a AND b AND PHEOPHYTIN (OR PHEOPHORBIDE) a AND b IN UNSEGREGATED PIGMENT MIXTURES*

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Abstract
Abstract— Simultaneous equations were derived for the determination of chlorophyllide a and b and pheophorbide a and b in unsegregated mixtures of the four tetrapyrroles. The same equations can also be used for the determination of chlorophyll a and b and pheophytin a and b in unsegregated mixtures, after separation of the phytylated tetrapyrroles from their unphytylated analogs by solvent extraction. The equations are capable of calculating the fluorescence Soret excitation amplitude contributed solely by any one of the four tetrapyrroles in the mixture, from two Soret excitation spectra recorded at emission wavelengths of 674 and 660 nm respectively. The Soret excitation amplitudes are then converted into concentrations by reference to standard calibration curves. The equations are usable with any excitation corrected spectrofluorometer. They are particularly suited for the study of Chl‡catabo‐lism with minimum generation of chemical artifacts.

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