SUMMARY Equations have been developed for calculation of the total quantity of pigments consisting of chlorophyll a and its derivatives (chlorophyllide a, pheophytin a, and pheophorbide a) and of chlorophyll b and its derivatives (chlorophyllide b, pheophytin b, and pheophorbide b) in a mixture of these pigments in diethyl ether. Calculations are based on conversion of the chlorophylls and chlorophyllides into the pheophytins and pheophorbides, respectively, by the addition of hydrochloric acid and reading absorbances at two wavelengths. After removal of chlorophyllides a and b and pheophorbides a and b from the diethyl ether solution of the pigment mixture by 0.01N KOH, the quantity of each of the eight components listed above is estimated by indicated experimental and mathematical procedures.

This study has been made to establish the molar absorbance coefficients of the zinc and copper(II) complexes of the pheophytins and pheophorbides. Spectral curves and metal analyses have been madefor each of the four mentioned chelates of zinc and copper(II) following purification by chromatography. Chelation was accomplished with ease in aqueous acetone solutions of the respective pheophytins and pheophorbides. Thin-layer chromatography procedures, developed during this study, provided reliable indication of the purity of the complexes. Zinc and copper(II) salts and their solutions promoted rapid allomerization of the pigment during chelation. Molar absorbance coefficients are presented.

The use of trade names in this publication does not imply endorsement by the North Carolina Agricultural Experiment Station of the products named, nor criticism of similar ones not mentioned.

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Procedures are described for the estimation of any one or more of the components present in mixtures in diethyl ether or 80% acetone comprised of the pigments chlorophylls a and b, pheophytins a and b, and the metallo-pigment complexes, zinc pheophytins a and b. Methods are described for component estimation from equations derived for spectrophotometric and fluorometric analyses. Fluorometric estimation may be made at concentrations of 1 X 10~2 of those required for spectrophotometric estimation.