Quantitative Measurement of Leaf Chlorophylls by Spectrophotometry of Their Pheophytins in Aqueous Alcoholic Extracts

Quantitative measurements of leaf chlorophylls generally utilize spectrophotometric or colorimetric assay following extraction of pigments from the tissue (3). However, all these methods require sampling of the leaf tissue, followed by extraction of the pigments. Both sampling and extracting introduce a variability into the results which may make the methods inadequate for detecting small changes in chlorophyll content. It is the purpose of this communication to evaluate the extent of the variability introduced into such methodology when assaying chlorophyll in leaves, as well as to estimate the precision of three types of instruments (2 spectrophotometers & a colorimeter) used in conjunction with these measurements. Ideally, it would be desirable to determine chlorophyll in situ, and both reflectance and transmission methods have been proposed (S. Aronoff. 1962. Dimerization of chlorophyll a in concentrated solutions. Unpublished (5). Unfortunately, these methods appear to lack the refinement necessary for detecting small differences. While spectrophotometry of solutions of chlorophylls a and b at two wavelengths permits the calculation of the concentrations of the two components individually, colorimetric measurements, using incident light of relatively broad spectral range, allows quantitative estimation of total chlorophyll only. Determination of the chlorophylls as phyllins may yield variable results arising from uncontrolled alteration of the pigments during processing of the sample and measurement. For example, pheophytinization and allomerization are encountered commonly. The method of determination of leaf chlorophylls reported here involves spectrophotometry of acidified 80 %-ethanolic leaf extracts at two wavelengths. Chlorophylls a and b are thereby measured as their pheophytins. Thus, the variability arising from manipulations involving separations, pheophytinization upon extraction, and allomerization of the chlorophylls, is minimized. Materials & Methods