A new procedure for the determination of chlorophylls a and b and its application to normal and regreening Chlorella

Abstract

A new method is deseribed for the extraction and determination of chlorophylls a and b. The method is suitable for use with both normal and regreening nitrogen-deficient Chlorella fusca. The assay involves extraction of chlorophylls by an alkaline pyridine reagent which converts the isocyclic ring of the pigment to a cyclic hydroxylactone. Millimolar extinetion coefficients for the hydroxylactone derivatives of both chlorophylls a and b have been determined at 419 and 454 nm. Using these coefficients, equations have been derived for the calculation of chlorophyll a and b concentrations. The new chlorophyll assay has been compared with other assays which involve the extraction of the pigments with 80% acetone or methanol. The new procedure extracts chlorophylls from rormally grown C. fusca more readily than methanol; the chlorophylls of normal Chlorella cannot be extracted with 80% acetone. The new assay is especially useful in the study of chlorophyll synthesis in regreening nitrogen-deficient C. fusca since the chlorophylls present in these deficient cells cannot be completely extracted with acetone, methanol, methanol-dimethylsulphoxide mixtures, or KOH-methanol.