APPLICATION OF ELUTION ANALYSIS TO THE STUDY OF CHLOROPHYLL TRANSFORMATIONS BY COLUMN CHROMATOGRAPHY ON SUCROSE

The transformations of chlorophyll (Chl) a b were studied by applying elution analysis to chromatographic separations on a sucrose column. When the Chl a-water adduct was heated in 1-propanol at 50° for 15 min, subsequent separation on sucrose revealed three principal products which were identical with Chl a according to their visible absorption spectra and yielded positive reactions to the Molisch phase test. Only minor amounts of oxidation products were observed in this instance. When water-free Chl a was healed under similar conditions, separation on sucrose yielded, in addition to Chl a, considerable amounts of Chl a′and oxidation products. On standing in 1-propanol at 25° for 4 days, Chl a was completely allomerized. Separation on sucrose revealed three pairs of oxidation products, each pair consisting of the 10(S) and (R) isomers. When a light petroleum solution of chloroplast pigments was extracted with 70% aqueous methanol, the separation of the precipitated chlorophylls on sucrose yielded three chlorophyll components in both series but virtually no oxidation products. As the most plausible interpretation, it is suggested that the most slowly migrating chlorphylls arose from the presence of considerable amounts of the enol of chlorophyll in the samples. When these samples were introduced on to the sucrose column, the hydrogen-bonded water molecules were removed from the enol. This rendered the enol susceptible to transformation during the separation on sucrose. As very little oxygen was present, principally Chl a and a′ were produced. Although no enol could be detected in the effluent, the lifetime of the enol was considered to be long enough to cause the appearance of shoulders corresponding to the slowly migrating chlorophylls. These results confirm the enol forms of chlorophyll play a key role in chlorophyll oxidation and epimerization. They also show that the use of methanol-water mixtures for the purification of chlorophyll preparations requires special precautions in order to prevent the enolization and epimerization reactions. © 1979.