INVESTIGATION OF THE ALLOMERIZATION REACTION OF CHLOROPHYLL-ALPHA - USE OF DIODE-ARRAY HPLC, MASS-SPECTROMETRY AND CHEMOMETRIC FACTOR-ANALYSIS FOR THE DETECTION OF EARLY PRODUCTS

The products of chlorophyll allomerization in methanol were isolated and analyzed by open column sucrose chromatography, liquid chromatography mass spectrometry (LCMS) and DAD-HPLC (diode-array high-performance liquid chromatography). Four main bands were found with molecular ions of(a) 908, (b) 938, (c) 938 and (d) 938, consistent with the structures(a) 13(2)-hydroxy-chlorophyll a (II), (b) and (c) Mg(II)-3(1),3(2)-didehydro-15(1)-hydroxy-15(1) -methoxy-rhodochlorin-15 acetic acid delta lactone 15(2)-methyl 17(3)-phytyl ester and its epimer (III) and (d) Mg(II)-3(1),3(2)-didehydro-rhodochlorin-15-glyoxylic acid 13(1),15(2)-dimethyl 17(3)-phytyl ester (IV), evidence enhanced by UV/visible spectroscopy, chromatographic coelutions and chemometrics. Chlorophyll a was degraded both in the dark and light, under O-2 and N-2. DAD-HPLC of the resultant degradation mixtures were analyzed using the chemometric heuristic-evolving latent projection method for resolution. Ultraviolet/visible spectra of II and III are reproducibly extracted from the mixtures after a short degradation time, whereas III and IV are the dominant compounds after longer degradation times. Changes in relative elution order of IV using open column chromatography and reverse-phase HPLC are established. A possible allomerization pathway is proposed.