SEPARATION OF CHLOROPHYLL COMPOUNDS AND THEIR POLAR DERIVATIVES BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

Chlorophyllides a and b were produced by incubation of spinach (Spinacia oleracea, cv. Melody) leaves in hot water (65-degrees-C for 30 min). Chlorophylls and chlorophyllides a and b and their degradation products were separated and identified by using a high-performance liquid chromatographic (HPLC) technique including a gradient solvent system and a reversed-phase C-18 column or a normal-phase separation with a silica column. Of the two separation methods, the reversed-phase technique was rapid (25-30 min) and capable of resolving 12 different chlorophyll derivatives. Peak identification was based on ultraviolet-visible absorption spectra by using a photodiode array detector and confirmed by using fast atom bombardment mass spectrometry.