Rapid separation of chlorophylls a and b and their demetallated and dephytylated derivatives using a monolithic silica C18 column and a pyridine-containing mobile phase

A monolithic C18-bonded silica rod column (Merck Chromolith) was applied to the separation of mixtures of chlorophylls a and b and their derivatives originated by hydrolysis of the phytyl ester linkage (chlorophyllides), loss of the central Mg atom (pheophytins), or both processes (pheophorbides). Mobile phases containing two different ion-pair reagents, ammonium acetate and buffered pyridine, were tested. Both eluents achieved the resolution of the eight pigments in less than 5 min. The method based on the pyridine-containing mobile phase was applied to the separation of chlorophylls and their green coloured degradation products in senescing leaves of deciduous trees, green fruits and a marine microalga.