Separation of chlorophylls and carotenoids from marine phytoplankton: a new HPLC method using a reversed phase C-8 column and pyridine-containing mobile phases

A high-performance liquid chromatographic (HPLC) method based on a reversed -phase C-8 column and pyridine-containing mobile phases was developed for t he simultaneous separation of chlorophylls and carotenoids. The method is s elective enough to resolve monovinyl (MV) and divinyl (DV) pairs of polar c hlorophylls and DV chlorophyll a (chl a) (the marker pigment for the prokar yote Prochlorococcus marinus) from chi a (the MV analogue). Only the pair D V chi a/chl b was not resolved. This resolution capability for chlorophylls was only previously achieved using polymeric C-18 columns in combination w ith ammonium acetate or pyridine-containing mobile phases. The proposed met hod also allows the separation of taxon-specific carotenoids belonging to 8 algal classes, including some critical pigment pairs for previous HPLC met hods using C-18 columns. The method employs a binary gradient, so it can be used with both low-pressure and high-pressure mixing instruments. Method t ransferability was tested using 3 HPLC systems. Only a slight adjustment of gradient profile was required to obtain similar results with HPLC equipmen t having different dwell volumes. The selectivity of the method towards som e recently discovered chlorophyll and carotenoid pigments makes it especial ly suitable for studying not only field samples, but also for re-examining the pigment composition of different algal classes.