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Chromatographic analysis of photosynthetic pigments - recent development and application in the marine science

Photosynthetic pigments (chlorophylls and carotenoids) comprise a numerous class of natural organic compounds present in all plants, in both terrestrial and marine environments. Chlorophyll a (chl a ) is the principal pigment of all photosynthetic organisms, while other chlorophyll and carotenoid pigments (carotens and xanthophylls), which serve as accessory photosynthetic and/or photoprotective pigments, may vary significantly among different plant taxa. In the marine science, analysis of phytoplankton pigments is central to studies of marine ecology and climate research. The concentration of chl a has been widely used for over 40 years to estimate the biomass of oceanic microscopic plants (phytoplankton) using a number of methods, ranging from simple spectrophotometric to remote sensing methods. However photosynthetic pigments of natural phytoplankton populations are rather complex mixtures and their separation has been a major challenge for analytical chemists for decades. Photosynthetic pigments cover a wide range of molecular structures, showing very different polarities (from the polar acidic chlorophylls, xanthophylls of the intermediate polarities and esterified chlorophylls to non-polar caroten hydrocarbons). Furthermore, some chlorophylls and carotenoids are difficult to separate as they differ only in the presence or position of a double bond (e.g. monovinyl (MV) and divinyl (DV) chlorophyll pairs, β , β -carotene and β , ε - carotene as well as their isomeric xanthophyll derivatives). A number of different chromatographic methods (TLC, HP TLC, HPLC) for individual chlorophyll and carotenoid pigment have been developed and employed in marine research (see Jeffrey et al. 1997 for reference and review). Although normal-phase HPLC was also applied, reversed-phase HPLC is presently considered as the method of choice. HPLC systems equipped with different columns, mobile phases and mobile phase modifiers were used in order to resolve as many different pigments as possible. Owing to the continuous advances in HPLC column technology, a significant progress has been made since the first method using C18 column was published in 1983, however the goal of finding one HPLC method capable of separating all significant phytoplankton pigments has not yet been achieved. The identification and quantification of all major photosynthetic pigments is easily achieved by spectrophotometric and spectrofluorimetric detectors, however, a coupling with mass spectrometry was shown to be essential in discovering novel types of phytoplankton pigments. [1] Jeffrey, S.W., R.F.C. Mantoura and S.W.Wright (eds) (1997) Phytoplankton pigments in oceanography: Guidlines to Modern Methods, UNESCO-Paris, pp 660.

Photosynthetic pigments (chlorophylls and carotenoids)

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