HPLC determination of phytoplankton and microphytobenthos pigments: comparing resolution and sensitivity of a C18 and a C8 method

Carlos R. Mendes, Paulo Cartaxana,Vanda Brotas

Limnol. Oceanogr. Methods 5:363-370 (2007) | DOI: 10.4319/lom.2007.5.363

ABSTRACT: The effectiveness of two HPLC methods (a monomeric C18 column with a high ion strength solvent gradient and a monomeric C8 column with a pyridine-containing mobile phase) in the separation, identification, and quantification of pigments are compared, using phytoplankton, microphytobenthos, and algal cultures. Although the two studied methods showed good analytical resolution, the C18 method presents a higher discrimination for several carotenoid pigments. On the other hand, the C8 method allows the separation of chlorophylls c1, c2, and Mg-2, 4-divinyl phaeoporphyrin a5 monomethyl ester as well as the pair chlorophyll a/divinyl Chl a. The C18 method had a shorter elution program and a lower solvent flow rate, making it cheaper and faster than the C8 method. Furthermore, the C18 method showed significantly lower detection and quantification limits, a particularly important advantage for pigments present in trace amounts in both phytoplankton and microphytobenthic samples. Differences in the sensitivity of the two studied methods were due mainly to differences in flow rate, with significantly higher peak areas for slower flow rates. No method is ideal for all pigments and sample types and the choice of method should be made in accordance with the objective of each study, taking into consideration the advantages and disadvantages identified for each method.