Improved separations of phytoplankton pigments using temperature-controlled high performance liquid chromatography

Varying the temperature of a polymeric C18 high performance liquid chromatography (HPLC) column caused significant changes in elution profiles of carotenoid and chlorophyll (chl) pigment mixtures. High temperature operation (60°C) was optimal for carotenoid separations, including the separation of zeaxanthin from lutein. Chlorophyll and chlorophyll derivative separations were optimal at a column temperature between 10 and 30 °C. A protocol is presented that achieves quantifiable resolution of all taxonomically important pigments tested by running the analysis at 2 temperatures: 10 and 60 °C. Of particular significance was the ability to separate divinyl chl a from monovinyl chl a, chl c1 from chl c2, and partial separation of chl c2 and MG 2,4-divinyl phaeoporphorin a5 monomethyl ester. An example is given of the utility in separating chl c pigments, and lutein and zeaxanthin in natural phytoplankton samples.