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

Abstract

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 (60C) 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 30C. A protocol is presented that achieves quantifiable resolution of all taxonomically important pigments tested by running the analysis at 2 temperatures: 10 and 60C. Of particular significance was the ability to separate divinyl chl a from monovinyl chl a, chl c, from chl c,, and partial separation of chl c, and MG 2,4-divinyl phaeoporphorin a, monomethyl ester. An example is given of the utility in separating chl c pigments, and lutein and zeaxanthin in natural phytoplankton samples.