Rapid and highly sensitive analysis of chlorophylls and carotenoids from marine phytoplankton using ultra-high performance liquid chromatography (UHPLC) with the first derivative spectrum chromatogram (FDSC) technique

Abstract

We developed a rapid and highly sensitive analytical method for chlorophylls and carotenoids derived from marine phytoplankton using ultra-high performance liquid chromatography (UHPLC). High-performance liquid chromatography (HPLC) has been widely used in phytoplankton pigment analysis since the 1980s for estimating the abundance, composition, and photosynthetic physiology of natural algal assemblages or laboratory cultures. However, the run-time of the HPLC analyses is generally ca. 30min or more, which is time-consuming for analysts. Our UHPLC technique enabled us to complete the separations of chlorophylls and carotenoids from marine phytoplankton within 7min with similar resolution as conventional HPLC methods. The analytical method was tested on authentic pigment standards, marine phytoplankton cultures, and field samples that were collected from the central tropical and subarctic Pacific plus the neritic Bering Sea. Critical pigment pairs that generally co-eluted as a single peak were successively resolved by obtaining the first derivative spectrum chromatograms (FDSCs) with a photodiode array (PDA) detector based on differences in pigment absorption spectra, e.g., chlorophyll (Chl) c2 and Mg 2,4 divinyl (DV) pheoporphyrin a5 monomethyl ester (MgDVP), as well as DVChl b and Chl b. Because the maximum injection volume of UHPLC is generally lower than that of HPLC to minimize the unwanted broadening of chromatographic peaks, the detection sensitivity needed to be increased, especially for oligotrophic seawater samples with low pigment concentration. To overcome this sensitivity issue, a PDA detector equipped with an 85mm path length capillary cell was used with a fluorescence detector. As a result, the limit of quantitation (LOQ) as determined by absorbance was of the order of 0.1ng for chlorophylls and carotenoids. Furthermore, a bead-beating technique using N,N-dimethylformamide (DMF) and zirconia beads was utilized to minimize the volume of the organic solvent used for pigment extraction. Our UHPLC method can replace the conventional HPLC techniques, and allows us to yield high-throughput data of the chlorophylls and carotenoids derived from marine phytoplankton.