The Development and Validation of a High Performance Liquid Chromatography Method to Quantify Chlorophylls a and b in Marine Phytoplankton and Identify Other Pigments of Interest

Scottish Marine and Freshwater Science Volume 1 No 12

1. High Performance Liquid Chromatography (HPLC) is an alternative method to light microscopy and fluorescence for characterising phytoplankton. The HPLC data provides valuable information about the contribution of different functional groups to the biomass of the phytoplankton community. The HPLC data should be supported by microscopic verification as many algal classes share the same pigments.

2. A quantative HPLC method was developed to quantify chlorophyll a and chlorophyll b. However, due to a lack of commercially available standards at suitable concentrations, only area % data is given for 19 hexanoyloxyfucoxanthin, alloxanthin, alpha-carotene, antheraxanthin, beta-carotene, chlorophyll c2, chlorophyll c3, diadinoxanthin, diatoxanthin, fucoxanthin, gyroxanthin-diester, lutein, neoxanthin, peridinin, prasinoxanthin, violaxanthin and zeaxanthin.

3. Ammonium acetate buffer (0.5 mol l-1) is added to the standards and samples immediately before injection, in order to increase the affinity of the pigments for the column during the loading step. This reaction is temperature dependant and must be carried out at ambient temperature to ensure adequate mixing.

4. The stability of the pigments, in the autosampler at ambient temperature, whilst awaiting injection for a typical analytical run, was investigated. All pigments were found to be stable in the autosampler for 18 hours. A typical analytical batch takes 15 hours of analytical time.

5. Replicate analysis for high standards and samples and low standards and samples on different days gave %CV of <11% for both chlorophyll a concentration and chlorophyll b concentration. This meets the limits for reproducibility given in the UK Clean Seas Environmental Monitoring Programme (CSEMP) Green Book. 2

6. The limit of detection (LOD) was calculated to be 0.1 µg l-1 for chlorophyll a and 0.02 µg l-1 for chlorophyll b. This meets the limits for LOD given in CSEMP Green Book.

7. Recovery for chlorophyll a was calculated to be 105%, and 110% for chlorophyll b, which meets the limits set by the United Kingdom National Marine Chemistry Advisory Group (NMCAG).