High Performance Liquid Chromatography HPLC and Fluorometric Analyses

High Performance Liquid Chromatography (HPLC) and Fluorometric Analyses of Phytoplankton Pigments at a Scottish Coastal Ecosystem Monitoring Site Kerry Smith, Eileen Bresnan, Lynda Webster & Pamela Walsham Marine Scotland Science, Aberdeen, AB 11 9 DB Background Phytoplankton are recognised as primary producers in the marine environment. Most phytoplankton contain the pigment chlorophyll a, which they use to harvest light energy from the sun, passing it up to higher trophic levels. Chlorophyll a is used as an estimate of the phytoplankton biomass to understand functioning in the marine ecosystem and respond to EU policy drivers. A coastal ecosystem monitoring site, 5 km offshore from Stonehaven, was established in 1997 to collect the data required to gain a better understanding of the marine ecosystem and provide a description of seasonal and inter-annual changes. Temperature, salinity, nutrients, phytoplankton and zooplankton are sampled on a weekly basis. Chlorophyll a has been determined by fluorometry at this site since 1997. Analysis of samples using HPLC methodology was introduced in 2009. 1 Methods Fluorometric analysis • Extractive, acidification method of Arar & Collins 1. • Determination of uncorrected chlorophyll a, corrected chlorophyll a and pheophytin a. HPLC analysis 2 • Tertiary gradient analysis using a C 18 column and UV detection at 442 nm. • Quantification of chlorophylls a & b. • Identification of chlorophylls c 2 & c 3, 19 hexanoyloxyfucoxanthin, alloxanthin, alpha-carotene, antheraxanthin, beta-carotene, diadinoxanthin, diatoxanthin, fucoxanthin, gyroxanthin-diester, lutein, neoxanthin, peridinin, prasinoxanthin, violaxanthin and zeaxanthin. 1

Comparison between Fluorometric and HPLC Methods ØA seasonal cycle is observed, where chlorophyll a concentrations are low in winter and increase in spring when growing conditions are more favourable. ØThe fluorometric method can overestimate the chlorophyll a concentration by as much as 320% during bloom periods. ØIt is reported that the fluorometric equations used to calculate corrected chlorophyll a are inaccurate when significant amounts of chlorophylls b and c are present in the samples 3. ØHPLC analysis shows that concentrations of photoprotective pigments such as chlorophyll c 2 and fucoxanthin are higher during the growing period than chlorophyll a. 2 ØOverestimated levels of fluorometric chlorophyll a correspond with elevated levels of chlorophyll c 2. ØIt is also reported that the only way to accurately quantify chlorophyll a is to use a separation technique such as HPLC 3. 2

Comparison between Light Microscopy and HPLC Methods Comparison of HPLC data with phytoplankton cell counts demonstrates that the behaviour of certain phytoplankton groups can be observed by monitoring their diagnostic pigments; Ø Peridinin is used to monitor thecate dinoflagellates ØGyroxanthin-diester is used to monitor the harmful dinoflagellate Karenia mikimotoi Conclusion Most laboratories taking part in monitoring programmes, such as the European Marine Strategy Framework Directive, use fluorometric or spectrophotometric techniques to determine chlorophyll a. These methods may overestimate chlorophyll a. This has implications for assessments using chlorophyll a concentrations as they have the potential to fail environmental quality standards. References 1. Arar, E. J. and Collins, G. B. 1997. Method 445. 0 In vitro determination of chlorophyll a and phaeophytin a in marine and freshwater algae by fluorescence. 2. Smith, K. , Webster, L. , Bresnan, E. and Moffat, C. F. 2010. 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. Vol 1, No 12, Scottish Marine & Freshwater Science. 3. Jeffrey, S. W. , Mantoura, R. F. C. and Wright, S. W. 1997. Phytoplankton pigments in oceanography, Monographs on oceanographic methodology. UNESCO Publishing. sbsdbdbd Acknowledgement This study has been funded by the Scottish Government Schedule of Service ST 03 p.