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, AB11 9DB
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 & Collins1.
•
Determination of uncorrected chlorophyll a, corrected
chlorophyll a and pheophytin a.
HPLC analysis2
• Tertiary gradient analysis using a C18 column and UV
detection at 442 nm.
• Quantification of chlorophylls a & b.
• Identification of chlorophylls c2 & c3, 19hexanoyloxyfucoxanthin, 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
A seasonal cycle is observed, where chlorophyll a
concentrations are low in winter and increase in spring when
growing conditions are more favourable.
The fluorometric method can overestimate the chlorophyll a
concentration by as much as 320% during bloom periods.
It 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 samples3.
HPLC analysis shows that concentrations of
photoprotective pigments such as chlorophyll c2 and
fucoxanthin are higher during the growing period than
chlorophyll a.
2
Overestimated levels of fluorometric chlorophyll a
correspond with elevated levels of chlorophyll c2.
It is also reported that the only way to accurately quantify
chlorophyll a is to use a separation technique such as HPLC3.
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;
 Peridinin is used to monitor thecate dinoflagellates
Gyroxanthin-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.
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3. Jeffrey, S. W., Mantoura, R. F. C. and Wright, S. W. 1997. Phytoplankton pigments in oceanography, Monographs on oceanographic methodology.
UNESCO Publishing.
Acknowledgement
This study has been funded by the Scottish Government Schedule of Service ST03p.