Phytoplankton pigment analysis by HPLC (High performance liquid chromatography)
Pigments in aquatic ecosystems
Three main groups of plant pigments occur in phytoplankton: The liposoluble Chlorophylls and Carotenoids, as well as water-soluble Phycobilins. The first two groups are found in all eukaryotic algae and in the prokaryotic Cyanobacteria and Prochlorophytes. The Phycobilins are unique to Cyanobacteria, Crypto- and Rhodophyceae.
All Chlorophylls, Phycobilins and most of the more than 100 Carotenoids have the task of absorbing light for photosynthesis. Some of the Carotenoids act as protecting mechanism of the cells against photo damage.
All pigments, except Chlorophyll-a, are referred to as "accessory" pigments.
The HPLC – system of the department (Fig. 1) is designed to analyze the Chlorophylls and Carotenoids as well as their derivatives. The system detects the pigments by means of their absorption and fluorescence. As result of the analysis one obtains a "chromatogram" on which each of the pigments present in the sample appears as a separate peak (Fig. 2). Characteristic for each pigment are both its "retention time" (the time it takes to elute at the end of the separation column) as well as its absorption spectrum (Fig. 3). By calibration of the system with pure standards the pigments can then be determined both quali - and quantitatively.
Pigments as chemotaxonomic markers
Each taxonomic class of phytoplankton owns its specific pigment composition (Fig. 4). With adequate ratios of Chlorophyll-a to the individual marker pigments and a calculation program (CHEMTAX; developed by MACKEY et al. (1996)) it is possible to calculate the taxonomic composition of a phytoplankton population and to determine the contribution of each class to total Chlorophyll by means of pigment analysis of a water sample. This is especially important for very small phytoplankton species (nano and pico plankton), which are not easily determined by light microscopic methods. Therefore, the microscopic method and the chemotaxonomic pigment HPLC analysis complement each other perfectly (Figure 5). The microscopic group summarized as μ-flagellates could be broken down by their marker pigments as Dinoflagellates, Prasino-, Chryso-, Prasino-, Crypto- and Chlorophyceae.
Pigment measurements of sediment samples provide information on the origin of the sunken material (for example, different trophic regimes etc.). The composition of the pigments in feces of zooplankton may show existing food preferences.
Pigments as markers of processes
The pheophorbides (these are the Chlorophyll molecules without Mg ion and phytol chain) arise when a Chlorophyll-a containing particle passes through the digestive tract of meso and makrozooplankton. Therefore, the ratio of pheophorbides to intact Chlorophyll-a in the water column can offer an idea of the grazing pressure experienced by phytoplankton community. The same ratio, determined in sunken material shows the influence of feces on sedimentation.
On the use of the degradation products of Carotenoids, and Phycobilins in this context is still little known.
Which main questions can be answered by pigment analysis?
1. Which classes of phytoplankton are involved in the composition of a given community and how much does each class contribute to total Chlorophyll-a?
2. How do the communities change over time?
3. Are there differences in the vertical distribution of the classes?
4. What classes contribute how much to the vertical flux?
5. What influence has the feeding activity of the zooplankton on phytoplankton communities and their decline?
6. Are there feeding preferences of zooplankton in relation to individual phytoplankton classes?
MACKEY, M.D., D.J. MACKEY, H.W. HIGGINS and S.W. WRIGHT (1996) CHEMTAX ‑ a program for estimating class abundances from chemical markers: application to HPLC measurements of phytoplankton. Mar. Ecol. Prog. Ser. 144: 265‑283