Our first pigment analyses

After all the long days of filtering water samples in our lab kunk back in March, the samples have been stored within a freezer ready for pigment analyses to begin. Filtration was carried out on the collected lake-water samples, to concentrate phytoplankton cells on a Whatman GF/F filter. All these samples have been run through the pigment analyser (High-Performance-Liquid-Chromatography system), to provide us with data on the algal community composition. This analysis can be performed as all phytoplankton groups produce pigments (chlorophylls and carotenoids) to enable cell photosynthesis, and some of these pigments can be used as important biomarkers for algal species presence and absence. The detection of these pigments provides information on the entire species assemblage within the samples, and not just a single group. This is an important aspect of the research, as phytoplankton respond very quickly to any changes within their environment, and provide excellent indicators of nutrient enrichment and climate change through species assemblage changes. Pigment records from across Lake Baikal, extending over natural timescales, will then enable the impact of recent nutrient loading on the ecosystem to be assessed.

To find out more about the application of algal biomarkers within this research, click HERE.

HPLC system: Agilent 1200 series

HPLC system: Agilent 1200 series

Before the samples are analysed on this HPLC system, sample preparation is carried out under special conditions, to enable the separation and detection of individual pigments. A mixture of organic solvents is used to extract the pigments from the cells, consisting of acetone, methanol and water. This sample preparation has to be carried out under dim light conditions, to avoid unnecessary photochemical degradation of pigments, which reduces their chemical stability. They are then stored for at least 12 hours within a freezer in the dark, to ensure pigment extraction from all the algal groups. This is important as algal species have different cell walls, and species with heavily silicified walls (such as large diatoms) require more time to fully extract their pigment composition. These samples are further filtered and HPLC-grade acetone is added as part of the final extraction step. Samples are then dried within the glass vials under nitrogen gas before analysis on the HPLC Agilent 1200 series.

Samples are placed in the autosampler within HPLC

Samples are placed in the auto-sampler tray within HPLC

Once dried, injection solvent is added and the glass vials are placed within the auto-sampler tray before being injected into the HPLC. As each sample is injected into the system, a diode-array detector then analyses the pigments. This produces a pigment chromatogram, which displays the pigment composition within the samples and individual pigments can be identified from their peak retention times and spectra.

Interpretation of these pigment chromatograms is now underway, and fortunately all the manual filtering of litres and litres of water paid off…as well as provoking some arm wrestling from all the bicep exercising…

Photo_on_14-03-2013_at_08.27

…Shortly our first pigment analyses on the sediment cores collected from the March 2013 field trip will be carried out, so more on this to follow soon…

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