Hawaii Ocean Time-series (HOT)
in the School of Ocean and Earth Science and Technology at the University of Hawai'i
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HPLC ANALYSIS OF ALGAL PIGMENTS
SUMMARY: Chlorophylls and carotenes are analyzed by high- performance liquid chromatography (HPLC). The diversity as well as their distributions and concentrations in the water column are used to describe the structure of the phytoplankton community.
Because different pigments are specific for individual phytoplankton taxa, the study of plant pigment diversity, concentration and distribution in the water column has become a useful tool when trying to describe the phytoplankton community. The method presented here is based on the protocol described by Mantoura and Lewellyn (1983) and modified according to Bidigare et al. (1989). The difference in polarity and molecular size between photosynthetic pigments is used to separate these molecules by high-performance liquid chromatography. The integration of the area under a particular peak in absorbance or fluorescence at 436 nm or 640 respectivelly is a measure of the amount of pigment injected in the column. Pigments routinely measured are: chlorophyll a, b and c and their degradation products, fucoxanthin, diadinoxanthin, β-carotene, zexanthin, lutein, alloxanthin, prasino- xanthin 19'-hexanoyloxyfucoxanthin and 19'-butanoyloxyfucoxanthin.
Light causes deterioration in pigments. Therefore, the samples should always be protected from exposure to light. Pigments can also decompose spontaneously, particularly when concentrated onto filters. Therefore, filters are stored in liquid nitrogen to minimize pigment degradation.
3. Sampling, Filtration, Extraction and Storage
4. Sample Analysis by HPLC
5. Data Reduction and Calculations
Pigment concentration in the concentrated sample is determined using the following equation:
(RF) * (Area of the peak) [P] = ------------------------- (Sample Loop Area)
The extraction volume is calculated as follows:
(AESP) * (volume ext. std. added to sample) vol. extract) = ------------------------------------------- (Area of ext. std. peak in sample)
where (AESP) = Area of the external standard when 1 ml external standard is mixed with 300 µl of IPS and 500 µl are injected in the HPLC.