HPLC ANALYSIS OF ALGAL PIGMENTS

SUMMARY: Chlorophylls, carotenes and other accessory pigments 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.

1. Principle

Because different pigments are specific to 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) and Jeffrey et al. (1997). 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 at 436 nm is used to calculate the amount of that pigment injected onto the column. Pigments routinely measured are: chlorophyll a, b and c and their degradation products, divinyl chlorophyll a, peridinin, fucoxanthin, diadinoxanthin, α-carotene, β-carotene, zeaxanthin, lutein, alloxanthin, prasinoxanthin, chlorophyllide, violaxanthin, 19'-hexanoyloxyfucoxanthin and 19'-butanoyloxyfucoxanthin.

2. Precautions

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 or at -80 °C to minimize pigment degradation.

3. Sampling, Filtration, Extraction and Storage

3.1. Seawater samples (4-10 liters) are collected in 12 liter Niskin bottles and transferred, via Tygon tubing, to opaque polyethylene filtration bottles. The filtration bottles and caps are rinsed three times with the sample before filling. Sampling depths correspond to those used for primary productivity. Four to six additional depths are distributed uniformly within the region of the chl maximum, as determined by the continuous profile of fluorescence (see Chapter 4).
3.2. After filling, the filtration bottles are placed upside down in the filtration rack and the contents pressure filtered (4-7 psi N2) through in-line 25 mm GF/F filters. All filtration procedures are done under subdued light conditions.
3.3. After the sample is filtered, each filter is folded and transferred to a cryotube. The tube is labeled, flushed with N2 gas, stored in liquid nitrogen and then at -80 °C until analysis.
3.4. Extraction and concentration of pigments
3.4.1. Filters are extracted for 24-48 hours in 3 ml 100% acetone at -20 °C. One hundred microliters of a known concentration of canthaxanthin are added as external standard to the extract to correct for changes in extraction volume resulting from the water content in the filter and the acetone evaporation. The sample is vortexed to ensure a homogeneous suspension and supernatant is decanted after centrifugation (1,500 rpm for 5 min.).
3.4.2. One milliliter of each extract is combined with 300 µl of deionized distilled water (DDW) in an autosampler vial kept at 5 °C during analysis by HPLC.

4. Sample Analysis by HPLC

4.1. Three eluant solutions (termed solvents A, B and C) are used for the chromatographic separation. Solvent A is a 0.5 M ammonium acetate solution of 20% DDW in methanol, with 0.01% BHT (2,6-di-tert-butyl-p-cresol, to prevent the formation of Chl a allomers). Solvent B is 10% DDW in acetonitrile, with 0.01% BHT. Solvent C is 100% ethyl acetate.
4.2. Samples are loaded in a temperature controlled autosampler kept at 5 °C. Injection volume is 100 µl. The separation of pigments is performed on a Waters C18 column (150 x 4.6 mm, 5 µm particle size) by linear gradient elution from 90% solvent A to 100% solvent B, followed by gradient elution to 90% C, ending analysis in 100% B at 30 minutes. The system is then returned to initial conditions (90% A, 10% B) to re-equilibrate, and stopped at 38 minutes. Flow rate is 1 ml min-1 and upper pressure limit for the column is 3000 psi.
4.3. Absorbance is read at 436 nm for chromatography. An absorbance spectrum for each pigment is saved from the response of the full spectrum photodiode array detector.
4.4. Peak areas are converted to concentration by the external standard calibration method using pigment standards provided by Sigma-Aldrich and DHI Lab Products.

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)
where: [P] = pigment concentration
(RF) = response factor (obtain from the calibration)
(RF) = [(calibration[P]) * (sample loop volume)]/(calibration [peak 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.

6. Equipment/Supplies

7. Reagents

8. References