Hawaii Ocean Time-series (HOT)
in the School of Ocean and Earth Science and Technology at the University of Hawai'i

HPLC ANALYSIS OF ALGAL PIGMENTS

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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.


1. Principle

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.

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 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 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 and stored in liquid nitrogen.
3.4. Extraction and concentration of pigments
3.4.1. Filters are extracted for 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 an homogeneous suspension and supernatant is decanted by centrifugation (1,500 rpm for 5 min.).
3.4.2. One milliliter of each extract is combined with 300 µl of an ion pairing solution (IPS) (50 mmol tetrabutyl ammonium acetate [95% purity] and 1 mol of ammonium acetate [analytical reagent grade] in 100 ml DDW) in an autosampler vial and store at 4 °C until analysis by HPLC.

4. Sample Analysis by HPLC

4.1. Two eluant solutions (termed solvent A and solvent B) are prepared for the chromatographic separation. One liter of solvent A is made by mixing 50 ml IPS and 150 ml DDW in 800 ml methanol. The solvent B is 100% methanol.
4.2. Samples are loaded in a temperature controlled autosampler equipped with a 500 µl injection loop. The temperature is kept at 4 °C. The separation of pigments is performed on a Radial-Pak C18 column (0.8 x 10 cm, 5µ particle size). by linear gradient elution from 100% eluant A to 100% eluant B in 12 min, followed by 20 min of 100% eluant isocratic hold. Flow rate is 6 ml min-1 and upper limit pressure for the column is 3000 psi.
4.3. Absorbance is read at 436 nm. The excitation wavelength of the fluorometer is 424 nm and the emission is read at 640 nm.
4.4. Peak areas are converted to concentration by the external standard calibration method using standards provided by Dr. Bidigare.

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

  • Niskin bottles and rosette/CTD unit

  • low pressure filtration apparatus

  • liquid nitrogen dewer

  • Spectra-Physics HPLC, Waters model 440 absorbance detector, Waters model 470 fluorescence detector, Radial-Pak C18 column (0.8 x 10 cm, 5 µm particle size), guard column

  • vortex

  • centrifuge

  • general laboratory glassware and supplies

  • N2 gas

  • freezer

7. Reagents

  • Deionized distilled water (DDW)

  • Acetone (HPLC grade)

  • Methanol (HPLC grade)

  • Tetrabutyl-ammonium acetate (95% purity)

  • Ammonium acetate (reagent grade)

  • Ion-pairing reagent (made of 1.5 g tetrabutyl ammonium acetate [95% purity] and 7.7 g of ammonium acetate [analytical reagent grade] in 100 ml DDW)

  • Solvent "A" (mix 50 ml of ion-pairing reagent, 150 ml DDW and 800 ml methanol)

  • Solution "B" (100% methanol)

8. References

  • Bidigare, R. R., O. Schofield and B. B. Prezelin. 1989. Influence of zeaxanthin on quantum yield of photosynthesis of Synechococcus clone WH7803 (DC2). Marine Ecology Progress Series, 56, 177-188.

  • Mantoura, R. F. and C. A. Lewellyn. 1983. The rapid determination of algal chlorophyll and carotenoid pigments and their breakdown products in natural waters by reverse-phase liquid chromatography. Analytica Chimica Acta, 151, 297-314.