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.
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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.
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