A high-performance liquid chromatographic (HPLC) method with a reverse phase C8 column is used to measure concentrations of chlorophylls and carotenoids in samples of particulate matter from the California Current Ecosystem (CCE). Concentrations of chlorophyll a are used as a proxy for phytoplankton biomass and concentrations of other taxon-specific pigments are used to determine contributions of phytoplankton taxa to total phytoplankton biomass (Wright et al. 1991).
The original description of the method was written Feb. 2010 by R. Goericke and M. Roadman. Changes to the method are listed below.
HPLC samples are collected at CalCOFI and SCCOOS (Southern California Coastal Ocean Observing System) stations located within the CCE. Samples are collected from three to eight different depths in the photic zone.
Samples are drawn from the niskin bottles using 0.5, 1.04 or 2.2 liter brown polypropylene bottles, depending on the chlorophyll concentration measured by the fluorometer on the CTD. For the higher chlorophyll values, typically the coastal stations, less water is sampled.
Samples (0.5, 1.04, or 2.2 L) are filtered onto 25 mm GF/F filters (Whatman) under a low vacuum pressure (≤ 40 mm Hg).
Once the sample has finished filtering, the filter is carefully folded in half, and blotted on a paper towel to remove excess water. The folded filter is placed in a labeled 2 ml cryovial and stored in liquid nitrogen for analysis ashore.
3. HPLC Sample Preparation
A small cooler, with a test tube rack, is filled with ice and the grinding tubes are placed inside.
The filters are removed from the cryovials and placed individually in a grinding tube. Each sample is extracted in the cold and dark for 30 minutes in 1.6 ml of a canthaxanthin /acetone mixture (~200 µl saturated solution of canthaxanthin in benzene added to ~250 ml acetone).
After 30 minutes, the grinding tube is placed in a small ice bath and the samples are ground for approximately 15 seconds (with pulses) using a pestle fitted to a power drill. Samples are returned to the cooler and extracted for 30 more minutes. Care must be taken to avoid spillage of the sample (this is a step that should be practiced beforehand).
Using a standard Pasteur pipette, the sample is removed from the grinding tube and returned to the original cryovial. The cryovials are centrifuged for 5 minutes at medium-high speed, and 300 µl of the supernatant is placed in the autosampler vial.
The autosampler vials are placed in the autosampler set at 4°C and the remaining sample in the cryovial is returned to the liquid nitrogen dewar for backup analysis.
4. HPLC Mobile Phase and Gradient
Two solvents, A and B, are used for the mobile phase in this method. (Zapata et al. 2000). Both are prepared separately, filtered under low pressure through a 47mm GF/F filter (Whatman), and stored in a 4 liter, glass amber bottle.
Solvent A is a mixture of methanol: acetonitrile: acidic 0.25 M pyridine solution (50:25:25 v:v:v). The aqueous pyridine solution is prepared by adding 10 ml acetic acid and 20 ml of pyridine to 900 ml Milli-Q water in a 1 L volumetric flask and mixed using a magnetic stirrer in the fume hood. The pH is checked and acetic acid added dropwise until the pH reaches 5.0. The flask is brought to 1 L volume and the pH rechecked.
Solvent B is a mixture of methanol: acetonitrile: acetone (20:60:20).
Gradient used with a flow rate of 1 ml min-1 (time; solvents, %A, %B): (0; 100, 0), (12; 60, 40), (36; 0, 100), (38; 0, 100), (40, 100, 0).
5. HPLC Sample Analysis
An Agilient 1200 series standard thermostatted autosampler, fitted with a 900 µl needle and sample loop, is used to prepare the sample prior to injection into the C8 reversed- phase silica column.
An injector program is used in overlapped injection mode to decrease sample run time.
The injector program is set up to mix water and the sample to a concentration of 60% acetone. The sample is prepared in the sample loop with segments of water, sample and air alternating to prevent the water and sample to mix until reaching the column to prevent the degradation of chlorophylls. After the injector program is complete, 160 µl of water, 255 µl of sample, and 23 µl of air are injected onto the column and the sample gradient (see 4.4) begins.
The canthaxanthin /acetone mixture (same as used to extract the samples) is mixed with water using a needle syringe to dilute the mixture to 85% acetone (300 µl of water and 1.7 ml of the canthaxanthin acetone mixture) and divided between three autosampler vials.
For each sample run, 100 µl of the standard is injected in triplicate at the beginning, middle and end of the samples.
6. HPLC Data Analysis
After the run is complete, the individual chromatograms are reviewed, checking baselines and peak labels. The peak areas with labels and retention times are exported from Agilent Chemstation.
|Chlorophyll c3Chlorophyllide aChlorophyll c2Chlorophyll c1Peridinin19′-butanoyloxyfucoxanthinFucoxanthin9′-cis-neoxanthinPrasinoxanthinViolaxanthin||19′-hexanoyloxyfucoxanthinDiadinoxanthinAlloxanthinDiatoxanthinZeaxanthinChlorophyll bDivinyl chlorophyll aChlorophyll aCarotene|
To calculate concentrations of pigments per L seawater (CP) the following needs to be known: Instrument response factor K for the pigment P (KP i.e. the units area per µg pigment injected, 1/µg), Area generated by the pigment (AreaP, no dim), the volume of the seawater filtered (VolSW, L), the volume acetone used for the extraction of the sample (VolEx, mL), the actual volume of extract injected on the HPLC column (VolInj, ml).
VolInj is calculated from the area of the canthaxanthin peak in the sample (CSa) and the area of the canthaxanthin peak in the canthaxanthin standard injection (CSt) and the volume canthaxanthin standard injected for the standard run (VolCanth, ml). This is calculated as:
VolInj = CSa / CSt * VolCanth* 0.85; dim (VolInj) = 1 / 1 * mL * 1
The factor of 0.85 corrects for the dilution of the 100% solution of canthaxanthin in acetone with water.
Pigment concentrations (CP) are calculated from:
CP = (AreaP / KP) / (VolSW * (VolExt / VolInj))
dim (CP) = 1 / (1/µg) / (L * (mL/mL)) = µg/L
- 25 mm GF/F filters (Whatman)
- 25 mm filter funnels and bases
- vacuum pump
- 2ml cryovials
- Wheaton Potter-Elvehjem Tissue grinders, 5 ml, and PTFE pestles
- Power drill
- Pasteur disposable pipet, Fisherbrand
- 500 µl needle syringe
- Autosampler vials – National Scientific Target DP RoboVial 9 mm screw thread
- Agilent 1100 Series HPLC (DAD detector, FLD detector, vacuum degasser, quaternary pump, and thermostatted autosampler with 900 µl injection loop and needle)
- Waters Symmetry C8 column (3.5 um particle size, 4.6 x 150 mm, silica, reverse-phase)
- Agilent’s ChemStation for LC software (Rev. B.04.01 SP1)
- Liquid Nitrogen
- Acetone, HPLC grade, Fisher Chemical
- Acetonitrile, HPLC grade, Fisher Chemical
- Methanol, HPLC grade, Fisher Chemical
- Acetic acid, glacial, HPLC grade, Fisher Chemical
- Pyridine, Certified ACS, Fisher Chemical
- Wright, S.W., S.W. Jeffery, R.F.C. Mantoura, C.A. Llewellyn, T. Bjornland, D. Repeta, and N. Welschmeyer. 1991. Improved HPLC method for the analysis of chlorophylls and carotenoids from marine phytoplankton. Marine Ecology Progress Series 77: 183-196.
- Zapata, M., F. Rodríguez, and J.L. Garrido. 2000. Separation of chlorophylls and carotenoids from marine phytoplankton: a new HPLC method using a reversed phase C-8 column and pyridine-containing mobile phases. Marine Ecology Progress Series 195: 83-102.