2.0 Definition
The concentration of all pigments is given as ng kg _-1 in seawater.

3.0 Principle of Analysis
The reverse phase high performance liquid chromatography method described here separates all the phytoplankton pigments listed below in order of polarity upon passage through a column. The most polar pigments are removed earlier than the less polar pigments.

Chlorophyllide b
Chlorophyllide a
Chlorophyll c₃
Chlorophyll c₁ + c₂ and Chlorophyll Mg 3,8DVP a₅
Peridinin
19' - Butanoyloxyfucoxanthin
Fucoxanthin
19' - Hexanoyloxyfucoxanthin
Prasinoxanthin
Pyrophaeophorbide a
Diadinoxanthin
Alloxanthin
Diatoxanthin
Lutein
Zeaxanthin
Chlorophyll b
Chlorophyll a
Phaeophytin b
Phaeophytin a
a Carotene
b Carotene

Picoplanktonic prochlorophytes are abundant in tropical and subtropical seas and oceans. They contain divinyl-chlorophyll a and divinyl-chlorophyll b (more appropriately called 8-desethyl, 8-vinyl Chlorophyll), both co-eluting with “normal” chlorophyll a and b with this reverse phase liquid chromatography technique.

4.0 Apparatus and Reagents
4.1 Filtration System and Whatman 47 mm GF/F filters

4.2 Liquid nitrogen and freezer for storage and extraction

4.3 Glass centrifuge tubes for extraction, 15 ml

4.4 High pressure liquid chromatograph capable of delivering three different solvents at a rate of 1 ml/minute.

4.5 High-pressure injector valve equipped with a 200 mL sample loop.

4.6 Guard Column (50 x 4.6 mm, ODS-2 C18 packing material, 5 mm particle size) for extending life of primary column.

4.7 Reverse phase HPLC Column (250 x 4.6 mm, 5 mm particle size, ODS-2 Spherisorb column).

4.8 Absorbance detector capable of monitoring ar 436 nm, or preferably, an on-line diode array spectrophotometer.

4.9 Data recording device: strip chart recorder or, preferably, an electronic integrator or computer equipped with hardware and software for chromatographic data analysis.

4.10 Glass syringe, 500 ml

5.0 Eluants
Eluant A (80:20, v:v, methanol: 0.5 M ammonium acetate, aq., pH=7.2), eluant B (90:10, v:v, acetonitrile:water), and eluant C (ethyl acetate). Use HPLC-grade solvents, measure volumes before mixing. Filter eluents through a solvent-resistant 0.4 mm filter before use and degas with helium. The gradient program is listed in Table 13-1.

6.0 Sample Collection and Storage
Water samples are collected from niskins into clean polyethylene bottles with Tygon Ò tubing. Samples are immediately filtered through 47 mm GF/F filters using polycarbonate in-line filter holders (Gelman) and a vacuum of less than 100 mm Hg. Filters are folded in half twice and wrapped in aluminum foil, labeled, and stored in liquid nitrogen (to avoid formation of degradation products) until on-shore analysis.Alternatively, filters can be immediately placed in acetone for pigment extraction if analysis is to be carried out onboard ship. Samples collected for HPLC analysis can also be used in the measurement of chlorophyll a and phaeopigments by fluorometric analysis.

Filtration volume will vary with sampling location. For oligotrophic waters, 4 liters are filtered, whereas in coastal regions a smaller volume (0.5-1.0 liters) may be appropriate. In
this case, a 25 mm GF/F filter is recommended.

7.0 Procedure
7.1 After removal from liquid nitrogen, the pigments are extracted by placing the filters in 5.0 ml 100% acetone. For 47 mm GF/F filters, 0.8 ml of water is retained on the filter, adjusting the final extraction solution to 86% acetone and the final extraction volume to 5.8 ml. In order to correct for any errors introduced by evaporation or experimental losses, 100 ml of an internal standard (canthaxanthin in acetone, Fluka)
is added to each sample which elutes after zeaxanthin and before chlorophyll b. The samples are covered with Parafilm to reduce evaporation, sonicated (0°C, subdued light) and allowed to extract for 4 hours in the dark at -20°C. Following extractionsamples are vortexed, filters are pressed to the bottom of the tube with a stainless
steel spatula, and centrifuged for 5 minutes to remove cellular debris. External standards are also run before each sample set for daily HPLC calibration.

The addition of 5.0 ml acetone for pigment extraction is necessary to completely submerge 47 mm GF/F filters in 15 ml centrifuge tubes. However, this volume can be altered depending on the sizes of the filter and the extraction tube.

7.2 The HPLC system is setup and equilibrated with solvent system A at a flow rate of 1 ml/min.

7.3 Samples and standards are prepared for injection by mixing a 1 ml aliquot of the pigment extract with 300 ml of distilled water in a 2 ml amber vial. Shake and allow to equilibrate for 5 minutes prior to injection.

7.4 Approximately 500 ml of a sample is injected into the 200 ml sample loop and the three-step solvent program initiated is on closure of the injection valve. The chromatogram is then collected on a recording device.

7.5 The identities of the peaks from the sample extracts are determined by comparing their retention times with those of pure standards and algal extracts of known pigment composition. Peak identities can be confirmed spectrophotometrically by collecting eluting peaks from the column outlet.

7.6 Calibration: The HPLC system is calibrated with pigment standards obtained commercially (chlorophylls a and b, and ß-carotene can be purchased from Sigma Chemical Co., and zeaxanthin and lutein from Roth Chemical Co.) and/or by preparative scale HPLC (collecting and purifying HPLC fractions and placing in standard solvents) standards. Concentrations of pigment standards should be determined using a monochromator-based spectrophotometer in the appropriate solvents prior to the calibration of the HPLC system. The recommended extinction coefficients for most of the common algal pigments are provided in Table 13-2 (Bidigare 1991). Pigment standard concentrations are calculated as follows:
        C_s= (A_max -   A_750nm) / (E * l) * 1000mg / 1g
where:
        Cs = pigment concentration (mg l _-1 )
        A _max = absorbance maximum (Table 2)
        A_{750 nm} = absorbance at 750 nm to correct for light scattering
        E = extinction coefficient (L g -1 cm -1 , Table 2)
        l = cuvette path length (cm)

Standards stored under nitrogen in the dark at -20°C are stable for approximately one month.

After determining the concentrations of the pigment standard they are injected onto an equilibrated HPLC system to calculate standard response factors (RF). Response factors are calculated as weight of standard injected (determined spectrophotometrically) divided by the area of the pigment standard plus areas of structurally related isomers, if present.

8.0 Calculation and expression of results
Concentration of the individual pigments in the sample are calculated using the following formula:
        C_i = ( A ) * ( RF ) * ( 1 / IV) * ( EV ) * ( 1 / SV )
where:

        C_i = individual pigment concentration (ng per liter)
        A = integrated peak area
        RF = standard response factor
        IV = injection volume
        EV = extraction volume with internal standard correction
        SV = sample volume
The units of ng kg ^-1 can be obtained by dividing this result by the density of the seawater.

9.0 References
Bidigare, R. (1991). in Spencer and Hurd (eds.). The analysis and characterization of marine particles. American Geophysical Union, Washington D.C.
Herbland, A., A. Le Bouteiller, and P. Raimbault. (1985). Size structure of phytoplankton biomass in the equatorial Atlantic Ocean. Deep-Sea Res. 32:819-836.
Holm-Hansen, O., and B. Riemann. (1978). Chlorophyll a determination: improvements in methodology. Oikos, 30: 438-447.
Wright, S.W., S.W. Jeffrey, F.C. Mantoura, C.A. Llewellyn, T. Bjørnland, D. Repeta, and N. Welschmeyer (1991). Improved HPLC method for the analysis of chlorophylls and carotenoids from marine phytoplankton. Mar. Ecol. Prog. Ser. 77:183-196.