Methodology for High Performance Liquid Chromatography (HPLC) phytoplankton pigment analysis at the LOV (Laboratoire d’Océanographie de Villefranche sur mer) from 1999 to 2004.
Sample collection and storage: 2 to 2.8 litres of water are sampled. They are filtered on 25 mm Whatman GF/F glass fibre filters (0.7µm size particle retention) and stored at -25°C until analysis.
Extraction and analysis (under dim light conditions): Filters are extracted at -20°C for half an hour in 3 mL of HPLC-grade methanol containing an internal standard. They are then ground by ultrasonication and returned to the freezer for another half an hour. The extracts are finally clarified through 25 mm Whatman GF/C filters (1.2 µm size particle retention) and analysed on the same day.
The use of an internal standard (beta-5,8-apocarotenal) was used to correct for possible losses during extraction as well as for the presence of water in the filter. At a later stage (since 2003, the internal standard was replaced with Vitamin E acetate.
The method is a modified version of Vidussi et al. (1996) using a reversed-phase C8 column. A complete resolution between DV Chl a and Chl a and partial resolution between zeaxanthin and lutein were obtained. It has been slightly modified in order to increase the sensitivity of the method (Claustre et al, 2004).
The HPLC system comprises:
o    an Agilent Technologies “Chemstation for LC” software (A.06.03)
o    a degasser (Agilent Technologies 1100)
o    a binary pump (Agilent Technologies 1100)
o    an autosampler (Agilent Technologies 1100 model) with temperature control (4°C) and, sample preparation and automatic injection.
o    A temperature-controlled column compartment
o    a Diode array detector (Agilent Technologies 1100).
o    a Fluorescence detector (Agilent Technologies 1100) with excitation and emission wavelengths respectively at 417 and 670 nm.

The analytical method is based on a gradient separation between a methanol:ammonium acetate (70:30) mixture and a 100% methanol solution, comparable to that described by Vidussi et al. (1996) but with a few differences allowing for improvement of sensitivity. The main characteristics of the method are summarized in the following Table 1:

Table 1 : Characteristics of the HPLC analytical method.
Flow rate    0.5 mL/min
Chromatographic Column    Reversed phase C8, Hypersil MOS, 3µm, 3mm internal diameter, length: 100 mm
Column temperature    25°C
Buffer solution    Ammonium acetate 1N, mixed with sample prior to injection
Time of analysis    22 minutes
Gradient solvents    Solvent A: 70% Methanol + 30% Ammonium acetate 0.5N
Solvent B: 100% Methanol (HPLC grade)
Gradient program    t=0 min: 80% A, 20% B         
t=4.7 min: 50% A, 50% B
t=18.7 min: 0% A, 100% B
t=22.7 min: 0% A, 100%B
Injection volume(sample + buffer)    125 µL + 125 µL
Quantification:
The Agilent Technologies Chemstation software is used for conducting the analysis as well as for post-analytical processing.
Array detection is achieved on all samples. Chlorophylls and carotenoids are detected and quantified (using peak area) from the absorption signal at 440 nm. Quantification of chlorophyll pigments are also done at 667 nm, especially for chlorophyllide a and phaeophorphide a which tend to coelute with carotenoids at 440 nm. The 667 nm signal is found to have a better signal to noise ratio for chlorophyll a detection. When two pigments tend to co-elute at a given wavelength, their identification is done spectrally, the quantification is done according to the spectrally dominant pigment and expressed as a sum. (Example: chlorophyll c1+c2; or Total chlorophyll b = chlorophyll b + divinyl chlorophyll b)
The different pigments that are quantified are recapitulated in Table 2.

PIGMENTS (IN ORDER OF RETENTION TIME)    DETECTION WAVELENGTH    OBSERVATIONS
1. Chlorophyll c3    440    
2. Chlorophyllide a    667    coelution with chlc1+c2
3. Chlorophyll c1+c2    440    
4. Phaeophorbide    667    coelution with peridinin
5. Peridinin    440    
6. 19’-butanoyloxyfucoxanthin    440    
7. Fucoxanthin    440    
8. 19’-hexanoyloxyfucoxanthin    440    Coelution with prasinoxanthin
9. Neoxanthin + violaxanthin    440    coelution
10. Diadinoxanthin    440    
11. Alloxanthin    440    
12. Diatoxanthin    440    
13. Zeaxanthin    440    
14. Lutein    440    
15. Non-polar chlorophyll c1    440    
16. Total chlorophyll b = chlorophyll b + divinyl chlorophyll b    440, 667    coelution
17. Crocoxanthin    440    
18. Divinyl chlorophyll a    440    
19. Chlorophyll a = chlorophyll a + allomers + epimers    440, 667    
20. Non polar chlorophyll c2    440    
21. Carotenes = α-caroten + β-caroten    440    coelution
22. Phaeophytin a    667    
Table 2: List of pigments detected by HPLC at the LOV, their detection wavelengths and their possible coelution with another pigment.
The limits of detection for chlorophyll a and fucoxanthin (based on a signal to noise ratio of 3 and a filtered volume of 2.8 L) were estimated to be 0.0014 mg.m-3 and 0.0023 mg.m-3 respectively.
References
Claustre H., Hooker S.B., Van Heukelem L., et al., 2004. An intercomparison of HPLC phytoplankton pigment methods using in situ samples: application to remote sensing and database activities. Marine Chemistry 85:41-61.
Vidussi, F., Claustre, H., Bustillos-Guzman, J., Cailliau, C., Marty, J.C., 1996. Determination of chlorophylls and carotenoids of marine phytoplankton : separation of chlorophyll a from divinyl-chlorophyll a and zeaxanthin from lutein. Journal of Plankton Research 18, 2377-2382.