Methodology for High Performance Liquid Chromatography (HPLC) phytoplankton pigment analysis at the LOV (Laboratoire d’Océanographie de Villefranche sur mer) until 1998.

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 (-5,8 apocarotenal) was used to correct for possible losses during extraction as well as for the presence of water in the filter.
The method used until 1993 was derived from that of Mantoura and Llewellyn (1983). The general procedure for HPLC pigment analysis, identification and quantification has been described by Claustre et al. (1994). This reversed-phase C18 method, only allowed for a partial resolution of divinyl-chlorophyll a (DV Chl a) from chlorophyll a (Chl a).
For samples from 1994 to 1998, the method described by Vidussi et al. (1996) using a reversed-phase C8 column was applied and a complete resolution between DV Chl a and Chl a and partial resolution between zeaxanthin and lutein were obtained. This method will be described in more detail in this report.
Before 1997, the HPLC system consisted of a LCD Milton Roy pump, a 10 cm (4.6 mm I.D) C18 Hypersil ODS 3 µm column and 2 spectrophotometers (LCD spectro monitor SM 3000 and SM 3100) set respectively at 440 nm and 667 nm. Gradient elutions were as in Williams and Claustre (1991) except that the flow rate was 1 ml.min-1. Peak areas were recorded and calculated using Nelson Analytical software.
Since 1997, the HPLC system comprises a degasser, binary pump, diode array detector set at 440nm and 667 nm (Agilent Technologies 1100 series), a refrigerated autosampler and a fluorescence detector (Thermoquest AS3000 series) and a reversed phase C8, Hypersil MOS, 3µm column (4.6mm x 100 mm), a refrigerated autosampler.  “Chemstation for LC” software (A.06.03) by Agilent Technologies was used.


The analytical method was based on a gradient separation between a methanol:ammonium acetate (70:30) mixture and a 100% methanol solution. The main features of the method (Vidussi et al, 1996) since 1994, are summarized as follows:

Flow rate    1 mL/min
Column temperature    ambient
Buffer solution    Ammonium acetate 1N, mixed with sample prior to injection
Time of analysis    19 minutes
Gradient solvents    Solvent A: 70% Methanol + 30% Ammonium acetate 0.5N
Solvent B: 100% Methanol (HPLC grade)
Gradient program    t=0 min: 75% A, 25 % B; t=1 min: 50% A, 50% B; t=15 min: 0% A, 100% B; t=18.5 min: 0% A, 100%B; t=19 min: 75% A, 25%B
Injection volume(sample + buffer)    200 µL

Quantification and Calibration: Chlorophylls and carotenoids were detected and quantified (using peak area) from the absorption signal at 440 nm. Identification of pigments was performed with retention-time data and comparison of on-line collected absorption spectra with those of a spectral library established from standards and reference cultures obtained from the Villefranche sur mer and Roscoff culture collections. Array detection was achieved on selected samples until 1993 and on all samples since 1994. Quantification of chlorophyll pigments was also done at 667 nm, especially for chlorophyllid a and phaeophorphid a which tend to coelute with carotenoids at 440 nm.  When two pigments tend to co-elute at a given wavelength, their identification is first done spectrally then they are summed. (Example: chlorophyll c1+c2; or Total chlorophyll b = chlorophyll b + divinyl chlorophyll b)
The different pigments that were quantified are recapitulated in Table 1.

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 possible effect of the change of analytical method (from RP-C18 to RP-C8) was tested by analyzing the same samples with the two procedures. The agreement between the two methods was good (+- 5%) of the same order than the agreement between 2 analyses of the same sample using the same method. Special attention was given to the quantification of DV Chl a, which is fully resolved from Chl a in the second part of the experiment. Although partial, the separation of these two compounds in the first phase of our study was sufficient for a good matching of the data from the two methods (equivalent to other pigments) except for low concentrations of DV Chl a (below 5 ng l-1) not detected in the first method.

Claustre, H., Kerhervé, P., Marty, J.C., Prieur, L., Videau, C., Hecq, J.H., 1994. Phytoplankton dynamics associated with a geostrophic front: ecological and biogeochemical implications. Journal of Marine Research 52, 711-742
Mantoura, R.F.C., Llewellyn, C.A., 1983. The rapid determination of algal chlorophyll and carotenoid pigments and their breakdown products in natural waters by reverse-phase high-performance liquid chromatography. Analytica Chimica Acta 151, 293-314.
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.
Williams R. and Claustre H., 1991. Photosynthetic pigments as biomarkers of phytoplankton populations and processes involved in the transformation of particulate organic matter at the Biotrans site(47°N, 20°W). Deep Sea Research 38(3): 347-355.


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.

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