Abstract
In the experiment reported the results obtained with two different columns for the RP-HPLC analysis of phytoplankton pigment components were compared. LiChroCART Hypersil ODS and LiChroCART LiChrospher 100 RP18e columns were used exchangeably. Two groups of high-purity pigment standards of reference phytoplankton strains (23 chlorophylls and carotenoids in the first case and 25 in the second case) were subjected to chromatographic analysis. All calibration parameters (retention times, calibration curves, spectrum shapes, detection thresholds) for qualitative and quantitative identification of pigments were determined. The results indicate that both columns used in the analysis ensure sufficient resolution and can be used interchangeably for the analysis of pigments present in naturally existing phytoplankton, even when present in only trace amounts.
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Introduction
Since the 1980s, reverse-phase high performance liquid chromatography (RP-HPLC) has been commonly used for the separation, identification and quantification of large groups of polar and non-polar pigments. Normal-phase HPLC systems are best suited for separating only a few groups of pigments with similar polarities and structures (Gieskes and Kraay 1983). The idea to employ the RP-HPLC in environmental studies was introduced by Mantoura (Mantoura and Llewellyn 1983) and has been developed by many researchers (Bidigare et al. 1985; Wright et al. 1991; Wright and Jeffrey 1997; Heukelem and Thomas 2001; Marty et al. 2002; Rodriguez et al. 2002; Brotas and Plante-Cuny 2003). The resolution of compounds is based on adsorption and desorption processes on the coating of chromatographic columns (stationary phase) in the presence of the suitable solvents’ composition (mobile phase).
There are many disciplines of environmental studies in which qualitative and quantitative characteristics of pigments obtained by the use of chromatographic techniques have found applications and are indispensable. The use of taxonomically important chlorophylls and carotenoids in the direct identification of phytoplankton classes, their biomass, condition and stage of development in seawater samples has increased due to advancements in these techniques (Wright and Enden 2000; Mackey et al. 2002). The knowledge of photosynthetic and photoprotectant pigment concentrations facilitates the determination of the absorption properties of phytoplankton (Woźniak et al. 2000) and of the rate of photosynthesis in natural waters (MacIntyre et al. 2000) approximated from satellites (Woźniak et al. 1997; Claustre et al. 2004). Additionally, the radioactively labeled taxon-specific pigments can be used to distinguish primary producers in the field (Gieskes and Kraay 1989) and for the mathematical analysis of pigment fingerprints (Gieskes et al. 1988; Rodriguez et al. 2002).
Solvents and their time-variable composition, temperature-controlled systems (ensuring stable retention times) and different columns were tested during years of investigations. Octadecylo silane (ODS) C18, C8 and even C30 chain columns with different diameters, lengths and particle sizes have, with different degrees of effectiveness, found application in the analysis of pigments (Schmid and Stich 1995; Zapata et al. 2000; Barlow et al. 2002; Rodriguez et al. 2002). Octadecyl silica C18 columns (with polymeric, as well as monomeric coating) have been commonly used for the determination of algal pigments. At present, over 50 phytoplankton pigments and their degradation products can be separated in a single analysis (Wright et al. 1991; Wright and Jeffrey 1997). Polymeric C18 columns were used to separate carotenoid pairs, such as lutein and zeaxanthin, 19′hexanoyloxyfucoxanthin and 9′cis-neoxanthin, and α- and β-carotene (Heukelem et al. 1992). The employment of C8 columns allowed separation of divinyl and monovinyl chlorophyll a and b (Goericke and Repeta 1992). In addition, the incorporation of a pyridine-containing mobile phase to the HPLC pigment analysis, either in combination with polymeric C18 columns or monomeric C8 columns, allows separation of chlorophyll c pigments (Zapata et al. 2000, 2001). The pigment resolution may also be enhanced by adjusting the temperature of the column (Heukelem et al. 1994). Also the solvent composition can be modified, for example, by the use of highly ionic solvents with chlorophylls c1, c2 and c3 separated on the C18 column (Kraay et al. 1992).
The RP-HPLC technique is highly accurate (±6%, Latasa et al. 1996; Claustre et al. 2004) in routine pigment measurements, even for trace amounts of <0.1 μg dm−3 (Roy et al. 1996; Stoń et al. 2002). The qualification and quantification of individual chlorophylls and carotenoids is mainly based on their properties in the visible light range. The identification relies on comparisons of the observed diode array spectroscopy during elution, or by transferring HPLC fractions to standard solvents and comparing their visible absorption spectra with reference standards or literature values, or by co-chromatography with authentic standards (Wright et al. 1991). Quantitative characterization of eluted pigments is based on peak area and excitation coefficients published in the literature (Mantoura and Llewellyn 1983; Latasa et al. 1996; Jeffrey 1997a; Bjørland et al. 2003) and on Lambert–Beer’s law. Concentrations of pigments can also be calculated using internal (e.g. canthaxanthin) and external standards (Andersen et al. 1996; Mantoura and Repeta 1997; Obayashi and Tanoue 2002; Qian et al. 2003).
A limited number of high-purity pigment standards exists for the calibration of spectrometers and HPLC detectors. Due to difficulties resulting from a very labile structure of pigment molecules, susceptible to degradation by light, heat, oxygen, acids and bases, the preparation of pigment standards is very complicated. Some synthetic chlorophyll and carotenoid standards are available from commercial sources, e.g. chlorophyll a and b, canthaxanthin, lutein, lycopene, zeaxanthin, α-carotene and β-carotene. In most cases, laboratories prepare and purify their own standards from reference phytoplankton monocultures with well-known pigment composition, or from other sources, such as tomatoes, leaves of maize, or other higher plants. Detailed information, full description and guidelines for the preparation of 17 chlorophyll and 29 carotenoid standards, chemotaxonomically significant in phytoplankton ecology, from reference strains of monocultures were given by Jeffrey (1997b) and Repeta and Bjørland (1997). In general, the approach is the same: pigments are extracted from suitable biological sources by organic solvents, purified by thin-layer or semi-preparative liquid chromatography, crystallized and transferred to an appropriate solvent (90% acetone in the case of chlorophylls; ethanol, diethyl ether, hexane, or petroleum ether for carotenoids) and characterized by spectrophotometry.
The aim of this paper is to present the results of comparative analysis of chromatographic resolution by the use of two types of columns: LiChroCART Hypersil ODS and LiChroCART LiChrospher 100 RP18e. Two groups of high-purity pigment standards from The International Agency for 14C Determination, DHI Institute for Water and Environment, Denmark, were subjected to chromatographic analysis in order to calibrate these two HPLC systems. The external calibration results obtained (such as spectrum shapes in appropriate solvent compositions, retention times, slopes of calibration curves, sensitivity, or detection thresholds) provide possibilities to use interchangeable analytical columns for separation, isolation and quantification of the pigments present in environmental samples.
Materials and methods
Pigment standards
Two groups of high-purity certified chlorophyll and carotenoid standards, isolated from reference strains of phytoplankton monocultures, were obtained from The International Agency for 14C Determination, DHI Institute for Water and Environment, Denmark. The first group of 23 pigments consisted of chlorophylls (a, b, c, c2, c3) and carotenoids (alloxanthin, antheraxanthin, aphanizophyll, 19′butanoyloxyfucoxanthin, canthaxanthin, α- and β-carotenes, diadinoxanthin, echinenone, fucoxanthin, 19′haxanoyloxyfucoxanthin, lutein, myxoxanthophyll, neoxanthin, peridinin, prasinoxanthin, violaxanthin, zeaxanthin). The second group consisted of 25 pigments, chlorophylls a, b, c2 and c3, chlorophyllide a, divinyl chlorophyll a, pheophythin a, alloxanthin, antheraxanthin, 19′butanoyloxyfucoxanthin, canthaxanthin, α- and β-carotenes, diadinoxanthin, diatoxanthin, echinenone, fucoxanthin, 19′haxanoyloxyfucoxanthin, lutein, myxoxanthophyll, neoxanthin, peridinin, prasinoxanthin, violaxanthin and zeaxanthin. Pigments were supplied in 2.5 cm3 sealed vials: the extracts were in 90% acetone for chlorophylls and in 100% ethanol for carotenoids. The parameters of reference pigments are given in Table 1.
These compounds were subjected to chromatographic analysis. Pigment standards were injected onto a chromatographic system, calibration curves were prepared and many factors (such as the response factor fp and detection thresholds) were established.
Apparatus
Pigments were isolated using the RP-HPLC technique. The chromatographic system was equipped with a Hewlett-Packard HP 1050 pump, diode array detector (model HP 1100), an HP 1046 fluorescence detector and a Rheodyne injector with a 100 μl sample loop. Two types of C18 analytical columns: LiChroCART Hypersil ODS (dimension: 250×4 mm, particle size: 5 μm; Merck) and LiChroCART LiChrospher 100 RP18e (dimension: 250×4 mm, particle size: 5 μm; Merck) were used for comparative analysis of analytical column responses and efficiency.
The diode array absorbance detector “dad” was set at λ=440 nm. During a single analysis, the full spectral range of 350–700 nm, with steps of 1 nm and a 0.4 s time resolution, was recorded. The fluorescence detector with the excitation wavelength λex=431 nm and emission λem=660 nm was only used to confirm the presence of chloropigments in the extract.
Solvents and chromatographic analysis
The solvents used for chromatography were filtered and degassed with helium before use. Mobile phases used in the gradient elution were composed of the primary eluant (A), consisting of methanol and 1 M ammonium acetate (80:20 v/v), and the secondary eluant (B), prepared from methanol and acetone (60:40 v/v). In addition, 1 M ammonium acetate was used as the ion-pairing reagent, and it is recommended that it be present in both the sample and the mobile phase, to improve pigment separation and suppress the dissociation of the isolated compounds (Mantoura and Llewellyn 1983). The separation was achieved by changing the solvent mixture composition (Mantoura and Llewellyn 1983; Barlow et al. 1993; Stoń and Kosakowska 2002). The solvent composition was changed linearly from 100% of solvent A to 100% of solvent B in 10 min after injection, and was isocratically held at a constant flow rate of 0.8 ml min−1 till the end of the analysis (25 and 32 min for first and second type of column, respectively). The equilibrium state was attained after 10 min, when the solvent composition returned to the initial conditions.
Statistical analysis
The repeatability of the chromatograms and the method’s precision were also checked. The analyses were based on monocultures grown in laboratory conditions and using pigment standards. The statistical analysis was based on 36 repetitions of the chromatographic resolution of the extracts. The margin of error of the adopted method ranged from 1.28% to 5.55%, with a mean value of ±2.9%.
Results
Chromatographic resolution
The separation of chlorophylls and carotenoid pigment standards by the use of two types of columns yielded satisfactory results. Sufficient resolution of all reference pigments in a single run was achieved. Representative absorbance chromatograms of the pigment standard mixture are shown in Fig. 1. The results obtained indicate that chlorophylls c1 and c2 elute as a single peak. Likewise, lutein and zeaxanthin were not completely resolved in either type of analytical column. A comparison of the results obtained with pigments separated on different columns showed some differences in the elution times of individual components of the mixtures. Although the sequence of retained and eluted pigments remained mostly the same in the two systems, in the case of LiChroCART Hypersil ODS, the retention times of pigments were shorter than those found with LiChroCART LiChrospher 100 RP18e. The greatest differences in elution times were observed for α-carotene and β-carotene (~7 to 8 min). This delay may have resulted from the molecular structure of these two non-oxygenated compounds. As a consequence, the time of a single analysis was prolonged from 25 min for LiChroCART Hypersil ODS to 32 min for LiChroCART LiChrospher 100 RP18e. The values of the chromatographic parameters obtained for each pigment in the two columns are given in Table 2.
Purity of pigments
The purity of chlorophyll and carotenoid standards (>95%) was confirmed by an analytical chem-station procedure. This procedure allows assessment of whether the eluting peak is spectrally pure, or contains impurities due to simultaneous retention of several compounds, or incomplete separation of peaks, and/or background absorption The peak shape and purity curves are shown in Fig. 2 for fucoxanthin.
Spectral properties of chlorophylls and carotenoids
Identification and quantification of pigments by HPLC is based on the characteristic absorption properties of these compounds in the visible spectral range. Pigments differ from each other in the shape and position of their absorption maxima. Carotenoids absorb light in the range 400–500 nm, displaying two or three distinct absorption maxima in this region. Chlorophylls additionally absorb in the red part of the light spectrum. The ability of carotenoids to absorb light is due to their system of regularly alternating single and double bonds (a chromophore). The chromophore structure in algal carotenoids consists of 9–11 aliphatic double bonds in conjunction with no, one, or two endocyclic double bonds. Spectral properties of carotenoids are not influenced by functional groups. Under given conditions (solvent, temperature) the spectrum is a characteristic feature of the absorbing molecules, and is therefore used not only in quantitative measurements but also to confirm the identity of the pigment.
Results of the comparative analysis of the spectra of two groups of reference pigments are presented in Fig. 3. The wavelengths of the absorption maxima differed between pigments and were generally shifted by about 1–5 nm relative to the same pigments separated on the other type of column. The spectra overlap only in the cases of neoxanthin and alloxanthin (Fig. 3, 9 and 15). Regarding other pigments, the shifts in the absorption maxima result from different retention times and, hence, different compositions of solvents during elution. The respective values of λmax of individual pigments are listed in Table 2.
Calibration parameters
Calibration curves were prepared based on a series of injections of known concentrations of pigment standards on the chromatographic system. Six to ten different concentrations were injected for each pigment. Such a method of external standard calibration allows establishment of the relationships between the weight of the pigment injected on the chromatographic column and its peak area.
The resultant chromatographic peak areas are related to pigment masses by the response factor (or calibration curve). Calibration factors for each pigment, designated “response factors” (fp, ng mAU−1 s−1) are obtained. The value of parameter fp is representative of an individual pigment isolated from the pigment mixture. The fp values and correlation coefficients obtained by the use of two types of chromatographic columns are given in Table 2. The calibration curves for two groups of standards are shown in Fig. 4.
Discussion
Within the framework of the experiment presented, the separation ability of two C18 chromatographic columns (LiChroCART Hypersil ODS and LiChroCART LiChrospher 100 RP18e) was compared. Different pigment standards were subjected to chromatography in order to obtain calibration parameters. The standard group encompassed 8 chlorophylls and 19 different carotenoids, with different concentrations (Table 1). For example, the concentration of lutein was equal to 0.659 mg dm−3 in the first group, but in the second group was almost twice as high (1.314 mg dm−3). Pigments originated from reference phytoplankton classes. The role of marker pigments in identifying and quantifying individual species has been well recognized. For example, fucoxanthin is considered to be a marker of diatoms; zeaxanthin-cyanobacteria; 19′-hexanoyloxyfucoxanthin-prymnesiophytes; 19′butanoyloxyfucoxanthin-pelagophytes; alloxanthin-cryptophytes; prasinoxanthin-prasinophytes; peridinin-dinoflagellates; and chlorophyll b and lutein-chlorophytes (Andersen et al. 1996; Jeffrey and Vesk 1997).
The chromatographic system and procedure presented herein can be used to separate chlorophylls and their derivatives, as well as carotenes and their oxygenated forms, called xanthophylls. Only in the case of zeaxanthin and lutein were the resolution results unsatisfactory. Complete resolution of the above pigment pair was accomplished only by the use of a polymeric-packed chromatographic column (Heukelem et al. 1992; Schmid and Stick 1995). A single analysis allowed isolation and resolution of a mixture of 25 pigments present in the extract. Only the duration of the analysis depends on the type of column used (25 min for LiChroCART Hypersil ODS and 32 min for LiChroCART LiChrospher 100 RP18e); the sequence of eluted pigments remains unchanged.
The duration of analysis depends on the isolation system applied (e.g. solvents, appropriate temperature) and on the type of column, its diameter, particle size, pore size and number of plates. The analysis may last as long as 60 min (Heukelem and Thomas 2001); 40 min when a C8 column (150×4.6 mm, 3.5 μm particle size) thermostated at 25°C is employed (Zapata et al. 2000), or 20 min when using 3 μm Hypersil MOS (100×4.6 mm, C8; Shandon) for separation of chlorophyll a from divinyl chlorophyll a and zeaxanthin from lutein (Vidussi et al. 1996).
The calibration parameters obtained during the analysis were different for two kinds of stationary phases. Spectral characteristics allowing qualitative identification of pigments were very close only in the case of alloxanthin (λmax=426, 454, 483 nm) and neoxanthin (λmax=414, 438, 466 nm). In other pigments, the absorption spectrum was shifted by 1–5 nm (all details are presented in Table 2). Spectral characteristics vary not only between individual pigments but also with temperature and solvent within the pigment itself. The data published by Jeffrey and Humphrey (1975) and Bjørland (1997) present data on how solvent effects the algal carotenoid chromofores. The different λmax values and different shapes of spectra presented in Fig. 3 may have resulted from not the same solvent composition during retention.
The slopes of the calibration curves differed not only between different pigments but also for the same pigment when different columns were used. Different response factors (fp, ng mAU−1 s−1) were observed in most cases. The values of factor fp are representative of individual pigments isolated from the pigment mixture. The linear approximation of fp, which resulted from a sequence of injections of fixed pigment amounts onto chromatographic systems, yielded satisfactory correlation (correlation coefficients ranged from 0.92 to 0.99 in the case of the first group and from 0.6 to 0.99 for the second group of pigments). Poor correlation (0.59, wide scatter of points) was noted for pheophythin a. This pigment also has a higher detection threshold (36.3 ng per 100 μl), which means that pheophythin a can only be determined in samples with a high content of degradation products. Other chlorophyll a derivatives, chlorophyllide a and divinyl chlorophyll a, revealed little scatter of points, and, thus, the values of fp were close to 1 (0.98 and 0.94, respectively).
Slopes of the calibration curves of chlorophyll c3, antheraxanthin, echinenone and chlorophyll a were similar for both column types. Slopes of the calibration curves of chlorophyll c2, peridinin, fucoxanthin, neoxanthin, prasinoxanthin, diadinoxanthin, alloxanthin, myxoxanthophyll, lutein, zeaxanthin, chlorophyll b and β-carotene were steeper for the second group of pigments, separated by LiChroCART LiChrospher 100 RP18e. In the other cases the situation was reversed.
The detection thresholds of isolated pigments were very low and in most of the cases did not exceed 1 ng per 100 μl. The comparison of the detection thresholds of chlorophylls and carotenoids in both columns indicated that the second type of columns was more sensitive: from 1.02 times for myxoxanthophyll up to 3.2, 4.06 and 4.34 times for 19′haxanoyloxyfucoxanthin, α-carotene and neoxanthin, respectively. Only prasinoxanthin, antheraxanthin, chlorophyll a and β-carotene could be detected at lower concentrations by the use of the LiChroCART Hypersil ODS analytical column. The minimum delectable concentration of β-carotene was similar in both systems ~3.3 and 3.35 ng per 100 μl. The results obtained also indicate that pheophythin a is difficult to detect because of its very high threshold concentration, 36.7 ng per 100 μl.
In conclusion, employment of the chromatographic systems discussed here allows precise determination of the pigments present in natural samples, even if only present in low amounts (<0.1 μg dm−3). Our experiments have provided unique calibration parameters for RP-HPLC systems equipped with two types of columns: LiChroCART Hypersil ODS and LiChroCART LiChrospher 100 RP18e. Sufficient resolution of all pigments present in the mixture was achieved. Both columns used provided similar results in the isolation of pigments. Except for two pigment pairs, the chlorophylls c1 and c2 and zeaxanthin and lutein, all other chlorophylls and carotenoids injected onto the chromatographic columns were separated to the baseline. Even though zeaxanthin and lutein are hard to separate, the absorption spectrum of combined peaks could allow a statement on which one dominated. Although both pigment groups analyzed differed in amount and type, it was possible to establish calibration parameters for the two HPLC systems. Unfortunately, only the group of compounds tested on LiChroCART LiChrospher 100 RP18e possessed chlorophyll a derivatives (chlorophyllide a and pheophythin a), which may exclude the use of the LiChroCART Hypersil ODS analytical column in certain environmental analyses, for example, of sediment samples. Pigment concentrations in samples of naturally living phytoplankton can be determined by the use of both types of columns. The pigments analyzed are characteristic biomarkers of different phytoplankton groups. The LiChroCART LiChrospher 100 RP18e column was tested on a wide group of pigments and in most chlorophylls and carotenoids yielded better detection results. However, in the case of prasinoxanthin, antheraxanthin, chlorophyll a and β-carotene, the use of LiChroCART Hypersil ODS allows detection of these pigments at lower concentrations. In conclusion, the columns used in the analysis are interchangeable.
References
Andersen RA, Bidigare RR, Keller MD, Latasa M (1996) A comparison of HPLC pigment signatures and electron microscopic observations for oligotrophic waters of the North Atlantic and Pacific Oceans. Deep-Sea Res II 43:517–537
Barlow RG, Mantoura RFC, Gough MA, Fileman TW (1993) Pigment signatures of the phytoplankton composition in the north-eastern Atlantic during the 1990 spring bloom. Deep-Sea Res II 40:459–477
Barlow RG, Aiken J, Holligan PM, Cummings DG, Maritorena S, Hooker S (2002) Phytoplankton pigment and absorption characteristics along meridional transects in the Atlantic Ocean. Deep-Sea Res I 49:637–660
Bidigare RR, Kennicutt II MC, Brooks JM (1985) Rapid determination of chlorophylls and their degradation products by high-performance liquid chromatography. Limnol Oceanogr 30:432–435
Bjørnland T (1997) UV-vis spectroscopy of carotenoids. In: Jeffrey SW, Mantoura RFC, Wright SW (eds) Phytoplankton pigments in oceanography: guidelines to modern methods. UNESCO, Paris, pp 578–594
Bjørnland T, Haxo FT, Liaaen-Jensen S (2003) Carotenoids of the Florida red tide dinoflagellate Karenia brevis. Biochem Syst Ecol 31:1147–1162
Brotas V, Plante-Cuny MR (2003) The use of HPLC pigment analysis to study microphytobenthos communities. Acta Oecol 24:109–115
Claustre H, Hooker SB, Heukelem L, Berthon JF, Barlow R, Ras J, Sessions H, Targa C, Thomas CS, Linde D, Marty JC (2004) An intercomparison of HPLC phytoplankton pigments methods using in situ samples: application to remote sensing and database activities. Mar Chem 85:41–61
Gieskes WWC, Kraay GW (1983) Dominance of Cryptophyceae during the phytoplankton spring bloom in the central North Sea detected by HPLC analysis of pigments. Mar Biol 75:179–185
Gieskes WWC, Kraay GW (1989) Estimating the carbon-specific growth rate of the major algal species groups in eastern Indonesian waters by 14C labeling of taxon-specific carotenoids. Deep-Sea Res 36:1127–1139
Gieskes WWC, Kraay GW, Nontji A, Setiapermana D, Sutomo (1988) Monsoonal alternation of a mixed and a layered structure in the phytoplankton of the euphotic zone of the Banda Sea (Indonesia): a mathematical analysis of algal pigment fingerprints. Neth J Sea Res 22:123–137
Goericke R, Repeta DJ (1992) The pigments of Prochlorococcus marinus: presence of divinyl chlorophyll a and b in marine prokaryotes. Limnol Oceanogr 37:425–433
Heukelem L, Thomas CS (2001) Computer-assisted high-performance liquid chromatography method development with applications to the isolation and analysis of phytoplankton pigments. J Chromatogr A 910:31–49
Heukelem L, Lewitus AJ, Kana TM (1992) High-performance liquid chromatography of phytoplankton pigments using a polymeric reverse-phase C18 column. J Phycol 28:867–872
Heukelem L, Lewitus AJ, Kana TM, Craft NE (1994) Improved separations of phytoplankton pigments using temperature-controlled high performance liquid chromatography. Mar Ecol Prog Ser 114:303–313
Jeffrey SW (1997a) Chlorophyll and carotenoid excitation coefficients. In: Jeffrey SW, Mantoura RFC, Wright SW (eds) Phytoplankton pigments in oceanography: guidelines to modern methods. UNESCO, Paris, pp 595–596
Jeffrey SW (1997b) Preparation of chlorophyll standards. In: Jeffrey SW, Mantoura RFC, Wright SW (eds) Phytoplankton pigments in oceanography: guidelines to modern methods. UNESCO, Paris, pp 207–238
Jeffrey SW, Humphrey GF (1975) New spectrophotometric equations for determining chlorophylls a, b, c1 and c2 in higher plants, algae, and natural phytoplankton. Biochem Physiol Pflanz (BPP) 167:191–194
Jeffrey SW, Vesk M (1997) Introduction to marine phytoplankton and their pigment signatures. In: Jeffrey SW, Mantoura RFC, Wright SW (eds) Phytoplankton pigments in oceanography: guidelines to modern methods. UNESCO, Paris, pp 37–84
Kraay GW, Zapata M, Veldhuis MJW (1992) Separation of chlorophylls c 1 , c 2 and c 3 of marine phytoplankton by reversed-phase C18 high-performance liquid chromatography. J Phycol 28:708–712
Latasa M, Bidigare RR, Ondrusek ME, Kennicutt II MC (1996) HPLC analysis of algal pigments: a comparison exercise among laboratories and recommendations for improved analytical performance. Mar Chem 51:315–324
MacIntyre HL, Kana TM, Geider RJ (2000) The effect of water motion on the short-term rates of photosynthesis by marine phytoplankton. Trends Plant Sci 5:12–17
Mackey DJ, Blanchot J, Higgins HW, Neveux J (2002) Phytoplankton abundances and community structure in the equatorial Pacific. Deep-Sea Res II 49:2561–2582
Mantoura RFC, Llewellyn CA (1983) The rapid determination of algal chlorophyll and carotenoid pigments and their breakdown products in natural waters by reverse-phase high-performance liquid chromatography. Anal Chim Acta 151:297–314
Mantoura RFC, Repeta DJ (1997) Calibration methods for HPLC. In: Jeffrey SW, Mantoura RFC, Wright SW (eds) Phytoplankton pigments in oceanography: guidelines to modern methods. UNESCO, Paris, pp 407–428
Marty JC, Chiavlerini J, Pizay MD, Avril B (2002) Seasonal and interannual dynamics of nutrients and phytoplankton pigments in the western Mediterranean Sea at the DYFAMED time-series station (1991–1999). Deep-Sea Res II 49:1965–1985
Obayashi Y, Tanoue E (2002) Growth and mortality rates of phytoplankton in the northwestern North Pacific estimated by the dilution method and HPLC pigment analysis. J Exp Mar Biol Ecol 280:33–52
Qian Y, Jochens AE, Kennicutt II MC, Biggs DC (2003) Spatial and temporal variability of phytoplankton biomass and community structure over the continental margin of the northeast Gulf of Mexico based on pigment analysis. Contin Shelf Res 23:1–17
Repeta DJ, Bjørland T (1997) Preparation of carotenoid standards. In: Jeffrey SW, Mantoura RFC, Wright SW (eds) Phytoplankton pigments in oceanography: guidelines to modern methods. UNESCO, Paris, pp 239–260
Rodriguez F, Varela M, Zapata M (2002) Phytoplankton assemblages in the Gerlache and Bransfield Straits (Antarctic Peninsula) determined by light microscopy and CHEMTAX analysis of HPLC pigment data. Deep-Sea Res II 49:723–747
Roy S, Chanut J, Gosselin M, Sime-Ngando T (1996) Characterization of phytoplankton communities in the lower St. Lawrence Estuary using HPLC—detected pigments and cell microscopy. Mar Ecol Prog Ser 142:55–73
Schmid H, Stich HB (1995) HPLC-analysis of algal pigments: comparison of columns, column properties and eluents. J Appl Phycol 7:487–494
Stoń J, Kosakowska A (2002) Phytoplankton pigment designation—an application of RP-HPLC in qualitative and quantitative analysis. J Appl Phycol 14:205–210
Stoń J, Kosakowska A, Łotocka M, Łysiak-Pastuszak E (2002) Pigment composition in relation to phytoplankton community structure and nutrient content in the Baltic Sea. Oceanologia 44:419–437
Vidussi F, Claustre H, Bustillos-Guzman J, Cailliau C, Marty J (1996) Determination of chlorophylls and carotenoids of marine phytoplankton: separation of chlorophyll a from divinyl-chlorophyll a and zeaxanthin from lutein. J Plankton Res 18:2377–2382
Woźniak B, Dera J, Majchrowski R, Ficek D, Koblentz-Mishke OI, Darecki M (1997) PAS initial model of marine primary production for remote sensing application. Oceanologia 39:377–395
Woźniak B, Dera J, Ficek D, Majchrowski R, Kaczmarek S, Ostrowska M, Koblentz-Mishke OI (2000) Model of the in vivo spectral absorption of algal pigments, part 1. Mathematical apparatus. Oceanologia 42:177–190
Wright SW, Enden RL (2000) Phytoplankton community structure and stocks in the eastern Antarctic marginal ice zone (BROKE survey, January–March 1996) determined by CHEMTAX analysis of HPLC pigment signatures. Deep-Sea Res II 47:2363–2400
Wright SW, Jeffrey SW (1997) High-resolution HPLC system for chlorophylls and carotenoids of marine phytoplankton. In: Jeffrey SW, Mantoura RFC, Wright SW (eds) Phytoplankton pigments in oceanography: guidelines to modern methods. UNESCO, Paris, pp 327–342
Wright SW, Jeffrey SW, Mantoura RFC, Llewellyn CA, Bjornland T, Repeta D, Welschmeyer N (1991) Improved HPLC method for the analysis of chlorophylls and carotenoids from marine phytoplankton. Mar Ecol Prog Ser 77:183–196
Zapata M, Rodriguez F, Garrido JL (2000) Separation of chlorophylls and carotenoids from marine phytoplankton: a new HPLC method using reversed phase C8 column and pyridine-containing mobile phases. Mar Ecol Prog Ser 195:29–45
Zapata M, Edvardsen B, Rodriguez F, Maestro MA, Garrido JL (2001) Chlorophyll c2 monogalactosyldiacylglyceride ester (chl c2-MGDG). A novel marker pigment for Chysochromulina species (Haptophyta). Mar Ecol Prog Ser 219:85–98
Acknowledgements
This work was carried out within the framework of the PAS Institute of Oceanology statutory activities, grant no. I.2.2, and was financed in part from the National Project funded by the Polish State Committee for Scientific Research no. PBZ-KBN 056/P04/2001 and grant no. 2 PO4 052 26/2004–2006.
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Stoń-Egiert, J., Kosakowska, A. RP-HPLC determination of phytoplankton pigments—comparison of calibration results for two columns. Marine Biology 147, 251–260 (2005). https://doi.org/10.1007/s00227-004-1551-z
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DOI: https://doi.org/10.1007/s00227-004-1551-z