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A. Pérez-Gálvez and J. Fontecha
families of pigments are biological markers of a wide array of essential indicators.
Consequently, their simultaneous analysis is a fundamental piece for acquisition of
useful data. However, the amount of chlorophyll derivatives and carotenoid types that
potentially may co-exist in cyanobacteria, as well as their coincident polarity properties complicate their simultaneous analysis by HPLC. Coelution is a latent source
of artifacts and equivocal identification and quantification. To avoid this problem
several alternatives are available, although the shortcut solution is to hydrolyze the
extract when the focus is solely the carotenoid profile (see Sect. 7.2.1). The analyst
faces a different context when the target of the study is the chlorophyll profile. In
that case, no technique has been developed so far to obtain a chlorophyll extract
free of carotenoids, and the HPLC method must finely resolve the separation of both
families of pigments (see Chap. 6).
The method developed by Wright et al. (1991) has been the benchmark in the
measurement of carotenoids and chlorophylls from phytoplankton materials and the
starting point for establishing alternative HPLC conditions with the same aim. Almost
50 different pigments may potentially separate in a C 18 column using a ternary
gradient system in a 30 min run. The quantification is made through the internal
standard method, adding canthaxanthin to the extract. This is a handicap of the
procedure as the internal standard co-elutes with zeaxanthin, when this xanthophyll
is present in the carotenoid profile. However, the ability of this method to separate
the substantial amount of pigments deserves the attention of the reader.
Table 7.1 contains the description of several HPLC approaches applied for separation of pigments from phytoplankton. These methods may be used by the reader as
a reference for direct application, or to introduce improvements in analytical studies.
The progress to be noted in this section is the progressive increasing number of procedures reporting the use of alternative columns to the classical octadecyl (C 18 ) packing
material. Thus, napthylethyl or cholesteryl bonded silica (Indriatmoko et al. 2015)
are recent stationary phases applied to the separation of photosynthetic pigments.
Different interactions between analytes and stationary phase in pentafluorophenyl
C 18 silica column have been used as an advantage in the simultaneous analysis of
chlorophylls and carotenoids, what it is very convenient in the analysis of green
plankton populations and cyanobacteria (Sanz et al. 2015). Methods based in octyl
silica (Zapata et al. 2000) and C 30 columns (Guarantini et al. 2009) are also remarked
considering their ability to produce chromatograms where carotenoid isomers and
other carotenoids with similar structural arrangements are separated. Peak resolution
of polar compounds is also improved with the application of a palmitamidopropylsilane bonded column that it is practical in the separation of complex pigment
profiles (Jayaraman et al. 2011). The reader is also referred to the optimized method
published by van Leeuwe et al. (2006), which was tested with a broad range of
phytoplankton species to obtain a high sensitivity and reproducibility. Improvement
of the chromatographic resolution was also the aim of the method published by
Airs et al. (2001), particularly useful in the resolution of complex pigment profiles.
Within all this milieu, the reader should clearly note that other methodological factors
beyond the chromatographic conditions have a direct consequence in the ability of
A. Pérez-Gálvez and J. Fontecha
families of pigments are biological markers of a wide array of essential indicators.
Consequently, their simultaneous analysis is a fundamental piece for acquisition of
useful data. However, the amount of chlorophyll derivatives and carotenoid types that
potentially may co-exist in cyanobacteria, as well as their coincident polarity properties complicate their simultaneous analysis by HPLC. Coelution is a latent source
of artifacts and equivocal identification and quantification. To avoid this problem
several alternatives are available, although the shortcut solution is to hydrolyze the
extract when the focus is solely the carotenoid profile (see Sect. 7.2.1). The analyst
faces a different context when the target of the study is the chlorophyll profile. In
that case, no technique has been developed so far to obtain a chlorophyll extract
free of carotenoids, and the HPLC method must finely resolve the separation of both
families of pigments (see Chap. 6).
The method developed by Wright et al. (1991) has been the benchmark in the
measurement of carotenoids and chlorophylls from phytoplankton materials and the
starting point for establishing alternative HPLC conditions with the same aim. Almost
50 different pigments may potentially separate in a C 18 column using a ternary
gradient system in a 30 min run. The quantification is made through the internal
standard method, adding canthaxanthin to the extract. This is a handicap of the
procedure as the internal standard co-elutes with zeaxanthin, when this xanthophyll
is present in the carotenoid profile. However, the ability of this method to separate
the substantial amount of pigments deserves the attention of the reader.
Table 7.1 contains the description of several HPLC approaches applied for separation of pigments from phytoplankton. These methods may be used by the reader as
a reference for direct application, or to introduce improvements in analytical studies.
The progress to be noted in this section is the progressive increasing number of procedures reporting the use of alternative columns to the classical octadecyl (C 18 ) packing
material. Thus, napthylethyl or cholesteryl bonded silica (Indriatmoko et al. 2015)
are recent stationary phases applied to the separation of photosynthetic pigments.
Different interactions between analytes and stationary phase in pentafluorophenyl
C 18 silica column have been used as an advantage in the simultaneous analysis of
chlorophylls and carotenoids, what it is very convenient in the analysis of green
plankton populations and cyanobacteria (Sanz et al. 2015). Methods based in octyl
silica (Zapata et al. 2000) and C 30 columns (Guarantini et al. 2009) are also remarked
considering their ability to produce chromatograms where carotenoid isomers and
other carotenoids with similar structural arrangements are separated. Peak resolution
of polar compounds is also improved with the application of a palmitamidopropylsilane bonded column that it is practical in the separation of complex pigment
profiles (Jayaraman et al. 2011). The reader is also referred to the optimized method
published by van Leeuwe et al. (2006), which was tested with a broad range of
phytoplankton species to obtain a high sensitivity and reproducibility. Improvement
of the chromatographic resolution was also the aim of the method published by
Airs et al. (2001), particularly useful in the resolution of complex pigment profiles.
Within all this milieu, the reader should clearly note that other methodological factors
beyond the chromatographic conditions have a direct consequence in the ability of