7 Analytical Protocols in Carotenoid Analysis
157
(Jeffrey and Humphrey 1975; Wellburn 1994; Porra 2006; Picazoet al. 2013). Basically, these methods determine the absorbance value at some wavelengths that are
pigment(s)-specific peaks and taking the zero value from the region at 700–750 nm
of the spectrum. The measured values are introduced in equations obtained empirically to determine the pigment content. Alternatively, some methods apply a refinedstrategy to rebuild the total spectrum as a weighted sum of all the individual spectra
(Naqvi et al. 2004; Küpper et al. 2000, 2007). This approach contains several biases
as the peak-width variations and pigment peak positions, as well as the contribution of the background spectra to the total determination. These approximations
could be improved by the application of alternative parametrization and fitting of
the boundaries, and Monte-Carlo based simulations of the pigment composition to
avoid aliasing of the samples, so that the predictions are closer to the real values
(Thrane et al. 2015). The reader should evaluate whether these methods are appropriate to solve the scientific question they face. Sometimes it is convenient to sacrifice
exhaustive description to gain in a fast comprehension and acquisition of analytical
data, while statistical power could be increased by introducing many replicates, and
still this technique is cost-effective. However, if the specific pigment composition
is required to answer questions regarding biosynthesis, new or unprecedent structural arrangements arising from environmental conditions, and exhaustive identification and quantification, liquid chromatographic techniques are the gold standard
procedures.
7.2.3 Liquid Chromatography (LC)
LC is the gold standard analytical technique for separation of the individual chlorophylls and carotenoids contained in the bulk pigment biomass, allowing their identification and quantification by examination of the data obtained from the observed
chromatographic, spectroscopic, and mass properties, once the chromatographic
method is coupled to UV-visible detection, and hyphenated with mass analyzers
and/or nuclear magnetic resonance spectrometers. The development of the chromatographic method is made according to the polarity range of the pigments aimed
to be individually isolated, so that a combination of stationary phase properties and
both eluent composition and elution program is made to achieve a clear-cut separation
and, subsequently, an unequivocal identification. Hence, reversed-phase stationary
phases, i.e., C 18 and C 30 and modern C 18 products (particles produced with “coreshell” morphology, or through organo-silica grafting processes, technologies that
increase the performance of the separation) are the common choice for carotenoid
analysis by HPLC. Indeed, a wide arrange of up-to-date column designs (combining
different internal diameter and length of the column, and particle size of the stationary
phase) allows the acquisition of chromatographic peaks with a high resolving power,
which is a tremendous feature for hyphenation of HPLC with mass spectrometry.
The reader should note that the extracts obtained from phytoplankton biomass, and
particularly from cyanobacteria, contain chlorophylls and carotenoids and that both
157
(Jeffrey and Humphrey 1975; Wellburn 1994; Porra 2006; Picazoet al. 2013). Basically, these methods determine the absorbance value at some wavelengths that are
pigment(s)-specific peaks and taking the zero value from the region at 700–750 nm
of the spectrum. The measured values are introduced in equations obtained empirically to determine the pigment content. Alternatively, some methods apply a refinedstrategy to rebuild the total spectrum as a weighted sum of all the individual spectra
(Naqvi et al. 2004; Küpper et al. 2000, 2007). This approach contains several biases
as the peak-width variations and pigment peak positions, as well as the contribution of the background spectra to the total determination. These approximations
could be improved by the application of alternative parametrization and fitting of
the boundaries, and Monte-Carlo based simulations of the pigment composition to
avoid aliasing of the samples, so that the predictions are closer to the real values
(Thrane et al. 2015). The reader should evaluate whether these methods are appropriate to solve the scientific question they face. Sometimes it is convenient to sacrifice
exhaustive description to gain in a fast comprehension and acquisition of analytical
data, while statistical power could be increased by introducing many replicates, and
still this technique is cost-effective. However, if the specific pigment composition
is required to answer questions regarding biosynthesis, new or unprecedent structural arrangements arising from environmental conditions, and exhaustive identification and quantification, liquid chromatographic techniques are the gold standard
procedures.
7.2.3 Liquid Chromatography (LC)
LC is the gold standard analytical technique for separation of the individual chlorophylls and carotenoids contained in the bulk pigment biomass, allowing their identification and quantification by examination of the data obtained from the observed
chromatographic, spectroscopic, and mass properties, once the chromatographic
method is coupled to UV-visible detection, and hyphenated with mass analyzers
and/or nuclear magnetic resonance spectrometers. The development of the chromatographic method is made according to the polarity range of the pigments aimed
to be individually isolated, so that a combination of stationary phase properties and
both eluent composition and elution program is made to achieve a clear-cut separation
and, subsequently, an unequivocal identification. Hence, reversed-phase stationary
phases, i.e., C 18 and C 30 and modern C 18 products (particles produced with “coreshell” morphology, or through organo-silica grafting processes, technologies that
increase the performance of the separation) are the common choice for carotenoid
analysis by HPLC. Indeed, a wide arrange of up-to-date column designs (combining
different internal diameter and length of the column, and particle size of the stationary
phase) allows the acquisition of chromatographic peaks with a high resolving power,
which is a tremendous feature for hyphenation of HPLC with mass spectrometry.
The reader should note that the extracts obtained from phytoplankton biomass, and
particularly from cyanobacteria, contain chlorophylls and carotenoids and that both