7 Analytical Protocols in Carotenoid Analysis
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profiles that potentially exist in phytoplankton biomass, and SFC may face the test
to resolve the separation of the individual carotenoids (including xanthophyll esters)
in such mixtures. Hence, some methods have been published dealing with SFC and
analysis of carotenoids in microalgae (Abrahamsson et al. 2012; Jumaah et al. 2016).
A suggestion for the reader is to consider the advantages of the SFC when coupling
this separation technique with mass analyzers and nuclear magnetic resonance spectroscopy. The evaporation of the supercritical mobile phase containing the analytes
is easily accomplished in the MS source, while the absence of protons in carbon
dioxide yields “clean” NMR spectra.
7.2.5 Detection Methods and Quantification
7.2.5.1 UV-Visible
Spectrophotometric detection means the first look at the pigment profile. Independently of whether the separation has been fully achieved (coelution), UV-visible
spectrophotometry is an elemental LC-based detection method. The features of the
carotenoid spectrum mainly arise from the polyenoic conjugated chain yielding
three peaks with characteristic λ max values that allow the annotation of some structural arrangements of the carotenoid molecule. Thus, the number of conjugated
double bonds is correlated with the location of the λ max values with shorter wavelengths for lower conjugated systems of double bonds and vice versa. Subsequently,
hypsochromic shift occurs when the spectra of acyclic and cyclic carotenoids with the
same configuration of the polyenoic system are compared. Additionally, deflection
of one of the maximum values arises with the consequent partial loss of fine structure
(peak definition of the spectrum). Further modifications of the carotenoid structure,
such as the introduction of hydroxyl groups, do not introduce changes in the spectra
of carotenoids with the same polyenoic system, so that the distinction of carotenoids
such as β-carotene (carotene), β-cryptoxanthin (monohydroxylated xanthophyll), and
zeaxanthin (dihydroxylated xanthophyll) are equivalent). The esterification of the
hydroxylated xanthophylls with fatty acids does not affect the spectrum of the parent
free xanthophyll. The same “no-effect” in the spectra of carotenoids with carbonyl
groups not conjugated with the polyenoic system is observed, but those structures
where the conjugation takes place show a bathochromic shift of the spectrum and
loss of the fine structure, so that a typical single broad curve is representative of
the carotenoids with conjugated-carbonyl group(s), such as the spectra of canthaxanthin or capsanthin. The introduction of epoxide or furanoid groups produce a
hipsochromic shift of the spectrum as the length of the polyenoic system is decreased.
Table 7.2 contains the absorption maxima for selected carotenoids that appear in the
pigment profile of phytoplankton biomass. The reader should take note that the UVvisible spectrum provides basic structural information but it does not assist in the
identification of geometrical isomers or carotenoids with the same configuration of
the polyenoic system, so that the information obtained from the UV-visible spectrum
165
profiles that potentially exist in phytoplankton biomass, and SFC may face the test
to resolve the separation of the individual carotenoids (including xanthophyll esters)
in such mixtures. Hence, some methods have been published dealing with SFC and
analysis of carotenoids in microalgae (Abrahamsson et al. 2012; Jumaah et al. 2016).
A suggestion for the reader is to consider the advantages of the SFC when coupling
this separation technique with mass analyzers and nuclear magnetic resonance spectroscopy. The evaporation of the supercritical mobile phase containing the analytes
is easily accomplished in the MS source, while the absence of protons in carbon
dioxide yields “clean” NMR spectra.
7.2.5 Detection Methods and Quantification
7.2.5.1 UV-Visible
Spectrophotometric detection means the first look at the pigment profile. Independently of whether the separation has been fully achieved (coelution), UV-visible
spectrophotometry is an elemental LC-based detection method. The features of the
carotenoid spectrum mainly arise from the polyenoic conjugated chain yielding
three peaks with characteristic λ max values that allow the annotation of some structural arrangements of the carotenoid molecule. Thus, the number of conjugated
double bonds is correlated with the location of the λ max values with shorter wavelengths for lower conjugated systems of double bonds and vice versa. Subsequently,
hypsochromic shift occurs when the spectra of acyclic and cyclic carotenoids with the
same configuration of the polyenoic system are compared. Additionally, deflection
of one of the maximum values arises with the consequent partial loss of fine structure
(peak definition of the spectrum). Further modifications of the carotenoid structure,
such as the introduction of hydroxyl groups, do not introduce changes in the spectra
of carotenoids with the same polyenoic system, so that the distinction of carotenoids
such as β-carotene (carotene), β-cryptoxanthin (monohydroxylated xanthophyll), and
zeaxanthin (dihydroxylated xanthophyll) are equivalent). The esterification of the
hydroxylated xanthophylls with fatty acids does not affect the spectrum of the parent
free xanthophyll. The same “no-effect” in the spectra of carotenoids with carbonyl
groups not conjugated with the polyenoic system is observed, but those structures
where the conjugation takes place show a bathochromic shift of the spectrum and
loss of the fine structure, so that a typical single broad curve is representative of
the carotenoids with conjugated-carbonyl group(s), such as the spectra of canthaxanthin or capsanthin. The introduction of epoxide or furanoid groups produce a
hipsochromic shift of the spectrum as the length of the polyenoic system is decreased.
Table 7.2 contains the absorption maxima for selected carotenoids that appear in the
pigment profile of phytoplankton biomass. The reader should take note that the UVvisible spectrum provides basic structural information but it does not assist in the
identification of geometrical isomers or carotenoids with the same configuration of
the polyenoic system, so that the information obtained from the UV-visible spectrum