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A. Pérez-Gálvez and J. Fontecha
C 18 columns coupled to C 30 , and different sequence of the columns for the first
and second dimension, respectively. The LC × LC set-up is also very convenient
for hyphenation with MS as demonstrated by Dugo et al. (2008b) whose were the
first authors to couple LC × LC with MS detection for the analysis of carotenoids.
Thus, the increased resolution of the LC × LC set-up is fully arranged with the
identification power of the mass spectrometry (Giuffrida et al. 2018a, b).
To finalize this section, it is challenging to recommend a LC method among the
different available alternatives. If the reader faces an unknown profile, the application
of the reference method described by van Leeuwe et al. (2006) is advised, with the first
screening of a pigments standard mix for an initial comparison between the known
carotenoid profile and the one of the target sample. Thereafter, the application of
a C 30 -based method is recommended to ascertain the presence of geometric and
cis-trans isomers.
7.2.4 Supercritical Fluid Chromatography (SFC)
Supercritical fluids have a pivotal role in different areas of chemistry, from operation units in sample preparation and isolation, to production processes and organic
synthesis, although their application in analytical chemistry has experimented a
discontinuous progress since the interest in the 80s decade of the last century and the
decline by the start of the new millennium. The commercially available equipment
capable of operating at high temperatures and pressures has not succeeded in routine
chromatography in most of the labs where liquid chromatography systems, either
GC, HPLC, or UHPLC are the standards for analytical separation. However, SFC has
found specific niches where it shows potential advantage over LC systems, considering the faster analysis and the universal coupling with detectors, particularly with
mass analyzers. Indeed, SFC has outstanding applications in the enantioseparation
of chiral analytes and isomers, and in the effective separation of complex mixtures of
structurally closely apolar compounds. Separation of cis/trans isomers of β-carotene
has been achieved with this technique (Lesellier et al. 2003). Epoxy-carotenoids
are difficult to isolate and distinguish from the corresponding hydroxy-carotenoids
isomers, and some references have reported the successful application of SFC with
that aim (Abrahamsson et al. 2012; Matsubara et al. 2012). Apocarotenoids also
deserve attention, as they represent a current challenge in chromatographic studies
(Zoccali et al. 2018). Last but not least, the reader should note the case of pigment
extracts containing xanthophyll esters. Although the esterification takes place with
different fatty acids for different xanthophylls, the amount of resulting combinations
does not yield a wide range of polarity but a very narrow one, so that coelution and
peak resolution are the absolute challenges for the analyst (Mariutti and Mercadante
2018; Zoccali et al. 2018). Indeed, experimental set-ups that include supercritical
fluid extraction coupled directly to SFC and analysis by spectrophotometry and mass
spectrometry have been developed for the analysis of carotenoids in complex matrices
(Giuffrida et al. 2018a, b). These issues are also related with the complex carotenoid
A. Pérez-Gálvez and J. Fontecha
C 18 columns coupled to C 30 , and different sequence of the columns for the first
and second dimension, respectively. The LC × LC set-up is also very convenient
for hyphenation with MS as demonstrated by Dugo et al. (2008b) whose were the
first authors to couple LC × LC with MS detection for the analysis of carotenoids.
Thus, the increased resolution of the LC × LC set-up is fully arranged with the
identification power of the mass spectrometry (Giuffrida et al. 2018a, b).
To finalize this section, it is challenging to recommend a LC method among the
different available alternatives. If the reader faces an unknown profile, the application
of the reference method described by van Leeuwe et al. (2006) is advised, with the first
screening of a pigments standard mix for an initial comparison between the known
carotenoid profile and the one of the target sample. Thereafter, the application of
a C 30 -based method is recommended to ascertain the presence of geometric and
cis-trans isomers.
7.2.4 Supercritical Fluid Chromatography (SFC)
Supercritical fluids have a pivotal role in different areas of chemistry, from operation units in sample preparation and isolation, to production processes and organic
synthesis, although their application in analytical chemistry has experimented a
discontinuous progress since the interest in the 80s decade of the last century and the
decline by the start of the new millennium. The commercially available equipment
capable of operating at high temperatures and pressures has not succeeded in routine
chromatography in most of the labs where liquid chromatography systems, either
GC, HPLC, or UHPLC are the standards for analytical separation. However, SFC has
found specific niches where it shows potential advantage over LC systems, considering the faster analysis and the universal coupling with detectors, particularly with
mass analyzers. Indeed, SFC has outstanding applications in the enantioseparation
of chiral analytes and isomers, and in the effective separation of complex mixtures of
structurally closely apolar compounds. Separation of cis/trans isomers of β-carotene
has been achieved with this technique (Lesellier et al. 2003). Epoxy-carotenoids
are difficult to isolate and distinguish from the corresponding hydroxy-carotenoids
isomers, and some references have reported the successful application of SFC with
that aim (Abrahamsson et al. 2012; Matsubara et al. 2012). Apocarotenoids also
deserve attention, as they represent a current challenge in chromatographic studies
(Zoccali et al. 2018). Last but not least, the reader should note the case of pigment
extracts containing xanthophyll esters. Although the esterification takes place with
different fatty acids for different xanthophylls, the amount of resulting combinations
does not yield a wide range of polarity but a very narrow one, so that coelution and
peak resolution are the absolute challenges for the analyst (Mariutti and Mercadante
2018; Zoccali et al. 2018). Indeed, experimental set-ups that include supercritical
fluid extraction coupled directly to SFC and analysis by spectrophotometry and mass
spectrometry have been developed for the analysis of carotenoids in complex matrices
(Giuffrida et al. 2018a, b). These issues are also related with the complex carotenoid