2 Carotenoids in Phototrophic Microalgae …
33
chemical structures of siphonaxanthin (Yoshii et al. 2005), loroxanthin, prasinoxanthin, and monadoxanthin, it could be considered that they are derived from lutein,
but the pathways and enzymes remain unknown (Figs. 2.1 and 2.2) (Takaichi 2011).
2.4 Identification Protocol of Carotenoids
Dr Liaaen-Jensen (1995), authorities on carotenoid chemistry, recommended a procedure to identify carotenoids; “For identification, the three minimum criteria, i.e. cochromatography with an authentic sample, UV/Vis spectrum, and mass spectrum of
a quality that allows identification of the molecular ion, must be fulfilled.” She also
recommends eight criteria for structure elucidation.
Other criteria have previously been suggested (Takaichi 2014), and here I
summarize some revisions to the general identification procedures.
(i) Extraction and purification
These processes should be carried out under dark, dim-light, or red-light conditions.
Carotenoids are easily isomerized by light; however, low temperature can prevent
isomerization and oxidation.
In some reports, crude extracts of pigments from phototrophs were analyzed by
C 18 HPLC, and some cis forms of carotenoids were indicated. This might be due to
photoisomerization during preparation. Usually, carotenoids in phototrophs are alltrans forms. Exceptionally, neoxanthin in chloroplasts is in the 9
-cis form (Takaichi
and Mimuro 1998).
(ii) Identification of chromophore (conjugated C=C and C=O double bond system)
The wavelength of absorption maximum shows the number of conjugated C=C
double bonds (Takaichi and Shimada 1992; Takaichi 2000; Britton et al. 2004). The
value of %III/II, which is the ratio of the peak heights of the longest and the middle
wavelength absorption bands from the trough between the two peaks, shows the
number of β-end groups, and that of %D B /D II shows the height of cis peak (Fig. 2.4).
A broad absorption spectrum indicates the presence of conjugated carbonyl group
(C=O). Note that the absorption spectrum depends on the solvent used.
In some reports, the absorption spectra of carotenoids did not fit their names,
since the authors depended only on LC-MS data and ignored absorption spectra.
Nostoxanthin and β-carotene have the same chromophores, and their absorption
spectra are the same (Takaichi et al. 1990).
(iii) Estimation of polar group(s) from C 18 HPLC retention time
Xanthophylls contain oxygenic groups, such as hydroxyl, keto, and epoxy groups.
The type and number of these polar group(s) may be estimated from the C 18 HPLC
retention times compared with those of authentic samples.
33
chemical structures of siphonaxanthin (Yoshii et al. 2005), loroxanthin, prasinoxanthin, and monadoxanthin, it could be considered that they are derived from lutein,
but the pathways and enzymes remain unknown (Figs. 2.1 and 2.2) (Takaichi 2011).
2.4 Identification Protocol of Carotenoids
Dr Liaaen-Jensen (1995), authorities on carotenoid chemistry, recommended a procedure to identify carotenoids; “For identification, the three minimum criteria, i.e. cochromatography with an authentic sample, UV/Vis spectrum, and mass spectrum of
a quality that allows identification of the molecular ion, must be fulfilled.” She also
recommends eight criteria for structure elucidation.
Other criteria have previously been suggested (Takaichi 2014), and here I
summarize some revisions to the general identification procedures.
(i) Extraction and purification
These processes should be carried out under dark, dim-light, or red-light conditions.
Carotenoids are easily isomerized by light; however, low temperature can prevent
isomerization and oxidation.
In some reports, crude extracts of pigments from phototrophs were analyzed by
C 18 HPLC, and some cis forms of carotenoids were indicated. This might be due to
photoisomerization during preparation. Usually, carotenoids in phototrophs are alltrans forms. Exceptionally, neoxanthin in chloroplasts is in the 9
-cis form (Takaichi
and Mimuro 1998).
(ii) Identification of chromophore (conjugated C=C and C=O double bond system)
The wavelength of absorption maximum shows the number of conjugated C=C
double bonds (Takaichi and Shimada 1992; Takaichi 2000; Britton et al. 2004). The
value of %III/II, which is the ratio of the peak heights of the longest and the middle
wavelength absorption bands from the trough between the two peaks, shows the
number of β-end groups, and that of %D B /D II shows the height of cis peak (Fig. 2.4).
A broad absorption spectrum indicates the presence of conjugated carbonyl group
(C=O). Note that the absorption spectrum depends on the solvent used.
In some reports, the absorption spectra of carotenoids did not fit their names,
since the authors depended only on LC-MS data and ignored absorption spectra.
Nostoxanthin and β-carotene have the same chromophores, and their absorption
spectra are the same (Takaichi et al. 1990).
(iii) Estimation of polar group(s) from C 18 HPLC retention time
Xanthophylls contain oxygenic groups, such as hydroxyl, keto, and epoxy groups.
The type and number of these polar group(s) may be estimated from the C 18 HPLC
retention times compared with those of authentic samples.