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enhances the system sensitivity and analysis of structural isomers (Su et al., 2002). The optical
spectroscopic analysis of carotenoids is based on the fact that the 0-0 energy of the i rst opti-
cally allowed transition is inversely correlated to the number of conjugated carbon double bonds
in the delocalized p-electrons (Kuhn, 1949). Therefore, in theory, violaxanthin, lutein, and zeax-
anthin, which have 9, 10, and 11 conjugated bonds should have all different 0-0 maxima positions.
Neoxanthin has the same number of these bonds as violaxanthin, 9. However, the
cis
-conformation
increases the energy of excited state leading to a slightly blueshifted 0-0 transition. In addition to
this shift, a
cis
-band emerges at around 310-330 nm, which can also be used for distinguishing
different isomers of the same carotenoid (Tsukida et al., 1982; Koyama et al., 1983).
Since the analytical approaches described above require the extraction of pigments from the liv-
ing tissues and the membranes and protein complexes with organic solvents, the elimination of all
structural and spectral features typical of the
in vivo
carotenoid state is lost. In addition, pigment
degradation during sample storage and extraction conditions can frequently take place (Su et al.,
2002; Feltl et al., 2005). It is also likely that the causes of some existing analytical discrepancies can
be found in the method of using standards and their extinction coefi cients. The hydrophobicity of
carotenoid molecules and the strong environmental dependency of the excited state energy (due
to high molecular polarizability) and oscillator strength could be the causes for signii cant varia-
tions in pigment quantii cation using UV-Vis detection. For example, in order to accurately separate
and quantify photosynthetic membrane xanthophylls, chlorophylls, and b-carotene, a three-solvent
system had to be employed (Snyder et al., 2004). All xanthophylls were separated using the polar
solvent acetonitrile mixed with a fraction of methanol, whereas, in order to run b-carotene some-
what more nonpolar solvent mixture hexane/ethyl acetate was required. Figure 7.1 displays a typical
HPLC proi le of all higher plant xanthophylls. The more oxygenated and polar xanthophylls such as
neoxanthin and violaxanthin elute much faster than the less polar lutein and zeaxanthin. In spite of
the identical molecular mass, the latter two have slightly different mobility because of coni guration
differences in the end-group orientation leading to the differences in the molecular polarity.
Solvents with different polarities and refractive indexes signii cantly affect carotenoid opti-
cal properties. Because the refractive index is proportional to the ability of a solvent molecule to
interact with the electric i eld of the solute, it can dramatically affect the excited state energy and
hence the absorption maxima positions (Bayliss, 1950). Figure 7.2a shows three absorption spec-
tra of the same xanthophyll, lutein, dissolved in isopropanol, pyridine, and carbon disuli de. The
solvent refractive indexes in this case were 1.38, 1.42, and 1.63 for the three mentioned solvents,
respectively.
1.0
0.8
490
505
383
535
0.8
0.6
480
0.6
0.4
473
0.4
J-type
0.2
1
2
3
H-type
0.2
Lutein
Zeaxanthin
0.0
350 375 400 425 450 475 500 525 550 575 600
0.0
400
420
440
460
480
500
520
540
(a)
Wavelength (nm)
(b)
Wavelength (nm)
FIGURE 7.2
(a) Absorption spectra of lutein dissolved in isopropanol (1), pyridine (2), and carbon disuli de
(3). (b) Absorption spectra of zeaxanthin (in ethanol) and zeaxanthin H- and J-type aggregates.
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