Chemistry Reference
In-Depth Information
1
150
1 (all-
E
) lutein
2 (all-
E
) zeaxanthin
3 (all-
E
) canthaxanthin
4 (all-
E
) β-carotene
5 (all-
E
) lycopene
3
100
2
50
0
4
-50
5
-100
-150
10
20
Retention time (min)
30
40
FIGURE 4.8
Capillary HPLC separation on a C
30
column of (all-
E
) lutein, (all-
E
7) zeaxanthin, (all-
E
)
canthaxanthin, (all-
E
) b-carotene, and (all-
E
) lycopene. (From Putzbach, K. et al.,
J. Pharm. Biomed. Anal
.,
910, 2005. With permission.)
(6.23 ppm) are slightly different from the
chemical shifts of the corresponding protons of (all-
E
) lutein. The main difference in the
1
H-NMR
spectra is found in the signals of protons 10/10
chemical shifts of the protons 12/12
′
(6.32 ppm) and 14/14
′
(6.07 ppm). The
centrosymmetric structure of (all-
E
) zeaxanthin leads to one signal for protons 7 and 7
′
(6.11 ppm), 8/8
′
(6.08 ppm), and 7/7
′
.
In comparison to the NMR spectra of (all-
E
) lutein and (all-
E
) zeaxanthin, the multiplet signal of
the protons 11/11
′
(6.65 ppm) of (all-
E
) canthaxanthin exhibits a slightly stron-
ger “low-i eld” shift. The doublets of the protons 12/12
′
(6.68 ppm) and 15/15
′
′
and 8/8
′
appear at 6.40 ppm and 6.36 ppm,
respectively. A multiplet of protons 14/14
′
(6.29 ppm), 10/10
′
(6.27 ppm), and 7/7
′
(6.25 ppm) is
shifted to lower i eld due to the shielding effect of the carbonyl group at C-4.
The
1
H NMR spectrum of (all-
E
) b-carotene shows the characteristic low-i eld multiplet at
6.75 ppm arising from protons 11/11
(6.74 ppm). Similar to the spectra
of (all-
E
) lutein and (all-
E
) zeaxanthin two doublets can be seen for protons 12/12
′
(6.76 ppm) and protons 15/15
′
′
(6.43 ppm) and
14/14
′
(6.34 ppm). Protons 7/7
′
(6.24 ppm) together with protons 10/10
′
(6.23 ppm) show a multiplet
(integration ratio four). The doublet of protons 8/8
is found at 6.18 ppm.
The pattern of the
1
H-NMR spectrum of lycopene differs from the spectra of the other carote-
noids because lycopene consists of conjugated double bonds. At 6.6 ppm the multiplet of protons
11/11
′
′
(6.63 ppm) and of proton pairs 15/15
′
(6.60 ppm) resonate adjacent to the doublet of proton
pair 7/7
′
(6.44 ppm), the doublet of proton pair 12/12
′
(6.29 ppm), the doublet of proton pair 14/14
′
(6.22 ppm), the doublet of proton pairs 8/8
′
(6.15 ppm), and i nally the doublet of proton pair 10/10
′
.
The resonance of proton pairs 6/6
′
and 2/2
′
are shifted to a higher i eld at 5.85 and 5.00 ppm due to
their position in the conjugated system.
In all recorded spectra the
3
J
HH
coupling constants between the olei nic protons are on the order
of 11-12 Hz, proving the all-
E
coni guration of the investigated carotenoids. Minor differences
between the reported chemical shifts and literature data are due to the effect of different solvent
compositions.
In addition to 1D
1
H-NMR spectroscopy, 2D NMR spectra recorded in the stopped-l ow mode
give valuable information of the homonuclear and heteronuclear scalar connectivities. Figure 4.10
shows the homonuclear correlated spectrum (
1
H
1
H-COSY) of (all-
E
) lycopene, proving all the
assignments shown in Figure 4.9e. An inverse detected spectrum (heteronuclear single quantum
coherence, HSQC) of tocopherol acetate is depicted in Figure 4.11. Here, the chemical shifts of the
proton signal can be directly correlated with the chemical shifts of the adjacent carbon atoms.
In contrast to mass spectroscopy, NMR spectroscopy reveals the effect of stereoisomerization.
One example is the isomerization of lutein to anhydroltutein induced by cooking (Hentschel et al.
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