Stoppedflow Experiments

In continuous-flow experiments only a short time is available for accumulation of the 1H NMR spectrum. Thus, this spectroscopic technique can only be used for high concentrations of the sample, since otherwise the signal to noise ratio is too low. The sample is transferred through the flow cell analogous to the continuous-flow experiment, but in the stopped-flow mode the valves of the sample unit switch and the chromatographic run is stopped as soon as the maximum of the peak reaches the flow cell (indicated by UV detector). At this point conventional NMR experiments can be performed and, therefore, the NMR experimental time can be adjusted to the sample concentration. Another advantage of the stopped-flow experiment is the possibility of multidimensional NMR spectra.

In Figure 6 a correlation spectroscopy (COSY) stopped-flow spectrum of the olefinic protons of the carotenoid all-trans zeaxanthin is shown, recorded on a Bruker AMX 400 spectrometer. The number of experiments in F1 dimension was 216, the number of accumulated transients was 256.

Due to the centrosymmetric molecule, the corresponding protons are identical. By means of the cross-peaks in the coupling system, the structure can be elucidated. Proton H11 couples with two other protons, H10 and H12, while proton H15 couples with

Figure 5 Continuous-flow 1H NMR spectra (600 MHz) of tocopherol isomers.

proton H14. The cross-peak at 6.1 p.p.m. can be assigned to the proton H7 and H8.

Figure 7 shows a stack plot of various zeaxanthin stereoisomers. Due to the cis-arrangements, the cen-trosymmetry of the molecule is repeated. Therefore, different resonance frequencies result for the shielded and unshielded protons. In Figure 7 the 1H NMR

spectra of all-trans, 13-cis and 9-cis zeaxanthin are depicted.

The assignment of cis/trans is adapted from the Karplus equation in general, with the difference that the coupling constants are available. In most cases the difference in the chemical shift values in systems with conjugated olefinic protons is more informative. They

Figure 6 COSY stopped-flow spectrum (400 MHz) of all-trans zeaxanthin.

Figure 6 COSY stopped-flow spectrum (400 MHz) of all-trans zeaxanthin.

can amount up to 0.6 p.p.m. Looking at the protons of the cis-bonding, the outer protons experience a shift to higher field, while the inner protons shift to lower field. As shown in Figure 7, the protons H12 and H'15 in 13-cis zeaxanthin shift to lower field and proton H14 to higher field. The protons H10 and H'15 also show a small influence. The other protons have the same chemical shift values as the all-trans zeaxanthin and the cis-bonding has no further influence. Analogous effects could be explained for the 9-cis zeaxanthin. Here the cis-bonding has no effect on the shielded protons and only the unshielded ones are affected.

Solar Panel Basics

Solar Panel Basics

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