Chemistry Reference
In-Depth Information
Thus the on-line coupling of capillary HPLC with NMR is the method of choice. Unambiguous
peak identii cation can be performed by using data obtained by HPLC-electrospray chemical ion-
ization (ESI) MS or HPLC-atmospheric pressure chemical ionization (APCI) MS coupled together
with results from on-line capillary HPLC-NMR. On-line capillary HPLC-NMR is conducted using
NMR l ow cells with detection volumes between 1.5 and 5.0 mL, enabling the use of deuterated sol-
vents. With small amounts of sample, higher concentrations of analyte in the nanoliter detection cell
are obtained leading to reasonable NMR acquisition times for 1D and 2D NMR spectra (Olson et
al. 1995, Webb 1997).
The on-line coupling of HPLC and NMR can either be performed in the stopped-l ow or in the
continuous-l ow mode (Krucker et al. 2004, Grynbaum et al. 2005, Putzbach et al. 2005, Albert
et al. 2006, Hentschel et al. 2006, Rehbein et al. 2007). Current sensitivity levels are in the lower
nanogram range for 1D
1
H NMR spectra and in the microgram range for 2D spectra.
Figure 4.5 shows the schematic design of a microcoil NMR probe. The horizontally oriented
radio frequency copper coil is directly attached to the glass with an internal diameter of 100 mL.
Thus an excellent i lling factor (ratio of sample volume versus detection coil volume) is guaranteed.
This newly designed probe with a microcoil shows signii cant improvements in the signal line shape
and an easy magnetic i eld homogenization. The obtained signal-to-noise ratio of 50:1 for the ano-
meric proton of a 0.2 M solution of sucrose in D
2
O is sufi cient to perform structure elucidation of
naturally occurring substances.
The instrumental setup for capillary HPLC-NMR coupling is shown in Figure 4.6. The capillary
pump is connected via 50 mm capillaries between the capillary HPLC pump, the UV detector, and
the NMR l ow probe.
Figure 4.7 shows the structures of important carotenoids: (all-
E
) lutein, (all-
E
) zeaxanthin, (all-
E
)
canthaxanthin, (all-
E
) b-carotene, and (all-
E
) lycopene. Employing a self-packed C
30
capillary
column, the carotenoids can be separated with a solvent gradient of acetone:water
80:20 (v/v) to
99:1 (v/v) and a l ow rate of 5 mL min
−1
, as shown in Figure 4.8 (Putzbach et al. 2005). The more
polar carotenoids (all-
E
) lutein, (all-
E
) zeaxanthin, and (all-
E
) canthaxanthin elute i rst followed by
the less polar (all-
E
) b-carotene and the nonpolar (all-
E
) lycopene. Figure 4.9 shows the stopped-
l ow
1
H NMR spectra of these i ve carotenoids. The chromatographic run was stopped when the
peak maximum of the compound of interest reached the NMR probe detection volume.
The spectrum of the noncentrosymmetric (all-
E
) lutein shows a multiplet (integration value
four) for the protons 11/11
=
′
(6.62 ppm) and 15/15
′
(6.59 ppm). The protons 12/12
′
(6.30 ppm) and
Transmitter/
receiver coil
Flow
capillary
Out
In
FIGURE 4.5
Schematic design of a microcoil NMR probe. (From Rehbein, J. et al.,
Characterization of
Bixin by LC-MS and LC-NMR
, John Wiley & Sons Ltd., 2387, 2007. With permission.)
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