HPLC-NMR
Introduction
In many fields of chemistry, biology, pharmacy and medicine, progress is often limited by the ability to resolve complex analytical problems. To this end analytical techniques have been developed in recent decades dealing with an integrated approach to the separation of mixtures together with structural elucidation of unknown compounds. High Performance Liquid Chromatography (HPLC), Gel Permeation Chromatography (GPC) and Supercritical Fluid Chromatography (SFC), as well as the capillary separation techniques Capillary HPLC (CHPLC), Capillary Electrophoresis (CE) and Capillary Electrochromatography (CEC) are the most powerful techniques within the group of chromatographic separation methods. Nuclear Magnetic Resonance (NMR) spectroscopy, in particular, is useful because of its powerful stereochemical information content but it has the disadvantage of the lower sensitivity in comparison to other methods, e. g. mass spectrometry.
The combination of chromatographic separation techniques with NMR spectroscopy is one of the most powerful and time-saving methods for the separation and structural elucidation of unknown compounds and mixtures. Especially for the structure elucidation of light- and oxygen-sensitive substances, for example hop bitter acids and carotenoid stereoisomers, on-line LC-NMR has important advantages. Here, structure elucidation with LC-MS is not possible, because the carotenoid isomers exhibit the same fragmentation pattern. Using a classical method with off-line separation, enrichment and transfer to a NMR sample tube, the isolated substances would be isomerized. A closed-loop LC-NMR flow-through system solves this problem. The on-line LC-NMR technique also allows the continuous registration of time changes as they appear in the chromatographic run. Unequivocal structural assignment of unknown chromatographic peaks is possible by two-dimensional stopped-flow HPLC-NMR experiments.
NMR flow cell design
Figure 1 shows the design of NMR cells employed for various coupling techniques. For on-line HPLC-NMR and GPC-NMR coupling, a vertically-oriented flow cell with a directly fixed double-saddle Helmholtz coil is used (Figure 1a). The whole arrangement is centered in the glass dewar of a conventional probe body in which a thermocouple is inserted, allowing temperature-dependent measurements to be made. The internal diameter of the glass tube is either 2, 3 or 4 mm, resulting in detection volumes of 60, 120 and 180 mL, respectively. The glass walls of the flow cell are parallel within the length of the proton detection coil, and taper at both ends to fit PTFE tubing (I. D. 0.25 mm). PTFE and glass tubing are connected by shrink-fit tubing. "Inverse" continuous-flow probes contain an additional coaxial coil (tuned to the 13C resonance frequency) surrounding the 1H detection coil for heteronuclear 1H/13C shift correlated experiments. This design leads to optimal NMR resolution values with a typical line width of chloroform at the height of the 13C satellites of 9-12 Hz, allowing the determination of coupling constants of 1 Hz in continuous-flow NMR spectra. The disadvantage of this design is the high detection volume, leading to a degraded chromatographic resolution. For analytical HPLC columns (250 x 4.6 mm I. D.) the plate height is increased for solutes with capacity factors less than 2.5 at detection volumes higher than 48 µL.
The probe design employed for SFC-NMR coupling is shown in Figure 1b. The inner glass tube of the originating HPLC-NMR probe is substituted with a sapphire tube (O. D. 5 mm, I. D. 3 mm, detection volume 120 µL) whereas the PEEK capillaries used in the HPLC-NMR probe are replaced by Titan tubings. A double-tuned proton deuterium coil is directly fixed to the sapphire flow cell. The whole arrangement is centered in the glass dewar of a conventional probe body, in which a thermocouple is inserted, allowing temperature-controlled experiments.
Figure 1c shows the schematic diagram of the capillary NMR detection probe. Here a fused-silica capillary is directly inserted within the NMR radiofrequency coil of a 2.0 mm microprobe. Within the area of the NMR detection coil the polyimide coating of the capillary is removed; either capillaries with an I. D. of 180 µm or bubble cell types with an increased I. D. of 220 µm are used.
Experimental set-up
For on-line HPLC-NMR, GPC-NMR and SFC-NMR experiments the chromatographic separation system consists of either HPLC or SFC pumps together with an injection valve, a separation column (250 x 4.6 mm I. D.) and an ultraviolet (UV) or refractive index (RI) detector. The system is located at a distance of 2.0 m from an unshielded cryomagnet (Figure 2). With shielded cryomagnets, the chromatographic separation system can be located at a distance of about 30 cm. The outlet of the UV (RI) detector is connected by stainless steel capillary tubing (0.25 mm I. D.) to a switching valve. The valve is used for the trapping of the desired peak in the NMR flow cell for stopped-flow experiments. For on-line experiments with continuous registration of NMR spectra in distinct time intervals (1 - 8 s), the switching valve is open for a continuous flow through the probe into the waste. Instead of the switching valve a peak sample unit (BPSU) can be used. This technique is advisable when long NMR experimental times are expected. Desired peaks from a separation can be stored in small capillary loops with the help of the peak sample unit. After the whole separation, every single peak can be transferred into the probe and the desired stopped-flow experiment can be conducted.
In SFC-NMR experiments the outlet of the high pressure SFC probe is connected to a back pressure regulator to guarantee supercritical conditions in the detection cell.
A feasible experimental set-up for on-line capillary HPLC-NMR coupling is outlined in Figure 3. The separation device either for pressure or electroosmotic flow driven separations is located at a distance of 2 m from the cryomagnet. Separation is performed on a packed fused silica capillary which is directly fixed in a micro probe. For capillary HPLC separations a T-piece in conjunction with a restriction column is used for flow rate adjustment of the eluent. The HPLC pump, the injection device and the packed separation capillary are connected by fused silica capillaries.
HPLC-NMR coupling
The coupling of HPLC and NMR requires the adjustment of both analytical systems. The flow of the mobile phase leads to a restricted exposure period m for the nuclei in the flow cell. The time m is defined as the proportion of the detection volume to the flow rate. This is the reason for a shorter transverse relaxation time T2, which includes larger NMR signals. On the other hand the equilibrium state will be reached in a shorter time due to the permanent flow non-excited nuclei than only through the longitudinal relaxation time T1. This allows a quicker repeat time rate for exposure of a spectrum and therefore a gain in the sensitivity.
HPLC-NMR spectroscopy is a relatively insensitive method requ
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