Sensitivity LC-NMR

Broadening of peaks in the flow system present yet another problem for sensitivity and can mean that even less of the analyte peak is used, even if it would appear narrow enough to be totally contained in the flow cell. Broadening of peaks during Ailing of the cell is much larger for peaks that are narrower than the volume of the cell compared with those that are larger than the cell volume [58]. This can lead to a further deterioration of LC-NMR sensitivity by a factor of 2 for narrow peaks [57]. 

In practice, if the NMR acquisition times are longer than 30 min then on-column diffusion dilutes subsequent peaks, thus reducing the sensitivity [63], Typically, LC peaks will be broadened by 30% and 50% after the flow is stopped for 5 and 10 h, respectively [74], significantly reducing the LC-NMR sensitivity. Such peaks are more efficiently analysed by repeating the chromatography. 

Equipment available (HPLC, automated PSU, SPE, flow probe, high-field spectrometer) will determine the overall LC-NMR sensitivity and levels of impurities that can be analysed. For low-level impurities the time required for multiple trapping on SPE cartridges may also need to be considered [84]. 

With current commercially available equipment the ideal set-up for online analysis would be an HPLC-SPE system, a cryogenic flow probe (30 p,l active volume) that is in permanent use within an actively shielded magnet operating at 500 MHz or higher. The system would offer the optimum LC-NMR sensitivity (no dependency on LC peak volumes), and complex impurities as low as 0.1% could be identified by one- and two-dimensional NMR experiments, provided that the impurities are sufficiently stable to permit isolation on the SPE cartridges,... 

The decision to use either NMR or LC-NMR for the analysis of mixtures in the pharmaceutical industry depends on factors related to their chromatographic separation and the ability of NMR to elucidate the structure of organic compounds whether hyphenated or not. The major technical considerations of LC-NMR, discussed below, are NMR sensitivity, NMR and chromatographi-cally compatible solvents, solvent suppression, NMR flow-probe design, and LC-NMR sensitivity or compatibility of the volume of the chromatographic peak with the volume of the NMR flow cell for better detection. Figure 20-1 shows the schematic setup of the LC-NMR connected to other devices, such as radioactivity detector and MS (see Section 20.4). 

LC-NMR can be operated continuously ( on-flow ) or discontinuously ( stopped-flow ). The optimum flow-rate in continuous-flow NMR is a compromise between best resolution and sensitivity. The sensitivity in NMR measurements has been increased significantly by ... 

Multiple hyphenation ( hypemation ) provides comprehensive spectroscopic information from a single separation. The first doubly hyphenated HPLC-NMR-MS appeared in 1995 [661], and its value is now accepted meanwhile fully integrated on-line LC-NMR-MS and MSn systems (QMS, QTTMS) are commercially available. On-line LC-NMR-MS coupling is by no means trivial. For example, the sensitivity of NMR is limited, while MS is incompatible with non-volatile buffers. The... 

In summary, NMR spectroscopy is an extremely versatile tool useful that enables researchers to understand the structure of natural products such as carotenoids. For a full structural assignment, the compound of interest has to be separated from coeluents. Thus, it is a prerequisite to employ tailored stationary phases with high shape selectivity for the separation in the closed-loop on-line LC-NMR system. For the NMR detection, microcoils prove to be advantageous for small quantities of sample. Overall, the closed-loop system of HPLC and NMR detection is very advantageous for the structural elucidation of air- and UV-sensitive carotenoids. 

Watanabe et al. published the first paper to appear in the literature dealing with the coupling of LC and NMR in 1978 [82], This early exploration of LC-NMR led to the modification of a standard NMR probe to include a flow cell comprised of a thin-wall Teflon tube with an inner diameter of 1.4 mm. The dimensions of this flow-cell were 1 cm in length and a total volume of 15 pi. This modification not only made the NMR spectrometer amenable to a flow system, but also overcame some of the inherent sensitivity issues associated with NMR as an LC... 

Of particular importance with the use of LC-NMR as an experimental technique is that it is suited for only a limited number of applications in reference to structure elucidation. As will be discussed in greater detail, the sensitivity issues that arise between the amount of compound one is able to load onto a particular chromatographic stationary phase, and hence elute into the flow-cell of an LC-NMR probe, limit what type of structural analysis that can be performed. It is this author s current opinion that most complete structure elucidations of unknown molecular entities are not amenable to LC-NMR. In these... 

It is appropriate at this time to discuss some of the limitations associated with LC-NMR. It is more accurate to say the limitations of the NMR spectrometer in an LC-NMR instrument. As compared to MS, NMR is an extremely insensitive technique in terms of mass sensitivity. This is the key feature that limits NMR in its ability to analyze very small quantities of material. The key limiting factor in obtaining NMR data is the amount of material that one is able to elute into an active volume of an NMR flow-probe. The quantity of material transferred from the LC to the NMR flow-cell is dependant on several features. The first being the amount of material one is able to load on an LC column and retain the resolution needed to achieve the desired separation. The second is the volume of the peak of interest. The peak volume of your analyte must be reasonably matched to the volume of the flow-cell. An example would be a separation flowing at lml/min with the peak of interest that elutes for 30 s. This corresponds to a peak volume of 500 pi, which clearly exceeds the volume of the typical flow-cell. This is the crux of the problem in LC-NMR. There is a balance that must be struck between the amount of compound needed to detect a signal in an... 

While the early days of LC-NMR and LC-NMR-MS were plagued by the poor sensitivity of the NMR spectrometer, the recent probe design advances have provided a means to potentially overcome this hurdle. As reported in the literature, it is possible to get both ID and 2D homo-nuclear and heteronuclear correlation data on sub micrograms of materials in quite complex mixtures utilizing cryogenic flow-probes in tandem with SPE peak trappings [98]. While these technologies are still in their infancy, they have the potential to revolutionize LC-NMR as a structure elucidation technique. 

The high sensitivity of hyphenated techniques such as HPLC-MS has also been exploited in the identification and structural studies of flavonoid glucosides [151] and the application of other hyphenated techniques such as LC-MS-MS and LC-NMR for the analysis of plant constituents has been discussed earlier [152],... 

When using NMR as a detector for online separations, additional consideration must be given to how the sensitivity is affected by the movement of nuclei past the detector cell. Aside from the physical hardware setup, the chromatographic and spectroscopic parameters also play a role in the quality of the resulting data. Flow rate, solvent composition, and residence and acquisition times can be optimized to provide optimal results. NMR sensitivity and chromatographic resolution tend to have an inverse relationship with respect to online LC-NMR experiments. By slowing the flow rate, more scans can be acquired for a particular analyte in the flow cell, but... 

As described in Section 7.4.2, one of the challenges of online LC-NMR is the need to match the chromatographic peak to the active volume of the CapNMR flow cell. An excellent discussion of the comparison of CapLC-NMR with other NMR probe types has been provided by Lewis et al. [17] Table 7.1 shows the sensitivity comparison of the CapNMR probe with larger volume probes. It is important to note that the experimental design used by these authors adjusted the concentration of analyte such... 

Xu et al. [39] used a semi-preparative LC-SPE-NMR system providing a sensitivity enhancement more than 30-fold greater than standard LC-NMR. 

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