HPLC-NMR Coupling

There exist in principle two general methods for carrying out HPLC-NMR on-flow and stopped-flow experiments. 

With the on-flow mode of operation the output of the chromatographic separation is recorded simultaneous with H NMR spectra. In most cases the flow of the mobile phase is decreased to allow a large number of NMR spectra to be recorded of one chromatographic peak. 

However if the quantity of compound is very small then it may be necessary to interupt the flow of the mobile phase as soon as the maximum of the peak reaches the flow cell (indicated by UV detector). This stopped-flow mode is used in order to accumulate hundreds of scans to improve the signal/noise ratio of the resultant spectrum. 

The next step is to set the same conditions for the HPLC system which is coupled with the NMR spectrometer. The field homogeneity of the probehead is first optimized (shimmed) using the same separation column and solvent mixture. 

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Our standard molecule is however not ideally suited for certain experiments (e.g. magnetic non-equivalence, NOE, HPLC-NMR coupling). In such cases other simple compounds of the same type, compounds 2-7, will be used ... 

The instrumental setup for capillary HPLC-NMR coupling is shown in Figure 4.6. The capillary pump is connected via 50 pm capillaries between the capillary HPLC pump, the UV detector, and the NMR flow probe. 

FIGURE 4.6 Instrumental setup for capillary HPLC-NMR coupling. (From Hentschel, P. et al., J. Chromatogr. A, 285, 2006. With permission.)... 

Strbhschein, S., Puisch, M., Handel, H., and Albert, K., Structure elucidation of beta-carotene isomers by HPLC-NMR coupling using a C-30 bonded phase, Fresenius J. Anal Chem., 357, 498, 1997. 

Figure 6.31 Schematic for HPLC-NMR coupling. ( ) direction of flow (—) electronic junctions (PSU) peak sampling unit (SPE) sohd phase extraction unit.

Santos, L.C. et al.. Application of HPLC-NMR coupling using C30 phase in the separation and identification of flavonoids of taxonomic relevance, Fresen. J. Anal. Chem., 368, 540, 2000. 

Figure 1.10 Schematic of the experimental set-up used for HPLC-NMR coupling BPSU, Bruker peak sampler unit (-) capillary junctions ( ) electronic junctions...

In principle, it would be possible to use fully deuterated solvents for HPLC-NMR coupling, and then solvent suppression techniques would be unnecessary however, due to the high costs of these solvents, only the use of D2O is economically acceptable. Solutions to this problem can be seen in the development of hyphenating capillary HPLC to NMR spectroscopy, and there, because of the reduced solvent consumption, the use of fully deuterated solvents would be reasonable. However, this approach is difficult and the development is still an on-going process (see Chapter 7.3 below). From the viewpoint of NMR spectroscopy, there could be a completely new situation, when separation techniques could be found which use proton-free solvents as the mobile phases. 

In on-line HPLC-NMR coupling, the commonly recorded nuclei are and 19F, because their natural abundances are 99.9 and 100%, respectively. Thus, a direct monitoring of chromatographic separations is possible, as outlined earlier in Chapter 1. Indirect access to the information content of 13C NMR spectra is obtained in the stop-flow mode, where inverse detected 1H, 13C correlation spectra can be recorded. The acquisition of these type of 2D-spectra relies on the fact that a direct proton carbon connectivity via scalar coupling is present. Quartenary carbons without any directly attached protons are not detected. Thus, it is of major interest to record 13C NMR spectra which reveal all possible information within a coupled LC experiment. 

S. Strohschein, C. Rentel,T. Lacker, E. Bayer, and K. Albert, Separation and identification of tocotrienol isomers by FIPLC-MS and HPLC-NMR coupling, Anal. Chem. 71 (1999), 1780-1785. 

Figure 15.26 An experimental result by HPLC-NMR coupling. Sqjaration of two dipeptides (5pg each) with identification of the signals. For more clarity the peaks of the solvents around 2 and 5 ppm (acetonitrile and water) have been eliminated (Sweeder R.D. et al. Anal. Chem. 1999, 71(23), 5335).

Figure 7-2 Experimental arrangement for on-line HPLC-NMR coupling.

The spectra were recorded at the corresponding peak maxima. Both display modes demonstrate the power of continuous-flow HPLC-NMR coupling. Whereas peaks 1 and 2 (11,13-di-cu and ll-c vitamin A acetate) are not fully separated, full discrimination of both compounds is possible in the two-dimensional contour plot (Figure 7-4). On the other hand, the quality of the recorded continuous-flow H NMR spectra (Fig. 5) easily allows the determination of characteristic chemical shift values, coupling constants and integration ratios. 

Behnke B, Schlotterbeck G, Tallarek U, et al. (1996) Capillary HPLC-NMR coupling high resolution NMR spectroscopy in the nanoliter scale. Analytical Chemistry 68 1110-1115. 

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