On-flow NMR

Applications Ideally, multiply hyphenated systems should be assembled rapidly in response to real need. Access to these means is restricted to a few laboratories only. Multiple LC hyphenations have been used to analyse test mixtures of polymer additives see Table 7.74. The relative ease with which SEC-UV using CDCI3 as a solvent can be coupled to on-line 1II NMR and an in series off-line FUR (Scheme 7.12b), has been shown for a mixture of polymer additives (BHT, Irganox 1076, DIOP) [666]. Figure 7.35 shows representative spectra for on-flow NMR and MS and off-line FTIR of 2,6-di-f-butyl-4-methoxyphenol. 

LC-NMR can also be run in an on-flow mode, where NMR acquisition is started at the chromatography injection and randomly divided into a series of time slices. However on-flow NMR measurement not only suffers from low sensitivity, but LC gradient elution also causes problems on account of the drift of signals relative to the signal lock, where the presaturation frequency of each time slice needs to be defined in a separate run. 

LC-NMR can be used to identify natural products in crnde plant extracts that usually consist of complex mixtnres. The crnde natural product extracts normally contain a great nnmber of closely related and difficult-to-separate compounds. The classical separation approach may become very tedious and time-consuming. The directly conpled HPLC-NMR presents an efficient separation techniqne together with a powerfnl spectroscopic method to speed up the identification process. LC-NMR has been nsed extensively for characterization of natnral prodncts. More recently, the combination of LC-NMR and LC-MS has been further developed in this field. Eor example, Wilson et al. have nsed combined on-flow NMR and electrospray ionization MS to characterize ecdysteroids in extracts of silene otites. After reversed-phase HPLC nsing D2O in acetonitrile-dj and UV detection, the LC flow was split 95 5 for the simnl-taneous detection by NMR and MS. The peaks of interest were analyzed by stop-flow NMR to give better quality spectra for structural assignment. 

In an on-flow NMR experiment, the excited nuclei leave the flow cell whereas fresh nuclei enter. Due to the decrease of the apparent Tmow rates, faster pulse repetition rates can be used and more transients in a distinct time-period can be accumulated (Figure 1.4). The theoretical maximum sensitivity is obtained... 

In natural products analysis, most frequently the stop-flow mode is chosen to acquire H spectra of the compounds of interest, or if further structural information is required to perform two-dimensional H NMR spectra, such as COSY, TOCSY, NOESY or ROESY. In many cases an on-flow NMR chromatogram (usually at flow rates between 0.3 and 1ml min-1) is recorded beforehand, either to screen for the presence of particular groups of compounds or to gain a general overview on the sample composition. (Heteronuclear LC-NMR experiments, such as HSQC and HMBC of a natural product, have been reported in the literature once [9] however, this was of a highly enriched fraction.) More recently, time-sliced stop-flow [14,16] and on-flow approaches at low flow rates [34,35] have been applied to natural product extracts in order to combine the advantages of both on-flow (a ready overview on the entire sample) and stop-flow (sufficient acquisition time for minor compounds) modes. 

As an example, Figure 5.1.6 shows a 2D WET-TOCSY spectrum of ruberoside A. In this figure, a correlation between a downfield methine proton at 3.67 ppm and both the aglycone methyl groups A-H3-26/27 (0.84/0.86 ppm) and A-H3-2I (0.90 ppm) confirms the 23-hydroxy function in the side-chain which was suggested by the on-flow NMR and MS-back-exchange data (see above). Ruberoside A was thus identified as a new asterosaponin and subjected to semipreparative isolation. 

Figure 6.3 Low-field part of the on-flow NMR chromatogram of a soil extract from the former ammunition site in Tanne (Germany) (see Table 6.1 for abbreviations). Conditions column, LiChrospher 100 RP18, 125 x 4 mm id, 5 pm spectrometer, Bruker AMX 600 probe head, 4 mm z-gradient LC probe eluent, acetonitrile/D20 (70/30), flow 0.05ml/min 48 scans per row were required...

On the other hand, the aromatic part of the on-flow NMR chromatogram is better resolved, both in the NMR chemical shift and along the chromatographic axis. As can be seen from the extracted rows of the NMR chromatogram... 

Figure 6.8 Contour plot of the on-flow NMR chromatogram of the leachate sample. Reprinted from Benfenati, E., Pierucci, P., Fanelli, R., Preiss, A., Godejohann, M., Astratov, M., Levsen, K. and Barcelo, D. J. Chromatogr., A, 831, 243-256, copyright (1999), with permission of Elsevier Science...

Figure 6.11 shows the pseudo-on-flow NMR chromatograms of a soil sample extracted with water. The soil sample was prepared by a Polish laboratory as part of the sixth proficiency test. It was spiked with five compounds, with two of them (compounds 3 and 4) being related to chemical weapons. 

The H NMR spectra of the soil extract were run in the time-slice mode, i.e. the chromatographic run was stopped every 20 s and the NMR spectrum measured twice, with (a) and without (b) 31P decoupling. Processing of the data files led to the pseudo-on-flow NMR chromatograms. 

Comparison of the two pseudo-on-flow NMR chromatograms reveals that compounds 1,2 and 3 are organophosphorus compounds (Table 6.5), but only compounds 2 and 3 are phosphonates and related to chemical weapons. In fact, compound 2 is a degradation product of compound 3... 

S/N for a flow cell of only 50 nL volume. The main drawback was rather wide NMR linewidths. Behnke etal. demonstrated capillary LC/NMR in 1996 [138], using flow cells of 50 nL and 900nL and a standard saddle coil, and achieved sensitivities approximately 10 times greater than conventional LC/NMR. The approach, however, did not incorporate a UV detection cell so that stopped-flow NMR measurements had to be nuule by reference to on-flow NMR measurements. 

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