Time-slice LC-NMR

Rgure 6 Comparison of the on-flow and time-slice LC/NMR contour plots of the MeOH extract of the aerial parts of Gnidia involucrata (Thymelaceae). As shown, the on-flow experiment allows the detection of four major compounds while the time-slice procedure reveals 20 components. Amount injected 20mg. HPLC conditions 018 column with radial compression, Waters RCM 8 X 10 (100 X 8 mm i.d.) MeCN-DaO gradient (5 95 to 20 80 in 50 min) 0.9 ml min k LC/NMR conditions 24 scans/increment (onflow) 1024 scans/increment (time-slice) 60 pi flow cell (3 mm i.d.) 500 MHz. (Reproduced with permission from Wolfender J-L, Ndjoko K, and Hostettmann K, (2001) The potential of LC-NMR in phytochemical analysis. Phytochemical Analysis 12 2-22 John Wiley Sons Ltd.)... 

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. 

There are four general modes of operation for LC-NMR on-flow, direct stop-flow, time-sliced and loop collection/transfer. The mode selected will depend on the level and complexity of the analyte and also on the NMR information required. All modes of LC-NMR can be run under full automation for LC peak-picking, LC peak transfer to storage loops or NMR flow cell, and NMR detection [46],... 

Applications of LC-NMR are still scarce but the technique will become more widely used. The main effort for efficient exploitation of LC-NMR needs to be made on the chromatographic side, where strategies involving efficient preconcentration, high loading, stop-flow, time slicing, or low flow procedures have to be developed. Microbore columns or capillary separation methods, such as capillary LC-NMR, CE-NMR, and CEC-NMR, will find increased application, one reason being that the low solvent consumption will allow the use of fully deuterated solvents. 

Figure 4.5 Typical H LC-NMR data obtained from a time-slice experiment. These data were obtained by stopping flow at 30 s intervals through a chromatographic run from 5.0min into the run through 14.0 min. Each spectrum was acquired for 128 transients...

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. 

Recently, NMR spectrometers directly coupled with LC systems have become commercially available. Spectra can be acquired in either of two modes, continuous or stopped flow. In continuous flow mode the spectrum is acquired as the analyte flows through the cell. This method suffers from low sensitivity since the analyte may be present in the cell for only a brief period of time, but it has the advantage of continuous monitoring of the LC peaks without interruption. Fig. 12A shows a contour plot of the continuous flow NMR analysis of a mixture of vitamin A acetate isomers.Fig. 12B shows the spectra taken from slices through the contour plot. These plots highlight the olefinic region of the spectra which provided ample information for the identification of each of the isomers. With very limited sample quantities, the more common method of LC-NMR analysis is stopped flow. Here the analyte peak is parked in the flow cell so any of the standard NMR experiments can be run. 

There are four general modes of operation for LC-NMR on-flow, stop-flow, time-sliced, and loop collection. 

Coupling of LC to NMR is relatively simple. The effluent from the column is delivered through a polyether-ether ketone (PEEK) transfer line to the NMR flow cell, which typically has a volume of 60 jA. The measurement can be carried out in one of four modes on-flow, stop-flow, time-sliced and loop collection. In the on-flow mode, the effluent from the column flows continuously through the NMR flow cell. Because of the very short time available for the measurement when peaks elute in real time, this approach is limited to major components of a mixture. In the stop flow mode, peaks detected with a UV detector are transferred to the NMR flow cell, and the run is automatically stopped. The NMR spectra can then be acquired over a period of several minutes, hours or even days. In the time-sliced mode, the elution is stopped several times during the elution of the peak of interest. This mode is usually used when two analytes are poorly resolved. In the loop collection mode, the chromatographic peaks are stored in loops for offline NMR study. This approach is therefore not a real online hyphenated technique. 

LC—NMR coupling can be realized by four operation modes on flow, stop flow, time sliced, and loop collection. Since, in the on-flow mode, only a relatively low number of NMR pulses can be acquired for a peak or... 

On-flow HPLC-NMR analysis can also be performed when sufficient material is available. It involves collecting the NMR data continuously as the sample passes through the probe. This is the most efficient method for stmcture evaluation by HPLC-NMR. The NMR data are represented in a 2-D plot where the x direction contains chemical shift information and they direction is representative of the LC retention time. The individual spectra can be extracted from the ID slices along the x axis if so desired. The resolutions in the individual spectra are of somewhat lower quality than in the stop-flow method however, the introduction of the second dimension allows for easy stmcture assignment even for overlapping peaks in the LC separation. As seen in Fig. 19, the on-flow HPLC-NMR characterization shows four distinct sets of resonances. 

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