Stop-Flow LC-NMR

If the retention times of the compounds to be separated are known, or if they can be detected by using UV (including diode arrays), radiochemical or fluorescence detectors, stop-flow LC-NMR becomes an option. Upon detection, the PC controlling the liquid chromatograph allows the pumps to continue running, moving the peak of interest into the NMR probe. Once the pumps have stopped, normal high-resolution NMR spectroscopy is possible. It could be 

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Figure 6.32 500 MHz H NMR spectra of a drug impurity (a) by stopped flow LC-NMR and (b) by a preparative approach. In addition to the improved signal to noise, the peak at 3.06 ppm is missing in the LC-NMR experiment owing to its close proximity to the pre-saturated water signal. Reproduced from [57] with permission of John Wiley and Sons Ltd.

Figure 6.36 500 MHz H NMR spectra obtained during a stop-flow LC-NMR experiment on a 1 mg injection of a crude sample of a drug compound, (a) LC chromatogram, (b) spectram corresponding to the parent bulk drug compound acquhed for 64 transients and (c) to the impurity peak RRT 0.87 (—3% by area), acquhed for 1024 transients. NOESY-type presaturation was used to suppress the solvent resonances. Bruker DRX500 H/ C 4-mm z-gradient probe with a 120 pi active cell volume.

The main peak at 35 min belongs to all-E lycopene (82%, peak 6). Furthermore, peak 1 can be assigned to all-E (3-carotene, whereas peaks 2-5 belong to various Z and ZZ lycopene stereoisomers, as summarised in Table 5.2.1. Thereby, the peak assignment can be proven by recording stopped-flow LC-NMR spectra as shown in Figures 5.2.5 and 5.2.6 (see below). 

Figure 6.15 presents the LC-UV chromatogram of the hydrolysis products from the first step of simulated waste water treatment of Remazol Black 5 (RB5), a commercially important textile dye, while Figure 6.16 shows a series of stop-flow LC-NMR spectra acquired in an LC-NMR-MS run. The NMR and MS data of the tentatively identified compounds are shown in Tables 6.6 and 6.7, respectively. These are only by- or degradation products which elute earlier than the hydrolysed Remazol Black. Peaks which elute later consist of coeluting dye components which have not yet been identified. 

LC-NMR data can be obtained using either a continuous- or stopped-flow method for acquisition of NMR spectra. Dachtler et ah (2001) used both techniques with H-NMR to separate and characterize zeaxanthin stereoisomers. Using stopped-flow they were able to identily (13-Z)-zeaxanthin with 800 ng of analyte in the flow cell, compared to 24 pg using continuous-flow. The higher sensitivity with stopped-flow LC-NMR arises because the chromatographic run is paused so that NMR data can be acquired at the peak maximum where the concentration of analyte is greatest (Albert, 1999). 

Figure 18-10. Stop flow LC-NMR of heptabarbitone after separation as in Figure 18-9 with D2O as the eluent at 200°C. (Reproduced from reference 59, with permission.)...

There are three main modes of operation for LC-NMR on-flow, peak collection, and stopped flow. On-flow LC-NMR involves acquisition of realtime NMR scans during the chromatographic run, but requires high sample concentration since only a small number of scans can be averaged for each NMR spectrum. Stopped-flow LC-NMR is normally used when a single peak of interest is to be analyzed. The peak of interest is selected either manually, by region of time, or by intensity threshold. In stopped-flow mode, the LC... 

The other methods of improving S/N and spectral resolution are to employ peak-directed stopped flow LC/NMR or loop-capture. The former is a... 

Figure 4, The stop-flow LC-NMR on the mixture of pMMA, polystyrene, and p(Sty-b-MMA), showing the proton signals for polystyrene and pMMA segments. (Reproduced with permission from Polym. Prepr. 2001,42.1, 23-24.

Hgure 2 Typical setup used for on-flow and direct stop-flow LC/NMR experiments. The control of the stop-flow valve is achieved by the computer of the LC/UV system which triggers both the valve of the HPLC pump and the NMR acquisition computer. A calibrated delay is used for parking the LC-peak of interest with precision at the center of the LC/NMR flow probe. 

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