NMR continuous-flow

The alternative method is continuous-flow , in which the reactants flow through the detection coil during data acquisition. Continuous-flow NMR techniques have been used for the direct observation of short-lived species in chemical reactions [4—6]. The main difference between stopped- and continuous-flow NMR is that in the latter the sample remains inside the detection coil only for a short time period, termed the residence time, x [7], which is determined by the volume of the detection cell and the flow rate. The residence time alters the effective relaxation times according to the relationship in Eq. (2.5.1) ... 

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 ... 

The specific constraints and requirements of continuous-flow NMR will be explained in the first chapter, whereas specific applications, such as biomedical and natural product analysis, LC-NMR-MS and LC-NMR in an industrial environment, together with polymer analysis, will be discussed separately. Thus, the reader will obtain a broad overview of the application power of LC-NMR and the benefits of its use. He/She will also be introduced to the pitfalls of this technique. Special attention will be given to the exciting newer coupled techniques such as SFC-NMR and capillary HPLC-NMR. However, new emerging future developments will also be discussed thoroughly. 

Table 1.1 Variation of residence time and line broadening as a function of detection cell volume and flow rate in continuous-flow NMR spectroscopy.

Figure 1.4 Acquisition scheme used for a continuous-flow NMR experiment...

Figure 1.6 System used for recording continuous-flow NMR spectra in a rotating NMR tube...

Figure 1.8 Schematics of (a) conventional and (b) continuous-flow NMR probes suitable for cryo magnets...

The analytical NMR flow-cell (see Figure 1.8) was originally developed for continuous-flow NMR acquisition, but the need for full structural assignment of unknown compounds led to major applications in the stopped-flow mode. Here, the benefits of the closed-loop separation-identification circuit, together with the possibilities to use all types of present available 2D and 3D NMR techniques in a fully automated way, has convinced a lot of application chemists [17-70], A detailed description of the different modes for stopped-flow acquisition (e.g. time-slice mode) is found in Chapters 2 und 3. 

The simplest of these is continuous-flow detection, but this is usually only practical when using H or 19F NMR for detection unless isotopically enriched compounds are available. However, there are examples of HPLC-NMR studies using 2H and 31P NMR detection in the drug metabolism field. Where continuous-flow NMR detection is used for gradient elution, the NMR resonance positions of the solvent peaks shift with the changing solvent composition. For effective solvent suppression, these solvent resonance frequencies must be determined as the chromatographic run proceeds. 

For the on-line SFE-NMR experiments, the set-up shown in Figure 7.2.17 can be used. A main pump serves an HP supercritical fluid chromatograph (G1205A), with analytical HPLC columns being used as extraction cells. The continuous-flow NMR cell is connected between the column outlet and the back-pressure regulator. 

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. 

Nuclear magnetic resonance spectroscopy (NMR) coupling to LC has seen significant progress in the past five years [11]. Continuous-flow NMR probes have been designed with a typical detection volume of 40-120 pi or smaller. The NMR spectmm is often recorded in stop-flow mode, although continuous-flow applications have been reported as well. 

A supercritical separation technique employing non-protonated solvents is supercritical fluid chromatography (SFC) using CO2. At a temperature of 323 K and a pressure of 161 bar, high-resolution, continuous-flow NMR spectra in supercritical CO2 can be obtained [15], [16], [17], [18], [19]. This is demonstrated in Figure 7-12, which shows the H NMR spectrum of ethylbenzene in supercritical CO2 [16]. 

For continuous-flow NMR measurements, two probe designs have become established. For LC-NMR with analytical HPLC columns (4.6 x 250 mm), probes with an active volume between 60 and 120 iL are used. In contrast to the conventional tube design, the double-saddle Helmholtz coil is arranged vertically, directly affixed to a U-shaped glass tube in a glass Dewar vessel. The inlet and outlet capillaries are coimected by shrinkable Teflon tubings (Fig. lb). 

There are special prerequisites for the solvents (mobile phase) used in continuous-flow NMR measurements. HPLC-grade solvents contain traces of impurities and often stabilizers. For continuous-flow NMR, solvents of higher purity are needed as these contaminants would give rise to additional signals in the NMR spectrum that may be coincident with the resonances of the analyte. 

This technique has been successfully applied to record continuous-flow NMR... 

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