Flow cell, NMR

Fig. 2.5.1 Schematic of an NMR flow cell used to introduce and mix reactants and follow product formation used in a conventional 5-mm diameter NMR probe [11].

Hyphenation of chromatographic separation techniques (SFC, HPLC, SEC) with NMR spectroscopy as a universal detector is one of the most powerful and time-saving new methods for separation and structural elucidation of unknown compounds and molecular compositions of mixtures [171]. Most of the routinely used NMR flow-cells have detection volumes between 40... 

LC-NMR hyphenation consists of a liquid chromatograph (autosampler, pump, column and oven) and a classical HPLC detector. The flow of the detector is brought via an interface to the flow-cell NMR probe. Using commercial NMR flow-cells with volumes between 40 and 180 p,L, in connection with microbore columns or packed capillaries, complete spectra have been provided from 1 nmol of sample. These micro-cells allow expensive deuterated solvents to be used, and thus eliminate solvent interference without excessive cost. The HPLC eluent can be split in order to allow simultaneous MS detection. 

It is appropriate at this time to discuss some of the limitations associated with LC-NMR. It is more accurate to say the limitations of the NMR spectrometer in an LC-NMR instrument. As compared to MS, NMR is an extremely insensitive technique in terms of mass sensitivity. This is the key feature that limits NMR in its ability to analyze very small quantities of material. The key limiting factor in obtaining NMR data is the amount of material that one is able to elute into an active volume of an NMR flow-probe. The quantity of material transferred from the LC to the NMR flow-cell is dependant on several features. The first being the amount of material one is able to load on an LC column and retain the resolution needed to achieve the desired separation. The second is the volume of the peak of interest. The peak volume of your analyte must be reasonably matched to the volume of the flow-cell. An example would be a separation flowing at lml/min with the peak of interest that elutes for 30 s. This corresponds to a peak volume of 500 pi, which clearly exceeds the volume of the typical flow-cell. This is the crux of the problem in LC-NMR. There is a balance that must be struck between the amount of compound needed to detect a signal in an... 

FIGURE 7.3 Peak tailing comparison illustrated by the UV profile (solid line) overlaid on the NMR flow cell profile. In (a) the HPLC peak volume is well matched to the CapNMR flow cell whereas in (b) considerably more tailing occurs on the analytical scale LC-NMR. (Reprinted from Lewis, R. J. et al. Magn. Reson. Chem. 2005, 43, 783-789, with permission from John Wiley Sons.)... 

The signal intensity and line shape can be significantly affected in continuous-flow detection and, in the case of CE, additional broadening is produced by the electrophoretic current. The amount of time that a nucleus spends in the NMR flow cell or the residence time, t, is related to the observe volume and the applied flow rate, F,... 

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],... 

Figure 6.45 Microbore LC-NMR layout. A Microbore HPLC system with a 0.5 mm X 150 mm C18 column is interfaced to a solenoidal microcoil probe. The transfer capfllary is connected to the NMR flow cell with a polyamide resin. Reproduced from [85] with permission. Copyright 1999 American Chemical Society.

For systems without an SPE unit (or other post-LC column sample concentrating device) the quality of the NMR data will depend on the volume of the chromatographic peak, volume of the NMR flow cell, probe sensitivity and the use of chromatography solvents that can be suppressed. For the analysis of impurities at <1% the overloading required to attempt to obtain sufficient analyte in the active volume tends to broaden peaks significantly. Indeed, many... 

Iggo and coworkers have recently developed a high pressure NMR flow cell for the study of homogeneous reactions and reported several interesting applications [245, 246]. In the reaction of [RuCp(p-CO)2(p-dcpm)RhCl2] (dcpm =... 

This type of high pressure NMR flow cell is specially convenient for mechanistic studies in gas-liquid reactions [61-69]. 

Figure 2.20 Exploded (left) and assembled (right) view of the high pressure NMR flow cell [59],...

Figure 7.3 Exploded view of the high pressure Figure 7.4 High pressure IR cell connected in situ NMR flow cell. (From J. A. Iggo, to an autoclave (constructed at ICCOM-CNR,...

LC-NMR can be operated in two different modes on-flow and stopped-flow. In the onflow mode, LC-NMR spectra are acquired continuously during the separation. The data are processed as a two-dimensional (2D) NMR experiment. The main drawback is the inherent low sensitivity. The detection limit with a 60 p.1 cell in a 500 MHz instrument for a compound with a molecular weight around 400 amu is 20 pig. Thus, on-flow LC-NMR runs are mainly restricted to the direct measurement of the main constituents of a crude extract and this is often under overloaded HPLC conditions. Typically, 1 to 5 mg of crude plant extract will have to be injected on-column.In the stopped-flow mode, the flow of solvent after HPLC separation is stopped for a certain length of time when the required peak reaches the NMR flow cell. This makes it possible to acquire a large number of transients for a given LC peak and improves the detection limit. In this mode, various 2D correlation experiments (COSY, NOESY, HSQC, HMBC) are possible. 

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. 

Figure 3.6 600 MHz 1H NMR spectrum obtained in stop-flow mode after the (3-4-O-acyl-glucuronide of 2-fluorobenzoic acid had been isolated in the NMR flow cell. This shows the successive formation of 3-O-acyl- and 2-O-acyl isomers as a function of time...

The extension of an LC-NMR system to include mass spectrometry has been in application for several years [35,36]. By directly coupling a mass spectrometer to an LC-NMR system it is possible to obtain valuable mass spectral data. Configuring the system to have the sample reach the mass spectrometer before it reaches the NMR flow-cell enables the mass spectrometer to be employed as an experimental control device for analysing complex mixtures. Mass spectrometry is an ideal detector, provided that the molecules of interest are ionizable. It provides data rapidly and can thus yield valuable information on parent or daughter ion masses prior to initiating time-consuming NMR experiments. This synergy is not possible when the instruments are not directly coupled. 

UV chromatogram after NMR flow cell UV chromatogram after NMR flow cell... 

Figure 6.1 UV chromatograms of the test mixture of four / -hydroxybenzoic acid esters (1, methyl 2, ethyl 3, propyl 4, butyl) after the column and after the NMR flow cell at flow rates of (a) 1.0 and (b) 0.1 ml/min conditions column, LiChrospher RP select B, 125 x 4 mm id, 5 Jim, spectrometer, Bruker DRX 600 probe head, 4 mm z-gradient LC probe, active volume 120 a1 eluents, acetonitrile (A) and D2O (B) gradient, t = Omin A/B (40/60), t = 8 min A/B (70/30) at a flow rate of 1.0 ml/min and t = 80 min A/B (70/30) at a flow rate of 0.1 ml/min...

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