Continuous-Flow LC-NMR

The simplest method of operation is continuous-flow detection. This mode of operation is generally only practical when using H or 19F NMR for detection 

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Watanabe and Niki introduced the coupling of NMR to LC as an on-line detector [20]. After these initial stopped-flow experiments, Bayer et al. [21] reported the first continuous-flow LC-NMR experiment. However, a number of impediments associated with LC-NMR hindered routine analytical application for a number of years. Since then, new instrumentation and analytical methodologies for LC-NMR have been developed and commercialized. The development of high-field-strength magnets, better solvent-suppression techniques, more sensitive small-diameter transmitter/receiver coils, on-column sample preconcentration, and expanded flow cells have improved the sensitivity of LC-NMR. 

Figure 1 Schematic of (A) conventional NMR probes, (B) saddle-type continuous-flow LC/NMR probes, and (C) solenoidal continuous-flow LC/NMR probes. (Reproduced with permission from Albert K (2002) On-line LC-NMR and Related Techniques. Chichester Wiley John Wiley Sons Ltd.)...

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. 

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... 

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). 

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 ... 

There are four main modes of operation of LC-NMR instruments, in terms of how the compound/fraction of interest is dealt with post chromatographic separation. These methods are continuous-flow, stop-flow, peak parking and peak trapping. There are several different variations within each of these modes (e.g. peak-slicing). Each one of these modes will be described in turn and advantages and disadvantages will be discussed. See Fig. 19.20. 

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. 

The main prerequisite for on-line LC-NMR, besides the NMR and HPLC instrumentation, are the continuous-flow probe and a valve installed before the probe for the registration of either continuous-flow or stopped-flow NMR spectra. 

Figure 4.1 Pseudo-2D plot of continuous-flow 1 H LC-NMR data obtained on human urine after dosing with paracetamol (1). The resonances from the glucuronide (2) and sulfate (3) conjugate metabolites of paracetamol and their ID slices are shown...

Continuous-flow 19F LC-NMR spectra were acquired for 16 transients using 60° pulses into 8192 data points over a spectral width of 11 364 Hz, giving an acquisition time of 0.36 s. A relaxation delay of 0.64 s was added to give a total acquisition time for each spectrum of 16 s. The data were multiplied by a line-broadening function of 3 Hz to improve the signal-to-noise ratio and zero-filled by a factor of two before Fourier transformation. The results are presented as a contour plot with 19F NMR chemical shift on the horizontal axis and chromatographic retention time on the vertical axis. 

Figure 4.10 Continuous-flow 19F LC-NMR-MS data displayed as a pseudo-2D plot with 19F chemical shift on the horizontal axis and retention time on the vertical axis. The peaks of interest are labelled with the relevant molecular weights...

The next phase of the experimental procedure was to collect in storage loops the peaks of interest as determined by the continuous-flow 19F LC-NMR-MS experiment. Storage in the loops was triggered by the UV response and desired M+D ion. 

LC-NMR data may be acquired in either onflow or stopped-flow modes. In the onflow mode, effluent flows directly from the column through the probe, where spectra are collected at fixed time intervals. Figure 4 shows a typical onflow LC-NMR spectrum.6 In the stopped-flow mode, several fractions are collected and analysed individually at a later time. Although mass spectrometry continues to be the favoured technique for the analysis of combinatorial libraries, the value of LC-NMR was demonstrated in a study by Chin et al.A1 in a stopped-flow study of four positional isomers of dimethoxybenzoylglycine. Iso-molecular-weight mixtures such as this can be problematic to analyse using mass spectrometry however, in this case assignment of the compounds was readily accomplished from the LC-NMR data (Fig. 5). 

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. 

The research group of Hostettmann significantly contributed to the current widespread use of LC-MS in the analysis, characterization, and dereplication of plant phenols including flavonoids. Initially, thermospray and continuous-flow FAB were applied. ESI and APCl were implemented in the mid 1990s [39]. The group evaluated both Q-TOF and multistage MS" with an ion-trap instrument for the dereplication of flavonoids in erode plant extracts [12], and pioneered the application of on-line LC-NMR in this field [39]. 

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. 

Detection schemes will also continue to evolve. While NMR is covered in another chapter in this volume, advances in NMR used as an LC detector have also been reported (25-28) that may eventually increase the utility of this tool for combinatorial chemistry. Used in both stopped flow and on line modes, LC/NMR can be extremely useful for structure elucidation provided the proper mobile phases or solvent suppression techniques are used (27). 

The standard LC-NMR detector is a saddle coil which is wound on the flow cell with a diameter of 2-4 mm and a length of 12-14 mm (Fig. 9.3.5) [Webl], The cell consists of a glass tube. Inflow and outflow are provided by tubings from FIFE with an inner diameter of typically 0.25 mm. The associated chromatography column is positioned outside the magnet. Because of time requirements 2D-NMR spectra can be acquired only in stopped flow mode, while in continuous flow mode ID-NMR spectra are acquired as a function of the elution time. 

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. 

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