LC-NMR analysis

For LC peaks at 0.1% level or less, a considerably long acquisition time may be required to achieve suitable signal-to-noise ratio. A couple of analytical techniques may be used to concentrate the degradants before the LC-NMR analysis, such as column switching or solid phase... 

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. 

Fig. 12 (A) Contour plot of the LC-NMR analysis of a mixture of vitamin A acetate isomers (400 MHz). (B) Slices taken through the contour plot showing spectra of the individual components of the mixture. (From Ref. l)...

When a new unknown impurity is observed, as a first experiment, the sample is analyzed by LC/MS. If the structure of the unknown impurity cannot be conclusively elucidated by LC/MS data, LC-NMR can be employed to analyze the sample. If the sample is not suitable for LC-NMR analysis, the impurity needs to be purified for NMR characterization. 

The advantages of directly interfacing HPLC separation with NMR analyses have so far been limited by the time and amount of material required to obtain various NMR spectra. However, two different types of HPLC interface have been developed for LC-NMR analysis. In stopped-flow techniques, the material of interest is directed into an NMR sample cell within the magnetic field after HPLC separation has occurred, and the pumps are then stopped to allow the necessary NMR experiments to be performed. The second type of... 

A general setup of the experimental configuration used for performing LC/UV/MS and LC/NMR analysis is presented in figure 2. 

Nuclear magnetic resonance (NMR) spectrometers offer spectral capabilities to elucidate polymeric structures. This approach can be used to perform experiments to determine comonomer sequence distributions of polymer products. Furthermore, the NMR can be equipped with pulsed-liied gradient technology (PFG-NMR), which not only allows one to determine self-diffusion coefficients of molecules to better understand complexation mechanisms between a chemical and certain polymers, but also can reduce experimental time for acquiring NMR data. Some NMR instruments can be equipped with a microprobe to be able to detect microgram quantities of samples for analysis. This probe has proven quite useful in GPC/NMR studies on polymers. Examples include both comonomer concentration and sequence distribution for copolymers across their respective molecular-weight distributions and chemical compositions. The GPC interface can also be used on an HPLC, permitting LC-NMR analysis to be performed too. Solid-state accessories also make it possible to study cross-linked polymers by NMR. 

A common drawback to LC-NMR analysis derives from the interference of the resonances of the mobile phase, usually much higher than those of the analyte itself. To overcome this problem, the mobile phase coming from the liquid chromatograph is efficiently suppressed with techniques such as water-suppression enhanced-through Ti effects (WET). This allows the separation of analytes in typical RP-HPLC conditions using common solvents such as MeOH or acetonitrile, yet usually replacing water by D2O to achieve better quality spectra however, in the case of analyte signals coincident with the suppressed solvent resonances, those of the analytes will also be suppressed. On the other hand, in spite of the usefulness of LC-NMR for stmcmral elucidation of flavonoids, this is not a sensitive LC detection technique. Therefore, to improve sensitivity, transients can be accumulated by operation in two different modes the on-flow and stop-flow modes. In both cases, the analytes from an LC column pass into an NMR microflow probe that typically has an active volume of 60-120 p.L, comparable with the conventional NMR sample size of 500-600 p,L. 

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