On-line liquid chromatography-NMR

Burton, K.I. Everett, J.R. Newman, M.J. Pullen, F.S. Richards, D.S. Swanson, A.G. On-line liquid chromatography coupled with high field NMR and mass spectrometry (LC-NMR-MS) A new technique for drug metabolite structure elucidation. J. Pharm. Biomed. Anal. 1997, 15, 1903-1912. 

Because carotenoids are light- and oxygen-sensitive, a closed-loop hyphenated technique such as the on-line coupling of high performance liquid chromatography (HPLC) together with nuclear magnetic resonance (NMR) spectroscopy can be used for the artifact-free structural determination of the different isomers. 

Hansen, S.H. et al.. High-performance liquid chromatography on-line coupled to high-field NMR and mass spectrometry for structure elucidation of constituents of Hypericum perforatum L., Anal. Chem., 71, 5235, 1999. 

Internal reflection spectroscopy is widely applied for on-line process control. In this type of application, the chemical reactor is equipped with an internal reflection probe or an IRE. The goal of this type of application is the quantification of reactant and/or product concentrations to provide real-time information about the conversion within the reactor. In comparison with other analytical methods such as gas chromatography, high-pressure liquid chromatography, mass spectrometry, and NMR spectroscopy, ATR spectroscopy is considerably faster and does not require withdrawal of sample, which can be detrimental for monitoring of labile compounds and for some other applications. 

Godjehann et al. [199] have applied on-line high performance liquid chromatography coupled to proton NMR to the determination of nitroaromatic compounds in ammunition plant ground waters. 

The direct coupling of liquid chromatography with proton NMR has been attempted numerous times. Early experiments of coupled HPLC- H-NMR were conducted in a stop-flow mode or with very low flow rates [193-195]. This was necessary to accumulate a sufficient number of spectra per sample volume in order to improve the signal-to-noise ratio. Other problems associated with the implementation of on-line HPLC-NMR have included the need for deuterated solvents. However, with the exception of deuterium oxide, the use of deuterated eluents is too expensive for routine analysis. Therefore, proton-containing solvents, such as acetonitrile or methanol, must be used. To get rid of the solvent signals in the spectra, the proton NMR signals of the solvents have to be suppressed. 

Often there is a need for structural identification of unknowns without available reference compounds and the identification can be done in connection with the chromatographic separation. One approach is to run measurements directly on-line using HPLC as the separation technique with UV-detection and monitoring at several wavelengths, but this is often not enough for safety identification. The last 15 years have seen a rapid development of combined liquid chromatography-mass spectrometry instrumentation, and this technique is the most valuable tool in qualitative analysis today (se below). In the absence of a reference compound some unknown substances e.g., isomers of the desired compound may require NMR for their definitive identification. 

The major advantage of on-line NMR detection as opposed to off-line detection for liquid chromatography (LC) are improved chromatographic resolution, consistent response, and rapid data acquisition. The disadvantages include poorer sensitivity due to limited measurement time and a flow-rate dependence of the NMR linewidth [Webl]. Following the first stopped flow design in 1978 [Wat 1 ], an on-line system had been developed within a year [Bayl], and the techniques have been improved continuously since then [Webl]. 

Recent developments in the sensitivity and resolution of NMR spectrometers permitted us to use one as a real-time detector for high-performance liquid chromatography as demonstrated in recent review articles.332,333 Recently we developed an on-line GPC/NMR system in which a 500-MHz 1H NMR spectrometer was used as a detector.334-339 By using on-line GPC/NMR the molecular weight of a polymer can be determined without a calibration curve if the polymer sample contains a known amount of end-group per polymer molecule. 

Liquid chromatography-nuclear magnetic resonance (LC-NMR) combines the separating power of LC with the structural elucidation capabilities of NMR. It is now possible to separate and elucidate the structures of components in a mixture on-line using this technique. It is most often used to identify natural products, novel compounds and metabolites. 

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