| Summary: | Mass spectrometry (MS), nuclear magnetic resonance (NMR) spectrometry and Fourier transform infrared (FTIR) spectrometry have for many years been three of the analyst's main tools for the characterisation of unknown compounds. Their complementary nature is well known - NMR can provide unequivocal data for structural isomers (something not possible by mass spectrometry) while mass spectrometry and infrared can often identify moieties which lack a proton NMR resonance. For mixtures, high performance liquid chromatography (HPLC) coupled with any of these spectrometric techniques would provide a better solution than analysing the sample mixture directly, where spectral deconvolution would be extremely difficult. Similarly this combination would avoid the time-consuming isolation of individual components followed by off-line spectrometry. This work addresses the problems of bringing chromatography together with these detectors simultaneously, to provide an extremely powerful tool for the characterisation of mixturesThe first combination used was HPLC-NMR-MS. The conflicting requirements of both detectors in terms of flow and solvent composition were investigated. While a working combination was achieved choice of eluent remained difficult, principally because of eluent signals in the NMR recordings. Deuterated eluents were expensive and still contained residual proton signals. In chapter 2 the feasibility of LC-NMR-MS is demonstrated, while some limits on the types of solvents required are established, with the need for a compromise of the ideal eluents necessary for either LC-NMR or LC-MS being noted. In an effort to seek a better eluent for the combination of multiple spectroscopic techniques, superheated water was investigated as an alternative eluent for LC-NMR-MS. This proved to be a good eluent for multiple hyphenation, especially for NMR, as few additional signals were introduced. The effects of superheated water on column lifetime were significant for silica based columns, and compound stability was also a potential problem for some compounds. It was found that superheated deuterated water could produce stable deuterated compounds in high yields, with deuteration possible at sites other than just those of labile protons. The introduction of FTIR to the LC-NMR-MS system added additional constraints in terms of eluent, but IR spectra of adequate quality for library searching were obtained with an ATR flow cell. An automated flow injection system was constructed and a working sensitivity of 150-200 mug for a range of pharmaceutical compounds established for the IR part of the system. Finally, a series of complex samples was investigated by LC-NMR-MS-IR with both conventional eluents and with superheated water. Good separations and good quality spectra were obtained for a range of compounds under different chromatographic conditions. The work generated 18 peer reviewed papers on both the practicalities and the application of multiple hyphenation. The work also generated great interest from instrument manufacturers and shortly after the completion of the work both Varian and Bruker launched multiply hyphenated systems for purchase.
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