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Interest in temperature as a modifier in HPLC separations has increased markedly recently due to miniaturisation, new stable hybrid stationary phases and the use of superheated water as an eluent. However, for decades temperature has generally been regarded as a parameter that should be kept constant in LC separations for retention reproducibility but there is now a mountain of journal papers supporting the use of this variable. Currently, the limit of implementation usually arises from the lack of LC ovens on the market capable of high temperature applications. This led to the development of a resistively heated LC oven which allowed rapid reproducible heating/cooling patterns of RP columns thus reducing equilibration times and realising high sample throughput. The main objective was to drive temperature programming to a new extreme by rapid column heating akin to GC rather than LC, the emphasis being to produce much sharper peaks very rapidly. It was hoped that temperature gradients could replace solvent gradients and extend the applicability of temperature-programming for hyphenation to other forms of detection. New column materials were sought and tested against high speed temperature gradients. This coupled with micro-column technology should reduce analysis time and appreciably limit the amount of solvent waste currently being generated by conventional LC techniques. The column heating was achieved by two system components, the oven and the eluent pre-heater unit both based on resistive heating. Post-column cooling prior to detection minimised baseline disturbances imposed by a temperature gradient and enabled the use of detection modes such as MS, RI and ELSD as well as UV spectroscopy.

Matrix-assisted laser desorption/ionisation (MALDI) is an established technique for the analysis, primarily, of large biomolecules, but is also widely applied to many other compounds as well. The development of atmospheric pressure MALDI (AP MALDI)has broadened the horizons for this technique in many ways. These include softer ionisation and simplicity of interfacing to a broader range of instruments on which MALDI can be applied. The study presented here is primarily concerned with the design, construction, development and testing of a novel atmospheric pressure MALDI source. Initially destined to be coupled to a time-of-flight instrument, circumstances dictated that the newly developed source be interfaced with a triple quadrupole instrument, of which this is the only known study. Differences between a pulsed ionisation source and a scanning instrument were overcome and the analyses of many types of compound are illustrated, demonstrating the flexibility of AP MALD!. Additionally, during the course of this study, the temporal pulse shape of the ion packets detected was observed to have a tail. Upon further investigation it was speculated that the information contained within these individual ion packets could provide valuable information concerning the ionisation mechanisms of the MALDI process. Similarities between the data gleaned from the pulse shape work and other ion mobility studies was observed. Finally, a preliminary investigation into the feasibility of capillary ion transport through fine glass tubes was undertaken. The transport properties of the capillaries was studied as a means of remotely transporting ions (or neutral species) created at atmospheric pressure to the vacuum of the mass analyser employed. Extending the length of the capillary could provide a practical method of remote sampling of the ions; an application which may become more important in the future of mass spectrometry.

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The use of lanthanide complexes for the detection of biologically relevant species such as anions, pH and metal ions has grown significantly over the past decade. Such probes offer significant advantages over conventional probes; sharp narrow emission lines encode detailed spectral information and allow ratiometric analysis, and their luminescence is long-lived allowing selective spectral acquisition. Many lanthanide-based probes operate in aqueous media, but few have been applied to intracellular measurements. The introductory chapter considers the design of lanthanide based probes for cellular applications. The fundamentals of lanthanide emission are discussed, and how the ligand structure needs to be carefully constructed to maximise emission efficiency. Reported bicarbonate- and pH-responsive probes, both lanthanide based and non-lanthanide based, are reviewed, leading to a set of proposed novel probe structures. The synthesis of these probes with further reasoning behind their design is described in chapter two. The chapter concludes with an overview of the complexes in terms of their emission spectral form, hydration number, HPLC and mass spectrometry properties. Chapter three presents work on bicarbonate-responsive probes. Through a series of spectral titrations, affinity constants for a range of anions were assessed. A high selectivity for bicarbonate was observed over other oxy-anions, and in the presence of protein. The complexes exhibited a mitochondrial localisation profile, and the europium luminescence intensity was modulated reversibly in response to pCO2. The terbium analogues showed negligible change under such conditions, and thus a europium to terbium emission intensity ratio could be used to calibrate pCO2. This principle was also applied to the analysis of bicarbonate levels in simulated biological fluid, and in a serum sample. Several pH-responsive complexes are described in chapter four. Key aspects of pH-probe design were highlighted in early examples, leading to the final set of probes based on reversible sulphonamide ligation. Spectral titrations were performed to assess pKa values, and the use of europium to terbium emission intensity ratios was again found to offer a convenient method for assessing pH. Unprecedented changes in CPL were found in response to pH in these systems, and gem values could be used to report upon pH. The complexes were observed to exhibit a lysosomal distribution. Finally, chapter five contains experimental procedures for each compound synthesised, as well as general experimental procedures. Both sets of complexes described in this thesis show great promise for use as well-defined intracellular probes of bicarbonate or pH.

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