No description available.

No description available.

Tyramide signal amplification is a technique used in microscopy experiments whereby phenol-bearing reporter molecules (containing, for example, fluorophore or heavy metal atoms, for use with fluorescence and electron microscopy respectively) are deposited in the vicinity of a target via an antibody-linked horseradish peroxidase-mediated coupling reaction. A paramagnetic lanthanide complex with a phenolic functionality was synthesised in order to adapt the method for use with MRI, and the process has been shown to be practicable both in model systems and in biological samples. A system has been developed so that chromatography experiments can be imaged with minimal disturbance to the column while it is in the MRI scanner and used to demonstrate measurements on ion exchange columns. Spin-labelled sugars were synthesised, with the intention of visualising the affinity chromatography process; these proved to be at the limit of detectability as relaxivity agents at the required concentrations. The affinity chromatography process was successfully imaged however, using the paramagnetic properties of Mn²⁺ ions bound to concanavalin A to provide the necessary image contrast. Stable nitroxide free radicals were synthesised to study the effect of charge-magnitude and -separation on proteoglycan affinity. It was demonstrated that species with greater positive charge had a greater affinity for articular cartilage. A brief investigation into the effect of nitroxides on the action of horseradish peroxidase was also carried out; they were found to act as non-competitive inhibitors.

The aim of this dissertation was to investigate and quantify the mass transfer processes occurring within a chromatography column by utilising Nuclear Magnetic Resonance (NMR). The dissertation describes the development of Magnetic Resonance Imaging techniques to enable the quantitative imaging of urea and lysozyme in one-, two- and three-dimensions, <i>in situ</i> and without chemical labelling. These images reveal the evolution of the distribution of both species simultaneously, and hence highlight that much of the distortion to the concentration distribution in the column arises from the inlet distributor. In addition to these novel imaging protocols, alternating pulsed gradient stimulated echo (APGSTE) NMR was used to obtain quantitative information on the microscopic motion of molecules over time periods from a few milliseconds up to two seconds. This enables quantitative measurement of eddy dispersion, mass transfer between mobile and stagnant liquid, and longitudinal diffusion. These effects can then be used to predict the microscopic (i.e. over displacements < 20 particle diameters) dispersion to within 25% over a range of flow rates. Furthermore, spatially resolved pulsed gradient stimulated echo NMR was used, in conjunction with the MRI techniques developed previously in this work, to isolate and quantify the effect of the inlet distributor and the packing heterogeneity on the macroscopic dispersion. Finally, a preliminary investigation of the influence of macroscopic flow heterogeneity on SEC protein refolding was performed. This confirmed an earlier hypothesis that sample distribution at the inlet of the column dominates the final refolding yield in SEC refolding reactors.

The interactions of CO, NO and O₂ with Pt{111}, pt{211}, and Pt{411} have been investigated with Single Crystal Adsorption Calorimetry (SCAC). Coverage-dependent heat of adsorption and sticking probability data for 300 K are presented, and are discussed with respect to the kinetics and thermodynamics of adsorption and desorption. CO adsorption proceeds molecularly on both Pt{111} and Pt{411}, with initial sticking probabilities of 0.78 and 0.80, and initial heats of adsorption of 176 kJ mol^-1 and 196 kJ mol^-1, respectively. Both adsorbate layers are found to be non-localised, having identical pre-exponential factors of 2x10¹⁵ s^-1. Discrepancies between isosteric and calorimetric heat of adsorption data at low coverage for CO/Pt{111} have been rationalised in terms of an entropy-driven localised/non-localised phase transition occurring above 300 K. Initial adsorption of NO occurs with a sticking probability of 0.84 on all three surfaces. The initial heat of adsorption for NO/Pt{411} indicates dissociative adsorption. The NO/Pt adsorbate layers are found to be highly localised, with pre-exponential factors in the range 10¹⁷ – 10²⁰ s^-1. This has been discussed in terms of desorption from highly compressed adsorbate layers in which co-operative effects are extant. O₂ adsorption proceeds dissociatively on all three surfaces, with the initial heats of adsorption all above 300 kJ mol^-1. Desorption occurs from localised adsorbate layers, with all systems having pre-exponential factors of 10¹⁸ s^-1. A kinetic compensation effect has been observed for the data of this thesis, as well as that of the SCAC literature, and is discussed within the context of the chemisorption entropy-enthalpy compensation.

No description available.

No description available.

Hybrid silica-based monoliths (prepared with tetramethoxysilane and methyltrimethoxysilane 3:1 v/v) were prepared in 100μm I.D. capillaries using a sol-gel technique. Although good electrochromatographic repeatability was shown on a single hybrid silica-based monolithic capillary poor reproducibility of the preparation was evidenced by the small number of good quality columns obtained in this study.

In recent years, non-electrolyte solution studies using chromatography have been extended to binary solvent systems. The results have not proved easy to reconcile with established theories of solution. An exciting spin-off of these studies has been the introduction of the so-called window analysis for chromatographic optimization. The thesis includes an exhaustive and critical survey of solution theories (some of which allow for complexation of one sort or another) as presented in the context of gas liquid chromatography. It is shown that in certain of these theories, misconceptions or algebraic infidelities have led to misleading results which, in some instances, have formed the basis of unnecessary polemic. In such cases, corrected versions of the theory are presented which allows securely based criticism and comparison. The experimental section comprises a comprehensive study of the squalane/dinonyl phthalate system. Retention data for ten solutes in five different liquid phase compositions, at each of eight temperatures in the range 0° to 700, have been determined with a high precision chromatograph designed and constructed in the laboratory. The results are compared with those of available static studies. It is shown that an approach by M.W.P. Harbison, R.J. Iaub, D.E. Martire, J.H. Purnell, and the author, provides an adequate account. Other theories (except, possibly, an extension of the so-called COPS theory of M.wa Muanda, J.B. Nagy and O.B. Nagy, which allows partial dimerization of dinonyl phthalate) are shown to fail, if only to an extent of a few percent in predictive power. The final section is devoted to the development of computer programs compatible with window-analysis optimization for binary solvents and for solvent mixtures of up to five components. Practical examples involving the complete baseline separation of mixtures of up to 40 components are provided. The very considerable power and potential of the window-analysis technique, and hence the computer programs, is discussed.

The major objective of these investigations was to develop a total assay for quantifying drugs of forensic interest in biological fluids that have presented problems with more conventional procedures. Supercritical fluid techniques in combination with mass spectrometry provided the main basis of the studies reported in this thesis. The practical aspects of the combination of supercritical fluid techniques with mass spectrometry was also investigated with a view to increasing sensitivity and specificity. Chapter 1 provides an overview of basic chromatographic techniques such as gas chromatography (GC) and high performance liquid chromatography (HPLC). The development of supercritical fluid chromatgraphy (SFC) and capillary zone electrophoresis (CZE) were discussed in greater depth. A method was developed for the separation of a number of beta-blockers using CZE. The basic theory of mass spectrometry was also reviewed together with an example of electrospray ionization of some small polar pharmaceutical compounds. The development of interfaces capable of coupling SFC systems to a mass spectrometer was also reviewed. Chapter 2 gives an overview of the beta-blockers which were used as test compounds during method development. Supercritical fluid separations of the beta-blockers using UV and mass spectrometric detection were compared. SFC/MS using the thermospray interface proved to be the most sensitive. The use of a triple quadrupole mass spectrometer was utilised to provide an increase in specificity needed when detecting trace amounts of drug in complex matrices. Chapter 3 gave an overview of some of the common problems with conventional sample preparation techniques and how supercritical fluid extraction (SFE) may be used to overcome them. On-line SFE/SFC/MS was developed for analysis of beta-blockers in horse and human blood fluids with quantitative results obtained from off-line SFE/HPLC/MS. Chapter 4 describes the use of double charge transfer (DCT) spectroscopy to gain information on ground and excited electronic states of doubly charged ions. The final chapter describes the instrumentation and experimental conditions used in these investigations.