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Accelerator mass spectrometry (AMS) has been used routinely for radiocarbon measurements for several years. During this period it has become evident neither the accuracy nor the range of the technique were as great as had originally been hoped. This thesis describes both theoretical work to understand the reasons for this and practical solutions to overcome some of the problems. The production and transport of the ions used in the measurements are found to be the most crucial stages in the process. The theories behind ion production by sputtering are discussed and applied to the specific case of carbon sputtered by caesium. Experimental evidence is also examined in relation to the theories. The phenomena of space charge and lens aberrations are discussed along with the interaction between ion beams and gas molecules in the vacuum. Computer programs for calculating phase space transformations are then described; these are designed to help investigations of the effects of space charge and aberrations on AMS measurements. Calculations using these programs are discussed in relation both to measured ion beam profiles in phase space and to the current dependent transmission of ions through the Oxford radiocarbon accelerator. Improvements have been made to this accelerator and these are discussed in the context of the calculations. There are many reasons for wishing to produce C^- ions directly from carbon dioxide. The most suitable type of source for achieving this is the Middleton High Intensity Sputter Source. Experiments to evaluate the performance of such a source are described and detailed design criteria established. An ion source designed and built specifically for radiocarbon measurements using carbon dioxide is described. Experiments to evaluate its performance and investigate the underlying physical processes are discussed. The source is found to have a high efficiency enabling small samples (<100 μg of carbon) to be measured. The cross contamination is measured to be low (<0.1%) and the background currents are small; the implications of these results are discussed.

Fundamental and applied aspects of ICP-MS have been investigated to gain an increased understanding of the technique and improve on its analytical capabilities. Dissociation temperatures of polyatomic ions were calculated using a double-focusing sector instrument, to obtain more reliable mass spectral data with controlled vapour introduction via a Dreschel bottle to allow accurate calculation of the ingredients in the plasma. The equilibrium temperature for the plasma, operated at 1280 W calculated using CO*, and as the thermometric probes, was c.a. 5800 - 7400 K, while using ArO* and ArC* as the thermometric probes the temperature calculated was c.a 2000 - 7000 K. Calculated dissociation temperatures were used to elucidate the site of formation of these ions. Results confirmed that strongly bound ions such as CO* and C2* were formed in the plasma whereas weakly bound ions such as ArO* and ArC* were formed in the interface region due to gross deviation of the calculated temperatures from those expected for a system in thermal equilibrium. The use of helium gas in a hexapole collision cell Attenuated the signals of ArH* Ar* ArO*, Arc*, ArCl* and Ara* allowing improved determination of ^^K*, *'Ca*, ^^e* ^^Cr*, ''As* and ^°Se*in standard solutions. The use of the hexapole collision cell also resulted in an enhancement of analyte signals due to the thermalisation of the ion beam. The ion kinetic energy of ions sampled from the plasma and those sampled from the skimmer cone were determined using a modified lens stack to assess the significance for memory effects of material deposited on the skimmer cone. The most probable kinetic energy of Be* ions sampled from the skimmer cone was found to be 2A eV, which was considerably lower than the most probable kinetic energy of Be* ions sampled from the plasma, which was found to be 9.5 eV. The low kinetic energy of the ions deposited on the skimmer cone means they will only contribute to the analytical signal under certain instrumental operating conditions. The feasibility of liquid sample introduction into a LP-ICP-MS system designed for gaseous sample introduction was investigated using a particle beam separator. The low signal was attributed to the low gas kinetic temperature of the plasma which was confirmed by the fact that the signal increased rapidly with increasing temperature of the transfer line between the particle beam separator and the LP-ICP torch. This was also supported by the fact that more volatile compounds gave mass spectra whereas less volatile compounds did not. A limit of detection of 30 mg 1'^ for chlorobenzene was achieved. Finally, silicon and phosphorus speciation was performed by HPLC coupled to sectorfield ICP-MS. Silicones ranging in molecular weight from 162 g mol'^ - 16500 g mol"^ were extracted from spiked human plasma and separated by size exclusion chromatography. Limits of detection ranged from 12 ng ml"' Si* for the 162 gmol'^ silicone to 30 ng ml'' Si* for the 16500 g mol' silicone. Organophosphate pesticides were extracted from spiked plasma and separated by reversed phase chromatography. Recoveries were between 55 - 81 %. Limits of detection were 0.9 ng ml'' P* 1.8 ng ml'' P* 1.6 ng ml"' P* and 3.0 ng ml'' P* for dichlorvos, methyl parathion, malathion and quinolphos respectively. Phosphates were extracted from various food products and separated by ion-exchange chromatography. Limits of detection were 1.0 ng ml"' P* 2.3 ng ml"' P*, and 39 ng ml"' P* for P04^", PaOy"^ and PsOio^" respectively.

Mass spectrometry has become an important tool for analysis of protein complexes. This study utilizes electrospray ionization (ESI) coupled to a Fourier transform ion cyclotron resonance mass spectrometer (FTICR-MS) to analyze noncovalent complexes in the gas phase. Binding of cucurbit[7]uril (CB7) to intact bovine insulin and the B-chain of insulin was investigated. Competition experiments involving the B-chain and a mutant B-chain were performed to probe the solution-phase binding site. Electron capture dissociation (ECD) of CB7 complexed to intact insulin and to the B-chain, produced a series of peptidic fragments of insulin in complex with CB7. Analysis of these fragments allowed the determination of the apparent gas-phase binding site, which appears different than the proposed solution-phase binding-site. These studies thus suggest that CB7 migrates when the complex is transferred from solution to gas phase. The results of this study caution against using ECD-MS as a stand-alone structural probe of solutionphase binding.

Mass spectrometry has become an important tool for analysis of protein complexes. This study utilizes electrospray ionization (ESI) coupled to a Fourier transform ion cyclotron resonance mass spectrometer (FTICR-MS) to analyze noncovalent complexes in the gas phase. Binding of cucurbit[7]uril (CB7) to intact bovine insulin and the B-chain of insulin was investigated. Competition experiments involving the B-chain and a mutant B-chain were performed to probe the solution-phase binding site. Electron capture dissociation (ECD) of CB7 complexed to intact insulin and to the B-chain, produced a series of peptidic fragments of insulin in complex with CB7. Analysis of these fragments allowed the determination of the apparent gas-phase binding site, which appears different than the proposed solution-phase binding-site. These studies thus suggest that CB7 migrates when the complex is transferred from solution to gas phase. The results of this study caution against using ECD-MS as a stand-alone structural probe of solutionphase binding.

No existent analytical method allowed the determination of the vicinal diketones (VDK), 2,3-butanedione (diacetyl) and 2,3-pentanedione, by liquid chromatography/mass spectrometry (LC/MS). An LC/MS method was developed for the simultaneous determination of diacetyl and 2,3-pentanedione in beer. A method allowing the determination of the amino acids (AA) related to the formation of VDK during fermentation was also developed. VDK were derivatized with o-phenylenediamine (OPDA) to form quinoxaline compounds. The reaction of VDK with OPDA was studied to optimize reaction time. Conversion of the diacetyl precursor, alpha-acetolactate, was tested using multiple oxidative decarboxylation techniques. Attempts were also made to determine simultaneously the AA, leucine, isoleucine and valine with the VDK. Simultaneous determination was unsuitable for the AA levels found in beer fermentation and separate methods for the determination of AA were developed. Total VDK were measured over a concentration range of 10 mug/L to 10 mg/L with less than 10% variation. These analytical methods were tested using a laboratory scale experiment to assess the impact of fermentation temperature on total VDK production and AA absorption. Samples collected in a local brewery were analyzed for total VDK using the developed method.

Formaldehyde cross-linking has been used to study protein-protein interactions in cells. Its short spacer arm, ability to permeate through cell membrane and the reversibility of the cross-linking reaction makes this a desirable cross-linker for in vivo studies. Although it has been widely used as a cross-linking reagent, the detailed chemistry behind protein cross-linking is not well understood. In vitro studies conducted under extended incubation periods (2 days) have shown that a multitude of amino acids are reactive to formaldehyde and that residue accessibility appears to play a role in reactivity. How applicable these findings are to formaldehyde cross-linking studies done under in vivo conditions (10-20 min incubations) is unclear. The chemistry of formaldehyde cross-linking was therefore investigated in model peptides under conditions similar to those used in in vivo studies. It was observed that only a subset of amino acids (amino termini and side chains of lysine and tryptophan) that were found reactive under extended incubation times was reactive in the much shorter incubation period. No cross-linking was detected between peptides, and elevating the peptide and formaldehyde concentrations resulted in only a minimal amount of cross-linked peptides. The relationship between residue accessibility and formaldehyde reactivity was assessed in model proteins that contain a more complex tertiary structure. It was shown that the extent of formaldehyde reactivity was dependent on the state of protein unfolding, i.e., solvent accessibility of reactive residues, and that an unfolded protein showed a significantly higher number of formaldehyde-induced modifications than a folded form, with lysine being the predominant reactive site. Formaldehyde treatment of proteins in their native form resulted in a low number of modifications even under an increased incubation time, suggesting that the protein remains folded during the course of the reaction. This is important for in vivo cross-linking studies where specificity and stability of protein-protein interactions is dictated by protein tertiary structure.

Contemporary proteomics studies require computational approaches to deal with both the complexity of the data generated, and with the volume of data produced. The amalgamation of mass spectrometry -- the analytical tool of choice in proteomics -- with the computational and statistical sciences is still recent, and several avenues of exploratory data analysis and statistical methodology remain relatively unexplored. The current study focuses on three broad analytical domains, and develops novel exploratory approaches and practical tools in each. Data transform approaches are the first explored. These methods re-frame data, allowing for the visualization and exploitation of features and trends that are not immediately evident. An exploratory approach making use of the correlation transform is developed, and is used to identify mass-shift signals in mass spectra. This approach is used to identify and map post-translational modifications on individual peptides, and to identify SILAC modification-containing spectra in a full-scale proteomic analysis. Secondly, matrix decomposition and projection approaches are explored; these use an eigen-decomposition to extract general trends from groups of related spectra. A data visualization approach is demonstrated using these techniques, capable of visualizing trends in large numbers of complex spectra, and a data compression and feature extraction technique is developed suitable for use in spectral modeling. Finally, a general machine learning approach is developed based on conditional random fields (CRFs). These models are capable of dealing with arbitrary sequence modeling tasks, similar to hidden Markov models (HMMs), but are far more robust to interdependent observational features, and do not require limiting independence assumptions to remain tractable. The theory behind this approach is developed, and a simple machine learning fragmentation model is developed to test the hypothesis that reproducible sequence-specific intensity ratios are present within the distribution of fragment ions originating from a common peptide bond breakage. After training, the model shows very good performance associating peptide sequences and fragment ion intensity information, lending strong support to the hypothesis.

The aim of this project was to investigate a range of medicinal herbs which have radical scavenging and antioxidant activities and then apply novel mass spectrometric techniques to investigate and analyse active components responsible for their pharmaceutical actions. A sensitive electron capture negative ionization of gas chromatography-mass spectrometry (ECNI-GC-MS) method was developed to assess hydroxyl radical production, as indicated by 3.4-dihydroxyphenylacetic acid (DOPAC) production, which allows excellent evaluation of hydroxyl radical scavenging and antioxidant activity of a number of medicinal Chinese herbs. Melatonin is an effective multiple radical scavenger and antioxidant and has been used in this study for the comparison of radical scavenging activity with medicinal herbs. To analyse active compounds from herbal extracts, mass spectrometric techniques were used to separate components that suppressed hydroxyl radical production from Dimocarpus longan Lour, determine known ginsenosides from ginseng extracts as well as to identify and quantify melatonin in ten herbal extarcts. The results obtained indicated that 1) the utilization of alumina in the ECNI-GC-MS method diminished interferences from ???noise??? products in a Fenton-type reaction, which allows obtaining pure final hydroxyl radical product and this method demonstrated optimal sensitivity and reliability; 2) Aqueous extracts of all herbs analysed showed different levels of hydroxyl radical scavenging activity. Dimocarpus longan Lour, Chrysanthemum morifolium Ramat, Lonicera hypoglauca Miq, Ginkgo biloba L, Rehmannia flutinosa and Libosch Cornus officinalis Sieb all exhibited stronger inhibitory effect on hydroxyl radical production than melatonin. 3) Aqueous extract of Dimocarpus longan Lour. showed the greatest inhibitory effect on hydroxyl radical production among the other herbs tested. The active fractions of this herb eluted just after the void volume using HPLC suggesting that the active compounds responsible for radical scavenging activity are polar and water soluble. They may belong to phenol group of chemicals. 4) Herbal extracts using non-polar solvents showed no effect on hydroxyl radical production suggesting active compounds in those herbs are water soluble. 5) Different species and origins of ginseng were compared for their radical scavenging activity. Chinese fresh ginseng (Oriental ginseng) showed higher activity than Korean ginseng tablet and American ginseng. Seven known active ginsenosides were identified using HPLC-MS-MS. 6) Melatonin was found at varying concentrations in ten herbs, which may contribute to the radical scavenging activity of herbs, on the other hand, it may provide the justification of clinical use and food resources, particularly for those herbs contain high level of melatonin.

Copies of author's previously published articles inserted. / Bibliography: leaves 195-196. / xv, 196 leaves : ill., maps ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / The aim of this research is to observe the evolutionary relationships within and between frog species, as well as to discover potentially useful medicinal peptides. The two main areas of research of this thesis are the characterisation of peptides from frogs belonging to the genus Litoria and the analysis of negative ion fragmentations from large peptides. / Thesis (Ph.D.)--University of Adelaide, Dept. of Chemistry, 1997?