The actual signal recorded with an analytical instrument is not always a true reflection of the analysed sample. In this thesis a further insight of the atmospheric pressure ionisation processes electrospray (ESI) and atmospheric pressure chemical ionisation (APCI) has been endeavoured, to provide a deeper understanding of and ways to minimize this bias. A response model for ESI has been modified and used to study the influence of solvent composition on the observed mass spectrometric signal. The response model divides the response into an analyte partitioning coefficient and an instrumental response factor. A number of experimental parameters influencing the response were investigated including spray position relative to the orifice, spray potential, nebulizer and curtain gas flow rates, ionic strength and organic content of the sprayed solution. The history of the generated droplets turned out to be of significant importance to both the partitioning coefficients and the instrumental response factor. Furthermore, it was found that the total ionic strength and not only the electrolyte concentration will influence the instrumental response factor. In addition, based on the importance of hydrophobicity and electrophoretic mobility, a model was proposed for the ion distribution within the electrosprayed droplets. The coupling of an electrochemical (EC) cell to a mass spectrometric (MS) system has been evaluated. The coupling of the EC cell to the MS was made to decouple the cell from the high voltage circuit of the ESI. The feasibility for analyte ionisation, sample pre-concentration and solvent exchange as well as studying redox reaction products was shown.

The analysis of pesticide residues in fruit, vegetables, rape seed and water has been improved using developments in sample handling and analytical techniques. The method development is associated with analytical difficulties, since pesticides currently used in agriculture represent a variety of chemical classes having very different physico chemical properties. The method development also encounters difficulties when many various commodity classes with different characteristics are studied. The main task in pesticide residue analysis has been to provide multi residue methods, and traditionally GC has been the main analytical technique. In order to regulate the use of hazardous pesticides, the EU commission introduces strict maximum residue levels (MRL). The need for improved sample handling and detection techniques are, however, high due to handling of lower detection limits, complex matrices and the need of more efficient sample throughput. Of the new techniques introduced as alternative techniques to the traditional extraction techniques, pressurised fluid extraction (PFE) has shown to be a promising technique in analysis of pesticide residues in fatty foodstuffs. In water analysis, large sample volumes are needed due to low MRLs. The solid phase extraction (SPE) technique allows a concentration of large sample volumes and simplifies the tedious laboratory work with traditional separation funnels. A new approach was to use non-polar solvents for the sample extraction from the earlier used polymeric column. Both these techniques provide low solvent consumption, short extraction times and ability to automate the manual steps. An LC-MS/MS multi residue method was finally developed for pesticide residues in fruit and vegetables. The technique is robust and sensitive and allows a simultaneous determination of 57 pesticides and metabolites in one single analysis and without any clean-up steps. The sensitivity was improved to achieve the maximum residue limits needed by EU. Several multi step methods, which involve more costly analysis, has been replaced by this technique.

Liquid chromatography-mass spectrometry (LC-MS) is a powerful technique for the analysis of endogenous compounds. The introduction of electrospray ionization (ESI) as an interface between LC and MS has contributed strongly to a trend towards miniaturization of LC, due to the possibility to perform ESI at low flow rates. In this thesis, several aspects regarding the design of miniaturized LC systems and electrospray emitters were investigated. In addition miniaturized LC-ESI-MS have been used for the qualitative and quantitative analysis of endogenous polar compounds, peptides and protein digests. The performance of miniaturized LC-MS was compared using different electrospray emitter configurations. The results indicated that the efficiency of the LC system is rather independent of the configuration of the emitter. The lifetime of gold-coated fused silica electrospray emitters based on vapor deposited adhesion layers of titanium were investigated. The long lifetime of the emitter facilitates the use in LC-MS experiments, exemplified LC-MS by analysis of neuropeptides. The ESI voltage is shown to interfere with liquid chromatographic separations performed in packed porous graphitic carbon capillary column. This interference is ascribed to the presence of an electric field over the conductive column in absence of a ground point between the column and the ESI emitter. The solid supported enhanced microdialysis for analysis of neuropeptides were compared with conventional microdialysis. The difference between the two methodologies were evaluated by LC-MS analysis of the microdialysates. The solid supported method gave in general higher relative recoveries. Finally, a method of standard addition was developed to determine total level of tryptophan and two of its metabolites in human plasma by capillary LC-ESI tandem mass spectrometry. The method was applied in a clinical study of multiple scleroses patients treated with cytokines (IFN Beta 1a, 1b). The results show that the intervention effects the tryptophan metabolism.

This thesis exemplifies the strength in the combination of inductively coupled plasma (ICP) spectrometry and electrospray ionization tandem mass spectrometry ESI-MS/MS as detection techniques for liquid chromatography (LC) in the search for and investigation of compounds that bind or can bind hetero-atoms. Furthermore, some aspects involved in the coupling of LC and ICP spectrometry and quantification without identical standards have been studied. The importance of using a separation step in combination with ICP spectrometry was shown for urine and blood plasma samples from patients treated with boron neutron capture therapy. In addition to the carrier molecule used in the therapy, one major and a few possible minor metabolites were found in the urine samples. One fragment mass of the major metabolite was obtained with LC-ESI-MS/MS. Liquid chromatography coupled to ICP-MS was also shown to be a valuable tool for fingerprinting metal-binding compounds in complex matrices, such as siderophores (iron-complexing compounds) in soil. The presence of at least two siderophores in a field soil solution sample could be revealed by LC-ICP-MS. Their identities could thereafter be determined by LC-ESI-MS/MS. The non-UV-absorbing o-carboranylalanine could be quantified in relation to its degradation products by LC-ICP-AES, which provided information about the mechanism behind the degradation. Moreover, LC-ICP spectrometry was shown to provide an accurate quantification of biomolecules (bias < 10 %) when evaluated from external calibration graphs based on inorganic elemental standards. Finally, the causes of the large decrease in boron signal seen when adding acetonitrile to the LC mobile phase in LC-ICP-MS was investigated in some detail. Space charge effects might explain a large part of the depression from carbon species on the boron signal.

This research studies the chemical nature of zirconia and the complex surface chemistry of zirconia in order to better comprehend its behavior under chromatographic conditions. This research shows how the physical and chemical properties of zirconia depend strongly on the thermal treatment during synthesis. The morphology of the samples was also studied. The absorption capability of Adenosine Triphosphate (ATP) on zirconia was also monitored and spectrally characterized. The results of this research showed how the properties of zirconia vary with thermal treatment. It was observed that the zirconia prepared at a higher temperature had lower surface area, lower pore size and pore volume as compared to the zirconia prepared at a lower temperature. The morphology studies showed the porosity of the zirconia. The results from the absorption experiments showed that zirconia prepared at a higher temperature absorbed more ATP than the zirconia prepared at a lower temperature. Significant changes were also observed on the pellets of zirconia pre and post absorption experiments. I hope that this research sheds more light on the complex properties of zirconia’s surface chemistry and the results of this study could better help in the application and use of zirconia in chromatography to separate proteins.

Electrospray ionisation (ESI) coupled to mass spectrometry (MS) is one of the most important detection techniques for chemical analysis of small drugs as well as large biomolecules in life science today. In this thesis, aspects on improved compatibility between liquid based separation systems and mass spectrometric detection were investigated regarding buffers, sample preparation and analysis of polar compounds as well as peptides and protein digests for enhanced ESI-MS performance. Capillary electrophoresis (CE) coupled to ESI-MS detection, was evaluated using both a sheath flow interface and a sheathless design. The separation of peptides and small, polar compounds was optimised for both CE-ESI interfaces. The effect of sheath liquid composition was also studied with the aim to improve sensitivity in the ESI-MS detection. Polar compounds were retained and separated by capillary ion-pair chromatography coupled to ESI-MS detection. Since commonly used ion-pairing reagents are detrimental to the ESI process they were effectively removed before the ionisation by the use of a trapping column after the separation. Alternatively, the ion-pairing reagents were exchanged to volatile constituents. A method for peptide mapping by liquid chromatography (LC)-ESI-MS was developed for lactate dehydrogenase. The method was further enhanced to involve the proteolysis on-line to the LC-ESI-MS. No manual sample handling was then needed and the total analysis time decreased from 7 to 1.5 hours. The amount of information was also shown to increase in the on-line system. Finally, the on-line concept was extended to an innovative interface for direct coupling of a pumped liquid flow to an electroosmotically driven flow. This provided a valve-free sample transfer between capillary LC and CE, aiming towards increased peak capacity per unit time for the analysis of complex peptide samples.

O presente trabalho, teve como objetivo, dar continuidade ao estudo apresentado na forma de dissertação de mestrado, utilizando a complexona EDTA, totalmente \"neutralizada\". na forma de Y4-. Na titulação de íons metálicos, a hidrólise acentuada deste ânion, Y4-, causa um aumento marcante de pH após o ponto estequiométrico. Esta variação de pH pode ser acompanhada potenciometricamente, utilizando um eletrodo de vidro, ou visualmente com um indicador ácido/base como a fenolftaleina, em substituição a indicadores metalocrômicos. Uma série de parâmetros foram considerados na preparação do reagente, sua armazenagem e padronização bem como os problemas ligados a percentagem de \"neutralização\". Vários equilíbrios foram considerados no desenvolvimento de um programa em linguagem BASIC, para interpretar a curva teórica da titulação de Mg2+ com EDTA \"tetraneutralizado\", na qual participam também espécies como MgHY-, antes do ponto estequiométrico. Um método complexométrico rápido e preciso foi desenvolvido com este titulante para determinar Mg2+ e SO42- simultaneamente (este por método indireto para o excesso de íons Ba2+) com viabilidade para ser aplicado em água do mar e em concentrados salinos. O método mostrou-se adequado, também, para a determinação da mistura Ca2+ e Mg2+, em substituição ao método clássico com tampão e indicador metalocrômico, eriocromo T. / This thesis has the purpose to give continuity to a former study from the master dissertation, about the use of the complexon EDTA, totally \"neutralized\", as the species Y4-. During the titration of metallic cations the marked hydrolysis of the y4- anion causes a marked pH increase after the stoichiometric point. This change of pH can be followed potentiometrically with the glass electrode or by visual end point indication by on acid/base indicator as phenolphtalein instead of metalochromic indicators. A serie of parameters were considered in the preparation of the titrant, its storage and standardization as well as problems related with the percent neutraIization\". Equilibria were considered and used in a computer program in BASIC language in order to interpret the theoretical titration curve. The species MgHY- was found in significant contribution before the end point. A fast and precise complexometric method was developed with this titrant for Mg2+ and SO42- simultaneously (this last by titration of excess of standard Ba2+ solution). The method has been found adequate to determine mixture of Ca2+ and Mg2+ instead of the classic method which uses a buffer and eriochrome T as indicator.

Quorum sensing (QS) is a process that allows bacteria to sense the population density of cells around them by communicating with each other via autoinducer molecules. This cross-communication is crucial in the regulation of bacterial processes such as bioluminescence, virulence, and biofilm formation. Previous research by Milburn and Makemson on Vibrio harveyi suggested that in addition of the known biosynthesis of three well-characterized autoinducers, dozens of unknown molecules are also produced and released to the environment by V. harveyi. This study was performed using electrospray tandem mass spectrometry with the purpose of detection and characterization of the extracellular molecules produced by V. harveyi, and assessment of their relationship to QS. A total of 11 molecules were characterized, from which three could be related to QS. These findings provide a glimpse of the nature of novel secondary metabolites produced by V. harveyi and provide the groundwork for further research.

<p>This thesis covers the four topics discussed in each of the following paragraphs. It is unified by the dual ability of ambient ionization mass spectrometry as a useful analytical tool allowing for monitoring of chemical reactions, in addition to its capability to accelerate reaction rates using the same equipment under accelerating or non-accelerating conditions. The ability to manipulate reactions and monitor the subsequent effects to the rate of the reactions can provide vital information for many industrial arenas. Current process analytical technology (PAT) is extremely time-consuming, and typically costly due to dependence on analysis conducted at the end stage of production. Additionally, many chemical reactions found to be useful in pharmaceutical or manufacturing industries are labor intensive and require harsh conditions such as heat or expensive catalysts. Several methods have been developed to overcome these current limitations, while providing vital information on short-lived intermediates, degradation products, and accelerated reaction rates. A sampling device was developed and coupled with nESI allowing for monitoring of heterogeneous chemical reactions by mass spectrometry without the additional requirement of separation (filters, chromatography, etc.) In addition, this technique maintains the high sensitivity, specificity, speed and structural elucidation provided by mass spectrometry analysis. The analysis provided kinetic profiles of all reactants, intermediates, products and coproducts throughout the course of the reaction.</p><p> The ability to effectively control chemical reactions and their rates is a priority across several fields of study. Several factors affecting reaction rates, such as heat and catalysts selected, have been well studied. However, there has been recent interest in exploring the capabilities for reaction acceleration in charged microdroplets. It is known that reaction rates on the surface of a droplet greatly differ from reactions occurring in the droplet. The Katritzky transamination reaction was used as a model to identify the effects of the air-solution interface on reaction acceleration by varying the air-liquid surface to volume ratio. The significant increase in reaction rate constants was further enhanced by solid–solution interfacial effects observed after addition of glass nanoparticles.</p><p> The effective degradation of non-polar hydrocarbons is an environmental concern as they are the main composition of waste generated from petroleum processing. Saturated alkanes are relatively stable molecules which present a challenge for analysis by mass spectrometry without the use of extreme experimental conditions. A rapid analysis method by paper spray ionization was developed that allows for the oxidation products of saturated alkanes to be monitored by MS in under two minutes. This method relies on the generation of a hydroxyl radical by reacting iron (III) chloride with aqueous hydrogen peroxide on the principle of Fenton’s chemistry. The presence of this radical in direct contact with an alkane produces several oxidation products which can be easily monitored by MS. The reagents are added to a paper triangle sequentially, creating a thin film which allows reaction acceleration in relatively small volumes analyzed directly from paper at atmospheric pressure.</p><p> The dimerization of 4-ethynylaniline derivatives in acetonitrile was monitored by nano electrospray ionization mass spectrometry. Dimer products formed by electrocyclization and radical processes were observed that are not detected as a corresponding bulk reaction. This gas-phase reaction has been interrogated in a solution phase analog with radical initiators and characterized by ¹H NMR. This work demonstrates that compounds can be synthesized by the electrospray process. Future studies may reveal how this observation affects the interpretation of the MS results involving electrospray.</p>

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