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El incremento del vertido de sustancias contaminantes al medio ambiente, debido al desarrollo industrial y los malos hábitos de consumo, han provocado la ruptura del equilibrio de muchos ecosistemas llegando afectar, en muchas ocasiones, a los humanos. Con el objetivo de controlar y disminuir estos efectos, los gobiernos han dirigido acciones para legislar y regular el vertido de sustancias tóxicas al medio. De esta manera el Gobierno Nacional de España ha financiado a nuestro grupo de investigación, el proyecto del Plan Nacional en Ciencia y Tecnología Química titulado “Desarrollo de métodos automáticos de análisis. Aplicación a planes de vigilancia ambiental”, con referencia CTQ 2010-15541. La presente tesis forma parte de este proyecto siendo su objetivo general el desarrollo de nuevos métodos automáticos para la determinación de especies químicas de interés ambiental. En la introducción de la tesis se realiza una revisión sobre la automatización del laboratorio químico mediante técnicas de análisis en flujo y se describen las dos utilizadas en esta tesis. Seguidamente se revisa la aplicación de estas técnicas a la automatización de los métodos electroquímicos, especialmente la conductimetría y la voltamperometría redisolución anódica. En el tercer apartado de la introducción se recogen los aportes de las técnicas de flujo en la automatización de procesos de tratamientos de muestras como la difusión gaseosa y la extracción líquido-líquido. De esta última técnica se resume su evolución histórica hasta los métodos más recientes descritos que logran la automatización total del proceso de extracción y que se pudieran clasificar dentro de la tercera generación de las técnicas en flujo. En el último apartado de la introducción se trata la automatización de métodos cinético-catalíticos mediante técnicas de análisis en flujo, destacando el elevado potencial que tienen estos métodos para el análisis de trazas y el bajo número de trabajos publicados donde se consigue la automatización de los mismos. Contenido de la investigación En esta tesis se han utilizado dos técnicas de análisis en flujo, el análisis por inyección en flujo multijeringa (MSFIA) y los sistemas en flujo multibombas (MPFS), acopladas a dos métodos de tratamiento de muestras, la difusión gaseosa y la extracción líquido-líquido, y tres técnicas de detección, la voltamperometría de redisolución anódica, la conductimetría y la espectrofotometría, para obtener cinco sistemas automáticos de análisis. Los sistemas desarrollados han sido aplicados a la determinación, fundamentalmente en agua de mar, de cinco analitos inorgánicos: cadmio, cromato, yoduro, amonio y carbono inorgánico total, en muestras ambientales. Durante el desarrollo de estos sistemas y el estudio de las técnicas utilizadas, se han solucionado problemas relacionados con la automatización de las técnicas en flujo, lo que ha mejorado significativamente las características analíticas de los métodos presentados en comparación con otros similares reportados en la literatura. Entre estas mejoras se encuentran por ejemplo el incremento de la reproducibilidad y sensibilidad de las determinaciones, la disminución de los volúmenes de reactivos y muestras utilizados, la reducción de las dimensiones del sistema y de los costes de la instrumentación, así como el aumento del grado de automatización del proceso analítico: tratamiento de la muestra, calibración y detección. Los trabajos presentados en esta tesis son los siguientes: 1- Un método simple, económico, portátil y automatizado mediante MSFIA que permite la determinación de cadmio mediante voltamperometria de redisolución anódica usando electrodos impresos con película de bismuto. (SPE- BiF-ASV- SFIA) 2- Un sistema automatizado mediante la técnica de MSFIA para la determinación de amonio en muestras de aguas naturales y marinas utilizando la difusión gaseosa y la detección conductimétrica. (C-GD-MSFIA) 3- Un analizador automático, basado en la técnica de MPFS, para la determinación de amonio y de carbono inorgánico total en muestras de aguas marinas usando difusión gaseosa y detección conductimétrica. (C-GD-MPFS) 4- Un sistema totalmente automatizado de microextraccion líquido-líquido dentro de la jeringa de una bureta automática para la determinación espectrofotométrica de cromo (VI) en muestras de aguas naturales. (In syringe-MSA-DLLME) 5- Un chip conductor de microfluidos para la automatización mediante MSFIA de un método cinético-catalíticos que permita el análisis de ioduro en agua de mar y fármacos con un consumo muy reducido de reactivos. (CHIP-MSFIA-Catalytic) Conclusiones Se han desarrollado nuevos sistemas automáticos para la determinación de analitos inorgánicos de interés ambiental. La utilización de técnicas de análisis en flujo multiconmutadas, como MSFIA y MPFS, han demostrado ser muy útiles en la automatización de sistemas de análisis que utilizan diferentes técnicas de detección (electroquímicas y espectrofotométricas), de pretratamiento de muestra y de métodos de determinación cinéticocatalíticos. Los sistemas desarrollados han demostrado su efectividad en la determinación de analitos inorgánicos (cadmio, cromato, yoduro, amonio y carbono inorgánico total) en muestras tan complejas como las aguas de mar, aguas residuales o preparados farmacéuticos. Bibliografía 1.Henríquez C, Laglera LM, Alpizar MJ, Calvo J, Arduini F, Cerdà V: Cadmium determination in natural water samples with an automatic multisyringe flow injection system coupled to a flow-through screen printed electrode. Talanta 2012, 96(0):140-146. 2.Henríquez C, Horstkotte B, Cerdá V: Conductimetric determination of ammonium by a multisyringe flow injection system applying gas diffusion Int J Environ Anal Chem 2012:1-17. 3.Henríquez C, Horstkotte B, Cerdá V: A highly reproducible and robust solenoid micropump system for the analysis of total inorganic carbon and ammonium using gas diffusion with conductometric detection. Talanta 2013, In review 4.Henríquez C, Horstkotte B, Solich P, Cerdá V: In-syringe magnetic stirring assisted liquid-liquid microextraction for spectrophotometric determination of Cr (VI) in waters. Analytical and Bioanalytical Chemistry 2013, Accepted/in press. 5.Abouhiat FZ, Henríquez C, Horstkotte B, El Yousfi F, Cerdà V: A miniaturized analyzer for the catalytic determination of iodide in seawater and pharmaceutical samples. Talanta 2013, 108(0):92-102. / Introducción The increasing discharge of pollutants into the environment due to the industrial development and consumer habits have caused the increasing unbalance of many ecosystems, already affecting in many occasions human species itself. In order to control and reduce these effects, many governments have initiated actions to control the discharge of toxic substances into the environment. In this way, the Spanish Government has funded, to our research group, the project of the National Science and Technology Chemistry entitled “Development of automated methods of analysis. Application to environmental monitoring plans”, with reference CTQ 2010- 15541. This PhD thesis is part of this overall project with the main objective of the development of new automated methods for the determination of some chemical species of environmental interest. In the first part of the introduction of this thesis, the automation of different procedures in a chemical laboratory by the use of flow techniques is reviewed and the two selected flow techniques are described. In the second section, the application of these techniques in the automation of electrochemical methods, especially conductometry and anodic stripping voltammetry, is reviewed. In the third section of the introduction, the contributions of flow techniques to the automation sample pretreatment are discussed, especially for gaseous diffusion and liquid-liquid extraction. Moreover, it was reviewed the historical evolution and the most recent reported methods to achieve the full automation of liquid-liquid extraction inside the syringe, which could be classified as the third generation of flow techniques. The last part of the introduction deals with the automation of kinetic-catalytic methods using flow techniques, emphasizing the high potential of these methods for trace analysis and the low number of published papers on this subject. Research content. In this thesis, two flow techniques, MSFIA and MPFS, coupled to two sample pretreatment methods, gas diffusion and liquid-liquid extraction, and three detection techniques, anodic stripping voltammetry, conductometry and spectrophotometry, have been used to develop five automatic analytical systems. These systems have been applied to the determination of five inorganic analytes: cadmium, chromate, iodide, ammonium, and total inorganic carbon and applied to different environmental water samples with main focus on seawater. Involved in the development of these systems were the study of the used techniques and the finding of solutions for the problems related to the automation using flow techniques. Significant improvements of the analytical characteristics of the presented methods in comparison with similar ones reported in the literature are obtained. These improvement were an increase of the reproducibility and sensitivity of measurement, the decrease of reagent and sample consumption, the reduction of the analyzers’ sizes and instrumentation costs, and further, an increase of the automation degree of the analytical procedure, including: sample pretreatment, calibration and detection. The presented contributions are listed below: 1- A simple, economic, automated, and portable analyzer system based on MSFIA, which allows the determination of cadmium with anodic stripping voltammetry using a bismuth film coated screen printed electrode. (SPE-BiF-MSFIA) 2- An automatic MSFIA system for the determination of ammonium in naturals and seawaters in a very wide concentration range using gas diffusion and conductometric detection. (C-GD-MSFIA) 3- An automated and highly reproducible analyzer based on MPFS for the determination of total inorganic carbon and ammonium in seawaters using gas diffusion and conductometric detection. (C-GD-MPFS) 4- A fully automated in-syringe magnetic stirred-assisted dispersive liquid-liquid microextraction system for the spectrophotometric determination of chromate in natural waters. (In syringe-MSA-DLLME) 5- A microfluidic “Chip” conductor to be used in combination with MSFIA for the automated quantification of iodide in seawaters and pharmaceutical preparations using a kinetic-catalytic method characterized by a very low reagent and sample consumption. (CHIP-MSFIA-Catalytic) Conclusions Five new systems have been developed for the automatic determination of inorganic analytes of environmental interest. The use of the multi-commutated flow techniques MSFIA and MPFS has proven to be very useful for the automation of the analytical systems, coupling to different detection techniques, as well as sample pretreatment and kinetic-catalytic determinations. The developed systems have been confirmed to be efficient for the determination of inorganic analytes in complex sample matrices such as seawater, wastewater, or pharmaceutical preparations.

Los lantánidos constituyen un grupo de metales fascinante, con unas propiedades químicas muy similares. Aunque no es posible encontrarlos como constituyentes principales de los productos usados a diario, están detrás de toda la revolución tecnológica de la miniaturización electrónica actual. Debido a la similitud de sus propiedades químicas y de los procesos de formación de sus minerales, la separación de estos elementos ha constituido desde su descubrimiento un gran reto. Nuestro trabajo se enfoca en la búsqueda de métodos de separación empleando la extracción líquido-líquido en un soporte polimérico en formato tubular. Se escoge el neodimio como representante de los lantánidos y D2EHPA como agente extractante. Se estudian los parámetros que influyen en la cinética de su extracción empleando una membrana líquida de volumen. Se calculan las constantes de velocidad del transporte de neodimio y holmio a través de la membrana líquida. Se estudia la influencia del pH de la disolución cargada, la concentración inicial de metal y la presencia de una sustancia tampón en la disolución cargada que luego serán aplicados en las diversas configuraciones de membrana líquida estudiados. Se desarrolla un modulo de vidrio y PVDF con una membrana líquida soportada en una fibra hueca. Se emplea para conseguir la concentración de neodimio y en la separación de éste del holmio. Se analizan las características hidrodinámicas del sistema para maximizar el transporte empleando la permeabilidad como parámetro de seguimiento del transporte del metal. Se encuentra que para la misma velocidad lineal el transporte se ve más favorecido cuando el metal se transporta desde el interior hacia el exterior de la fibra hueca. Se observa que en las condiciones estudiadas el neodimio efectivamente se transporta mientras que el holmio se queda retenido en la membrana líquida. Con los parámetros anteriores se desarrolla un módulo concentrador en línea para determinar cantidades reducidas de neodimio en un sistema FIA. Se emplea el mismo lado de la membrana líquida soportada para la realización de los fenómenos de extracción y reextracción. Se alcanzan límites de detección del orden de 100 veces los límites atribuibles al sistema de determinación por sí solo. Es posible determinar neodimio en presencia de lantánidos pesados y algunos elementos como calcio, níquel, zinc, cobre y magnesio que también son extraidos habitualmente con D2EHPA. No es posible hacerlo cuando se trata de lantánidos del grupo de los ligeros y de hierro que presentan una interferencia muy elevada. Para poder evaluar la concentración de metal en las disoluciones acuosas, se emplearon dos métodos de análisis. Por una parte un sistema FIA con un método colorimétrico con Arsenazo III que permite el análisis de muestras con un pH muy diferente entre ellas, gracias al uso de un canal neutralizador con un tampón. Por otra un sistema HPLC con una columna modificada dinámicamente con DDS y utilizando una fase reversa con HIBA, que permite el análisis de todos los elementos lantánidos en muestras que los contienen a todos ellos. Se aplica al análisis de muestras reales de mineral de bastnasita. Se desarrolla un módulo de vidrio con una membrana líquida de volumen, contenida en una fibra hueca, que permite la concentración y separación en continuo de tierras raras empleando D2EHPA como extractante. Se produce la recuperación efectiva en un alto grado de eficacia de neodimio incluso con tiempos cortos de contacto. En las condiciones estudiadas no es posible la separación de los diferentes lantánidos. / The lanthanides are a fascinating group of metals, with very similar chemical properties. Even it is not possible to find them as main constituents of products used on a daily basis, they are behind all the technological revolution of the nowadays electronic miniaturization. Due to the similarity of their chemical properties and the processes of formation of their ores, separation of these elements has constituted a great challenge since its discovery. Our work focuses on the search for methods of separation using the liquid-liquid extraction in a polymeric support in tubular form. As representative of the lanthanides neodymium and D2EHPA as extraction agent is chosen. We study the parameters influencing the kinetics of extraction using a bulk liquid membrane. The speed constants of the transport through the liquid membrane of neodymium and holmium are calculated. It is discussed the influence of the pH, the initial concentration of metal and the presence of a buffer substance in feed solution that then will be applied in the different configurations of liquid membrane studied. It is developed a glass and PVDF module with a hollow fibre supported liquid membrane inside. It is used to achieve the concentration of neodymium and its separation from holmium. The hydrodynamic characteristics of the system are analyzed to maximize transport using the permeability as a parameter of the transport of metal tracking. It is found that, for the same linear speed, transport is favoured when metal goes from the lumen towards the shell of the hollow fibre. It is observed that in the studied conditions neodymium is actually transported while the holmium is retained in the liquid membrane. With the previous parameters an in line concentrator module is developed for determining small amounts of neodymium with a FIA system. It employs the same side of the supported liquid membrane to carry out extraction and re-extraction phenomena. It reaches limits of detection of the order of 100 times attributable to the system determination limits itself only. It is possible to determine neodymium in the presence of heavy lanthanides and some elements like calcium, nickel, zinc, copper and magnesium which are also commonly extracted with D2EHPA. It is not possible to do so with iron and the light lanthanides group that present a very high interference. Two methods of analysis were used to evaluate the concentration of metal in aqueous solutions. On the one hand a FIA system with a colorimetric method with Arsenazo III that allows the analysis of samples with a very different pH between them, thanks to the use of a buffer neutralization channel. On the other hand an HPLC system with a column dynamically modified with DDS and using a reversed phase with HIBA, which allows the analysis individual lanthanide elements in samples containing all of them. That is applied to the analysis of real samples of bastnasite ore. It is developed a glass module with a bulk liquid membrane, contained in a hollow fibre, which allows the concentration and continuous separation of rare earths using D2EHPA as extracting agent. It is achieved the effective recovery in a high degree of neodymium even with short contact times. The separation of the different lanthanides is not possible under the conditions studied.

El disseny i la caracterització de plataformes analítiques i miniaturitzades, modificades amb diferents nanomaterials (tals com nanotubs de carboni o nanocables), s’ha portat a terme per a la seva posterior aplicació en la detecció de diversos analits d’interès (com per exemple fenol, glucosa, o la captura d’oli). Cal destacar que en alguns casos les plataformes sensores s’han modificat prèviament amb entitats biològiques que aporten selectivitat i sensitivitat a l’estudi analític. Referent als processos en absència de flux (mode batch), el elèctrodes serigrafiats de capa gruixuda (screen-printed electrodes, SPE) s’han modificat amb nanotubs de carboni, demonstrant un comportament analític excepcional per a aplicacions mediambientals. La detecció de fenol, modificat amb l’enzim de Tyrosinasa, va ser caractertizat mitjançant diferents tècniques de microscòpia com SEM, TEM o microscòpia confocal (CSLM), les quals van permetre obtenir una sèrie de dades estadístiques referents a la morfologia del material, alhora que van permetre determinar la distribució de la Tyrosinasa en la matriu sensora. Es van estudiar diferents mètodes d’immobilització, a més a més de ser implementat en un sistema de d’anàlisi per injecció en flux. A més a més, els SPE modificats amb nanocables d’or i plata, donant lloc a una plataforma sensor de glucosa que treballa en absència d’enzim, mitjançant la mesura de corrent produïa per l’oxidació directa de la glucosa sobre la superfície de l’elèctrode. Els estudis es van portar a terme a partir de la implementació de tècniques de cronoimpedància. El desenvolupament de noves plataformes nanoestructurades preparades a partir de la deposició alternada de nanopartícules de Prussian Blue, prèviament modificades amb ciclodextrina, amb polyallylamine (PAH) va permetre la creació d’una estructura supramolecular microcúbica. Aquestes plataformes presenten un comportament molt interessant electroquímicament, ja que els seus pics de corrent catòdics i anòdics augmenten amb el número de bicapes, indicant que efectivament les nanopartícules estan connectades elèctricament entre les multicapes. Cal destacar també la combinació de partícules paramagnètiques en un microchip d’electroforesis. Mitjançant la combinació d’un camp magnètic extern, l’electroforesis i tècniques electroquímiques, es va aconseguir la detecció de fenol (com a indicació de la immunodetecció). En aquest cas concret la detecció de fenol es va portar a terme per a seguir la hidròlisis de la phenol phosphate portat a terme per l’enzim alkaline phosphatase (AP) (immobilitat en les partícules magnètiques). El sistema requereix molt poc volum de mostra, alhora que presenta molts avantatges per a la seva versatilitat i per ser un sistema miniaturitzat que permet la renovació del la plataforma immunoespecífica. Finalment, s’ha desenvolupat una plataforma dinàmica a partir de la implementació de motors a la nano/microescala per a aplicacions mediambientals, a partir de la creació d’una superfície exterior superhidrofòbica que interactua amb substàncies olioses capturant-les. El desenvolupament de diferents plataformes, a més a més de la posterior integració de nano/micromaterials que han millorat significativament el seu comportament a nivell analític, ha mostrat resultats excel·lents en la detecció d’analits d’interès ambiental i mediambiental, projectant-se com a un element clau en sistemes monitoritzats i altres plataformes analítiques d’interès. / The design and characterization of novel miniaturized (bio)sensing platforms and devices modified with different nanomaterials, such as carbon nanotubes or nanowires, have been achieved. These platforms and devices have been applied in the detection of various analytes (e.g. phenol, glucose, oil droplets), in some cases closely related to the previously immobilization of biological entities that confer selectivity and sensitivity to the analytical platform. Regarding batch platforms, screen-printed electrodes (SPE) modified with carbon-nanotubes has demonstrated to be a potential analytical platform for environmental purposes by being applied for phenol detection in case of being modified by tyrosinase enzyme. The characterization of modified SPE by SEM, TEM and Confocal Scanning Laser Microscopy (CSLM) has brought the necessary statistical data over the morphological properties of the nano/micromaterials treated and the distribution of tyrosinase enzyme along the sensor matrix. Several immobilization approaches have been developed and the present system has been integrated in a Flow Injection Analysis (FIA). Additionally, carbon nanotubes modified SPE with gold-silver nanowires has resulted in a non-enzymatic electrochemical glucose sensor, based on the measurement of the current response during the direct glucose oxidation on the electrode surface by chronoimpedance technique (CIT) studies. The development of novel assembled nanoplatform taking advantage of layer by layer depositions from Prussian blue (PB) - cyclodextrin (CD) nanoparticles (NPs) with polyallylamine (PAH) was designed, showing a microcubic supramolecular like organitzation. Such platforms present interesting electrochemical behaviour, as the cathodic and anodic peak currents increased with the number of bilayers, indicating that PB nanoparticles are electrically connected within multilayers. Moreover, the combination of paramagnetic particles with microchip electrophoresis, external magnetic field manipulations, and coupled to EC detection permitted the detection of phenol (as indicator for immunodetection) by using low reagent consumption, in addition to the inherent miniaturization and versatility of such miniaturized system. Phenol detection was achieved by following the hydrolysis of phenyl phosphate by alkaline phosphatase (AP) enzyme linked to the magnetic beads (MBs), which resulted in a very interesting system due to the possibility to renew the immuno-specific support. Finally, an alternative and dynamic analytical platform was developed through the implementation of artificial nano/microscale machines for environmental remediation applications based on the creation of a superhydrophobic outer surface to strongly interact and capture oily liquids has been achieved. The development of different platforms, along with the wide range of nano/micromaterials integrated on them as to improve their analytical performance, have shown good results towards the detection of analytes of interest in health and environmental issues, being envisioned future key components of monitoring systems and other analytic platforms of interest.

The aim of this project was to investigate EELS from two-layer systems and relate the results to the existing theory. Two systems were investigated, magnesium silicide platelets within an aluminium matrix and silicon precipitates within an aluminium matrix. Both systems were prepared through thermal treatment of a 6061 A1 alloy. The majority of the data presented in this thesis was acquired using EELS. However, energy dispersive x-ray spectroscopy (EDX) and electron microscopy were also used. EELS was performed on two different electron microscopes, the VG HB5 STEM and the FEI Tecnai TF20 (S)TEM. The bulk of the results were acquired on the HB5. To facilitate the comparison of theoretical and experimental results, the data was separated into bulk and interface components. The component amounts were then plotted against distance from the interface. Bessel functions were then fitted to this plot to give characteristic values. These values represented how well the optimal interface position had been chosen, the comparative decay of the interface plasmon on each side of the interface and the relative thickness of the bulk material. The experimental data from most of the interfaces examined indicated significant variations in the thickness of the sample. Despite this, the experimental results were found to follow the trend suggested by the theoretical equations. Analysis of the characteristic values indicated that the data from the HB5 and Tecnai for an interface showed a strong correlation. However, comparison of the experimental values with the theoretical reference showed a deviation of ~20%. Though the source of this deviation was not clear, a number of possible causes were investigated. Theoretical models were generated of systems with a variety of thickness profiles. In addition, systems containing steps, wide and narrow bulk plasmons and a thin interfacial layer of a third material were all considered. The deviation between the results from experiment and the simple theoretical model was believed to be consistent with the factors affecting EELS from a real interface. In particular, thickness variations and imperfections at the interface were found to be the most likely cause of the discrepancy between theory and experiment.

Time-of-flight secondary ion mass spectrometry (ToF-SIMS) is a widely used surface chemical analysis technique that is traditionally employed to characterise the first few molecular layers of a material interface. The ability of this technique to accurately reflect the surface chemistry of polymers, biomaterials and many other solid materials is well documented. However, the majority of research that utilises this technique is based upon a qualitative rather than quantitative assessment of the material under investigation. The qualitative analysis of a range of traditional tablet and bead formulations containing drug and multiple excipients was performed in order to identify key diagnostic ions for all the different components. The lateral distributions of the ions across the surfaces of these formulations were imaged. Two different methods were then used to perform a qualitative analysis of the surfaces and results from these experiments were compared to the bulk composition. The effect of surface roughness on the ability to produce reproducible quantitative analyses from ToF SIMS ion yield data was investigated. A range of samples with different topographies were studied including polytetrafluoroethylene (PTFE), glass microscope slides, gold coated abrasive papers and gold coated precision measurement samples. The surface roughness was assessed by Atomic Force Microscopy and Laser Profilometry. Samples were analysed in imaging mode and the variance in ionization across the total image was measured for each sample. Evidence is presented that there is a relationship between ion yield and surface roughness, and that the surface roughness of the analysed surfaces will effect on any quantification approach in the processing of ToF-SIMS data. In addition, the presence of any orientation/directionality in surface features also needs to be evaluated when considering use of a quantitative approach. To investigate the effect of chemical environment on the ability to derive quantitative data from ToF SIMS analysis of pharmaceutical materials, drug loaded spun cast polymer films with low surface roughness were studied. ToF SIMS data were obtained for two chemically similar drugs in two different polymer matrices. In the majority of the samples there was no quantitative relationship between drug ion intensity and nominal bulk composition. Due to the large sample set, the multivariate technique, Principal Component Analysis (PCA) was employed to look at variance in secondary ion yields from the different samples. PCA is becoming more prevalent in ToF-SIMS data interrogation as it allows for a mathematically un-biased analysis of sample variables through the identification of the ions that account for the majority of the variance in the sample set. PCA successfully highlighted the impact of the chemical environment, showing secondary ion yields of drugs can be dependent on the surrounding matrix. PCA was also used to look at variance in two of the tablet samples and was successfully able to differentiate between the tablet samples with the lowest and highest concentrations of paracetamol. This thesis has demonstrated that surface topography and surface chemical environment or matrix will have a significant impact on ion yields in the ToF-SIMS experiments. These findings suggest caution in the use of ToF-SIMS for the quantitative analysis of complex chemically heterogeneous and topographically diverse pharmaceutical formulations.

A state-of-the-art gas electron diffraction (GED) apparatus has been reassembled in the school of chemistry at the University of Edinburgh. This combines molecularbeam and telefocus-electron-gun technologies and the alignment of the electron beam produced by the latter has been discussed. A new custom-made CCD detector has also been installed and electron diffraction patterns for a few small molecules have been recorded. In analogy to the rotating sector in a conventional GED apparatus, the new camera contains an optical filter and a procedure for its calibration is outlined and followed step by step to produce an estimate of the filter transmittance. The data have been shown to be of less than ideal quality and the probable root of the problem is discussed. GED refinements of two pairs of compounds (arachno-6,9-decaboranes, and a covalent sulfonate and thiosulfonate) are presented, using data collected with the conventional Edinburgh GED apparatus.

Distinctive fine-structure due to ³⁵Cl/³⁷Cl isotopologue and isotopomer effects is resolved at high magnetic fields for ¹⁹⁵Pt and ¹⁰³Rh NMR signals, resulting in a unique NMR “finger-print”, with which it is possible to uniquely identify all chlorido containing Pt(IV) and Rh(III) complexes. In this study, these isotope shifts are computed from first principles in order to provide a solid theoretical framework for the empirical observations. Use is made of DFT to calculate the ³⁵/³⁷Cl and ¹⁶/¹⁸O induced isotope shifts in the ¹⁹⁵Pt NMR spectra of [Pt³⁵Cl₆]²⁻ and [Pt³⁷Cl₆]²⁻, for the [Pt³⁵Cl n ³⁷Cl₅₋ n(H₂O)]⁻ (n=0-5), cis-Pt³⁵Cln³⁷Cl₍₄₋n₎(H₂O)₂ (n=0-4), and fac-[Pt³⁵Cl n³⁷Cl₍₃₋ n₎(H₂O)₃]⁺ (n=0-3) series. The computational protocol is extended to calculate the ³⁵/³⁷Cl and ¹⁶/¹⁸O induced isotope shifts in [Pt³⁵Cl n³⁷Cl₍₅₋ n₎(OH)]²⁻ (n=0-5), cis- [Pt³⁵Cln³⁷Cl₍₄₋ n₎(OH)₂]²⁻ (n=0-4), fac-[Pt³⁵Cln³⁷Cl₍₃₋ n₎(OH)₃] ²⁻ (n=0-3), cis- [Pt³⁵Cl n³⁷Cl₂₋n(OH)₄]²⁻ (n=0-2) and [Pt³⁵Cl n³⁷Cl₍₁₋ n₎(OH)₅]²⁻ (n=0-1). For Rh(III), the ³⁵Cl/³⁷Cl isotope shifts in the ¹⁰³Rh NMR spectra of [Rh³⁵Cl n³⁷Cl₅₋ n(H₂O)] ²⁻ (n=0-5), cis-[Rh³⁵Cl n³⁷Cl₍₄₋ n₎(H₂O)₂]⁻ (n=0-4), and fac-Rh³⁵Cl n³⁷Cl₍₃₋ n₎(H₂O)₃ (n=0-3) were calculated. The ¹⁹⁵Pt and ¹⁰³Rh NMR chemical shifts computed for these species reproduce the order of magnitude of the observed effect reasonably well, up to ca. 1 ppm. In most cases, general trends are also captured qualitatively, thus providing the first theoretical basis for the origin of subtle isotope shifts in ¹⁹⁵Pt NMR spectra. Neither simple polarizable continuum models nor small, microsolvated complexes lead to improved isotope shifts for the series investigated, however, valuable insight into the degree of solvent interaction was gained. Using the polarized continuum model to calculate shielding/bond-length derivatives together with gas-phase zero-point corrections to estimate shieldings, it was shown that the contraction of the coordination sphere in the hydroxide complexes cause the Pt-Cl bonds to become magnetically equivalent, justifying why their isotopomers aren't resolved. In this study, theoretical modelling of structural effects on NMR parameters extends to the smallest scale, distance changes of a few femtometers upon isotopic substitution.

The proportion of antibiotic resistant Gram-positive strains in the clinic and community continue to rise, despite the number of new antibiotics continuing to fall with time. At the intersection of this problem is the established challenge of working with what has ultimately been both nature’s and humanity’s favoured and most successful antibiotic target, the biosynthesis of the bacterial cell wall. The challenge lies in the predominately membrane/lipid linked habitat that the enzymes and substrates of this complex biosynthetic pathway function within. Membrane protein science remains non-trivial and often difficult, and as such remains undeveloped despite its hugely important role in the medical and biological sciences. As a result, there is a paucity of understanding for this pathway, with limited methods for assay of the activity of the biosynthetic enzymes. These enzyme include the monofunctional transglycosylases, monofunctional transpeptidase penicillin-binding proteins (PBPs) and bifunctional PBPs capable of both transglycosylation and transpeptidation. A number of these enzymes were expressed and purified, with the intention of obtaining novel kinetic and catalytic characterisation of their activities. The more complex of these enzymes could not be proven to be active, and so the comparatively simpler enzyme, an S. aureus monofunctional transglycosylase called MGT, was taken as a model enzyme and used to help design novel assay methods for its transglycosylase activity. The assays developed in this work gave access to novel time-course data and will help demonstrate other interesting mechanistic/catalytic information about the MGT enzyme and of transglycosylation in general. Mathematical modelling was performed around the experimental work. Novel and unique models were designed to define the mechanism of the MGT and generic transglycosylation, as this had not been performed before. The mathematical concepts of structural identifiability and structural indistinguishability were used to analyse these models. Data from experiments were then used to attempt data fitting with the models, and information about the underlying unknown kinetic parameters were collected. Together, a new framework of understanding of the MGT and transglycosylation can be made, which may hopefully be a small step towards answering the challenge now posed by widespread antibiotic resistance.