This thesis explores a new technique for investigating the photochemical mechanisms of femtosecond laser inscription of permanent photonic structures in Poly(methylmethacrylate) (PMMA). The refractive index (RI) structures were fabricated with a direct writing method without ablation, and analysed using a non-invasive method - namely: Raman mapping spectrometry. The writing conditions for the photonic structures under investigation are mainly represented by 800nm and 400nm wavelength with 44fs and 100fs pulse length and a low repetition rate in the kHz domain. The mass percentage of the induced monomer and end groups modification (MMA) as a measure of the modification of the ratio of C=C and C=O Raman transition varies linearly with the total fluence (total). The mass percentage of the induced monomer and end groups change is defined by the modification of normalised ratio of the Raman intensity of C=C bond (I(C=C)) and the Raman intensity of C=O bond (I(C=O)) which is denoted by I(C=C/C=O)n. The modification of this ratio is denoted by I (C=C/C=O)n and also by MMA. MMA varies linearly with total with a positive slope for both writing conditions due to the induced main chain scission and unzipping. If total increases by 1J/cm2, it is predicted an increase in MMA, by (1.550±0.11)x10-2 (cntsxcm2)/J, for the near infrared (NIR) irradiated samples that is higher than the increase of MMA for the ultraviolet (UV) irradiated sample that show a value of (1.9200.274)x10-3 ( (cntsxcm2)/J). The same trend was found for the variation of MMA with diffraction efficiency () for NIR irradiated structures and also for UV irradiated structures. If  increases by 1cnt, it is predicted that there will be an increase in MMA, by (4.233±0.383) cnts for NIR irradiated samples that is lower than the increase of MMA for the UV irradiated sample that shows a value of (14.3922.477) cnts. The variation of MMA with  is higher for UV irradiated samples than for NIR irradiated samples, and this indicates that the nonlinear absorption of two photons produces a larger percentage of the monomer and end groups than the nonlinear absorption of three photons. Gel Permeation Chromatography (GPC), which is a destructive analytical method, was applied only for the investigation of the time dependent behaviour of the molecular weight of the photonics structures which were written with the parallel writing technique using 775nm wavelength and 160fs pulse length that shows an increase of 66 in  after seven days from the laser irradiation. Twenty-four hours after laser irradiation, the GPC results show that the weighted average molecular weight (Mw) of the exposed sample of 28,610,000 Daltons is about thirty times higher than the MW of the unexposed sample of 963,425 Daltons. This is an indication of the photo-cross-linking reaction. As a result of this reaction, the polymer chains link together through intermolecular forces to form a 3D network which produces an increase of molecular weight. It was also observed that there was a further decrease of molecular weight after three days to 437,441 Daltons due to main chain scission and unzipping. The main chain scission is actually the breaking of C-C bonds between structural units and the formation of radicals which further produce the monomer and end groups (MMA) through the unzipping reaction which leads to a decrease of the molecular weight. The main chain scission occurred with the greatest efficiency after three days following the end of irradiation, when the number of the main chain scissions (Ns) reached the maximum value of 1.193. An increase of molecular weight signifies an increase of the refractive index since the optical density has increased. The mechanical properties of PMMA optical fibres (e.g., Young's modulus) and of bulk PMMA (e.g., glass transition temperature) were investigated using Dynamical Mechanical Analysis (DMA) tests (e.g., stress-strain test and temperature ramp/frequency sweep test). These measurements were performed to study the effect of the manufacturing process that involves stretching and heating or cooling on the mechanical properties of PMMA optical fibres and unmodified PMMA material. T he ultimate aim of this section was to see the effect of the laser irradiation on the strain properties of an optical fibre sensor with gratings. The stress strain results show an increase of Young's modulus of the PMMA optical fibre of 5%, and this is an indication of decreased elasticity which is induced during the fabrication process. For a femtosecond laser irradiated region with UV wavelength, it is expected that there will be an increase of Young's modulus to 65%. This variation was obtained inthe research group from The Photon Science Institute by measuring Young's modulus for a diffraction grating which was written in PMMA with 180fs pulse length and 387nm wavelength and which was subjected to a strain. The elasticity was measured using the displacement of the first order diffracted beams as a result of a modification due to the applied strain [ ]. The temperature ramp/frequency sweep test shows an increase of glass transition temperature of the bulk PMMA of 54.12% which is also an indication of decreased elasticity induced during the fabrication process. A further increase in this temperature is expected for UV irradiated samples.

<p>Confocal microscopy has been used as a tool for studying adsorption of biomolecules to individual chromatographic adsorbent particles. By coupling a fluorescent dye to protein molecules, their penetration into single adsorbent particles could be observed visually at different times during batch uptake. By relating the relative fluorescence intensity obtained at different times to the value at equilibrium, the degree of saturation versus time could be constructed. The use of two different fluorescent dyes for protein labeling and two independent detectors, allowed direct observation of a two-component adsorption process. The confocal technique was also applied for visualization of nucleic acids. Plasmid DNA and RNA were visualized with fluorescent probes that binds to double stranded DNA and RNA respectively. Confocal measurements following single component adsorption to ion exchange particles, revealed an interesting phenomenon. Under certain experimental conditions, development of "inner radial concentration rings" (i.e. adsorbed phase concentrations that are higher at certain radial positions within the particle) were observed. Some examples are given that show how such concentration rings are formed within a particle.</p><p>Methods were also developed for measurement of the spatial distribution of immobilized functional groups. Confocal microscopy was used to investigate the immobilization of trypsin on porous glycidyl methacrylate beads. Artefacts relating to optical length differences could be reduced by use of "contrast matching". Confocal microscopy and confocal micro-Raman spectroscopy, were used to analyze the spatial distribution of IgG antibodies immobilized on BrCN-activated agarose beads. Both these measurement methods indicate an even ligand distribution. Finally, confocal Raman and fluorescence spectroscopy was applied for measurement of the spatial distribution of iminodiacetic- and sulphopropyl groups, using Nd3+ ions as fluorescent probes. Comparison of different microscope objectives showed that an immersion objective should be used for measurement of wet adsorbent particles.</p><p><i>Direct experimental information from the interior of individual adsorbent particles will increase the scientific understanding of intraparticle mass transport and adsorption mechanisms, and is an essential step towards the ultimate understanding of the behaviour of chromatographic adsorbents.</i></p>

Confocal microscopy has been used as a tool for studying adsorption of biomolecules to individual chromatographic adsorbent particles. By coupling a fluorescent dye to protein molecules, their penetration into single adsorbent particles could be observed visually at different times during batch uptake. By relating the relative fluorescence intensity obtained at different times to the value at equilibrium, the degree of saturation versus time could be constructed. The use of two different fluorescent dyes for protein labeling and two independent detectors, allowed direct observation of a two-component adsorption process. The confocal technique was also applied for visualization of nucleic acids. Plasmid DNA and RNA were visualized with fluorescent probes that binds to double stranded DNA and RNA respectively. Confocal measurements following single component adsorption to ion exchange particles, revealed an interesting phenomenon. Under certain experimental conditions, development of "inner radial concentration rings" (i.e. adsorbed phase concentrations that are higher at certain radial positions within the particle) were observed. Some examples are given that show how such concentration rings are formed within a particle. Methods were also developed for measurement of the spatial distribution of immobilized functional groups. Confocal microscopy was used to investigate the immobilization of trypsin on porous glycidyl methacrylate beads. Artefacts relating to optical length differences could be reduced by use of "contrast matching". Confocal microscopy and confocal micro-Raman spectroscopy, were used to analyze the spatial distribution of IgG antibodies immobilized on BrCN-activated agarose beads. Both these measurement methods indicate an even ligand distribution. Finally, confocal Raman and fluorescence spectroscopy was applied for measurement of the spatial distribution of iminodiacetic- and sulphopropyl groups, using Nd3+ ions as fluorescent probes. Comparison of different microscope objectives showed that an immersion objective should be used for measurement of wet adsorbent particles. Direct experimental information from the interior of individual adsorbent particles will increase the scientific understanding of intraparticle mass transport and adsorption mechanisms, and is an essential step towards the ultimate understanding of the behaviour of chromatographic adsorbents.

<p>Chromatography is a powerful technique, essential in chemical analyses and preparative separation in industry and research. Many different kinds of chromatographic material are needed, due to the large variety of applications. Detailed methods of characterisation are needed to design new chromatographic materials and understand their properties. In this thesis, confocal Raman spectroscopy and surface enhanced Raman spectroscopy (SERS) have been applied to micrometer-size chromatographic beads, for which these techniques have not been used earlier. New methodology, optimized for use with the chromatographic beads, has been developed and evaluated. </p><p>Confocal spectroscopy has been used to determine distributions of functional groups within single chromatographic beads. This distribution is of great importance in determining the chromatographic properties, since the material is porous and the solute molecules can diffuse inside the beads. Most of the confocal experiments have been performed with Raman spectroscopy; fluorescence spectroscopy, using Nd³⁺ ions or dye-labelled proteins as fluorescence probes, has been used for comparison. </p><p>The concentration of adsorbed analytes is very low within the beads. SERS was therefore used to enhance the Raman signal. SERS-active surfaces were prepared by incorporating gold nano-particles into the interior of the bead. TEM measurements showed that the gold nano-particles could be observed throughout, and it was possible to record analyte spectra from different positions within the bead. Enhanced spectra could be obtained both for small test molecules and for larger bio-molecules, although the spectra for the smaller analytes were much more intense.</p>

Chromatography is a powerful technique, essential in chemical analyses and preparative separation in industry and research. Many different kinds of chromatographic material are needed, due to the large variety of applications. Detailed methods of characterisation are needed to design new chromatographic materials and understand their properties. In this thesis, confocal Raman spectroscopy and surface enhanced Raman spectroscopy (SERS) have been applied to micrometer-size chromatographic beads, for which these techniques have not been used earlier. New methodology, optimized for use with the chromatographic beads, has been developed and evaluated. Confocal spectroscopy has been used to determine distributions of functional groups within single chromatographic beads. This distribution is of great importance in determining the chromatographic properties, since the material is porous and the solute molecules can diffuse inside the beads. Most of the confocal experiments have been performed with Raman spectroscopy; fluorescence spectroscopy, using Nd3+ ions or dye-labelled proteins as fluorescence probes, has been used for comparison. The concentration of adsorbed analytes is very low within the beads. SERS was therefore used to enhance the Raman signal. SERS-active surfaces were prepared by incorporating gold nano-particles into the interior of the bead. TEM measurements showed that the gold nano-particles could be observed throughout, and it was possible to record analyte spectra from different positions within the bead. Enhanced spectra could be obtained both for small test molecules and for larger bio-molecules, although the spectra for the smaller analytes were much more intense.

Ce travail combine l'électrochimie à l'interface liquide - liquide avec le procédé sol - gel pour la modification interfaciale avec de la silice mésoporeuse. Dans la première partie de ce travail, l’interface liquide – liquide macroscopique a été utilisée pour séparer la solution aqueuse de l'espèce de précurseur de silice hydrolysées (tétraéthoxysilane (TEOS)) de l'agent tensioactif cationique (cethyltrimethylammonium (CTA+) qui a agi comme un template et a été dissous dans le dichloroéthane. Le dépôt de matériau de silice a été déclenchée par le transfert du CTA+ à partir de la phase organique vers la phase aqueuse. CTA+ qui a transféré à la phase aqueuse a catalysé la réaction de condensation de la silice sur l’interface liquide – liquide. Le dépôt de silice à des interfaces liquide – liquide miniaturisées était la deuxième partie de ce travail. Les dépôts stables sur le côté de l'interface ont été synthétisés in situ par voie électrochimique. La stabilité mécanique des dépôts de silice permis un traitement thermique de la silice. Basé sur les techniques d’imagerie (par exemple SEM) il a été constaté que les dépôts forment des hémisphères pour des temps plus long. La réaction interfaciale a également été suivie in situ par spectroscopie Raman confocale. Caractéristiques moléculaires de l'interface ont été modifiées de manière spectaculaire une fois les espèces CTA+ ont été transférés à la phase aqueuse. Les interfaces liquide – liquide miniaturisés et modifiés ont également été évaluée avec le transfert voltampérométrique / This work combines the electrochemistry at the interface between two immiscible electrolyte solutions (ITIES) with the Sol – Gel process of silica leading to an interfacial modification with mesoporous silica using soft template. In the first part of this work the macroscopic liquid – liquid interface was employed to separate the aqueous solution of the hydrolyzed silica precursor species (tetraethoxysilane (TEOS)) from the cationic surfactant (cethyltrimethylammonium (CTA+)) dissolved in the dichloroethane. The silica material deposition was controlled by the electrochemical CTA+ transfer from the organic to the aqueous phase. Template transferred to the aqueous phase catalyzed the condensation reaction and self-assembly resulting in silica deposition at the interface. Silica deposition at the miniaturized ITIES (membranes supporting array of micrometer in diameter pores were used in this regard) was the second part of this work. Silica interfacial synthesis performed in situ resulted in stable deposits growing on the aqueous side of the interface. Mechanical stability of the supported silica deposits allowed further processing – silica material was cured. Based on imaginary techniques (e.g. SEM) it was found that deposits forms hemispheres for longer experimental time scales. Interfacial reaction was also followed with in situ confocal Raman spectroscopy. Molecular characteristics of the interface were changed dramatically once CTA+ species were transferred to the aqueous phase. Array of microITIES modified with silica was also assessed by ion transfer voltammetry