Biofilms of Actinobacillus succinogenes, grown in a biofilm reactor system, were investigated for structure and cell viability, through microscopic visualisation with a confocal scanning laser microscope (CSLM) and a scanning electron microscope (SEM). Biofilms were sampled and visualised at steady state conditions with the broth containing succinic acid titres between 15 and 21 g/L. All sampled biofilm was 6 days old. Six-day-old biofilms of A. succinogenes showed a heterogeneous biofilm architecture composed of cell micro-colony pillars which varied considerably in thickness, area and shape. Microcolony pillars consisted of a densely packed entanglement of sessile cells. Quantitative analysis revealed that the pillars were mostly large, with a mean pillar diameter of 170 m and a mean thickness of 92 m, although pillar diameter and thickness were variable as they ranged from 25 – 500 m and 30 – 300 m, respectively. In the regions close to the substratum surface, pillars were characterised by having defined borders with a network of channels ranging from 40 – 200 m in width separating them. However, towards the middle of the biofilm depth some of the pillars coalesced. For this reason low cross sectional area coverage of biofilm consistently occurred at the bottom portion of the biofilm whilst the highest coverage was in the middle portion of the biofilm. Regarding cell morphology, very large differences were observed. Planktonic cells were rod-shaped, whereas sessile cells expressed an elongated rod morphology and thus were much longer and thinner compared with planktonic cells. Planktonic cells were 1 – 2 m thick and 4 – 5 m long, while sessile cells were 0.5 – 1 m thick and 5 – 100 m long. Long sessile cells resulted in extensive tangling in microcolony pillars, which may have contributed to the structural stability of the pillars. Fibre-like connections of constant diameter were observed between cells, and between the cells and surface. The diameter of these connections was approximately 20 – 30 nm. Viability stains showed that in the bottom portion (from 0 - 20 m above the substratum surface) of the biofilm, most of the cells were dead. However, the portion of covered area attributed to living cells increased past the middle of the biofilm towards the top part of the biofilm. A high percentage of living cells was thus found towards the top part of the biofilm. Overall, 65% (with 2% standard deviation) of the entire biofilm was composed of dead cells. In this way, the results show that operation at high acid conditions comes at a cost of low overall biomass productivity due to decreased active biomass. / Dissertation (MEng)--University of Pretoria, 2017. / Chemical Engineering / MEng / Unrestricted

A proposta deste trabalho foi analisar um produto experimental (VIPI LTDA, Pirassununga, SP), que através da tecnologia sol-gel, modifica a superfície de resinas acrílicas para base de próteses e forma uma camada de nanopartículas de sílica (NPS) visando diminuir o acúmulo e facilitar a remoção de microrganismos. Dessa forma, inicialmente, confirmou-se a deposição de NPS e formação do revestimento cerâmico em polimetilmetacrilato (PMMA) através de espectroscopia no infravermelho por Transformada de Fourier (FTIR); e posteriormente, quantificou-se o biofilme de Candida albicans nesta superfície através da contagem de unidades formadoras de colônia (UFC/mL) e microscopia confocal (MC). Um total de 51 espécimes (10x10x3mm) de PMMA foi confeccionado e distribuído aos experimentos designados. Para a análise da composição dos espécimes em FTIR, foram avaliados 3 grupos (n=1): CN- espécime que não recebeu tratamento algum; CP- espécime que recebeu a aplicação do primer do produto; CL- espécime que passou tanto pela aplicação do primer como pelo processo sol-gel. Na etapa seguinte, foram utilizados 48 espécimes divididos em 3 grupos (n=16), de acordo com o tipo de polimento: PM3- mecanicamente polido com 3&#x3BC;m de rugosidade média; PM03- mecanicamente polido com 0,3&#x3BC;m de rugosidade; PL- polido quimicamente pelo líquido conforme instruções do fabricante. Anteriormente aos experimentos, os espécimes foram esterilizados por óxido de etileno e, então, imersos em saliva artificial por 2hs para a formação da película adquirida. Em seguida, foram secos e inoculados com 2mL de suspensão de C. albicans (1.107 cel/mL) para adesão das células fúngicas durante 90min. Após esta fase, as amostras foram lavadas em solução salina e imersas em meio estéril (RPMI) para crescimento do biofilme em estufa sob agitação (12hs a 37oC). Metade do número das amostras de cada grupo (n=8) foi destinada a contagem de UFC/mL e a outra metade dos espécimes (n=8), foi designada ao método de MC, que com o auxílio de um software (BioImageL v.2), permitiu a determinação do biovolume total (&#x3BC;m3), biovolume de células viáveis (&#x3BC;m3), biovolume de células não-viáveis (&#x3BC;m3) e área de cobertura do campo pelo biofilme (%). Os dados obtidos pelo FTIR foram analisados através da estatística descritiva. Os resultados obtidos pelos experimentos de quantificação após teste de normalidade Kolmogorov-Smirnov foram analisados através do teste paramétrico ANOVA, seguido do teste de Tukey para comparações entre grupos (p<0,05). Através do FTIR, observou-se a deposição satisfatória da camada de NPS, permitindo assim, o desempenho dos experimentos de quantificação. Os resultados do UFC/ml e MC demonstraram semelhança na quantificação do biofilme entre os grupos PL e PM3, e diferença quando comparados ao grupo de superfícies mais lisas, PM03. Dessa forma, observou-se que o polimento líquido experimental não foi efetivo para a diminuição da colonização de biofilme deC. albicans em superfícies de PMMA. Entretanto, maiores investigações sobre as propriedades de superfície deste revestimento devem ser realizadas, já que o processo sol-gel permite uma facilidade na modificação dessas características, podendo levar ao desenvolvimento de um material de revestimento ideal. / This study investigates an experimental coating (VIPI LTDA, Pirassununga, SP) by sol-gel process that modifies acrylic resin denture base with silicon dioxide nanoparticles (SNP) to decrease C. albicans biofilm growth. Therefore, it was first investigated the presence of sol-gel ceramic coating on polymethylmethacrylate (PMMA) by Fourier Transform Infrared Spectroscopy (FTIR). Then C. albicans biofilms were quantified by colony forming units (CFU/mL) and confocal scanning laser microscopy (CSLM). Fifty-one PMMA specimens were manufactured (10x10x3mm) and assigned to the experiments. To evaluate specimens composition, it was analyzed three groups (n=1): CN- the specimen did not receive any surface treatment; CP- it was applied the coating primer on the specimen surface CL- the specimen was treated with the whole sol-gel process. In the following stage, 48 samples were divided into 3 groups (n=16) according to the polish type: PM3- 3&#x3BC;m of roughness mechanical polish; PM03- 0,3&#x3BC;m of roughness mechanical polish; PL- liquid polish. Samples of experimental group were coated according to manufacturers instructions and all the samples were sterilized with ethylene oxide. After that, they were dipped in artificial saliva for 2hs to acquire the salivary pellicle, and then, dried and inoculated with 2 mL suspension of C.albicans (1.107 cel/mL) for 90 min. Then, specimens were washed and immersed in sterile RPMI solution (37oC for 12h). Half of the samples of each group (n=8) was assigned to each quantification test (UFC/mL and CSLM). By CSLM and software (BioImageL v.2) analysis was possible to obtain the total biovolume (&#x3BC;m3), viable biovolume (&#x3BC;m3), non-viable biovolume (&#x3BC;m3), and covered area (%) by C. albicans biofilm. The data obtained by FTIR were analyzed by descriptive statistic. Whereas the records acquired by the quantification experiments were first analyzed by Kolmogorov-Smirnov normality test and then by one way ANOVA followed by Tukeys test to assess difference between groups (p<0,05). FTIR results showed an adequate SNP deposition, allowing the quantification tests to be performed. UFC/mL and CLSM records showed similarity between PL and PM3 groups and difference when comparing these groups to smoother surfaces group (PM03). Therefore, in this study, the experimental coating was not effective to reduce colonization by C. albicans biofilm on PMMA surfaces. Nevertheless, further investigations are required since sol-gel ceramic coating process eases the features modification of the material, possibly leading to an ideal coating development.

A proposta deste trabalho foi analisar um produto experimental (VIPI LTDA, Pirassununga, SP), que através da tecnologia sol-gel, modifica a superfície de resinas acrílicas para base de próteses e forma uma camada de nanopartículas de sílica (NPS) visando diminuir o acúmulo e facilitar a remoção de microrganismos. Dessa forma, inicialmente, confirmou-se a deposição de NPS e formação do revestimento cerâmico em polimetilmetacrilato (PMMA) através de espectroscopia no infravermelho por Transformada de Fourier (FTIR); e posteriormente, quantificou-se o biofilme de Candida albicans nesta superfície através da contagem de unidades formadoras de colônia (UFC/mL) e microscopia confocal (MC). Um total de 51 espécimes (10x10x3mm) de PMMA foi confeccionado e distribuído aos experimentos designados. Para a análise da composição dos espécimes em FTIR, foram avaliados 3 grupos (n=1): CN- espécime que não recebeu tratamento algum; CP- espécime que recebeu a aplicação do primer do produto; CL- espécime que passou tanto pela aplicação do primer como pelo processo sol-gel. Na etapa seguinte, foram utilizados 48 espécimes divididos em 3 grupos (n=16), de acordo com o tipo de polimento: PM3- mecanicamente polido com 3&#x3BC;m de rugosidade média; PM03- mecanicamente polido com 0,3&#x3BC;m de rugosidade; PL- polido quimicamente pelo líquido conforme instruções do fabricante. Anteriormente aos experimentos, os espécimes foram esterilizados por óxido de etileno e, então, imersos em saliva artificial por 2hs para a formação da película adquirida. Em seguida, foram secos e inoculados com 2mL de suspensão de C. albicans (1.107 cel/mL) para adesão das células fúngicas durante 90min. Após esta fase, as amostras foram lavadas em solução salina e imersas em meio estéril (RPMI) para crescimento do biofilme em estufa sob agitação (12hs a 37oC). Metade do número das amostras de cada grupo (n=8) foi destinada a contagem de UFC/mL e a outra metade dos espécimes (n=8), foi designada ao método de MC, que com o auxílio de um software (BioImageL v.2), permitiu a determinação do biovolume total (&#x3BC;m3), biovolume de células viáveis (&#x3BC;m3), biovolume de células não-viáveis (&#x3BC;m3) e área de cobertura do campo pelo biofilme (%). Os dados obtidos pelo FTIR foram analisados através da estatística descritiva. Os resultados obtidos pelos experimentos de quantificação após teste de normalidade Kolmogorov-Smirnov foram analisados através do teste paramétrico ANOVA, seguido do teste de Tukey para comparações entre grupos (p<0,05). Através do FTIR, observou-se a deposição satisfatória da camada de NPS, permitindo assim, o desempenho dos experimentos de quantificação. Os resultados do UFC/ml e MC demonstraram semelhança na quantificação do biofilme entre os grupos PL e PM3, e diferença quando comparados ao grupo de superfícies mais lisas, PM03. Dessa forma, observou-se que o polimento líquido experimental não foi efetivo para a diminuição da colonização de biofilme deC. albicans em superfícies de PMMA. Entretanto, maiores investigações sobre as propriedades de superfície deste revestimento devem ser realizadas, já que o processo sol-gel permite uma facilidade na modificação dessas características, podendo levar ao desenvolvimento de um material de revestimento ideal. / This study investigates an experimental coating (VIPI LTDA, Pirassununga, SP) by sol-gel process that modifies acrylic resin denture base with silicon dioxide nanoparticles (SNP) to decrease C. albicans biofilm growth. Therefore, it was first investigated the presence of sol-gel ceramic coating on polymethylmethacrylate (PMMA) by Fourier Transform Infrared Spectroscopy (FTIR). Then C. albicans biofilms were quantified by colony forming units (CFU/mL) and confocal scanning laser microscopy (CSLM). Fifty-one PMMA specimens were manufactured (10x10x3mm) and assigned to the experiments. To evaluate specimens composition, it was analyzed three groups (n=1): CN- the specimen did not receive any surface treatment; CP- it was applied the coating primer on the specimen surface CL- the specimen was treated with the whole sol-gel process. In the following stage, 48 samples were divided into 3 groups (n=16) according to the polish type: PM3- 3&#x3BC;m of roughness mechanical polish; PM03- 0,3&#x3BC;m of roughness mechanical polish; PL- liquid polish. Samples of experimental group were coated according to manufacturers instructions and all the samples were sterilized with ethylene oxide. After that, they were dipped in artificial saliva for 2hs to acquire the salivary pellicle, and then, dried and inoculated with 2 mL suspension of C.albicans (1.107 cel/mL) for 90 min. Then, specimens were washed and immersed in sterile RPMI solution (37oC for 12h). Half of the samples of each group (n=8) was assigned to each quantification test (UFC/mL and CSLM). By CSLM and software (BioImageL v.2) analysis was possible to obtain the total biovolume (&#x3BC;m3), viable biovolume (&#x3BC;m3), non-viable biovolume (&#x3BC;m3), and covered area (%) by C. albicans biofilm. The data obtained by FTIR were analyzed by descriptive statistic. Whereas the records acquired by the quantification experiments were first analyzed by Kolmogorov-Smirnov normality test and then by one way ANOVA followed by Tukeys test to assess difference between groups (p<0,05). FTIR results showed an adequate SNP deposition, allowing the quantification tests to be performed. UFC/mL and CLSM records showed similarity between PL and PM3 groups and difference when comparing these groups to smoother surfaces group (PM03). Therefore, in this study, the experimental coating was not effective to reduce colonization by C. albicans biofilm on PMMA surfaces. Nevertheless, further investigations are required since sol-gel ceramic coating process eases the features modification of the material, possibly leading to an ideal coating development.

No description available.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / O conhecimento da morfologia e ultraestrutura dos helmintos permite a correta classificação destes organismos, bem como fornece subsídios que poderão ser utilizados para diagnóstico e controle. A microscopia laser confocal é uma ferramenta para estudar a organização estrutural de várias espécies de helmintos, possibilitando acesso a detalhes morfológicos não evidenciados pela microscopia óptica. Echinostoma paraensei é um trematódeo, digenético, hermafrodita parasito de numerosos hospedeiros vertebrados. Neste trabalho foi investigado o desenvolvimento dos órgãos reprodutivo e a morfometria de E. paraensei, desde a fase jovem até a adulta, como contribuição ao conhecimento do desenvolvimento reprodutivo desta espécie. Os trematódeos foram recuperados aos 3, 4, 5, 6, 7, 10, 14 e 21 dias posterior à infecção (dpi) experimental em hamsters. Estes foram corados em carmim clorídrico, desidratados em série alcoólica e montados em lâmina permanente em bálsamo do Canadá, fotografados e medidos usando microscopia de luz de campo claro (MCC) e microscopia de varredura laser confocal (MVLC). Entre 3 e 4 dpi, os primórdios genitais estavam presentes e nenhuma organização do sistema reprodutivo foi visualizada por MCC e MVLC. Os primórdio do ovário, dos testículos e da bolsa do cirro foram visualizados por MCC aos 5 e 6 dpi, no entanto, MVLC dos helmintos aos 5dpi mostra que estes primórdios, o ootipo e o útero estavam presentes, como estruturas individualizadas. A bolsa do cirro apresenta metratermo e o ovário com primórdio do oótipo adjacente aos 7dpi por MVLC. A vesícula seminal, receptáculo seminal, células diferenciadas nos testículos, ducto e reservatório vitelínico e oviducto foram visualizados após 10 dias, enquanto os espermatozóides na vesícula seminal, ovos e oócitos, células vitelínicas, poro e canal de Laurer aos 14 dias. A morfometria evidencia um acelerado crescimento dos órgãos reprodutores a partir do 7 dia. Os testículos apresentam aumento significativo no comprimento do 7 ao 21 dia e o ovário durante o período de 7 à 10 dpi. Aos 21 dpi, todos os helmintos apresentaram glândulas vitelínicas, útero contendo ovos e espermatozóides no oviducto enquanto outros ovos estão sendo formados. As mudanças morfológicas acentuadas durante a gametogênese consistem no aumento do comprimento do helminto, maturação das gônadas, desenvolvimento e maturação das glândulas vitelínicas. O desenvolvimento do helminto como um todo está relacionado à maturação dos órgãos reprodutivo masculino e feminino indicando o investimento deste trematódeo em garantir a produção e eliminação dos ovos ao meio exterior. / The knowledge about morphology and ultra structure of helminthes are great importance in correct classification these organisms. The Scanning Laser Microscopy (LSM) is an important tool to study the structural organization of several helminthes species. Echinostoma paraensei is a trematode, digenetic, hermaphroditic parasite of several hosts. In this study, the development of reproductive organs and the morphometry of E. paraensei from young stage to adult worm were investigated, to contribute knowledge of the reproductive development of this specie. The trematodes were recovered on 3, 4, 5, 6, 7, 10, 14 and 21 days post infection (dpi) from experimental hamsters. It were dehydrated in alcohol series, stained with hydrochloric carmine, mounted on permanent slide using Canada balsam, photographed and measured using light microscopy (LM) and Scanning Laser Microscopy (LSM). Between 3 and 4 dpi the genital anlage were present and were not observed reproductive system organization by either LM and LSM. The anlage of ovary, testes and Cirruss sac were seen at 5 and 6dpi by LM, however LSM from 5dpi image shows theses anlage, ootype and uterus are present as individualized structure. Cirrus sac showed metraterm and ootype adjacent to primordial ovary were seen at 7 dpi by LSM. The seminal vesicle, seminal receptacle, differentiated cells in the testes, viteline ducts and oviduct were visualized after 10 days infection, while sperms in seminal vesicle, eggs, oocyte, Laurer canal and pore from 14 dpi by LSM. The morphometry shows a rapid growth of reproductive organs from 7th day. The testes have significantly increased length from 7 until 21dpi and ovary from 7 until 10dpi. All helminthes showed vitellines glands, uterus contained eggs and sperm in oviduct while another eggs were forming at 21 dpi. The marked morphologic changes during gametogenesis are increase of body length of helminthes, gonad maturation and development and maturation of vitelline glands. The development of helminthes as a whole is related to maturation of female and male organs of reproductive system showing the investment this trematode taken to ensuring the production, maintenance and delivery the eggs to external environment.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / O conhecimento da morfologia e ultraestrutura dos helmintos permite a correta classificação destes organismos, bem como fornece subsídios que poderão ser utilizados para diagnóstico e controle. A microscopia laser confocal é uma ferramenta para estudar a organização estrutural de várias espécies de helmintos, possibilitando acesso a detalhes morfológicos não evidenciados pela microscopia óptica. Echinostoma paraensei é um trematódeo, digenético, hermafrodita parasito de numerosos hospedeiros vertebrados. Neste trabalho foi investigado o desenvolvimento dos órgãos reprodutivo e a morfometria de E. paraensei, desde a fase jovem até a adulta, como contribuição ao conhecimento do desenvolvimento reprodutivo desta espécie. Os trematódeos foram recuperados aos 3, 4, 5, 6, 7, 10, 14 e 21 dias posterior à infecção (dpi) experimental em hamsters. Estes foram corados em carmim clorídrico, desidratados em série alcoólica e montados em lâmina permanente em bálsamo do Canadá, fotografados e medidos usando microscopia de luz de campo claro (MCC) e microscopia de varredura laser confocal (MVLC). Entre 3 e 4 dpi, os primórdios genitais estavam presentes e nenhuma organização do sistema reprodutivo foi visualizada por MCC e MVLC. Os primórdio do ovário, dos testículos e da bolsa do cirro foram visualizados por MCC aos 5 e 6 dpi, no entanto, MVLC dos helmintos aos 5dpi mostra que estes primórdios, o ootipo e o útero estavam presentes, como estruturas individualizadas. A bolsa do cirro apresenta metratermo e o ovário com primórdio do oótipo adjacente aos 7dpi por MVLC. A vesícula seminal, receptáculo seminal, células diferenciadas nos testículos, ducto e reservatório vitelínico e oviducto foram visualizados após 10 dias, enquanto os espermatozóides na vesícula seminal, ovos e oócitos, células vitelínicas, poro e canal de Laurer aos 14 dias. A morfometria evidencia um acelerado crescimento dos órgãos reprodutores a partir do 7 dia. Os testículos apresentam aumento significativo no comprimento do 7 ao 21 dia e o ovário durante o período de 7 à 10 dpi. Aos 21 dpi, todos os helmintos apresentaram glândulas vitelínicas, útero contendo ovos e espermatozóides no oviducto enquanto outros ovos estão sendo formados. As mudanças morfológicas acentuadas durante a gametogênese consistem no aumento do comprimento do helminto, maturação das gônadas, desenvolvimento e maturação das glândulas vitelínicas. O desenvolvimento do helminto como um todo está relacionado à maturação dos órgãos reprodutivo masculino e feminino indicando o investimento deste trematódeo em garantir a produção e eliminação dos ovos ao meio exterior. / The knowledge about morphology and ultra structure of helminthes are great importance in correct classification these organisms. The Scanning Laser Microscopy (LSM) is an important tool to study the structural organization of several helminthes species. Echinostoma paraensei is a trematode, digenetic, hermaphroditic parasite of several hosts. In this study, the development of reproductive organs and the morphometry of E. paraensei from young stage to adult worm were investigated, to contribute knowledge of the reproductive development of this specie. The trematodes were recovered on 3, 4, 5, 6, 7, 10, 14 and 21 days post infection (dpi) from experimental hamsters. It were dehydrated in alcohol series, stained with hydrochloric carmine, mounted on permanent slide using Canada balsam, photographed and measured using light microscopy (LM) and Scanning Laser Microscopy (LSM). Between 3 and 4 dpi the genital anlage were present and were not observed reproductive system organization by either LM and LSM. The anlage of ovary, testes and Cirruss sac were seen at 5 and 6dpi by LM, however LSM from 5dpi image shows theses anlage, ootype and uterus are present as individualized structure. Cirrus sac showed metraterm and ootype adjacent to primordial ovary were seen at 7 dpi by LSM. The seminal vesicle, seminal receptacle, differentiated cells in the testes, viteline ducts and oviduct were visualized after 10 days infection, while sperms in seminal vesicle, eggs, oocyte, Laurer canal and pore from 14 dpi by LSM. The morphometry shows a rapid growth of reproductive organs from 7th day. The testes have significantly increased length from 7 until 21dpi and ovary from 7 until 10dpi. All helminthes showed vitellines glands, uterus contained eggs and sperm in oviduct while another eggs were forming at 21 dpi. The marked morphologic changes during gametogenesis are increase of body length of helminthes, gonad maturation and development and maturation of vitelline glands. The development of helminthes as a whole is related to maturation of female and male organs of reproductive system showing the investment this trematode taken to ensuring the production, maintenance and delivery the eggs to external environment.

Magister Chirurgiae Dentium (MChD) / Purpose: To evaluate and compare the in-vitro shear bond strength and micro leakage of two compomers with their adhesive systems and to examine the dentine-restorative interface under confocal scanning laser microscopy (CSLM). Matoiats and Methods: For shear bond strength (SBS) testing thirty non-carious human molars were used of which fifteen molars were restored with Dyract AP using Non-Rinse Conditioner (NRC) and Prime&Bond NT (PBNT) and fifteen were restored with F2000 and Scotchbond Multi-Purpose Plus (SBMP). For the microleakage evaluation cavity preparations were made on the facial surfaces of thirty non-carious premolars. These were then restored with the respective compomer system. The specimens were thermocycled, sectioned and examined for dye penetration. The dentine-restorative interface was examined through a confocal scanning laser microscope. The primers of the bonding agents were labelled with rhodamine B and the adhesive resins were labelled with fluorescein and examined under CSLM in fluorescent mode. Results: The mean SBS for PBNT and SBMP were 12.8 and 18.1 MPa, respectively. The microleakage scores showed Dyract with PBNT leaked on the dentine side in 13 of the 15 specimens examined. On the enamel side 2 of the 15 specimens showed microleakage. With F2000 and SBMP no micro leakage was observed on either enamel or dentine sides. The CSLM images show clear resin tag and hybrid layer formation for both the materials examined, although SBMP showed deeper penetration into the dentine with longer resin tags. The length of the resin tags and thickness of the hybrid layer for PBNT was found to be approximately 10 um and 2 um respectively. SBMP showed resin tags measuring about 100 um while the hybrid layer measured about 5 um. Conclusion: This study demonstrates that the acid-etch technique ofSBMP with F2000 produces higher bond strength and no micro leakage when compared to the self-etching/self-priming "non-rinse technique" of NRC with PBNT and Dyract.

<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.