Neurological and neuroendocrine disorders result from signaling dysregulation at the molecular, cellular, and multi-cellular levels. This dissertation presents the development of separation methods, using capillary zone electrophoresis (CZE) and capillary liquid chromatography (CLC), for detecting and quantifying small molecules, peptides, and proteins involved in cellular signaling. CZE is a rapid separation technique, making it ideal for monitoring cellular dynamics with high temporal resolution. An ultraviolet - light emitting diode was used for photolytic optical gating of caged fluorophore-labeled biogenic amines, common functional groups in neurotransmitters. Additionally, a novel caged fluorophore with faster reaction kinetics than commercially available dyes was used to label reduced thiols and primary amines in the presence of o-phthalaldehyde. Together this light source and novel caged dye illustrate the utility of these methods for monitoring chemical dynamics during continuous sampling. Many cellular second messengers, including inositol phosphates, are known to exist within the cell, but their dynamics and intermolecular interactions are poorly understood since they lack chromophores or electroactive functional groups making direct detection difficult. Utilizing CZE with capacitive coupled contactless conductivity detection (C4D), biological phosphates were separated and detected based on their high anionic charge, suggesting the utility of C4D in label-free detection of biological molecules. The techniques described above require higher sensitivity to monitor physiologically relevant analyte concentrations; therefore, Hadamard transform capillary electrophoresis (HTCE) was used as a multiplexing method in which multiple separations were performed simultaneously. HTCE resulted in increased sensitivity by decreasing the random background noise. Peptides and proteins propagate signals within or between cells; yet, they are difficult to separate and detect by CZE since their highly charged surfaces result in non-specific adsorption to the capillary wall. To minimize these interactions, stable hybrid phospholipid bilayers were prepared as capillary coatings for CZE separations of cationic proteins. Additionally, stabilized phospholipid bilayer coatings were formed on silica particles through redox polymerization of synthetic, polymerizable lipids. These bilayers were stable after exposure to surfactant, organic solvents, and after storage for one month, suggesting their value as lipid chromatography stationary phases for future incorporation of transmembrane proteins to analyze binding interactions with small molecules.

Apesar de seu início promissor, a miniaturização da cromatografia líquida tem se desenvolvido de forma lenta devido à dificuldade em adquirir colunas e equipamentos adequados à técnica. Nos últimos anos, o interesse nesta área da cromatografia tem sido crescente em decorrência das inúmeras vantagens proporcionadas pelo uso de colunas com diâmetro interno reduzido. Ainda que as colunas capilares se encontrem disponíveis comercialmente, sua produção em laboratório pode reduzir custos. Vários parâmetros de grande importância na cromatografia, como o tempo de retenção dos analitos e a pressão do sistema, sofrem variações quando a temperatura da coluna é alterada. Em cromatografia líquida capilar, a aplicabilidade da programação de temperatura é uma alternativa viável, uma vez que a distribuição de calor no interior das colunas ocorre de maneira rápida e uniforme. Os detectores espectrométricos de massas são considerados os mais adequados a esta técnica, pois permitem o monitoramento de compostos presentes em amostras pouco concentradas. Em decorrência do alto custo e indisponibilidade destes em alguns laboratórios, as pesquisas instrumentais nesta área têm empregado detectores mais simples, como os baseados na absorção de luz ultravioleta. Contudo o diâmetro interno reduzido das colunas capilares e, por consequência, o baixo volume de injeção da amostra tornam necessário que as celas de detecção sejam apropriadamente dimensionadas para que não ocorra excessivo alargamento dos picos cromatográficos. Desta maneira, o presente trabalho visa o desenvolvimento de colunas empacotadas e monolíticas, bem como a produção de celas de detecção adequadas ao uso em sistemas destinados à cromatografia líquida capilar. O estudo de algumas variáveis de empacotamento permitiu a obtenção de colunas com excelente eficiência. As fases estacionárias monolíticas foram confeccionadas pelo método sol-gel e utilizadas para promover separações bastante rápidas. Uma das celas de detecção produzidas obteve ótimo desempenho quando comparada a duas celas comerciais comumente empregadas em sistemas miniaturizados. A programação de temperatura foi aplicada durante a separação de tetraciclinas e esteróides, sendo estas análises obtidas com maior rapidez quando comparadas às realizadas sob condições isotérmicas. / In spite of its promising start, the miniaturization of liquid chromatography has developed slowly due to the difficulties in obtaining appropriate columns and equipments suitable to this technique. In recent years, interest in this area of chromatography has been increasing due to the several advantages afforded by the use of columns with reduced internal diameter. Although the capillary columns are commercially available, its in-house production can reduce costs. Several parameters of great importance in chromatography, such as the retention time of analytes and the pressure of the system vary widely when the column temperature is changed. In capillary liquid chromatography the applicability of temperature programming is a viable alternative since the heat distribution across the column occurs quickly and uniformly. Mass spectrometric detectors are considered the most suitable for this technique since they allow the monitoring of compounds in low concentrated samples. Due to the high cost and unavailability of these instruments in some laboratories, instrumental research in this area has been using the simpler detectors, such as those based on the absorption of ultraviolet light. However, the reduced internal diameter of capillary columns and, consequently, the low injection volume of sample imply an appropriate dimensioning of the detection cell, so that excessive broadening of chromatographic peaks is avoided. Thus, this work aims with the development of monolithic and packed columns, as well as the production of suitable cells for the use in detection systems for capillary liquid chromatography. The study of some packing variables allowed to obtain columns with excellent efficiency. Monolithic stationary phases were prepared by sol-gel process and were used to promote very fast separations. One of the obtained detection cells produced good performance when compared to two commercial cells commonly used in miniaturized systems. Temperature programming was applied for the separation of tetracyclines and steroids, at higher speed when compared to those obtained under isothermal conditions.

Desde que foi introduzida por Tswett no começo do século XX, a cromatografia vem sofrendo contínuos avanços. Entretanto, a miniaturização da cromatografia líquida, apesar de seu inicio promissor, ainda continua bastante lenta e até o presente não alcançou ampla difusão, sendo que o número de grupos trabalhando nesta área é ainda bastante restrito. O motivo deste lento avanço se encontrava na dificuldade em se desenvolver equipamentos adequados, colunas apropriadas e sistemas de tratamento de dados suficientemente rápidos para os sistemas miniaturizados. Apesar de muitos desses problemas atualmente serem de fácil resolução, ainda não se dispõe de equipamentos comercialmente disponíveis que supram satisfatoriamente às condições impostas pelas micro-colunas. Tal carência deve ser suprida antes de nos beneficiarmos de todas as vantagens intrínsecas à miniaturização da cromatografia líquida. Deste modo, o presente projeto visa o desenvolvimento de instrumentação totalmente dedicada aos sistemas miniaturizados de cromatografia líquida incluindo o desenvolvimento de uma bomba de alta pressão, um sistema de injeção a base de tempo, um forno capaz de realizar programação de temperatura e o software de controle dos mesmos. Os equipamentos desenvolvidos foram então aplicados em separações de estatinas demonstrando um excelente desempenho. / Since the chromatography was introduced by Tswett, in the beginning of the 20th century, the technique has suffered a constant progress. However, the miniaturization of liquid chromatography, instead of its promising start, it is still too slow and this technique has not reached a wide divulgation so far. It is important to mention that the number of groups working in this area is very limited yet. The reason of this slow progress was due to the difficulty in developing suitable equipments, appropriated columns and data treatment systems that are quick enough for the miniaturized systems. Currently, many of these problems are easy to be solved, however, there are no equipments available commercially that supply the conditions imposed by microcolumns satisfactorily. Such lack should be filled before benefiting from all the advantages concerned to the miniaturization of liquid chromatography. In this way, this study aims at the development of instrumentation totally dedicated to the miniaturized systems of liquid chromatography, including the development of a high pressure pump, an time-based injector, an oven that sets the temperature programming and the software that can control all these devices. The developed equipments were then applied in the separation of statins, demonstrating an excellent performance.

Preeclampsia (PE) is a potentially fatal complication of pregnancy characterized by an increase in blood pressure (>140/90 mmHg) and proteinuria (>300 mg/24 hrs), often accompanied by edema. Symptoms of PE start after 20 weeks of gestation. If PE remains untreated, it can lead to eclampsia, grand-mal seizures responsible for most fatalities. PE is believed to affect 2-10% of pregnancies worldwide, and claims the lives of over 75,000 mothers and 500,000 newborns yearly. No therapeutic agents have been developed to prevent or cure PE. Part of the reason for this is the absence of a complete understanding of the pathogenesis of this disease. PE has long been regarded as a “disease of theories”, and the pathophysiology of PE continues to be the subject of debate. Nonetheless, several abnormalities have been observed to precede established, clinical PE and have in turn been proposed to be involved in the causation of this disease, all with involvement of the mother's placenta as a central feature. Removal of placenta is the only cure for PE and results in a rapid resolution of the symptoms. Thus, the placenta remains an organ of substantial interest and many research groups have attempted to identify abnormal placental features occurring in PE. None of these studies have focused on less abundant, low molecular weight (LMW) biomolecules, which play important roles in the pathophysiology of many diseases. There are a number of alterations that are believed to affect the placenta and contribute to the pathogenesis of PE. The most widely accepted ones include hypoxia, oxidative stress, and an increase of pro-inflammatory mediators in the mother's placenta. The goal of my initial study was to identify which of these hypothesized causative pathways has a significance in the etiology of this syndrome as well as to investigate which less abundant, low molecular weight biomolecules change in response to these abnormalities. For this purpose, we first adapted and optimized a previously developed methodology that studied LMW biomolecules in tissue specimens to study placental biomolecules. This approach involved a tissue homogenization step followed by protein depletion using acetonitrile. We compared two regions of human placenta: the chorionic plate and the basal plate to find differences in the LMW fraction. We discovered 16 species with statistically significant differences between the two sides, and identified 12 of them using tandem mass spectrometry. In the second study we collected normal human term placentas from elective C-section deliveries and exposed explants to each of the above-mentioned provocative agents or stress conditions for 48 hrs. Other explants without any stressors were cultured in parallel for the same amount of time. The processing of explants was divided into five steps: 1) explant culture; 2) tissue homogenization; 3) acetonitrile precipitation to remove high abundance, high molecular weight proteins; 4) injection of the protein-depleted specimen into a capillary liquid chromatography–mass spectrometer; 5) analysis of MS data to identify quantitative differences between cases (stressed explants) and controls (normal explants). In total, we observed 146 molecules changed in abundance between the treated explants and the controls with 75 of these molecules changed in response to hypoxic treatment, 23 changed due to hypoxia-reoxygenation, a process generating reactive oxygen species, and 48 changed due to tumor necrosis factor–alpha (TNFα), a pro-inflammatory cytokine. We were successful in identifying 45% of all these molecules by tandem MS. Statistical modeling that applied LASSO analysis allowed for the development of a model that used 16 of the 146 differentially expressed biomolecules to accurately classify and differentiate each of the 4 stressed conditions. In my third study, I then submitted actual preeclamptic and non-diseased placental tissue to our established homogenization and acetonitrile precipitation protocol to see if any of the differences in LMW biomolecules produced under stress conditions in normal placenta were recapitulated in actual diseased placenta. In a preliminary statistical analysis, 8 of the original 146 differentially expressed species, displayed significant or near significant changes in the actual disease placenta. After applying two stringent statistical tests that eliminated any potential influence of gestational age, four out of the 146 biomarkers previously studied, continued to be differentially expressed in both stringent analyses. Of the four, 1 biomarker (m/z 649.49 (+1)) showed an increased abundance in hypoxic placental explants as well as in PE placenta; 2 (461.06 (+1), 476.24 (+1)) were increased in response to TNFα-exposed placental explants and in these PE placentas and 1 (426.35 (+1)) increased in response to hypoxia-reoxygenation-treated placental explants was also increased in PE placenta. We have chemically characterized 2 of the 4 biomarkers. One was a phospholipid (m/z 476.24) while the other was an acyl-carnitine (m/z 426.35). This suggests that features of PE appear to arise from the predicted early abnormalities that affect the placenta. In conclusion, I was successful in developing an ‘omics’ approach to study less abundant, low molecular weight biomolecules in human placenta as well as investigate which biomarkers show differential expression in human placenta when exposed to proposed abnormalities of PE and have data to suggest that these same responses are present in PE placenta.

Apesar de seu início promissor, a miniaturização da cromatografia líquida tem se desenvolvido de forma lenta devido à dificuldade em adquirir colunas e equipamentos adequados à técnica. Nos últimos anos, o interesse nesta área da cromatografia tem sido crescente em decorrência das inúmeras vantagens proporcionadas pelo uso de colunas com diâmetro interno reduzido. Ainda que as colunas capilares se encontrem disponíveis comercialmente, sua produção em laboratório pode reduzir custos. Vários parâmetros de grande importância na cromatografia, como o tempo de retenção dos analitos e a pressão do sistema, sofrem variações quando a temperatura da coluna é alterada. Em cromatografia líquida capilar, a aplicabilidade da programação de temperatura é uma alternativa viável, uma vez que a distribuição de calor no interior das colunas ocorre de maneira rápida e uniforme. Os detectores espectrométricos de massas são considerados os mais adequados a esta técnica, pois permitem o monitoramento de compostos presentes em amostras pouco concentradas. Em decorrência do alto custo e indisponibilidade destes em alguns laboratórios, as pesquisas instrumentais nesta área têm empregado detectores mais simples, como os baseados na absorção de luz ultravioleta. Contudo o diâmetro interno reduzido das colunas capilares e, por consequência, o baixo volume de injeção da amostra tornam necessário que as celas de detecção sejam apropriadamente dimensionadas para que não ocorra excessivo alargamento dos picos cromatográficos. Desta maneira, o presente trabalho visa o desenvolvimento de colunas empacotadas e monolíticas, bem como a produção de celas de detecção adequadas ao uso em sistemas destinados à cromatografia líquida capilar. O estudo de algumas variáveis de empacotamento permitiu a obtenção de colunas com excelente eficiência. As fases estacionárias monolíticas foram confeccionadas pelo método sol-gel e utilizadas para promover separações bastante rápidas. Uma das celas de detecção produzidas obteve ótimo desempenho quando comparada a duas celas comerciais comumente empregadas em sistemas miniaturizados. A programação de temperatura foi aplicada durante a separação de tetraciclinas e esteróides, sendo estas análises obtidas com maior rapidez quando comparadas às realizadas sob condições isotérmicas. / In spite of its promising start, the miniaturization of liquid chromatography has developed slowly due to the difficulties in obtaining appropriate columns and equipments suitable to this technique. In recent years, interest in this area of chromatography has been increasing due to the several advantages afforded by the use of columns with reduced internal diameter. Although the capillary columns are commercially available, its in-house production can reduce costs. Several parameters of great importance in chromatography, such as the retention time of analytes and the pressure of the system vary widely when the column temperature is changed. In capillary liquid chromatography the applicability of temperature programming is a viable alternative since the heat distribution across the column occurs quickly and uniformly. Mass spectrometric detectors are considered the most suitable for this technique since they allow the monitoring of compounds in low concentrated samples. Due to the high cost and unavailability of these instruments in some laboratories, instrumental research in this area has been using the simpler detectors, such as those based on the absorption of ultraviolet light. However, the reduced internal diameter of capillary columns and, consequently, the low injection volume of sample imply an appropriate dimensioning of the detection cell, so that excessive broadening of chromatographic peaks is avoided. Thus, this work aims with the development of monolithic and packed columns, as well as the production of suitable cells for the use in detection systems for capillary liquid chromatography. The study of some packing variables allowed to obtain columns with excellent efficiency. Monolithic stationary phases were prepared by sol-gel process and were used to promote very fast separations. One of the obtained detection cells produced good performance when compared to two commercial cells commonly used in miniaturized systems. Temperature programming was applied for the separation of tetracyclines and steroids, at higher speed when compared to those obtained under isothermal conditions.

Desde que foi introduzida por Tswett no começo do século XX, a cromatografia vem sofrendo contínuos avanços. Entretanto, a miniaturização da cromatografia líquida, apesar de seu inicio promissor, ainda continua bastante lenta e até o presente não alcançou ampla difusão, sendo que o número de grupos trabalhando nesta área é ainda bastante restrito. O motivo deste lento avanço se encontrava na dificuldade em se desenvolver equipamentos adequados, colunas apropriadas e sistemas de tratamento de dados suficientemente rápidos para os sistemas miniaturizados. Apesar de muitos desses problemas atualmente serem de fácil resolução, ainda não se dispõe de equipamentos comercialmente disponíveis que supram satisfatoriamente às condições impostas pelas micro-colunas. Tal carência deve ser suprida antes de nos beneficiarmos de todas as vantagens intrínsecas à miniaturização da cromatografia líquida. Deste modo, o presente projeto visa o desenvolvimento de instrumentação totalmente dedicada aos sistemas miniaturizados de cromatografia líquida incluindo o desenvolvimento de uma bomba de alta pressão, um sistema de injeção a base de tempo, um forno capaz de realizar programação de temperatura e o software de controle dos mesmos. Os equipamentos desenvolvidos foram então aplicados em separações de estatinas demonstrando um excelente desempenho. / Since the chromatography was introduced by Tswett, in the beginning of the 20th century, the technique has suffered a constant progress. However, the miniaturization of liquid chromatography, instead of its promising start, it is still too slow and this technique has not reached a wide divulgation so far. It is important to mention that the number of groups working in this area is very limited yet. The reason of this slow progress was due to the difficulty in developing suitable equipments, appropriated columns and data treatment systems that are quick enough for the miniaturized systems. Currently, many of these problems are easy to be solved, however, there are no equipments available commercially that supply the conditions imposed by microcolumns satisfactorily. Such lack should be filled before benefiting from all the advantages concerned to the miniaturization of liquid chromatography. In this way, this study aims at the development of instrumentation totally dedicated to the miniaturized systems of liquid chromatography, including the development of a high pressure pump, an time-based injector, an oven that sets the temperature programming and the software that can control all these devices. The developed equipments were then applied in the separation of statins, demonstrating an excellent performance.

(EN) This dissertation thesis is focused on physicochemical and chromatographic characterization of polyaniline-coated stationary phases. In the first part, surfaces of bare silica and octadecyl silica sorbents were modified by in-situ chemical polymerization of aniline hydrochloride and their subsequent systematic characterization was performed by using the linear solvation energy relationship approach in the HILIC mode of capillary LC. In addition, several common physicochemical techniques were used to characterize properties of these altered materials. The modified sorbents were then packed into capillary columns. The retention interactions taking place between solute and the separation system were evaluated on the basis of retention data of a number of various solutes. The results showed that polyaniline coating had a significant effect on the retention promoting interactions of both polyaniline-coated stationary phases. The assumed mixed-mode retention mechanism was proven for both the stationary phases. The second part dealt with investigation of the separation potential of polyaniline- coated silica stationary phase in different chromatographic modes. The retention factor curves of structurally similar solutes were constructed as a function of organic modifier portion in the mobile phase....

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal.

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal