This Master's thesis deals with determination of nucleobases in formamide samples (pure or catalyzed form) after their initiation by high-power density energy events. The theoretical part states the reader to the problems of prebiotic chemistry and acquaints him with the various theories of the origin of the nucleobases. The experimental part deals with the analysis of the samples initiated by Laser System PALS, which was used for the simulation high-power density energy events. For the identification and determination of the final products of laser plasma initiated chemical reactions in the gaseous phase of the samples was used the Fourier Transform Infrared Spectroscopy. The liquid part of the samples and the nucleobases in this part were analyzed by the gas chromatography with the mass detection.

A apramicina é um antibiótico aminoglicosídeo produzido por uma cepa de Streptomyces Tenebrarius, utilizada na medicina veterinária na forma de sulfato para tratar e prevenir doenças infecciosas produzidas por bactérias gram-negativas em porcos, bezerros e aves domésticas. As preparações comercialmente disponíveis são injetável, premix para adição em rações e pó oral solúvel, sendo esta última objeto de estudo deste trabalho. A Farmacopéia Britânica é o único compêndio oficial que descreve a apramicina (tanto a matéria-prima quanto as preparações) e recomenda o método microbiológico turbidimétrico para o seu doseamento. Embora os métodos microbiológicos ainda sejam os métodos de escolha na determinação da potência de antibióticos, há uma tendência crescente em substituí-los pelos métodos físico-químicos, sobretudo a cromatografia líquida de alta eficiência (CLAE), a qual proporciona sensibilidade, especificidade, exatidão e precisão; ela já vem sendo usada com sucesso na determinação do teor de antibióticos, embora a sua potência possa estar sujeita a confirmação por um método microbiológico. Considerando-se as vantagens da CLAE, bem como a inexistência de métodos físico-químicos oficiais para a análise de apramicina, o objetivo do presente trabalho foi desenvolver e validar um método empregando a cromatografia líquida de alta eficiência para a quantificação de apramicina em pó oral solúvel, bem como correlacioná-lo com o método turbidimétrico. Foi desenvolvido um método por CLAE empregando fase reversa com o uso de uma coluna C18 de 150 mm x 4,6 mm x 4 &#181;m, derivatização pré-coluna com o-ftalaldeído (OPA) e detecção por UV em 332 nm. A metodologia proposta foi validada de acordo com normas dos compêndios oficiais e apresentou: boa robustez; linearidade no intervalo de 0,02 a 0,05 mg/mL com obtenção de uma curva de calibração de equação igual a y = 132887304,980x + 127223,837 e coeficiente de correlação linear de 0,9999; boa precisão, sendo o DPR<1,0% para a precisão intra-dia e DPR = 1,1% para a precisão inter-dia; 99,33% de recuperação média, demonstrando exatidão; sensibilidade, com limites de detecção e de quantificação de 0,08 e 0,25 &#181;g/mL, respectivamente. Três lotes de apramicina pó oral solúvel foram analisados pelo método por CLAE e pelo método microbiológico oficial e a comparação estatística entre os resultados obtidos através do Teste T demonstrou que não houve diferença significativa entre ambos no nível de &#945; = 0,05. / Apramycin is an aminoglycoside antibiotic produced by Streptomyces tenebrarius strain, widely used in veterinary medicine under sulfate form to treat and prevent infectious diseases induced by gram-negative bacteria in swines, calves and poultry. The commercially available preparations consist of injections, additive feed (premix) and soluble oral powder, being the latter the aim of this work. British Pharmacopoeia is the only official compendium that describes apramycin (bulk pharmaceutical and its formulations) recommending the microbiological turbidimetric method for its dosage. Although microbiological methods are still of chosen for the determination of antibiotics potency, there is an increasing tendency of replacing them by physico-chemical methods, mainly high performance liquid chromatography (HPLC), which offers sensitivity, specificity, accuracy, and precision; it has been successfully used in the determination of antibiotic assay, although its potency may be subjected to confirmation by a microbiological method. Taking into account the advantages of HPLC as well as the absence of official physicochemical methods to analyze apramycin, the aim of the present work was to develop and validate a method employing high performance liquid chromatography to the quantification of soluble oral powder, as well as to compare it with the turbidimetric method. A HPLC method was developed employing reverse phase with a C18 column of 150 mm x 4,6 mm x 4 &#181;m, pre-column derivatization with OPA (o-phthalaldehyde) and UV detection at 332 nm. The proposed method was validated according to official compendia guidelines, having demonstrated: good robustness; linearity in the concentration range of 0,02 to 0,05 mg/mL with linear equation of y = 132887304,980x + 127223,837 and correlation coeficient of 0,999; good precision, being RSD<1,0% for intra-day precision and = 1,1% for inter-day precision; average recuperation of 99,33%, what demonstrates accuracy; sensitivity, with quantification and detection limits of 0,08 and 0,25 &#181;L/mL, respectively. Three batches of apramycin soluble oral powder were analyzed by HPLC method and by the official microbiological method having the statistical comparison between the obtained results by T test demonstrated that there are no significant differences between both of them at &#945; = 0,05 level.

In der vorliegenden Arbeit wurden Einsatzmöglichkeiten der Headspace-Festpahsenmikroextraktion (HS-SPME) in Kombination mit der GC-MS für forensische und toxikologische Fragestellungen, insbesondere in der Haaranalytik, untersucht. Für basenstabile Drogen- und Medikamentenwirkstoffe wurden Nachweismethoden nach alkalischer Hydrolyse der Haare mit Natriumhydroxid und anschließender HS-SPME entwickelt. Für das Lokalanaesthetikum Lidocain und für das Heroinsubstitutionsmittel Methadon wurden optimierte Verfahren auf Haarproben von Drogentodesfällen angewandt. Um die Einsatzmöglichkeiten der HS-SPME gerade für Wirkstoffmetaboliten zu erweitern, wurden drei Derivatisierungsmöglichkeiten überprüft: (i) Umsetzung von primären und sekundären Aminen im alkalischen Haarhydrolysat mit Alkylchloroformiaten zu Carbamaten ("in sample" Derivatisierung), (ii) "On Fiber" Derivatisierung primärer und sekundärer Amine nach erfolgter HS-SPME mit N-Methyl-bis-trifluoracetamid (MBTFA) sowie (iii) Reaktion von Monofluoressigsäure mit 1-Pyrenyldiazomethan (PDAM) an einer vorher mit dem Derivatisierungsreagenz benetzten SPME-Faser. Fettsäureethylester konnten nach einer Flüssigextraktion der Haare von Alkoholikern mit einem DMSO/n-Hexan-Gemisch extrahiert und nach optimierter HS-SPME nachgewiesen werden. Im Vergleich zu Haarproben von Abstinenzlern und Gelegenheitstrinkern ergaben sich deutliche Unterschiede in den Konzentrationen. Für die schnelle Bestimmung von Valproinsäure und Trichlorethanol wurde eine Quantifizierungsmethode aus Blut- und Urinproben erarbeitet. Die Anwendung der HS-SPME ermöglichte eine schnelle, einfache und lösungsmittelfreie Extraktion bei geringem Probenverbrauch. Sie führte zu sauberen Extrakten und für dieses Verfahren hohen absoluten Extraktionsausbeuten. Bessere Reproduzierbarkeiten, ein hoher Probendurchsatz und eine verlängerte Einsetzbarkeit der SPME-Faser wurden durch die Verwendung eines automatischen Probengebers erreicht. / In the presented thesis possible fields of applications of the headspace solid-phase microextraction (HS-SPME) in combination with gas chromatography-mass spectrometry (GC-MS) for forensic and toxicological problems were investigated with special focus on hair analysis. Detection methods after alkaline hydrolysis of hair with sodium hydroxid and following HS-SPME were developed for basic stable drugs. For the local anaesthetic lidocaine and for the heroine substitute methadone optimized methods were applied to hair of drug fatalities. In order to enhance the fields of application of the HS-SPME especially for drug metabolites three derivatization procedures were tested: (i) conversion of primary and secondary amines in the alkaline hair extract with alkylchloroformates to carbamates ("in sample" derivatization), (ii) "on fibre" derivatization of primary and secondary amines after HS-SPME with N-methyl-bis-trifluoroacetamide (MBTFA) and (iii) reaction of monofluoroacetic acid (compound 1080) with 1-pyrenyldiazomethane (PDAM) on a SPME-fibre previously occupied with the derivatization reagent. Fatty acid ethyl ester (FAEE) could detected after liquid extraction from hair of alcoholics with a mixture of DMSO/n-hexane and optimized HS-SPME. In comparison to hair samples of teetotalers and social drinkers significant differences in the concentrations of the FAEE were found. A quantification method were developed for the fast determination of valproic acid and trichloroethanol from blood and urine. The application of HS-SPME allows a fast, simple and solvent free extraction from a small sample amount. HS-SPME lead to clean extracts and high absolute extraction yields for this kind of extraction. Better reproducibilities, a high sample throughput and extended lifetime of the SPME-fibre were obtained using an automatic sampler.

Le développement de nouveaux donneurs d’oxyde nitrique (NO) dans le traitement chronique des maladies cardiovasculaires nécessite l’étude de leur biodisponibilité après administration par voie orale. Les S-nitrosothiols (RSNOs) apparaissent d’intéressants candidats médicaments pour ce faire, et l’étude de leur perméabilité intestinale est une première étape indispensable. Il est nécessaire de disposer d’une méthodologie analytique suffisamment sensible et sélective, en particulier permettant de différencier entre la production endogène de NO, l’apport alimentaire en ions nitrites et nitrate et le médicament lui-même. Nos travaux de thèse ont consisté à utiliser le S-nitrosoglutathion (GSNO) comme modèle après son marquage par l’isotope stable 15 de l’azote (15N). La dérivation du 15NO libéré par deux méthodes conventionnelles (méthode de Griess conduisant à la formation d’un adduit azoïque ; réaction avec le 2,3-diaminonaphtalène (DAN) formant l’adduit 2,3-naphtotriazole (NAT)) et l’étude de la fragmentation en spectrométrie de masse tandem (MS/MS) des deux adduits correspondants ont mené à sélectionner la dérivation par le DAN comme étant la plus sensible. Une transition originale résultant de la fragmentation du NAT en mode Higher-energy Collisional Dissociation (HCD) au lieu du mode conventionnel Collisionally Induced Dissociation (CID) a été mise en évidence ; elle permet d’atteindre une limite de quantification de 5 nM (soit 20 fois plus basse que celle offerte par la fluorescence). La méthode LC-MS/MS a été validée et appliquée à l’étude de la perméabilité intestinale du GS15NO par deux modèles : l’un in vitro (monocouche de cellules épithéliales type Caco-2), l’autre ex vivo (intestin de rat isolé (ileum) dans une chambre de Ussing). Les valeurs de perméabilité apparente calculées à partir des concentrations des métabolites du GS15NO (ions nitrites, nitrates et RSNOs) le classent comme un médicament de perméabilité intermédiaire. En outre, des études sur les mécanismes de dénitrosation du GSNO ont été menées sur intestin isolé, démontrant en particulier le rôle d’enzymes telles que la γ-glutamyltransférase et la protein disulfide isomerase / The development of innovative nitric oxide (NO) donors for the chronic treatment of cardiovascular diseases implies their bioavailability studies after oral administration. S-nitrosothiols (RSNOs) look interesting drug candidates for this purpose and evaluating their intestinal permeability appears the first step to be realized. Thus, an analytical method offering high sensitivity is needed; moreover this method should be selective by differentiating between the endogenous production of NO, the intake of nitrite and nitrate ions via the diet, and the drug itself. Our work consisted in using S-nitrosoglutathione (GSNO) labeled with the stable nitrogen isotope 15 (15N) as a model. Released 15NO species were derivatized by two conventional methods: Griess method leading to the formation of an azo adduct; reaction with 2,3-diaminonaphthalene (DAN) producing 2,3-naphtotriazole (NAT); fragmentation studies of the two adducts by tandem mass spectrometry (MS/MS) allow the selection of DAN method because it provides the highest sensitivity. An original transition resulting from the NAT fragmentation in Higher-energy Collisional Dissociation (HCD) mode instead of the conventional Collisionally Induced Dissociation (CID) mode was pointed out and permitted to reach a limit of quantification of 5 nM (20 fold less than when using fluorescence). The LC-MS/MS method was validated and applied to the GS15NO intestinal permeability studies with two models: in vitro (a monolayer of Caco-2 epithelial cells), and ex vivo (isolated intestine of rat (ileum) in an Ussing chamber). The apparent permeability values calculated with concentrations of GS15NO metabolites (nitrite, nitrate ions and RSNOs) classify it as a middle permeable drug. Studies on GSNO denitrosating processes using isolated rat intestine demonstrate that the enzymes γ-glutamyltransferase and protein disulfide isomerase play a pivotal role

O para-cresol (4-metilfenol) e as guanidinas são compostos envolvidos em uma série de processos bioquímicos e fisiológicos. Em condições normais esses compostos são eliminados do organismo pelos rins. Entretanto, em pacientes com doença renal crônica (DRC), esses compostos acumulam nos fluidos biológicos e tecidos, e são de difícil remoção, gerando alta taxa de mortalidade em pacientes humanos hemodialisados. Cromatografia gasosa (GC) e cromatografia líquida de alta eficiência (HPLC) são as técnicas analíticas mais utilizadas para a separação e quantificação de p-cresol e guanidinas. A derivatização pré ou pós-coluna geralmente é empregada para aumentar a volatilidade, absorbância ou fluorescência desses compostos, permitindo a detecção e quantificação. A ninidrina é um dos reagentes mais utilizados para a derivatização das guandinas pois apresenta alta sensibilidade, solubilidade em água e o procedimento aplicado para a derivatização é simples, permitindo a automação do sistema de preparo de amostras quando necessário. Embora haja vários estudos sobre a concentração e os efeitos desses compostos no sangue de pacientes humanos com DRC, a concentração de p-cresol e das guanidinas no sangue de cães com DRC e os efeitos associados a tais compostos foram pouco estudados. Assim, são necessários estudos similares aos realizados em humanos, para o desenvolvimento de novas estratégias de tratamento veterinário para cães. Para isso, é importante o desenvolvimento de uma metodologia analítica para a quantificação desses compostos no sangue de cães. Nesse trabalho desenvolveu-se, validou-se e aplicou-se um método para análise de p-cresol e guanidinas em plasma de cães urêmicos com detecção por fluorescência. A reação de derivatização das guanidinas com a ninidrina foi revisada, caracterizada por espectrometria de massas (MS) e otimizada utilizando planejamento fatorial. Foi analisado um total de 63 amostras de plasma de cães incluindo grupo controle e urêmico. A concentração média de guanidina, metilguanidina e p-cresol livre determinada foi maior no grupo urêmico do que no grupo controle, a exemplo do que ocorre em humanos, sugerindo um comportamento metabólico similar desses compostos em cães. O método desenvolvido possibilitou as análises, sendo relativamente simples, evitando um preparo de amostras complexo. Por sua vez, a otimização por planejamento fatorial da reação de derivatizacão com ninidrina resultou em ganhos na área dos picos cromatográficos que chegaram a 1000%. / Para-cresol (4-methylphenol) and guanidino compounds are involved in a series of biochemical and physiological cycles. Under ordinary conditions, these compounds are excreted from the body by healthy kidneys. However, in uremic patients, the concentration of these compounds is highly increased in biological fluids and tissues and is of hard removal, increasing the mortality rate in dialysis patients. Gas chromatography (GC) and high performance liquid chromatography (HPLC) are the most used techniques for separation and quantification of p-cresol and guanidino compounds. For the guanidines determination, derivatizing reactions are usually employed to enhance the absorbance, fluorescence or volatility of these compounds, allowing the quantification. Ninhydrin is one of the most used reagents for derivatization because it shows high sensitivity and presents water solubility. The procedure employed for the derivatization reaction is simple, allowing the automation of sample preparation system, when it is required. There are few studies quantifying the concentration of guanidino compounds and p-cresol in blood of uremic dogs, therefore, a better understanding of the role of these compounds in the metabolism of dogs are needed. For this reason, it is important to develop an analytical method for the quantification of these compounds in dog\'s blood. The development of a method for simultaneous determination of these compounds, which is not reported in the literature, can improve the analytical productivity, decreasing the time spent with sample preparation and analysis. Here, a method based in reversed-phase HPLC for simultaneous quantification of guanidino compounds and free p-cresol in plasma of uremic dogs with fluorescence detection was developed, validated and applied. For this purpose, the ninhydrin derivatization reaction was reviewed, characterized by mass spectrometry (MS) and optimized using central composite design (CCD). A total of 63 samples of dog plasma, including healthy and uremic groups, were analyzed. The mean concentration of guanidine, methylguanidine and free p-cresol determined by the validated method, for the uremic group, were higher than the healthy group, alike usual for humans, suggesting a similar metabolic behavior of these compounds in dogs. The obtained method is relatively simple avoiding complex sample preparation and the experimentally designed optimization of the ninhydrin reaction by CCD yielded a gain of area as high as 1000% for the chromatographic peaks.

Esta tese tem como objetivo estudar a derivatização da celulose sob condições homogêneas, utilizando líquidos iônicos (LIs), em micro-ondas, através de um método simples e reprodutível. Foram estudadas três etapas: i) Síntese dos LIs, buscando otimizar seu preparo; ii) Efeitos da estrutura dos LIs sobre a dissolução de celulose; iii) Otimização da derivatização da celulose em LIs. Foi realizado um planejamento experimental para a otimização da síntese dos LIs, mostrando-se que o uso de solvente molecular como diluente não traz vantagens. Na dissolução de celuloses fibrosas em LIs, observou-se a importância das estruturas dos cátions e ânions destes. Estudou-se a influência de um solvente molecular, DMSO, na reação de derivatização de celulose em LIs e percebeu-se que o uso do mesmo não é vantajoso. Por fim, realizou-se um planejamento experimental da acetilação da celulose, mostrando-se quais fatores mais afetam esse processo: tempo de reação, razão molar de anidrido acético/celulose e temperatura / The work reported has the target of studying the acylation of cellulose under homogeneous reaction conditions. Imidazolium-based ionic Liquids (ILs) were employed as solvents, and the reaction was assisted by a microwave. These following processes have been studied: Optimization of the synthesis of ILs; ii) Effects of the structure of ILs on the dissolution of cellulose; iii) Optimization of cellulose acylation. Experimental design has been applied in order to optimize the synthesis of ILs. The use of DMSO as a diluent decreased the efficiency of the synthesis. Dissolution of cellulose (eucalyptus, mercerized eucalyptus and mercerized cotton) in several ILs has shown the importance of the counter-ion and the structure of the cation side-chain. The addition of DMSO decreased the amount of dissolved cellulose. Experimental design has been applied in order to optimize the acetylation of cellulose. Reaction time, molar ratio acetic anhydride/anhydroglucose unit, and cellulose concentration influenced the reaction outcome.

Esta tese tem como objetivo estudar a derivatização da celulose sob condições homogêneas, utilizando líquidos iônicos (LIs), em micro-ondas, através de um método simples e reprodutível. Foram estudadas três etapas: i) Síntese dos LIs, buscando otimizar seu preparo; ii) Efeitos da estrutura dos LIs sobre a dissolução de celulose; iii) Otimização da derivatização da celulose em LIs. Foi realizado um planejamento experimental para a otimização da síntese dos LIs, mostrando-se que o uso de solvente molecular como diluente não traz vantagens. Na dissolução de celuloses fibrosas em LIs, observou-se a importância das estruturas dos cátions e ânions destes. Estudou-se a influência de um solvente molecular, DMSO, na reação de derivatização de celulose em LIs e percebeu-se que o uso do mesmo não é vantajoso. Por fim, realizou-se um planejamento experimental da acetilação da celulose, mostrando-se quais fatores mais afetam esse processo: tempo de reação, razão molar de anidrido acético/celulose e temperatura / The work reported has the target of studying the acylation of cellulose under homogeneous reaction conditions. Imidazolium-based ionic Liquids (ILs) were employed as solvents, and the reaction was assisted by a microwave. These following processes have been studied: Optimization of the synthesis of ILs; ii) Effects of the structure of ILs on the dissolution of cellulose; iii) Optimization of cellulose acylation. Experimental design has been applied in order to optimize the synthesis of ILs. The use of DMSO as a diluent decreased the efficiency of the synthesis. Dissolution of cellulose (eucalyptus, mercerized eucalyptus and mercerized cotton) in several ILs has shown the importance of the counter-ion and the structure of the cation side-chain. The addition of DMSO decreased the amount of dissolved cellulose. Experimental design has been applied in order to optimize the acetylation of cellulose. Reaction time, molar ratio acetic anhydride/anhydroglucose unit, and cellulose concentration influenced the reaction outcome.

O para-cresol (4-metilfenol) e as guanidinas são compostos envolvidos em uma série de processos bioquímicos e fisiológicos. Em condições normais esses compostos são eliminados do organismo pelos rins. Entretanto, em pacientes com doença renal crônica (DRC), esses compostos acumulam nos fluidos biológicos e tecidos, e são de difícil remoção, gerando alta taxa de mortalidade em pacientes humanos hemodialisados. Cromatografia gasosa (GC) e cromatografia líquida de alta eficiência (HPLC) são as técnicas analíticas mais utilizadas para a separação e quantificação de p-cresol e guanidinas. A derivatização pré ou pós-coluna geralmente é empregada para aumentar a volatilidade, absorbância ou fluorescência desses compostos, permitindo a detecção e quantificação. A ninidrina é um dos reagentes mais utilizados para a derivatização das guandinas pois apresenta alta sensibilidade, solubilidade em água e o procedimento aplicado para a derivatização é simples, permitindo a automação do sistema de preparo de amostras quando necessário. Embora haja vários estudos sobre a concentração e os efeitos desses compostos no sangue de pacientes humanos com DRC, a concentração de p-cresol e das guanidinas no sangue de cães com DRC e os efeitos associados a tais compostos foram pouco estudados. Assim, são necessários estudos similares aos realizados em humanos, para o desenvolvimento de novas estratégias de tratamento veterinário para cães. Para isso, é importante o desenvolvimento de uma metodologia analítica para a quantificação desses compostos no sangue de cães. Nesse trabalho desenvolveu-se, validou-se e aplicou-se um método para análise de p-cresol e guanidinas em plasma de cães urêmicos com detecção por fluorescência. A reação de derivatização das guanidinas com a ninidrina foi revisada, caracterizada por espectrometria de massas (MS) e otimizada utilizando planejamento fatorial. Foi analisado um total de 63 amostras de plasma de cães incluindo grupo controle e urêmico. A concentração média de guanidina, metilguanidina e p-cresol livre determinada foi maior no grupo urêmico do que no grupo controle, a exemplo do que ocorre em humanos, sugerindo um comportamento metabólico similar desses compostos em cães. O método desenvolvido possibilitou as análises, sendo relativamente simples, evitando um preparo de amostras complexo. Por sua vez, a otimização por planejamento fatorial da reação de derivatizacão com ninidrina resultou em ganhos na área dos picos cromatográficos que chegaram a 1000%. / Para-cresol (4-methylphenol) and guanidino compounds are involved in a series of biochemical and physiological cycles. Under ordinary conditions, these compounds are excreted from the body by healthy kidneys. However, in uremic patients, the concentration of these compounds is highly increased in biological fluids and tissues and is of hard removal, increasing the mortality rate in dialysis patients. Gas chromatography (GC) and high performance liquid chromatography (HPLC) are the most used techniques for separation and quantification of p-cresol and guanidino compounds. For the guanidines determination, derivatizing reactions are usually employed to enhance the absorbance, fluorescence or volatility of these compounds, allowing the quantification. Ninhydrin is one of the most used reagents for derivatization because it shows high sensitivity and presents water solubility. The procedure employed for the derivatization reaction is simple, allowing the automation of sample preparation system, when it is required. There are few studies quantifying the concentration of guanidino compounds and p-cresol in blood of uremic dogs, therefore, a better understanding of the role of these compounds in the metabolism of dogs are needed. For this reason, it is important to develop an analytical method for the quantification of these compounds in dog\'s blood. The development of a method for simultaneous determination of these compounds, which is not reported in the literature, can improve the analytical productivity, decreasing the time spent with sample preparation and analysis. Here, a method based in reversed-phase HPLC for simultaneous quantification of guanidino compounds and free p-cresol in plasma of uremic dogs with fluorescence detection was developed, validated and applied. For this purpose, the ninhydrin derivatization reaction was reviewed, characterized by mass spectrometry (MS) and optimized using central composite design (CCD). A total of 63 samples of dog plasma, including healthy and uremic groups, were analyzed. The mean concentration of guanidine, methylguanidine and free p-cresol determined by the validated method, for the uremic group, were higher than the healthy group, alike usual for humans, suggesting a similar metabolic behavior of these compounds in dogs. The obtained method is relatively simple avoiding complex sample preparation and the experimentally designed optimization of the ninhydrin reaction by CCD yielded a gain of area as high as 1000% for the chromatographic peaks.

A eletroforese capilar (CE) é uma técnica poderosa de separação que explora as diferenças na mobilidade de espécies iônicas sob efeito do campo elétrico. Não permite, contudo, separação de misturas de moléculas neutras, possível mediante formação de complexos com carga, derivatização química ou cromatografia eletrocinética micelar (MEKC). A derivatização eletroquímica tem sido usada em combinação com HPLC, entre outras técnicas, mas não ainda com CE e para fins quantitativos, como proposto e demonstrado nesta dissertação, em que se enfoca, como sistemas modelo, álcoois primários de cadeia curta e se recorre a sistema em fluxo designado de EC-CE-C4D, que consiste de uma célula eletroquímica acoplada com equipamento de eletroforese capilar provido de detector de condutividade sem contato direto com os eletrodos. O sistema EC-CE-C4D, inicialmente concebido para efetuar pré-concentração e redissolução eletroquímica de metais seguida de separação eletroforética, possibilitou também o monitoramento de produtos com carga, formados em processos eletrocatalíticos, fato que inspirou a investigação da aplicabilidade, também do sistema à derivatização eletroquímica de analitos neutros em iônicos ou ionizáveis. Inicialmente, a oxidação de etanol foi avaliada por voltametria cíclica em eletrodos de ouro, paládio e platina policristalinos, nos meios ácido, neutro e alcalino. Maior formação de ácido acético se deu sobre platina em meio ácido (pH = 2,3). Entre os tampões avaliados por simulação (PeakMaster 5.1) para a separação dos carboxilatos, o TRIS/HCl (pH = 8,6) foi favorável em termos de resolução, rapidez e sensibilidade. Após a eletrooxidação da amostra sobre Pt a 1,6 V vs. Ag/AgCl por 60 s seguida de transferência de nanolitros dos produtos formados para o capilar por injeção hidrodinâmica, o meio ácido é trocado pelo tampão de corrida com auxílio de microbombas controladas por computador. A eficiência da conversão eletroquímica dos álcoois em carboxilatos, de 16% para etanol e propanol nas condições adotadas, apesar de não muito eficiente, proporcionou boa repetibilidade e limites de quantificação ao redor de 5 10-5 mol L-1 para etanol, 1-propanol, 1-butanol e 1-pentanol, ou seja, cerca de 20 vezes melhores que os reportados para MEKC. As curvas analíticas para os 4 álcoois apresentaram boa linearidade (R > 0,996) e a boa separação entre os picos permitindo determinações quantitativas simultâneas. Como exemplo de aplicação a amostras reais, demonstrou-se a determinação quantitativa de etanol em cerveja Pilsen convencional (teor alcoólico 4,5% vol.) e sem álcool (teor alcoólico <0,5% vol.), diluídas de 1000 vezes e 100 vezes, respectivamente. A repetibilidade das análises foi muito boa (~6% DPR, n = 40), indicando a estabilidade do sistema automático e ausência de envenenamento da superfície do eletrodo. Resultados ainda melhores foram obtidos utilizando 1-propanol como padrão interno para a determinação de etanol. Comparação dos resultados da dosagem alcoólica obtida com o sistema EC-CE-C4D e por CG-FID não apresentou diferença significativa utilizando um intervalo de confiança de 95 %, demonstrando a efetividade e concordância da nova combinação de técnicas. Este trabalho pioneiro, aliando derivatização eletroquímica com a eletroforese para a quantificação de espécies neutras, abre perspectivas de pesquisa não só para aprofundamento e otimização da análise de álcoois, como para a derivatização de outras espécies neutras na faixa de pH comum em CE, como aldeídos e açúcares. / Capillary electrophoresis (CE) is a powerful separation technique which exploits the differences in the mobility of ionic species under the effect of electric field. However, it does not allow the separation of mixtures of neutral molecules, and alternatives like formation of charged complexes, chemical derivatization and micellar electrokinetic chromatography (MEKC) have been proposed. Electrochemical derivatization has been combined before with HPLC, among other techniques, but not yet with CE and for quantitative purposes, as proposed and demonstrated in this Masters Thesis, focusing as model systems, short chain primary alcohols, using a flow system, designated EC-EC-C4D, composed of an electrochemical cell coupled with a capillary electrophoresis equipment provided with conductivity detector without direct contact with the electrodes. The EC-CE-C4D system, originally proposed to perform electrochemical pre-concentration and stripping of metals followed by electrophoretic separation, also enabled the monitoring of charged products formed in electrocatalytic processes, inspiring the application of the system to electrochemical derivatization of uncharged analytes into ionic or ionizable ones. Initially, the ethanol oxidation was evaluated by cyclic voltammetry on gold, palladium and platinum polycrystalline electrodes, in acid, neutral and alkaline medium. Larger formation of acetic acid was observed on platinum in acid medium (pH = 2.3). Among the buffers evaluated by simulation (PeakMaster 5.1) for separation of carboxylates, aiming to compromise between resolution, speed and sensitivity, TRIS / HCl (pH 8.6) was favorable. After the sample electrooxidation on Pt at 1.6 V vs. Ag/AgCl for 60 s is replaced automatically (by computer controlled micropumps) by running buffer, followed by the hydrodynamic injection into the capillary of products formed, the acid medium is replaced by running buffer with help of computer controlled micropumps. The efficiency of the electrochemical conversion of alcohols into carboxylates, 16% for ethanol and propanol at the given conditions, although not very efficient, but showed good repeatability and rendered quantification limits around 5 10-5 mol L-1 for ethanol, 1-propanol, 1-butanol and 1-pentanol the 4 alcohols, about 20 times better for MEKC. The analytical curves for the four alcohols, showed good linearity (r > 0.996) and the separation between peaks allowed simultaneous quantitative determinations. As an example of application to real samples, the quantitative determination of ethanol in Pilsen conventional beer (alcohol content 4.5% vol.) and non-alcoholic beer (alcohol content <0.5% vol.) was carried out after 1000-fold and 100 fold dilution, respectively. The repeatability of the analysis was very good (~6% RSD, n = 40), indicating the stability of the system and no poisoning of the electrode. Even better results were obtained using 1-propanol as an internal standard for the determination of ethanol. Comparison of results obtained with the CE-CE-C4D system and by GC-FID showed no significant difference for a confidence interval of 95%, demonstrating the effectiveness and consistency of the new combination of techniques. This proof of concept work of the combination of electrochemical derivatization with electrophoresis for quantification of neutral species opens perspectives not only for the optimization of the analysis of alcohols, but for research on the derivatization of other neutral species in the pH range typical for CE, like aldehydes and sugars.

Vor dem Hintergrund des weltweit wachsenden Bedarfes an Bodenverbesserungsmitteln, durch die Humus-, Nährstoff- und Wassermangel auf Problemstandorten kompensiert werden sollen, war es das Ziel der vorliegenden Arbeit, Bodenwasser-speicher auf Basis der nachwachsenden Biopolymere Stärke und Lignin zu entwickeln. Durch Derivatisierung der Stärke wurde deren Hydrophilie deutlich gesteigert, was Voraussetzung für die Bildung von Hydrogelen war. Es konnte gezeigt werden, dass durch Vernetzung der wasserlöslichen Stärkederivate Carboxymethylstärke und Monostärkemonophosphat mit Di-/Tricarbonsäuren quellfähige Hydrogele erzeugt werden, deren Quellungsvermögen und rheologische Eigenschaften über die Wahl des Vernetzers und die Vernetzerstoffmenge gezielt eingestellt werden können. Die Modifizierung von Lignin wurde durch Oxidation mit Wasserstoffperoxid, z.T. in Kombination mit Fe(II)- bzw. Mn(II)-Chloriden realisiert. Dadurch wurde die Vernetzbarkeit von Lignin deutlich verbessert, was auf oxidativ bedingte Strukturänderungen des Lignins zurückzuführen war. Diese bestanden im Wesentlichen in der Spaltung und Oxidation der Lignin-Seitenkette sowie der Hydroxylierung der Seitenkette und aromatischer Strukturen. Die Vernetzung von Lignin mit Poly-(ethylenglycol)-diglycidylether ergab quellfähige Hydrogele, deren Wasseraufnahmevermögen und rheologische Materialfunktionen von der eingesetzten Vernetzerstoffmenge abhängig sind. Es konnte gezeigt werden, dass durch den Einsatz von Stärkephosphat- und Lignin-Hydrogelen das Wasserspeichervermögen erhöht und die Evaporationsraten eines entsprechend behandelten Sandbodens verringert werden. Im Wachstumsversuch wurden die Frischmasseerträge von Gelbsenf (Sinapis alba) durch Behandlung eines Sandbodens mit Hydrogelen gesteigert. / Soil degradation leading to a lack of humus, nutrients and water especially on exploited sites causes the worldwide need in soil amendments. Aim of the work was the development of hydrogels from renewable biopolymers starch and lignin improving water retention especially in degraded soils. A significant increase of hydrophilic properties of starch was obtained by chemical modification with the objective of forming starch based hydrogels. Swellable hydrogels were formed by cross-linking of water soluble starch derivatives like carboxymethyl starch and monostarch monophosphates with di- and tricarboxylic acids. Swelling capacity and rheological properties of the starch gels were selective adjusted by variation of cross linking agent and whose amounts. Modification of lignin was realized by oxidation with hydrogen peroxide partly in combination with ferrous and manganese chlorides, respectively. In consequence of oxidative structural changes which were cleavage and oxidation of side chain as well as aliphatic and aromatic hydroxylation, gelation of lignin was improved significant. Lignin hydrogels with different swelling capacities and rheological functions were formed by cross-linking lignin with different amounts of poly (ethylene glycol) diglycidyl ether. Application of hydrogels based on starch and lignin causes increased water storing capacity/field capacity and decreased evaporation of a sandy soil as well as an increased biomass yield of yellow mustard (Sinapis alba).