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.

Os monoalquil carbonatos podem ser vistos como produtos da hidrólise parcial de carbonatos orgânicos. Apesar de serem estudados desde a década de 1920, a literatura apresenta poucas evidências de sua formação em meio aquoso. Recentes estudos apontaram a eletroforese capilar (CE) com detecção condutométrica sem contato (C4D) como uma técnica versátil para a detecção e quantificação dessas espécies, a partir da qual foi possível evidenciar a formação de monoalquil carbonatos pela reação direta entre o bicarbonato e o álcool correspondente. A presença de monoetil carbonato (MEC) em amostras de cerveja foi demonstrada pela primeira vez no presente trabalho, bem como sua formação em drinks preparados a partir de uma bebida alcoólica destilada e um refrigerante. Um equipamento de CE com dois detectores do tipo C4D foi utilizado para a identificação e a quantificação dessa espécie, principais objetivos do presente trabalho. Uma propriedade intrínseca à detecção condutométrica - o fato de a resposta do detector estar, sob certas condições, exclusivamente relacionada às mobilidades dos compostos presentes - permitiu aquantificação do MEC apesar da impossibilidade de fazê-lo por meio de calibração externa, dada a sua instabilidade em meio aquoso. O método de quantificação, que utiliza como padrões de calibração do detector cinco soluções de espécies estáveis com mobilidades eletroforéticas próximas à do MEC, foi aplicado na determinação de tricloroacetato em solução aquosa e os resultados obtidos condizem com a concentração da espécie determinada por titulação. As concentrações de MEC encontradas em uma amostra de cerveja do tipo lager e em um drink de rum com refrigerante de cola foram 1,2 mmol.L-1 e 4,1 mmol.L-1, respectivamente. Esses valores concordam com as quantidades esperadas de MEC nessas bebidas com base em suas concentrações de álcool e bicarbonato. Apesar dos estudos prévios sugerirem a formação lenta de MEC após a mistura do bicarbonato com o etanol, o preparo dos três drinks estudados mostrou rápida formação de MEC logo após a mistura dos ingredientes. Estudos envolvendo a cinética de formação da espécie em diferentes valores de pH foram realizados, evidenciando maiores velocidades de formação em condições ácidas (pH 4) quando comparadas com condições básicas (pH 8). Pôde-se concluir que, apesar de as condições ácidas favorecerem o desprendimento de CO2 do sistema, deslocando o equilíbrio no sentido da decomposição do MEC, a formação da espécie também ocorre em sistemas ácidos. Além disso, o tempo necessário para a sua formação nesses sistemas condiz com a escala de tempo necessária para o preparo e o consumo dos drinks. Apesar de o presente trabalho ser o primeiro relato da existência de MEC em alimentos, bebidas como a cerveja são consumidas há milênios, sugerindo a baixa probabilidade de a espécie estudada ser nociva. Pouco se sabe, no entanto, a respeito do seu papel no sabor das bebidas estudadas e na absorção do álcool pelo organismo. / The monoalkyl carbonates can be treated as products of the partial hydrolysis of organic carbonates. Despite they are studied since late 1920\'s, the literature shows little evidence about their formation in aqueous media. Recent studies point the capillary electrophoresis (CE) with capacitively coupled contactless conductivity detection (C4D) as a versatile technique for the identification and determination of these species, showing their formation from the direct reaction between bicarbonate and the corresponding alcohol. The presence of monoethyl carbonate (MEC) in beer is demonstrated for the first time, as well as the formation of this species in drinks prepared with a distilled beverage and a carbonated soft drink. A CE equipment with two C4D detectors was used to identify and quantify this species in cited beverages, the main objectives of the present work. An intrinsic property of the conductometric detection made possible the determination of MEC in spite of the impossibility to quantitate it by external calibration due to its stability issues in aqueous media. The quantitation method, which uses five solutions of stable species with similar electrophoretic mobilities to the analyte, was applied in the determination of trichloroacetate in aqueous solution and the results were in agreement with the concentration obtained by titration. The concentrations of MEC in samples of lager beer and rum and cola drink were, respectively, 1.2 mmol.L-1 and 4.1 mmol.L-1, which agree with the levels of ethanol and bicarbonate available in these products. Although the previous studies suggests the formation of a small amount of MEC right after the mixing of bicarbonate with ethanol - that grows over time - the three studied cases showed a fast formation of MEC after the mixing of the ingredients of the drinks. Studies about the formation kinetics in two different pH values were made showing higher formation rates for acidic media when compared with alkaline media. One can conclude that although the acidic conditions favor the loss of CO2, shifting the equilibrium toward the decomposition of MEC, the species formation also occurs in acidic media. Besides, the required time for its formation in these systems matches the time scale needed for the preparation and the consumption of the drink. Although the present study is the first report of MEC existence in food, beverages like beer have been consumed for a long time, suggesting the low probability of its harmful potential. There are, however, little knowledge about its role in flavoring and alcohol absorption in the body.

Em função da potente atividade antifúngica de piperolídeos (1 e 2) isolados do extrato das folhas de Piper malacophyllum foram sintetizados diversos análogos visando estabelecer indicativos sobre relações estrutura-atividade frente à Cladosporium cladosporioides e C. sphaerospermum e frente à forma epimastigota de Trypanosoma cruzi. Outros produtos naturais foram investigados nos extratos e foi constatada a predominância dos butenolídeos 1 e 2 no extrato metanólico de folhas. Foram isolados e caracterizados ainda dois sesquiterpenos [acetato de shizuka-acoradienolila (4) (inédito) e shizuka-acoradienol (5)] e dois compostos alquenilfenílicos [5-[(3E)-oct-3-en-1-il]-1,3- benzodioxol (3) (inédito) e gibbilimbol B (6)]. Esses dois ultimos foram observados como compostos predominantes nos extratos das raízes, caules e frutos. O alquenilbenzeno 3 foi também o produto majoritário no óleo essencial das folhas. Foi desenvolvido e validado um método em cromatografia eletrocinética micelar (MEKC) para a análise dos butenolídeos em extratos de folhas de P. malcophyllum. Os compostos sintéticos foram preparados partir da condensação da 4-metoxifuran-2(5H)-ona (24) com diversos benzaldeídos que resultaram na obtenção de butenolídeos 7-20, 22 e 23. Os piperolídeos naturais e sintéticos mostraram-se como classes promissoras como modelos para antifúngicos em ensaios contra C. sphaerospermum e C. cladosporioides, mas não foram promissores como tripanocidas. / Based on the powerful antifungal activity of piperolides (1 e 2) isolated from Piper malacophyllum leaves extracts, several analogues were synthesized aiming at establishing preliminary structure-activity relationship against Cladosporium cladosporioides, C. sphaerospermum and against epimastigote form of Trypanosoma cruzi. Phytochemical studies carried out on methanolic extract from leaves indicated butenolides 1 e 2 as major compounds in addition to two sesquiterpenes [shizukaacoradienol acetate (4) (new compound) and shizuka-acoradienol (5)] and two alkenylphenyl compounds [5-[(3E)-oct-3-en-1-yl]-1,3-benzodioxole (3) (new compound) and gibbilimbol B (6)]. The last two were detected as major compounds in the root, stem and fruit extracts. The alkenylbenzene 3 was also found as the major product in the essential oil from leaves. One validated method based on micellar electrokinetic chromatography (MEKC) was developed to analyse and validated the amount of butenolides the in P. malcophyllum leaves extracts. The butenolides 7-20, 22 e 23 were prepared by condensation of 4-methoxyfuran-2(5H)-one (24) with several benzaldehydes. The natural and synthetic piperolides were found as a promissing antifungal class against C. sphaerospermum and C. cladosporioides, although no trypanocidal activity was observed.

<p>Liquid Chromatography (LC) and Capillary Electrophoresis (CE) are modern analytical techniques that play very important roles in many areas of modern science such as life science, biotechnology, biomedicine, environmental studies, and development of pharmaceutics. Even though these two techniques have existed and been subjected to studies for several decades, the developments of new separation materials for them are still very important till now in order to meet the different new demands for improvement from other disciplines in science.</p><p>In this doctoral thesis, several novel covalently bonded sulfobetaine type zwitterionic separation materials are synthesized for the application in LC and CE. These materials carry both positively charged quaternary ammonium groups and negatively charged sulfonic groups, which result in a very low net surface charge compared to conventional separation materials with only anionic or cationic functional groups. Consequently, it is possible to employ these materials for separation of different ionic species under mild conditions. The surface properties have also been characterized, mainly by elemental analysis, sorption isotherm, ζ-potential measurements, and spectroscopic methods.</p><p>By using packed zwitterionic columns for liquid chromatography, small inorganic anions or cations, and acidic or basic proteins can be independently and simultaneously separated in a single run using optimal sets of separation conditions. This is a unique property compared to conventional ionic separation material for LC. When fused silica capillaries coated with zwitterionic polymer are used for capillary electrophoresis, good separations can be achieved for solutes as different as inorganic anions, peptides, proteins, and tryptically digested proteins.</p>

<p>This thesis describes the current status and novel aspects of the analysis of the molecules of life, i.e. peptides and proteins, using capillary electrophoresis (CE) coupled to mass spectrometry (MS) via (sheathless) electrospray ionization (ESI). Early reports of sheathless CE-ESI-MS were plagued by limited lifetimes of the electrospray emitter. In this thesis, two new approaches, the Black Dust and the Black Jack methods, utilizing polymer-embedded graphite instead of noble metals are presented. These emitters have shown improved long-term stability and proven excellent for sheathless electrospray operation. Failure of an emitter is often caused by electrochemical reactions occurring at the emitter-liquid interface. The electrochemical properties of the graphite coated emitters were therefore evaluated by classical electrochemical methods, such as cyclic voltammetry and chronoamperometry. The graphite coated emitters showed excellent electrochemical stability and properties compared to noble metal and polymer configurations.</p><p>Analyte-wall interactions have long been known to cause problems in the CE analysis of biomolecules. This can be circumvented by internal modification of the capillary walls. Additionally, it is of outermost importance to have a stable and sufficiently high electroosmotic flow (EOF) to sustain the electrospray, when using a sheathless approach. New monomer and polymer coatings are presented for rapid and high-efficient CE-ESI-MS separations of peptides and proteins.</p><p>Furthermore, the use of CE-ESI coupled to Fourier transform ion cyclotron resonance mass spectrometry (FTICRMS) shows great potential for rapid proteomic probing of human cerebrospinal fluid. The results are comparable with more established techniques, such as liquid chromatography and two-dimensional gel electrophoresis coupled to MS. However, the CE-ESI-FTICRMS analysis has significantly lower sample consumption and faster analysis time compared to the other techniques. The applications and use of CE-ESI-MS is expected to have a bright future with continued growth as current trends of multidimensional hyphenation and microfabricated devices are further developed and explored.</p>

<p>Proteins have such structural features that they may interact with different types of surfaces by all possible forces, i.e., electrostatic, hydrogen bonding, hydrophobic. In this thesis two different types of coatings for fused silica capillaries aimed to eliminate such interactions have been studied. The first is a covalent, electroosmosis-free coating with polyacrylamide (PAA) and the second involves a non-covalent coating with the quaternary ammonium compound N, N-didodecyl –N, N- dimethylammonium bromide (DDAB) with a strong anodic electroosmosis. Optimal conditions regarding efficiency and resolution were established by variations of the composition and ionic strengths of buffers at pH below the isoelectric point of the proteins. To achieve high efficiency and resolution the choice of buffer constituents was extremely important. </p><p>The PAA coating was very stable at neutral and acidic conditions. Ammonium acetate (0.12 M) and ammonium hydroxyacetate (0.15 M) both at pH 4 provided the best separations with plate numbers up to 1 700 000 plate/m that is among the highest reported in the literature. Capillaries coated with DDAB were stable enough to, without recoating, permit consecutive separations of the proteins up to 9 hours (90 injections). High apparent efficiencies (over 1 million plates/m) were achieved with ammonium acetate (0.07 M), ammonium hydroxyacetate (0.08 M) and sodium phosphate (0.1 M) at pH 4. </p><p>Zone broadening was studied by determination of the variance contributions from all main parameters. Significant variances were contributions from longitudinal diffusion, capillary curvature, injection plug, detector time response and detector slit width while other variances, e.g., variances for Joule heat and vertical sedimentation were negligible. The remaining undetermined variance may have its origin in all types of relatively slow interactions including adsorption onto the capillary surfaces and protein-buffer component interactions. The results indicate that the latter is the main cause to zone broadening in protein separations.</p>

This thesis describes the current status and novel aspects of the analysis of the molecules of life, i.e. peptides and proteins, using capillary electrophoresis (CE) coupled to mass spectrometry (MS) via (sheathless) electrospray ionization (ESI). Early reports of sheathless CE-ESI-MS were plagued by limited lifetimes of the electrospray emitter. In this thesis, two new approaches, the Black Dust and the Black Jack methods, utilizing polymer-embedded graphite instead of noble metals are presented. These emitters have shown improved long-term stability and proven excellent for sheathless electrospray operation. Failure of an emitter is often caused by electrochemical reactions occurring at the emitter-liquid interface. The electrochemical properties of the graphite coated emitters were therefore evaluated by classical electrochemical methods, such as cyclic voltammetry and chronoamperometry. The graphite coated emitters showed excellent electrochemical stability and properties compared to noble metal and polymer configurations. Analyte-wall interactions have long been known to cause problems in the CE analysis of biomolecules. This can be circumvented by internal modification of the capillary walls. Additionally, it is of outermost importance to have a stable and sufficiently high electroosmotic flow (EOF) to sustain the electrospray, when using a sheathless approach. New monomer and polymer coatings are presented for rapid and high-efficient CE-ESI-MS separations of peptides and proteins. Furthermore, the use of CE-ESI coupled to Fourier transform ion cyclotron resonance mass spectrometry (FTICRMS) shows great potential for rapid proteomic probing of human cerebrospinal fluid. The results are comparable with more established techniques, such as liquid chromatography and two-dimensional gel electrophoresis coupled to MS. However, the CE-ESI-FTICRMS analysis has significantly lower sample consumption and faster analysis time compared to the other techniques. The applications and use of CE-ESI-MS is expected to have a bright future with continued growth as current trends of multidimensional hyphenation and microfabricated devices are further developed and explored.

Proteins are a highly diversified group of molecules, andfor their study, advanced analytical tools are required. Inparticular, a need for high-throughput techniques has emergedin order to enable the characterization of large sets ofproteins. In this thesis, improved techniques for proteinseparations as well as new tools for the mass spectrometricanalysis of proteins are described. In the work, presented in the first part of the thesis, arefined extract containing proteases from Antarctic krill (Euphausia superba) was separated and characterized bymeans of capillary electrophoresis (CE) and mass spectrometry(MS). Tailored CE separations of the krill extract revealed thepresence of approximately 50 components. In addition, adetailed CE and MS analysis of fractions, containing individualkrill proteases has been carried out. Trypsin-like proteasesfrom krill exhibited a 12-fold and a 60-fold higher digestionefficiency at 37 °C and 2 °C respectively compared todigests performed with bovine trypsin. Furthermore, thecleavage specificity of the trypsin-like proteases wasstudied. In the last part of the thesis, novel concepts forchip-based nanoelectrospray (nanoESI) and matrix-assisted laserdesorption/ionization (MALDI) mass spectrometry are described.First, a micromachined silicon chip with a two-dimensionalmatrix of out-ofplane nanoESI needles for high-throughputanalysis was fabricated. A two-fold improvement insignal-to-noise reproducibility was obtained. Second, achip-based target for MALDI was developed, which featured pairsof elevated 50×50 µm anchors in close proximity. Theanchors were individually addressable with sample solution. Theminiaturized sample preparations at close distance to eachother allowed a simultaneous ionization of a physicallyseparated sample and standard by one single laser pulse. Thisresulted in a twofold reduction of relative mass errors.Moreover, ion suppression of the analyte was significantlyreduced. The effective utilization of the sample resulted in adetection limit of ca 200 zeptomole of angiotensin I. Key words:Proteins, peptides, proteases, Antarctickrill,Euphausia superba, capillary electrophoresis,fluorosurfactants, mass spectrometry, nanoelectrospray, ESI,MALDI, chip, high-throughput, reproducibility, sensitivity andmass accuracy.

No description available.

Liquid Chromatography (LC) and Capillary Electrophoresis (CE) are modern analytical techniques that play very important roles in many areas of modern science such as life science, biotechnology, biomedicine, environmental studies, and development of pharmaceutics. Even though these two techniques have existed and been subjected to studies for several decades, the developments of new separation materials for them are still very important till now in order to meet the different new demands for improvement from other disciplines in science. In this doctoral thesis, several novel covalently bonded sulfobetaine type zwitterionic separation materials are synthesized for the application in LC and CE. These materials carry both positively charged quaternary ammonium groups and negatively charged sulfonic groups, which result in a very low net surface charge compared to conventional separation materials with only anionic or cationic functional groups. Consequently, it is possible to employ these materials for separation of different ionic species under mild conditions. The surface properties have also been characterized, mainly by elemental analysis, sorption isotherm, ζ-potential measurements, and spectroscopic methods. By using packed zwitterionic columns for liquid chromatography, small inorganic anions or cations, and acidic or basic proteins can be independently and simultaneously separated in a single run using optimal sets of separation conditions. This is a unique property compared to conventional ionic separation material for LC. When fused silica capillaries coated with zwitterionic polymer are used for capillary electrophoresis, good separations can be achieved for solutes as different as inorganic anions, peptides, proteins, and tryptically digested proteins.