Made available in DSpace on 2014-10-09T12:27:38Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:04:21Z (GMT). No. of bitstreams: 0 / Resveratrol (3, 4, 5-trihidroxiestilbeno) é um polifenol produzido por uma grande variedade de plantas em resposta ao estresse e encontrado predominantemente em cascas de uvas. Este princípio ativo apresenta vários benefícios à saúde, como a capacidade antioxidante, relacionada à prevenção de diversos tipos de câncer e do envelhecimento precoce da pele. No entanto, apresenta baixa biodisponibilidade quando administrado por via oral, o que torna interessante sua aplicação tópica. O principal objetivo deste trabalho foi a incorporação de resveratrol em hidrogéis poliméricos para obtenção de um sistema de liberação utilizado topicamente contra o desenvolvimento de desordens cutâneas, como o envelhecimento cutâneo e o câncer de pele. As matrizes poliméricas compostas por poli(N-vinil-2-pirrolidona) (PVP), poli(etileno glicol) (PEG) e ágar ou PVP e propano-1,2,3-triol (glicerina) e irradiadas a 20 kGy foram caracterizadas pelos ensaios de fração gel e intumescimento; sua biocompatibilidade preliminar foi avaliada in vitro por meio do ensaio de citotoxicidade utilizando o método de incorporação do vermelho neutro. Devido à baixa solubilidade do resveratrol em água, verificou-se o efeito da adição de 2% de etanol às matrizes. Todas as matrizes estudadas, contendo ou não álcool, apresentaram alto grau de reticulação, capacidade de intumescimento e não apresentaram toxicidade em ensaio preliminar de biocompatibilidade. Os dispositivos foram obtidos pela incorporação de resveratrol nas matrizes poliméricas, realizada de forma direta e indireta, ou seja, antes e após irradiação, respectivamente. Os dispositivos obtidos pelo método direto foram submetidos aos ensaios de fração gel, intumescimento e citotoxicidade e apresentaram-se semelhantes às respectivas matrizes. Os dispositivos contendo 0,05% de resveratrol obtidos pelo método direto e os dispositivos contendo 0,1% de resveratrol obtidos pelo método indireto foram submetidos ao ensaio de cinética de liberação durante 24 h. A quantificação do resveratrol liberado foi realizada por cromatografia líquida de alta eficiência (HPLC). Apenas os dispositivos obtidos pelo método indireto apresentaram capacidade de liberar o resveratrol incorporado, que apresentou capacidade antioxidante após liberação. / Dissertacao (Mestrado) / IPEN/D / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

Doctor of Philosophy / Department of Chemistry / Christopher T. Culbertson / High-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) are two well-established analytical separation techniques that are continuously being adapted for performing distinctive separations and analyses of multitudes of complex and/or unique samples. Since their introduction, these techniques have been pivotal in the discovery, analysis, and understanding of a variety of samples and still prove to be key analytical tools for biological investigation. Using these techniques, one can obtain a wide-range of valuable sample information from the hydrophobicity and molecular weights to size and charge distributions. Furthermore, these techniques allow for sample analysis, purification, and collection for additional sample analysis, such as mass spectrometry analysis. My doctoral dissertation encompasses the full scope of these two techniques and novel approaches for the investigation of distinct, relevant samples. Described herein is the fabrication of glass microfluidic devices used for CE and their diversity for numerous investigations. Chapter 2 shows that the resolution of the photomasks used in microchip fabrication does not alter the separation efficiency of the devices, as the separations remain diffusion-limited. Using an in-house built capillary electrophoresis system, wheat proteins were separated more than 25% faster than previously reported in literature, and the electropherograms used for sample varietal identification. The fabrication of a robust, portable CE system capable of performing biological analysis in microgravity and hypergravity environments is also discussed. The need for and features necessary to achieve a reliable, robust, automated system is further described in Chapter 4. Isolation and analysis of the pea aphid (Acyrthosiphon pisum) salivary secretions was completed for the first time using HPLC. By altering the aphid environment and the sample treatment parameters, sample concentrations were increased above the limit of detection. Coupled with mass spectrometry, identification of pea aphid salivary proteins such as exopeptidase, angiotensin converting enzyme, and Buchnera proteins has been achieved. Finally, a simplified contact conductivity detection system for the detection of jurkat cells was developed that surpasses current, complex optical systems. The experiments described in this dissertation demonstrate novel approaches for the preparation, separation, analysis, and identification of a wide variety of common, and uncommon, samples.

Thesis (PhD)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Complex polymers are defined by their distributive properties with respect to molecular weight, chemical composition, functionality and molecular topology. As a result, polymer properties are very frequently determined not only by one of these entities but by the correlation of two or more distributions. Aliphatic polyesters are industrially implemented in high performance coatings, paints and varnishes. However, it is typically difficult to correlate the resulting properties with the synthesis parameters as these polymers vary in reactivity and application properties. Copolyester synthesis by direct polyesterification is often assumed to produce randomized products due to the mechanisms involved in stepwise polymerization. The formation of cyclic products by intramolecular reactions of hydroxyl (OH) and carboxylic (COOH) functional groups, sidereactions such as transesterification, alcoholysis, and ester-ester interchange allow even further randomization, enabling a highly complex system. Therefore, in addition to molecular weight distribution, polyesters exhibit chemical composition, functionality type as well as branching distributions, classifying them as complex polymeric systems. The different methods of polymer chromatography in combination with sophisticated spectrometry techniques are useful tools for enabling the full description of the molecular heterogeneity of these complex polyesters. The present study entails method development of different modes of chromatography and mass spectrometry along with their combination, to facilitate the analysis of the various distributions of two model polyester systems, phthalic and maleic anhydride, respectively, in combination with propylene glycol. Gradient HPLC analysis enabled an oligomeric separation based on chemical composition of the respective anhydride/propylene glycol samples. Its off-line coupling to MALDITOF MS and ESI-QTOF MS revealed the presence of several distributions of varying endgroup functionality type and molecular weight distributions at different intervals throughout the polymerization. In addition, online gradient HPLC x size exclusion chromatography (2D-LC) was conducted to obtain the dual chemical composition-molecular weight (CCD-MWD) distribution. The combination of the different coupling techniques provided the opportunity to a more in-depth analysis of the structure-property relationships. / AFRIKAANSE OPSOMMING: Komplekse polimere word gedefinieer deur hul verdelings eienskappe ten opsigte van molekulêre massa, chemiese samestelling, funksionaliteit en molekulêre topologie. Gevolglik, word hul eienskappe dikwels bepaal deur nie net een van hierdie entiteite nie, maar ‘n korrelasie van twee of meer verdelings. Alifatiese poliësters word industrieel geϊmplimenteer in hoë werkverrigting bestrykings, verwe en politoere, dog is dit tipies moeilik om die uiteinde eienskappe met die verwante sintese parameters te korrelleer, aangesien die polimere varieer in reaktiviteit en toepassingseienskappe. Ko-poliëster sintese vanaf direkte poliësterivering word dikwels aanvaar om willekeurige produkte op te lewer as gevolg van die meganismes wat betrokke is tydens trapgroei polimerisasie. Die produsering van sikliese produkte weens intra-molekulêre reaksies van hidroksiel(OH) en karboksiel (COOH) verwante funksionele groepe, newereaksies soos transverestering, alkoholise en ester-ester verwisseling, het verdere ewekansigmaking tot gevolg wat ‘n hoog gekomplekseerde sisteem tot gevolg het. Benewens die molekulere massa verdeling, vertoon poliësters dus chemiese samestelling, funksionaliteit tipe so wel as vertakkings verdeling wat hul as komplekse polimeer sisteme klassifiseer. Die verskillende metodes van polimeer chromatografie in kombinasie met gesofistikeerde spektrometriese tegnieke dien as nuttige bronne vir die volledige beskrywing wat betref die molekulêre heterogeniteit van komplekse poliesters. Die huidige studie stel metode ontwikkeling van verskillende modus van chromatografie, massa spektrometrie sowel as hul aaneenvoeging bekend, om die die verskillende verdelings van twee model poliester sisteme, ftaal- en maleϊensuuranhidried onderskeidelik in kombinasie met propileenglikol, suksesvol te analiseer. Gradiënt hoë-druk vloeistof chromatografie (HPLC) analise het ‘n oligomeriese skeiding, gebaseer op die chemiese samestelling van die verskeie anhidried /propileenglikol monsters, opgelewer. Die nie-gekoppelde skakeling met matriks-assisteerdelaser/ desorpsie-ionisasie tyd-van-vlug (MALDI-TOF) en elektron-sproei-ionisasie kwadrupool-tyd-van-vlug (ESI-QTOF) massa spektrometrie het die teenwoordigheid van verskeie verdelings van varieërende endgroep funksionaliteit tipe en molekulêre verdelings by verskillende intervalle tydens die polimerisasie aan die lig gebring. Gekoppelde skakeling van gradient HPLC en grootte uitsluitings chromatografie is ook uitgevoer om die tweedelige chemiese samestelling-molekulere massa verdeling te bepaal. Aaneenvoeging van die verskeie skakelings tegnieke het die geleentheid gebied om ‘n deeglike studie van die struktuureienskappe verhoudinge suksesvol uit te voer.

Thesis (MSc)--Stellenbosch University, 2000. / ENGLISH ABSTRACT: The phenolic content of wine is responsible for determining characteristics such as the organoleptic qualities, colour stability, ageing properties and health-beneficial effects associated with wine. The aim ofthis study was to investigate the possibilities offered by capillary electrophoresis (CE) as an alternative separation technique to high performance liquid chromatography (HPLC) for the analysis of polyphenols in wine. The complexity of wine samples was the cause that neither technique was capable of a satisfactory singlestep analysis of wine. Suitable sample preparation techniques such as Sephadex- and Sep- Pak fractionation and ether extraction of wine polyphenols were investigated. These techniques did not, however, prove to be universal. A novel form of sample preparation namely a process analogous to lyophylization used to separate wine volatiles from nonvolatiles was introduced. The versatility of CE was further investigated in an attempt to eliminate the need for sample preparation. The use of polyvinylalcohol (PVA) coated capillaries, micellar electrokinetic chromatography (MEKC) and capillary gel electrophoresis (CGE) were investigated in this regard. Although none of these techniques could offer conclusive results, useful applications were forthcoming and routes for further investigation were outlined. Liquid chromatography coupled to electro spray ionisation mass spectroscopy (LC-ESI-MS) and capillary electrophoresis coupled to electro spray ionisation mass spectroscopy (CE-ESI-MS) were compared for the analysis of polyphenols in wine. While the latter technique could not produce sufficient separation compared to the former, future development ofCE-ESI-MS should make it a powerful technique for these analyses. / AFRIKAANSE OPSOMMING: Die fenoliese komponente in wyn speel 'n bepalende rol by eienskappe soos die organoleptiese karakter, kleur stabiliteit, verouderingspotensiaal en gesondheids-voordele wat met wyn geassosieër word. Die doel van hierdie projek was om ondersoek in te stel na die potensiaal wat kapillêre elektroforese (CE, "capillary electrophoresis") as 'n alternatiewe skeidingstegniek teenoor hoë druk vloeistof chromatografie (HDVC) vir die analise van die polifenole in wyn bied. Die kompleksiteit van wyn monsters is van so 'n aard dat 'n bevredigend enkelstap analise met geeneen van die tegnieke moontlik is nie. Gepaste monster-voorbereidingstappe soos Sephadex- en Sep-Pak fraksionering asook eter ekstraksie van die polifenole in wyn is ondersoek. Geeneen van die tegnieke was egter universeel toepaslik nie. 'n Nuwe metode van monster-voorbereiding, naamlik 'n proses analoog aan liofilisasie wat gebruik word om die wyn te skei in vlugtige en nievlugtige komponente is gedemonstreer. Die veelsydigheid van CE was gevolglik ondersoek in 'n poging om monstervoorbereiding uit te skakel. Die gebruik van polyvinielalkohol-(pVA) bedekte kapillêre, missellêre elektrokinetiese chromatografie (MEKC) en kapillêre gel elektroforese (CGE, "capillary gel electrophoresis) is in hierdie verband ondersoek. Alhoewel geeneen van hierdie tegnieke onweerlegbare resultate gelewer het nie, het bruikbare toepassings hieruit voortgespruit en is die grondslag vir verdere navorsing gelê. Vloeistof chromatografie gekoppel aan eIektrosproei ionisasie massaspektroskopie (LCESI- MS) en kapillêre elektroforese gekoppel aan elektrosproei ionisasie massaspektroskopie (CE-ESI-MS) is vergelyk vir die analise van polifenole in wyn. Alhoewel laasgenoemde tegniek onvoldoende skeiding lewer vergeleke met eersgenoemde, behoort toekomstige ontwikkelinge op die gebied van CE-ESI-MS dit 'n kragtige tegniek vir die analise van hierdie monsters te maak.

Thesis (MSc)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: In this study, the analysis of ammo acids usmg High Performance Liquid Chromatography (HPLC) with pre-column derivatisation was optimised. The derivatisation reagents include o-phthaldialdehyde (OPA), 9- fluorenylmethylchloroformate (FMOC) and iodoacetic acid (IDA). Detection was performed using UV and fluorescence in series. The developed method was utilised for the analysis and quantitation of amino acids in eighteen wines. The application of chemometric data evaluation was initiated. / AFRIKAANSE OPSOMMING: Hierdie ondersoek behels die optimisering van die aminosuuranalise deur gebruik te maak van Hoë Druk Vloeistof Chromatografie (HDVC) in kombinasie met pre-kolom derivatisering. Die derivativatiserings reagense sluit in o-phthaldialdehied (OPA), 9- fluorenielmetielchloroformaat (FMOC) en jodoasynsuur (IDA). Deteksie is gedoen deur gebruik gemaak van 'n ultraviolet (UV) en 'n fluorosensie detektor in serie. Die metode sodoende ontwikkel is gebruik vir die analise en kwantifisering van aminosure in agtien wyne. Die toepassing van chemometriese data evaluasie is ook geïnisieer.

Thesis (MSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: See item for full text / AFRIKAANSE OPSOMMING: Sien item vir volteks

published_or_final_version / abstract / Chemistry / Doctoral / Doctor of Philosophy

The Supercritical Fluid Extraction (SFE) technique was used to fractionate Suwannee River reference fulvic acid (FA). The fractions were characterized by gas chromatography (GC) and reversed-phase high performance liquid chromatography (RP-HPLC). A SFE fractionation method was developed using stepwise gradient of supercritical CO₂ and methanol. Three FA fractions were separated. The average mass recovery was 102% with the coefficient of variation of 2.8%. The fractionation dynamics and the difference in the ratios of UV absorption to fluorescence emission indicate the real fractionation of the FA. The HPLC chromatographic peak patterns and the spectra of the corresponding peaks were almost indistinguishable. The overall results of this research support the argument that FA exhibits polymer-like molecular structure.

High performance liquid chromatography was used to measure ribonucleoside triphosphates in microbial samples. Anion exchange columns in a radial compression module were used to separate and quantify purine and pyrimidine ribonucleotides. Endogenous ribonucleoside triphosphates were extracted from Escherichia coli and pseudomonas aeruginosa using three different solvents, namely trifluorocetic acid (TFA; 0.5M), trichloroacetic acid (TCA; 6 per cent w/v) and formic acid (1.0M) Extracts were assayed for uridine 5'-triphosphate (ATP), and guanosine 5'-triphosphate (GTP) by using anion exchange radial compression high performance (pressure) liquid chromatography. The three extraction produres were compared for yield of triphosphates. E. coli, the TFA extraction procedure was more sensitive and reliable than TCA and formic acid extraction procedures, but , in P. aeruginosa, the best yields of ATP and GTP were obrained following extraction with TFA. Yields of UTP and CTP increased when extraction was performed in TCA. These data illustrate that different extraction produres produce different measures for different triphosphates, a point often overlooked.

High Performance Liquid Chromatography (HPLC) was used to investigate the utility of this technique for the analytical and preparative separation of components of aquatic fulvic acids (FA). Three modes of HPLC namely adsorption, anion exchange and reversed phase were evaluated. Aquatic fulvic acids were either extracted from surface water and sediment samples collected from the Southwest of the U.S., or were provided in a high purity form from the USGS. On the adsorption mode, a major fraction of aquatic fulvic acid was isolated on a semipreparative scale and subjected to Carbon-13 NMR and FAB Mass Spectroscopy. Results indicated that (1) The analyzed fraction of fulvic acid contains more aliphatic than aromatic moieties; (2) Methoxy, carboxylic acids, and esters are well-defined moieties of the macromolecule; (3) Phenolic components of the macromolecules were not detected in the Carbon-13 NMR spectrum possibly because of the presence of stable free radicals. Results of the anion exchange mode have shown that at least three types of acidic functionalities in aquatic fulvic acid can be separated. Results also indicated that aquatic fulvic acid can be progressively fractionated by using subsequent modes of HPLC. Results of reversed phase mode have shown that (1) The fractionation of aquatic fulvic acid by RP-HPLC is essentially controlled by the polarity and/or pH of the carrier solvent system; (2) Under different RP-HPLC conditions aquatic fulvic acid from several locations are fractionated into the same major components; (3) Fulvic acid extracted from water and sediment from the same site are more similar than those extracted from different sites; (4) Cationic and anionic ion pair reagents indicated the presence of amphoteric compounds within the polymeric structure of fulvic acid. Each mode of HPLC provided a characteristic profile of fulvic acid. The results of this research provided basic information on the behavior of aquatic fulvic acids under three modes of HPLC. Such informations are prerequisite for further investigation by spectroscopic methods.