Cancer cells have been known for some time to have very different metabolismas compared to that of normal non proliferating cells. As metabolism is involvedin almost every aspect of cell function, there has been a recent resurgence ofinterest in inhibiting cancer metabolism as a therapeutic strategy. Inhibitors thatspecifically target altered metabolic components in cancer cells are being developedas antiproliferative agents. However, many such inhibitors have not progressedinto the clinic due to limited efficacy either in vitro or in vivo. In this study weexplore the hypothesis that this is often due to the robustness of the metabolicnetwork and the differences between individual cancer cell lines in their metaboliccharacteristics. We take a systems biology approach. We investigate the cellular bioenergetic profiles of a panel of five non-small celllung cancer cell lines before and after treatment with a novel inhibitor of theglutaminase-1 (GLS1) enzyme. Additionally, we explore the effects of this inhibitoron intracellular metabolism of these cell lines as well as on the uptake and secretionof glucose, lactate and amino acids. To be able to do the latter robustly, wehad to modify the experimental assay considerably from procedures that seemto be standard in the literature; using these earlier procedures the metabolicenvironment of the cells was highly variable, leading to misleading results onthe metabolic effects of the inhibitor. We reduced cell density, altered mediumvolume and changed the time-window of the assay. This led to the cells growingexponentially, appearing indifferent to the few remaining changes. In this newassay, the metabolic effects of the glutaminase inhibitor became robust. One of the most significant results of this study is the metabolic heterogeneitydisplayed across the cell line panel under basal conditions. Differences in themetabolic functioning of the cell lines were observed in terms of both theirbioenergetic and metabolic profile. The amount of respiration attributed tooxidative phosphorylation differed between cell lines and respiratory capacity wasattenuated in most cells. However, the rate of glycolysis was similar betweencell lines in this assay. These results suggest that the Warburg effect arisesthrough a greater diversity of mechanisms than traditionally assumed, involvingvarious combinations of changes in the expression of glycolytic and mitochondrialmetabolic enzymes. The effects of GLS1 inhibition on cellular bioenergetics and metabolism alsodiffered between cell lines, even between resistant cell lines, indicating that theremay also be a diversity of resistance mechanisms. The metabolomic response ofcell lines to treatment suggests potential resistance mechanisms through metabolicadaptation or through the prior differences in the metabolic function of resistantcell lines. Part of the metabolome response to GLS1 inhibition was quite specificfor sensitive cells, with high concentrations of IMP as the strongest marker. Our results suggest that the metabolome is a significant player in what determinesthe response of cells to metabolic inhibitors, that its responses differ between cancercells, that responses are not beyond systems understanding, and that thereforethe metabolome should be taken into account in the design of and therapy withanti-cancer drugs.

<p>Liquid chromatography coupling with mass spectrometry (LC/MS) plays an important role in pharmaceutical characterization because of its ability to separate, identify, and quantify individual compounds from the mixture. Polymer brush layer bonded to the silica surface is designed as a novel stationary phase to improve the LC resolution and MS compatibility. The polymer thickness can be controlled to shield the analyte from interacting with the active silanol on the surface and reduce peak tailing. The functional group of the polymer can be changed to tune the selectivity in different separation modes. </p><p> </p><p>Two projects on LC/MS method development for biomolecule characterization using polymer-shell column are discussed in this work. In the first project, a polymer-shell column is used for disulfide bonds and free thiol subspecies identification, which is a major type of structural heterogeneities in IgG1. Compared with commercial columns, the polymer-shell column is able to resolve the free thiol variants without the presence of trifluoroacetic acid and greatly improve the MS signal. In the second project, a polymer-shell column is used for characterizing the drug-loading profile for antibody-drug-conjugates (ADC) via online LC/MS. The separation employs a mobile phase of 50 mM ammonium acetate to keep the ADC intact, and a gradient of water/isopropanol for ADC elution. MS data show that all ADC species remained intact and native on the column. Positional isomers can be separated and identified with the new method as well. Furthermore, to understand the surface chemistry and protein separation behavior quantitatively, a chromatographic simulation study is performed. The result shows that protein separation in RPLC can be described by a bi-Langmuir adsorption isotherm with mixed-mode retention of strong and weak sites. Smaller fractions and lower equilibrium constant of the strong site, which is the active silanol, give less tailing for protein separation.</p>

No description available.

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.

Vascular risk factors inducing a state of chronic cerebral hypoperfusion and metabolic stress are thought to influence the onset and progression of Alzheimer’s disease (AD). To investigate the complex molecular changes underpinning cellular adaptation to metabolic stress, the first aim of this thesis was to define the proteomic response of the SH-SY5Y human neuroblastoma cell line after exposure to the metabolic challenge of oxygen glucose deprivation (OGD). 958 proteins across multiple subcellular compartments were detected and quantified by label-free liquid chromatography mass spectrometry (LC-MS). The levels of 130 proteins were significantly increased (P<0.01) after OGD and the levels of 63 proteins were significantly decreased (P<0.01) while expression of the majority of proteins (765) was not altered. Ingenuity Pathway Analysis identified novel protein-protein interactomes involved with mitochondrial energy production, protein folding, and protein degradation, indicative of coherent and integrated proteomic responses to the metabolic challenge. Approximately one third (61) of the differentially expressed proteins were associated with the endoplasmic reticulum and mitochondria. Electron microscopic analysis of these subcellular structures showed morphologic changes consistent with the identified proteomic alterations. Pertinent to AD research, amyloid binding alcohol dehydrogenase (ABAD) was found to be significantly increased in response to OGD. ABAD is emerging as a key player in mitochondrial dysfunction in AD, yet full understanding of the biochemical pathways in which this protein is involved remain elusive. Using immunoprecipitation coupled to LC-MS (IP-MS), the second aim of the thesis was to characterise the ABAD protein interactome in SH-SY5Y cells and its response to metabolic stress. 67 proteins were identified as potential ABAD interactors under control conditions, and 69 proteins were identified as potential ABAD interactors under OGD conditions. The Database for Annotation, Visualization and Integrated Discovery (DAVID) was used to determine the subcellular locations and biological functions of the ABAD interacting proteins in control and OGD conditions. DAVID identified the nuclei and mitochondria to contain the greatest number of changes in ABAD interacting proteins following OGD. “Glucose Metabolic Process” (GO:0006006) was the top functional cluster for ABAD interacting proteins in both control and OGD conditions. Independent immunoprecipitations, western blotting, immunohistochemistry and electron microscopy were used to validate specific protein interactions. OGD was found to initiate a novel interaction between ABAD and glucose-regulated protein 75 (GRP75), a finding confirmed in human AD tissue. GRP75 is a mitochondrial protein and marker of the mitochondrial associated membrane (MAM), a specialised region between the mitochondria and the ER. The MAM is known to be enriched with presenilin proteins, involved in the proteolytic cleavage of amyloid precursor protein (APP). These data were used to generate an “ABAD-GRP75-MAM hypothesis of mitochondrial dysfunction in AD”, which might provide a novel link between chronic metabolic stress, ABAD, mitochondrial dysfunction and the onset / progression of AD. The third aim of the thesis was to test this novel hypothesis. Western blotting revealed APP to be significantly decreased following OGD, concurrent with an increase in ABAD protein levels. Over-expression of ABAD protein in SH-SY5Y cells was used to test whether the increased levels of ABAD following OGD were the driving force behind APP down-regulation. ABAD over-expression in SH-SY5Y cells was found to have no detectable effect on APP. Conversely, electron microscopy revealed a dynamic response of the MAM to metabolic stress. This result, along with the interaction of ABAD with GRP75, and the enrichment of presenilins at the MAM, suggests that this specialised membrane region may have an important role to play in AD.

Thesis (MSc)--Stellenbosch University, 2011. / ENGLISH ABSTRACT: Block copolymers are very interesting materials but they are quite complex. During polymer synthesis only a certain amount of control can be enforced. As copolymers are made up of two or more different homopolymer segments, and therefore have different end group possibilities, varying block lengths and block sequences, they have complex structures and are therefore difficult to analyse. Different techniques exist by which polymers can be analysed to determine the aforementioned distributions. In order to achieve a complete characterisation of a polymer structure, it is best to first use a separation technique to fractionate the polymer into more homogeneous fractions, and then use identification techniques to analyse these fractions. Polystyrene-block-poly(ethylene oxide) (PS-b-PEO) copolymers were investigated using liquid chromatography at the critical conditions (LCCC) of the copolymers' corresponding homopolymers, two-dimensional liquid chromatography (2D-LC) and FTIR. The block copolymers were analysed using the established LCCC of PS but it was found that even though separation of PS homopolymer and copolymer was obtained, PS blocks of the copolymers contributed to some extent to the retention of the PEO blocks. Some of the block copolymer samples were fractionated at the established critical conditions of PS. These fractions were qualitatively and quantitatively analysed using FTIR spectroscopy. The settings for the 2D-LC analysis were established, using LCCC of PS as the first dimension and as the second dimension SEC, using DMF as eluent. DMF was a suitable solvent to be used for the second dimension because PS, PEO and PS-b-PEO exhibited good solubility in this solvent. THF did not dissolve the block copolymers completely. The same solvent system as used for LCCC of PS was used for LCCC of PEO, but the critical conditions correspond to a different solvent composition. The block copolymers were analysed using the established LCCC of PEO but it was found that even though separation of PEO homopolymer and copolymer was obtained, the PEO blocks of the copolymers contributed to some extent to the retention of the PS blocks. Some of the block copolymer samples were fractionated at the established critical conditions of PEO. These fractions were qualitatively and quantitatively analysed using FTIR spectroscopy. The settings for the 2D-LC analysis were established, using LCCC of PEO as the first dimension and as the second dimension SEC using DMF as eluent was used. Lastly, qualitative and quantitative analyses of the block copolymers were carried out using FTIR spectroscopy. / AFRIKAANSE OPSOMMING: Alhoewel blokkopolimere baie interessante verbindings is, is hulle redelik ingewikkeld. Gedurende die kopolimerisasiereaksie kan daar net 'n sekere mate van kontrole behaal word. Aangesien kopolimere uit twee of meer homopolimeersegmente, met verskillende end-groep moontlikhede, bloklengtes en blokvolgordes bestaan, is dit baie moeilik om hierdie verbindings te analiseer. Verskillende tegnieke kan gebruik word vir die analise van polimere en die bepaling van bogenoemde verspreidings. Ten einde 'n polimeerstruktuur volledig te karakteriseer is die beste manier om eers 'n skeidingstegniek te gebruik om die polimeer in meer homogene fraksies te fraksioneer en dan daarna hierdie fraksies te analiseer. Polistireen-blok-poli(etileenoksied) (PS-b-PEO) kopolimere is ondersoek deur gebruik te maak van vloeistofchromatografie by kritiese kondisies (LCCC) van die kopolimeer se ooreenkomstige homopolimere; twee-dimensionele vloeistofchromatografie (2D-LC) en FTIR. Die blokkopolimere is gekarakteriseer deur gebuik te maak van bevestigde LCCC van PS. Daar is egter gevind dat alhoewel skeiding van die PS homopolimeer en die kopolimeer behaal is, PS blokke van die kopolimere in 'n mate bygedra het tot die retensie van die PEO blokke. Sommige van die blok-kopolimeermonsters is gefraksioneer by die bepaalde kritiese kondisies van PS. Hierdie fraksies is kwalitatief en kwantitatief geanaliseer deur gebruik te maak van FTIR spektroskopie. Die stellings vir die 2D-LC analise is bepaal deur gebruik te maak van LCCC van PS as die eerste dimensie en SEC as die tweede dimensie, met DMF as elueermiddel. DMF was 'n geskikte oplosmiddel vir die tweede dimensie aangesien PS, PEO en PS-b-PEO goed oplosbaar is daarin. Die blokkopolimere was nie volledig oplosbaar in THF nie. Dieselfde oplosmiddelsisteem soos gebruik vir die LCCC van PS is gebruik vir die LCCC van PEO, maar die kritiese kondisies stem ooreen met 'n ander oplosmiddelsamestelling. Die blokkopolimere is geanaliseer deur gebruik te maak van die bevestigde LCCC van PEO, maar daar is bevind dat alhoewel skeiding van die PEO homopolimeer en kopolimeer behaal is, die PEO blokke van die kopolimere in 'n mate bygedra het tot die retensie van die PS blokke. Sommige van die blokkopolimeermonsters is gefraksioneer by die bevestigde kritiese kondisies van PEO. Hierdie fraksies is kwalitatief en kwantitatief geanaliseer deur gebruik te maak van FTIR spektroskopie. Die stellings vir die 2D-LC analise is bepaal deur gebruik te maak van LCCC van PEO as die eerste dimensie en SEC as die tweede dimensie, met DMF as elueermiddel. Laastens is kwalitatiewe en kwanitatiewe analises van die blokkopolimere m.b.v. FTIR spektroskopie uitgevoer.

Thesis (PhD)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: The wine industry is an important sector of agriculture and wine analysis forms the basis of assessing compliance of its commodities with regulatory standards and research in this field. Liquid chromatography (LC) is extensively used for the determination of a wide range of nonvolatile wine components, but conventional detectors impose performance limitations on the technique that prevents its application to sophisticated analytical problems. In particular, conventional detectors for LC often lack the sensitivity and specificity for the determination of many wine compounds, especially trace level analytes, and furthermore, do not possess spectral capabilities for compound identification or structure elucidation. The hyphenation of mass spectrometry (MS) to LC has led to the introduction of a range of detectors that confers high levels of sensitivity and selectivity to the technique. In addition, a wide variety of MS architectures are available that are inherently suited for targeted analysis or structure elucidation studies. In this dissertation, the potential benefits of liquid chromatography – tandem quadrupole mass spectrometry (LC-MS/MS) to solve analytical problems relevant to the wine industry are explored. LC-MS/MS is a particularly versatile analytical technique because both mass analysers can be operated in full-spectrum mode or selected-ion monitoring, which, together with optional fragmentation, gives rise to four modes of operation that may be used for highly specific and sensitive targeted analysis or spectral investigations. In multiple reaction monitoring (MRM) mode, both analysers are set at single ion frequencies specific for the compound under investigation and one or more of its product fragments, respectively. MRM mode is ideally suited for trace level analysis in complex mixtures, even in cases where the target components are not resolved from interferences. In this study, MRM detection was used to solve challenges relevant to the wine industry for the selective quantitation of target analytes that could not be analysed by conventional LC methods. The application of this approach for the analysis of natamycin, ethyl carbamate (EC) and 3-alkyl-2- methoxypyrazines (MPs) in wine is demonstrated. Natamycin is an antimicrobial preservative that is not permitted in wine in the European Union. A rapid and sensitive method for the determination of natamycin was developed, and has been used since 2009 to regulate this vitally important sector of the South African wine export industry. EC is a natural carcinogen that occurs at trace level amounts in alcoholic products. It also has the potential to accumulate in wines and can occur in very high concentrations in some fruit brandies. The determination of EC is complicated by its physicochemical properties, and available analytical methods suffer from drawbacks such as the requirement for elaborate extraction procedures and high solvent consumption. A novel method for the determination of EC in wines, fortified wines and spirits is described and it was applied to perform an audit of the South African industry as well as to investigate factors responsible for its accumulation in alcoholic beverages. This work forms an integral part of the food safety mandate of the State and it ensures that export products comply with international norms for trade. MPs are ultra-trace-level aroma compounds that contribute to the varietal character of Sauvignon blanc wines. Their analytical determination is challenging due to their low levels of occurrence. The loading capacity of LC combined with the sensitivity and resolving power of MS was exploited to analyse concentrated extracts, in order to achieve very low limits of detection. The performance of the LC-MS/MS method enabled the quantitation of these compounds at their natural levels of occurrence, including the first quantitation and spectral confirmation of 3- ethyl-2-methoxypyrazine in wine. Extensive data pertaining to South African Sauvignon blanc wines are reported and statistical analysis is performed, reporting the correlation of variables such as vintage and origin as well as wine parameters such as malic acid with wine MPs. Furthermore, the application of LC-MS/MS for structural elucidation and screening of target classes of analytes was demonstrated for the analysis of red wine anthocyanins. The anthocyanidin-glycosides are responsible for the colour of red grapes and wine, contribute to the sensory properties of wine, and are also of interest due to their beneficial biological properties. Their determination is complicated by their large numbers and structural diversity, further exacerbated by diverse reactions during wine ageing as well as the lack of reference standards for most members of this class of compounds. Tandem MS in scan mode was used for the highly selective detection of glycosylated anthocyanins and derivatives, exploiting the predictable elimination of the sugar moiety in neutral loss mode. Concurrent survey scan experiments were used to unambiguously identify neutral loss detected compounds. The method therefore follows a simplified and structured approach for unambiguous peak identification based on elution order and mass spectral information to impart a high level of certainty in compound identification. In summary, the work presented in this dissertation demonstrates that LC-MS/MS is a versatile and powerful analytical approach for the analysis of diverse compounds of relevance to the wine industry. The sensitivity and specificity of MRM mode, and the selectivity and spectral capabilities of neutral loss and survey scan modes of MS/MS detection, is amply demonstrated by the applications presented in the dissertation. / AFRIKAANSE OPSOMMING: Die wynbedryf is ‘n belangrike komponent van landbou en wyn-analise vorm ‘n integrale deel van gehalteversekering ten opsigte van toepaslike wetlike standaarde. Wyn-analise is ook belangrik in navorsing oor die samestelling van wyn. Vloeistofchromatografie word dikwels aangewend vir die bepaling van ‘n wye verskeidenheid nie-vlugtige wynkomponente, maar konvensionele detektors plaas beperkinge op die aanwending van die tegniek tot gesofistikeerde analitiese toepassings. Meer spesifiek, konvensionele detektors vir vloeistofchromatografie beskik nie oor die sensitiwiteit en selektiwiteit vir die bepaling van baie wynkomponente nie, veral in die geval van spoorvlakanalise, en beskik boonop ook nie oor spektrale vermoëns vir identifikasie van komponente en struktuurbepaling nie. Die koppeling van vloeistofchromatografie met massaspektrometrie het ‘n reeks detektors tot die tegniek toegevoeg wat hoë vlakke van sensitiwiteit en selektiwiteit bied. Verder bied die verskeidenheid van massaspektrometrie-konfigurasies ook instrumente wat inherent geskik is vir geteikende analise of struktuurbepaling, afhangende van die doel van die ondersoek. In hierdie dissertasie word die voordele ondersoek wat verbonde is aan die aanwending van vloeistofchromatografie – tandem kwadrupool massaspektrometrie om relevante analitiese vraagstukke in die wynbedryf op te los. Hiedie tegniek is besonder toepaslik aangesien beide massa-analiseerders in geselekteerde-ioon modus of in volle skandering gebruik kan word. Tesame met opsionele fragmentasie, gee hierdie uitleg aanleiding tot vier funksionaliteite wat vir hoogs sensitiewe geteikende analise of spektrale onledings gebruik kan word. Eerstens word beide massa analiseerders vir enkel-ioon frekwensies opgestel, spesifiek tot die teikenkomponent en een of meer van sy produkfragmente, wat verkry word deur komponentspesifieke fragmentasie. Hierdie modus is by uitstek geskik vir spoorvlakontleding van komplekse monsters, selfs wanneer die teikenkomponente nie chromatografies van die matriks geskei is nie. In hierdie studie is die tegniek aangewend vir die hoogs sensitiewe bepaling van spoorvlak komponente wat nie met konvensionele detektors gemeet kon word nie. Die aanwending van hierdie tegniek word gedemonstreer vir die spoorvlakbepaling van natamycin, etielkarbamaat en 3-alkiel-2-metoksiepierasiene in wyn. Natamycin is ‘n antimikrobiese preserveermiddel wat ontoelaatbaar is in wyn in die Europese Unie. ‘n Vinnige en sensitiewe metode vir die bepaling van natamycin is ontwikkel, en word reeds sedert 2009 aangewend om hierdie uiters belangrike sektor van die Suid-Afrikaanse wyn uitvoerbedryf te reguleer. Etielkarbamaat is ‘n karsinogeen wat natuurlik voorkom in spoorhoeveelhede in alkoholiese produkte. Dit kan ook onder sekere omstandighede akkumuleer in wyn en in hoë konsentrasies voorkom in vrugtebrandewyne. Die bepaling van etielkarbamaat word bemoeilik deur sy chemiese eienskappe, en gevolglik word analitiese metodes gekenmerk deur uitgebreide, arbeidsintensiewe monstervoorbereiding en die gebruik van groot hoeveelhede, meestal giftige, oplosmiddels. ‘n Nuwe metode vir die bepaling van etielkarbamaat in wyn, gefortifiseerde wyn en spiritualië word beskryf en word aangewend om die faktore vir vorming daarvan te ondersoek. Die metode word aangewend om die Suid-Afrikaanse bedryf te ouditeer in terme van die voedselveiligheid mandaat van die Staat, en om te verseker dat uitvoere voldoen aan standaarde vir internasionale handel. Metoksiepierasiene is vlugtige, ultraspoorvlak wynaromakomponente wat verantwoordelik is vir die kenmerkede kultivarkarakter van Sauvignon blanc wyne. Hul analitiese bepaling word bemoeilik deur hulle lae konsentrasies in wyn. Die ladingskapasiteit van vloeistofchromatografie tesame met die sensitiwiteit en selektiwiteit van massaspektrometrie was benut om hoogs gekonsentreerde ekstrakte te ontleed. Baie hoë vlakke van sensitiwiteit word sodoende verkry. Die verrigting van die metode was voldoende om hierdie komponente teen hulle natuurlike konsentrasies te kwantifiseer, insluitende die eerste kwantifisering en spektrale bevestiging van 3-etiel-2-metoksiepierasien. Omvattende data van die vlakke van hierdie komponente in Suid- Afrikaanse Sauvignon blanc wyne word getoon en statistiese ontleding is gedoen om korrelasies tussen veranderlikes soos oorsprong en oesjaar sowel as basiese wyn veranderlikes soos byvoorbeeld appelsuur, met metoksiepierasienvlakke te ondersoek. Verder was die toepassing van vloeistofchromatografie – tandem massaspektrometrie tot struktuurbepaling en skandering vir groepe van komponente gedemonstreer vir die ontleding van rooiwyn antosianiene. Die antosianien-glukosiede is verantwoordelik vir die kleur van rooi druiwe en wyn, dra by tot die sensoriese eienskappe daarvan, en is ook relevant as gevolg van die voordelige biologiese eienskappe daarvan. Die bepaling van hierdie komponente word gekompliseer deur hulle groot getalle en strukturele diversiteit, verder bemoeilik deur die wye verskeidenheid van reaksies wat hulle ondergaan tydens veroudering. Daar is ook ‘n gebrek aan beskikbaarheid van standaarde vir die meeste van die lede van hierdie klas van komponente. Tandem massaspektrometrie was in skanderingsmodus gebruik vir hoogs selektiewe deteksie van die antosianien-glukosiede deur die voorspelbare eliminasie van die suiker komponent in neutrale verliesskandering te benut. Gelyktydige skanderings van die komponente wat met neutraleverliesskandering waargeneem word, is gebruik vir ondubbelsinnige komponent identifikasie. Die metode volg daarom ‘n eenvoudige en gestruktureerde benadering vir piek identifikasie wat gebaseer is op chromatografiese orde, sowel as massaspektrale inligting, om ‘n hoë vlak van sekerheid aan die identifikasie van komponente te verleen. Samevattend, word daar getoon deur die werk wat in hierdie dissertasie uiteengesit is dat vloeistofchromatografie – tandem massaspektrometrie ‘n veelsydige en kragtige tegniek bied vir chemiese analise relevant tot die wynbedryf. Die sensitiwiteit, selektiwiteit en spektrale vermoëns van die tegniek word duidelik deur toepassings in die dissertasie getoon.

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 (PhD)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Complex polymer systems have multiple distributions with regard to molecular parameters such as molar mass, functionality, chemical composition, molecular architecture and microstructure. These distributions affect the properties of the polymers making it necessary to develop separation methods to be able to correlate structure to property. A single onedimensional chromatographic method is usually not sufficient to separate these complex polymers with respect to all the distributions. Hence, multidimensional liquid chromatography is necessary for the complete analysis of complex polymers using two or more chromatographic techniques before detection. In this work, two novel liquid chromatographic methods were developed to separate complex polymers according to microstructure. Comprehensive two-dimensional liquid chromatography (LC x LC) was carried out to observe the correlation between microstructure and molar mass. The separation according to microstructure was coupled to NMR (LC-NMR) to observe, identify and quantify the different microstructural components during chromatographic elution. The first chromatographic method separated hydrogenated and deuterated polystyrene homopolymers with respect to the isotope effect. For the LC x LC experiments, liquid chromatography at critical conditions (LCCC) was employed as the first dimension separating according to the isotope effect and size exclusion chromatography (SEC) as the second dimension separating according to molar mass. The LC x LC results of the blends showed that there was an improvement in isotopic separation with an increase in molar mass. The LCNMR coupling using both 1H and 2H NMR detection allowed for the identification of low molar mass blend components which were not sufficiently separated by liquid chromatography. The second chromatographic method separated stereoregular poly(methyl methacrylate)s (PMMAs) with respect to tacticity. The LC x LC experiments of stereoregular PMMAs utilised solvent gradient liquid chromatography as the first dimension to separate according to tacticity and size exclusion chromatography (SEC) as the second dimension to separate according to molar mass. The LC x LC results showed a change in the triad composition with elution of the stereoregular PMMAs with a slight influence of molar mass. The LC-NMR coupling allowed the observation of the triad composition during chromatographic elution. / AFRIKAANSE OPSOMMING: Komplekse polimeriese sisteme het meervoudige verspreidings ten opsigte van molekulêre parameters, soos byvoorbeeld, molêre massa, funksionaliteit, chemiese samestelling, molekulêre argitektuur en mikrostruktuur. Hierdie verspreidings beïnvloed die eienskappe van die polimere en dus is dit nodig om skeidingsmetodes te ontwikkel ten einde polimeerstruktuur met polimeereienskappe te kan korreleer. ‘n Enkele een-dimensionele chromatografiese metode is gewoonlik nie voldoende om hierdie komplekse polimere te skei met betrekking tot al die verspreidings nie. Multidimensionele vloeistofchromatografie, met die insluiting van twee of meer chromatografiese tegnieke, is dus nodig om polimere te skei voor waarneming kan plaasvind. Twee nuwe chromatografiese metodes is ontwikkel om komplekse polimere volgens mikrostruktuur te skei. Twee-dimensionele vloeistofchromatografie (LC x LC) is uitgevoer ten einde die korrelasie tussen mikrostruktuur en molêre massa te ondersoek. Daarna is die skeiding wat op mikrostruktuur gebasseer is, gekoppel aan KMR (LC-KMR) om die verskillende mikrostrukturele komponente gedurende chromatografiese eluering waar te neem, te identifiseer en te kwantifiseer. Die eerste chromatografiese metode het die gehidrogeneerde en gedeutereerde polistireen geskei met betrekking tot die isotoopeffek. Hier het die LC x LC skeiding bestaan uit vloeistofchromatografie onder kritiese kondisies (LCCC) as die eerste dimensie, wat skeiding bewerkstellig het gebasseer op die isotoopeffek, en grootte-uitsluitingschromatografie (SEC) as die tweede dimensie, wat skeiding bewerkstellig het gebasseer op die molêre massa. Die LC x LC resultate van die vermengings het ‘n verbetering in isotopiese skeiding met ‘n toename in molêre massa getoon. Deur gebruik te maak van die LC-KMR koppeling, waar beide 1H en 2H KMR waarneming gebruik is, was dit moontlik om die lae-molêre-massakomponente van vermengings wat nie volledig d.m.v. LC geskei kon word nie, te identifiseer. Die tweede chromatografiese metode het stereoreëlmatige polimetielmetakrilate (PMMAs) m.b.t. taktisiteit geskei. Die LC x LC skeiding van stereoreëlmatige PMMAs het bestaan uit oplosmiddel -gradiënt-LC as eerste dimensie om volgens taktisiteit te skei, en SEC as tweede dimensie om volgens molêre massa te skei. Die LC x LC resultate het ‘n molêre massa afhanklikheid van stereoreëlmatige PMMAs op taktisiteit getoon. Die LC-KMR koppeling het dit moontlik gemaak om die triade-samestelling gedurende chromatografiese eluering waar te neem.

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.