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Thesis (MSc (Chemistry and Polymer Science))--University of Stellenbosch, 2009. / ENGLISH ABSTRACT: The separation of apple, cocoa and green tea phenolic compounds by comprehensive 2-dimensional liquid chromatography (2-D-LC) has been studied. In the first dimension, phenolic compounds were separated according to polarity by hydrophilic interaction chromatography (HILIC) on a diol stationary phase with a mobile phase containing acetonitrile, methanol, acetic acid and water. Gradient reversed-phase (RP) LC using a C18 column with fluorescence detection was employed in the second dimension to separate compounds according to hydrophobicity. Compounds were identified using negative electrospray ionisation mass spectrometry (ESI-MS) coupled to both HILIC and RP separations. The coupling of HILIC and RP separations proved to be especially beneficial since this provided simultaneous information on both the polarity and hydrophobicity of phenolics. The low degree of correlation (r2 < 0.21) between the two LC modes afforded peak capacities in excess of 3000 for the off-line method. An on-line method was also developed utilizing a short, small particle-packed column to provide fast separation in the second dimension. A 1 mm i.d. column was used in the first dimension for the on-line system to reduce injection volumes onto the second dimension column. A significantly lower practical peak capacity was measured for the on-line system, due largely to the reduction in second dimension peak capacity. On the other hand, analysis could be performed in an automated fashion using the online system reducing the risk of sample alteration and guaranteeing better operation reliability and reproducibility. Especially the off-line comprehensive HILIC × RP-LC method developed demonstrated its utility in the analysis of various groups of phenolic compounds including proanthocyanidins, phenolic acids, flavonols and flavonol conjugates in a variety of natural products. / AFRIKAANSE OPSOMMING: Die skeiding van fenoliese komponente in appel, kakao en groen tee is deur middel van ‘comprehensive’ 2-dimensionele vloeistof chromatografie (2-D-LC) bestudeer. Hidrofiliese interaksie chromatografie (HILIC) is gebruik om die fenoliese komponente in die eerste dimensie te skei op grond van polariteit, deur gebruik te maak van ‘n diol stationêre fase en mobiele fase bestaande uit asetonitriel, metanol, asynsuur en water. ‘n Gradiënt omgekeerde fase (RP) LC analisie op ‘n C18 kolom met fluorosensie deteksie is in die tweede dimensie gebruik om fenole volgens hidrofobisiteit te skei. Negatiewe elektrosproei-ionisasie massa spektometrie (ESIMS) gekoppel aan HILIC en RP skeidings is gebruik vir identifikasie van fenole. Die koppeling van HILIC en RP skeidings veral voordelig deurdat dit gelyktydige informasie verskaf het oor die polariteit sowel as die hidrofobisiteit van die fenoliese komponente. Die lae graad van korrelasie (r2 < 0.21) tussen die twee LC metodes was verantwoordelik vir piek kapasiteite bo 3000 vir die af-lyn metode. ‘n Aanlyn metode was ontwikkel deur gebruik te maak van ‘n kort, klein partikel gepakte kolom om vinnige skeiding in die tweede dimensie te verseker. 1 mm i.d. kolom was gebruik in die eerste dimensie vir die aanlyn sisteem om die inspuit volume op die tweede dimensie kolom te verminder. Aansienlike laer praktiese piek kapasiteit was gemeet vir die aanlyn sisteem, grootliks toegeskryf aan die reduksie in die tweede dimensie piek kapasitiet. Aan die ander kant, analise kan geoutomatiseerd uitgevoer word deur gebruik te maak van die aanlyn sisteem, wat monster alterasie, beter betroubaarheid en reproduseerbaarhied verseker. Veral die ontwikkelde af-lyn ‘comprehensive’ HILIC × RP-LC metode toon demonstreerbare voordele vir die analiese van verskeie groepe fenoliese komponente, insluitende proantosianiede, fenoliese sure, flavonole en gekonjugeerde flavonole in ‘n verskeidenheid natuurlike produkte.

Thesis (PhD)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Phenolic chemistry is quite complex; natural phenolic compounds vary widely in terms of size and chemical properties. The high structural diversity within this family presents severe analytical challenges. High performance liquid chromatography (HPLC) is the preferred method for phenolic analysis; however, conventional HPLC methods offer limited separation power and often provide incomplete separation of the large number of components present in natural phenolic extracts. Multi-dimensional chromatographic techniques have proven much more effective in the analysis of complex samples. The current study explored the potential of comprehensive two-dimensional liquid chromatography (LC×LC) for the characterisation of phenolic compounds in complex natural products, with the emphasis on proanthocyanidins (PACs). Initial work focused on the evaluation of the state of the art in phenolic analysis, to allow information which was used in the development of optimal 1-D separations for use in LC×LC. The combination of hydrophilic interaction chromatography (HILIC) in the first dimension with reversedphase liquid chromatography (RP-LC) in the second dimension afforded an orthogonal and powerful separation system for phenolics, providing separation on the basis of hydrophilicity and hydrophobicity, respectively. A detailed and systematic procedure was therefore developed to allow the optimisation and evaluation of on-line, off-line and stop-flow HILIC×RP-LC methods. Results showed that all three approaches provide much better separation performance than conventional one-dimensional LC (1-D LC) techniques. On-line HILIC×RP-LC offers automation, shorter analysis times, better reproducibility and minimal sample exposure. The off-line and stopflow methods are characterised by much higher peak capacities, but relatively long analysis times. It was also demonstrated that stop-flow operation results in negligible additional band broadening for procyanidins (PCs), implying that this method is an attractive alternative to the off-line method as it offers automation and minimal sample handling. Experimental verification of the predictions based on fundamental principles confirmed the validity of the optimisation procedure for cocoa PCs. The hyphenation of on-line HILIC×RP-LC separation with fluorescence (FL) and mass spectrometry (MS) detection methods provided enhanced resolution in a practical analysis time with the added benefit of selective detection and greater certainty in compound identification. This strategy proved much more powerful, as demonstrated by the identification of the highly complex PACs in grape seeds based on chromatographic retention data in two dimensions and accurate mass information. It was further shown that on-line coupling of HILIC×RP-LC separation with an optimised radical scavenging assay provides an improved approach for screening of individual radical scavengers in complex phenolic fractions, as demonstrated for cocoa, grape seed and green tea extracts. / AFRIKAANSE OPSOMMING: Fenoliese chemie is baie kompleks; natuurlike fenoliese verbindings varieer in terme van beide grootte en chemiese eienskappe. Hierdie hoë strukturele diversiteit binne die familie bied daadwerklike analitiese uitdagings. Hoëverrigtingvloeistofchromatografie (HPLC) is die voorkeurmetode vir fenoliese analises, maar konvensionele HPLC metodes bied egter 'n beperkte skeidingsvermoë en verskaf dikwels onvolledige skeiding van die groot aantal komponente teenwoordig in natuurlike fenoliese ekstrakte. Multi-dimensionele chromatografiese tegnieke is bewys om baie meer effektief te wees met betrekking tot die ontleding van komplekse monsters. Hierdie studie ondersoek die potensiaal van omvattende twee-dimensionele vloeistof chromatografie (LC×LC) vir die karakterisering van fenoliese verbindings in komplekse natuurlike produkte, met die fokus op pro-antosianidiëne (PAC’s). Aanvanklike werk het gefokus op die evaluering van moderne tegnieke vir fenoliese analise – inligting wat in die ontwikkeling van optimale 1-D skeidings vir die toepasing in LC×LC gebruik is. Die kombinasie van hidrofiliese interaksie chromatografie (HILIC) in die eerste dimensie met omgekeerde-fase vloeistof chromatografie (RP-LC) in die tweede dimensie verleen 'n ortogonale en kragtige skeidingsisteem vir fenoliese komponente en verskaf skeiding op grond van onderskiedelik hidrofiliteit en hidrofobiteit. ‘n Gedetailleerde en sistematiese prosedure is dus ontwikkel om die optimisering en evaluering van aan-lyn, af-lyn en stop-vloei HILIC×RP-LC metodes uit te voer. Resultate het getoon dat al drie benaderings baie beter skeidingsvermoë bied as konvensionele een-dimensionele LC (1-D LC) tegnieke. Aan-lyn HILIC×RP-LC bied outomatisering, korter ontledingstyd, beter herhaalbaarheid en minimale monster blootstelling. Die af-lyn en stop-vloei metodes word gekenmerk deur 'n veel hoër piekkapasiteit, maar relatief lang ontledingstye. Daar is ook getoon dat die stop-vloei prosedure geringe bykomende bandverbreding vir prosianodiniëne (PC’s) tot gevolg het, wat beteken dat hierdie metode 'n aantreklike alternatief is vir die af-lyn metode aangesien dit outomatisering bied en minimale monster hantering behels. Eksperimentele verifiëring van die voorspellings gebaseer op fundamentele beginsels bevestig die geldigheid van die optimalisering proses vir kakao PCs. Die koppeling van aan-lyn HILIC×RP-LC skeiding met fluoressensie (FL) en massaspektrometrie (MS) deteksie verskaf verbeterde resolusie binne 'n praktiese ontledingstyd saam met die bykomende voordeel van selektiewe opsporing en groter sekerheid betreffende die verbindings se identifikasie. Hierdie strategie was baie meer kragtig, soos gedemonstreer deur die identifisering van die hoogs komplekse PAC’s in druiwepitte gebaseer op chromatografiese behoud van die integriteit van die data in twee dimensies tesame met akkurate massa inligting. Daar is verder getoon dat aanlyn koppeling van HILIC×RP-LC skeiding met 'n geoptimiseerde radikale vangers deteksie-metode 'n beter benadering bied om die gedrag van individuele radikale vangers in komplekse fenoliese fraksies te bestudeer, soos bewys is vir kakao, druiwepitte en groen-tee ekstrakte.

published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

This study was conducted to set up a systematic approach utilizing advantages of both wet lab and bioinformatic methodologies to study biodegradation abilities and microbial bacterial-functional relationship within bioremediation communities. Firstly, 11pharmaceuticals and personal care products (PPCPs)were selected as target chemicals for establishing an effective determination process in analyzing trace-level concentrations in the environment, and understanding the removal routes during pollutants removal process in wastewater treatment process using activated sludge. Ultra performance liquid chromatography-tandem mass spectrometry was utilized to develop a rapid, sensitive and reliable method without solid phase extraction pre-concentration for trace analysis of 11 PPCPs in influent and effluent from municipal wastewater treatment plants. Shorten the detection time and significant reduction of detection cost were achieved due to the omitting usage of solid phase extraction (SPE)process and avoiding the consumption of hydrophiliclipophilic balancced (HLB)cartridge. Research on removal routes of ten selected PPCPs in activated sludge found activated sludge hardly removed carbamazepine. Biodegradation was the sole route to remove acyclovir, metronidazole, benzylparaben, ethylparaben, methylparaben and propylparaben. Both adsorption and biodegradation were involved in the removal of ranitidine and benzophenone-3, while fluoxetine could be totally removed by adsorption in activated sludge. Secondly, as the target microbial community, activated sludge community was used to set up the global bioinformatic analysis process. Both metagenomic and metatranscriptomic approaches were processed to characterize microbial structure and gene expression of activated sludge community. The taxonomic profile showed thatactivated sludge was dominated by Proteobacteria, Actinobacteria, Bacteroidetes, Firmicutes and Verrucomicrobiaphyla. Gene expression annotation of nitrogen removal revealed that denitrification-related genes sequences dominated in both DNA and cDNA datasets while nitrifying genes were also expressed in relative high levels. Specially, ammonia monooxygenase and hydroxylamine oxidase demonstrated the high cDNA/DNA ratios, indicating strong nitrification activity. Ammonia-oxidizing bacteria present mainly belonged to Nitrosomonas and Nitrosospira species. A fast method to construct local sub-databases has been established for the quick similarity search and annotation of huge metagenomic datasets. The conducted tests showed sub-database annotation pipeline achieved a speedup of ~150-385 times, and got exactly the same annotation results with those of the direct NCBI-nr database BLAST-MEGAN method. This approach provides a new time-efficient and convenient annotation similarity search strategy for laboratories without access to high performance computing facilities. Thirdly, bisphenol A(BPA), which has a partially known biodegradation pathway and relevant bioremediating genes, was chosen as a model to establish a pipeline for systematical understanding the pathways and gene/bacteria relationships in an enriched microbial community. 11 new metabolites were detected during BPA degradation. Thereby, a novel pathway of degrading BPA metabolite was proposed. Sphingomonas strains were dominant taxa in initial degradation of BPA, while the other taxa were competing BPA metabolites during degradation. Metagenomic binning results showed a cytochrome P450 monooxygenase system, which was previously reported BPA mediator, was sharing by two Sphingomonas strains, showing the undergoing mechanism of competition of the two strains. The observations suggested bacterial specialization may occur in that community that each taxon was selected to degrade certain metabolite in a community economical way. / published_or_final_version / Civil Engineering / Doctoral / Doctor of Philosophy

Several phenyl alkyl bonded phases for liquid chromatography were synthesized and characterized by liquid chromatography, gas chromatography and ¹³C nuclear magnetic resonance (NMR) spectroscopy. The chromatographic physical parameters investigated include a quantitative determination of the mobile phase volume and the stationary phase volume. The stationary phase volume was determined to be a function of the bonded moiety chain length and the chromatographic solvent system employed. The interpretation of the stationary phase volume is discussed in terms of the porous nature of the silica gel support. The chemical parameters determined include a quantitative determination of the mobile phase and stationary phase composition (which were different from each other). The selectivity of the chromatographic separations was dependent on the chemical composition and the volume of both the stationary and mobile phases. Carbon 13 NMR spectroscopy provided information about the environment of the bonded moiety in the stationary phase. The liquid-like nature of the bonded moiety was influenced by the chain length of the attached species, the choice of organic modifier, and the chemical composition of the solvents. Temperature did not appear to play a role in the line widths under the experimental conditions examined. The separation of several peptide diastereoisomers on different commercially available hydrocarbon bonded adsorbents is also reported. Select diastereoisomers of arginine vasopressin and oxytocin are extremely sensitive to differences in the composition of the stationary phase. The selectivity and elution order were dependent upon the choice of adsorbent and solvent system employed. The addition of a second organic modifier provided a method for the dynamic modification of the stationary phase. The ability to dynamically modify the stationary phase can enhance the selectivity for the separation of selected peptide diastereoisomers.

The effect of the physical state and chemical composition on molecular interactions has been studied for a number of chemically modified adsorbents. During the course of the study a reaction scheme was put together for the synthesis of a particular substituted crown ether. The synthesized allyl-benzo-15crown-5, which is not reported in the literature to date, was silylated and immobilized on a silica surface. The bonded phase was characterized by UV spectroscopy and by chromatography under both "normal" and "reverse" phase conditions. UV spectroscopy was also used to elucidate the physical state of several other phenyl alkyl bonded phases. Chromatographically, the bonded crown ether phase was found to be more polar than a C₈ stationary phase. A comparison of the selectivity of the two phases revealed that the former has a better selectivity towards a homologous series of alkyl benzenes under different reverse phase conditions. The selectivity of the crown ether phase was found to be dependent on the nature of the organic modifier in the mobile phase. This dependence was considered to be added evidence for the universality of the dynamic solvated stationary phase model. Both normal and reverse phase chromatographic conditions indicated an acid-base type of interaction between the crown ether and a number of substituted phenols. This was reflected in an increase in the retention of these probes as a function of their increasing acidity. A dramatic temperature effect observed on the crown ether stationary phase under aqueous THF mobile phase, but not under aqueous MeOH, was attributed to a temperature and/or solvent-induced phase change. A hysteresis effect, also seen only with aqueous THF, indicated that the crown ether phase undergoes a solvent-assisted conformational change. Further evidences for such a change was found spectroscopically in the abrupt break in the UV absorbance of these molecules as a function of temperature, as well as the irreversibility of the absorbance of the n- π* band on cooling. UV spectroscopy of bonded phenyl alkyls showed that there are about two monolayers of water molecule strongly adsorbed to the surface and totally impermeable to lypophilic species. Evidence for the existence of a solvated crystal, or liquid crystal, like clusters was rationalized with a cooperative sorption effect which may be dependent on the reaction conditions during immobilization. Despite a significant increase in the liquid character observed as the chain length is increased to 4-7 methylene groups, the bonded clusters still appear to preserve a fairly ordered environment. The physical state of the immobilized species was found to change with the experimental conditions and the change was reflected on the selectivity of the system.

A new theory has been proposed for stationary phase formation of chemically modified chromatographic adsorbents. This theory consists of a model in which the bonded hydrocarbon moiety, silica substrate, and their respective solvation layers all participate in stationary phase formation. Stationary phase formation was found to be dependent on three parameters: (1) Solvent strength of the mobile phase components for the bonded organic moiety and the silica substrate; (2) the type of organic moiety covalently bound to the surface; and (3) the bound moiety density or surface coverage. Binary aqueous-organic mobile phases were investigated for LiChrosorb RP-8 and RP-18. For RP-8 the solica substrate played a more important role in stationary phase formation. Whereas, for RP-18 the longer bound hydrocarbon chain dominated stationary phase formation. With different organic modifiers in the mobile phase, the modifier with the larger solvent strength for the bound hydrocarbon was selectively enriched in the stationary phase solvation layer for RP-18. Ternary mobile phase systems were also investigated for RP-18. The second modifier was found to exert a large influence on stationary phase formation. Temperature's role in stationary phase formation was studied with a ternary mobile phase of 40/45/15 methanol, water, THF with RP-18. In this specific case, changing the temperature of the system did not impact on stationary phase formation. A new type of column structure was investigated. This structure involved a totally porous silica gel as compared to a column packed with totally porous silica microparticles. These silica gel columns were characterized both thermodynamically and kinectically. Under Normal Phase chromatographic conditions the silica gel column was found to have a higher selectivity but poorer efficiency for the separation of aniline from nitrobenzene than a packed column. The silica gel can be chemically modified by silane reaction and its bonded phase characteristics were investigated. The gel also showed ion-exchange properties which were investigated using sodium nitrite.

A mini-dissertation in partial compliance with the requirements for a Master's Degree in Technology: Homoeopathy, Durban Institute of Technology, 2002. / The purpose of this study was to compare the amount of caffeine extracted from triturated samples and ethanolic samples of Coffea tosta using high pressure liquid chromatography (HPLC) as a method of analysis. The study wanted to expand on homoeopathic pharmaceutical knowledge, specifically looking at the two methods of remedy preparation of plant materials. From the same batch of ground roasted coffee beans, using the decimal scale of dilution, the mother tincture (bill) and the first triturated (bill) samples were prepared. The subsequent 2xH and 3xH triturated and ethanolic potencies were then made in accordance with homoeopathic methodology. Each group contained three different dilution levels (bill, 2xH and 3xH), 18 samples per group giving a total of36 samples that were analysed using HPLC. Three samples were analysed from the three dilution levels in each Group, in total there were 18 samples from the triturated group and 18 from the ethanolic group. . The samples were analysed quantitatively using the highly accurate and advanced method of high pressure liquid chromatography. This method gives accurate readings of the caffeine concentrations of a sample compared to a caffeine standard. This allowed for quantification of the caffeine concentration of each sample. The percentage caffeine was calculated from each sample. The aim of the study was to evaluate the difference in each method of preparation by measuring the amount of caffeine extracted from the samples. The results obtained from the inter-Group Mann-Whitney and ANOVA tests showed that there was a significant difference between the ethanolic dilutions and triturated dilutions with regards to the 1xH and 2xH dilutions. In the 1xH dilution the ethanolic method retained / M

Heparin is a highly sulfated glycosaminoglycan with potent anticoagulant, antimetastatic, and anti-inflammatory effects. Polymeric and polyanionic nature of heparin makes dosing and side effects a nightmare for healthcare professionals. Our laboratory has proposed appropriately designed, small, highly sulfated aromatic molecules as potential mimetics of heparin. These easier-to-synthesize small molecules have been shown to possess interesting pharmacological and improved toxicological profiles. However, the detection and characterization of these highly sulfated molecules is challenging. A robust RP-IP UPLC-MS method was developed to successfully retain, resolve and quantify sulfated non-saccharide GAG mimetics without the requirement of pre- or post- column derivatization. Comparative analysis reveals intricate dependence of resolution and ionization on the structure of ion-pairing agents. This is the first report showing systematic use of MS cone voltage to fingerprint sulfated GAG mimetics, perhaps eliminating the need for tandem MS techniques.