Doctoral Degrees (Chemistry and Polymer Science)
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Browsing Doctoral Degrees (Chemistry and Polymer Science) by browse.metadata.advisor "De Villiers, Andre J."
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- ItemAnalysis of complex tannins by multidimensional techniques(Stellenbosch : Stellenbosch University, 2019-03) Venter, Pieter; De Villiers, Andre J.; Pasch, Harald; Stellenbosch University. Faculty of Science. Dept. of Chemistry and Polymer Science.ENGLISH ABSTRACT: Hydrolysable tannins are plant derived (poly)phenolics which for centuries have been used in tanning, the transformation of animal hides into leather. In addition to their industrial applications, epidemiological evidence suggests that the consumption of hydrolysable tannins can be linked to disease prevention and treatment, although their mode of action at a molecular level is still unknown. In order to investigate their biological and chemical properties, the determination of the molecular composition of hydrolysable tannins in plant material is a critical step. Liquid chromatography hyphenated to mass spectrometry (LC-MS) is the preferred analytical approach to obtain this information, although conventional methods often provide insufficient performance for highly complex mixtures of hydrolysable tannins. The goal of the work reported in this thesis was, therefore, to develop improved multidimensional methods for the analysis of hydrolysable tannins. In the first part of the study, a methodology for the comprehensive analysis of ellagitannins and gallotannins in chestnut and tara using reversed phase liquid chromatography (RPLC) and hydrophilic interaction chromatography (HILIC) combined with ultraviolet (UV), ion mobility (IM) spectrometry and high-resolution mass spectrometry (HR-MS) detection is reported. Good chromatographic performance was achieved using both HILIC and RP-LC for chestnut tannins, with the complementary separation mechanisms proving useful for particular compound classes also pointing to the potential of the combination of HILIC and RP-LC in a comprehensive two-dimensional separation. In contrast, RP-LC provided much better separation of tara gallotannins than HILIC. Incorporation of IM into the LC-MS workflows offered several advantages for such non-targeted screening analyses, including improved mass spectral quality by filtering MS data according to IM arrival time, the availability of an additional identification criterion in the form of collisional cross section (CCS) values obtained from arrival times, and an improved MS duty cycle and, therefore improved sensitivity. Furthermore, additional isomeric species were resolved by IM and, in combination with UV spectral data, IM provided a simple methodology to differentiate between positional isomers. In the second part of the study, an on-line comprehensive two-dimensional HILIC×RPLC method was developed for the analysis of hydrolysable tannins by kinetic optimisation of the relevant experimental parameters. The use of a weak make-up flow to dilute the HILIC effluent and avoid second dimension injection band broadening resulted in excellent performance, with practical peak capacities in excess of 1000 determined for both samples. Despite on-column dilution and the short second dimension analysis times ultimately limiting the number of compounds detected in chestnut and tara, the group-type separations obtained show promise for the fast fingerprint analysis of hydrolysable tannins. Building on previous findings, the final part of the work evaluated the potential of IM spectrometry as a third dimension in a comprehensive three-dimensional HILIC×RPLC×IM configuration. Apart from the inherent advantages of IM alluded to above, the additional separation step offered by IM contributed to an increase in practical peak capacity by a factor of 13 when integrated into the LC×LC workflow. Certain instrumental constraints which limit the ultimate performance of LC×LC×IM-HR-MS are highlighted. Despite these, this approach shows significant promise for the high resolution separation of highly complex mixtures.
- ItemComprehensive multidimensional gas chromatography for the analysis of Fischer-Tropsch products(Stellenbosch : Stellenbosch University, 2011-12) Van der Westhuizen, Katriena Elizabet; Sandra, Pat; De Villiers, Andre J.; University of Stellenbosch. Faculty of Science. Dept. of Chemistry and Polymer Science.ENGLISH ABSTRACT: The analysis of Fischer–Tropsch–derived (FT–derived) synthetic crude and derived products is very challenging because of the highly complex nature of these products. In this study, the use of comprehensive multidimensional gas chromatography (GCxGC) with time-of-flight mass spectrometry (TOF-MS) and flame ionisation detection (FID) was investigated for the analysis of these products and the technique was found to be invaluable for the analysis of these complex mixtures. The compositions of FT synthetic crude, produced at low temperature (LT–FT) and high temperature (HT–FT) processes were compared and the effect that changes in FT reaction temperature has on product formation was investigated. Results for conventional onedimensional GC (1D-GC) and GCxGC were compared. It was found that conventional 1D–GC does not have sufficient peak capacity to separate the thousands of compounds in the HT FT products. GCxGC provides a huge peak capacity of tens-of-thousands to separate highly complex mixtures. Structured chromatograms, where groups of compounds with similar properties are grouped together, aid in peak identification. Moreover, sensitivity at low microgram per milliliter levels is obtained. These attributes enabled accurate analysis of various complex feed and product streams in the FT refinery, and also various final fuel products. The use of GCxGC alone was demonstrated, and also combined with high performance liquid chromatography (HPLC), supercritical fluid chromatography (SFC) and nuclear magnetic resonance (NMR) when even more separation power was needed. HPLC–GCxGC enabled the separation of alkene and cyclic alkane compound classes in oligomerisation products. These compound classes have similar mass spectra, elute in adjacent regions and co–elute even to some extent on the GCxGC contour plot, making differentiation difficult. SFC is a good replacement for HPLC for these applications because it does not use solvents as mobile phases. CO2 is easily evaporated after the separation and does not interfere with the GCxGC separation of the analytes. SFC is also a very good technique to separate the compound classes of alkanes, alkenes, aromatics and oxygenates, and is therefore highly complementary to GCxGC. The combination of GCxGC with NMR data was also found to be very valuable for the identification of branched alkane isomers in LT–FT diesels. GCxGC provides excellent separation of individual compounds but the identification of isomers (except for mono–methyl branching) is difficult because the mass spectra of most of these isomers are similar and not all compounds are in the mass spectral libraries. NMR, on the other hand, is able to distinguish between the individual types of branched isomers but has limited separation power for the complex mixtures. By combining the two techniques, the best of both was obtained. The study found GCxGC to be invaluable for the analysis of the highly complex FT–derived products, while its combination with other techniques such as HPLC, SFC and NMR provided even more separation power.
- ItemComprehensive two-dimensional liquid chromatographic analysis of anthocyanins(Stellenbosch : Stellenbosch University, 2015-04) Willemse, Chandre Monique; De Villiers, Andre J.; Stander, Maria A.; Stellenbosch University. Faculty of Science. Dept. of Chemistry and Polymer Science.ENGLISH ABSTRACT: Anthocyanins are naturally occurring pigments responsible for the colour of many natural products, including grapes and wine. These pigments are important to the food industry and have been recognised for their nutritional value since they play an important role in the reduced risk of various chronic diseases in humans. Anthocyanins also play an important role in the aesthetic perception and quality of red wine. However, due to the large structural diversity of grape-derived anthocyanins and the many derivatives formed from these during wine ageing, the accurate analysis of wine pigments is extremely challenging. Reversed-phase liquid chromatography (RP-LC) is mostly used for anthocyanin analysis, although the technique often provides insufficient resolving power for complex mixtures of anthocyanins. In addition, the lack of commercially available standards and identical mass spectral characteristics hampers identification of these compounds. The coupling of multiple orthogonal separation systems in comprehensive 2-dimensional liquid chromatography (LC×LC) offers a more powerful approach for the separation of complex mixtures. The current work therefore focussed on exploring the potential of LC×LC for the improved analysis of anthocyanins and derived pigments in natural products and wine. The first part of this work focussed on developing a hydrophilic interaction chromatography (HILIC) method as an alternative to RP-LC for the anthocyanin analysis. Following extensive optimisation, the method proved suitable for the analysis of a diverse range of anthocyanins in natural products. Significantly, it also showed alternative selectivity compared to RP-LC. The optimised HILIC method was then used in combination with RP-LC to develop an off-line LC×LC approach for anthocyanins. For this purpose, half-minute fractions of the HILIC effluent were collected and reinjected onto a RP-LC column. The off-line HILIC×RP-LC method demonstrated exceptionally high resolving power, as measured in terms of the practical peak capacity, with many compounds separated in two dimensions that co-eluted in 1-dimensional HPLC. Interestingly, group-type separation was also observed based on the degree and/or nature of glycosylation and acylation of anthocyanins. In the final part of the work, a systematic approach was used for the development and optimisation of and on-line HILIC×RP-LC method by using a 10-port switching valve to automatically transfer fractions between the two columns. This method was then coupled to high resolution mass spectrometry (MS) to allow the detailed investigation of anthocyanins and derived products in wine. Ninety four pigments were identified in one- and six-year old Pinotage wines based on HILIC×RPLC separation in combination with accurate mass MS data and fragmentation information. Significant differences in especially the content of derived pigments were observed between the wines. In summary, the methods developed in this work provide the means to improve anthocyanin analysis, and therefore also show promise for the detailed investigation of these important compounds and their alteration in natural products and their derived commodities.
- ItemComprehensive two-dimensional liquid chromatographic analysis of phenolics(Stellenbosch : Stellenbosch University, 2013-12) Kalili, Kathithileni Martha; De Villiers, Andre J.; Stellenbosch University. Faculty of Science. Dept. of Chemistry and Polymer science.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.
- ItemIdentification of the putative urinary intraspecific recognition pheromone of the caracal caracal caracal(Stellenbosch : Stellenbosch University, 2017-03) Hailemichael Goitom, Aron; Burger, B. V.; De Villiers, Andre J.; Stellenbosch University. Faculty of Science. Dept. of Chemistry and Polymer Science.ENGLISH ABSTRACT: Sheep husbandry makes a considerable contribution to the economy of South Africa. However, it has been pressure during the last few decades due to the rapidly growing numbers of predators such as the caracal, Caracal caracal, and the red jackal, Canis mesomelas. Currently, sheep farming is hardly viable in the arid southern parts of the country. When experimenting with various methods of controlling caracal numbers, sheep farmers found that this problem animal could be effectively lured into traps using the urine of another male or female caracal. The main disadvantage here is that a sheep farmer could incur serious stock losses before he is able to obtain a starting sample of urine from another source (e.g., another farmer). It was hypothesised that caracal urine contains a volatile organic substance, or substances (VOCs), that are involved in the semiochemical communication between members of this species. The objective of this investigation was to identify these putative attractants in the urine, for the subsequent formulation of a caracal lure composed of synthetic analogues of the natural VOCs. As sheep farmers reported that male and female urine were equally effective attractants, it was considered unlikely that the attracting agent could be a sex pheromone; rather, it could have an intraspecific signalling function. Efforts were made to identify all the VOCs present in caracal urine samples donated by farmers from different sheep farming areas of the country. A total of 191 VOCs, including five steroid hormones, were identified. The identification of 86% of these VOCs was corroborated by gas chromatographic-mass spectrometric (GC-MS) comparison of the natural substances with authentic synthetic analogues. Obligate proteinuria is known in certain animals, in which it is not an indicator of renal abnormality. In the house mouse, Mus domesticus, for example, so-called major urinary proteins (MUPs) bind dehydro-exo-brevicomin and 2-(sec-butyl)-4,5-dihydrothiazole elicit male aggression. The carboxylesterase-like urinary excreted pheromone-binding protein, also known as cauxin, has been identified in the urine of several felids, but was not found in caracal urine. However, the pheromone-binding proteins ‘Transgelin fragment (M3WJ37)’ and ‘Uncharacterized protein (M3XEJ0)’, both previously identified in Felis catus and Felis silvestris catus, were identified in caracal urine. The macrocyclic C15 ketone, cyclopentadecanone, was identified as one of the VOCs present in the headspace gas of caracal urine. The headspace concentration of this ketone increased when the urine was heated at 95 ºC for 10 min. A similar, but more pronounced increase in cyclopentadecanone concentration was observed when urinary protein present in caracal urine was denatured by subjecting it to similar treatment. This was interpreted as an indication that this ketone is a ligand of caracal urinary protein. The C13, C14, C16, and C17 macrocyclic ketones were subsequently also identified as urinary protein ligands. In bioassays, a mixture of synthetic analogues of a large number of the other ketones identified in caracal urine, including cyclopentadecanone, elicited typical feline behaviour in two male caracal in captivity. Similar behavioural patterns were observed when a mixture of the C13, C14, C15, and C16 macrocyclic ketones were tested. It was concluded that these ketones, that are ligands of the caracal’s urinary protein, could play an essential role in the intraspecific communication of the caracal.