An LSI-11 micro-processor and a Spectra-Physics model 3500B gradient elution liquid chromatography were interfaced for the purpose of automating the chromatograph, incorporating it into the laboratory data network system and collecting data for Michael Starlings' project. The automation involved the construction of hardware for the control of the chromatograph and for data acquisition and display and the writing of software to operate the interface. The LSI-11 collects data by clock interrupts while displaying the current data buffer on the oscilloscope. It stores all data files on a floppy disk storage device, accessed through the main host computer. An analysis of the data is performed by a peak processing routine, calculating peak area and retention time. The network is the DEC Rr-11/REMOTE system. It operates on a PDP-11-03 with a floppy disk system for mass storage. The laboratory system has three satellites operating under it. They can utilize all of the facilities of the main computer while maintaining a minimal operational configuration. The project also had the purpose of being assimilated into Michael Starlings' data correlation system. The LSI-11 will collect data and send it to the NOVA computer system where it will be stored. The system was tested with several mixtures and the results were found to be accurate and easily obtainable, / Master of Science

Microbore columns were investigated for their application to High Performance Liquid Chromatography. Microbore columns consist of stainless steel tubing, 1/16¹¹ OD x 1mm ID, packed with silica gel or other suitable material. These columns were evaluated in terms of extra-column variance effects, mass sensitivity, and solvent consumption. Three methods of extracting the extra-column variance effects were evaluated. When used with an instrument whose contribution to total peak variance is small compared to the column, microbore columns provide the best performance for analyses where sample is limited or where reduction of solvent usage is desirable. A method from the literature for extracting extra column variance effects was found to be easy and reliable. It is proposed as a useful way of allowing column performance to be compared from instrument to instrument and for determining whether a particular instrument is suitable for use with microbore columns. / M.S.

The separation of basic nitrogen-containing compounds has been investigated via supercritical CO₂ and 1% methanol modified CO₂. Packed columns with the following stationary phases were employed: silica, octadecyl (C₁₈), propylamino (NH₂), and polystyrene-divinyl benzene (PRP). Without modifier the range of basicities which could be eluted increased in the order of silica < PRP < C₁₈ = NH₂. Chromatographic peak shapes and selectivity were much better with propylamino column. Different aromatic amines and azaarenes were successfully separated on both analytical scale and microbore propylamino bonded phase packed columns with 100% supercritical CO₂. Separation is compared with both reversed phase and normal phase high performance liquid chromatography (HPLC). The retention mechanism study for these aromatic amines and azaarenes shows that the elution order not only depends on basicity and steric hindrance, but also on the solubility of the solute in CO₂. New cross-linked cyanopropyl and phenyl bonded phases are studied as stationary phases for packed column SFC, as well as for separation of nitrogen-containing compounds. The cross-linked bonded phase impedes access to uncapped silanol sites, thereby giving rise to better peak shapes, and more rapid elution without the necessity of a polar modifier in the mobile phase. Experiments both at elevated temperature and in the presence of methanol modifier revealed that there is no short or long term deleterious effect on the column. The separation of model mixtures of nitrated diphenylamine and nitrated anilines via SFC employing cyanopropyl packed and capillary columns is described. Peak identification and peak purity were performed by on-line Fourier transform infrared spectrometric detection. Supercritical CO₂ is employed with cyanopropyl packed columns for separation of non-polymeric components in double-base rocket propellants. Both supercritical CO₂ and CH₂Cl₂ were compared as a solvent for extraction of nonpolymeric components in "good" and "bad" double-base propellant. Finally, twenty-four phenylthiohydantion amino acids (PTH-AA) have been rapidly and efficiently separated on a cyanopropyl packed column by gradient elution of supercritical CO₂ and tetramethyl ammonium hydroxide-modified methanol. Complete or partial resolution of 22 derivatives is observed with only valine co-eluting with norleucine and lysine co-eluting with asparagine. No modifier was required for elution of neutral PTH-AA's from the cross·linked stationary phase. The addition of base plays a major role in elution of acidic and basic PTH-AA's. / Ph. D. / incomplete_metadata

The Variable Temperature Size Exclusion Chromatography (VTSEC) was used to study the dilute solution properties of various homo- and block copolymers as well as polymer blends, focus being primarily on the configuration-dependent properties such as average chain dimensions and hydrodynamic behavior of polymers. The study constituted of three parts. In the first part, the dilute solution properties of a series of poly(alkyl methacrylates) with varying side alkyl(ester) group were investigated as to the effect of the size of the side group on the average chain dimensions at various temperatures. The VTSEC results were found to reveal that the effect of the side group depends on the extent of chain flexibility imparted by the side group. In the second part, VTSEC was employed to investigate the applicability and validity of the various models for block copolymers in solution. For this purpose, several series of samples (all anionically synthesized) with various composition and molecular weight were used: i) poly(alkyl methacrylates), their diblock copolymers and blends and ii) diblock and triblock copolymers of poly(styrene/substituted styrene) and poly(butadiene/isoprene). VTSEC results showed that the applicability of each model to a block copolymer in solution depends on the compatibility among the components, the composition of the samples, the solvating power of the solvent used and the operating temperature. Micellization in the hydrocarbon diblock copolymer solutions was found to occur in selective solvent, its extent depending on the solubility parameter difference between the components, molecular weight, composition and solvent power. In the third part, poly(methyl methacrylate) samples with varying tacticities were studied as to the effect of stereochemical configuration on their dilute solution behavior and found to indicate that both syndiotactic and isotactic stereosequences are required for stereoassociation. Two new VTSEC data reduction procedures were devised in order to correct for the temperature drifts due to instrumentation in the first and to neutralize the effect of molecular weight in the second. In the second, termed as the Molecular Weight-Elution Volume Superposition Procedure, the effect of molecular weight alone was enutralized leaving only the effect of the intrinsic character (such as the size of the side group) of the sample in the elution behavior. Numerical computations, using the Matxrix Generation method based on the Rotational Isomeric State approximation, were carried out for stereoregular chains in order to evaluate their chain dimensions at various temperatures. / Ph. D.

Very early in the history of atomic emission spectroscopy (AES) it was understood to be a powerful analytical tool. Until the 1930's the usefulness of atomic spectroscopy was not utilized very extensively even though its fundamental power was understood. The breakthrough that placed it in the standard chemistry laboratory was the discovery and implementation of the photoelectric effect. Since this discovery there has been a revolution in atomic spectroscopy which has brought it from the role of a humble servant used for primary elemental screening to an outstanding leader in applications of elemental analysis. Atomic emission spectroscopy of complex samples has long suffered from matrix effects which result in overlapping of spectral lines, fluctuating backgrounds and changing conditions in the source. Investigations employing an echelle polychromator with a two dimensional solid state array detector show great promise in minimizing the effects of these interferences on multielement analyses of complex samples. The Charge Injection Device (CID) detector used exhibits many characteristics which make it uniquely qualified for simultaneous, multielement detection in AES. With only slight modifications to the optics of a commercial spectrometer and the employment of a CID detector, detection limits for a number of elements are quite favorable. Dynamic ranges of over seven orders of magnitude are obtainable with this experimental system. The reduction of matrix effects by utilizing the huge wealth of information available from over 60,000 individual detector elements are demonstrated through results from several complex matrix standards. This CID-polychromator system was also employed for the element selective detection of gas chromatographic (GC) effluents. A microwave-induced plasma (MIP) based on the Surfatron design was built. A helium plasma from this device has shown to have resilience to organic samples and give good emission response to non-metallic atoms. A number of studies with this GC-AES-polychromator system are presented. This system is capable of monitoring atomic emissions from C, H, F, Cl, Br, I, O, N and S all simultaneously, and the selectivity of this system is unsurpassed. Elemental ratios for separated compounds are also presented as a precursor to empirical formula prediction.

Thesis (MSc)-- Stellenbosch University, 2012. / ENGLISH ABSTRACT: Over the past two decades, field flow fractionation (FFF), as a polymer characterization technique, has become cutting edge technology. The demand for molar mass and size characterisation of complex polymer systems has increased, especially in cases where classical calibration techniques such as size exclusion chromatography (SEC) has shown several shortcomings. FFF is a technique resembling chromatography. It has several significant advantages over SEC, especially for the characterisation of ultrahigh molar mass (UHMM), branched and gel-containing polymers. In this study, polybutadienes, which often contain the abovementioned species, were analysed by SEC and asymmetric flow field flow fractionation (AF4). Both separation techniques were coupled to refractive index and multi-angle laser light scattering detection. Similarly, polyrotaxanes, which are polymers with complex and unique molecular architectures, were also investigated. Results showed that AF4 can explicitly be used as a superior tool over SEC. In the case of UHMM polybutadienes, much higher molar masses could be detected by AF4, due to the absence of shear degradation which is often encountered in SEC. Gel-containing species could be detected by AF4 as no filtering is required prior to injection. Abnormal retention behaviour, a phenomenon often encountered in UHMM branched polymers, was observed in SEC analysis of the polyrotaxanes materials. AF4 provided sufficient separation from low to high molar masses, without out any irregularities. / AFRIKAANSE OPSOMMING: Gedurende die afgelope twee dekades het veldvloeifraksionering (FFF) as ‘n polimeerkarakteriseringstegniek groot veld gewen. Die aanvraag na molekulêre massa en groottekarakterisering van komplekse polimeersisteme het toegeneem, veral in die gevalle waar klassieke kalibrasietegnieke soos grootte-uitsluitingschromatografie (SEC) etlike tekortkominge getoon het. FFF is ‘n tegniek soortgelyk aan chromatografie, en het voorheen bewys dat dit oor ‘n redelike aantal voordele bo SEC beskik, veral in die geval van ultrahoë molekulêre massa- (UHMM-), vertakte- en jelbevattende spesies. In die huidige studie is polibutadieenpolimere, wat dikwels bogenoemde spesies bevat, geanaliseer met behulp van SEC en onsimmetriese vloei-veldvloeifraksionering (AF4). Beide skeidingstegnieke is gekoppel aan ‘n brekingsindeks en multihoek-laserligverstrooiingsdetektors. Op dieselfde wyse is polirotaksane (polyrotaxanes) met komplekse molekulêre argitektuur bestudeer. Daar is bewys dat AF4 uitsluitlik gebruik kan word as ‘n meer geskikte tegniek bo SEC. Baie hoër molekulêre massas kon deur middel van AF4 vir UHMM polibutadieenpolimere raakgesien word as gevolg van die verminderde afbrekende degradasie wat dikwels voorkom met SEC. Jel-bevattende spesies is suksesvol geïdentifiseer met behulp van AF4 waartydens geen filtrering vir analise nodig was nie. Abnormale retensie was sigbaar tydens SEC analise van monsters van polirotaksane, wat dikwels voorkom in vertakte polimere. In teenstelling het AF4 bewys dat ‘n bevredigende skeiding van klein na groot molekulêre massas, sonder enige tekortkominge, moontlik is.

Thesis (MSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: In traditional medicine, there is a long-standing culture of combining herbal drugs to increase the therapeutic efficacy. The improved medical action is thought to be due to synergistic interactions between different plant bioactive components. The aim of this study was to test the pharmacological interactions in a medicinal plant combination which consisted of Agathosma crenulata, Dodonaea viscosa and Eucalyptus globulus. The rationale for the analysis of this particular mixture is that it had noteworthy antibacterial activity and exhibited the highest activity out of seven medicinal plant mixtures previously investigated. Using chromatographic analysis, the phytochemistry of the plants was also assessed. The chloroform: methanol (1:1; v/v) extracts or hydo-distilled essential oils (A. crenulata and E. globulus) were screened individually and in combinations (double and triple plant combination) for activity against five respiratory pathogens using a microdilution assay. The antimicrobial interactions in combinations were assessed with the fractional inhibitory concentration (FIC) and the isobolograms. The organic extracts generally showed the highest antimicrobial activity with E. globulus having the highest activity with MIC values below 1 mg ml-1 representing noteworthy activity. The overall activity of the aqueous extracts was poor. The essential oil activity of E. globulus was mostly noteworthy (0.5 to 2 mg ml-1) while A. crenulata essential oil displayed moderate activity (1 to 4 mg ml-1). The ΣFIC values for double combinations (1:1) of A. crenulata with D. viscosa, A. crenulata with E. globulus and D. viscosa with E. globulus were calculated from the minimum inhibitory concentration (MIC) data and the interactions were classified as synergistic, additive, indifferent and antagonistic. The highest synergistic interactions observed were for a 1:1 combination of A. crenulata with E. globulus against K. pneumoniae, S. aureus and B. subtilis with ΣFIC values of 0.07. There was only one incident of antagonism noted in the study for D. viscosa with E. globulus (1:1) against C. neoformans with ΣFIC value of 4.25. The double combinations against selective pathogens (K. pneumoniae, S. aureus and E. coli) were further analysed for interactions using isobolograms. Mostly, the antimicrobial interactions as presented by the isobolograms were congruent with FIC results which further validated the occurrence of relevant antimicrobial interactions in those combinations. The ΣFIC values for triple combinations (1:1:1) revealed mostly synergistic interactions. When the triple combinations were analysed further against certain pathogens based on the predictions of the Design of Experiments software program (MODDE 9.1®), the MIC values remained the same despite the different combinations that were tested Thin layer chromatography (TLC) was used for a quick chemical fingerprinting of the plant extracts. This was followed by a bio-autographic assay. The chemical profiles of the organic extracts and essential oils from two of the study aromatic plants (A. crenulata and E. globulus) were further analysed with liquid chromatography mass spectrometry (LC-MS) and gas chromatography mass spectrometry (GC-MS) respectively. For combined plant extracts, a multivariate data analysis using principal component analysis (PCA) and hierarchical clustering analysis (HCA) was used to determine the relationship of the chemical make-up of combinations with that of individual plant extracts. According to the TLC analysis, E. globulus extracts had more compounds than the other two plants in the study. For the bio-autographic assay, E. globulus and combinations that included this plant showed greater inhibition zones than A. crenulata and D. viscosa. For the LC-MS analysis, PCA and HCA showed a close relationship between A. crenulata with D. viscosa, D. viscosa with E. globulus and the triple combination. Twenty one components were identified in the essential oil of A. crenulata representing 88.83% of the total oil composition. The oil was dominated by oxygen-containing monoterpenes (46.25%). In the essential oil of E. globulus, twenty six compounds were identified making up to 95.62% of the oil composition. Oxygen-containing monoterpenes (32.98%) also dominated the E. globulus essential oil. There was no great variation in essential oil metabolites of the individual plants and their combination as shown by both PCA and HCA. The enhanced in vitro antimicrobial activity and pharmacological interactions (synergy and additivity) in some of the combinations (double and triple) that were tested in this study adds scientific support to the use of medicinal plant combinations in Western Cape traditional medicine. The metabolic profiles of plants in combination might be unique due to interaction of the different plant bioactive molecules and thus result into defined antimicrobial activity. / AFRIKAANSE OPSOMMING: In tradisionele geneeskunde is dit ’n lank bestaande kultuur om kruiemiddels te kombineer om die terapeutiese werking daarvan te verhoog. Dié verbeterde mediese werking word toegeskryf aan die oënskynlik sinergistiese interaksies tussen verskillende bioaktiewe plantkomponente. Die doel van hierdie studie was om die farmakologiese interaksies in medisinale plantkombinasies van Agathosma crenulata, Dodonaea viscosa en Eucalyptus globulus te bestudeer. Daar is op die ontleding van hierdie spesifieke mengsel besluit omdat dit oor beduidende antibakteriese waarde beskik en omdat dit uit sewe medisinale plantmengsels wat voorheen bestudeer is, as die doeltreffendste een aangewys is. Die fitochemie van die plante is ook met behulp van chromatografiese ontleding beoordeel. Deur middel van ’n mikroverdunningstoets is die chloroform:metanol- (1:1; v/v-)ekstrakte of hidrogedistilleerde vlugtige olies (A. crenulata en E. globulus) individueel sowel as in kombinasie (dubbele en drievoudige plantkombinasies) nagegaan vir hul werking met betrekking tot vyf respiratoriese patogene. Die gekombineerde antimikrobiese interaksies is met behulp van fraksioneel stremmende konsentrasie (FIC) en isobologramme ondersoek. Die organiese ekstrakte het oor die algemeen die meeste antimikrobiese aktiwiteit by E. globulus getoon, met MIC-waardes onder 1 mg ml-1 wat as noemenswaardige aktiwiteit beskou is. Die algehele aktiwiteit van die waterekstrakte was swak. Die vlugtige-olieaktiwiteit van E. globulus was merendeels noemenswaardig (0,5 tot 2 mg ml-1), terwyl die vlugtige olie van A. crenulata matige aktiwiteit getoon het (1 tot 4 mg ml-1). Die ΣFIC-waardes vir dubbelkombinasies (1:1) van A. crenulata en D. viscosa, A. crenulata en E. globulus, en D. viscosa en E. globulus is uit die minimum stremmende konsentrasie (MIC) bereken en die interaksies is as sinergisties, additief, neutraal en antagonisties geklassifiseer. Die sterkste sinergistiese interaksies is by ’n 1:1-kombinasie van A. crenulata en E. globulus met betrekking tot K. pneumoniae, S. aureus en B. subtilis opgemerk, met ΣFIC-waardes van 0,07. Die studie het slegs een geval van antagonisme opgelewer, naamlik by D. viscosa en E. globulus (1:1) met betrekking tot C. neoformans, wat ’n ΣFIC-waarde van 4,25 geregistreer het. Die werking van die dubbelkombinasies met betrekking tot gekose patogene (K. pneumoniae, S. aureus en E. coli) is voorts met behulp van isobologramme vir interaksies nagegaan. Die antimikrobiese interaksies wat uit die isobologramme geblyk het, was meestal in pas met FIC-resultate, wat die bestaan van tersaaklike antimikrobiese interaksies in daardie kombinasies verder bevestig het. Die ΣFIC-waardes vir die drievoudige kombinasies (1:1:1) het meestal sinergistiese interaksies aan die lig gebring. Toe die drievoudige kombinasies verder op grond van die voorspellings van die sagteware Design of Experiments (MODDE 9.1®) met betrekking tot sekere patogene ontleed is, het die MIC-waardes onveranderd gebly, ondanks verskillende toetskombinasies. Dunlaagchromatografie (TLC) is vir ’n vinnige chemiese ontleding van die plantekstrakte gebruik en is gevolg deur ’n bio-outografiese toets. Die chemiese profiele van die organiese ekstrakte en vlugtige olies van twee van die aromatiese plante in die studie (A. crenulata en E. globulus) is verder met vloeistofchromatografie-massaspektrometrie (LC-MS) en gaschromatografie-massaspektrometrie (GC-MS) onderskeidelik ontleed. Vir gekombineerde plantekstrakte is veelveranderlike-ontleding in die vorm van hoofkomponentontleding (PCA) en hiërargiese groepsontleding (HCA) gebruik om die verhouding van die chemiese samestelling van kombinasies in vergelyking met dié van individuele plantekstrakte te bepaal. Volgens die TLC-ontleding beskik E. globulus-ekstrakte oor meer verbindings as die ander twee plante in die studie. Vir die bio-outografiese toets het E. globulus en kombinasies daarmee groter stremmingsones as A. crenulata en D. viscosa getoon. In die LC-MS-ontleding het PCA en HCA op ’n hegte verhouding tussen A. crenulata en D. viscosa, D. viscosa en E. globulus, en die drievoudige kombinasie daarvan gedui. Een-en-twintig komponente is in die vlugtige olie van A. crenulata gevind, wat 88,83% van die algehele oliesamestelling uitmaak. Die olie is deur suurstofhoudende monoterpene (46,25%) oorheers. Die vlugtige olie van E. globulus het 26 verbindings opgelewer, wat 95,62% van die oliesamestelling uitmaak. Suurstofhoudende monoterpene (32,98%) het ook die vlugtige olie van E. globulus oorheers. Nóg PCA nóg HCA het op enige beduidende variasie in die metaboliete van die vlugtige olies van die individuele plante en hul kombinasies gedui. Die verhoogde in vitro- antimikrobiese aktiwiteit en farmakologiese interaksies (sinergie en additiwiteit) in van die kombinasies (dubbel én drievoudig) wat in hierdie studie getoets is, bied wetenskaplike stawing vir die gebruik van medisinale plantkombinasies in Wes-Kaapse tradisionele geneeskunde. Die metaboliese profiele van plantkombinasies kan verander weens die interaksie van die verskillende bioaktiewe plantmolekules, en kan baie bepaalde antimikrobiese aktiwiteit tot gevolg hê.

Thesis (PhD (Chemistry and Polymer Science))--University of Stellenbosch, 2007. / In this thesis the main capillary electrodriven chromatographic techniques (i.e. Capillary Electrochromatography CEC, Micellar Electrokinetic Chromatography MEKC and Microemulsion Electrokinetic Chromatography MEEKC) were compared in terms of column manufacturing, fundamental chromatographic performance, and some applications were developed. The first stage of this thesis aimed at developing improved packed and open tubular CEC columns. For the manufacturing of packed CEC columns, the frit-burning step proved of critical importance, together with the slow build-up of the packed bed. The making of open tubular columns is a relatively simple, "one pot" sol-gel reaction taking place in mild conditions. The nature of the gel and the resulting selectivity of the column could easily be changed by changing the precursors. In a second stage of this thesis the packed and open tubular CEC columns were evaluated chromatographically and compared with the results obtained by MEKC and MEEKC. All electrodriven separation techniques showed high efficiencies. The selectivity proved easier to tune with sol-gel chemistry for the making of open tubular columns. Resolution is acceptable for packed CEC, MEKC and MEEKC. For peak capacity, CEC has the advantage of a practically non-limited elution time, while MEKC and MEEKC suffer of the drawback of the existence of an elution window which is limited in time by the elution of the micelles. Some applications were developed in this study on open tubular CEC columns and for the packed CEC columns. Various sugars derivatized with 9- aminopyrene-1,4,6-trisulfonic acid (APTS) could be separated with open tubular CEC, using an octyl, amino or cyano stationary phase. Open tubular columns containing α, β and γ cyclodextrins attached to the stationary phase were developed. This approach proved promising for the separation of positional isomers. A method was developed for the analyses of a mixture of carbamates and for several steroids with packed column CEC directly coupled with MS.

Thesis (PhD)--Stellenbosch University, 2015. / 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. / AFRIKAANSE OPSOMMING: Antosianiene is natuurlike pigmente wat verantwoordelik is vir die kleur van baie natuurlike produkte, insluitende dié van druiwe en wyn. Hierdie pigmente is belangrik vir die voedsel industrie en word gereken vir hul voedingswaarde aangesien hulle 'n belangrike rol speel in die verlaagde risiko van verskeie chroniese siektes onder die mens. Antosianiene speel ook 'n belangrike rol in die estetiese persepsie en kwaliteit van rooiwyn. Desnieteenstaande, as gevolg van die groot strukturele diversiteit van druifgeproduseerde antosianiene en die vele chemiese afgeleides wat uit hulle gevorm kan word tydens wyn bereiding en veroudering, is die akkurate analise van natuurlike wyn-pigmente uiters uitdagend. Omgekeerde-fase vloeistofchromatografie (RP-LC) word meestal gebruik vir die analise van antosianiene. Dié tegniek bied egter dikwels onvoldoende skeidingsvermoë vir komplekse mengsels van antosianiene en verwante molekules. Verder belemmer die onbeskikbaarheid van kommersiële standaarde en identiese massa spektrale eienskappe die identifikasie van hierdie verbindings. Die kombinasie van verskillende ortogonale skeidings meganismes in omvattende 2- dimensionele vloeistofchromatografie (LC×LC) bied egter 'n baie kragtiger benadering vir die skeiding van komplekse mengsels. Die huidige werk fokus dus op die ontginning van die potensiaal van LC×LC vir die verbeterde ontleding van antosianiene en verwante afgeleide pigmente in natuurlike produkte en wyn. Die eerste deel van hierdie werk het gefokus op die ontwikkeling van 'n hidrofiliese interaksie chromatografiese (HILIC) metode as ʼn alternatief vir RP-LC analise van antosianiene. Na uitgebreide optimisering, is gevind dat die metode geskik is vir die ontleding van 'n verskeidenheid van antosianiene in natuurlike produkte. Van groot belang is dat dit ook alternatiewe selektiwiteit in vergelyking met RP-LC demonstreer. Hierdie geoptimiseerde HILIC metode word dan voorts gebruik in kombinasie met RP-LC vir die ontwikkeling van ʼn af-lyn LC×LC benadering vir die analise van antosianiene. Hiervoor is half-minuut fraksies van die HILIC uitvloei opgevang en her-ingespuit op 'n RP-LC kolom. Dié af-lyn HILIC×RP-LC metode toon buitengewoon hoë skeidingsvermoë, gemeet in terme van die bereikbare praktiese piek kapasiteit, met baie verbindings wat geskei is in die twee dimensies wat saam elueer in 1-dimensionele HPLC. Interessant genoeg is groep-tipe skeiding ook waargeneem gebaseer op die graad en / of aard van glukosilasie en asilering van die antosianiene. In die laaste deel van die werk, is 'n sistematiese benadering gevolg vir die ontwikkeling en optimisering van ʼn aan-lyn HILIC×RP-LC deur gebruik te maak van 'n 10-poort oorskakelingsklep wat fraksies outomaties oordra tussen die twee kolomme. Die bogenoemde metode is ook verder gekoppel aan hoë resolusie massaspektrometrie (HR-MS) om ʼn gedetailleerde ondersoek van antosianiene en hulle afgeleide verbindings in wyn moontlik te maak. Vier en negentig pigmente is in een- en ses jaar oue Pinotage wyne geïdentifiseer gebaseer op HILIC×RP-LC skeiding in kombinasie met akkurate massa MS data en fragmentasie inligting. Beduidende verskille in veral die inhoud van antosianien-afgeleide pigmente is tussen die wyne waargeneem. Ter samevatting, die metodes ontwikkel in hierdie werk baan die weg om antosianien ontleding te verbeter en stel gevolglik die moontlikheid van selfs meer gedetailleerde studies van hierdie belangrike verbindings in natuurlike produkte in die vooruitsig.

published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy