Comprehensive two-dimensional liquid chromatographic analysis of anthocyanins
| dc.contributor.advisor | De Villiers, Andre J. | en_ZA |
| dc.contributor.advisor | Stander, Maria A. | en_ZA |
| dc.contributor.author | Willemse, Chandre Monique | en_ZA |
| dc.contributor.other | Stellenbosch University. Faculty of Science. Dept. of Chemistry and Polymer Science. | en_ZA |
| dc.date.accessioned | 2015-05-20T09:13:42Z | |
| dc.date.available | 2015-05-20T09:13:42Z | |
| dc.date.issued | 2015-04 | en |
| dc.description | Thesis (PhD)--Stellenbosch University, 2015. | en_ZA |
| dc.description.abstract | 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. | en_ZA |
| dc.description.abstract | 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. | af_ZA |
| dc.format.extent | x, 30, 8 pages : colour illustrations | en_ZA |
| dc.identifier.uri | http://hdl.handle.net/10019.1/96675 | |
| dc.language.iso | en_ZA | en_ZA |
| dc.publisher | Stellenbosch : Stellenbosch University | en_ZA |
| dc.rights.holder | Stellenbosch University | en_ZA |
| dc.subject | Colour pigments, food industry, chronic diseases in humans | en_ZA |
| dc.subject | Anthocyanins | en_ZA |
| dc.subject | Chromatographic analysis | en_ZA |
| dc.subject | Food -- Analysis | en_ZA |
| dc.subject | UCTD | en_ZA |
| dc.title | Comprehensive two-dimensional liquid chromatographic analysis of anthocyanins | en_ZA |
| dc.type | Thesis | en_ZA |