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.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/96675 |
| Date | 04 1900 |
| Creators | Willemse, Chandre Monique |
| Contributors | De Villiers, Andre J., Stander, Maria A., Stellenbosch University. Faculty of Science. Dept. of Chemistry and Polymer Science. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | Unknown |
| Type | Thesis |
| Format | x, 30, 8 pages : colour illustrations |
| Rights | Stellenbosch University |
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