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.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/85749 |
| Date | 12 1900 |
| Creators | Kalili, Kathithileni Martha |
| Contributors | De Villiers, Andre, 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 | English |
| Type | Thesis |
| Format | 1 v. (various pagings) : ill. (some col.) |
| Rights | Stellenbosch University |
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