Comprehensive two-dimensional liquid chromatographic analysis of phenolics

Kalili, Kathithileni Martha (2013-12)

Thesis (PhD)--Stellenbosch University, 2013.

Thesis

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.

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