Comprehensive two-dimensional liquid chromatographic analysis of phenolics

[摘要] ENGLISH ABSTRACT: Phenolic chemistry is quite complex; natural phenolic compounds vary widely in terms of size andchemical properties. The high structural diversity within this family presents severe analyticalchallenges. High performance liquid chromatography (HPLC) is the preferred method for phenolicanalysis; however, conventional HPLC methods offer limited separation power and often provideincomplete separation of the large number of components present in natural phenolic extracts.Multi-dimensional chromatographic techniques have proven much more effective in the analysis ofcomplex samples. The current study explored the potential of comprehensive two-dimensionalliquid chromatography (LC×LC) for the characterisation of phenolic compounds in complex naturalproducts, with the emphasis on proanthocyanidins (PACs).Initial work focused on the evaluation of the state of the art in phenolic analysis, to allowinformation which was used in the development of optimal 1-D separations for use in LC×LC. Thecombination of hydrophilic interaction chromatography (HILIC) in the first dimension with reversedphaseliquid chromatography (RP-LC) in the second dimension afforded an orthogonal andpowerful separation system for phenolics, providing separation on the basis of hydrophilicity andhydrophobicity, respectively. A detailed and systematic procedure was therefore developed toallow 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 thanconventional 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 stopflowmethods 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 broadeningfor procyanidins (PCs), implying that this method is an attractive alternative to the off-line methodas it offers automation and minimal sample handling. Experimental verification of the predictionsbased on fundamental principles confirmed the validity of the optimisation procedure for cocoaPCs.The hyphenation of on-line HILIC×RP-LC separation with fluorescence (FL) and massspectrometry (MS) detection methods provided enhanced resolution in a practical analysis timewith the added benefit of selective detection and greater certainty in compound identification. Thisstrategy proved much more powerful, as demonstrated by the identification of the highly complexPACs in grape seeds based on chromatographic retention data in two dimensions and accuratemass information. It was further shown that on-line coupling of HILIC×RP-LC separation with anoptimised radical scavenging assay provides an improved approach for screening of individualradical scavengers in complex phenolic fractions, as demonstrated for cocoa, grape seed andgreen tea extracts.

[发布机构] Stellenbosch University