Application of comprehensive 2-dimensional liquid chromatography for the analysis of complex phenolic fractions

[摘要] ENGLISH ABSTRACT:The separation of apple, cocoa and green tea phenolic compounds by comprehensive2-dimensional liquid chromatography (2-D-LC) has been studied. In the firstdimension, phenolic compounds were separated according to polarity by hydrophilicinteraction chromatography (HILIC) on a diol stationary phase with a mobile phasecontaining acetonitrile, methanol, acetic acid and water. Gradient reversed-phase (RP)LC using a C18 column with fluorescence detection was employed in the seconddimension to separate compounds according to hydrophobicity. Compounds wereidentified using negative electrospray ionisation mass spectrometry (ESI-MS) coupledto both HILIC and RP separations.The coupling of HILIC and RP separations proved to be especially beneficialsince this provided simultaneous information on both the polarity and hydrophobicityof phenolics. The low degree of correlation (r2 < 0.21) between the two LC modesafforded peak capacities in excess of 3000 for the off-line method. An on-line methodwas also developed utilizing a short, small particle-packed column to provide fastseparation in the second dimension. A 1 mm i.d. column was used in the firstdimension for the on-line system to reduce injection volumes onto the seconddimension column. A significantly lower practical peak capacity was measured forthe on-line system, due largely to the reduction in second dimension peak capacity.On the other hand, analysis could be performed in an automated fashion using the onlinesystem reducing the risk of sample alteration and guaranteeing better operationreliability and reproducibility. Especially the off-line comprehensive HILIC × RP-LCmethod developed demonstrated its utility in the analysis of various groups ofphenolic compounds including proanthocyanidins, phenolic acids, flavonols andflavonol conjugates in a variety of natural products.

[发布机构] Stellenbosch University