Comprehensive Two-dimensional Liquid Chromatography Coupled to the ABTS Radical Scavenging Assay: a Powerful Method for the Analysis of Phenolic Antioxidants

The on-line combination of comprehensive two-dimensional liquid chromatography (LC × LC) with the 2,2'-azino-bis(3-ethylbenzothiazoline)-6 sulphonic acid (ABTS) radical scavenging assay was investigated as a powerful method to determine the free radical scavenging activities of individual phenolics in natural products. The combination of hydrophilic interaction chromatography (HILIC) separation according to polarity and reversed-phase liquid chromatography (RP-LC) separation according to hydrophobicity is shown to provide much higher resolving power than one-dimensional separations, which, combined with on-line ABTS detection, allows the detailed characterisation of antioxidants in complex samples. Careful optimisation of the ABTS reaction conditions was required to maintain the chromatographic separation in the antioxidant detection process. Both on-line and off-line HILIC × RP-LC-ABTS methods were developed, with the former offering higher throughput and the latter higher resolution. Even for the fast analyses used in the second dimension of on-line HILIC × RP-LC, good performance for the ABTS assay was obtained. The combination of LC × LC separation with an on-line radical scavenging assay increases the likelihood of identifying individual radical scavenging species compared to conventional LC-ABTS assays. The applicability of the approach was demonstrated for cocoa, red grape seed and green tea phenolics.