Hydrophilic interaction chromatographic (HILIC) analysis of anthocyanins — ASN Events

Hydrophilic interaction chromatographic (HILIC) analysis of anthocyanins (#225)

Chandré M. Willemse 1 , Maria A. Stander 2 , André de Villiers 1
  1. Stellenbosch University, Stellenbosch, WC, South Africa
  2. Department of Microbiology and Biochemistry, Hochschule Geisenheim University, Geisenheim, Germany

Hydrophilic interaction chromatography (HILIC) provides an alternative separation mechanism to reversed phase liquid chromatography (RP-LC) for the analysis of phenolic compounds, in which a aqueous-organic mobile phases with polar stationary phases are used. In this contribution we present an evaluation of the applicability of HILIC for anthocyanin analysis. Anthocyanins are important natural phenolic pigments which are of interest because of their chromophoric properties and a range of health benefits associated with their consumption. While RP-LC is currently the method of choice for anthocyanin analysis, the unique chromatographic behaviour of these molecules, coupled to their large structural diversity, is responsible for the fact that complete chromatographic separation of complex anthocyanin mixtures by RP-LC is not always possible.

In the development of HILIC methods for anthocyanin analysis, several stationary phases and mobile phase combinations were evaluated. A 1.7 µm amide phase provided the best results, while the mobile phase composition and pH proved critically important to reduce on-column inter-conversion between different anthocyanin species. Optimised gradient HILIC methods were developed for the analysis of anthocyanins in blueberries, grape skins, black beans, red cabbage and red radish. Selective detection was performed using diode array detection, while high resolution quadrupole-time-of-flight mass spectrometry (Q-TOF-MS) was used for compound identification. A wide range of anthocyanins varying in the nature of anthocyanidin base as well as degree of glycosylation and acylation were tentatively identified. HILIC is shown to be a complementary separation method to RP-LC for anthocyanin analysis.