Effects of the operation parameters on HILIC separation of flavonoids on zwitterionic column

The hydrophilic interaction chromatography (HILIC) coupled to mass spectrometry was employed to study retention behavior of several flavonoids from their different groups using the polymeric zwitterionic stationary phase (ZIC-pHILIC). It contains sulfobetaine-bonded ligand with an inner positively charged quaternary ammonium and an outer negatively charged sulfonate functional groups. Two organic solvents - acetonitrile (ACN) and methanol (MeOH) - were compared as a component of mobile phase. Separation parameters such as a content of organic modifier, pH of an eluent and a column temperature were studied. Retention of flavonoids is controlled primarily by a partition between the mobile phase eluent and a water-enriched layer on the hydrophilic stationary phase with some contribution from hydrogen bonding formation. Using MeOH, in contrast to ACN, strongly retained compounds (myricetin, morin, rutin and quercetrin) could be eluted under isocratic conditions. A better sensitivity was achieved with MeOH as mobile phase component, particularly for quercetin, naringenin and kaempferol. The method was applied to the determination of flavonoids in fruit juices.