Separation of minor steviol glycosides using hydrophilic interaction liquid chromatography (HILIC) and off-line two-dimensional reversed-phase liquid chromatography/HILIC methods

Steviol glycosides (SGs) are increasingly used as noncaloric sweeteners. In this study, hydrophilic interaction liquid chromatography (HILIC) methods and an off-line environmentally friendly two-dimensional reversed-phase liquid chromatography/HILIC (2D-RPLC/HILIC) for the separation of SGs were developed. The retention behavior on the polyacrylamide-based silica (PA) column was investigated by separating twelve SGs and comparing with three HILIC columns (Diol, XAmide and Unitary NH 2 columns). Ethanol instead of acetonitrile as the weak eluent in HILIC mode was investigated. The PA column exhibited superior hydrophilicity, higher selectivity and better resolution. Two HILIC-UV methods for separation of SGs on a PA column with water/acetonitrile (LOD, 0.0049–0.013 mg/mL; RSD <3.73%; recovery, 95.02–102.5%) or water/ethanol (LOD, 0.010–0.016 mg/mL; RSD <4.52%; recovery, 91.90–97.76%) as elution system were developed. The preparative separation of minor SGs from stevia extracts was investigated by 2D-RPLC/HILIC using a preparative C18 column with the methanol/water mobile phase in the first dimension and a preparative PA column with the ethanol/water mobile phase in the second dimension. Two SGs were isolated and identified. In summary, safer and cheaper ethanol/water binary eluent system increased the diversity of HILIC retention and improved the solubility of SGs, which is greatly beneficial at a preparative scale in the food industry. • Twelve steviol glycosides were separated on four kinds of HILIC column. • EtOH/H 2 O eluent system was feasible on a polyacrylamide-based silica column. • Two HILIC-UV methods were developed and validated for steviol glycosides analysis. • 2D-RPLC/HILIC were used for the preparative separation of polar steviol glycosides.