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 reversedphase 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 NH2 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.

Automated summary

In this study, hydrophilic interaction liquid chromatography (HILIC) methods and a two-dimensional reversed-phase liquid chromatography/HILIC (2D-RPLC/HILIC) for the separation of steviol glycosides (SGs) were developed. The PA column exhibited superior hydrophilicity, higher selectivity, and better resolution. HILIC-UV methods with water/acetonitrile or water/ethanol as elution systems were developed for the separation of SGs. The preparative separation of minor SGs from stevia extracts was successfully achieved using 2D-RPLC/HILIC.