Separation of Steviol Glycosides by Hydrophilic Liquid Interaction Chromatography

Stevia rebaudiana Bertoni contains several steviol glycosides with sweet flavour. They all are sweeter than sucrose (up to factor 450). The various steviol glycosides are difficult to separate by reversed-phase chromatography. In this paper, five different hydrophilic liquid interaction chromatography columns are characterized using isocratic elution (5-20% water in acetonitrile with buffer or formic acid). Separation of the steviol glycosides is possible with all but one of the tested columns, but the robustness of the separation against changes of buffer concentration and percentage of water differ. Aqueous percentage and ion strength of the eluent are the main factors to be optimized in method development.