A ternary eluent strategy to tune the peak shape of steviol glycosides in reversed-phase liquid chromatography.

In this paper, an effective strategy of using acetonitrile-methanol-water as mobile phase was developed to achieve acceptable peak shape of steviol glycosides in reversed-phase liquid chromatography (RPLC). The change of elution profiles of rebaudioside A (RA) was systematically investigated. Two classical distributions, namely, tailing and fronting peaks resulting from injections of RA solution in range of 0.5-5 mg were both observed in a ternary eluent of acetonitrile-methanol-water (21:43:36, v/v). Next, a three-phase diagram of tailing factor (Tf) was illustrated, showing high dependence of elution profile of RA on the ternary composition. The peak shape of RA can be adjusted mainly based on the additive effect, that is, acetonitrile is more strongly adsorbed to the stationary phase than RA in the pure weak solvent (H2O). Therefore, with the increase of acetonitrile concentration in the ternary eluent, the RA band profiles went from being tailing to fronting shapes. At the same time, due to the large RA-RA interactions, there was anti-Langmuir adsorption isotherm in acetonitrile-water mobile phase, which is the reason for the fronting peaks of RA. It could be concluded that the way of using the ternary eluent of acetonitrile-methanol-water does control and tune the peak shape of steviol glycosides in RPLC separation.