Retention of stevioside polar compounds on a sulfonic acid-functionalized stationary phase

To explore the retention and separation of stevioside polar compounds on a sulfonic acid-functionalized cation exchange column, the effects of different organic solvent-water mobile phases on the retention behavior of polar rebaudioside A (RA) and its analogues on the column were investigated over a wide range of organic solvent contents. The obtained U-shape curves hinted that the retention of the compounds on the same column transitioned from a reversed-phase liquid chromatography (RPLC) mode to a hydrophilic interaction liquid chromatography (HILIC) mode when the water-rich state in the mobile phases changed to an organic solvent-rich state. Under the RPLC mode, no separation of RA from its analogues was observed. The HILIC mode was beneficial to the retention and separation of RA and its analogues. Compared with polar protic solvents, aprotic solvents were more conducive to the retention and separation of the polar compounds based on the HILIC mode in organic solvent-rich mobile phases. Three models were used to evaluate and discuss the HILIC retention and separation of the compounds on the column. In the aprotic solvent-rich mobile phase, the HILIC retention of RA and its analogues was effectively described by a mixed-mode model; in the polar proton solvent-rich mobile phase, the retention of analytes was best described by an linear solvation strength (LSS) model. The content and composition of the organic solvent in the mobile phase were determined to be important influencing factors that regulated the retention time for the RA and its analogues, and even the separation mechanism for HILIC. The present work provides a theoretical basis for guiding one to prepare high-purity RA from its analogues by predicting the retention time.