HPLC separation of sulfanamide drugs with C12-hydroxy mixed-mode column, investigation of mixed mode separation mechanism with quantitative parameters

Sulfonamides exhibit broad-spectrum antibacterial effects. However, their extensive use poses health risks, such as antimicrobial resistance. Therefore, advanced and accurate analytical techniques to monitor the presence of sulfonamide residues are needed. In this study, a mixture of eight sulfonamides was separated on a C12-diol mixed-mode column containing both a hydrophobic C12 chain and a hydrophilic diol group. The influence of various experimental parameters, including pH, buffer concentration, mobile phase composition, temperature, and flow rate, on the separation efficiency of the column was investigated. All eight sulfonamides were separated within 20 min via gradient elution with an acetonitrile/phosphate buffer (pH 6.5, 40 mM KH 2 PO 4 /K 2 HPO 4 ) solution at 15 °C and a detection wavelength of 250 nm. Examination of the retention times of the analytes at different pH values ranging from 3 to 7 revealed that the sulfonamides were significantly affected by the acidity of the medium, thus enabling a detailed discussion of the mixed-mode separation mechanism. The interaction mechanism between the eight sulfonamides and the C12-diol mixed-mode column was explored using quantitative structure–retention relationship parameters such as logD, logS, and pKa. Although the interaction of the sulfonamides with the C12-diol mixed-mode column partly showed the classic reverse-phase feature, polar interactions played a major role, and the mixed-mode interaction mechanism was dominated by polar and apolar interactions. The obtained results were compared with a commercial ODS-C18 column in terms of both separation performance and retention behavior.