Separation of sulphonamides on a C12-diol mixed-mode HPLC column and investigation of their retention mechanism

Abstract Sulphonamides exhibit broad-spectrum antibacterial effects. However, their extensive use poses health risks, such as antimicrobial resistance. Therefore, advanced and accurate analytical techniques are needed to monitor the presence of sulphonamide residues. In this study, a mixture of eight sulphonamides 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 sulphonamides 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 sulphonamides 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 sulphonamides and the C12-diol mixed-mode column was explored using quantitative structure–retention relationship parameters such as log D , log S , and p K a . Although the interaction of the sulphonamides 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 results obtained were compared with a commercial ODS-C18 column in terms of both separation performance and retention behaviour.