A 1,4-diazabicyclo[2.2.2]octane-based amphiphilic sulfobetaine stationary phase for versatile mixed-mode liquid...

A novel amphiphilic sulfobetaine-type zwitterionic stationary phase, designated as Sil-C8-DSB, was fabricated via a facile thiol-ene click strategy by anchoring sulfobetaine-functionalized 1,4-diazabicyclo[2.2.2]octane ligands onto thiol-functionalized silica through hydrophobic octyl spacers. ¹H and ¹³C NMR spectroscopy supported the proposed structures of the mono-quaternized intermediate and the C8-DSB ligand, while Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, and elemental analysis supported the surface functionalization of Sil-C8-DSB. The retention behavior was investigated using polar, ionizable, and non-polar probe compounds. The effects of acetonitrile content, mobile-phase pH, salt concentration, and temperature indicated combined hydrophilic-interaction, ion-exchange, and reversed-phase retention contributions. Sil-C8-DSB resolved a four-component mixture containing neutral, basic, and acidic probes, whereas co-elution occurred on the commercial HILIC-Z and VP-ODS reference columns under the respective tested conditions. Application to a commercial Sprite sample yielded a sodium benzoate content of 140.2 mg L⁻¹, with recoveries of 97.05-99.14%. Same-column intra-day and inter-day retention-time RSDs were 0.21-0.52% and 0.05-0.76%, respectively, while a 30-day storage test showed no progressive loss of column efficiency. Following a 4 h stop-flow treatment in 50 mM aqueous ammonium acetate, retention times and theoretical plate numbers changed by <1%, with baseline separation maintained. These results support the mixed-mode selectivity, analytical applicability, and operational stability of Sil-C8-DSB under the evaluated conditions.