Development of a new HPLC column based on tris (hydroxymethyl) aminomethane, mixed-mode HPLC applications and investigation of structure-interaction relationships

A novel mixed-mode stationary phase functionalized with tris(hydroxymethyl)aminomethane was developed for the efficient separation of structurally diverse analytes through the combined retention mechanisms of reversed-phase liquid chromatography (RPLC), hydrophilic interaction liquid chromatography (HILIC), and ion-exchange interactions. The material was synthesized via a straightforward two-step surface modification using epoxy silanes, resulting in a chemically stable phase containing both hydrophilic and hydrophobic domains. Its structural integrity was confirmed by Fourier-transform infrared spectroscopy (FTIR), solid-state nuclear magnetic resonance (NMR), scanning electron microscopy (SEM), Brunauer–Emmett–Teller (BET) surface area analysis, and elemental analysis. The column exhibited excellent separation performance for a broad range of analytes, including polycyclic aromatic hydrocarbons (PAHs), alkylbenzenes, benzoic acids, phenols, anilines, Sudan dyes, and nucleobases/nucleosides. Remarkably, a nine-component nucleobase/nucleoside mixture was baseline-resolved within 18 minutes under HILIC conditions and also successfully separated under reversed-phase conditions using a water-rich mobile phase (95% H₂O, 5% ACN). This unexpected RPLC compatibility for highly polar compounds emphasizes the column's dual retention capability and highlights its versatility as an effective mixed-mode platform. Furthermore, eight benzoic acid derivatives were completely resolved in under 7 minutes, demonstrating the column's capacity for rapid and efficient separations. Column efficiency values (N/m) exceeded 40,000 for several analytes, and peak asymmetry factors (As) consistently approached 1.0, indicating high chromatographic performance. Retention mechanisms were further elucidated using quantitative structure–retention relationship (QSRR) models based on logP, logD, and logS parameters, which revealed that hydrophobic interactions, hydrogen bonding, and electrostatic forces collectively governed analyte retention. These findings validate the multifunctionality of the functionalized stationary phase and underscore its strong potential for pharmaceutical, biochemical, and environmental HPLC applications.