Tris(hydroxymethyl)aminomethane functionalized a novel stationary phase as a versatile mixed-mode HPLC platform for complex analyte separation and retention mechanism elucidation

A novel mixed-mode stationary phase functionalized with tris(hydroxymethyl)aminomethane was developed for efficient separation of structurally diverse analytes through combined reversed-phase liquid chromatography (RPLC), hydrophilic interaction chromatography (HILIC), and ion-exchange mechanisms. The material was synthesized via a simple two-step surface modification using an epoxy silane, resulting in a robust phase containing both hydrophilic and hydrophobic domains. Comprehensive characterization by FTIR, SEM, BET, and elemental analysis confirmed the successful functionalization and high structural integrity of the phase. The column exhibited excellent separation performance for various classes of compounds, including polycyclic aromatic hydrocarbons, alkylbenzenes, benzoic acids, phenols, anilines, Sudan dyes, and nucleobases/nucleosides. Notably, a nine-component nucleobase/nucleoside mixture was baseline resolved within 18 min under HILIC conditions and could also be effectively separated under RPLC conditions with a water-rich mobile phase (95 % H2O, 5 % ACN), demonstrating remarkable dual-mode retention for highly polar compounds. Eight benzoic acids were also completely separated within 7 min, indicating rapid and efficient analysis. The column provided high efficiency (over 40,000 plates/m) and excellent peak symmetry (As values near 1.0). Retention mechanisms were elucidated using quantitative structure-retention relationship (QSRR) modeling based on logP, logD, and logS descriptors, highlighting the synergistic contributions of hydrophobic, hydrogen bonding, and electrostatic interactions. Comparative experiments with a commercial ACE C18 column (RPLC) and a Kromasil-amino column (HILIC) further validated the superior mixed-mode performance. Overall, this study demonstrates the versatility and high separation capability of the developed stationary phase, indicating its strong potential for complex pharmaceutical, biochemical, and environmental analyses.