Synthesis of a novel diethanolamine-derived stationary phase, mixed-mode HPLC applications, and investigation of the interaction mechanism

Mixed-mode chromatography offers a versatile separation approach by integrating multiple interaction mechanisms between analytes and the stationary phase. Unlike traditional single-mode techniques, this method allows for the simultaneous separation of compounds with varying functional groups—such as hydrophobic and ionic moieties—within a single chromatographic run. In this study, a novel silica-based stationary phase was synthesized using diethanolamine as the functional ligand. This ligand introduces both polar and nonpolar characteristics, enabling a mixed-mode retention mechanism that combines hydrophilic interaction (HILIC), reversed-phase (RPLC), and ion-exchange (IEC) chromatography. The retention behaviour of this phase was assessed using quantitative structure-retention relationship (QSRR) parameters, including logP, logD, logS, and pKa values. The stationary phase was prepared through a straightforward and cost-efficient procedure, utilizing [2-(3,4-epoxycyclohexyl)ethyl]trimethoxysilane as the coupling agent. To evaluate the effect of ligand density, three variants with differing carbon loadings were synthesized and packed into standard HPLC columns. Performance testing was conducted to identify the most effective composition, followed by comprehensive chromatographic evaluations. Characterization of the synthesized phases was carried out using elemental analysis, Fourier-transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), and Brunauer–Emmett–Teller (BET) surface area measurements. The optimized column demonstrated excellent performance across a broad spectrum of analytes, from highly polar to strongly hydrophobic compounds. It achieved baseline separation of nine polar nucleobase and nucleoside analytes within 15 minutes. Furthermore, it efficiently resolved complex mixtures, including alkylbenzenes (5 compounds), polyaromatic hydrocarbons (PAHs) (4 compounds), Sudan dyes (7 compounds), benzoic acids (8 compounds), phenols (8 compounds) and anilines (9 compounds). The column exhibited a high theoretical plate number (N/m) exceeding 49000 and a peak asymmetry factor close to 1.00, indicating excellent chromatographic efficiency and peak shape. In order to examine the retention mechanism in detail, the effect of experimental conditions such as pH, mobile phase content, temperature, flow rate, buffer concentration on separation was studied for nucleobases/nuclesides in the HILIC phase and benzoic sites in the RPLC/IEC phase. These results highlight the strong potential of the developed mixed-mode stationary phase for comprehensive analytical applications involving diverse analyte classes.