Synthesis of a novel multifunctional stationary phase derived from diethanolamine, HILIC/RPLC/WAX mixed-mode HPLC applications, and investigation of the retention mechanism

Abstract

Mixed-mode chromatography is increasingly valued for retaining analytes with diverse polarity and charge by integrating hydrophilic interaction (HILIC), reversed-phase (RPLC), and ion-exchange mechanisms. However, designing stationary phases that are both easy to synthesize and chromatographically versatile remains challenging. This study presents DEA-Mix-SP, a novel silica-based stationary phase functionalized with diethanolamine via [2-(3,4-epoxycyclohexyl)ethyl]trimethoxysilane, offering a simple synthetic route for broad-spectrum separation. Three variants with different ligand densities were characterized by elemental analysis, FTIR, SEM, and BET. The columns showed excellent separation for various analytes: nine nucleobases/nucleosides were resolved within 13 min, achieving up to 35,000 theoretical plates per meter, and eight benzoic acid derivatives were fully separated in 9 min with efficiencies up to 50,000 N/m. Alkylbenzenes, PAHs, Sudan dyes, phenols, and anilines were also effectively separated. Retention mechanisms were studied using quantitative parameters such as logD, logS, and pK_a, confirming significant anion-exchange effects. Performance was compared with ACE C18 (RPLC) and aminopropyl (HILIC) columns: DEA-Mix-SP showed superior separation performance in HILIC over the aminopropyl column and better separation of phenols, benzoic acids, and anilines than ACE C18, while achieving comparable results for fully non-polar analytes like alkylbenzenes, Sudan dyes, and PAHs. DEA-Mix-SP thus combines cost-effectiveness and high mixed-mode selectivity, providing reliable separation for analytes with wide polarity and charge ranges, making it a strong option for pharmaceutical, environmental, and food analyses.

Graphical Abstract