Separation of polar and nonpolar compounds using HILIC/RPLC mixed-mode chromatography: evaluation of separation performance and retention mechanism of the Acclaim Mixed-Mode HILIC-1 column

Abstract The Acclaim Mixed-Mode HILIC-1 column, combining hydrophilic interaction (HILIC) and reversed-phase liquid chromatography (RPLC) mechanisms, enables efficient separation of diverse analytes. This study examined its separation performance and mixed-mode retention behavior using both polar (e.g., nucleobases, nucleosides, water-soluble vitamins), and nonpolar compounds (e.g., alkylbenzenes, PAHs, Sudan dyes, substituted phenols, aniline derivatives, plant growth hormones, chlorinated herbicides). Mobile phase composition was tailored to analyte polarity: high acetonitrile for HILIC and high water content for RPLC. Detailed optimization for nucleobase and nucleoside separation was conducted by varying pH, buffer concentration, solvent ratio, temperature, and flow rate under HILIC conditions. Polar analytes showed increased retention with higher acetonitrile, while nonpolar ones retained strongly in aqueous-rich conditions. The column demonstrated excellent selectivity tuning by mobile phase adjustment and provided sharp, symmetrical peaks across all analyte groups. Under optimized conditions, resolution and peak quality were high. Comparative analysis with literature confirmed that this column’s performance is on par with or superior to existing systems. The results affirm the column’s versatility in handling a broad analyte spectrum and elucidate the dual-mode retention mechanism it employs, offering valuable insights for complex mixture analyses.