Preparation and investigation of two-component silica-modified hydrophobic layer for minimizing retention loss of reversed-phase chromatographic columns using fully aqueous mobile phases

Abstract

Chromatographers often employ fully aqueous mobile phases to retain highly polar compounds in reversed-phase liquid chromatography (RPLC). However, when the flow rate is interrupted, either accidentally or intentionally, a substantial loss in retention occurs due to the spontaneous dewetting of water from the hydrophobic surface of conventional RPLC-C18 particles. Previous studies have shown that maintaining a low C18 surface coverage (approximately 1.5 μmol/m2) can mitigate water dewetting by increasing chain disorder, facilitating the intercalation of water clusters between the C18-bonded chains, and keeping the mesopores wetted. In this research, we explore the potential and additional benefits of using two-component surface bonding materials (C8/C18 and PhenylHexyl (PhHx)/C18) at a constant and low total surface coverage of 1.51 ± 0.15 μmol/m2. We synthesized seven one- and two-component modified silica particles with a volume average particle size of 5.22 μm and an average mesopore size of 104 Å. The surface coverage was increased from 0 to 0.54, 1.00, and to 1.66 μmol2 for C8 chains and from 0 to 0.52, 0.70, and to 1.65 μmol2 for PhHx ligands. To prevent interactions between water and any unreacted silanols, all seven derivatized particles were heavily endcapped with trimethylsilane (TMS) reagent. The fraction of the surface area remaining in contact with water was determined by measuring the retention times of weakly (thiourea) and strongly (thymine) retained compounds at intervals of 1, 2, 4, 8, 16, 32, and 64 minutes following the cessation of flow. Two distinct column temperatures, 24°C and 60°C, were employed in the experiments. Retention losses were found to be minimized in the presence of a small quantity of C8 chains (less than 40% of the total surface coverage). Additionally, it is essential to consider substantial fractions of PhHx chains, as long as the presence of the PhHx ligand does not significantly impact retention and selectivity. Combining mixed RPLC bondings with a low total surface coverage of approximately 1.5 μmol/m2 emerges as a viable solution for further minimizing retention loss in standard C18-bonded RPLC columns, particularly within the surface coverage range of 2.5-3.0 μmol/m2.