Insights from molecular simulations about dead time markers in reversed-phase liquid chromatography

Among the most popular compounds to estimate the hold-up time in reversed-phase liquid chromatography (RPLC) are acetone and uracil, which are considered as too small and too polar, respectively, for retention by the hydrophobic stationary phase, although their observed elution behavior does not fully support this assumption. We investigate how acetone and uracil as solutes interact with the chromatographic interface through molecular dynamics simulations in an RPLC mesopore model of a silica-supported, endcapped, C₁₈ phase equilibrated with a water (W)‒acetonitrile (ACN) mobile phase. The simulation results provide a molecular-level explanation for the observed elution behavior of acetone and uracil, but also question whether true dead time markers for RPLC exist. Both solutes have a density maximum in the interfacial region in addition to a low presence in the bonded-phase region, but these density peaks clearly differ from the adsorption and partitioning peaks of true analytes. Acetone partially behaves like a co-solvent of ACN and partially like the analyte acetophenone. Like ACN, acetone can be found in the first and second layer of solvent molecules at the silica surface; like acetophenone, acetone adsorbs to the bonded-phase chains by orienting its polar group to the bulk region to sustain hydrogen bonds with W molecules. Uracil behavior is governed by a need for extensive hydrogen-bond coordination by W molecules. Uracil adsorbs to the very edge of the bonded-phase chains, on the bulk-region side of the ACN density maximum in the interfacial region. Further penetration into the chains is prevented by the absence of W molecules, which are not found deeper in the bonded phase, except at the silica surface. Contrary to true analytes, accumulation of uracil and acetone in the interfacial region ceases at an equimolar presence of W and ACN in the mobile phase (at 70‒80% ACN volume fraction). Uracil achieves a closer approximation of the stationary-phase limit than acetone, but carries the risk of HILIC retention at high ACN fraction in the mobile phase.