Organic-solvent ditch overlap in reversed-phase liquid chromatography: A molecular dynamics simulation study in cylindrical 6-12 nm-diameter pores.

Mass transport through the mesopore space of a reversed-phase liquid chromatography (RPLC) column depends on the properties of the chromatographic interface, particularly on the extent of the organic-solvent ditch that favors the analyte surface diffusivity. Through molecular dynamics simulations in cylindrical RPLC mesopore models with pore diameters between 6 and 12 nm we systematically trace the evolution of organic-solvent ditch overlap due to spatial confinement in the mesopore space of RPLC columns for small-molecule separations. Each pore model of a silica-based, endcapped, C18-stationary phase is equilibrated with two mobile phases of comparable elution strength, namely 70/30 (v/v) water/acetonitrile and 60/40 (v/v) water/methanol, to consider the influence of the mobile-phase composition on the onset of organic-solvent ditch overlap. The simulations show that, as the pore diameter decreases from 9 to 6 nm, the bonded-phase density extends and compacts towards the pore center, which leads to increased accumulation of organic-solvent excess and thus enhanced organic-solvent diffusivity in the ditch. Because the acetonitrile ditch is more pronounced than the methanol ditch, acetonitrile ditch overlap sets in at less severe spatial confinement than methanol ditch overlap. The pore-averaged methanol and acetonitrile diffusivities are considerably raised by ditch overlap in the 6 nm-diameter pore, but also benefit from the ditch (without overlap) in the 7 to 12 nm-diameter pores, whereby local and pore-averaged effects are generally larger for acetonitrile than methanol.

The Solvation Shell of Small Solutes in Aqueous-Organic Solvent Mixtures and Its Implications for Reversed-Phase Liquid Chromatography.

Reversed-phase liquid chromatography (RPLC) operates with water-organic solvent (W-OS) mobile phases where preferential solvation (PS) of solutes is likely. To investigate the relevance of the solute solvation shell in the mobile phase for RPLC retention, we combine data from molecular dynamics simulations of small, neutral solutes (six analytes and two dead time markers) in W-methanol (MeOH) and W-acetonitrile (ACN) mixtures with corresponding retention data obtained on an RPLC column over a wide range of W/OS ratios. Data derived from Kirkwood-Buff integrals show PS by the OS for analytes vs low or negative PS for dead time markers. W-ACN mixtures generate a higher amount of PS than W-MeOH mixtures, which contributes to the higher eluent strength of ACN in RPLC. Difference spatial distribution functions reveal anisotropic solvation shells with OS excess at hydrocarbon elements and W excess at functional groups, predicting that retention by the hydrophobic stationary phase is favored by hydrocarbon elements and limited by functional groups. Analysis of solute-solvent hydrogen bonds pinpoints the hydrogen-bond requirements toward W as the retention-limiting factor. The relation between the solute solvation shell and retention confirms the importance of W-OS and solute-W hydrogen bonding for RPLC retention.

Mobile-Phase Contributions to Organic-Solvent Excess Adsorption and Surface Diffusion in Reversed-Phase Liquid Chromatography.

Fast transport of retained analytes in reversed-phase liquid chromatography occurs through surface diffusion in the organic-solvent (OS)-enriched interfacial "ditch" region between the hydrophobic stationary phase and the water (W)-OS mobile phase. Through molecular dynamics simulations that recover the OS excess adsorption isotherms of a typical C18-stationary phase for methanol and acetonitrile, we explore the relation between OS properties, OS excess adsorption, and surface diffusion. The emerging molecular-level picture attributes the mobile-phase contribution to surface diffusion to the hydrogen-bond capability and the eluting power of the OS. The higher affinity of methanol for the formation of W-OS hydrogen bonds at the soft, hydrophobic surface presented by the bonded-phase (C18) chains reduces the OS excess and the related viscosity drop in the ditch. The lower eluting power of methanol, however, translates to increased bonded-phase contacts for analytes, which can increase their mobility gain from surface diffusion above the gain observed with acetonitrile.

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.