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.

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.

Prediction of surface excess adsorption and retention factors in reversed-phase liquid chromatography from molecular dynamics simulations.

An alternative method to the classical fit of semi-empirical, statistical, or artificial intelligence-based models to retention data is proposed to predict surface excess adsorption and retention factors in liquid chromatography. The approach is based on a fundamental, microscopic description of the liquid-to-solid adsorption of analytes taking place at the interface between a bulk liquid phase and a solid surface. Molecular dynamics (MD) simulations are performed at T=300 K in a 100 Å wide slit-pore model (ß-cristobalite-C18 surface in contact with an acetonitrile/water mobile phase) to quantify a priori the retention factors of small molecules expected in reversed phase liquid chromatography (RPLC). Uracil is chosen as the reference "non-retained" marker, whereas benzyl alcohol, acetophenone, benzene, and ethylbenzene are four selected retained, neutral compounds. The MD simulations allow to determine the pore-level density profiles of these five compounds, i.e., the variation of the analyte concentration as a function of distance from the silica surface. The retention factors of the retained analytes are expressed using their respective calculated surface excess adsorption relative to uracil. By definition, the retention factors are proportional to the surface excess adsorbed and the proportionality constant is directly scaled to the retention time of the "non-retained" marker. Experimentally, a 4.6 mm × 150 mm RPLC-C18 column packed with 5 µm 100 Å High Strength Silica (HSS)-C18 particles is used and the retention times of these five compounds are measured. The volume fraction of acetonitrile in water increases from 20 to 90% generating a wide range of retention factors from 0.15 to 183 at T=300 K. The results demonstrate very good agreement between the MD-predicted surface excess adsorption data and measured retention factors (R2> 0.985). A systematic error is observed as the proportionality constant is not exactly scaled to the retention time of uracil. This is most likely caused by the differences between the chemical and morphological features of the slit-pore model adopted in the MD simulations and those of the actual HSS-C18 particles: the average surface coverage with C18 chains, the geometry of the mesopores, and the pore size distribution. Specifically, the impact on RPLC retention of slight, local variations in surface chemistry (e.g., functional group density and uniformity) and how this aspect is affected by the pore space morphology (e.g., pore curvature and size) is worth investigating by future MD simulations.

Confinement Effects on Distribution and Transport of Neutral Solutes in a Small Hydrophobic Nanopore.

Molecular dynamics simulations are used to study confinement effects in small cylindrical silica pores with extended hydrophobic surface functionalization as realized, for example, in reversed-phase liquid chromatography (RPLC) columns. In particular, we use a 6 nm cylindrical and a 10 nm slit pore bearing the same C18 stationary phase to compare the conditions inside the smaller-than-average pores within an RPLC column to column-averaged properties. Two small, neutral, apolar to moderately polar solutes are used to assess the consequences of spatial confinement for typical RPLC analytes with water (W)-acetonitrile (ACN) mobile phases at W/ACN ratios between 70/30 and 10/90 (v/v). The simulated data show that true bulk liquid behavior, as observed over an extended center region in the 10 nm slit pore, is not recovered within the 6 nm cylindrical pore. Instead, the ACN-enriched solvent layer around the C18 chain ends (the ACN ditch), a general feature of hydrophobic interfaces equilibrated with aqueous-organic liquids, extends over the entire pore lumen of the small cylindrical pore. This renders the entire pore a highly hydrophobic environment, where, contrary to column-averaged behavior, neither the local nor the pore-averaged sorption and diffusion of analytes scales directly with the W/ACN ratio of the mobile phase. Additionally, the solute polarity-related discrimination between analytes is enhanced. The consequences of local ACN ditch overlap in RPLC columns are reminiscent of ion transport in porous media with charged surfaces, where electrical double-layer overlap occurring locally in smaller pores leads to discrimination between co- and counterionic species.

Consequences of Cylindrical Pore Geometry for Interfacial Phenomena in Reversed-Phase Liquid Chromatography.

The interfacial phenomena behind analyte separation in a reversed-phase liquid chromatography column take place nearly exclusively inside the silica mesopores. Their cylindrical geometry can be expected to shape the properties of the chromatographic interface with consequences for the analyte density distribution and diffusivity. To investigate this topic through molecular dynamics simulations, we introduce a cylindrical pore inside a slit pore configuration, where the inner curved and outer planar silica surface bear the same bonded phase. The present model replicates an average-sized (9 nm) mesopore in an endcapped C18 column equilibrated with a mobile phase of 70/30 (v/v) water/acetonitrile. Simulations performed for ethylbenzene and acetophenone show that the surface curvature shifts the bonded phase and analyte density toward the pore center, decreases the solvent density in the bonded-phase region, increases the acetonitrile excess in the interfacial region, and considerably enhances the surface diffusivity of both analytes. Overall, the cylindrical pore provides a more hydrophobic environment than the slit pore. Ethylbenzene density is decidedly increased in the cylindrical pore, whereas acetophenone density is nearly equally distributed between the cylindrical and slit pore. The cylindrical pore geometry thus sharpens the discrimination between the apolar and moderately polar analytes while enhancing the mass transport of both.

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.

Organometallic ring vs. chain formation beyond kinetic control: steering their equilibrium in two-dimensional confinement.

Control over the competition between an organometallic hexamer macrocycle and oligomer chains formed from the non-alternant aromatic 1,3-dibromoazulene (DBAz) precursor has been achieved in surface-assisted synthesis on a copper(111) surface. In contrast to kinetic reaction control via the high-dilution principle, the ring formation is achieved here by thermodynamic control, which is based on two-dimensional (2D) confinement and reversible bonds.