Comprehensive analysis of the effective and intra-particle diffusion of weakly retained compounds in silica hydrophilic interaction liquid chromatography columns.

A detailed analysis of intra-particle volumes and layer thicknesses and their effect on the diffusion of solutes in hydrophilic interaction liquid chromatography (HILIC) was made. Pycnometric measurements and the retention volume of deuterated mobile phase constituents (water and acetonitrile) were used to estimate the void volume inside the column, including not only the volume of the mobile phase but also part of the enriched water solvent acting as the stationary phase in HILIC. The mobile phase (hold-up) volume accessible to non-retained components was estimated using a homologous series approach. The joint analysis of the different approaches indicated the formation of enriched water layers on the hydrophobic silica mesopore walls with a thickness varying significantly with mobile phase composition. The maximal thickness of the enriched water layers, which corresponded to the minimum void volume accessible to unretained solutes, marked a transition in the retention behavior of the studied analytes. Discrepancies between deuterated solvent measurements and pycnometry were explained by the existence of an irreplaceable water layer adsorbed on the silica surface. Regarding the diffusion behavior in HILIC, peak parking experiments were used to interpret the influence of the acetonitrile content on the effective diffusion coefficient Deff. A systematic decrease in Deff and molecular diffusion Dm was observed with decreasing acetonitrile concentration, primarily attributed to variations in mobile phase viscosity. Notably, Deff/Dm remained nearly unaffected by variations in mobile phase composition. Finally, the effective medium theory was used to make a comprehensive analysis of Dpart/Dm to study the contribution to band broadening when the solute resides in the mesopores. The obtained data unveiled a curvature with a minimum corresponding to conditions of maximum water-layer thickness and retention. For the weakly retained compounds (k' < 0.5) the Dpart/Dm-values were found to be relatively high (order of 0.35-0.5), which directly reflects the high γsDs/Dm-values that were observed (order 0.35-7).

Characterization of solute-solvent interactions in liquid chromatography systems: A fast method based on Abraham's linear solvation energy relationships.

The Abraham's solvation parameter model, based on linear solvation energy relationships (LSER), allows the accurate characterization of the selectivity of chromatographic systems according to solute-solvent interactions (polarizability, dipolarity, hydrogen bonding, and cavity formation). However, this method, based on multilinear regression analysis, requires the measurement of the retention factors of a considerably high number of compounds, turning it into a time-consuming low throughput method. Simpler methods such as Tanaka's scheme are preferred. In the present work, the Abraham's model is revisited to develop a fast and reliable method, similar to the one proposed by Tanaka, for the characterization of columns employed in reversed-phase liquid chromatography and particularly in hydrophilic interaction liquid chromatography. For this purpose, pairs of compounds are carefully selected in order to have in common all molecular descriptors except for a specific one (for instance, similar molecular volume, dipolarity, polarizability, and hydrogen bonding basicity features, but different hydrogen bonding acidity). Thus, the selectivity factor of a single pair of test compounds can provide information regarding the extent of the dissimilar solute-solvent interactions and their influence on chromatographic retention. The proposed characterization method includes the determination of the column hold-up volume and Abraham's cavity term by means of the injection of four alkyl ketone homologues. Therefore, five chromatographic runs in a reversed-phase column (four pairs of test solutes and a mixture of four homologues) are enough to characterize the selectivity of a chromatographic system. Tanaka's method is also analyzed from the LSER point of view.

Evaluation of Hold-Up Volume Determination Methods and Markers in Hydrophilic Interaction Liquid Chromatography.

Common methods for hold-up time and volume determination in Reversed-Phase Liquid Chromatography (RPLC) have been tested for Hydrophilic Interaction Liquid Chromatography (HILIC). A zwitterionic ZIC-HILIC column has been used for the testing. The pycnometric determination method, based on differences in column weight when filled with water or organic solvent, provides the overall volume of solvent inside the column. This includes the volume of eluent semi-sorbed on the packing of the column, which acts as the main stationary phase. The homologous series approach, based on the retention behavior of homologues in relation to their molecular volume, allows the determination of accurate hold-up volumes. However, the application of this method is time-consuming. In some cases, large neutral markers with poor dipolarity/polarizability and hydrogen bonding interactions can be used as hold-up volume markers. This is the case of dodecylbenzene and nonadecane-2-one in clearly HILIC behaving chromatographic systems, the use of decanophenone as a marker can be even extended to the boundary between HILIC and RPLC. The elution volume of the marker remains nearly unaffected by the concentration of ammonium acetate in the mobile phase up to 20 mM. The injection of pure solvents to produce minor base-line disturbance as hold-up markers is strongly discouraged, since solvent peaks are complex to interpret and depend on the ionic strength of the eluent.

Volume and composition of semi-adsorbed stationary phases in hydrophilic interaction liquid chromatography. Comparison of water adsorption in common stationary phases and eluents.

Pycnometric and homologous series retention methods are used to determine the volume and mean composition of the water-rich layers partially adsorbed on the surface of several hydrophilic interaction liquid chromatography (HILIC) column fillings with acetonitrile-water and methanol-water as eluents. The findings obtained in this work confirm earlier studies using direct methods for measuring the stationary phase water content performed by Jandera's and Irgum's research groups. Water is preferentially adsorbed on the surface of the HILIC bonded phase in hydroorganic eluents containing more than 40% acetonitrile or 70% methanol, and a gradient of several water-rich transition layers between the polar bonded phase and the poorly polar bulk mobile phase is formed. These layers of reduced mobility act as HILIC stationary phases, retaining polar solutes. The volume of these layers and concentration of adsorbed water is much larger for acetonitrile-water than for methanol-water mobile phases. In hydroorganic eluents with less than 20-30% acetonitrile or 40% methanol the amount of preferentially adsorbed water is very small, and the observed retention behavior is close to the one in reversed-phase liquid chromatography (RPLC). In eluents with intermediate acetonitrile-water or methanol-water compositions a mixed HILIC-RPLC behavior is presented. Comparison of several HILIC columns shows that the highest water enrichment in the HILIC retention region for acetonitrile-water mobile phases is observed for zwitterionic and aminopropyl bonded phases, followed in minor grade for diol and polyvinyl alcohol functionalizations. Pentafluorophenyl bonded phase, usually considered a HILIC column, does not show significant water adsorption, nor HILIC retention.

HILIC characterization: Estimation of phase volumes and composition for a zwitterionic column.

A methodology for the estimation of the different phase volumes in HILIC is presented. For a ZIC-HILIC column the mobile phase volume (hold-up volume) is determined in several acetonitrile- and methanol-water compositions by a Linear Free Energy Relationships (LFER) homologous series approach involving n-alkyl-benzenes, -phenones, and -ketones. We demonstrate that the column works as a HILIC column when the mobile phase contains high and medium proportions of methanol or acetonitrile. However, for acetonitrile contents below 20%, or 40% for methanol, same column works in RPLC. In between, a mixed HILIC-RPLC behavior is observed, and solutes of low molecular volume are retained as in HILIC mode, but the largest ones show RPLC retention. From the homologous series retention data and pycnometric measurements involving the pure organic solvents and their mixtures with water, the mean solvent composition of the water-rich transition layers between column functionalization and the bulk mobile phase, which act as stationary phase, is estimated. Finally, the phase ratio between stationary and mobile phases is also estimated for each eluent composition, allowing the calculation of the corresponding stationary phase volumes. All volumes are strongly dependent on the water content in the eluent, especially when acetonitrile is selected as mobile phase constituent. In HILIC mode, when the water content in the hydroorganic mobile phase increases, the volumes of mobile phase decrease, but the volumes of stationary phase (mainly the water layer adsorbed onto the bonded-phase and the water-enriched interface) increase. However, at high water concentrations, where the column works in RPLC mode, the mobile phase volume increases and the stationary phase (which is now the bonded zwitterion) volume decreases when increasing the water percentage in the mobile phase.