Advancing HIC method development: Retention-time modeling and tuning selectivity with ternary mobile-phase systems.

The use of a ternary mobile-phase system comprising ammonium sulphate, sodium chloride, and phosphate buffer was explored to tune retention and enhance selectivity in hydrophobic interaction chromatography. The accuracy of the linear solvent-strength model to predict protein retention with the ternary mobile-phase system based on isocratic scouting runs is limited, as the extrapolated retention factor at aqueous buffer conditions (k) cannot be reliably established. The Jandera retention model utilizing a salt concentration averaged retention factor (k¯) in aqueous buffer for ternary systems overcomes this bottleneck.

Detailed analysis of the effective and intra-particle diffusion coefficient of proteins at elevated pressure in columns packed with wide-pore core-shell particles.

To determine the efficiency that can be obtained in a packed-bed liquid-chromatography column for a particular analyte, a correct determination of the molecular and effective diffusion coefficients (D and D) of the analyte is required. The latter is usually obtained via peak parking experiments wherein the flow is stopped. As a result, the column pressure rapidly dissipates and the measurement is essentially conducted at ambient pressure.

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.

On-column modification for the creation of temperature-responsive stationary phases.

Temperature-responsive liquid chromatography (TRLC) offers an alternative for retention and selectivity optimisation in HPLC. This approach thereby exploits temperature gradients on stimuli-responsive stationary phases and forfeits the necessity for solvent gradients, allowing analyses to be performed using aqueous mobile phases. Consequently, it can be employed as a green alternative to reversed-phase separations.