Abstract
Polar columns used in the HILIC (Hydrophilic Interaction Liquid Chromatography) systems take up water from the mixed aqueous–organic mobile phases in excess of the water concentration in the bulk mobile phase. The adsorbed water forms a diffuse layer, which becomes a part of the HILIC stationary phase and plays dominant role in the retention of polar compounds. It is difficult to fix the exact boundary between the diffuse stationary and the bulk mobile phase, hence determining the column hold-up volume is subject to errors. Adopting a convention that presumes that the volume of the adsorbed water can be understood as the column stationary phase volume enables unambiguous determination of the volumes of the stationary and of the mobile phases in the column, which is necessary for obtaining thermodynamically correct chromatographic data in HILIC systems. The volume of the aqueous stationary phase, Vex, can be determined experimentally by frontal analysis combined with Karl Fischer titration method, yielding isotherms of water adsorbed on polar columns, which allow direct prediction of the effects of the composition of aqueous–organic mobile phase on the retention in HILIC systems, and more accurate determination of phase volumes in columns and consistent retention data for any mobile phase composition. The n phase volume ratios of 18 columns calculated according to the new phase convention strongly depend on the type of the polar column. Zwitterionic and TSK gel amide and amine columns show especially strong water adsorption.
Highlights
High Performance Liquid chromatography (HPLC) has become one of the most powerful tools for the separation and determination of samples containing non-polar, moderately or strongly polar and ionic compounds, either simple species or complex highmolecular synthetic polymers or biopolymers
The volume of the aqueous stationary phase, Vex, can be determined experimentally by frontal analysis combined with Karl Fischer titration method, yielding isotherms of water adsorbed on polar columns, which allow direct prediction of the effects of the composition of aqueous–organic mobile phase on the retention in Hydrophilic Interaction Liquid Chromatography (HILIC) systems, and more accurate determination of phase volumes in columns and consistent retention data for any mobile phase composition
The issue was significantly alleviated by introduction of Hydrophilic Interaction Liquid Chromatography (HILIC) employing polar columns and mobile phases with high concentrations of polar organic solvents in water, i.e., aqueous normal-phase systems
Summary
High Performance Liquid chromatography (HPLC) has become one of the most powerful tools for the separation and determination of samples containing non-polar, moderately or strongly polar and ionic compounds, either simple species or complex highmolecular synthetic polymers or biopolymers. Non-ionic compounds are usually separated on the basis of differences in polarities, most often in reversed-phase (RP) systems with a non-polar stationary phase and a mixed aqueous–organic mobile phase. For the separation of moderately polar compounds, the conventional HPLC employs a polar adsorbent such as silica gel and a less polar mixed organic solvent mobile phase in organic normal-phase (NP) systems. The issue was significantly alleviated by introduction of Hydrophilic Interaction Liquid Chromatography (HILIC) employing polar columns and mobile phases with high concentrations of polar organic solvents in water, i.e., aqueous normal-phase systems. HILIC has been found especially useful in () pharmaceutical, biomedical and clinical analysis
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