Due to complexity of retention mechanisms in HILIC system, detailed examination of experimental space assessing the influence of important factors (acetonitrile content in the mobile phase, buffer concentration and pH of the mobile phase) and their interactions was done by applying 33 experimental design.
Factors independent variables (acetonitrile content, pH of the mobile phase and concentration of sodium heptane sulfonate in water phase) were extracted from the preliminary study and as dependent variables five responses (retention factor of ropinirole, retentin factor of its impurity, resolution, symmetry of ropinirole peak and symmetry of impurity peak) were selected.
Chromatographic conditions, especially analytical columns and mobile phase compositions (concentration of buffer, pH value of the buffer and percentage of the organic modifiers), were optimized to achieve good sensitivity and peak shape, as well as a relatively short run.
The effects of operational parameters such as mobile phase modifier concentrations, mobile phase flow rate, heating block temperature and drying gas flow rate were evaluated by the 24 − 1 fractional factorial experimental design, resolution IV, in the screening phase and by Doehlert experimental design.
The linear adsorption isotherm and the equilibrium-dispersive model were adopted to approximatively describe the chromatographic separation behaviors of liquiritin and liquiritigenin in the raw material under different column temperatures, ethanol contents and flow rates of the mobile phase, sample concentrations and feeding times.
The effect of changing the ionic strength of phosphate buffer on the selectivity and retention times of the test solutes was investigated using mobile phases containing concentration of 0.0075- 0.04 M of phosphate buffer.
The four independent variables studied simultaneously were: acetonitrile content of the mobile phase, pH and concentration of buffer and column temperature each at three levels.
The effects of gradient steepness, mobile phase pH, salt concentration and type, as well as organic modifier were evaluated for tuning selectivity and retention in HIC.
For that purpose, the gradient, the mobile phase composition (buffer concentration, buffer pH, and elution salt), and the temperature were optimized.
