SYSTEMATIC METHOD DEVELOPMENT IN HYDROPHOBIC INTERACTION CHROMATOGRAPHY .1. CHARACTERIZATION OF THE PHASE SYSTEM AND MODELING RETENTION

In order to rationalize the selection of the phase system (the station ary phase and the mobile phase) applicable for the separation of some proteins, gradient measurements were carried out on three columns of d ifferent types using three salts as eluent constituents. The slope and intercept of the In k-salt concentration relationship for proteins va ried with the different phase systems. The hydrophobicity index (c()) showed how the alteration of the type of phase system affects the sel ectivity of separation. Two-dimensional mapping along the parameters o f the slope-intercept relationship revealed the extent of the differen ce among the phase systems. In addition, the effect of solutions of bi nary and ternary salt mixtures on the retention of proteins was invest igated. Measurements were carried out according to a mixture design an d the retention time was modelled with the predictive method of Jander a and by fitting a polynomial to the data according to the Sentinel me thod. The accuracy of the former method were dependent on the type of phase system. The fit of second-order polynomial gave very good result s but a first-order model was also acceptable.