Separation of potentially therapeutic peptide hormones by liquid chromatography. Optimisation of the composition and pH of the mobile phase.

The aim of this work is to optimise the proportion of the organic modifier and the pH of the mobile phase, in order to separate a series of peptide hormones with therapeutic interest in the molecular mass range from 500 to 6000. The composition of the mobile phase was optimised by establishing relationships between retention parameters and either the scale of solvent polarity, or the Kamlet-Taft multiparameter solvent scale of the eluent, using linear solvation energy relationships. Likewise, linear correlations between the chromatographic retention and Reichardt's E(T)N parameter were obtained. These relationships allowed an important reduction of the experimental retention data needed for developing a given separation. In addition, a model describing the effect of the correctly measured pH of the mobile phase on retention in LC was established and tested for the series of selected peptides using an octadecylsilica column. The proposed equations permit the prediction of the optimum pH and also permit the determination of the acidity constants of the peptides in the hydro-organic mixtures using a minimum number of measurements.