Microparticulate non-polar stationary phases, such as octadecyl-silica offer a rapid and efficient means for the separation of peptides and amino acids by high-performance liquid chromatography. Retention is attributed to hydrophobic interaction between the solutes and the hydrocarbonaceous functions covalently bound to the stationary phase surface. Consequently the species are eluted in the order of increasing hydrophobicity. Various peptide mixtures were analyzed by using gradient elution with increasing acetonitrile concentration in the eluent and monitoring the column effluent at 200 or 210 nm with an UV detector. The separation of angiotensins and enzymic digest of polypeptides illustrates the speed of the method which can be used to assay the purity of peptide hormones such as α-melanotropin and gramicidin or to analyze the composition of reaction mixtures involving peptides. The efficiency of the method is superior to that obtained on the conventionally used ion-exchanger columns, except for hydrophilic amino acids and peptides that are poorly retarded. Nevertheless, with a suitable ionic surfactant in the mobile phase, non-polar stationary phases can be used for the separation of these species as well.

The effect of solute ionization on the retention of weak acids, bases, and ampholytes on octadecylsilica was investigated both theoretically and experimentally. The retention was attributed to a reversible association of the stationary phase. A phenomenological treatment of the corresponding equilibria was developed for various types of ionogenic substances. The energetics of the association process was analyzed in a rigorous fashion in the light of the solvophobic theory and a semi-empirical extension of the Debye-Hückel theory to high ionic strength. The predicted effect of solute ionization on the capacity factors was substantiated by experimental data. The observed dependence of the capacity factors on the ionic strength of the eluent and the hydrophobic surface of the solute molecules showed good agreement with the theory. The advantages of the technique in the separation of biological substances are illustrated.

Charcoal, a nonpolar sorbent, had been widely used for the separation of peptides1 before the advent of ion-exchange chromatography. Recent developments in high performance liquid chromatography revived the interest in the use of nonpolar stationary phases for the separation of biological substances by “reversed phase” chromatography, which employs columns packed with 5 or 10µm porous silica particles having hydrocarbonaceous functions covalently bound to the surface.

This report illustrates the potential of this type of chromatography for the rapid analysis of minute quantities of peptide mixtures. The results suggest that octadecyl-silica columns can be used for fast separation of a wide variety of peptides. By monitoring the column effluent with a UV-detector at 200 nm, the sample components can be analyzed at the subnanomole level without the formation of UV absorbing or fluorescent derivatives.