RELATIONSHIP BETWEEN HYDROPHOBICITY AND RPLC RETENTION BEHAVIOR OF AMPHOTERIC COMPOUNDS

Abstract

In this paper, we systematically investigate the regulation of retention behavior of amphoteric compounds, amino acids, and small peptides in the whole pH range in reversed-phase liquid chromatography (RPLC) using methanol-ammonium acetate buffer solution as a binary mobile phase. According to Snyder-Soczewinski equation, logarithm of hypothetical retention factor corresponding to neat aqueous fraction of hydroorganic mobile phase (logk w) is obtained by extrapolation from different compositions of the mobile phase. Then, we establish the relationship between octanol-water partition coefficient (P) and k w of these amphoteric compounds and discuss the dependence of retention behavior on mobile phase pH. The experimental results reveal that the linear correlation between logarithm of apparent octanol-water coefficient (logD) and logk w is better than that between logP and logk w, as well as that between logD and logk, the logarithm of retention factor at a specific organic modifier fraction for common amphoteric compounds. The linear relationships of logD–logk w approximate those of logP–logk w, and they are superior to those of logD–logk for amino acids and small peptides. In addition, we provide an insight into the fact that isoelectric point (pI), dissociation constants (p K a1 and p K a2 ) of amphoteric compounds affect their RPLC retention behavior significantly.