Mechanistic Studies of the Separation of an HIV Protease Inhibitor from Its Piperazine Diastereomer by Reversed Phase High Performance Liquid Chromatography

Abstract

The HPLC separation and retention behavior of the piperazine diastereomers of an Human Immunodeficiency Virus (HIV) protease inhibitor on a commercially available C₁₈ chromatographic phase is discussed. The pH and mobile phase composition have the greatest effect on the selectivity of this system. Evidence is presented that the selective interaction involves hydrogen bonding. As the mobile phase pH was varied, a reversal in the elution order corresponding to the change in the protonation state of the molecule is observed. It is proposed, that the selectivity is governed by coordination of the active piperazine amine as a proton donor or proton acceptor with the organic modifier, while interacting with the stationary phase. It is shown, that longer chain alcohols produce poorer selectivities than shorter chain alcohols. It is proposed, that this is because it is more difficult for the longer hydrophobic chains to form stable complexes with the solute through hydrogen bonding. The hydrogen bonding moiety of the solute may, thus, coordinate with water and not selectively interact with the stationary phase. The effects of stationary phase chemistry, ionic strength, and modifier strength are also described.

Keywords:

• Diastereomer selectivity
• HIV protease inhibitors
• Hydrogen bonding
• pH effects
• Piperazine
• Reverse phase HPLC
• Solvation effects

Acknowledgments

The authors wish to acknowledge Drs. A. Clausen, M. Trone, and F. Wang for helpful discussions.