Thermodynamic studies of pressure-induced retention of peptides in reversed-phase liquid chromatography

and temperature at both low(19 bar) and high (318 bar) pressures using a homologous series of hydrophobic poly-l-phenylalanine (n = 2–7) as the model compound. Based on van’t Hoff plots under different organic solvent compositions and pressures, the enthalpy change for the solute (H) was determined. Moreover, both the enthalpy and entropy change for each phenylalanine residue (H and S), which corresponds to solute retention on a microenvironment along the depth of C18 chain, were also calculated by direct subtractions. Results indicate that under acetonitrile (ACN) compositions above 35%, the pressure caused S value to change from a negative to a positive value and both H and H to change from a negative to a less negative value, all leading to a thermodynamic state closer to those under 35% acetonitrile composition. This implies that the pressure-induced retention observed in this study was an entropy-favored but enthalpy-unfavored process and was explained by pressure-induced desorption of solvent molecules that were associated with the stationary phase or with the peptide solute. Under 35% acetonitrile composition, however, itwas found that neitherHnorS valuewas significantly changed by the pressure. Whereas, both H value and the intercept of van’t Hoff plots under 35% acetonitrile composition were increased by pressure. This indicates that under low organic solvent composition, 35%, most of the acetonitrile molecules adsorbed on the surface of the stationary phase and only little solvent molecules were dissolved in the bulk stationary phase where the phenylalanine residues were partitioned. This study has provided new thermodynamic insights to the pressure-induced retention for peptides and proteins.