Comparison between the isocratic and gradient retention behaviour of polypeptides in reversed-phase liquid chromatographic environments

The isocratic and gradient elution behaviour of beta-endorphin and glucagon, two polypeptides known to exist in amphipathic alpha-helical conformations in lipophilic environments, have been examined under reversed-phase high-performance liquid chromatographic (RP-HPLC) conditions with low pH, aquo-acetonitrile mobile phases. The effects of changes in the volume fraction, psi, of the organic solvent modifier and temperature, T, on the magnitudes of the S and log k(o) values of these two polypeptides, obtained from the plots of logarithmic capacity factor (log k) vs. psi using isocratic elution conditions have been determined. These data have then been compared to the corresponding (S) over bar and log k(o) values, obtained from the plots of logarithmic median capacity factor (log (k) over bar) versus the median volume fraction of the organic solvent modifier (<(psi)over bar>) derived from the linear gradient elution data, using the same n-butyl silica sorbent and related aquo-acetonitrile mobile phase conditions. As apparent from these studies, substantial differences occur in the temperature-dependent trends and magnitudes of the corresponding S and (S) over bar values, or the log k(o) and log (k) over bar(o) values, when these parameters are derived from experimental data acquired by these two different elution methods. Moreover, when gradient elution data for beta-endorphin and glucagon are utilised, the extrapolated values of the intercept and slope of the plots of log (k) over bar vs. 1/T (corresponding to an apparent change in the median enthalpy of association, Delta (H) over bar(assoc)degrees, or an apparent change in the median entropy of association, Delta (S) over bar(assoc)degrees) substantially deviated from the values obtained for the thermodynamic parameters, Delta H(assoc)degrees and Delta S(assoc)degrees, derived from the log k' vs. 1/T plots using the corresponding isocratic data. These findings thus have important implications for biophysical and thermodynamic investigations when gradient elution data are employed to assess the molecular basis of the interaction of polypeptides with non-polar ligates. (C) 1999 Elsevier Science B.V. All rights reserved.