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

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Abstract

The isocratic and gradient elution behaviour of β-endorphin and glucagon, two polypeptides known to exist in amphipathic α-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, ψ, of the organic solvent modifier and temperature, T, on the magnitudes of the S and logk(o) values of these two polypeptides, obtained from the plots of logarithmic capacity factor (log k) vs. ψ using isocratic elution conditions have been determined. These data have then been compared to the corresponding S and logk(o) values, obtained from the plots of logarithmic median capacity factor (logk) versus the median volume fraction of the organic solvent modifier (ψ) 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 values, or the logk(o) and logk(o) values, when these parameters are derived from experimental data acquired by these two different elution methods. Moreover, when gradient elution data for β-endorphin and glucagon are utilised, the extrapolated values of the intercept and slope of the plots of logk vs. 1/T (corresponding to an apparent change in the median enthalpy of association, ΔH(assoc)(o), or an apparent change in the median entropy of association, ΔS(assoc)(o)) substantially deviated from the values obtained for the thermodynamic parameters, ΔH(assoc)(o) and ΔS(assoc)(o), derived from the logk' 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. Copyright (C) 1999 Elsevier Science B.V.

Original languageEnglish
Pages (from-to)43-57
Number of pages15
JournalJournal of Chromatography A
Volume852
Issue number1
DOIs
Publication statusPublished - 6 Aug 1999

Keywords

  • Endorphin
  • Glucagon
  • Gradient elution
  • Mobile phase composition
  • Peptides
  • Retention behaviour
  • Thermodynamic parameters

Cite this

@article{f28dff380b384719a05027a5fc38e379,
title = "Comparison between the isocratic and gradient retention behaviour of polypeptides in reversed-phase liquid chromatographic environments",
abstract = "The isocratic and gradient elution behaviour of β-endorphin and glucagon, two polypeptides known to exist in amphipathic α-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, ψ, of the organic solvent modifier and temperature, T, on the magnitudes of the S and logk(o) values of these two polypeptides, obtained from the plots of logarithmic capacity factor (log k) vs. ψ using isocratic elution conditions have been determined. These data have then been compared to the corresponding S and logk(o) values, obtained from the plots of logarithmic median capacity factor (logk) versus the median volume fraction of the organic solvent modifier (ψ) 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 values, or the logk(o) and logk(o) values, when these parameters are derived from experimental data acquired by these two different elution methods. Moreover, when gradient elution data for β-endorphin and glucagon are utilised, the extrapolated values of the intercept and slope of the plots of logk vs. 1/T (corresponding to an apparent change in the median enthalpy of association, ΔH(assoc)(o), or an apparent change in the median entropy of association, ΔS(assoc)(o)) substantially deviated from the values obtained for the thermodynamic parameters, ΔH(assoc)(o) and ΔS(assoc)(o), derived from the logk' 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. Copyright (C) 1999 Elsevier Science B.V.",
keywords = "Endorphin, Glucagon, Gradient elution, Mobile phase composition, Peptides, Retention behaviour, Thermodynamic parameters",
author = "Purcell, {A. W.} and Zhao, {G. L.} and Aguilar, {M. I.} and Hearn, {M. T.W.}",
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Comparison between the isocratic and gradient retention behaviour of polypeptides in reversed-phase liquid chromatographic environments. / Purcell, A. W.; Zhao, G. L.; Aguilar, M. I.; Hearn, M. T.W.

In: Journal of Chromatography A, Vol. 852, No. 1, 06.08.1999, p. 43-57.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Comparison between the isocratic and gradient retention behaviour of polypeptides in reversed-phase liquid chromatographic environments

AU - Purcell, A. W.

AU - Zhao, G. L.

AU - Aguilar, M. I.

AU - Hearn, M. T.W.

PY - 1999/8/6

Y1 - 1999/8/6

N2 - The isocratic and gradient elution behaviour of β-endorphin and glucagon, two polypeptides known to exist in amphipathic α-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, ψ, of the organic solvent modifier and temperature, T, on the magnitudes of the S and logk(o) values of these two polypeptides, obtained from the plots of logarithmic capacity factor (log k) vs. ψ using isocratic elution conditions have been determined. These data have then been compared to the corresponding S and logk(o) values, obtained from the plots of logarithmic median capacity factor (logk) versus the median volume fraction of the organic solvent modifier (ψ) 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 values, or the logk(o) and logk(o) values, when these parameters are derived from experimental data acquired by these two different elution methods. Moreover, when gradient elution data for β-endorphin and glucagon are utilised, the extrapolated values of the intercept and slope of the plots of logk vs. 1/T (corresponding to an apparent change in the median enthalpy of association, ΔH(assoc)(o), or an apparent change in the median entropy of association, ΔS(assoc)(o)) substantially deviated from the values obtained for the thermodynamic parameters, ΔH(assoc)(o) and ΔS(assoc)(o), derived from the logk' 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. Copyright (C) 1999 Elsevier Science B.V.

AB - The isocratic and gradient elution behaviour of β-endorphin and glucagon, two polypeptides known to exist in amphipathic α-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, ψ, of the organic solvent modifier and temperature, T, on the magnitudes of the S and logk(o) values of these two polypeptides, obtained from the plots of logarithmic capacity factor (log k) vs. ψ using isocratic elution conditions have been determined. These data have then been compared to the corresponding S and logk(o) values, obtained from the plots of logarithmic median capacity factor (logk) versus the median volume fraction of the organic solvent modifier (ψ) 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 values, or the logk(o) and logk(o) values, when these parameters are derived from experimental data acquired by these two different elution methods. Moreover, when gradient elution data for β-endorphin and glucagon are utilised, the extrapolated values of the intercept and slope of the plots of logk vs. 1/T (corresponding to an apparent change in the median enthalpy of association, ΔH(assoc)(o), or an apparent change in the median entropy of association, ΔS(assoc)(o)) substantially deviated from the values obtained for the thermodynamic parameters, ΔH(assoc)(o) and ΔS(assoc)(o), derived from the logk' 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. Copyright (C) 1999 Elsevier Science B.V.

KW - Endorphin

KW - Glucagon

KW - Gradient elution

KW - Mobile phase composition

KW - Peptides

KW - Retention behaviour

KW - Thermodynamic parameters

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U2 - 10.1016/S0021-9673(99)00440-9

DO - 10.1016/S0021-9673(99)00440-9

M3 - Article

VL - 852

SP - 43

EP - 57

JO - Journal of Chromatography A

JF - Journal of Chromatography A

SN - 0021-9673

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