Role of conformational changes in the elution of proteins from reversed-phase HPLC columns

To test the hypothesis that conformational alterations might be involved in the elution of proteins from reversed-phase HPLC columns, the conformations of proteins bound onto a C-8 alkyl-bonded silica surface have been examined in the presence of increasing concentrations of the commonly employed eluent, 1-propanol. Using a combination of photoacoustic, diffuse reflectance deconvolution Fourier transform infrared and front face fluorescence spectroscopic techniques (to minimize interference from light scattering), the existence of surface-associated protein conformational changes induced by propanol is unequivocally demonstrated. The linear relationship found between the amount of propanol needed to elute proteins from C-8 columns and the midpoint of spectrally observed structural transitions is consistent with a role for conformational changes in the elution process.