Salt concentration effects in high performance hydrophobic interaction chromatography in comparison with NMR of proteins in solution

The effects of salt concentration on the chromatographic behavior of cytochrome C, ribonuclease A, and alpha-chymotrypsinogen A in hydrophobic interaction chromatography (HIC) has been examined by isocratic elutions on a Bio-Gel TSK Phenyl 5 PW column. In some cases, conformational variations were manifest chromatographically by reproducible changes in peak shape and appearance of multiple peaks as a function of sodium sulfate concentration in the mobile phase. A parallel study by proton nuclear magnetic resonance (NMR) spectroscopy on the salt concentration dependence of the spectral property of these proteins is in agreement with the possible contribution of the mobile phase composition to the observed chromatographic behavior.