Use of the surfactant 3-(3-cholamidopropyl)-dimethyl-ammoniopropane sulfonate in hydrophobic interaction chromatography of proteins.
Isocratic hydrophobic interaction chromatography of five proteins has been carried out using mobile phases containing the surfactant 3-(3-cholamidopropyl)-dimethylammoniopropane sulfonate (CHAPS). Linear relationships were found between log k' and ammonium sulfate concentrations for all the proteins with CHAPS in the submicellar concentration range. The slope of such a plot decreases monotonically as CHAPS concentration is increased. To a first approximation, the effect of CHAPS on protein retention can be explained in terms of a competitive binding model. However, CHAPS does show differential effects on the elution of proteins, substantially altering selectivity. The use of a normalized capacity factor, k'/k'o, proves useful for comparing retention times of different proteins as a function of CHAPS concentration. The magnitudes of k'/k'o were found to be inversely correlated with the slopes of plots of log k' vs. ammonium sulfate concentration in the absence of CHAPS. Adsorption isotherms for CHAPS were determined over the working range of ammonium sulfate. The binding of CHAPS to the SynChropak Propyl stationary phase and its effects on retention were found to be readily reversible. For each protein, plots of k'/k'o vs. surface concentration of CHAPS were superposable for data obtained at different salt concentrations. These findings support a competitive binding model. A simple geometric argument for stationary phase occupancy provides a qualitative explanation for the observed surfactant selectivity.
Buckley
J J
JJ
University of Delaware, Department of Chemistry and Biochemistry, Newark 19716.
Wetlaufer
D B
DB
D002793:Cholic Acids; D002853:Chromatography, Liquid / Q000379:methods; D007202:Indicators and Reagents; D011506:Proteins / Q000032:analysis*
D002793:Cholic Acids; D007202:Indicators and Reagents; D011506:Proteins; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate