Adsorption kinetics and isotherms of proteins on chromatographic surfaces: A total internal reflectance fluorescence study

Hydrophobic interaction chromatography (HIC) is increasingly used in the area of biotechnological separations despite the limited quantitative knowledge of the adsorption process and the factors governing it. This study is aimed at obtaining quantitative measurements characteristic of sorption of proteins on chromatographic surfaces under HIC conditions. The adsorption behavior was investigated in terms of adsorption isotherms and adsorption kinetics. The two aspects of adsorption were studied as a function of the nature of the adsorbing surface (i.e., butylated or aminopropylated surface), the type of salt (i.e., NaCl or (NH{dollar}sb4{dollar}){dollar}sb2{dollar}SO{dollar}sb4{dollar}) and the salt concentration, for two different proteins (i.e., lysozyme and {dollar}alpha{dollar}-chymotrypsinogen A).; Due to its sensitivity, total internal reflectance fluorescence (TIRF) spectroscopy was used; it allows real-time fluorimetric measurements of protein fluorescence in the immediate vicinity of the adsorbing surface. With the aid of a calibration technique, the total fluorescence intensity detected was related to surface concentration.; The adsorption of lysozyme and chymotrypsinogen A on butylated slides was found to be largely irreversible, while on the aminopropylated slides, adsorption was reversible. Both proteins were found to adsorb on aminopropylated surfaces despite the expected electronic repulsion between the positively-charged surface and the proteins, which bear a net positive charge at pH 7.0. The effects of varying salt type and concentration on the extent of adsorption were found to correlate with the salting-in and salting-out behavior of the protein and salts in aqueous solution.; The analysis of experimental data using a detailed model of the adsorption process allows a rate constant to be estimated from TIRF adsorption curves. This approach accounts for mass transfer limitations occurring during the adsorption process. Three different types of transport models were considered: (1) a diffusion-limited model, (2) an irreversible kinetic model, and (3) a reversible kinetic model with a linear isotherm.; The rate of adsorption of lysozyme and chymotrypsinogen A on butylated and aminopropylated surfaces appeared to be affected by the salt type and salt concentration. The rate of adsorption was found to correlate with the extent of adsorption.