Non-linear elution effects in split-peak chromatography II. Role of ligand heterogeneity in solute binding to columns with adsorption-limited kinetics

The split-peak effect is a useful phenomenon in studying the kinetic behavior of chromatographic supports. This work examined the combined role of ligand heterogeneity and non-linear elution conditions (i.e., sample load dependence) on the solute free fractions that are measured during split-peak studies. Exact expressions were derived to describe the effects of ligand heterogeneity under linear elution conditions, and simulation models were developed to specifically examine the combined effects of ligand heterogeneity and non-linear elution in systems with adsorption-limited rates for solute binding. The simulations showed that ligand heterogeneity increased the amount of free solute seen at any flow-rate or sample size, with this being most noticeable when using low flow-rates or large samples. One application in which these increases were examined in detail concerned the use of the split-peak effect for association rate constant measurements. It was found that linear extrapolation methods developed for homogeneous systems (as a correction for non-linear elution conditions) could successfully be applied to columns containing heterogeneous ligands. Columns containing immobilized protein A and/or protein G were used as experimental models to test the validity of the simulations; the behavior of these columns showed good quantitative and qualitative agreement with the predicted theoretical results.