Non-linear elution effects in split-peak chromatography. I. Computer simulations for the cases of irreversible diffusion- and adsorption-limited kinetics

The split-peak effect, in which even a small sample of pure analyte elutes in both non-retained and retained fractions, has been shown to be useful chromatographic tool for such applications as the determination of protein adsorption rate constants and diffusion coefficients. To evaluate such parameters, it is necessary to obtain data independent of sample size, or to work under linear elution conditions. In this paper, computer simulations were used to determine how non-linear elution conditions affect such measurements. The two cases studied were those in which the rate of analyte retention was limited either by diffusion or adsorption of analyte on the column. The simulation data were then compared to results obtained with two experimental systems: the retention of hemoglobin on reversed-phase columns and the binding of immunoglobulin G to protein A affinity columns. From the simulations, guidelines were developed for minimizing or eliminating non-linear elution effects in both of the cases studied.