Numerical simulation of the chromatographic process for direct ligand-macromolecule binding studies

As a continuous dissociation of the complex may occur during its migration, the apparent binding curve and the theoretical binding isotherm coincide at extremely low dissociating rates. At larger dissociation rates (0.001 s−1 < kd < 0.1 s−1, for a first peak eluting in 1 min) the simulations were used to test various chromatographic conditions. The flow rate (or column volume) is the major effect which influences the on-column dissociation process as an exponential decay was found when the apparently bound fraction is plotted against the analysis time. The apparent equilibrium coefficient is close to the theoretical one for a binding curve generated with an initial solution containing a relatively low total concentration of binding sites (≤KD). The apparent stoichiometric term is largely underestimated as its value decreases exponentially at increasing dissociation rates. An extrapolation at extremely short analysis times could be used to determine the stoichiometric coefficient characterizing the binding interaction.