Experimental and theoretical studies of rate constant evaluation for the solute-matrix interaction in affinity...

In an investigation of the problem of determining kinetic parameters for the interaction of a solute with immobilized ligand sites on an affinity matrix, a combination of experimental studies and numerical simulations of frontal chromatography of methyl orange on Sephadex G-25 has yielded a simpler method than existing procedures for characterizing solute-matrix kinetics. A significant change in approach has entailed the direct evaluation of the kinetic contribution to boundary spreading from the flow-rate dependence of boundary variance under conditions of concentration-independent chromatographic migration (linear kinetics). This kinetic contribution is then interpreted in terms of an experimentally more appropriate form of a quantitative relationship for diffusion-free chromatographic migration (H. W. Hethcote and C. DeLisi, 1982, J. Chromatogr. 240, 269-281). Finally, the results of numerical simulations of concentration-dependent chromatographic migration (Langmuir kinetics) have indicated that rate constants should also be determinable under these conditions by extrapolation of their apparent values obtained by the above procedure to infinite dilution.