Theoretical Description of Nonlinear Chromatography, with Applications to Physicochemical Measurements in Affinity Chromatography and Implications for Preparative-Scale Separations

The theory of nonlinear chromatography Is developed further, and the behavior of chromatographic peaks obtained under nonlinear conditions Is described by extensive computations of peak moment values. It Is shown that when adsorption/ desorption rates are slow, band broadening Is Insensitive to, or may actually be decreased by, a moderate degree of column overload. The linear model of Horvath and Lin and the nonlinear model developed here are used to characterize the retention behavior of p nitrophenyl-α-D-mannopyranoside on silica-bound Concanavalln-A affinity columns. The two theories are compared In terms of their general applicability and the significance of the thermodynamic and kinetic constants derived from each. For heterogeneous affinity adsorbents such as Concanavalln-A (Con-A) more meaningful physical constants are derived by applying nonlinear theory to data obtained under the most severe nonlinear conditions. The analysis presented here yields adsorption/desorption rate constants and the binding site density for the more populous binding site in Immobilized Con-A.