Kinetic modeling for preparative chromatography is a topic of present interest in the fine chemicals and pharmaceutical industries. In this study, chromatographic separation of the two nucleotides CMP and UMP was simulated by the equilibrium-dispersive (ED) model, and the adsorption isotherms in the ED model were determined by the inverse method. Prediction performance of the model was validated under three different kinds of conditions and the importance of selecting isotherms was discussed in detail. Excellent agreement was achieved with the experimental band profiles and the prediction of the ED model. The ED model with bi-Langmuir isotherm was especially suitable for simulating chromatographic separation of CMP and UMP. The error of prediction by the ED model with bi-Langmuir isotherm was about 9.4 times smaller than that with Langmuir isotherm.