Cytidine 5'-monophosphate (CMP) and guanosine 5'-monophosphate (GMP) were separated based on hypercross-linked resins. The adsorption performances of different resins to the two 5'-ribonucleotides were compared. The adsorption equilibria, kinetics, and dynamic separation process of CMP and GMP with pH gradient elution were studied experimentally and theoretically. The nonpolar resin XDA-1 was selected as the best adsorbent for separating CMP and GMP. A pH-dependent Langmuir isotherm model was developed. The model predicted the adsorption isotherms of CMP and GMP at different pH levels satisfactorily. The surface diffusion model can describe the adsorption kinetics curves of CMP and GMP better than the pseudo-first-order rate model and the pseudo-second-order rate model. CMP and GMP could be both separated with a high separation resolution when the loading amount is 0.031 or 0.093 bed volume (BV). A novel pH-dependent film-surface diffusion model was developed and the model predicted the dynamic separation process of CMP and GMP with pH gradient elution satisfactorily.