Separation of guanine and cytosine base pairs in nucleotide is an interesting topic for investigation of DNA structure. Therefore, an understanding of nucleotide separation by chromatography is necessary to prepare DNA molecules. Guanine and cytosine separation in SMB was simulated by Aspen chromatography and it was experimented by assembled 3-zone simulated moving bed (SMB) with change of stream flow rates, sample concentration, and desorbent flow rate. The simulation of batch chromatography was also confirmed by HPLC experiments. Based on these, good operating conditions of SMB chromatography were determined. Three-zone SMB equipment was set up by connecting three C18-HPLC columns, four HPLC pumps, and six multiposition valves. Batch chromatography of cytosine and guanine was conducted to determine the isotherms of the two nucleotides. The outlet streams of SMB, raffinate and extract were sampled and analyzed by analytical HPLC system. The adsorption isotherms of cytosine and guanine were HC=0.5 and HG=1.05. The highest experimental purity of cytosine and guanine in SMB was obtained as 94.9% and 89.8% with operating parameters of Qfeed=0.2 mL/min, Qdesorbent=0.6 mL/min, Qextract=0.2 mL/min, Qraffinate=0.6 mL/min, and switching time=7 min.