DNA structure studies attract many interests in pharmaceutical, biochemical and medical disciplines. Among them, base pairs play a vital role in biological information transfer. Therefore, they need to be analyzed in various ways and the pair of guaninine and cytosine is the present analytical object. Separation of guanine and cytosine was researched by Aspen chromatography simulator and HPLC(High Performance Liquid Chromatography) experiments. Aspen chromatography simulation resulted in various chromatograms with changes of sample concentration, eluent flow rate and number of plate. The resolutions and yields of guanine and cytosine were calculated to obtain a best separation condition. C18 HPLC column and water/methanol/acetic acid mixture(90/10/0.2) were used for separation of guanine and cytosine. HPLC parameters(resolution and number of theoretical plate) were calculated under different flow rates and sample concentrations. Aspen chromatography simulation and HPLC experimental results were compared with fair agreement.