The purpose of this study was to investigate the effects of different pH and ion concentration of mobile phase on separating purine and pyrimidine bases by reversed phase high performance liquid chromatography (RP-HPLO). The 7 nucleobases could be separated perfectly in18minutes by RP-HPLO at a 0.02M, pH 4.0KOAc mobile phase with 0.1% triethylamine. The purine bases in soybean (0.05g) were liberated from nucleic acids, nucleotides or nucleosides by a acid hydrolization with 2.75mL trifluoroacetic acid (TFA) formic acid (FA): deionized water(5:5:1) at 120℃ for 30 minutes. The preparation of sample with Sep-pak C18 cartridge could protect ODS column effectively, because the nonpolar hydrolates would be eliminated.