Abstract: Objective To study the effect of different mobile phase systems,analytical chromatography columns of different tampons,pH,and column temperature on separation of compound antigenic peptide and optimize the conditions for reversed phase chromatographic separation. Methods The effect of analytical chromatography columns of different tampons（ZorbaxSB C18 4.6×250mm 5u,Zorbax XDB C8 4.6×250mm 5u,Sephsil C4 4.6×100mm 5u） and different mobile phase systems（velocity of acetonitrile flow=1.0ml/min,test wavelength=214nm,and injection dose=20μl） on separation of compound antigenic peptide was detected. Results The analytical chromatography columns of different tampons and different mobile phase systems significantly influenced the pH value of heptanesulfonic acid sodium.The pH value of heptanesulfonic acid sodium could be adjusted to 0.05mol/L KH2PO4 and 0.05mol/L TEA/0.1% with H3PO4. Conclusion This method is simple with good results and can thus be used in separation of compound antigenic peptide.