Abstract: An ideal bactericide benzalkonium chloride which can effectively kill the bacteria infected in affinity chromatography gel and not affect the protein adsorption capacity was eventually selected from several bactericides by quantitative bactericidal test on bacterial suspension and affinity chromatography gel protein adsorption quantity determination. An ion pair reversed phase high performance liquid chromatography (RP-IPHPLC) method was developed for the determination of benzalkonium chloride residue in affinity chromatography gel. Separation was carried out on C18 column, using acetonitrile and 0.2mol/L sodium heptanesulfonate solution as the mobile phase in an isocratic elution．The detection wavelength was 210 nm; the detection limit was 0.000 25 mg/mL; relative standard deviation of benzalkonium chloride determination test in practical samples was 0.63%. This method presents excellent sensitivity and precision.

Key words: benzalkonium chloride, protein adsorption capacity, detection limit, ion pair revered phase high-performance liquid chromatography