Abstract: AIM: The generalized HCRF (gHCRF) was proposed as a new optimization parameter based on heterogeneous chromatographic response function(HCRF), and was first used in the HPLC separation of chrolobenzylidine and its related compounds. METHODS: By use of Spherical Symmetric Design, optimization was made on the two factors (x₁: methanol concentration;x₂: perchloric acid concentration) which affected the chromatographic separation. The equations simulating the relationship between response values and factors were setup. RESULTS: The theoretical optimal condition from gHCRF equation is x₁=76% andx₂=0.01 mol·L^-1 , which is very much close to the experimental optimal condition (x₁=75% andx₂=0.01mol·L^-1). Therefore, the prediction from the equation is reasonable. CONCLUSION: The separation of chrolobenzylidine and its related compounds showed that the gHCRF parameter can be satisfactorily applied to the chromatographic optimization of complex samples.