Significant performance differences in the separation of desamido-insulin from insulin and of amitriptyline from imipramine have been found for five, macroscopically similar, preparative C18 silica materials. Structure-performance relations have been used to understand this difference. Physical measurements on the unpacked silica gels as well as chromatographic measurements have been carried out. The latter measurements comprise: hydrophobicity, metal impurity, steric selectivity and silanol activity. The performance difference could not be explained by the difference in hydrophobicity, steric selectivity or metal impurity between the materials. Instead a correlation between the silanol ion exchange activity and the selectivity could be found. The highest performance was found under conditions where the silanol groups have a moderate activity. For the separations considered in this work, the silanol groups have a beneficial effect.