Abstract Separations of various classes of pharmaceuticals using several HPLC retention modes simultaneously are described. The drugs including barbiturates, anti-inflammatories, cephalosporins and sulfa antibiotics are separated by a combination of reversed phase and ion exchange on a new multi-phase HPLC column. This column contains two types of particles, a macroporous substrate particle, which has adsorptive properties, and a monolayer of ion exchange latex particles which is attached to the substrate. The reversed phase and ion exchange retention modes operate independently and therefore can be controlled independently through selection of eluant components and gradient conditions. This approach to multi-mode chromatography is applied to accomplish assay reduction and on-line sample preparation. The determination of drugs in biomedical matrices such as urine, serum and saliva are discussed. Advantages of multi-mode chromatography over ion-pair reversed phase chromatography are discussed.