The polarity, pH, ion concentration and polarity of the buffer ions of the mobile phase were modified systematically in order to find optimal conditions for the separation of nucleobases, nucleosides and nucleotides by ion-exchange chromatography. The effects of these mobile phase parameters on the retardation of 26 nucleobases, nucleosides and nucleotides on the cation exchanger Partisil-10 SCX and the anion exchanger Partisil-10 SAX were examined and resulted in the formulation of the following simple rules: (1) These ion exchangers also have reversed-phase and normal-phase properties; their occurrence is determined by the polarity of the mobile phase. (2) The ion concentration has an effect only on ion-exchange properties. (3) The pH of the mobile phase determines the degree of protonation of the solutes, and therefore the charge and polarity. Many solutes have different pK values, thus providing a tool for the variation of selectivity. (4) The polarity of the buffer ions has an indirect effect on ion-exchange chromatography through differential distribution of the buffer ions over the two phases. This differential distribution is caused by reversed-phase or normal-phase characteristics. The integration of these three types of chromatography has been demonstrated by some examples.