A new method for the qualitative analysis of adenosine nucleotides (AMP, ADP, and ATP) and synthetic oligonucleotides has been proposed, utilizing a pH- and temperature-responsive polymer of N-isopropylacrylamide (NIPAAm), butyl methacrylate (BMA) and N,N-dimethylaminopropylacrylamide (DMAPAAm) as the stationary phase of HPLC. In the chromatographic system using the copolymer with ionizable groups of modified packing materials, we investigated how to separate adenosine nucleotides and oligonucleotides by temperature. The properties of the surface of the copolymer-grafted stationary phase altered from hydrophilic to hydrophobic and from charged to non-charged due to changes in the temperature and in the pH, respectively. In addition, it is possible to exhibit and hide ion-exchange groups on the polymer chain surface by temperature changes. These phenomena result from changes in the charge and hydrophobicity of the pH- and temperature-responsive polymer on the stationary surface with the controlling temperature. A pH- and temperature-responsive chromatography would be greatly useful for biopolymer and nucleotide separation and purification.