To understand the mechanism of interaction between peptides and peptides with hydrophobic ligands, the oligomers (GWG, GWWG, GWWWG) were designed and synthesized to study adsorption behavior with octyl sepharose and CM-octyl sepharose. By batch equilibrium binding analysis and dilution heat of peptide solution measurement, the binding isotherm and adsorption enthalpy were obtained and the binding thermodynamics parameters were calculated and analyzed. In the isotherm analysis, we reveled that the affinity of GWG for both adsorbents is stronger than that of GWWG and GWWWG. The results demonstrate that the cation-pi interaction between the peptides and the buffer molecules is significant for solutions of peptides with tryptophan residues, and the solvation is competitive with the hydrophobic interaction between the peptides and the hydrophobic ligands. From the dilution heat measurements, we observed an endothermic dilution heat for GWG and exothermic for GWWG and GWWWG. All these results indicate that the increased tryptophan chain length can promote the solvation behavior of the peptides by the peptide-buffer interaction in this buffer system. Comparing the types of ligands reveals that the binding affinities of each peptide for the two adsorbents are similar. However, the mechanism of adsorption for peptides with hydrophobic ligands might be quite different with respect to the binding enthalpy between peptides and adsorbents. The adsorption of the peptides on octyl sepharose is an entropy-driven process for all the peptides. In contrast, the adsorption of CM-octyl sepharose with GWG and GWWG is an enthalpy-driven process, whereas that with GWWWG is entropy-driven. These findings indicate that the amount of tryptophan controls the characteristics of the peptides and the interaction mechanism in the binding procedure. This study of the adsorption mechanism of the designed peptide could provide fundamental information for peptide purification and amino acid residue behavior in peptide drug design.