Poly(N-isopropylacrylamide) (PNIPA)-based sorbents have been successfully used as sorbents in temperature-sensitive chromatography. Yet, the mechanisms controlling the binding of biochemicals to these sorbents and, therefore, the separation process are not fully understood. In the current work, the role of hydrophobic interactions in the binding of amino acids of different hydrophobicities to PNIPA microgels was studied. Binding experiments were conducted both below (25 degreesC) and above (37 degreesC) the volume-phase transition temperature of the gel. At 25 degreesC, no straightforward correlation between the partition coefficient and the hydrophobicity could be suggested for low hydrophobicity values. Contrary, at higher hydrophobicities the partition coefficient increases with increasing hydrophobicity. This correlation holds for the whole hydrophobicity range at 37 degreesC; however, the binding data suggests two different binding mechanisms of the hydrophilic amino acids and the hydrophobic ones. Isothermal titration calorimetry measurements confirmed this suggestion: The binding of hydrophobic amino acids seems to be driven by hydrophobic interactions, as evident from the positive binding enthalpy and the clear correlation between the amino acid's hydrophobicity and the binding entropy. Contrary, the binding of the hydrophilic amino acids was exothermic, implying a binding mechanism based on specific interactions, most probably hydrogen bonding.