The Gibbs free energy of transfer of a methylene group, Delta G*(CH2), is reported as a measure of the relative hydrophobicity of the equilibrium phases. Furthermore, Delta G*(CH2) is a characteristic parameter of each tie-line, and for that reason can be used for comparing different tie-lines of a given aqueous two-phase system (ATPS) or even to establish comparisons among different ATPSs. In this work, the partition coefficients of a series of four dinitrophenylated-amino acids were experimentally determined, at 23 degrees C, in five different tie-lines of PEG8000-(sodium or potassium) citrate ATPSs. Delta G*(CH2) values were calculated from the partition coefficients and used to evaluate the relative hydrophobicity of the equilibrium phases. PEG8000-potassium citrate ATPSs presented larger relative hydrophobicity than PEG8000-sodium citrate ATPSs. Furthermore, the results obtained indicated that the PEG-rich phase (top phase) has higher affinity to participate in hydrophobic hydration interactions than the salt-rich phase (bottom phase). 2015 Elsevier B.V. All rights reserved.