The relative hydrophobicity of the phases of several {polyethylene glycol (PEG) 8000 + sodium sulfate (Na(2)SO(4))} aqueous two-phase systems (ATPSs), all containing 0.01 mol . L(-1) sodium phosphate buffer (NaPB, pH 7.4) and increasing concentration of a salt additive, NaCl or KCl, up to 1.0 mol . L(-1), was measured by the free energy of transfer of a methylene group between the phases, Delta G(CH(2)). The Delta G(CH(2)) of the systems was determined by partitioning of a homologous series of five sodium salts of dinitrophenylated (DNP) - amino acids with aliphatic side chains in three different tie-lines of each biphasic system. The relative hydrophobicity of the phases ranged from -0.125 to -0.183 kcal . mol(-1), being the NaCl salt the one to provide the more effective changes. The results show that, within each system, there is a linear relationship between the Delta G(CH(2)) and the tie-line length (TLL), and biphasic systems with high salt additive concentration present the most negative Delta G(CH(2)) values. Therefore, the feasibility of establishing a relationship between the relative hydrophobicity of the phases in a given TLL and the ionic strength of the salt additive was investigated and a satisfactory correlation was found for each salt. (C) 2011 Elsevier Ltd. All rights reserved.