PEG-based ATPSs have been successfully used for the separation of not only biomolecules but also inorganic compounds as an environmentally friendly platform for liquid–liquid extraction. However, a fundamental understanding of the structure of aqueous PEG solutions and its effect on the partitioning of solute compounds in the ATPS remains elusive. In this chapter, spectrophotometric studies on the formation equilibria of the complexes of Co(II) with thiocyanate ion in aqueous PEG solutions are described. From the dependence of the apparent formation constants of the tetrahedral and octahedral Co(II) thiocyanate complexes on the concentration of ethylene oxide (EO) and terminal hydroxyl (OH) groups in the solutions, a micro-heterogeneous structure consisting of pseudo-phases formed by hydration of the EO chains and OH groups in PEG molecules is estimated. It is revealed that the intrinsic formation constant of tetrahedral Co(NCS)42− complex in the EO pseudo-phase is much larger than those in the OH phase and the bulk water phase. An important conclusion is drawn from these studies that weakly hydrated anions such as Co(NCS)42− distribute into the hydrophobic outer region in the helices of the EO chains and the partitioning of solute molecules and ions in PEG-based ATPSs is governed by the difference in the fractions of the EO and OH phases between the upper and lower phases of the systems.