The synthesis route was investigated and optimized for the preparation of iminodiacetic acid-polyethylene glycol (IDA-PEG) for immobilized metal ion affinity partitioning in aqueous two-phase systems. IDA-PEG was synthesized from PEG in two steps by the reaction of iminodiacetic acid with a monosubstituted derivative of epichlorohydrin-activated PEG. The Cu2+ content combined with IDA-PEG was determined by atomic absorption spectrometry as 0.5 mol.mol(-1) (PEG). Furthermore, the affinity partitioning behavior of lactate dehydrogenase in polyethylene glycol/hydroxypropyl starch aqueous two-phase systems was studied to clarify the affinity effect of the Cu(II)-IDA-PEG.