The behaviour of two structural isomers of the copper(II) complex of 1,4,8,11-tetraazacyclotetradecane-1,8-bis(methylphosphonic acid) (H 4 dipon), penta- and hexacoordinated species, was studied by capillary electrophoresis. The separation and determination of both the isomers with the same molecular weight and charge and the free H 4 dipon ligand were achieved in 75mM disodium tetraborate aqueous solution (pH 9.5) as background electrolyte and, simultaneously, magnitudes of their effective mobilities were determined. The influence of both variables (pH and buffer composition/concentration) on the separation was established and the principle of the separation was proposed. The method of separation was applied to the study of the reaction mechanism of the isomerization. It was proved that the reaction can be explained by several mechanisms with the same fit through experimental data. The first step of the isomerization reaction proceeds relatively slowly and is the rate-determining step (rds) of overall isomerization reaction. Since no intermediate was detected during the reaction, it was concluded that the next step(s) is/are much faster than the rds (at least 10 times). The magnitudes of the rate constant at two different temperatures (k 80°C (1.23±0.09)×10 −3 min −1 , k 90°C (2.45±0.06)×10 −3 min −1 ) and activation energy (E a =72±8kJmol −1 ), enthalpy (ΔH # =69±8kJmol −1 ) and entropy (ΔS # =−106±23JK −1 mol −1 ) were determined.