The 13C isotope fractionations in the exchange reaction were experimentally determined for a chemical system consisting of H2O and CH3OH solvents and NH4Cl, NH4OH and HCl solutes at ambient temperature under a pressure of 9.5×104 Pa. The heavy isotope component, 13C, was found to be enriched in the CO–Cu(I) complex in the solution phase. Both CO solubility and the isotope separation factor α were found to increase with decreasing temperature in all Cu(I)Cl solutions. An isotope separation factor of 1.023 and concentration of CO absorption of 1.2 M were obtained for an aqueous solution composition of 1.5 M Cu(I)Cl, 0.75 M NH4Cl, 7.0 M NH4OH at a temperature of 8.5°C. The enthalpy and entropy changes in isotope exchange reactions were calculated from the temperature dependence of the isotope separation factor. In addition, the experimentally determined isotope separation coefficients (e=ln α) were found to be well fitted by the equation e=a/T+b/T2, and the values of a and b were obtained for both experimental systems of aqueous solutions and methanol–water mixed solutions. The isotope exchange reaction was seen to reach an equilibrium state within 1 min at a stirring speed of 4000 rpm.