MEASUREMENT OF ASSOCIATION CONSTANTS FOR COMPLEXES BY REVERSED-PHASE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

Association constants for metal-binding by various nucleotides, crown ethers and nitroso-napththolsulfonic acids in solution were measured by high-performace liquid chromatography employing appropriate eluents and non-polar bonded stationary phases. The results are in excellent agreement with pertinent literature data. The procedure is applicable to the measurement of stability constants of complexes which contain one solute molecule and are formed in the solution used as the eluent, provided species bound to the stationary phase are not involved in the complex formation and the chromatographic system is linear. The concentration of the metal ion in the appropriate mobile phase was varied and small quantities of the above substances were injected into the column. The hyperbolic dependence of the retention factgor, k, on the concentration of Na+, K+, Mg2+ or Zn2+ in the eluent allowed the use of various linear plots for the evaluation of the stability constants for complexes formed by such ions with substances mentioned above. The effect of complexation on retention is conveniently measured by the retention modulus, ν, which is given for the complex, νc, by the ratio of the retention factors of the complex and the uncomplexed solute, both measured under otherwise identical conditions. With nucleotides chromatographic retention increases upon metal binding due to reduction of electronic charge on the molecule and η > 1. On the other hand nitroso-naphtholsulfonates and crown ethers eluted faster in the form of a complex with metal ions so that η < 1. Theoretical and practical limitations of the chromatographic approach to evaluation of stability constants are extensively discussed and tests are proposed to assure the applicbility of the method to a given system. © 1979.