Stability constants or equilibrium constants for metal complex formation have long been employed as an effective measure of the affinity of a ligand for a metal ion in solution. The role of trace metals in biological systems is well recognized in recent years. Some of the trace metal ions like Co(II), Ni(II) and Cu(II) are essential and any variation in their biological concentrations leads to metabolic disorders. So in the present study 2-meracptosuccinic acid (MSA) and 1,10-Phenanthroline (Phen) are chosen as a model compounds to investigate the binding effect of biomolecules with the trace metal ions Co(II), Ni(II) and Cu(II) in DMF- and AN-water mixtures under conditions comparable to those of biological systems. The aim of the present study is to probe into the nature of interactions and speciation of the complexes of Co(II), Ni(II) and Cu(II) with MSA and Phen in DMF- and AN-water mixtures of varying compositions at an ionic strength of 0.16 mol dm-3 by monitoring the change in the free hydrogen ion concentration by glass electrode.