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A study of the interaction of chromium with O-donor humic-like ligands war performed using electrospray-ionization mass spectrometry (ESI-MS) and ultraviolet/visible (UV/Vis) spectrophotometry. The heterogeneity of the functional groups justifies the use of model compounds of humic substances. For studying the interaction of chromium with humic substances, benzoic, salicylic, phthalic and citric acid, which include O-donor atoms also present in heterogeneous and polydispersed ligands, such as humic and fulvic acids, were used as model substances. The intensity of the interaction is correlated with the acid-base and electron-donor properties, geometric and steric characteristics, and the number and the positions of the O-donor atoms in the investigated ligands. UV/Vis data describing chromium interaction with humiclike ligands was placed in correlation with ESI-MS data of the complexes, both in quantitative and in qualitative terms. UV/Vis spectroscopy and ESI-MS quantification showed a large difference in the information they yield in describing the interaction of chromium(III) with the ligand, the ESI-MS technique being more informative. ESI-MS Technique can be used for quantitative analysis of the system Cr(III)-ligand. ESI-MS ion current chromatograms of 20 μL loop injections of systems Cr(III)-ligand, indicate a stabile peak and signal integrity.