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Dissolved organic matter (DOM) is known to form strong complexes with heavy metals and thus governs the distribution, toxicity, bioavailability, and ultimate fate of heavy metals in the environment. The relevant aspects of metal–organic interactions remain unclear because the metal binding functionalities in DOM are substantially nonuniform and the availability of the models is limited. In this work, two-dimensional correlation spectroscopy (2DCOS) integrated with synchronous fluorescence and infrared absorption spectroscopy was used to explore the binding process of copper to DOM. A series of heterogeneous binding sites in humic acid (HA), a representative DOM, and the subsequent subtle changes of these sites within the molecular interactions were elucidated by the 2DCOS method. The band assignments and the correspondence between the results obtained by two spectral probes (synchronous …