2.2. UV–Vis Metal Binding Studies

The synthesized compounds were tested for their metal chelating effect through spectroscopic measurements [59,60]. The UV–Vis absorbance of compounds was recorded in the absence and presence of metal ions using a UV–Vis spectrometer (a SPECTROstarNano (220–1000 nm), Ortenberg, Germany. The experiment was performed in 96-well plates and each experiment was made in triplicates. A fixed amount of investigated ligand (20 μM) solubilized in MeOH was mixed with increasing concentration of Cu²⁺ (CuCl₂), Fe²⁺ (FeSO₄) and Zn²⁺ (ZnCl₂) from 0 to 80 μM and incubated for 30 min at room temperature (r.t.). The absorbance change was monitored at wavelengths ranging from 220 to 700 nm. The stoichiometry of studied compounds for Cu²⁺ and Fe²⁺ complexes was determined by employing the mole ratio method.

Due to a non-measurable UV absorbance for compounds 4a and 5c, a different protocol using pyrocatechol violet (PV) was applied [61]. The compounds (200 µM) incubated for 10 min with CuSO₄ (200 µM), after 200 µM PV was added and the plate was incubated at 25 °C for 20 min. The absorption spectra were recorded. The same protocol was used for Fe²⁺ (FeSO₄) in presence of ferrozine.