Fig. 2. Absorbance response of ligands 2c and 3c with Cu²⁺. (a) Ultraviolet (UV)-visible titration of 2c (50 μM) with CuCl₂ (0–60 μM) in buffer. Black arrows indicate spectral changes upon addition of Cu²⁺. The inset shows the plot of absorbance at 420 nm versus concentration of Cu²⁺. (b) UV-visible titration of 3c (50 μM) with CuCl₂ (0–60 μM) in buffer. Black arrows indicate spectral changes upon addition of Cu²⁺. The inset shows the plot of absorbance at 450 nm versus concentration of Cu²⁺. (c) Visible-near infra-red (NIR) absorbance spectra for titration of 2c with Cu²⁺. The inset shows visible-NIR absorbance spectra for titration of 3c with Cu²⁺. (d) Area normalized absorbance spectra representing titration of CuCl₂ to 3c. Black arrows indicate isosbestic points. (e) Job's plot depicting absorbance versus mole fraction for the Cu²⁺–3c system. (f) Absorption binding plot for titration of Cu²⁺ to 2c (50 μM, black squares) with simulated fits (fitted to equation: conditional stability constant β = [ML₂]/[M][L]²), using log β as 9 (green dashed line), 11 (purple dashed line), 12 (blue dashed line), 13 (red dashed line), 14 (light green dashed line). Buffer used 20 mM HEPES (100 mM NaCl, pH 7.0).