Ferrozine Assay for Determining Fe(II) Concentrations in Solutions Containing Coumarins

Potential spectral interferences of Fe–coumarin complexes with the Ferrozine assay for determining Fe(II) concentrations were examined by adding Ferrozine to solutions containing Fe(II), Fe(III), Fe(II)–coumarin complexes, Fe(III)–coumarin complexes, and a mixture of both Fe(II)– and Fe(III)–coumarin complexes. For treatments involving Fe–coumarin complexes, Fe and coumarin stock solutions were mixed first in order to allow Fe–coumarin complexes to form (2 h), before addition of Ferrozine. The analyzed samples contained reactants in the following concentrations: 10 μM Fe(II), 10 μM Fe(III), 83 μM coumarin, 3 mM Ferrozine, 0.01 M NaCl, and 5 mM of the pH buffers (pH 6, 7, and 8.5). All experimental solutions and samples were prepared under anaerobic conditions. Samples were taken out of the anaerobic chamber just before analysis. The spectra of the samples were analyzed by UV–vis spectrophotometry over the wavelength range of 200 to 800 nm. Total Fe concentrations were measured by inductively coupled plasma mass spectrometry (ICP–MS, Agilent-7700; limit of quantification: 0.8 μg L^–1 Fe (0.014 μM)).

Changes in Fe redox speciation due to reduction of Fe(III) in Fe(III)–coumarin complexes by Ferrozine were investigated under anoxic conditions at pH 6, 7, and 8.5. Absorbance was measured repeatedly for the wavelength range from 400 to 800 nm, and Fe(II) concentrations were determined using the Ferrozine assay (563 nm; ε = 2.86 × 10⁴ M^–1 cm^–1). Reduction rates were calculated from the slopes of linear regression lines of the Fe(II) concentration as a function of time; the time interval used for the regression was from 0 to 4 h (for treatments in which more than 50% of the Fe(III) was reduced within 4) or from 0 to 24 h (for treatments in which less than 50% of the Fe(III) was reduced within 24 h) (Figure S26).