Revisiting the phenanthroline and ferrozine colorimetric methods for quantification of Fe(II) in Fenton reactions

Colorimetric methods for Fe(II) determination based on complexation of heterocyclic amines, such as phenanthroline (Phen) and ferrozine (FZ), have been widely used for analysis of iron species in solution, which is critical to discuss the mechanism of Fenton and related reaction systems. However, the reliability of both methods in the background of hydrogen peroxide has never been addressed. In this study, we demonstrate that serious Fe(II) overestimation possibly occurs for both methods in Fenton systems, particularly for the Phen method. A systematic variation of incubation conditions reveals that the available Fe(II) concentration depends greatly on the incubation time after mixing the colorimetric reagents with the sample. Also, it is highly sensitive to pH and temperature as well as the initial concentrations of Fe(III), H2O2, and acetate buffer. The experimental results combined with simulations indicate that Fe(II) overestimation mainly results from the reduction of Fe(III)-Phen or Fe(III)-FZ complexes by residual H2O2 during analysis, a reaction that is facilitated by the ligand field effect of the heterocyclic amine. An effective remedy for the instability and inaccuracy of the methods in the presence of H2O2 is the addition of NH4F to the incubation mixture (55 mM for Phen and 35 mM for FZ), which converts residual Fe(III) to a colorless and reduction-insensitive fluoride complex.