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Interference of ferric ions with ferrous iron quantification using the ferrozine assay

https://doi.org/10.1016/j.mimet.2013.10.005Get rights and content

Highlights

  • Ferric ions (Fe3 +) interfere with ferrous iron (Fe2 +) measurements.

  • Fe3 + interference increases linearly with incubation time.

  • Incubation time must be carefully controlled to avoid erroneous measurements.

Abstract

The ferrozine assay is a widely used colorimetric method for determining soluble iron concentrations. We provide evidence for a heretofore unrecognized interference of ferric ions (Fe3 +) on ferrous iron (Fe2 +) measurements performed in the dark. Fe3 + concentrations affected the absorbance measurements, which linearly increased with incubation time.

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Acknowledgements

Financial support for this work was provided by the U.S. Department of Energy, Office of Biological and Environmental Research, Subsurface Biogeochemical Research Program.

References (14)

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    It is generally assumed that ferrozine selectively react with Fe(II) and therefore, it is possible to quantify Fe(II) in a mixture of Fe(II) and Fe(III) without any interference from Fe(III) [9,29,31]. Even though Fe(III) does not make any stable complexes with ferrozine, reduction of Fe(III) to Fe(II) in the presence of ferrozine has been reported [32–34]. It has been observed that longer incubation periods and exposure of the reagents to sunlight could increase the amount of Fe(III) reduced to Fe(II) in the presence of ferrozine and hence overestimating the concentration of Fe(II) [33,34].

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    Although ferrozine is not a physiologically relevant ligand, the ferrozine assay has been widely used in the detection of ferrous ions in a variety of samples, including in mineral and biological systems. The accurate quantification of Fe2+ ions is affected by several factors including pH, temperature, concentration of reagents, incubation time, anions such as oxalate, cyanide and nitrite, and also the presence of Fe3+ cations [7,13–20]. Because of the rather high binding affinity of ferrozine for Fe2+ ions [15], many studies have used stoichiometric amounts of ferrozine, or a slight excess of chelator, to make sure that Fe2+ ions are fully bound.

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

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    The slopes at this Fe(III) concentration are 6.16 × 10-5 and 5.76 × 10-5 absorbance units min−1, corresponding to 5.4 × 10-3 and 2.0 × 10-3 μM Fe(II) min−1 for the Phen and FZ method, respectively. Previous studies using the FZ method attributed the growing absorbance at 562 nm to photoreduction of Fe(III)-FZ complex by ambient light or reduction of Fe(III) by FZ itself [21,22,28]. For the Phen method, the increased absorbance at 510 nm has been attributed to photoreduction of the dimers, [(Phen)2Fe(OH)2Fe(Phen)2)]4+ or [(Phen)2Fe-O-Fe(Phen)2]4+ [17,29,30].

  • Measuring total dissolved Fe concentrations in phytoplankton cultures in the presence of synthetic and organic ligands using a modified ferrozine method

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    For example, Stookey (1970) identified several alkali metals other than Fe and alkaline earths which form colored complexes with FZ, as well as some anions (e.g. NO2−, CN− and C2O42−) that could interfere with Fe(II) measurements when present at concentrations over 500 mg/L. Dawson and Lyle (1990) and Luther et al. (1996) reported an incomplete reduction step, depending on the speciation of Fe(III) in the sample. Some studies have reported reduction of incubated Fe(III) with FZ over time (Im et al., 2013) where Fe(III)-FZ complexes were photosensitive (Anastácio et al., 2008). The presence of NOM in samples can also retard Fe(II)-FZ complexation, resulting in development of an asymptotic absorbance over time (Box, 1984; Rose and Waite, 2003).

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