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Catalog No.: GC47346

Ferrozine reacts with divalent iron to form a stable magenta complex species and used for the direct determination of iron in water with maximum absorbance at 562 nm.

Ferrozine Chemical Structure

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1 g
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5 g
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10 g
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Sample solution is provided at 25 µL, 10mM.

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Ferrozine reacts with divalent iron to form a stable magenta complex species and used for the direct determination of iron in water with maximum absorbance at 562 nm [1,2]. The visible absorption spectrum of the ferrous complex of ferrozine exhibits a single sharp peak at 562 nm. At this wavelength, the molar absorptivity is 27,900 and the Beer-ambert law is obeyed to approximately 4 mg/L of Fe [1].

Ferrozine assay of ISE6 cells[3]

The ferrozine assay for measuring non-haem iron was adapted to determine the concentration of iron in ISE6 cells. After knockdown and/or iron exposure, cells were collected, and cell lysates were collected using the method described above. Concentrated HCl (99.5%) was added and then heated to 95 °C. After cooling to room temperature, the mixture was centrifuged, and the supernatant was obtained, to which was added 75 mM ascorbate or water. Afterward, 10 mM ferrozine was added. Saturated ammonium acetate was added to facilitate colour development. Absorbance was measured at 550 nm, and the iron concentration was calculated based on a molar extinction coefficient of the iron-ferrozine complex of 27,900 M-1cm-1 and based on the protein concentration. The protein concentration was measured using a Micro BCA Protein Assay Kit. The total iron concentration is computed from samples with ascorbate. The ferrous iron concentration was computed from samples without ascorbate (reducing agent), while the ferric iron concentration is computed from the difference between the total iron and ferrous iron concentration.

[1]. Stookey L L. Ferrozine---a new spectrophotometric reagent for iron[J]. Analytical chemistry, 1970, 42(7): 779-781.
[2]. Jeitner T M. Optimized ferrozine-based assay for dissolved iron[J]. Analytical biochemistry, 2014, 454: 36-37.
[3]. Hernandez E P, Kusakisako K, Talactac M R, et al. Induction of intracellular ferritin expression in embryo-derived Ixodes scapularis cell line (ISE6)[J]. Scientific reports, 2018, 8(1): 1-12.

Chemical Properties

Cas No. 69898-45-9 SDF
Canonical SMILES OS(C1=CC=C(C2=C(C3=CC=C(S([O-])(=O)=O)C=C3)N=C(C4=CC=CC=N4)N=N2)C=C1)(=O)=O.[Na+]
Formula C20H13N4O6S2.Na M.Wt 492.5
Solubility PBS (pH 7.2): 1 mg/ml Storage Store at 2-8°C
General tips For obtaining a higher solubility , please warm the tube at 37 ℃ and shake it in the ultrasonic bath for a while.Stock solution can be stored below -20℃ for several months.
Shipping Condition Evaluation sample solution : ship with blue ice
All other available size: ship with RT , or blue ice upon request

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Research Update

Colorimetric ferrozine-based assay for the quantitation of iron in cultured cells

Anal Biochem2004 Aug 15;331(2):370-5.PMID: 15265744DOI: 10.1016/j.ab.2004.03.049

The ferrozine-based colorimetric assay described here permits the quantitation of iron in cultured cells in amounts ranging between 0.2 and 30 nmol. Ferrous and ferric iron were detected equally well by the assay and the accuracy was unaffected by other divalent metal cations. This colorimetric assay was used to study iron accumulation in brain astrocytes that had been cultured in 24-well dishes. Iron complexed to cellular proteins was made accessible to ferrozine by treatment of cell lysates with acidic KMnO(4) solution. The basal amounts of iron in untreated astrocyte cultures were approximately 10 nmol iron per mg protein. Incubation of the cells with ferric ammonium citrate caused the total cellular iron content to increase in a concentration-dependent manner. The estimates of cellular iron content that were obtained with the ferrozine-based assay did not differ from those determined by atomic absorption spectroscopy. The colorimetric assay described here provides a sensitive, cheap, and reliable method for the quantitation of intracellular iron and for the investigation of iron accumulation in cultured cells.

Optimized ferrozine-based assay for dissolved iron

Anal Biochem2014 Jun 1;454:36-7.PMID: 24632099DOI: 10.1016/j.ab.2014.02.026

The following report describes a simple and optimized assay for the detection of iron in solution based on the binding of this metal by ferrozine. This assay accurately measures between 1 and 200 ¦̍ sample iron concentrations within 2? hours.

Complexation of ferrous ions by ferrozine, 2,2'-bipyridine and 1,10-phenanthroline: Implication for the quantification of iron in biological systems

J Inorg Biochem2021 Jul;220:111460.PMID: 33866045DOI: 10.1016/j.jinorgbio.2021.111460

Iron is an essential nutrient for virtually all forms of life. Because of its redox properties and involvement in a wide range of biological processes, a number of qualitative and quantitative chemical tools have been developed to detect reduced (Fe2+) and oxidized (Fe3+) forms of iron in biomolecules. These types of measurements are not only important in detecting iron species in solution, but also in understanding iron distribution, accumulation, and role in physiological and pathological processes. Here, we use UV-vis spectrophotometry and three common chromogenic reagents, ferrozine, 2,2'-bipyridine, and 1,10-phenanthroline to detect and quantify the concentration of ferrous ions in aqueous solutions, owing to the unique absorption spectra, specific molar absorptivity, and characteristic colors of these Fe2+-chelator complexes. Our results show that the kinetics of the formation of the {Fe2+-(ferrozine)3} complex, but not the{Fe2+-(bipyridine)3} or the {Fe(II)-(phenanthroline)3} complexes depend on the concentration of the iron chelator, requiring up to 20 min to complete when close to stoichiometric ratios are employed. The molar absorptivity values of these complexes under excess chelator concentrations were ~ 10% to 15% higher than reported literature values (i.e. 31,500 ¡À 1500 M-1 cm-1 for ferrozine at 562 nm, 9950 ¡À 100 M-1 cm-1 for 2,2'-bipyridine at 522 nm, and 12,450 ¡À 370 M-1 cm-1 for 1,10-phenanthroline at 510 nm). Our results have important implications when quantifying iron in biological systems and reveal optimal experimental conditions that must be employed for the accurate measurements of ferrous ions, whether free in solution, or after reduction of protein-bound ferric ions.

A Production-Accessible Method: Spectrophotometric Iron Speciation in Wine Using Ferrozine and Ethylenediaminetetraacetic Acid

J Agric Food Chem2019 Jan 16;67(2):680-687.PMID: 30561197DOI: 10.1021/acs.jafc.8b04497

Wine oxidation is reported to be linked to the iron species present in the wine, but spectrophotometric speciation is plagued by unstable measurements due to alterations to the reduction potential of iron by complexing agents. Ferrozine raises the reduction potential of iron by complexing preferentially to iron(II), inducing the reduction of iron(III) during analysis; here, EDTA is added to chelate iron(III) and to stabilize the forms of iron. Bisulfite addition allows the use of ferrozine for red wine analysis by mitigating color interference. Measurements agree with values from a previous method for iron(II) and from FAAS for total iron. Spike recoveries were in the range of 103.5-110.1%. The method is linear for iron concentrations in the range of 0.10-6.00 mg L-1 and offers good precision (CV 0.4-10.1%) and low limits of detection (0.02 mg L-1) and quantification (0.06 mg L-1). The method demonstrated changes to iron speciation during the oxygenation of red wines.

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

J Microbiol Methods2013 Dec;95(3):366-7.PMID: 24140574DOI: 10.1016/j.mimet.2013.10.005

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 (Fe(3+)) on ferrous iron (Fe(2+)) measurements performed in the dark. Fe(3+) concentrations affected the absorbance measurements, which linearly increased with incubation time.


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