Ferrozine Research Articles

Development of an Fe2+ sensing system based on the inner filter effect between upconverting nanoparticles and ferrozine.

The ferrozine (FZ) assay is a vital oxidation state-specific colorimetric assay for the quantification of Fe2+ ions in environmental samples due to its sharp increase in absorbance at 562 nm upon addition of Fe2+. However, it has yet to be applied to corresponding fluoresence assays which typically offer higher sensitivites and lower detection limits. In this article we present for the first time its pairing with upconverting luminescent nanomaterials to enable detection of Fe2+via the inner filter effect using a low-power continuous wave diode laser (45 mW). Upon near infra-red excitation at 980 nm, the overlap of the upconversion emission of Er3+ at approximately 545 nm and the absorbance of the FZ:Fe2+ complex at 562 nm enabled measurement in the change of UCNP emission response as a function of Fe2+ concentration in a ratiometric manner. We first applied large, ultra-bright poly(acrylic acid) (PAA)-capped Gd2O2S:Yb3+,Er3+ UCNPs upconverting nanoparticles (UCNPs) for the detection of Fe2+ using FZ as the acceptor. The probe displayed good selectivity and sensitivity for Fe2+, with a low limit of detection (LoD) of 2.74 μM. Analogous results employing smaller (31 nm) PAA-capped hexagonal-phase NaYF4:Yb3+,Er3+ UCNPs synthesised in our lab were achieved, with a lower LoD towards Fe2+ of 1.43 μM. These results illustrate how the ratiometric nature of the system means it is applicable over a range of particle sizes, brightnesses and nanoparticle host matrices. Preliminary investigations also found the probes capable of detecting micromolar concentrations of Fe2+ in turbid solutions.

Facile liquid colorimetric sensor using high-density deep eutectic solvent for trace detection and speciation of iron in milk

An efficient colorimetric sensor was developed using a high-density deep eutectic solvent (HD-DES) for the trace detection and speciation of iron in various milk samples. A liquid colorimetric probe was fabricated by dissolving ferrozine (FZ) in HD-DES prepared from TBABr and PBA. The prederivatization of Fe2+ via complexation with FZ on the HD-DES/FZ probe provided the [Fe(FZ)3]4- complex, which led to a color change from pale yellow to purple before it was simultaneously extracted by HD-DES. The Fe3+ content was calculated by subtracting the amount of Fe2+ from the total Fe content following the reduction of Fe3+ to Fe2+ by L-ascorbic acid in an acid buffer. Under the optimized conditions, the proposed colorimetric sensor exhibited appreciable linearity in the concentration range of 0.003–0.04 mg L-1, a low limit of detection (0.95 µg L-1), high enrichment factor (50), and outstanding repeatability. The liquid colorimetric probe was successfully applied for the determination and speciation of iron in milk samples, and the results were compared with those obtained using the standard atomic absorption spectrometry method. Moreover, quantitative analysis was performed on a smartphone using the Image J application to estimate the color intensity change, which eliminated the requirement of sophisticated scientific instruments.

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

Abstract 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.

Solid-Phase Extraction Spectrophotometric Determination of Total Antioxidant Capacity in Antioxidant-poor Samples by Using the Ferric-Ferrozine Method.

Ferrozine (FZ) preferentially stabilizes Fe(II) over Fe(III) to raise the ferric reduction potential and oxidize antioxidants. The advantages of the ferric-ferrozine method over other iron-based total antioxidant capacity assays were: (i) higher molar absorptivity and enhanced sensitivity, (ii) lower interference from foreign ions, (iii) wide pH tolerance (iv) additivity of the absorbances for mixtures. Solid-phase extraction (SPE) could be combined with spectrophotometry, because the magenta-colored anionic Fe(II)-FZ complex was quantitatively sorbed on Sephadex QAE A-25 resin. The sensitivity enhancement using the resin enabled us to conduct total antioxidant capacity (TAC) measurements of antioxidant-poor samples. The apparent molar absorptivity, linear concentration range and trolox equivalent antioxidant capacities (TEAC) of certain antioxidants were found. The calibration curves (lines) of trolox, rutin, and rosmarinic acid individually and in herbal infusions-by using the method of standard additions-were parallel, confirming that the added antioxidants did not interact with herbal constituents to cause chemical deviations from Beer's law.

Limitation of ferrozine method for Fe(II) detection: reduction kinetics of micromolar concentration of Fe(III) by ferrozine in the dark

ABSTRACTThe dark reduction kinetics of micromolar concentrations of Fe(III) in aqueous solution were studied in the presence of millimolar concentrations of ferrozine (FZ) over the pH range 4.0–7.0. A pseudo-first-order kinetics model was used to describe Fe(III) reduction at pH 4.0 and 5.0, and the reduction rate decreased with increasing pH or initial Fe(III) concentration. A more molecular-based kinetics model was developed to describe Fe(III) reduction at pH 6.0 and 7.0. From this model, the intrinsic rate constants (k1) of Fe(III) reduction by FZ in the dark were obtained as 0.133 ± 0.004 M−1 s−1 at pH 6.0 and 0.101 ± 0.009 M−1 s−1 at pH 7.0. It was also found in this model that a higher pH, a higher concentration of Fe(III), a lower concentration of FZ and less incubation time led to a lower fraction of Fe(III) reduction by FZ in the dark.

Determination of Labile Fe(II) Species Complexed with Seawater Extractable Organic Matter Under Seawater Conditions Based on the Kinetics of Ligand-exchange Reactions with Ferrozine

A fertilizer, comprised of a mixture of steel slag and compost, was used to restore seaweed beds in barren coastal areas. Complex Fe(II) species, supplied by steel slag, play a significant role in supplying Fe(II) to coastal areas and stimulating seaweed growth. Seawater extractable organic matter (SWEOM) from compost is generally assumed to serve as a chelator of Fe(II) in the fertilizer. It is considered that the bioavailability of Fe(II)-SWEOM complexes is higher in the dissociable (labile) species. In the present study, a method for determining labile species of Fe(II)-SWEOM complexes in seawater (pH 8.0, I = 0.7) was developed. The method is based on a ligand-exchange reaction between SWEOM and ferrozine (FZ). Because Fe(II) is readily oxidized to Fe(III) under normal seawater conditions, ascorbic acid was added as an antioxidant. The coloring for the Fe-FZ complex in the presence of SWEOM was retarded. This retarding can be attributed to a ligand-exchange reaction between FZ and labile Fe(II)-SWEOM complexes. Conditional binding constants for the labile Fe(II)-SWEOM complexes and binding capacities of labile sites in SWEOM to Fe(II) were evaluated for a variety of total Fe(II) concentrations.

The measurement of organically complexed FeII in natural waters using competitive ligand reverse titration

Whilst there is increasing evidence for the presence of stabilized FeII associated with organic matter in aquatic environments, the absence of a reliable method for determining FeII speciation in solution has inhibited the study of this aspect of Fe biogeochemistry. A technique is described here for the determination of FeII organic complexation in natural waters that is based on competitive ligand reverse titration and a model fit to experimental results, from which ligand concentration and a conditional stability constant can be obtained. Spectrophotometry was used to detect the Ferrozine (FZ) complex with reactive FeII, which in combination with a liquid waveguide capillary cell (LWCC) enabled high sensitivity and precision measurements of FeII to be made. A series of samples was collected in the Itchen River in Southampton, UK to test the method at a wide range of salinities including river water. Levels of FeII and total dissolved Fe were within previously reported values for this system. FeII was found to occur organically complexed with values for log K′FeIIL (conditional stability constant for FeII-natural ligand complexes) of ≈8 at salinities between 0 and 21, whilst no measurable complexation was detected at a salinity of 31. This work demonstrates that spectrophotometry can be used in combination with ligand competition to investigate metal speciation in natural waters.

In situ speciation of trace Fe(II) and Fe(III) in atmospheric waters by the FZ method coupled to GFAAS analysis

A quasi-on-line method of measurement of the oxidation states of iron coupled with a GFAAS analysis is reported for the trace conditions found in atmospheric waters. This technique is based on the formation of a specific complex [Fe(FZ)3]4− between Fe(II) and ferrozine (FZ). tC18 solid-phase extraction cartridges (Sep-Pak) are used to separate the [Fe(FZ)3]4− and Fe(III) so as to limit the risk of redox evolution of the sample. The adaptation to dilute aqueous media, via acidification at pH = 2 of rainwater sample, and atmospheric interferences are discussed, and the Fe(II) recovery in rainwaters is determined. This method coupled with a quasi-on-line sampling protocol has been tested on rains collected in Guadeloupe Island (Caribbean Sea, 16°N, 61°W) during a field campaign in May 2005. Our results show that the proposed method can satisfactorily be applied to the determination of Fe(II) and Fe(III) in atmospheric waters under in situ conditions.

On the use of iron radio-isotopes to study iron speciation kinetics in seawater: A column separation and off-line counting approach

The determination of unidirectional fluxes of Fe in the seawater Fe-pools requires the use of Fe (radio)isotopes, which in turn necessitates off-line iron counting. To implement new off-line counting approaches, methods by King et al. (1991) [King DW, Lin J, Kester DR. Spectrophotometric determination of iron(II) in seawater at nanomolar concentrations. Anal Chim Acta 1991;247:125–32] were adapted for use with radio-iron for Fe(II) and total Fe analysis. The approach consists of passing samples through preloaded SepPak® C 18 cartridges with either ferrozine (FZ) for Fe(II) measurement or 1-nitroso-2-naphthol (1N2N) for total iron analysis. Cartridges are afterwards eluted with methanol and the eluate subsequently counted for radio-iron. A series of experiments was carried out to investigate the new method's applicability. This included loading and extraction behaviour, selectivity for the targeted iron species, iron species recovery, memory effects and the influence of iron concentration. The FZ-preloaded cartridge showed 85% Fe(II) recovery in seawater by direct determination. Recoveries of about 60% for Fe(II) in seawater were found when including 10–15 min waiting time and sample loops, which was probably caused by the instability of Fe(II) due to oxidation. Using 1N2N cartridges for total dissolved iron measurement, recovery was obtained as nearly 100%, but with high variability which was probably caused by the interaction time of Fe with the 1N2N-preloaded cartridges. The cartridges showed only small memory effects. Remaining radio-iron could only be removed from the cartridges with 0.5 M HCl, which destroyed the functionality of the cartridge. This result suggests that the Fe fraction, which could not be eluted by methanol, was irreversibly bound to the cartridge. Desferrioxamine B was applied as a model ligand to test for interference from naturally occurring organic ligands; the results indicate that interferences may occur and should be considered very carefully. The described approaches are tested for seawater iron concentrations in a 1.4–2.3 nM Fe range; further testing is necessary for other Fe concentration ranges.

Simultaneous determination of iron reduction and uptake by phytoplankton

We have developed a new method to assess simultaneously the rates of biological reduction of Fe(III)‐ligand complexes at nanomolar concentrations and iron uptake in marine phytoplankton. Other methods for measuring iron reduction require micromolar iron concentrations, which can saturate iron uptake in these organisms. In the present study, high sensitivity was obtained by combining radiometric techniques, ligand competition, and selective retention on solid phase extraction columns (C18 Sep‐Pak©). Following the addition of the Fe(II) specific ligand ferrozine (FZ) to seawater spiked with nanomolar concentrations of radiolabeled iron, the rate of iron uptake is determined by filtering aliquots of cultures, and the rate of Fe(II) binding to FZ by passing the filtrate through a column, which retains the Fe(II)FZ3 complex. The method was calibrated using 55Fe(II) standards in NaCl solution, yielding high recovery efficiency and linear behavior across the entire range tested from 3 × 10−11 M to 2 × 10−6 M Fe(II). Special attention was given to possible artifacts caused by reduction of Fe(III) in the presence of FZ and Fe(III) adsorption onto the column, which are critical when measuring picomolar concentrations of Fe(II). The method was successfully applied to Thalassiosira weissflogii cultures and to natural phytoplankton populations in the Bering Sea using ethylenediaminetetraacetic acid as a model ligand.

Iron analysis in atmospheric water samples by atomic absorption spectroscopy (AAS) in water-methanol.

To distinguish between Fe(II) and Fe(III) species in atmospheric water samples, we have adapted an analytical procedure based on the formation of a specific complex between Fe(II) and ferrozine (FZ) on a chromatographic column. After elution of Fe(III), the Fe(II) complex is recovered with water-methanol (4:1). The possibility of trace iron measurements in this complex medium by graphite-furnace atomic-absorption spectrometry has been investigated. A simplex optimization routine was required to complete the development of the analytical method.

Interactions Between Cadmium Uptake and Phytotoxic Levels of Zinc in Hard Red Spring Wheat

Wheat grown on cadmium (Cd)‐uncontaminated soils can still potentially translocate unacceptable levels of Cd to grain. The effect of zinc (Zn) and Cd levels on Cd uptake and translocation in “Grandin” hard red spring wheat (HRS‐wheat) (Triticum aestivum L.) was investigated using a double chelator‐buffered nutrient solution [EGTA used to buffer Cd, Zn, copper (Cu), manganese (Mn), and nickel (Ni); and Ferrozine (FZ) used to buffer Fe2+]. In the Zn level series of treatments, Cd2+ activity was held constant at 10−10.7 M, and the Zn2+ activity was varied from 10−7.6 to 10−5.2 M. As Zn2+ activity increased, the translocation of Cd to the shoots decreased. The shoot : root Cd concentration ratio decreased from 0.20 to 0.03 as pZn2+ went from 7.6 to 5.2, indicating that adequate to high levels of Zn are effective in reducing Cd translocation to the shoots of “Grandin” HRS‐wheat. In the Cd series, the Zn activity was at 10−6.6 M, while Cd activity was increased from 10−10.7 to 10−9.2 M. High levels of Cd did not significantly affect the uptake and translocation of Zn in the roots and shoots. While at pCd2+ of 9.2, the root and shoot Cd concentrations significantly increased, there was not a significant increase in the shoot : root Cd ratio. This would indicate that even at high Cd2+ activities, Zn is effective in regulating Cd uptake and translocation in “Grandin” HRS‐wheat.

Determination of Fe(II) and total iron in natural waters with 3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine (PDT)

A method for the determination of Fe(II) and total iron in natural waters using the chelating agent 3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine (PDT) is reported. PDT is a ferroin-type ligand and forms a strongly coloured Fe(II) complex, whose concentration can be measured colourimetrically. PDT is the non-sulfonated precursor to Ferrozine (FZ), a commonly used reagent for Fe(II). Solvent extraction of the Fe(II)(PDT) 3 complex into 1,2-dichloroethane with the counter ion tetrabromophenolphthalein ethyl ester (TBPE) was found to be a most sensitive technique for measuring Fe(II) in natural waters using PDT. However, due to the rapid oxidation of Fe(II) in ambient seawater (pH 7.7–8.2) this method is better employed for measuring total iron, with the addition of a reducing agent. Attempts to pre-concentrate Fe(II) onto PDT loaded C18 Sep-Paks is hindered in seawater by the loss of PDT from the Sep-Pak. However, in low ionic strength solutions, PDT is retained on the Sep-Pak and quantitatively trapped Fe(II). This method can be utilised for measuring nanomolar concentrations of Fe(II) in lakes and rivers.

Iron bound to ferritin catalyzes ascorbate oxidation: effects of chelating agents

Ferritin is the main intracellular iron storage protein. Ferritin iron may be released by many reducing agents including ascorbate. In this work we report ferritin to catalyze the oxidation of ascorbate. The kinetics of this process were studied in detail in phosphate buffer (pH 7.40), at 37°C by using the Clark electrode technique and ESR. The catalytic effect of ferritin manifested itself as the increase both in the rate of oxygen uptake and steady-state concentration of the ascorbate radical. The ferritin catalytic activity was found to be modified by iron chelators, EDTA, Desferal (DFO) as well as by ferrozine (FRZ) which is widely used in kinetic studies on ferritin iron release thanks to the formation of a coloured complex with Fe(II). While EDTA promotes the catalytic action of ferritin, DFO and FRZ diminished it. From the comparison of the kinetics of ascorbate oxidation obtained in the current work and data on the kinetics of ferritin iron release reported by Boyer and McCleary ((1987) Free Rad. Biol. Med. 3, 389–395), we conclude that iron bound to ferritin rather than the iron released is likely responsible for ferritin catalytic action. In addition, it has been concluded that the use of FRZ as an analytical reagent in kinetic studies of reductive ferritin iron release requires taking into account the competitive character of the formation of the Fe(II)-FRZ complex.

Application of chelator-buffered nutrient solution technique in studies on zinc nutrition in rice plant (Oryza sativa L.)

It has been difficult to impose different degrees of Zn deficiency on Poaceae species in nutrient solution because most chelators which would control Zn to low activities also bind Fe3+ so strongly that Poaceae species cannot obtain adequate Fe. Recently, a method has been developed to provide buffered Fe2+ at levels adequate for rice using Ferrozine (FZ), and use of other chelators to buffer the other micronutrient cations. The use of Fe2+ buffered with FZ in nutrient solutions in which Zn is buffered with HEDTA or DTPA was evaluated for study of Zn deficiency in rice compared to a conventional nutrient solution technique. The results showed that growth of rice plants in FZ+HEDTA-buffered nutrient solution was similar to that in the conventional nutrient solution. Severe zinc deficiency symptoms were observed in 28-day-old rice seedlings cultured with HEDTA-buffered nutrient solution at Zn2+ activities < 10-10.6 M. With increasing free Zn2+ activities, concentrations of Zn, Fe, Cu, and Mn in shoots and roots were quite similar for the FZ+HEDTA-buffered nutrient solution and the conventional nutrient solution techniques. The percentages of water soluble Zn, Fe, Cu and Mn in shoots with HEDTA-buffered nutrient solution were also similar to those with the conventional solution. However, with DTPA-buffered nutrient solution, the rice seedlings suffered severe Fe deficiency; adding more FeFZ3 corrected the Fe-chlorosis but shifted microelement buffering. Further, much higher total Zn concentrations are required to provide adequate Zn2+ in DTPA-buffered solutions, and the contents of Mn and Cu in shoots and roots cultured with DTPA-buffered solutions were much higher than those with the conventional or HEDTA-buffered solutions. In conclusion, DTPA-buffered nutrient solutions are not suitable but the FZ/HEDTA-buffered nutrient solution technique can be used to evaluate genotypic differences in zinc efficiency in rice.

The kinetics of Fe(II) complexation by Ferrozine in seawater

The kinetics of Fe(II) complexation by Ferrozine (FZ), the disodium salt of 3-(2-pyridyl)-5,6-bis(4-phenylsulfonic acid)-1,2,4-triazine, in seawater was studied over a range of pH, salinity and temperature, and the following rate equation was observed: − d[Fe(II) dt =k c[ Fe(II)][ FZ] 3 The overall complexation rate constant, k c , was determined from pH 6.0 to 8.4 (at S = 36‰ and T = 24°C), for S = 0–36‰ (at 24°C and pH 7.0) and for T = 15–32°C (at S = 36‰ and pH 7.0). The results have been fitted to an equation of the form log k c= logk c−[ 1.064I 1 2 (1+I 1 2 )] +0.1106I where logK O c=16.184−[ 792.2 T( K) ] ( T (K) = 273.15 + T (°C)) at pH 7.0 with a standard deviation of 0.013. The energy of activation for the overall rate constant is 15.2 ± 0.6 kJ mol −1. A possible mechanism of complexation is proposed. The slight decrease in k c with increasing pH can be explained by the decrease in the percentage in free Fe 2+ as a result of hydrolysis of Fe(II) with increasing pH. The change in ionic strength is the major component causing the decrease of k c with increasing salinity. The oxidation rate constant of Fe(II) above pH 8 has been determined simultaneously with the complexation rate constant. This provides an approach to determine the Fe(II) oxidation rate in natural waters, especially at high pH. There is competition between the complexation and oxidation of Fe(II) during the formation and dissociation of the Fe(II)(FZ) 3 complex. The dissociation rate constant was (2.16 ± 0.02) × 10 −3 min −1 at S = 36‰, pH 8.1 and T = 24°C. This kinetic study leads to an improved understanding of Fe(II) analysis based on the Fe(II)(FZ) 3 complex.

Diferric transferrin reduction by K562 cells. A critical study

This paper critically examines the redox activity of K562 cells (chronic myelogenous leukemia cells) and normal peripheral blood lymphocytes (PBL). Ferricyanide reduction, diferric transferrin reduction, and ferric ion reduction were measured spectrophotometrically by following the time-dependent changes of absorbance difference characteristic for ferricyanide disappearance and for the formation of ferrous ion:chelator complexes. Bathophenanthroline disulfonate (BPS) and ferrozine (FZ) were used to detect the appearance of ferrous ions in the reaction mixtures when diferric transferrin or ferric reduction was studied. Special attention was devoted to the analysis of time-dependent absorbance changes in the presence and absence of cells under different assay conditions. It was observed and concluded that: (i) FZ was far less sensitive and more sluggish than BPS for detecting ferrous ions at concentrations commonly used for BPS; (ii) FZ, at concentrations of at least 10-times the commonly used BPS concentrations, seemed to verify the results obtained with BPS; (iii) ferricyanide reduction, diferric transferrin reduction and ferric ion reduction by both K562 cells and peripheral blood lymphocytes did not differ significantly; and (iv) earlier values published for the redox activities of different cells might be overestimated, partly because of the observation published in 1988 that diferric transferrin might have loosely bound extra iron which is easily reduced. It is suggested that the specific diferric transferrin reduction by cells might be considered as a consequence of (i) changing the steady-state equilibrium in the diferric transferrin-containing solution by addition of ferrous ion chelators which effectively raised the redox potential of the iron bound in holotransferrin, and (ii) changing the steady-state equilibrium by addition of cells which would introduce, via their large and mostly negatively charged plasma membrane surface, a new phase which would favor release and reduction of the iron in diferric transferrin by a ferric ion oxidoreductase. The reduction of ferricyanide is also much slower than activities reported for other cells which may indicate reduced plasma membrane redox activity in these cells.

Mixed reagents in multicomponent flow-injection analysis: Simultaneous determination of iron and copper in blood serum with mixed bathocuproinedisulphonate and bathophenanthrolinedisulphonate or ferrozine

Mixtures of 0.6m M bathocuproinedisulphonate (BC) and 0.2m M bathophenanthrolinedisulphonate (BP) or 2m M BC and 0.2m M ferrozine (FZ) were used for a rapid determination of iron and copper in deproteinated blood serum in the presence of 0.1 M formate buffer (pH 3.5), 10m M ascorbic acid and 0.3 M trichloroacetic acid, by two variants of multicomponent FIA with a diode-array detector (MC—FIA). Merging zones MC-FIA is especially suitable for the determination of 0.7–33μ M Fe and 0.4–35μ MCu, for Cu:Fe concentration ratios from 10:1 to 1:10and with RSD <3 or 2% for Fe or RSD <6 or 5% for Cu in artificial mixtures and deproteinated standard blood sera, respectively, and relative errors of less than 3–5%. The concentrations of both elements were calculated according to a simple computer program (ORTHO) for overdetermined systems (10 or 11 wavelengths), but evaluation at the absorption maxima for the individual chelates (at 2 or 3 wavelengths) also gave satisfactory results.