I have been using Ferrozine reagent (0.050 % w/w in 50 mM HEPES buffer) for determining the total Fe and Fe2+ ion concentration (and therefore the Fe3+ ion concentration from the difference). Ferrozine in the presence of ferrous ions, gives a pink-purple color which can be measured in a spectrophotometer. I use a saturated solution of hydroxyl amine hydrochloride as the reducing agent (so that all Fe is in the form of Fe2+) for total Fe estimation. The concentration range for Fe is 0-10 mM.
However, sometimes in the presence of chelating agents such as EDTA and NTA, the results are different. An equimolar Fe-EDTA and Fe-NTA solution at similar pH range (5-6) gives different color intensities using the same protocol.
Why is that so?
The answer lies with the stability constants of Fe-EDTA or Fe-NTA complexes and that of Fe-Ferrozine complex.
The stability constant (log k) of Fe(II)-EDTA is 14.3 while for Fe(III)-EDTA it is 25.7.
If the stability constant of Fe(II)-Ferrozine complex is lesser than the stability constant of Fe(II)-EDTA complex, then EDTA shall have more attraction towards Fe(II) than Ferrozine. The net effect is a reduction in the measured concentration of Fe(II) using Ferrozine method.
Since Fe(III)-EDTA complex is having higher stability constant than Fe(II)-EDTA complex, the Fe(III) complexed with EDTA shall find it difficult to get reduced by the hydroxylamine hydrochloride.
Such a kind of argument also applies to NTA.
Also, if you have equimolar Fe-EDTA and Fe-NTA solution and measure iron concentration using Ferrozine method, you shall get different results for iron concentration - since the quantum of Fe(III) available for reduction from EDTA and NTA complex shall be different due to their different stability constants.
Under such circumstances, increasing the concentration of hydroxylamine hydrochloride or Ferrozine shall help you to some extent. Again, this depends on their relative stability constants. Some times, adjusting the pH of the solution may solve the problem.
If you need a total Fe estimation, the Atomic Absorption method (or ICP method) is better for that. You can search the pK (complex formation constant) in order to explain the differences when you make the estimation in presence of EDTA or NTA, I think this complex are very strong interferences.
Thank you for the answer Dr. Daza. But I do not have an atomic absorption spetrometer. Anyways, will AAS differeniate between ferric and ferrous?
I would also like to know if there is any easy method to separate Fe3+ or Fe2+ from EDTA, If we can esterify EDTA or NTA and then analyze Ferrous or ferric in the solution using the method?
X-ray absorption spectroscopy may help - like any other method using hard X-rays it is possible to investigate the liquid in a small container with suited polymer windows. Furthermore, each iron species has its own fingerprint in the X-ray absorption spectrum near the Fe K-edge at 7112 eV, so it is feasible to differentiate Fe2+ and Fe3+ by different edge energies, and different Fe2+/Fe3+ species give different near edge structures. See the attachement for a comparison of different Fe species. This should also apply to your system!
Best regards, Dirk
No, the AAS method is for Fe total estimation and no Fe3+ and Fe2+. have you make an experiment at lower pH? Maybe you can shift the equilibrium of EDTA to H4EDTA.
The answer lies with the stability constants of Fe-EDTA or Fe-NTA complexes and that of Fe-Ferrozine complex.
The stability constant (log k) of Fe(II)-EDTA is 14.3 while for Fe(III)-EDTA it is 25.7.
If the stability constant of Fe(II)-Ferrozine complex is lesser than the stability constant of Fe(II)-EDTA complex, then EDTA shall have more attraction towards Fe(II) than Ferrozine. The net effect is a reduction in the measured concentration of Fe(II) using Ferrozine method.
Since Fe(III)-EDTA complex is having higher stability constant than Fe(II)-EDTA complex, the Fe(III) complexed with EDTA shall find it difficult to get reduced by the hydroxylamine hydrochloride.
Such a kind of argument also applies to NTA.
Also, if you have equimolar Fe-EDTA and Fe-NTA solution and measure iron concentration using Ferrozine method, you shall get different results for iron concentration - since the quantum of Fe(III) available for reduction from EDTA and NTA complex shall be different due to their different stability constants.
Under such circumstances, increasing the concentration of hydroxylamine hydrochloride or Ferrozine shall help you to some extent. Again, this depends on their relative stability constants. Some times, adjusting the pH of the solution may solve the problem.
Fe+2 can be determined by o-phenanthroline by Spectrophotometric method. Fe+3 will not interfere. If concentration is in mg/litre order Fe+2 can be determined by Red-Ox titration also.Total iron can be determined by AAS/ICP or the by Spectrophotometric methods by reducing Fe+3 to Fe+2 by ascorbic acid.
If you have, you could use electrochemical method to determine them separately.
Addition of Zinc sulphate (7mM) to commercial iron determination regents has been proposed for dealing with samples containing EDTA; however - I have not personally tried this. The original publication may worth checking out? [ Walmsley, T. A., P. M. George, et al. (1992). "Colorimetric measurement of iron in plasma samples anticoagulated with EDTA." J Clin Pathol 45(2): 151-4.]
Dear Dr. Aakash
I could not find any accurate and simple method as ferrous oxidizes very rapidly. I am using the quicker ferrozine based spectrophotomteric method for determining the concentration of ferrous and ferric ions in the presence of EDTA. In my experiments, I assume that all iron is bound to EDTA (Fe:EDTA ratio being 1:1.2).
Anyway, if you have any method, please share it.
Regards
Hi
How to determine the concentration of Fe+3 in an ethanol solution? Any recommendation with espesctroscopy, thank you very much. Regards
This can be done by a simple titration of two steps!
Step 1, take a solution containing Fe2+ and Fe3+ ions. Add 10 ml sulphuric acid to it followed by 2 drops of indicator N phenyl anthranilic acid. Titrate it with standard K2Cr2O7 solution. This titration will provide you the extent of Fe2+ in the solution.
Step 2, now add a pich of Zn dust and ~ 5 ml of conc H2SO4 in this solution. This will reduce Fe3+ of the solution to Fe2+. Now total iron present in the solution is as Fe2+. Further repeat the above titration and this end point would provide extent of total iron: sum of Fe2+ and Fe3+. By subtracting the value of Fe2+ received in step 1 would provide extent of Fe3+ in the solution. there are other mathods also, but this is the most authentic and simple method to get the extent of ferrous and ferric ions in the solution.
Ion Chromatography will be the better option:
Refer following link for PDF (Page 15)
http://tools.thermofisher.com/content/sfs/manuals/4342-31188-06_CS5A_V17.pdf
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