Ion-Pair HPLC Analysis of EDTA-Complex

[GavinCarp]

Hello,

I am trying to get started analysing two EDTA-complexes by HPLC (Fe(III) and Cu(II) EDTA complexes to be precise) and I am experiencing problems.

I don't know how to work out what pH range is best for the analysis using ion-pair chromatography. I.e Can i just base my selection on the pKa's of EDTA as a free molecule or do i have to take into account the Metal ion in the complex somehow. What's the best way to go about this? Basically, what i need help with is understanding when the EDTA-complexes will be protonated/deprotonated etc and is it the same for both Cu and Fe.

I read in a piece of literature that the Fe(III)-EDTA complex will be fully deprotonated between pH 3.5-5.0 ..why is this?

Can you help?

Thank you in advance.

[Bryan Evans]

Here is an application for Fe (III)-EDTA, EDTA disodium salt, and Cu (II)-EDTA separated on Unison UK-Phenyl:

http://www.imtakt.com/TecInfo/TI261E.pdf

[Noser222]

We do a method where we complex the EDTA with Iron and then use ion-pair reverse phase. The mobile phase is 20 mM Acetic Acid/Sodium Acetate, pH 3.6, with 1 mM Tetrabutylammonium Bromide, and 5% Methanol. Detection is at 254 nm

Here is a reference that is similar:

Loyaux-Lawniczak, S., Douch, J., Behra, P. Fresenius J Anal Chem 364, 727-731 (1999)

I don't know where the Cu(II)-EDTA will elute, but the Fe(III)-EDTA has a -1 charge at this pH.

[GavinCarp]

Thank you for the help so far.

I have a general idea about the approach that i wish to take with the chromatography but am really looking to understand the EDTA complexes more thoroughly.

How do you calculate that the Fe(III)-EDTA complex has a -1 charge at pH3.6? Are there tables that i can use to calculate charge at various pH (for Cu(II)-EDTA as well: is a pH of 3.6 optimum for Cu-EDTA determination as well?)

And so the mechanism would be that the anion complex Fe-EDTA (-1) would interact with the TBA cationic ion pair?

Thanks in advance.

[GavinCarp]

To add to my above comment and make things a little clearer:

I want to know what happens to the Fe(III)-EDTA complex and the Cu(II)-EDTA complex as pH is changed from 1.5-10. Unless i understand this i cannot understand the ion-pair reverse phase reactions taking place.

Bryan Evans,

I am interested in your Unison UK Phenyl column sheet. How have you calculated that the Cu-EDTA compelx is four coordinate leaving two unprotonated carboxyl groups?

Thanks

[Noser222]

I guess at some point if you reduce the pH you may protonate the nitrogens and maybe break up the chelate, but I don't know whether that is at 2, 1.5,....

[Bryan Evans]

Gavin -

According to Imtakt's data, both carboxyl groups on Cu (II) EDTA complex are unprotonated using 1:1 20mM NH4AcOH:AcOH.
This should be around pH 5.

The ip reagent used was a tetrabutylammonium acetate - a volatile ip reagent that works well for anionic compounds.

[GavinCarp]

Hi Bryan,

I was under the impression that Cu-EDTA was a six-coordinate complex but this is clearly not the case in the Imtakt data??

I just really need to find some data/calculations so i can work out what the complexes look like/what charges they have at say pH3, pH5, pH7, pH9, pH11. I.e would they be 4/5/6/7 coordinate? Protonated/unprotonated? Would the complexes even form at some of these pH's (in a decent time)? Would precipitates form?

And I need to find answers to these questions for Cu(II)-EDTA and Fe(III)-EDTA complexes..

Also,
For the Fe(III)-EDTA complex how does the complex interact with the ion pair reagent since there are no free unprotonated carboxyl groups in the complex? do you know the answer to this?

[Bryan Evans]

This was done with EDTA disodium salt.

You'll have to do some research to find the answers you're looking for.
If you go to Google Scholar, do EDTA (and the EDTA complexes you are looking for) - that might be helpful.

[zokitano]

I just really need to find some data/calculations so i can work out what the complexes look like/what charges they have at say pH3, pH5, pH7, pH9, pH11. I.e would they be 4/5/6/7 coordinate? Protonated/unprotonated?

You can find the answers for your questions if you firstly find the pKa values of the 4 COOH groups in the molecule of EDTA. Thus, you can predict how many -COOH groups will ionize at specific pH.

Regards

[HW Mueller]

He will also have to know the equilibrium constants for the complexes, nice messy math. By this time a lit search on HPLC of these, and some good empirical work would have long since yielded useable conditions.

[GavinCarp]

Thanks for your comments.

I've done a little research and found a useful paper (J Chrom A, Baraj, 695, 103-111) ..

It contains figures showing the EDTA species distribution at various pHs so i can use these.

I'll have a go and provide an update soon.

Thanks,

[Einar Ponten]

A textbook on complexiometric titration will probably also be helpful.

An option that you could explore is HILIC. Then both the complexes and free EDTA (and free metal ions?) could be separated in a similar way as done in a recent paper.

Separation and Identification of Phytosiderophores and their Metal Complexes in Plants by Zwitterionic Hydrophilic Interaction Liquid Chromatography Coupled to Electrospray Ionization Mass Spectrometry
Y. Xuan, E. B. Scheuermann, A. R. Meda, H. Hayen, N. von Wirén, G. Weber
J. Chromatogr. A, 1136 (2006) 73-81