Chromatography Forum

Hello,
I found that JP pharmacoepial method for analysing Cefdinir (RSMethod) uses EDTA in combination with Ion pairing Agent. I think that the EDTA will complex with Cefdinir molecule which inturn will be seperated by ion pairing agent. If we analyse Cefdinir in normal mode ( ie without any ion pair reagent being used) cefdinir peak is very broad . So will anyone clarify what is the mechanism of this type of analysis



thanking you
Santosh Gandhi

The EDTA in the mobile phase should bind with the heavy metals
in the silica base packing material. It reduces the secondary interactions
that can contribute to peak distortion.

Normal phase separation of Cephem antibiotics on Unison UK-Amino (no EDTA):
http://www.imtakt.com/TecInfo/TI372E.pdf

I just wonder: Why should anyone choose to utilize Normal Phase Chrom. for separation of these compounds, when the Reversed Phase mode (below in the same text/poster) demonstrates superior resolution and big acetonitrile savings?

Best Regards

Hey Danko -

Thanks for your comments.

I suppose it's all about options:
a). RP (no ion pairing)
b). RP with EDTA & ion pairing
c). Normal phase

There's advantages and disadvantages to all 3 methods.

Maybe other modes of separation can be used as well.

Actually, I am glad you raised this issue. I have wondered about this myself.

As I understand it, metals in the base silica make the silanols more acidic and, therefore, more likely to cause tailing problems. But does EDTA in the mobile phase help deal with metals that are in the stationary phase: this doesn't make sense to me. Is this really what is happening?

If it is I guess it's not that effective since this approach isn't used much.

Can anyone offer anything to clarify this.

Thanks

I have wondered about the same thing (Hajdaei's post above). Even if the EDTA in the mobile phase somehow did complex with the metals in the stationary phase (which seems very unlikely); I would think that the metals would still make the silanols more acidic.

Does anyone know how this works - or if it really does work.

The situation is a bit more complex. Your question however is very legitimate, since even writers of textbooks show some confusion about this aspect.

It is not the surface metals on the silica that make silanols acidic. The metal impurities inside the matrix of the silica are responsible for acidifying the silanols. Metal ions on the surface of the silica from the stainless steel in the instrument or the brown solvent bottles can be removed again with EDTA or other complexing agents. Metal ions in the matrix of low purity silicas, mostly alumina and iron, are not accessible to EDTA, nor to any acid treatment in the preparation of the silica. Dispite the fact that they are buried in the matrix, or maybe because of that, they acidify the surface silanols.

A high purity modern silica like Symmetry has a pKa around 7. An older silica like Spherisorb has two distinct silanol populations, one of them being rather acidic. I do not recall the pKa's out of the top of my head, but they can be found in the literature.

Hmm. Your answer makes sense to me. But still one question: what harm comes from the surface metals. As you pointed out these are not the ones that give the acidic silanols. Is there some characteristic problem that is associated with these.

I'm just wondering since - in many years doing HPLC - I have never added EDTA to the mobile phase. But now I'm thinking it may be something to add at least to "wash out" solutions: i.e. solutions we use to try and regenerate columns (often something like 30% THF in ACN or MeOH).

I have never needed to worry about surface metals either. However, there is a publication by Euerby that demonstrates that surface metals can influence the chromatography of sensitive analytes. I need to dig out the publication to be more specific.
But as you say: this case is rather rare...

Uwe, it would be very much appreciated if you could post the ref. of this article if you find it. Especially interesting: how much Fe ions are floating around or are attached anywhere in relation to "sensitive" compound, and how do they interact. I also have never seen a problem due to metal ions, but maybe all my substances were not "sensitive".

Investigations into the epimerisation of tipredane ethylsulphoxide diastereoisomers during chromatographic analysis on reversed-phase silica II. The involvement of metals in commercially available C18 silicas
Journal of Chromatography A, Volume 705, Issue 2, 30 June 1995, Pages 229-245
Melvin R. Euerby, Christopher M. Johnson, Ian D. Rushin, D. A. S. Sakunthala Tennekoon

Some solutes can form "loose complexes" with heavy metals
(which can come from the instrument, tubing, packing material, ect.).

If you are using phosphate buffer at low pH - you may not
notice it. Volatile salts have poor buffer capacity,
and it will be more noticeable under those conditions.

There's a nice little chapter about it here:

Practical Problem Solving in HPLC, pg. 87
S. Kromidas

(important information for when evaluating columns for LC-MS, ELSD, ect.)

I don't have any hands-on exp with EDTA, but I know some South American Fuel Ethanol plant labs were adding it to their mobile phase as a preservative, apparently without problems? But ion exchange for Fermentation samples only needs to ID Sugars, Acids and Alcohols... Sorry I couldn't be more help.