Chromatography Forum

Currently working on a method for the determination of disodium edatate in eye drops. The problem Is the poor chromatography. The peaks are not completely split but consist of more like two very closely spaced humps. The method consists of Acetonitrile/0.006m tetrabutylammonium hydroxide as mobile phase ph 6.5. Two ml of sample is diluted with two ml of cupric nitrate. I have been told in the lab that the analysis of edta is very difficult at times with some formulations taking weeks to be accepted for there chromatography.obviously not very robust for a validated method. I'm thinking this peak splitting has something to do with ionic interactions between the sample and mobile phase. Could someone give me some suggestions?

here is our UV-based method for analysis of EDTA (top chromatogram). It is validate in few pharma companies for cleaning validation. Copper sulphate is used as visualization agent:
http://www.sielc.com/Compound-EDTA-Ethy ... -Acid.html

LOD is about 100 ppb, as far as I know.

Thank you for the reply but I need to make it clear that this is not method development that I am involved in. This method has been used for years and is approved for use by this particular pharmaceutical company.

We don't have any business need to validate our procedure, but our sample matrices (surfactants or finished consumer products) are definitely more complicated than eye drops. We add copper acetate to the samples, and also have copper in our mobile phase. We use a C8 column and UV detection at 290nm, isocratic.

It's possible that your multiple peaks reflect EDTA with different counterions. They would differ in their polarity and so elute at different times. The solution would be to increase the concentration of electrolyte in your mobile phase and in the sample solvent to a concentration that is sure to supply all of the carboxyl groups in the EDTA with the same anion to serve as the counterion. Now, is the 6 mM concentration of your N(Bu)4OH the concentration in the mobile phase overall, or just in the aqueous portion of the mobile phase? Either way, it's almost certainly too low to serve to knock all of a polyelectrolyte like EDTA into a single ion-counterion form. I'd try 40 mM overall. If that works, then you can experiment with lower concentrations.
This is not a problem that's unique to reversed-phase HPLC. It's also a factor in HILIC and ERLIC. See the example with arginine in Fig. 14 of the paper via the following link: pubs.acs.org/doi/pdf/10.1021/ac070997p
A more extreme case is aminoglycoside antibiotics (kanamycin, etc.). Those don't afford symmetrical peaks in HILIC unless you have > 100 mM salt present.