Chromatography Forum

We need to analyze Hydrogen peroxide (in a pharmaceutical) and I think that a reversed phase separation will be problematic:

- minimal retention
- reactivity in the mobile phase and diluents
etc

I am thinking Normal phase or HILIC would be a better approach.

Any thoughts or suggestions on which one would be preferred.

Thanks

Adam,

What is the concentration of H2O2 in the pharmaceutical product? If there are no other oxidizing agents beside H2O2 in the product you could try cheaper analysis of H2O2 by redox titration with potassium permanganate for instance (again depends on the concentration in the pharmaceutical).

Regarding your question, a simple googling revealed several HPLC methods for H2O2 determination which include derivatization of H2O2 and subsequent RP HPLC analysis or employing cation-exchange resin column to do the separation. I didn't find any NP or HILIC methods.

Hope this helps

There are also published methods that derivatise trace H2O2, eg using Triphenylphosphine, followed by reverse phase HPLC.

I would suggest a good literature search will offer several alternatives, depending on the matrix and concentration you have.

Please keep having fun,

Bruce Hamilton

I've been trying to determine the concentration of hydrogen peroxide in pure water solutions, no substrates or enzymes involved.
It seems to be a small retention of the hydrogen peroxide peak when I'm using an ion-exchange column ( anion 50mm ).
the problem has been to separate the H2O2 from the front, but it seems to work out when I adjusts the pH of the mobile phase ( water ) to ~12.
pKa of the hydrogen peroxide is ~11.25 ( 30 % ).

I would use enzyme assays or special dyes for hydrogen peroxide. They are very sensitve and can be more sensitive - example http://www.chromeon.com/catalog/assays/peroxide

Cheers!

Dave

I've seen the enzyme approaches.

What do you mean by special dyes. Is this something that can be combined with an HPLC assay (the enzyme approaches can not).

Thanks

These dyes are very specific for hydrogen peroxide - hence very sensitive for determining concentration. They are UV-Vis or Fluorescent assays.

Adam,

HILIC, NP, or any other chromatography based technique, is not an advisable route for hydrogen peroxide determination. You will experience stability issues and detectability problems.

One very sensitive alternative could be to employ peroxyoxalate chemiluminescence detection. In the 1980's to late 1990's, there was quite a few academic research groups pursuing this topic. I have attached two suitable references and a link to a thesis.

Peroxyoxalate Chemiluminescence for Miniaturized Analytical Flow Systems
http://www.diva-portal.org/diva/getDocu ... lltext.pdf

Emteborg M., Ponten E., Irgum K.
Influence of imidazole and bis(trichlorophenyl)oxalate in the oxalyldiimidazole peroxyoxalate chemiluminescence reaction.
Anal. Chem. 1997, 69, 2109-2114.

Stigbrand M., Karlsson A., Irgum K.
Direct and selective determination of atmospheric gaseous hydrogen peroxide by diffusion scrubber and 1,1'-oxalyldiimidazole chemiluminescence detection.
Anal. Chem. 1996, 68, 3945-3950.