Chromatography Forum

Hi,

I found an article about the determination of hydrogen peroxide by LC, by using triphenylphosphine (TPP) as the reagent. TPP reacts with hydrogen peroxide in the sample and triphenylphosphineoxide (TPPO) is formed.

TPP and TPPO are easily separated on a short C18 column. So far so good. I have tried to use the method for samples with a lower H2O2 conc than described in the article and I have run into problems. The oxidation never stops! The longer I wait to analyse my samples, the higher are my results. I added 0.1 mg/ml of EDTA to the samples, and the results got more stable but are still increasing over time.

Can it be air that is causing this? What is the best way to prevent unwanted oxidation. He-sparging the samples will be hard since they contain acetonitrile.

We have developed several analysis methods for peroxides, mainly lipid peroxides(Not HPLC methods, just normal spectrofotometrical methods) but every single one has the same problem... Namely that the methods are very time sensitive... The longer we wait to meassure the higher values we get.

We have tried almost everything to prevent this but nothing has succeded. We have ultrasonicatet solutions to remove oxygen, we have done the complete analysis in a glovebox in N2 atmosphere.. Nothing had the slightest effect.

The only solution we came upwith was to do the analysis very precisly at a given time..

Kind regards

Thanks for your reply leadazide!

That got me on the track to test to mix the sample and reagent by using the autosampler. And it seems to work!

The time for how long the sample and reagent is mixed can be controlled within seconds (and I can still go home for the evening). So far the results look very reproducible.

My experience from water samples is that hydrogen peroxide forms spontaneously - especially when exposed to light, when stored in glass vials, and when not degassed with He.

We used peroxyoxalate chemiluminescence to determine H2O2 at nM levels - mainly in water but also in acetonitrile and other organic solvents, also selectively in the presence of other peroxides.

it is interesting to me. I do not have experience about this. just some comments:
1) can you try blank control prepared in the same way as the sampls? if so, will you expect the oxidation rate stablized after certain time?
If so, could you do a subtract from the sample results?

2) is the oxygen from air causing the non-stopping TPP oxidation?

3)

He-sparging the samples will be hard since they contain acetonitrile

.
can you QS after He-sparging?

4) did you try other reductant such as NaHSO3, or KI?