Chromatography Forum

Here's a question. When you do forced degradation with hydrogen peroxide, is there a simple way to stop (or quench) the reaction, at a given time.

I am wondering what is the common approach that people are using.

Much thanks in advance.

Any of a number of mild reducing agents such as bisulfite, thiosulfate, or hydroxylamine. If the reaction is slow enough, just take an aliquot and inject it; a little H2O2 won't hurt your column.

One of my colleagues has suggested that dilute NaOH will do the trick. I wonder if this may be a wise approach because the product of the reaction is water - which, of course, will not react any further with the analytes.

On a related question, would the peroxide oxidation continue after the solution is diluted up in something like methanol or acetonitrile. For example, many times we do the following: we add 1 mL of aqueous peroxide solution to the sample, then after a certain interval of time, we bring to final volume with methanol or acetonitrile. I'm wondering if - in cases like this - the organic solvent would quench the reaction.

Dilution with organic will not quench the reaction, but it will cause it to slow down.

NaOH will not react with H2O2 to form water, but since many of the reactions are acid-catalyzed, it will effectively halt oxidation. Unfortunately, many oxidizable substrates are more susceptible as high pH, for instance phenolics and mercaptans.

In a couple of pharma companies where I have been, the reaction is not quenched.

What is usually done is to make the initial forced degradation preparation, store it for the desired time interval and then dilute with mobile phase (which did contain some organic solvent).

We realized that the dilution step did not stop the oxidation reaction, but we knew it would be slowed, as Mark noted, and we felt that was good enough. If a little additional degradation occurred, we felt it would be a minor amount and that was acceptable.

If there was no dilution step, then we just time the reaction time interval from the intial preparation to the time of injection.

We figured that a little H2O2 would not hurt the column just as Mark has noted.

There may be a need to run a H2O2 blank as, on occasion, we did see a peak (or peaks) near the beginning of the chromatogram that is attributed to excess H2O2. You don't want to misinterpret this peak as a possible degradant peak. This peak is similar to what you might see for a solvent/injection peak, so it is kind of obvious as to what it is but, in the regulated world, you just want to show what it defeinetly is; so a blank injection was used. (Excess HCl or NaOH in your acid and base forced degradation samples can also give peaks in the chromatogram.)

Regards,
Dan

Hi

You can also quench the reaction by add methionine (may be u have to optimize the amount to be added, certain E.P has this method)..also u can use the enzyme catalase…plz check if this substance interfere with your analysis

Rick

Thanks for your comment. Can you provide a specific EP method that uses this approach. I would be very interested to look it up.

Thanks!
Adam

One simple way to decompose hydrogen peroxide is with a bit of platinum. If you add some platinum wool or foil to your sample, this will quantitatively decompose hydrogen peroxide back to water and oxygen. Of course, this requires an investment in some platinum but it's reusable as it only plays a catalytic role in the decomposition. It's not a superfast process, though, so you need to give it at least an hour until all the hydrogen peroxide is gone as evidenced by no additional bubble formation.

Another choice to quench peroxide is adding some MnO2 into your sample. Hydrogen peroxide will decompose to water and oxygen immediately. When the bubble is stopping, remove the MnO2 by filtration. It is easy and quick. MnO2 in here just a catalyst.

And we have a winner.

I think I like the MnO2 idea the best because:

- it won't likely react with anything else (other than the peroxide)

- it won't cause a problem chromatographically (in fact I don't imagine it would even dissolve.

- and it is cheap and easy to use: just throw in a small amount of powder.

Good suggestion

Dear adam,

MnO2 reacts with peroxide and converts to soluble Mn++ ions, which may damage your column, so as per me Ptatinum degradation is the best idea of all. It is not too costly to purchase pt strip/wire/loop and you can reuse it many time.