Chromatography Forum

Hello,

I'm trying to evaluate an RPC method for detection of oxidized products of a certain protein, so, I have the profile of the sample in it's native unoxidized form, and need to treat the sample somehow with an oxidizing agent (eg. hydrogen peroxide) and re-inject to determine the retention time of the oxidized by-products.
Clearly my problem is that it is usually said that no oxidizing agents be used with RPC resins as it could be damaged.. is there a certain limited % allowed? is there any other way to go around this problem? I don't know any other way to oxidize my sample other than adding an oxidizing agent!

Your input will be appreciated

The easiest approach might be to simply use a guard cartridge between injector and column.

Isn't 3% H2O2 used in forced degradation studies?
Maybe if it's a concern, you can use 10X (or more), let it sit and react,
and than dilute it in "gentle" solvent

(of course as Tom said, guard cartridge will protect the column)

I use to place a small amount of the protein of interest (spread in a small Petri dish or something) and another small Petri dish containing some (e.g. 1 mL) concentrated H2O2 solution in a closed container (box or larger Petri dish or whatever) and let the oxidation take place. A couple of hours should be enough – at room temperature. You can draw a sample every 10 – 15 min and dissolve it, thus make a series of sample solutions, which will give you the possibility of choosing the fittest degree of oxidation and that will be your future method for preparing a SST for instance.
One of the good things is that you won’t have any considerable amounts of H2O2 in your sample (solution).
Ever since I thought of and shared that procedure with colleagues, many of them are using it extensively and are quite pleased by the obtained results.

Best Regards

Thank you all for your helpful replies, definetely will try Danko's idea, I'm just wondering will it be enough that the sample is in the same atmosphere of the H2O2 to be oxidized without direct contact/mixing?

I can guarantee you, it works perfectly. I got this idea some 10 years ago and I’ve used it with many proteins since then. Some of my colleagues have tried the procedure as well and were impressed.

But let me understand you: You are working with a protein powder – don’t you? Because if it is a solution the effect is weaker and some of the H2O2 will dissolve in the solvent and stay there. So, the result would be less impressive.

Best Regards

Dear Danko, unfortunately the protein is already dissolved in a certain formulation buffer but we have it also in a lyophilized form, should that work better? and do I still need a guard column?

Hi Miro,

You need to use the lyophilised form, otherwise it doesn’t make sense. For the liquid in which you’ve dissolved the protein will dissolve some of the H2O2 from the “micro atmosphereâ€