methionine oxidation during reverse phase hplc

[Nicola]

I am purifying some peptides. During the reverse-phase hplc and lyophilisation process oxidation of methionine occurs. Is there any thing I can do or add to stop it?

[AdrianF]

You could try adding an anti-oxidant eg ascorbic acid.

[danko]

Or methionine

[Nicola]

Dear AdrianF and Danko, Have you tried using ascorbic acid or methionine in reverse phase? Do you have any methods?

Thanks very much for your help.

[danko]

Hi Nicola,

I haven’t tried it, but I would gladly develop a working method for a symbolic reward – joke
Here is a suggestion, you can try as a starter:

Prepare a 5 - 10 mM methionine solution and adjust pH to 2.4 - 2.5 with phosphoric acid (this would be your eluent A)
Prepare 60 – 70 % Acetonitile mixture with water (this would be your eluent B)

The stationary phase could be any C18 column – not too long (10 – 15 cm)

Equilibrate the column with 95 % A / 5 % B. Inject sample and run a gradient from 5 to 80 % B in 20 min.

Note the mane peak elution-mobile-phase-composition and refine the gradient (make it shallower) or try isocratic mode.

The detection wavelength could be anything from 215 to 230 nm.

Good luck.

P.S. If you examine this suggestion, please get back with your experience, because I really haven’t tried it – just an idea.

[XL]

Here is an example of analysis of water-soluble vitamins (including Ascorbic acid) on a polar-embedded reversed-phase column - Acclaim PolarAdvantage:
http://www1.dionex.com/en-us/acclaim_li ... 25327.html

If the retention of ascorbic acid is not sufficient, the Figure 7 in the following document provides an alternative method using a RP/WAX mixed-mode column - Acclaim Mixed-Mode WAX-1:
http://www1.dionex.com/en-us/webdocs/48 ... 021407.pdf

[Kostas Petritis]

If you are trying to purify a peptide, the problem of using anything non-volatile in your mobile phase is that you will end up with it at the end. Furthermore, Methionine (I am not sure about ascorbic acid but maybe as well) will be retained in the column at 100% aqueous solution. So XL's post is a little bit irrelevant as ideally you would like to have such chromatographic conditions that ascorbic acid would be unretained...

A pretty effecient reducing agent used in proteomics (it brakes the disulfur bonds) is DTT (Dithiothreitol) which is pretty hydrophylic. The best case scenario would be to use something volatile and the only thing I can think of is mercaptoethanol but probably this will create some safety issues (in addition of everybody in the lab hating you due to the awful smell). But if you can overcome both issues, maybe it can be a good solution... 1 mM should be enough...