Why are peptides oxidizing on my BEH C18, CSH C18 or HSS T3 column? - WKB92876

ENVIRONMENT

• Peptide oxidation
• Bridged ethylene hybrid (BEH)
• BEH C18 columns

ANSWER

There are several possible reasons. See the Additional Information section.

ADDITIONAL INFORMATION

For easily oxidized peptides:

• Waters has observed that the use of stainless steel tubing showed more peptide oxidation than did use of a Bio system that was set up with inert MP35N tubing.

Too much dissolved oxygen in the mobile phase can lead to excess peptide oxidation.

• Degassing the mobile phase with vacuum sonication is effective in reducing dissolved oxygen in the mobile phase.
• Vacuum sonication should be performed before adding volatile ion-pairing agents such as FA (Formic Acid), TFA (Trifluoracetic acid) and DFA (Difluoroacetic acid).
• Slowly sparging helium after vacuum sonication can help to eliminate re-dissolving of atmospheric gases by creating positive pressure inside the mobile phase bottle.