Chromatography Forum

Hello,
I have a problem with oxidated forms in my analysis. We are using H2O + 0.1% TFA as solvent A and ACN + 0.1% TFA as solvent B, C18 waters symetry column. I'm analysing a specific protein all the time, and when I do my system routine check with reference standart, oxidised protein forms should be around 0.25 - 0.40 area%, but something has changed now and it's around 0.5-0.6%. I have rechecked everything, solvents, precolumn, column what causes may result this?

Apreciate your help.

Have you considered storage conditions of samples? Temperature, pH, buffer composition, time?

Yes these factors have been check already, something is deeper than that

A detailed investigation of peptide oxidation in autosampler vials is given in the thread below. Many oxidation sources were tested. Maybe this will give you some ideas.

viewtopic.php?f=19&t=13785&hilit=peptide+oxidation