Hello!
I am working with some materials that require desalting of the peptide samples prior testing. My samples contain a salt of potassium. With a standard sample, I would use R3 or C18 resins with low pH solvents for desalting (Formic acid + MeCN, for example). However, I am working with a modification that is unstable at low pH but completely stable at high pH.
Can you de-salt peptide digest using C18 or similar with basic pH solvents as if you were doing high pH RPLC fractionation? In our lab we always desalt prior high pH fractionation but we do it with acidic conditions. But with high pH, would the peptides be retained in the C18 stationary phase? And if it's possible, what kind of solvents would be good for it? (Keep in mind that the peptides are analyzed by LC-MS/MS).
Thank you for your advice.
Ignacio
Check this:
https://www.agilent.com/cs/library/applications/5991-6525EN.pdf
Some C18 material is not stable above pH 7.5 to pH 8 because the silica to which the C18 is bonded dissolves. Read the information which came with your column, or contact the manufacturer to determine if the column can be run in basic mobile phases for an extended period.
As mentioned above, some C18 columns have been modified to be stable when used with basic mobile phases and these columns are also available from Waters and Phenomenex. Also consider polymeric reverse phases, which may be run at any pH. You may use the same solvents as for the acidic purifications (methanol or acetonitrile).
As for whether the peptides would be retained, it depends on the peptide. Under acid conditions, the carboxylic acid is mainly neutral but the amine portion is ionized. Under basic conditions, the acidic end is ionized while the amine is mainly neutral, depending on the pH of the mobile phase and the pKa of your peptides. They will probably be retained, but the elution order is likely to be different then under acidic conditions.
What were you planning to use to keep the mobile phase basic? I tend to use ammonium hydroxide since it is volatile and can be removed.
Please look at the following below links which may help you in your analysis:
https://www.tandfonline.com/doi/pdf/10.1586/epr.10.46
https://www.researchgate.net/publication/223991405_High-pH_reversed-phase_chromatography_with_fraction_concatenation_for_2D_proteomic_analysis
Thanks
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