I am wondering how important pH is here. I have a sample tryptic sample in 50mM TEAB, wondering if I need to add in TFA to ~2% before desalting. I understand the need to acidify samples with ion exchange.
In general, no, because you should already have a purified compound. Since there should not be impurities eluting with the compound, peak shape is less important. The C18 is only being used for catch-and-release, as in a solid-phase extraction cartridge.
Hi,
Do not think TFA as only acidity serving additive.It is also increase the protein/peptide solubility but most important one is ion pairing agent which is related to your question. It increases peptide retention in a wide variety of polarity...Thereby if you need to desalt your peptide mixture and ensure the retention of hydrophilic peptides on c18 columns, it could be chosen. Also.consider that for online desalting purposes at ms systems, TFA is not compitable due to ionisation suppression effect
Hello Law Mo ,
I'd recommend acidifying peptide samples prior to C18 desalting to pH 3 (you can check by spotting a small amount on universal indicator pH paper).
I use a minimum of 0.1 % TFA final concentration. This assists in proper peptide binding, but also ensures any residual protein material is denatured & less likely to bind to the column non-specifically. In addition, use 0.1 % TFA as the washing buffer and add it to the elution buffer (up to 60 % acetonitrile with 0.1 % TFA).
Good luck!
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