In this article, from Issue 12 of the Analytix Reporter, produced by Merck, difluoroacetic acid (DFA) is discussed as a suitable alternative to TFA and FA for LC-UV/MS analysis.
Trifluoroacetic acid (TFA) is typically the mobile phase modifier used in protein analysis by reversed-phase HPLC, especially when combined with UV detection. However, TFA can cause ion suppression in mass spectrometric (MS) detection. Formic acid (FA) is generally the modifier of choice with MS, but it often results in less efficient chromatographic separation. In this article, difluoroacetic acid (DFA) is discussed as a suitable alternative to TFA and FA in the LC-UV/MS analysis of proteins.
The analysis of intact proteins and protein digests by reversed-phase high performance liquid chromatography (RP-HPLC) provides researchers with
valuable information not only about a protein’s identity, but also the post-translational modifications that affect its properties.
This article explores the use of DFA as an attractive acid modifier alternative in the LC-UV/MS analysis of proteins. DFA is a strong ion-pairing agent and provides the desired low pH (it has a lower pKa compared to FA).
The article concludes that in the HPLC analysis of proteins where combining UV and MS detectors is sometimes necessary, DFA is an attractive alternative to TFA and FA as mobile phase modifier. This reagent provides better separation efficiency than FA, and it does not cause strong ion suppression like TFA. However, DFA causes reduced MS ion yield compared to FA and slightly broader peaks than obtained with TFA. Therefore, users need to do an assessment regarding a possible and acceptable compromise for their application.
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*The life science business of Merck operates as MilliporeSigma in the U.S. and Canada.