Comparing Mobile Phase Additives for the Separation of mAb Tryptic Peptides: A Case Study on Formic, Difluoroacetic, and Trifluoroacetic Acid

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Jennifer M. Nguyen, Xiaoxiao Liu, and Matthew A. Lauber
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Application Notes
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Application Notes
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ACQUITY UPLC H-Class Bio System

This application note expands upon the noted advantages of DFA. With this work, we have explored the use of purified DFA (IonHance DFA) for peptide mapping and found value in its unique selectivity and ability to afford low adduct mass spectra. Moreover, we have compared the performance of DFA peptide mapping against traditional approaches based on FA and TFA.

Peptide mapping separations are typically run using FA- or TFA-modified mobile phases, with FA providing better MS sensitivity than TFA and TFA producing higher peak capacities than FA. However, an alternative mobile phase modifier, DFA, has been developed that can give a better balance between chromatographic and mass spectrometric performance. IonHance DFA, a stringently purified form of DFA containing less than 100 ppb of sodium and potassium, offers unique selectivity for peptide RPLC separations while also giving strong MS sensitivity gains over TFA and improved peak capacities over FA. Its low metal content is essential to minimizing adduct formation in peptide mass spectra. Ultimately, IonHance DFA provides analysts with a valuable ion pairing option for improving LC-MS separations of peptides given that it confers unique selectivity and sensitivity gains that can prove advantageous for method development.


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