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

■ ■ MS-grade purity to facilitate the acquisition of high-quality mass spectra INTRODUCTION Peptide mapping is an important technique for the characterization of biopharmaceuticals. Peptide mapping can report on the primary structure of a protein through an examination of its amino acid sequence. The separations underlying this approach are based upon reversed-phase liquid chromatography and are readily coupled to mass spectrometry (RP LC-MS). Separations using spectroscopic detection often employ trifluoroacetic acid (TFA) in the mobile phase, a strong, hydrophobic acid and an effective ion pair for minimizing chromatographic secondary interactions. Conversely, to avoid ion suppression, LC-MS analyses are often performed with a weaker ion pairing modifier, like formic acid (FA), but with a compromise to chromatographic resolution.