Peptide Mapping and Small Protein Separations with Charged Surface Hybrid (CSH) C18 and TFA-Free Mobile Phases

■ ■ CSH130 C18 is QC tested with a tryptic digest of cytochrome c IN T RODUC T ION Peptide mapping of a biopharmaceutical, when employed for quality control, has traditionally involved detection by UV absorbance. However, to characterize the species in a peptide map, LC separations often must be coupled with ESI-MS. Mobile phases containing trifluoroacetic acid (TFA) have been almost exclusively used for peptide mapping, likely because the performance of many C18 columns is highly dependent on strong ion pairing agents to improve peak shape. For LC/MS applications, it is desirable to avoid strong ion pairing agents such as TFA due to ion suppression, which can be more than an order of magnitude in MS intensity. Weaker acid modifiers with reduced ion pairing properties, such as formic acid (FA), are preferred for LC/MS as they permit more sensitive detection.