I would like to know the role of the TFA (trifluoroacetic acid) used on reverse phase chromatography c-18 column. It's is often used with a concentration of 0.1% on the mobile phase.
When analyzing peptides by liquid chromatography (LC) and mass spectrometry (MS), it is useful to introduce additives into the mobile phase to facilitate ionization and improve the analyte signal . Additionally, these additives (i.e., strong acids or buffers) are commonly used in CL to improve analyte retention and peak shape. But for MS separations, high purity mobile phase additives are required to reduce background contribution and ensure data quality.
TFA is generally the additive of choice for the mobile phase in cases where UV detection is used due to its high solubility and resolving power as a strong acid. In contrast, TFA performs poorly when using MS detection because deprotonated TFA forms ion pairs with the peptides or analytes of interest, decreasing the abundance of the analyte delivered to the detector. This phenomenon is called “ion suppression”. Additionally, TFA is known to contaminate mass spectrometry systems and to be virtually impossible to remove. It is ideal for UV detection, but not for MS detection.
Trifluoroacetic acid (TFA) is commonly used in reverse phase chromatography, particularly with C-18 columns, for several important reasons:
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