The use of trifluoroacetic acid (TFA) as an ion-pairing agent is a common practice in the reverse phase chromatography of peptides and proteins. The trifluoroacetic acid in the mobile phase responds to the peak broadening and the tailing problem by interacting with the hydrophobic bonding phase and the residual polar surface in a variety of modes. Trifluoroacetic acid binds to the positive and polar groups on the polypeptide to reduce polarity retention and bring the polypeptide back to the hydrophobic reverse phase. In the same way, trifluoroacetic acid shields the residual polar surface on the stationary phase. The behavior of trifluoroacetic acid can be understood as that it remains on the surface of the inverted stationary phase while acting with the polypeptide and the bed.

Trifluoroacetic acid is superior to other ionic modifiers because it is easy to volatilize and can be easily removed from the prepared sample. On the other hand, the maximum UV absorption peak of trifluoroacetic acid is less than 200 nm, and the detection of the polypeptide at low wavelength is very small