Trifluoroacetic acid performance of the Vanquish Flex Binary UHPLC system

Trifluoroacetic acid performance of the Vanquish Flex Binary UHPLC system

Introduction

Trifluoroacetic acid (TFA) is the most common ion-pairing agent used in reversed-phase (RP-) UHPLC for peptide and protein separations. It lowers the pH and modifies the interaction of the molecules with the stationary phase to control selectivity and thus enhance separations. Common conditions for peptide and protein separations include linear and shallow, low organic to high organic, LC gradients where the mobile phase is composed of water and acetonitrile containing approximately 0.1% TFA. Typically, the analytes are detected with a UV detector at 210–220 nm for peptide bonds, as well as at 280 nm for aromatic amino acid residues.

However, under these analytical LC conditions TFA shows some undesirable effects. TFA strongly absorbs UV light below 250 nm, depending on the water/acetonitrile ratio,¹ resulting in a strong shift in baseline during gradient elution. In addition, TFA is retained on RP columns causing the TFA concentration of the mobile phase within the column to fluctuate with varying organic solvent concentration. In the case of incomplete mixing or fluctuating mobile phase content, the dynamics of TFA equilibrium in the column are disturbed causing a strong amplification of mixing noise. Because TFA absorbs 50–100 times stronger than water or acetonitrile in the UV range, significant baseline ripples are observed.¹