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

As a consequence, the TFA associated baseline ripples can significantly increase the limit of detection (LOD) for analytes. The LOD is defined as the lowest analyte concentration that can be detected over baseline noise and is usually expressed as the concentration at a signal-to-noise ratio of at least 3:1. Those baseline ripples can mask the detection of low concentrated and harmful impurities. A solution to reduce baseline ripples is to use larger mixer volumes,² which also increase the gradient delay volume (GDV) of a LC system. However, by increasing the mixer volume, the separation is delayed, which translates into longer LC run times and therefore limits sample throughput per day. When throughput is a concern, UHPLC systems with small GDVs, and therefore with small mixer configurations, are the preferred option.³ When faced with a challenging TFA application that requires high throughput (“small mixer volume required”) and low LOD (“large mixer volume required”), one fundamental requirement is that the pump flow must be extremely consistent to avoid fluctuations of TFA concentration.⁴

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