Optimize your HPLC-UV system for applications with trifluoroacetic acid (TFA)

Significance of the Topic

Trifluoroacetic acid (TFA) is a widely used ion-pairing agent in reversed-phase HPLC, especially for peptide and protein analysis. Its strong UV absorption below 250 nm, however, can introduce baseline ripple and noise that impair limits of detection (LOD) and quantification (LOQ). Understanding and mitigating these effects is essential for reliable, high-sensitivity analyses in pharmaceuticals, proteomics, and quality control.

Study Objectives and Overview

This work investigates how pump mixer volume influences baseline stability when TFA is used as a mobile phase modifier. The primary goals are:

• Characterize baseline ripple induced by TFA absorption and pump pulsation.
• Compare mixer volumes (100, 200, 400, 600 µl) under a representative peptide gradient.
• Provide practical guidance on selecting mixer size to optimize sensitivity and run time.

Methodology and Instrumentation

A shallow gradient from 5 % to 55 % acetonitrile (both phases containing ~0.1 % TFA) was run at 0.6 ml/min over 30 min. Baseline noise and ripple amplitude were recorded on a UV detector at 220 nm for four mixer volumes. Key instrumentation included:

• Pump: AZURA® P 6.1L high-pressure dual-piston pump
• Autosampler: AZURA® AS 6.1L HPLC autosampler
• Column: Thermo Fisher Accucore aQ C18 (100 × 2.1 mm, 2.6 µm)
• Detector: AZURA® DAD 2.1L with 10 mm LightGuide UV flow cell (2 µl volume)
• Mixers: AZURA® microfluidic mixers at 100, 200, 400, and 600 µl volumes
• Software: ClarityChrom 9.0 with PDA extension

Main Results and Discussion

Baseline ripple amplitude decreased progressively as mixer volume increased. The smallest mixer (100 µl) exhibited pronounced positive and negative spikes due to incomplete mixing and pulsation-induced TFA concentration fluctuations. Mixers of 400 µl and 600 µl provided the smoothest baselines, although they added modest gradient delay volume. The trade-off between baseline stability and analysis time must be balanced according to method sensitivity requirements.

Benefits and Practical Applications

Optimizing mixer volume when using TFA enables:

• Improved LOD/LOQ through reduced baseline noise
• More consistent retention times and peak shapes
• Enhanced reproducibility in peptide and protein assays
• Flexibility to tailor analysis time versus sensitivity

Future Trends and Potential Applications

Emerging microfluidic mixer designs offer even lower dead volumes and faster mixing, further minimizing ripple without excessive gradient delay. Alternate detector technologies (e.g., MS-compatible flow cells) and novel mobile phase modifiers could reduce reliance on strongly absorbing acids. Integration of real-time pump feedback and adaptive mixing may offer automated baseline optimization.

Conclusion

Baseline disturbances from TFA absorption and pump pulsation can be effectively mitigated by selecting an appropriate mixer volume. While larger mixers smooth out ripple, they also increase gradient delay. Users must balance sensitivity and run time when configuring HPLC-UV systems for TFA-containing methods. Modern microfluidic mixers, such as the AZURA® series, provide a flexible solution for high-precision applications.

References

• Choikhet et al., The Physicochemical Causes of Baseline Disturbances in HPLC, Part I – TFA-Containing Eluents, LCGC Europe, 2003.
• Strobl et al., TN-72505, KNAUER Application Note, 2017.