Intelligent UHPLC pump stroke control - Ensures sensitive peptide analysis by ripple-free baseline in TFA applications

Significance of the Topic

Trifluoroacetic acid (TFA) is a key mobile phase additive in reversed-phase UHPLC for peptide and protein analysis thanks to its ion-pairing ability and selectivity modulation. However, TFA’s strong UV absorbance below 250 nm and its adsorption/desorption dynamics on the stationary phase induce baseline drift and ripples that increase noise and limit detection of low-abundance species. Addressing these artifacts is crucial for high-sensitivity UV detection in proteomics and biopharma applications.

Objectives and Study Overview

This technical note evaluates how an advanced UHPLC pump algorithm (SmartStroke) implemented on the Thermo Scientific Vanquish Horizon system can virtually eliminate TFA-induced baseline ripples without increasing gradient delay volume. Performance is compared between Vanquish Horizon, Vanquish Flex Binary, and a competitive UHPLC system under identical peptide mapping conditions.

Methodology and Used Instrumentation

This study analyzed a lyophilized cytochrome C digest reconstituted in water with 0.1% TFA. The UHPLC gradient employed solvent A (water + 0.1% TFA) and solvent B (acetonitrile + 0.085% TFA) on an Accucore C18 column (2.1×100 mm, 2.6 µm) at 0.6 mL/min and 25 °C with UV detection at 214 nm. Chromatograms were acquired in Thermo Scientific Chromeleon CDS. Key instrumentation:

• Vanquish Horizon UHPLC system with Binary Pump H, split sampler HT, column compartment, DAD detector, and 200 µL mixer.
• Vanquish Flex Binary UHPLC system with Binary Pump F, split sampler FT, column compartment, DAD detector, and 400 µL mixer.
• Competitor A UHPLC system with 100 µL mixer per manufacturer recommendation.

Main Results and Discussion

TFA baseline ripples originate from micro-fluctuations in acetonitrile compressibility and TFA desorption/adsorption on the reversed-phase surface. Large mixers can smooth solvent strokes, but increase gradient delay volume. SmartStroke dynamically optimizes piston stroke volume to match the mixer’s volume period, smoothing transient composition variations without sacrificing GDV. Activation of SmartStroke on Vanquish Horizon reduced baseline ripple amplitude and noise (0.06 mAU vs. 0.15 mAU in competitor) and enhanced signal-to-noise for low-level peptides (S/N 5.2 vs. 1.9 at 1 µL injection). Comparative runs confirmed superior UV baseline stability on the Horizon system even with a smaller mixer volume.

Benefits and Practical Applications

By eliminating TFA-related baseline ripples, SmartStroke on Vanquish Horizon enables:

• Lower limits of detection and quantitation in peptide mapping workflows.
• Consistent, ripple-free UV baselines without increasing gradient delay volume.
• Improved confidence in identifying and quantifying low-abundance peptides for proteomics and QC applications.

Future Trends and Potential Applications

Future advancements may include integration of real-time pump performance monitoring with AI-driven method optimization, expansion of smart pumping algorithms to other solvent modifiers, development of ultra-low-volume mixers, and broader application of ripple-elimination technology in biomolecule characterization and clinical testing environments.

Conclusion

SmartStroke on the Vanquish Horizon UHPLC system effectively removes TFA-induced UV baseline ripples while maintaining a low gradient delay volume. This innovation substantially enhances sensitivity and reliability in peptide and protein analyses, making it an ideal solution for demanding analytical and QC workflows.

Reference

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