Improved Sensitivity for Trifluoroacetic Acid Gradients on the Alliance™ iS HPLC Systems

Significance of the Topic

Trifluoroacetic acid (TFA) is widely used as an ion-pairing reagent in reversed-phase bioseparations of peptides and proteins. At low UV wavelengths (<220 nm), TFA’s strong absorbance and partial retention on the column lead to baseline disturbances that reduce sensitivity and impair reproducible quantitation. Stabilizing the gradient and lowering baseline noise are critical for regulated methods such as the USP monograph for Tryptophan Organic Impurities.

Aims and Overview of the Study

This work evaluates the performance of TFA gradients on two HPLC platforms (Alliance™ iS HPLC System and Alliance iS Bio HPLC System) in their default configuration and with an optional diffusion bonded mixer. The study applies the USP monograph for Tryptophan Organic Impurities (Procedure 1), assessing sensitivity, baseline noise, and peak area precision under varying mixer and pump settings.

Methodology and Used Instrumentation

The systems were configured with:

• Alliance iS HPLC System (stainless steel flow path, 675 µL bead mixer, full stroke 132 µL)
• Alliance iS Bio HPLC System (biocompatible MP35N and titanium flow path, 680 µL bead mixer, default half-stroke 66 µL)
• Optional diffusion bonded titanium mixer (690 µL) for both systems

Chromatographic conditions included an XBridge C18 column (4.6 × 250 mm, 5 µm), flow rate 1 mL/min, column 30 °C, detection at 220 nm, and gradient of 0.1% TFA in water/acetonitrile (80:20) at 1.7% B per minute. System suitability solution comprised 1 mg/L Tryptophan Related Compound B, six replicate injections, 20 µL volume, data acquired in Empower™ 3.

Main Results and Discussion

Using the standard bead mixer on both systems, the method met USP precision criteria (≤5% RSD), but baseline ripples were evident, especially on the stainless steel system with full stroke. The Alliance iS Bio half-stroke setting yielded slightly smoother baselines. Replacing the bead mixer with the diffusion bonded mixer reduced baseline noise substantially on both systems. Peak area RSD improved from 1–2% to below 0.5%, and signal-to-noise ratios increased 8–18×. The combination of half-stroke pumping and diffusion bonding delivered the highest sensitivity, nearly doubling S/N compared to the stainless-steel system.

Benefits and Practical Applications

• Reduced baseline fluctuations in TFA gradients at low UV wavelengths
• Enhanced sensitivity (up to 18× improvement) for low-level impurities
• Improved peak area precision (RSD <0.5%) for regulated methods
• Compatibility with existing quaternary solvent managers in HPLC labs

Future Trends and Opportunities

Advancements in microfluidic mixer designs and pump control strategies will further stabilize mobile phase mixing for UV-active additives. Integration of inline sensors to monitor gradient composition in real time may provide adaptive compensation for baseline drift. There is also potential to extend diffusion bonded mixer technology to UHPLC and capillary systems for bioanalysis workflows.

Conclusion

The diffusion bonded mixer significantly enhances mixing efficiency in TFA-based gradients, reducing baseline noise and boosting sensitivity on both Alliance iS HPLC platforms. When combined with optimized pump stroke settings, the system achieves superior peak precision and meets stringent USP requirements for Tryptophan impurity analysis.

References

• Jennifer Simeone, Paula Hong. Peptide Mapping using Binary Biocompatible LC Systems. Waters Application Note. 2021.
• United States Pharmacopeia. USP Monographs Tryptophan Organic Impurities, USP-NF. 2023.
• Ti Diffusion Bonded Mixer – 690 µL Kit Installation Guide. Waters Corporation. 2024.
• F. Gritti. Thermodynamic Interpretation of Gradient Baseline Drift with Mobile Phase Additives. J. Chromatogr. A, 2020.