COMPARISON OF MIXER PERFORMANCE FOR HPLC METHODS UTILIZING TFA GRADIENTS

Significance of the Topic

Efficient mixing of mobile phases is essential for achieving high-quality separations in gradient reversed-phase HPLC, especially when using trifluoroacetic acid (TFA) as an ion-pairing reagent. At low detection wavelengths, TFA–acetonitrile gradients often produce baseline ripples that compromise sensitivity and peak integration. This study addresses the impact of mixer design and volume on baseline stability, retention time precision, and signal-to-noise performance.

Objectives and Study Overview

The primary goal was to compare mixer performance in two HPLC configurations: a high-pressure binary system (System X) and a low-pressure quaternary system (Waters Alliance iS). Mixers evaluated included a 400 µL static mixer (System X standard), a 675 µL packed-bead stainless steel mixer, and a 690 µL titanium microfluidic channel mixer. The USP tryptophan impurities method was employed to assess retention precision and signal-to-noise for Tryptophan Related Compound B.

Methodology

Mixer evaluation involved:

• Gradient elution with TFA–acetonitrile at low UV wavelength (<250 nm).
• Blank injections to characterize baseline ripple and noise.
• System suitability runs measuring retention time repeatability and USP signal-to-noise for a standard solution.

This approach quantified how mixer volume and design influence mobile phase composition fluctuations.

Instrumentation

The study utilized two HPLC platforms and three mixer types:

• System X: competitor high-pressure binary system with 400 µL static mixer.
• Waters Alliance iS: low-pressure quaternary system with standard 675 µL packed-bead stainless steel mixer.
• Waters Alliance iS: same system fitted with an optional 690 µL titanium diffusion-bonded microfluidic channel mixer.

Key Results and Discussion

Blank injection traces revealed pronounced baseline ripples for the 400 µL and 675 µL mixers, whereas the 690 µL titanium mixer delivered a notably smoother baseline. Retention time precision was comparable across all configurations (RSD <0.1%). Signal-to-noise analysis for Tryptophan Related Compound B showed:

• System X (400 µL mixer): baseline performance.
• Alliance iS with 675 µL mixer: lower S/N than System X.
• Alliance iS with 690 µL mixer: S/N improved 7-fold over the 675 µL mixer and doubled relative to System X.

These improvements stem from reduced baseline noise rather than increased peak height.

Benefits and Practical Applications

Selection of an appropriately designed mixer can:

• Enhance method sensitivity by minimizing noise in TFA gradients.
• Facilitate more reliable peak integration through a stable baseline.
• Maintain tight retention time reproducibility for quality control assays.

This has direct relevance for pharmaceutical impurity profiling, bioanalytical workflows, and any low-wavelength gradient separations.

Future Trends and Applications

Emerging directions include:

• Advanced microfluidic mixer architectures using novel materials to further reduce dispersion and dead volume.
• Integration of on-line sensors to monitor mixing efficiency in real time.
• AI-driven optimization of gradient profiles coupled with mixer selection for targeted applications.
• Expansion into high-throughput and ultrahigh-pressure systems to meet the demands of next-generation separations.

Conclusion

This comparative study underscores the critical role of mixer geometry and volume in achieving optimal performance for TFA-based gradient HPLC. The 690 µL titanium microfluidic channel mixer on the Waters Alliance iS system provided the best combination of smooth baseline, enhanced signal-to-noise, and precise retention times. Careful mixer selection is recommended when running low-wavelength ion-pairing methods.

References

1. Hong, P. and Simeone, J. (2019) Deciding Between Quaternary and Binary Systems. How to Select the Best UPLC System for Your Application Needs? Waters Corporation.
2. United States Pharmacopeia (2024). USP Monographs, Tryptophan. USP-NF. Rockville, MD: USP.
3. Ti Diffusion Bonded Mixer – 690 µL Kit Installation Guide, 715009251 V00, Waters Corporation, Milford, MA, USA, 2024.