Performance of the ACQUITY UPLC I-Class PLUS System for Methods which Employ Long, Shallow Gradients

Importance of the Topic

Liquid chromatography methods that use long, shallow gradients are essential for resolving complex biomolecules such as peptides and glycans.
Reliable gradient delivery ensures consistent retention times, which is vital for accurate identification, quantitation, and peak tracking in proteomics and natural product profiling.

Objectives and Study Overview

This application note evaluates the retention time reproducibility of the ACQUITY UPLC I-Class PLUS System when employing long, shallow binary gradients.
The focus is on peptide mapping using a standard enolase digest over multiple injections and extended time periods.

Methodology and Instrumentation

• Sample: Waters MassPREP™ Enolase Digestion Standard reconstituted in 0.1% TFA.
• System: ACQUITY UPLC I-Class PLUS with high-pressure binary mixing.
• Detector: ACQUITY UPLC TUV Detector at 214 nm.
• Column: ACQUITY UPLC Peptide BEH C18, 130 Å, 1.7 µm, 2.1 × 100 mm at 65 °C.
• Mobile Phases: A – 0.1% TFA in water; B – 0.1% TFA in acetonitrile; flow rate 0.2 mL/min.
• Gradient: 1% to 90% B over ~90 min and re-equilibration, generating 0.66% organic change per column volume.
• Software: Empower 3 for data acquisition and analysis.

Main Results and Discussion

• Eight replicate injections over 17 hours yielded an average retention time standard deviation of 0.013 min (0.78 s) across 48 peptide peaks, demonstrating exceptional short-term precision.
• Extended testing over seven days spanning 135 days showed maximum relative retention time variability of 0.004 min (0.24 s) for representative peaks, indicating long-term stability.
• High-pressure binary mixing minimized compositional variance, preserving selectivity and resolution even at very shallow gradient slopes and low flow rates.

Benefits and Practical Applications

The system’s reproducible gradient formation enhances confidence in peptide mapping, glycan analysis, and complex natural product profiling.
Consistent retention times support robust process validation, quality control, and comparative studies in pharmaceutical and biotechnological laboratories.

Future Trends and Opportunities

• Integration with advanced mass spectrometry for deeper proteome and glycome characterization.
• Automation and high-throughput workflows for large-scale biomarker discovery.
• Application to novel separation media and multidimensional liquid chromatography approaches.

Conclusion

The ACQUITY UPLC I-Class PLUS System delivers outstanding reproducibility for methods employing long, shallow gradients, ensuring reliable retention times over prolonged periods.
This performance supports accurate identification and quantitation in complex separations, making it a valuable tool in analytical laboratories.

Reference

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3. Nikles S, Monschein H, Zou Y, Liu X, He D, Fan A, Lu K, Yu G, Isaac R, Bauer R. Metabolic Profiling of the Traditional Chinese Medicine Formulation Yu Ping Feng San for the Identification of Constituents Relevant for Effects on Expression of TNF-α, IFN-γ, IL-1β, and IL-4 in U937 cells. J Pharm Biomed Anal. 2017;145:219–229.