Waters ACQUITY UPC2

Importance of the Topic

Convergence chromatography using compressed liquid CO2 offers a powerful, green alternative to traditional normal-phase and reversed-phase separations. By combining broad polarity coverage, tunable selectivity, and mass spectrometry compatibility, the ACQUITY UPC2 System addresses persistent challenges in chiral and achiral analysis across pharmaceuticals, natural products, DMPK, lipidomics, and QC laboratories.

Objectives and Study Overview

The key goals are to demonstrate how the ACQUITY UPC2 platform leverages UltraPerformance Convergence Chromatography™ to:

• Enable high-speed chiral and achiral separations
• Cover a wide range of compound classes from steroids and vitamins to lipids and synthetic drug candidates
• Integrate seamlessly with various detection modes including PDA, mass spectrometry, and light scattering

Methodology and Instrumentation

Convergence chromatography uses liquid CO2 as the primary mobile phase with precisely metered organic co-solvents. Retention is controlled by modulating pressure, temperature, and co-solvent percentage in 0.1 % increments. Both supercritical and subcritical CO2 states are supported, expanding the retention window from least polar to most polar compounds in a single system.

Applied Instrumentation

• ACQUITY UPC2 System with binary solvent manager and two-stage back pressure regulator
• Sample Manager with partial-loop injection (0.1–50 µL)
• Column Manager capable of 8 or 15 column positions with active temperature control and pre-heating
• Photodiode Array (PDA) Detector for low-noise UV detection
• Optional detectors: QDa MS, Xevo TQ-S/MS/MS, Xevo G2-XS QTof, and ELSD for mass-blind analytes

Main Results and Discussion

• Steroid and vitamin panels separated with Torus 1-AA columns in under 6 minutes, using <5 % co-solvent for improved sensitivity
• Enantiomeric separation of propranolol and its three hydroxy metabolites in under 15 minutes with Trefoil CEL1 and MS/MS detection
• Direct analysis of free fatty acids and intact triacylglycerols by UPC2-MS without derivatization, reducing analysis time by more than an order of magnitude compared to GC-MS
• Synthetic chemistry applications: rapid reaction monitoring, chiral ee determination, and orthogonal selectivity relative to reversed-phase LC
• Chiral method development in as little as 10 minutes using four blended co-solvent steps on Trefoil AMY1, CEL1, and CEL2 columns
• Achiral Torus columns (2-PIC, DEA, DIOL, 1-AA) deliver robust retention with minimal drift over weeks of use

Benefits and Practical Applications

• Up to 30× faster separations than legacy normal-phase methods
• 75× reduction in solvent consumption and 100× lower per-run cost
• Broad orthogonal selectivity complementing reversed-phase LC
• Mass spectrometry-compatible mobile phases for definitive compound identification
• Streamlined method development with permanent column banks and pre-defined co-solvent screening
• Reduced toxic waste through non-chlorinated, non-toxic CO2 use

Future Trends and Opportunities

Anticipated developments include:

• Expanded column chemistries for even greater chiral and achiral selectivity
• Automated high-throughput screening workflows integrating AI-driven method optimization
• Deeper integration with high-resolution MS for lipidomics, metabolomics, and environmental analysis
• Continued shift toward green analytical protocols leveraging CO2-based mobile phases

Conclusion

The ACQUITY UPC2 System unifies the advantages of normal-phase selectivity and reversed-phase ease-of-use in a single, robust platform. Its modular design, rapid method development capabilities, and broad detection compatibility make it an essential tool for modern analytical laboratories seeking high-speed, high-confidence chiral and achiral separations.

Reference

1. C. Hudalla et al., Method Development for the Analysis of Endogenous Steroids Using Convergence Chromatography with Mass Spectrometric Detection. Waters Application Note 720004692EN, 2013.
2. K. Ebinger and H. N. Weller, Comparative assessment of achiral stationary phases for high-throughput analysis in supercritical fluid chromatography. Journal of Chromatography A, doi:10.1016/j.chroma.2014.01.060, 2014.