Effects of Aqueous Sample Content and Aqueous Co-Solvent Composition on UPC2 Separation Performance

Importance of the Topic

The screening of highly polar metabolites in aqueous biological samples such as urine and plasma is essential for biomarker discovery and clinical diagnostics. Conventional reversed-phase liquid chromatography (RPLC) struggles with very polar species, while hydrophilic interaction liquid chromatography (HILIC) suffers from long re-equilibration times and limited tolerance for high aqueous content. UltraPerformance Convergence Chromatography (UPC²) addresses these limitations by combining supercritical CO₂ with small proportions of polar co-solvents, enabling rapid, high-resolution separation of polar analytes with minimal sample pre-treatment.

Objectives and Study Overview

This work evaluates how aqueous sample composition and co-solvent makeup affect UPC² separation performance. Using caffeine as a model polar analyte, the study explores:

• The impact of different injection solvents (water, various water-miscible organics, non-polar heptane:isopropanol) on chromatographic efficiency.
• The benefits of adding trace amounts of water to the co-solvent.
• The tolerance limits for aqueous injections and the interplay between injection volume and aqueous content.
• The influence of key system parameters (co-solvent percentage, flow rate, back-pressure, column temperature) on separation efficiency.

Methodology and Instrumentation

Sample solutions of caffeine (0.1 mg/mL) were prepared in solvents spanning polar to non-polar character. UPC² separations employed an isocratic mixture of supercritical CO₂ and methanol (or methanol:water) at 95:5 (v/v), with a flow rate of 2.5 mL/min, column temperature 55 °C, and back-pressure 2 000 psi. Injection volumes ranged from 1 to 7 µL. Chromatographic performance was measured by relative plate number (N_rel) against a heptane:IPA control.

Used Instrumentation

• ACQUITY UPC² Binary Solvent Manager
• ACQUITY UPC² Sample Manager
• Automatic Back-Pressure Regulator (ABPR)
• ACQUITY Photo Diode Array Detector (273 nm)
• ACQUITY UPC² BEH Column (1.7 µm, 3.0×100 mm)

Main Results and Discussion

Injection solvent polarity strongly influenced peak efficiency. Non-polar heptane:IPA matched the mobile phase and maintained stable efficiency (N_rel ~1). Among organics, acetonitrile yielded the best aqueous diluent performance. Pure water injections unexpectedly doubled efficiency at 2 µL (N_rel ≈2.05) before efficiency declined and splitting occurred above 5 µL. Incorporation of 5% water into the co-solvent boosted tolerance: efficiencies peaked at 1.72 and remained stable up to 6 µL water injections. A maximum of ~2 µL aqueous content per injection was identified to preserve chromatographic performance. Systematic variation of co-solvent percentage (2–10% MeOH), flow rate (1–3 mL/min), ABPR pressure (1 500–3 000 psi), and temperature (35–75 °C) showed that optimal conditions (95:5 CO₂:MeOH, 2.5 mL/min, 3 000 psi, 55 °C) applied equally to aqueous samples, though aqueous injections were more sensitive to parameter deviations.

Benefits and Practical Applications

• Direct analysis of polar metabolites in aqueous matrices reduces sample prep and analyte loss.
• High chromatographic efficiency and rapid re-equilibration accelerate method throughput.
• Wide tolerance for aqueous injection simplifies workflow for biomarker screening.

Future Trends and Potential Applications

Further refinements may include automated gradient methods combining water-rich modifiers for broader polarity ranges, coupling UPC² with high-resolution mass spectrometry for comprehensive metabolomics, and extending this approach to chiral separations and pharmaceutical QC in aqueous formulations.

Conclusion

UPC² demonstrates robust capability for direct injection and high-efficiency separation of polar analytes from aqueous samples. Careful control of injection volume, aqueous content, co-solvent composition, and system parameters yields reproducible, high-resolution chromatographic performance, offering a valuable tool for metabolite profiling and pharmaceutical analysis.

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