Stereoselective Separation of Triazole Fungicides Using the ACQUITY UPC2 System and ACQUITY UPC2 Trefoil Chiral Columns

Importance of the Topic

The enantioselective analysis of chiral pesticides is critical in agrochemical research and environmental monitoring.
Different stereoisomers can exhibit distinct biological activity and environmental fate, making accurate resolution of enantiomers and diastereomers essential.
Rapid and reliable separation of these isomers supports optimized pesticide efficacy and reduced environmental impact.

Objectives and Overview

This study presents fast and reproducible analytical methods for stereoselective separation of twelve triazole fungicides using supercritical fluid chromatography (SFC) on the Waters ACQUITY UPC2 system coupled with Trefoil chiral columns.

Methodology and Instrumentation

• Instrument: Waters ACQUITY UPC2 System with Photodiode Array Detector and Empower 3 Software
• Columns: ACQUITY UPC2 Trefoil AMY1 and CEL1 (3.0 × 150 mm, 2.5 µm)
• Mobile phase: Supercritical CO2 with methanol or mixed alcohol co-solvents (ethanol, 2-propanol)
• Operating conditions: Pressure 110–207 bar, flow rates 1.5–3.5 mL/min, temperatures 10–45 °C
• Sample preparation: Racemic standards in methanol or acetonitrile at 1 mg/mL (100 µg/mL for selected compounds)

Main Results and Discussion

• Twelve triazole fungicides were resolved with baseline separation for most compounds in under 1.5 minutes on AMY1 with methanol modifier.
• Uniconazole, penconazole, and hexaconazole achieved baseline separation in less than 1.2 minutes using a 50:50 2-propanol/ethanol mixture.
• Propiconazole (two chiral centers) was resolved in under 2.8 minutes on AMY1, while cyproconazole and bromuconazole were separated in under 4.5 minutes on CEL1.
• Resolution factors (Rs) exceeded 1.75 for all stereoisomers, reaching values above 10 and up to 27 for some transitions.
• Reproducibility (n=8) showed percent RSDs ≤ 0.60% for retention time, peak area, height, and resolution for bromuconazole.
• Compared to normal-phase HPLC and conventional SFC, UPC2 delivered 3–40 times faster separations with reduced solvent consumption.

Benefits and Practical Applications

• Enhanced sample throughput with run times often below two minutes.
• Reduced consumption of organic solvents and lower hazardous waste.
• Reliable enantiomeric and diastereomeric quantification for quality control and environmental studies.

Future Trends and Opportunities

Integration of advanced chiral stationary phases and mass spectrometric detection could extend this approach to a broader range of chiral agrochemicals and complex matrices.
Automation and online fraction collection may further increase throughput and facilitate preparative separations.
Enantioselective analyses will support regulatory compliance and environmental fate investigations.

Conclusion

The combination of UPC2 and Trefoil chiral columns offers a rapid, robust, and green method for stereoselective separation of triazole fungicides, enabling high-resolution enantiomeric analysis with minimal solvent use and improved laboratory productivity.

Instrumentation

• Waters ACQUITY UPC2 System
• ACQUITY UPC2 Photodiode Array Detector
• Empower 3 Software
• ACQUITY UPC2 Trefoil AMY1 and CEL1 Columns (3.0 × 150 mm, 2.5 µm)

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