ULTRA PERFORMANCE LC FOR ONLINE MONITORING OF A CONTINUOUS PROCESS

Significance of the Topic

This study addresses the growing demand for rapid, high-resolution analytical techniques in continuous manufacturing and process analytical technology (PAT). By leveraging Ultra Performance Liquid Chromatography (UPLC), real-time monitoring of reactive streams becomes feasible, enabling tighter process control, faster decision-making, and improved product quality while reducing batch failures and manual sampling risks.

Objectives and Study Overview

The primary aim was to demonstrate the application of an integrated UPLC-based PAT system for online analysis of a two-step continuous flow synthesis of 5-amino-benzyl-1H-1,2,3-triazole-4-carboxamide. Key goals included:

• Implementing a Design of Experiments (DoE) to study reagent concentration and temperature effects.
• Monitoring conversion and clearance of potential genotoxic impurities (benzyl bromide and benzyl azide).
• Assessing system performance in terms of speed, sensitivity, and automation.

Methodology and Instrumentation

An advanced Pilot Process Analysis System was deployed, comprised of:

• ACQUITY UPLC Quaternary Solvent Manager for gradient delivery.
• ACQUITY UPLC Process Sample Manager (PSM) enabling online and at-line sampling with programmable dilution and barcode operation.
• ACQUITY UPLC Column Manager and PDA eλ detector for high-resolution separation and spectral selectivity.
• Empower 3 Chromatography Data Software for real-time data integration and reporting.

The continuous reactor platform used a Chemtrix KiloFlow system with four feed lines (benzyl bromide, sodium azide, sodium hydroxide, cyanoacetamide) held at 70–90°C and 1 mL/min flow each. The UPLC method employed a BEH C18 1.7 µm column at 40°C, with a water/acetonitrile/formic acid gradient over 2.5 minutes for sub-minute cycle times.

Main Results and Discussion

Compared with conventional HPLC, UPLC reduced cycle times by over 70%, delivering results in under one minute per injection. Seven DoE conditions (varying reagent concentration and temperature) were monitored online at two reactor ports, yielding:

• Clear calibration curves from a single standard vial, enabling accurate quantitation across dilutions (1:4 to 1:50).
• Progressive increase in 1,2,3-triazole yield with rising temperature and reagent concentration.
• Effective removal of benzyl bromide; residual benzyl azide detected at lower conversion conditions.

Benefits and Practical Applications

Implementation of UPLC PAT provides:

• Automated, high-throughput analysis of continuous processes without manual intervention.
• Enhanced sensitivity for low-level genotoxic impurities, supporting regulatory compliance.
• Programmable dilution and DCS integration for streamlined control and data management.
• Reduced reaction volumes and safety risks, notably lower explosion hazards for azide intermediates.

Future Trends and Opportunities

Advancements may include integration with artificial intelligence for predictive control, miniaturized microfluidic sampling interfaces, in-line purification modules, and cloud-based data analytics. Expansion to multi-analyte monitoring and real-time feedback loops will further accelerate process optimization and scale-up efforts.

Conclusion

This work highlights the transformative potential of UPLC in continuous flow PAT, delivering fast, sensitive, and automated monitoring of complex synthesis pathways. The approach enhances process understanding, ensures impurity control, and supports agile manufacturing in chemical and pharmaceutical industries.

References

• Phoebe A.D., Tinder R.J., Phoebe C.H. Jr. Ultra Performance LC® for Online Monitoring of a Continuous Process. Waters Corporation, 2014.