Overlapping Reaction Sampling and Analysis for Online LC Reaction Monitoring of Fast Processes

Significance of the Topic

Reaction monitoring of fast chemical transformations is critical in process optimization and control. Conventional analytical methods often cannot match the rapid kinetics of small-molecule or large-biomolecule reactions, leading to information gaps and delayed decision-making. The Agilent InfinityLab Online LC Monitoring solution addresses this by enabling overlapping sampling and analysis, ensuring comprehensive data capture in real time.

Objectives and Study Overview

The primary aim of this study was to demonstrate the capability of automated, parallel sampling and analysis for a fast reaction between 1,1-carbonyl-di-1,2,4-triazole (CDT) and benzylamine. Using Agilent InfinityLab Online LC Monitoring Software, the experiment sought to sample at high frequency while a slower chromatographic gradient runs, ensuring no sampling points are lost and initial data are available promptly for reaction control.

Methodology and Instrumentation

• Instrumentation:
  • Agilent 1290 Infinity II High-Speed Pump and 1260 Infinity II Online Sample Manager with external valve
  • 1290 Infinity II Multicolumn Thermostat and Diode Array Detector (DAD)
  • InfinityLab Poroshell 120 EC-C18 column (2.1×50 mm, 1.9 µm)
  • OpenLab CDS and Agilent Online LC Monitoring Software
• Reaction conditions:
  • 82 mg CDT in 45 mL anhydrous DMSO (10 mmol), stirred at 20 °C
  • Initiated by adding 120 µL benzylamine in 5 mL DMSO
• Analytical method:
  • Gradient from 3% to 95% acetonitrile with 0.1% formic acid over 5 min
  • Flow rate 0.5 mL/min, column temperature 50 °C, 3 µL injection
  • Sampling to deep-well plates every 1.5 min, dilution factor 5, automated needle wash

Main Results and Discussion

The software successfully scheduled and executed overlapping sampling for 14 time points over 19.5 min while running chromatographic analysis in parallel. Delays for individual samples were limited to a few seconds, with no sampling points lost. Trending plots showed immediate formation of the intermediate benzyl triazole urea (BTU), which decreased from 81.7% area at the first point to 4.7% by the tenth, while the product 1,3-dibenzylurea (DBU) rose from 18.3% to 95.3%. Chromatographic overlays of early, mid and late samples confirmed separation at 1.81 min (BTU) and 2.33 min (DBU). Feed injection mode allowed loop removal from the flow path, facilitating uninterrupted sampling.

Benefits and Practical Applications

• High-frequency, unattended sampling with complete data capture
• Near real-time monitoring for rapid decision-making in reaction control
• Economic and time-efficient workflow suitable for QA/QC and process development

Future Trends and Potential Applications

Advancements may include integration with chemometric algorithms and AI-driven process control, coupling with mass spectrometry for enhanced selectivity, and expansion into continuous flow and bioprocess monitoring. Such systems can further accelerate development cycles and improve quality assurance in pharmaceutical and industrial settings.

Conclusion

The study demonstrates that Agilent InfinityLab Online LC solutions effectively handle fast reaction kinetics by overlapping sampling and analysis without data loss. Automated scheduling yields high-resolution kinetic profiles in a single experiment, enhancing productivity and decision-making in analytical labs.

References

• Naegele E. Overlapping Reaction Sampling and Analysis for Online LC Reaction Monitoring of Fast Processes; Agilent Technologies; 2022.