Automated Reaction Monitoring by the Agilent 1260 Infinity II Prime Online LC

Significance of the Topic

The ability to monitor chemical reactions in real time is critical for pharmaceutical and biopharmaceutical production. Automated online sampling and analysis minimizes manual interventions, reduces waste of valuable reagents, and ensures optimal yield and quality of active pharmaceutical ingredients. Continuous feedback on reaction progress supports precise control, prevents overreaction or degradation, and accelerates process development and scale-up.

Objectives and Overview of the Study

This application describes an automated online monitoring experiment of an aldol condensation between p-anisaldehyde and acetone. The goals were:

• To demonstrate direct, automated sampling from a reaction vessel using the Agilent 1260 Infinity II Prime Online LC system.
• To apply inline dilution or quenching to prepare samples for immediate chromatographic analysis.
• To generate real-time quantitative trends of reactant consumption and product formation using integrated software alerts.

Methodology

The reaction mixture (p-anisaldehyde in acetone/water) was circulated through an online sampling interface. Initial samples were taken prior to base addition to establish a calibration reference. Sodium hydroxide was then added to initiate the aldol condensation. At three-minute intervals, 600 µL aliquots were drawn, diluted 1:100 with formic-acid–modified water/acetonitrile, and injected (1 µL) for chromatography. A rapid gradient from 40% to 90% organic in under one minute enabled sub-minute cycle times and continuous data acquisition over 90 minutes.

Used Instrumentation

• Agilent 1260 Infinity II Prime Online LC with Online Sample Manager set and external valve.
• Agilent 1290 Infinity II Multicolumn Thermostat and Diode Array Detector.
• Agilent 1260 Infinity II Flexible Pump and Isocratic Pump for sample delivery.
• InfinityLab Poroshell 120 EC-C18 column (2.1 × 30 mm, 1.9 µm).
• OpenLab CDS and Agilent Online LC Monitoring Software for control, data acquisition, and display.

Main Results and Discussion

Real-time trending showed equal reactant and product at approximately 17 minutes. After 82 minutes, p-anisaldehyde residual was 6% and product reached 94% area percent. Overlayed chromatograms confirmed decreasing reactant peak (RT 0.25 min) and increasing product peak (RT 0.35 min). Relative quantification, corrected for dilution, provided more accurate monitoring than raw area percent due to differing UV responses.

A sample withdrawn at 18 minutes was reanalyzed after 24 hours, yielding retention-time RSDs below 0.3% and area RSDs below 1%, demonstrating effective quenching and sample stability. An integrated limit alarm flagged when educt fell below 5%, illustrating real-time process control capabilities.

Benefits and Practical Applications

• Fully automated sampling, dilution/quenching, and analysis reduces manual labor and error.
• Rapid UHPLC cycles enable high-frequency monitoring of fast reactions.
• Integrated software provides on-demand reprocessing, trend alerts, and real-time decision support.
• Preserved diluted samples allow follow-up analysis or orthogonal methods.

Future Trends and Potential Applications

The integration of mass spectrometric detectors (e.g., LC/MSD iQ) will enhance sensitivity for low-level impurities. Advanced data analytics and machine learning could predict optimal reaction endpoints and adapt process parameters dynamically. Applying this platform to flow-chemistry and multistep syntheses offers further opportunities for closed-loop process intensification and accelerated route development.

Conclusion

The Agilent 1260 Infinity II Prime Online LC system with Online Sample Manager enables reliable, high-throughput online reaction monitoring. Automatic sampling, inline dilution/quenching, and fast UHPLC analysis deliver accurate, real-time insight into reaction progress. Integrated software supports calibrated quantification and threshold alerts, facilitating enhanced process control and efficiency.

References

1. Viviano M. et al. A Scalable Two-Step Continuous Flow Synthesis of Nabumetone and Related 4-Aryl-2-Butanones. Org. Process Res. Dev. 2011, 15, 858–870.
2. Naegele, E.; Kutscher, D. Online Reaction Monitoring by the Agilent InfinityLab Online LC Solutions. Agilent Technologies Application Note, 5994-3528EN, 2021.
3. Performance Characteristics of the Agilent 1260 Infinity II Online Sample Manager. Agilent Technologies Technical Overview, 5994-3529EN, 2021.
4. Reactor Sample Dilution and Mixing Performance of the Agilent 1260 Infinity II Prime Online Sample Manager. Agilent Technologies Technical Overview, 5994-3679EN, 2021.
5. Naegele, E.; Kutscher, D. Sensitive Detection of Low-Level By-Products in a Small Molecule Reaction by the Agilent 1260 Infinity II Prime Online LC System. Agilent Technologies Application Note, 5994-3981EN, 2021.