Chiral Multicolumn Method Development on the Agilent 1260 Infinity II SFC System

Importance of the Topic

The separation of enantiomers is critical in pharmaceutical analysis because mirrored molecular structures can exhibit vastly different biological activities. Supercritical fluid chromatography (SFC) offers substantial advantages over conventional normal-phase high-performance liquid chromatography (HPLC), including faster run times, reduced solvent consumption, and lower waste disposal costs. Automating chiral method development accelerates discovery and scale-up of enantiomerically pure compounds in both research and quality-control environments.

Study Objectives and Overview

This application study aimed to demonstrate how the Agilent ChemStation Method Scouting Wizard, coupled with the Agilent 1260 Infinity II SFC system, can automate the screening and optimization of chiral separations. The case study focused on propranolol enantiomers, employing four different chiral columns under four distinct isocratic mobile-phase compositions. The goal was to identify the fastest condition delivering baseline resolution within minimal run time.

Methodology and Instrumentation

The experiments used an Agilent 1260 Infinity II SFC Control Module integrated with:

• Agilent 1260 SFC Binary Pump
• Agilent 1260 Multisampler
• Agilent 1260 Diode Array Detector with SFC flow cell
• Agilent 1260 Multicolumn Thermostat and a Quick Change 4-position/10-port selection valve
• Capillary kit enabling automated column switching

Four chiral stationary phases (Chiralpak IA, IB, IC, ID; 4.6×250 mm, 5 μm) were tested. The mobile phase comprised supercritical CO₂ (solvent A) and methanol containing 0.1 % diethylamine (modifier B). Screening conditions included isocratic modifier ratios of 15, 20, 25, and 30 % at 2.0 mL/min, 60 °C backpressure regulator, 30 °C column temperature, and 5 μL injection. Data collection used diode array detection at 230 nm.

Main Results and Discussion

Column IA achieved enantiomer resolution only at low modifier content (15 % methanol) with long retention times (>7 min) and compromised peak shape. Column IC failed to resolve the enantiomers under all conditions. Column ID showed partial separation but insufficient baseline resolution. In contrast, column IB yielded clear baseline separation at 30 % modifier in about 4.5 and 5.4 min, with acceptable peak shapes and shorter runs.
Further optimization of column IB by increasing flow rate to 2.5 mL/min and temperature to 40 °C reduced retention to 3.0–3.7 min, achieving a total run time of 4 min with retention-time RSDs below 0.2 %. Preparative potential was demonstrated by injecting 80 μL of a concentrated propranolol solution, achieving baseline separation at 5.9 and 6.8 min under 25 % modifier conditions suitable for fraction collection.

Benefits and Practical Applications

• Run times nearly tenfold faster than normal-phase HPLC chiral methods.
• Automated method scouting reduces development effort and errors.
• Lower solvent usage and simpler waste handling support greener operations.
• Flexible scale-up from analytical screening to preparative fractionation.

Future Trends and Possibilities

Advances in software-driven method development are likely to integrate machine-learning algorithms to predict optimal chiral separations. Emerging stationary phases and modifiers will expand the toolbox for challenging enantiomer pairs. Further miniaturization and flow-path enhancements will enable ultrafast screening campaigns. Preparative SFC workflows will increasingly adopt continuous-flow collection and in-line monitoring for real-time fraction management.

Conclusion

The Agilent 1260 Infinity II SFC system with the Method Scouting Wizard provides a streamlined, reproducible platform for rapid chiral separation development. By screening multiple columns and conditions automatically, the workflow delivered a four-minute baseline separation of propranolol enantiomers and demonstrated preparative capability under high-load injection. This approach accelerates analytical and preparative tasks in pharmaceutical research and quality control.

References

1. Naegele E. Supercritical Fluid Chromatography with Flexible Injection Volumes at Highest Precision. Agilent Technologies Technical Overview; publication 5991-7623EN, 2017.