Demonstrating Chiral Scale-up and Purification Using the ACQUITY UPC2 and Prep 80q SFC Systems

Importance of the Topic

Chiral separations are critical in pharmaceutical and fine chemical industries because enantiomers often exhibit distinct biological activities. Supercritical fluid chromatography (SFC) offers a fast, efficient, and environmentally friendly alternative for chiral purification. Demonstrating reliable scale-up from analytical screening to preparative isolation streamlines method development, reduces solvent use, and accelerates time to pure enantiomer fractions for downstream processing.

Objectives and Study Overview

This application study used racemic bucetin to illustrate a complete workflow for chiral SFC purification. The goals were to:

• Develop and optimize a chiral separation at the analytical scale.
• Convert the gradient method into an isocratic purification protocol.
• Scale the method to preparative dimensions using geometric scaling rules.
• Perform stacked injections to maximize throughput and collection efficiency.
• Assess final enantiomer purity after isolation.

Methodology and Instrumentation

Analytical method development, screening, and post-purification analysis:

• ACQUITY UPC 2 System with PDA detector, controlled by MassLynx software.
• Chiralpak IA analytical column (4.6 × 150 mm, 5 µm).
• Mobile phases: supercritical CO₂ (A) and methanol (B), flow 3 mL/min, 120 bar, 35 °C.

Preparative scale-up and purification:

• Prep 80q SFC System with 2489 UV/Vis detector, controlled by ChromScope software.
• Chiralpak IA preparative column (21 × 150 mm, 5 µm).
• Flow 62 mL/min, 120 bar, 40 °C, methanol as modifier.

The racemic sample was prepared at 10 mg/mL in HPLC-grade methanol. Waters Prep Calculator was used for geometric scaling of flow rate and injection volume between analytical and preparative columns.

Main Findings and Discussion

Initial gradient screening (5–40% methanol over 5 min) on the ACQUITY UPC 2 achieved baseline separation but limited loading capacity. Retention data were used to calculate a starting isocratic condition at 22% methanol. Reducing modifier to 12% improved loading, and a final isocratic composition of 11% methanol balanced resolution and sample throughput at a 10 µL injection.

Geometric scale-up yielded equivalent chromatographic profiles on the Prep 80q SFC System. Using 200 µL stacked injections, approximately 10 mg of racemate was processed in 14 min. Timed collection windows captured individual enantiomer fractions without cross-contamination. Post-purification analysis confirmed purities of 100% for the first enantiomer and 95% for the second.

Benefits and Practical Applications

Key advantages demonstrated include:

• Rapid transition from analytical screening to preparative purification.
• Reduced solvent consumption and sample usage during method development.
• High collection efficiency using stacked injections in SFC.
• Consistent chromatographic performance across scales by matching column chemistry, particle size, and system pressure.
• Minimal downstream workup due to high enantiomer purities.

Future Trends and Potential Uses

Advances in chiral stationary phases, detector technologies, and automation software will further streamline SFC purifications. Integration with mass-directed fraction collection and online analytics can enable real-time monitoring. Emerging green solvents and pressure-modulated techniques may enhance selectivity and sustainability. Wider adoption is expected in pharmaceutical development, natural product isolation, and chiral agrochemical manufacturing.

Conclusion

This study illustrates a robust workflow for chiral method development, scale-up, and purification using SFC. Starting from analytical gradient screening, conversion to an isocratic method enabled efficient preparative separations with stacked injections. Geometric scaling and matching chromatographic conditions ensured reproducible performance, delivering high-purity enantiomers with minimal solvent use and cycle time.

Reference

1. Aubin A, Jablonski J. Prep 150 LC System: Considerations for analytical to preparative scaling. Waters Application Note 720005458EN. July 2015.
2. Zulli S, Rolle D, Wang Z, Martin T, Chen R, Sidhu H. Chiral purification with stacked injections and collections using the Prep 100 SFC MS directed system. Waters Application Note 720003773EN. November 2010.
3. Jablonski J, Aubin A. Peptide isolation using the Prep 150 LC system. Waters Application Note 720005455EN. July 2015.