UPC2 Strategy for Scaling SFC Methods: Applications for Preparative Chromatography

Importance of the Topic

Supercritical fluid chromatography (SFC) offers rapid separations, reduced solvent usage and high resolution, making it valuable for both analytical screening and preparative purification of pharmaceuticals and fine chemicals. Scaling methods between analytical and preparative SFC can be challenging because retention depends on mobile phase density as well as temperature. A systematic strategy to match density profiles across scales enables predictable method transfer, reduces development time, and lowers consumable costs.

Objectives and Study Overview

This work illustrates a density-modulation approach for scaling SFC methods using the Waters ACQUITY UPC² platform. Two case studies are presented: (1) achiral analysis and preparative purification of an Imatinib synthesis intermediate and final API, and (2) chiral purification of a proprietary water-labile compound. Both examples demonstrate fast analytical method development followed by direct transfer to preparative scale while maintaining chromatographic integrity.

Methodology

The key concept is to simulate and match average mobile phase densities between analytical and preparative columns. Retention in CO₂-rich mobile phases is strongly influenced by density, which can be modulated by adjusting back-pressure (ABPR), flow rate or column dimensions (L/d_p). Density profiles were calculated using NIST REFPROP software, assuming a linear pressure gradient along the column under gradient elution. Scaling by maintaining the column length‐to‐particle size ratio provides a useful starting point; further ABPR adjustments refine density matching.

Instrumentation Used

• Analytical system: ACQUITY UPC² with PDA detector
• Preparative system: Prep 100q SFC with PDA and MS detection
• Columns: ACQUITY UPC² BEH 2-Ethylpyridine, BEH, CSH Fluoro-Phenyl (1.7 µm); Viridis BEH 2-Ethylpyridine OBD Prep (5 µm, 19 × 150 mm); chiral cellulose tris(3,5-dimethylphenylcarbamate) (5 µm, 21 × 250 mm)
• Mobile phases: CO₂ with modifiers (methanol ± 0.3 % NH₄OH or acetonitrile)

Main Results and Discussion

Case Study 1 (Imatinib): Initial analytical screening with a 4–40 % methanol gradient showed severe tailing for the amine intermediate and no product elution on most columns. Adding 0.3 % ammonium hydroxide improved peak shape, and BEH 2-Ethylpyridine chemistry was selected. Density simulations indicated nearly identical average densities (0.857 vs. 0.847 g/mL) for 1.7×50 mm vs. 5 × 150 mm columns at 1.5 and 80 mL/min, enabling direct geometric scaling without extra ABPR tuning. Focused gradients (9–19 % for intermediate, 24–34 % for product) delivered high-purity fractions (> 99 % purity, ~120 mg per injection) via mass-directed collection.

Case Study 2 (Chiral Compound): A water-labile chiral analyte required acetonitrile modifier. Analytical method development evaluated column chemistry, temperature, pressure and flow. Geometric scaling alone produced mismatched densities, so ABPR was adjusted to achieve comparable average densities (0.856 vs. 0.859 g/mL). The resulting preparative separation reproduced analytical resolution, demonstrating the robustness of density modulation for chiral scale-up.

Benefits and Practical Applications

• Rapid method development on analytical UPC² with direct transfer to preparative scale
• Predictable chromatographic performance by matching mobile phase density profiles
• Reduced solvent consumption and waste disposal costs
• Focused gradients target individual analytes, improving throughput and purity
• Adaptable to achiral and chiral separations, including water-sensitive compounds

Future Trends and Potential Applications

Advancements may include automated density simulation and ABPR optimization, integration of machine learning for predictive scale-up, exploration of new modifiers and stationary phases, and continuous SFC for higher throughput. Expansion into green solvents and coupling with multi-dimensional separations will further enhance the utility of preparative SFC in pharmaceutical and chemical manufacturing.

Conclusion

A systematic density-modulation approach, combined with geometric scaling and focused gradients, enables efficient and predictable transfer of SFC methods from analytical screening to preparative purification. This strategy preserves chromatographic integrity, accelerates development timelines, and reduces operational costs across both achiral and chiral applications.

References

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