The Effects of SFC Preparative Scale-up on Throughput, Purity and Recovery of an Impurity in an API Mixture

Significance of the Topic

Supercritical fluid chromatography (SFC) offers high efficiency and selectivity for isolating low-level impurities in active pharmaceutical ingredient (API) mixtures. Scaling analytical SFC methods to preparative scale enables laboratories to produce gram-level purified compounds rapidly, supporting quality control, process development, and regulatory compliance in pharmaceutical research.

Objectives and Overview

This study demonstrates the scale-up of an analytical SFC method for isolating a 0.1% impurity (4-nitrophenol) from an acetaminophen API mixture. The goals were to evaluate throughput, purity, and recovery at milligram to gram scales, and to perform a cost-time analysis comparing 19 mm and 30 mm preparative columns.

Methodology and Instrumentation

• Analytical SFC: Waters ACQUITY UPC2 with Torus 2-PIC column (4.6 × 100 mm, 5 µm), mixed-stream injection, 3.5 mL/min (80% CO₂/20% co-solvent), ambient temperature, PDA detection at 247 nm and 306 nm.
• Preparative SFC: Waters Prep SFC 150 Mgm with Torus 2-PIC column (19 × 100 mm, 5 µm), modifier-stream injection, 500 µL volume, 35 °C; UV detector at 247 nm and 306 nm; CO₂ pump at 137 bar, ABPR at 120 bar.
• Orthogonal validation: Waters ACQUITY H-Class UPLC with CORTECS C18 column (2.1 × 150 mm, 1.7 µm), gradient 20→80% ACN/0.1% formic acid, 0.5 mL/min, PDA at 247 nm and 306 nm.
• Scale-up calculations: geometric scaling of flow rate and column volume, adjustment of system pressure or temperature to match chromatographic density profiles.

Main Results and Discussion

• Retention times and resolution of 4-nitrophenol matched between analytical and preparative systems by adjusting temperature and pressure.
• First purification cycle (19 mm column): recovery ~92%, purity ~52% due to API carryover at high sample concentration.
• Second cycle on reconstituted isolate improved purity to 99% with 89% recovery.
• Scaling to a 30 mm column increased load capacity 2.5-fold and reduced purification time by the same factor, with comparable material costs per milligram.
• Cost analysis showed mobile phase and CO₂ consumption proportional to column diameter, with larger columns offering significant time savings for multi-gram isolation.

Benefits and Practical Applications

Preparative SFC scale-up enables efficient isolation of trace impurities, streamlines API purification workflows, and supports timely delivery of purified standards for analytical method validation, toxicity studies, and regulatory submissions.

Future Trends and Potential Applications

Advances may include automated SFC platforms integrated with real-time analytics, AI-driven method optimization, expanded use of trifluoromethane modifiers, continuous-flow preparative systems, and broader adoption in multi-kilogram API manufacturing.

Conclusions

• SFC preparative scale-up can effectively isolate low-level impurities with high recovery and purity via two sequential purification cycles.
• Geometric scaling rules validated for column diameter selection facilitate predictable throughput gains.
• ChromScope™ 2.0 software and modifier-stream injection reliably track narrow peaks in preparative mode.
• Column diameter is a key parameter balancing throughput, cycle time, and material consumption.

Reference

• Runco J. Beginners Guide to Preparative Chromatography. Library of Congress 2017933625; Waters Corporation; 2017.