SFC Analytical to Preparative Scale Up of Similar Compounds with Torus DIOL 1.7 μm and 5 μm Columns: Separation of Benzoic Acid and Derivatives

Importance of the Topic

Supercritical fluid chromatography (SFC) offers high efficiency and speed for separating small organic molecules, making it attractive for analytical and preparative workflows. Scaling SFC methods from analytical screening to prep-scale purifications is challenging due to changes in mobile phase density when column geometry or particle size varies. Addressing this issue enables reliable method transfer, reduces development time, and supports isolation of structurally similar compounds in pharmaceutical, agrochemical, and fine chemical industries.

Objectives and Study Overview

This study aimed to demonstrate a straightforward scale-up strategy for SFC separations of benzoic acid and nine structurally related derivatives, including an impurity. By employing Torus DIOL stationary phases in 1.7 µm and 5 µm formats and matching the average column pressure (ACP), the authors transferred an analytical method on small-particle columns to larger-particle analytical and preparative columns while preserving separation performance.

Methodology and Instrumentation

The separation utilized carbon dioxide with 20 mM ammonium hydroxide in a 1:1 mixture of isopropanol and methanol as co-solvent. Analytical experiments were performed on an ACQUITY UPC2 system with PDA detection and Torus DIOL 1.7 µm (3.0 x 50 mm) and 5 µm (3.0 x 50 mm) columns at 1.6 mL/min. Preparative runs employed a Prep 100q SFC system with PDA detection and a Torus DIOL OBD Prep 5 µm column (19 x 150 mm) at 100 mL/min. Column temperature was maintained at 30 °C and UV detection set at 220 nm. ACP was harmonized to 3073 ± 10 psi by adjusting the active backpressure regulator (ABPR) to 1800, 2600, and 1740 psi for the respective columns.

Key Results and Discussion

Chromatograms showed near-identical retention times and peak shape for all benzoic acid derivatives across column formats. The impurity 3-hydroxybenzoic acid was baseline resolved. Fraction collection on the prep scale was effectively triggered by UV absorbance, allowing isolation of each component. Matching ACP maintained consistent mobile phase density and elution strength, validating the scale-up approach.

Benefits and Practical Applications

• Reliable transfer of SFC methods between analytical and preparative scales.
• Preservation of chromatographic selectivity and resolution for structurally similar analytes.
• Efficient isolation of minor impurities without additional method optimization.
• Reduced development time by avoiding re-optimization of mobile phase conditions.

Future Trends and Perspectives

Expanding this ACP-matching strategy to other stationary phases and compound classes can broaden its impact. Integration with automated fraction collection and real-time monitoring will streamline prep workflows. Further exploration of greener co-solvent systems and continuous SFC could enhance sustainability and throughput in large-scale purifications.

Conclusion

Matching average column pressure provides a simple yet powerful rule to scale SFC separations from analytical screening to preparative purification. Torus DIOL columns in 1.7 µm and 5 µm formats deliver consistent performance, enabling rapid method transfer and reliable isolation of benzoic acid derivatives.

Reference

• Hudalla CJ, Tarafder A, Jablonski J, Fountain KJ. UPC 2 Strategy for Scaling from Analytical to Preparative SFC Separations. Waters Application Note 720004818EN.
• Hudalla CJ, Tarafder A, Jablonski J, Roshchin R, Fountain KJ, Patel MA. UPC 2 Strategy for Scaling SFC Methods: Applications for Preparative Chromatography. Waters Application Note 720005064EN.
• Tarafder A, Hill J. Scaling rule in SFC. II. A practical rule for isocratic systems. J Chromatogr A. 2017;1482:65–75.