An Analytical and Scale-Up Separation by HPLC and SFC of the Flavonoid Naringenin on a RegisPack® Chiral Stationary Phase

Ted Szczerba¹, Zahid Ali¹, Raffaella Gaggeri², Daniela Rossi², Simona Collina², Barbara Mannucci³, Stefanie Abel⁴, Doris Frühauf⁵, and Markus Juza⁵, ¹Regis Technologies, Inc., ²Department of Pharmaceutical Chemistry, University of Pavia, Pavia, Italy, ³Centro Grandi Strumenti, University of Pavia, Pavia, Italy, ⁴Chromatographic Services, CarboGen-Amcis, and ⁵Analytical Development SIL, DSM Nutritional Products

Naringenin, a chiral flavonoid found in several fruit juices, is well known for its beneficial health-related properties, including antioxidant, antimicrobial, anti-inflammatory, and immunomodulating activities. The HPLC and supercritical fluid chromatography (SFC) enantioselective separation of (R,S)-Naringenin has been performed on both analytical and semi-preparative scale using an amylose derived RegisPack® CSP. The developed preparative method proved to be suitable for obtaining quick access to the desired enantiomers with high enantiomeric excess and amounts sufficient for biological investigations. Scale–up options by SFC and simulated moving bed (SMB) chromatography were also evaluated.

Figure 1: Column: 5 μ, 25 cm × 4.6 mm RegisPack®. Mobile Phase: 100% CH3OH. Flow Rate: 1.0 mL/min.

Two 5 μ RegisPack® columns (25 cm × 4.6 mm and 25 cm × 10.0 mm), which are based on the tris-(3,5-dimethylphenyl) carbamoyl amylose support, were used for the study.

Figure 2: Column: 5 μ, 25 cm × 10.0 mm RegisPack®. Mobile Phase: 100% CH3OH. Flow Rate: 4.0 mL/min.

Conclusion

A comparison of three chromatographic techniques; HPLC, SFC, and SMB (Charts #1 and #2) illustrates the advantages and drawbacks for the preparative separation of Naringenin on a RegisPack® column. Although HPLC does an excellent job in scale-up, the solvent consumption is high. Preparative SFC seems to be better suited for the separation of multi-kilo amounts of Naringenin due to substantial savings in solvent usage. An SMB process will necessitate a higher solvent consumption when compared to SFC, but can lead to an industrial approach for producing multi-ton quantities of Naringenin enantiomers (1).

Charts 1 & 2: *SFC: CO2/CH3OH (75/25;v/v), i.e. 932 L CH3OH/KgRac.

Reference

(1) R. Gaggeri, D. Rossi, S. Collina, B. Mannucci, M. Baierl, M. Juza, J. Chromatogr. A, submitted.

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