1Isolation of Flavonoids from Ginkgo Biloba Leaf using the Waters Prep 150 LC System

Significance of the Topic

Ginkgo biloba leaves have been used for centuries in traditional medicine, owing to their rich content of bioactive flavonoids such as quercetin, kaempferol, and isorhamnetin. Rapid and efficient isolation of these compounds at high purity is critical for preparing analytical standards, conducting bioassays, and supporting clinical investigations in pharmaceuticals and nutraceuticals.

Objectives and Study Overview

This study aimed to develop and demonstrate a robust preparative RP-HPLC workflow using the Waters Prep 150 LC System for the isolation of flavonoids from Ginkgo biloba leaf powder. Key goals included method development at the analytical scale, seamless scale-up to preparative dimensions, and evaluation of purity and yield of target compounds.

Methodology and Instrumentation

Extraction Procedure:

• 20 g of Ginkgo biloba leaf powder sonicated in methanol for 60 min.
• Hydrolysis with 3 N HCl under reflux for 90 min to convert glycosides into aglycones.
• Filtration and direct use of the crude extract for HPLC analysis.

Analytical Method Development:

• System: ACQUITY UPLC H-Class with PDA detection at 371 nm.
• Gradient and isocratic runs on a 2.1 × 100 mm BEH C18 column (1.7 µm, 130 Å).
• Quantitation of pre-purification extract: quercetin (0.088 mg/mL), kaempferol (0.104 mg/mL).

Preparative Scale-up:

• Prep System: Waters Prep 150 LC with 19 × 100 mm XBridge BEH C18 OBD column (5 µm, 130 Å).
• Autoscaling: ChromScope Analytical-to-Prep Gradient Calculator for flow rate and timing adjustments.
• Isocratic mobile phase: 73% 0.2% formic acid in water / 27% acetonitrile at 24.8 mL/min, detection at 371 nm.
• Fraction collection via threshold-based windows in ChromScope software.

Key Results and Discussion

Analytical UPLC showed initial purity of target flavonoids at approximately 11% (quercetin), 14% (kaempferol), and 5% (isorhamnetin). Following preparative HPLC and threshold-based fraction collection, UV area–based purities improved to 87%, 96%, and 85% respectively. The ChromScope software enabled precise timing and threshold control for peak collection, ensuring minimal cross-contamination and efficient recovery.

Benefits and Practical Applications

• High‐throughput isolation of pure flavonoid standards for pharmacological and analytical studies.
• Reduced solvent consumption and sample usage during method optimization through analytical‐scale trials.
• Intuitive software control for method flexibility and reproducibility.

Future Trends and Opportunities

Advances in preparative chromatography anticipate integration with mass spectrometry for real-time compound identification, adoption of greener solvents to minimize environmental impact, and automation of scale-up processes through enhanced software algorithms. Expanding this approach to other plant extracts can widen the portfolio of isolated natural products for research and commercial applications.

Conclusion

The Waters Prep 150 LC System combined with ChromScope software provides a streamlined, reliable platform for isolating high-purity flavonoids from Ginkgo biloba. By coupling analytical method development with straightforward scale-up and automated fraction collection, the workflow meets the demands of modern natural product research.

References

1. PDR for Herbal Medicines, 4th Ed., Thompson Healthcare Inc., Montvale NJ, USA, 2007.
2. Sarker SD, Latif Z, Gray AI (Eds.), Natural Products Isolation, 2nd Ed., Humana Press Inc., 2006.
3. Aubin A, Cleary R., Analytical HPLC to Preparative HPLC: Scale Up Techniques using a Natural Product Extract, Waters App. Note 720003120en, 2009.