Purification of Cannabidiol from Hemp Oil Using the Prep150 LC System

Purification of Cannabidiol from Hemp Oil Using the Prep150 LC System

Importance of the Topic:
Cannabidiol (CBD) is a non-psychoactive compound with potential therapeutic benefits including anti-inflammatory, neuroprotective, and immunomodulatory effects. Efficient purification of CBD from hemp extracts is essential for producing high-purity standards, supporting clinical studies, and enabling reliable bioassays in pharmaceutical and research settings.

Objectives and Study Overview:
This study demonstrates a streamlined workflow for isolating CBD from a high-CBD hemp paste. Key steps include rapid UPLC assay development for quantification, analytical HPLC method optimization, scale-up calculations, and preparative HPLC purification using the Waters Prep 150 LC System.

Instrumentation:

• Waters ACQUITY UPLC H-Class with PDA Detector (UPLC assays and analytical HPLC)
• Waters Prep 150 LC System with 2489 UV/Visible detector and WFC III fraction collector
• SunFire C18 and SunFire C18 OBD Prep columns
• ChromScope™ Version 1.4.1 and Empower® 3 Software

Methodology:
A 500 mg portion of decarboxylated hemp paste was sonicated in methanol, filtered, and assayed by UPLC (228 nm detection) to confirm CBD content (8.7 mg/mL). An analytical HPLC method (85 % to 100 % methanol gradient) established resolution and sizing parameters. Scale-up gradients and flow rates were calculated with ChromScope’s Analytical to Prep Gradient Calculator. Preparative injections (320 µL) on a 19 mm SunFire Prep column collected fractions when UV response exceeded 1,000 mAU.

Main Results and Discussion:
The preparative method produced fractions containing 0.56 mg/mL CBD in ~5 mL volumes, corresponding to ~2.8 mg per injection. Five successive injections yielded a combined 12.3 mg of purified CBD. Chromatograms at each stage showed consistent retention times and resolution between CBD and impurities, confirming the validity of the analytical-to-prep scaling approach.

Benefits and Practical Applications:
• Rapid assay to purification workflow reduces development time and solvent use
• Reproducible scale-up ensures predictable yields and purity
• Software-guided fraction collection simplifies method setup and operation

Future Trends and Applications:
Advances in preparative chromatography software and hardware will further streamline natural product isolation. Emerging detector technologies (e.g., mass-directed fractionation) and continuous purification platforms may increase throughput. Integration with automation and in-line analytics will support large-scale production of pharmaceutical-grade cannabinoids.

Conclusion:
This work outlines a robust, scalable protocol for CBD purification from hemp oil. Combining high-speed UPLC assays, analytical HPLC development, and software-driven preparative chromatography delivers high-purity CBD with minimal development overhead. The approach can be adapted for other natural products requiring high-purity isolation.

References:

• American Herbal Pharmacopoeia. Cannabis Inflorescence. 2013.
• El-Alfy AT, et al. Antidepressant-like effect of Δ9-THC and other cannabinoids. Pharmacol Biochem Behav. 2010;95(4):434–442.
• Aubin A, Cleary R. Analytical HPLC to Preparative HPLC: Scale Up Techniques using a Natural Product Extract. Waters Application Note 720003120en; 2009.