Purify CBD and other cannabinoids by preparative HPLC

Importance of the topic

Cannabidiol (CBD) and other cannabinoids are in growing demand for medical, industrial and research applications. High-purity fractions of CBD, Δ9-THC and minor cannabinoids such as CBC enable reliable formulation of therapeutic products, compliance with regulatory limits and deeper investigation of individual compound activities.

Objectives and study overview

This study aimed to develop and demonstrate a single preparative HPLC method capable of purifying CBD, Δ9-THC and cannabichromene (CBC) from a commercial CBD-rich oil. Key goals included analytical profiling of the crude extract, scale-up of an optimized analytical method to preparative dimensions, and assessment of purity, recovery and fraction composition.

Methodology and instrumentation

Sample preparation

• Dilution of CBD oil in methanol (5–50 mg/ml), sonication and filtration to remove undissolved lipid.

Analytical screening

• Acetonitrile-water gradient HPLC on a C18 column, UV detection at 228 nm and 306 nm, five-point calibration for seven cannabinoids (R²>0.999).

Preparative method development

• Methanol-water step gradient in analytical scale (150×4.6 mm, 3 µm C18), followed by linear scale-up to 150×20 mm, 10 µm C18 using a ScaleUp Converter.
• Injection volumes up to 2 ml of 50 mg/ml sample, flow rates increased from 1.2 ml/min to 23 ml/min.

Instrumentation

• AZURA P 6.1L LPG pump, AZURA AS 6.1L autosampler, AZURA DAD 2.1L detector, PurityChrom® Software.
• Columns: Eurospher II C18 in 150×4.6 mm and 150×20 mm formats.

Key results and discussion

Analytical profiling revealed the CBD oil contained ~72% CBD, 5.7% CBC, 2.2% Δ9-THC, 2.5% CBG and ~15.6% unidentified peaks. The preparative run produced five fractions:

• Fraction 1: CBD/CBG mix, CBD purity increased to 93% (area%) and full recovery of CBD and CBG.
• Fraction 4: Δ9-THC enriched to ~80% purity and ~90% recovery from the sample.
• Fraction 5: CBC fraction at ~97% purity and complete CBC recovery.

Other fractions contained mixtures of minor cannabinoids and impurities. Linear scale-up preserved resolution and allowed efficient collection of target compounds in a single run.

Benefits and practical applications

• Simultaneous purification of multiple cannabinoids from a single sample in one chromatographic run.
• Analytical-to-preparative scale-up saves development time, sample volume and solvents.
• High recovery and purity suitable for downstream formulation, quality control and research.

Future trends and potential applications

Adaptation of this workflow can include alternative solvents (ethanol, acetonitrile or normal phase) and target additional minor cannabinoids. Higher-concentration feed streams and continuous preparative systems may further improve yields. Integration with online analytics and automation will support large-scale production and compliance with evolving regulatory requirements.

Conclusion

A methanol-based preparative HPLC method was successfully developed for simultaneous purification of CBD, Δ9-THC and CBC from a commercial CBD oil. Analytical screening guided a fast gradient method, which was linearly scaled to preparative dimensions with high resolution and recovery. CBD purity increased from 72% to 93%, and separate fractions of Δ9-THC (~80% purity) and CBC (~97% purity) were obtained. The approach underscores the value of analytical-scale optimization and linear scale-up for efficient cannabinoid purification.

Reference

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