Determination of retention behavior of cannabis extract by column screening

Importance of the Topic

The global demand for high-purity cannabinoids, especially cannabidiol (CBD), continues to grow due to their therapeutic potential. Efficient and selective purification methods are essential to remove psychoactive Δ9-tetrahydrocannabinol (Δ9-THC) and other impurities from crude hemp extracts, ensuring product safety and compliance with regulatory standards.

Objectives and Study Overview

This study evaluated the retention behavior of six major cannabinoids in a crude CBD extract on six analytical stationary phases under normal-phase conditions. The goal was to identify optimal column and mobile-phase combinations to inform subsequent preparative purification steps.

Methodology

A supercritical CO2 hemp extract containing ~4 % CBDA and 6 % CBD (w/w) was prepared. Decarboxylation was performed at 120 °C in open reactors, balancing conversion rate and CBD degradation. Analytical reversed-phase HPLC with a C18 column and UV detection at 228 nm identified ten cannabinoids and established five-point calibration curves (R² > 0.999). Normal-phase screening employed six columns (NH2, CN, Diol, Si, C18, Phenyl) and four mobile-phase mixes (heptane/isopropanol, heptane/ethanol, hexane/isopropanol, hexane/ethanol) at 90:10 (v/v).

Instrumentation

• HPLC pump: AZURA P6.1L
• Autosampler: AZURA AS 6.1L
• Detector: AZURA DAD 2.1L (UV 228 nm, 10 Hz)
• Column oven: AZURA CT 2.1 (25 °C)
• Columns tested (150 × 4.6 mm): Eurospher II NH2, CN, Diol, Si, C18, Phenyl

Key Results and Discussion

Reversed-phase analysis quantified CBDA, CBD, Δ9-THC, CBN, CBC and other cannabinoids in original and decarboxylated samples (CBD purity increased from ~30 % to ~65 % area). In normal phase screening, C18 and Phenyl columns showed no separation; Diol and Si offered poor resolution. NH2 and CN columns achieved good separation of six target cannabinoids, with heptane/isopropanol reversing elution order of CBD, CBN, CBC beneficial for downstream purification. Optimal mobile-phase ratios were 92:8 (heptane/isopropanol) on NH2 and 95:5 on CN columns.

Benefits and Practical Applications

• The screening strategy guides the selection of normal-phase columns and solvent systems for cannabinoid purification.
• NH2 and CN stationary phases with heptane/isopropanol enable efficient separation of CBD from Δ9-THC and minor cannabinoids.
• Data support development of preparative HPLC protocols for high-purity CBD production.

Conclusion

This work demonstrates that NH2 and CN normal-phase columns combined with heptane/isopropanol mobile phases offer superior retention selectivity for key cannabinoids in crude CBD extracts. The findings provide a practical foundation for scaling to preparative purification.

Future Trends and Applications

• Scale-up of optimized conditions to preparative HPLC for large-scale CBD purification.
• Investigation of alternative greener solvents and novel stationary phases.
• Integration with mass-spectrometric detection for enhanced compound identification.
• Application of similar screening approaches to other phytocannabinoid-rich matrices.

References

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