Separation of Cannabinoids Using UltraPerformance Convergence Chromatography

Importance of the Topic

With the growing legalization and scientific interest in cannabis, accurate separation and quantification of cannabinoids have become essential for forensic toxicology, clinical diagnostics, quality control, and regulatory compliance. Conventional reversed-phase liquid chromatography (RPLC) methods often face challenges such as co-elution of structurally related cannabinoids and high solvent consumption. UltraPerformance Convergence Chromatography (UPC2) provides an orthogonal, rapid, and greener alternative by utilizing supercritical carbon dioxide as the primary mobile phase, enabling improved selectivity across a broad polarity range and reduced organic solvent waste.

Aims and Overview of the Study

This application brief describes the development of a UPC2-PDA method for the separation of seven key cannabinoids (Δ9-THC, Δ8-THC, CBC, CBD, CBN, CBG, THCA) in under ten minutes. The study aims to demonstrate high chromatographic resolution, reproducible quantification, and a complementary approach to traditional RPLC analysis. By leveraging a Viridis BEH 2-ethylpyridine column and a CO2-organic modifier gradient, the method seeks to achieve baseline separation of closely eluting isomers and enhance throughput in routine cannabinoid testing.

Methodology

The separation was carried out using a gradient elution of compressed CO2 (mobile phase A) and a 1:1 mixture of methanol and ethanol (mobile phase B) over a 10-minute run time. Key parameters included:

• Column temperature: 50 °C
• Sample temperature: 5 °C
• Automatic Back Pressure Regulator (ABPR): 2000 psi
• PDA detection at 228 nm
• Injection volume: 1 µL

Instrumentation Used

• ACQUITY UPC2 System
• ACQUITY UPC2 PDA Detector
• Empower 3 Chromatography Data Software
• Viridis BEH 2-EP Column, 100 × 3.0 mm, 1.7 μm, 130 Å

Results and Discussion

The method achieved clear chromatographic separation of all seven cannabinoids within 10 minutes. Key performance metrics included:

• Resolution >1.40 between Δ9-THC and Δ8-THC
• Resolution >1.65 for other cannabinoid pairs
• Injection-to-injection retention time precision within ±0.05 min
• Area reproducibility with RSD between 0.3 % and 0.7 %

The orthogonal retention behavior relative to RPLC helps confirm analyte identity in complex matrices and minimizes risks of co-elution. The supercritical CO2-based mobile phase significantly reduces organic solvent consumption, aligning with green analytical chemistry principles.

Benefits and Practical Applications

• Orthogonal separation to conventional RPLC for confirmation of analyte identity
• Fast, high-resolution analysis of a wide polarity range of cannabinoids
• Reduced solvent usage and waste generation
• High reproducibility and throughput for forensic and QC laboratories

Future Trends and Opportunities

UPC2 technology is poised for further integration with mass spectrometry, enhancing selectivity and sensitivity in complex sample analyses. Emerging stationary phases and modifier systems may widen the scope of detectable analytes, including novel synthetic cannabinoids. Continued development will focus on high-throughput workflows, automation, and deeper coupling with data analytics to support regulatory and clinical applications.

Conclusion

An ACQUITY UPC2-PDA method has been successfully developed for rapid, reproducible separation of seven major cannabinoids. The combination of a Viridis BEH 2-ethylpyridine column and supercritical CO2 gradient elution delivers high resolution, short run times, and reduced solvent consumption. This orthogonal approach provides an effective complementary tool to RPLC-PDA, supporting accurate cannabinoid profiling in forensic, pharmaceutical, and research settings.

References

1. Regulation of Cannabis and Cannabis-Derived Products, Including Cannabidiol (CBD), U.S. FDA.
2. UPLC Separation for the Analysis of Cannabinoid Content in Cannabis Flower and Extracts, Waters Application Note.
3. Separation of 16 Cannabinoids in Cannabis Flower and Extracts Using a Reversed-Phase Isocratic HPLC Method, Waters Application Note.
4. Screening for Cannabinoids Using the Waters Forensic Toxicology Application Solution with UNIFI, Waters Application Note.