LIQUID CHROMATOGRAPHIC METHOD MODERNIZATION OF AN ISOCRATIC CANNABINOID HIGH PRESSURE LIQUID CHROMATOGRAPHIC SEPARATION

Importance of the Topic

Cannabis testing laboratories require efficient, cost-effective analytical techniques to quantify cannabinoid content in complex plant and extract matrices. High-pressure liquid chromatography (HPLC) remains a widely adopted method due to its balance of performance and accessibility. By modernizing legacy HPLC platforms, laboratories can achieve enhanced throughput, reduced solvent usage, and greater operational flexibility.

Study Objectives and Overview

This study compares an established isocratic HPLC separation of 16 cannabinoids on a legacy system with a modernized method on an upgraded platform. The objectives include scaling the column dimensions and flow conditions, maintaining chromatographic integrity, and quantifying improvements in run time, solvent consumption, and daily sample throughput.

Methodology

A mixed reference standard of 16 cannabinoids was prepared with major analytes at 0.150 mg/mL and minors at 0.015 mg/mL in methanol. Samples of hemp and cannabis flower and extracts were extracted according to a validated application note. The mobile phase consisted of 0.1% trifluoroacetic acid in water (A) and acetonitrile (B) under isocratic conditions with detection at 220 nm. Geometric scaling formulas were applied to adjust flow rate and injection volume when transitioning from a 4.6 × 150 mm column to a 3.0 × 150 mm column while preserving L/dp ratios.

Instrumentation Used

• Alliance HPLC System with CORTECS Shield RP18, 4.6 × 150 mm, 2.7 µm.
• Alliance iS HPLC System with CORTECS Shield RP18, 3.0 × 150 mm, 2.7 µm.
• Empower Chromatography Data System (CDS).

Results and Discussion

• Geometric scaling maintained chromatographic resolution of all 16 cannabinoids in both flower and extract matrices.
• Run time decreased from ~26 to ~17 minutes, representing a 35% reduction.
• Flow rate was reduced from 2.00 to 1.30 mL/min, lowering solvent consumption per injection by 63% (2,860 mL to 1,806 mL) and increasing injections per day by 45% (55 to 85).
• The modernized system’s higher backpressure tolerance (10,000 psi) enabled efficient operation at increased flow rates on smaller columns.

Practical Benefits and Applications

Transitioning to a modernized HPLC platform offers laboratories significant gains in productivity and cost savings. Reduced solvent use decreases consumable expenses and environmental impact. Faster analysis times support high-volume testing environments such as quality control in the cannabis and hemp industries.

Future Trends and Opportunities

Ongoing advances in column technology and UHPLC platforms promise further miniaturization, faster separations, and enhanced sensitivity. Integration with mass spectrometry, automated sample handling, and data processing will expand analytical capabilities for cannabinoid profiling and related natural product analyses.

Conclusion

The application of geometric scaling rules to modernized isocratic HPLC systems effectively preserves separation quality while delivering shorter run times, solvent savings, and higher throughput. Laboratories adopting upgraded instrumentation can achieve improved operational efficiency and cost-effectiveness.

References

1. Neue UD, McCabe D, Ramesh V, Pappa H, DeMuth J. Transfer of HPLC Procedures to Suitable Columns of Reduced Dimensions and Particle Sizes. Pharmacopeial Forum. 2009;35(6):1622.
2. Aubin AJ, Layton C, Helmueller S. Separation of 16 Cannabinoids in Cannabis Flower and Extracts Using a Reversed-Phase Isocratic HPLC Method. Application Note, Waters Corporation; 2018:720006426EN.
3. Waters. Preparative OBD Column Calculator. www.waters.com. Accessed January 15, 2024.
4. Waters. Columns Calculator 2.0. www.waters.com. Accessed January 15, 2024.