The High-Resolution Reversed-Phase HPLC Separation of Licorice Root Extracts Using Long Rapid Resolution HT 1.8-μm Columns

Significance of the Topic

High-resolution reversed-phase HPLC is essential for the detailed analysis of complex natural extracts such as licorice root. Enhanced chromatographic resolution and faster throughput enable more comprehensive profiling of bioactive components, supporting quality control in food additives, pharmaceuticals, and traditional medicine research.

Study Objectives and Overview

This application study compared conventional 5 µm ZORBAX SB-C18 columns with 1.8 µm Rapid Resolution HT (RRHT) columns for separating licorice root extracts. Key goals included:

• Evaluating chromatographic resolution and peak capacity.
• Reducing analysis time while maintaining or improving separation of glycyrrhizin (G) and its hydrolysis product glycyrrhetinic acid (GA).
• Demonstrating applicability to two commercial licorice extracts.

Methodology and Instrumentation

An Agilent 1200 Series Rapid Resolution HPLC system with a multiple wavelength detector (MWD) was used. Mobile phases consisted of 1% acetic acid in water (A) and in acetonitrile (B). Gradient elution ranged from 5% to 100% B over 50 min (5 µm column) and 30 min (1.8 µm column) at 1.0 mL/min, ambient temperature. Standards of glycyrrhizic acid ammonium salt and 18-β-glycyrrhetinic acid were prepared in mobile phase. Two licorice root extracts (HERB FARM and Newark Natural Foods) were vortexed, filtered (0.2 µm), and injected (5 µL).

Main Results and Discussion

Comparison of the two column formats revealed:

• Resolution: The 1.8 µm RRHT column achieved peak capacities of 442–426 versus 290–306 for the 5 µm column (Rs=1.0).
• Analysis Time: Separation of G and GA was reduced by ~40%, from ~42 min to ~25 min.
• Complex Extract Profiling: The RRHT column produced finer chromatographic detail, facilitating detection of minor components in both extracts.
• Relative Concentrations: GA levels were ~0.5% of G in extract A and ~1% in extract B, consistent with hydrolysis product expectations.

Benefits and Practical Applications

The use of small-particle, high-resolution columns offers:

• Greater analytical throughput, reducing instrument time per sample.
• Improved separation of closely eluting natural product constituents.
• Enhanced sensitivity to trace components for quality control and research.
• Scalability to other complex botanical or traditional medicine matrices.

Future Trends and Opportunities

As natural-product research and quality assurance demand more detailed profiling, trends include:

• Adoption of sub-2 µm and core-shell column technologies for faster, higher-resolution separations.
• Integration with high-resolution mass spectrometry for structural elucidation of unknown compounds.
• Automation and online extraction–HPLC workflows to streamline sample preparation.
• Application to broader panels of traditional medicines and complex food matrices.

Conclusion

This study demonstrated that 1.8 µm RRHT columns significantly outperform conventional 5 µm columns in resolution and speed for licorice root extract analysis. The improved peak capacity and reduced run times enable more comprehensive profiling of bioactive constituents and support evolving demands in natural-products analytics.

References

1. Fanali S., Aturki Z., D’Orazio G., Raggi M. A., Quaglia M. G., Sabbioni C., Rocco A. (2005) Journal of Separation Science 28, 982–986.
2. Kitagawa I. (2002) Pure and Applied Chemistry 74(7), 1189–1198.