Gradient Manipulation and 1.8 μm LC Columns for High Resolution Analysis of Herbal Supplements

Importance of the Topic

The high diversity of natural products in herbal supplements demands chromatographic methods capable of resolving complex mixtures with high peak capacity and resolution. UHPLC systems with sub-2 µm particles allow operation at elevated pressures, generating narrower peaks and enhanced separation performance.

Study Objectives and Overview

This study investigates gradient manipulation and key chromatographic parameters—column length, gradient time, flow rate, and temperature—using 1.8 µm ZORBAX RRHD Eclipse Plus C18 columns on an Agilent 1290 Infinity UHPLC. The goal is to maximize peak capacity and resolution for licorice, goldenseal, and echinacea root extracts.

Used Instrumentation

• Agilent 1290 Infinity UHPLC system with DAD detection at 280 nm, 4 nm bandwidth, 60 µm flow cell
• ZORBAX RRHD Eclipse Plus C18 columns (2.1×50, 100, 150 mm; 1.8 µm particles)
• Column thermostat maintained at 26 °C via 1.6 µL TCC heat exchanger
• Mobile phase A: water with 0.1% formic acid v/v; mobile phase B: acetonitrile with 0.1% formic acid v/v

Methodology

Herbal supplement samples were diluted 1:100 in deionized water, filtered 0.2 µm RC, and analyzed under various conditions. Peak capacity was calculated as 1 plus gradient time divided by average peak width based on three select peaks (early, mid, late eluters) measured at baseline. Parameters varied included column length at fixed gradient time, gradient time at fixed column length, flow rate adjustments with scaled gradient time, and elevated temperature.

Main Results and Discussion

• Column length effect: Increasing length from 50 to 150 mm with a 30 min gradient at 0.2 mL/min reduced average peak width from 0.088 to 0.055 min and raised peak capacity from 115 to 183.
• Flow rate influence: Higher flow rates narrowed peaks but required proportional gradient time scaling, enabling threefold faster analyses with minimal loss of resolution.
• Gradient time impact: Extending from 10 to 30 min improved peak capacity by about 20%, significantly enhancing separation of closely eluting compounds.
• Temperature effect: Raising temperature to 60 °C decreased mobile phase viscosity, lowered peak width (for example from 0.25 to 0.17 min) and increased peak capacity up to 228, though selectivity changes were analyte dependent.

Benefits and Practical Applications

This optimized UHPLC approach enables high-throughput and high-resolution profiling of herbal supplement constituents, beneficial for QA/QC, natural product research, and regulatory compliance by balancing analysis time and separation performance.

Future Trends and Potential Applications

Future developments may include coupling UHPLC with high-resolution mass spectrometry for comprehensive compound identification, novel column chemistries tailored for complex natural products, automated software-driven gradient optimization, multidimensional LC workflows, and AI-assisted data processing.

Conclusion

Four variables—column length, gradient time, flow rate, and temperature—play pivotal roles in maximizing peak capacity and resolution in gradient liquid chromatography using 1.8 µm UHPLC columns. The Agilent RRHD columns and 1290 Infinity UHPLC platform provide the high-pressure capabilities needed to exploit these parameters effectively for complex herbal analyses.

Reference

• Henderson Jr J W, Faye T, Witteg U, Joseph M Gradient Manipulation and 1.8 µm LC Columns for High Resolution Analysis of Herbal Supplements Agilent Technologies Application Note RAFA2009
• Neue U D Theory of peak capacity in gradient elution Journal of Chromatography A 1079 2005 153–161