Purification of epigallocatechin gallate and other related polyphenols from green tea by mass-triggered fractionation

Significance of the topic

Epigallocatechin gallate (EGCG) is a predominant polyphenolic metabolite in green tea renowned for its antioxidant capacity and positive health effects. Efficient isolation of EGCG and related catechins is vital for natural product research, nutraceutical development, and quality control in food and pharmaceutical industries.

Objectives and overview of the study

This work aimed to establish a rapid, high-purity preparative HPLC protocol using mass-triggered fractionation for isolating EGCG along with three other catechins from a crude green tea extract. Method transfer from analytical to preparative scale was demonstrated on an AZURA® system to minimize fraction numbers and total purification time.

Methodology and instrumentation used

The approach combined analytical method development with preparative mass-directed purification:

• Analytical development: AZURA Analytical HPLC Plus with Eurospher II C18 column (150 × 4.6 mm), step gradient (10 – 15% acetonitrile), UV detection at 220 nm.
• Preparative system: AZURA P 2.1L pump with LPG module, 5 mL injection loop, AZURA UVD 2.1S UV detector, 4000 MiD mass spectrometer in negative SIM mode monitoring m/z 289.2, 305.2, 441.4, 457.4, Foxy R1 fraction collector, Eurospher II 100-5 C18 column (150 × 20 mm).
• Mobile phases: 0.1% formic acid in water (A) and acetonitrile (B) with a 50 min step gradient at 18.9 mL/min.
• Sample preparation: Sonication of ground green tea leaves in 75% ethanol, followed by filtration and dilution.

Main results and discussion

Mass-triggered fractionation enabled selective collection of four fractions corresponding to epigallocatechin, epicatechin, epicatechin gallate, and EGCG. Analytical re-analysis confirmed purities >95% for EGCG and >90% for the other catechins. Targeted fraction collection dramatically reduced unnecessary fractions and shortened overall purification time compared to conventional fractionation.

Benefits and practical applications of the method

• High selectivity: SIM-based detection ensures precise isolation of target compounds.
• Time efficiency: Fewer fractions accelerate processing and reduce solvent consumption.
• Scalability: Seamless transfer from analytical to preparative scale maintains performance.
• Versatile application: Suitable for natural product research, nutraceutical manufacturing, and QA/QC workflows.

Future trends and possibilities

Further expansion of mass-triggered preparative HPLC to other plant metabolites could streamline natural product discovery. Integration with automated sample preparation and real-time data analytics may enhance throughput. Advances in column technologies and ionization techniques will likely extend sensitivity and applicability in complex matrices.

Conclusion

Mass-triggered preparative HPLC on the AZURA® platform offers a rapid, selective, and scalable strategy for isolating EGCG and related catechins from green tea. The approach achieves high purities and reduced processing times, supporting its adoption in academic and industrial settings.