Antioxidant Isolation Using the Prep 150 LC System

Significance of the topic

Preparative isolation of antioxidant compounds such as chlorogenic acid from natural sources is critical for research in pharmaceuticals, nutraceuticals and food sciences. Efficient separation conserves valuable samples and solvents, while delivering high-purity isolates for biological evaluation and product development.

Objectives and study overview

This application note demonstrates the development, scale-up and fraction collection of a chromatographic method to purify chlorogenic acid from Aronia berry extract using the Waters Prep 150 LC System. The focus is on rapid gradient scouting, formation of a focused gradient to maximize resolution, and scaling from analytical to preparative columns to enhance throughput and purity.

Methodology and Instrumentation

Fresh, lyophilized Aronia berries were ground and extracted with acidified water, then centrifuged and filtered. Analytical scouting utilized a SunFire C18 4.6×50 mm column with a 5 µm particle size at 1.46 mL/min, detecting at 324 nm. After identifying optimal conditions, a focused gradient was calculated based on column volume and system delay. Scale-up employed a SunFire C18 OBD Prep 19×50 mm column at 24.9 mL/min with a 1.7 mL injection volume. The system comprised:

• Waters Prep 150 LC System with 2545 Binary Gradient Module
• 2489 UV/Visible Detector (UV 324 nm)
• FlexInject Manual Dual Injector and Fraction Collector III
• ChromScope™ v1.6 software and OBD Prep Calculator

Key results and discussion

• Rapid scouting gradients (2→25, 50, 75, 95% B over 5 min) indicated the 2→25% run provided the best separation of chlorogenic acid from impurities.
• A focused gradient was designed to elute the target at 15% B over an 8.7 min shallow ramp, significantly enhancing peak resolution.
• An abbreviated focused gradient reduced cycle time by ~6 min by immediately washing the column after elution of chlorogenic acid.
• Analytical purity of chlorogenic acid in crude extract was 32% by area; prep-scale isolation achieved 99% purity relative to a reference standard.
• Scale-up calculations based on column diameter squared and volume yielded prep flow of 24.9 mL/min and injection volume of 1706 µL, with consistent retention times.
• ChromScope absorbance-triggered fraction collection efficiently isolated the purified compound.

Benefits and practical applications

• The modular Prep 150 LC System enables seamless transition from method development to prep isolation on one platform.
• Focused gradient design maximizes resolution while conserving time and solvents.
• High-purity isolates support downstream biological assays and product formulation.
• Optimized workflow allows multiple daily purifications, boosting laboratory throughput.

Future trends and potential applications

• AI-driven gradient optimization could further streamline method development.
• Integration with mass spectrometry for real-time fraction guidance and compound identification.
• Continuous flow and inline detection to increase throughput and automation.
• Application of prep-scale techniques to a broader range of natural products and biomolecules.

Conclusion

The Waters Prep 150 LC System provided a streamlined workflow for isolating chlorogenic acid from Aronia berries. Strategic gradient scouting and focused gradient design, combined with accurate scale-up, resulted in high-purity isolation with reduced run times. This approach exemplifies efficient preparative chromatography for natural product purification.

Reference

1. Aronia: The North American super berry with cancer-fighting properties. foxnews.com/health/2013/06/07/aronia-north-american-super-berry-with-cancer-fighting-benefits.html. July 1, 2016.