Small Scale Purification of Constituents from Complex Natural Product Extracts Using ACQUITY H-Class and Waters Fraction Manager-Analytical Systems

Significance of the topic

The isolation of individual constituents from complex natural product extracts is critical for bioactivity studies, quality control, and further chemical characterization. Ultra-high performance liquid chromatography (UPLC) equipped with sub-2-µm columns delivers sharp, narrowly eluting peaks but also poses challenges for conventional fraction collectors due to band broadening and carryover. The Waters Fraction Manager – Analytical (WFM-A) is specifically designed to address these issues, enabling efficient small-scale purification from highly complex mixtures.

Study objectives and overview

This work aimed to demonstrate the performance and versatility of the WFM-A in conjunction with an ACQUITY UPLC H-Class System for the targeted isolation of compounds from three supercritical fluid extracts: rosemary, schisandra berry, and angelica root. Specific goals included evaluating time-based, threshold-triggered, and mixed-mode fraction collection methods and assessing resulting fraction purity.

Methodology and instrumentation

Supercritical fluid extraction (SFE) was performed on ~25 g of each dried plant material using 99% CO₂/1% isopropanol at 40 °C, 200 bar and 50 g/min for 60 min. Chromatographic separations employed an ACQUITY UPLC H-Class System with BEH C18 (1.7 µm, 2.1 × 100 mm) column, PDA detection at 254 nm, and a 0.1% formic acid water/acetonitrile gradient at 0.5 mL/min and 50 °C. Fraction collection was controlled by Empower 3 software via the WFM-A.

Used instrumentation

• Waters SFE 500 System
• ACQUITY UPLC H-Class System
• ACQUITY UPLC PDA Detector
• Waters Fraction Manager – Analytical
• Empower 3 Software
• ACQUITY BEH C18 Column, 1.7 µm, 2.1 × 100 mm

Key results and discussion

• Time-based collection (rosemary): Two major peaks were pooled across eight injections, yielding 558 µL and 479 µL of fractions with purities of 94.0% and 97.8%, respectively.
• Threshold-triggered collection (angelica root): Two closely eluting peaks were isolated with start/end UV thresholds, achieving purities of 89.4% and 94.1% based on UV detection.
• Mixed-mode collection (schisandra berry): One target peak was isolated by time window and two by threshold within a time window, yielding purities of 70.6%, 85.9%, and 98.9% for peaks known to co-elute.
• Real-time monitoring and method simulation: The ACQUITY console provided live bed-status updates, and Empower 3’s simulator enabled rapid method optimization using previously acquired chromatograms.

Benefits and practical applications

The WFM-A system minimizes band broadening and sample carryover, facilitating the reliable collection of narrow UPLC peaks. Its integration with Empower 3 allows seamless automation, flexible collection strategies, and pre-run simulation, which collectively enhance throughput and fraction purity for natural product research, bioassay-guided isolation, and QA/QC workflows.

Future trends and potential applications

Advances may include coupling the WFM-A with mass spectrometry–based fraction screening, expanded automation for high-throughput natural product libraries, and application to other challenging matrices such as food, environmental samples, and metabolites. Data-driven method development using machine learning could further refine collection parameters and reduce development time.

Conclusion

The Waters Fraction Manager – Analytical, combined with ACQUITY UPLC H-Class technology, effectively isolates narrow, closely eluting peaks from complex natural extracts. Time-based, threshold, and mixed-mode collection strategies produced high-purity fractions, demonstrating the system’s utility in small-scale purification tasks where traditional preparative HPLC collection is inadequate.

Reference

No additional references were provided in the original text.