Principles of fraction collection using the Vanquish HPLC and UHPLC systems

Principles of Fraction Collection Using Vanquish HPLC and UHPLC Systems

Importance of the Topic

Automated fraction collection in liquid chromatography enables isolation of target compounds, removal of impurities, and preparation of pure samples for downstream analyses. Precise coordination of sample introduction, column separation, detection and fraction handling is critical to preserve chromatographic resolution and achieve high purity and recovery. Minimizing dead volume and dispersion between detector and collection vessels, as well as rapid valve switching and needle handling, are key to translating on-column resolution into discrete, high-quality fractions.

Objectives and Study Overview

This technical note focuses on the design and optimization of the Thermo Scientific™ Vanquish™ Fraction Collector (VFC) in analytical-scale HPLC and UHPLC workflows (0.05–10 mL/min). It describes the fluidic path, quantifies delay volume components, introduces an automated delay-volume determination, and explains flush and wash strategies. Performance metrics such as delay time, dispersion, carry-over and recovery are demonstrated with a model oligonucleotide mixture, comparing time-based and peak-based fractionation modes.

Methodology and Instrumentation

• System: Vanquish Horizon/Flex LC with Quaternary Pump, Split Sampler FT, Column Compartment H, VWD detector, and Integral Fraction Collector FT.
• Detection: Variable-wavelength UV at 260 nm; Semimicro flow cell (2.5 µL).
• Delay path: Delay capillary (various IDs/lengths matched to 0.05–10 mL/min), fraction-collection valve, needle capillary, and stainless-steel needle (or drop former on non-integrated modules).
• Software: Chromeleon 7.3.1 CDS with fraction-collection wizard; automated delay-volume determination (DVD) using an injected air bubble.
• Model analytes: Mixed oligonucleotide standards (12 mer to 40 mer) separated on a DNAPac RP column with TEAA/acetonitrile gradient.

Main Results and Discussion

• Delay Volume and Time: Delay volume contributions from capillaries, valve, and needle were measured by Automated DVD. Accurate delay-time settings in the CDS ensured alignment of detector peaks and collection windows.
• Dispersion: Use of low-ID, short capillaries and an optimized flow cell minimized post-column band broadening, preserving peak resolution in collected fractions.
• Carry-over and Recovery: Active flush loops plus internal/external needle washes reduced cross-contamination below detectable limits. Recoveries of > 95% were demonstrated by comparing source and fraction-reanalysis peak areas, accounting for dilution and flow volumes.
• Fractionation Modes: Time-based collection enabled simple slicing of the chromatogram but may include small tail-diffusion artifacts. Peak-based collection, using threshold and slope criteria, delivered superior purity by aligning vessel switching precisely with peak boundaries.

Benefits and Practical Applications

• High-throughput purification of biomolecules, pharmaceuticals, and complex mixtures for analytical and preparative workflows.
• Automated, reproducible fraction collection with minimal user intervention and method development time.
• Compatibility with downstream analyses (MS, NMR, bioassays) due to high sample integrity and low carry-over.

Future Trends and Potential Uses

• Integration with high-resolution MS for targeted fraction collection based on real-time mass detection.
• Micro- and nano-scale fractionation platforms for single-cell and omics applications demanding ultra-low dead volumes.
• Advanced software algorithms employing machine learning to predict optimal fraction windows from complex chromatograms.
• Automated multi-step purification workflows coupling fraction collection with online desalting or sample concentration.

Conclusion

The Vanquish Fraction Collector, when combined with optimized fluidics and Chromeleon CDS control, effectively translates UHPLC resolution into discrete fractions with high purity, minimal dispersion, and excellent recovery. Automated delay-volume calibration, active flushing/washing and flexible fractionation modes allow robust, reproducible isolation of analytes across a wide flow-rate range, meeting the needs of analytical and preparative workflows.

References

1. Gamache, P.H. Nebulization. In Charged Aerosol Detection for Liquid Chromatography and Related Separation Techniques; Wiley, 2017; pp. 12–14.
2. Thermo Scientific Vanquish Integral Fraction Collector Operating Manual, v1.0, July 2022.
3. Thermo Scientific Chromatography Consumables Catalog – Sample Handling.
4. Kromidas, S.; Steiner, F.; Lamotte, S. Detection Limit, Peak Capacity, Resolution: Possibilities for Gradient Optimization. In The HPLC Expert; Wiley-VCH, 2016; pp. 156–158.
5. Dittmann, M. The Issue of External Band Broadening in HPLC/UHPLC Devices. In The HPLC Expert II; Wiley-VCH, 2017; pp. 89–94.
6. Thermo Scientific Vanquish Fraction Collector Product Specification Sheet, PS000922.