Reduce Your Purification Footprint: Scouting, Collection, and Reanalysis in Well Plates on a Single System

Significance of the Topic

Purification of small molecules via HPLC typically requires separate analytical and preparative systems, leading to complex workflows, manual transfers and calculations. Integrating scouting, method development, fraction collection and reanalysis into a single system streamlines operations, reduces bench space, errors and processing time.

Objectives and Study Overview

• Demonstrate a unified LC system for analytical scouting, preparative purification and reanalysis.
• Leverage Agilent InfinityLab LC Purification Solutions to automate method development, gradient scaling and fraction handling.
• Test performance on six diverse samples to assess throughput, purity and reproducibility.

Methodology and Instrumentation

The study employed the Agilent 1290 Infinity II Autoscale Preparative LC/MSD system controlled by Automated Purification software for OpenLab ChemStation. Key hardware modules included:

• 1290 Infinity II Preparative Binary Pump and Quaternary Pump
• 1290 Infinity II Open-Bed Sampler/Collector with dual loops and fraction collector
• 1290 Infinity II MS Flow Modulator and Valve Drive
• 1260 Infinity II detectors (variable wavelength and diode array) and LC/MSD XT

Solvents comprised LC-grade acetonitrile, methanol, DMF, DMSO and ultrapure water. Six test mixtures of varying polarity were processed using sample-specific dilution and sandwich injection schemes. Automated Purification software handled scouting gradient calculation, focused gradient generation, scale-up by factor 10 000 and mass-based fraction collection.

Main Results and Discussion

• Analytical scouting runs successfully identified target peaks by overlaying UV and mass signals.
• Software-generated focused gradients optimized resolution around each target under preparative conditions, reducing run times and solvent use.
• Mass-based fraction collection on a 48-well plate enabled simultaneous handling of samples and fractions, minimizing cross-contamination and errors.
• Scale-up achieved without manual recalculation; sample load increased 10 000-fold between scouting and preparative steps.
• Automated fraction reanalysis accurately assessed purity, with fractions reaching >98% purity and revealing unexpected impurities for further processing.

Benefits and Practical Applications

• Fully integrated workflow reduces manual interventions, bench footprint and error sources.
• Automated method development and scale-up accelerate development timelines in pharmaceutical and chemical research.
• Mass-based collection enhances specificity and traceability in fraction handling.
• Reinjection of fractions for quality assessment ensures high confidence in compound purity.

Future Trends and Applications

The integration of intelligent software with flexible LC hardware paves the way for further automation, including AI-driven method optimization and real-time data analytics. High-throughput platforms may expand to parallel purification, and coupling with advanced detectors (e.g., high-resolution MS) could enhance impurity profiling. Such progress will benefit drug discovery, synthetic chemistry and quality control environments.

Conclusion

Combining analytical scouting, preparative purification and fraction reanalysis in a single LC/MSD system significantly streamlines workflows, reduces manual handling and improves data reliability. The Agilent InfinityLab solution demonstrated efficient scale-up, targeted fraction collection and robust purity assessment across diverse samples.

Reference

Spuling I, Rieck F. Spotting Fraction Impurities with More Confidence Using the Agilent 1290 Infinity II Preparative Open-Bed Sampler/Collector. Agilent Technologies Technical Overview. 2019;5994-1643.