Injection of Highly Concentrated Samples Using an Agilent 1290 Infinity II Preparative LC System with Independent Flow Channels

Importance of the Topic

Reversed-phase preparative liquid chromatography often faces limitations in sample loading due to solubility constraints. Highly concentrated samples in aqueous mobile phases can precipitate, causing pressure spikes, clogging, and downtime. An injection strategy that only exposes the sample to the organic phase until it reaches the column head can overcome these challenges, boosting throughput and reducing maintenance.

Objectives and Study Overview

This study demonstrates how to modify an Agilent 1290 Infinity II Preparative LC System to inject samples via the organic solvent channel. By separating aqueous and organic mobile-phase flowpaths and rerouting the autosampler to the organic channel, the work aims to:

• Test the feasibility of organic-phase injection for high-concentration samples
• Evaluate chromatographic performance, reproducibility, and fraction purity
• Quantify potential gains in sample throughput

Methodology and Instrumentation

The modified flowpath uses independent pump channels for aqueous (A) and organic (B) solvents. Key hardware and software components include:

• Agilent 1290 Infinity II Preparative Binary Pump with independent A/B channels and pressure sensors
• Agilent 1260 Infinity II Preparative Autosampler with multidraw loop kit, connected to channel B
• Agilent 1260 Infinity II Column Organizer and Variable Wavelength Detector (274 nm, 10 Hz)
• Agilent 1290 Infinity II Preparative Open-Bed Fraction Collector
• Agilent OpenLab CDS ChemStation software

Solvents were LC-grade water (channel A) and acetonitrile (channel B). A T-piece located just upstream of the column head mixes the two phases with minimal dwell volume. The sample loop and capillaries (1 mm id) were optimized to maintain flow continuity and avoid precipitation.

Main Results and Discussion

A test mixture of five compounds (caffeine, ethylparaben, biphenyl, phenanthrene, pyrene) dissolved in DMSO at concentrations up to 250 mg/mL was injected at 1,500 µL (522 mg load, 2.5% of stationary phase mass). Chromatographic separation (10–80% B gradient) yielded well-resolved peaks despite overloading. Six consecutive runs showed retention time RSD ≤0.07%. Fraction collection triggered by a 30 mAU UV threshold produced fractions with >99% purity and >91% recovery. No pressure spikes or clogging events were observed, confirming stable operation.

Benefits and Practical Applications

Implementing organic-phase injection provides:

• Increased sample throughput by enabling higher concentration loads
• Reduced risk of precipitation-induced instrument downtime
• High reproducibility and fraction purity for preparative workflows
• Simplified maintenance by avoiding clogged valves and lines

Future Trends and Potential Applications

The organic-phase injection concept can be extended to other preparative platforms and coupling strategies. Future developments may include automated optimization of flowpath switching, integration with mass-directed fraction collection, and adaptation to continuous-flow purification formats. These advances could further accelerate compound purification in pharmaceutical and chemical research.

Conclusion

The modified Agilent 1290 Infinity II Preparative LC system with independent flow channels successfully addresses sample solubility limits in preparative HPLC. By injecting samples in the organic stream and mixing just before the column, the method permits overloading to 2.5% of stationary phase while maintaining peak integrity, high recovery, and minimal downtime.

References

• Wheat T.E.; Phoebe C.H.; Neue U.D. Mass-directed Autopurification: Optimization of Separation and Detection Chemistry. 48th ASMS Conference on Mass Spectrometry and Allied Topics, June 11–15, 2000.
• Blom K.F. Two-Pump at-Column-Dilution Configuration for Preparative Liquid Chromatography-Mass Spectrometry. J. Combinatorial Chem. 2002, 4(4), 295–301.
• Neue U.D. et al. At-column Dilution for Improved Loading in Preparative Chromatography. Chromatographia 2003, 57(1), S121–S127.