Performance Comparison to Determine LC Method Compatibility

Significance of the Topic

Liquid chromatography addressing both small molecules and fragile biomolecules requires robust and biocompatible hardware capable of handling aggressive mobile phases and cleaning protocols. The introduction of an iron-free fluid path in the Agilent 1260 Infinity II Prime Bio LC System is key to expanding the application space to native proteins, nucleic acids, and conventional reversed-phase assays while maintaining precision and reliability.

Goals and Overview of the Study

This work compares the performance of the stainless-steel–based 1260 Infinity II Prime LC System with the iron-free 1260 Infinity II Prime Bio LC System. Three typical chromatographic modes were assessed: isocratic separation of a small-molecule sample, a rapid 6-minute gradient on sub-2.7 µm particle columns, and a shallow gradient for a USP Ganoderma Lucidum extract.

Methodology and Instrumentation

• Pump: 800 bar quaternary pump with iron-free (Bio) or stainless-steel fluid path.
• Autosampler: Multisampler with sample thermostat and needle wash.
• Column thermostat: Biocompatible heat exchanger for Bio vs. standard unit.
• Detector: Diode-array (DAD) with Max-Light biocompatible cell.
• Columns: ZORBAX Eclipse Plus C18 5 µm; Poroshell 120 SB-C18 2.7 µm.
• Mobile phases and gradients tailored per application (isocratic, fast gradient, shallow gradient).

Results and Discussion

In isocratic and fast gradient modes, both systems delivered excellent repeatability and retention time precision (RT deviations <2%), confirming method compatibility. Mobile-phase mixing precision met RSD specifications (<0.15%). In the shallow gradient assay, RT shifts up to 5% were observed, driven by minor differences in isocratic hold composition (±0.2% B), underlining the sensitivity of long holds to pump accuracy.

Benefits and Practical Applications

The Bio LC system’s corrosion-free fluid path extends compatibility to high-salt buffers and harsh cleaning solvents without sacrificing performance. Users can transfer existing methods seamlessly for routine QA/QC in pharmaceutical, food, and biotechnology laboratories.

Future Trends and Application Possibilities

Emerging developments include tighter integration with mass spectrometry, high-throughput sample handling, AI-driven method optimization, and advanced biocompatible column chemistries for intact protein and complex biomolecule analysis.

Conclusion

Both Agilent 1260 Infinity II Prime platforms achieve comparable performance in isocratic and steep-gradient workflows. The iron-free Bio LC system offers a robust solution for bioanalytical needs, while shallow gradient methods require precise mobile-phase control to ensure transferability.

References

1. Ganoderma lucidum Fruiting Body Monograph. USP, 2015.
2. Ganoderma lucidum Fruiting Body Dry Extract Lot Certificate F012B0. USP, 2014.