Agilent 1290 Infinity II LC with ISET – Emulation of Waters Alliance 2695 LC Through Waters Empower Software

Importance of the topic

Instrument-to-instrument method transfer is a key challenge in regulated laboratories, impacting data consistency, method validation and operational efficiency.
Overcoming differences in system delay volume and solvent mixing behavior is crucial for maintaining retention times and chromatographic resolution during equipment changes.

Objectives and Study Overview

This study demonstrates a seamless transfer of an LC method for paracetamol and its impurities from a Waters Alliance 2695 system to an Agilent 1290 Infinity II LC.
The impact of Agilent Intelligent System Emulation Technology (ISET) with and without fine-tuning is evaluated under Waters Empower software control.

Methodology

A reversed-phase gradient method using 0.1% TFA in water and 0.09% TFA in acetonitrile was applied. Chromatographic conditions included a 4.6×100 mm, 3.5 μm C18 column at 30 °C, a 1.2 mL/min flow, 3 μL injection, and UV detection at 270 nm.
Sample solution comprised paracetamol (5 mg/mL) and six impurities at 10 µg/mL each.

Instrumentation

• Waters Alliance 2695 LC with Dual Absorbance Detector VWD 2487
• Agilent 1290 Infinity II LC system: Flexible Pump, Multisampler, Multicolumn Thermostat, Diode Array Detector
• Milli-Q ultrapure water system
• Waters Empower 3 and Agilent ICF control software

Main Results and Discussion

Enabling ISET aligned retention times within ±5% or ±0.3 min of the original method, compared with significant deviations when ISET was disabled.
Fine-tuning of added delay volume improved correlation to within ±0.1 min or 2% for all analytes.
Resolution values increased on the UHPLC system, exceeding the 5% loss limit and enhancing peak separation.

Benefits and Practical Applications

• Rapid method transfer without hardware or protocol modifications
• Preservation of validated conditions for regulated workflows
• Improved resolution and throughput using UHPLC technology

Future Trends and Opportunities

Advancements may include broader emulation across different LC vendors, automated parameter optimization, and integration with cloud-based chromatographic data systems.
Further development could extend ISET to multidimensional separations and emerging detector technologies.

Conclusion

Agilent ISET streamlines the migration of established HPLC methods to UHPLC platforms, preserving retention time and resolution. Employing fine-tuning capabilities ensures high fidelity emulation, enabling efficient, validated method transfers in regulated environments.

References

• Huesgen AG. Agilent 1290 Infinity Binary LC with ISET: Emulation of Waters Alliance 2695 LC system analyzing aromatic acids. Application Note 5991-2019EN; 2013.
• Agilent. Agilent 1290 Infinity with ISET. User Manual G4220-90313; 2014.
• Huesgen AG. Agilent 1290 Infinity Binary LC with ISET: Emulation of Waters Alliance 2695 LC system analyzing analgesics. Application Note 5991-2792EN; 2013.