Agilent 1290 Infinity II LC with ISET–Emulation of the Waters Acquity H-Class for Ternary Gradients

Significance of the Topic

The accurate replication of chromatographic methods across different high performance liquid chromatography platforms is critical in pharmaceutical and biopharmaceutical analysis. Small changes in modifier concentration or solvent mixing behavior can lead to significant shifts in retention times and peak shapes. Intelligent System Emulation Technology plays a key role in enabling seamless method transfer without the need for revalidation or method redevelopment.

Objectives and Study Overview

This study aims to demonstrate how ISET within the Agilent 1290 Infinity II LC can emulate a ternary gradient method originally developed on a Waters Acquity H-Class system for peptide analysis. Key goals include:

• Comparing retention times and peak precision for five peptides using Waters Acquity H-Class, Agilent 1290 Infinity II LC without ISET, and Agilent 1290 Infinity II LC with ISET
• Quantifying retention time deviations and evaluating agreement
• Assessing the feasibility of direct method transfer for ternary gradients

Methodology and Instrumentation

• Instrumentation Used
  • Waters Acquity Bio H-Class system with BioQuaternary Solvent Manager, BioSample Manager FTN, Column Manager, and TUV Detector
  • Agilent 1290 Infinity II LC comprising Flexible Pump, Multisampler, Multicolumn Thermostat, Diode Array Detector
• Chromatographic Conditions
  • Stationary phase: Poroshell 120 EC-C18, 3.0 × 50 mm, 2.7 μm
  • Mobile phases: A water; B acetonitrile; C 1 % trifluoroacetic acid
  • Flow rate: 0.5 mL/min; column temperature: 50 °C; injection volume: 0.2 μL
  • Ternary gradient profile over 6.5 minutes with 10 % TFA delivered via solvent channel C
  • Detection at 214 nm with reference at 360 nm, data rate 20 Hz

Main Results and Discussion

Retention time comparisons showed that the 1290 Infinity II LC without ISET exhibited deviations up to 6 % compared to the Waters system, exceeding typical tolerance limits. After enabling ISET, deviations were reduced to below 2.3 % for all five peptides, corresponding to over 97 % agreement. Precision across six consecutive injections showed relative standard deviations below 0.2 % with or without ISET, confirming robust reproducibility.

Benefits and Practical Applications of the Method

• Enables direct transfer of ternary gradient methods without reoptimization
• Maintains consistency of legacy methods across different vendor platforms
• Optimizes laboratory workflow by reducing method development time
• Applies to peptide and biopharmaceutical analyses where modifier precision is critical

Future Trends and Opportunities

The extension of system emulation technologies may include multi-platform digital method repositories, automated gradient optimization using independent solvent channels, and integration with laboratory information management systems. Further applications are anticipated in metabolomics, proteomics, and high-throughput quality control workflows.

Conclusion

ISET within the Agilent 1290 Infinity II LC successfully emulates ternary gradient methods from Waters Acquity H-Class systems, achieving retention time agreement above 97 %. This capability streamlines method transfer, supports legacy protocols, and enhances laboratory efficiency in peptide analysis.

Reference

• Gratzfeld-Huesgen A. Fast and flexible optimization of buffer modifier concentrations using ternary gradients with the Agilent 1260 Infinity LC Quaternary System. Agilent Technologies Application Note 5990-6864EN, 2010.
• Gratzfeld-Huesgen A. Agilent 1290 Infinity Binary LC with ISET emulation of the Waters Alliance 2695 LC System analyzing analgesics. Agilent Technologies Application Note 5991-2792EN, 2013.
• Gratzfeld-Huesgen A. Seamless instrument to instrument method transfer from an Agilent 1100 1200 Series LC to an Agilent 1290 Infinity LC using Intelligent System Emulation Technology. Agilent Technologies Application Note 5990-9113EN, 2011.
• Agilent Technologies. Agilent 1290 Infinity with ISET User Manual, document G4220-90314, 2015.