Automated LC Method Development and Robustness Tests

Significance of the Topic

The automated development of liquid chromatography methods accelerates the creation of reliable and robust analytical protocols, especially for complex mixtures such as pharmaceutical samples. By replacing manual trial‐and‐error with systematic and intelligent automation, laboratories can achieve higher productivity, reproducibility, and data quality while minimizing resource consumption and analyst effort.

Objectives and Study Overview

This technical overview illustrates how the Agilent Instrument Control Framework (ICF) integrates with ChromSwordAuto 5 and an Agilent 1290 Infinity II UHPLC system to support automated and intelligent method development. Key goals include establishing software and hardware prerequisites, demonstrating scouting and design of experiments (DoE) workflows, and evaluating system performance in method scouting, fine optimization, and robustness testing of a 16‐component pesticide standard.

Used Instrumentation

• Agilent 1290 Infinity II Flexible Pump (G7104A)
• Agilent 1290 Infinity II Multicolumn Thermostat (MCT) with Quick Change valves (G7116B, G4239C)
• Agilent 1290 Infinity II Multisampler (G7167B)
• Agilent 1290 Infinity II Diode Array Detector (DAD) (G7117B)
• Two Agilent 1290 Infinity Valve Drives (G1170A) with Quick Change valve (G4235A)
• ChromSwordAuto 5.1 suite: Scout, Developer, AutoRobust, ReportViewer
• Software prerequisites: ICF A.02.05 with LC drivers A.02.18; all LC module firmware ≥ A.06.50/B.06.75/D.06.75; CAN and LAN connectivity

Methodology

An initial scouting phase screened multiple reversed‐phase columns (ZORBAX Bonus-RP, RRHD C18, StableBond C8, Eclipse Plus C18) and organic solvents using rapid gradient runs (3–4 per condition). Users could select a DoE approach for detailed evaluation of method variables. ChromSwordAuto’s AutoRobust module then executed full factorial or Plackett–Burman designs to assess flow rate, temperature, and solvent composition. The workflow combined robotic process automation for routine tasks and intelligent automation for multistep gradient optimization with advanced data processing and reasoning.

Main Results and Discussion

Rapid optimization identified ZORBAX Bonus-RP (100 × 2.1 mm, 1.8 µm) with an optimized gradient starting at 20% acetonitrile to achieve full elution of 16 pesticides in under 22 minutes with baseline separation (resolution ≥ 2.03). Fine optimization refined gradient slopes and tracking of critical peak pairs. Robustness testing via a full factorial DoE revealed an optimal operating window at 29.5 °C, 0.28 mL/min flow, and 25.9% acetonitrile. Two‐ and three‐dimensional resolution maps enabled visual identification of robust regions and informed selection of method conditions that withstand small variations in parameters.

Benefits and Practical Applications

• Substantial reduction in method development time (from days to hours)
• Minimal manual intervention—analyst effort of only 1–2 hours for scouting and data review
• Comprehensive exploration of variable space via DoE for robust method performance
• Automated generation of reports and design spaces for regulatory submissions and quality assurance
• Improved reproducibility and confidence in critical separations through systematic optimization and robustness assessment

Future Trends and Applications

Advances in machine‐learning algorithms promise to enhance prediction of separation outcomes, further reducing screening experiments. Integration with real‐time spectral deconvolution and on‐line mass spectrometry will expand automated workflows to complex matrices and bioanalytical applications. Continued development of greener solvent systems and miniaturized UHPLC hardware will drive sustainable high‐throughput method creation.

Conclusion

The combination of Agilent ICF, ChromSwordAuto 5, and a 1290 Infinity II UHPLC system provides a powerful platform for automated LC method development. It delivers rapid scouting, fine‐tuning, and robustness evaluation with minimal manual effort, enabling laboratories to generate high‐quality methods efficiently and reproducibly.

References

1. Galushko S, et al. ChromSword Software for Automated and Computer-Assisted Development of HPLC Methods. In HPLC Made to Measure: A Practical Handbook for Optimization; Kromidas S, Ed.; Wiley-VCH: Weinheim, 2006; pp 557–570.
2. Hewitt EF, Lukulay P, Galushko S. Implementation of a Rapid and Automated High Performance Liquid Chromatography Method Development Strategy for Pharmaceutical Drug Candidates. J Chromatogr A. 2006;1107(1–2):79–87.
3. Xiao KP, et al. Efficient Method Development Strategy for Challenging Separation of Pharmaceutical Molecules Using Advanced Chromatographic Technologies. J Chromatogr A. 2007;1163(1–2):145–156.
4. Vogel F, Galushko S. Application of ChromSword Software for Automatic HPLC Method Development and Robustness Studies. Separation of Terbinafine and Impurities. Chromatography Today. 2013;3–6.
5. Zhuang J, Kumar S, Rustum A. Development and Validation of a Normal Phase Chiral HPLC Method for Analysis of Afoxolaner Using a Chiralpak AD-3 Column. J Chromatogr Sci. 2016;54(10):1813–1819.