Orthogonal Chromatographic Separations using the Agilent 1260 Infinity II SFC/UHPLC Hybrid System

Importance of the topic

Combining supercritical fluid chromatography and ultra high performance liquid chromatography in one hybrid system addresses the growing need for complementary analytical separation techniques in complex sample analysis. Orthogonal separations enable the resolution of coeluting compounds that would remain hidden under a single mode of chromatography. This capability supports comprehensive profiling in pharmaceutical, environmental and food safety laboratories where detailed compound characterization is critical.

Objectives and Overview of the Study

This technical overview describes the configuration and performance evaluation of the Agilent 1260 Infinity II SFC/UHPLC Hybrid System. The main goals were to demonstrate that a single instrument can switch rapidly between SFC and UHPLC, maintain performance equivalent to stand-alone systems, and achieve orthogonal separation of a complex pesticide mixture.

Methodology and Instrumentation

The hybrid system shares a common autosampler, column compartment and detector while using a two-position ten-port valve for mode switching.

• SFC method – CO2 mobile phase with methanol modifier, flow 2.5 mL/min, gradient from 5 to 25 % modifier in 6 min, backpressure 140 bar, column ZORBAX RX-SIL or NH2 at 40 °C, injection volumes 0.1–10 µL by Feed Injection
• UHPLC method – water with 0.1 % formic acid and acetonitrile with 0.1 % formic acid, flow 2.5 mL/min, gradient 15 to 70 % organic in 6 min, column ZORBAX SB-C18 at 40 °C, injection volumes up to 100 µL
• Optional single quadrupole MS – positive ESI with make-up flow for SFC, full flow for UHPLC, scan range 180–450 m/z

Main Results and Discussion

The hybrid system achieved peak area RSDs below 0.3 % for injection volumes above 0.5 µL and retention time RSDs around 0.08 % in both SFC and UHPLC modes. Linearity across the injection volume range exhibited R2 values of 0.9999. Rapid switching between modes within a single sequence required no manual replumbing and showed stable performance. Orthogonal separation of a 15-pesticide mixture demonstrated that compounds coeluting under SFC could be resolved under reversed-phase UHPLC and vice versa.

Benefits and Practical Applications

• Cost-effective integration of two separation techniques in one instrument
• High reliability and reproducibility across both modes
• Ability to uncover hidden analytes through orthogonal workflows
• Flexible injection volumes for sensitivity or high-throughput requirements
• Seamless coupling to mass spectrometry for structural identification

Future Trends and Potential Applications

Advances may include full automation of mode switching and column selection, expanded compatibility with high-resolution MS detectors, greener supercritical fluid methods with reduced solvent consumption and broader adoption in quality control, metabolomics and natural product research. Integrating data analytics and AI-driven method development could further streamline orthogonal workflows.

Conclusion

The Agilent 1260 Infinity II SFC/UHPLC Hybrid System delivers standalone-level performance in both SFC and UHPLC modes, rapid mode switching, and true orthogonal separations. This versatility enhances laboratory efficiency and analytical depth while reducing instrument footprint and cost.

Reference

1. Naegele E Supercritical Fluid Chromatography with Flexible Injection Volumes at Highest Precision Agilent Technologies Technical Overview 5991-7623EN 2017
2. Naegele E Feed Speed and Overfeed Volume New Parameters for SFC Injection Agilent Technologies Technical Overview 5991-7626EN 2017
3. Huesgen AG Schneider S Performance Characteristics of the Agilent 1290 Infinity II Multisampler Agilent Technologies Technical Overview 5991-5348EN 2014