ADD A NEW DIMENSION TO YOUR RESEARCH WITH THE AGILENT 6560 ION MOBILITY Q-TOF LC/MS

Importance of the Topic

Ion mobility spectrometry coupled with high-resolution Q-TOF mass spectrometry adds a third, orthogonal separation dimension to traditional LC/MS workflows. By measuring gas-phase ion mobility and collision cross section (CCS), researchers gain enhanced confidence in compound identification, structural characterization, and quantitation, even in highly complex mixtures.

Objectives and Study Overview

This white paper introduces the Agilent 6560 Ion Mobility Q-TOF LC/MS system and its capability to combine UHPLC, uniform-field drift tube ion mobility, and high-resolution mass analysis. The goals are to demonstrate how this platform resolves structural isomers, increases peak capacity and specificity, detects trace-level components, and preserves native protein conformations.

Methodology and Instruments Used

The Agilent 6560 system integrates:

• An Infinity II UHPLC for chromatographic separation.
• A uniform-field drift tube with Agilent’s iFunnel ion-sampling technology.
• A quadrupole time-of-flight mass spectrometer (Q-TOF) with >40,000 mass resolution and five orders of dynamic range.

Drift tube conditions (electric field, buffer gas pressure, temperature) are optimized for accurate CCS determination without external calibrants. Advanced MassHunter software performs 4-D feature finding, CCS calculation, and mobility-filtered data extraction.

Main Results and Discussion

Applications across small molecules, lipids, carbohydrates, and proteins illustrate the system’s capabilities:

• Separation of isobaric pesticides differing by <0.2 mDa, achieving ~2,000,000 resolving power through combined LC-IM-MS.
• Clear resolution of citrate/isocitrate and methylmalonic acid/succinic acid isomers without lengthy HPLC methods.
• Differentiation of isomeric tri-saccharides and human milk oligosaccharides by drift time and CCS trends.
• Enhanced peak capacity for complex matrices such as crude bacterial extracts, native glycans, and petroleum or ink dispersant samples by mobility-filtered spectral cleanup.
• Improved sensitivity for trace analytes in urine with superior signal-to-noise in IM-Q-TOF mode.
• Preservation and separation of native and denatured protein conformers (cytochrome C, IgG-1/IgG-2 isoforms), demonstrating low ion-heating and high conformational fidelity.

Benefits and Practical Applications

By adding an ion mobility separation dimension, users achieve:

• Unambiguous isomer and conformer discrimination.
• Higher throughput through shorter gradients and all-ions MS/MS workflows.
• Lower chemical background and improved quantitation accuracy.
• Expanded proteomics, glycomics, metabolomics, lipidomics, and biopharmaceutical characterization.

Future Trends and Potential Applications

Ongoing developments will further enhance IM-MS utility:

• Extension of CCS libraries and machine-learning prediction for rapid annotation.
• Coupling with GC-APCI and MALDI sources for broader compound coverage.
• Integration with cloud-based informatics and AI-driven data mining.
• High-throughput screening in omics and real-time process monitoring.
• Advanced native MS for detailed structural biology and biopharmaceutical comparability.

Conclusion

The Agilent 6560 Ion Mobility Q-TOF LC/MS platform delivers a powerful combination of UHPLC, uniform-field ion mobility, and high-resolution MS. It overcomes key analytical challenges by providing unmatched separation of isomers, enhanced specificity in complex samples, sensitive quantitation of trace analytes, and preservation of native biomolecular conformations.

References

1. Imatani K., Wong D. Applications Highlights: Agilent 6560 Ion Mobility Q-TOF LC/MS. Agilent Technologies; 2015.
2. Agilent Technologies. Technical Overview: Uniform Field Drift Tube Ion Mobility Q-TOF LC/MS (5991-3244EN). 2015.