AN IMPROVED CALIBRATION APPROACH FOR TRAVELLING WAVE ION MOBILITY SPECTROMETRY: ROBUST, HIGH-PRECISION COLLISION CROSS SECTIONS

Importance of the Topic

Travelling wave ion mobility spectrometry (TWIM) is widely used to separate ions by mobility and derive collision cross section (CCS) values, which inform on molecular structure and conformation. Reliable CCS calibration is essential for consistent, high‐precision analyses of small molecules, peptides, proteins and complex mixtures. Enhanced calibration strategies increase confidence in structural assignments, support cross‐platform comparisons and broaden TWIM applications in proteomics, metabolomics and quality control.

Study Objectives and Overview

This work introduces novel calibration functions for TWIM that incorporate velocity relaxation and radial field effects to improve accuracy and precision across diverse molecules and operating conditions. The main goals were to:

• Develop analytical approximations of ion motion in a travelling wave field including relaxation and off‐axis effects
• Validate new calibration functions on Waters Synapt G2 and SELECT SERIES Cyclic IMS instruments
• Compare all‐ion versus class‐specific calibrations and evaluate overfitting risks
• Identify minimal calibrant sets for routine application

Methodology and Instrumentation

A detailed ion optical model was implemented in SIMION 3D to simulate discrete travelling wave stepping, anharmonic fields, RF confinement and diffusion using an SDS collision model. Calibration functions evaluated included:

• Traditional power‐law expressions
• An expanded mobility‐based form accounting for velocity relaxation coefficients (α, γ)
• A blended calibration combining power‐law and analytical approximations
• A radial correction factor to address off‐axis field variations

Experimental calibration data were acquired on:

• Waters Synapt G2 with nitrogen at ~3 mbar, wave heights 20–50 V and speeds up to 1000 m/s
• Waters SELECT SERIES Cyclic IMS quadrupole time‐of‐flight with a 98 cm path and velocity ramping

Custom scripts processed arrival time distributions and fitted calibration parameters against published drift‐tube CCS reference values.

Key Results and Discussion

New blend calibration functions incorporating radial corrections achieved all‐ion calibration root‐mean‐square errors below 1.3% across molecular classes, wave amplitudes and velocities. In cross‐validation tests, the two‐ and three‐parameter blended approaches outperformed six‐parameter expansions by reducing overfitting and residual variability, particularly for native proteins and small multiply charged ions. Minimal calibrant sets containing a single native protein and a few peptides provided robust CCS extrapolation over a 45–220 nm2 range with deviations under 4%, compared with >15% errors using power‐law methods.

Benefits and Practical Applications

• High‐precision CCS values across a wide molecular weight and charge range
• Robust all‐ion calibrations suitable for routine QC in pharmaceutical and food analysis
• Reduced calibrant complexity supports streamlined sample preparation
• Improved comparability of CCS databases and structural models

Future Trends and Potential Applications

Ongoing developments will focus on:

• Designing standardized minimal calibration mixtures for commercial distribution
• Extending calibration functions to emerging high‐resolution IMS platforms
• Integrating machine learning to optimize calibration parameter selection
• Expanding applications in conformational dynamics and native mass spectrometry

Conclusion

Advanced calibration strategies that incorporate ion mobility theory, velocity relaxation and radial field effects deliver robust, high‐precision TWIM CCS measurements across diverse molecules and instrument configurations. Blended calibration functions with few free parameters minimize overfitting and enable simplified all‐ion or class‐specific workflows, paving the way for standardized, cross‐platform CCS libraries and broader adoption in analytical science.

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