Scaling of the Resolving Power and Sensitivity for Planar FAIMS and Mobility-Based Discrimination in Flow- and Field-Driven Analyzers

Continuing development of the technology and applications of field asymmetric waveform ion mobility spectrometry (FAIMS) calls for better understanding of its limitations and factors that govern them. While key performance metrics such as resolution and ion transmission have been calculated for specific cases employing numerical simulations, the underlying physical trends remained obscure. Here we determine that the resolving power of planar FAIMS scales as the square root of separation time and sensitivity drops exponentially at the rate controlled by absolute ion mobility and several instrument parameters. A strong dependence of ion transmission on mobility severely discriminates against species with higher mobility, presenting particular problems for analyses of complex mixtures. While the time evolution of resolution and sensitivity is virtually identical in existing FAIMS systems using gas flow and proposed devices driven by electric field, the distributions of separation times are not. The inverse correlation between mobility (and thus diffusion speed) and residence time for ions in field-driven FAIMS greatly reduces the mobility-based discrimination and provides much more uniform separations. Under typical operating conditions, the spread of elimination rates for commonly analyzed ions is reduced from >5 times in flow-driven to 1.6 times in field-driven FAIMS while the difference in resolving power decreases from ∼60% to ∼15%

Optimization of the design and operation of FAIMS analyzers

Field asymmetric waveform ion mobility spectrometry (FAIMS) holds significant promise for post-ionization separations in conjunction with mass-spectrometric analyses. However, a limited understanding of fundamentals of FAIMS analyzers has made their design and operation largely an empirical exercise. Recently, we developed an a priori simulation of FAIMS that accounts for both ion diffusion (including anisotropic components) and Coulomb repulsion, and validated it by extensive comparisons with FAIMS/MS data. Here it is corroborated further by FAIMS-only measurements, and applied to explore how key instrumental parameters (analytical gap width and length, waveform frequency and profile, the identity and flow speed of buffer gas) affect FAIMS response. We find that the trade-off between resolution and sensitivity can be managed by varying gap width, RF frequency, and (in certain cases) buffer gas, with equivalent outcome. In particular, the resolving power can be approximately doubled compared to “typical” conditions. Throughput may be increased by either accelerating the gas flow (preferable) or shortening the device, but below certain minimum residence times performance deteriorates. Bisinusoidal and clipped-sinusoidal waveforms have comparable merit, but switching to rectangular waveforms would improve resolution and/or sensitivity. For any waveform profile, the ratio of two between voltages in high and low portions of the cycle produces the best performance

Recommendations for reporting ion mobility Mass Spectrometry measurements

Here we present a guide to ion mobility mass spectrometry experiments, which covers both linear and nonlinear methods: what is measured, how the measurements are done, and how to report the results, including the uncertainties of mobility and collision cross section values. The guide aims to clarify some possibly confusing concepts, and the reporting recommendations should help researchers, authors and reviewers to contribute comprehensive reports, so that the ion mobility data can be reused more confidently. Starting from the concept of the definition of the measurand, we emphasize that (i) mobility values (K0) depend intrinsically on ion structure, the nature of the bath gas, temperature, and E/N; (ii) ion mobility does not measure molecular surfaces directly, but collision cross section (CCS) values are derived from mobility values using a physical model; (iii) methods relying on calibration are empirical (and thus may provide method‐dependent results) only if the gas nature, temperature or E/N cannot match those of the primary method. Our analysis highlights the urgency of a community effort toward establishing primary standards and reference materials for ion mobility, and provides recommendations to do so. © 2019 The Authors. Mass Spectrometry Reviews Published by Wiley Periodicals, Inc

Recommendations for reporting ion mobility mass spectrometry measurements

© 2019 The Authors. Mass Spectrometry Reviews Published by Wiley Periodicals, Inc. Here we present a guide to ion mobility mass spectrometry experiments, which covers both linear and nonlinear methods: what is measured, how the measurements are done, and how to report the results, including the uncertainties of mobility and collision cross section values. The guide aims to clarify some possibly confusing concepts, and the reporting recommendations should help researchers, authors and reviewers to contribute comprehensive reports, so that the ion mobility data can be reused more confidently. Starting from the concept of the definition of the measurand, we emphasize that (i) mobility values (K0) depend intrinsically on ion structure, the nature of the bath gas, temperature, and E/N; (ii) ion mobility does not measure molecular surfaces directly, but collision cross section (CCS) values are derived from mobility values using a physical model; (iii) methods relying on calibration are empirical (and thus may provide method-dependent results) only if the gas nature, temperature or E/N cannot match those of the primary method. Our analysis highlights the urgency of a community effort toward establishing primary standards and reference materials for ion mobility, and provides recommendations to do so. © 2019 The Authors. Mass Spectrometry Reviews Published by Wiley Periodicals, Inc

Ultrahigh-Resolution Differential Ion Mobility Separations of Conformers for Proteins above 10 kDa: Onset of Dipole Alignment?

Biomacromolecules tend to assume numerous structures in solution or the gas phase. It has been possible to resolve disparate conformational families but not unique geometries within each, and drastic peak broadening has been the bane of protein analyses by chromatography, electrophoresis, and ion mobility spectrometry (IMS). The new differential or field asymmetric waveform IMS (FAIMS) approach using hydrogen-rich gases was recently found to separate conformers of a small protein ubiquitin with the same peak width and resolving power up to ∼400 as for peptides. The present work explores the reach of this approach for larger proteins, exemplified by cytochrome <i>c</i> and myoglobin. Resolution similar to that for ubiquitin was largely achieved with longer separations, while the onset of peak broadening and coalescence with shorter separations suggests the limitation of the present technique to proteins under ∼20 kDa. This capability may enable one to distinguish whole proteins with differing residue sequences or localizations of post-translational modifications. Small features at negative compensation voltages that markedly grow from cytochrome <i>c</i> to myoglobin indicate the dipole alignment of rare conformers in accord with theory, further supporting the concept of pendular macroions in FAIMS

Isomer-resolved ion spectroscopy

We demonstrate the isomer-resolved spectroscopy of gas-phase ions, with different geometries separated prior to spectroscopic probe using ion mobility techniques. Specifically, ring and chain isomers of carbon cluster anions with 10̄ 12 atoms have been separated by ion mobility/mass spectrometry and examined by photoelectron spectroscopy. This methodology should also apply to other ion spectroscopies, including IR photodissociation