Ultra-Fast Ion Mobility Spectrometer for High-Throughput Chromatography.

Fast chromatography systems especially developed for high sample throughput applications require sensitive detectors with a high repetition rate. These high throughput techniques, including various chip-based microfluidic designs, often benefit from detectors providing subsequent separation in another dimension, such as mass spectrometry or ion mobility spectrometry (IMS), giving additional information about the analytes or monitoring reaction kinetics. However, subsequent separation is required at a high repetition rate. Here, we therefore present an ultra-fast drift tube IMS operating at ambient pressure. Short drift times while maintaining high resolving power are reached by several key instrumental design features: short length of the drift tube, resistor network of the drift tube, tristate ion shutter, and improved data acquisition electronics. With these design improvements, even slow ions with a reduced mobility of just 0.94 cm2/(V s) have a drift time below 1.6 ms. Such short drift times allow for a significantly increased repetition rate of 600 Hz compared with previously reported values. To further reduce drift times and thus increase the repetition rate, helium can be used as the drift gas, which allows repetition rates of up to 2 kHz. Finally, these significant improvements enable IMS to be used as a detector following ultra-fast separation including chip-based chromatographic systems or droplet microfluidic applications requiring high repetition rates.

Permethylation and Metal Adduction: A Toolbox for the Improved Characterization of Glycolipids with Cyclic Ion Mobility Separations Coupled to Mass Spectrometry.

Lipids are an important class of molecules involved in various biological functions but remain difficult to characterize through mass-spectrometry-based methods because of their many possible isomers. Glycolipids, specifically, play important roles in cell signaling but display an even greater level of isomeric heterogeneity as compared to other lipid classes stemming from the introduction of a carbohydrate and its corresponding linkage position and α/ß anomericity at the headgroup. While liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) remains the gold standard technique in lipidomics, it is still unable to characterize all isomeric species, thus presenting the need for new, orthogonal, methodologies. Ion mobility spectrometry-mass spectrometry (IMS-MS) can provide an additional dimension of information that supplements LC-MS/MS workflows, but has seen little use for glycolipid analyses. Herein, we present an analytical toolbox that enables the characterization of various glycolipid isomer sets using high-resolution cyclic ion mobility separations coupled with mass spectrometry (cIMS-MS). Specifically, we utilized a combination of both permethylation and metal adduction to fully resolve isomeric sphingolipids and ceramides with our cIMS-MS platform. We also introduce a new metric that can enable comparing peak-to-peak resolution across varying cIMS-MS pathlengths. Overall, we envision that our presented methodologies are highly amenable to existing LC-MS/MS-based workflows and can also have broad utility toward other omics-based analyses.

The dependence of reduced mobility, ion-neutral collisional cross sections, and alpha values on reduced electric field strengths in ion mobility.

As ion mobility spectrometry (IMS) is used with mass spectrometry in more applications, increased emphasis is placed on the ion-neutral collisional cross sections (CCS) to identify unknown analytes in complex matrices. While CCS values can provide useful information about relative analyte size, several critical assumptions are inherent in the most common method of calculating CCS values, the Mason-Schamp equation. The largest source of error in the Mason-Schamp equation originates from not accounting for higher reduced electric field strengths, which are present in low-pressure instruments that require calibration. Previous corrections based on field strength have been proposed in literature, but their data used atomic ions in atomic gases, whereas most applications examine molecules measured in nitrogen. Here, we use a series of halogenated anilines measured in air and nitrogen between 6-120 Td on a first principles ion mobility instrument (HiKE-IMS). With this series of measurements, the average velocity of the ion packet is known allowing for direct calculation of reduced mobilities (K0), alpha functions, and finally, a detailed examination of CCS as a function of E/N. In the worst-case scenario, there is over a 55% difference in CCS values for molecular ions measured at high fields depending on the method used. When comparing CCS values to those in a database for unknown identification, this difference can lead to misidentification. To immediately alleviate some of the error in calibration procedures, we propose an alternative method using K0 and alpha functions that simulate first principles mobilities at higher fields.

Implementation of an Open-Source Multiplexing Ion Gate Control for High Kinetic Energy Ion Mobility Spectrometry (HiKE-IMS).

With ion mobility spectrometry increasingly used in mass spectrometry to enhance separation by increasing orthogonality, low ion throughput is a challenge for the drift-tube ion mobility experiment. The High Kinetic Energy Ion Mobility Spectrometer (HiKE-IMS) is no exception and routinely uses duty cycles of less than 0.1%. Multiplexing techniques such as Fourier transform and Hadamard transform represent two of the most common approaches used in the literature to improve ion throughput for the IMS experiment; these techniques promise increased duty cycles of up to 50% and an increased signal-to-noise ratio (SNR). With no instrument modifications required, we present the implementation of Hadamard Transform on the HiKE-IMS using a low cost, high-speed (600 MHz), open source microcontroller, a Teensy 4.1. Compared to signal average mode, 7- to 10-bit pseudorandom binary sequences resulted in increased analyte signal by over a factor of 3. However, the maximum SNR gain of 10 did not approach the theoretical 2n-1 gain largely due to capacitive coupling of the ion gate modulation with the Faraday plate used as a detector. Even when utilizing an inverse Hadamard technique, capacitive coupling was not completely eliminated. Regardless, the benefits of multiplexing IMS coupled to mass spectrometers are well documented throughout literature, and this first effort serves as a proof of concept for multiplexing HiKE-IMS. Finally, the highly flexible Teensy used in this effort can be used to multiplex other devices or can be used for Fourier transform instead of Hadamard transform.

A Comparison of the Performance of Modular Standalone Do-It-Yourself Ion Mobility Spectrometry Systems.

As the spectrum of ion mobility spectrometry (IMS) applications expands and more experimental configurations are developed, identifying the correct platform for an experimental campaign becomes more challenging for researchers. Additionally, metrics that compare performance (Rp, for example) often have nuanced differences in definition between platforms that render direct comparisons difficult. Here we present a comparison of three do-it-yourself (DIY) drift tubes that are relatively low cost and easy to construct, where the performance of each is evaluated based on three different metrics: resolving power, the ideality of resolving powers, and accuracy/precision of K0 values. The standard PCBIMS design developed by Reinecke and Clowers (Reinecke, T.; Clowers, B. H. HardwareX 2018, 4, e00030) provided the highest resolving power (>90) and the highest ideality of resolving power ratios (>90% at best) of the three systems. However, the flexible tube (FlexIMS) construction as described by Smith et al. (Smith, B. L. Anal. Chem. 2020, 92 (13), 9104-9112) exhibited the highest degree of precision of K0 values (relative standard deviation of <0.42%). Depending on the application, the drift tube variants presented and evaluated here offer a low-cost alternative to commercial drift-tube systems with levels of performance that approach theoretical maxima.

The origin of isomerization of aniline revealed by high kinetic energy ion mobility spectrometry (HiKE-IMS).

Although aniline is a relatively simple small molecule, the origin of its two peaks observed in ion mobility spectrometry (IMS) has remained under debate for at least 30 years. First hypothesized as a difference in protonation site (amine vs. benzene ring), each ion mobility peak differs by one Dalton when coupled with mass spectrometry where the faster mobility peak is the molecular ion peak, and the slower mobility peak is protonated. To complicate the deconvolution of structures, some previous literature shows the peaks as unresolved and thus proposes these species exist in equilibrium. In this work, we show that when measured with high kinetic energy ion mobility spectrometry (HiKE-IMS), the two peaks observed in spectra of both aniline and all n-fluoroanilines are fully separated (chromatographic resolution from 2-7, Rp > 110) and therefore not in equilibrium. The HiKE-IMS is capable of changing ionization conditions independently of drift region conditions, and our results agree with previous literature showing that ionization source settings (including possible fragmentation at this stage) are the only influence determining the speciation of the two aniline peaks. Finally, when the drift and reactant gas are changed to nitrogen, a third peak appears at high E/N for 2-fluoroaniline and 4-fluoroaniline for the first time in reported literature. As observed by HiKE-IMS-MS, the new third peak is also protonated showing that the para-protonated aniline and resulting fragment ion, molecular ion aniline, can be fully separated in the mobility domain for the first time. The appearance of the third peak is only possible due to the increased separation of the other two peaks within the HiKE-IMS.

Implications of Blanc's Law for Use in Trapped Ion Mobility Spectrometry.

Blanc's Law has served as a way to predict the mobilities of ions in mixed drift gases for over 100 years yet has remained largely unexplored using newer ion mobility spectrometry (IMS) configurations, including traveling wave and trapped IMS (TIMS) systems. Here, we evaluate a drift-tube IMS (DTIMS) and compare it to a similar set of experiments performed in TIMS. We found that Blanc's Law can be applied in a DTIMS to determine the mobility of an analyte in the minor gas component of a ternary mixed drift gas system within 2% error. Additionally, the calibration procedure for TIMS to convert elution voltages into a mobility value corrects for significant deviations (>4%) from Blanc's Law in the elution voltage domain. For the range of gas identities probed in this effort, up to an 11% error in calibrated mobilities was observed when using a gas mixture in the TIMS that differed from the gas used for the reference mobility. However, when the gas mixture within the TIMS was the same as the respective calibrant mobilities, calibration errors within the TIMS were as low as 0.01%. Interestingly, when probing the behavior of ions with argon-containing mixtures within the TIMS, the current accepted paradigm of elution voltage being proportional to inverse mobilities in TIMS calibrations procedures was shown to deviate substantially from the trends observed with DTIMS measurements. With this initial effort, foundations for future mixed drift gas measurements in TIMS are set for expanded analyte classes and larger molecules.

Reevaluating the Role of Polarizability in Ion Mobility Spectrometry.

With the expanding commercial availability of gas-phase separation systems that incorporate gas-phase mobility, there is a concurrent rise in efforts to cast the gas-phase mobility coefficient in terms of an ion-neutral collision cross-section (CCS). The motivating factors for this trend are varied, but many aim to complement experimental results with computationally generated CCS values from in silico structural approximations. Unfortunately, the current paradigm for relating experimental mobility results to computationally derived structures relies upon empirical approaches, including a myriad of variables that do not realistically bound the comparison. In this Critical Insight, we advocate for the development of a self-consistent experimental and computational framework that uses laboratory results to constrain the scope of the modeling effort. This paper aims to prompt discussion, challenge assumptions, and promote the development of more efficient, accurate computational techniques within the gas-phase ion measurement community. Specifically, we postulate whether experimental deviations from Langevin's polarization limit (Kpol) are suitable to estimate the relative contributions of hard-sphere collisions and long-range interactions within CCS values. Not surprisingly, different molecule classes exhibit different trends in the K/Kpol ratio when normalized for reduced mass, and the most common IMS calibrants (e.g., tune mix, polyalanine, tetraalkylammonium salts) follow different polarizability trends than many of the analytes probed in the literature. Succinctly, if gas-phase ion structure is largely invariant based upon the colliding neutral and newly developed experimental efforts can quantitatively capture ion polarizability, then modeling efforts describing a target analyte must be self-consistent as the collision neutral is changed in silico.

Evaluation of Trapped Ion Mobility Spectrometry Source Conditions Using Benzylammonium Thermometer Ions.

A key aspect of reduced pressure ion mobility spectrometry (IMS) experiments is to identify experimental conditions that minimize the role of collisional energy transfer that allows for assessing effective ion-neutral collision cross sections of metabolites, peptides, and proteins in "native-like" or compact states. Across two separate experimental campaigns using a prototype trapped ion mobility spectrometer (TIMS) coupled to a time-of-flight mass spectrometer, we present independent findings that support the results recently published by Morsa et al. using a different set of thermometer ions (Morsa et al. Anal. Chem. 2020, 92 (6), 4573-4582). First, using five para-substituted benzylammonium ions, we conducted survival yield experiments to assess ion internal energy across different experimental settings. Results from the present set of experiments illustrate that greater ion heating occurs at lower pressures and higher voltage settings applied to the TIMS. At the "softest" settings where the benzylammonium thermometer ions have an effective average energy of 1.73 eV, we observe the majority of bradykinin in the compact state. Under more extreme operating conditions where the energy of the benzylammonium ions varies from 1.83 to 1.86 eV, the bradykinin transitions from the compact to the elongated state. In addition to independently confirming the findings of Morsa et al., we also report the mobilities for the benzylammonium parent and fragment ions using the tandem drift-tube-TIMS calibration procedure described by Naylor et al. ( J. Am. Soc. Mass Spectrom. 2019, 30 (10), 2152-2162).

Assessing the Impact of Drift Gas Polarizability in Polyatomic Ion Mobility Experiments.

Due to the core assumptions of kinetic theory and the drive toward realizing reproducible gas-phase measurements, ion mobility experiments are commonly conducted in the presence of an inert, neat buffer gas, usually nitrogen or helium. Mixing drift gases in defined, static ratios can provide useful information not only for optimizing the separation of analytes but also for defining the interaction between the ion and neutral particle. In a foundational effort, we seek to validate the role of the drift gas polarizability on the observed mobility of the ions by systematically mixing drift gases to discretely access a range of bulk gas polarizabilities not given by pure drift gases. Compared to historical efforts to probe the role of polarizability on the ion-neutral collisional cross section where a linear relationship was assumed, the data collected in the present effort clearly illustrate a quadratic dependency of the ion-neutral particle collision cross section and polarizability (R2 > 0.999). When translating these data into the mobility dimension, we illustrate that the gas-phase mobility of polyatomic ions conforms to Blanc's law. These observations combined with considerations related to Langevin's polarization limit provide an experimental mechanism to estimate to what degree an ion-neutral interaction conforms to either the hard-sphere or induced-dipole model. To support these observations, additional comparisons are made with the respective reduced masses, polarizabilities, and mobilities of ions in mixtures where different degrees of hard-sphere interactions are present.

Validation of Calibration Parameters for Trapped Ion Mobility Spectrometry.

Using contemporary theory for ion mobility spectrometry (IMS), gas-phase ion mobilities within a trapped ion mobility-mass spectrometer (TIMS) are not easily deduced using first principle equations due to non-linear pressure changes and consequently variations in E/N. It is for this reason that prior literature values have traditionally been used for TIMS calibration. Additionally, given that verified mobility standards currently do not exist and the that the exact conditions used to measure reported literature values may not always represent the environment within the TIMS, a direct approach to validating the behavior of the TIMS system is warranted. A calibration procedure is presented where an ambient pressure, ambient temperature, two-gate, printed circuit board drift-tube IMS (PCBIMS) is coupled to the front of a TIMS allowing reduced mobilities to be directly measured on the same instrument as the TIMS. These measured mobilities were used to evaluate the TIMS calibration procedure which correlates reduced mobility and TIMS elution voltages with literature values. When using the measured PCBIMS-reduced mobilities of tetraalkyl ammonium salts and tune mix for TIMS calibration of the alkyltrimethyl ammonium salts, the percent error is less than 1% as compared with using the reported literature K0 values where the percent error approaches 5%. This method provides a way to obtain accurate reference mobilities for ion mobility techniques that require a calibration step (i.e., TIMS and TWAVE).