These techniques include, but are not limited to, gas and liquid chromatography, capillary electrophoresis, and ion mobility spectrometry. These advances will push the field of ion mobility to the forefront of the mass spectrometry community, increasing the peak performance of existing workflows and creating additional confidence in building collision cross section libraries for untargeted analysis.
Introduction
Regardless of the chosen analytical approach to chiral selectivity, the general mechanics of chiral separations are thought to be related at a molecular level to the so-called "three-point model" known as Pirkle's Rule (see Figure 1.2 A).13,14 To distinguish chiral molecules in a chromatographic system, both isomers must interact with the chiral selector in such a way that one analyte interacts more strongly with the chiral selector than the other and is thus more retained. This preferred retention mechanism is based on the stereochemistry at the chiral center and subsequent neighboring atoms, which interact more strongly with the chiral selector in one enantiomeric form than the other (for example by hydrogen bonding), leading to enantioselectivity in chiral separations.
Chromatography and Mass Spectrometry
Liquid Chromatography
Briefly, several chiral selectivity mechanisms have been proposed for LC chiral separation, including dipole–dipole interactions, π–π stacking, hydrogen bonding, inclusion complexation, and steric hindrance (see Figure 1.3 A).33 Many modern CSPs use a combination of these strategies to improve chiral recognition and selectivity. While uncovering the intricacies of the splitting interaction in chiral LC is important from a fundamental standpoint, for an in-depth explanation of the chemistry behind CSP selectivity, the reader is directed to several comprehensive literature reviews that have been devoted to this topic.30,34.
Electrophoresis-Mass Spectrometry
Isomer Separations by IM-MS
The current hypothesis for this IM separation is that the change in chirality at Arg 8 causes a large structural shift in the conformation between the tail and ring systems of the molecule, thereby facilitating the separation of both isomers into a two-component mixture (see Figure 1 ,7B). However, because the addition of a chiral modifier inherently changes the composition of the drift gas (an integral part of the Mason-Schamp relationship), the collision cross sections measured in these experiments will be specific to the exact gas composition used, and thus the Adding modifiers loses structural context and makes cross-platform comparisons difficult.
Conclusions
Therefore, the push for higher resolution IM instruments with separation capabilities that rival traditional chromatographic approaches is a principle goal for future instrument design, and these new technologies may be more suitable for chiral separations of IM.
Acknowledgements
Groessl, M.; Graf, S.; Knochenmuss, R., High-resolution ion mobility-mass spectrometry for the separation and identification of isomeric lipids. ISOMERIC AND CONFORMATIONAL ANALYSIS OF SMALL AND SIMILAR DRUG MOLECULES BY ION MOBILITY-MASS SPECTROMETRY (IM-MS).
Introduction
Isomers
For example, rotamers are small molecule conformers where multiple three-dimensional molecular structures can form as a result of rotation around a single bond. Over the past 15 years, IM-MS has made major contributions to the analysis of constitutional and conformational isomers.22,23,52 Due to the commercialization and rapid adoption of IM-MS instruments24,33,37, the Web of Science database cites more than 3,500 articles over the past decade.
Instrumentation and Theory
Drift Tube Ion Mobility
As ions are introduced into the drift tube, they are drawn through the drift region as a result of an applied electric field along the ring electrodes. Mathematically, the CCS of the analyte can be calculated using the Mason-Schamp equation36,37, where K0 is the measured mobility of the ion, z is the charge of the ion, T is the temperature of the propellant gas, and N0 is the number density of the propellant gas at standard temperature and pressure.
Traveling Wave Ion Mobility
This mechanism is almost identical to that experienced in DTIMS, but with the exception that larger ions are slower in TWIMS as a result of "dropping". The drift times are converted to collision cross sections through a calibration procedure which takes into account the drift times and CCS of known internal standards.40,41 The ion mobility spectra obtained by TWIMS and DTIMS are qualitatively similar.
Current Work in Isomer Structural Separations
Separation of Constitutional Isomers
To illustrate the usefulness of the above equation, consider two interesting isomers that possess cross-sectional area differences of 1.0% (e.g., 200 Å2 and 202 Å2). In this way, the study by Dodds and colleagues can predict how efficiently two isomers of interest will separate on a specific instrument platform, provided that the CCS of each analyte is previously known and the resolving power of the ion mobility instrument is well characterized.
Separation of Conformational Isomers
A recent example of the applicability of IM to study carbohydrates was performed by Li and co-workers.53 In their study of sugars, TWIMS was used to measure the drift time profiles of ions of the monosaccharide glycolaldehydes and disaccharides of various simple sugars. While this work is another example of the ability of ion mobility to separate constitutional isomers, there were occasions when the presence of multiple conformations appeared even for what was believed to be a single analyte.
Materials
In the following sections, we provide the materials and methods needed to obtain an IM-MS spectrum for small molecules (here S-thalidomide, 258 Da) using a commercially available IM-MS platform (Agilent IM-MS 6550 ). The flow rate was as described in the methods section to follow. d) IM-MS data acquisition was obtained using Agilent Mass Hunter Acquisition Software (v.7.00).
Methods
Preparing the Instrument
Source temperatures and voltages will be preset for the Agilent Tune mixture (Figure B.1), and the ion polarity and scan mode will be selected as a function of the molecules of interest for each experiment (low mass mode (50–250 m /z), conventional mode (50–1700 m/z) or high mass m/z range). If not, the user may want to repeat the automatic setup process. and "Apply". g) Once the instrument is set to the desired ion mode, switch to acquisition mode under the context menu [Figure B.1 (A)] to start data collection.
Data Acquisition
Once sample reaches the instrument, analyte ion peaks begin to appear in the mass window. In the sample run mode, name the file [Figure B.2 (D)] and select the path directory [Figure B.2 (E)] for your file. e) To obtain data, click the forward arrow [Figure B.2 (F)] in the sample run screen.
Data Workup
In sample run mode, name the file [Figure B.2 (D)] and select the path directory [Figure B.2 (E)] for your file. e). To get data, click the forward arrow [Figure B.2 (F)] on the sample run screen. all ions in the sample.
Acknowledgements
The views expressed in this document are solely those of the authors and do not necessarily reflect the views of the Agency. Furthermore, the content is solely the responsibility of the authors and does not necessarily represent the official views of the granting agencies and organizations.
A commercial uniform field ion mobility mass spectrometer (6560, Agilent Technologies, Santa Clara, CA, USA) was used to acquire high-resolution mass spectrometry and ion mobility data (nominally 15,000 and 60, respectively).
Assessing Ion Mobility Resolving Power Theory for Broadscale Mobility Analysis with a
Introduction
The single peak resolving power can be used to directly compare the achievable resolution of different IMS instruments using the same ion mobility technique. The theoretical drift time of an ion in a uniform electric field is described by a rearrangement of the ion mobility proportionality equation:15.
Experimental
- Uniform Field IM-MS Instrument
- Analytical Precision
The instrumentation used in this study is a commercial IM-MS (6560 Ion Mobility-QTOF, Agilent) and has been previously described in detail.22 A conceptual diagram showing the main components of the instrument is included in Figure 3.1. The instrumentation used in this work is considered a “high precision” ion mobility instrument because it is capable of obtaining reproducible measurements of the mobility drift time to better than 0.1 ms.
Data Acquisition and Analysis
- Semi-Empirical Fitting Procedure
The α coefficient is the slope of the best-fit line to the data plotted as Ttd2/V (the diffusion parameter) versus Δt2 for a range of ions. The β coefficient is determined from the slope of the best-fit line to the data plotted as tg2 versus Δt2.
Results and Discussion
- Factors affecting semi-empirical coefficients
- Empirical Correlation of Theoretical Resolving Power
- Broadscale Validity of Semi-Empirical Results
Empirical resolving power curves (data points) compared to theory (solid lines) as a function of the ion mobility cutoff field (drift field) for three molecular ions (nominal m/z 322, 622, and 922). The semi-empirical resolution power (RSE) from equation (7) shows a more quantitative correlation than the conditional resolution power for all gate widths examined in this study and 500 μs).
Conclusions
A plot of the resolving power required to separate two compounds for a given percent difference in CCS for uniform field ion mobility.
Acknowledgments
Investigation of the Complete Suite of the Leucine and Isoleucine Isomers: Towards
Introduction
Ion mobility mass spectrometry (IM-MS) has recently gained interest as a rapid separation technique that can be applied to the separation and characterization of isomers. separation of distinct isomeric classes, IM-MS operates on a time scale that is several orders of magnitude faster and can be used in conjunction with condensed phase separations and sequential MS/MS techniques.21,22 IM-MS is a particularly useful analytical combination in that mass spectrometry separates molecules based on their intrinsic mass, while ion mobility provides a complementary separation based on molecular size and shape based on the gas phase collision cross section (CCS). Although nonlinear field IM-MS techniques (eg, FAIMS) have been shown to improve the separation of these types of isomers, drift tube ion mobility spectrometry (DTIMS) was specifically chosen for this work, as this uniform technique of ground-based facilitates quantity. comparison of separations through empirical measurement of CCS.
Experimental Methods
- Preparation of Standards
- Experimental Parameters
- Collision Cross Section Measurements
Details of the instrument have been previously described.34,35 Briefly, the instrument consists of a 78.1 cm uniform field drift tube coupled to a tandem quadrupole time-of-flight (QTOF) mass spectrometer. The drift field also strongly affects instrument resolution, and separations were performed at drift voltages that maximize resolution in the mobility and mass range of the analytes, corresponding to 14.7 V/cm (see Figure D.2, Supporting Information).
Results and Discussion
- Isomer Classifications and Separations
- Peak Shape Modeling and Ion Mobility
- Resolving Power and Separations
Predicted two peak resolution is within an approx. 5% error from the experimental solution of the two-component mixture. For half-height separations, a resolving power of 200 is sufficient to separate 74% of isomer pairwise matches.
Conclusions
The correlation between CCS and resolution developed in this and ongoing work is expected to benefit the development of computational approaches that can predict the dissociation of any two compounds with known CCS, given that the resolving power of the ion mobility instrument used is well characterized . This in turn will allow for the prediction of ion mobility separation behavior as long as a high-precision experimental measurement of CCS (or other transport property) exists.
Acknowledgements
However, larger molecular systems will also possess significantly more possible isomeric forms than the relatively small system investigated in this current work22 and may exist as conformers that are expected to place greater demands on the required ion mobility solvation capacity. The broad-scale applicability of equation 4 and Figure 4.4 to other ion mobility techniques (eg FAIMS/DMS, TWIMS and TIMS) is currently being investigated.
Separation of leucine and isoleucine by electrospray ionization - asymmetric waveform high field ion mobility spectrometry - mass spectrometry.
Correlating Resolving Power, Resolution, and Collision Cross Section: Unifying Cross-
Introduction
In ion mobility and mass spectrometry, the resolving power (Rp) is quantitatively defined from a single peak as a ratio of the location of the peak divided by its width (eq. 1).17. For a mobility separation of two analytes, Rp should be calculated as the average resolving power of both peaks, while the percentage difference in cross section (ΔCCS%) is based on the average CCS, as defined in equation 4.
Experimental Methods
- Chemical Standards
- Instrumentation and Methods
- Selection of Published Spectra
- Evaluation of Separation Efficiency
It is also important to note that when resolving power is measured as CCS/ΔCCS, ΔCCS is the FWHM of a given IMS peak and is not related to the percentage difference in cross sections of two peaks (ΔCCS%). Using the average resolving power and the calculated percent difference in CCS, the predicted resolution (Rpp) is subsequently calculated via Equation 3.
Results and Discussion
- Gaussian Distributions
- CCS-Based Resolving Power
- Cross-Platform Assessment
- Traveling Wave Resolving Power
- Cross-Platform Assessment of Separation Capabilities
- How Much Resolving Power is Necessary
- Mass Analysis
- Ion Mobility Analysis
- Ion Mobility-Mass Spectrometry
The plot in Figure 2 depicts boundary regions representing different levels of separation efficiency calculated through Equation 3, covering a wide range of percent difference in CCS and resolving power. Plot showing the resolving power required to separate two compounds in ionic mobility with a known percentage difference in cross section.
Conclusions
However, the primary features (II and III) have almost identical CCS values and cannot be resolved by ion mobility alone, whereas the mass spectrometer separates these features as two distinct peaks. Nevertheless, routine ion mobility resolution powers in excess of 300 now being demonstrated will be essential to address chemical separations in highly demanding studies such as synthetic biology, medicine, and omics.
Acknowledgements
Ion mobility experiments therefore provide the greatest analytical benefits when combined with mass spectrometry, as well as other analytical dimensions (eg LC-IM-MS), which together act to extend the analytical selectivity of the chemical separation. Furthermore, the content is solely the responsibility of the authors and does not necessarily represent the official views of the granting agencies and organizations.
Investigation of the full range of leucine and isoleucine isomers: towards prediction of ion mobility separation capacities. Anal. Improving the separation of isomers in ion mobility mass spectrometry. Quantitative Chemical Biology Conference, Nashville, TN (Fall 2016) POSTER.
Supplementary Materials for Chapter II
Supplementary Materials for Chapter III
Supplementary Materials for Chapter IV
Supplementary Materials for Chapter V
Summary of Results from Semi-Empirical Fitting
Predicted Resolving Power for Leucine Isomer Separations
Various Ion Mobility Techniques and Dispersive Dimensions
Separation Parameters for Published Ion Mobility Separations
Increasing Structural Complexity for One Molecular Formula
Three Point Model for Chiral Separations
Mechanisms of Chiral Separations
Instrument Schematic for Agilent 6560
Timescale of Analytical Separations
Skeletal Structure and Separation of Leucine Isomers
Desmopressin Structure and Chiral IM-MS Separation
Structures of Acetaminophen Constitutional Isomers
Block Diagrams of Drift Tube and Traveling Wave Instrumentation
Skeletal Structure and Separation of Leucine Isomers
Complexity of Carbohydrate Isomers
Drift Time Profiles for Thalidomide Chiral Isomers
Instrument Schematic for Agilent 6560
Resolving Power Curves as a Function of Gate Width
Resolving Power as a Function of Reduced Mobility
Resolving Power Curves as a function of Drift Gas Composition
Isomer Classification Hierarchy
Skeletal Structure and Separation of Leucine Isomers
Theoretical Resolution as a Function of Resolving Power and Concentration
Plot of Increasing Separation as a Function of Resolving Power
Experimental and Theoretical Separations of Leucine Isomers
Selected Ion Mobility Separations from Literature Publications
Ion Mobility Separations as a Function of Resolving Power and CCS
Biological Separation of Peptides from Ion Mobility and Mass Spectrometry
