Mass Spectrometry: Electron Ionization Concepts

Analysis of Complex Chemical Systems

• This is a Master's degree curriculum in Chemistry, specifically focusing on Complex and Data-Driven Chemistry, offered at the University of Padua.

Non-targeted Analysis Workflow

• Define the aims of the analysis (the problem to be solved).
• Collect and store samples (quantity and characteristics).
• Perform analysis.
• Process data (compound identification, statistics).
• Validate findings.

Sample Pre-treatment

• Extraction
• Pre-concentration
• Protein precipitation
• Desalting
• Filtration

Analysis (Techniques)

• HPLC (High-Performance Liquid Chromatography)
• GC (Gas Chromatography)
• IC (Ion Chromatography)
• MS (Mass Spectrometry)
• HRMS (High-Resolution Mass Spectrometry)
• NMR (Nuclear Magnetic Resonance)

Data Analysis

• Data processing
• Compound identification
• Statistical analysis

Focus on MS (Mass Spectrometry)

• Incorrectly calling mass spectroscopy 'mass spectrometry'.
• Spectroscopy involves electromagnetic radiation absorption.
• Mass spectrometry involves separating ions by mass-to-charge ratio (m/z).

Basic Principle of MS

• Ions generate from inorganic/organic compounds.
• Ions separate based on their mass-to-charge ratio.
• Ions detect qualitatively and quantitatively by their m/z and abundance.

Mass Spectrum

• X-axis: m/z (atomic mass per number of elementary charges).
• Y-axis: Relative abundance.
• Molecular ion
• Base Peak
• Isotopic peaks
• Fragment peaks

Profile vs. Centroid

• Profile:
• Larger number of data points.
• More complete data.
• Useful for high-mass analytes and high-resolution MS analysis
• Centroid:
• Single point per peak.
• Data points represent the center of the peak.
• Useful for peak area interpretation.
• Better suitable for data visualization.

Ionization

• ESI/nano-ESI/MALDI: Ion source methods
• APCI another ionization source method
• GC-MS: Gas Chromatography-Mass Spectrometry ionization source
• LC-MS: Liquid Chromatography-Mass Spectrometry ionization source
• Importance of analyte polarity for ionization methods
• Necessary derivatization for polar or low-volatility compounds.

Gas Phase Ionization by Electrons (EI)

• Analytes are in a gas phase.
• Electrons accelerate to gain kinetic energy.
• Even-electron ions produced.
• Negative ions can also be produced under certain conditions.

EI - Produced Ions

• Excitation
• Ionization
• Dissociative ionization
• Dissociative rearrangement
• Electron capture
• Dissociative electron capture
• Ion-pair formation

EI - Ionization Energies

• Charge is delocalized.
• Molecular ions are represented as localized at the lowest energy ionization points.
• Electrons are easily removed from the molecule.

EI - Efficiency and Fragmentation

• Low ionization yield (10⁻⁴ to 10⁻³).
• Hard ionization.
• Information on the molecular structure of molecules.
• Fragmentation, and ionization, are reproducible.

EI - The EI Source

• Source is heated (250-300°C) for low memory effect.
• Ion volume: 1 mm.
• Ionization yield: 1:800 molecules

EI - Lower Electron Energies

• Loss of sensitivity.
• Higher "memory" effect.
• Increased probability to observe molecular ion.

Other Ion Sources

• Accelerator mass spectrometry (AMS).
• Electron ionization (EI).
• Chemical ionization (CI).
• Atmospheric pressure chemical ionization (APCI).
• Atmospheric pressure photoionization (APPI).
• Inductively coupled plasma (ICP).
• Field ionization (FI).
• Secondary ion mass spectrometry (SIMS).
• Laser desorption/ionization (LDI).
• Fast atom bombardment (FAB).
• Liquid secondary ion mass spectrometry (LSIMS).
• Field desorption (FD).
• Matrix-assisted laser desorption/ionization (MALDI).
• Electrospray ionization (ESI).

Ion Source vs. Chemical Properties

• Relationship between ionization source and compound properties.

From Solution to Gas Phase, HPLC-MS

• Oxidation and reduction processes.
• Electric field.
• Flow rate and spray voltage.

Electrospray

• Flow (10-200 µL/min).
• Sheath gas (1-20 L/min, nitrogen, heated).
• Compatibility with HPLC/UPLC flows.
• Spray needle (3-6 kV).
• Taylor cone
• Excess charge on the surface, solvent and neutralized ions.
• Metal plate (~100V).

ESI Geometries

• Robustness.
• Longer analytical sequences.
• Less maintenance.

Ions Formed by ESI

• Positive and Negative Ions
• Analyte polarity and resulting ions.
• Clusters, adducts, and exchange reactions in different polarities.

Lactose - Ions in ESI

• Data visualization of mass spectrum for Lactose.
• Identifying ions like [M+Na]⁺, [M+NH₄]⁺, and [M+H]⁺.

Summary for ESI

• Ionization occurs after application of electric field.
• Ionization aided by inert gas flow.
• MW information, low fragmentation.
• Requires acid-base and electrochemical properties.
• High matrix effect.
• Coupling with HPLC/capillary electrophoresis.

Mass Analyzer

• Physical property measured: Mass-to-charge ratio (m/z).
• Mass range limit.
• Analysis speed (scan speed).
• Transmission.
• Sensitivity.
• LOD, S/N
• Mass accuracy.
• Resolving power.

Mass Range Limit

• Highest singly charged ion mass with peak height significantly above the instrumental noise level

Scan Speed

• Rate at which the analyzer measures mass over a specified range, often measured in u/s.

Transmission Efficiency

• Fraction of ions successfully transmitted to the detector.

Sensitivity

• Overall response of analyzer for specific analyte under controlled conditions.
• Measured in C/µg.

Signal-to-Noise Ratio (S/N)

• Ratio of signal intensity to noise intensity.

Limit of Detection

• Lowest analyte concentration detectable with a given instrument.

Mass Resolution

• Smallest difference in m/z (m/z) that can be resolved by an instrument.
• Calculation relating resolving power to differences in mass values.

Resolving Power

• Ability of an instrument to separate neighboring peaks.
• Calculation with peak width at specific percentages of peak height (often 50%, also known as FWHM).

Mass Accuracy

• Difference between theoretical and measured m/z.
• Accuracy ensured by calibration (internal and/or external).
• Relationship between resolution and mass accuracy.

Mass Analyzers at a glance

• Analyzers tabulated by type, dynamic mass range and mass resolution.

Time of Flight (TOF)

• Ions of different m/z are dispersed in time during their flight.
• Principle is quite simple: ions are dispersed in time based on their mass-to-charge ratio.
• M/Z (m/z) range of a TOF analyzer is unlimited.
• TOF analyzers offer high ion transmission leading to high sensitivity.
• TOF mass spectral acquisition rate is high, essentially above 103 Hz.
• TOF instrument design is straightforward.
• Modern TOF instruments provide accurate mass measurement and tandem MS experiments.
• Calculation of time of flight based on the ion´s mass to charge ratio and electric potential.
• External mass calibration is needed.

Reflectron

• Acts as an ion mirror to focus ions with different kinetic energies.
• Increases mass resolution.
• Limitation affecting sensitivity and mass range.

Orthogonal TOF

• High sensitivity due to good duty cycle and high transmission.
• High rate of spectra per second.
• High mass resolution (e.g., >10K).
• Mass accuracies (e.g., up to 1ppm).
• Compact design.
• Coupling with continuous ion sources (ESI, EI).

Linear Quadrupole

• High transmission.
• Light-weight, compact, comparatively low-priced.
• Low ion acceleration voltages.
• High scan speeds.
• Mass scanning; U and V voltages varied in magnitude.
• a/q ratio remains constant.
• Scan speed consistent, e.g., 1000 u/s.

Linear Quadrupole- Mass Scanning

• U and V voltages varied to scan through different masses.
• Constant a/q ratio.
• Scan speed is uniform (e.g., 1000 u/s).
• Relationship between mass, m/z, U and V voltages, and stability regions for a given a/q ratio.

Quadrupole Mass Resolution (mass resolving power)

• Quadrupoles generally operated with mass resolution which increases linearly with increasing mass (constant width).
• Variable transmission efficiency.
• Relationship between m/z and DC/RF voltage settings, and their influence on transmission efficiency.

Ion Guide

• Transports ions efficiently and simultaneously.
• Good transmission efficiency across a large mass range and energy conservation.
• Minimizes m/z dependence related to transmission efficiency.
• For instance, the U=0 RF-only mode, where q=0.908 determines minimum transmitted m/z.
• High mass ions are poorly focused and RF voltage V can be modulated to focus different m/z ranges.

Ion Guide (Types)

• Quadrupole
• Hexapole
• Octapole
• Transmission efficiency and mass range for simultaneous transmission of ions

Orbitrap

• Electric field from the complex shape of the Orbitrap electrodes = quadrologarithmic field
• Ions combine rotation around the central axis and axial oscillations
• Frequency depends on charge q, mass m and curvature of the field
• Accurate measurement of m/z values by image current detection and fast Fourier transform
• Important parameters for orbitrap mass resolution and their relationship to experimental settings

Hybridisation

• Diagram of a combined mass spectrometer
• The Agilent 6540 Q-TOF supports the Agilent Jet Stream technology
• Components such as Octopole, Quad Mass Filter (Q1), Lens 1 and 2, Single 3-stage Turbo Pump, Collision Cell, and Ion Beam Shaper, Ion Mirror, Ion Pulser and Detector.