🌈Spectroscopy Unit 13 – Mass Spectra: Interpretation and Applications

Fundamentals of Mass Spectrometry

• Analytical technique measures the mass-to-charge ratio (m/z) of ionized molecules or fragments
• Consists of three main components: ionization source, mass analyzer, and detector
• Ionization source converts sample molecules into gas-phase ions
• Mass analyzer separates ions based on their m/z values using electric or magnetic fields
• Detector records the abundance of each ion and generates a mass spectrum
• Mass spectrum plots the relative abundance of ions against their m/z values
• Provides information about the molecular mass, elemental composition, and structure of the analyte
• Requires a high vacuum environment to minimize ion-molecule collisions and maintain ion trajectory

Ionization Techniques

• Electron Ionization (EI) is a hard ionization technique that uses high-energy electrons to ionize molecules
  • Operates at 70 eV to ensure reproducibility and comparability of mass spectra
  • Causes extensive fragmentation, providing structural information but may not preserve molecular ion
• Chemical Ionization (CI) is a soft ionization technique that uses a reagent gas (methane, ammonia) to ionize molecules
  • Produces protonated or deprotonated molecules with minimal fragmentation
  • Useful for determining molecular mass and analyzing thermally labile compounds
• Electrospray Ionization (ESI) is a soft ionization technique for liquid samples
  • Applies high voltage to create charged droplets that evaporate, leaving multiply charged ions
  • Suitable for large, polar molecules (proteins, peptides, nucleic acids)
• Matrix-Assisted Laser Desorption/Ionization (MALDI) is a soft ionization technique for solid samples
  • Uses a laser to desorb and ionize analytes co-crystallized with a matrix
  • Generates singly charged ions with minimal fragmentation
  • Applicable to large biomolecules (proteins, polymers, oligonucleotides)

Mass Analyzers and Detectors

• Quadrupole mass analyzer uses oscillating electric fields to selectively stabilize or destabilize ion trajectories based on their m/z
  • Consists of four parallel rods with alternating DC and RF voltages
  • Acts as a mass filter, allowing only ions with a specific m/z to pass through at a given voltage ratio
• Time-of-Flight (TOF) mass analyzer separates ions based on their velocities in a field-free drift tube
  • Ions with the same kinetic energy but different masses travel at different velocities
  • Lighter ions reach the detector faster than heavier ions
  • Provides high mass accuracy and resolution
• Ion Trap mass analyzer confines ions in a three-dimensional electric field
  • Ions are trapped, selectively ejected, and detected based on their m/z values
  • Allows for multiple stages of mass spectrometry (MS/MS) for structural elucidation
• Fourier Transform Ion Cyclotron Resonance (FT-ICR) mass analyzer traps ions in a strong magnetic field
  • Ions circulate at frequencies inversely proportional to their m/z values
  • Ion frequencies are measured and converted to m/z using Fourier transform
  • Offers ultra-high resolution and mass accuracy
• Electron multiplier detects ions by amplifying the signal generated when ions strike a dynode surface
  • Produces a cascade of secondary electrons, resulting in a measurable current
  • Provides high sensitivity and fast response time

Interpreting Mass Spectra

• Molecular ion peak (M+) represents the intact ionized molecule and provides the molecular mass
  • May be absent in hard ionization techniques due to extensive fragmentation
  • Isotope peaks (M+1, M+2) can help confirm the molecular ion and elemental composition
• Base peak is the most intense peak in the mass spectrum, assigned a relative abundance of 100%
  • Other peaks are reported as a percentage of the base peak intensity
  • Not always the molecular ion peak, especially in EI mass spectra
• Fragment ions result from the dissociation of the molecular ion or other precursor ions
  • Provide structural information based on the mass differences between fragments
  • Common fragmentations include the loss of neutral molecules (H2O, CO, NH3) and bond cleavages
• Isotope distribution reflects the natural abundance of isotopes in the sample
  • Helps determine the elemental composition and confirm the molecular formula
  • Characteristic patterns for elements with multiple stable isotopes (Cl, Br, S)
• Nitrogen rule states that odd-electron ions (M+) have an odd nominal mass for molecules containing an odd number of nitrogen atoms
  • Helps distinguish between molecules with different nitrogen content
  • Exceptions include molecules with an odd number of nitrogen atoms and an even number of charges

Fragmentation Patterns and Rules

• Even-electron rule states that even-electron ions (protonated or deprotonated molecules) preferentially fragment to form even-electron ions
  • Odd-electron ions (radical cations) tend to form odd-electron fragment ions
  • Helps predict the type of fragments observed in different ionization techniques
• Alpha-cleavage occurs at the bond adjacent to a heteroatom (N, O, S) or unsaturated system
  • Produces stable even-electron ions and neutral fragments
  • Common in EI and CI mass spectra
• McLafferty rearrangement is a specific type of beta-cleavage that involves the transfer of a hydrogen atom through a six-membered transition state
  • Occurs in molecules with a carbonyl group and a gamma-hydrogen
  • Results in the formation of an unsaturated neutral fragment and an even-electron ion
• Retro-Diels-Alder (RDA) fragmentation occurs in molecules containing a cyclohexene ring with a double bond
  • Involves the concerted cleavage of two sigma bonds, forming two unsaturated fragments
  • Helps identify the presence of certain structural motifs
• Ortho effect describes the enhanced fragmentation adjacent to an ortho-substituted aromatic ring
  • Stabilization of the resulting fragment ion through resonance or hydrogen bonding
  • Aids in the identification of ortho-substituted aromatic compounds

Isotope Patterns and Molecular Formula Determination

• Isotope patterns arise from the natural abundance of stable isotopes in the sample
  • Most common isotope patterns involve carbon (13C), chlorine (35Cl, 37Cl), bromine (79Br, 81Br), and sulfur (32S, 33S, 34S, 36S)
  • Relative intensities of isotope peaks depend on the number of atoms of each element in the molecule
• Carbon isotope pattern shows a small M+1 peak due to the presence of 13C (1.1% natural abundance)
  • Intensity of the M+1 peak relative to the monoisotopic peak (M) increases with the number of carbon atoms
  • Helps determine the number of carbon atoms in the molecule
• Chlorine and bromine isotope patterns are distinct due to the presence of two stable isotopes with similar abundances
  • Chlorine: 35Cl (75.8%), 37Cl (24.2%); Bromine: 79Br (50.7%), 81Br (49.3%)
  • Molecules containing one chlorine or bromine atom show a characteristic M+2 peak with a specific intensity ratio
  • Multiple chlorine or bromine atoms result in more complex isotope patterns
• Sulfur isotope pattern is less pronounced but can help confirm the presence of sulfur in the molecule
  • Main isotopes: 32S (95.0%), 33S (0.8%), 34S (4.2%), 36S (0.02%)
  • Presence of a small M+2 peak suggests the presence of sulfur
• Molecular formula determination involves comparing the observed isotope pattern with theoretical patterns
  • Generate possible molecular formulas based on the nominal mass and ring/double bond equivalents (RDBE)
  • Calculate the theoretical isotope pattern for each candidate formula
  • Compare the theoretical and observed patterns to find the best match
  • High-resolution mass spectrometry provides accurate mass measurements, narrowing down the possible formulas

Advanced MS Techniques (MS/MS, High-Resolution MS)

• Tandem mass spectrometry (MS/MS) involves multiple stages of mass analysis with an intermediate fragmentation step
  • Precursor ion is selected in the first mass analyzer, fragmented, and the resulting product ions are analyzed in the second mass analyzer
  • Provides detailed structural information and helps identify specific compounds in complex mixtures
• Collision-Induced Dissociation (CID) is a common fragmentation technique used in MS/MS
  • Selected precursor ions collide with neutral gas molecules (He, N2, Ar), causing fragmentation
  • Collision energy can be varied to control the extent of fragmentation
• High-resolution mass spectrometry (HRMS) offers high mass accuracy and resolving power
  • Time-of-Flight (TOF) and Fourier Transform Ion Cyclotron Resonance (FT-ICR) are common HRMS analyzers
  • Accurate mass measurements help determine the elemental composition and distinguish between isobaric compounds
• Orbitrap is a high-resolution mass analyzer that traps ions in an electrostatic field
  • Ions oscillate around a central electrode with frequencies proportional to their m/z values
  • Fourier transform is used to convert the oscillation frequencies to m/z
  • Provides high mass accuracy and resolving power, comparable to FT-ICR
• Data-dependent acquisition (DDA) automatically selects precursor ions for MS/MS based on predefined criteria
  • Typically selects the most intense ions in each full-scan mass spectrum
  • Helps identify unknown compounds in complex samples without prior knowledge of their presence
• Data-independent acquisition (DIA) simultaneously fragments all ions within a given m/z range
  • Provides comprehensive fragmentation data for all analytes in the sample
  • Requires advanced data processing algorithms to deconvolute the complex spectra and identify individual compounds

Applications in Various Fields

• Proteomics: MS is used to identify and quantify proteins in biological samples
  • Bottom-up approach involves digesting proteins into peptides, which are then analyzed by MS/MS
  • Top-down approach analyzes intact proteins, providing information on post-translational modifications and isoforms
• Metabolomics: MS helps identify and quantify small-molecule metabolites in biological systems
  • Untargeted metabolomics aims to comprehensively profile all detectable metabolites in a sample
  • Targeted metabolomics focuses on specific metabolites of interest, often using isotope-labeled standards for quantification
• Drug discovery and development: MS is used in various stages, from compound screening to pharmacokinetic studies
  • High-throughput screening (HTS) identifies active compounds from large libraries using MS-based assays
  • Pharmacokinetic studies monitor drug absorption, distribution, metabolism, and excretion using MS
• Environmental analysis: MS detects and quantifies pollutants, pesticides, and other contaminants in environmental samples
  • Gas chromatography-mass spectrometry (GC-MS) is commonly used for volatile and semi-volatile compounds
  • Liquid chromatography-mass spectrometry (LC-MS) is suitable for non-volatile and thermally labile compounds
• Forensic science: MS aids in the identification of drugs, explosives, and other substances in forensic investigations
  • Helps determine the presence and concentration of illicit drugs in biological samples
  • Identifies trace evidence such as fibers, paint chips, and gunshot residue
• Food safety and quality control: MS monitors food contaminants, adulterants, and natural toxins
  • Detects pesticide residues, mycotoxins, and other harmful substances in food products
  • Verifies the authenticity and origin of food ingredients using isotope ratio mass spectrometry (IRMS)
• Petroleum and biofuel analysis: MS characterizes the composition of crude oil, refined products, and biofuels
  • Helps optimize refining processes and ensure product quality
  • Monitors the production and quality of biofuels derived from renewable sources