🌈Spectroscopy Unit 12 – Mass Spectrometry: Principles & Instruments

What's Mass Spectrometry?

• Analytical technique used to measure the mass-to-charge ratio (m/z) of ions
• Determines the molecular mass and elemental composition of a sample
• Provides structural information about molecules
• Can identify unknown compounds in a sample
• Highly sensitive technique detects trace amounts of analytes
• Widely used in various fields (chemistry, biochemistry, pharmacology, forensics)
• Requires the conversion of sample molecules into gaseous ions
• Separates ions based on their m/z ratios in the presence of electric or magnetic fields

Key Principles of Mass Spectrometry

• Ionization converts sample molecules into gaseous ions
  • Positive or negative ions formed depending on the ionization technique
• Ion separation occurs based on the mass-to-charge ratio (m/z)
  • Lighter ions with higher charge are deflected more than heavier ions with lower charge
• Detection of separated ions generates a mass spectrum
  • Plots relative abundance of ions against their m/z ratios
• Fragmentation of ions provides structural information
  • Specific bond cleavages produce characteristic fragment ions
• Isotopic distribution helps determine elemental composition
  • Relative abundances of isotopes create a unique pattern for each element
• High vacuum conditions are essential for efficient ion transport and analysis
• Calibration using known compounds ensures accurate mass measurements

Main Components of a Mass Spectrometer

• Inlet system introduces the sample into the mass spectrometer
  • Direct insertion probe, gas chromatograph, or liquid chromatograph
• Ionization source converts sample molecules into gaseous ions
  • Electron ionization (EI), chemical ionization (CI), electrospray ionization (ESI), matrix-assisted laser desorption/ionization (MALDI)
• Mass analyzer separates ions based on their m/z ratios
  • Quadrupole, time-of-flight (TOF), ion trap, Fourier transform ion cyclotron resonance (FT-ICR)
• Detector records the abundance of separated ions
  • Electron multiplier, Faraday cup, or photomultiplier
• Data system processes and displays the mass spectrum
  • Converts detector signal into a digital format and provides data analysis tools
• Vacuum system maintains high vacuum conditions throughout the instrument
  • Turbomolecular pumps and rotary pumps create a pressure gradient

Ionization Techniques

• Electron Ionization (EI) uses a beam of high-energy electrons to ionize molecules
  • Produces highly fragmented ions and reproducible mass spectra
  • Limited to volatile and thermally stable compounds
• Chemical Ionization (CI) uses a reagent gas to ionize molecules through ion-molecule reactions
  • Softer ionization technique produces less fragmentation than EI
  • Suitable for analyzing polar and thermally labile compounds
• Electrospray Ionization (ESI) generates ions from a liquid sample sprayed through a charged capillary
  • Ideal for large biomolecules (proteins, peptides, nucleic acids)
  • Can produce multiply charged ions, extending the mass range
• Matrix-Assisted Laser Desorption/Ionization (MALDI) uses a laser to desorb and ionize molecules from a solid matrix
  • Suitable for large, non-volatile, and thermally labile compounds
  • Commonly used for analyzing proteins, polymers, and oligonucleotides
• Atmospheric Pressure Chemical Ionization (APCI) uses a corona discharge to ionize molecules in a heated vaporizer
  • Applicable to less polar compounds than ESI
  • Useful for analyzing small to medium-sized molecules

Mass Analyzers: Types and How They Work

• Quadrupole mass analyzer uses oscillating electric fields to selectively stabilize or destabilize ion trajectories
  • Consists of four parallel rods with alternating DC and RF voltages
  • Allows ions with a specific m/z ratio to pass through to the detector
• Time-of-Flight (TOF) mass analyzer separates ions based on their velocities in a field-free drift region
  • Measures the time taken by ions to travel a fixed distance
  • Lighter ions reach the detector faster than heavier ions
• Ion Trap mass analyzer confines ions in a three-dimensional space using electric fields
  • Quadrupole ion trap (QIT) uses a ring electrode and two endcap electrodes
  • Linear ion trap (LIT) uses four parallel rods to trap ions radially
  • Ions are selectively ejected based on their m/z ratios by varying the electric fields
• Fourier Transform Ion Cyclotron Resonance (FT-ICR) mass analyzer traps ions in a strong magnetic field
  • Ions circulate at a frequency proportional to their m/z ratios
  • Excitation and detection of ion frequencies using electrodes
  • Fourier transform of the time-domain signal yields a mass spectrum
• Orbitrap mass analyzer traps ions in an electrostatic field between two electrodes
  • Ions orbit around a central spindle-shaped electrode
  • Axial oscillations of ions are detected and converted into a mass spectrum using Fourier transform

Detectors and Data Output

• Electron Multiplier detects ions by amplifying the signal through a series of dynodes
  • Incoming ions strike the first dynode, releasing secondary electrons
  • Cascade of electrons amplifies the signal at each subsequent dynode
• Faraday Cup directly measures the ion current by collecting ions in a metal cup
  • Suitable for detecting high ion currents without amplification
  • Provides a more quantitative measurement compared to electron multipliers
• Photomultiplier detects ions by converting them into photons using a scintillator
  • Photons strike a photocathode, releasing electrons
  • Electrons are amplified by a series of dynodes, similar to an electron multiplier
• Data system processes the detector signal and generates a mass spectrum
  • Analog-to-digital converter (ADC) converts the analog signal into digital data
  • Data analysis software identifies peaks, calculates m/z ratios, and performs data interpretation
• Mass spectrum displays the relative abundance of ions as a function of their m/z ratios
  • Base peak represents the most abundant ion and is assigned a relative intensity of 100%
  • Other peaks are normalized relative to the base peak intensity

Applications and Real-World Uses

• Protein identification and characterization in proteomics
  • Peptide mass fingerprinting and tandem mass spectrometry (MS/MS)
• Drug discovery and development in the pharmaceutical industry
  • Identification of metabolites and impurities
  • Pharmacokinetic and pharmacodynamic studies
• Environmental monitoring and analysis
  • Detection of pollutants, pesticides, and contaminants in air, water, and soil samples
• Forensic analysis and toxicology
  • Identification of drugs, explosives, and other trace evidence
  • Confirmation of chemical warfare agents and toxic substances
• Food safety and quality control
  • Detection of adulterants, contaminants, and allergens in food products
  • Authenticity testing and origin determination of food ingredients
• Metabolomics and biomarker discovery
  • Identification of metabolites in biological fluids and tissues
  • Biomarker discovery for disease diagnosis and prognosis
• Petroleum and biofuel analysis
  • Characterization of crude oil and its fractions
  • Quality control and optimization of biofuel production processes

Tips for Interpreting Mass Spectra

• Identify the molecular ion peak (M+) to determine the molecular mass of the compound
  • Highest m/z peak in the spectrum, unless fragmentation is extensive
• Recognize characteristic isotopic patterns for elements with multiple stable isotopes (Cl, Br, S)
  • Helps confirm the presence of these elements in the molecule
• Look for common neutral losses (H2O, CO, NH3) to identify functional groups
  • Water loss (18 Da) suggests the presence of hydroxyl or carboxyl groups
  • Carbon monoxide loss (28 Da) indicates the presence of carbonyl groups
• Compare the observed fragmentation pattern with reference spectra or databases
  • National Institute of Standards and Technology (NIST) mass spectral library
  • Wiley Registry of Mass Spectral Data
• Consider the ionization technique used and its impact on the fragmentation pattern
  • EI typically produces more extensive fragmentation than soft ionization techniques (ESI, MALDI)
• Use high-resolution mass spectrometry (HRMS) for accurate mass measurements and elemental composition determination
  • Time-of-flight (TOF) and Fourier transform (FT) mass analyzers provide high mass accuracy
• Interpret tandem mass spectrometry (MS/MS) data for structural elucidation
  • Precursor ion selection followed by fragmentation and analysis of product ions
  • Helps identify specific functional groups and connectivity within the molecule