Mass spectrometry is a powerful tool for figuring out what molecules are made of. It measures the mass of ions, helping scientists determine a compound's formula and structure. This technique is crucial for identifying unknown substances and understanding their properties.

In this section, we'll learn how to use mass spectrometry to determine molecular formulas and distinguish between isomers. We'll also explore how to identify functional groups and piece together molecular structures from fragmentation patterns.

Molecular Formula and Isomer Determination

Molecular Formula Calculation and Nitrogen Rule

• Molecular formula determination involves calculating the elemental composition of a compound from its mass spectrum
• Mass spectrometers measure the mass-to-charge ratio (m/z) of ions, allowing precise molecular mass determination
• Accurate mass measurements combined with isotopic abundance patterns help deduce molecular formulas
• Nitrogen rule states even-mass molecules contain zero or an even number of nitrogen atoms, while odd-mass molecules contain an odd number of nitrogen atoms
• Applying the nitrogen rule narrows down possible molecular formulas for unknown compounds

High-Resolution Mass Spectrometry for Formula Determination

• High-resolution mass spectrometry provides precise mass measurements with accuracy up to four decimal places
• Exact mass measurements allow differentiation between compounds with similar nominal masses but different elemental compositions
• Resolving power of high-resolution instruments enables separation of closely spaced peaks (isobaric species)
• Fourier transform ion cyclotron resonance (FT-ICR) and Orbitrap instruments offer ultra-high resolution for complex mixture analysis
• High-resolution data combined with isotope pattern analysis significantly reduces the number of possible molecular formulas

Structural Isomers and Mass Spectrometry

• Structural isomers are compounds with the same molecular formula but different atomic arrangements
• Mass spectrometry can differentiate between some structural isomers based on their fragmentation patterns
• Constitutional isomers often produce distinct mass spectra due to different bond cleavages
• Stereoisomers (geometric isomers, optical isomers) may have similar mass spectra but can sometimes be distinguished by specialized techniques
• Ion mobility spectrometry coupled with mass spectrometry (IMS-MS) separates isomers based on their collision cross-sections

Functional Group and Structural Analysis

Functional Group Identification Using Mass Spectrometry

• Mass spectrometry identifies functional groups through characteristic fragmentation patterns and molecular ions
• Alcohols often show loss of water (M-18) and tend to have weak molecular ion peaks
• Carboxylic acids frequently exhibit loss of OH (M-17) and COOH (M-45) fragments
• Aldehydes and ketones display prominent M-1 peaks due to loss of hydrogen from the molecular ion
• Aromatic compounds typically have stable molecular ions and characteristic fragmentation patterns
• Halogenated compounds show distinct isotope patterns due to the natural abundance of halogen isotopes

Fragmentation Patterns and Structural Elucidation

• Fragmentation patterns provide crucial information about molecular structure and bonding
• Alpha cleavage occurs adjacent to functional groups, producing characteristic fragment ions
• McLafferty rearrangement involves hydrogen transfer and bond cleavage, common in ketones and esters
• Retro-Diels-Alder fragmentation helps identify cyclic compounds with conjugated double bonds
• Aromatic compounds often undergo stepwise loss of small neutral molecules (CO, HCN)
• Fragmentation rules and common neutral losses aid in predicting and interpreting mass spectra

Mass Spectral Libraries and Database Matching

• Mass spectral libraries contain reference spectra for thousands of compounds
• Library searching algorithms compare unknown spectra with reference spectra to identify compounds
• Probability-based matching scores help assess the reliability of spectral matches
• NIST (National Institute of Standards and Technology) and Wiley are widely used mass spectral libraries
• Specialized libraries exist for specific compound classes (metabolites, drugs, environmental contaminants)
• In-house libraries can be created for frequently analyzed compounds or custom synthesized molecules

Advanced Mass Spectrometry Techniques

Isotope Ratio Analysis and Applications

• Isotope ratio mass spectrometry (IRMS) measures the relative abundance of stable isotopes in samples
• Carbon isotope ratios (13C/12C) help determine the origin of organic compounds and track carbon sources
• Nitrogen isotope analysis (15N/14N) aids in studying food webs and nitrogen cycling in ecosystems
• Oxygen and hydrogen isotope ratios provide information about water sources and paleoclimate conditions
• Sulfur isotope analysis (34S/32S) assists in tracing pollution sources and studying biogeochemical cycles
• Multi-element isotope analysis combines multiple isotope systems for comprehensive sample characterization

High-Resolution Mass Spectrometry Techniques

• Fourier transform ion cyclotron resonance (FT-ICR) offers ultra-high resolution and mass accuracy
• Orbitrap mass analyzers provide high resolution and sensitivity without the need for superconducting magnets
• Time-of-flight (TOF) instruments with reflectrons achieve high resolution and fast acquisition rates
• High-resolution MS enables accurate mass measurements for elemental composition determination
• Resolving power of high-resolution instruments separates isobaric species and complex mixtures
• Applications include proteomics, metabolomics, and analysis of environmental and petroleum samples

Tandem Mass Spectrometry (MS/MS) for Structural Analysis

• Tandem MS involves multiple stages of mass analysis to obtain structural information
• Precursor ion selection followed by fragmentation and analysis of product ions
• Collision-induced dissociation (CID) fragments ions through collisions with neutral gas molecules
• Electron transfer dissociation (ETD) and electron capture dissociation (ECD) provide complementary fragmentation patterns
• MS/MS techniques include product ion scanning, precursor ion scanning, and neutral loss scanning
• Applications in proteomics for peptide sequencing and post-translational modification analysis
• Structural elucidation of complex molecules and mixture analysis in metabolomics and environmental studies