Mass Spectrometry

Introduction to Mass Spectrometry

  • Purpose:

    • Provides high selectivity and specificity.

    • Combines retention time with mass for identification.

    • Information on unknown compounds is accessible.

  • Key Features:

    • Determines molecular weight (m/z ratio).

    • Provides fragmentation patterns for structural information.

    • Offers data other spectroscopic methods cannot.

  • Units:

    • Molecular weight measured in Daltons (Da).

  • Concentration Required:

    • Very low: 10⁻⁹ to 10⁻¹⁵ mol/mL.

  • Five Main Components of a Mass Spectrometer:

    • Sample introduction

    • Ion formation

    • Ion separation (by m/z)

    • Ion detection

    • Data recording and processing

3.2 Electron Ionisation (EI)

  • Technique:

    • Ideal for small (<700 Da), non-polar molecules.

    • Vaporisation followed by electron bombardment (70 eV).

  • Ionisation Process:

    • Loss of one electron → radical cation (M⁺•).

  • Fragmentation:

    • Absorbed energy causes molecular fragmentation.

  • Drawbacks:

    • Molecular ion sometimes absent.

    • Limited to low MW, thermally stable molecules.

3.3 Chemical Ionisation (CI)

  • Technique:

    • Softer method, suitable for small, more polar molecules.

    • Ionisation via reaction with a reagent gas (e.g., methane, ammonia).

  • Ionisation Products:

    • Formation of [M+H]⁺ ions, fewer fragments.

  • Advantages:

    • Good for molecular weight determination.

    • Good for quantitation (clearer spectra).

  • Disadvantages:

    • Possible confusion over adducts ([M+H]⁺, [M+CH₅]⁺, etc.).

    • Limited to similar sample types as EI.

3.4 Interpretation of Mass Spectra

  • Strategy:

    • Identify the molecular ion (M⁺•).

    • Rationalise fragment ions.

    • Nitrogen Rule:

      • Odd m/z → odd number of nitrogen atoms.

      • Even m/z → even number or zero nitrogen atoms.

  • Isotope Patterns:

    • Look for patterns from elements like Cl, Br.

  • Reasonable Losses:

    • E.g., CH₃ (15 Da), H₂O (18 Da).

    • Avoid irrational losses (e.g., 3–14 Da).

3.5 Isotopes in Mass Spectrometry

  • Common Isotopes Observed:

    • C, Cl, Br, S, Si.

  • Characteristic Patterns:

    • Intensity patterns indicate presence of elements.

    • No higher isotopes: H, F, P, I.

  • Key Isotopic Gaps:

    • 1 Da gap: C, N.

    • 2 Da gap: O, Si, S, Cl, Br.

3.6 Carbon Isotopes

  • Carbon Ratio:

    • ¹²C:¹³C = 100:1.1.

  • Determining Carbon Count:

    • Normalise M⁺• to 100%.

    • Calculate [M+1]⁺ relative intensity.

    • Divide by 1.1 to estimate number of carbons.

3.7 Isotope Ratio Mass Spectrometry

  • Application:

    • Detects synthetic steroid doping.

  • Principle:

    • Natural vs synthetic steroids differ in ¹³C/¹²C ratios.

3.8 Chlorine Isotopes

  • Chlorine Ratio:

    • ³⁵Cl:³⁷Cl = 3:1.

  • Isotope Pattern:

    • Molecules with chlorine → two peaks, 2 Da apart, 3:1 intensity.

    • More Cl atoms → complex series (grid method for calculations).

3.9 Characteristic Ions

  • Typical Ions:

    • Amines → m/z 30, 44, 58.

    • Benzoyl compounds → m/z 51, 77, 105.

    • Benzyl compounds → m/z 91 (Tropylium ion).

3.10 Fragmentation Mechanisms

  • Common Processes:

    • α-Cleavage

    • β-Cleavage

    • McLafferty Rearrangement

    • Decarbonylation

  • Fragmentation:

    • Only charged fragments detected, not radicals.

3.11 General Hints for Spectrum Interpretation

  • General Advice:

    • Identify M⁺• carefully.

    • Consider the Nitrogen Rule.

    • Inspect isotope patterns and high-mass fragments.

    • Avoid overinterpreting low m/z fragments.

    • Sequential losses are rare – direct losses are more likely.