MS Spectra — Lecture Notes & Visualizer
Mass spectrometry (MS) is an analytical technique that measures the mass-to-charge ratio (m/z) of ions. Unlike IR or NMR, it does not use electromagnetic radiation to probe bond vibrations; instead, it physically separates ions by their mass. The result is a mass spectrum: a plot of relative intensity (%) versus m/z, showing which ionic masses are present and how abundant they are.
A mass spectrometer has five essential components working in sequence:
| Stage | Component | Function |
|---|---|---|
| 1. Introduction | Sample inlet | Vaporises the sample under vacuum |
| 2. Ionisation | Ion source | Converts neutral molecules into ions (see below) |
| 3. Separation | Mass analyser | Separates ions by m/z (magnetic, quadrupole, TOF, etc.) |
| 4. Detection | Detector | Measures ion abundance at each m/z |
| 5. Output | Data system | Produces the mass spectrum |
The entire system operates under high vacuum (~10⁻⁵ to 10⁻⁷ torr) to prevent ions from colliding with gas molecules before detection.
The choice of ionisation method depends on the sample type. The most common methods in organic chemistry are:
| Method | Abbreviation | Principle | Best For |
|---|---|---|---|
| Electron Ionisation | EI | 70 eV electron beam strips one electron: M → M⁺• + e⁻. Gives extensive fragmentation. | Volatile small organics; library matching |
| Chemical Ionisation | CI | Reagent gas (CH₄, NH₃) protonates the analyte: M + H⁺ → [M+H]⁺. Softer than EI. | Labile molecules; molecular mass confirmation |
| Electrospray Ionisation | ESI | Solution sprayed through high-voltage needle; produces multiply-charged ions. | Proteins, peptides, polymers; LC-MS |
| Matrix-Assisted Laser | MALDI | Laser desorption from matrix co-crystal; singly charged ions. | Large biomolecules, polymers |
Mass spectrometry is arguably the most information-rich single analytical technique. It provides:
It requires only nanogram to microgram quantities of sample, making it ideal for trace analysis, forensics, environmental monitoring, and pharmaceutical quality control.
| Feature | Symbol | Definition |
|---|---|---|
| Molecular ion | M⁺ (or M⁺•) | Intact molecule minus one electron; gives the molecular mass |
| Base peak | 100% | Most abundant ion; all others expressed relative to it |
| Fragment ions | m/z < M⁺ | Bond cleavage products — reveal structural elements |
| Isotope peaks | M+1, M+2 | Natural isotope contributions (¹³C, ³⁷Cl, ⁸¹Br, etc.) |
| Metastable ions | Broad, low | Ions that fragment in the analyser; broad diffuse peaks |
In EI-MS, the molecular ion M⁺• is formed by removal of one electron from the intact molecule. It appears at the highest m/z value in the spectrum (ignoring isotope peaks) and equals the nominal molecular mass (sum of most abundant isotope masses of all atoms).
1. It is the highest-mass peak (excluding M+1, M+2 isotope peaks).
2. It must be a reasonable mass loss from fragments: the difference M⁺ − fragment must correspond to a stable neutral (e.g., •CH₃ = 15, H₂O = 18, CO = 28, HCl = 36/38).
3. Losses of 3–14 and 21–25 Da from M⁺ are chemically unreasonable — these usually indicate M⁺ has been misidentified.
4. Some compound classes give weak or absent M⁺ (branched alkanes, alcohols, amines). Use CI or ESI to confirm MW in these cases.
A useful heuristic for EI spectra of compounds containing only C, H, O, N, S, and halogens:
Example: Ethylamine (C₂H₇N, MW = 45) — odd M⁺ ✓ | Acetone (C₃H₆O, MW = 58) — even M⁺ ✓
Every element has naturally occurring heavy isotopes. This means every molecular ion has accompanying isotope peaks at M+1, M+2, etc. Their relative intensities follow predictable patterns and are diagnostic for the elemental composition.
| Isotope | Natural Abundance | Effect on Spectrum |
|---|---|---|
| ¹³C | 1.1% per C atom | M+1 grows with number of carbons: ~1.1 × n(C) % |
| ²H (D) | 0.015% | Negligible contribution |
| ¹⁵N | 0.37% per N | Small M+1 contribution |
| ¹⁷O / ¹⁸O | 0.04% / 0.20% | Small M+1 and M+2 contributions |
| ³³S / ³⁴S | 0.75% / 4.25% | Visible M+2 peak (~4%) if one S present |
| ³⁷Cl | 24.5% (³⁵Cl 75.5%) | M+2 ≈ 1/3 × M⁺; characteristic 3:1 doublet pattern |
| ⁸¹Br | 49.3% (⁷⁹Br 50.7%) | M+2 ≈ M⁺; characteristic 1:1 doublet pattern |
The most visually striking isotope effects arise from chlorine and bromine. Their patterns are immediately recognisable:
| Chlorine Pattern | M : M+2 |
|---|---|
| 1 × Cl | 3 : 1 |
| 2 × Cl | 9 : 6 : 1 |
| 3 × Cl | 27 : 27 : 9 : 1 |
| Bromine Pattern | M : M+2 |
|---|---|
| 1 × Br | 1 : 1 |
| 2 × Br | 1 : 2 : 1 |
| 1 × Br + 1 × Cl | 3 : 4 : 1 : ··· |
Low-resolution MS gives nominal (integer) masses. High-resolution MS (HRMS) measures m/z to 4–5 decimal places, allowing exact molecular formula determination from a single measurement. This is possible because each element's exact isotopic mass is unique.
Example: C₂H₆O (ethanol, MW = 46) has exact mass 46.0419, while CH₂O₂ (formic acid, MW = 46) has exact mass 46.0055. HRMS distinguishes these instantly; unit-resolution MS cannot.
Fragmentation in EI-MS follows predictable chemical rules. The most important are:
| Pathway | Notation | Description | Example Loss |
|---|---|---|---|
| α-Cleavage | α | Bond adjacent to heteroatom or C=O breaks homolytically | Ketones lose •CH₃ (–15) or •CₙH₂ₙ₊₁ |
| Inductive cleavage | i | Charge-directed; bond between charge site and leaving group | Halides lose X• giving [M−X]⁺ |
| McLafferty rearrangement | McL | γ-H migration through 6-membered TS; requires γ-H and C=O | Aldehydes, ketones, esters: even-electron rearrangement |
| Retro-Diels-Alder | RDA | Cyclohexene systems fragment as reverse DA reaction | Terpenes, steroids |
| Benzyl/tropylium | Ar | Benzylic cleavage gives stable tropylium (C₇H₇⁺, m/z 91) | Benzyl alcohol, ethylbenzene → m/z 91 |
MS is the primary tool for determining molecular mass and formula of an unknown compound. Combined with IR, NMR, and UV data, a mass spectrum enables full structure elucidation. The systematic approach is: identify M⁺ → apply nitrogen rule → calculate degree of unsaturation → interpret key fragments → propose structure → confirm with other spectral data.
GC-MS couples gas chromatography (which separates a mixture) with EI-MS (which identifies each component). It is the gold-standard technique for identifying volatile organic compounds in complex mixtures. Each separated peak is matched against MS library databases (NIST, SDBS) containing hundreds of thousands of reference spectra.
Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) is the dominant technique in the pharmaceutical industry. It identifies and quantifies drugs, metabolites, and biomarkers in biological fluids (plasma, urine, CSF) at pg/mL concentrations — far beyond the reach of other methods.
In drug development, LC-MS/MS is used for pharmacokinetic studies (tracking how a drug moves through the body), metabolite identification, and impurity profiling during synthesis. Regulatory agencies require MS data for drug approval dossiers.
ESI and MALDI mass spectrometry revolutionised biology by enabling rapid analysis of proteins and peptides. In "bottom-up" proteomics, a protein is digested with trypsin, and the resulting peptides are identified by their MS/MS fragmentation patterns (b and y ions). Software reconstructs the protein sequence and identifies post-translational modifications.
MS is the confirmatory technique in forensic chemistry. A positive immunoassay drug screen must be confirmed by GC-MS or LC-MS/MS before it can be used as legal evidence. MS provides unambiguous molecular identification because the combination of retention time and full mass spectrum is essentially unique to each compound.
Isotope ratio mass spectrometry measures the precise ratio of stable isotopes (e.g., ¹³C/¹²C, ²H/¹H, ¹⁸O/¹⁶O) in a sample. Because isotope ratios vary predictably with geography, biosynthetic pathway, and growth conditions, IRMS can determine the geographic origin of food, authenticate vintage wines, detect performance-enhancing drug use (testosterone doping), and verify the botanical source of natural products.
New ambient ionisation techniques (DESI, DART, REIMS) allow MS analysis directly from surfaces, intact tissue, or even living organisms — with no chromatographic separation or sample preparation. Applications include real-time surgical margin assessment during cancer operations (the "iKnife"), rapid food authentication, and in-field screening of counterfeit pharmaceuticals.
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