Mass Spectrometry
Mass spectrometry (MS) measures the mass-to-charge ratio (m/z) of ions produced from a sample. In electron impact (EI) mode, a high-energy electron beam ionizes and fragments the sample molecules, producing a distinctive pattern of ion peaks. The intact molecule’s mass (the molecular ion, M⁺) gives the molecular weight. Fragment peaks reveal structural features.
The Molecular Ion (M⁺)
The largest peak in the mass spectrum that corresponds to the intact molecule is the molecular ion. It is produced when one electron is knocked off the neutral molecule, leaving a radical cation: M → M⁺• + e⁻.
The m/z of the molecular ion equals the molecular weight of the compound (for a singly charged ion).
Practical note: the M⁺ peak may not be the tallest peak (that is the base peak - see below). The M⁺ peak is the highest m/z peak representing the whole molecule.
The Base Peak
The base peak is the tallest peak in the spectrum, representing the most abundant ion. It is set to 100% relative abundance, and all other peaks are scaled relative to it.
The base peak is usually a stable fragment ion, not the molecular ion. Its identity reveals something about the molecule’s preferred fragmentation.
The Nitrogen Rule
If the molecular ion has an ODD m/z, the molecule contains an odd number of nitrogens.
- No N: molecular weight is even.
- 1 N: molecular weight is odd.
- 2 N: even.
- 3 N: odd.
- etc.
This is because nitrogen has valence 3 (odd) but atomic mass 14 (even), an unusual combination among organic elements. Carbon (mass 12, valence 4) and oxygen (mass 16, valence 2) give even contributions.
The nitrogen rule quickly identifies nitrogen-containing compounds from mass spec data. An odd M⁺ is a strong clue.
Isotope Patterns
Some elements have multiple naturally occurring isotopes:
- Chlorine: ³⁵Cl (75%) and ³⁷Cl (25%). A compound with one chlorine shows TWO M⁺ peaks, separated by 2 mass units, in a 3:1 intensity ratio.
- Bromine: ⁷⁹Br (50%) and ⁸¹Br (50%). One bromine gives two M⁺ peaks separated by 2, in 1:1 ratio.
- Carbon: ¹²C (98.9%) and ¹³C (1.1%). A compound with n carbons shows an M+1 peak with intensity approximately (1.1% × n) of the M⁺ peak.
Seeing the Cl/Br patterns is diagnostic. If you see M⁺ and M+2 peaks in a 3:1 ratio, there is one chlorine. If 1:1, one bromine.
Common Fragmentation Losses
Fragmentation typically loses small, stable neutral molecules. Key losses:
| Loss from M⁺ | Likely fragment | Suggests |
|---|---|---|
| 1 | H | (common for aldehydes) |
| 15 | CH₃ | methyl group |
| 17 | OH | alcohol (but less common) |
| 18 | H₂O | alcohol (dehydration) |
| 28 | C₂H₄ or CO | ethylene or carbonyl |
| 29 | CHO or C₂H₅ | aldehyde or ethyl |
| 31 | OCH₃ | methyl ester |
| 35 | Cl | chloride |
| 43 | CH₃CO or C₃H₇ | acetyl or propyl |
| 45 | OEt | ethyl ester |
Alpha-cleavage next to a carbonyl, loss of water from alcohols, and loss of halogen from alkyl halides are the most common fragmentation pathways.
McLafferty Rearrangement
A classic fragmentation in ketones with a gamma-hydrogen: the gamma-H migrates to the carbonyl O via a 6-membered transition state, and the alpha-beta bond cleaves. Products: an enol (the neutral fragment) and an alkene (the observed ion, or vice versa). This gives characteristic mass losses.
Example: MS of Pentan-2-one (CH₃COCH₂CH₂CH₃, MW 86)
- Molecular ion (M⁺): m/z 86.
- Alpha-cleavage at the methyl side → m/z 43 (CH₃CO⁺, acylium) plus m/z 43 (C₃H₇⁺) - overlap.
- Alpha-cleavage at the propyl side → m/z 71 (M-15 for methyl loss) and m/z 15 (CH₃⁺).
- McLafferty fragmentation → m/z 58 (C₃H₆O loss of CH₂=CH₂).
The base peak is often m/z 43 (acetyl cation) for methyl ketones.
Electrospray Ionization (ESI)
For biological macromolecules, electrospray ionization (ESI) is used instead of EI. ESI produces [M+H]⁺ ions (protonated molecules) with minimal fragmentation. Multiply-charged ions ([M+2H]²⁺, [M+3H]³⁺) let you measure molecules with MW up to 100,000 or more (proteins, large oligonucleotides).
MALDI (matrix-assisted laser desorption/ionization) is similar - used for proteins and larger biomolecules. Both are “soft” ionization methods that preserve the molecular ion better than EI.