Mass spectrometry is a measurement technique that identifies and quantifies molecules by converting them into charged particles, sorting those particles according to their mass-to-charge ratio, and counting how many arrive at each value. The output is a mass spectrum, a plot of signal intensity against mass-to-charge ratio, from which the composition of a sample can be inferred. This article explains the physics and chemistry of how the measurement is produced. It is a methods explainer about the technique and its research uses.
The three core stages
Every mass spectrometer, whatever its design, performs three operations in sequence: ionisation, separation and detection. The sample molecules are first turned into ions, because the instrument manipulates particles using electric and magnetic fields, which act only on charged particles. The ions are then separated according to their mass-to-charge ratio, written m/z, the mass of the ion divided by its number of charges. Finally a detector records the ions arriving at each m/z value, producing the spectrum.
| Stage | Function | Physical basis |
|---|---|---|
| Ionisation | Convert neutral molecules into ions | Adding or removing charge so fields can act on them |
| Separation | Sort ions by mass-to-charge ratio | Response to electric or magnetic fields depends on m/z |
| Detection | Count ions at each m/z | Charge collected and amplified into a signal |
Ionisation
The ion source converts neutral molecules into gas-phase ions. The choice of method depends on the sample. Some methods impart a great deal of energy and tend to fragment molecules into characteristic pieces, which is informative for small, robust compounds. Others are gentle and produce intact ions of large, fragile molecules such as proteins, which would otherwise break apart. Soft ionisation methods made it possible to apply mass spectrometry to large biomolecules, vastly extending its reach. Whichever method is used, the result is a population of charged particles ready to be sorted.
Separation by mass-to-charge ratio
Once charged, ions are separated according to m/z. Different analyser designs achieve this through different physics. In a time-of-flight analyser, all ions are given the same kinetic energy and allowed to drift down a tube; lighter ions travel faster and arrive sooner, so arrival time encodes m/z. In a quadrupole, oscillating electric fields allow only ions of a selected m/z to pass through at a given moment, and scanning the fields sweeps through the range. Other designs trap ions and measure the frequency at which they orbit or oscillate, which depends on m/z. In all cases the separating principle is that an ion’s motion in a controlled field depends on its mass and charge.
Detection and reading a spectrum
The detector converts arriving ions into an electrical signal, typically amplifying the tiny charge of each ion into a measurable pulse and counting the pulses at each m/z. The accumulated counts form the mass spectrum. Reading it, each peak corresponds to an ion of a particular mass-to-charge ratio, and the height of the peak reflects how many such ions were detected, which relates to abundance. The pattern of peaks, including any fragment peaks, acts as a fingerprint that can identify a compound or, with calibration, quantify it. This conversion of a physical property into a counted signal places mass spectrometry alongside techniques such as PCR and ultrasound in the family of controlled-condition measurement methods.
Common configurations and research uses
Mass spectrometers are frequently coupled to a separation stage that feeds the sample in over time, such as a chromatography column that releases different components at different moments, so that the spectrometer analyses a mixture component by component. Tandem mass spectrometry chains two analysers so that a selected ion is fragmented and its pieces measured, giving structural detail. These configurations underpin major research fields. In proteomics, mass spectrometry identifies and quantifies the proteins in a sample by measuring peptide masses and fragments. In metabolomics, it profiles the small molecules of a biological system. It is equally central to environmental analysis, materials characterisation and many other areas.
Because results depend heavily on instrument settings and calibration, reproducibility requires careful documentation, the subject of our guide on reporting analytical methods reproducibly. Standard terminology is held in the CASRAI dictionary, and the wider context appears across our research lifecycle coverage.
Frequently asked questions
Why must molecules be ionised first?
A mass spectrometer separates particles using electric and magnetic fields, and those fields exert force only on charged particles. Neutral molecules would be unaffected and impossible to sort, so ionisation, adding or removing charge, is the necessary first step.
What is mass-to-charge ratio and why is it the axis?
Mass-to-charge ratio, m/z, is an ion’s mass divided by the number of charges it carries. The instrument separates ions by how they respond to fields, which depends on this combined quantity rather than mass alone, so m/z is the natural axis of a mass spectrum.
How does a peak’s height relate to the sample?
The height of a peak reflects how many ions of that mass-to-charge ratio reached the detector, which in turn relates to the abundance of the corresponding species. With appropriate calibration, peak intensities can be used to quantify amounts.
What does tandem mass spectrometry add?
Tandem mass spectrometry selects a specific ion, fragments it, and measures the fragments. The fragmentation pattern gives structural information that a single mass measurement cannot, which is valuable in fields like proteomics. Reproducibility of such workflows is discussed in our reproducibility coverage and the author guidance.
