Mass spectrometry

Mass spectrometry separates and detects ions according to mass-to-charge ratio. It can distinguish isotopes which belong to the same chemical element but have different atomic masses. The instrument measures ions: interpreting the result as a neutral atomic mass requires accounting for electron removal and its ionization energy. For a singly positive ion in its ground state, $m_{+}=m_{\mathrm{a}}-m_e+I/c^2$, where $m_e$ is the electron rest mass, $I$ is the ionization energy, and $c$ is the speed of light.

For a simple illustrative analyzer, positive ions of mass $m$ and electric charge $q=ze$ gain kinetic energy $qV=m{v}^2/2$ across an electric potential difference $V$ before entering a transverse uniform magnetic field $B$. The Lorentz force supplies centripetal acceleration, soAt fixed $V$ and $B$, the measured bending radius $r$ separates mass-to-charge ratios, not masses independently of electric charge. This ideal model assumes negligible initial kinetic energy and speeds small compared with the speed of light; the historical focusing mass spectrograph used the geometry described below.

The experimental achievement is to turn invisible differences between individual ions into separated, calibrated positions. Aston velocity focusing explains how an early mass spectrograph sharpened its images; the velocity-selected magnetic mass analyzer provides an independent model whose equations can be checked directly. The magnesium isotope mass spectrum shows why position, width, and intensity carry different information.

A combined analytical method separating compounds chromatographically before mass-spectrometric characterization. Peptide identification in a gel region can suggest a protein identity, but comigration requires independent validation of an assigned band.

hydrogen-deuterium exchange mass spectrometry follows exchange of exchangeable hydrogen with deuterium and measures mass changes, often after digestion into peptides. Protection can reflect hydrogen bonds, burial or restricted structural opening; altered exchange is not uniquely diagnostic of a binding interface.

chemical cross-linking mass spectrometry identifies residue pairs joined by a chemical reagent and uses them as spatial constraints on protein structures or assemblies. A distance limit depends on linker chemistry, reactive residue positions and accessible conformations.

co-fractionation mass spectrometry quantifies proteins across fractions from a native separation. Similar elution profiles suggest shared assemblies; coincident migration and assemblies that exchange partners are alternative explanations.

native mass spectrometry transfers intact biomolecular assemblies into the gas phase under conditions intended to preserve noncovalent association sufficiently for mass analysis. Charge-state assignment and possible loss of subunits or bound lipids must be considered when inferring stoichiometry.

tandem mass spectrometry selects precursor ions, fragments them, and measures the fragments to constrain precursor identity or structure. In proteomics, fragmentation of peptides provides sequence evidence that can be matched against a database.

Fragmentation of molecular ions induced through electron transfer, useful for preserving and localizing some labile modifications in protein analysis.

Accelerator mass spectrometry uses ion acceleration and particle identification to count rare isotopes relative to abundant ones. For radiocarbon dating, it measures carbon-14 directly rather than waiting to count its beta decays.

In the International Union of Pure and Applied Chemistry (IUPAC) mass spectrometry convention, a molecular ion is formed by removing or adding one or more electrons to a molecule without fragmentation of its molecular structure.^[ref] For example, removing one electron from intact water gives ${\mathrm{H}}_2{\mathrm{O}}^{\bullet +}$: two hydrogen and one oxygen chemical atom, total electric charge $+e$, and an unpaired electron, hence a radical cation. Atom inventory, net electric charge, and electronic state are separate pieces of information. Hydronium, ${\mathrm{H}}_3{\mathrm{O}}^{+}$, has a different inventory; it is a polyatomic ion, rather than the molecular ion obtained by electron removal from water.

The H3+ ion. Two electrons occupy a fully bonding three-centre orbital at a triangular geometry; the filled nondegenerate shell differs from the open degenerate shell of neutral ground-state H3.

A mass spectrometry instrument with a photographic readout. In the 1919 apparatus described in Chapter V: The Mass-Spectrograph, a gas discharge produced positive ions. Two narrow slits formed a ribbon beam in a vacuum; plates applied an electric field to spread it, an aperture selected part of it, and a magnetic field bent it oppositely. Focusing reduced the spread caused by differing ion speeds, concentrating a given mass-to-charge ratio into a line on a photographic plate.

The observed quantity was line displacement from a fiducial spot. Known reference lines calibrated displacement against mass-to-charge ratio; repeated exposures with changed magnetic field tested the calibration. Evacuation limited collisions. Comparing line shapes with narrow and wide apertures helped distinguish geometric broadening from photographic spreading. Finite slit widths, plate response, and overlapping molecular or multiply charged ions limited interpretation. See Chapter V: The Mass-Spectrograph (§§33–40: apparatus and photographic procedure; §§43 and 45–46: calibration, aperture comparisons, and accuracy).

In Mass spectra and isotopes by Francis William Aston (Nobel lecture, 12 December 1922), the reported neon lines at 20 and 22 have companions at 10 and 11 from doubly charged ions. That paired pattern and comparison with reference lines support different atomic masses within one chemical element, rather than an atomic mass concentrated at the bulk average. The lecture also describes an earlier failed separation by distillation and partial separation by diffusion, showing why the focused mass spectrum supplied stronger evidence. These numbers used a historical oxygen-based reference scale. The lecture's whole-number rule was an empirical approximation at its stated accuracy, not the modern exact definition $A=Z+N$.^[ref]

In Mass spectra and isotopes by Francis William Aston, printed pp. 10–12, Fig. 1, Francis William Aston describes successive electric and magnetic deflections in one plane, with opposite senses. This geometry focuses a range of ion speeds; it does not require the straight-through cancellation of a velocity selector.

The reason compensation is possible follows from the Lorentz force. In a short region of length ${\ell }_E$, a small electric deflection angle has magnitude ${\theta }_E\simeq qE{\ell }_E/(m{v}^2)$: the transverse acceleration acts for a time ${\ell }_E/v$, and the deflection angle is the acquired transverse velocity divided by $v$. A short magnetic region of length ${\ell }_B$ gives ${\theta }_B\simeq qB{\ell }_B/(mv)$, since the magnetic force already contains one factor of speed. Slower ions therefore receive a disproportionately larger electric deflection.

An original local approximation makes the focusing idea explicit. At fixed mass-to-charge ratio, replace each region by a small angular kick and include the drift distances in two positive constants $a$ and $b$. With $t=v/v_0$, write the detector coordinate asChoosing the geometry and fields so that $b=2a$ eliminates the first-order speed sensitivity. Indeed,A fractional speed error $\epsilon =t-1$ then contributes only at order ${\epsilon }^2$ in this approximation. This is a local thin-kick explanation, not Aston's exact pole geometry, focal-plane equation, or a prediction of its measured mass resolution. Finite slit widths, initial directions, and field boundaries still matter. Unlike a velocity selector, focusing can collect nearby speeds into a narrow image instead of discarding all but a narrow accepted range.

Reuse the velocity selector analysis in the Thomson charge-to-mass experiment, but now send positive ions with $q=ze$ into a separate magnetic analyzer. The selector fields $E_s$ and $B_s$ give $v_0=E_s/B_s$. If the analyzer field $B_a$ is perpendicular to the selected velocity, its gyroradius givesfor a semicircular return to the entrance plane. The displayed impact separation $D$ is a diameter. This formula is linear in $r$ at fixed selected speed; the fixed-electric potential difference model above instead gives $m/q\propto r^2$. They impose different experimental conditions.

For a chosen example, take $E_s=2.00\times 10^{4}V/m$, $B_s=0.200\mathrm{T}$, and $B_a=0.500\mathrm{T}$. Thus $v_0=1.00\times 10^{5}m/s$. Approximating an ion mass by $24\mathrm{u}$ and setting $q=e$ gives $r\approx 49.75\mathrm{mm}$ and $D\approx 99.50\mathrm{mm}$. Here $\mathrm{u}$ denotes the unified atomic mass unit. These are stipulated teaching inputs, not a reported Aston measurement or an exact magnesium atomic mass.

A finite accepted speed range broadens the radius distribution. At fixed $m/q$ and $B_a$, $\delta r/r=\delta v/v$. At fixed electric charge and mass, $K=m{v}^2/2$ gives $\delta v/v\simeq \delta K/(2K)$ for a small kinetic energy spread. A one-percent kinetic energy spread therefore produces roughly a half-percent radius spread before other broadening is included. Its width convention must be specified before converting that statement into a mass resolving power.

For a modern chosen example, the National Institute of Standards and Technology lists neutral magnesium atomic masses $23.985041697\mathrm{u}$, $24.985836976\mathrm{u}$, and $25.982592968\mathrm{u}$, with reference isotopic abundances $0.7899$, $0.1000$, and $0.1101$. They describe the isotopes with mass numbers 24, 25, and 26, respectively.^[ref] These modern inputs are not a reconstruction of historical observations.

Use the dimensionless mass-to-charge ratio coordinate $x=(m/\mathrm{u})/z$. For the illustration only, approximate ion masses by the neutral atomic masses. The singly charged positions are then near $23.98504$, $24.98584$, and $25.98259$; the doubly charged companions are near $11.99252$, $12.49292$, and $12.99130$. The neutral-to-ion correction already given above is essential at higher accuracy. A peak near $x=12$ alone need not identify a chemical element: another ion with a different electric charge can occupy the same mass-to-charge ratio interval.

For the narrow singly charged peak at $x\approx 24.98584$, the chosen full width at half maximum $\Delta x=0.12$ gives mass resolving power $R\approx 208.2$; widening to $\Delta x=1.20$ gives $R\approx 20.82$. Adjacent isotope spacings here are approximately one unit in $x$. Peaks are visibly separated in the first chosen profile and overlap strongly in the second. A small uncertainty in a fitted peak centre would not by itself make the overlapping components resolved.

Even integrated peak areas need a response model. For isotope fraction $f_i$, let ${\eta }_{i,z}$ collect the probability of ionization into electric charge state $z$, transmission through the analyzer, and the detection response. In a linear instrument with overall exposure factor $C$, the component area isEqual ${\eta }_{i,z}$ at a fixed $z$ makes normalized areas equal the fractions; otherwise it does not. In the chosen example, doubling only the mass-25 response changes the normalized areas to $0.71809$, $0.18182$, and $0.10009$, although the neutral sample is unchanged. Photographic darkening introduces another possible nonlinearity: neither line darkness nor peak height is a universal measure of neutral isotopic abundance. Calibration of position and calibration of intensity answer different questions. Matched standards, a known electric charge assignment, and a response correction make isotope analysis useful; a visually impressive set of peaks alone does not supply those controls.

The ratio of an ion's mass to its electric charge; in SI it has units $kg/C$. A convenient numerical mass spectrum coordinate is $x=(m/\mathrm{u})/z$, with $q=ze$ and positive integer $z$ here. This $x$ is dimensionless. Doubling $z$ at fixed mass halves $x$, so the electric charge state must be identified before assigning an isotope.

A mass spectrum displays detected signal against mass-to-charge ratio. Peak positions identify candidate ions; peak areas give isotopic abundances only after the response and overlapping contributions have been accounted for.

Broadening can hide a weak component without changing its area in this ideal model. An actual mass spectrum can also contain molecular ions, different electric charge states, background, and unequal detection responses. A missing visible peak therefore cannot by itself prove the absence of an isotope. Conversely, a fractional atomic weight is a property of a population: there is no requirement for an ion peak at the weighted mean.

Mass resolving power compares a peak coordinate $x$ with a stated width or separation criterion $\Delta x$. With $\Delta x$ taken as full width at half maximum, a Gaussian function of standard deviation $\sigma$ hasAt the neon-20 coordinate in the synthetic mass spectrum, $\sigma =0.04$ gives $\Delta x\approx 0.0942$ and $R\approx 212$, while $\sigma =0.70$ gives $\Delta x\approx 1.648$ and $R\approx 12.1$. The width criterion and relative peak areas both matter when judging whether a small neighboring isotope can be distinguished. Mass resolution also differs from the accuracy of a calibrated peak position.