Category: Live Forensic

Sample Introduction

The analyte (the substance to be analysed) is introduced into the mass spectrometer through the inlet system. The entire instrument operates under high vacuum (10⁻⁵ to 10⁻⁸ torr) to prevent ions from colliding with air molecules. Samples can be introduced as a gas directly, as a liquid via a syringe or LC inlet (ESI), or as a solid using laser desorption (MALDI). The sample must be vaporised before it can be ionised.

Ionisation

Neutral molecules must be converted into ions before they can be separated by the mass analyser. There are several ionisation methods:

Electron Ionisation (EI): A beam of high-energy electrons (70 eV) bombards the sample vapour. An electron is knocked out of the molecule, creating a positively charged radical cation called the molecular ion (M⁺•). This is a “hard” ionisation method that causes significant fragmentation.

Electrospray Ionisation (ESI): The liquid sample is sprayed through a charged needle, creating multiply-charged ions. This is a “soft” method that keeps large molecules (like proteins) intact.

Ion Acceleration

The newly formed positive ions are accelerated through a high electric potential (a few hundred to several thousand volts). This gives all ions of the same charge the same kinetic energy. Ions then enter the mass analyser as a focused beam. The key relationship here is:

Where m = mass, v = velocity, z = charge, V = accelerating voltage. Lighter ions move faster than heavier ones at the same kinetic energy, which allows them to be separated.

Mass Analysis — Separation by m/z

This is the heart of mass spectrometry. The mass analyser separates ions according to their mass-to-charge ratio (m/z). Different analysers use different principles:

Quadrupole: Four parallel rods with alternating DC and RF voltages. Only ions of a specific m/z have stable trajectories and pass through. Others are deflected and lost. By scanning the voltages, a full mass spectrum is recorded.

Time-of-Flight (TOF): Ions are accelerated over the same distance. Lighter ions arrive at the detector first. The time of flight is used to calculate m/z precisely.

Magnetic Sector: A magnetic field deflects ions — heavier ions are deflected less than lighter ones at the same velocity.

Ion Detection

Ions exiting the mass analyser strike the detector. The most common detector is the Electron Multiplier (EM). When a single ion hits the first dynode, it releases secondary electrons. These cascade through a series of dynodes, each releasing more electrons — amplifying the signal by a factor of 10⁵ to 10⁸. The final electrical current is proportional to the number of ions arriving, giving a relative intensity reading. Faraday cup detectors are also used for accurate quantitation of high-abundance ions.

Data Processing — Mass Spectrum

The detector signals are sent to a data system which plots the mass spectrum — a graph of relative intensity (%) on the Y-axis versus m/z on the X-axis. Each vertical bar (peak) represents an ion of that particular m/z. The tallest peak is called the base peak (set to 100%). Key peaks to identify:

Molecular Ion Peak (M⁺): Appears at the highest m/z and corresponds to the intact molecule. Its m/z value gives the molecular weight of the compound directly.

Fragment Ion Peaks: Produced when the molecular ion breaks apart. The pattern of fragmentation is characteristic of the compound’s structure — like a fingerprint.

M+1 / M+2 Isotope Peaks: Small peaks one or two units above M⁺ due to naturally occurring ¹³C, ²H, ³⁷Cl, ⁸¹Br isotopes. Their relative heights help identify elemental composition.