Mass Spectroscopy: Principle, Types, Applications & Instrumentation Explained

Mass Spectrometry

Mass Spectrometry (MS) is an analytical technique used to identify the composition, structure, and molecular weight of compounds by measuring their mass-to-charge ratio (m/z).

It is widely used in pharmaceuticals, forensics, environmental studies, and biological research to analyze unknown substances and verify sample purity.

Basic Principles of Mass Spectrometry

Organic Molecules are Bombarded with electron → Converted into highly energetic +ve charged ions → breakup into smaller ions → formed ions are separated by deflection in magnetic field a/c to mass/charge ratio.

The fundamental principle of MS involves:

1. Ionization – The sample is converted into gaseous ions.
2. Acceleration– Ions are accelerated by an electric field, imparting them with kinetic energy.
3. Separation – Ions are separated based on their mass-to-charge ratio (m/z).
4. Detection – Separated ions are detected and analyzed based on their intensity.

1. Ionization:

• The sample (solid, liquid, or gas) is introduced into the mass spectrometer and converted into ions.

• Different ionization techniques (e.g., Electron Ionization, Electrospray Ionization, MALDI) are used based on the nature of the sample.

• Ionization can be soft (minimal fragmentation, e.g., ESI, MALDI) or hard (extensive fragmentation, e.g., EI, CI).

• The formed ions can be single-charged or multiply charged, which affects their mass spectrum.

2. Acceleration:

• The generated ions are accelerated using an electric field, ensuring they have uniform kinetic energy.

• The focusing system (electrostatic lenses) directs these ions into the mass analyzer for separation.

3.Mass Analysis (Separation of Ions):

• The ions travel through a mass analyzer, which separates them based on their m/z ratio.

• Different analyzers operate on different principles: 

• Time-of-Flight (TOF): Ions with lower mass travel faster than heavier ions.
• Quadrupole: Uses oscillating electric fields to selectively filter ions based on m/z.
• Magnetic Sector: Ions bend in a magnetic field, and the curvature depends on their mass.
• Ion Trap: Traps ions using electric/magnetic fields and releases them sequentially.
• Orbitrap & Fourier Transform (FT-MS): Measures ion oscillation frequencies in an electric field to determine their mass with ultra-high resolution.

4. Detection & Signal Processing:

• The separated ions reach the detector, where their arrival is recorded based on intensity and time.

• The detector generates a mass spectrum, which represents the relative abundance of ions as a function of m/z.

• The obtained data is processed to determine molecular weight, isotope patterns, fragmentation pathways, and structural information.

From basic physics, the motion of an ion in an electric/magnetic field follows the equation:

Instrumentation of Mass Spectrometry

1. Sample Introduction System

• Direct Insertion Probe (DIP): Solid or liquid samples are placed on a probe and inserted into the ionization chamber.
• Gas Chromatography (GC-MS): Used for volatile and thermally stable compounds.
• Liquid Chromatography (LC-MS): Used for non-volatile and thermally labile compounds.

Capillary Electrophoresis (CE-MS): Used for charged biomolecules.   

2. Ionization Source

Ionization is the process of converting neutral molecules into charged ions so they can be manipulated in an electric/magnetic field. Different ionization techniques are used depending on the nature of the sample.

(a) Hard Ionization Techniques (Produce extensive fragmentation)

Electron Ionization (EI):
→ High-energy electrons (70 eV) bombard the sample, causing ionization and fragmentation.
→ Commonly used in GC-MS.
Chemical Ionization (CI):
→ A reagent gas (e.g., methane, ammonia) interacts with the sample to produce protonated or deprotonated ions.
→ Produces softer fragmentation compared to EI.

(b) Soft Ionization Techniques (Minimal fragmentation, useful for biomolecules) –

Electrospray Ionization (ESI):
→ Sample is dissolved in a solvent and passed through a charged capillary, creating fine droplets that undergo desolvation, producing gas-phase ions.
→ Used in LC-MS for proteins, peptides, and pharmaceuticals.

Matrix-Assisted Laser Desorption/Ionization (MALDI):
→ A laser pulse ionizes the sample, which is embedded in a matrix.
→ Commonly used for large biomolecules like proteins and polymers.

Fast Atom Bombardment (FAB):
→ Uses a beam of high-energy neutral atoms (e.g., argon or xenon) to ionize the sample.
→ Used for non-volatile and thermally labile compounds.

3. Mass Analyzer

Once the ions are generated, they are separated based on their m/z ratio by the mass analyzer. Different mass analyzers have different principles and resolving powers.

(a) Quadrupole Mass Analyzer
• Uses four parallel rods with oscillating electric fields to selectively allow ions of a specific m/z to pass through.
• Used in LC-MS and GC-MS due to its compact design and rapid scanning ability.

(b) Time-of-Flight (TOF) Mass Analyzer
• Ions are accelerated and travel through a flight tube; lighter ions reach the detector faster than heavier ones.
• Provides high mass accuracy and is commonly used with MALDI-MS.

(c) Magnetic Sector Mass Analyzer
• A magnetic field bends the path of ions based on their m/z ratio.
• Used for high-resolution mass spectrometry (HRMS).

(d) Ion Trap Mass Analyzer
• Traps ions using electric/magnetic fields and sequentially ejects them for detection.
• Includes 3D ion traps and linear ion traps.

(e) Orbitrap Mass Analyzer
• Ions are trapped in an electric field and oscillate around a central electrode.
• Offers ultra-high resolution and accuracy.

(f) Fourier Transform Ion Cyclotron Resonance (FT-ICR) Mass Analyzer
• Uses a strong magnetic field to trap ions and detect their frequencies.
• Provides extremely high resolution and mass accuracy.

4. Detector

The detector records the abundance of separated ions and converts the signal into a mass spectrum.

• Faraday Cup: Simple, low-sensitivity detector that collects ions.
• Electron Multiplier: Detects ions by amplifying their signal through secondary electron emissions.
• Microchannel Plate Detector: Provides high sensitivity and fast response time.
• Photomultiplier Tube: Used for high-speed detection in TOF-MS.

5. Data Processing System

Once the detector records the ion signal, the data is processed using software to generate a mass spectrum.

This spectrum provides information about the molecular weight, isotope distribution, and structural fragmentation of the sample.

Application

1. Molecular weight, Formula and element composition.
2. Determination, identification of unknown compound.
3. Distinction between cis & trans – isomers.
4. Identification of fragmentation pattern.
5. Impurity detection.
6.  Analysis of Protein.