DR.

CATHERINE NGAMBI

FACULTY OF SCIENCE

DEPARTMENT OF BIOCHEMISTRY AND MOCELULAR BIOLOGY

BIOC 203: INTRODUCTION TO LABORATORY TECHNIQUES I

CONTINUOUS ASSESMENT TEST II

NAME: LINOX OCHIENG WASONGA

REGISTRATION NUMBER: S15/03116/23

COLLECTION DATE: 4TH MARCH,2025

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DESCRIBE MASS SPECTROMETRY IN TERMS OF;
1. PRINCIPLE

2. INSTRUMENTATION

3. IONIZATION TECHNIQUES APPLIED IN MASS SPECTROMETRY

4. APPLICATIONS

Mass spectrometry

Mass spectrometry (MS) is an analytical technique used to identify and quantify compounds based on
their mass-to-charge ratio (m/z). It measures the masses of individual molecules and atoms, allowing for
the determination of molecular structure, composition, and chemical properties.

1.Principle of Mass Spectrometry (MS)

Mass spectrometry (MS) is an analytical technique that identifies and quantifies molecules based on
their mass-to-charge ratio (m/z). The fundamental principle involves converting molecules into ions,
separating them based on their masses, and detecting them to generate a mass spectrum that provides
structural and compositional information.

1. Sample Ionization (Formation of Ions)

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

Different ionization methods are used depending on the nature of the sample:

Electron Ionization (EI): A high-energy electron beam knocks off an electron from the molecule,
producing positively charged ions.

Electrospray Ionization (ESI): A liquid sample is passed through a fine needle with a high voltage,
generating charged droplets that evaporate, leaving ions in the gas phase.

Matrix-Assisted Laser Desorption/Ionization (MALDI): A laser beam strikes a sample embedded in a

special matrix, causing desorption and ionization.

2. Mass Analysis (Separation of Ions by Mass-to-Charge Ratio)

The produced ions are accelerated in an electric field and enter a mass analyzer, where they are
separated based on their m/z ratio.

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The mass analyzer determines how the ions travel depending on their mass and charge. Common
analyzers include:

Quadrupole: Uses oscillating electric fields to filter ions based on m/z.

Time-of-Flight (TOF): Measures the time it takes for ions to reach the detector; lighter ions travel faster
than heavier ones.

Magnetic Sector: Uses a magnetic field to bend ion paths; their deflection depends on their m/z ratio.

3. Ion Detection (Measuring Ion Abundance)

After separation, the ions strike a detector that converts their signal into an electric current.

The signal intensity corresponds to the number of ions detected, providing a mass spectrum.

Common detectors:

Electron Multipliers: Amplifies the ion signal by generating secondary electrons.

Faraday Cup: Measures total ion current but has lower sensitivity.

4. Data Processing and Interpretation

The mass spectrometer generates a mass spectrum, which is a plot of m/z values vs. signal intensity.

Peaks in the spectrum represent different ions, with the most abundant ion being the base peak.

The molecular ion peak (M⁺) corresponds to the intact molecule's mass.

Fragmentation patterns help determine molecular structure and composition.

Key Concepts in Mass Spectrometry

1. Mass-to-Charge Ratio (m/z):

The mass (m) of an ion divided by its charge (z).

Most ions are singly charged (z = 1), so their m/z value corresponds to their actual mass.

2. Molecular Ion Peak (M⁺):

The peak representing the intact molecule with one lost electron.

Provides the molecular weight of the compound.

3. Fragmentation:

Some ions break apart into smaller fragment ions, producing a characteristic pattern.
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Used to determine structural features of the molecule.

4. Isotopic Peaks:

Some elements have multiple isotopes, leading to peaks at slightly different masses (e.g., chlorine and
bromine show distinct isotope patterns).

2. Instrumentation of Mass Spectrometry (Components of a

Mass Spectrometer)
A mass spectrometer consists of several key components that work together to analyze a sample based
on its mass-to-charge ratio (m/z).

1. Sample Introduction System

Introduces the sample into the ionization chamber in a controlled manner.

Methods depend on the state of the sample (solid, liquid, or gas).

Common sample introduction techniques:

Direct Insertion Probe: Used for solid samples.

Gas Chromatography (GC-MS): Separates volatile compounds before ionization.

Liquid Chromatography (LC-MS): Introduces liquid-phase samples.

2. Ionization Source (Ionizer)

Converts neutral sample molecules into gas-phase ions.

Different ionization techniques are used depending on the sample type:

Hard Ionization Techniques (High Energy, More Fragmentation)

Electron Ionization (EI): A high-energy electron beam knocks electrons off molecules, forming positively
charged ions (M⁺).

Chemical Ionization (CI): Uses reagent gases (e.g., methane) to ionize the sample through proton
transfer.

Soft Ionization Techniques (Less Fragmentation, Larger Molecules)

Electrospray Ionization (ESI): Creates charged droplets from a liquid sample, allowing large biomolecules
(proteins, peptides) to be analyzed.

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Matrix-Assisted Laser Desorption/Ionization (MALDI): Uses a laser to ionize large biomolecules
embedded in a matrix.

3. Mass Analyzer (Separates Ions by Mass-to-Charge Ratio, m/z)

Determines how ions travel based on their m/z values.

Types of mass analyzers:

Quadrupole Mass Analyzer

Uses four parallel metal rods with oscillating electric fields to filter ions.

Only ions of a specific m/z ratio reach the detector at a time.

Common in GC-MS and LC-MS instruments.

Time-of-Flight (TOF) Mass Analyzer

Ions are accelerated into a flight tube, and their speed depends on their mass (lighter ions move faster).

Used in MALDI-TOF MS for analyzing proteins and large biomolecules.

Magnetic Sector Analyzer

Uses a magnetic field to bend ion paths; ions of different masses deflect differently.

Provides high-resolution mass spectrometry (HRMS) but is expensive and large.

Orbitrap Mass Analyzer

Uses an electrostatic field to trap ions in circular orbits and measures their frequencies.

Provides ultra-high resolution and is commonly used in proteomics.

4. Ion Detector (Measures Ion Abundance)

Converts ion impacts into an electric signal for analysis.

Types of detectors:

Electron Multiplier Detector

Most common detector.

Ions strike a surface, causing electrons to be emitted, which are multiplied to create a stronger signal.

Faraday Cup Detector

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Simple but less sensitive than electron multipliers.

Measures the total ion current.

Photomultiplier Tube (PMT)

Used for detecting light emitted from excited ions in some MS systems.

5. Data System and Mass Spectrum Interpretation

The mass spectrometer generates a mass spectrum (a graph of m/z values vs. intensity).

Software analyzes peak patterns, molecular weights, and isotopic distributions.

Used to determine the identity and structure of compounds.

3. Ionization Techniques in Mass Spectrometry

Ionization is the process of converting neutral molecules into charged ions, which can then be analyzed
in a mass spectrometer. The choice of ionization technique depends on the sample type, molecular size,
and desired fragmentation pattern.

Categories of Ionization Techniques:

1. Hard Ionization Techniques – Provide high energy, causing extensive fragmentation.

2. Soft Ionization Techniques – Provide lower energy, minimizing fragmentation and preserving the
molecular ion peak.

1. Hard Ionization Techniques (High Energy, More Fragmentation)

A. Electron Ionization (EI)

Principle: A high-energy electron beam (70 eV) collides with sample molecules, ejecting an electron and
producing positively charged ions (M⁺·).

Fragmentation: Extensive fragmentation, generating multiple product ions.

Common Applications:

Small organic molecules, pharmaceuticals, volatile compounds.

Used in Gas Chromatography-Mass Spectrometry (GC-MS).

Advantages:

Produces reproducible fragmentation patterns.

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Useful for structural analysis.

Limitations:

Destructive (molecular ion peak may be weak or absent).

Not suitable for large biomolecules.

B. Chemical Ionization (CI)

Principle: A reagent gas (e.g., methane, ammonia) undergoes ionization first, and the charged species
transfer protons to the analyte, producing protonated molecular ions (M+H)⁺.

Fragmentation: Less fragmentation than EI.

Common Applications:

Small organic molecules, pharmaceuticals.

Complementary to EI in GC-MS.

Advantages:

Provides a strong molecular ion peak.

Less fragmentation than EI.

Limitations:

Requires a reagent gas.

Not suitable for nonvolatile or large biomolecules.

2. Soft Ionization Techniques (Low Energy, Less Fragmentation)

A. Electrospray Ionization (ESI)

Principle: The sample is dissolved in a solvent and passed through a fine capillary at high voltage,
forming charged droplets. The solvent evaporates, leaving behind gas-phase ions.

Fragmentation: Minimal fragmentation; produces multiply charged ions ([M+nH]ⁿ⁺).

Common Applications:

Large biomolecules (proteins, peptides, nucleotides, lipids).

Used in Liquid Chromatography-Mass Spectrometry (LC-MS).

Advantages:
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Suitable for large, polar, and thermally unstable molecules.

Allows analysis of intact biomolecules.

Limitations:

Requires a liquid-phase sample.

Can produce complex charge states.

B. Matrix-Assisted Laser Desorption/Ionization (MALDI)

Principle: The sample is mixed with a matrix and irradiated by a laser. The matrix absorbs energy,
leading to desorption and ionization of the sample molecules.

Fragmentation: Minimal fragmentation; produces singly charged ions ([M+H]⁺).

Common Applications:

Proteins, peptides, polymers, large biomolecules.

Used in MALDI-TOF MS.

Advantages:

High sensitivity.

Suitable for large biomolecules and complex mixtures.

Limitations:

Requires a specialized matrix.

Not ideal for small molecules.

C. Atmospheric Pressure Chemical Ionization (APCI)

Principle: The liquid sample is nebulized and heated to form gas-phase molecules, which are ionized by
proton transfer from ionized reagent gases.

Fragmentation: Moderate fragmentation, but stronger than ESI.

Common Applications:

Medium-polarity compounds, lipids, steroids.

Used in LC-MS.

Advantages:
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Works well with nonpolar and semi-volatile compounds.

High sensitivity.

Limitations:

Requires volatile samples.

Cannot handle large biomolecules.

D. Field Desorption Ionization (FDI) & Field Ionization (FI)

Principle: The sample is deposited on a high-voltage emitter, causing ionization through electron
tunneling.

Fragmentation: Very minimal; produces intact molecular ions.

Common Applications:

Nonvolatile, thermally unstable compounds.

Advantages:

Minimal fragmentation.

Good for nonvolatile molecules.

Limitations:

Low sensitivity.

Complex setup.

E. Fast Atom Bombardment (FAB) Ionization

Principle: The sample is mixed with a liquid matrix and bombarded with high-energy atoms (Xe, Ar),
leading to ionization.

Fragmentation: Moderate fragmentation.

Common Applications:

Polar and nonvolatile compounds.

Advantages:

Suitable for polar compounds.

Limitations:
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Requires a liquid matrix.

Low sensitivity.

4. Applications of Mass Spectrometry (MS)

Mass spectrometry (MS) is a powerful analytical technique used in various fields for qualitative and
quantitative analysis. It helps determine the molecular weight, structure, and composition of
compounds.

1. Biological and Biomedical Applications

A. Proteomics & Protein Analysis

Used to identify and characterize proteins in biological samples.

Helps in post-translational modification (PTM) analysis.

Techniques: MALDI-TOF MS, ESI-MS.

Applications:

Disease biomarker discovery.

Drug target identification.

Vaccine development.

B. Metabolomics & Lipidomics

Used to study metabolites in biological systems.

Identifies metabolic changes in diseases.

Applications:

Understanding metabolic disorders (e.g., diabetes).

Studying lipid profiles in cardiovascular diseases.

C. Clinical Diagnostics

Used for disease diagnosis through biomarker detection.

Helps in detecting inborn errors of metabolism (e.g., phenylketonuria, PKU).

Detects drugs and toxins in forensic toxicology.

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Common techniques: LC-MS, MALDI-TOF MS.

D. Pharmaceutical Drug Analysis

Used in drug discovery and development.

Ensures drug purity and stability.

Helps in pharmacokinetics and pharmacodynamics studies.

Detects drug metabolites in bioavailability studies.

2. Environmental and Food Safety Applications

A. Environmental Analysis

Detects pollutants in air, water, and soil.

Identifies pesticides, herbicides, and heavy metals in the environment.

Used for monitoring industrial emissions.

Common techniques: GC-MS, LC-MS.

B. Food Safety & Quality Control

Detects food contaminants, such as pesticides, toxins, and additives.

Used in adulteration detection (e.g., identifying fake honey or olive oil).

Ensures compliance with food safety regulations.

Common techniques: GC-MS, LC-MS.

3. Forensic Science Applications

A. Drug and Poison Detection

Identifies illicit drugs, toxins, and poisons in biological samples.

Helps in post-mortem toxicology.

Common techniques: GC-MS, LC-MS.

B. Explosives and Chemical Warfare Agent Detection

Detects explosive residues at crime scenes.

Identifies chemical warfare agents (e.g., nerve gases).

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Used in homeland security and defense.

4. Industrial and Material Science Applications

A. Petrochemical Industry

Analyzes crude oil composition.

Identifies impurities in fuels and lubricants.

Helps in developing better fuel formulations.

B. Polymer and Material Analysis

Determines polymer composition and degradation.

Used to characterize synthetic materials.

5. Space Exploration and Geochemistry

Used to analyze extraterrestrial materials (e.g., Martian soil, meteorites).

Helps in studying the chemical composition of planets.

Examples:

NASA's Curiosity Rover uses MS for Martian soil analysis.

Mass spectrometers are used in space probes like Rosetta.

6. Isotope Ratio Analysis

Determines isotopic composition of elements in geological and biological samples.

Used in radiocarbon dating (C-14 dating).

Helps in studying climate change through ice core analysis.

Types of Mass Spectrum in Mass Spectrometry

Mass spectra can be classified based on different factors, such as ionization techniques, scan modes, and
fragmentation patterns.

1. Based on Ionization Mode

A. Positive Ion Mode Spectrum

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Detects positively charged ions (⁺, ⁺, etc.).

Common in Electrospray Ionization (ESI) and Matrix-Assisted Laser Desorption/Ionization (MALDI).

Used for small molecules, peptides, and proteins.

B. Negative Ion Mode Spectrum

Detects negatively charged ions (⁻, ⁻, etc.).

Useful for acidic compounds (e.g., fatty acids, nucleotides, and phospholipids).

Common in Electrospray Ionization (ESI) and Chemical Ionization (CI).

2. Based on Scan Type

A. Full-Scan Mass Spectrum

Detects all ions within a specified mass range (e.g., m/z 50–1000).

Provides an overview of all detected compounds in a sample.

Used in unknown compound identification.

B. Selected Ion Monitoring (SIM) Spectrum

Detects only specific ions of interest (e.g., m/z 182 for caffeine).

More sensitive than full-scan mode.

Used in quantitative analysis of drugs, metabolites, and contaminants.

C. Tandem Mass Spectrum (MS/MS or MSⁿ)

Obtained by fragmenting a selected ion to study its structure.

Common in triple quadrupole and ion trap mass spectrometers.

Used in structural elucidation, peptide sequencing, and targeted analysis.

3. Based on Fragmentation Pattern

A. Molecular Ion Spectrum

Shows the molecular ion peak (M⁺ or ⁺), representing the intact molecule.

Useful for determining molecular weight.

Common in soft ionization techniques like ESI, MALDI, and CI.

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B. Fragmentation Spectrum

Shows peaks of fragment ions after the molecular ion breaks apart.

Common in Electron Ionization (EI) and Tandem Mass Spectrometry (MS/MS).

Used for structural analysis of molecules.

4. Based on Isotopic Distribution

A. Isotopic Mass Spectrum

Shows the isotopic pattern of elements (e.g., Cl, Br, S, Si).

Used to determine the elemental composition of a compound.

B. High-Resolution Mass Spectrum (HRMS)

Obtained from high-resolution instruments (e.g., Orbitrap, TOF-MS).

Measures mass-to-charge ratio with high precision (up to 4–6 decimal places).

Used for exact mass determination and compound identification.

Advantages of mass spectrometry

1. High Sensitivity

Can detect and analyze substances at very low concentrations (picogram or femtogram levels).

Useful for trace analysis in forensic, environmental, and clinical applications.

2. High Specificity

Differentiates compounds based on their exact mass-to-charge ratio (m/z).

Allows identification of isotopes, molecular structures, and complex mixtures.

3. Fast and Efficient Analysis

Provides rapid results, often in seconds to minutes.

Coupled with chromatography (GC-MS, LC-MS), it enables high-throughput screening.

4. Versatile Applications

Used in pharmaceuticals, proteomics, metabolomics, environmental science, and forensics.

Can analyze a wide range of molecules, from small organic compounds to large biomolecules.

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5. Accurate Mass Determination

High-resolution mass spectrometers (e.g., Orbitrap, TOF-MS) provide exact mass measurements.

Essential for identifying unknown compounds and structural elucidation.

Limitations of mass spectrometry

1. Expensive Instrumentation

High initial cost and maintenance expenses.

Requires specialized equipment and expertise.

2. Complex Data Interpretation

Requires advanced knowledge of ionization techniques, fragmentation patterns, and isotopic

distributions.

Data processing and spectral analysis can be time-consuming.

3. Limited Analysis of Some Compounds

Some molecules, especially non-volatile or thermally unstable compounds, may not ionize efficiently.

May require derivatization or special ionization techniques.

4. Destructive Technique

Sample is often consumed or altered during analysis, making repeated measurements difficult.

5. Interference and Matrix Effects

Presence of contaminants, salts, or complex biological matrices can suppress ionization.

May require sample cleanup or use of internal standards for accurate quantification.

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