PRINCIPLE:

Matrix Application:

A sample is mixed with a suitable organic matrix material, often a small organic acid, such as sinapinic
acid or alpha-cyano-4-hydroxycinnamic acid. The matrix plays a crucial role in the process by absorbing
the laser energy and transferring it to the analyte molecules.

Sublimation of Matrix:

As the solid matrix absorbs the laser energy during irradiation, it undergoes sublimation—directly
transitioning from a solid to a vapor phase without passing through the liquid phase. This sublimation
process generates a plume of vaporized matrix molecules, carrying the embedded analyte molecules
with them.

Sample Desorption/Ionization:

The mixture of sample and matrix is deposited on a solid support, such as a metal plate.

A pulsed laser is used to irradiate the sample-matrix mixture. The matrix absorbs the laser energy and
becomes highly excited, causing the analyte molecules to be desorbed and ionized in the process.

Ionization Mechanism:

The ionization mechanism in MALDI is primarily attributed to the photochemical and thermal processes
that occur upon laser irradiation.

The high-energy laser pulse causes the vaporized matrix molecules to become ionized, generating a
plume of ions. These ions then transfer their charge to the analyte molecules, leading to the formation
of analyte ions.

Ion Detection:

The resulting ions, now carrying a positive charge, are accelerated into the mass spectrometer.

The mass spectrometer separates these ions based on their mass-to-charge ratio (m/z) and detects
them, generating a mass spectrum that represents the distribution of ions in the sample.

In summary, the sublimation of the matrix is a crucial step in MALDI, enabling the desorption of analyte
molecules from the solid phase and contributing to the overall ionization process. This combination of
sublimation, desorption, and ionization makes MALDI a powerful technique for the mass spectrometric
analysis of large biomolecules.

ESI

1. Sample Preparation:
 The sample is dissolved in a volatile solvent, typically a mixture of water and
organic solvents like methanol or acetonitrile.
2. Formation of Taylor Cone:
 The sample solution is introduced into a capillary tube or a metal needle.
  A high voltage (typically in the range of 1-5 kV) is applied to the solution, leading
to the formation of a Taylor cone at the tip of the capillary.
3. Spraying of Charged Droplets:
 As the electric field becomes stronger, the surface tension of the solution
overcomes, and charged droplets are formed at the tip of the Taylor cone.
 These charged droplets are usually positively or negatively charged, depending
on the polarity of the applied voltage.
4. Evaporation of Solvent:
 As the charged droplets move towards the mass spectrometer inlet, the solvent
in the droplets begins to evaporate.
 The process of evaporation leaves behind highly charged ions of the analyte.
5. Formation of Gas-Phase Ions:
 Further evaporation and reduction in droplet size result in the formation of gas-
phase ions.
 The gas-phase ions generated are typically multiply charged due to the removal
of several protons or other charged species during the evaporation process.
6. Ion Detection:
 The resulting gas-phase ions are introduced into the mass spectrometer.
 Inside the mass spectrometer, these ions are separated based on their mass-to-
charge ratio (m/z) and are detected to generate a mass spectrum.

Types of Mass Analyzers

A Magnetic Sector Analyzer (MSA) is a type of mass spectrometer that uses a magnetic
field to separate ions based on their mass-to-charge ratio (m/z). It is one of the earliest
and fundamental types of mass analyzers used in mass spectrometry.

Principle of Magnetic Sector Analyzer:

1. Ionization:
 The sample is ionized, often through techniques like electron impact ionization or
laser ionization, depending on the instrument.
2. Acceleration:
 The resulting ions are accelerated by an electric field, giving them kinetic energy.
3. Magnetic Field Deflection:
 The ions then enter a magnetic field that is perpendicular to their direction of
motion.
 According to the Lorentz force law, the force experienced by a charged particle
moving in a magnetic field is perpendicular to both the particle's velocity and the
magnetic field. This force causes the ions to move in a circular or helical path.
4. Centripetal Force Equilibrium:
 The centripetal force required to keep the ions in their circular or helical path is
provided by the magnetic field. This force is balanced by the kinetic energy of the
ions.
5. Radius of Curvature:
 The radius of curvature of the ion's path is directly proportional to the mass-to-
charge ratio (m/z) of the ion. Heavier ions or ions with higher charge states have
larger radii.
6. Ion Detection:
 Ions of different masses are spatially separated based on their radii of curvature.
 A detector is positioned at different points along the ion trajectory, allowing ions
of specific masses to be detected at specific locations.
7. Data Analysis:
 The data collected from the detector is used to generate a mass spectrum, which
represents the abundance of ions at different mass-to-charge ratios.

The Magnetic Sector Analyzer provides high resolution and precision in mass analysis,
making it suitable for accurate determination of mass and isotopic composition.
However, it tends to be slower compared to more modern mass analyzers, such as time-
of-flight (TOF) or quadrupole mass analyzers. Despite this, magnetic sector analyzers are
still utilized in certain applications where high resolution and accuracy are critical.

SLIDE 39:
SLIDE 40:
TOF:
SLIDE 46:
 A Quadrupole Analyzer, specifically a quadrupole mass spectrometer, is a type of mass
spectrometer that uses radiofrequency (RF) and direct current (DC) electric fields to
selectively transmit ions based on their mass-to-charge ratio (m/z). The quadrupole
mass analyzer is a widely used and versatile instrument in mass spectrometry.

Principle of Operation:

1. Ionization:
 The sample is ionized using methods such as electron impact, chemical
ionization, or laser ablation, depending on the application.
2. Ion Acceleration:
 Ions are accelerated into the quadrupole mass filter.
3. Quadrupole Configuration:
 The quadrupole mass analyzer consists of four parallel metal rods arranged in a
square or rectangular configuration.
 These rods are connected to both RF and DC power supplies.
4. Radiofrequency (RF) Field:
 The RF field is applied to the rods, creating a rapidly oscillating electric field.
5. DC Voltage:
 A DC voltage is superimposed on the RF field, creating a combined electric field.
6. Ion Motion:
 The combined electric field acts as a filter, allowing only ions with specific mass-
to-charge ratios to traverse the quadrupole rods.
 The oscillating RF field causes ions to undergo a stable trajectory through the
quadrupole while other ions are ejected.
7. Stability Region:
 Ions with a particular mass-to-charge ratio, within a stability region determined
by the settings of the RF and DC voltages, pass through the quadrupole and
reach the detector.
8. Mass Scan:
 By scanning the RF and DC voltages over a range, different mass-to-charge ratios
are selectively transmitted at different times, allowing for a mass spectrum to be
generated.

Key Points:

 The quadrupole mass analyzer selectively transmits ions with specific mass-to-charge
ratios based on their stability in the oscillating electric field.
 Adjusting the RF and DC voltages allows for scanning through different mass-to-charge
ratios, enabling the measurement of a complete mass spectrum.
 Quadrupole mass spectrometers are versatile, offering good mass resolution and are
commonly used in various analytical applications, including environmental analysis,
pharmaceuticals, and proteomics.

Fourier Transform Ion Cyclotron Resonance (FT-ICR) mass spectrometry is a high-

resolution mass spectrometry technique that uses a strong magnetic field and
radiofrequency (RF) pulses to analyze ions. It is known for its exceptional mass accuracy
and resolving power. Here's an overview of the principle of FT-ICR:

Principle of FT-ICR:

1. Ion Cyclotron Motion:

 Ions generated by ionization methods like electrospray or laser ablation are
injected into a strong, homogeneous magnetic field.
2. Cyclotron Motion in Magnetic Field:
 The magnetic field causes ions to move in circular or helical paths, a
phenomenon known as cyclotron motion.
3. RF Excitation:
 A radiofrequency (RF) electric field is applied perpendicular to the magnetic field,
causing the ions to oscillate or "cyclotron resonate."
4. Induced Electric Current:
 The cyclotron motion induces an electric current in detection electrodes
orthogonal to both the magnetic and RF fields.
Induced Electric Current:
 The circular motion (cyclotron motion) of ions in the magnetic and
radiofrequency fields induces an electric current in detection electrodes.
 This current flows in a direction perpendicular to both the magnetic and
radiofrequency fields. (FORUNDERSTANDING)

5. Signal Detection:
 The induced current is detected as a time-domain signal.
 The signal is a superposition of individual frequencies corresponding to different
ions in the sample.

Signal Detection:
 The electric current generated by the ions' motion is detected as a signal that varies
over time (time-domain signal).
 This signal is like a musical chord, combining different frequencies.
 Each frequency in the signal corresponds to a different type of ion present in the
sample. (FORUNDERSTANDING)

6. Fourier Transform:
 The time-domain signal is then subjected to Fourier Transform (FT).
 Fourier Transform converts the time-domain signal into a frequency-domain
spectrum.
7. Mass Calculation:
 The resulting frequency spectrum provides information about the masses of ions
present in the sample.
 High-resolution and mass accuracy are achieved by the precise measurement of
the frequencies of ion motion.

Key Features:

 High Resolution: FT-ICR mass spectrometers can achieve very high mass resolution,
often exceeding 1 million.
 High Mass Accuracy: The method allows for extremely accurate determination of the
mass-to-charge ratio of ions.
 Versatility: FT-ICR is versatile and can be used for various ionization techniques, making
it suitable for analyzing a wide range of compounds.