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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5647_Библиотеки_им_академика_М_И_Перельмана.pdf
9.3 Instrumentation 381
9.3.2.10.4 Disadvantages of MALDI
• Matrix interference: The choice of matrix can impact the ionization efficiency
and detection of the analyte, potentially leading to matrix interference.
• Limited structural information: While MALDI provides mass information, it
does not offer detailed structural insights. Complementary techniques such as
tandem mass spect rometry (MS/MS) may be required for structural elucidation.
• Not suitable for volatile compounds: MALDI is not suitable for small volatile
molecules due to their tendency to evaporate during the sample preparation
process.
9.3.2.10.5 Applications of MALDI
• Proteomics: MALDI is extensively used in proteomics
for protein
identification,
characterization, and quantification through techniques such as peptide mass
fingerprinting and protein profiling.
• Metabolomics: The technique is utilized for
analyzing
metabolites in biological
samples, aiding in the understanding of metabolic pathways and disease
mechanisms.
• Glycomics: MALDI facilitates
the study of
glycans and glycoproteins, providing
insights into glycosylation patterns and their biological significance.
• Clinical diagnostics: MALDI is increasingly applied in clinical diagnostics,
including the identification of pathogens and biomarkers associated with diseases.
Figure 9.4 briefl
Fig. 9.4 Schematic representation of mode of action of MALDI in mass spectrometry. This figure
illustrates the working mechanism of MALDI in mass spectrometry. The process begins with the
analyte (orange spots) co-crystallized with a matrix (green spots) on a sample plate. When a laser
beam is applied to the matrix–analyte mixture, it causes desorption, releasing both matrix and
analyte molecules into the gas phase. Next, the matrix molecules absorb the laser energy, leading to
desolvation and ionization of the analyte molecules. A proton transfer occurs from the matrix to the
analyte, resulting in the formation of positively charged analyte ions. These ions are then
accelerated toward the mass analyzer for detection and further analysis. This technique is widely
used due to its ability to ionize large, nonvolatile biomolecules without significant fragmentation
y explai
ns the mechanism of action of MALDI.
382 9 Comprehensive Insights into Mass Spectrometry
9.3.2.10.6 Mechanism of Ionization in MALDI
The mechanism of ionization in MS involves converting a sample into ions, which
are
then analyzed based on their mass-to-charge ratio (m/z). There are several
ionization techniques in MS, each with its own mechanism.
• Protonation: When a proton is added to a molecule then it increases the positive
charge.
M þ H
þ
→ MH
þ
:
It is used in MALDI, electrospray ionization, and APCI. The samples used are
carbohy
• Deprotonation: A proton is removed from a molecule, which causes
drates.
production
of cations.
M - H
þ
→ M - HðÞ
þ
:
It can be used in MALDI, APCI, and electrospray ionization. The sample used in
this
process is salicylic acid.
• Cationization: This can be done by
the addition of cation into the molecule along
with an ammonium or alkali. This is a very stable method as compared to that of
protonation.
M cation → Mcation
þ
þ
:
It is used in MALDI, APCI, and electrospray ionization. The sample used is
D-galact
ose.
• Charge transfer: This is also called as desorption. In this method the sample
solution
is converted into gas state. It is particularly used for charged complexes.
This method cannot be used for many other compounds.
It is used
phosphine.
þ
solution → Mþ gas :
ðÞðÞ
M
in MALDI and electrospray ionization. The sample used are tetraphenyl
9.3 Instrumentation 383
• Electron ejection: This can be done by removing the electron from molecule to
form the positively charged molecule. It is used in process of electron ionization.
The sample used are anthracene.
M
- e
→ M
þ
• Electron capture: Addition of electrons to the sample molecule by absorption or
capture.
M
þe
→ M
-
• Electrostatic system: The cations produced from the ionization source are allowed
to
pass through the electric field. Electric field is produced between the repeller
plate and the accelerator plate, which causes the acceleration of the ions of masses
, m2, and m3 to their final velocities.
m
1
Energy eV = ½m
2 2 2
= ½m2v2 = ½m3v
1v1
3
• Ion separator: This is commonly called as analyzer in which sample molecules
are separated on the basis of their masses.
9.3.3 Mass Analyzer
The mass analyzer is a crucial part of the instrument, responsible for separating ions
based on their m/z ratio. Mass spectrometers use various types of analyzers to
separate and measure ions based on their mass-to-charge ratio (m/z). The following
sections provide some common types of analyzers used in mass spectrometry.
9.3.3.1 Single Focusing Analyzer (FSA)
A single focusing analyzer (SFA) is a type of magnetic sector mass analyzer. In this
system, ions are accelerated by an electric field and then pass through a magnetic
field that separates them based on their m/z. The magnetic field bends the trajectory
of the ions, with lighter ions being deflected more than heavier ones. Only ions of a
specific m/z follow a curved path to reach the detector.
9.3.3.1.1 Components
• Ion source: Produces the ions that will be analyz ed.
• Magnetic sector:
Separates the ions based on their mass-to-charge ratios by
deflecting them according to their momentum.
• Detector: Measures the ions that successfully pass through the magnetic sector.
9.3.3.1.2 Advantages:
• Good resolution for relatively simple mixtures.
• Reliable for precise
mass determination.
384 9 Comprehensive Insights into Mass Spectrometry
9.3.3.1.3 Limitations
Not as high in resolution or sensitivity as modern mass analyzers such as quadrupole
or TOF analyzers.
9.3.3.1.4 Applications:
• Used in early mass spectrometry systems for elemental analysis and isotopic
studies.
• Suitable for simple organic compounds and isotope ratio determinations.
9.3.3.2 Double Focusing Analyzer (DFA)
The double focusing analyzer (DFA) combines both an electrostatic field and a
magnetic field to achieve higher resolution in mass spectrometry. This design
compensates for differences in ion energy, leading to more accurate separation of
ions based on their mass-to-charge ratio (m/z). The electrostatic field corrects energy
dispersion, while the magnetic field deflects ions according to their momentum, thus
achieving “double focusing” for precise mass determination.
9.3.3.2.1 Components
• Ion source: Produces the ions.
• Electrostatic sector: Focuses ions with varying kinetic energies to ensure they
travel at the same velocity.
• Magnetic sector: Separates ions b
ased on thei
r m/z by deflecting them according
to their momentum.
• Detector: Collects and measures the ions that are successfully focused.
9.3.3.2.2 Advantages
• High resolution due to the combination of energy and momentum focusing.
• Precise mass measurement, especially for complex mixtures.
• Greater accuracy than single focusing analyzers.
9.3.3.2.3 Limitations
• More complex and expensive than single focusing analyzers.
• Slower scanning speed compared to time-of-flight (TOF) or quadrupole mass
analyzers.
9.3.3.2.4 Applications
• High-resolution mass spectrometry
etailed molecular structure analysis.
for d
• Used in isotopic and elemental analysis, especially in research settings.
• Ideal for applications requiring precise mass measurements, such as organic and
inorganic compound studies.
9.3.3.3 Time-of-Flight (TOF) Analyzer
The time-of-flight (TOF) analyzer operates by measuring the time it takes for ions to
travel a fixed distance to the detector. Ions are accelerated by an electric field to the
same kinetic energy, and their velocities depend on their m/z. Lighter ions travel
9.3 Instrumentation 385
faster, while heavier ions take longer to reach the detector, allowing separation based
on their mass.
9.3.3.3.1 Components
• Ion source: Generates ions from the sample.
• Accelerating region: An electric field accelerates ions to the same kinetic energy.
• Flight tube: A field-free region where ions travel based on their m/z ratio.
• Reflectro n (optional): A device used to reflect
dispersio
n, increa
• Detector: Measures the time it
sing resolution.
takes for
ions to reach it and converts that
ions and
correct for energy
information into mass data.
9.3.3.3.2 Advantages
• High-sp eed analysis with a wide mass range.
• Suitable for large biomolecu
les such as
proteins and peptides.
• High sensitivity and can be coupled with other ionization methods such as
MALDI or ESI.
9.3.3.3.3 Limitations
• Limited mass resolution compared to more advanced analyzers such as Orbitrap
or FT-ICR.
• Accuracy depends on precise calibration and consistent ion velocities.
9.3.3.3.4 Applications
• Widely used in proteomics and metabolomics for large biomolecule analysis.
• Commonly paired with MALDI for protein and peptide identification.
• Environmental monitoring and trace
detection
due to its high sensitivity.
Figure 9.5 reflects the schematic representation of TOF. Matrix-assisted laser
desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry: MALDI
involves laser bombardment of sample molecules for ionization. Samples are
premixed with a highly absorbing matrix compound, which converts laser energy
into excitation energy, leading to sputtering of analyte and matrix ions from the
mixture’s surface. MALDI-TOF mass spectrometry combines MALDI as the ion
source with a time-of-flight (TOF) analyzer (Fig.
9.6).
9.3.3.3.5 Comparison Between MALDI and TOF mass spectrometry
Aspect MALDI TOF
Purpose Ionization technique for generating
Ionization
method
ions from large biomolecules and
polymers
Utilizes a matrix to absorb laser
energy, leading to the desorption and
ionization of analytes
Mass analysis technique that
measures the time it takes ions to
travel a speci fic distance
Ions are accelerated by an electric
field and then travel through a
vacuum
(continued)
386 9 Comprehensive Insights into Mass Spectrometry
Fig. 9.5 Schematic representation of TOF analyzer. This figure demonstrates the key components
and working mechanism of a time-of-flight (TOF) mass analyzer, commonly used in mass spectrometry. The process begins
The ions are then accelerated by an electrical field (Step 2) into the flight tube. Inside the flight tube,
ions travel based on their mass-to-charge ratio (m/z), with lighter ions moving faster and reaching
the detector first (Step 3). A laser beam is used to initiate the ionization process, and a beam splitter
directs a portion of the laser to a clock, triggering a timer as soon as ionization begins (Step 4). This
time measurement, along with the distance traveled by the ions, allows for the calculation of the
mass of the ions, which is recorded by the transient recorder. The detector collects the ions,
completing the process of analysis
with the ionization of the protein sample in the ion source (Step 1).
Aspect MALDI TOF
Sample
preparation
Type of ions
produced
Analyte size
range
Resolution Moderate to high resolution depending
Sensitivity High sensitivity, especially for large
Fragmentation Minimal fragmentation occurs during
Typical
applications
Requires mixing the analyte with a
matrix material before analysis
Primarily produces singly charged
ions from large molecules
Effective for large biomolecules (e.g.,
proteins, peptides)
on the TOF setup used after MALDI
ionization
biomolecules
ionization
Proteomics, biomolecule
polymer characterization
analysis,
Ions generated from any source,
including MALDI, can be analyzed
using TOF
Can analyze both singly and
multiply charged ions
Capable of analyzing a wide range of
molecule sizes
Very high resolution due to the time
measurement of ion travel
Generally, very sensitive for a wide
range of analytes
Provides options for tandem MS
(MS/MS) for fragmentation analysis
(continued)
9.3 Instrumentation 387
Fig. 9.6 Schematic representation of MALDI-TOF. This schematic illustrates the working principle of MALDI-TOF mass spectrometry. The sample is placed on a sample slide, and a laser beam,
guided by a focusing lens, strikes the matrix on the sample, causing desorption and ionization of the
analytes. The resulting ions are accelerated through an ion acceleration field into the TOF analyzer.
In the TOF region, ions travel based on their mass-to-charge ratios (m/z), with lighter ions reaching
the detector faster than heavier ones. The detector records the ions’ arrival time, generating a
time vs. intensity spectrum that is used to identify the mass of the analytes. This technique is widely
used for analyzing biomolecules such as proteins and peptides (Source: Google images)
Aspect MALDI TOF
Wide applications in small molecule
analysis, environmental testing, and
proteomics
9.3.3.4 Quadrupole Analyzer
The quadrupole analyzer separates ions based on their m/z ratio using oscillating
electric fields. It consists of four parallel rods, with alternating radio frequency
(RF) and direct current (DC) voltages applied. Only ions with a specific m/z ratio
can pass through the quadrupole and reach the detector, while others are filtered out.
9.3.3.4.1 Components
• Ion source: Generates ions from
• Quadrupol e rods: Four
paral
lel metal rods with alternating RF and DC voltages
create a dynamic electric field (Fig.
• Ion detector: Detects the
ions that successfully traverse the quadrupole, allowing
the samp
le (e.g., ESI, APCI).
9.7).
for mass analysis.
388 9 Comprehensive Insights into Mass Spectrometry
Fig. 9.7 Schematic representation of quadrupole analyzer in mass spectroscopy. This diagram
illustrates the working mechanism of a quadrupole mass analyzer used in mass spectrometry. Ions
generated from the source enter the quadrupole, where they are subjected to a combination of
oscillating radiofrequency (RF) and direct current (DC) electric fields applied to four cylindrical
rods. The resonant ions, which have specific m/z ratios, follow a stable trajectory and reach the
detector. In contrast, non-resonant ions experience unstable oscillations and are filtered out. By
varying the RF and DC voltages, the quadrupole can selectively allow ions of a particular m/z ratio
to pass through to the detector, enabling mass analysis of the sample
9.3.3.4.2 How it Works
• The RF/DC voltages create an oscillating electric field that selectively stabilizes
ions with certain m/z values.
• Ions with stable trajectories pass through the quadrupole, while others are
destabilized and filtered out.
• By adjusting the RF/DC ratio, different m/z values can be scanned sequentially.
9.3.3.4.3 Advantages
• Fast scanning speeds for real-time analysis.
• Capable of filtering specific ions for targeted analysis.
• Compact and cost-effective compared to other mass analyzers.
9.3.3.4.4 Limitations
• Lower mass resolution and accuracy compared to TOF, Orbitrap, or FT-ICR
analyzers.
• Limited to detecting ions within a narrower mass range.
9.3.3.4.5 Applications
• Commonly used in
tandem mass spectrometry (MS/MS) for fragment ion
analysis.
9.3 Instrumentation 389
• Ideal for quantitative analysis in drug discovery, pharmacokinetics, and clinical
diagnostics.
• Widely employed in environmental and food safety testing for contaminant
detection.
9.3.3.5 Fourier-Transform Ion Cyclotron Resonance (FT-ICR) Analyzer
The Fourier-transform ion cyclotron resonance (FT-ICR) analyzer operates on the
principle of ion motion in a magnetic field. Ions are trapped in a magnetic field and
move in circular (cyclotron) paths. The ir cyclotron frequencies are directly related to
their m/z ratios. By detecting these frequencies, FT-ICR can precisely determine the
m/z values of ions.
9.3.3.5.1 Components
• Ion source: Generates ions (e.g., from MALDI, ESI, or other ionization sources).
• Magnetic field: A strong magnetic field traps ions, causing them to move in
cyclotron orbits.
• Trapping electrodes: Confining ions in the magnetic field for
precise
measurement.
• Detection
via Fourier transform to generate mass spect
system: Measures the ion cyclotron frequency, which is transformed
ra.
9.3.3.5.2
How it Works
1. Ions are introduced into the magnetic field, where they follow circular orbits due
to the Lorentz force.
2. The frequency of their cyclotron motion is inversely proportional to the m/z ratio.
3. A radiofrequency pulse excites the ions, and their cyclotron frequencies are
detected.
4. Fourier transformation of the frequency data provides the ion masses, generating
a mass spectrum.
9.3.3.5.3 Advantages
• High mass resolution: FT-ICR offers one of the highest resolutions among mass
analyzers, enabling the distinction of ions with very close m/z values.
• Mass accuracy: It provides extremely precise m/z determinations, often to within
a few parts per billion (ppb).
• Broad mass range: Can detect ions over a wide mass range, from small molecules
to large biomolecules such as proteins and peptides.
• Multiple ion trapping: Capable of trapping and analyzing multiple ions
simultaneously.
9.3.3.5.4 Limitations
• Cost and complexity: FT-ICR systems are expensive and require strong magnetic
fields (often superconducting magnets).
390 9 Comprehensive Insights into Mass Spectrometry
• Slow analysis time: Compared to quadrupoles or TOF analyzers, FT-ICR is
slower due to the complex ion trapping and detection process.
• Size: The requirement of large magnets makes FT-ICR systems bulky.
9.3.3.5.5 Applications
• Proteomics and metabolomics: Provides high-resolution analysis of proteins and
metabolites.
• Complex mixtures: Ideal for resolving complex mixtures, such as in petroleum
analysis or environmental studies.
• Structura l elucidation: Used for detailed structural information, especially in
biomolecular research.
• Isotopic analysis: Effective in studying isotopic distributions and elemental
compositions.
9.3.3.6 Ion Trap Analyzer
The ion trap analyzer operates by trapping ions in a three-dimensional electric field,
where they oscillate in stable orbits. By systematically altering the elect ric field, ions
of specific m/z ratios are sequentially ejected for detection, enabling mass analysis.
9.3.3.6.1 Types of Ion Traps
1. Quadrupole ion trap (3D ion trap): Uses a combination of radiofrequency
(RF) and direc t current (DC) electric fields to trap ions.
2. Linear ion trap: An extension of the quadrupole ion trap, where ions are trapped
along the axis of a linear RF field.
3. Orbitrap: Traps ions in a harmonic electric field between electrodes, where ions
oscillate and generat
e freque
ncies directly related to their m/z ratios.
9.3.3.6.2 Components
• Ring electrode: Generates the electric field that traps ions.
• End-cap electrodes: These are located at both
ends of
the ion trap and help control
ion movement.
• RF generator: Provides the radiofrequency field to maintain ion trapping.
9.3.3.6.3 How it Works
1. Ions generated from the ion source are introduced into the ion trap.
2. An RF voltage is applied, creating an oscillating electric field that traps the ions in
stable orbits.
3. The RF field is incrementally adjusted, destabilizing ions of specific m/z ratios,
causing them to be ejected from the trap.
4. Ejected ions are detected sequentially, producing a mass spectrum based on their
m/z ratios.
9.3.3.6.4 Advantages
• High sensitivity: Can
trap and analyze ions over extended periods, increasing
detection sensitivity.