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2. EM spectrum and
Interaction of
EMR with matter
What is
spectroscopy?
What is Mass
spectroscopy?
Instrumentation
of MS.
What is
Chromatography?
What is Gas
chromatography?
What is GC-MS? Case study
Contents:
3. What is
Spectroscopy?
Spectroscopy is basically an experimental
subject and is concerned with the
absorption, emission or scattering of
electromagnetic radiation by atoms or
molecules.
Spectroscopic techniques are one of the
main sources of molecular geometries, that
is, bond lengths, bond angles, and torsion
angles, and can also yield, as will be seen,
significant information about molecular
symmetry, energy level distributions,
electron densities, or electric and magnetic
properties
4. Electromagnetic Radiation
Electromagnetic radiation includes, in addition to what we commonly refer to as ‘light’, radiation of longer and
shorter wavelengths. As the name implies it contains both an electric and a magnetic component which are
perpendicular to each other and to the direction of propagation of the wave.
The distance between two points of the same phase in
successive waves is called the "wavelength”, λ.
1 Å = 10−8
𝑐𝑚 = 10−1
𝑛𝑚
The frequency, ν, is the number of waves in the distance
light travels in one second
𝜈 =
𝐶
𝜆
(𝑠𝑒𝑐𝑜𝑛𝑑−1
)
The third parameter, which is most common to vibrational
spectroscopy, is the "wavenumber," 𝝂.
𝜈 =
𝜈
𝑐
=
1
𝜆
(𝑐𝑚−1
)
5. From the early work of Bohr on atomic spectra, it could be established that absorption or emission of
radiation is possible because of the quantization of atomic and molecular energy levels. If a molecule
interacts with an electromagnetic field, a transfer of energy from the field to the molecule can occur
only when Bohr's frequency condition is satisfied. Namely,
Δ𝐸 = ℎ𝜈 = ℎ
𝑐
𝜆
= ℎ𝑐𝜈
Here Δ𝐸 is the difference in energy between two quantized states, h is Planck's constant (6.626 ×
10−27
erg s) and c is the velocity of light. Thus, 𝜈 is directly proportional to the energy of transition.
Suppose that,
Δ𝐸 = 𝐸2 − 𝐸1
where 𝐸2 and 𝐸1 are the energies of the excited and ground states, respectively. Then, the molecule
"absorbs" Δ𝐸 when it is excited from 𝐸1 to 𝐸2 , and "emits" Δ𝐸 when it reverts from 𝐸2 to 𝐸1 .
Finally, we have
Δ𝐸 = 𝐸2 − 𝐸1 = ℎ𝜈 = ℎ
𝑐
𝜆
= ℎ𝑐𝜈
𝐸2 (Excited state)
𝐸1 (Ground state)
Δ𝐸
Absorption Emission
6. Interaction of Electromagnetic Radiation with
Matter:
Electromagnetic
radiation can
interact with
matter in
various ways,
including
absorption,
reflection, and
transmission.
Absorption of electromagnetic radiation increases particle energy, causing heating or chemical reactions.
Absorption amount depends on material properties like density, conductivity, and structure. Metals are
good absorbers; glass is not.
Reflection is electromagnetic radiation bouncing off a material. Reflectivity, the fraction reflected, depends
on material properties and angle. Mirrors have high reflectivity; paper reflects poorly.
Transmission is radiation passing through a material without absorption. Transmittance, the fraction
transmitted, depends on material properties and thickness. Air and glass are good transmitters; metal is
poor.
Emission of secondary radiation includes fluorescence (immediate re-emission) and phosphorescence
(delayed re-emission). Ionization occurs when radiation removes electrons, forming ions. Excitation matches
energy levels, leading to emission.
Scattering, deflection of radiation, occurs in forms like elastic, inelastic, and Raman scattering. Elastic
conserves energy; inelastic results in different energy. Raman involves energy transfer to/from material
vibrational energy.
Creation or destruction of particles, e.g., pair production, happens with high-energy radiation generating
particle-antiparticle pairs from the vacuum.
7. Electromagnetic Spectrum
• The electromagnetic spectrum, or EM spectrum, is the name given to the
collection of all electromagnetic radiation in the universe. This is a type of
energy that pervades the cosmos in the form of electric and magnetic waves,
allowing for the transfer of energy and information. The EM spectrum is a range
of frequencies that corresponds to all different forms of electromagnetic
radiation in the universe. It begins at the highest frequencies, where the waves
are most stretched out (low frequency) to very tightly packed waves (high
frequency).
• These frequencies correspond to different levels of radiation, which is the
transmission of energy through the universe in the form of waves and particles.
Lower frequency radiation has much longer wavelengths, meaning the distance
between the waves of radiation is long, up to many kilometers. At the other
end, higher frequency radiation has very short wavelengths, trillionths of a
meter long.
• The type of radiation emitted by an object depends on its temperature. Things
that are colder emit radiation at lower frequencies and thus longer
wavelengths. Conversely, things that are hotter emit radiation at higher
frequencies and shorter wavelengths.
8. What is Mass Spectroscopy ?
• Mass spectroscopy is a crucial technique in analytical
chemistry, requiring minimal samples for microanalysis (few
nanomoles).
• Involves ionization, separation, and measurement of ionized
molecules and their products.
• Despite its destructive nature, it provides unique mass
spectra, serving as chemical fingerprints for sample
characterization.
• Used to determine molecular mass, quantify known
compounds, and elucidate compound structures.
• Converts samples into gaseous ions, characterizing them by
mass and charge ratios (m/z) and relative abundance.
• Applied for both pure samples and complex mixtures.
This Photo by Unknown Author is licensed under CC BY-SA-NC
9. Mass Spectrometry: Basics
Principle:
• Molecules bombarded with energetic electrons.
• Ionization and fragmentation occur, producing positive ions.
• Each ion has a specific mass-to-charge ratio (m/e ratio).
• For most ions, the charge is one, making the m/e ratio equal to the
molecular mass.
• Ions traverse magnetic and electric fields.
• Detected at the detector, signals recorded for mass spectra.
Theory:
• Mass spectroscopy examines characteristic fragments (ions) from the
breakdown of organic molecules.
• A mass spectrum plots ion abundance against the mass/charge ratio.
• In organic mass spectrometry, energetic electrons bombard vapor to form
positively charged molecular ions.
10. • Electron energy breaks molecular bonds, causing fragmentation into neutral or positively
charged species.
• Fragmentation may result in the formation of even electron ions and radicals.
• Positive ions formed are accelerated and deflected by magnetic or electric fields based on mass,
charge, and velocity.
• Multiple beams with the same m/z values are obtained.
• The resulting beams strike a photographic plate, creating separate lines with recorded intensity.
• Mass spectra visually present m/z values against relative abundance, with the most abundant ion
as 100% (base peak).
• Unlike IR, NMR, and UV, mass spectrometry involves no selective absorption of radiation.
• Mass spectrometry induces irreversible chemical changes, unlike the reversible physical
changes in other methods.
• Mass spectral reactions are more drastic than typical chemical reactions.
11. The Mass
Spectrometer
Parts of a mass spectrometer:
1. Inlet device
2. Ion Source
3. Analyzer system
4. Detector
5. Vacuum system
12. Types of Mass Spectrometer
Ionisation
Ion Source
Ion separation
Mass Analyzer
Ion Detection
Detector
Depending upon the methods of ionization, ion separation and detection, there are various types of mass
spectrometers as shown.
• Electron Ionisation (EI)
• Chemical Ionisation (CI)
• Fast Atom
Bombardment (FAB)
• Electrospray Ionisation
(ESI)
• Matrix-Assisted Laser
Desorption/Ionisation
(MALDI)
• Quadrupole
• Magnetic Sector Field
• Electric Sector Field
• Time of Flight (TOF)
• Ion Trap
• Electron multiplier
• Faraday Cup
• Multichannel plate
• Tandem MS
13. Ion Sources
• Electron Ionization.
• Chemical Ionization.
• Field Ionization/Desorption.
• Plasma Desorption.
• Fast Atom Bombardment/Secondary
Ion Mass Spectrometry.
• Matrix-Assisted Laser
Desorption/Ionization.
• Electrospray Ionization.
• Thermospray Ionization.
• Atmospheric Pressure Chemical and
Photo-Ionization.
14. Electron Ionization (EI)
• Heated filament emits electrons towards an anode.
• Optimal wavelength for organic molecules: 70 eV kinetic
energy, 1.4 Å.
• Energy transfer during interaction with analyte molecules leads
to ionization.
• Compact source design with sample input, electron
inlet/outlet, and ion ejection gap.
• Magnetic field guides electrons for enhanced analyte
interaction.
• Vacuum required for ionization; samples heated for gas phase
transition.
• Negative ions generated through electron capture with voltage
reversal.
• Electrons are associated with a wave whose wavelength is
given by;
𝜆 =
ℎ
𝑚 ⋅ 𝑣
15. Chemical Ionization
(CI)
• Chemical ionization (CI): low-energy technique,
minimizes fragmentation, aids easy recognition
of molecular species.
• Complementary to electron ionization, CI
produces ions through collisions with primary
ions in the source.
• Ionization plasma forms through subsequent
reactions, generating positive and negative ions
of the substance.
• Produced ions, termed ions of the molecular
species or pseudomolecular ions, facilitate
molecular mass determination.
• Molecular ions in CI refer to M•+
𝑜𝑟 M•−
ions.
In the first step, the reagent gas is ionized, followed by a proton transfer to
the analyte:
16. Field Ionization/desorption (FI/FD)
Field Ionization (FI)
High positive electric potential at a pointed electrode
creates a potential gradient, causing molecular orbitals
distortion and electron tunneling, leading to the formation
of a positive ion [M]•+
The formed positive ion is repelled by the positive
electrode (emitter) and enters the mass spectrometer.
Field Desorption (FD)
FD is closely related to FI and is employed for studying
non-volatile compounds. The sample is directly coated on
the emitter via solution evaporation, and the application
of a field in high vacuum induces desorption of intact
molecular ions from regions of high field strength.
FD spectra are dominated by the [M]^+ ion for neutral
compounds, while singly charged salts provide
[𝐶𝑎𝑡𝑖𝑜𝑛]+
𝑜𝑟 [𝐴𝑛𝑖𝑜𝑛]−
as base peaks in positive and
negative ion modes, respectively.
Both FI & FD are ‘soft’ ionization techniques, minimizing
energy imparted to the molecule, leading to reduced
fragmentation, and often producing an abundant [M]•+
peak.
17. Fast Atom Bombardment & Secondary
Ion MS (FAB/SIMS)
FAB and SIMS employ high-energy atoms for one-step sputtering and
ionization, focusing either a rare gas beam (FAB) or an ion beam (SIMS)
on liquid or solid samples.
Effective for compounds with molecular weights up to a few 10,000 Da,
especially useful for thermally labile compounds.
FAB requires analyte dissolution in a liquid matrix, while SIMS, used for
surface species and solid samples, doesn't use a matrix.
SIMS is sensitive for surface chemistry and materials analysis but can be
challenging to quantify results.
FAB: Inert gases, e.g., Ar or Xe, are ionized and accelerated to 5 keV, with
radicals neutralized at the exit, maintaining momentum as neutral
species.
SIMS: Primary ion beam: 𝐶𝑠+ ions generated by heating a cesium salt
pellet, accelerated to 30 keV, and focused by lenses onto the sample-
containing target.
The fast-moving beam of atoms/ions blasts the matrix and analyte into
the gas phase. The secondary ions that are mass analysed either are
originally charged or acquire a positive charge from protonation (or
association with another charged species such as 𝑁𝑎+
) or a negative
charge by deprotonation
18. Plasma
desorption (PD)
• Plasma Desorption (PD)
introduced by Mcfarlane and
Torgesson utilizes a small
aluminized nylon foil.
• In PD, the sample on the foil is
exposed to the fission
fragments of 𝐶𝑓252
, inducing
shock waves that desorb
neutrals and ions, enabling
observation of ions above
10,000 Da. Nowadays, it has
limited use and is primarily
replaced by matrix-assisted
laser desorption ionization.
19. Matrix-Assisted Laser Desorption/Ionization
(MALDI)
• MALDI efficiently produces intact gas-phase
ions from large compounds (over 300 kDa)
using a matrix for both desorption and
ionization.
• The mechanism involves dissolving analyte
with a matrix, forming crystals, and ablating
them with intense laser pulses.
• The laser energy induced heating of the solid
matrix forms a plume that experiences a
phase transition. This process results in the
desorption and ionization of the embedded
analyte, generating ions through gas-phase
ion/molecule reactions.
• Advantages include high sensitivity, minimized
sample damage, and universality in
wavelength adjustments.
20. Electrospray Ionization
(ESI)
• Electrospray ionization (ESI) creates highly
charged microdroplets in a strong electric
field.
• A charged capillary produces an electrospray,
generating charged droplets.
• Desorption occurs through thermal and
pneumatic methods.
• Solvent evaporation causes Coulomb
explosion, resulting in bare analyte ions.
• The electrospray ion source operates at
atmospheric pressure, interfacing with a
mass analyzer via skimmer cones.
• Electrochemical processes at the capillary
influence observed ions in ESI mass spectra.
21. Thermospray (TSP)
• Thermospray (TSP) involves pumping a solution
with salt and sample into a heated steel capillary.
• The heated liquid forms a supersonic beam,
generating a fine-droplet spray containing ions,
solvent, and sample molecules.
• Ions are extracted, accelerated towards the
analyzer, and desorbed from droplets without
vaporization.
• To enhance ion extraction, droplets can be charged
by a corona discharge.
• Continuous pumping of droplets prevents freezing
under vacuum, and liquid heating during injection is
controlled by a feedback-controlled thermocouple.
22. Spark Sources
• Spark sources employ RF voltage for pulsed
electrical discharges in a vacuum to desorb
and ionize analytes.
• For non-metallic samples, mixing with
graphite is done, and atomization is
achieved through electron-heated
discharge and plasma.
• Ionization primarily occurs due to plasma
heating by accelerated electrons.
• Positive ions produced include singly and
multiply charged atomic ions, polymer ions,
and heterogeneous compound ions.
23. Direct Analysis in Real-Time (DART)
• DART allows chemical detection in various states (surfaces, liquids,
gases) without sample preparation, under ambient conditions,
avoiding alterations from high voltage or vacuum exposure.
• In the DART source, helium or nitrogen is introduced, and a high-
voltage potential generates a plasma that interacts with the sample
and atmosphere.
• Ionization mechanisms include Penning ionization and proton
transfer for positive ions, while negative ions result from electron
capture or reactions with atmospheric ions.
• Desorption mechanisms involve thermal desorption and energy
transfer to the surface by metastable atoms and molecules.
• DART produces simple mass spectra with ions like M•+ or [M + H]+
(positive mode) and M•− or [M − H]− (negative mode).
• Fragmentation is common, similar to DESI, but DART lacks multiply
charged ions and is less versatile for a broad analyte range.
• DART is not suitable for spatial surface analysis.
24. Atmospheric Pressure Chemical Ionization (APCI)
• APCI operates at atmospheric pressure, akin to CI, and
is widely used in GC–MS for polar and non-polar
compounds with moderate molecular weight.
• APCI involves the introduction of the analyte in
solution, converting it into a thin fog in a pneumatic
nebulizer.
• A heated desolvation/vaporization chamber facilitates
vaporization of the mobile phase and sample in the
gas flow.
• Ionization occurs via corona discharge, with primary
ions colliding with vaporized solvent molecules to form
secondary reactant gas ions.
• The ionization process is efficient due to high collision
frequency at atmospheric pressure.
• The produced ions enter the mass spectrometer
through a tiny inlet or heated capillary using
differential pumping.
25. Atmospheric Pressure Photoionization (APPI)
• APPI is an atmospheric pressure source using photons to ionize gas-
phase molecules.
• It vaporizes the sample with a heated nebulizer, and photons from a
discharge lamp induce ionization.
• Different APPI sources exist, including direct APPI and dopant APPI,
using molecules like toluene or acetone.
• Direct APPI results in radical cation M•+ and protonated molecule [M +
H]+ ions.
• Dopant APPI involves additional ionization processes, including charge
exchange and proton transfer.
• APPI is effective in both positive and negative ion modes, depending
on the analyte's properties and the presence of a dopant.
• APPI complements APCI and ESI, offering an alternative for ionizing
non-polar compounds.
• Its efficiency is notable for various compound classes, such as
flavonoids, steroids, drugs, pesticides, and polyaromatic hydrocarbons.
26. Mass Analysers
Single Focussing
Sector Analysers
Magnetic Sector Analyser
Electrostatic Sector Analyser
Double Focussing
Sector Analysers
Quadrupole Analyser
Time-of-Flight (TOF)
system
Fourier Transform Ion
Cyclotron Resonance
Orbitrap Analyser
Ion Trap Analyser
27. Magnetic
Sector Analyser
In these, ions leaving the ion
source are accelerated to a high
velocity. The ions then pass
through a magnetic sector in
which the magnetic field is
applied in a direction
perpendicular to the direction
of ion motion. Therefore, these
sector(s) follow an arc; the
radius and angle of the arc vary
with different instrument
designs.
Mass to Charge ratio under the influence of a
magnetic field:
𝑚
𝑧
=
𝐵2𝑟2
2𝑉
Momentum analyzer: The magnetic sector does not directly separate ions by
mass. Rather it effects ion separation by their momentum and this feature can
can be used as a measure of mass provided all ions possess equal kinetic
28. Electrostatic Sector
Analyser
• An electrostatic sector analyzer
comprises two curved plates with
equal and opposite potential.
• As ions move through the electric
field, they experience deflection.
• The force on the ion in the electric
field equals the centripetal force,
focusing ions with the same kinetic
energy.
• Ions with different kinetic energies are
dispersed in this process.
Energy filter: The ESA affects energy dispersion. Thus, the kinetic
energy distribution of an ion beam can be reduced. The ESA does
not allow for mass separation among monoenergetic ions.
29. Double Focussing
Sector Analyzers
These employ both Magnetic and Electrostatic Sectors, usually one
each but nowadays alternative multiple sector arrangements are being
used like BEB/EBE/EBEB types.
• EB analyser
• BE analyser or Reverse EB analyser
30. Quadrupole
• These consist of two pairs of metal rods spaced
equidistantly and biased with equal and opposite
potentials.
• These twin potentials contain a fixed DC and an
alternating RF component, with adjustable RF
strength.
• Ion trajectories are deflected proportionally to their
m/z values upon entering the quadrupole.
• At specific RF values, only ions with a particular m/z
value resonate and reach the end for detection.
• Ions with different m/z values collide with the
quadrupoles, lose charge, and go undetected.
31. Time-of-Flight (TOF) Analyzer
• Time-of-flight (TOF) mass analyzers utilize a flight tube to separate ions based on
their travel time.
• They feature a straightforward design with fixed voltages, eliminating the need
for a magnetic field.
• During operation, ions are swiftly generated by a rapid ionization pulse and then
accelerated into the flight tube through an electric field.
• Ions traverse a field-free region within the flight tube, with the time taken to
reach the detector determined by factors such as drift region length, mass-to-
charge ratio, and acceleration voltage.
• Mass spectrum acquisition involves the detection of low m/z ions reaching the
detector first, enabling the measurement of the detector signal over time for
each ion pulse.
• TOF instruments exhibit high transmission efficiency, leading to an improved
signal-to-noise ratio and absence of an upper m/z limit, low detection limits, and
rapid scan rates.
32. Linear TOF:
The analyte on a sample holder is pulsed with a laser, and
ions desorbed during this pulse are continuously extracted
and accelerated into the gas phase using an applied
acceleration voltage (U) between the target and a grounded
counter electrode.Upon leaving the acceleration region (𝑠0),
ions possess equal kinetic energies.
Ions drift down a field-free flight path (s) of about 1–2 meters
before hitting the detector.
Reflector TOF:
Reflectron TOF focuses ions with varying kinetic energies,
often using multistage designs.
Allows linear mode operation with a detector behind
when the reflector voltage is off.
Basic design includes ring-shaped electrodes creating a
retarding electric field, with 𝑈𝑟 set slightly higher than U.
Ions enter, reach zero kinetic energy, and are expelled,
improving resolving power and focusing ions.
33. Fourier Transform Ion Cyclotron Resonance
• The cyclotron employs perpendicular magnetic fields in a high vacuum
to continuously orbit and accelerate ions.
• In FT-ICR, ions are injected into a modified cyclotron cell, generating a
mass spectrum through fast Fourier transform.
• FT-ICR instruments trap ions in circular orbits, utilizing cyclotron
frequency for precise mass measurements.
• A short-duration radio frequency 'chirp' excites ions, increasing their
orbital radius, and induced electrons create a sinusoidal image current.
• The complex image current is converted into a mass spectrum using fast
Fourier transform.
• The orbit frequency in a magnetic field depends on the charge and mass
of ions, not velocity. Constant magnetic field allows determination of
charge-to-mass ratio (m/z) through angular velocity (ωc): high ωc
corresponds to low m/z, and low ωc corresponds to high m/z. Opposite
charge signs share the same ωc, differing only in orbit direction.
34. Orbitrap Mass Analyzer
• Orbitrap mass analyzers have cup-shaped outer electrodes and a spindle-
like central electrode.
• Voltage creates a linear electric field, attracting ions strongly to the central
electrode.
• Ions are tangentially injected through a slot in one outer electrode.
• Increasing inner electrode voltage squeezes ions toward the inner
electrode.
• When ions reach the desired orbit, the electric field becomes static.
• Injected ions form rings with different rotational frequencies but the same
axial frequency.
• Voltage between central and outer electrodes creates a radial electric
field, bending ion trajectories.
• Tangential velocity opposes centrifugal force, keeping ions in a nearly
circular orbit.
• Ion rotation frequency is directly related to their mass-to-charge ratio
(m/z).
Axial Oscillation frequency:
35. Quadrupole Ion Trap
• The Quadrupole Ion Storage Trap Mass Spectrometer
(QUISTOR) is a recently developed mass analyzer with
high sensitivity, affordability, and fast scan capabilities.
• QUISTOR utilizes a doughnut-shaped ring electrode and
two endcap electrodes. A combination of RF and DC
voltages creates a quadrupole electric field, trapping
ions in a potential energy well at the center of the
analyzer.
• The mass spectrum is acquired by scanning RF and DC
fields to destabilize low mass-to-charge ions.
Destabilized ions are ejected through a hole in an
endcap electrode and detected. Scanning fields eject
ions of increasing m/z values, generating the mass
spectrum.
• The trap is refilled with a new batch of ions for the next
mass spectrum. Mass resolution is enhanced by
introducing a small amount of Helium as a bath gas (0.1
Pa or 10 torr), which dampens ion motion through
collisions and increases trapping efficiency.
37. Photographic Plate
• The first mass spectrometers used photographic
plates located behind the analyser as detectors.
• Ions sharing the same m/z ratio all reach the plate
at the same place and the position of the spots
allows the determination of their m/z values after
calibration.
• The darkness of the spots gives an approximate
value of their relative abundance.
• This detector, which allows simultaneous detection
over a large m/z range, has been used for many
years but is obsolete today
38. Electron Multiplier Detectors
Discrete Dynode EMD:
A type of electron multiplier with 12 to
20 dynodes, each with good secondary
emission properties. Held at decreasing
negative potentials, secondary electrons
cascade through the dynodes, producing
amplified electric current. The first
dynode is at a high negative potential (-1
to -5 kV), while the output remains at
ground potential.
39. Continuous Electron
Multiplier Detector:
Channeltron:
An electron multiplier replacing discrete dynodes
with a lead-doped glass tube. The tube has uniform
electric resistance, creating a continuous
accelerating field. Secondary particles collide with
the tube's inner wall, generating secondary
electrons that cascade, and a metal anode collects
the stream at the detector exit.
Microchannel plate (MCP)
This has Parallelly running cylindrical channels. The
electrons are multiplied by a semiconductor
material that coats each channel. The plate input
side is at a negative potential of around 1 kV.
Amplification ranges from102
𝑡𝑜 105
, with
numerous plates achieving up to 108
.
40. Faraday Cup
• A Faraday cup is made of a metal cup or cylinder with a
small orifice. It is connected to the ground through a
resistor.
• Ions reach the inside of the cylinder and are neutralized
by either accepting or donating electrons as they strike
the walls. This leads to a current through the resistor.
• The discharge current is then amplified and detected.
• It provides a measure of ion abundance. Because the
charge associated with an electron leaving the wall of the
detector is identical to the arrival of a positive ion at this
detector.
• Secondary electrons emitted by ions can cause errors in
detectors. To improve accuracy, devices like coating
carbon on inner walls capture ions efficiently and
minimize secondary electron losses.
41. Photomultiplier Detector or Daly
Counter
• In a photomultiplier (or scintillation counter) the
ions initially strike a dynode which results in
electron emission.
• These electrons then strike a phosphorous screen
which in turn releases a burst of photons.
• A photomultiplier detects these photons,
converting them into an electric current, which is
then amplified.
• - The detector's phosphorescent screen has a thin
aluminum conductor layer to prevent charge
buildup.
• Amplification ranges from 104
𝑡𝑜 105
.
• Photomultipliers are now probably the most
common detectors in modern mass spectrometers.
42. Focal Plane Detectors
• Focal Plane Detectors (FPD) or array detectors can detect a
small m/z range (about 2–5% of the center mass) at a time.
• Ions hitting the focal plane on an MCP (Chevron plate) are
converted to electrons, and those emitted from the
backside of the MCP are turned into photons by a
phosphor screen.
• The light image is directed onto a photodiode array or CCD
detector through a fiber optic device in a multichannel
electro-optical detection system.
• This system typically enhances sensitivity or signal-to-noise
ratio by a factor of 20–100, compensating for ion current
losses and fluctuations.
43. Charge Coupled Detector (CCD)
• CCD imagers have a photoactive region (usually an epitaxial layer of
silicon) and a transmission region (shift register).
• When a particle hits the sensor, it generates a charge proportional to
its energy, charging a capacitor within the pixel.
• In a CCD chip, this charge is transported row-by-row along the
column to a shift register row at the bottom of the pixel matrix.
• The charge is read out pixel-by-pixel from the shift register row, and
the last pixel transfers the charge to an amplifier and a charge-to-
voltage converter.
• Analog buffering may be included, and the information is sent off the
chip as an analog signal.
• Sampling and digitization are performed off-chip.
• Sequential readout results in relatively long readout times, scaling
with the pixel array's area.
• CCD readout pixels can be small (e.g., 10 µm × 10 µm) but achieve full
area coverage.
To Amplifier
44. Vacuum System
Mass spectrometers operate at high vacuum
(10−2
𝑡𝑜 10−5
Pa) to minimize ion collisions.
Ion collisions can lead to undesirable reactions,
neutralization, scattering, or fragmentation,
affecting mass spectra.
A two-stage pumping system is employed for high
vacuum: a mechanical pump for rough vacuum
(down to 0.1 Pa) and diffusion or turbomolecular
pumps for high vacuum.
In some cases, like ICR instruments, a third
pumping stage with a cryogenic pump is used for
even higher vacuum requirements.
45.
46. What is Chromatography?
Chromatography is a separation method where components are
distributed between a stationary phase and a mobile phase. This
technique utilizes the size, shape, chemical properties, or charge of
molecules to separate them in a sample.
By exploiting the varied affinities and interactions of components with
the stationary and mobile phases, chromatography enables their
differential migration, resulting in spatial separation.
Analysis of the separated components allows for identification and
quantification, making chromatography essential in chemistry,
biochemistry, pharmaceuticals, and environmental science.
47. Gas Chromatography
Gas chromatography (GC) is a widely used analytical technique. It utilizes a gas mobile
phase, typically helium or nitrogen, to carry volatile analytes through a column filled with
inert packing material. This separation process is essential for identifying and quantifying
components in complex mixtures.
Principle:
In GC, separation occurs mainly according to two principles: adsorption and partition
chromatography.
• In adsorption chromatography, separation is obtained when the analytes have different
adsorptivity to a solid stationary phase. Gas adsorption chromatography, also called gas–
solid chromatography (GSC), is mainly used for separation of permanent gases.
• In partition chromatography, also called gas–liquid chromatography (GLC), the stationary
phase is a nonvolatile liquid and separation is obtained if the analytes have different
distribution between the mobile and the stationary phases.
48. Gas Chromatograph
1) Gas flask with carrier gas
2) Reduction valve
3) Injection system
4) Column oven
5) Column
6) Detector(s).
49. Mobile Phase/Carrier Gas
• The mobile phase must be an inert gas, reacting
with neither the stationary phase nor the
sample components.
• The gas used must be of high purity.
• The gas should be easily available and
inexpensive, provide high safety at use, and give
good detector response for the analytes.
• For the chromatographic separation, a constant
gas flow rate is required.
• The most common carrier gases are Helium
(He), Hydrogen (𝑯𝟐), and Nitrogen (𝑵𝟐).
• Reduction valves are used to reduce the pressure
of carrier gas from the gas flask, with the help of
pressure meters attached to the gas flask; in
addition, pressure control is provided at the gas
chromatograph.
Reduction valve
50. Injection System
Different sample introduction methods in GC:
• Liquid or solid samples dissolved in a solvent are introduced by a syringe into the
injector.
• Choice of injection system depends on column type and sample composition.
In packed columns:
• Sample is injected directly into the column inlet.
For smaller inner diameter columns:
• Liquid samples use split injection, splitless injection, or on-column injection techniques.
Manual or mechanical injection methods:
• Injections can be done manually with a handheld syringe.
• Autoinjectors, now standard for GC instrumentation, enable mechanical injection.
For gas samples:
• A loop injector, similar to HPLC, or a gas-tight syringe using split or split-less injectors can
be employed.
51. Packed Column Injector
• The packed column injector, or evaporation injector,
involves introducing a sample (2–10 ml) into a heated
metal block just before the column inlet.
• The injection temperature, typically kept 50°C above the
boiling point or column temperature, ensures rapid
evaporation of both solvent and sample components.
• A synthetic rubber septum facilitates sample transfer via a
syringe needle, maintaining gas tightness by closing
puncture holes through septum elasticity.
• Periodic septum replacement is required due to permanent
holes formed after multiple injections.
52. Split/Split-less Injection
• In split injection, a liquid sample (typically 1–2 ml) is introduced through a septum
into a heated zone with a glass tube (liner). The carrier gas is introduced at the
liner's inlet, and a valve allows flushing of the septum. To prevent overwhelming a
capillary column, the injection volume is split, with only a portion directed to the
column while the majority is directed to waste.
• In split-less injection, the entire injected sample (1–2 ml of gas) is introduced into
the column for trace determination. Unlike split injection, the splitter outlet valve
is closed. The sample, brought into the gas phase in a heated liner, is transferred
to the column. The column inlet temperature is kept lower than the solvent
boiling point, causing solvent condensation on the column wall. After transfer, the
column temperature is increased for separation. The splitter valve is opened to
remove remaining sample before the next injection. This technique is used for
trace determinations and requires temperature programming.
53. On-Column & PTV Injection systems
• On-Column Injector: This technique for introducing a
liquid sample at room temperature directly into the
column entrance or through a retention gap,
particularly suited for temperature-labile analytes. It
provides repeatable injections without discrimination,
making it suitable for high-boiling compounds.
• Programmed Temperature Vaporizing (PTV) Injector:
Allows the introduction of larger sample volumes
using a vaporizing chamber (liner) that can be rapidly
heated and cooled. The PTV injector is flexible,
accommodating various injection methods, including
cold and hot split/splitless injections, and cold
splitless solvent vent injection. It minimizes
discrimination and thermal degradation associated
with conventional split/splitless injection.
54. Headspace Techniques (Static and
Dynamic)
Common methods for determining volatile
analytes from aqueous samples.
• Static Headspace: Equilibrium is established by
thermostating the sample vial; a quick transfer
of the headspace volume to a trap column
follows pressurization.
• Dynamic Headspace (Purge-and-Trap): A gas
passes over or through the sample, transporting
volatile compounds to a cryogenic or sorbent
trap before GC separation. Internal standards
are essential for both techniques.
55. Chromatographic columns
• A chromatographic column provides a
location for physically retaining the
stationary phase. The column’s construction
also influences the amount of sample that
can be handled, the efficiency of the
separation, the number of analytes that can
be easily separated, and the amount of time
required for the separation.
• The two types of columns used in GC are:
• Packed columns.
• Capillary Columns.
• The column is connected directly with the
injector and the detector by nuts and
ferrules.
56. Packed Column
• A packed column is constructed from glass, stainless
steel, copper or aluminum and is typically 2–6 m in
length, with an internal diameter of 2–4 mm.
• The column is filled with a particulate solid support,
with particle diameters ranging from 37–44 µm to
250–354 µm.
• The most widely used particulate support is
diatomaceous earth, which is composed of the silica
skeletons of diatoms. These particles are quite
porous, with surface areas of 0.5–7.5 m2/g, which
provides ample contact between the mobile phase
and stationary phase.
• When hydrolyzed, the surface of a diatomaceous
earth contains silanol groups (–SiOH), providing
active sites that absorb solute molecules in gas–solid
chromatography. Packed Column
57. Capillary Columns
Capillary, or open tubular columns are constructed from fused silica
coated with a protective polymer. Columns may be up to 100 m in
length with an internal diameter of approximately 150–300 µm.
Larger bore columns of 530 µm, called megabore columns, also are
available. There are 3 types of OTCs:
Wall-Coated Open Tubular (WCOT) Column: In these, the liquid
stationary phase is coated as a thin film (typically 0.1–0.5 mm) on
the inner wall of the capillary. Primarily used for gas-liquid
chromatography (GLC) in partition chromatography.
Support-Coated Open Tubular (SCOT) Column: Here the liquid
stationary phase is coated on the porous layer or porous particles
at the inner wall of the capillary. Allows for GLC separations with a
higher sample capacity compared to WCOT columns. However, it
has lower efficiency.
Porous-Layer Open Tubular (PLOT) Column: These contain a porous
layer or porous particles on the inner wall of the capillary.
Specifically designed for gas adsorption chromatography (GSC),
where the porous layer serves as the stationary phase.
58. Stationary
Phase
Selectivity in gas chromatography is influenced by
the choice of stationary phase. Elution order in
GLC is determined primarily by the solute’s boiling
point and, to a lesser degree, by the solute’s
interaction with the stationary phase.
Criteria for choosing a stationary phase:
• Chemical inertness and Thermal stability
• Low volatility and Suitable polarity
• Liquid stationary phases may face challenges
like "bleeding," which can be managed by
adhering to specified temperature limits.
• Bonded or cross-linked phases, offer enhanced
stability.
• The stationary phase: thinner films (0.25 µm)
improve separation efficiency, while thicker
films suit highly volatile solutes.
59. Detectors
• The final part of a gas chromatograph is the detector. The
ideal detector has several desirable features, including low
detection limits, a linear response over a wide range of
solute concentrations (which makes quantitative work
easier), responsiveness to all solutes or selectivity for a
specific class of solutes, and an insensitivity to changes in
flow rate or temperature.
• Thermal Conductivity Detector
• Flame Ionization Detector
• Electron Capture Detector
• Nitrogen–Phosphorus Detector
• Other Detectors
60. Thermal Conductivity Detector (TCD)
• TCD is a heated metal block with two channels equipped with
filaments.
• The filaments are connected to a Wheatstone bridge.
• The filament temperature depends on the heat conductivity of
the gas passing.
• The TCD detects compounds with a conductivity less than the
carrier gas.
• It is nondestructive and suitable for preparative separations.
• Commonly used for determining light and permanent gases in
packed or PLOT columns.
• Ideal for portable gas chromatographs due to its
miniaturization and lack of extra gases.
61. Flame Ionization Detector
• Comprises a heated zone with a mixture of carrier gas
and H2 before entering the detection compartment.
• Air enters the compartment through a separate
channel, maintaining a flow rate ratio of 1:1:10 (for
Carrier gas, H2 & Air).
• A flame is started by an electrical discharge and
sustained in excess air.
• A potential (300 V) is applied between the flame tip
and collector, generating a small current proportional
to the amount of compound combusted.
• Can detect all organic compounds containing C and H,
except formic acid and methane.
• Mass-sensitive, with a minimum detectable mass of
0.01–0.1 ng and a large dynamic range of 107
.
When a hydrocarbon compound from the column enters the
flame, the following happens in the reducing zone:
62. Nitrogen–Phosphorus Detector (NPD)
• Also known as the alkali flame ionization detector (AFID).
• Like the FID but with an additional unit, usually a rubidium silicate bead heated
by an electrical current.
• Ion current increases when a compound enters the detection compartment.
• Mechanism for N detection:
• Neutral Rb atoms react with cyano radicals, neutralizing 𝑅𝑏+
ions on the
negatively charged rubidium salt, and 𝐶𝑁−
ions on the positively charged
collector.
• Optimized detector gas flow rates differ for N and P detection.
• Suppresses formation of 𝐶𝐻+
radicals.
• Selective detector for N- and P-containing compounds.
• Used for pesticide determination in food and environmental applications.
63. Electron Capture Detector
• It is an example of a selective detector.
• The detector consists of a 𝛽 − emitter (a beta (𝛽) particle is an
electron) such as 63Ni.
• The emitted electrons ionize the mobile phase, which is usually
𝑁2, resulting in the production of additional electrons that give
rise to an electric current between a pair of electrodes.
• When a solute with a high cross section for the capture of
electrons elutes from the column, the electric current decreases.
This decrease in electric current serves as the signal.
• The ECD is highly selective toward solutes with electronegative
functional groups, such as halogens, and nitro groups and is
relatively insensitive to amines, alcohols, and hydrocarbons.
65. Flame Photometric Detector (FPD):
Mass-sensitive and specific for sulfur and phosphorous.
Emission measurement from combustion products.
Detection limit: 5 pg/s for S, 50–100 pg/s for P
Linearity: 103
− 104
Main application: Specific detection of sulfur in
petroleum, petrochemical samples, and phosphorus in
pesticides.
Electrolytic Conductivity Detector (ELCD):
Mass-sensitive, selective for halogens (X),
sulfur (S), and nitrogen (N)
Converts compounds to ionizable gas (HX) at
high temperatures
Detection limit: Low pg/s range
Linearity: 105 - 106
Main application: Official methods for halogen-
containing compounds.
Other Detectors:
Atomic Emission Detector (AED):
Mass-sensitive, both universal and selective
Atomization of organic compounds
Excitation of atoms in high-energy microwave plasma
(helium)
Emitted light's wavelengths specific for each element
Detection limit: About 1–150 pg/s (element-dependent)
Linearity: 103
- 104
66. Photoionization Detector (PID): Non-destructive
Concentration-sensitive detector. Universal/selective for
organic compounds based on ionization potential.
Formation of molecular ions using high-energy photons.
Current needed for neutralization measures compound
concentration
Detection limit: 10 pg/s for C
Linearity: 106
Main application: Measurement of trace levels of
aromatic compounds in environmental or health
concerns.
Fourier Transform Infrared Detector (FTIR):
Non-destructive and concentration-sensitive
Universal detection
Detection limit: About 150 pg (compound-dependent)
Linearity: 103
Other Detectors:
Chemiluminescent Detector:
Mass-sensitive, highly selective for sulfur (SCD) or
nitrogen (NCD). Two-step process involving combustion
and reaction with ozone
Characteristic light emission measured
Detection limit: 0.5 pg/s for S, 3 pg/s for N
Linearity: 104
Main application: Determination of sulfur compounds in
petrochemical products.
67. Gas chromatography-Mass spectrometry
(GC-MS) is one of the so-called hyphenated analytical techniques. It is actually two
techniques that are combined to form a single method for analyzing mixtures of organic
chemicals.
Gas chromatography separates the components of a mixture, and mass spectrometry
characterizes each of the components individually.
The combination of the two techniques allows for both qualitative and quantitative
evaluations of a sample containing a number of organic compounds. The uses for GC-MS
are numerous, including chemical, geological, environmental, and forensic research.
69. GC-MS Analysis Process
Sample introduction in
GC can be manual or
automated with an
autosampler.
Liquid samples are
vaporized in the heated
GC inlet and transferred
to the analytical column.
Analytes in the sample
are separated based on
partitioning between
the mobile and liquid
stationary phase.
In GC-MS, a common
setup uses a liquid
stationary phase in a
narrow, short column.
Neutral molecules elute
through a heated
transfer line into the
mass spectrometer.
Ionization, often by
electron ionization (EI),
leads to ion formation.
Ions are separated
based on mass-to-
charge ratio (m/z) in the
mass analyzer.
Common mass analyzers include
quadrupoles ( used in full scan or
selected ion monitoring modes)
and Time-of-flight (ToF) mass
analyzers separate ions based on
their travel time down the flight
tube.
Ion detectors, such as
electron multipliers or
multi-channel plates,
amplify signals after ion
separation.