Mass spectrometry

IN 504 Analytical Instruments
Module 5
1
Presented by;
Anju Sunny
CUSAT
Reference Text: R S Khandpur
“Handbook of Analytical Instrumentation”

Introduction
 Mass spectrometers is an analytical instrument which can be used
for all kind of chemical analysis like;
 To measure the elemental composition of matter
 To analyze the structures of inorganic, organic and biological
molecules
 To find the isotopic ratios of atoms in the sample
 For the detection of poisons in a sample

General Block Diagram – Mass Spectrometer

Analogy between Optical Spectrophotometer
& Mass Spectrometer

Components of a Mass Spectrometer
Ionisation Ion DetectionIon Separation
Ion Source Mass Analyser Detector
Electron Ionisation (EI)
Chemical Ionisation (CI)
Fast Atom Bombardment (FAB)
Electrospray Ionisation (ESI)
Matrix-Assisted Laser Desorption
Ionisation (MALDI)
Quadrupole
Magnetic deflection type
Electrostatic type
Time-Of-Flight (TOF)
Ion Trap
Electron Multiplier
Multichannel plate
Faraday Cup

IONIZATION SOURCES
1) Electron Ionization (EI)
- Commonly used for analysis of organic samples
- Electrons are emitted from a heated tungsten filament cathode.
- Electrons are accelerated towards the anode with a potential
of about 50 – 100 V
- Electrons meet at right angles with the sample molecules
- Interaction with the high energy electrons causes ionization of
sample molecules and fragmentation into smaller ions.

IONIZATION SOURCES
2) Chemical Ionization (CI)
- A large excess of reagent gas (1000 – 10000 times) is introduced
into the ionization region
- Pressures in source are typically higher than EI
- Electrons are allowed to bombard the gas-sample mixture
Examples of reagent gas
- Methane, ammonia, isobutane

IONIZATION SOURCES
3) Atmospheric Pressure Ionization (API) Sources
- Two major types
- Electrospray Ionization (ESI)
- Atmospheric Pressure Chemical Ionization (APCI)
- Operate at atmospheric pressure
- Modified version of ESI is the Ion Spray Source
- Used for mixtures of nonvolatile high molecular weight compounds

IONIZATION SOURCES
4) Desorption Ionization
- For direct ionization of solids
- Excellent tool for analysis of large molecules
- Solid samples are placed on a support and then bombarded with
ions or photons
- Different types are available

IONIZATION SOURCES
a) Laser Desorption Ionization
- Uses pulsed laser
- Provides selective ionization by choosing appropriate λ
- Laser is focused on a solid surface to ionize material
Examples of Lasers
- IR laser: CO2 laser
- UV laser: Nd:YAG (yttrium aluminum garnet)

IONIZATION SOURCES
b) Matrix-Assisted Laser Desorption Ionization (MALDI)
- Matrix disperses large amounts of energy absorbed by the laser
- Minimizes fragmentation of the molecule
- Used for study of polymers, proteins, peptides

IONIZATION SOURCES
c) Fast Atom Bombardment (FAB)
- Employs fast moving neutral inert gas atoms (Ar) to ionize large
molecules
- Sample is dissolved in glycerol and spread in a thin layer on a
metal probe
- Probe is then inserted into the mass spectrometer and a beam of
fast moving atoms probe the surface

Types of Mass Spectrometers
 Various types of Spectrometers are available in the means of
separating the ions according to their mass-to-charge ratio or
simply, on the basis of Mass Analyzers used. They are;
 1) Magnetic Deflection Mass Spectrometer
 2) Electrostatic Mass Spectrometer
 3) Time-of-Flight Mass Spectrometer
 4) Quadrupole Mass Spectrometer

1) Magnetic Deflection Mass Spectrometer

Magnetic Deflection Mass Spectrometer
 In a mass spectrometer, the sample to be analyzed is first
bombarded with an electron beam to produce ionic fragments of the
original molecule.
 These ions are accelerated in a high vacuum into a magnetic field ,
which deflects them into circular paths.
 Since the deflection for light ions is greater than that for heavy ions,
the ion stream separates into beams of different molecular weight.
 A suitably placed slit allows a beam of selected molecular weight to
pass through to a collection electrode or detector.

2) Electrostatic Mass Spectrometer
 Electrostatic sector analyzer consists of 2 curved plates of equal and
opposite potential.
 As the ion travels through the electric field, it is deflected and the force on
the ion due to the electric field is equal to the centripetal force on the ion.
 Here the ions of the same kinetic energy are focused, and ions of different
kinetic energies are dispersed.
 Electrostatic and magnetic sector analyzers when employed individually
are single focusing instruments. When both techniques are used together,
it is called a double focusing instrument, because in this instrument both
the energies and the angular dispersions are focused.

3)Time of Flight (ToF) Mass Spectrometer
 In ToF Mass Spectrometer, ions of different mass/charge ratio are
separated by the difference in time they take to travel over an identical path
from the ion source to the collector at the detector.
 Here ions generated by bombardment of the sample with a brief pulse of
electrons, secondary ions or laser-generated photons.
 Ions accelerated by electric field and they enter into field-free drift tube.
 Ions enter tube with same kinetic energy and ion velocity vary inversely
with mass.
 Lighter particles arrive at detector before heavier particles and ions are
separated in the drift tube according to their velocities (v)

 V = accelerating voltage
 If L is the length of tube (typically 1-2 m) and t is the flight
time of ion, then v = L/t
 Implies mass-to-charge ratio and flight time can be found from
1/2
m
2eV
v 






2
2
L
2Vt
e
m

2eV
m
Lt 

Advantages of ToF
 High speed operation
 Ability to record entire mass spectrum at one time
 Accuracy only depend on electronic circuits
21
Disadvantages of ToF
 Poor resolution due to display on an Oscilloscope screen

Applications of ToF
 Qualitative Analysis
 Molecular Weight Determination
 Structure Determination
 Quantitative Analysis
 Biotechnology
 Analysis of proteins & peptides
 Pharmaceutical
 drug discovery
22

4) Quadrupole Mass Spectrometer
23

4) Quadrupole Mass Spectrometer
Four parallel cylindrical rods serve as electrodes.
 Opposite rods are connected electrically
- One pair attached to positive side of variable dc source
- One pair attached to negative side of variable dc source
 Variable radio-frequency ac potential (180o out of phase) applied
to each pair of rods.
 The result is an ac potential superimposed on a dc potential and
this creates an electric field.
Ions accelerated through the electric field between rods.
 ac and dc voltages increased simultaneously with ratio being
constant
 All ions without specific m/e strike rods and become neutral
 Only ions having a limited range of m/e reach detector. 24

Features of Quadrupole Mass Spectrometer
 The quadrupole acts as a filter so is often called the
mass filter.
 Sample must be ionized and it should be in gas phase.
 Has smaller range and lower resolution than magnetic
sector but faster.
 More compact
 less expensive
 High scan rate
25

DETECTORS
1) Multichannel plate
- Detector which measures one m/e value at a time called
single channel detectors.
- Multichannel plate detectors are used for the detection
of multiple ion beams with different m/e value.
- High resolution magnetic sector instruments use
multichannel detectors (called multicollectors)

DETECTORS
2) Electron Multiplier (EM)
- The most common detector in Mass spectrometer for ions.
- Similar to PMT.
- Very sensitive and has fast response.

DETECTORS
3) Faraday Cup
- A metal or carbon cup serves to capture ions and
store the charge.
- Cup shape decreases loss of electrons.
- Least expensive detector for ions.
- Has long response time.

DETECTORS
4) Array Detectors
- Used in ToF Mass spectometers.
- Employs a focal plane camera (FPC) consisting of an
array of 31 Faraday Cup
- Up to 15 m/e values can be measured simultaneously.
- Exhibits improved precision compared with single
channel detectors.

 Molecular weight can be obtained from a very small sample.
 It does not involve the absorption or emission of light.
 Detection limits better by 3 orders of magnitude of other
techniques.
 Remarkably simple spectra that are unique and easily
interpreted.
 Ability to measure isotopic ratios.
Mass Spectrometry : Advantages
Mass Spectrometry : Disadvantages
• Instrument costs are 2 to 3 times higher than others.
• Instrument drift that can be as high as 5-10% per hour.
• Interference effects may occur.

Principle
 It is a separation technique based on the different
interactions of compounds with two phases, a mobile
phase and a stationary phase, as the compounds travel
through a supporting medium.
 Components:
 Mobile phase: a solvent that flows through the supporting
medium.
 Stationary phase: a layer or coating on the supporting
medium that interacts with the analyte.
 Supporting medium: a solid surface on which the
stationary phase is bound or coated.
32

Types of Chromatography
 The primary division of chromatographic techniques is
based on the type of mobile phase used in the system.
Type of Chromatography Type of Mobile Phase
 Gas chromatography (GC) Gas
 Liquid chromatography (LC) Liquid
33

 Further divisions can be made based on the type of stationary
phase used in the system:
Gas Chromatography
Name of GC Method Type of Stationary Phase
 Gas-solid chromatography(GSC) solid support
 Gas-liquid chromatography(GLC) liquid-coated support
 Bonded-phase gas chromatography chemical support
34

Liquid Chromatography
Name of LC Method Type of Stationary Phase
 Adsorption chromatography - solid support
 Partition chromatography - liquid-coated support
 Ion-exchange chromatography - support containing fixed charges
 Size exclusion chromatography - porous support
 Affinity chromatography - support with immobilized material
35

Gas Chromatography- Equipments
37
 Carrier source with pressure and flow regulators.
(Eg: Ar, He, N2, H2 )
 Injector or sample application system.
 Chromatographic column with oven for temperature control.
(Eg: 2-50 m, coiled stainless steel/glass/Teflon tube)
 Detector & computer or recorder.
Eg: Thermal Conductivity Detector (TCD), Flame Ionization
Detector (FID), Electron Capture Detector (ECD)

38
 Mobile Phase or Carrier gas
 Main purpose of carrier gas in GC is to move the solutes along the
column.
 Common carrier gas include He, Ar, H2, N2 etc.
 Carrier Gas or Mobile phase does not affect solute retention time.
(Time required for the sample to travel from the injection port
through the column to the detector).
 But does affect:
 Desired efficiency for the GC System
 Stability of column and solutes
 Response of the detector

39
 Stationary Phase
 It is the main factor determining the selectivity and retention of
solutes.
 There are three types of stationary phases used in GC:
 Solid adsorbents (GSC)
 Liquids coated on solid supports (GLC)
 Bonded-phase supports

How Does Chromatography Work?
 In all chromatographic separations, the sample is transported
in a mobile phase. The mobile phase can be a gas or a liquid.
 The mobile phase is then forced through a stationary phase
held in a column or on a solid surface. The stationary phase
needs to be something that does not react with the mobile
phase or the sample.
 The sample then has the opportunity to interact with the
stationary phase as it moves past it. Samples that interact
greatly, then appear to move more slowly. Samples that
interact weakly, then appear to move more quickly. Because
of this difference in rates, the samples can then be separated
into their components.
40

Chromatography is based on a physical equilibrium
that results when a solute is transferred between the mobile
and a stationary phase.
K = distribution coefficient or partition ratio
K 
CS
CM
Where CS is the molar concentration of the solute in the
stationary phase and CM is the molar concentration in the
mobile phase.

Gas-Solid Chromatography (GSC)
 Here same material is used as both the stationary phase
and support material
 Common adsorbents include:
 Alumina
 Molecular sieves (crystalline aluminosilicates [zeolites] and
clay)
 Silica
 Active carbon
42

Gas-Liquid Chromatography (GLC)
 Here stationary phase is some liquid coated on a solid
support.
 Over 400 liquid stationary phases available for GLC.
 Material range from polymers (polysiloxanes, polyesters,
polyethylene glycols) to fluorocarbons, molten salts and
liquid crystals .
 Disadvantage is the liquid may slowly bleed off with time.
43

Bonded Phase Gas Chromatography
 Stationary phase is covalently attach to the solid support.
 So it avoids column bleeding in GLC.
 Bonded phases are prepared by reacting the desired phase
with the surface of a silica-based support.
 Commonly recommended bonded-phases:
 Dimethylpolysiloxane
 Methyl(phenyl)polysiloxane
 Polyethylene glycol (Carbowax 20M)
44

Liquid Chromatography (LC)
Introduction:
 A chromatographic technique in which the mobile phase is a
liquid.
 LC is a much older technique than GC, but was
overshadowed by the rapid development of GC in the
1950’s and 1960’s.
 LC is currently the dominate type of chromatography and is
even replacing GC in its more traditional applications.
45

Advantages of LC compared to GC
1) Separation of any compound that is soluble in a liquid phase is
possible. (Eg: biological compounds, synthetic or natural
polymers, inorganic compounds etc.)
2) Can be used at lower temperatures than GC.
3) Retention of solutes in LC depend on their interaction with both
the mobile phase and stationary phase, where GC retention based
on volatility and interaction with stationary phase.
4) Most LC detectors are non-destructive and most GC detectors are
destructive.
46

Classification of LC based on overall
efficiency or performance
 Low-performance Liquid Chromatography (LPLC)
 High-performance Liquid Chromatography (HPLC)
47

Low-performance liquid chromatography
(LPLC)
 LC methods that use large, non-rigid support material.
 Poor system efficiency.
 Such systems have the following characteristics:
 broad peaks
 poor limits of detection
 long separation times
 columns can only tolerate low operating pressures
48

Low-performance liquid chromatography (LPLC)
 Column chromatography – an example of the equipment
used in low-performance liquid chromatography .
49
Solvent reservoir
Column head
Column
Column packing
Porous glass plate
• Sample is usually
applied directly to the
top of the column.
• Detection is by fraction
collection with later
analysis of each fraction

High-performance liquid chromatography
(HPLC)
 LC methods that use small, uniform, rigid support material.
 Good system efficiencies
 Such systems have the following characteristics:
 narrow peaks
 good limits of detection
 short separation times
 columns can tolerate high operating pressures and faster flow-
rates
50

High-performance liquid chromatography (HPLC)
 It requires higher operating pressures for mobile phase delivery
and uses special pumps and other system components.
 Sample applied using closed system (i.e., injection valve)
 Detection uses a flow through detector.
51

High-performance liquid chromatography (HPLC)
 Advantages:
 fast analysis time
 ease of automation
 good limits of detection
 preferred choice for analytical applications
 popular for purification work
 Disadvantages:
 greater expense
 lower sample capacities
52

Types of Liquid Chromatography
Techniques in LC are classified according to the method of solute
separation.
1) Adsorption chromatography: Separates solutes based on their
adsorption to the stationary phase.
2) Partition chromatography: Separates solutes based on their
partitioning between a liquid mobile phase and a liquid stationary
phase coated on a solid support.
3) Ion-exchange chromatography: Separates solutes by their
adsorption onto a support containing fixed charges on its surface.
4) Affinity chromatography: Separates based on the use of
immobilized biological molecules as the stationary phase.
5) Size-exclusion chromatography: separates molecules according to
differences in their size.

Types of Liquid Chromatography

Chromatographic Detectors
 After the components of a mixture are separated using
chromatography, they must be detected as they exit the
chromatographic column.
 Most commonly used detectors are:
 Thermal Conductivity Detector (TCD)
 Flame Ionization Detectors (FID)
 Electron Capture Detector (ECD)
 Atomic Emission Detector (AED)
 Mass spectrometer (MS)
56

Thermal Conductivity Detector (TCD)
 A TCD detector consists of an electrically-heated wire or
thermistor.
 The temperature of the sensing element depends on the thermal
conductivity of the gas flowing around it.
 Changes in thermal conductivity, such as when organic
molecules displace some of the carrier gas, cause a temperature
rise in the element which is sensed as a change in resistance.
 The TCD is not as sensitive as other detectors but it is non-
specific and non-destructive.
57

Thermal Conductivity Detector (TCD)
58

Flame Ionization Detectors (FID)
 The effluent from the column is mixed with hydrogen and air and
then ignited electrically.
 Most organic compounds, when pyrolyzed at the temperature of a
hydrogen/air flame, produce ions and electrons that can conduct
electricity through the flame.
 The ions are collected on a biased electrode and the resulting
current (~10-12 A) is then measured.
 Extremely sensitive and have large dynamic range.
 Disadvantage of FID is that it is destructive of sample.
59

Flame Ionization Detectors (FID)
60

Electron Capture Detectors(ECD)
 The ECD uses a radioactive source such as Ni63 which produces
Beta particles, which react with the carrier gas producing free
electrons.
 These electrons flow to the anode & producing an electrical signal .
 When electrophillic molecules are present, they capture the free
electrons, lowering the signal. The amount of lowering is
proportional to the amount of analyte present.
 It is widely used detectors for environmental samples, because it
detects halogen containing compounds, such as pesticides and
polychlorinated biphenyls. 61

Electron Capture Detectors(ECD)
62
63

Atomic Emission Detector (AED)
 One of the newest gas chromatography detector & it is quite
expensive compared to other chromatographic detectors.
 The strength of the AED lies in the detector's ability to
simultaneously determine elements.
 It uses microwave energy to excite helium molecules (carrier gas)
which emit radiation & which breaks down molecules to atoms such
as S, N, P, Hg, As, etc.
 These excited molecules emit distinctive wavelengths which can be
separated by a grating and sent to the detector (typically a photodiode
array) which produces the electrical signal. 63

Atomic Emission Detector (AED)
64

Mass Spectrometer (MS)
 Uses the difference in mass-to-charge ratio (m/e) of ionized atoms
or molecules to separate them from each other.
 Molecules have distinctive fragmentation patterns that provide
structural information to identify structural components.
 The general operation of a mass spectrometer is:
1. Create gas-phase ions
2. Separate the ions in space or time based on their mass to charge ratio
3. Measure the quantity of ions of each mass-to-charge ratio.
65

 Centrifugal force is the apparent force that draws a
rotating body away from the centre of rotation.
 Centripetal force is a force that makes a body follow a
curved path.
67
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Mass spectrometry

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