Mass spectroscopy, Ionization techniques and types of mass analyzers

SpectroscopySpectroscopy
MASS SPECTROSCOPY
1
-: Presented By :-
M Asif shaheen
Lecturer
KEMU Lahore

Basic principle
Mass spectroscopy is the most accurate method for
determining the molecular mass of the compound and its
elemental composition.
In this technique, molecules are bombarded with a beam of
energetic electrons.
The molecules are ionised and broken up into many fragments,
some of which are positive ions.
Each kind of ion has a particular ratio of mass to charge, i.e.
m/e ratio(value). For most ions, the charge is one and thus, m/e
ratio is simply the molecular mass of the ion.
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SpectroscopySpectroscopy Principle and Instrumentation
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Ionisation
The atom is ionised by knocking one or more electrons
off to give a positive ion. (Mass spectrometers always
work with positive ions).
The particles in the sample (atoms or molecules) are
bombarded with a stream of electrons to knock one or more
electrons out of the sample particles to make positive ions.
10

Most of the positive ions formed will carry a charge
of +1.
These positive ions are persuaded out into the rest of the
machine by the ion repeller which is another metal plate
carrying a slight positive charge.
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SpectroscopySpectroscopy  Acceleration
 The ions are accelerated so that they all have the
same kinetic energy.
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The positive ions are repelled away from the positive
ionisation chamber and pass through three slits with voltage
in the decreasing order.
The middle slit carries some intermediate voltage and the
final at ‘0’ volts.
All the ions are accelerated into a finely focused beam.
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 Deflection
The ions are then deflected by a magnetic field
according to their masses. The lighter they are, the
more they are deflected.
The amount of deflection also depends on the
number of positive charges on the ion -The more the
ion is charged, the more it gets deflected.
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Different ions are deflected by the magnetic field by
different amounts. The amount of deflection depends
on:
The mass of the ion: Lighter ions are deflected more
than heavier ones.
The charge on the ion: Ions with 2 (or more)
positive charges are deflected more than ones with only
1 positive charge.
15

 Detection
The beam of ions passing through the machine is detected
electrically.
When an ion hits the metal box, its charge is neutralised
by an electron jumping from the metal on to the ion.
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Only ion stream B makes it right through the machine to
the ion detector.
The other ions collide with the walls where they will
pick up electrons and be neutralised.
They get removed from the mass spectrometer by the
vacuum pump.

That leaves a space amongst the electrons in the
metal, and the electrons in the wire shuffle along to fill
it.
A flow of electrons in the wire is detected as an
electric current which can be amplified and recorded.
The more ions arriving, the greater the current.
17

Inlet
Ion
source
Mass
Analyzer Detector
Data
System
High Vacuum System
Mass Spectrometer Block Diagram

Inlet
Ion
source
Mass
Analyzer Detector
Data
System
High Vacuum System
Mass Spectrometer Block Diagram
Turbo molecular
pumps

Inlet
Ion
Source
Mass
Analyzer Detector
Data
System
High Vacuum System
HPLC
Flow injection
Sample plate
Sample Introduction

Inlet
Ion
Source
Mass
Analyzer Detector
Data
System
High Vacuum System
MALDI
ESI
FAB
LSIMS
EI
CI
Ion Source

Inlet
Ion
source
Mass
Analyzer Detector
Data
System
High Vacuum System
Time of flight
(TOF)
Quadrupole
Ion Trap
Magnetic Sector
Mass Analyzer

The Sample Inlet System
 Batch Inlets
 The batch inlet system is considered the most
common and simplest inlet system. Normally, the
inside of the system is lined with glass to elude losses
of polar analyte by adsorption.
 This system externally volatizes the sample which
leaks into an empty ionization region. Boiling points up
to 500 degrees C of gaseous and liquid samples can
be used on typical systems.
 The system's vacuum contains a sample pressure of
10-4
to 10-5
Torr. Liquids are introduced using a
microliter syringe into a reservoir; gases are enclosed
in a metering area that is confined between two valves
before being expanded into a reservoir container.

 Liquids that have boiling points lower than 500
degrees C can not be used in the system because
the reservoir and tubing need to be kept at high
temperatures by ovens and heating tapes. This is
to ensure that the liquid samples are transformed
to the gaseous phase and then leaked through a
metal or glass diaphragm containing pinholes to
the ionization area.

 The Direct Probe Inlet:
 A direct probe inlet is for small quantities of
sample, solids, and nonvolatile liquids. Solids and
nonvolatile liquids are injected through a probe, or
sample holder.
 The probe is inserted through a vacuum lock.
Unlike the batch inlet, the sample will need to be
cooled and/or heated on the probe.
 The probe is placed extremely close (a few
millimeters) to the ionization source, where the slit
leads to the spectrometer,

SpectroscopySpectroscopy Chromatographic and Capillary
Electrophoretic Inlets
 Chromatographic systems and Capillary
Electrophoretic units are often coupled with mass
spectrometers in order to allow separation and
identification of the components in the sample. If
these systems and units are linked with a mass
spectrometer, then other specialized inlets,
 Electrokinetic and
 Pressure injection, are required.
 Electrokinetic and pressure injection controls the
amount of volume injected by the duration of the
injection, which typically range between 5 to 50 nL.

ION SOURCE
 Since the mass analyzer utilizes only gaseous ions i.e.,
starting point of mass spectrometric analysis is
formation of gaseous analyte ions.
 • Non –Volatile solids are first converted in to gases and
from the gaseous sample the ions are produced in a Box
like enclosure called Ion Source.
 Function
 Produces ion without mass discrimination of the
sample.
 Accelerates ions into the mass analyzer.

 Desorption
 A phenomenon whereby a substance is released from or
through a surface.
 Sorption
 A process whereby one substance attached to another. It
can be of two types
 1 Adsorption
 Adhesion of atoms ions or molecules from a gas liquid
or dissolved solid to a surface. This process create a
adsorbate on the surface of adsorbent.
 2 Absorption
 A process in which atoms ions or molecules are taken up
by a bulk phase i.e solid liquid or gas. Molecules are
taken up by the volume not by the surface.

Catogories of Ion sources
 Gas Phase Sources
 Electron Impact Ionization (EI)
 Chemical Ionization (CI)
 Field Ionizations (FI)
 Desorption Sources
 Field Desorption (FD)
 Electrospray Ionization (ESI)
 Matrix assisted desorption/Ionisation (MALDI)
 Plasma desorption (PD)
 Fast Atom Bombardment (FAB)
 Thermospray Ionization (TS)
 Secondary Ion Mass Spectrometry (SIMS)

• Electron impact (EI) ionization
 Electron impact (EI) is the classical ionization method
in mass spectrometry.
 • It is the most widely used and highly developed
method.
 • It is also known as Electron bombardment or
Electron Ionization.

CONSTRUCTION & WORKING:
 Electron impact ionization source consists of a ionizing chamber
which is maintained at a pressure of 0.005 torr and temperature of
200 ± 0.25 degrees.
 Electron gun is located perpendicular to chamber.
 Electrons are emitted from a glowing filament (tungsten or
rhenium) and accelerated by a potential of 70 V applied between
the filament and anode.
 These electrons are drawn in the ionization chamber through
positively charged slits.
 • The number of electrons is controlled by filament temperature
and energy of energy is controlled by filament potential.
 The sample is brought to a temperature high enough to produce
molecular vapors.
 • The gaseous Neutral molecules then pass through the molecular
leaks and enter the ionization

 The gaseous sample and the electrons collide at right
angles in the chamber and ions are formed by exchange
of energy during these collisions between electron
beam and sample molecules
 Since the ionization energy of most of the organic
molecules is 15eV an electron is expelled to produce a
radical cation.
 At hard ionization event i.e at 70 e V molecule ions are
fragmented.
 The positive ions formed in the chamber are drawn out
by a small potential difference (usually 5eV) between
the large repeller plate (positively charged) and first
accelerating plate (negatively charged).

 ADVANTAGES
 Gives molecular mass and also the fragmentation
pattern of the sample.
 Extensive fragmentation and consequent large number
of peaks gives structural information.
 Gives reproducible mass spectra.
 DISADVANTAGES
 Sample must be thermally stable and volatile.
 A small amount of sample is ionized (1 in 1000
molecules).
 Unstable molecular ion fragments are formed so readily
that are absent from mass spectrum.

•EI works well only for thermally stable and
volatile samples;

Schematic representation of an electron
ionization ion source.
sample pressure in the ion source
is about 10-5
torr
about every 1/1000
molecule is ionized
only cationsthe sample is heated up
until a sufficient vapour
pressure is obtained

Chemical ionization
 In chemical ionization, the ionization of the analyte is
achieved by interaction of it’s molecules with ions of
a reagent gas in the chamber or source.
 Chemical ionization is carried out in an instrument
similar to electron impact ion source with some
modifications such as:-
 Addition of a vacuum pump.
 Narrowing of exit slit to mass analyzer to maintain
reagent gas pressure of about 1 torr in the ionization
chamber.
 Providing a gas inlet.

 It is a two part process.
 • In the first step
 A reagent gas is ionized by Electron Impact ionization
in the source.
 The primary ions of reagent gas react with additional
gas to produce stabilized reagent ions.
 In the second step, the reagent ions interact with
sample molecules to form molecular ions.
 • In this technique the sample is diluted with a large
excess of reagent.
 Gases commonly used as reagent are low molecular
weight compounds such as Methane, tertiary Isobutane,
Ammonia, Nitrous oxide, oxygen and hydrogen etc.

 TYPES OF CI:
 Depending upon the type of ions formed CI is
categorized as:-
 1. Positive Chemical Ionization
 2. Negative Chemical Ionization
 1. Positive Chemical Ionization
 In this technique positive ions of the sample are
produced.
 In positive chemical ionization, gases such as Methane,
Ammonia, Isobutane etc are used.

 For example,
 Ammonia is used as reagent gas.
 First ammonia radical cations are generated by electron
impact and this react with neutral ammonia to form
ammonium cation (reactive species of ammonia CI).
 NH4+
reacts with the sample molecules by proton transfer
to produce sample ions

 Negative Chemical Ionization
 Negative chemical ionization is counterpart of Positive
chemical ionization.
 In this technique, negative ions of the sample are
formed.
 Oxygen and Hydrogen are used as reagent gasses.
 This method is used for ionization of highly
electronegative samples.

 ADVANTAGES
 Used for high molecular weight compounds.
 Used for samples which undergo rapid fragmentation in
EI.
 LIMITATIONS
 Not suitable for thermally unstable and non-volatile
samples.
 Relative less sensitive then EI ionization.
 Samples must be diluted with large excess of reagent gas
to prevent primary interaction between the electrons and
sample molecules.

Field Ionization
 FI is used to produce ions from volatile compounds
that do not give molecular ions by EI.
 It produces molecular ions with little or no
fragmentation.
 Application of very strong electric field induces
emission of electrons.
 FI utilizes 10-micron diameter tungsten emitter wires
on which carbon whiskers, or dendrites, have been
grown.
 A high electric field gradient (1010
V/cm) at the tips of
the whiskers produces ionization

 ADVANTAGES
 As fragmentation is less, abundance of molecular ions
(M+
) is enhanced, hence this method is useful for
relative molecular mass and empirical formula
determination.
 DISADVANTAGES
 Not suitable for thermally unstable and non volatile
samples.
 Sensitivity is les than EI ion source.
 No structural information is produced as very little
fragmentation occurs.

Field Desorption
 Also known as offspring of field ionization.
 In field desorption method, a multitipped emitter
(made up of tungsten wire with carbon or silicon
whiskers grown on its surface) similar to that used in FI
is used.
 The sample solution is deposited on the tip of the
emitter whiskers either by
 dipping the emitter into analyte solution or
 using a microsyringe.
 Then the sample is ionized by applying a high voltage
to the emitter.

 ADVANTAGES
 Works well for small organic molecules, low
molecular weight polymers and petrochemical fractions.
 DISADVANTAGES
 Sensitive to alkali metal contamination.
 Sample must be soluble in a solvent.
 Not suitable for thermally unstable and non volatile
samples.
 Structural information is not obtained as very little
fragmentation occurs.

Electrospray ionization
 Electrospray ionization is a technique used in mass
spectrometry to produce ions from macromolecules
such as proteins, polypeptides and oligonucleotides
having molecular weights of 10,000 Da or more.
 The method generates ions from solution of a sample by
creating fine spray of charged droplets.
 • A solution of sample is pumped through a fine,
charged stainless steel capillary needle at a rate of few
microlitres/minute.
 The needle is maintained at a high electric field
(several kilovolts) with respect to cylindrical electrode.
 • The liquid pushes itself out of the capillary as a mist
or aerosol of fine charged droplets.

 These charged droplets are then passed through
desolvating capillary where the solvent is evaporated in
the vacuum and attachment of charge to the analyte
molecules takes place.
 Desolvating capillary uses warm nitrogen as nebulising
gas.
 The desolvating capillary is maintained under high
pressure.
 • As the droplets evaporate the analyte molecules comes
closer together.

 These molecules become unstable as the similarly
charged molecules comes closer together and the
droplets explode once again. This is referred as
Coulombic fission.
 • The process repeats itself until the analyte is free
from solvent and is alone ion.
 • The ion then moves to the mass analyzer.

 ADVANTAGES
 Most important techniques for analysis of high
molecular weight biomolecules such as polypeptides,
proteins, oligonucleotides and synthetic polymers.
 Can be used along with LC and capillary
electrophoresis.

High voltage applied
to metal sheath (~4 kV)
Sample Inlet Nozzle
(Lower Voltage)
Charged droplets
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MH+
MH3
+
MH2
+
Pressure = 1 atm
Inner tube diam. = 100 um
Sample in solution
N2
N2 gas
Partial
vacuum

Plasma Desorption
 Plasma desorption produces molecular ions from the
samples coated on a thin foil when a highly energetic
fission fragments from the Californium-252 “blast
through” from the opposite side of the foil.
 The fission of Californium-252 nucleus is highly
exothermic and the energy is released.

 When such a high energy fission fragments passes
through the sample foil, extremely rapid localized
heating occurs, producing a temperature in the range of
10000K.
 •Consequently, the molecules in this plasma zone are
desorbed.

Laser Desorption
 Laser desorption methods involves interaction of
pulsed laser beam with the sample to produce both
vaporization and ionization.
 Laser beam is usually of different wavelengths from far
U.V to far IR depending upon the sample to be
analyzed.
 REQUIREMENTS
 Laser wavelength must be at absorption wavelength of
the molecule.
 In order to avoid decomposition absorbed energy must
be quickly dispersed in the molecules.

 IONIZATION TECHNIQUE:
 • Ionization is carried out by two techniques :-
 Microprobe techniques
 Laser beam is focused to a very small spot on the back
side of a thin metal foil that holds a thin film of sample.
 Ions emerge out on the front side from a small cratered
hole in the foil.
 Bulk analysis techniques
 The technique uses a less focused beam and larger
samples.
 The laser beam produces microplasma that consists of
neutral fragments with elementary and fragment ions.

SpectroscopySpectroscopy Matrix assisted laser desorption
(MALDI)
 Matrix assisted laser desorption is a technique in mass
spectrometry for ionization of biomolecules (polymers
such as proteins, polypeptides and sugars) and synthetic
polymers that are more fragile and form fragments
when ionized by conventional methods.
 It consist of two components
 1 Matrix : Matrix is used in MALDI to
 Absorb the laser energy.
 Prevent analyte agglomeration.
 Protect analyte from being destroyed by direct laser
beam

 Matrix consists of a crystallized molecules of which the
most commonly used are
 Sinapinic acid)
 – cyano cinnamic acid ( –cyano or – matrix)α α α
 Dihydroxy benzoic acid (DHB)
 Nicotinic acid
 Matrix solution is then mixed with the analyte to be
investigated.
 The solution is then spotted in a air tight chamber on
the tip of the sample probe.

SpectroscopySpectroscopy  With a vacuum pump the air is removed and vacuum is
created which leads to evaporation of the solvent leaving
behind a layer of recrystalized matrix containing analyte
molecules.
 2 Laser
 The solid mixture is then exposed to pulsed laser
beam.
 The matrix absorbs the laser energy and transfers some
of this energy to the analyte molecules which results in
the sublimation of sample molecules as ions or the
matrix after
 Absorbing the laser energy gets ionized and transfer
part of this charge to the sample molecules and ionize
it.

 When the polymers form cations the cathode is placed
right behind the sample and anode in front of the
sample.
 The cations get attracted towards the negatively charged
anode. This acceleration is used to move the ion to the
detector.
 When the polymer forms anions the electrodes are
interchanged.

 Fast atom bombardment (FAB) is an ionization
technique used in mass spectrometry in which a beam
of high energy atoms strikes a surface to create ions.
 When a beam of high energy ions is used instead of
atoms (as in secondary ion mass spectrometry), the
method is known as liquid secondary ion mass
spectrometry (LSIMS)

Quadrupole mass analyzer
 A quadrupole mass spectrometer contains four parallel
cylindrical rods which can scan or filter sample ions
based on their mass-to-charge ratio.
 Opposing rods are connected electrically and a radio
frequency voltage is applied between the pairs of rods.
 Ions travel between the rods and only ions with a
specific mass-to-charge ratio will exit the quadrupole;
other ions will collide with the rods.
 The desired mass-to-charge ratio can be altered by
changing the applied voltage.

 Triple quadrupole mass spectrometry makes use of the
same technology, but uses a linear series of three
quadrupoles to improve sensitivity and selectivity.
 This type of spectrometry is useful when studying
particular ions of interest since it is able to stay tuned to
a single ion for extended periods of time.

Ion trap mass analyzer
 This analyzer employs similar principles as the quadrupole
analyzer mentioned above, it uses an electric field for the
separation of the ions by mass to charge ratios.
 The analyzer is made with a ring electrode of a specific voltage and
grounded end cap electrodes.
 The ions enter the area between the electrodes through one of the
end caps. After entry, the electric field in the cavity due to the
electrodes causes the ions of certain m/z values to orbit in the
space.
 As the radio frequency voltage increases, heavier mass ion orbits
become more stabilized and the light mass ions become less
stabilized, causing them to collide with the wall, and eliminating
the possibility of traveling to and being detected by the detector.

TOF Analyzers
 TOF Analyzers separate ions by time without the use of
an electric or magnetic field.
 In a crude sense, TOF is similar to chromatography,
except there is no stationary/ mobile phase, instead the
separation is based on the kinetic energy and velocity of
the ions.

 Ions are accelerated by an electric field of known
strength.This acceleration results in an ion having the
same kinetic energy as any other ion that has the same
charge. The velocity of the ion depends on the
mass-to-charge ratio (heavier ions of the same charge
reach lower speeds)
 The time that it subsequently takes for the ion to reach a
detector at a known distance is measured.
 This time will depend on the velocity of the ion, and
therefore is a measure of its mass-to-charge ratio. From
this ratio and known experimental parameters, one can
identify the ion.

Magnetic sector analyzers
 Similar to time of flight (TOF) analyzer mentioned
earlier,
 In magnetic sector analyzers ions are accelerated
through a flight tube.
 Where the ions are separated by charge to mass ratios.
The difference between magnetic sector and TOF is that
a magnetic field is used to separate the ions.
 As moving charges enter a magnetic field, the charge is
deflected to a circular motion of a unique radius in a
direction perpendicular to the applied magnetic field

THANK UUUUUUUUUUUU

Mass spectroscopy, Ionization techniques and types of mass analyzers

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