Mass Spectrometry (MS)

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Mass Spectrometry (MS)
The mass spectrometer (MS) is an unrivaled
analytical tool which provides information for
species identity.
Coupling an MS to a chromatographic system for
use as a detector can provide sample information
of species quantity and identity. Massive
libraries of compounds for species identification
make MS the most powerful analytical tools ever!
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Mass Spectrometry uses the differences in
molecular masses (weights), to identify a
molecules structure.
Fragmentation Molecular Ions and Daughter Ions
HOW?
The difference in the mass of ions is used to
isolate and identify these fragments.
Ions with a different masses are sorted by a
mass analyser
The modern Mass Spectrometer consists of a number
of components
Signal Processing
Detection
Separation?
Mass Analyser
Ionisation (EI/CI)
Sample Inlet
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Detection of Ions in Mass Spectrometry.
Remember Photo-Multiplier Tubes?
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• There are many different types of mass analyser
• Some are fast and compact with low mass
  resolution
• e.g. measure masses to within 1 atomic mass unit
  Carbon for example 12
• Others large and expensive but with very high
  mass accuracy
• e.g. carbon measured as 12.0124

• Some common types of mass analyser
• ION TRAP
• QUADRUPOLE
• TIME-OF-FLIGHT
• MAGNETIC SECTOR

ALL Mass spectrometers require very high vacuums
inside the mass analysers and flight tubes
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Time-of-Flight Mass Spectrometry
The first commercial linear TOF analysers were
produced in the late 1950s.
There has been renewed interest in TOFs since
the late 80s mainly because progress in
electronics has simplified the handling of the
large data flow
Linear time-of-flight mass spectrometer
Ions are expelled from the source in bundles that
are either produced by an intermittent process
such as laser desorption or by pulsing the
potentials of extraction plates.
The ions are accelerated by a potential Vs and
fly a distance d before reaching the detector
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Detection of ions of different m/z ratios
Mass-to-charge (m/z) ratios are determined by
measuring the time that ions take to move through
a field-free region between the source and the
detector.
As it leaves the source, an ion with mass m and
total charge q ze has a kinetic energy EK of
(1)
EK mv2 z e VS
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where e elementary charge
The time needed to fly the distance d is given by
t d / v
(2)
Replacing v by its value in (1) gives
t2 m d2 z 2VS e
(3)
Equation (3) shows that (m/z) can be calculated
from a measurement of t2, since the terms in
parentheses are constant
i.e. the time taken for the ion to reach the
detector is proportional to the square root of
its m/z ratio
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Time of Flight Mass Spectrometry - Speed of
Detector.
Each pulsed ion packet reaching the detector will
consist of the complete mass range of ions, with
the lighter ions arriving first and the heaviest
ions arriving last. The detector electronics
must have extremely fast time resolution to
resolve all ions.
Example The source is pulsed at 10kHz (10,000
times per second) with each pulse having a
duration of 50 ms. i.e. each ion packet of 50 ms
is separated by 50 ms. If the TOF measures in
the range 0-300 Daltons, the detector has less
than 200 ns (167 ns) to measure 1 Da. To achieve
a mass resolution of 0.01 Da would therefore
require the detector to have a 1.5 ns resolution.
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Magnetic Sector Mass Spectrometry.

• Rather than measuring free flight times, a
  magnetic sector instrument differentiates between
  ions of different masses by their trajectories
  under the influence of a magnetic field.
• Very high mass resolution
• Used extensively in areas such as pesticides,
  food safety etc - generally coupled to GC

Before influence of the magnets all four ions
travel with the same trajectory.
On passing across the magnetic field the
trajectory of each ion is changed.
Light ions are bent more than heavy ions.
After passing through the field all four ions
travel in a different direction.
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The magnetic sector mass analyser uses bent
magnets to form the magnetic field.
In this format ions of a particular mass make it
through to the detector.
The magnetic field is also adjustable, and can be
increased to bend heavier ions toward the
detector or decreased allowing the lightest ions
through the field.
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It is very important that the volume inside the
analyser is under a strong vacuum as we only want
to measure the analyte molecules.
The initial trajectory of the ions is created by
an accelerating voltage across metal plates with
an inlet slit.
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The curvature of an ions path through a magnetic
field is dependant on its momentum, but is also
dependant on the charge it carries.
higher charged ions react to a field more an so
bend in a tighter arc, lower charged ions in a
wider arc
B field strength q ions charge in electron
volts
m mass of the ion v velocity of the ion r
radius of the arc
So how do we know if we are detecting a heavy ion
with a high charge or a lighter ion with a lower
charge?
The detector can identify the magnitude of the
charge on an ion from the current it produces,
using the ionic charge number z (1, 2..etc)
the spectrometer now gives a reading for the
masscharge ratio (m/z) for the molecule or
fragment ion.
m/z is related to magnetic field strength and the
accelerating voltage and the radius through which
the ion is bent
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How is the analyte ionised?
There are many ways which have been developed for
the ionisation of samples in MS.
These range from hard techniques which split
analyte molecules into several ionised fragments.

• To soft techniques which try to ionise the
  analyte keeping the molecule intact.

We will focus on two of the most common
ionisation methods, electron impact (a hard
technique) and chemical ionisation (a soft
technique).
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Ions created in the chamber accelerate to the
charged plates.
Most strike the first plate with some passing
through a slit.
Having more than one plate/slit produces a narrow
stream of ions with a uniform trajectory.
The voltage across the accelerating plates can be
changed to give ion streams of different
velocities, so changing the momentum and kinetic
energy of the ions.
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Electron impact ionisation (EI)
Analyte molecules are introduced into a chamber
which is kept under high vacuum, here they are
vaporised to a gas.
Electrons are formed from a heated filament,
these are accelerated toward a positive cathode
as a beam, the beam passing through the chamber
containing analyte molecules.
Impact with high energy electrons cause molecules
to expel one or more of their own electrons to
become positive ions.
This process often forms unstable ions which
fragment into smaller more stable constituents.
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Like the voltage across the accelerator plates,
the voltage across the chamber can be changed to
control the energy of the electron beam.
Electron energies ten times that required to
ionise the analytes are often used.
The excess energy causes fragmentation and
changing the energy of the beam produces a
different set of fragments.
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Three interesting points!
The ionisation process also produces some ions
with a negative charge, these are repelled by the
accelerating plates and so are not analysed.
Ionisation by EI results mainly in ions with a 1
charge.
Only about one in every thousand analyte
molecules are ionised and therefore analysed, the
rest being sucked out of the chamber by the
vacuum pump.
The fragments formed by EI give a consistent
pattern for a given analyte molecule, which aids
identification.
However, as the molecular ion does not survive
analysis it is difficult to determine the
molecular weight of the analyte.
For this reason soft ionisation techniques
which preserve the molecular ion are sometimes
preferred.
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Chemical ionisation (CI)
The format for CI is very similar to EI but along
with the sample a reagent gas is also added to
the chamber such that for every analyte molecule
100000 reagent molecules exist.
In this situation the any molecules ionised by
the electron beam will be those of the reagent
gas.
These ions interact with other reagent gas
molecules but also with the dilute number of
analyte molecules.
Common gases to be used as the reagent are
methane, water and ammonia.
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Reagent gas ionised to form ion --gt Reaction of
ion with analyte molecule.
The simplest of the reactions which take place in
the chamber to create analyte ions is
CH4 e- ? CH4 2e- CH4 M ? CH4 M
Electron beam quenched by reagent gas - difficult
to predict chemistry Much higher probability of
ionisation, dense cloud of ions (CH4)
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Obviously the resulting mass spectrum using CI
will contain the spectrum of reagent ions as well
as analyte ions, but this can be ignored when
interpreting spectra.
Ionisation of analyte ions through the reaction
with reagent ions is an efficient process, much
more so than by electron impact.
So CI give more analyte ions but less
fragmentation than EI resulting in fewer peaks
and better sensitivity.
Like EI the ionisation also produces negative
analyte ions, however with CI techniques the
polarity of the accelerating plates is sometimes
reversed giving the ability for negative ion
analysis.
Many environmental compounds give strong negative
spectra.
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How is the sample introduced?
The inlet for MS depends on in what state your
sample exists.
The majority of environmental analysis by MS is
done when the spectrometer is coupled to a
chromatography system.
The sample is therefore already in the form of a
gas (GC) or in a liquid solution (HPLC).
HPLC and GC separate samples into their
components and determine the quantity of each
constituent, coupling this to MS takes each
constituent and identifies it - the complete
analytical tool!
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Coupled GC and MS (GC-MS)
The sample eluting from a GC column is gaseous
and if the bore of the column and mobile phase
flow rate are low enough the eluent may be
allowed to flow directly into the chamber.
This can only be done if the vacuum inside the
chamber is not altered by addition of the eluting
mobile phase and analytes.
If the bore of the column is too wide and the
flow rate high such that too much sample be
introduced to the chamber an open split interface
is used.
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The split interface consists of a split between
the column end and the MS inlet with a make up
gas flushing over the periphery.
Most of the eluent is vented away with the make
up gas but some is diluted and passes through to
the chamber.
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Coupled HPLC and MS (LC-MS)
There are two things which make coupling LC to MS
more complex than GC-MS
In GC-MS the mobile phase gas is much lighter and
volatile than the analtyes it carries making it
easily removed from the sample.
In LC-MS the mobile phase is a liquid and of a
similar volatility to any analyte molecules,
making removal of the solvent difficult.
When a liquid is introduced to a vacuum it
expands very rapidly, so raising pressure and
causing the vacuum to fail.
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Two interfaces which bridge these problems
The particle beam interface which removes the
mobile phase from the eluent but leaves analyte
molecules behind.
Thermospray interface which vaporises the
eluent forming analyte molecular ions which can
be deflected from the mobile phase vapor straight
into the MS.
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The particle beam interface allows the flow from
an LC column to enter a nebuliser, here a spray
of helium gas breaks up the liquid into droplets
containing the analyte but mostly mobile phase
solvent and dissolved helium.
Passing through the desolvation chamber solvent
and helium molecules start to evaporate from the
droplets which pass through the exit nozzle in a
particle beam.
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The droplets pass from the nozzle through a
section under vacuum. Here solvent and helium
molecules evaporate from the surface of the
droplets and are removed through a series of
vacuum vents.
The first of these skimmers removes about 90
of the helium and mobile phase solvent and after
the second skimmer any such remaining particles
are removed by heating the gaseous beam.
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With this interface the beam of analyte molecules
entering the chamber of the MS will be ready for
EI or CI ionisation.
The result is a beam of analyte particles
directly in to the MS.
Why do analyte molecules not get sucked away
through the vents?
Analyte molecules are usually far heavier than
solvent molecules and He atoms and their momentum
carries them through the skimmers.
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The thermospray interface introduces the eluent
from LC through a capillary the end of which is
strongly heated.
When the liquid passes this region it is rapidly
vaporised forming a supersonic jet which
creates a mist of fine droplets.
Due to the speed of vaporisation the droplets are
left with a residual positive charge.
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Like the particle beam interface the droplets
pass through a vacuum where the solvent molecules
start to evaporate from the surface.
The droplets become smaller but the positive
charge on them stays the same.
Eventually the droplets become unstable so that
along with the solvent, analyte ions evaporate
from the surface.
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The resulting stream of solvent molecules and
analyte ions pass an opening to the MS chamber
which is also under vacuum.
The ions are deflected by an electric field
directly into the MS for analysis, while the
neutral solvent molecules flow past and are
removed by the vacuum.
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Detection
Separation?
Mass Analyser
Ionisation (EI/CI)
Sample Inlet
Interpreting Mass spectra
Signal Processing
A mass spectrum is a plot of relative abundance
against M/Z and appears as a series of bars each
representing a specific M/Z with the height of
each its abundance.
As M/Z is used and not mass of the detected ion,
we must realise that an M/Z of 28 could be an ion
of weight 56 and a charge of 2 or an ion of 28
charged 1.
Most commonly though the ionic charge is 1 and
M/Z is simply the mass of the detected ion
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This is the Mass spectrum for air of which the
main components are Nitrogen (atomic mass 14) and
oxygen (atomic mass 16).
However we also see the presence of the 15N which
exists naturally.
The other small peaks correspond to water and
carbon dioxide.
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There are features of simple mass spectra which
are a fingerprint for the type of compound
present.
The general shape of spectra is governed by
fragmentation patterns.
Alkanes only split at the C-C bond so making very
regular clusters of peaks.
The smaller peaks of a cluster are due to the
presence of the 13C isotope.
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If a hydrocarbon chain has a branch, each side of
this branch will be the most likely point for
fragmentation.
We see larger quantities of the four resulting
fragments.
Here the fragment with weight 71 is from
splitting of the 141 chain.
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A similar fragmentation pattern can be seen with
alkenes but the presence of a double bond reduces
the weights by 2 x H (2 units).
For cycloalkanes the degree of fragmentation is
considerably less.
Hydrocarbon rings are not easily fragmented as
this would require the breaking of two C-C bonds.
Hydrocarbon compounds with similar weights are
quickly identified by their fragmentation
patterns.
This is seen with some C6 compounds, hexanes and
hexenes.
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For n-hexane we see normal hydrocarbon
fragmentation.
Addition of a double bond reduces ion weights by
2.
Addition of two double bonds reduces ion weights
by a further 2 mass units.
With cyclohexene we see a very large molecular
ion peak, reduced fragmentation and reduction of
ion weights by 2 mass units.
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Identifying spectra is becoming easier as more
instruments incorporate compound libraries.
These libraries scan through mass spectra
information gathered for thousands of compounds
looking for matching peaks, relative intensities
and clusters.
A list of several matches are produced for a
recorded spectra along with a measure of the
match.
A match index of 1000 would be considered a
perfect match, however it is more common for the
operator to decide between matches with a score
of around 600-800.
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Below is the mass spectrum of a hydrocarbon, but
what is it?
Regular peaks at intervals of 14, perhaps a chain
alkane! No peak at 71? Large peaks at 57 and 43
mass units! Molecular ion at 100 with next
largest at 85 (after loosing 15!)
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The fact that we do not see an ion with a mass of
71 means that the alkane is not a straight chain.
We also see the most common ions have mass of 57
and 43 suggesting that C3H7 and C4H9 are joined
across a branched group.
The peak with mass 29 is caused by secondary
splitting of the fragments, otherwise we would
see the opposite peak at 71.
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When interpreting mass spectra it is useful to
know the most commonly lost fragments.
In practice we may know from elemental analysis,
that the composition of a compound is C5H12O and
has the mass spectra below, so what is the
compounds structure?
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We know that no double bonds occur as the 12
Hydrogen atoms would saturate 5 Carbon atoms.
The molecular weight is 88 with the smallest
fragment lost weighing 88-73 15 (CH3).
Another loss occurs to give an ion at 70 relating
to a loss of 18, probably OH.
The largest peak of weight 45 corresponds to a
loss of 43 units, this could not be either of the
ions OCH2CH3 or COOH as we only have one O and it
is probably in an OH formation.
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We have accounted for 4 carbon, 11 hydrogen and
the oxygen atom.
The remaining C and H must be a link to the OH
fragment suggesting the structure
Fragmentation at the three points around the
branch would give the observed spectra, the
sample is 2-pentanol.
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AromaticCompounds

• In organic chemistry, the tropylium ion is an
  aromatic species with a formula of C7H7. Its
  name derives from the molecule tropane (itself
  named for the molecule atropine).
• It is a heptagonal, planar, cyclic ion as well,
  it has 6 p-electrons (4n 2, where n1), which
  fulfills Hückel's rule of aromaticity. It can
  coordinate as a ligand to metal atoms.
• The structure shown is a composite of seven
  resonance contributors in which each carbon
  carries part of the positive charge.
• The tropylium ion is frequently encountered in
  mass spectrometry in the form of a signal at m/z
  91 (see mass spectrum analysis). This fragment
  is often found for aromatic compounds containing
  a benzyl unit. On ionization, the benzyl fragment
  is cleaved off. It (PhCH2) rearranges to the
  highly stable tropylium cation (C7H7).

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