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MASS SPECTROMETRY
Mass Spectrometry
Structure
13.1 Introduction
Objectives
13.5 Summary
13.6 Terminal Questions
13.7 Answers
13.1 INTRODUCTION
In this course, you have so far learnt about a number of spectroscopic methods and
their analytical applications. You would have understood by now that besides
quantitative analysis, one important aspect of the spectroscopic methods is that they
help in structure elucidation of the molecules being analysed. In the context of
structure determination we take up mass spectrometry in this unit. The mass
spectrometry is not a spectroscopic method in true sense of the word because here we
do not make use of any electromagnetic radiation; yet it is no less significant. In mass
spectrometry we study the consequences of bombardment of the analyte in vapour
phase with high energy electrons.
We will begin the unit by explaining the principle of mass spectrometry, describe a
typical mass spectrum and discuss its characteristics. The discussion on the
information available from the mass spectrum will be followed by a detailed account
of the instrumental components of mass spectrometers. We will then discuss different
applications of mass spectrometry.
Objectives
After studying this unit, you should be able to:
define and explain the terms like base peak, molecular ion, fragments, etc.,
+
+
explain the importance of [M+1] . and [M+2] . ion peaks,
determine the molecular formula of the compound on the basis of its mass
spectrum,
identify the common fragmentation patterns in the mass spectra and interpret
them to make preliminary leads into the structure of the molecule,
enlist different methods of ionisation and explain the principle behind them,
37
Miscellaneous Methods
discuss various fragmentation types and the factors affecting them, and
Have you ever wondered what would happen if we take a molecule and bombard it
with a beam of high energy electrons? Taking clue from ionisation of atoms to give
ions you may be prompted to say that they would ionise; and you are absolutely right.
This is what exactly is done in mass spectrometry. Here the molecule in the vapour
phase is bombarded with a beam of high energy (~ 70 eV) electrons. These electrons
knock off an electron from the molecule leading to the formation of a positively
+
+.
NH3 + 2 e
NH 3 + e
The radical ions so obtained have a large amount of extra energy which is much more
than that required for breaking the covalent bonds. Therefore, the highly energetic
molecular ions undergo fragmentation yielding smaller fragments. For example,
+.
NH3
m/z 17
NH2 + H
m/z 16
+
NH . + H
m/z 15
+
N + H
m/z 14
m z denotes a
dimensionless quantity
obtained by dividing the
mass of the ion in unified
atomic mass units, u by
its charge number.
As the molecular ion can fragment in a number of ways depending on the nature of the
molecule, the fragments provide clue to the structure of the molecule. The radical ion
and the fragments obtained from the molecule are then separated according to their
m z value (where m is the mass and z is the charge on the ion) using magnetic and/or
electrostatic fields. The record of m z values of these species against their relative
amounts or intensities is known as mass spectrum of the sample.
38
The x-axis in the spectrum represents the m z value of the fragment ions and the
y-axis gives the relative intensity or abundance of different fragments. For y-axis the
intensity of the peak representing the most abundant fragment (CH2OH+ ; m z =31, in
this spectrum) is arbitrarily assigned an intensity of 100% and the peak intensities of
other ions are measured relative to it. This most intense peak is called as the base
peak and it need not necessarily be the molecular ion peak which is at m z = 32. Are
Mass Spectrometry
you wondering, why do we observe a small peak at m z = 33? What is the origin of
this peak? Let us learn about it.
[M+1] . peak. The origin of this peak can be understood if we take into account the
natural abundance of the isotopes of constituent atoms of a molecule. You know that,
most of the elements exist in nature predominantly as a single entity i.e., as a
collection of identical atoms. However, some elements have isotopes i.e., they exist as
a mixture of atoms having same atomic number but different mass numbers. For
example, carbon exists in nature as a mixture of 126 C as well as 136 C atoms. The
natural abundance of
13
6C
13
6C
12
6C
13
6C
12
6C
33 gmol as against the normal value of 32 and hence the peak at m z = 33. Further,
since oxygen ( 168 O and 188 O ) and hydrogen ( 11 H , and 21 H ) can also exist as isotopes,
they would also influence the spectrum and we may expect a very small signal at
+
+
+
m z = 34 i.e., at [M + 2] . in addition to the M . and [M + 1] . peaks. The other
peaks in the spectrum appear due to the fragmentation of the molecular ion. The
natural abundance of some common elements is compiled in Table 13.1.
Table 13.1: Relative isotopic abundance* of some common elements
Element
Isotope
Relative
abundance
Isotope
Relative
abundance
Isotope
Relative
abundance
Carbon
12
100
13
1.11
Hydrogen
100
0.016
Nitrogen
14
100
15
0.38
Oxygen
16
100
17
0.04
18
Sulphur
32
100
33
0.78
34
4.40
Chlorine
35
Cl
100
37
Cl
32.5
Bromine
79
Br
100
81
Br
98.0
N
O
S
These contribute to
[M+1]
+.
ion peak
0.20
These contribute to
[M+2]
+.
ion peak
39
+.
Miscellaneous Methods
The presence and the intensity of peaks at m z values greater than that of the
molecular ion can provide important information about the elemental composition of
the molecule. As an example let us have a look at the mass spectrum given in Fig.13.2.
What do you observe in this case? Have a look at the Table 13.1 again, what do you
infer? Yes it is a typical case of a molecule containing a bromine atom; the intensities
+
SAQ 1
The mass spectral data shows the following intensity pattern in the region of highest
m z values.
m z
78
79
80
Intensity
(% of base peak)
24
0.8
8.0
What do you infer about the nature of the compound? You may use Table 13.1 for
answering this question.
.
.
.
.
Inlet device
Analyser
Detector
40
Mass Spectrometry
The inlet device loads the sample into the ionisation chamber where the analyte is
ionised by a suitable method and the molecular ion and/or the fragment ions obtained
by fragmentation of the molecular ion are directed towards the analyser. In the
analyser these ions obtained by the fragmentation of the molecular ion are sorted out
on the basis of their m z value by using one of the many available techniques and are
sent to the detector (transducer). In the detector the ion flux generates an electrical
current proportional to the number of ions reaching it. The processing unit records the
magnitude of these electrical signals as a function of m z and gives an output in the
form of a mass spectrum. Let us learn about the components of mass spectrometer.
Desorption sources
Gas phase sources: In these sources the sample is first vaporised and then ionised.
These are used for low molecular weight (< 1000 Da) samples which are thermally
stable i.e., do not decompose on heating and have low boiling points (< 500 oC). There
are three methods that belong to this category. These are:
41
Miscellaneous Methods
i)
ii)
iii)
Let us learn about the first two of these as the principle of the third method is beyond
the scope of this course.
In most of the mass
spectrometers the electrons
are emitted from a heated
tungsten or rhenium
filament and are accelerated
by applying potential.
Electron ionisation: This is the oldest and probably the best-characterized of all the
ionisation methods. In this method, a high energy beam of electrons passes through the
sample in the gas-phase. These electrons generally have energy of 10-150 eV. The
electrons on colliding with the neutral analyte molecule knock off an electron from it
and generate a positively charged ion. This process produces either a molecular ion or
one of its fragments. This method is good for volatile compounds but the molecular
ion peak is either weak or absent.
Chemical ionisation: The chemical ionisation method uses ion/molecule reactions to
produce ions from the analyte. For this purpose a reagent gas such as methane, isobutane, or ammonia is passed into the ionisation chamber where it gets ionised by
electron ionisation. For example, methane gas gives mainly CH +4 and CH 3+ ions as
follows
CH4 + e
+.
CH4 +
2e
.
+
CH3 + H
+.
CH4
These reagent gas ions then react with the neutral molecules of the reagent gas as
follows
+.
CH4 +
CH4
+
CH5 + CH3
+
CH3 +
CH4
+
C2H5 +
H2
The products of these ion-molecule reactions react with the analyte molecules (M) to
produce analyte ions. The reactions with the above ions can be shown as
M +
CH5+
M +
C2H5
MH +
+
MH +
CH4
C2H4
These give an ion at [M+1]. For the analyte M of RH type, we may have reaction that
can be represented as
RH +
CH5
R+ +
CH4 + H2
In this case the ions would be obtained at [M 1]. Thus, the mass spectrum resulting
from chemical ionisation method generally contains well defined [M+1]+ and [M 1]+
ion peaks. Further since the (M+1)+ ions do not undergo significant fragmentation, the
spectra are simpler as compared to the ones obtained in EI method.
Desorption sources: In these sources the solid or liquid sample is directly converted
into the gaseous ions. These are used for high molecular weight (> 1000 Da) samples
which are thermally unstable (i.e., decompose on heating) and are non volatile. A
number of sources belonging to this category are available but we shall take up just
one of them, namely the fast atom bombardment (FAB) method.
42
Fast atom bombardment (FAB): In this method the analyte is dissolved in a liquid
matrix like glycerol and a small amount of this is placed on a target. The target is then
bombarded with a beam of fast atoms (e.g., xenon or argon atoms at several keV).
These desorb positive and negative analyte ions from the sample.
+
A B
Fast
A +
Atoms
Mass Spectrometry
As the sample heating is achieved very quickly the fragmentation of the analyte ions is
greatly reduced.
Thus, the ionisation chamber generates a stream of ions (primarily positive ions)
which is accelerated by applying suitable potential and is sent to the analyser. Let us
understand how the analyser separates these ions.
13.3.3 Analysers
The analysers in the mass spectrometers are like the grating in the spectroscopic
instruments. In a way, these disperse the fragment ions according to their mass to
charge ( m z ) ratios. Several types of analysers are available; we shall take up only
two such analysers that are commonly employed in the mass spectrometers. These are:
Magnetic sector analyser: As the name suggests, these analysers make use of a
permanent magnet (or an electromagnet) to separate the fragment ions. It consists of
an evacuated curved metallic tube through which the fragment ions pass on their way
from the ion source to the detector. The electromagnets are mounted perpendicular to
the tube and provide a stable and uniform magnetic field (Fig 13.4).
As the ions entering the analyser have approximately the same kinetic energy, they
have different velocities depending on their masses; heavier ions would be slower. The
field of the magnet makes these ions to travel in a circular path generally of 60, 90 or
180 degrees. The mass to charge ratio of the fragment ions is related to the parameters
of the instrument as per the equation.
43
Miscellaneous Methods
m B2 r 2e
=
z
2V
where,
B = magnetic field strength,
r = radius of curvature of the magnetic sector (metallic tube) ,
V= the accelerating voltage applied on the ion in the ionisation chamber and
e = the electronic charge.
According to this equation the mass spectrum can be obtained by varying any of the
three parameters viz., B,V or r. In most of the modern spectrometers, the separation is
achieved by varying the field strength of the electromagnet keeping V and r constant.
At a given magnetic field strength, only the fragments of a certain m z value would
be able to travel through the circular path in the analyser tube; the ions of larger or
smaller m z values would strike the tube and get destroyed. Thus, with the gradual
increasing strength of the electromagnet, the fragment ions of increasing m z values
successfully pass through the analyser and get detected by an ion collector.
Double focussing analysers: In describing the magnetic sector analyser we
mentioned that the ions entering the analyser have the same kinetic energy. In fact
these fragments have a small range of kinetic energies and when passed through the
magnetic field these get focussed at the same position. Thus, the spread of kinetic
energies cause a broadening of the beam reaching the detector leading to a loss in
resolution.
For a better resolution and more accurate mass determinations double focussing
instruments are used which employ an electrostatic field prior to the magnetic sector.
This combination of electrostatic and magnetic fields causes the energy focusing as
well as the directional focusing respectively. A schematic diagram of the double
focussing mass analyser is given in Fig. 13.5.
The electrostatic analyser consists of two curved metallic plates as shown in Fig. 13.5
and a dc potential is applied across them. This allows the fragment ions with only a
limited range of kinetic energy to go over to the magnetic sector. As in case of the
magnetic sector, here also the ions with greater and smaller kinetic energies are
destroyed at the plates.
44
Mass Spectrometry
In the mass spectrometers the ions after passing through the analyser are generally
detected by a suitable electron multiplier. The electron multipliers are capable of
providing quick response times and high current gains. The electrical signal so
obtained can be processed, stored or suitably displayed.
SAQ 2
Arrange the following fragment ions in the order of their detection in the mass
spectrometer.
+
.
.
.
.
.
SAQ 3
List three ion sources used in mass spectrometers. Give one characteristic feature of
each one of them.
.
.
.
.
45
Miscellaneous Methods
specialised areas but be contented with the most common qualitative and quantitative
applications.
Example 1
The mass spectrum of a compound containing only carbon, hydrogen and oxygen gave
the following intensity pattern in the region of molecular ion.
46
Mass Spectrometry
Intensity
(% of molecular ion peak)
100 (M)
100
101 (M+1)
6.67
102 (M+2)
0.41
m z
Solution
Since the molecular ion peak is at m z of 100 we need to check for the possible
molecular formulae from the tables referred above. The following table gives the
possible molecular formulae containing C, H and O and having a molar mass of 100.
M =100
C4H4O3
4.50
0.68
100.0160
C5H8O2
5.61
0.53
100.0524
C6H13O
6.71
0.39
100.0888
It is quite obvious that the given mass spectrum is for the compound with a molecular
formula as, C6H13O; the compound is cyclohexanol; as the (M+1) is 6.71 and
(M+2) = 0.39 which matches with 6.67 and 0.41 given in the data.
If you look at the table given in the solution to the example 1 you can appreciate that
these tables can be used in a yet another way. Suppose we take a high resolution mass
spectrum of the compound and find that the molar mass obtained from molecular ion
peak is 100.0890 we are quite sure that the compound is C6H13O.
Thus, the molecular formula of an analyte can be obtained either from the intensity
pattern of the isotopic peaks or by accurate molar mass determination from high
resolution mass spectrum. Let us learn how to determine the structure of the analyte
molecule once the molecular formula is known?
A simple cleavage of the single bond in the molecule is quite common and important
fragmentation mechanism. In such a cleavage of a radical cation we get a radical and a
47
Miscellaneous Methods
cation. For example, the simple cleavage of C-C single bond in molecular ion radical
of propane can be represented as follows.
+
CH3CH2
.
CH3
m/z = 29
+.
CH3CH2CH3
.
CH3CH2
CH3
m/z = 15
In this cleavage we get two cations with the m z values of 29 and 15 respectively and
both of them are observed in the mass spectrum of propane. However, the intensity of
the peak at m z 29 is much more than that of the peak at m z 15. In fact the m z 29
peak is the base peak which is the most intense peak. The intensities of the peaks is
determined by the stability of the fragment ions; the secondary ethyl carbocation
(CH3CH2 +) is much more stable than the primary methyl carbocation (CH3 +).
For the same reason the cleavage is favoured at more alkyl substituted carbons in
branched alkanes.
Fragmentation by -cleavage in Molecules with Heteroatoms
The bond on the carbon atom next to a herteroatom (N, O, halogen) in a molecule is
easily cleaved. This is called -cleavage and in such a cleavage the charge goes with
the fragment containing the heteroatom. For example, two such cleavages are shown
below in case of an alcohol
(i)
R
+.
CH
OH
(i)
+
CH
(ii)
H
.
..
.. + R
OH
..
H
(ii)
CH
+
CH
.
..
.... + H
OH
+
OH
....
m/z= M-1
CH
+
. .OH
..
The resulting cations are resonance stabilised due to the participation of the
nonbonding pair of electrons on the heteroatom as shown above.
Fragmentation due to Intramolecular Rearrangement: McLafferty
Rearrangement
Sometimes the origin of the fragments observed in a mass spectrum can not be
attributed to a formal cleavage; instead it involves some kind of an intramolecular
rearrangement. Such rearrangements often explain the existence of prominent
characteristic peaks in the mass spectrum and are therefore very useful in structure
elucidation. One of the most important rearrangements, called McLafferty
rearrangement is observed in the molecules containing a carbonyl group having
abstractable hydrogen at a position to the carbonyl group.
48
Mass Spectrometry
. .O .
+
H
CHR
C
Y
. .O .
+
CH2
C
H2
H
+
RCH=CH2
C
Y
CH2
at m z 72 is the molecular ion peak and the ones at 57 (M 15) and 29 (M 43) can
be explained in terms of methyl and propyl radicals respectively. The peak observed at
m z 44 (M 28) can be explained in terms of McLafferty rearrangement as shown
below.
. .O .
+
H
CH2
C
H
.. .
O
+
CH2
C
H2
H
+
CH2=CH2
C
H
CH2
m/z = 44
m/z = 28
49
Miscellaneous Methods
Table 13.2: Some commonly lost fragments and the stable fragments in the mass
spectrum
Commonly lost fragments
Fragment lost
Peak obtained
CH3
M.
OH
M.
CN
M.
H 2C
15
17
CH 2
. CH2CH3
M.
M.
Fragment lost
Peak obtained
. OCH
+
M.
- 31
+
M . - 35
Cl
.
O
M +. - 43
OCH2CH3
+
M . - 45
26
CH 3C
28
29
.
CH2
M.
- 91
m/z = 43
Ion
CH 3
+
C
O
.
+
m/z = 91
CH2
O.
m/z = M . - 1
CH
SAQ 4
The mass spectrum of butanoic acid gives a characteristic peak at m z = 60. Justify
the same.
.
.
.
.
.
50
instrumental parameters like, ionisation method, temperature, pressure, etc. It may not
always be possible to have reference spectra under the conditions of the sample being
analysed.
Mass Spectrometry
Now-a-days most of the modern mass spectrometers are equipped with computerised
library search systems. The identification of the analyte can be achieved by
performing the library search on the expected group of compounds. Once a reasonable
match is obtained the spectra of the analyte and the reference compound can be plotted
simultaneously and compared.
Very often we deal with mixtures of substances. The complexity of the spectra for
mixtures necessitates that the components be separated prior to their mass spectral
analysis. Such a separation is usually achieved by gas chromatography and the gas
chromatograph is coupled with the mass spectrometer. In this hyphenated technique,
the components from the GC are allowed to directly enter the spectrometer in the
vapour phase one by one and get analysed. Recent advances have allowed even the
liquid chromatographs, supercritical fluid (SCF) chromatographs, and capillary
electrophoresis (CE) devices to be coupled directly to mass spectrometers.
SAQ 5
How can you analyse a mixture of substances using mass spectrometry?
.
.
.
.
.
51
Miscellaneous Methods
13.5 SUMMARY
In this unit, you have learnt about mass spectrometry which is an analytical tool with
wide ranging applications. In mass spectrometry a molecule in the vapour phase is
bombarded with a beam of high energy electrons that knock off an electron from the
+
52
1.
2.
3.
4.
What are isotopic peaks and in what way are these useful?
Mass Spectrometry
5.
6.
7.
The mass spectrum of hexane is given below. Explain the origin of important
fragment ion peaks observed in the spectrum.
13.7 ANSWERS
Self Assessment Questions
+
1.
Since the intensity ratio of the M and [M + 2] ion peaks is 24:8 :: 3:1, we
can infer (using Table 13.1) that the molecule contains a chlorine atom.
2.
In mass spectrometer the fragment ions are detected in the increasing order of
their m z values. Since there is unit charge in all the given species, these will
be detected in the increasing order of their masses. The order of their detection
would therefore be:
+
3.
The three ion sources used in mass spectrometers and their characteristic
features are as follows:
Electron ionisation: The method is good for volatile compounds but the
molecular ion peak is either weak or absent.
Chemical ionisation: This method uses ion-molecule reactions to produce ions
from the analyte.
Fast atom bombardment: This method is good for polar high molecular
weight species and uses a beam of fast atoms that desorb positive and negative
analyte ions from the sample.
4.
The butanoic acid has a carbonyl group with abstractable hydrogen at position;
therefore it can easily undergo McLafferty rearrangement. Accordingly the peak
at m z =60, [M-28] can be explained as follows.
. .O .
+
H
CH2
C
HO
. .O .
+
CH2
C
H2
H
+
CH2=CH2
C
HO
CH2
m/z = 60
m/z = 28
53
Miscellaneous Methods
5.
Terminal questions
1.
In mass spectrometry, the analyte in the vapour phase is bombarded with a beam
of high energy which knock off an electron from it to form a positively charged
+
radical or molecular ion, M . The radical ions so obtained undergo
fragmentation yielding smaller fragments. The radical ion and the fragments
obtained from the molecule are then separated according to their m z values
using magnetic field analysers or double focussing mass analysers. These are
then detected by a suitable transducer or analyser and an output is generated
after suitable processing.
2.
3.
The mass spectrum gives a plot of the intensities (proportional to the amounts)
of different fragments as a function of their m z values. The most intense peak
in the spectrum is given an intensity of 100 and is called the base peak; the
intensity of other peaks is given with respect to the base peak in fragmentation
pattern. Another characteristic of the spectrum is the presence of peaks called
isotopic peaks, observed due to the natural abundance of the isotopes of
constituent atoms of a molecule.
4.
5.
In double focussing mass spectrometer, the molecular and the fragment ions
coming out of the ionisation chamber are passed through an electrostatic
analyser. This allows the fragment ions with only a limited range of kinetic
energy to go over to the magnetic sector. In other words this analyser causes the
energy focusing of the ions. In the magnetic sector analyser these ions are then
dispersed according to their m z ratios and sent to the detector.
6.
. .O .
+
H
CH2
CH2
C
H2
54
.. .
O
+
H
+
C
CH2
m/z = 120
CH2=CH2
7.
The molecular ion peak is observed at m/z = 86 and the prominent fragment ions
are at m/z = 71, 57, 43 and 29. A little observation reveals that these fragment
ions differ by a m/z of 14 units. This indicates that the different fragments differ
by CH2 units. Accordingly, the possible fragmentation pattern leading to the
observed peaks can be depicted as:
Mass Spectrometry
+
CH3CH2CH2CH2CH2 + .CH3
m/z= 71
+
CH3CH2CH2CH2 + .CH2CH3
m/z= 57
CH3CH2CH2CH2CH2CH3
+.
+ .
CH3CH2CH2 + CH2CH2CH3
m/z= 43
+
CH3CH2 + .CH2CH2CH2CH3
m/z= 29
+
CH3 + .CH2CH2CH2CH2CH3
m/z= 15
55