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Mass Spectrometry

By: Bijaya Kumar Uprety


Introduction and principle:

Mass spectrometry is a technique used for measuring the
molecular weight and determining the molecular formula of an
organic compound.
Note: Atoms can be deflected by magnetic fields – provided the atom is first
turned into an ion. Electrically charged particles are affected by a magnetic field
although electrically neutral ones aren’t. This is why in mass spectrometer
atoms are ionized.

In a mass spectrometer, a molecule is vaporized under vacuum
and then ionized by bombardment with a beam of high-energy
electrons causing the loss of an electron.

The energy of the electrons is ~ 1600 kcal (or 70 eV).
Since it takes ~100 kcal of energy to cleave a typical o bond,
1600 kcal is an enormous amount of energy to come into contact
with a molecule.


¢ When the electron beam ionizes the molecule, the
species that is formed is called a radical cation, and
symbolized as M*’.

¢ The radical cation M* is called the molecular ion or
parent ion and the mass of M* represents the
molecular weight of M.

¢ Because M is unstable, some ions decompose to
form fragments of radicals and cations that have a
lower molecular weight than M*’.

¢ The ions are then accelerated through a potential of
about 10,000 volts so that they all have the same
kinetic energy.



¢ 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 – in other
words, on how many electrons were knocked off in
the first stage. The more the ion is charged, the more
it gets deflected.

¢ The beam of ions passing through the machine is
detected electrically.


Mass Spectrometry
Figure 13.1 Schematic of a mass spectrometer


accelerating and

desir plate focusing plates


mass spectrum

In a mass spectrometer, a sample is vaporized and bombarded by a beam of electrons to form an
unstable radical cation, which then decomposes to smaller fragments. The positively charged ions
are accelerated toward a negatively charged plate, and then passed through a curved analyzer tube
in a magnetic field, where they are deflected by different amounts depending on their ratio of mass
to charge (m/z). A mass spectrum plots the intensity of each ion versus its m/z ratio.


Schematic diagram of mass spectrometer.


ann i



to Yacuum

Yaporised pt pee
sample [ @









The essential components of a mass spectrometer are:

¢ Inlet device

¢ [onisation chamber or Ion Source

¢ Analyser

¢ Detector

¢ Processing and output devices

The arrangement of these components of the mass
spectrometer is schematically represented in Fig. 13.3.


— Sorting Detection |

lonisation of ions of ions [——|

—» Gaseous Mass lon | E wet
ion source analyzer transduce = & |

10°to 10° torr gal |

3 Mass
al + Spectrum


Fig.13.3: A schematic diagram showing the components of a mass spectrometer



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

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.


1. Inlet devices
¢ The purpose of the inlet device is to load the sample into the ionisation
chamber. The device used depends on the nature of the sample.

¢ The solid samples are placed on the tip of a rod called direct insertion probe
which is inserted into the evacuated chamber having a vacuum-tight seal. This
is then heated to evaporate or sublime the sample to get the molecules in the gas

¢ The gases and heat volatile liquids, on the other hand are generally
introduced through specially designed devices with controlled flow. The liquid
samples are also suitably evaporated.

¢ Once the sample is evaporated the gaseous molecules are then ionised by a
suitable technique; this usually is accompanied by fragmentation also.

¢ When the analyte is thermally labile i.e., it can decompose upon heating, then
we need to use other methods like desorption or desolvation methods to bring
the analyte into the vapour phase.



Ionization Chamber or Ion Source

e Since mass spectrometer works by sorting out the charged particles by using
magnetic and/or electric fields. Therefore, a compound/molecule must be
charged or ionized to be analyzed by a mass spectrometer.

¢ In the ionization chamber (also called ion source) the molecule is ionized by
using one of the many methods available for the purpose. The ion sources fall
into two categories as follows:

¢ Gas phase sources

¢ Desorption sources

Gas phase sources: In these sources the sample is first vaporized and then
ionized. These are used for low molecular weight (< 1000 Da) samples which are
thermally stable i1.e., do not decompose on heating and have low boiling points
(< 500 °C). There are three methods that belong to this category. These are:



1) Electron ionisation (EI)

11) Chemical ionisation (CI)

111) Field ionisation (FI)

¢ 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.





eS Y :

Molecules (M) e”” mw M Ions (M~
Injected Into Source M | Accelerated

to Mass Analyzer



Figure 2. Electron Ionization Source.



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, iso-butane, or ammonia 1s
passed into the ionisation chamber where it gets ionised by
electron ionisation. For example, methane gas gives mainly CH,*
and CH,* ions as follows

= ¥
CH, + e€ —ws» CH+, 2 e

+ – ,
CH, ——— CH, + H

«These reagent gas ions then react with the neutral molecules of the
reagent gas as follow


as HH —. CG”, oe

CH,+ CH, ——> CH.+ H,



¢ 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

M + CH.+ > MH*+ + CH,
M +(C,H.* > MH* + C,H,

” These give an ion at [M+1]. For the analyte M of RH type, we may have

reaction that can be represented as

RH + CH; > R’+ CH, + H,

* 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. 15


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 (1.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.

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.

_- -— Fast + _
A B ———> + B


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 ts sent to the analyser. Let us
understand how the analyser separates these tons.



Beam Source
keV Atom <



Sample Sputtered Ions

Figure 6. Fast Atom Bombardiment Source.



¢ There are four general types of mass analyzers that can be

used for the separation of ions in a mass spectrometry.

1. Quadrupole Mass Analyzer

¢ A Quadrupole is a mass analyzer that uses an electric field
to separate ions. The Quadrupole consists of 4 parallel
rods/ poles, where adjacent rods have opposite voltage
polarity applied to them. The voltage applied to each rod is
the summation of a constant DC voltage (U) and a varying
radio frequency (V,,cos(wt)), where w = angular frequency
of the radio frequency field. The electric force on the ions
causes the ions to oscillate/orbit in the area between the 4
rods, where the radius of the orbit is held constant.



¢ The ion moves in a very complex motion that is directly
proportional to the mass of the ion, voltage on the quadrupole, and
the radio frequency. The ions will remain orbiting in the area
between the poles with no translation along the length of the poles
unless the ions have a constant velocity that is created as the ions
enter the quadrupole. Before entering the analyzer, the ions travel
through a potential of a certain voltage, usually created by ring
electrode, in order to give the ions a constant velocity so they can
transverse along the center of the quadrupole.

¢ While in the quadrupole, the trajectories of the ions change
slightly based on their masses. Ions of specific mass have a certain
frequency by which they oscillate. The greater the mass, the
greater the frequency. A certain limit is associated with each
quadrupole and it selects ions which are within the desirable
frequency range.





2. TOF (Time of Flight) Mass Analyzer

¢ 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.

¢ Jons of the same charges have equal kinetic energies; kinetic
energy of the ion in the flight tube is equal to the kinetic energy of
the ion as it leaves the ion source:

KE = my2/2 = zV

The time of flight, or time it takes for the ion to travel the length
of the flight tube is:

T, = Ldength of tube)/v(velocity of ion)



Substituting the equation for kinetic energy in equation for time of flight:

T, = Lam/z)!7(1/2V)!? = (Constant)*(m/z)!”

During the analysis, L, length of tube, V, Voltage from the ion source, are all
held constant, which can be used to say that time of flight is directly
proportional to the root of the mass to charge ratio.

Unfortunately, at higher masses, resolution is difficult because flight time is
longer. Also at high masses, not all of the ions of the same m/z values reach
their ideal TOF velocities. To fix this problem, often a reflectron is added to the
analyzer. The reflectron consists of a series of ring electrodes of very high
voltage placed at the end of the flight tube. When an ion travels into the
reflectron, it is reflected in the opposite direction due to the high voltage.

The reflectron increases resolution by narrowing the broadband range of flight
times for a single m/z value. Faster ions travel further into the reflectrons, and
slower ions travel less into the reflector. This way both slow and fast ions, of
the same m/z value, reach the detector at the same time rather then at different
times, narrowing the bandwidth for the output signal.






TO terre goes Oy. The ton ere separated




3. Sector: Magnetic Sector Mass Analyzer

¢ Similar to time of flight 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. Ions in the magnetic field experience two equal
forces; force due to the magnetic field and centripetal force.

F,= ZVB =F.= mv*/r
The above equation can then be rearranged to give:

v = Bzr/m

If this equation is substituted into the kinetic energy

KE= zV=mv,/2



Basically the ions of a certain m/z value will have a unique path radius
which can be determined if both magnetic field magnitude B, and
voltage difference V for region of acceleration are held constant. when
similar ions pass through the magnetic field, they all will be deflected
to the same degree and will all follow the same trajectory path. Those
ions which are not selected by V and B values, will collide with either
side of the flight tube wall or will not pass through the slit to the
detector. Magnetic sector analyzers are used for mass focusing, they
focus angular dispersions.



Deflected ions, varying
ame! radi

ion beam



4. Sector: Electrostatic Sector Mass Analyzer

¢ Is similar to time of flight analyzer in that it separates the ions while in flight,
but it separates using an electric field. 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.

KE = zV =mv2/2

F,= ZE= F =mv?/R

Electrostatic sector analyzers are energy focusers, where an ion beam is focused
for energy.

Electrostatic and magnetic sector analyzers when employed individually are single
focusing instruments. However 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.



Detector or Ion Collector

¢ 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.



Processing and Output Devices

A typical mass spectrum contains a large amount of structural data in terms of
the m/z values and the relative intensities of all the fragments obtained from
the molecule.

Further, for the data to be dependable and useful a number of instrumental
parameters need to be monitored and controlled. This means a large amount
of data and its manipulation. It is achieved with the help of microprocessors
and microcomputers that are an integral part of all mass spectrometers. The
mass spectrometer data systems also include softwares for quantification,
interpretation, and identification of the molecules using on-line spectral

In the processing units the ion-current signals obtained from the detector is
digitalised and extensively processed before being displayed in terms of a
mass spectrum. The spectrum displays the m/ z values of all the fragments
and their intensities relative to that of the most intense peak called base
peak. Sometimes, the data is also displayed in the form of a table wherein
the m/z values are listed in an increasing order and the corresponding
relative peak intensities are given in numbers.



Mass spectrogram

¢ The mass spectrometer analyzes the masses of

¢ A mass spectrum is a plot of the amount of each
cation (its relative abundance) versus its mass to
charge ratio (m/z, where m is mass, and z is charge).

¢ Since in most of cases, z Is almost +1 (since it is
much more difficult to remove further electrons from
an already positive ion or in simple terms in most of
the cases only one electron is lost during the
bombardment so ‘z’ is equivalent to +1), m/z actually
measures the mass (m) of the individual ions.


¢ The tallest peak in the mass spectrum is called the
base peak.

¢ The base peak may also be the M peak, although
this may not always be the case.

¢ Isotopes:

¢ Though most C atoms have an atomic mass of
12, 1.1% have a mass of 13. Thus, ‘CH, is
responsible for the peak at m/z = 87 in hexane.
This is called the M + 1 peak.

– Some isotopes show M + 2 peaks.



Characteristics of mass spectrum

The mass spectrum of methanol (molar mass = 32 gmol’) is shown as a typical mass
spectrum in Fig. 13.1.

100 7 100
~ 90- Mi! ~ 90

©. 80 – – 80
a 1- – 70
€0- 29 32 – 60
5 50- 50
se 40- – 40
<< 30

@ 20- –

_ 20
© | – 10

_ a I —— 0

0 10 20 30 40 50 60 70 80 90
Mass/charge ratio (m/z)

Fig. 13.1: Mass spectrum of methanol (CH,OH)


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 (CH;OH”; m/z=31, in
this spectrum) 1s arbitrarily assigned an intensity of 100% and the peak intensities of
other tons are measured relative to it. This most intense peak 1s called as the “base
peak’ and it need not necessarily be the mole cular ion peak which is at m/z=32, Are

you wondering, why do we observe a small peak at m/z= 33? What is the origin of
this peak? Let us learn about tt



13.2.2 Isotopic Peaks

The peak at m/z 33 in the case discussed above is an isotopic peak and is called as

[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 Le., as a
collection of identical atoms. However, some elements have isotopes 1.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 ‘2C as well as’3C atoms. The

natural abundance of ‘2C is 1.1% as compared to’2C. This means that if we have a
thousand atoms of carbon (having ‘<C isotope) then there would be 11 atoms that

would be of the ‘2C isotope. In case of the above example it amounts to saying that

for every 1000 molecules of methanol containing a ‘2C isotope, there would be 11

methanol molecules with a ‘2C in them. These molecules would have a molar mass of

33 gmol’ as against the ‘normal’ value of 32 and hence the peak at m/z = 33. Further,

since oxygen (‘$Oand’$O ) and hydrogen (}H, and*+H_) can also exist as isotopes,
they would also influence the spectrum and we may expect a very small signal at

m/z = 34i.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.


Mass Spectrometry
Figure 13.2 Mass spectrum of hexane (CH,CH,CH,CH.CH.CH,), C,H,,


molecular weight = 86 molecular ion
© 7 m/z = 86

= . base peak ——————>
S= 2 m/=z 5 7

2 M + 1 peak
@ 90 m/=z 8 7


| | ll et alll, —L [oT 4
0 10 20 30 40 50 60 70 80 90 100

The molecular ion for hexane is at m/z = 86.
The base peak occurs a m/z = 57.
A small M + 1 peaks occurs at m/z = 87.



Mass Spectrometry
A Branched alkane:



80 t –

£2 60 :
= i
& 40 ss ae a

– I
90 }—- ial eet ll Mh ane eee ae | ene ee eh

Te $7 7M 3

r 86
9 I e eeeemnes met iemnivs i tepiei
10 20 30 40 $50 60 7 8 © 100 110 120 130 140 150 160


¢ Note that alkyl substituted benzenes generate a peak at
91 m/z due to the tropylium ion.



Mass Spectrometry
An Alcohol:

Alcohols are easily dehydrated in the injection chamber
under full vacuum. As a result the molecular ion does
not appear and one observes an M-18 peak.



(M —18—15)




10 20 30 40 50 6 70 80 9 100 tlo 120 130 140 1SO 160



NI a

So oS a] S 30


Mass Spectrometry

Carbonyl Cleavage:

te @ a
CH,CH,CH.-CHO _—_ CH,CH,CH. + H-C= O|

McLafferty Rearrangement:

¢ This fragmentation pattern is typically seen in
carbonyl compounds that have a y hydrogen.

vH te CH H_ +.
CH. 2 O

. (0 — Te
CH eae _ H CH. cn? _C-H



Mass Spectrometry

Alkyl Halides and the M + 2 Peak:

¢ Most elements have one major isotope.
¢ Chlorine has two common isotopes: °*Cl and ?/Cl,
which occur naturally in a 3:1 ratio.

» Thus, there are two peaks in a 3:1 ratio for the
molecular ion of an alkyl chloride.

» The larger peak, the M peak, corresponds to the
compound containing the *°Cl. The smaller peak, (M + 2
peak), corresponds to the compound containing °’Cl.

»®» Thus, when the molecular ion consists of two peaks (M
and M + 2) ina 3:1 ratio, a Cl atom is present.

– Br has two isotopes: “Br and ®’Br, in a ratio of ~1:1.
Thus, when the molecular ion consists of two peaks
(M and M + 2) in a1:1 ratio, a Br atom is present.



Mass Spectrometry

Alkyl Halides and the M + 2 Peak:
Figure 13.3 Mass spectrum of 2-chloropropane [(CH,),CHCI]

al (CH.,),CHCI

molecular weight = 78, 80

= i
& two molecular ions

& 50- —
$ 7 height an 3

& 7 m/z=78 m/z=80

9 ee ali aa



Mass Spectrometry

Alkyl Halides and the M + 2 Peak:
Figure 13.4 Mass spectrum of 2-bromopropane [(CH,),.CHBr]

2 (CH3)oCHBr

molecular weight = 122, 124

= two molecular ions

height bower! 1 <a

m/z=122 m/z=12 4


0 ] a | = | | A ———
0 20 40 60 80 100 120


Relative abundance




Mass Spectrometry
The Nitrogen Rule:

¢ Hydrocarbons like methane (CH,) and hexane
(C,H,,), as well as compounds that contain only C,
H, and O atoms, always have a molecular ion with
an even mass.

¢ An odd molecular ion indicates that a compound
has an odd number of nitrogen atoms.

¢ The effect of N atoms on the mass of the
molecular ion in a mass spectrum is called the
nitrogen rule: A compound that contains an odd
number of N atoms gives an odd molecular ion. A
compound that contains an even number of N
atoms (including zero) gives an even molecular



Mass Spectrometry
High Resolution Mass Spectrometers:

– Low resolution mass spectrometers report m/z
values to the nearest whole number. Thus, the
mass of a given molecular ion can correspond to
many different molecular formulas.

¢ High resolution mass spectrometers measure
m/z ratios to four (or more) decimal places.

| Exact Masses of Some Common Isotopes

Isotope Mass

eG 12.0000

‘H 1.00783

6 15.9949

“SN 14.0031 42


Mass Spectrometry
High Resolution Mass Spectrometers:

» This is valuable because except for ‘*C whose
mass is defined as 12.0000, the masses of all
other nuclei are very close—but not exactly—
whole numbers.

» Table 13.1 lists the exact mass values for a few
common nuclei. Using these values it is
possible to determine the single molecular
formula that gives rise to a molecular ion.



Mass Spectrometry

High Resolution Mass Spectrometers:

¢ Consider a compound having a molecular ion at m/z = 60
using a low-resolution mass spectrometer. The molecule
could have any one of the following molecular formulas.

Formula Exact mass

CaHeO 60.0575

CoH,0O> 60.071 1

CoHeN> 60.0688