Mass spectroscopy

MASS
SPECTROSCOPY
Seminar presentation
Submitted by :
Akshay
M.pharm 1st sem
Dept. of pharmaceutical chemistry

CONTENTS
 Introduction to spectroscopy
 Introduction to mass spectroscopy
 Basic principle
 Instrumentation
 Ionization techniques
 Types of ions
(Cont.)
Spectroscopy ; Donald L.pavia , George S.kriz
2

 Types of mass analyzers
 Fragmentation Rules
 Application of mass spectroscopy
3

INTRODUCTION TO
SPECTROSCOPY
Spectroscopy is the study of interaction of matter with
the electromagnetic radiation. Historically, spectroscopy
originated through the study of visible light dispersed
according to its wavelength and later concept was
expanded to interaction with the frequency. Spectroscopic
data is often represented by the spectrum.
4

INTRODUCTION TO MASS
SPECTROSCOPY
Mass spectrometry (MS) is an analytical chemistry
technique that helps identify the amount and type of
chemicals present in a sample by measuring the mass-to-
charge ratio and abundance of gas-phase ions. A mass
spectrum (plural spectra) is a plot of the ion signal as a
function of the mass-to-charge ratio.
5

BASIC PRINCIPLE
 In this technique, molecules are bombarded with a beam
of energetic electrons.
 The molecules are ionized 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.
 The ions passes through magnetic and electric fields to
reach detector where they are detected and signals are
recorded to give mass spectra.
6

INSTRUMENTATION
(Cont.)
7

 Sample Inlet : Sample stored in large reservoir from which
molecules reaches ionization chamber at low pressure in
steady stream by a pinhole called “Molecular leak”.
 Ionization : Atoms ionized by knocking one or more
electrons off to give positive ions by bombardment with a
stream of electrons. Most of the positive ions formed will
carry charge of +1.
 Acceleration : Ions accelerated so that they all have
same kinetic energy. +ve ions pass through 3 slits with
voltage in decreasing order. Middle slit carries
intermediate and finals at zero volts. All ions are(Cont
8

 Deflection : Ions are deflected by a magnetic field due to
difference in their masses. The lighter mass, more they
are deflected. It also depends upon the no. of +ve charge
an ion is carrying; the more +ve charge, more it will be
deflected.
 Detection : The beam of ions passing through the mass
analyzer are detected by detector on the basis of m/z
ratio. When an ion hit the metal box, Charge is
neutralized by an electron jumping from metal on to the
ion.
9

IONIZATION TECHNIQUES
 Electron Ionization (EI-MS)
 Chemical Ionization (CI-MS)
 Desorption Technique (FAB)
10

ELECTRON IONIZATION (EI-
MS)
 Beam of high energy electrons (~70eV) is emitted from
filament which is pre heated to several thousand
degrees.
 High energy electrons strike stream of molecules to strip
and electron from molecule and form a cation.
 Repeller is +vely charged plate, directs newly formed
ions to the accelerating plates.
 Acceleration is provided with potential of 1-10(kV)
 Focusing slits directs the beam
 Ionization potential required ~15eV and for bond
cleavage, required potential is ~3eV to 10eV
(Cont.)
11

12

 Advantage : Fragmentation pattern are predictable due
to large databases.
 Disadvantages :
(a) Loss of the molecular ion peak.
(b) Sample must be volatile to come in contact with
electron in chamber.
(c) High MW compounds cannot be analyzed.
13

CHEMICAL IONIZATION
 It is a soft ionization technique.
 Sample molecules are combined with ionized reagent
gas which is present in excess than the sample.
 Due to collision, Various molecules are ionized by
different mechanisms like proton transfer, electron
transfer, adduct formation etc.
 Any readily available gas or highly volatile liquid can be
used as CI-MS ionized reagent gas. For ex = Methane,
Ammonia, Isobutane and methanol.
 In case of methane, proton transfer takes place.
(Cont.)
14

(Cont.)
15

 Varying reagent in CI-MS allow selectivity of degree of
ionization and degree of ion fragmentation.
 Choice of gas should be made closest to proton affinity
of reagent gas with that of sample to ensure efficient
ionization of sample without excess of fragmentation.
 Using Reagent gas with proton affinity matched closely
to that of sample will keep molecular ion peak intact and
smaller no. of fragments.
16

DESORPTION TECHNIQUE
 Allows analysis of larger molecular weighted molecules.
 Includes FAB – Fast atom bombardment method.
 Sample to be analyzed is dispersed in the matrix and
placed in the path of high energy neutral atoms.
(Cont.)
17

 Xenon and Argon neutral atoms are used in this
technique.
 Collision of neutral atoms takes place with molecule of
sample to form ions and are forwarded to accelerating
plates and finally to mass analyzer.
 In FAB, It uses neutral atoms to ionize sample which
means both +ve and –ve ions are formed and could be
analyzed.
 Used for sample weights up to 20000
 These molecules basically includes polypeptides,
nucleotides, oligonucleotides etc.
 Matrix used in this technique should be non-volatile and
inert.
18

TYPES OF IONS
 Molecular ion/ Parent ion
 Fragment ion
 Rearrangement ions
 Multi-charged ions
 Negative ions
 Meta stable ions
19

MOLECULAR ION/PARENT
ION
 When a molecule is bombarded with electrons in high
vacuum in Mass spectrometer, it is converted into positive
ions by loss of an electron. These ions are called as
Molecular or Parent ions.
M + e  M+° + 2e—
Where,
M – represents the Molecule;
M+°– represents the Molecular or Parent ion.
 The order of energy required to remove electron is as
follows :
 electrons > non-conjugated  > conjugated  > non
bonding or lone pair of electrons. (Cont.)
20

 Most molecules show a peak for the molecular ion, and
the stability order is as follows :
 Aromatic > Conjugated acyclic polyenes > Alicyclic > n-
hydrocarbons > ketones > ethers> Branched chain
hydrocarbons > Alcohols.
 The molecular ion M+° has mass, corresponding to the
molecular weight of the compound from which it is
generated. Thus the mass of a Molecular ion M+° is an
important parameter in the identification of the compound
i.e. m/z of molecular ion = molecular weight of the
compound.
Ex: C2H5
+ (m/e=29) gives the molecular weight of Ethane.
21

FRAGMENT IONS
 When the energy is given to Molecular ion during
electron impact, further cleavage takes place and ions of
lower mass number known as Fragment ions are
produced.
M+°  M+
1 + M2
Ex: CH3OH
+°
 CH2OH
+
m/z 32 m/z 31
22

REARRANGEMENT IONS
 Rearrangement ions are the fragments whose origin
cannot be described by simple cleavage of bonds in the
parent ion, but are result of intra-molecular atomic
rearrangement during fragmentation
 These are probably due to recombination of fragment
ions and known as rearrangement peaks.
 Ex: Prominent peak in spectrum of diethyl ether occurs
at m/e 31. This is due to the ions CH3O+, which is
formed by rearrangement of C2H5O+ ions.
23

MULTI CHARGED IONS
 Some times ions may also exist with two or three
charges instead of usual single charge in the mass
spectrum. These are known as doubly or triply charged
ions. They are created as follows:
M+° + e-  M++ + 3e-
 But under normal operating conditions, most of the ions
produced are single charged. The doubly or triply
charged ions are recorded at a half or one third of the
m/e value of the single charged ions. Formation of these
multiple charged ions is more common in hetero aromatic
compounds. They are also common in inorganic mass
spectrum. Gases such as CO, N2,CO2 and O2 have
measurable peaks corresponding to CO+2,N+2,and O+2.Spectroscopy ; Donald L.pavia , George S.kriz
24

NEGATIVE IONS
 The positive ions predominate in electronic impact
ionization because of greater stability. The Negative ions
are not very useful in structural determinations. The
formation of Negative ions is very rare but these can be
produced in three ways:
1. AB + e  A+ + B—
2. AB + e  AB—
3. AB + e  A+ + B— + e—
25

META STABLE IONS
 Fragment of a parent ion will give rise to a new ion
(daughter) plus either a neutral molecule or a radical.
M1
+ M2
+ + non charged particle
 An intermediate situation is possible; M1
+ may decompose
to M2
+ while being accelerated. The resultant daughter ion
M2
+ will not be recorded at either M1 or M2, but at a position
M* as a rather broad, poorly focused peak. Such an ion is
called a metastable ion.
 Metastable ions are useful in helping to establish fragments
routes. Metastable ion peak can also be used to distinguish
between fragmentation Processes, which occur in
microseconds.
26

TYPES OF MASS ANALYZERS
 Magnetic sector mass analyzer
 Double focusing mass analyzer
 Quadra pole mass analyzer
27

MAGNETIC SECTOR MASS
ANALYZER
 Kinetic energy of accelerated ion is :
1
2
𝑚𝑉2
= zV
Where, m = Mass of ion
v = Velocity
z = Charge
V = Potential diff b/w ion and accelerating plates
 In magnetic sector, charged particle in magnetic field
describes a curved flight path.
 𝑟 =
𝑚𝑣
𝑧𝐵
( B = Magnetic field) ( r = Radius of
curvature)

𝑚
𝑧
=
𝐵2 𝑟2
2𝑉
(Cont.
28

 Greater the value of m/z , greater the radius of curvated
path.
 Analyzer tube has fixed radius of curvature and correct
m/z can negotiate and pass to detector.
 Varying magnetic field, so that all ions could have
correct m/z to reach the detector.
29

DOUBLE FOCUSING TYPE
 For many applications, much higher resolution is needed
and achieved by modification.
 An electric field region is present before or after the
magnetic field region.
 In presence of electric field, all particles travels at same
velocity and resolution of mass analyzer which improves
by a factor of 10 or more over magnetic sector alone.
30

QUADRA POLE ANALYZER
 It has 4 rods that are electrodes with electric fields
around them.
 Electrodes have both DC and AC (RF) voltages applied
to them.
 Each pair of rod have exactly the same voltage as the
one directly opposite.
Cont
31

 Depending upon the ratio of DC and AC, Ions acquire or
affected by magnetic field.
 Ions of correct m/z ratio don’t collide with the rods and
travels to detector and other with laws or high m/z are
unstable and doesn’t pass.
 If DC is +ve = high mass ions will have stable path
 If DC is –ve = Low mass ions will have stable path.
32

FRAGMENTATION RULES
 The relative height of the molecular ion peak is greatest for
the straight chain compound and decreases as the degree of
branching increases.
 The relative height of the Molecular ion peak usually
decreases with increasing molecular weight in a
homologous series.
 Cleavage is favored at alkyl substituted carbon atoms; the
more substituted, the more likely is cleavage. This is
consequence of the increased stability of a tertiary carbon
atom over a secondary, which in turn is more stable than a
primary.
 CH3
+ < RCH2
+ < R2CH+ < R3C+
(Cont.)
33

 Double bonds favor allylic cleavage and give the resonance
Stabilized allylic carbonium.
 Saturated rings tend to lose alkyl side chains at the 
carbon atom. This positive charge tends to stay with the
ring fragment.
 Mass spectrum of n-propyl cyclohexene
(Cont.)Spectroscopy ; Donald L.pavia , George S.kriz
34

 In alkyl substituted aromatic compounds, cleavage is
very probable at the bond  to the ring, giving the
resonance stabilized benzyl ion or more likely, the
tropylium ion:
Ex: mass spectra of n-butyl benzene.
35

McLafferty REARRANGEMENT
 Fragmentation due to rearrangement of Molecular or Parent
ion:
Here cleavage of bonds in Molecular ion is due to the intra
molecular atomic rearrangement. This leads to
fragmentation whose origin cannot be described by simple
cleavage of bonds. When fragments are accompanied by
bond formation as well as bond for breaking, a
rearrangement process is said to have occurred.
 Such rearrangement involves the transfer of hydrogen from
one part of the molecular ion to another via, preferably, a six-
membered cyclic transition state. This process is favored
energetically because as many bonds are formed as are
broken.
36

 Compounds containing hydrogen atom at position
gamma to carbonyl group have been found to a relative
intense peak. This is probably due to rearrangement and
fragmentation is accompanied by the loss of neutral
molecule. This rearrangement is known as McLafferty
rearrangement.
37

 To undergo McLafferty rearrangement, a molecule must
posses
a. An appropriately located heteroatom (ex. oxygen)
b. A double bond
c. An abstractable Hydrogen atom which is γ (gamma)
to
C=O system.
38

APPLICATIONS
 Environmental Monitoring and Analysis (soil, water
and air pollutants, water quality, etc.)
• Geochemistry – age determination, Soil and rock
Composition, Oil and Gas surveying
• Chemical and Petrochemical industry – Quality
control
 Applications in Biotechnology
• Identify structures of biomolecules, such as
carbohydrates, nucleic acids
• Sequence biopolymers such as proteins and
oligosaccharides
39

40
 Thank you 

Mass spectroscopy

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