BY: RAGHAV DOGRA

M. PHARM (PHARMACEUTICAL ANALYSIS)

1ST SEMESTER
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 INTRODUCTION.
 PRINCIPLE.
 INSTRUMENTATION AND DIAGRAMS.
 SAMPLE INTRODUCTION.
 ION SOURCES.
 MASS ANALYZERS.
 DETECTORS.
 APPLICATIONS.
 NITROGEN RULE.
 Ring rule.
 RULE OF 13.
 FRAGMENTATION.
 RETRO DIELS ELDER REACTION.
 MC LAFFERTY RE ARRANGEMENT.
 Types of peaks. 2
 As the name itself gives the idea that it weighs a
molecule or in other words, measures the molecular
mass of the molecule.

 It is destructive technique of analysis.

 It differs from other spectroscopic technique by the fact

that it does not include absorption or emission of any
kind of EMR(such as UV, IR, or Radio waves).

 It is the most accurate method of determination of the

molecular mass and its molecular composition.

3
 The principle includes the ionization of the sample and
breaking of sample into small fragments or ions by various
technique( electron impact, chemical ionization etc).

 Some of these are the positive ions, set of these ions are
separated and analyzed in such a way, signal is obtained for
each value of m/z or m/e (mass to charge ratio)

 Ion thus formed by eliminating only 1 e- from the

compound have approx. the same molecular mass as that of
the compound and is designated as M.+ or the parent ion
peak having largest m/z value.

 The intensity of each signal represents the relative

abundance and the most abundant signal is called as base
peak having relatve intensity 100. 4
 Sample introduction and vacuum.

 Ion source.

 Electrostatic accelerator .

 Magnetic field.

 Mass separator or analyzer.

 Detector or Collector .

 Recording or Read out.

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 The sample introduction system basically depends upon the
physical state of the sample and several system must be
available if variety of sample are to be analyzed.

 A)Batch inlet: Commonly sample introduced as gas with 1-5 liter

reservoir having pressure greater 1 to 2 greater magnitude
than of ionization chamber. To flow through a pinhole with
0.01 torr pressure.

 For low boiling liquid boiling below 150° C, certain quantity

are evaporated in evacuated reservoir at room temperature.

 For less volatile sample reservoir can be externally heated if

sample is thermo-stable if not than directly introduced into
ionization chamber for which special equipment is required.
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 B)The Direct Probe Inlet: Non volatile or thermally
unstable introduced directly into the ion chamber
by sample probe via vacuum lock.

 Consist of small capillary tube or cup containing

µg of sample.

 Heater is their to volatilize the sample at low

pressure.

 The probe is used to study carbohydrates, steroids

and low molecular weight polymers.

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PROBE INLET

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 Gas chromatographic inlet system: volatile sample condition is
their .

 Effluent from chromatographic system act as sample.

 The sensitivity can be increased by separating the

sample molecule from large amount of carrier gas
present in effluent.

 The separation is achieved by passing effluent into the

narrow chromatographic column of porous glass or
Teflon which is permeable to carrier gas .

 The later is then passed to the ionization chamber

through pinhole.

 The combine technique is a power tool for analyst

known as GS-MS. 12
  Several methods are there for converting the sample
into the gaseous ionic phase these are as under :

IONIZATION TECHIQUES

GAS PHASE SOURCES DESORPTION SOURCES

-Electron Impact Ionization -Atmospheric Pressure

(EI). ionization(API).
-Chemical Ionization (CI). -Fast Atom
Bombardment(FAB).

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 It is the type of hard ionization technique due the
high energy of Electron Impact.

 Ions are accelerated at the voltage of ~104 V.

 Ionization method as name includes the impact of

beam of high energetic electron to a gaseous phase
or the volatile organic sample.

 Due to the electron impact the sample is broken into

positive or negative ions.

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 The energetic electron beam is emitted by a
electrically heated tungsten or rhenium which are
then accelerated by the potential difference of 70eV.

 Collision between ions and molecules may also result

in ion with higher m/z values than the molecular
ion.
M(g) + e-  . + 2e-
M+(g)

 Where M+. Is a radical cation which gives molecular

weight

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 EI is not appropriate for certain compounds due to
the excessive fragmentation

 Chemical ionization includes the ionization of

reagent gas in high volume approx 1000 times more.

 Typically used reagent gas is methane, ammonia,

isobutane.

 Firstly at high pressure the reagent gas is ionized and

subsequently this ionized gas molecule collide with
sample as gaseous phase and bring about
fragmentation.
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 For Example - Methane CI
1. electron ionization of CH4:
 CH4 + e-  CH4+ + 2e-
 Fragmentation forms CH3+, CH2+, CH+
2. ion-molecule reactions create stable reagent
ions:
 CH4+ + CH4  CH3 + CH5+
 CH3+ + CH4  H2 + C2H5+
 CH5+ and C2H5+ are the dominant
methane CI reagent ions
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 Several Types of Reactions May Occur
 Form Pseudomolecular Ions (M+1)
 CH5+ + M  CH4 + MH+
 M+1 Ions Can Fragment Further to Produce a
Complex CI Mass Spectrum
 Form Adduct Ions
 C2H5+ + M  [M + C2H5]+ M+29 Adduct
 C3H5+ + M  [M + C3H5]+ M+41 Adduct
 Molecular Ion by Charge Transfer
 CH4+ + M  M+ + CH4
 Hydride Abstraction (M-1)
 C3H5+ + M  C3H6 + [M-H]+
 Common for saturated hydrocarbons 19
 Contd…
 it is a soft ionization technique.

 Generally have less fragmentation and molecular

ion is abundant.

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 It operates at the atmospheric pressure.

 It is used for a mixture of high molecular weight non

volatile compound

 It is of various types which are:

+Matrix Assisted Laser Desorption Ionization(MALDI).
+Electrospray Ionization(ESI).
+Atomic Pressure Chemical Ionization.(APCI).
+Atomic Pressure Photon Ionization(APPI).

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 It operates at atmospheric pressure.

 A sample solution is sprayed from a small pore into

electric field in the presence of flow of warm nitrogen
to assist desolvation.

 The droplets thus formed evaporates in the region of

vacuum maintained at high pressure to form ions.

 The increased pressure causes the charge to increase

in the ion thus formed
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 Generally used for molecule such as peptides,
proteins, organometallic and polymers.
 but cannot be used for buffer of phosphates as the
trace level of this can interfere with ESI process

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 A mixture of the analyte and the solvent i.e. a liquid
solution is first vaporized with the help of nebulizing gas
N2.

 the mixture enters the ionization chamber at

atmospheric pressure.

 The mixture is then exposed to the UV source of

krypton lamp. The photon emitted from this lamp has a
specific energy level i.e. 10eV.

 It is high enough to ionize sample excluding the

unwanted species. Hence analyte molecule is analyzed
or measured.
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 The corona discharge produces primary ions in this
technique.

 The nebulized sample via high speed nitrogen gas is

displaced to a quartz tubing called as desolvation
chamber.

 In desolvation chamber these droplets are converted

to mixture of compound which are subsequently
carried to a corona discharge electrode.

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 Due to these molecule are thus ionized in two ways
or modes :

 Positive mode: proton transfer or charge exchange

occurs .

 Negative mode: proton abstraction or electron

capture or adduct formation is their.

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 It produces singly charged species.

 Generally employed for large biomolecules and

polymers.

 It is a high mass pulsed technique hence it is

generally combined with TIME OF FLIGHT .

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 For polar molecules such as peptides with molecular
weight up to 10000 can be analyzed by soft ionization
technique called as Fast Atom Bombardment.

 Thermally unstable molecule it works well as it

works at room temperature.

 The beam for bombardment is generally consist of

Xenon or Argon gas atom of high energy, the beam is
produced by ionizing xenon atom by the electrons
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 The sample is dissolved in glycerol and fine layer is
formed over metal probe which is then ionized by
fast beam of xenon or argon striking the sample.

 Generally it causes less fragmentation and molecular

ion is obtained.

 Hence sample mass is analyzed in this way.

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 To separate the ions produced in the ion source acc.
to their mass/charge ratio.

 Ideally mass analyzer should be capable of

distinguishing small mass differences.

 It should also allow passage of a sufficient number of

ions to yield radially measurable ion current.

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 Scanning (Filter)
 Linear Quadrupole ( Separation in Space)

 Sector
 Pulsed (Batch)
( Separation in Time)
 Ion Trap
 FT-ICR
 Time-of-Flight
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 SECTOR ANALYZERS :
a) Magnetic field only - Single Focusing Mass Analyzer

b) Magnetic and electrostatic field – Double Focusing

Mass Analyzer
2) TIME OF FLIGHT ANALYZER ( TOF )
3) QUADRUPOLE ANALYZERS:
a) Quadrapole filter

b) b) Ion trap analyzer

4) FT – ICR ( Ion cyclotron Resonance – Mass

Spectrometer ).

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 The cations from the ion source are passed through a
magnet that is located outside the tube.

 The magnetic force deflects the ions toward the

detector at the end of the tube.

 Lighter ions are deflected too much and heavier ions

are deflected too little.

 Only ions that match the small mass range reach the
detector .

 A 10‐7 vacuum is applied to the metal analyzer tube.

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THEORY:
KE = ½ mv2 = zV
Where m is the mass of the ion , v is its velocity
z is the charge on the ion and V is the applied voltage of
the ion optics.
 The ions enter the flight tube and are deflected by the
magnetic field, B.
 Only ions of mass-to-charge ratio that have equal
centripetal and centrifugal forces pass through the
flight tube:
mv2 /r = Bzv,
where r is the radius of curvature

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 By rearranging the equation and eliminating the
velocity term using the previous equations
r = mv/zB
 Therefore,
m/z = B2r2/(2V) •
This equation shows that the m/z ratio of the ions
that reach the detector can be varied by changing
either the magnetic field (B) or the applied voltage of
the ion optics (V)
m/ z =B2 r2 /2V

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 Consists of 4 parallel metal rods, or electrodes.

 The ions are accelerated by a potential of 5‐15 V and

injected into the area between the 4 rods .

 Opposite electrodes have potentials of the same sign .

 One set of opposite electrodes has applied potential of

[U+Vcos(ωt)] .

 Other set has potential of ‐ [U+Vcos(ωt)] .

 U= DC voltage, V=AC voltage, ω= angular velocity of

alternating voltage
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 Voltages applied to electrodes affect trajectory of ions
with the m/z ratio of interest as they travel down the
center of the four rods.

 These ions pass through the electrode system.

 Ions with other m/z ratios are thrown out of their

original path and these ions are filtered out or lost to
the walls of the Quadrupole, and then ejected as
waste by a vacuum system.

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 The ion trap is a variation of the Quadrupole mass
filter, and consequently is sometimes refer to as a
Quadrupole Ion Trap.

 The trap contains ions in a 3-dimensional volume

rather than along the center axis.

 Helium gas is added to the trap causing the ions

to migrate toward the center.

 After trapping, the ions are detected by placing

them in unstable orbits, causing them to leave the
trap.
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 Consisted of ring electrode and a pair of end‐cap
electrodes .
 Radio‐frequency voltage is applied and varied to the
ring electrode .
 As radio‐frequency voltage increases, heavier ions
stabilize and lighter ions destabilized and then
collide with the ring wall.
 Ion trap analyzer forms positive or negative ions and
holds them for a long period of time by electric
and/or magnetic fields.
 It can be used as a detector for GS-MS
 It is cheap, more compact and more rugged then
magnetic sector and Quadrupole.
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 TOF Analyzers separate ions by time without the use
of an electric or magnetic field.
 Analyzation is based on the kinetic energy and
velocity of the ions.
 Separates ions based on flight time in drift tube.

 Positive ions are produced in pulses and accelerated

in an electric field (at the same frequency), all
particles have the same kinetic energy

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 K.E. = zV = 1/2 mv2
Solving for velocity (v)
v = (2zV/m)1/2

 The transit time (t) through the drift tube is L/v

where L is the length of the drift tube (usually 1‐3
meters).

 t =L (m/z)1/2 (1/ 2V)1/2

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 ICR is an ion trap that uses a magnetic field in order to
trap ions into an orbit inside of it.

 In this analyzer there is no separation that occurs rather

all the ions of a particular range are trapped inside, and
an applied external electric field helps to generate a
signal.

 when a moving charge enters a magnetic field, it

experiences a centripetal force making the ion orbit.
Again the force on the ion due to the magnetic field is
equal to the centripetal force on the ion.
zvB=mv2/r

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 Angular velocity of the ion perpendicular to the
magnetic field can be substituted here
ωc=v/r
zB=m ωc
ωc=zB/m
 Frequency of the orbit depends on the charge and mass
of the ions, not the velocity. If the magnetic field is held
constant, the charge to mass ratio of each ion can be
determined by measuring the angular velocity ωc.

 The relationship is that, at high ωc, there is low m/z

value, and at low ωc, there is a high m/z value. Charges
of opposite signs have the same angular velocity, the
only difference is that they orbit in the opposite
direction.
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 Electron multiplier.

 Faraday cups .

 Micro-channel plate detectors.

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 Continuous dynode electron multiplier .

 An electron multiplier (continuous dynode electron

multiplier) is a vacuum-tube structure that multiplies
incident charges.

 In a process called secondary emission, a single electron can,

when bombarded on secondary emissive material, induce
emission of roughly 1 to 3 electrons.

 If an electric potential is applied between this metal plate

and yet another, the emitted electrons will accelerate to the
next metal plate and induce secondary emission of still more
electrons.

 This can be repeated a number of times, resulting in a large

shower of electrons all collected by a metal anode, all having
been triggered by just one. 54
 A Faraday cup is a metal (conductive) cup designed to catch
charged particles in vacuum.

 The resulting current can be measured and used to determine

the number of ions or electrons hitting the cup.

 When a beam or packet of Ions hits the metal it gains a small

net charge while the ions are neutralized.

 The metal can then be discharged to measure a small current

equivalent to the number of impinging ions.

 By measuring the electrical current (the number of electrons

flowing through the circuit per second) in the metal part of
the circuit the number of charges being carried by the ions in
the vacuum part of the circuit can be determined. 55
 It is a planar component used for detection of particles
(electrons or ions) and impinging radiation
(ultraviolet radiation and X-rays).

 It is closely related to an electron multiplier, as both

intensify single particles or photons by the
multiplication of electrons via secondary emission.

 However, because a micro channel plate detector has

many separate channels, it can additionally provide
spatial resolution.
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 A micro-channel plate is a slab made from highly
resistive material of typically 2 mm thickness with a
regular array of tiny tubes or slots (micro-channels)
leading from one face to the opposite, densely
distributed over the whole surface.

 The micro-channels are typically approximately 10

micrometers in diameter (6 micrometer in high
resolution MCPs) and spaced apart by
approximately 15 micrometers; they are parallel to
each other and often enter the plate at a small angle
to the surface (~8° from normal).
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 Mass spectrometry has both qualitative and
quantitative uses.
1.Structure elucidation
2.Detection of impurities
3.Quantitative analysis
4.Drug metabolism studies
5.Clinical, toxicological and forensic applications
6.GC-MS-MS is now in very common use in analytical
laboratories that study physical, chemical, or
biological properties of a great variety of
compounds.
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1.Determination of molecular weight: Mass spectrometry serves as
the best possible technique for the determination or confirmation of
molecular weight of compounds.

2.Determination of molecular formula : For the determination of

molecular formula by mass spectrometry, it is essential to identify
the molecular ion peak as well as its exact mass.

3. Determination of partial molecular formula: Generally, atoms are

poly-isotopic. In mass spectrometer, the ions are selected according
to their actual mass. Exact information about the atomic
composition of the selected ions is furnished by the mass
distribution of molecular ions

4.Determination of structure of compounds: Bombardment of

vaporized sample molecules with a high beam of electrons results
in their fragmentation producing a large number of ions with
varying masses.
59
1.Determination of isotope abundance: Although
differences in the masses of isotopes of an element are
very small, the isotope abundance i.e., the isotopic
composition of molecules within an easily vaporizable
sample can be determined with mass spectrometry. The
information so obtained may be useful for:
(a) tracer studies with isotopes
(b) determination of atomic weights of compounds
(c) determination of age rocks and minerals
(d) study of origin as well as nature of solar system

2 Determination of isotope ratio : Mass spectroscopy is

used to determine isotope ratio which in turn helps to
determine the concentration of individual components
present in complex mixture from which it cannot be
separated quantitatively.
60
3.Differentiation between Cis and Trans isomers: Mass
spectrometry may be used to differentiate between cis and
trans isomers. Both the isomers yield similar spectra but are
differentiated from the intensity of the molecular ion peaks.
The molecular ion peak of trans isomer is more intense than
that of cis isomer.

4.Mass spectrometry in thermodynamics:

(a) Determination of heat of vapourization To determine the
heat of vapourization of high temperature compounds, data
from the spectrum is collected and a graph is plotted by
taking ion intensities on Y- axis and temperature on X-axis.

(b) Determination of heat of sublimation To determine the heat

of sublimation of a compound, vapours of the sublimed solids
are passed into the ionization chamber of the mass
spectrometer. The spectrum is recorded in which the obtained
peak intensities are directly proportional to the vapour
pressure (VP) of the sample in the ionization chamber. 61
5.Measurement of ionization potential : Ionization
potential is the minimum energy required by the
bombarding electrons to produce the molecular ions
from a molecule of an atom.

6.Determination of ion-molecule reactions: Mass

spectrometry finds its use in the study of ion molecule
reaction i.e., the reactions in between the fragment ion
and the unionized molecules. The rate of these reactions
directly depend on the operating pressure.

7.Detection of impurity: The impurities present sample

even in low concentration (parts per million) can be
detected by spectrometry, provide the molecular weights
of the impurities differ considerably from the major
components. 62
8.Identification of unknown compounds: Mass
spectrometry can be used to identify the unknown
compounds. this can be achieved by recording the
spectrum of the unknown compounds and comparing it
with the spectrum of the standard compound recorded
under identical conditions.

9.Identification of proteins: Mass spectrometry serves

as valuable tool in the study of structure and functions
of proteins (proteomics). Electro spray ionization (ESI)
and matrix-assisted laser desorption/ionization
(MALDI) are the widely used ionization methods for
this purpose. Mass spectrometry in the proteomics
particularly deals with the analysis of protein digested
by protease like trypsin.
63
• Molecules containing atoms limited to C,H,O,N,S,X,P of
even-numbered molecular weight contain either NO
nitrogen or an even number of N and similarly odd
molecular weight compound contains odd no of
nitrogen.
• This is true as well for radicals as well.
• Not true for pre-charged, e.g. quats, (rule inverts) or
radical cations.
• In the case of Chemical Ionization, where [M+H]+ is
observed, need to subtract 1, then apply nitrogen rule.
64
 If molecular formula of a compound is known the
number of unsaturation can ne calculated by ring
rule .
 The no. of unsaturated sites R , is equal to the no. of
rings in the molecule + the no. of double bond + 2x
no. of triple bond.
 The ring rule for molecule CwHzNyOx may be stated
as follows
R = w+1+(y-z)/2
The ring rule for diethyl ether C2H5OC2H5.
R = 4+1+ (0-10)/2
= 4+1+5
= 0
65
 The Rule of 13 is a simple procedure for tabulating
possible chemical formula for a given molecular mass. The
first step in applying the rule is to assume that only carbon
and hydrogen are present in the molecule and that the
molecule comprises some number of CH "units" each of
which has a nominal mass of 13. If the molecular weight of
the molecule in question is M, the number of possible CH
units is n:
M/13 = n + r /13
 where r is the remainder. The base formula for the molecule is
CnH {n+r} .
 the degree of unsaturation.
µ =( n-r+2 )/2

66
 A negative value of u indicates the presence of hetero-
atoms in the molecule and a half-integer value
of u indicates the presence of an odd number of
nitrogen atoms.

 On addition of hetero-atoms, the molecular formula is

adjusted by the equivalent mass of carbon and
hydrogen. For example, adding N requires removing
CH2 and adding O requires removing CH4.

67
 Pattern of fragmentation of molecule depends
upon :
 Energy of electron beam.
 Temperature of ion source.
 Pressure in the ion source.
 Geometry of the molecule.

Molecular ion breaks into 2 parts one of which is

positive ion and other is an uncharged free
radical.

68
1. The relative height of the M+ peak is greatest for straight
chain molecules and decreases as the branching
increases.
2. The relative height of the M+ peak decreases with
increasing molecular weight.
3. Cleavage is favored at alkyl-substituted carbons, with the
probability of cleavage increasing as the substitution
increases.
These rules mostly arise from the fact that carbocation and
radical stability show the following trend:
Most Stable Benzylic > Allylic > Tertiary > Secondary
>>Primary Least Stable
“Stevenson’s Rule” At the point of breakage, the larger
fragment usually takes the radical to leave the smaller
cation.
69
4. Double bonds, cyclic structures, and especially
aromatic rings will stabilize the molecular ion and
increase its probability of appearance.
5. Double bonds favor Allylic cleavage to give a
resonance stabilized Allylic carbocation, especially
for cycloalkenes.

6. For saturated rings (like cyclohexanes), the side

chains tend to cleave first leaving the positive charge
with the ring . 70
 7. Unsaturated rings can also undergo retro-Diels-
Alder reactions to eliminate a neutral alkene.

 8. Aromatic compounds tend to cleave to give

benzylic cations, or more likely tropylium cations.

71
 9. C-C bonds next to heteroatoms often break leaving
the positive charge on the carbon with the
heteroatom.

 10. Cleavage is often favoured if it can expell small

stable molecules like water, CO, NH3, H2S, etc. In
addition to bond fragmentation, various
intramolecular rearrangements can take place to give
sometimes unexpected ion.
72
 Cyclohexenes, with favorable 6-membered transition
state. Can include heteroatoms (N,O, driven by keto-
enol like stability.

 More stable cation will be predominate

 Also works for hetero-substituted (e.g. make enol)
 Both EI (shown) and in Chemical Ionization.
(protonated molecular ion, cleave, then reprotonation
73
74
 Fragmentation is due to the rearrangement of parent
or molecular ion. Here cleavage of bonds in the
molecule is due to the intramolecular atomic re
arrangement.

 This leads to the fragmentation whose origin cannot

be described by simple cleavage of bonds.

 When the fragments are accompanied by bond

formation as well as bond for breaking, a
rearrangement process have said to been occurred.

 This process is energetically favored as many bonds

are formed are broken. 75
 It involves the migration n-γ hydrogen followed by the
cleavage of the β bond the re arrangement leads to the
elimination of neutral molecule of aldehyde ketone,
unsaturated compound etc. the rearragment proceeds
through steady kinesis.

 for McLaffertty re arrangement a molecule must posses a

appropriately located hetero-atom, a double bond and
abstractable hydrogen which is γ hydrogen in nature. 76
77
78
 Parent ion or molecular ion peak : the molecular ion is formed by
loss of one electron also called parent ion peak.

 M+1 peak: In organic compound, there is generally a small

peak appearing 1 mass unit higher than the parent peak
due to the small but observable, natural abundance of
13C and 2H.

 M+2 PEAK: The same molecule have 2 heavy isotopes, there

is an even smaller 2 peaks of the isotopes ne , e.g.
chlorine , bromine etc

 Base peak: The highest peak in the spectra is called the base
peak and its intensity is taken 100, and the height of
other peaks are measured with respect to base peak.
79
 Rearrangement ion peak: Sometimes fragments of peak are
observed which are not a part of original molecule these
peak are formed due to rearrangement of molecule at
same instance of decomposition. In specific heteroatom
case the peak observed may be very intense.

 Multiple charged : Sometimes multiple charged ions are

formed in mass spectra generally these are doubly or
triply less intense than singly charged m/z peak.

 Metastable ions: The ions resulting from the decomposition

between the source region and the magnetic analyzer are
called metastable ions which appear as broad peak as
non integral mass number.
80
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