ST.

JOSEPH COLLEGE OF ENGINEERING

DEPARTMENT OF ELECTRICAL AND ELECTRONICS ENGINEERING

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OBT751 - ANALYTICAL METHODS AND
INSTRUMENTATION

UNIT - 3
LECTURE - 9

Presented by
J.Jayashree AP/EEE
SJCE
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 UNIT – 3 NMR AND MASS SPECTROMETRY
3.8 Applications of Molecular Mass Spectrometry
• The applications of molecular mass spectrometry are so numerous
and widespread.
• Table below lists several of these applications to provide some idea of
the capabilities of mass spectrometry.

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Table : Applications of Molecular Mass Spectrometry
 UNIT – 3 NMR AND MASS SPECTROMETRY
3.8 Applications of Molecular Mass Spectrometry
Identification of Pure Compounds:
• The mass spectrum of a pure compound provides several kinds of
data that are useful for its identification.
• The first is the molecular mass of the compound, and the second is its
molecular formula.
• In addition, study of fragmentation patterns revealed by the mass
spectrum often provides information about the presence or absence
of various functional groups.
• Finally, the actual identity of a compound can often be established by
• comparing its mass spectrum with those of known compounds.
Molecular Masses from Mass Spectra:
• For compounds that can be ionized to give a molecular ion the mass
spectrometer is an unsurpassed tool for the determination of
molecular mass. 4
 UNIT – 3 NMR AND MASS SPECTROMETRY
3.8 Applications of Molecular Mass Spectrometry
• This determination, requires the identification of the molecular ion
peak, the (M + 1)+ or the (M - 1)+ peak.
• The location of the peak on the abscissa then gives the molecular
mass with an accuracy that cannot be found out easily by any other
method.
Molecular Formulas from Exact Molecular Masses :
• Molecular formulas can be determined from the mass spectrum of a
compound, provided the molecular ion peak can be identified and its
exact mass determined.
• This application, however, requires a high-resolution instrument
capable of detecting mass differences of a few thousandths of a
dalton.

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 UNIT – 3 NMR AND MASS SPECTROMETRY
3.8 Applications of Molecular Mass Spectrometry
Molecular Formulas from Isotopic Ratios:
• The data from a low-resolution instrument that can discriminate only
between ions differing in mass by whole mass numbers can also yield
useful information about the formula of a compound, provided that the
molecular ion peak is sufficiently intense that its height and the heights
of the (M + 1)+ and (M + 2)+ isotope peaks can be determined accurately.
Compound Identification from Comparison Spectra :
• Generally, after determining the molecular mass of the analyte and
studying its isotopic distribution and fragmentation patterns, the
experienced mass spectroscopist is able to narrow the possible
structures.
• When reference compounds are available, final identification is then
based on a comparison of the mass spectrum of the unknown with
spectra for authentic samples of the suspected compounds.
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 UNIT – 3 NMR AND MASS SPECTROMETRY
3.8 Applications of Molecular Mass Spectrometry
• The procedure is based on the assumptions that
(1) mass fragmentation patterns are unique and
(2) experimental conditions can be sufficiently controlled to produce
reproducible spectra.
• The first assumption often is not valid for spectra of stereo- and
geometric isomers and occasionally is not valid for certain types of
closely related compounds.
• The probability that different compounds will yield the same spectrum
becomes markedly smaller as the number of spectral peaks increases.
• Assumption 2 can also be problematic.
• EI spectra are fairly reproducible from laboratory to laboratory.
• However, spectra from other sources can vary significantly.
• For this reason, electron-ionization is the method of choice for
spectral comparison and for building spectral libraries. 7
 UNIT – 3 NMR AND MASS SPECTROMETRY
3.8 Applications of Molecular Mass Spectrometry
Analysis of Mixtures by Hyphenated Mass Spectral Methods :
• Although ordinary mass spectrometry is a powerful tool for the
identification of pure compounds, its usefulness for analysis of all but
the simplest mixtures is limited because of the immense number of
fragments of differing m/z values produced.
• It is often impossible to interpret the resulting complex spectrum.
• For this reason, chemists have developed methods in which mass
spectrometers are coupled with various efficient separation devices in
so-called hyphenated methods.
Chromatography–Mass Spectrometry :
• Gas chromatography–mass spectrometry (GC/MS) has become one of
the most powerful tools available for the analysis of complex organic
and biochemical mixtures.
• In this application, spectra are collected for compounds as they exit
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from a chromatographic column.
 UNIT – 3 NMR AND MASS SPECTROMETRY
3.8 Applications of Molecular Mass Spectrometry
• These spectra are then stored in a computer for subsequent
processing.
• Mass spectrometry has also been coupled with liquid
chromatography (LC/MS) for the analysis of samples that contain
nonvolatile constituents.
• A major problem that had to be overcome in the development of
both of these hyphenated methods is that the sample in the
chromatographic column is highly diluted by the gas or liquid carrying
it through the column.
• Thus, methods had to be developed for removing the diluent before
introducing the sample into the mass spectrometer.
Capillary Electrophoresis–Mass Spectrometry :
• This hyphenated method is the analysis of large biopolymers, such as
proteins, polypeptides, and DNA species. 9
 UNIT – 3 NMR AND MASS SPECTROMETRY
3.8 Applications of Molecular Mass Spectrometry
• In most of the applications reported to date, the capillary effluent is
passed directly into an electrospray ionization device, and the
products then enter a quadrupole mass filter for analysis.
• Continuous flow FAB has also been used for ionization in some
applications.
Applications of Tandem Mass Spectrometry :
• Dramatic progress in the analysis of complex organic and biological
mixtures began when the mass spectrometer was first combined with
gas chromatography and subsequently with liquid chromatography.
• Tandem mass spectrometry offers some of the same advantages as
GC/MS and LC/MS but is significantly faster.
• Separations on a chromatographic column are achieved in a time
scale of a few minutes to hours, but equally satisfactory separations
in tandem mass spectrometers are complete in milliseconds. 10
 UNIT – 3 NMR AND MASS SPECTROMETRY
3.8 Applications of Molecular Mass Spectrometry
• In addition, the chromatographic techniques require dilution of the
sample with large excesses of a mobile phase and subsequent
removal of the mobile phase, which greatly enhances the probability
of introduction of interferences.
• Consequently, tandem mass spectrometry is potentially more
sensitive than either of the hyphenated chromatographic techniques
because the chemical noise associated with its use is generally
smaller.
• A current disadvantage of tandem mass spectrometry with respect to
the two chromatographic procedures is the greater cost of the
required equipment; this gap appears to be narrowing as tandem
mass spectrometers gain wider use.

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 UNIT – 3 NMR AND MASS SPECTROMETRY
3.9 Electron paramagnetic resonance
• Electron Paramagnetic Resonance(EPR) is also called Electron Spin Resonance(ESR)
.
• This absorption spectroscopy is similar to NMR. It is possible only with molecules
having unpaired electrons.
• Instead of Radiowaves in NMR. Micowaves are used in ESR.
• A molecule or atom has discrete (or separate) states, each with a corresponding
energy.
• Spectroscopy is the measurement and interpretation of the energy differences
between the atomic or molecular states.
• With knowledge of these energy differences, you gain insight into the identity,
structure, and dynamics of the sample under study.
• We can measure these energy differences, ΔE, because of an important
relationship between ΔE and the absorption of electromagnetic radiation.
• According to Planck's law, electromagnetic radiation will be absorbed if:

where h is Planck's constant and v is the frequency of the radiation12.

 UNIT – 3 NMR AND MASS SPECTROMETRY
3.9 Electron paramagnetic resonance
The Zeeman Effect :
• An isolated electron, all alone in space without any outside forces,
still has an intrinsic angular momentum called "spin",S̅.
• Because an electron is charged, the angular motion of this charged
particle generates a magnetic field.
• In other words, the electron due to its charge and angular
momentum, acts like a little bar magnet, or magnetic dipole, with a
magnetic moment , ̅.

Fig: Free, unpaired electron in space: electron spin – magnetic

moment 13
 UNIT – 3 NMR AND MASS SPECTROMETRY
3.9 Electron paramagnetic resonance
• The energy differences are predominately due to the interaction of
unpaired electrons in the sample with a magnetic field produced by a
magnet in the laboratory.
• This effect is called the Zeeman Effect.
• The magnetic field, B0, produces two energy levels for the magnetic
moment,  ̅, of the electron.
• The unpaired electron will have a state of lowest energy when the
moment of the electron is aligned with the magnetic field and a stage of
highest energy whe  ̅ is aligned against the magnetic field.

Fig: Minimum and maximum energy

orientations of  ̅ with respect to the
magnetic field B0

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 UNIT – 3 NMR AND MASS SPECTROMETRY
3.9 Electron paramagnetic resonance
• The two states are labeled by the projection of the electron spin, ms,
on the direction of the magnetic field.
• Because the electron is a spin ½ particle, the parallel state is
designated as ms = -½ and the antiparallel state is ms = +½.
• The energy of each orientation is the product of μ and B0.
• For an electron μ = msgeβ
where
  is a conversion constant called the Bohr magneton
 ge is the spectroscopic g-factor of the free electron and equals
2.0023192778 (≈ 2.00).
• , the energies for an electron with ms = +½ and ms = -½ are,
respectively
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 UNIT – 3 NMR AND MASS SPECTROMETRY
3.9 Electron paramagnetic resonance
• Therefore , the energies for an electron with ms = +½ and ms = -½
are, respectively

As a result there are two energy levels for the electron in a magnetic field.

Fig. : Induction of the

spin state energies as a
function of the
magnetic field B 0.

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 UNIT – 3 NMR AND MASS SPECTROMETRY
3.9 Electron paramagnetic resonance
Spin-Orbit Interaction:

• When we take an electron in space with no outside forces on it and

place it on to a molecule, its total angular momentum changes
because, in addition to the intrinsic spin angular momentum (S ̅), it
now also possesses some orbital angular momentum ( ̅).
 An electron with orbital angular momentum is in effect a circulating
current, and so there is also a magnetic moment arising from the
orbital angular momentum.
These two magnetic moments interact, and the energy of this spin-17
 UNIT – 3 NMR AND MASS SPECTROMETRY
3.9 Electron paramagnetic resonance
Electron in space

Electron in a molecule

In general, the orbital angular momentum is approximately zero for an

electron in the ground state (s electron).

It is common practice to assume that the spin-orbit coupling term is

proportional to S ̅ which means we can simply combine both terms on
the right and just change the value of ge to g, or

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 UNIT – 3 NMR AND MASS SPECTROMETRY
3.9 Electron paramagnetic resonance
• The magnitude of the spin-orbit coupling contribution depends on
the size of the nucleus containing the unpaired electron.
• Therefore, organic free radicals, with only H, O, C and N atoms, will
have a small contribution from spin-orbit coupling, producing g
factors very close to ge while the g factors of much larger elements,
such as metals, may be significantly different from ge.
• A simpler alternative way of thinking about the spin-orbit coupling is
that a virtual observer on the electron would experience the nucleus
(nuclei) as an orbiting positive charge producing a second magnetic
field, δB, at the electron.

Since only the spectrometer value of B is known we can rewrite this as:

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 UNIT – 3 NMR AND MASS SPECTROMETRY
3.9 Electron paramagnetic resonance
• The quantity ‘ge + δg’ or ‘g’ contains the chemical
information on the nature of the bond between the
electron and the molecule, the electronic structure
of the molecule.
• The value of g can be taken as a fingerprint of the
molecule.

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 UNIT – 3 NMR AND MASS SPECTROMETRY
3.10 g-Factor
• When a paramagnetic sample is placed in a large uniform
magnetic field, splits the energy levels of the ground state
by an amount ΔE where
----------------(1)
• Since  is a constant and the magnitude of B0 can be measured, all
we have to do to calculate g is determine the value of ΔE, the energy
between the two spin levels.
• This is done by irradiating the sample with microwaves with a set
frequency and sweeping the magnetic field

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UNIT – 3 NMR AND MASS SPECTROMETRY
3.10 g-Factor
Absorption of energies will occur
when the condition in Equation -1
is satisfied.
The value of g can then be
calculated from ν (in GHz) and B0
(in gauss) using,

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 UNIT – 3 NMR AND MASS SPECTROMETRY
3.10 g-Factor
• Two facts are apparent from equations and the graph.
• Firstly, the two spin states have the same energy in the absence of a
magnetic field.
• Secondly, the energies of the spin states diverge linearly as the
magnetic field increases.
• These two facts have important consequences for spectroscopy:
1) Without a magnetic field, there is no energy difference to measure.
2) The measured energy difference depends linearly on the magnetic
field
• Because we can change the energy differences between the two spin
states by varying the magnetic field strength, we have an alternative
means to obtain spectra.
• We could apply a constant magnetic field and scan the frequency of
the electromagnetic radiation as in conventional spectroscopy. 23
 UNIT – 3 NMR AND MASS SPECTROMETRY
3.10 g-Factor
• Alternatively, we could keep the electromagnetic radiation frequency
constant and scan the magnetic field.
• A peak in the absorption will occur when the magnetic field “tunes” to
the two spin states so that their energy difference matches the energy of
the radiation.
• This field is called the “field of resonance”.
• A radiation source for radar waves produces only a very limited spectral
region.
• In EPR such a source is called a klystron.
• A so-called X-band klystron has a spectral band width of about 8.8-9.6
GHz.
• This makes it impossible to continuously vary the wavelength similarly to
optical spectroscopy.
• It is therefore necessary to vary the magnetic field, until the quantum of
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the radar waves fits between the field-induced energy levels.
 UNIT – 3 NMR AND MASS SPECTROMETRY
3.11 Instrumentation
KLYSTRONS
• Klystron tube acts as the source of radiation.
• It is stabilized against temperature fluctuation by
immersion in an oil bath or by forced air cooling.
• The frequency of the monochromatic radiation is
determined by the voltage applied to klystron.
• It is kept a fixed frequency by an automatic control
circuit and provides a power output of about 300
milli watts.
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 UNIT – 3 NMR AND MASS SPECTROMETRY
3.11 Instrumentation
WAVE GUIDE OR WAVEMETER
• The wave meter is put in between the oscillator and
attenuator.
• To know the frequency of microwaves produced by
klystron oscillator.
• The wave meter is usually calibrated in frequency
unit (megahertz) instead of wavelength.
• Wave guide is a hollow, rectangular brass tube. It is
used to convey the wave radiation to the sample
and crystal.
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 UNIT – 3 NMR AND MASS SPECTROMETRY
3.11 Instrumentation
• ATTENUATORS
• The power propagated down the wave guide may
be continuously decreased by inserting a piece of
resistive material into the wave guide. This piece is
called variable attenuator.
• It is used in varying the power of the sample from
the full power of klystron to one attenuated by a
force 100 or more.

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 UNIT – 3 NMR AND MASS SPECTROMETRY
3.11 Instrumentation
ISOLATORS
• It’s device which minimizes vibrations in the
frequency of microwaves produced by klystron
oscillator.
• Isolators are used to prevent the reflection of
microwave power back into the radiation source.
• It is a strip of ferrite material which allows micro
waves in one direction only.
• It also stabilizes the frequency of the klystron.
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 UNIT – 3 NMR AND MASS SPECTROMETRY
3.11 Instrumentation
SAMPLE CAVITIES
• The heart of the ESR spectrometer is the resonant
cavity containing the sample.
• Rectangular TE120 cavity and cylindrical TE011 cavity
have widely been used.
• In most of the ESR spectrometers, dual sample cavities
are generally used This is done for simultaneous
observation of a sample and a reference material.
• Since magnetic field interacts with the sample to cause
spin resonance the sample is placed where the
intensity of magnetic field is greatest. 29
 UNIT – 3 NMR AND MASS SPECTROMETRY
3.11 Instrumentation
COUPLERS AND MATCHING SCREWS
• The various components of the micro wave
assembly to be coupled together by making use of
irises or slots of various sizes.
CRYSTAL DETECTORS
• Silicon crystal detectors, which converts the
radiation in D.C has widely been used as a detector
of microwave radiation.

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 UNIT – 3 NMR AND MASS SPECTROMETRY
3.11 Instrumentation
MAGNET SYSTEM
• The resonant cavity is placed between the poles
pieces of an electromagnet.
• The field should be stable and uniform over the
sample volume.
• The stability of field is achieved by energizing the
magnet with a highly regulated power supply.
• The ESR spectrum is recorded by slowly varying the
magnetic field through the resonance condense by
sweeping the current supplied to the magnet by the
power supply. 31
 UNIT – 3 NMR AND MASS SPECTROMETRY
3.11 Instrumentation
MODULATION COIL
• The modulation of the signal at a frequency consistent with good signal
noise ratio in the crystal detector is accomplished by a small alternating
variation of the magnetic field.
• The variation is produced by supplying an A.C. signal to modulation coil
oriented with respect the sample in the same direction as the magnetic
field.
• If the modulation is of low frequency (400 cycles/sec or less), the coils
can be mounted outside the cavity and even on the magnet pole pieces.
• For higher modulation frequencies, modulation coils must be mounted
inside the resonant cavity or cavities constructed of a non-metallic
material e.g., Quartz with a tin silvered plating.
DISPLAY DEVICES
• In order to observe the signal a system is connected different devices
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can be used.
UNIT – 3 NMR AND MASS SPECTROMETRY
3.11 Instrumentation

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UNIT – 3 NMR AND MASS SPECTROMETRY
3.11 Instrumentation

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THANK YOU
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