MASS SPECTROMETRY

Mass spectrometry is perhaps the most widely applicable of all the analytical tools available because
the technique can provide information about the

1. Elemental composition of a sample of matter

2. Structures of inorganic, organic and biological molecules
3. The qualitative and quantitative composition of complex mixtures
4. The structure and composition of solid surfaces
5. The isotopic ratio of atoms in the sample

Principle of Mass Spectrometry [The first part is for understanding purposes in exam it is
best to explain the principle with the example given in the second part]
A beam of electrons will be bombarded in the analyte compound and it leads to the removal of one
electron from the molecule. [Start referring to the picture] The removal of one electron from the
analyte gives rise to a positively charged ion known as a molecular ion. However, the energy of the
electron which is responsible for the bombardment is sufficiently high compared to the ionisation
enthalpy of the molecular ion so even after the emission of one electron. This excess energy leads to
the formation of fragment ions, also known as daughter ions. The + sign indicates over the molecular
ion denotes the positive structure while the ‘dot’ indicates the single electron left after emission of
the electron [Stop referring to the picture]. The ions are sorted according to their mass-to-charge
ratios by the Mass Analyzer, detected by a Detector and displayed in the form of a mass spectrum.
(basically mass vs relative abundance graph is generated)

Example
The analyte was ethylbenzene, which has a nominal molecular mass of 106 daltons (Da). To obtain
this spectrum, ethyl benzene vapour was bombarded with a stream of electrons that led to the loss
of an electron by the analyte and the formation of the ion as shown in the reaction

The charged species C6H5CH2CH+ is the molecular ion. The collision between energetic electrons and
analyte molecules usually imparts enough energy to the molecules to leave them in an excited state.
Relaxation then often occurs by fragmentation of part of the molecular ions to produce ions of lower
masses. For example, a major product in the case of ethylbenzene is C6H5CH2+, which results from the
loss of a CH3 group. Other smaller positively charged fragments are also formed in lesser amounts.

The positive ions produced in electron ionization (EI) are attracted through the slit of a mass
spectrometer where they are sorted according to their mass-to-charge ratios and displayed in the
form of a mass spectrum. Note that the plot shown in Figure 20-1 is in the form of a bar graph that
relates the relative intensity of mass peaks to their mass-to-charge ratio. The largest peak in a
spectrum termed the base peak, is arbitrarily assigned a value of 100. The heights of the remaining
peaks are then computed as a percentage of the base-peak height. Modern mass spectrometers are
programmed to automatically recognize the base peak. They then normalize the remaining peaks in
the spectrum relative to the base peak.

Instrumentation
A mass spectrometer consists of several key components:

1. Sample Inlet: The Sample Inlet System is a crucial component of a mass

spectrometer as it serves the purpose of introducing the sample into the ionization
chamber of the mass spectrometer while ensuring minimal disturbance of the high
vacuum system. The selection of the inlet system is highly dependent upon the type of
sample and sample matrix which will be introduced into the ionization chamber. The
sample molecules undergo ionization, and the ionization process prefers sample ions
in the gaseous state. Samples of different types such as solids, liquids, or gases,
depending on their vapour pressure, are injected through various inlet systems.

A. Direct Vapor Inlet Method: If the sample to be injected is highly volatile with
high vapor pressure, it can be introduced directly into the ionization chamber. This
process is called the Direct Vapor Inlet method. The sample is injected into the
mass spectrometer through a needle valve.
B. Other Inlet Source: There are also other inlet sources such as Direct Insertion
Probe and Direct Condensed Vapor Ionization, which are very useful while
injecting specific samples depending on the pressure and temperature sensitivity
of the sample.

2. Ionization Source: The starting point for a mass spectrometric analysis is the
formation of gaseous analyte ions, and the scope and the utility of a mass
spectrometric method are dictated by the ionization process. The appearance of mass
spectra for a given molecular species strongly depends on the method used for ion
formation. Ionization sources fall into three major categories: gas-phase sources,
desorption sources, and ambient desorption sources. (only first two in syllabus)

GAS-PHASE SOURCES DESORPTION SOURCES

With a gas-phase source, the sample is Desorption and ambient sources, which
first vaporized and then ionized. With a do not require volatilization of analyte
desorption source, however, the solid- or molecules, apply to analytes having
liquid-state sample is converted directly molecular masses as large as 105 Da.
into gaseous ions.
An advantage of desorption sources is
Gas-phase sources are generally that they apply to non-volatile and
restricted to the ionization of thermally thermally unstable samples.
stable compounds that have boiling
points less than about 500°C. In most
cases, this requirement limits gaseous
sources to compounds with molecular
masses less than roughly 103 Da.
Ion sources are also classified as hard sources or soft sources. Both hard- and soft-source
spectra are useful for analysis.

Hard ionization sources impart enough energy to analyte molecules to leave them in a
highly excited energy state. Relaxation then involves the rupture of bonds, producing
fragment ions that have mass-to-charge ratios less than that of the molecular ion. The many
peaks in a hard-source spectrum provide useful information about the kinds of functional
groups and thus structural information about analytes.

Soft ionization sources cause little fragmentation. Thus, the mass spectrum from a soft
ionization source often consists of the molecular ion peak and only a few, if any, other peaks.
Soft-source spectra are useful because they supply accurate information about the molecular
mass of the analyte molecule or molecules.
Figure 20-2 illustrates the difference in spectra obtained from a hard ionization source
and a soft ionization source. (detailed explanation provided at the end of Chemical
ionization)

3. Mass Analyzer: Once ionized, the ions are sorted and divided according to their
mass-to-charge (m/z) ratio. Several devices are available for separating ions with
different mass-to-charge ratios. Ideally, the mass analyzer should be capable of
distinguishing minute mass differences. In addition, the analyzer should allow passage
of a sufficient number of ions to yield readily measurable ion currents. These two
properties are not entirely compatible, and design compromises must be made.

Resolution: The capability of a mass spectrometer to differentiate between masses is usually

stated in terms of its resolution R, which is defined as

R= m/Δm

Where Δm is the mass difference between two adjacent peaks of equal intensity that are just
resolved and m is the nominal mass of the first peak (the mean mass of the two peaks is
sometimes used instead). Two peaks are considered to be separated if the height of the valley
between them is no more than a given fraction of their height (often 10%). Thus, a
spectrometer with a resolution of 4000 would resolve peaks at m/z values of 400.0 and 400.1
(or 40.00 and 40.01).

The resolution required in a mass spectrometer depends greatly on its application. For
example, discrimination among ions of the same nominal mass such as C2H4+, CH2N+, N2+,
and CO+ (all ions of nominal mass 28 Da but exact masses of 28.0313, 28.0187, 28.0061, and
27.9949 Da, respectively) requires an instrument with a resolution of several thousand. On
the other hand, low-molecular-mass ions differ by a unit of mass or more such as NH3+ (m=
17) and CH4+ (m= 16), for example, can be distinguished with an instrument having a
resolution smaller than 50.

4. Detector: At different deflections, a detector counts the number of ions. Detectors

function by recording the induced charge or current generated by an ion hitting or
passing through a surface. Electron Multipliers, Faraday cups, and Photomultiplier
Conversion Dynodes are some of the common detectors used.

These components work together to measure the mass-to-charge ratio (m/z) of one or more
molecules in the sample. Such measurements may also often be used to determine the precise
molecular weight of the sample components. Mass spectrometry is an analytical method to
find the molecular mass of a compound and indirectly helps to prove the identity of isotopes.

IONIZATION SOURCES

Gas Phase Ionization Sources:

Gas phase ionization sources are used when the sample can be volatilized before it enters the
mass spectrometer’s inlet. Two common types of gas-phase ionization sources are:
 Electron Ionization (EI): In this process, the sample is brought to a temperature high
enough to produce a molecular vapour, which is then ionized by bombarding the resulting
molecules with a beam of energetic electrons.

Electrons are emitted from a heated tungsten or rhenium filament and accelerated by
applying approximately 70 V between the filament and the anode. As shown in the figure,
the paths of the electrons and molecules are at right angles and intersect near the center of
the source, where collision and ionization occur. The primary product is singly charged
positive ions formed when the energetic electrons approach molecules closely enough to
cause them to lose electrons by electrostatic repulsion.

M + e- M.+ + 2e-

M represents the analyte molecule, and M.+ is its molecular ion. The positive ions
produced by electron ionization are attracted through the slit in the first accelerating plate
by a small potential difference (typically 5 V) that is applied between this plate and the
repellers. High voltages are applied to the accelerator plates, which give the ions their
final velocities before they enter the mass analyzer.

Electron Ionization Spectrum

To form a significant number of gaseous ions at a reproducible rate, electrons from the
filament in the source must be accelerated by a voltage of greater than about 50 V. The
low mass and high kinetic energy of the resulting electrons cause little increase in the
translational energy of impacted molecules. Instead, the molecules are left in highly
excited vibrational and rotational states. Subsequent relaxation then usually takes place by
extensive fragmentation, giving a large number of positive ions of various masses that are
less than (and occasionally, because of collisions, greater than) that of the molecular ion.
These lower-mass ions are called product or fragment ions.

Advantages

• EI sources are convenient to use.

• They produce high ion currents, which lead to good sensitivities.
• The extensive fragmentation and resulting large number of peaks is also an
advantage because it often makes unambiguous identification of analytes possible.
Disadvantages

• Extensive fragmentation can also be a disadvantage, however, when it results in

the disappearance of the molecular ion peak so that the molecular mass of analytes
cannot be easily established.
• Another limitation of the EI source is the need to volatilize the sample, which may
result in thermal degradation of some analytes before ionization can occur.
• EI sources apply only to analytes having molecular masses smaller than about 103
Da.

Chemical Ionization (CI): In CI, gaseous atoms of the sample (from either a batch inlet
or a heated probe) are ionized by collision with ions produced by electron bombardment
of an excess of a reagent gas. Usually, positive ions are used, but negative ion CI is
occasionally used with analytes that contain very electronegative atoms.

To carry out CI experiments, it is necessary to modify the electron beam ionization area
shown in Figure 20-3 by adding vacuum pump capacity and by reducing the width of the
slit to the mass analyzer. These measures allow a reagent pressure of about 100 Pa (0.75
torr) to be maintained in the ionization area while maintaining the pressure in the analyzer
below 10-7 Pa. With these changes, a gaseous reagent is introduced into the ionization
region in an amount such that the concentration ratio of reagent to sample is 103 to 104.
Because of this large concentration difference, the electron beam reacts nearly exclusively
with reagent molecules

One of the most common reagents for CI is methane, which reacts with high-energy
electrons to give several ions such as CH4+, CH3+, and CH2+. The first two predominate
and represent about 90% of the reaction products. These ions react rapidly with additional
methane molecules as follows:

CH4+ + CH4 CH5+ + CH3

CH3+ + CH4 C2H5+ + H2

Generally, collisions between the analyte molecule M and CH5+ or C2H5+ are highly
reactive and involve proton or hydride transfer.

For example,

CH5+ + M MH+ + CH4 [proton transfer]

C2H5+ + M MH+ + C2H4 [proton transfer]

C2H5+ + MH M+ + C2H6 [hydride transfer]

Comparison of the spectrum obtained from Hard ionization (EI source) and Soft
ionization (CI source) [Not important for exam but important for understanding]
 Figure 20-2 contrasts the CI and EI spectra for 1-decanol. The EI spectrum (Figure 20-2a)
shows evidence for rapid and extensive fragmentation of the molecular ion. Thus, no
detectable peaks are observed above mass 112, which corresponds to the ion C8H16+. The
base peak is provided by the ion C3H5+ at mass 41. Other peaks for C3 species are
grouped around the base peak. A similar series of peaks, found at 14, 28, and 42 mass
units greater, correspond to ions with one, two, and three additional CH2 groups.

Relative to the EI spectrum, the CI spectrum shown in Figure 20-2b is simple indeed,
consisting of the (MH - H)+ peak, a base peak corresponding to a molecular ion that has
lost an OH group, and a series of peaks differing from one another by 14 mass units. As
in the EI spectrum, these peaks arise from ions formed by the cleavage of adjacent
carbon-carbon bonds. As we have just noted, chemical ionization spectra generally
contain well-defined (M 1 H)+ or (MH - H)+ peaks resulting from the addition or
abstraction of a proton in the presence of the reagent ion.

Desorption Ionization Sources:

The ionization methods discussed so far require that the ionizing agents act on gaseous
samples. Such methods do not apply to nonvolatile or thermally unstable samples. Several
desorption ionization methods have been developed for dealing with this type of sample.
These methods have enabled mass spectra to be obtained for thermally delicate
biochemical species and species having molecular masses of greater than 100,000 Da.
Desorption methods dispense with volatilization followed by ionization of the gaseous
analyte molecules. Instead, energy in various forms is introduced into the solid or liquid
sample in such a way as to cause direct formation of gaseous ions. As a consequence,
spectra are greatly simplified and often consist of only the molecular ion or the protonated
molecular ion.

Matrix-Assisted Laser Desorption/Ionization (MALDI): Matrix-assisted laser

desorption/ionization (MALDI) spectrometry is an ionization method that can be used to
obtain accurate molecular mass information about polar biopolymers ranging in
molecular mass from a few thousand to several hundred thousand Da.
In the MALDI technique, a low concentration of the analyte is uniformly dispersed in a
solid or liquid matrix deposited on the end of a stainless steel probe or placed on a metal
plate. The plate is then placed in a vacuum chamber and a laser beam is focused onto the
sample. The MALDI matrix must strongly absorb the laser radiation. The matrix and
analyte are then desorbed and ionized, creating an ion plume. The most common type of
mass analyzer used with MALDI is the time-of-flight (TOF) analyzer.

Absorption of the laser beam by the matrix, followed by transfer of the energy from the
matrix to the analyte is followed by desorption of the analyte and the matrix. The analyte
is thought to desorb as neutral molecules and then to be ionized by proton-transfer
reactions with protonated matrix ions in a dense phase over the surface containing the
matrix. A series of photochemical reactions may produce the protonated matrix ions.

The most common sample-preparation method is the dried-droplet technique in which a

droplet of the matrix containing the analyte is deposited on the metal plate and then dried.
Typically, the ratio of analyte to matrix is 1:103 to 1:105.

Fast Atom Bombardment (FAB): Fast atom bombardment (FAB) sources involve
samples in a condensed state, usually in a viscous solution matrix that are ionized by
bombardment with energetic (several keV) xenon or argon atoms.

This treatment provides very rapid sample heating, which reduces sample fragmentation.
The liquid matrix helps to reduce the lattice energy, which must be overcome to desorb an
ion from a condensed phase and provides a means of “healing” the damage induced by
bombardment.

A beam of fast atoms is obtained by passing accelerated argon or xenon ions from an ion
source, or gun, through a chamber containing argon or xenon atoms at a pressure of about
10-3 Pa (10-5 torr). The high-velocity ions undergo a resonant electron-exchange reaction
with the atoms without substantial loss of translational energy. Thus, a beam of energetic
atoms is formed. The lower-energy ions from the exchange are readily removed by an
electrostatic deflector (EXTRACTION GRID IN IMAGE).
 The many drawbacks of FAB have led to its decrease in popularity. These include the
limited molecular mass range of FAB, the need for larger sample quantities than many
other ionization techniques and the necessity for finding an appropriate matrix in which
the analyte is soluble.

MASS ANALYZERS

Quadrople Mass Analyzers:

The most common type of mass spectrometer used in atomic mass spectroscopy is the
quadrupole mass analyzer. This instrument is more compact, less expensive, and more rugged
than most other types of mass spectrometers. It also has the advantage of high scan rates so
that an entire mass spectrum can be obtained in less than 100 ms.

The heart of a quadrupole instrument is the four parallel cylindrical (originally hyperbolic)
rods that serve as electrodes. Opposite rods are connected electrically, one pair being attached
to the positive side of a variable dc source and the other pair to the negative terminal. In
addition, variable radio-frequency ac voltages, which are 180° out of phase, are applied to
each pair of rods. To obtain a mass spectrum with this device, ions are accelerated into the
space between the rods by a potential difference of 5 to 10 V. Meanwhile, the ac and dc
voltages on the rods are increased simultaneously while maintaining their ratio constant. At
any given moment, all of the ions except those having a certain m/z value strike the rods and
are converted to neutral molecules. only ions having a limited range of m/z values reach the
transducer.

Scanning with a Quadrupole Filter

The resolution of a quadrupole is determined by the ratio of the ac to dc voltage and becomes
a maximum when this ratio is just slightly less than 6. Thus, quadrupole spectrometers are
operated at a constant voltage ratio of this value. To scan a mass spectrum with a quadrupole
instrument, the ac voltage V and the dc voltage U are increased simultaneously from zero to
some maximum value while their ratio is maintained at slightly less than 6. Quadrupole mass
spectrometers with ranges that extend up to 3000 to 4000 m/z are available from several
instrument manufacturers. These instruments easily resolve ions that differ in mass by one
unit.

Time-of-Flight Mass Analyzers:

TOF instruments, positive ions are produced periodically by bombardment of the sample with
brief pulses of electrons, secondary ions, or laser-generated photons. The ions produced in
this way are then accelerated into a field-free drift tube by an electric field pulse of 103 to
104 V. Separation of ions by mass occurs during the transit of the ions to the detector located
at the end of the tube. Because all ions entering the tube have the same kinetic energy, their
velocities in the tube vary inversely with their masses (Equation 20-4), with the lighter
particles arriving at the detector earlier than the heavier ones. The flight time tF is given by

Advantages and Disadvantages

TOF instruments offer several advantages over other types of mass spectrometers, including
simplicity and ruggedness, ease of accessibility of the ion source, and virtually unlimited
mass range. They suffer, however, from limited resolution and sensitivity. TOF instruments
also require fast electronics because ions often arrive at the transducer only fractions of
microseconds apart. Several instrument manufacturers offer TOF instruments, but they are
less widely used than magnetic sector and quadrupole mass spectrometers.

FRAGMENTATION ( NOT VERY IMPORTANT FOR EXAM)

Molecular fragmentation patterns can be categorised into the following types

a. Homolytic and heterolytic alpha-cleavage

b. Cleavage with proton transfer
c. Fragmentation of Ring Structure

Homolytic and heterolytic alpha-cleavage

Cleavage with proton transfer

Fragmentation of ring structure

It is a complex type of fragmentation which involves rearrangement of the structure of a

molecule before fragmentation and this is more likely to occur if the molecule has a ring in
the structure.
 NMR SPECTROSCOPY
SYLLABUS

Principles of H-NMR and C-NMR, chemical shift, factors affecting chemical shift, coupling constant,
Spin - spin coupling, relaxation, instrumentation and applications

PRINCIPLE
1. Any spinning charged body (nucleus) generates a magnetic field thereby behaving as a
small bar magnet. When an external magnetic field is applied to the bar magnet (nucleus),
it can either align with the external field (same direction) i.e. the lower energy state or
opposed to the external field (opposite direction). Under the influence of an external
magnetic field, a magnetic nucleus can take up different orientations with respect to that
field; the number of possible orientations is given by (2I + 1), so that for nuclei with spin ½
(1H, 13C,) only two orientations are allowed.
2. Along with aligning itself in the above-mentioned way the nucleus also shows a waltz-like
motion known as precessional motion. The angular velocity of this motion ω0, in radians
per second, is given by
ω0=γB,
where B is the magnetic field. γ is the gyromagnetic ratio.

3. The precessional frequency is directly proportional to the strength of the external magnetic
field. The angular velocity (stated above) can be converted to the frequency of precession
v0, known as the Larmor frequency, by dividing by 2π

4. If a proton is precessing in the aligned orientation, it can absorb energy and pass into the
opposed orientation [see the diagram]; subsequently it can lose this extra energy and relax
back into the aligned position. If we irradiate the precessing nuclei with a beam of
radiofrequency energy of the correct frequency [from the external source], the low-energy
nuclei may absorb this energy and move to a higher energy state.
5. The precessing proton will only absorb energy from the radiofrequency source if the
precessing frequency is the same as the frequency of the radiofrequency beam; when this
occurs, the nucleus and the radiofrequency beam are said to be in resonance; hence the
term nuclear magnetic resonance.

SUMMARY

The simplest NMR experiment consists in exposing the proton (H-NMR) in an organic molecule to
a powerful external magnetic field; the protons will precess, although they may not all precess at
the same frequency. We provide these precessing protons with radiofrequency energy of the
appropriate frequencies (external source), and promote protons from the low-energy (aligned)
state to the high-energy (opposed) state. We record this absorption of energy in the form of an
NMR spectrum.

RELAXATION
When a nucleus is exposed to radiation of a suitable frequency, absorption occurs because of the
slight excess of lower-energy state nuclei present in the strong magnetic field. This excess is
small, so there is always danger that the absorption process will equalize the number of nuclei in
the two states and cause the absorption signal to decrease and to approach zero. When this
occurs, the spin system is said to be saturated. To reduce saturation and produce a readily
detectable absorption signal, relaxation should occur as rapidly as possible; that is, the lifetime of
the excited state should be small. A second factor—the inverse relationship between the lifetime
of an excited state and the width of its absorption line—negates the advantage of very short
lifetimes. Thus, when relaxation rates are high, or the lifetimes low, line broadening prevents
high-resolution measurements.

These two opposing factors cause the optimal half-life for an excited species to range from about
0.1 to 10 s. Two types of relaxation processes are important in NMR spectroscopy: (1) spin-
lattice, or longitudinal, relaxation and (2) spin-spin, or transverse, relaxation.

CHEMICAL SHIFT

[1st Part for Understanding]

The spectra for ethyl alcohol, shown in Figure 19-

12, illustrate two types of environmental effects.
The spectrum in Figure 19-12a, obtained with a
lower-resolution instrument, shows three proton
peaks with areas in the ratio 1:2:3 (left to right).
the small differences that occur in the resonance
frequency of the proton depend on the group to
which the hydrogen atom is attached. This effect
is called the chemical shift. The breaking of the
peaks in Figure 19-12b, is the spin-spin splitting
phenomenon (discussed later in detail)

The chemical shift is caused by small magnetic

fields generated by electrons as they circulate
nuclei. These fields usually oppose the applied
field. As a consequence, the nuclei are exposed
to an effective field that is usually somewhat smaller than the external field and is determined by
the electron density and its spatial distribution around the nucleus (shielding effect). The
shielding constant for protons in a methyl group is larger than the corresponding constant for
methylene protons, and it is even smaller for the proton in an iOH group. For an isolated
hydrogen nucleus, the shielding constant is zero. Thus, to bring any of the protons into resonance
at a given frequency n, it is necessary to apply a stronger electric field to overcome shielding.
Alternatively, if the applied field is held constant, the frequency must be increased to bring about
the resonance condition.

Theory (for exam)-

Chemical shifts arise from the secondary magnetic fields produced by the circulation of electrons
in the molecule. These so-called local diamagnetic currents are induced by the fixed magnetic
field and result in secondary fields that may either decrease or enhance the field to which a
given proton responds. Under the influence of the magnetic field, electrons bonding the proton
tend to precess around the nucleus in a plane perpendicular to the magnetic field. A
consequence of this motion is the development of a secondary field, which opposes the primary
field, analogous to what happens when electrons pass through a wire loop. The nucleus then
experiences a resultant field that is smaller, so the nucleus is said to be shielded from the full
effect of the primary field. As a consequence, the external field must be increased to cause
nuclear resonance.

Measurement of Chemical shift (for exam)

To measure the precessional frequency of a group of nuclei in ab solute frequency units is not
difficult but is rarely required. More commonly th e differences in frequency are me asured with
respect to some reference group of nuclei. For protons and C13, the universally accepted
reference is tetramethylsilane, TMS:

TMS is chosen because it gives an intense sharp signal even at low concentrations (having 12
protons in magnetically equivalent positions) ; the signal ari ses on the NMR spectrum well clear
of mo st common organic protons; ; it is chemically inert and has a low boiling point, so that it is
easily removed from a recoverable sample of a valuable organic compound ; it is soluble in most
organic solvents, and can be added to the sample solution (0 .01-1.0 per cent) as an internal
standard.

In recording an NMR spectrum, we measure the differences in chemical shift position between
the reference (TMS) and the signals from the compound being examined. By international
convention, an NMR spectrum is always plotted with TMS on the right-hand side, at low
frequency, and with high frequency on the left-hand side.

Chemical shift positions are normally expressed in delta units, which are defined as proportional
differences, in parts per million (ppm), from an appropriate reference standard (TMS in the case
of proton and carbon-13 NMR). Since the delta unit is a proportionality, it is a dimensionless
number so doesn’t vary from instrument to instrument
Factors affecting chemical Shift
1. Electronegativity- Shielding and Deshielding

Hydrogen nuclei are surrounded by electron density, which to some extent shields the nucleus
from the influence of the applied field Bo, and the extent of this shielding will influence the
precessional frequency of the nucleus-the greater the shielding effect, the lower the
precessional frequency. In a magnetic field the electrons around the proton are induced to
circulate, and in so doing they generate a small secondary magnetic field, which acts in
opposition (that is, diamagnetically) to the applied field. The greater the electron density
circulating around the proton, the greater the induced diamagnetic shielding effect and the
lower the precessional frequency of the proton. Electronegative groups, such as fluorine in CH3F,
withdraw electron density from the methyl group (inductive effect) and this deshielding effect
means that the methyl protons experience a greater net magnetic field, and, hence, precess with
higher frequency . Since fluorine is more electronegative than chlorine, its deshielding influence
is greater and, hence, the attached protons have higher precessional frequencies (higher delta
values).

Silicon is electropositive, and the opposite effect operates in, for example, TMS; silicon pushes
electrons into the methyl groups of TMS by a +I inductive effect, and this powerful shielding
effect means that the TMS protons come to resonance at low frequency (low delta value, defined
as zero). The effect of charged species on chemical shift values is very marked; protons adjacent
to N+ (as in quaternary ammonium ions, R4N+) are very strongly deshielded (high delta values),
while carbanionic centers act as powerful shielding influences (low delta values).

2. Vanderwaals Deshielding

Ina rigid molecule it is possible for a proton to occupy a sterically hindered position, and in
consequence the electron cloud of the hindering group will tend to repel, by electrostatic
repulsion, the electron cloud surrounding the proton. The proton will be deshielded and appear
at higher delta values than would be predicted in the absence of the effect.

3. Anisotropic Effects

The chemical shift positions (delta) for protons attached to C=C in alkenes is higher than can be
accounted for by electronegativity effects alone. The same is true of aldehydic protons and
aromatic protons, whereas alkyne protons appear at relatively low delta.

The explanation is again collated with the manner in which electrons, in this case 'IT electrons,
circulate under the influence of the applied field . The effect is complex, and can lead to shifts to
higher frequency (downfield shifts , or paramagnetic shifts) or to lower frequency (upfield shifts,
or diamagnetic shifts). In addition, the effects are paramagnetic in certain directions around the
'IT clouds, and diamagnetic in others, so that these effects are described as anisotropic, as
opposed to isotropic (operating equally through space).

• For Alkenes and Carbonyl Compounds

We can divide the space around a double bond into two categories, as shown in figure 3.6.
Deshielding occurs in the cone shaped zones, and in these zones delta values will tend to be
higher. Shielding is found outside the cones and protons in these zones are shielded (lower delta
values).

• For Alkynes

Electron circulation around the

triple bond occurs in such a way
that the protons experience a
diamagnetic shielding effect.
Figure 3.7 shows how this arises,
when the axis of the alkyne
group lies parallel to the
direction of Bo.

• For Aromatic Compounds

In the molecule of
benzene (and aromatic
compounds in general) 1T
electrons are delocalized
cyclically over the
aromatic ring . These loops
of electrons are induced to
 circulate in the presence of the applied field, Bo, producing a substantial electric current, called
the ring current. The magnetic field associated with this electric field has the gedmetry and
direction shown in figure 3.8. The induced field is diamagnetic (opposing Bo) in the center of the
ring , but the returning flux outside the ring is paramagnetic (augmenting Bo).

Correlation table (no need to remember but just for reference)

Spin-Spin Coupling and Spin-Spin Splitting

Splitting of the spectral lines arises because of a coupling interaction between neighbouring protons,
and is related to the number of possible spin orientations that these neighbours can adopt. The
phenomenon is called either spin-spin splitting or spin-spin coupling.

Rules of Splitting

The signal from proton Ha appears as a triplet, while that from protons Hx is a doublet.

1. The number of lines (multiplicity) observed in the NMR signal for a group of protons is not
related to the number of protons in that group; the multiplicity of lines is related to the
number of protons in neighbouring groups. For example, protons Hx in figure 3.12 have only
one neighbouring proton, and Hx appears as a two-line signal (doublet); proton HA has two
neighbours and the signal is split into three lines (triplet).
2. (n + 1) rule. The simple rule is: to find the multiplicity of the signal from a group of protons,
count the number of neighbours (n) and add 1.
The diagram in Figure 3.13 represents two vicinal protons similar to the alkene protons in cinnamic
acid, HA and Hx. These protons, having different magnetic environments, come to resonance at
different positions in the NMR spectrum; they do not give rise to single peaks (singlets) but doublets.
The separation between the lines of each doublet is equal: this spacing is called the coupling
constant, J

Explanation of
Doublet: The
resonance position for
A depends on its total
magnetic environment;
part of its magnetic
environment is the
nearby proton X, which
is itself magnetic, and
proton X can have its
nuclear magnet either
aligned with proton A
or opposed to proton
A. Thus, proton A can
either increase the net
magnetic field
experienced by A (X
aligned) or decrease it
(X opposed); in fact, it
does both. The two
spin orientations of X
create two different magnetic fields around proton A: in roughly half of the molecules the spin
orientation of X creates a shielding field around proton A, and in the other half a deshielding field.
Therefore, proton A comes to resonance, not once, but twice, and proton A gives rise to a doublet.
Similarly, proton A is a magnet having two
spin orientations with respect to X, and A
creates two magnetic fields around X. Proton
X comes to resonance twice in the NMR
spectrum.

Explanation of Triplet (refer to left image)

When proton A 'sees ' the two neighbouring

protons X and X', A can 'see' three different
possible combinations of spin: (1) the
nuclear spins of X and X' can both be parallel
to A; (2) both can be antiparallel to A; : (3)
one can be parallel and the other antiparallel
and this can arise in two ways-X parallel with
X' antiparallel or X antiparallel with X'
parallel. Three distinct energy situations, (1),
(2) and (3), are created, and therefore
proton A gives rise to a triplet. The
probability of the first two energy states arising is equal, but since the third state can arise in two
different ways, it is twice as likely to arise; the intensity of the signal associated with this state is
twice that of the lines associated with the first two states, and we see in the spectrum of 1,1,2-
trichloroethane that the relative line intensities in the triplet are 1:2:1.

Coupling constant
In Nuclear Magnetic Resonance (NMR) spectroscopy, the coupling constant, often denoted as J, is
the measure of the distance between the split lines (or peaks) in the NMR spectrum. This splitting is
a result of the spin-spin interaction between adjacent, non-equivalent NMR-active nuclei. The value
of the coupling constant gives us information about the strength of this interaction.

The coupling constant in NMR spectra can also be defined as a measure of the magnetic interaction
between neighbouring, non-equivalent NMR-active nuclei. It is expressed in hertz (Hz) and reflects
the strength of this interaction. Here are the key points about coupling constants:

• Magnetic Interactions: Coupling constants arise from the magnetic interactions between
neighbouring nuclei in a molecule.

• Measured in Hz: The value of J is measured in hertz and does not depend on the frequency
of the NMR machine or the solvent used.

• Splitting of Signals: It causes the splitting of NMR signals into multiple sub-peaks, known as
multiplets (e.g., doublets, triplets).

• Structural Information: The magnitude and pattern of splitting provide valuable information
about the molecular structure, such as the number and arrangement of neighboring
protons1.

INSTRUMENTATION

There are two types of Instruments CWNMR AND FTNMR

1. The Continuous Wave Nuclear Magnetic Resonance (CW-NMR) spectrometer

operates on the principle of field sweep, where the magnetic field strength is varied
while the radiofrequency (RF) signal remains constant. Here’s a detailed explanation
of its instrumentation:
 • Magnet: The instrument uses a powerful magnet to create a strong and uniform
magnetic field, which is essential for the alignment of nuclear spins.
• Sweep Coils: These are variable electromagnet coils that adjust the magnetic field
strength across the sample, allowing for the resonance condition to be met for
different nuclei at different times.
• Radio Frequency Source: A constant RF signal is applied to the sample. This RF
signal has a frequency that matches the Larmor precession frequency of the nuclei
being observed.
• Sample: The sample is placed in a sample holder or tube, which is then situated
within the magnetic field.
• Detector: As the magnetic field is swept, nuclei in the sample come into resonance
and absorb energy from the RF source. This absorption is detected and converted into
an electrical signal.
• Recorder: The electrical signal from the detector is recorded as a spectrum, with
peaks corresponding to different nuclear environments in the sample.

Continuous Wave (CW) Mode: In CW-NMR, each transition is induced in succession by a

continuous scan from low field to high field, with the RF signal remaining constant1. This
mode requires several minutes to record a spectrum.

CW-NMR is characterized by its simplicity and was widely used before the advent of Fourier
Transform NMR (FT-NMR), which allows for faster data acquisition and better sensitivity.
CW-NMR is still useful for certain applications, particularly in educational settings and for
analyzing simple spectra. It provides valuable information about the chemical environment of
nuclei, such as hydrogen and carbon, within a molecule.

2. Pulsed or FT-NMR

The instrumentation of Fourier

Transform Nuclear Magnetic Resonance
(FTNMR) spectroscopy involves several
key components:

• Magnet: The core of an FTNMR

instrument is a powerful magnet,
typically a superconducting magnet,
which generates a strong and uniform
magnetic field. This field is necessary to
align the nuclear spins of the sample.
• Sample Probe: A sample probe
holds the sample and is placed within the
magnetic field. It contains a
radiofrequency (RF) coil that can both
transmit RF pulses to excite the nuclear
spins and receive the resulting NMR
signal.
• RF Transmitter: The RF
transmitter generates the precise RF
pulses required for the specific NMR
experiments being conducted. These
 pulses are sent to the probe to excite the sample.
• RF Receiver: After the RF pulses are applied, the nuclei in the sample emit an NMR
signal. The RF receiver detects this signal, which is then digitized for processing.
• Computer and Software: A computer equipped with specialized software controls
the instrument, processes the digitized NMR signals using Fourier Transform
algorithms, and converts them into spectra for analysis.
• Fourier Transform: The key feature of FTNMR is the use of Fourier Transform
algorithms to convert the time-domain NMR signals into frequency-domain spectra.
This process allows for the rapid acquisition and processing of data, enabling the
observation of all resonances simultaneously.
• Automation and Data Handling: Modern FTNMR instruments often include
automated sample changers and advanced data handling capabilities, allowing for
high-throughput analysis and sophisticated spectral interpretation.

FTNMR spectroscopy provides detailed information about the molecular structure, dynamics,
and environment of the nuclei within a sample, making it a powerful tool in organic
chemistry and materials science. The technology’s ability to provide high-resolution data
quickly and efficiently has made it the method of choice for many applications in research
and industry.

Advantages of Fourier Transform Nuclear Magnetic Resonance (FTNMR):

• Speed: FTNMR is significantly faster than traditional continuous wave (CW) NMR,
allowing for rapid data acquisition.
• Sensitivity: The technique is highly sensitive, making it possible to detect signals
from samples with very low concentrations.
• Resolution: FTNMR provides high resolution, which is essential for distinguishing
between closely related frequencies and for detailed structural analysis.
• Multipulse Techniques: The use of multipulse sequences in FTNMR can provide
additional information about the sample, such as relaxation times and molecular
dynamics.
• Quantitative Analysis: FTNMR allows for quantitative analysis of sample
components without the need for separate calibration standards.

These advantages make FTNMR a powerful tool for the structural determination and analysis
of organic compounds.

APPLICATION OF NMR

• Molecular Structure Elucidation: NMR spectroscopy is instrumental in determining

the structure of organic molecules by revealing the different magnetic environments
of nuclei within the molecule. It can identify the number and types of atoms in each
environment, their neighboring groups, and the quantity of atoms in each
environment.
• Chemical Shift Analysis: The chemical shift in NMR spectra provides insights into
the electronic environment of magnetic nuclei. Factors like electronegativity, van der
Waals deshielding, and anisotropic effects influence chemical shifts, allowing for the
analysis of molecular structures and interactions.
• Spin-Spin Coupling: NMR spectra exhibit splitting patterns due to spin-spin
coupling, which is the interaction between magnetic nuclei through bonding electrons.
 This splitting provides information about the number of neighboring nuclei and their
distances, aiding in structural analysis.
• Quantitative Analysis: NMR can be used for quantitative analysis by measuring the
area under the signals in the spectrum. This allows for the determination of the
relative number of nuclei contributing to each signal, which is useful in mixture
analysis and concentration determination.
• Dynamic Studies: Variable-temperature NMR enables the study of molecular
dynamics, such as rotational barriers and conformational changes. It helps in
understanding the behavior of molecules under different temperature conditions.
• Multinuclear and Multidimensional NMR: Advanced NMR techniques involve
observing nuclei other than protons, like carbon-13, fluorine-19, and phosphorus-31.
Multidimensional NMR provides more detailed structural information, including
spatial relationships between atoms.
• Medical Applications: NMR is the basis for Magnetic Resonance Imaging (MRI), a
medical diagnostic technique that produces images of soft tissues in the body,
complementing X-ray imaging of bone structures2.

These applications highlight the versatility and power of NMR spectroscopy in both scientific
research and medical diagnostics. <|/tool_invocations|>