5-Spectroscopic techniques & applications

Spectroscopic
Techniques and
Applications
Spectroscopic Techniques and Applications 1

• Matter/ molecules can interact with electromagnetic radiation (light/
photons) to produce a change in the molecular structure.
• Spectroscopy is the study of the interaction between matter and
electromagnetic radiation as a function of the wavelength or frequency of
the radiation.
• A spectrum is simply a chart or a graph that shows the intensity of light
being emitted over a range of energies/ wavelengths.

• Electromagnetic radiation is a form of energy that is transmitted
through space with huge velocity in a straight line unless it is
refracted or reflected.
• It has electric and magnetic components, which oscillate in planes
perpendicular to each other and perpendicular to the direction of
propagation of the radiation.

y
(λ)

Electromagnetic spectrum: It is an ordered arrangement of
electromagnetic radiation over the entire range of wavelength.

• An electromagnetic radiation exhibits dual nature, i.e., it displays both
wave as well as particle characteristics.
• Wave properties of an electromagnetic radiation:
• Wavelength: (λ) It is defined as the distance between two successive
maxima of either electric or magnetic components of the wave.
• Unit: meters (m), centimeters (cm), nanometers (nm), angstrom (A○)
• Wave number: ( ) It is the reciprocal of wavelength.
• Unit: reciprocal of distance (generally cmand-1Applications

Spectroscopic Techniques ) 6
An electromagnetic radiation exhibits dual nature, i.e., it displays both wave as well as particle characteristics.
Wave properties of an electromagnetic radiation:
• Frequency: (v) It is the number of waves that pass through a
fixed point in unit time.
• Unit: cycles per second or Hertz (Hz)
• 1 Hz = 1 cps
• 1 MHz = 103 kHz = 106 Hz

Wave properties of an electromagnetic radiation:
• Velocity:
• Product of wavelength and frequency of the wavelength in the given
medium.
• Wavelength x Frequency = Velocity
λ x v = v
• Velocity of light in a vacuum is denoted by ‘c’ and is (3 x 108 m/s).

• Particle properties of an electromagnetic radiation:
• The electromagnetic radiation consists of a stream of discrete
packets (particles) of pure energy, called photons
• They have definite energy and travel in the direction of
propagation of the radiation beam with the velocity equal to that
of the light. Spectroscopic Techniques and Applications 9

• The energy of the photon is proportional to the frequency of radiation given

𝒉.𝒄
by the relationship: 𝑬 = 𝒉𝝂 = =hc
𝝀
Where
‘E’ is the energy of photon in Joules.
‘v' is frequency of photon, is the wave number
‘h’ is the Planck’s constant (6.624 x 10-34 Joule-second or 6.624 x 10-27 erg-seconds
‘c’ is the velocity of light in vacuum
‘λ’ is the photon's wavelength Spectroscopic Techniques and Applications 10

UV-Visible Spectroscopy
• The technique is useful in the structural determination of organic molecules.
• A molecule absorbs ultraviolet (UV/ VIS) light (or photons of energy from the
UV/VIS region of electromagnetic radiation)
• As a result of the absorption of radiation, a transition of valence electrons in
the molecule occurs from lower to higher electronic energy level, leading to
changes in the different electronic energy level of the molecule.

• In absorption spectroscopy, the intensity of absorption of different
wavelengths is recorded against a range of wavelengths in the uv/vis region.
• The plot thus obtained, is called an absorption spectrum.
• In emission spectroscopy the molecules undergo transition from excited to
ground state. The plot of emission against wavelength is called emission
spectrum.
• The electronic transitions involved in organic molecules, can be:
• σ σ* Transition from bonding into antibonding orbitals
• π π* Transition from bonding into antibonding orbitals
• n σ* Transition from non-bonding into antibonding orbitals,
• n π*. Transition from non-bonding into antibonding orbitals

• Of the possible electronic transitions, the π π* , n σ* and n π*
transitions require energy which falls in uv/vis region.
• Therefore, unsaturated compounds and compounds containing atoms like
nitrogen, oxygen, sulphur or halogens (which have lone electron pairs) only
can be analysed by uv/vis spectroscopy.

λmax
• The wavelength (λmax) of maximum absorption for an electronic
transition is the characteristic of the molecular group involved.
• The intensity of absorption will be dependent on concentration of
compound.
Molar attenuation coefficient

• More the conjugation, more/ higher the λmax (lower energy), hence 1,3 -
butadiene has greater λmax (217 nm) than ethylene (180 nm)
Ethylene 1,3 -butadiene

• Chromophore is the functional group, which is responsible for
imparting colour to a compound. Examples: Groups containing
unsaturated linkage such as >C=C<, >C=O, -N=N- etc.

• Auxochrome is the substituent, attached to the chromophore, which
enhances colour-imparting property of the chromophore but it itself does not
impart colour.
• Examples: >C-OH, >C-NH2 etc.
• Auxochromes have one or more non-bonding pair of electrons.
• Auxochrome helps in extending the conjugation by sharing its non-bonding
pair of electrons. Spectroscopic Techniques and Applications 19

Beer Lambert’s Law: (for absorbing behaviour of solutions in uv/vis range )
When a beam of monochromatic radiation (Electromagnetic radiation of only
one frequency or wavelength) is passed through a solution of an absorbing
substance, the rate of decrease in intensity of transmitted radiation with path
length of the absorbing solution is proportional to the intensity of incident
radiation as well as the concentration of the solution.

Mathematical expression of the Beer Lambert’s Law: (for
absorbing behaviour of solutions in uv/vis range )
𝑰𝟎 𝟏
A = log (𝑰 ) = ɛ c l = log (𝑻)
𝒕

𝑰𝟎 𝟏
A = log ( ) = ɛ c l = log ( )
𝑰𝒕 𝑻
Where ɛ = Absorption coefficient (Molar attenuation coefficient)
I0 = Intensity of incident radiation
It = Intensity of transmitted radiation
A = Absorption
T = Transmittance
c = Concentration absorbing material in mol dm-3
l = Path length in ‘cm’
The law is useful in studying the absorbing behaviour of solutions containing relatively
low concentration (< 0.01M).
Changes in the position and the intensity of absorption:
• Bathochromic shift or red shift: It involves the shift of absorption maxima
(λmax) towards longer wavelength due to presence of auxochromes.
• Bathochromic shift is also produced when two or more chromophores are
present in conjugation in a molecule.
• Secondly, the increase in polarity of the solvent gives rise to
Bathochromic shift. Spectroscopic Techniques and Applications 23

• Bathochromic shift or red shift. Ex.
Compound λmax (nm)

Ethylene 180
1,3-butadiene 217
β-carotene 450
lycopene Spectroscopic Techniques and Applications 471 24

Hypsochromic shift or blue shift: It involves the shift of
absorption maxima (λmax) towards shorter wavelength by
removal of conjugation in a system.

• Hypsochromic shift or blue shift. Ex.
Compound λmax (nm)

Lycopene 471
β-carotene 450
1,3-butadiene 217
Ethylene Spectroscopic Techniques and Applications 180 26

• Hyperchromic shift: This effect involves an increase in the intensity of
absorption which is usually brought about by introduction of an
auxochrome.
• For example, the B-band for pyridine having εmax 2750 is shifted to εmax
3560 for 2-methyl pyridine.

Pyridine 2-methylpyridine
• Introduction of an auxochrome
Spectroscopicusually increases intensity of absorption.
Techniques and Applications 27
• Hypochromic shift: This effect involves a decrease in the intensity of
absorption
• For example, biphenyl absorbs with εmax 19000 whereas 2-methyl biphenyl
absorbs with εmax 10250 (εmax decreases).

Biphenyl 2-methylbiphenyl
• Introduction of the methyl group distorts the geometry of the molecule
thus, causing hypochromic Spectroscopic

effect.Techniques and Applications 28

Nature of Shift Descriptive Term
To Longer Wavelength Bathochromic
To Shorter Wavelength Hypsochromic
To Greater Absorbance Hyperchromic
To Lower Absorbance Hypochromic

Applications of UV spectroscopy:
• Quantitative analysis: The λmax of the compound is initially found. A
calibration curve is constructed to determine the unknown concentration of the
compound.
• Chemical kinetics: As absorbance is directly proportional to the
concentration of the solute, the change in concentration of either a reactant or
product with time is measuredSpectroscopic

to study the
Techniques kinetics of the reaction.
and Applications 31
• Detection of cis and trans isomerism: This technique is advantageous to
distinguish between geometrical isomers (cis and trans isomers) of a
compound as the trans isomer displays longer λmax than the corresponding cis
isomer.
• Geometry of metal complexes: This technique is useful for predicting the
geometry of metal complexes by comparing λmax values of complexes of
unknown geometry with those of complexes with known geometry

Infra Red or IR Spectroscopy/ Rotation-vibrational spectroscopy
• This technique is used in the detection of functional groups in pure
compounds and mixtures.
• Molecules absorb infra red light (or photons of energy from the IR region of
electromagnetic radiation)
• The IR region can be divided into near, mid and far IR radiation. The most
commonly used region are 4000-1000 cm-1 known as the functional group
region, and < 1000 cm-1 is known as theand fingerprint

Spectroscopic Techniques Applications region. 34
IR Spectroscopy / Rotational-vibrational spectroscopy
• IR radiation does not have sufficient energy to cause electronic excitation,
however it causes atoms or group of atoms in a molecule to vibrate faster about
the covalent bonds
• The absorption causes fluctuation in the bond, which increases the vibrational
energy, thus causing an excitation of molecules from a lower to the higher
vibrational level.
• Since each vibrational level is associated with a number of closely
spaced rotational levels, the IR spectra is considered as vibrational-
rotational spectra.
• Different forms of vibrations, displayed by a molecule, depend upon
the functional groups present in the molecule.

IR Spectroscopy
Homonuclear molecules do not possess a net dipole moment and hence
inactive in IR, as energy absorption does not cause any changes in vibrations.
IR Spectroscopy
Non-polar molecules like N2, Cl2 do not absorb in IR region as they
do not have permanent dipole moment and also do not produce a
temporary dipole moment during vibrations.
Homonuclear molecules do not possess a net dipole moment and hence
inactive in IR, as energy absorption does not cause any changes in vibrations.
The fundamental vibrations are of two types:
(1) Stretching vibrations
(2) Bending vibrations
(1) Stretching vibrations: The atoms move along the bond axis so that the
bond length increases or decreases periodically i.e interatomic distance
changes periodically
(1)Stretching vibrations: Stretching vibrations are of two types,
(i)Symmetrical
(ii) Unsymmetrical

Stretching vibrations are of two types:
(i) Symmetrical: in which all the bonds are either
stretched or contracted at the same time.
(ii) Unsymmetrical: in which one of the bonds is
stretched and others are contracted or one of the bonds
is contracted and others are stretched.

(2) Bending vibrations: Here there is a change in bond angle between
bonds with a common atom
Bending vibrations are of two types,
(i) In-plane bending vibrations
(ii)Out of plane bending vibrations

(2) Bending vibrations:
(i)In-plane bending vibrations – these include scissoring and rocking
vibrations.
Scissoring vibrations: The two bonds
move in the opposite directions similar
to the movement of the two blades of a
pair of scissor Spectroscopic Techniques and Applications 43

(2) Bending vibrations:
(i)In-plane bending vibrations – these include scissoring and rocking
vibrations.
Rocking vibrations: The two bonds move in
the same directions.

(ii) Out-of-plane bending vibrations: These include twisting and wagging
vibrations.
Twisting vibrations : The two bonds turn on
opposite sides, i.e., one bond turns on backside
of the plane and the other turns on front side of
the plane

(ii) Out-of-plane bending vibrations: These include twisting and wagging
vibrations.
Wagging vibrations : The bonds turn
out of the mean position but on the same
side of the plane, i.e., both the bonds turn
to either the back side of the plane or the
front side of the plane. Spectroscopic Techniques and Applications 46

The molecules like CO 2 has no permanent dipole moment but unsymmetrical
vibrations give rise to the change in dipole moment. Hence, IR active.

IR Spectroscopy
• IR spectrum can be usually divided into two regions for the purpose of its
interpretation.
• Functional group region which ranges from 4000 cm-1 to 1450 cm-1 and
• Fingerprint region which ranges from 1450 cm-1 to 400 cm-1.
• The functional group region usually consist of fewer peaks associated with
the stretching vibrations of functional groups.

IR Spectroscopy
• The finger print region usually consist of bending vibrations within the
molecule.
• The pattern of peak is much more complex and it is difficult to assign
individual bond in this region.
• The importance of this region is that, every compound has a unique
pattern and hence it is called as the finger print region.

IR Spectroscopy

IR Spectroscopy
Characteristic
Functional Group Type of Vibration Intensity
Absorptions (cm-1)
Alkane -C-H bending 1350-1480 variable
Alkane -C-H stretch 2850-3000 strong
-C-X stretch 500-1400 strong
Alkyne C-H stretch 3300 strong, sharp
Alkyne stretch 2100-2260 variable

IR Spectroscopy
Characteristic
Functional Group Type of Vibration Intensity
Absorptions (cm-1)
3300-3500
N-H stretch medium
N-H bending 1600 medium
-C=O stretch 1670-1820 strong
-C-O stretch 1000-1300 strong

Applications of IR Spectroscopy
• Identification of functional groups: Since most of the functional
groups have characteristic IR absorption frequencies, the functional
groups present in an unknown compound can be identified by
correlating the absorption frequency with the data.

• Study of chemical reactions: IR spectroscopy is suitable to detect
the progress of organic reactions that involve changes in functional
groups. The rate of disappearance of a characteristic absorption band
of a reactant group or the rate of increasing absorption band due to
the formation of product helps in studying the progress of the
reaction. Spectroscopic Techniques and Applications 54

• Detection of cis and trans isomerism: Since trans isomer is more
symmetric than cis isomer, trans isomer has little change in dipole
moment in the presence of IR radiation, showing a simpler
spectrum than cis isomer. Thus, geometrical isomers (cis - trans)
can be easily distinguished by means of IR spectroscopy.

NMR spectroscopy
• Nuclear magnetic resonance: A condition when the frequency of
the rotating magnetic field becomes equal to the frequency of the
processing nucleus
• Hydrogen atoms are able to absorb and emit radio frequency energy
when placed in an external magnetic field.

NMR spectroscopy

NMR spectroscopy
Equivalent and non-equivalent protons:
• The protons with different environments absorb at different applied field
strengths whereas the protons with identical environments absorb at the
same field strength.
• A set of protons with identical environments are known as equivalent
protons while protons with different environments are known as non-
equivalent protons. Spectroscopic Techniques and Applications 58

NMR spectroscopy
Equivalent and non-equivalent protons:
• Examples:
2, 2- Dibromopropane Isopropyl chloride n-Propyl chloride

one set of equivalent protons two sets of equivalent protons three sets of equivalent protons

NMR spectroscopy
Chemical shifts:
• The shifts in the positions of NMR signals (compared with a standard
reference) resulting from the shielding or deshielding by electrons are
referred to as chemical shifts.
• It is expressed as delta (δ) in ppm.
• The compound generally used as reference for proton resonance is
δ scale,
tetramethylsilane (TMS). OnSpectroscopic the position of TMS is taken as 0 ppm.
Techniques and Applications 60
NMR spectroscopy
Chemical shifts:
• Different nuclei in a molecule experience different magnetic fields as a result
of the secondary magnetic fields associated with the molecule.
• Example: The protons in CH3-F (δ = 4.26) are more deshielded than CH3 -Cl
(δ = 3.05) owing to the higher electronegativity of F than Cl. Similarly, in
CH3-CH2-Cl, there is greater shielding of CH3 protons than CH2 protons.

NMR spectroscopy
Upfield and downfield shift:
Upfield shift:
• When the secondary field produced by the circulating electrons around the
nucleus, opposes the applied field, the effective field experienced by the
nucleus is less than the applied field.
• This is known as positive shielding and the resonance position moves
upfield (i.e. toward δ = 0) inSpectroscopic

NMRTechniques
spectrum.
and Applications 62
NMR spectroscopy
Upfield shift:
• The presence of an electron donating group increases electron
density around the proton and thus shields the proton
• Example functional groups such as the hydroxy and the amino
group shield the neighbouring protons.

NMR spectroscopy
Downfield shift:
• When the secondary field produced by the circulating electrons around the
nucleus reinforces the applied field, the effective field experienced by the
nucleus is more than the applied field.
• This is known as negative shielding and the resonance position moves
downfield (i.e. away from δSpectroscopic

= 0) in NMR
Techniques spectrum.
and Applications 64
NMR spectroscopy
Downfield shift:
• The presence of an electron withdrawing group reduces electron
density around the proton and thus deshields the proton.
• Example functional groups such as nitro, carboxyl, carbonyl
groups deshield the neighbouring protons.

NMR spectroscopy
Spin-Spin Coupling or Splitting of Signals:
• The splitting of signal lines in the NMR spectrum due to the
interaction between the spins of the neighbouring nuclei in a
molecule is known as spin-spin coupling.

NMR spectroscopy
Spin-Spin Coupling or Splitting of Signals:
• It occurs as the magnetic field of the protons in a group (which are in the
same environment) is influenced by the spin arrangements of the protons in
the adjacent group (which are in a different environment).
• A signal of a proton with ‘n’ equivalent protons on the neighboring carbon
atoms will split into (n + 1) peaks in the NMR spectrum.

a

a
Acetone
a a

a
Diethyl ketone OR 3-pentanone
a b b a
b

a
Diethyl ether
H3C . CH2 . O . CH2 . CH3

b b a
a b

Applications of NMR spectroscopy
1) Number of signals: The number of main NMR signals are equal to the
number of sets of equivalent protons in a compound, thus giving the
information of the number of sets of equivalent protons present in the
compound.
2) Position of signals: The chemical shift indicates the type of hydrogen atoms
present in a compound, i.e., the electronic environment of protons.

3) Splitting of signals: The spin-spin splitting specifies the arrangement of
atoms in a group.
4) Intensities of signals: Since peak area or intensity is directly proportional to
the number of protons, the number of protons present in each group of a
compound can be revealed from peak area.

• Magnetic resonance imaging is a medical application of nuclear magnetic
resonance (NMR).
• Hydrogen atoms are naturally abundant in humans and other biological
organisms, particularly in water and fat.
• Magnetic Resonance Imaging (MRI) helps to visualize the structures of the
body that include water and fat molecules.

• MRI does not involve X-rays or the use of ionizing radiation,
which distinguishes it from CT or CAT scans.
• The technique is used in Neuroimaging, Cardiovascular,
Musculoskeletal, Liver and Gastrointestinal imaging,
Angiography etc.

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Spectroscopic

Spectroscopic Techniques and Applications 1

photons) to produce a change in the molecular structure.

• Spectroscopy is the study of the interaction between matter and

electromagnetic radiation as a function of the wavelength or frequency of

• A spectrum is simply a chart or a graph that shows the intensity of light

being emitted over a range of energies/ wavelengths.

through space with huge velocity in a straight line unless it is

• It has electric and magnetic components, which oscillate in planes

perpendicular to each other and perpendicular to the direction of

propagation of the radiation.

Spectroscopic Techniques and Applications 4

electromagnetic radiation over the entire range of wavelength.

Spectroscopic Techniques and Applications 5

wave as well as particle characteristics.

• Wave properties of an electromagnetic radiation:

• Wavelength: (λ) It is defined as the distance between two successive

maxima of either electric or magnetic components of the wave.

• Unit: meters (m), centimeters (cm), nanometers (nm), angstrom (A○)

• Wave number: ( ) It is the reciprocal of wavelength.

• Unit: reciprocal of distance (generally cmand-1Applications

Wave properties of an electromagnetic radiation:

• Frequency: (v) It is the number of waves that pass through a

fixed point in unit time.

• Unit: cycles per second or Hertz (Hz)

• 1 MHz = 103 kHz = 106 Hz

Wave properties of an electromagnetic radiation:

• Product of wavelength and frequency of the wavelength in the given

• Wavelength x Frequency = Velocity

• Velocity of light in a vacuum is denoted by ‘c’ and is (3 x 108 m/s).

• Particle properties of an electromagnetic radiation:

• The electromagnetic radiation consists of a stream of discrete

packets (particles) of pure energy, called photons

• They have definite energy and travel in the direction of

propagation of the radiation beam with the velocity equal to that

of the light. Spectroscopic Techniques and Applications 9

• The energy of the photon is proportional to the frequency of radiation given

‘E’ is the energy of photon in Joules.

‘v' is frequency of photon, is the wave number

‘c’ is the velocity of light in vacuum

‘λ’ is the photon's wavelength Spectroscopic Techniques and Applications 10

• The technique is useful in the structural determination of organic molecules.

UV/VIS region of electromagnetic radiation)

• As a result of the absorption of radiation, a transition of valence electrons in

changes in the different electronic energy level of the molecule.

• In absorption spectroscopy, the intensity of absorption of different

wavelengths is recorded against a range of wavelengths in the uv/vis region.

• The plot thus obtained, is called an absorption spectrum.

• In emission spectroscopy the molecules undergo transition from excited to

ground state. The plot of emission against wavelength is called emission

• The electronic transitions involved in organic molecules, can be:

• σ σ* Transition from bonding into antibonding orbitals

• π π* Transition from bonding into antibonding orbitals

• n σ* Transition from non-bonding into antibonding orbitals,

• n π*. Transition from non-bonding into antibonding orbitals

Spectroscopic Techniques and Applications 13

transitions require energy which falls in uv/vis region.

• Therefore, unsaturated compounds and compounds containing atoms like

can be analysed by uv/vis spectroscopy.

5-Spectroscopic techniques & applications

5-Spectroscopic techniques & applications

5-Spectroscopic techniques & applications