Unit -II

SPECTROSCOPIC TECHNIQUES
AND
APPLICATIONS
Introduction

Spectroscopy is the branch of science dealing with the study of interaction of electromagnetic radiation
with matter like atoms and molecules. The interaction of EMR with matter gives rise to two types of spectra
namely atomic spectra and molecular spectra.

Atomic spectrum arises from the transition of electrons from one energy level to another due to changes
of energy in the atom.

Molecular spectrum involves transition of electrons between rotational and vibrational energy levels in
addition to electronic transition. Therefore molecular spectrum is much more complicated than the atomic
spectrum.

Molecular Spectroscopy provides a clear image of how diatomic and polyatomic molecules interact by
looking at the Frequency, Wavelength, Wave number, Energy, and molecular process also provides most useful
information regarding the shape and size of molecules, the bond angles, bond lengths, strength of bonds and bond
dissociation energies.

Hence molecular spectroscopy is of great use in determining the structure and constitution of compounds.

Electromagnetic Radiations (EMR)

EM radiation is created when a subatomic particle, such as an electron, is accelerated by an electric field. The
movement produces oscillating electric and magnetic fields, which travel at right angles to each other in a bundle
of light energy called a photon. Photons travel as harmonic waves at the fastest speed possible in the universe:
186,282 miles per second (299,792,458 meters per second) in a vacuum, also known as the speed of light.

The EM waves are characterized by frequency, wavelength, wave number and energy.

Electromagnetic (EM) radiation is a form of energy that is all around us and takes many forms, such as radio
waves, microwaves, X-rays and gamma rays.

Visible light is only a small portion of the EM spectrum, which contains a broad range of electromagnetic
wavelengths.
Electromagnetic waves are formed when an electric field (shown in red arrows) couples with a magnetic field
(shown in blue arrows). Magnetic and electric fields of an electromagnetic wave are perpendicular to each other
and to the direction of the wave.

The four main electromagnetic interactions:

The force of attraction or repulsion between electric charges is inversely proportional to the square of the distance
between them.

Magnetic poles come in pairs that attract and repel each other, similar to that of electrical charges.

An electric current in a wire produces a magnetic field, the direction of which depends on the direction of the
current.

A moving electric field produces a magnetic field, and vice versa.

Characterization of EMR

Wavelength (λ) : It is the distance between two consecutive peaks of a wave (crests). Unit = m. Frequency (√):

It is the number of waves that are formed in a given length of time. Unit =
number of wave cycles per second or hertz (Hz).

A short wavelength means that the frequency will be higher and a longer wavelength has a lower frequency.

Wavenumber (ῡ ): It is the number of waves per unit distance. Unit = cm-1 ῡ = 1/λ

Energy of EMR (E):Electromagnetic radiations consists of particles having small packets of energies called
quanta or photons. Photons possess the characteristic of wave and travel with the speed of light. The amount of
energy corresponding to one photon is expressed by Planck’s equation as

E = hν or E = ℎ
where h = Planck’s constant (6.62x10-34Js) ν -

frequency in Hz

λ - wavelength in cm/m.

Energy (E) = hc/λ E

= h√

E = hcῡ

Interaction of EMR with matter

EMR interacts with matter only when the matter has some electric and magnetic effect and are influenced
by the electric and magnetic components of the EM radiation.

The net change in the electric/magnetic dipole moment in the molecule or nuclear spin, interact with the
magnetic/electrical component of the EMR by either absorption or emission of the EMR.

Total energy of molecules = Translational + rotational + vibrational + electronic Absorption or

emission of EMR causes a change in any of these types of energies. In molecular spectroscopy,

we measure the change in these energy states.

Translational energy – It is due to the overall movement of the molecule. Energy levels are not quantized .
Hence no spectroscopy.

Rotational energy – It is due to spinning of molecules about the axis passing through the centre of gravity -
Rotational Levels are quantized – Rotational spectroscopy (Microwave spectroscopy)
Vibrational energy – It is due to vibrations in molecules – Vibrational Levels are Quantized – IR Spectroscopy
(Vibrational spectroscopy)

Electronic energy – Consists of electronic levels which are quantized – UV/visible spectroscopy (Electronic
spectroscopy)

If E is the total energy of a molecule, it can be expressed as the sum of translational, rotational, vibrational
and electronic contributions. E = Etrans +Erot + Evib + Eelec

Spectra

Molecular spectra result from either the absorption or the emission of electromagnetic radiation as molecules
undergo changes from one quantized energy state to another.

• The electrons in a molecule possess kinetic energy due to their motions and potential energy arising
from their attraction by the positive nuclei and their mutual repulsion. These two energy factors, along
with the potential energy due to the mutual electrostatic repulsion of the positive nuclei, constitute the
electronic energy of a molecule.
• Molecules are not rigid structures, and the motion of the nuclei within the molecular framework gives
rise to vibrational energy levels.
• In the gas phase, where they are widely separated relative to their size, molecules can undergo free
rotation and as a result possess quantized amounts of rotational energy.
• In theory, the translational energy of molecules through space is also quantized, but in practice the
quantum effects are so small that they are not observable, and the motion appears continuous.
 • The interaction of electromagnetic radiation with these molecular energy
levels constitutes the basis for Electronic, IR and Microwave spectroscopy.

Absorption and Emission Spectra

Absorption of electromagnetic radiation by compounds gives absorption spectrum and spectrum obtained by the
emission of absorbed radiation is called emission spectrum.

Ground level/state is the lowest energy state. Higher energy levels/states are called excited states.

QUANTIZATION OF ENERGY LEVELS

Relative Population : Boltzmann Distribution Formula Relative

population in different energy levels is given by N2/N1 N2 = number of

molecules in the higher energy state E2

N1 = number of molecules in the lower energy state E1

N2/N1 = e - (E2 – E1)/kT

k = Boltzmann constant, T = temperature

Thermodynamic equilibrium condition: N1 > N2 ; There is observed absorbance of electromagnetic radiation

For emission spectra : N2 > N1 ; There is population inversion

Saturation condition : N1 = N2 ; There is no absorption or emission of radiation

Selection Rule in Spectroscopy

Selection rules describe the allowed transitions between energy states in quantum systems. They are
determined by which final states are accessible given an initial state and perturbing potential.

Atomic and molecular species contain a very large number of states in which energy can be distributed,
although generally only the states lying lowest in energy will be thermally populated.

If electromagnetic radiation could effectively stimulate transitions between any one of these states,
atomic and molecular spectra would be complex.

Selection rules, limit the possible transitions and render these spectra amenable to analysis. Some transitions

are “allowed” by the selection rules, while others are “forbidden”.

• The selection rules may differ according to the technique used to observe the transition.
• In quantum mechanics the basis for a spectroscopic selection rule is the value of the transition
moment integral
 • where Ψ1 and Ψ2 are the wave functions of the two states, "state 1" and "state 2", involved in the
transition, and µ is the transition moment operator. This integral represents the propagator (and
thus the probability) of the transition between states 1 and 2; if the value of this integral is zero then
the transition is “forbidden".
• In practice, to determine a selection rule the integral itself does not need to be calculated: It is sufficient
to determine the symmetry of the transition moment function. If the transition moment function is
symmetric over all of the totally symmetric representation of the point group to which the atom or
molecule belongs, then the integral's value is (in general) not zero and the transition is allowed.
Otherwise, the transition is “forbidden”.
• The transition moment integral is zero if the transition moment function, is anti-symmetric or odd.

Width of Spectral lines

Spectral lines are broadened because of two reasons:

1. Energy levels are not sharp.

2. Atoms are moving relative to observer.

Three mechanisms determine the line profile –

1. Quantum mechanical uncertainty in the energy E of levels with finite lifetimes. Determines the natural
width of a line (generally very small).
2. Collisional broadening: Collisions reduce the effective lifetime of a state, leading to broader lines. High
pressure gives more collisions (eg stars).
3. Doppler or thermal broadening, due to the thermal (or large-scale turbulent) motion of individual
atoms in the gas relative to the observer.

Signal-to-Noise Ratio (SNR)

The ability of the spectrometer to make accurate measurements depends on the quality of the signal obtained
from the detector and the subsequent electrical circuits. The signal-to-noise ratio (SNR) provides a measure of the
signal quality. The SNR compares the average power available in
the signal to the average power contained in the noise, which includes any signal from sources other than the
target signal source. In a spectrometer, the desired signal consists of the optical power at a given wavelength
directed by the diffraction grating (and by the DMD, in a DLP-based system) to the detector. The noise signal
arises from a number of sources, both electrical and optical.

In order to improve the SNR in a spectrometer, the design choices must increase the power in the measurement
signal while at the same time minimize the noise sources as much as possible.

• Thermal energy provided by ambient heating generates the additional carriers that contribute to the noise
current.
• Additional electronic noise, sometimes referred to as the readout noise, arises from the circuitry directly
behind the detector that provides the initial filtering and scaling of the signal.
• Fixed pattern noise arises from the variation in the response to incident light of the detectors in a detector
array. The variation originates primarily from differences in quantum efficiency caused by differences in
the aperture area and the thickness of the detectors that occurred during fabrication. Only spectrometers
employing a linear detector array for discriminating between wavelengths suffers from this source of
noise.

Improving SNR

Several methods of spectrometer design and measurement, based on the nature of the noise sources, can improve
the SNR of the spectrometer and lead to higher quality measurements.

• The use of holographic gratings, which have much fewer imperfections than ruled gratings, can reduce
the stray light generated by the optical system. For this reason, holographic gratings are commonly used
in UV spectrometers that suffer signal losses in the optical system due to absorption and are thus more
susceptible to noise from stray light.
• A thermo-electric cooler (TEC) attached to any detector reduces the effective temperature of the detector
and thus can reduce the impact of on the SNR.
Question Bank
Part A

1. What is electromagnetic radiation?

2. Mention the important characteristics of electromagnetic radiation.

3. Define wavelength, frequency and wavenumber.

4. Give the equation relating energy and frequency of EMR.

5. Relate energy and wavelength of EMR.

6. What are the different quantized energy levels?

7. Compare absorption and emission spectra.

8. What is SNR in spectroscopic techniques?

9. List out the important conditions for spectral line broadening.

10. Give any two methods of improving SNR.

11. Give the expression for Boltzmann Distribution Law.

Part B

1. Explain EMR and how are they characterized?

2. Elaborate on energy level quantization and the different spectroscopic method of analysis.

3. Write short note on Boltzmann Distribution Law and give its significance.

4. Discuss the signal to noise ratio related to spectroscopy.

5. Explain the various factors causing spectral line broadening.

6. Elaborate on selection rule in spectroscopic method of analysis.

Electromagnetic Spectrum– UV-Visible range

The UV-Visible range in the electromagnetic spectrum is from 200 – 800nm. The range 200 – 400 nm is the UV
range and from 400 – 800 nm is the Visible range.

The electronic transitions are accompanied by vibrational and rotational transitions. Therefore the electronic
absorption bands are broad in the spectrum.

Characteristics of electronic transitions

• Electronic transitions are appeared as broad bands due to the accompanying vibrational and rotational
transitions.

• During electronic transition there should be change in the dipole moment of the molecule (i.e. Ground
state and Excited state dipole moment should not be the same)

• During electronic transition change in angular momentum should be zero or +/- 1

Types of Electronic Transitions

Examples of molecules exhibiting various electronic transitions

Types of electronic transitions

• σ → σ * transition: The energy required for this transition is high (i.e. very short wavelengths; 150nm).
The saturated alkanes will undergo this type of transition.
• n → σ * transition: The saturated hydrocarbons attached to hetero atoms will undergo this type of
transition. Example: Alcohols, amines, ether and water. The energy required for this transition is 180-190
nm.
• π → π * transition: The unsaturated hydrocarbons, carbonyl compounds, cyanides and azo compounds
will undergo this type of transition around 250 nm. Ex: Alkenes, Alkynes, Aldehyde and Ketone. The
energy required for this transition is very low (i.e. very longer wavelengths).
• n → π * transition: The carbonyl compounds will undergo this type of transition around 275 nm. Ex:
Aldehydes, Ketone and cyanide.
Differences between π → π * and n → π * transitions

S.No. π→π* n→π*

1 Allowed transition Forbidden transition

2 High energy transition Lower energy transition

3 Molar extinction coefficient (ε) Molar extinction coefficient (ε)

value lies between 100 to 10000 value is < 100

4 More intense than n → π * More intense than π → π *

λmax is the wavelength corresponding to maximum absorbance and εmax is the molar absorption coefficient
which is a constant for a particular molecule and proves the identity of the molecule.
Effect of Substitutents on Absorption Spectra

• Bathochromic shift :- Absorption maximum of a compound shift to longer wavelength, it is known as

Bathochromic shift or red shift.

• Hypsochromic shift:- Absorption maximum of a compound shifts to a shorter wavelength it is called a

Hypsochromic shift or Blue shift.

• Hyperchromic shift :- Intensity of the absorption band increases.

• Hypochromic shift :- Intensity of the absorption band decreases.

UV-Visible spectroscopy – Instrumentation

A spectrophotometer is an instrument which uses monochromatic light. It measures the absorbance of various
solutions at different wavelenths. This instrument scans the entire UV-visible regions.

In a double beam spectrophotometer, radiation from hydrogen lamp or tungsten lamp enters the monochromator, which
produces very narrow band widths. The beam is then passed through a
reference cell and the sample cell by means of rotating mirrors. The photomultiplier tube is used as a detector
which receives alternate pulses of radiation from the reference and sample beam.

Sources: Commonly used sources of UV radiation are the hydrogen lamp and the deuterium lamp. Tungsten
filament lamp is used for the visible range.

Filters and Monochromators: Tinted glass filters are used to produce monochromatic radiations. Filters resolve
polychromatic light into a relatively wide bandwidth to produce monochromatic radiations. Monochromator is a
device which resolves polychromatic radiation into its individual wavelength and isolates them into very narrow
bands.

Prism: A prism disperses polychromatic light from a source into its constituent wavelengths. Cornu quartz and
littro prisms are used. Glass prisms are used for visible range while silica, fused silica or quartz prisms are used
for the UV range.

Sample Holder (Cells / Cuvette) : The sample container should be transparent to the UV-visible radiation.
Cuvettes are made ordinary glass or quartz. Fused silica cells are used for the UV range. The path length of these
cuvettes is usually 1 cm.

Solvents: A solvent is selected in such a way that it does not absorb in the UV-visible range. The solvents that are
frequently used are water, methanol, ethanol, hexane, chloroform etc.

Detectors: The commonly used detectors are photocells, phototubes and photomultiplier tubes.

Amplification and Read out: The transmitted radiation is converted into electrical signal and the electrical
signals are interpreted using ammeters, amplifiers and potentiometers.

Mathematical Derivation of Beer- Lambert’s Law:

When a beam of monochromatic radiation passes through a transparent absorbing medium, the rate of decrease of
intensity of radiation with the thickness of the absorbing medium is proportional to the intensity of the incident
radiation and concentration of the medium.

dI
  cI dx

dI
where  = decrease of intensity of radiation with the thickness of the absorbing medium
dx

c = concentration of the absorbing medium

dI = Change in intensity of incident light (range is from I0 to I)

 dx = Change in thickness of medium (range is from 0 to b)

dI
  cdx I

 Molar extintion coefficient

dI
  cdx
I

I xb
dI
    cdx
Io I x0

log[I]II  c[x]b 0
o

log I  log Io  bc

I
log  bc
I
o

T  bc , T  Transmittance

I
log o  bc
I

A  bc , A  Absorbance or Optical density

Transmittance (T) : It is the fraction of the incident light that is transmitted by a sample. T = I/Io Absorbance

(A) : It is the negative logarithm to the base 10 of the transmittance of the solution. A = -log T (no unit)

Molar absorptivity (ε): It the absorbance of a one molar solution placed in a cell of one cm path length. A =
εbc (unit = dm3mol-1cm-1)

Chromophore

It is a covalently unsaturated group which is responsible for absorption of UV or visible radiation and may or may
not have an impact on the colour to the compound.
A compound which contains chromophore it is called chromogen. In unsaturated linkage such as – C=C-,-N=N-,
the π electron are loosely bound. These loosely bound electron required less energy for electronic transition and
the absorption band occur in near UV region.

Example: Acetylene possess -C=C- in structure its λmax is 175-180 nm.

Auxochrome

It is saturated and unsaturated group which consists of one or more pair of non-bonded electron. This group
when attached to a Chromophore help in altering the wavelength by increasing the intensity of absorption and
increases λmax.

Examples of Auxochrome are: -OH, -NH2, -OR etc.

Problems and Solutions

Problem 1

If the transmittance of a solution is 19%, what is its absorbance or optical density? Solution
%T = 19.4
T = 0.194
Absorbance = - log T
= log 1/T
= 0.712

Problem 2

A solution of a transmittance of 20% when taken in a cell of 2.5cm thickness. Calculate its concentration if the
molar absorption coefficient is 12,000 dm2mol-1cm-1

Solution:

log 1/T = εbc T = I/Io

%T = 20, T = 0.2, b = 2.5 cm log T = log I/Io ε

= 12000 dm3mol-1cm-1 - log T = log Io/I

log 1/0.2 = 12000 x2.5xc - log T = εbc c

= 2.33 x 10-5 mol dm-3

Problem 3: A solution of thickness 2 cm transmits 40% incident light. Calculate the concentration of the solution
given that ε = 6000 dm3mol-1cm-1

Log 1/T = εbc

%T = 40 , T = 0.4 , b = 2 cm

Log 1/0.4 = 6000 x 2 x c C =

3.31x10-5 mol/dm3

Problem 4: The transmittance of a 2x10-4 M solution of a substance was found to be 76.2% at a wavelength of
360nm, when placed in a cell of 1 cm length. Calculate A and ε.

%T = 76.2 , T = 0.762

A = -log T or A = log 1/T A

= log 1/0.762 = 0.118

A = εbc or ε = A/bc ε

= 0.118/1 x 2x10-4

ε = 0.059 x104 dm3mol-1cm-1

Problem 5: The molar extinction coefficient of a solute is 1.4x104 dm3mol-1cm-1. If a solution of a substance has
an absorbance of 0.85, in a cell of 1cm path length, calculate T and c of the solution.

Log 1/T = A = 0.85

1/T = 7.0794

T = 0.1412

A = εbc c

= A/εb

c = 0.85/1x1.4x104 c =

6.07x10-5 M
Question Bank

Part A

1. What is electronic spectroscopy?

2. Mention the different types of electronic transitions in UV-visible spectroscopy.

3. Differentiate chromophore and auxochrome.

4. Write on solvent selection in electronic spectroscopy.

5. Give the important parts of an UV-visible spectrophotometer.

6. Write the principle behind UV-visible spectroscopy.

7. What are the limitations of electronic spectroscopy?

8. What is Beer-Lambert’s Law?

9. Compare bathochromic and hypsochromic shifts.

10. Differentiate hyperchromic and hypochromic shifts.

11. Give one example of a molecule showing σ → σ * and π → π * transitions.

12. Differentiate π → π * and n → π * transitions in electronic spectroscopy.

13. Define λmax and εmax in electronic spectroscopy.

Part B

1. Write short on the different types of electronic transitions in molecules.

2. Explain the instrumentation of UV-visible spectroscopy in a block diagram

3. Derive the expression for Beer – Lambert’s Law.

4. Explain the effect of substituents on λmax and εmax values in electronic spectroscopy.

5. Discuss the effect of chromophoric and auxochromic groups in molecules.

VIBRATIONAL SPECTROSCOPY (IR spectroscopy)

Vibrational spectroscopy is due to the interaction of matter with the Infra red region of the electromagnetic
spectrum.

Spectral Range of IR Radiation:

• Near IR: 12000 cm-1 to 4000 cm-1

• MID IR: 4000 cm-1 to 620 cm-1

• Far IR: 300 cm-1 to 10 cm-1

The mid IR region is used for the sample analysis in IR spectroscopy.

Quantum Approach
Irradiation of sample with IR radiation brings about vibrational changes in molecules. The transition of molecule
is from lower vibrational energy level to higher vibrational energy level. The transition is induced by absorption
of photon of the IR radiation of appropriate frequency, which matches with energy gap between the two levels.

IR absorption by molecules happens only when there is a change in the dipole moment of the molecule.

Dipole Moment (µ) = Charge (Q) * distance of separation (r)

Measured in Debye units denoted by ‘D’.

1 D = 3.33564 × 10-30 Cm, where C is Coulomb and m is meter.

The bond dipole moment that arises in a chemical bond between two atoms of different electronegativities can be
expressed as follows:

μ = .d

where μ is the bond dipole moment, is the magnitude of the partial charges +
and ,
–
 and d is the internuclear distance between +
and .
–

The bond between the atoms is considered as a spring which can undergo stretching and bending vibrations.
During vibration, there is a change in the dipole moment of the molecule as a result of the change in the distance
between the atoms. When an atom in a molecule is stretched over a certain distance keeping the other atom
stationary, the atom gets displaced from its equilibrium position by distance “x” . To attain equilibrium, the atom
tries to move back to the original position, by applying a restoring force which is equal the displaced distance but
will be in the opposite direction and has a negative value. According to Hook’s Law:
-restoring force α x

-F α x

-F = kx k = force constant

Harmonic Oscillator – vibrating pendulum

ωosc = vibrational frequency =

VIBRATIONS OF POLYATOMIC MOLECULES

Degrees of freedom:

The number of degrees of freedom is equal to the sum of coordinates necessary to locate all the atoms of a
molecule in space. Each atom has three degrees of freedom corresponding to the three Cartesian coordinates (X,
Y, Z) which is necessary to describe its position on relative to other atoms in a molecule.

The total number of degrees of freedom in a molecule containing N-atoms is equal to 3N which includes
rotational, vibrational and translation degrees of freedom.

Total number of degrees of freedom (3N) = Translational + Vibrational + Rotational The

number of degrees of freedom for H atom = 3
The number of degrees of freedom for Methane (CH4) molecule = 3N = 3x5 = 15
Vibrational Degrees of freedom for Linear molecule
Total degrees of freedom for a polyatomic molecule = Translational + Rotational + Vibrational 3N =
3 + 2 + Vibrational

• Vibrational Degrees of freedom = 3N-5

• For example: CO2, CO, HCl, Acetylene

• Vibrational Degrees of freedom for CO = 3N-5 = 1

• Vibrational Degrees of freedom for C2H2 = 3N-5 = 7

• Vibrational Degrees of freedom for CO2 = 3N-5 = 4

Degrees of freedom of vibration for Non-linear molecule

Total degrees of freedom for polyatomic molecule = Translational + Rotational + Vibrational 3N =

3 + 3 + Vibrational
Vibrational Degrees of freedom = 3N-6
Vibrational Degrees of freedom for H2O = 3N-6 = 3
Vibrational Degrees of freedom for CH4 = 3N-6 = 9
Vibrational Degrees of freedom for NH3 = 3N-6 = 6
Vibrational Degrees of freedom for C6H6 = 3N-6 = 30

Types of Vibrations
Vibration is periodic displacement of atoms or nuclei from their equilibrium position.
Regions of the Infrared spectrum
 • Most of the bands that indicate what functional group is present are found in the region from 4000 cm-1
to 1300 cm-1. Their bands can be identified and used to determine the functional group of an unknown
compound.

• Bands that are unique to each molecule, similar to a fingerprint, are found in the fingerprint region,
from 1300 cm-1 to 400 cm-1. These bands are only used to compare the spectra of one compound to
another.

IR – Sampling Techniques
• The samples used in IR spectroscopy can be either in the solid, liquid, or gaseous state.

• Solid samples can be prepared by crushing the sample with a mulling agent (KBr) which has an oily
texture. A thin layer of this mull can now be applied on a salt plate to be measured.

• Liquid samples are generally kept between two salt (NaCl) plates and measured since the plates are
transparent to IR light. Salt plates can be made up of sodium chloride, calcium fluoride, or even
potassium bromide.

• Since the concentration of gaseous samples can be in parts per million, the sample cell must have a
relatively long pathlength, i.e. light must travel for a relatively long distance in the sample cell.

• Thus, samples of multiple physical states can be used in Infrared Spectroscopy.

IR Spectroscopy – Instrumentation

• A beam of IR light from the source (Nernst Glower / Globar) is split into two and passed through the
reference and the sample respectively.
 • Now, both of these beams are reflected to pass through a splitter and then through a detector. Finally,
the required reading is printed out after the processor deciphers the data passed through the detector.

Vibrational Frequencies

Selection rules for Infrared transitions

For a particular vibration to be infrared active there must be a change in the dipole moment of the molecule
during the vibration. In other words transition dipole moment must not be zero.

Homonuclear diatomic molecules are inactive in the infrared spectrum. They do not have a dipole moment and
during the vibration also the dipole moment is zero. eg: H2, O2, N2 etc.

Heteronuclear diatomic molecules such as CO, NO are active in IR.

Symmetrical polyatomic molecules such as CO2, the symmetric stretching vibration is infrared inactive where as
the asymmetric stretching vibration is IR active ∆ν = ± 1, transition can take place between Adjacent vibrational
levels, 0 to 1, 1 to 2 etc.

• IR spectrum shows bands rather than line spectrum due to coupling of various rotational transitions
within a given vibrational transition. IR spectrum is generally complex and contains many bands in
addition to the ones corresponding to fundamental vibrational transitions

• Overtones: Bands corresponding to integral multiple of fundamental vibration. They are due to transition
from ground state to higher vibrational states. They are very weak bands. An absorption band at 1050 cm-
1
may well have an accompanying band at 2100 (2 ν) and 3150 (3 ν) cm-1.
 • Combination bands: Two vibrational frequencies in a molecule couple to give a new frequency
within the molecule. This band is a sum of the two interacting bands.

• Difference bands: Similar to combination bands. The observed frequency is the difference between the
two interacting frequencies.

• Fermi resonance: When a fundamental vibration couples with overtone or combination Band, the
coupled vibration is called a Fermi resonance.

Raman Spectroscopy

Raman spectroscopy is an analytical technique where scattered light is used to measure the vibrational energy
modes of a sample. It is named after the Indian physicist C. V. Raman who, together with his research partner K.
S. Krishnan, was the first to observe Raman scattering in 1928.

Raman spectroscopy can provide both chemical and structural information, as well as the identification of
substances through their characteristic Raman ‘fingerprint’. Raman spectroscopy extracts this information
through the detection of Raman scattering from the sample.

Raman Scattering

When light is scattered by molecule, the oscillating electromagnetic field of a photon induces a polarisation of
the molecular electron cloud which leaves the molecule in a higher energy state with the energy of the photon
transferred to the molecule. This can be considered as the formation of a very short-lived complex between the
photon and molecule which is commonly called the virtual state of the molecule.
The virtual state is not stable and the photon is re-emitted almost immediately, as scattered light.

Rayleigh scattering
In the vast majority of scattering events, the energy of the molecule is unchanged after its interaction with the
photon; and the energy, and therefore the wavelength, of the scattered photon is equal to that of the incident
photon. This is called elastic (energy of scattering particle is conserved) or Rayleigh scattering and is the
dominant process.

Stokes and Antistokes lines

In a much rarer event (approximately 1 in 10 million photons) Raman scattering occurs, which is an inelastic
scattering process with a transfer of energy between the molecule and scattered photon. If the molecule gains
energy from the photon during the scattering (excited to a higher vibrational level) then the
scattered photon loses energy and its wavelength increases which is called Stokes Raman scattering (after G. G.
Stokes). Inversely, if the molecule loses energy by relaxing to a lower vibrational level the scattered photon gains
the corresponding energy and its wavelength decreases; which is called Anti-Stokes Raman scattering.
Quantum mechanically Stokes and Anti-Stokes are equally likely processes. However, with an ensemble of
molecules, the majority of molecules will be in the ground vibrational level (Boltzmann distribution) and Stokes
scatter is the statistically more probable process. As a result, the Stokes Raman scatter is always more intense than
the anti-Stokes and for this reason, it is nearly always the Stokes Raman scatter that is measured in Raman
spectroscopy.

Differences between IR and Raman methods

RAMAN IR
It is due to the scattering of light by the vibrating It is the result of absorption of light by vibrating
molecules. molecules.

The vibration is Raman active if it causes a change in Vibration is IR active if there is change in dipole
polarisability. moment.

The molecule need not possess a permanent dipole The vibration concerned should have a
moment. change in dipole moment due to that vibration.

Water can be used as a solvent. Water cannot be used due to its intense absorption of
IR.

Sample preparation is not very elaborate, it can be in Sample preparation is elaborate Gaseous
any state. samples can rarely be used.

Gives an indication of covalent character in the Gives an indication of ionic character in the
molecule. molecule.

Cost of instrumentation is very high Comparatively inexpensive.

Mutual Exclusion Principle

In molecules with a center of symmetry it is observed that vibrations that are Raman active are IR inactive and
vice-versa, this is called the Principle of mutual exclusion (eg: CO2). In molecules with different elements of
symmetry, certain bands may be active in IR, Raman, both or neither. For a complex molecule that has no
symmetry except identity element, all of the normal modes are active in both IR and Raman. In both types the
neighbouring strong bands may obscure weak bands, while others may be intrinsically too weak to be observed
even if they are theoretically “allowed”.

In general the strong bands in the IR spectrum of a compound corresponds to weak bands in the Raman and vice
versa. This complimentary nature is due to the electrical characteristic of the vibration. If a bond is strongly
polarised, a small change in its length such as that occurs during a vibration, will have only a small additional
effect on polarisation. Vibrations involving polar bonds (C-O , N-O , O-H) are therefore, comparatively weak
Raman scatterers. Such polarised bonds, however, carry their charges during the vibrational motion, (unless
neutralised by symmetry factors), which results in a large net dipole moment change and produce strong IR
absorption band. Conversely, relatively neutral bonds ( C-C , C-H , C=C ) suffer large changes in polarisability
during a vibration. But the dipole moment is not similarly affected and vibrations that predominantly involve this
type of bond are strong Raman scatterers but weak in the IR.

In molecules having inversion center, none of the normal modes of vibrations will be both Raman and IR active.
This is known as “mutual exclusion principle”. A simple molecule which obeys this principle is CO2.
Carbondioxide has an inversion center or center of symmetry. The following are its normal modes of vibrations.
The IR and Raman active modes are indicated below each type of vibration.
Question Bank
Part A

1. Mention the use of IR radiation in vibrational spectroscopy.

2. Write the important conditions for IR absorption by molecules.

3. Calculate the number of vibrational degree of freedom in CO2, H2O molecules

4. Give the expression relating forcr constant and wavenumber used in IR spectroscopy.

5. Differentiate functional and finger print regions in vibrational spectroscopy.

6. What is Rayleigh scattering?

7. What is Raman scattering?

8. Compare stokes and antistokes lines in IR spectroscopy.

9. What is mutual exclusion principle?

10. Compare Raman and IR spectroscopic techniques. Part B

1. Discuss the different types of vibrational transitions.

2. Explain the instrumentation of IR spectroscopy with a neat block diagram.
3. Elaborate on the structural determination of molecules by Raman spectroscopy.
4. Write short note on mutual exclusion principle.
5 .Compare and contrast IR and Raman spectroscopic methods of analysis.

6. Write briefly on the selection rule in IR spectroscopy.

Introduction
Nuclear Magnetic Resonance (NMR) spectroscopy takes advantage of the magnetic properties of
certain nuclei and records the absorption of energy between quantized nuclear energy levels. In an NMR
experiment, the spectrometer is tuned to the frequency of a particular nucleus and the spectrum reveals all
such nuclei in the molecule being investigated. It is thus a very powerful technique, the closest analogy being a
powerful microscope that allows the chemist to "see" the structure of molecules in solution.

Theory

NMR is possible owing to the magnetic properties of certain nuclei. In addition to charge and mass,
which all nuclei have, various nuclei also possess a property called nuclear spin, which means that they
behave as if they were spinning. Since nuclei have a charge, they generate a magnetic field with an associated
magnetic moment.
There are useful empirical rules relating mass number, atomic number (Z) and nuclear spin quantum
number (I):

Mass Number Z I
even even 0
odd even or odd 1/2, 3/2, 5/2, ...
even odd 1, 2, 3, ...
Since NMR depends on the existence of a nuclear spin, nuclei with I = 0 have no NMR spectrum (e.g., 12C, 16O,
18O). From standpoint of generating NMR spectra, the most important class of nuclei are those with I = 1/2.

Nuclei with I >1/2 (e.g., B, I = 3/2; 14N, I = 1) have quadrupole moments, a non-spherical distribution of
11

nuclear charge, which results in broad absorption lines and makes observation of spectra more difficult. The
quadrupole moment can even affect the lineshape of neighbouring nuclei. For example, resonances of protons
bonded to nitrogen or boron atoms are generally broad in 1H NMR spectra. We shall thus be primarily
concerned with nuclei where I = 1/2, but the effect that quadrupolar nuclei can have on the NMR spectra of I =
/2 nuclei should be remembered. A listing of isotopes with I = 1/2 is provided in Table 1.
1

Table 1: Natural abundances of isotopes with I = 1/2.

Isotope Natural Isotope Natural Isotope Natural

Abundance (%) Abundance (%) Abundance (%)
1H 100 107Ag 51.35 129Xe 26.44
13C 1.108 109Ag 48.65 169Tm 100
15N 0.365 111Cd 12.75 183W 14.4
19F 100 113Cd 12.26 187Os 1.64
29Si 4.71 115Sn 0.34 195Pt 33.8
31P 100 117Sn 7.57 199Hg 16.84
57Fe 2.17 119Sn 8.58 203Tl 29.50
 77Se 7.58 123Te 0.87 205Tl 70.50
89Y 100 125Te 6.99 207Pb 21.7
103Rh 100

In an NMR experiment, the sample is placed in a strong magnetic field, Bo. Since the spins of the
magnetic nuclei are quantized, they can have only certain well-defined values. If we have nuclei with I = 1/2
(e.g., 1H, P), the spins can orient only in two directions: either with (mI = +1/2, α) or against (mI = -1/2, β)
31

the applied field. NMR transitions are allowed for cases where ∆mI = ±1. There is an energy difference, ∆E,
between the two states, and this is given by
ΔE = hν = (h/2π γ) Bo or ν = (1/2π γ) Bo
 mI = +1/2 -1/2
hν
-1/2 β
Energy
(E)
Bo α β

applied
E
nuclear +1/2 α
field spin states

where h is Planck’s constant, γ is the gyromagnetic ratio (a constant characteristic of each nucleus)*, and Bo is
the applied magnetic field. When the energy of the incoming radiation matches (is in resonance with) the
energy difference between the spin states, energy is absorbed and the nucleus is promoted from the lower +1/2
to the higher -1/2 spin state. Since the sign of mI changes, this is sometimes referred to as a "spin flip". NMR
transitions occur in the radio frequency (rf) range of the electromagnetic spectrum. The absorption of rf energy
is electronically detected and is displayed as an NMR spectrum.
The above equation is very important since it shows that ΔE depends only on γ and Bo. The
gyromagnetic ratio, γ, is an intrinsic property of the magnetic nucleus. Therefore, each type of nucleus has a
distinct and characteristic value of γ. Accordingly, the NMR experiment must be tuned for a specific nucleus
and one must record a different NMR spectrum for each NMR active nucleus of interest. Conversely, you do
not have to worry about observing signals from different nuclei on the same NMR spectrum. In order to gather
all NMR knowledge about a molecule such as PH3, we would record two different NMR spectra - a 1H NMR
spectrum to observe the 1H nuclei and a 31P NMR spectrum to observe the 31P nucleus. We would not observe
the 31P nucleus in a 1H NMR spectrum and vice-versa.
The above equation also reveals that ΔE is directly proportional to Bo, the external magnetic field.
The higher the external field, the greater is the energy separation between the α (mI = +1/2) and β (mI = -1/2)
spin states. Recalling that E = hν, another way of saying this is that the resonance frequency of the nucleus
increases with increasing Bo since if E increases, so does ν. This is shown in the following table.

Bo Resonance Frequency (ν, MHz)

(tesla)‡ 1H 13C 11B 19F 31P

2.35 100 25.2 32.1 94.1 40.5

4.70 200 50.4 64.2 188.2 81.0
‡a tesla is a unit describing magnetic field strength

.
Note that all I = 1/2 nuclei behave according to the same theoretical principles - although 1H NMR
spectroscopy is the most commonly practiced, 19F and 31P NMR spectra are generated in exactly the same way
as a 1H NMR spectrum. The main difference between the different I = 1/2 nuclei is that the resonance
frequency is changed when recording the spectrum.

Chemical Shift
The resonance frequency is determined only by γ and Bo, therefore, all atoms of a given nucleus in a
molecule (e.g., all 1H nuclei) should resonate at the same frequency. If this were the case, the only thing NMR
could tell us is whether a molecule contains NMR active nuclei (1H, 31P, 13C, etc.). Fortunately, the frequency
of the NMR absorptions of a given nucleus also depends on the chemical environment of the nucleus. The
variation of the resonance frequency with chemical environment is termed the chemical shift, and herein lies
the power of the NMR method.
The origin of the chemical shift can be traced to the electrons surrounding the nucleus, and the
interaction of the electron cloud with the applied field, Bo. The reason for this is that circulating electrons also
generate a magnetic field that orients itself in the opposite direction to the applied field.

The actual field (Blocal) “felt” by a nucleus is thus less than Bo, and the ability of the electrons to alter the
field felt at the nucleus can be expressed by θ, the shielding constant.
Blocal = Bo (1-θ)

Nuclei are said to be shielded or deshielded depending on the presence or absence of electron density
surrounding them. For example, the introduction of an electron withdrawing group (e.g., halogen, O, etc.) will
reduce the electron density around a nucleus (deshielding; δ is small) and the resonance frequency will increase.
Conversely, an electron donating substituent (e.g., CHx, SiHx) will cause increased shielding (δ is large) and
lower the resonance frequency.

In reporting chemical shifts, one could use absolute field or absolute frequency, but this would be
cumbersome and would result in the chemical shift being dependent upon the applied field. A simpler scale for
chemical shifts has been devised. Chemical shifts (δ) are expressed in units of parts per million (ppm) of the
spectrometer frequency with respect to a reference material whose position is arbitrarily assigned a value of
0.0 ppm.
When expressed in such dimensionless units (δ in ppm), the chemical shifts are invariant of the frequency of
the spectrometer and can be used as molecular parameters. For example, 1.0 ppm at 60 MHz is equal to a
separation of 60 Hz, and at 200 MHz, 1.0 ppm equals 200 Hz. Thus, the same two resonances that are
separated by 1 ppm at 60 MHz are still 1 ppm apart at 200 MHz, because δ = 60 Hz/60 MHz = 200 Hz/200
MHz = 1 ppm. Therefore, if the same sample is run at two different spectrometer frequencies, the chemical
shifts of the resonances will be identical. Naturally, this statement is only true if the same reference material is
used for each spectrum. Different references are used for different nuclei. The most widely accepted
reference for 1H and 13C NMR is tetramethylsilane (Si(CH3)4 = TMS). For 11B NMR, F3B•OEt2 is
commonly used, as are CFCl3 for 19F NMR and 85% H3PO4 for 31P NMR spectroscopy.
In the past, NMR spectra were obtained by varying the applied field and measuring the chemical
shift as a function of the field strength. This gave rise to the terminology of a downfield shift for
nuclei that were deshielded (as they required a lower applied field to bring the nucleus into resonance) and
upfield shift for shielded nuclei. For example, one would say that a resonance at δ 8.0 ppm is downfield of
one at δ 2.0 ppm, and conversely that the signal at δ 2.0 ppm was upfield of the signal at δ 8.0 ppm.
More modern NMR spectrometers generate spectra by varying the frequency, γ, while
keeping the magnetic field strength, Bo, constant. Nevertheless, the upfield/downfield terminology remains in
common use. Unfortunately, this results in the confusing situation that δ is positive in the downfield direction
(to the left of the standard on spectra) where resonance
frequencies are higher. Resonances that are upfield of the reference appear at lower frequencies

and have negative δ values.

TMS (δ = 0.0 ppm)

Low High
shielding shielding
Low
High
Frequency
Upfiel
Frequency d

10 9 8 7 6 5 4 3 2 1 0 -1 -2 -3
δ (ppm)

The concept of chemical shift is illustrated in Figure 8. As the hydrogens of methane are increasingly
substituted by electron withdrawing chlorine atoms, the chemical shift of the remaining hydrogens shifts
further downfield as the hydrogens become increasingly deshielded. Substitution of the methyl groups of
tetramethylsilane (TMS) by chlorine has similar, but far less dramatic, results. In this case, the electron
withdrawing chlorine atoms are separated from the hydrogens by carbon and silicon, resulting in less
significant deshielding of the 1H nuclei.

ClSi(CH
3)3

Cl2Si(CH3)2

1. 0.
CHCl CH CH 0 CH 0
2Cl 2 3Cl

Si(CH3)4

9 8 7 6 5 4 3 2 1 0
(ppm)

1H NMR Spectra of CHxCly and ClySi(CH3)x.

One important consequence of chemical shift is that each chemically different type of NMR-
active nucleus in a molecule will give rise to its own signal in an NMR spectrum. Nuclei
are thus referred to as chemically equivalent or chemically inequivalent in determining how many signals will
be observed in an NMR spectrum. For example, both CH3Cl and CH2Cl2

provided one resonance each in the 1H NMR spectrum in the figure. From this, we can infer that the individual
hydrogens in each of these molecules are chemically equivalent. From the viewpoint of chemical structure, the
reason for this is that hydrogens are related by symmetry elements (reflection through a mirror plane or
rotation about an axis) and are thus identical.
Cl Cl Cl

Ha Cl
C
Hb H C Hc C Hb C
Cl
b Ha
a H
c Ha Hc
H
Hb

mirror in the plane of the three-fold rotation axis demonstrates Ha =

page renders Ha = Hb Hb = Hc

Sometimes, determining chemical equivalence or inequivalence is straightforward. The methyl

hydrogens in ethanol (CH3CH2OH)are different from the methylene hydrogens and that both of these are
different than the hydroxyl hydrogen; we would thus anticipate three signals in the 1H NMR spectrum. Upon
further reflection though, why should the hydrogens of the methyl group all be equivalent? The answer is
simple when it is recognized that methyl groups rotate freely and rapidly, with the result that each hydrogen
experiences the same overall chemical shift as it completes one rotation, a situation analogous to CH3Cl
described above. Therefore, all methyl groups generally give rise to one signal in 1H NMR spectra. This
concept can generally be applied to analogous groups such as tert-butyl, C(CH3)3, trimethylsilyl, Si(CH3)3,
and trifluoromethyl, CF3 (in 19F NMR spectra).
The most general method of determining whether nuclei are chemically equivalent to other nuclei in a
molecule is to determine whether they are in the same environment, and whether one nucleus can be related to
the other through a symmetry transformation such as rotation or reflection through a mirror plane. Some
examples are provided below for illustration.

CH3CH2 O CH2CH3 CH3CH2 O CH3

The CH2 groups are equivalent and the CH3 The CH3 groups are inequivalent. 3 signals
groups are equivalent. 2 signals in either the in either the 1H or 13C NMR spectra
1
H or 13C NMR spectra
CH3CH2CH2Cl ClCH2CH2CH2CH2Cl

The CH2 groups are inequivalent. 3 signals There are two distinct sets of CH2 groups. 2
in either the 1H or 13C NMR spectra signals in either the 1H or 13C NMR spectra
 F Fax
F F Feq
S S
F F Feq
F
Fax

SF6 is a highly symmetrical octahedral The axial (ax) and equatorial (eq) fluorines
molecule. 1 signal in the 19F NMR spectrum are chemically inequivalent 2 signals in the
19
F NMR spectrum

Fapical O
F F F F
Cl Xe
F F F F
The apical fluorine is chemically distinct The four fluorine nuclei in the square base are
from the four fluorines in the square base 2 chemically equivalent 1 signal in the 19F
signals in the 19F NMR spectrum NMR spectrum

Integration
The area under each NMR absorption peak can be electronically integrated to determine the relative
number of nuclei responsible for each peak. The integral of each peak can be provided numerically, and is
often accompanied by a line that represents the integration graphically. Intensities of signals can be compared
within a particular NMR spectrum only. For example, 1H intensities cannot be compared to those of 19F or 31P
nuclei. It is important to note that the integration of a peak is a relative number and does not give the absolute
number of nuclei that cause the signal. Thus, the 1H NMR spectrum of H3C–SiH3 will show two peaks in a
1:1 ratio, as will the 1H NMR spectrum of (H3C)3C– Si(CH3)3. This is simply because the ratios 3:3 = 9:9 =
1:1. Nonetheless, the integrated intensities of the signals in an NMR spectrum are a vital piece of the puzzle.
The concept of integration, and also that of chemical shift, is illustrated by Figure 9. Determining
integration ratios is an exercise in finding the greatest common divisor for the series of peaks (the largest whole
number divisor that will produce a whole number ratio). In the above example, this value is either 1.4 cm or
9.9 integration units. It should be remembered that integration is a measurement that is subject to error; it is
common for the error in integrated intensity to approach 5 - 10 %. The ratio of the integrated peak intensities is
1:3 = 3:9, allowing us to assign the resonance at δ 3.21 to the methyl group and that at δ 1.20 to the (CH3)3C
group. It is important to note that the hydrogens of the (CH3)3C group are more shielded than the CH3
group. This occurs because the CH3 group is directly adjacent to the electron withdrawing oxygen, but the
corresponding methyl protons in the (CH3)3C group are separated from oxygen by a second intervening
carbon center.

4 3 (ppm) 2 1 0

Figure 1. 1H NMR Spectrum of CH3OC(CH3)3.

At this stage, we can begin to appreciate how NMR resembles a molecular microscope. For example,
at one frequency we could "see" the various protons, while the carbons, fluorines, phosphorus, and even certain
metal nuclei could be observed at other frequencies. Within one spectrum, we can make use of the position
(chemical shift) and integrated intensity of the different signals to assign particular molecular fragments
responsible for them, and to build up a model of the molecule. There is one more aspect of NMR that is
extremely helpful in determining how to connect the parts together.

Spin-Spin Splitting (Coupling)

The appearance of a resonance may be very different when there are other neighbouring magnetic
nuclei. The reason for this is that the nucleus under observation will interact with the magnetic spins of the
different neighbouring nuclei.
 The simplest case is that of two protons having significantly different chemical shifts (designated A
and X). Considering chemical shift and integration only, we could represent the spectrum as:
HA HX

Both protons have a spin of 1/2, and both can exist in the +1/2 and -1/2 spin states. Now, it turns out that the
magnetic environment of HA is slightly different when HX is in the +1/2 state than when it is in the -1/2 state.
This can be represented pictorially with arrows (pointing either up or down) representing the two spin states of
HX.

and
H X X
A
As a result, HA will split into two lines, each half the intensity of the unperturbed signal. Similarly, HA will
influence HX which becomes a doublet also. The splitting, or coupling, is symmetrical about the unperturbed
resonances HA and HX, and is described by the means of a coupling constant, JAX, which has units of Hz.

unpertur
be
JAX dsignals JAX

A X

Note that the magnitude of JAX is identical at both signals - coupled nuclei must share the same
coupling constant.
In a similar way, the resonance of a proton attached to phosphorus will be a doublet, since the
phosphorus nucleus has I = 1/2 and may be in the +1/2 or -1/2 state. However, the key distinction here is that
we are dealing with two different nuclei, and thus two different NMR spectra. Each NMR spectrum (1H and
31
P) will show one doublet with a JPH coupling constant that is identical in magnitude. Recall that we cannot
"see" a 31P nucleus in a 1H NMR spectrum and vice-versa. Nonetheless, the splitting of the peaks into
doublets in each spectrum tells us that the 1H and 31P nuclei are interacting.
 JPH JPH

e.g., HPCl2

H P

1
H NMR Spectrum P NMR Spectrum
31

To review, the influence of the neighbouring spins is called spin-spin coupling and NMR peaks are split into
multiplets as a result. The separation between the two peaks is called the coupling constant, J, which is
expressed in Hz. Spin-spin coupling has the following characteristics:
• the magnitude of J measures how strongly the nuclear spins interact with each other.
• coupling is normally a through-bond interaction, and is proportional to the product of the gyromagnetic
ratios of the coupled nuclei. For example, 1JCH = 124 Hz for 1H-13C coupling in CH4, and 1JSnH =
1931 Hz for 119Sn-H coupling in SnH4. This happens because δ (119Sn) is much larger than δ (13C).

• since coupling occurs through chemical bonds, the magnitude of J normally falls off rapidly as the
number of intervening bonds increases. e.g., 1JPH ~700; 2JPH ~20 Hz in

2J
PH
H

H2C P
X
1J H
PH

Coupling constants are thus labeled to show the types of nuclei and the number of bonds separating the
nuclei that give rise to spin-spin splitting.

number of bonds x labels

eparating the describing
J AB thetwo
coupled
• since spin-spin coupling is a through-bond interaction, it is sensitive to the orientation of the bonds
between two interacting nuclei. This is particularly important for two-bond coupling constants. The
influence of the orientation of the two coupled nuclei can occasionally render 2J < 3J. For example,
10 Hz
Ha Hc 2J a b << 3J 3
2 Hz
H Ha c < J HbHc
b H H
H R 17

Hz
1J is not affected by the orientation of the coupled nuclei, so it is generally true that 1J

>> 2J or 3J, but it is not always true that 2J > 3J.

• spin-spin interactions are independent of the strength of the applied field. The spacing (in Hz) between
lines at two different field strengths will be the same if it is due to coupling, but will be proportional to
the field strength if it is due to a difference in chemical shift.

Table 2: Typical Coupling Constant Ranges (in Hz)

Coupled Nuclei (AB in xJAB)

x HH CH PHb PCb
1 – 115 - 250 630 - 710 120 - 180
2a 2 - 30 5 - 60 7 - 13 5 - 40
3 2 - 17 2 - 20 6 - 11 5 - 11
a 4 bond couplings
Two – are particularly sensitive
– 0 - 1 arrangement of the
to the geometrical – nuclei,
which in some cases may render 2JAB < 3JAB. b
Restricted to acyclic compounds.

Cases involving more than two nuclei with I = 1/2 are direct extensions of the above. However,
because there are more nuclear spins interacting, the pattern of lines observed in the NMR spectrum becomes
-
more complicated. For example, let’s consider the 1H NMR spectrum of the HF2 anion (i.e., [F--H--F]-). We
are observing the 1H nucleus, but it is coupled to two chemically equivalent 19F (I = 1/2) nuclei. There are four
ways that we can arrange the nuclear spins of the two fluorine nuclei, but only three different energy states are
created, as is explained below:

1J 1J

intensity ratio: 1 HF 2 HF 1

Extending what we learned about the generation of a doublet, we can clearly see that the 1H environment
where both 19F spins are “up” is different from that where both 19F spins are “down”. However, we can also
arrange things so that one 19F spin is “up” and the other is “down”. The latter case is degenerate; that is, there
is more than one way of accomplishing an “up/down” arrangement of nuclei, but each “up/down” arrangement
has the same energy. As a result, a pattern of three peaks (or triplet) with an intensity pattern of 1:2:1 is
generated as shown above. It is important to note that each line in the triplet is separated by the same 1JHF
coupling
constant. As we would expect, the F NMR spectrum of HF2- would show a doublet because the fluorine
19

nuclei are chemically equivalent and couple to one 1H nucleus.

Another way of looking at this is to begin with a singlet for the 1H nucleus and then couple each 19F
nucleus one step at a time. The coupling of the first 19F nucleus generates a doublet. When each line in this
doublet is split again into a doublet, they overlap identically at the center of the signal, generating a single line
of intensity two relative to each outer line of intensity one:

absence of coupling

J
coupled to one I = 1/2 nucleus (doublet)
J J
coupled to a second I = 1/2 nucleus with identical J
intensity ratio: 1 2
1

When a similar exercise is undertaken for the 31

P NMR spectrum of PF3, the nuclear spins of the
three equivalent F nuclei can be arranged in four ways to generate a quartet
19

1J 1J 1J

PF PF PF

intensity ratio: 1 3 3 1

or we can split a singlet into doublets three times to accomplish the same transformation:

absence of coupling

J coupled to one I = 1/2 nucleus (doublet)

J J coupled to a second I = 1/2 nucleus with identical J coupled to a

J J J third I = 1/2 nucleus with identical J
intensity ratio: 1 3 3 1

In this case, when each line at the triplet stage is split again into doublets, the intensity of the overlapping
peaks is not identical; a signal of relative intensity two (from the middle peak) overlaps with a signal of
intensity one (from the outer peak) to create a peak of intensity three.
 Fortunately, the pattern of peaks generated by the interaction of I = 1/2 nuclei can be easily generated
by remembering that one nucleus is split by (n) equivalent nuclei into (n+1) peaks, each separated by the
coupling constant, xJAB. The number of peaks is referred to as the multiplicity. The intensity pattern is a direct
consequence of the number of combinations of the various nuclear spins that are possible and is described by a
series of binomial coefficients. In practice, it is easiest to determine the intensity pattern by use of a mnemonic
device such as Pascal's triangle.

n n+1 Intensity Multiplicity Pattern Example

0 1 1 singlet (s) CH4

1 2 1:1 doublet (d) (CH3)2CHCl

2 3 1:2:1 triplet (t) CH3CH 2Cl

3 4 1:3:3:1 quartet (q) CH3CH2Cl

4 5 1:4:6:4:1 quintet 29SiF

4

5 6 1 : 5 : 10 : 10 : 5 : 1 sextet PF5

6 7 1 : 6 : 15 : 20 : 15 : 6 : 1 septet (CH3)2CHCl

etc.

The phenomenon of spin-spin coupling and its effect on the appearance and interpretation of NMR
spectra is best described by example, several of which appear on the following pages.

Analyzing NMR Spectra and Reporting Results

NMR spectra contain a wealth of information and must be analyzed in a methodical way. Much like a
jig-saw puzzle, all of the pieces (i.e., chemical shift, integration, multiplicity, and coupling constants) must fit
together properly. As with a puzzle, you may find that your initial conclusion is incorrect because several
“pieces” are out of place. It is important to approach the problem in a creative way and investigate alternate
solutions. The most straightforward method for analyzing NMR spectra is:

1) identify signals by chemical shift and determine their relative integration

2) identify the multiplicity of the peaks and calculate coupling constants.
Many students are tempted to “leap in” and attempt to analyze coupling patterns first, but the coupling pattern
may not correlate if the integration ratio of the coupled multiplets has not already been deduced. Above all else,
remember to double-check that the assignments make sense. It is often a good practice to use your results
to generate a simple stick-diagram of the
NMR spectrum. If the stick-diagram matches the actual spectrum exactly, then you have correctly analyzed
the NMR spectrum.
Chemical shifts should generally be reported to two decimal places. Multiplicities may be written out
(e.g., “triplet”) or expressed in terms of common abbreviations (e.g., “t”). Coupling constants are commonly
reported as whole numbers, but may be expressed to one decimal place if the spectrum is of sufficiently high
resolution. The coupling constants should be properly labeled (i.e., xJAB) to show the nuclei that are coupled;
if there is more than one NMR active isotope for a nucleus (e.g., 117Sn/119Sn), it should be clearly defined
which is involved in the coupling interaction you are
describing.
Example : 1
H NMR spectrum of (CH3CH2O)4Si.

CH2 CH3

1 3 3 1 1 2 1
3J = 7 Hz 3
JHH = 7 Hz
HH

1. On the basis of chemical shift and integration, a CH2 signal of intensity two appears downfield of a
CH3 signal of intensity three.
2. The CH3 signal will be split into a triplet by interaction with the two equivalent methylene protons (n
= 2 and thus n+1 = 3). The CH2 signal is split into quartet by the three equivalent CH3 protons (n = 3
and thus n+1 = 4).
3. The spacing is 3JHH = 7 Hz, and is the same in both regions.
4. The relative peak heights in the methyl triplet will be 1 : 2 : 1 and will be 1 : 3 : 3 : 1 for the
methylene quartet. Recalling the overall integration, the methyl absorption must be 3/2 as intense as
methylene absorption as the total signal intensity is proportional to the number of nuclei; the
integration ratio is 3.03÷2.01 ~ 3/2.
Question Bank Part
A

1. What type of EMR is used in NMR. Why?

2. Differentiate shielding and deshielding in NMR.
3. Mention the use of chemical shift in NMR.
4. Define spin-spin coupling in NMR spectroscopy.
5. Give the significance of splitting of signals in NMR.
6. Relate the effect of spin quantum number in NMR spectroscopy.
7. What is spin flip in NMR spectroscopy?
8. What is shielding constant and bring out its role in NMR spectroscopy.
9. What is the principle of NMR spectroscopy?
 Part B

1. Discuss shielding and deshielding effects in NMR spectroscopy.

2. Explain chemical shift in NMR.
3. Elaborate on spin spin coupling in NMR with examples.
4. Explain the role of reference compounds in NMR.
5. Explain the significance of sin quantum number values in NMR spectroscopy.

FLUROSCENCE SPECTROSCOPY

Fluorescence spectroscopy is a rapid, sensitive method for characterizing molecular environments and events samples.
Fluorimetry is chosen for its extraordinary sensitivity, high specificity, simplicity and low cost as compared to other
analytical techniques. It is widely accepted and powerful technique that is used for a variety of environmental, industrial,
medical diagnostics, DNA sequencing, forensics, genetic analysis and biotechnology applications. It is a valuable analytical
tool for both quantitative and qualitative analysis. This article presents a brief overview of the theory of fluorescence
spectroscopy, together with examples of applications of this technique in organic and inorganic chemistry, medical
diagnosis, medical science etc. Keywords: Fluorimetry, DNA sequencing.

Fluorophores play the central role in fluorescence spectroscopy. Fluorophores are the components in molecules that cause
them to fluorescence. Majorly fluorophores are the molecule which contains aromatic rings such as Tyrosine, Tryptophan,
and Fluorescein etc. Luminescence is emission of light by a substance not resulting from heat is thus a form of cold body
radiation. It can be caused by chemical reactions, electrical energy, subatomic motions, or stress on a crystal. There are two
pre-requisites for luminescence: The luminescent material must have a semiconductor structure with a nonzero band gap.
[Metals do not provide luminescence if they have no band gap]. The energy must be imparted to this material before
luminescence can take place.

Principle of fluorescence spectroscopy:

Absorption of UV or visible radiation causes transition of electrons from singlet ground state to the singlet excited state. As
this state is not stable, it emits energy in the form of UV or visible radiation and returns to singlet ground state.
Fluorescence emission occurs as the fluorophore decay from the singlet electronic excited states to an allowable vibrational
level in the electronic ground state.

Instrumentation

The fluorescence spectroscopy instrumentation is similar to UV-Visible spectroscopy. It has the following

1. Light source: Xenon arc lamp, mercury vapor lamp and tungsten lamp

2. Monochromators: These help to separate light bands.

3. Sample cells: The sample cell is mostly quadrilateral. It is made of glass.

4. Detectors: This a photomultiplier tube that helps to detect radiation with low intensity.
Fluorimetric instrumentation is of two types like filter fluorometer and spectrofluorometer.

Of them, the spectrofluorometer is advanced with high sensitivity and efficiency. But both of them have
similar instrumentation as below.

Light source: The source should provide stable radiation with sufficient intensity over an entire range.

Xenon lamp: This is widely used in spectrofluorometers. Here the light is produced by passing electricity
through ionized Xenon gas under high pressure. It provides a bright light with intense radiation over a wide
range. Hence, most compounds can be analyzed.

Mercury vapor lamp: Here, mercury vapor is subjected to the high pressure of 8 atmospheres. It gives
intense radiation over 350nm. At low pressures, this vapor also provides one more radiation at 254nm. But,
this is preferred in inexpensive filter type fluorimeters.

Tungsten lamp: This lamp has radiation with low intensity. It is used when the excitation radiation is in the
visible region.

Filters and monochromators: The light source produces polychromatic light. So, filters or
monochromators are needed to convert them into monochromatic light. The filters have less resolution
capacity and hence the light radiation has a bandpass of ±30nm. Consequently, they are inexpensive and
used in filter fluorometers. In comparison, the monochromators have a very high-resolution capacity with a
bandpass of just ±0.1nm. But, they are expensive and hence in spectrofluorometers.

Sample cells: The fluorometer cuvettes are small tube-like structures used to hold the sample for analysis.
They are made of color corrected fused glass. All the sides of the cell have polished surfaces. The path
length of the sample holder is 10mm.

Detectors: Photo-multiplier tubes are employed as detectors. These are efficient and accurate for radiations
with low intensity.

Advantages of fluorescence spectroscopy

Sensitivity: Fluorimetry is a highly efficient spectroscopic with its ability to measure samples of small
quantities. Samples concentrations as low as μg and ng/ml are determined.

Precision: The accuracy of the result is also high in this fluorimetry spectroscopy. A margin of up to 1% is
achievable.

Specificity: UV Vis spectroscopy relies on just the excitation wavelength of the substance. In fluorescence
spectroscopy, both excitation and emission wavelengths are characteristic. If two compounds could have
the same excitation wavelength, they would differ in emission wavelength. Hence, the specificity for a
compound is enhanced.

A wide range of compounds: This method is used to analyze many compounds. Even those compounds
which do not have fluorescent property can be made by chemical conversion.

Disadvantages of Fluorimetry

Despite the many benefits of this method, some limitations exist. These are related to fluorescent intensity.

1. pH changes and the presence of oxygen can affect fluorescent intensity.

2. When a sample is subjected to UV absorption, it can undergo chemical changes due to the high energy
of radiation.

3. Not all substances can be converted to be fluorescent. So, not all compounds can be analyzed by this
method.

4. Presence of heavy metals and electronegative halides can change the fluorescent intensity.

Applications of fluorimetry

1. To analyze nucleic acids like DNA and proteins.

2. It is used to measure inorganic compounds containing metals or ions like beryllium, lithium, aluminum, zinc, etc.

2. It helps to analyze organic compounds like steroids, proteins, alkaloids, etc.

3. It is also a specified method to analyze medicines like morphine, quinine, indomethacin and vitamins like riboflavin, etc.

Fluorescence has a wide range of applications in medicine, including:

Medical Diagnostics: Fluorescence imaging is used to detect and diagnose diseases such as cancer, cardiovascular diseases,
and skin conditions.
. Techniques like fluorescence lifetime imaging (FLIM) and fluorescence spectroscopy help in identifying abnormal tissues
and cells.
.

Surgical Guidance: Fluorescence imaging can guide surgeons during operations by highlighting specific tissues or blood
vessels
. This helps in precise removal of tumors and reduces damage to healthy tissues
.

Drug Delivery and Monitoring: Fluorescent markers are used to track the delivery and distribution of drugs within the
body
. This helps in understanding how drugs interact with tissues and their effectiveness.

Research and Development: Fluorescence spectroscopy is a valuable tool in biomedical research for studying cellular
processes, protein interactions, and gene expression
DIFFRACTION AND SCATTERING

Diffraction happens when a wave encounters an obstacle or a slit, causing it to bend and spread
out. You can see this with light, sound, and even water waves.

For light, when it passes through a small opening or around an object, it doesn't just go straight.
Instead, it bends and creates patterns of light and dark bands. This is why you see those patterns
when light shines through a tiny slit.

This principle is important in many fields:

In optics: Diffraction limits the resolving power of microscopes and telescopes.

In everyday life: The way sound spreads when you hear someone around a corner.

In technology: It's used in devices like diffraction gratings to separate light into its component
wavelengths for analysis.

The Physics Behind Diffraction

When a wave (like light or sound) encounters an obstacle or a slit, it doesn't just bounce off or
pass straight through; it bends around the edges. This bending and spreading out of waves is
called diffraction. The extent of diffraction depends on the wavelength of the wave and the size
of the obstacle or slit.

Diffraction Gratings

One of the most significant applications of diffraction is in the creation of diffraction gratings.
These are tools that have many closely spaced slits or grooves. When light passes through or
reflects off these slits, it diffracts and creates an interference pattern of light and dark bands.
These patterns can be analyzed to determine the wavelength of the light, making diffraction
gratings essential in spectroscopy.

Applications in Everyday Life

Optics and Astronomy: Diffraction limits the resolving power of optical instruments like
microscopes and telescopes. This means there's a limit to how much detail we can see. In
astronomy, diffraction patterns help in understanding the properties of light from stars and other
celestial bodies.

Sound Waves: Diffraction allows sound to bend around corners, which is why you can hear
someone calling you from another room. It also plays a role in the design of speakers and
hearing aids.

Medical Imaging: Techniques like X-ray diffraction are used to study the structures of complex
molecules, including proteins and DNA, which is crucial for understanding many biological
processes.
Diffraction Patterns

When light waves pass through a single slit, they produce a central bright fringe with alternating
dark and bright fringes on either side. This pattern is called a diffraction pattern. The spacing and
intensity of these fringes depend on the wavelength of the light and the width of the slit.

Practical Example: Holography

Holography, the technique to create three-dimensional images (holograms), relies on the

principles of diffraction and interference. A hologram captures the light waves scattered from an
object, and when it's illuminated appropriately, it recreates the light field, giving a 3D effect.

Scattering is when particles or waves are forced to deviate from a straight trajectory due to non-
uniformities in the medium they pass through.

Types of Scattering

Rayleigh Scattering: Occurs when particles are much smaller than the wavelength of the
radiation. This is why the sky is blue; shorter blue wavelengths are scattered more than longer
red wavelengths by the tiny molecules in the air.

Mie Scattering: Happens when the particles are about the same size as the wavelength of the
radiation. This is why clouds appear white, as water droplets scatter all wavelengths equally.

Tyndall Effect: Visible when particles in a colloid scatter light, making the path of a beam of
light visible in fog or dust.

Applications
Medical Imaging: Techniques like ultrasound imaging rely on scattering to produce images of
internal body structures.

Environmental Monitoring: Scattering is used in LIDAR technology to measure atmospheric

particles.

Astrophysics: Helps in studying the properties of interstellar dust and gases.

Real-World Example

Think of shining a flashlight in a foggy night. The light beams scatter in all directions due to the
tiny water droplets in the fog, creating a glow that makes the beam visible.