B.

Tech First Year Course

Course Title- Chemistry-I (Concepts in Chemistry for Engineering)
Course Code- BTCH101-18
Scheme & Credit – L-3, T-1, P-0, C-4

-Dr. Sanjeeb Sutradhar

 Spectroscopic Techniques and Applications (L-8)

Principles of spectroscopy and selection rules. Electronic spectroscopy. Fluorescence and

its applications in medicine. Vibrational and rotational spectroscopy of diatomic
molecules. Applications. Nuclear Magnetic Resonance and Magnetic Resonance Imaging.
surface characterization techniques. Diffraction and scattering.

References
 Principles of Spectroscopy
Spectroscopy: Deals with transitions occurring in a molecule when it interacts with
electromagnetic (EM) radiations

Principles of spectroscopy : If matter is exposed to EM radiation (e.g. infrared light) the

radiation can be absorbed, transmitted, reflected, scattered or undergo
photoluminescence.

Photoluminescence is a term used to designate a number of effects, including

fluorescence, phosphorescence, and Raman scattering.
Electromagnetic Spectrum

Atomic Spectra
– Electronic transition

Molecular Spectra
-Rotational, Vibrational, and
Electronic transitions
 Selection Rules
Atomic and molecular spectral transitions are governed by selections rule

 Dl=±1 (l=azimuthal quantum number) sp, pd

 DJ=±1 (J=rotational quantum number)

 Dn=±1 (n=vibrational quantum number)

Allowed transition – Intense strong

Forbidden transition- Weak
 Electronic (UV-Visible) Spectroscopy
Electronic spectroscopy: Electromagnetic radiation of UV-visible interacts with the
molecules and brings about the electronic transition from lower electronic energy level to
higher energy level.

Principles of Absorption Spectroscopy:

When an electronic component of EMR interact with sample and it changes its electrical
component (dipole moment) to gives a simple plot of absorbance vs. wavelength.

Region of UV and Visible:

Far UV – Vacuum UV – 1-100 nm; Near UV -Applicable UV – 200-400 nm
Visible (VIBGYOR)- 400-750 nm
 Electronic Spectroscopy
Beer-Lambert Law:
More effectively a molecule absorbs light of a given wavelength,
the greater the extent of light absorption. From this idea,
Beer-Lambert Law may be formulated as follows

A = absorbance (Optical density); l0 = intensity of light incident upon sample cell

l = intensity of light leaving sample cell; c = molar concentration of solute
b = length of sample cell (cm); e = molar absorptivity
 Limitations of Beer-Lambert Law
The linearity of the Beer-Lambert law is limited by chemical and instrumental factors.

Causes of nonlinearity include:

 Deviations in absorptivity coefficients at high concentrations (>0.01M) due to
electrostatic interactions between molecules in close proximity
 Interaction with solvent: hydrogen bonding
 Scattering of light due to particulates in the sample
 Fluorescence or phosphorescence- a positive deviation in % T and negative deviation
for A
 Changes in refractive index at high analyte concentration
 Shifts in chemical equilibria as a function of concentration
 Non-monochromatic radiation, deviations can be minimized by using a relatively flat
part of the absorption spectrum such as the maximum of an absorption band
 Stray light
Problem
 Basic Components of Spectrophotometer
Spectrophotometer/spectrometer: Instruments use to measure the amount of the EM
radiation absorbed by the molecules/compounds
 Electronic Transitions
Electronic transition for organic molecules are

p-p* transition (K-band): Observed in conjugated double bond molecules with higher
molar extinction coefficient.
e.g.-Aldehyde-ketone- lmax=170-190 nm and Ehtylene=180 nm

Benzenoid system shows B (Benzenoid) and E (Ethylenenic) bands due to p-p* transition
e.g.- Benzene, lmax(E1 &E2)=180 & 200 nm
 Electronic Transitions
n-p* transition (R-band): Observed with lower molar extinction coefficient (<100).
e.g.-Acetone- lmax=277 nm and Nitrosobutane=665 nm

Chromophores: Groups or atoms are responsible for electronic transition and the
wavelength of radiation is absorbed. e.g.- C=C, -CN, -CO, -N=N-, -C=S, -N=O

Auxochromes: Groups or atoms which don’t impart color to the compound but increases
the coloring of the compound. e.g.- -OH, -NH2, -OR, -CH3

Bathochromic shift (red shift) a shift to lower energy or longer wavelength.

Hypsochromic shift (blue shift) a shift to higher energy or shorter wavelength.
Hyperchromic effect an increase in intensity.
Hypochromic effect a decrease in intensity.
 Effect of Conjugation and H-bonding on lmax and e
Effect of conjugation: Increase in the extent of conjugation in double bond systems
increases the bathochromic shift and hyperchromic shift.

e.g.
CH3-(CH=CH)n-CH3
n=3
n=4
n=5

Phenol
Effect of H-bonding: Solute-solvent interaction and
bathochromic shift

cis-isomer compounds loose coplanarity due to steric hindrance

and cis-isomer will have lower lmax than corresponding
trans-isomer
 Application of ES and Franck-Condon Principle
cis-isomer compounds loose coplanarity due to steric hindrance and cis-isomer will have
lower lmax than corresponding trans-isomer.

lmax = 268 nm lmax = 272 nm

Franck-Condon Principle: Intensity of vibrational-
Electronic spectra
An electronic transition takes place so rapidly that a
vibrating molecule does not change its internuclear
distance appreciably during the transition
 Fluorescence Spectroscopy
Luminescence: Emission of light from any substance, and occurs from electronically
excited states.
Photoluminescence: Emission of light from the substance, when the substances excite by
using photon.
Photoluminescence two types:
 Fluorescence: Emission rates of fluorescence are typically 108 s–1, so that a typical
fluorescence lifetime is near 10 ns
 Phosphorescence: Emission rates of phosphorescence are slow (103 to 100 s–1), so that
phosphorescence lifetimes are typically ms to s.

Fluorescence spectroscopy (fluorometry or spectrofluorometry), is a type of

electromagnetic spectroscopy which analyzes fluorescence from a sample.
Principle: It involves using a beam of light, usually ultraviolet light, that excites the
electrons in molecules of certain compounds and causes them to emit light of a lower
energy, typically, but not necessarily, visible light. This shift to longer wavelength is called
the Stokes shift.
Devices that measure fluorescence are called fluorometers or fluorimeters
Jablonski Diagram
 First Fluorescence Spectrum
The first observation of fluorescence from a quinine solution
in sunlight was reported by Sir John Frederick William
Herschel in 1845.

The quinine in tonic water is excited by the ultraviolet light

from the sun. Upon return to the ground state the quinine
emits blue light with a wavelength near 450 nm.

Factors interfering with fluorescence intensity

 Concentration
 Transition type in fluorescence
 Structure
 Temperature and solvent
 Impurities present in the solution
 Quantum Yield
Quantum Yield = FF is a measure of the efficiency of photon emission
• FF = number of fluorescence quanta emitted divided by number of quanta absorbed to
a singlet excited state
• FF = ratio of photons emitted to photons absorbed
 Application of Fluorescence in Medicine
Fluorescence uses in medical field in the following way
 Time-resolved fluorescence spectroscopy
 Confocal fluorescence microscopy images

Application in medical field

 Diagnosis of cancer of the gastrointestinal (GI) tract, bronchi/lung, skin, head and
neck, and brain
 Ophthalmic pathologies
 Atherosclerotic cardiovascular disease
 Vibrational (IR) Spectroscopy of Diatomic Molecules
Vibrational (IR) Spectroscopy:
Those molecules give IR spectra who can change their dipole moment during their
vibrational transition.

All molecules are IR active except homo diatomic molecules.

e.g. CO, HCl, O2, N2, CO2, NO2

Fundamental vibration modes:

Stretching Bending
Asymmetric and Symmetric In plane Out of Plane
Rocking and Scissoring Wagging and Twisting

nsym> nasym> nbend

 Vibrational (IR) Spectroscopy of Diatomic Molecules
Calculation of fundamental modes:

3n = vibrational mode + translational mode + rotational mode

Vibrational mode = 3n – (translational mode + rotational mode)

For linear: 3n-5 (stretching=n-1 and bending = 2n-4)

For non-linear: 3n-6 (stretching=n-1 and bending = 2n-5)

Where n = # of atoms present in the molecule

 Vibrational (IR) Spectroscopy of Diatomic Molecules
Vibrational frequency range:
IR Region

Near IR Mid IR Far IR

125000 cm-1 4000 cm-1 667 cm-1 50 cm-1

Applicable region

0.8 mm 2.5 mm 15 mm 200 mm

Single bond region = 2500-4000 cm-1

Triple bond region = 2000-2500 cm-1
Double bond region = 1500-2000 cm-1
Finger print region = 600-1500 cm-1
Vibrational (IR) Frequency Range in a Corresponding Molecules
 Vibrational (IR) Spectroscopy of Diatomic Molecules
Types of transition:

Fermi resonance: Intermixing of fundamental frequency and adjacent overtone to give

equi-energetic two peaks
 Factors Effect the IR Frequency
Types of transition:
Bond Strength: Higher the bond strength, higher would be the vibrational frequency

Mass of atom: Higher the mass of atom, lower would be the vibrational frequency

Hybridization: more s character increases the vibrational frequency

Resonance effect: Resonance has the effect of reducing the force constant K, and the
absorption moves to a lower frequency.

H-bond: Decreases the vibrational frequency.

 Rotational (Microwave) Spectroscopy of Diatomic Molecules
Rotational (Microwave) Spectroscopy:

Heavier diatomic or polyatomic molecules

e.g. Cl2, Br2, I2

Gaseous molecules with permanent dipole moment can give rotational spectra

Solid and liquid: Absence of free rotation, so can not give rotational spectra
 Introduction of NMR

NMR-Spectroscopic technique based on absorption of electromagnetic radiation in

the radio frequency region 4-900 MHz by nuclei of the atoms

Study for nuclei – 1H, 13C, 15N, 19F, 31P

Condition- Should have non-zero value spin quantum number (I)

Elements with odd atomic number or odd mass have the property of nuclear spin
Eg. 1H (I=1/2), 2H (I=1), 12C(I=0), 13C(I=1/2)
 Principle of NMR
Many nuclei have spin and all nuclei are electrically charged. If an external magnetic field
is applied, an energy transfer is possible between the base energy to a higher energy level
(generally a single energy gap). The energy transfer takes place at a wavelength that
corresponds to radio frequencies and when the spin returns to its base level, energy is
emitted at the same frequency. The signal that matches this transfer is measured in many
ways and processed in order to yield an NMR spectrum for the nucleus concerned.

Bo = strength of the applied magnetic field

g= Magnetogyric ratio (ratio of Nuclear magnetic moment to angular moment)
For H (I=1/2) possible orientation 2I+1 = 2
NMR Spectrum and Solvent used for Acquiring Spectrum

Deuterated solvent –
D2O
CDCl3
C6D6

Plot of intensity of NMR signal vs.

magnetic field (frequency) in reference
to TMS
Chemical shift, Shielding, Deshielding, and Chemical Equivalence

Chemical shift-Difference in ppm between the resonance frequency of the observed

frequency and H of TMS

Shielding- Higher electron density around the nucleus shields the nucleus from the
external magnetic field and signals are upfield

Deshielding- Lower electron density around the nucleus shields the nucleus from the
external magnetic field and signals are downfield
 Factors Affecting Chemical shift

Electronegative groups- deshield

Magnetic anisotropy of p-systems- Both shield and deshield

Hydrogen bonding- deshield

Simplified Correlation Chart for Proton Chemical Shift Values
Spin-spin Splitting (n + 1) Rule
Spin-spin Splitting (n + 1) Rule
Spin-spin Splitting (n + 1) Rule
Intensity Ratio in NMR

Pascal’s Triangle
Analysis of NMR Spectrum
 Application of NMR in Medicine

MRI is specialist application of multidimensional Fourier transformation NMR

 Surface Characterization Techniques

Surfaces may be characterized with respect to their topography (i.e. roughness),

chemistry, surface orientation, and thickness of chemically homogeneous regions
at the surface. Typically, mean free path of probe into sample is low or sample is
comprised only of surface atoms.

Contact Methods vs. Non-contact Methods

 Surface Characterization Techniques
Microscopy Spectroscopy
 Scanning Electron Microscope  Surface Enhanced Raman Spectroscopy

 Scanning Tunneling Microscopy  TOF-Secondary Ion Mass Spectrometry

(TOF-SIMS)
 Transmission Electron Microscopy
 Near-IR Spectroscopy
 Atomic Force Microscopy
 X-ray Photoelectron Spectroscopy
 Low Energy Electron Diffraction (XPS)/Electron Spectroscopy for
(LEED) Chemical Analysis (ESCA)

 Auger Electron Spectroscopy (AES)

Classification of Characterization Techniques based upon ‘Probe’
used on sample
 Scanning Electron Microscope (SEM)
Optical microscope- Low resolution (~ mm)
SEM – High resolution (100 nm)

Information provide ----

Images of external morphology,

A finely focused beam of electrons impinges on the surface of the solid sample. In
analog instruments, the beam of electrons is scanned across the sample in a raster
scan by scan coils. The resulting raster scanning pattern is similar to that used in
the cathode-ray tube (CRT) of a television set in which the electron beam is
(1) swept across the surface linearly in the x direction,
(2) returned to its starting position-
(3) shifted downward in the y direction by a standard increment.

Bombarding electrons- primary electrons

Dislodge electrons -secondary electrons
Instrumentation
SEM Images
 Surface Enhanced Raman Spectroscopy (SERS)

 Raman spectroscopy: inelastic scattering of photons from an atom or

molecule in chemical entities is utilized to analyze the composition of solids,
liquids and gases.

 However, the low cross-section limits its applications.

 The introduction of surface-enhanced Raman spectroscopy in 1974 has

attracted a lot of attention from researchers due to the large enhancement of
weak Raman signal, which facilitates identification in chemical and biological
systems

 A thin film of colloidal metal particles is deposited on a glass slide and a drop
or two of the sample solution spotted on the film
 Raman Spectroscopy: Overview
 A vibrational spectroscopy
- IR and Raman are the most common vibrational spectroscopies for
assessing molecular motion and fingerprinting species
- Based on inelastic scattering of a monochromatic excitation source
- Routine energy range: 200 - 4000 cm–1

 Complementary selection rules to IR spectroscopy

- Selection rules dictate which molecular vibrations are probed
- Some vibrational modes are both IR and Raman active

 Great for many real-world samples

- Minimal sample preparation (gas, liquid, solid)
- Compatible with wet samples and normal ambient
- Achilles Heal is sample fluorescence
Origins of Rayleigh and Raman Scattering
 SERS of Rhodamine 6G

774 cm-1
C-C stretches (A term
Raman scattering?)
614 cm-1

1650 cm-1

Reason for enhancement

Bend vibrations
 electrochemically roughened surface
 electromagnetic effect

Frequency (cm-1)
 Diffraction and Scattering

Scattering Diffraction
Interaction with Interaction with
a single particle a crystal

A process in which a parallel beam of

A process in which light is deviated from radiation is bent as it passes by a sharp
the straight path on interaction with a barrier or through a narrow opening
single particle

Diffraction- X-ray diffraction (XRD)

Scattering – Dynamic light Scattering (DLS)
 X-ray diffraction (XRD)

X- ray diffraction : atomic planes of a crystal cause an incident

beam of X- rays to interfere with one another as they leave the
crystal

 non-contact and non-destructive technique

 used to understand the crystalline phases, different polymeric
forms and the structural properties of the materials

 Every crystalline substance gives a pattern; the same

substance always gives the same pattern; and in a mixture of
substances each produces its pattern independently of the
others”

 XRD pattern of a pure substance like a fingerprint of the

substance.
 Bragg’s equation
Path difference between Ray-1 and Ray-2
= AB+ BC
= (d Sin + d Sin) = (2d.Sin)

For constructive interference, this path

difference should be an integral multiple of l:
nl = 2d Sin

Information provide
 Measure the average spacing's between layers or rows of atoms
 Determine the orientation of a single crystal
 Find the crystal structure of an unknown material
 Measure the size, shape and internal stress of small crystalline regions
 Dynamic Light Scattering (DLS)

 Particle size can be determined by measuring the random change in intensity of light
scattered from suspension.

 It measure and interpolate the light scattering up to microsecond

 So it measure real time intensity, thus measuring the dynamic properties size
distribution, hydrodynamic radius, and diffusion coefficient

Applications
 measure hydrodynamic Size of nanoparticle, protein and biomaterial
 Study stability of nanoparticles as function of time
 Good for detecting the aggregation of the particles
 Experimental Set up

 Brownian motion of the particle is random motion due to

the bombardment by the solvent molecule surround them.
 Stokes-Einstein equation
 Limitation of DLS

 We measure the hydrodynamic radius of the particle, not able to measure the actual
size of the particle

 The particles having size greater than 1000 nm are not measured by this method

 Size of Solid particles are not measured by DLS