Analytical Techniques and

Instrumentation
 Analytical Techniques and Instrumentation

1. Spectroscopic methods of analysis – Introduction to spectroscopy – Energy concepts,

properties of EMR, General features of spectroscopy, Types of molecular spectra,
Interaction of EMR with matter, Instrumentation, Applications 6

2. Microwave spectroscopy – Introduction, Diatomic rigid rotor model, Non-rigid

diatomic molecules, Instrumentation, Applications, Advantages and disadvantages 4

3. Raman spectroscopy – Basic principles, Properties of Raman lines, Mechanism of

Raman effect- Classical and quantum theories, Techniques and instrumentation,
Applications 2

4. Infrared spectroscopy – Background and theory, Diatomic molecule as a harmonic

oscillator, Numerical problems, Diatomic molecule as anharmonic oscillator, Diatomic
vibrating rotator, Vibrations of polyatomic molecules, Instrumentation and techniques
of dispersive and FT-IR, Double beam IR spectrometer, Uses of IR spectrum,
Advantages and limitations 5
5. UV-visible spectroscopy – Theory of electronic spectra of diatomic molecules,
Types of transitions in organic molecules, Instrumentation, Double beam
spectrophotometer, Applications in qualitative and quantitative analysis,
Spectrophotometry, Beer’s law and its derivation, Deviations from Beer’s Law,
Numerical problems 5

6. NMR spectroscopy – Basic principles of NMR, Chemical shift, Spin-spin coupling,

Instrumentation, Applications of proton NMR spectroscopy 3

7. Chromatographic Techniques – General concepts, Classification, Column

chromatography, HPLC, Instrumentation, Applications, Thin layer chromatography,
Experimental techniques, Applications, Advantages and disadvantages, Gas
chromatography, principles, instrumentation and applications 9

8. Electroanalytical methods- Conductometric titrations – basic principles,

Applications in titrations involving weak and strong acids and bases, Potentiometric
titrations- Fundamental principles, Applications involving neutralization, redox,
precipitation and complexation types 4
 References

1. D.A. Skoog, J. Holler, F.T.A. Nieman, Principles of Instrumental Analysis, 5th Edn,
Saunders, Philadelphia, 1992

2. D. A. Skoog, D. M. West and F. J. Holler, Fundamentals of Analytical Chemistry, 5th

Edn, Saunders College Publishing, Philadelphia, 1988

3. Vogel's Textbook of Quantitative Chemical Analysis, GH Jeffery, John Wiley & Sons Inc,
5th Edn, 1989

4. G. Chatwal, S. Anand , Instrumental Methods of Chemical Analysis, Himalaya

Publishers, Bombay, 1996

5. G.W. Ewing, Instrumental Methods of Chemical Analysis, Mc-Graw-Hill, 1989

6. C.N. Banwell, C.N. McCash, Fundamentals of Molecular Spectroscopy, McGraw-Hill,

London, 1972
Spectroscopic methods of
analysis
Advantages of spectroscopic methods
over chemical methods
• Easier and faster
• Provide more information – all functional
groups and structural features can be
detected with small amounts of sample
• Non – destructive; entire sample can be
recovered

Spectroscopic methods used in organic chemistry –

ultraviolet – visible, infrared, nuclear magnetic
resonance and mass spectroscopy
Spectroscopy
“Seeing the Unseeable” Using electromagnetic
radiation as a probe to obtain information about
atoms and molecules that are too small to see.
Electromagnetic radiation is propagated at the
speed of light through a vacuum as an oscillating
wave.
 Electromagnetic Radiation &
Electromagnetic spectrum
• The radiations which travels with speed of light
are called Electro magnetic radiations.
• It vibrates perpendicular to the direction of
propagation with a wave motion.
• It is broken in to several regions called as Electro
Magnetic Spectrum
• Different regions of the electromagnetic spectrum
provide different kinds of information as a result
of interactions.
• Light may be considered to have both wave-like
and particle-like characteristics.
 Y

Electronic
vector
Wavelength
λ Electronic
component

X Direction of
propagation

Z
Magnetic component

Representation of Electromagnetic Wave

Electromagnetic relationships:
λν = c λ 1/ ν
E=hν E ν
E = hc/λ E 1/λ
Wave number = 1/λ (Kaiser – K)
λ = wave length
ν = frequency λ
c = speed of light c
E = kinetic energy
h = Planck’s constant
Two oscillators will strongly interact when their
energies are equal.
E1 = E2
λ1 = λ2
ν 1= ν 2
If the energies are different, they will not strongly
interact!
We can use electromagnetic radiation to probe
atoms and molecules to find what energies they
contain.
Some electromagnetic radiation ranges
The Spectrum and Molecular Effects

=>
 Calculate the frequency, wave number and energy for
typical uv radiation of wave length 2000 Å.
Frequency = 1.5x1015 cycles sec-1
Wave number = 5x104 cm-1
Energy = 9.93 x 10-19 J

Calculate the wave number for IR radiation with

wavelength 5 μm.
Wave number = 2000 cm-1

Calculate the range of wavelength in nanometers that

corresponds to every transitions of 1.5 to 8 eV.
Ans = 825 nm
 Properties of Light
1.) Refraction: change in direction in the travel of a light beam when it comes at
an angle to a boundary (interface) between two transparent media with
different densities.

Pencil appears to bend at water/air

interface due to refraction of light
a.) Refractive Index (hi): medium/substance specific
hi = c/vi
c = speed of light in a vacuum
vi = speed of light in medium of interest at the
specified frequency

Note: hi ≥ 1 since vi ≤ c
Typical values for
refractive index:
Refractive
Material
Index
Air 1.0003 Values of h are wavelength dependent
Water 1.33 (useful for design of prisms)
Glycerin 1.47
Immersion Oil 1.515
Glass 1.52
Flint 1.66
Zircon 1.92
Diamond 2.42
Lead Sulfide 3.91
2.) Reflection: when radiation crosses an interface between media that differ in refractive
index, some or all of the light travels back into the medium from where it travel

normal

Ir

Io

• Reflected light comes out at same angle as incident beam, but on other side of normal.
- Reflection occurs at each interface (when enters and exit)
3.) Diffraction: the bending of a parallel beam of light (or other electromagnetic
radiation) as it passes a sharp barrier or through a narrow opening.

a.) most pronounced when size of slit or opening is approximately the same
size as the frequency of light.

Radiation of a point source of light in

all directions on other side of slit.
b.) Interference – diffraction is a consequence of interference

i.) Two types of interference.

1) constructive – waves “in-phase” electric fields are additive

When two light waves of the same

wavelength (color) combine exactly
in phase (in step) their amplitudes
add to produce a large (brighter)
wave of maximum intensity.

2) destructive – waves “out of phase” electric fields subtract

If the light waves combine out of

phase (out of step) their combined
amplitudes are less, and may even
totally cancel each other!
 Principle of Spectroscopy
All Organic molecules interact with EMR – they
absorb energy
While absorbing energy a transformation occurs
(temporary / permanent)
Lower energy radiation may cause a molecular
rotation or a bond vibration
Higher energy radiation may cause the promotion
of electrons to higher energy levels or bond
cleavage
 Principle of Spectroscopy
Whatever may be the transformation, the molecule
absorbs only the wavelength of radiation with exactly
the energy necessary for the transition
Absorption of wavelengths of radiation is selective for a
particular transition which depends on the structure of
the molecule
By measuring the absorption spectra of known
compounds, we can correlate the wavelengths of
energy absorbed with characteristic structural features
This information can be used to determine the
structure of unknown compounds
Gamma-rays - Radioactive materials can emit
gamma-rays. Big particle accelerators that
scientists use to help them understand what
matter is made of may sometimes generate
gamma-rays. But the biggest gamma-ray
generator of all is the Universe! It makes gamma
radiation in all kinds of ways.
• Wave length range 0.02 – 1Å
• Energy – 109 – 1011 Joules/gram atom
X-rays - Doctor uses them to look at bones. Hot
gases in the universe emit X-rays.
• Wave length range 1 - 10Å
• Energy – 103 k Joules/gram atom
Ultraviolet - We know that the Sun is a source of
ultraviolet (or UV) radiation, because it is the UV
rays that cause our skin to burn! Stars and other
"hot" objects in space emit UV radiation.
• Wave length range – 1 – 400 nm
• Energy – 1907 – 71.5 kJ/mol
Visible - This is the part that our eyes see. Visible
radiation is emitted by everything from fireflies
to light bulbs to stars and also by fast-moving
particles hitting other particles.
• Wave length range – 400 - 750 nm
• Energy – 35.7 – 71.5 kJ/mol
Infrared - we often think of this as being the
same thing as 'heat', because it makes our
skin feel warm. In space, IR light maps the
dust between stars.
• Wave length range – 0.7 – 200 μ
• Energy – 35 – 143 kJ/mol
Microwaves - they will cook your popcorn in just
a few minutes! In space, microwaves are used by
astronomers to learn about the structure of
nearby galaxies, including our own Milky Way.
• Wave length range – 0.1 mm – 1 cm
• Energy – 0.57 – 9.4 10-5 kJ/mol
• Corresponds to changes in the rotation of the
molecules
Radio waves - Kind of energy that radio stations emit
into the air. But radio waves are also emitted by other
things such as stars and gases in space. Radio waves
emitted by these objects can be used to learn what
they are made of.
• Wave length range – 10 m – 1 cm
• Energy – 0.001 – 10 Joules/mole
• Used in NMR
The study of the interaction between ELECTROMAGNETIC
RADIATION and MATTER
The study of spectroscopy deals with absorption as well as emission
spectra.

Emission

An emission spectra is produced by the emission of radiant energy by

an exited atom.
The excitation of atoms can be brought about thermally like heating
the substrate strongly or electrically like passing electric Discharge
through the vapours of the substrate at a very low pressure.
When the electric discharge is passed through the vapours of the
substrate, energy is absorbed and electrons in the ground state are
promoted to meta – stable states.
When the electrons from the meta stable state jump to the lower
energy state, then some energy of definite frequency is released as a
radiation.
If this radiation is analysed with the help of spectroscope, an
emission spectrum is observed.
 Discrete Spectrum Again, the
pattern of the
Emission lines
determines the
identity of the
element.

Ex: comets, nebula

& certain stars

Cold, Thin,hot
empty Emission
gas
space spectrum

Spectra
Absorption

If electromagnetic radiation (of certain wavelength range ) are

passed through the substrate under analysis of sometime, then
radiation of certain wavelengths are absorbed by the substrate.
The wavelength which is absorbed characterize some
particular functional groups are present in the compound. This
dark pattern of lines which corresponds to the wavelengths
absorbed is called absorption spectra.
After absorption, the transmitted light is analyzed by the
spectrometer relative to the incident light of a given frequency.
In absorption spectra energy may heat up the sample or is re-
emitted.
 Discrete Spectrum

Absorption

Each element
has a unique
signature of
absorption lines.
Ex: stars, That pattern
planets w/ helps scientists
atmospheres, & identify the
galaxies element(s).

Spectra Cool,
thin gas Absorption
spectrum
Hot object
Atomic and molecular spectroscopy
Atomic spectroscopy:
 Atomic spectroscopy deals with the interaction of
electromagnetic radiation with atoms (which are
most commonly in their lowest energy state – ground
state)
 The electrons in the atomic orbitals can move from
one energy level to another by either absorbing or
releasing the energy they have.
 The energy that the electron absorbs or emits should
equal the energy difference between the two
quantized energy levels, ΔE = hν
 Since each and every chemical element has a unique
number of electrons at their ground state, an atom will
absorb or release energy in a pattern unique to its
elemental identity.
 Therefore, they will absorb/emit photons in a
correspondingly unique pattern.
 We can determine the elemental composition of a
sample by measuring the changes in light wavelength
and light intensity.
Molecular Spectroscopy
 Molecular Spectroscopy deals with the interaction of
electromagnetic radiations with molecules.
 This results in transitions between the rotational and vibrational
levels in addition to the electronic transitions.
 As a result the spectra is more complicated than those of atoms.
 The sample will absorb particular wavelengths but not all,
depending on the chemical composition of the sample.
 Therefore, the non-absorbed wavelengths pass on through the
sample.
 Depending on the absorbed wavelengths and the intensity of
absorption, we can determine the nature of the energetic
transitions that a molecule is able to undergo, and therefore,
gather the information about its structure.
Types spectra given by molecular species
a) Rotational (microwave spectra) – Arises due to the
transitions between the rotational energy levels of a gaseous
molecule on the absorption of radiation in the microwave
region.
 Molecules possessing permanent dipole moment show this
spectra (HCl, CO, H2O etc)
 Homonuclear diatomic molecules like H2, N2, Cl2 etc and
linear polyatomic molecules like CO2 which do not have
dipole moment, do not show microwave spectra.
b) Vibrational rotational (infrared) spectra – Arises due the
transitions between the vibrational energy levels of a molecule
on the absorption of IR radiation.
 IR spectra is shown by the molecules when vibrational
motion is accompanied by change in the dipole moment of
the molecule.
C) Raman Spectra – Raman spectra is related to
vibrational and vibrational-rotational transitions.
 In this case the scattering of light is measured (not
the absorption).
 The scattered with different frequencies than the
incident radiation is called Raman scattering.
 It is observed in the visible region.
d) Electronic spectra – Arises due to the electronic
transitions in a molecule by absorption of radiations in
the visible and UV region.
 Electronic transitions in a molecule is invariably
accompanied by vibrational and rotational
transitions, the electronic spectra of molecules are
highly complex.
e) Nuclear Magnetic Resonance (NMR) spectra – Arises
due to the transitions induced between the nuclear spin
energy levels of a molecule in an applied magnetic field.

In addition to these, Electron Spin Resonance (ESR)

Spectra, Mossbauer Spectra etc. are also widely used.
Different types of Molecular energies:
a) Internal energy of the molecule, Eint
= Eelectronic + Evib + Erot
b) Translational energy, Et – due to transitional
motion
According to quantum mechanical model, Et is
given by
ℎ2 𝑛𝑥 2 𝑛𝑦 2 𝑛𝑧 2
Et = + + where M is the
8𝑀 𝑎 𝑏 𝐶
mass of the molecule, a, b and c are the dimensions
of the rectangular box in which the molecule is
considered and the values of n are integers.
c) Rotational energy, Erot
Rotational energy, Erot = ½ Iω2
Where, I is the moment of inertia and ω is the
angular velocity of the molecule.
However, according to the quantum mechanical
ℎ2
model, Erot = 2 J(J+1); where, I is the moment
8𝜋 𝐼
of inertia of a simple linear molecule J is
rotational quantum number, = 0 or +ve integer.
Note: Only polar molecules can rotate in an EM
field.
d) Vibrational energy, Evib = hν (v + ½); ν is
vibrational frequency, v is vibrational quantum
number 0 or positive integer.
e) Electronic energy –
Associated with motion of electrons
considering the nuclei of the atoms/molecules
as fixed points.
Increase in the electronic energy occurs due to
increase in KE and PE of electrons of the
molecules.
Energy needed to move the electron from the
ground state to excited state varies with the
type of the molecular bond in which the
electron exists.
Born – Oppenheimer approximation:
Total energy of the molecule, E = Ee + Ev + Er
Note: Ee > Ev > Er
Note: Translational energy is not quantized and negligible.
According to Uncertainty principle, uncertainty is of the order of
ℏ/𝑅 where R is the dimension of the molecule which is assumed
linear. Thus,
𝑝2 ℏ2
𝐸𝑒 ~ ~ 2 ~𝑠𝑒𝑣𝑒𝑟𝑎𝑙 𝑒𝑉
2𝑚 2𝑚𝑅
1 1 𝑘 1/2
𝐸𝑣 = v + ℏ𝑤 = 𝑣 + ℏ ;
2 2 𝑀
Where, Ev = 0, 1, 2 3,……; k is the force constant, M is the mass of
the nucleus.
𝑘 1/2
For fairly low vibrational mode, 𝐸𝑣 ∼ ℏ ∼ 0.1 eV
𝑀
 ℎ2
Er = 2 𝐽(𝐽 + 1); for lower rotational modes,
8𝜋 𝐼
Er ∼ 0.001 eV
Thus, Eelectronic > Evib > Erot
Interaction of EMR with matter
 If the photons possess appropriate energies, they may
be absorbed by the matter resulting in electronic
transition, vibrational changes or rotational changes or
combinations of these.
 After the absorption, atoms and molecules become
excited.
 They give out energy quickly either by losing energy
in the form of heat or by re-emitting EMR.
 Radiation passing through the matter may not be
absorbed completely, it may undergo scattering or
reflection and may be re-emitted with same or
different wavelength.
 When the EMR is neither absorbed nor
scattered, it may undergo changes in orientation
or polarization.
 In some cases fluorescence or phosphorescence
also may occur.
 Molecular spectra

 Molecule absorbs EMR, gets excited and when it

returns to the ground state, it emits bands.
 Total energy state of a molecule includes Ee, Evib and
Erot.
 Rotational transitions involve far infra red and micro
waves.
 Vibrational transitions involve IR waves.
 Electronic transitions involve UV-Vis waves.
Types of Molecular spectra

Excited States
 Types of Molecular spectra
Absorption spectra:
• In absorption spectroscopy a photon is absorbed by an
atom or molecule, which undergoes a transition from a
lower-energy state to a higher energy, or excited state.
• The type of transition depends on the photon’s energy.
• The absorbing a photon of visible light promotes one of
the atom’s or molecule’s valence electrons to a higher-
energy level.
• When an molecule absorbs infrared radiation, on the other
hand, one of its chemical bonds experiences a change in
vibrational energy.
 When it absorbs electromagnetic radiation the number of photons
passing through a sample decreases.
 The measurement of this decrease in photons, which we call
absorbance, is a useful analytical signal.
 Absorption occurs only when the photon’s energy, hν, matches the
difference in energy, ΔE, between two energy levels.
 A plot of absorbance as a function of the photon’s energy is called an
absorbance spectrum.

Visible absorbance spectrum for cranberry

juice. The anthocyanin dyes in cranberry juice
absorb visible light with blue, green, and
yellow wavelengths. As a result, the
juice appears red.
Emission Spectra:
• When an atom or molecule in an excited state
returns to a lower energy state, the excess energy
often is released as a photon, a process we call
emission.
• There are several ways in which an atom or
molecule may end up in an excited state, including
thermal energy, absorption of a photon, or by a
chemical reaction.
• Emission following the absorption of a photon is
also called photoluminescence, and that following a
chemical reaction is called chemiluminescence.
Photoluminescence spectrum of the dye coumarin. The broad band
centered at approximately 500 nm is the dye’s emission band.
 Characteristics of Photoluminescence

 Fluorescence is short-lived with luminescence ending

almost immediately.

 Phosphorescence involves change in electron spin and

may endure for several seconds.

 In most cases, photoluminescent radiation tends to be

at longer wavelengths than excitation radiation.

 Chemiluminescence is based on an excited species

formed by a chemical reaction.
 Molecular Multiplicity, M

M = 2S + 1 S = spin quantum number of the molecule

= Σ net spin of the electrons in the molecule

 Most organic molecules have S = 0 because molecules

have even number of electrons thus the ground state
must have all electrons paired.
 M = 2 X (0) + 1 = 1; Molecules in the ground state
mostly have a singlet state; So, S1 and S2 for first and
second excited states.
 While molecules in the excited state, one e- may reverse its
spin
 S = (+1/2) + (+1/2) = 1 M = 2(1) + 1 = 3 = Triplet State=
T1
 A molecule with an even number of e- can not have a
ground triplet state because the spins of all electrons are
paired
 Molecules with one unpaired electron are in doublet state
(organic free radicals)
Electronic States
 Singlet State: electron spins paired, no splitting of energy
level. May be ground or excited state.
 Doublet State: free radical (due to odd electron).
 Triplet State: one electron excited to higher energy state,
spin becomes unpaired (parallel).
 Difference between triplet and singlet states
1. Molecule is paramagnetic in the T excited state and
diamagnetic in the S excited state.

2. S → T transitions (or reverse) are less probable

than S → S transitions.
Thus average lifetime of T excited state is longer than
the S excited state.
Also absorption peaks due to S-T transitions are less
sensitive than S-S transitions.

When an excited triplet state can be populated from

an excited S state of certain molecules, a
phosphorescence process will be the result.
Emission and Fluorescence
 Spectrophotometer
Spectrophotometer or spectrometer - The instrument
used to measure the amount of electromagnetic radiation
absorbed by an organic molecule

The recording is called the spectrum of the

compounds
 Numericals
1. Calculate the energy in Joules per quantum, calories per mole and
electron volts of photons of wavelength 3000 Aº
Solution : 6.62 x 10 -34 J.s x 3 x 108 m s-1
E= hν = h c /λ =
3000 x 10-10 m
= 6.62 x 10 -19 Joule
Energy per mole = Nhν = (6.02 x 1023 mol-1) x( 6.62 x 10 -19 Joule)
= 3.98 X 105 Joule/mol
3.98 X 105 Joule/mol
Calories per mole = = 95,300 cal mol-1
4.184 Joule/cal
95,300 cal mol-1
Ev of photons =
23, 060 cal mol-1 eV-1
= 4.13 eV
2. Calculate ν, ν and E for typical ultraviolet radiation of
wavelength 2000 A
6.62 x 10-27erg.s x 3 x 10 10cm s-1
Solution: E= hν = h c /λ =
2000 x10 -8 cm
= 9.93 x 10 -12 erg

ν = c /λ = 3 x 1010 cm s-1 / 2000 x 10-8 cm = 1.5 x1015 cycles. s-1

ν= 1/ λ = ν/ c = 1.5 x1015cycles.s-1 = 5 x 104 cm-1

3 x 1010 cm s-1
3. For infrared radiation of 5μm, what is the
wave number in cm-1 ?

Solution. Wave number = ν= 1/ λ

= 1/ (5 x10-4 cm )=2000 cm-1