BP701T IMA-I B.Pharmacy 7th Semester 2025 (2024-2025) Previous Year's Question Papers/Notes Download - HK Technical PGIMS

Dr. Minaketan Sahoo, IPT, Salipur B Pharm 7th Semester
BP701T. INSTRUMENTAL METHODS OF ANALYSIS
This subject deals with the application of instrumental methods in qualitative and quantitative analysis
of drugs. This subject is designed to impart a fundamental knowledge on the principles and
instrumentation of spectroscopic and chromatographic technique. This also emphasizes on theoretical
and practical knowledge on modern analytical instruments that are used for drug testing.
UNIT -I
UV Visible spectroscopy
Electronic transitions, chromophores, auxochromes, spectral shifts, solvent effect on absorption
spectra, Beer and Lambert’s law, Derivation and deviations.
Instrumentation - Sources of radiation, wavelength selectors, sample cells, detectors- Photo tube,
Photomultiplier tube, Photo voltaic cell, Silicon Photodiode.
Applications - Spectrophotometric titrations, Single component and multi component Analysis
Fluorimetry
Theory, Concepts of singlet, doublet and triplet electronic states, internal and external conversions,
factors affecting fluorescence, quenching, instrumentation and applications1
UV VISIBLE SPECTROSCOPY
Introduction to Electro Magnetic Radiation (EMR):
Light travels in a straight line, but phenomenon like interference, refraction, diffraction, etc. could
not explain this. To explain these phenomena, light is supposed to travel in waves. Light or EMR is
a form of energy that is transmitted through space at a constant velocity of 3 × 10଼ 𝑚𝑒𝑡𝑒𝑟/𝑠𝑒𝑐𝑜𝑛𝑑.
These radiations are said to have dual nature exhibiting both:
 Wave character
 Particle character or corpuscular theory
According to wave theory, light travels in the form of waves. This wave motion consists of oscillating
electric (E) & magnetic (H) fields (vectors) directed perpendicular to each & perpendicular to the
direction of the propagation of wave (Fig. 1).
Fig. 1: Wave nature of light
Quantum theory of EMR:
 Quantum theory describes the EMR as consisting of a stream of energy packets, called
Photons or Quanta, which travel in the direction of propagation of the beam with the velocity
of light.
 Thus, during emission or absorption of light by chemical species, the energy changes take
place only discretely always as integral multiples of small units of energy i.e. photon.
 The energy of the photon is proportional to the frequency of radiation, i.e. E α ν, or, E = hν ,
Where, h = Plank’s constant = 6.626 x 10-27 erg.sec.
The energy of a photon is called quantum of energy & this depends only on the frequency but
not on the intensity of radiation.
 The waves are characterized by their wavelengths or frequencies or wave numbers.
 The energy carried by an EMR is directly proportional to the frequency.
 All types of radiations travel with the same velocity & no medium is required for their
propagation. They can travel through vacuum also.
 When visible light (a group of EMR) is passed through a prism, it is split up into seven colours
which correspond to definite wavelengths. This phenomenon is called ‘dispersion’.2
Spectroscopy:
 The word spectroscopy is derived from spectrum which means a band of different colours
formed due to difference in wavelength and skopin means examination or evaluation. Thus,
spectroscopy is the branch of science that deals with the examination or evaluation of
spectrum. It is defined as the interaction between the matter & EMR. It deals with emission
as well as absorption spectra.
 It is used to measure the energy difference between various molecular energy levels & to
determine the atomic & molecular structures. The instruments used in such studies are called
spectrophotometer.
 If EMR (of certain wavelength range) are passed through the substance under analysis for
sometimes, then radiations of certain wavelengths are absorbed by the substance. The
wavelengths which are absorbed characterize some practical functional groups present in the
compound or the compound itself. This dark pattern of lines which corresponds to the
wavelengths absorbs is called Absorption spectrum. After absorption, the transmitted light is
analyzed by the spectrometer relative to the incident light of a given frequency. The absorbed
energy may heat up the sample or is re-emitted.
 An emission spectrum is produced by the emission of radiant energy by an excited atom. The
excitation of atoms can be brought about thermally (by heating the substance strongly) or
electrically (by passing electric discharge through the vapours of the substance at a very low
pressure). When an electric discharge is passed through the vapours of the substance, energy
is absorbed & electrons in the ground state are promoted to Meta-stable states. When electrons
from the Meta-stable state jump to the lower energy state, then some energy of definite
frequency is released as radiation. If this radiation emitted is analyzed with the help of a
spectroscope, an emission spectrum is observed.
Characteristics/Units of wave (Fig.2):
a. Wavelength: It is the distance between the adjacent crests or troughs in a particular wave. It is
denoted by ‘λ’ (lambda). It can be expressed in Angstrom (0A) or nanometer (nm) or
millimicrons (mμ) or centimeter (cm) or micrometer (μm).
1 nm = 10-9 m = 10-3 μm = 10 0A = 10-7 cm = 1 mμ
Nanometer is frequently used in UV-Visible technique.
b. Wave Number: It is the reciprocal of wavelength & it is expressed in per centimeter; or it is
defined as the total number of waves which can pass through a space of 1 cm. It is expressed as
‘ū (nu bar)’. It is frequently used in IR technique.
c. Frequency: it is defined as the number of waves which can pass through a point in one second.
It is expressed as ν (nu) in cycles per second or in Hertz (Hz).
1 Hz = 1 cycle sec-1
Frequency α ଵ
௪௔௩௘௟௘௡௚௧ i.e., greater the wavelength, smaller is the frequency.
Frequency, ν = ௖
஛ Where, c = velocity of EMR (light) = 2.998 x 10-8 cm/sec
d. Energy: Energy of a particular wave is calculated as
E = hν = ௛௖
஛ = hcū Where, h = Plank’s constant = 6.626 x 10-27 erg. sec.3
Fig. 2: Characteristics of wave
Electromagnetic spectrum:
 The electromagnetic spectrum, for most spectroscopic purposes, is considered to be consisting
of region of radiant energy ranging from wavelengths of 10 m to 1 x12-12 cm.
 When a molecule absorbs EMR, it can undergo various types of excitation. This excitation
may be
 Electronic excitation,
 Rotation excitation,
 Excitation leading to a change in nuclear spin,
 Excitation resulting in bond deformation & so on.
 If the energy available approaches the ionization potential of the molecule, an electron may
be ejected & ionization may occur.
 Since each mode of excitation requires a specific quantity of energy, the different absorptions
appear in different regions of the electromagnetic spectrum.
 The various regions of electromagnetic spectrum are set out (Table:1 & Fig.3) & are labeled
either according to the wavelength/ wave no. range used, or according to the type of the
molecular energy levels involved, e.g. UV (electronic) spectra, IR (vibrational) spectra or RF
(NMR) spectra.
Table-1: Various regions of electromagnetic spectrum
Type of
Radiation Wavelength Wave no. Type of molecular spectrum
RF > 100 mm < 3 x 109 Hz NMR (Spin orientation)
Microwave 1 – 100 mm 10 – 0.1 cm-1 Rotational
Far IR 50 μm – 1mm 200 – 10cm-1 Vibrational fundamental or rotational
Mid IR 2.5 μm – 50 μm 4000 – 667 cm-1 Vibrational fundamental
Near IR 780nm – 2.5 μm (13 – 4) x 103 cm-1 Vibrational (overtones)
Visible 380nm – 780nm (2.6 – 1.3) x 104 cm-1 Electronic (valence orbital)
Near UV 200nm – 380nm (5 – 2.6) x 104 cm-1 Electronic (valence orbital)
Vacuum UV 10nm – 200nm (102 – 5) x 104 cm-1 Electronic (valence orbital)
X-rays 10pm – 10nm 109 - 106 cm-1 Electronic (core orbitals)
Gamma rays 10-10 cm 1010cm-1 Mossbauer effect (Nuclear transitions)
excited states of nucleiCosmic rays 10-12 cm 1012cm-14
Photons of electromagnetic radiation of different energies (frequencies or wavelengths) interact
with molecules in a variety of ways:
1. X-rays can excite and eject inner shell electrons, causing ionization and bond fragmentation.
2. Ultraviolet (UV) radiation causes high energy electronic transitions and visible radiation
induces low energy electronic transitions in molecules. The resulting excited states may
relax via bond breakage or various radiative and non-radiative pathways.
3. Infrared (IR) radiation causes vibrations in molecular bonds, such as bond stretching, bond
bending etc. Therefore, IR spectroscopy is often called vibrational spectroscopy.
4. Microwaves can cause the molecules to rotate in the gas phase. This is the subject matter of
microwave or rotational spectroscopy.
5. Radio waves can induce resonance of atomic nuclei rotation in a strong magnetic field. This
is the subject of nuclear magnetic resonance (NMR) spectroscopy.
Fig.3: Electromagnetic radiation of different frequencies or wavelengths5
Absorption of EMR by organic molecules:
 When a molecule absorbs radiation, its energy increases in proportion to the energy of the
photon as E = hν
 Since the energy absorbed by a molecule is quantized, there will not be continuous absorption
by a molecule throughout a particular spectral range; instead the molecule absorbs those
frequencies which will provide it with the exact quantity of energy (quantum theory) necessary
to raise its normal energy level to a higher level or levels.
 Thus, when light radiations are passed through an organic compound or, when an organic
molecule interacts with EMR, then electrons of the component atoms are excited. It may change
its energy from E1 to E2 by absorption of radiation of frequency ‘ν’ so that, E2 - E1 = ∆E = nhν,
when, n = an integer.
 The lowest state of energy of an atom or molecule is called ground state. By absorbing one
quantum of energy ‘hν’, the molecule is promoted to the next higher level & is said to be in the
‘b’. Similarly, absorption of more energy in integral multiples of hν, will result in further
excitation to next higher energy levels i.e. excited state.
 Study of absorbed radiations from a continuous source that are utilized in raising the internal
energy of a molecule constitutes absorption spectroscopy.
 In general, an absorption spectrum curve will consist of a series of peaks, each peak coinciding
with a value of ‘ν’ which satisfies the relation (E2 - E1 = nhν).
 After absorption of energy, the excited species returns to the ground state by emitting the
energies radiations. The study of this emitted radiation constitutes emission spectroscopy.
 The portions of EMR which do not satisfy relation may either simply pass through the matter
or undergo scattering or reflection with or without change of wavelength.
When molecules absorb energy and get excited, then either of the following energy changes occurs.
a. Transition of an electron to high energy level,
b. Change in the intermolecular vibrations of the molecule,
c. Change of the moment of inertia of the molecule around its center of gravity,
d. Transitions between electronic levels are found in the UV & visible regions,
e. Transitions between vibrational levels, but within same electronic level in mid & near IR region,
f. Transitions between neighboring rotational levels in far IR & microwave regions.6
UV-Visible Spectroscopy
UV wavelength region  200nm – 380nm, Visible wavelength region 380nm – 780nm
 It is also known as Electronic spectroscopy since it involves the promotion of electrons (σ,
π, n- electrons) from the ground state to the higher energy state.
 It very useful to measure the number of conjugated double bonds & also aromatic conjugation
within the various molecules.
 It distinguishes between conjugated & non-conjugated systems, α, β- unsaturated carbonyl
compounds from β, γ-analogues, homo annular & hetero annular conjugated dienes, etc.
Principle:
Since the energy levels of a molecule are quantized, the energy required to bring about the excitation
is a fixed quantity. Thus, the EMR with only a particular value of frequency will be able to cause
excitation. If the substance is exposed to radiation of some different value of frequency, energy will
not be absorbed & thus, light radiation will not suffer any loss in intensity.
 When UV or Visible radiation is passed through a substance, absorption of energy results in
the promotion of electron from the ground electronic state to the excited electronic state. The
amount of absorption of energy depends upon wavelength of the radiation & the structure
of compound.
 During the process of absorption, a large number of photon-molecule collisions are possible
but only those collisions will cause absorption of energy in which the energy of the photon
matches the energy difference between the ground & the excited electronic state of the
molecule. The absorption of energy is quantized.
 The wavelength at which maximum absorption of radiation takes place is called λmax.
This λmax is characteristic or unique for every substance & this is a qualitative aspect, useful
in identifying the substance (Fig.4).
 λmax is not usually affected by concentration of the substance. The absorbance of a solution
increases with concentration of a substance, but there is no change in λmax when concentration
changes.