Unit-5

Instrumental Methods and Applications

Electromagnetic radiation: Electromagnetic radiation is an electric and magnetic disturbance
travelling through space at the speed of light (2.998 × 108 m/s). It contains neither mass nor charge
but travels in packets of radiant energy called photons, or quanta.

Electromagnetic Spectrum: The Electromagnetic Spectrum is the range of all types of

Electromagnetic radiation.

Radio waves: A radio basically captures radio waves that are transmitted by radio stations. Radio
waves can also be emitted by gases and stars in space. Radio waves are mainly used for TV/mobile
communication.

Microwave: This type of radiation is found in microwaves and helps in cooking at home/office. It
is also used by astronomers to determine and understand the structure of nearby galaxies and stars.

Infrared: It is used widely in night vision goggles. These devices can read and capture the infrared
light emitted by our skin and objects with heat. In space, infrared light helps to map interstellar
dust.

X-ray: X-rays can be used in many instances. For example, a doctor can use an X-ray machine to
take an image of our bones or teeth. Airport security personnel use it to see through and check
bags. X-rays are also given out by hot gases in the universe.

Gamma-ray: It has a wide application in the medical field. Gamma-ray imaging is used to see
inside our bodies. Interestingly, the universe is the biggest gamma-ray generator of all.

Ultraviolet: The Sun is the main source of ultraviolet radiation. It causes skin tanning and burns.
Hot materials that are in space also emit UV radiation.

Visible: Visible light can be detected by our eyes. Light bulbs, stars, etc., emit visible light.
Absorption of radiation:

When ground state electrons or lower energy state electrons (E1) absorbs sufficient energy from
photons, they jump into the next higher energy level or higher energy state (E 2). In other words,
when the ground state electrons absorb energy which is equal to the energy difference between the
two energy states (E2 – E1), the electrons jumps from ground state (E1) to the excited state or
higher energy level (E2). The electrons in the higher energy level are called excited electrons.

The light or photons energy applied to excite the electrons can be mathematically written as

hv = E2 – E1

Where h = Planck’s constant

V = Frequency of photon

E1 = Lower energy level electrons or ground state electrons

E2 = Higher energy level electrons or excited state electrons

Beer-Lambert law: It states that the concentration of the solution and path length is directly
proportional to the absorption of light.

It can also be defined as the intensity of transmitted light decreases exponentially as the
concentration of the substance and distance travelled through the substance increases.
Applications of Beer-Lambert law:

 This law is important in the field of physics, chemistry and meteorology.

 The law is used in chemistry to measure the concentration of chemical solutions, analyze
oxidation, and measure polymer degradation.
 The law also explains the attenuation of radiation through the Earth’s atmosphere.

UV-VIS Spectroscopy:

Ultraviolet-visible spectroscopy refers to absorption spectroscopy in the ultraviolet-visible

spectral region. Ultraviolet-visible (UV-VIS) spectroscopy is an analytical method that can
measure the analyte quantity depending on the amount of light received by the analyte.

When the interaction between incident radiation and the electron cloud in a chromophore results in
an electronic transition involving the promotion of one or more of the outer shell or the bonding
electrons from a ground state into a higher energy state, ultraviolet-visible (UV-Vis) spectra are
derived.

Generally, the UV and visible spectral bands of substances are large and may not exhibit a high
degree of compound recognition accuracy. Nonetheless, they are sufficient for quantitative assays
and are useful as an alternate means of detection for several substances.

Electronic Transitions:

Electronic transition is that when the energy from UV or visible light (higher energy radiation in
the UV (200-400 nm) and visible (400-700 nm)) is absorbed by a molecule, one of its electrons
jumps from a lower energy to a higher energy molecular orbital.
σ → σ* Transition:
 Example: Molecular hydrogen, H2, the molecular orbital picture consists of one
bonding σ MO (HOMO), and a higher energy antibonding σ* MO (LUMO).

 If the molecule is exposed to light of a wavelength with energy equal to ΔE=HOMO-

LUMO energy gap, this wavelength will be absorbed and the energy used to jump one of
the electrons from the HOMO to the LUMO – in other words, from the σ to the σ* orbital.
This is referred to as a σ - σ* transition. ΔE for this electronic transition is 258 kcal/mol,
corresponding to light with a wavelength of 111 nm.

π -π* Transition:
 Where UV-vis spectroscopy becomes useful to most organic and biological chemists is in
the study of molecules with conjugated π systems.

 In these groups, the energy gap for π -π* transitions is smaller than for isolated double
bonds, and thus the wavelength absorbed is longer.

 Molecules or parts of molecules that absorb light strongly in the UV-vis region are
called chromophores.

 Example: MO diagram for 1,3-butadiene, the simplest conjugated system contains two
bonding orbitals, and two antibonding orbitals.
n - π* Transition:
The conjugated pi system in 4-methyl-3-penten-2-one gives rise to a strong UV absorbance at 236
nm due to a π - π* transition. However, this molecule also absorbs at 314 nm. This second
absorbance is due to the transition of a non-bonding (lone pair) electron on the oxygen up to a π*
antibonding MO

This is referred to as an n - π* transition. The nonbonding (n) MO’s are higher in energy than the
highest bonding p orbitals, so the energy gap for an n→π∗ transition is smaller than that of a π - π*
transition – and thus the n - π* peak is at a longer wavelength. In general, n - π* transitions are
weaker (less light absorbed) than those due to π - π* transitions.

The instrumentation of UV-Visible spectroscopy:

Light Source: UV-Visible spectrophotometers have a light source that emits a broad spectrum of
light covering the ultraviolet and visible regions. Tungsten filament lamps are commonly used for
the visible region (350-800 nm), while deuterium lamps or xenon arc lamps are used for the
ultraviolet region (190-350 nm).

Monochromator: The monochromator is a crucial component that selects a specific wavelength of

light from the broad spectrum emitted by the light source. It consists of prisms or diffraction
gratings that disperse light into its component wavelengths, and then a slit or another device is used
to select the desired wavelength.

Sample Compartment: The sample compartment holds the sample that is being analyzed. It
typically consists of a cuvette transparent to the wavelength or a sample holder through which light
passes.

Detector: The detector measures the intensity of light that passes through the sample.
Photomultiplier tubes (PMTs) or charge-coupled devices (CCDs) are commonly used detectors in
UV-Visible spectrophotometers. The detector converts the light intensity into an electrical signal,
which is then used to calculate the absorbance of the sample.

Amplifier and Signal Processor: The electrical signal generated by the detector is often weak,
and an amplifier is used to strengthen it.

Wavelength Selector/Controller: The wavelength selector or controller allows the user to choose
the desired wavelength for the analysis. It controls the monochromator to select specific
wavelengths of light.
Applications of UV-VIS Spectroscopy:

Quantitative Analysis of Concentrations: UV-Visible spectroscopy is widely used for

quantitative analysis of the concentration of a substance in a solution. The Beer-Lambert Law
relates the absorbance of light to the concentration of the absorbing species.

Identification of Functional Groups: UV-Visible spectroscopy can provide information about the
electronic transitions within a molecule, allowing for the identification of functional groups.

Kinetic Studies and Reaction Monitoring: is employed in kinetic studies to monitor the progress
of chemical reactions over time, by measuring the changes in absorbance during a reaction.

Quality Control in Pharmaceuticals: widely used in the pharmaceutical industry for quality
control purposes. It is employed to determine the concentration of active pharmaceutical
ingredients (APIs) in formulations.

Environmental Analysis: applied in environmental science for the analysis of water, air, and soil
samples to detect and quantify pollutants, such as heavy metals, organic compounds, and other
environmental contaminants.

IR Spectroscopy:

Infrared spectroscopy (IR spectroscopy or vibrational spectroscopy) is the measurement of the

interaction of infrared radiation with matter by absorption, emission, or reflection. It is used to
study and identify chemical substances or functional groups in solid, liquid, or gaseous forms.

IR radiations lie in the wavelength range of 0.7 to 400 µm. IR Spectroscopy is based upon the
selective absorption of IR radiations by the molecule which induces vibration of the molecules of
the compound
Principle of Infrared Spectroscopy:

 Infrared (IR) radiation lies in the electromagnetic spectrum between visible light and
microwave radiation.
 The technique is based on the interaction of infrared radiation with chemical bonds in
molecules, causing them to vibrate.
 Different bonds absorb specific frequencies of IR radiation, resulting in characteristic
absorption patterns that can be used for identification and analysis.

Fundamental modes:

There are two types of fundamental molecular vibrations viz., stretching (change in bond length)
and bending (change in bond angle).

Stretching vibrations: It involves a rhythmic movement along a bond axis with a subsequent
increase and decrease in bond length.

Bending vibrations: A change in the angle occurring between two bonds is known as a bending
vibration. Four bending vibrations exist namely, wagging, twisting, rocking and scissoring.
Selection rules:

A molecule will absorb Infrared radiation if the change in vibrational states is associated with a
change in dipole moment of the molecule

Vibrations which do not change the dipole moment are Infrared inactive.

Homonuclear diatomic molecules are IR inactive. Examples: O2, N2, H2, Cl2

Heteronuclear diatomic molecules are IR active. Examples: HF, HCl

Instrumentation of Infrared Spectroscopy:

Infrared Radiation Source:

Infrared spectrometers require a suitable source of infrared radiation, which is typically provided
by a heated filament or a globar, a silicon carbide rod. These sources emit a broad range of infrared
wavelengths, covering the mid-infrared (MIR) and near-infrared (NIR) regions.

Sample Compartment:

It may consist of a sample holder, such as a transparent window or an attenuated total reflection
(ATR) accessory, depending on the sample type and measurement technique.

Interferometer:

The interferometer splits the radiation into two beams that travel different paths before
recombining.

Optical Components:

Various mirrors and beam splitters are used within the spectrometer to manipulate and direct the
infrared radiation.

Beam splitters divide the radiation into reference and sample beams, while mirrors ensure proper
alignment and optical efficiency.
Detector:

Detectors include thermal detectors (such as pyroelectric or thermopile detectors) and photo
detectors (such as photodiodes or photoconductive detectors).

Different detectors offer varying levels of sensitivity, speed, and spectral range.

Data Analysis System:

Spectral analysis software is used to analyze and interpret the obtained spectrum, enabling
identification of functional groups and compounds.

Applications of Infrared Spectroscopy:

 Infrared spectroscopy finds applications in diverse fields such as pharmaceuticals, forensics,

polymers, environmental analysis, and material science.
 It is used for structural elucidation, identification of unknown compounds, quantification of
components, and monitoring chemical reactions.
 IR spectroscopy is also utilized in quality control and authenticity assessment of various
substances.

Chromatography: Chromatography is a technique for separating mixtures into their components

in order to analyze, identify, purify and quantify the mixture or components.

Principles of Chromatography:

 Chromatography is based on the differential migration of components in a mixture between

a stationary phase and a mobile phase.
 It exploits the principles of adsorption, partition, ion exchange, or size exclusion to
achieve separation.
 The sample is introduced into the system, and as it interacts with the stationary and mobile
phases, the components are separated.
Classification of Chromatography:

Gas Chromatography (GC): In GC, the mobile phase is a gas, and separation is based on the
different volatilities and partition coefficients of components.

Liquid Chromatography (LC): In LC, the mobile phase is a liquid, allowing separation based on
factors such as polarity, size, or ion exchange properties.

Thin-Layer Chromatography (TLC): TLC employs a thin layer of adsorbent coated on a solid
support, enabling rapid and convenient separations.

High-Performance Liquid Chromatography (HPLC): HPLC is a powerful form of liquid

chromatography that utilizes high pressure to enhance separation efficiency.

High Performance Liquid Chromatography:

High-performance liquid chromatography (HPLC) is a powerful analytical technique used for the
separation, identification, and quantification of components in a liquid mixture.

Principle:

 HPLC is based on the principle of liquid-solid or liquid-liquid chromatography, where the

separation occurs on a stationary phase.
 The sample is dissolved in a liquid mobile phase and passed through a chromatographic
column packed with a high-performance packing material (porous silicon materials).
 Separation is achieved based on the differential interactions between the analytes, stationary
phase, and mobile phase.
Components and Instrumentation of HPLC:

HPLC involves several key components:

 HPLC Pump: Provides a continuous flow of the mobile phase at a constant pressure.
 Injector: Introduces the sample into the mobile phase stream, typically using an injection
valve.
 Chromatographic Column: Contains the stationary phase, typically packed with small
particles for efficient separation.
 Detector: Measures the concentration of analytes exiting the column and generates a signal.
Eg. UV-Visible (UV-Vis) Detector, Electrochemical Detector:
 Data System: Collects and processes the detector signal, displaying and analyzing
chromatographic data.

Modes of Operation:

HPLC can be operated in different modes

Normal Phase HPLC: The stationary phase is polar, and the mobile phase is nonpolar. Eg. Silica
hydride, Diol: Diol-based stationary phases contain hydroxyl (-OH) functional groups, Silica
gel.

Reverse Phase HPLC: The stationary phase is non polar, and the mobile phase is polar. Eg:
Phenyl Phases, Bi phenyl phase.

Applications of HPLC:

 HPLC has wide-ranging applications in various fields, including pharmaceuticals,

chemicals, food and beverage, environmental analysis, and forensic science.
 It is used for drug quality control, impurity analysis, quantification of active
pharmaceutical ingredients (APIs), and pharmacokinetic studies.
 HPLC is employed in environmental analysis to detect pollutants, analyze water quality,
and monitor pesticide residues.
 Food and beverage industry relies on HPLC for quality assurance, flavor analysis, and
identification of additives and contaminants.