Lecture 1 Chromatography

• Chromatography is an analytical technique that is widely used for the separation, isolation and
identification of closely related chemical components for the separation organic compounds like
amino acids, sugars, vitamins, hormones, plant pigments etc.
• Chromatography is a technique of separation and purification of components of a mixture by
their differing affinities for two phases (states) of matter with which they come into contact.
• A Russian botanist Mikhail Tswett in 1986 invented chromatographic method to separate various
plant pigments such as chlorophylls and Xanthophyll's and several other substances by passing
solution of these compounds (vegetable extracts) through a glass column packed with finely
divided calcium carbonate. His column developed bands of color, and he named this separation
technique chromatography, which in Greek means "written in color".
• Chromatography is the process of separating the components of mixtures (solutes) that are
distributed between a stationary phase and a flowing mobile phase according to the rate at which
they are transported through the stationary phase.
• According to A.I.M. Keulemans, "Chromatography is a physical method of separation, in which the
components to be separated are distributed between two phases, one of these phase
constituting a stationary bed of large surface area, the other being a fluid that percolates through
along the stationary bed."
Principle of Chromatography
• Chromatography is based on the ability of a column of finely powdered solid material (such as
Al2O3), the stationary phase, to adsorb substances from a solution (the mobile phase) that is
allowed to trickle through it.
• In general, the attractive force of the solid surface differs for different species in solution. The
substance that solid absorbs most strongly moves through the stationary phase much more
slowly than do those that are not so strongly adsorbed. This means that although the various
components present in the mobile phase start out together, when they first come in contact with
the stationary phase, they soon become separated.
• Chromatography is based on the principle of selective distribution of the different components of
a mixture between two phases, namely stationary phase and mobile (or moving) phase. The
stationary phase can be a solid or liquid; while the mobile phase is a liquid or gas. When the
stationary phase is solid, the selective distribution is based on adsorption, while when it is a
liquid, the basis of selective distribution is partition.
• The stationary phase may be either a solid or liquid; and the moving phase may be either a liquid
or a gas.
• In all the chromatographic techniques, the solutes to be separated migrate along a column, and of
course, the basis of the separation lies in different rates of migration for the different solutes.
Steps of Chromatographic methods
• 1) Adsorption or retention of substances on the stationary
phase.
• 2) Separation of the adsorbed substances by the mobile
phase.
• 3) Recovery of the separated substances by a continuous flow
of mobile phase, the method is called elution.
• 4) Qualitative and quantitative analysis of the eluted
substances.
No other separation process can match chromatography for
simplicity, efficiency, and range of applications. Therefore,
chromatography is a major analytical tool for chemists,
biologists, and workers in the related field.
Classification of Chromatographic Techniques
A) Mobile Phase arrangement
1) Liquid Chromatography (LC) - The mobile phase is liquid
2) Gas Chromatography (GC) - The mobile phase is gas
Classification of Chromatographic Techniques
B) Stationary Phase arrangement
1) Column Chromatography – The stationary phase is placed in a column.
2) Planar technique
a) Paper Chromatography (PC) – Stationary Phase is a special paper
b) Thin Layer Chromatography (TLC) – Stationary Phase is spread on solid flat support (like glass
plate or aluminum foil)
Classification of Chromatographic Techniques
C) Interaction of analyte with the stationary phase
1) Adsorption Chromatography: It is a technique in which small differences in the adsorption
behavior of substances between a moving solvent (liquid or gas) and a stationary solid phase are
utilized to achieve the separation.
When the mobile phase is a liquid, it is called liquid-solid chromatography
When the mobile phase is a gas, it is called gas-solid chromatography
2) Partition Chromatography: It is a technique in which mixtures of substances are separated using a
partition between a moving solvent and a stationary liquid held on a suitable solid support.
When a solvent (mobile phase) is a liquid, it is called liquid-liquid chromatography.
When a solvent (mobile phase) is gas, it is called vapor or gas-liquid chromatography.
3) Ion Exchange Chromatography
4) Size Exclusion Chromatography
5) Affinity Chromatography
Paper Chromatography

• Paper chromatography is a simple and widely used chromatographic technique. It was developed
in 1865 by Schonbein. Later on, it was further developed by Martin and Synge in 1944.
• In 1952, A. Martin and R. Synge won the Nobel Prize for Chemistry in 1952 for the development of
chromatographic techniques.
• This is a powerful method of liquid-liquid chromatography, used widely in separating free amino
acids (produced as a result of hydrolysis of protein material), sugars, peptides, and sugar
derivatives.
Principle of Paper Chromatography
• In paper chromatography, both the stationary phase and mobile phase are liquid. Specially designed
filter paper, composed primarily of cellulose materials, serves as the stationary phase, while an
appropriate immiscible organic solvent is the mobile phase.
• An ideal filter paper for chromatography is composed of 99% alpha-cellulose, 0.3% beta-cellulose,0.5%
pentasans, and 0.07% ash.
• The mixture of different components can pass through the paper using appropriate solvent systems.
The organic solvent moves in the filter paper by capillary action and adsorption on the filter paper.
• The components of the mixture exhibit differential adsorption due to their different partition
coefficients (the ratio of the concentration of a substance in one medium to the concentration of
another substance in which these concentrations are at equilibrium). The ratio of the distance traveled
by the solute to the distance traveled by the solvent is expressed in terms of the retardation factor (Rf).
The Rf value can be regarded as a function of the partition coefficient. Its value is always less than one.

• The retardation factor (Rf) is calculated as follows: Rf = Distance traveled by solute

Distance traveled by the solvent

• The Rf value can be the basis of the separation of components of mixtures, as this value for a particular
substance is always the same under the given set of conditions.
 Experiment: an example of separation of amino acids

A thin pencil line is drawn near one edge of a rectangular piece of filter paper and parallel to it.

The solid mixture, present in solution, is now applied as a small drop from a fine glass syringe along
the pencil line, and near one vertical edge of the paper.
If the problem is to identify the amino acids in a mixture, then small drops of separate solutions of
pure amino acids (thought to be present in the mixture) are also placed at intervals along the pencil
line.
Experiment continue

The filter paper is now hung vertically in a glass tank, which contains the developing solvent and
the bottom edge of the filter paper is so positioned in the solvent that the pencil line is just clear
of it.
The tank is sealed with a lid to prevent the evaporation of solvent.
As the solvent travels up (due to capillary action) the paper, it moves the components in the
mixture, and the pure substances, at different rates.
Experiment continue

The paper is removed from the tank, and allowed to dry, once the solvent has almost reached
the top of the paper.
Since amino acids are colorless, their positions on the paper are located by spraying the whole
paper with a solution of ninhydrin in propanone.
After warming the paper in an oven set at a temperature of about 373 K the individual amino
acids show up as blue-lilac spots.
Experiment continue

• Under carefully controlled conditions, it is possible to characterize a particular compound separated from
a mixture by its so-called Rf value.
• This is defined to be
• Rf value = The distance moved by the pure substance /The distance moved by the solvent front.
• Its value for a particular compound depends upon the solvent used and the temperature. From its
definition above, it is clear that all Rf values are less than unity.
Experiment continue

Rotate +90°

Solvent A Solvent B

In some cases, the use of one solvent may fail to separate two or more of the constituents present in
the mixture, ie, their Rf, values under these conditions are almost identical. The answer to this
problem is simple. The paper is turned through 90° and the whole process is repeated with an end
solvent. The logic being that it is most unlikely that two or more components will have identical Rf
values in a different solvent. This technique is called two-way chromatography.
 Steps involved in the paper chromatography
a. Preparation of solution of organic compounds or analyte:
Proper solvent is selected for the preparation of solution. Commonly, concentrated solution prepared for paper chromatography to avoid diffusion process.
It is preferable to remove the impurities to nullify the effect of interfering entities.
b. Application of the sample on the paper
Usually, it is preferable to use a rectangular filter paper with dimension (15 cm x 30 cm or different as per requirement). A pencil line is drawn above 2.5 cm
form one end. However small dimension of filter paper piece can also be prepared. A drop of about 1-2 µL of solution of sample is spotted from a capillary
tip for the study of paper chromatography. On the sample lines, other standard references can be spotted for comparative study.
c. Development of the chromatogram
Appropriate solvent is chosen for the development of chromatogram. Commonly, ple solvents are used for this purpose. If required, a mixture of solvent
can be used for preparation of a chromatogram. Commonly used solvents are propanol, n-butanol and furan. The
Solvent is allowed to move upward by ascending through the filter paper in the solvent tank.
d. Drying of the chromatogram
After passing the solvent up to certain distance and the separation of components is over, the chromatogram is removed from the solvent and dried by
blowing hot air.
e. Locating the components of the mixture on the of chromatogram:
The separated components are located by physical (eg, fluorescence) or chemical spraying with reagents) methods.
f. Elution of the spots:
The identified area of each spots are cut and extracted with a suitable solvent.
Applications of paper chromatography
• Identify and separate different components from an analyte.
• Check the pharmaceutical compounds.
• Check food adulteration.
• Analysis of cosmetics.
• Forensic studies (DNA and RNA sequences).
Thin Layer Chromatography
• propounded by Lamaiter and Schwelt.
• developed by Stahl in 1956.
• In this chromatography, the filter paper is replaced by a thin layer of an inert adsorbent (like
Sephadex, cellulose, alumina, or silica gel) spread over a square plate of glass or plastic.
• The method is used to identify the components in a tiny sample of a solid mixture.
• Its advantage over paper chromatography arises from the fact that a variety of thin films (eg,
alumina, Sephadex, silica gel) can be used depending upon the nature of the substances used.
• The thin film is more compact than filter paper, and the sample spots are smaller and more
concentrated.
• The process is quite rapid. This rapid method is useful in isolating and identifying amino acids,
nucleotides, triglycerides, other lipids, sterols, sugars, and sugar derivatives.
Experimental details for carrying out thin-layer
chromatography

A slurry of silica gel, for

example, is made in
trichloromethane contained in
an ordinary screw-cap bottle.
A previously cleaned
microscope slide is immersed in
the slurry and then carefully
withdrawn using a pair of
tweezers.
The coated glass plate
is now allowed to dry
in an upright position
and is then ready for
use.
Experimental details continue

Then, with the help of a syringe (or

At a distance of about 2.0-2.5 cm from the bottom of the
micropipette), spots of the sample
plate, a horizontal line is drawn.
solution and standards are applied.
Experimental details Continue

The chromatogram is developed by the ascending technique in which the plate is immersed in the
developing solvent to a depth of 0.5 cm.
Development is allowed to proceed, until the solvent front has traveled the required distance (usually
10-15 cm).
The plate is removed from the chamber and the solvent front reached is marked with a pointer object.
The plate is then allowed to dry in a fume cupboard (or in air. oven).
Experimental details Continue

Rf, values of different

components and pure substance
are measured

The positions of the separated solutes can be located by various methods.

Colored sub- stances can be seen directly when viewed against the stationary phase; whereas
colorless species usually detected by spraying the plate with an appropriate reagent that
produces colored areas in the regions which they occupy.
Some compounds may be located without spraying if they fluoresce under ultraviolet light.
 Principle of TLC

• Thin layer chromatography is performed in glass slides or sheet of glass of aluminum

coated with thin layer of adsorbent guard intents could be silica gel of aluminum oxide.
• The layer of adsorbent acts as stationary phase and liquid serves as mobile phase.
• Different analytes ascent onto the TLC plate with different rates and the separation of
either component is possible in this type of chromatography.
• In TLC, components of mixture partitioned between adsorbent (eg, silica gel on glass or
alumina support) and solvent (mobile phase).
• Moisture or water affects the development of adsorption chromatography. Therefore,
should be removed by putting the chromatoplates in an oven at about 100-105 °C for
about half an hour. This step is termed as activation of chromatoplates.
Various steps involved in the separation of
compounds by TLC
• Preparation of sample: The analyte sample is dissolved in appropriate solvent.
• Preparation of TLC plate: A rectangular TLC plate of appropriate dimension (e.g., 15 cm x 20 cm
or as per requirement) is prepared and a baseline is drawn above some distance (say 2.5 cm) from
one end. The sample under test is spotted on a plate followed by dried with hot air.
• Development of chromatogram: The chromatoplate is placed in the TLC chamber vertically such
that the spots of the plates remain just above the solvent surface. The atmosphere of the tank is
saturated with the vapor of the solvent. The lid of the tank is tightly closed. When the solvent
rises sufficiently 10-12 cm) abonents of the mixture are taken out from the tank and dried.
• Locating the components of the moisture on the mixture of chromatogram: The separated
components are located by physical method or chemical method.
• Elution of the spots: The identified areas of each spot are cut and the components are extracted
with a suitable solvent.
Advantage of TLC
• It is simple and quick method relative to other methods of chromatography.
• Varieties of organic and inorganic compounds can be separated by this technique.
• Even a corrosive substance can be analyzed by this method.
• The chromatogram can be heated without damaging it.
• Thin layers of adsorbents are better adsorbed than paper.
Appropriateness of TLC

• When a substance to be separated or purified is nonvolatile and polar.

• If sample used for analysis is likely to damage the column of liquid chromatography or gas
chromatography.
• If substance in the material being analyzed cannot be detected by other methods of
chromatography like liquid chromatography (LC) or gas chromatography (GC).
Applications of TLC

• It is used to check the purity of substance.

• It is used to identify the compounds.
• It is used to purify the compounds.
• It is used to separate the components from the mixture.
• It is used to monitor the progress of reaction in research.
References
• Compiled by : NA nalecture@gmail.com
• References:
• P.C. Jain, M. Jain, Engineering Chemistry, (16th Edition), 2013.
• Bhishma Raj Pandey, An Easy Approach to ANALYTICAL CHEMISTRY, (1st Edition), 2015.
• Hem Raj Pant, Tanka Mukhiya, Deval Prasad Bhattarai, Prakash Chandra Lohani, Engineering
Chemistry, (1st Edition), 2080.
• 2080/11/10
Lecture 2 Mass Spectrometry
• Mass spectroscopy is an analytical tool that measures mass to charge
ratio of ions in the gas phase. It provides qualitative as well as
quantitative information. In this method, the material being analyzed
is vaporized and vapor is bombarded with a high-energy electron
beam whereby many molecular ions and fragments are formed.
M+ e  M+ + 2 e
• Fragmentations are guided by their relative stabilities. The stability of
a carbocation is in the order
CH3+ <1° carbocation < 2 ° carbocation < 3 ° carbocation
Principle of Mass Spectroscopy
• In a mass spectrometer, organic molecules are vaporized and bombarded with a
beam of very high-energy electrons.
• The resulting collisions impart considerable energy to the molecule, emitting
electrons to produce positively charged ions.
• These ions possess so much energy that they often fragment through various
bond cleavages to produce new positively charged ions.
• ABC electron beam  ABC+ (Molecular ion) + AB+ + C+ + A+ + BC+
• The positive ions are accelerated toward a negatively charged plate by passing
them through a magnetic field, which deflects the ions.
• The ions of lighter mass are deflected more than heavier ions.
• Each kind of ion has a particular mass to charge (m/e) ratio.
• For most ions produced in the fragmentation of the molecule, the charge is +1 so,
that m/e usually represents the mass of the ion.
 Principle Continue
• Base Peak: The set of ions produced from a molecule can be analyzed since
each has its own m/e ratio, and produces a signal whose intensity is due to
the relative abundance of that ion. The largest peak found in a mass
spectrum (that of the highest intensity) is called the base peak and is given
the numerical value of 100. The intensities of all other peaks are expressed
relative to the height of the base peak. A mass spectrum is highly
characteristic of a particular compound.
• Molecular Ion: The ion formed by removing one electron from the parent
molecule is called a molecular ion or parent ion. The molecular ion peak is
usually represented as M +. It may or may not be the peak of the highest
intensity. The molecular ion is the most important since its mass is the
Molecular Weight of the parent molecule.
Instrumentation of mass spectrometry

Schematic diagram of a Mass spectrometer.

Instrumentation of mass spectrometry
a. Ion Source: It produces gaseous ions from the given sample.
b. Analyzer: It is used to analyze and separate the ions into their
characteristic mass according to their mass-to-charge ratio.
c. Detector: Detectors in mass spectrometry detect the ions and
maintain their relative abundance.
Apart from these, a sample introduction system is required to add the
sample to the ion source.
Instrumentation of mass spectrometry

Gas phase ion Ion sorting Ion detection

Input Source Analyzer Detector

Output
Block diagram of a Mass spectrometer.
Mass Spectrum : Examples
A mass spectrum is a graphical representation of the distribution of ions as a function of their mass-to-charge
ratio (m/z). It is generated through a technique called mass spectrometry.
 m/z 16 (Molecular ion, CH4+​): Usually the most intense peak,
with a relative abundance set at 100% for normalization
purposes.
m/z 15 (Methyl cation, CH3+​): Typically, less intense than the
molecular ion peak but still significant.
m/z 14 (Methylene cation, CH2+​): Usually present but with
lower intensity compared to CH3+​.
m/z 13 (Methyl cation radical, CH+): Typically, a minor peak
compared to CH3+​ and CH2+​
m/z 12 (Carbon cation, C+): Usually present but with low
intensity.
m/z 17 (Methyl cation with hydrogen radical, CH3++ H2​):
Typically, less intense than the main fragment ions.

Mass Spectrum of methane

Mass Spectrum : Examples
CH3+ (Methyl cation): m/z 15
C2H4+ (Ethylene cation): m/z 28
CH3CH2+ (Ethyl cation): m/z 29
CH2OH+ (Methoxy cation): m/z 31 Base Peak
CH3OH+: M + (Molecular ion peak, Methanol): m/z 32
Mass Spectrum : Examples
Applications of mass spectroscopy

a. Mass spectrum is useful to establish structure of a new compound.

b. It helps to find molecular formulas or at least a narrow list of possible compounds.
c. Mass spectrum is used for the analysis of gas, test of impurities in semiconductors.
d. Mass spectrum is used for the analysis of gas, test of impurities in understanding
reaction mechanisms.
e. Mass spectroscopy is the most reliable tool to determine accurate molecular mass.
f. Mass spectrum is highly characteristic of a particular compound. Therefore, the mass
spectrum is used to prove the identity of two compounds.
g. It can be used as a fingerprint in forensic science.
Drawback of mass spectrometry

a. One major drawback of mass spectrometry is that samples under

investigation are destroyed.
b. Installation cost of the mass spectrometer is high.(A high vacuum is
maintained (10-5-10-8 torr) and a computer system is needed to store
the data well and to compare the obtained spectrum with the
reference one)
References
• Compiled by : NA nalecture@gmail.com
• References:
• Arun Bahl, B.S. Bahl, The Essentials of Physical Chemistry.
• Hem Raj Pant, Tanka Mukhiya, Deval Prasad Bhattarai, Prakash Chandra Lohani, Engineering
Chemistry, (1st Edition), 2080.
Lecture 4: UV- Visible Spectroscopy
The UV -visible spectroscopy is an important analytical tool for analyzing
organic and inorganic chemical species. The method is based on the
absorption of light by the analyte. Therefore, UV-visible spectroscopy is an
example of absorption spectroscopy.
Spectrophotometry is a special technique of absorption photometry where
the use of a spectrophotometer is made for absorption measurement.
Ultraviolet-visible (UV-vis) spectroscopy involves the absorption of
ultraviolet/visible light by a chemical substance causing the promotion of an
electron from a ground electronic state to an excited electronic state.
In this spectroscopy, the wavelength region of 200-400 nm in the UV
spectrum and 400-750 nm in the visible spectrum are used to get some
qualitative and quantitative information about given chemical species.
 Principle
The fundamental principle of UV-visible spectroscopy is the interaction of radiant energy with matter.
The basic principle of this spectroscopy depends on the absorption of UV-vis radiation that causes
electrons within molecules to be promoted from the ground or lower energy level to a higher
electronic energy level.
UV-visible radiation is sufficiently energetic to cause the promotion of loosely held electrons, such as
nonbonding electrons or electrons involved in a π-bond of organic molecules to higher energy levels.
When an organic molecule absorbs UV-vis radiation, it means that the compound contains a carbonyl
group or conjugated double bonds. For example, carbonyl compounds, conjugated dienes, and
aromatic compounds can absorb UV-vis radiation.
Radiation of UV and Visible waves is sufficiently energetic to cause the promotion of loosely held
electrons, such as nonbonding electrons or electrons involved in a π-bond to higher energy levels.
For absorption in this particular region of the ultraviolet spectrum, the molecule must contain
conjugated double bonds. If the conjugation is extensive, the molecule will absorb in the visible region.
The wavelength of maximum absorbance is referred to as λmax.
A diagram showing the bonding, non-bonding, and antibonding orbital Molecules
Lambert-Beer Law: Basic Principle of UV-
visible spectrophotometer
The basic principle of the UV-visible spectrophotometer is based on the Lambert-Beer Law.
The effect of the thickness and concentration of the sample on the absorption is measured by
Lambert-Beer Law.
According to this law, the absorbance of light by a solution is directly proportional to its molar
concentration and path length.
According to this law, Absorbance (A)= Ɛbc.
Where,
A=absorbance,
Ɛ=molar absorption coefficient
b =path length (the path length is usually 1 cm)
c =molar concentration
Instrumentation
Lamp
The UV-Visible spectrophotometer can be equipped with a tungsten filament lamp (350-2500 nm) Deuterium
lamp (200-400 nm) or a xenon Arc lamp (200-1000 nm) which cover the whole range for UV-visible reasons.
Mirror
The radiation emanating from tungsten lamps and deuterium lamps are reflected to the mirror whereby these
get incident onto the monochroismator.
Monochromator
It is composed of prisms and slits. It selects the wavelength of radiation reflected from the mirror. The beam
selected by the slit is a monochromator.
Exit slit
It is the opening through which the monochromatic radiation passes into the beam splitter.
Beam splitter
It splits the beam emanating from the monochromator and causes it to pass into the reference sample and
trust sample in a double-beam UV-Visible spectrophotometer. One of the two divided beams is passed through
the reference sample and another is passed through the test sample.
Sample
The test sample as well as the reference sample are put into the cuvette and the absorbance of the test sample
is recorded by computer after processing. Quartz cuvettes are referred to over a plastic of cuvette for sample
analysis as glass cuvettes can absorb radiation.
Detector
Two photocells act as detectors in UV-vis spectroscopy. One photocell receives the radiation from The
reference solution and another photocell receives the radiation from the test solution. The intensity of
radiation obtained from the reference solution is stronger than that from the test sample. The difference in
intensity of radiation generates the alternating current.
Amplifier
The current generated from the detector is too small and is amplified by the amplifier which can generate
signals.
Recorder
All the generated data is recorded and stored in the computer. In UV-visible spectroscopy, molecules undergo
electronic transition involving n and n electrons. The o electrons are present in saturated compounds, t
electrons are present in unsaturated compounds and n electrons are present in non-bonded electrons.
UV-Visible spectrophotometer
Instrumentation of UV-Visible spectrophotometer
 Sample
Source Monochromator
Detection Unit Recorder

Reference
Beam
Splitter

Block Diagram of UV-Visible spectrophotometer

 UV-Vis Spectrum
A UV-Vis spectrum graph shows the absorption of light by a sample at various wavelengths in the ultraviolet-
visible region, providing insights into the electronic transitions occurring within molecules or atoms.
Chromophore
Most of the absorption of organic compounds results from the presence of π -bonds. Such a bond containing
a molecule is called chromophore which makes a compound with absorption between 185 and 800 nm.
Auxochrome
Conjugation of the double bond with additional double bonds increases both the intensity and the
wavelength of the absorption band. A group that extends the conjugation of a chromophore by sharing
nonbonding electrons is called auxochrome.
An auxochrome is a group of atoms with one or more lone pairs of electrons attached to a chromophore
which can alter both the wavelength and intensity of absorption. Some examples of auxochrome are, -OH, -
OR, -NH2 , -NHR, -SH
Auxochromes can affect the molecular structure of a compound by influencing the electron density of the
chromophore.
For example, in aniline, the amino group can increase the electron density of the benzene ring which leads to
the electron shifting of absorption spectra towards longer wavelength and increase the intensity of color. The
presence of substituents in a molecule causes the UV absorbance band.
If the absorbance band shifts to a longer
wavelength (lower energy) region, it is termed a
bathochromic shift (redshift).
If the absorbance band shifts to a shorter
wavelength (higher energy) region, it is termed a
hypsochromic shift (blue shift).
If the intensity of the absorbance band gets
increased, it is termed a hyperchromic shift.
If the intensity of the absorbance band gets
decreased, it is termed a hypochromic effect.
Qualitative Application of uv-visible
spectroscopy
• The qualitative applicability of the absorption method is based on the
fact that the wavelength of absorbed light depends on the properties
of absorbing atoms, ions, or molecules.
• For a given species, an absorption spectrum (a single band) is
observed for a qualitative identification of compounds, λ max value is
calculated.
• λ max is the wavelength (in nm) in which maximum absorbance
occurs by the chemical species. For a chemical species, the λ max
value is always unique.
 What is this?

Qualitative analysis from UV Visible spectroscopy

Quantitative Application of uv-visible
spectroscopy
• The quantitative applicability of the absorption of the number of
photons is directly proportional to the number of concentrations of
atoms, ions, and molecules.
• For Quantitative analysis, a calibration curve is obtained for the
known concentration vs Absorbance.
• From the Graph based on Beer-Lambert law, the concentration of the
unknown sample is calculated.
 How much is
this?

Quantitative analysis from UV Visible spectroscopy

Strength of UV-Vis Spectroscopy
• Instruments are easy to handle, requiring little user training before
use.
• The technique allows the sample to be reused or proceed to further
processing.
• Measurements can be made easily and quickly.
• Data analysis generally requires minimal processing even completed
with little work braining.
Weakness of UV-Vis Spectroscopy
• Light may come from the environment and may cause errors.
• Light scattering can be termed out by suspended solids in liquid
samples, which may cause undesired measurement errors.
• Misaligned positioning of any one of the instrument's components,
especially the cuvette holding the sample, may yield inaccurate
results.
 Applications of UV-vis spectroscopy
Qualitative analysis
Quantitative analysis
Structural analysis
UV-Visible spectroscopy is applied for structural analysis of compounds. It is possible
to measure the presence or absence of unsaturation in an organic compound.
Chromophore group analysis
It is applicable to ensure the presence of chromophore groups in molecules.
Photometric analysis
It is applicable in photometry
Detection of impurities
The presence of impurities can be detected by UV-Visible spectrometers. The
presence of impurities gives some additional peaks in UV-Visible spectra.
DNA and RNA analysis
UV-Visible spectrometer is used in the analysis of DNA and RNA. It is applicable in
protein analysis
Study of kinetics of photochemical reaction
The Kinetics of photochemical reactions can be studied using UV-Visible
spectroscopy.
Pharmaceutical analysis
This technique is widely used to ensure the purity of pharmaceutical raw materials.
Beverage Analysis
Visible spectroscopy can give a wide range of information regarding food and
beverage
.
Bacterial and cell culture
Bacteria and cell culture studies can be carried out by the UV-Vis method
Pollution control
absorbance can be useful in the study of the presence of contaminants like
dyes and heavy metal ions like Cr(VI) response to the UV-Vis light and their
absorbance can be ng UV-Vis spectroscopy. However, some metal ions like
iron need color complexing for its absorbance measurement.
Study the properties of nanoparticles of novel metals and quantum dots
Some nanoparticles have their characteristic absorbance band in the UV-Vis
region. For example, silver nanoparticles exhibit surface plasmon resonance
(SPR) phenomena at around 450 Particle size, and UV-Vis absorbance bands
are related in the case of many nanoparticles, and quantum dots.
• Compiled by: NA nalecture@gmail.com
• References:
• Hem Raj Pant, Tanka Mukhiya, Deval Prasad Bhattarai, Prakash
Chandra Lohani, Engineering Chemistry, (1st Edition), 2080.
• P.C. Jain, M. Jain, Engineering Chemistry, (16th Edition), 2013.
• Bhishma Raj Pandey, An Easy Approach to ANALYTICAL CHEMISTRY,
(1st Edition), 2015.
• 2080/11/27
Lecture 5: IR Spectroscopy
• Infrared spectroscopy is absorption spectroscopy that deals with the
recording of the absorption of the electromagnetic radiations of the
infrared region of the electromagnetic spectrum. The IR region ranges
from 780-1000 nm.
• This technique is widely used for the detection of functional groups,
characterization of proteins, analysis of various liquids, and
identification of molecules and their composition.
Principle
• Compounds that contain functional groups having single and double bonds always stay in
a state of vibration motion. They are stretching and bending vibrations. They absorb only
a specific frequency of IR radiation and the rest unwanted frequencies get transmitted
from the compound without any absorption. That transmitted radiation is detected by
the detector and a graph is plotted.
• Absorption of infrared radiation causes covalent bonds within the molecule to be
promoted from one vibrational energy level to a higher vibrational energy level.
• Stronger bonds require greater energy to vibrate (stretch or bend). Therefore, such
bonds absorb infrared radiation of shorter wavelengths.
• When the molecule is subjected to radiation in the IR range, it absorbs only those
possessing exactly the energy required to cause the corresponding vibration.
• Different functional groups absorb infrared radiation at different wavelengths, and their
presence or absence in a molecule can be determined by examination of an IR spectrum.
• No two compounds have identical infrared spectra.
Principle
For example, carbon-carbon double bond is weaker than carbon-
carbon triple-bond and requires a longer wavelength (lesser energy) to
stretch.
C=C 1620-1680 cm^-1
C≡C 2100-2200 cm^-1
Modes of Vibrations in IR Spectroscopy (youtube.com)
Types of Molecular vibration caused by IR radiation
Instrumentation
1. Radiation source
The radiation source should emit IR electromagnetic radiations that are steady, intense and extended over the
desired wavelength. That's why radiation sources like Nernst glower, globar source, incandescent lamp, and
mercury arc lamp.
2. Monochromator
It helps to select desired frequencies of IR radiations because our sample will only absorb some specific
frequencies of IR radiations. Prism and grating are the most commonly used monochromators.
3. Sample cells
IR spectroscopy is used for the characterization of different samples like solids, liquids, and gases. The sampling
of different phases should be done in a material that is transparent to IR radiation.
4. Detector
The detector helps to detect the transmitted IR radiation by the sample. The detector should be placed and
designed in such a way that it can detect even a low-frequency signal. For that reason, thermal detectors are
widely used in IR spectrometry.
5. Recorder
The IR Spectra are recorded with the help of a recorder and this is printable.
 IR
Radiation Monochromator Sample Detector Recorder
source

Block Diagram of IR Spectrometer

In an IR spectrometer, a sample is irradiated with frequencies of IR radiation, and the

frequencies that pass through (that are not absorbed by the sample) are detected.
A plot is then constructed showing which frequencies were absorbed by the sample.
The most commonly used type of spectrometer, called a Fourier transform (FT-IR)
spectrometer, irradiates the sample with all frequencies simultaneously and then utilizes
a mathematical operation called a Fourier transform to determine which frequencies
passed through the sample.
IR Spectrum
The infrared (IR) spectrum is a graphical representation of the absorption of infrared radiation by a
sample as a function of wavenumber “ν”.
In an IR spectrum:
The x-axis typically represents the wavenumber (cm^-1) of the infrared radiation.
The y-axis represents the absorbance, transmittance, or percentage transmittance of the sample at
each wavelength or wavenumber.
Absorbance indicates the amount of infrared radiation absorbed by the sample at a particular
wavelength, while transmittance measures the fraction of incident radiation that passes through
the sample.
Peaks or bands in the spectrum correspond to specific molecular vibrations or transitions within the
sample.
Each functional group within a molecule absorbs infrared radiation at characteristic wavelengths,
allowing for the identification of functional groups and the elucidation of molecular structure.
An infrared spectrum is usually studied in two sections :
(1) Functional Group Region: The area from 4000 cm-1 to 1300 cm-1 is
called the functional group region. The bands in this region are
particularly useful in determining the type of functional groups in the
molecule.
(2) Fingerprint Region: The area from 1300 cm-1 to 400 cm-1 is called
the fingerprint region. A peak-by-peak match of an unknown spectrum
with the spectrum of the suspected compound in this region can be
used, much like a fingerprint, to confirm its identity.
Reference Table
• A reference table of IR spectroscopy typically includes characteristic
absorption frequencies (wavenumbers or wavelengths) associated with
various functional groups present in organic molecules.
• This table provides a general overview of typical absorption frequencies for
common functional groups encountered in organic molecules. However, it's
important to note that variations in molecular structure, chemical
environment, and instrument calibration can lead to slight shifts in
absorption frequencies. Therefore, reference tables should be used as
guides rather than strict rules, and interpretation should consider
additional factors such as sample purity and experimental conditions.
Advanced reference tables may also include more detailed information and
additional functional groups.
Hydroxyl Group (OH):
The stretching vibration of the hydroxyl group
typically appears as a broad peak around 3200-
3600 cm^-1.
C-H Stretching:
C-H stretching vibrations in the methyl and
methylene groups occur around 2800-3000 cm^-
1.
Alcohol C-O Stretching:
The C-O stretching vibration of the alcohol group
occurs around 1000-1300 cm^-1.
C-O-H Bending:
Bending vibrations of the C-O-H bond typically
occur around 1300-1400 cm^-1.
Hydroxyl Group (OH):
The stretching vibration of the hydroxyl group typically appears as a broad
peak around 3200-3600 cm^-1. This broad peak is often the most
prominent feature in the IR spectrum of benzyl alcohol.
C-H Stretching:
C-H stretching vibrations in the benzene ring occur around 3030-3100
cm^-1. These peaks are typically weaker compared to the OH stretching
peak.
Aromatic C=C Stretching:
Aromatic C=C stretching vibrations usually appear as medium to strong
peaks around 1500-1600 cm^-1.
Alcohol C-O Stretching:
The C-O stretching vibration of the alcohol group occurs around 1000-1300
cm^-1. This peak is often observed as a medium to strong band.
Aromatic C-H Bending:
Bending vibrations of aromatic C-H bonds in the benzene ring appear
around 680-900 cm^-1.
 Ethyl Alcohol and Benzyl Alcohol IR Spectra Comparison
Hydroxyl Group (OH) Stretching:
Both ethyl alcohol and benzyl alcohol contain hydroxyl groups, but the environment and bonding of
these groups differ. In ethyl alcohol, the OH stretching peak typically appears as a broad peak around
3200-3600 cm^-1. In benzyl alcohol, this peak is also present but may exhibit differences in intensity
and shape depending on the environment of the hydroxyl group.
Aromatic C-H Stretching:
Benzyl alcohol contains an aromatic benzene ring, so it exhibits peaks associated with aromatic C-H
stretching vibrations, usually around 3030-3100 cm^-1. Ethyl alcohol lacks an aromatic ring, so it does
not display significant peaks in this region related to aromatic C-H stretching.
C-O-H Bending:
Both compounds exhibit bending vibrations of the C-O-H bond, typically around 1300-1400 cm^-1.
However, the precise position and intensity of this peak may vary between the two compounds due to
differences in molecular structure.
Additional Functional Groups:
Benzyl alcohol contains an aromatic ring, leading to additional characteristic peaks associated with
aromatic vibrations, such as aromatic C=C stretching vibrations around 1500-1600 cm^-1. Ethyl alcohol
lacks these additional functional groups.
Overall Spectrum Shape:
The overall shape and intensity of the IR spectra may differ due to the presence of aromatic groups in
benzyl alcohol, leading to additional peaks and variations in peak intensity compared to the simpler
structure of ethyl alcohol.
https://osf.io/5s7wm/download
 Application of IR spectroscopy
1. Identification of functional group present in the organic compound
IR spectroscopy helps us to identify the different organic compounds as it is a highly characteristic property.
Identification of substances like hydrocarbons like alkenes, cycloalkanes, and aromatic hydrocarbons is done
by IR spectroscopy.
2. Determination of the molecular structure
Prediction of the molecular structure of the unknown samples can be done using IR spectroscopy. It is done by
assessment of the positions of absorption bands in the electromagnetic spectrum.
3. Studying the progress of reactions
Progress of a chemical reaction can be readily followed by examining spectra of small portions of the reaction
mixture withdrawn from time to time.
4. Detection of impurities in compound
It is likely to determine whether a given sample of a compound is pure or not provided the reference spectrum
of the pure compound is available.
5. Isomerism in organic chemistry
IR spectra are useful for identifying isomers. This distinction between isomers may not be possible by chemical
methods.
• Compiled by: NA nalecture@gmail.com
• References:
• Arun Bahl, B.S. Bahl, The Essentials of Physical Chemistry.
• David Klein, Organic Chemistry, 3rd Edition.
• Hem Raj Pant, Tanka Mukhiya, Deval Prasad Bhattarai, Prakash
Chandra Lohani, Engineering Chemistry, (1st Edition), 2080.
• P.C. Jain, M. Jain, Engineering Chemistry, (16th Edition), 2013.
• https://osf.io/5s7wm/download
• Modes of Vibrations in IR Spectroscopy (youtube.com)
Lecture 5: NUCLEAR MAGNETIC
RESONANCE (NMR) SPECTROSCOPY
• Nuclear magnetic resonance spectroscopy involves the absorption of
electromagnetic radiation in the radio frequency region.
• Absorption of radio waves in the presence of a magnetic field is accompanied by a
special type of nuclear transition, and for this reason, we call this type of
spectroscopy nuclear magnetic resonance (NMR) spectroscopy.
• Magnetic resonance (NMR) is a spectroscopic technique that deals with the study
of molecules based on the interaction of radio frequency of electromagnetic
radiations with nuclei of molecules in a strong magnetic field.
• NMR spectroscopy is useful to study the physical and chemical properties of
atoms or molecules. Initially, NMR was experimentally detected by 1945.
• The first NMR spectrum published was in 1946.
• Bloch and Purcell were jointly awarded the 1952 Nobel Prize in Physics for their
research on NMR spectroscopy.
Principle
• The nuclei of certain atoms behave as if they are spinning charges. Any
spinning charge creates a magnetic field and behaves like a tiny bar magnet.
Of the three nuclei most common in organic compounds (1H, 12C, and 16O)
only the hydrogen nucleus proton behaves in this manner.
• When a proton in an organic molecule is placed in a strong magnetic field,
it can align with the field or against it.
• In the more stable low-energy state, it is aligned with the magnetic field.
• If energy is supplied in the form of radiowaves of exactly the right
frequency, radiation will be absorbed and the nucleus will "flip" and align
against the applied magnetic field in the higher energy state.
Like electrons, the nucleus of an atom is
also spinning on its axis.
The spinning of charge particles generates
magnetic moments.
Therefore, nuclei of atoms act as a tiny bar
magnet.
Normally, nuclear magnetic fields are
randomly oriented.
When a sample is placed in an external
magnetic field (B0 or H0), the nuclear
magnetic field can be aligned or opposed to
the external magnetic field.
• The stronger the field the greater the tendency to lined up with it and
the higher the frequency or energy of the radiation. The frequency
value is given by
Frequency = γ × H0​​/2π
Frequency is the resonant frequency in Hertz (Hz),
γ (gamma) is the gyromagnetic ratio of the nucleus,
H0 is the strength of the magnetic field in Tesla (T),
π (pi) is a mathematical constant approximately equal to 3.14159.

• In practice, either the magnetic field can be held constant and the radio
frequency varied, or more commonly, the radio frequency can be held
constant and the magnetic field varied.
• The main purpose of NMR is not to detect the presence of protons in a
molecule. It can distinguish between protons in different chemical
environments within the molecule.
• Protons on the benzene ring, or on a carbon bearing a chlorine, or on a
carbon adjacent to a carbonyl group absorb radio frequency energies at
different applied magnetic fields, and appear at different locations (chemical
shifts) on the recording paper.
• Also, the position of absorption is relatively constant for protons in a
particular chemical or structural environment. Hence, the number of signals
recorded on the NMR chart paper indicates the number of different types of
protons in a molecule.
• The position of the peak can give information about the molecular structure
in the vicinity of the proton.
Instrumentation
NMR spectrometers are available with different megahertz ratings, commonly in 400
MHz and 600 MHz configurations. A typical NMR spectrometer comprises:
a. Magnet: Many NMR spectrometers utilize a superconducting magnet to generate
the magnetic field. This magnet is enclosed within an outer stainless steel or
aluminum casing in contact with liquid nitrogen to maintain low temperatures. The
inner casing houses superconducting coils immersed in liquid helium.
b. Radio frequency transmitter: It emits precise radio waves at a specific frequency.
c. Radio frequency receiver: This component measures the absorption of radio
frequency by the sample.
d. Recorder: During an experiment, a sample solution is placed in a uniform 5-mm
glass tube and allowed to spin while radio frequency radiation is applied. The output
from the radio frequency receiver is plotted against the applied magnetic field.
e. Computer: NMR instruments are typically controlled via a PC connection or
workstation.
Instrumentation of NMR Spectroscopy
 Sample Tube

Magnet

Display

Radio Detector
Frequency and
Generator Amplifier

Block Diagram of NMR Spectroscopy

NMR Spectra
• While passing radiation into an analyte at varied magnetic field
strength, the energy required to flip the proton matches the energy of
radiation at a particular value of magnetic field strength, where an
absorption signal is observed. This is the NMR spectra.
• When subjecting an analyte to radiation at various magnetic field
strengths, the energy needed to flip the proton signal is observed.
This phenomenon, occurring at a specific magnetic field value, results
in the absorption signal detected, forming the NMR spectra.
• The magnetic field strength experienced by a proton is referred to as
the effective field strength, influenced by the proton's surrounding
environment or electron density.
• NMR spectroscopy provides insights into the location and quantity of
protons within a molecule.
• The number of signals indicates the different types of protons
present, while their positions reveal the electronic environment of
each type.
• Signal intensity correlates with the number of protons of each type,
and signal splitting elucidates the proton's environment relative to
neighboring protons.
• The magnetic field strength felt by a proton is termed effective field
strength.
• The effective field strength of a proton depends upon the
environment of a proton or the electron density of the proton.
• The signal obtained from NMR spectroscopy gives information
regarding the location and number of protons.
• The number of signals informs how many types of protons are there
in a molecule.
• The position of the signal gives information about the electronic
environment of each kind of proton. The intensity of the signal gives
the information about the number of protons of each kind and
splitting of the signal gives the information of the environment of a
proton concerning other nearby protons.
• However, not all nuclei are suitable for NMR; notable NMR-active
nuclei include 1H (proton NMR) and 13C (C-13 NMR).
• Various nuclei absorb electromagnetic radiation at distinct wavelengths,
resonating at different energies based on their chemical and electrical
surroundings.
• The NMR signal's position is determined by its chemical shift (δ) value.
• Tetramethylsilane (TMS) serves as a reference with an δ value arbitrarily
set at zero.
• The δ value typically ranges from 1 to 10. The δ value is calculated using
the formula:
• δ = Chemical shift in Hz/ Operating frequency in MHz​
• Chemically and magnetically equivalent nuclei resonate at the same energy
level, producing a single signal in NMR spectra.
In 1H NMR spectrum, the absorption of the protons
of TMS (tetramethylsilane) is defined as “zero” on the
chemical shift (δ) scale, and the absorption of other
protons is reported as a relative shift compared with that
of TMS.
TMS is chosen as a reference compound and defined as
“zero” for several reasons. Since silicon is less
electronegative than carbon, the hydrogens of TMS are in
the high electron-density environment and, therefore are
highly shielded with very low resonance frequency and
rarely interfere with the signals of other compounds.
Delta value: In NMR spectroscopy, the delta value (denoted as δ) refers to
the chemical shift of a signal on the NMR spectrum, measured in parts per
million (ppm). It indicates the relative position of a signal compared to a
reference standard, typically tetramethylsilane (TMS), which is assigned a
chemical shift of 0 ppm.
Downfield: A signal is said to be downfield if it appears at a higher
chemical shift value on the NMR spectrum compared to the reference
standard (TMS). Downfield shifts indicate that the protons experiencing
these signals are in deshielded environments, often due to the presence of
electron-withdrawing groups or atoms nearby.
Upfield: Conversely, a signal is considered upfield if it appears at a lower
chemical shift value on the NMR spectrum compared to the reference
standard (TMS). Upfield shifts indicate that the protons experiencing these
signals are in shielded environments, often due to the presence of electron-
donating groups or less electronegative atoms nearby.
NMR Spectra of Ethanol

• There are 3 types of proton in ethanol

(CH3CH2OH). Hence low resolution NMR shows
3 peaks.
• The hydrogen atoms on the methyl group (-CH3)
appear as a triplet (1:2:1) at around 1.26 ppm.
CH3 protons are split by CH2 proton into 2+1

Intensity
splitting.
• The hydrogen atoms on the methylene group (-
CH2-) appear as a quartet (1:3:3:1) at around 3.69
ppm. CH2 protons are split by CH3 proton into
TMS 3+1 splitting.
• The hydrogen atom on the hydroxyl group (-OH)
appears as a broad singlet (1) at around 2.61 ppm.
OH proton is not split by adjacent proton.
Therefore there is no splitting.
Chemical Shift ppm
NMR Spectra of Acetaldehyde low resolution

There are 2 types of proton in

H acetaldehyde (CH3CHO) Hence low
H O resolution NMR shows 2 peaks.
| || • The hydrogen atoms on the methyl
H— C — C — H group (-CH3) typically appear as a

Intensity
| doublet (1:1) at around 2.21 ppm.
H CH3 protons are split by CHO proton
into 1+1 splitting.
• The hydrogen atom on the aldehyde
H TMS group (-CHO) typically appears as a
quartet (1:3:3:1) at around 9.79 ppm.
CHO protons are split by CH3 proton
into 3+1 splitting.

Chemical Shift
NMR Spectra of Acetaldehyde high resolution
Applications of NMR:
a. NMR provides valuable insights into molecular structure and
aids in assessing compound purity.
b. NMR techniques, such as magnetic resonance imaging (MRI),
are crucial in advanced medical imaging.
c. NMR spectroscopy plays a significant role in protein studies.
d. Solid-state NMR is utilized to determine the molecular
structure of solid substances.
• Compiled by: NA nalecture@gmail.com
• References:
• Arun Bahl, B.S. Bahl, The Essentials of Physical Chemistry.
• David Klein, Organic Chemistry, 3rd Edition.
• Hem Raj Pant, Tanka Mukhiya, Deval Prasad Bhattarai, Prakash
Chandra Lohani, Engineering Chemistry, (1st Edition), 2080.
• P.C. Jain, M. Jain, Engineering Chemistry, (16th Edition), 2013.
Lecture 3: X ray Diffraction (XRD)
X-ray diffraction (XRD) is a powerful analytical technique used to study the
structure and properties of materials at the atomic and molecular levels.
In 1895, Wilhelm Röntgen's accidental discovery of X-rays paved the way for
the development of X-ray spectroscopy, initially applied in medicine for bone
imaging.
In 1912, Max Laue and colleagues conducted groundbreaking experiments at
the University of Munich, demonstrating the wave-like nature of X-rays and
revealing the atomic lattice structure of crystals through diffraction patterns.
H.G.J. Moseley's investigations into emitted X-ray spectra established the
relationship between characteristic radiation wavelength and atomic number
(Z).
Basic Principle of X-ray Diffraction
• X-ray diffraction relies on the phenomenon of diffraction, where X-
rays interact with the periodic arrangement of atoms in a crystal
lattice.
• Different methods of X-ray diffraction, such as powder and single-
crystal diffraction, are employed to analyze the atomic structure of
materials.
• The diffraction pattern produced by X-rays scattered by atoms in a
crystal lattice follows Bragg's law, providing information about
interatomic spacings.
Bragg's Law
Bragg's Law is a fundamental principle in X-ray crystallography, explaining
how X-rays are diffracted by a crystal lattice.
It is expressed mathematically as:
Nλ =2d sin(θ)
Where:
n is an integer representing the order of the diffraction.
λ is the wavelength of the incident X-ray.
d is the spacing between the crystal lattice planes.
θ is the angle of incidence of the X-ray beam.
Bragg's Law provides a fundamental understanding of how X-rays interact with
crystalline materials, forming the basis for X-ray diffraction techniques used in
the structural analysis of crystalline solids.
 Derivation Bragg's Law
Suppose that a plane wave (of any type) is incident on I1 R1
R2
planes of lattice points, with separation d, at an angle θ as I2
shown in the Figure. θ O
There will be a path difference between the ray that gets
reflected (I1 R1) and the ray that gets transmitted and A
d
C
then reflected (I2 R2) .
This path difference is AB+BC B
The two separate waves will arrive at a point (infinitely far
from these lattice planes) with the same phase, and hence O
undergo constructive interference, if and only if this path In △AOB
Sin θ = AB/OB θ
difference is equal to any integer value of the wavelength, Sin θ = AB/d
i.e nλ AB = d Sin θ A
Now B
Similarly
nλ= AB+BC BC = d Sin θ
nλ= d sin θ+ d sin θ
nλ= 2dsin θ
X-ray Diffraction Analysis Technique
• XRD is a non-destructive technique based on the constructive
interference of monochromatic X-rays.
• In XRD analysis, X-rays are directed onto a sample, and the resulting
diffraction pattern is detected and processed.
• XRD characterization is commonly used to examine the crystallinity
and grain size of nanomaterials.
Instrumentation
X ray Source Detector

Sample

Goniometer

Block Diagram of XRD Instrumentation

X-ray Source: The X-ray source emits X-rays of a specific wavelength. Commonly used sources include
copper (CuKα), cobalt (CoKα), and iron (FeKα) anodes. CuKα radiation at a wavelength of 1.5418 Å is the
most widely used source in XRD.
Sample Holder: The sample holder securely holds the sample in place during analysis. Samples are often
prepared as finely powdered or solid compact forms and mounted on sample holders for analysis.
Goniometer: The goniometer is a precision instrument that rotates the sample about various axes, allowing the
angle of incidence (θ) to be controlled accurately. This rotation is crucial for collecting diffraction data at
different angles.
Detector: The detector records the intensity of X-rays diffracted by the sample at various angles. There are
several types of detectors used in XRD, including scintillation counters, proportional counters, and
semiconductor detectors. Modern XRD instruments often use area detectors, such as CCD (charge-coupled
device) or CMOS (complementary metal-oxide-semiconductor) detectors, which allow for rapid data
collection.
Analyzer: The analyzer is responsible for selecting the desired X-ray wavelength and filtering out any
unwanted X-ray radiation. This ensures that only the desired wavelength is used for diffraction.
XRD spectra
• X-ray diffraction (XRD) spectra, also known as diffraction patterns, are
graphical representations of the intensity of X-rays diffracted by a sample as
a function of the diffraction angle (2θ).
• Crystalline solids exhibit well-defined diffraction peaks in their XRD patterns
due to the ordered arrangement of atoms in their crystal lattice. These
diffraction peaks are sharp and discrete, providing information about the
crystal structure, lattice parameters, and phase composition of the material.
• Amorphous solids lack long-range order in their atomic arrangement,
resulting in diffuse scattering in their XRD patterns. Amorphous solids
typically do not produce sharp diffraction peaks but instead exhibit a broad,
featureless background signal. However, they may still show some broad
features related to short-range ordering or local structural motifs.
XRD Spectra

Representative XRD Spectra of Amorphous and Crystalline substance

 Grain Size Determination from XRD Pattern
Debye Scherer equation is used to determine the crystallite size of nanophase materials from XRD data.
The equation relates the average crystallite size (D) to the wavelength of X-rays (λ) and the line broadening
(β). D = K λ/ β cos θ
Where: D: Average crystallite size.
K: Scherrer constant, which varies depending on the crystallite size and crystal symmetry. For the given conditions:
If using the full width at half maximum (FWHM) of spherical crystals with cubic symmetry, K=0.94.
If using the integral breadth of spherical crystals with cubic symmetry, K=0.89.
λ: X-ray wavelength of CuKα radiation, typically given as 1.5418 A˚.
β: Line broadening in radians, usually measured as the full width at half maximum (FWHM) of a diffraction peak. If
given in degrees, it should be converted to radians by multiplying by π/180​.
θ: Bragg angle, denoting the angle at which X-rays are diffracted by the crystal lattice.
Now the equation becomes D = 0.94 λ/ β cos​ θ
 β

2θ
Example
The most intense peak of a crystalline substance is observed at 2θ values of 31.8 upon passing the wavelength of
1.5418A˚. If the full width at half maximum for the peak is 0.5, calculate the grain size of NP.
We have
θ = 31.8/2 ˚
= 15.9 * π / 180 [radians = degree * π / 180] β = 0.5
= 0.27765 radians
λ = 1.5418 Å
= 1.5418 / 10 [1 Å = 0.1 nm.]
= 0.15418 nm
β = 0.5 * π/180 [radians = degree * π / 180]
≈ 0.00872665 radians
Now, plug in the values and perform the calculation as shown in the previous response.
D = 0.94 * λ / (β * cos(θ))
Now, plug in the values:
D = 0.94 * 0.15418 / (0.00872665 * cos(0.27765)) [cos(0.27765) ≈ 0.964631]
D ≈ 0.94 * 0.15418 / (0.00872665 * 0.964631)
D ≈ 0.14478932 / 0.00842461
D ≈ 17.192 nm
So, the average crystallite size is approximately 17.192 nanometers.
Applications of XRD
• XRD is widely used in chemical analysis for studying crystallinity,
structure, composition, and phase identification of materials.
• It finds applications in mineral analysis, purity testing, and
determining the percentage crystallinity of substances.
• XRD spectra are utilized for measuring particle size, including
nanoparticles, using Scherrer's equation.
• It is used for the identification of unknown crystalline substances with
respect to reference
• It can be used to measure the size of nanoparticles.
• Compiled by : NA nalecture@gmail.com
• References:
• Bunaciu, Andrei A., Elena Gabriela UdriŞTioiu, and Hassan Y. Aboul-
Enein. "X-ray diffraction: instrumentation and applications." Critical
reviews in analytical chemistry 45.4 (2015): 289-299.
• Hem Raj Pant, Tanka Mukhiya, Deval Prasad Bhattarai, Prakash
Chandra Lohani, Engineering Chemistry, (1st Edition), 2080.
• 2080/12/21
 Thank You
Best Wishes for your exam
NA nalecture@gmail.com