MODULE IV ANALYTICAL TECHNIQUES AND

SOFTWARE SOLUTIONS (7)

Characterization tools – UV-Visible (DRS), FT-IR, SEM,
TEM, AFM, TG-DTA and XRD (Principle and applications
only). Introduction to softwares - ChemOffice, Image
J, Origin - Molecular modelling, comparison of old drug
structures with new, drug designing-drug for COVID-19
and drug delivery. Molecular docking (drug interaction in
a human body).
Principle and Application of ultraviolet spectroscopy
Ultraviolet-visible (UV-vis) spectroscopy is used to obtain the absorbance
spectra of a compound in solution or as a solid.
What is actually being observed spectroscopically is the absorbance of
light energy or electromagnetic radiation, which excites electrons from
the ground state to the first singlet excited state of the compound or
material.
The UV-vis region of energy for the electromagnetic spectrum covers 1.5 -
6.2 eV which relates to a wavelength range of 800 - 200 nm.
The Beer-Lambert Law is the principle behind absorbance spectroscopy.
 Principle of Ultraviolet Spectroscopy

UV spectroscopy follows the Beer-Lambert law: Principle

when a beam of monochromatic light is passed through a
solution of an absorbing substance, the rate of decrease of
intensity of radiation with thickness of the absorbing solution
is proportional to the incident radiation as well as the
concentration of the solution.
The expression of Beer-Lambert law
A = log (I0/I) = ЄCL; Where, A = absorbance; I0 = intensity of
light incident upon sample cell; I = intensity of light leaving
sample cell; C = molar concentration of solute; L = length of
sample cell (cm.); Є = molar absorptivity or molar extinction
co-efficient.
It is clear that greater the number of molecules capable of
absorbing light of a given wavelength, the greater the extent of
light absorption.
 Ultraviolet Spectroscopy

Observed electronic transitions:

 The lowest energy transition is typically an electron in the
Highest Occupied Molecular Orbital (HOMO) to the
Lowest Unoccupied Molecular Orbital (LUMO).
 For any bond (pair of electrons) in a molecule, the
molecular orbitals are a mixture of the two contributing
atomic orbitals; for every bonding orbital “created” from
this mixing (s, p), there is a corresponding anti-bonding
orbital of symmetrically higher energy (s*, p*).
 The lowest energy occupied orbitals are typically the s;
likewise, the corresponding anti-bonding s* orbital is of the
highest energy
 p-orbitals are of somewhat higher energy, and their
complementary anti-bonding orbital (p*) somewhat lower
in energy than s*.
 Principle of Ultraviolet Spectroscopy

 Unshared pairs lie at the energy of the original atomic orbital,

most often this energy is higher than p or s (since no bond is
formed, there is no benefit in energy)

s*
s s* alkanes
Unoccupied levels
p*
s p* carbonyls

Atomic orbital p p* unsaturated compds.

n
Atomic orbital
n s* O, N, S, halogens
Occupied levels
p n p* carbonyls

s
Molecular orbitals
Principle of Ultraviolet Spectroscopy

The limits the transitions that can be observed:

s s* alkanes 150 nm

s p* carbonyls 170 nm

p p* unsaturated cmpds. 180 nm √ - if conjugated!

n s* O, N, S, halogens 190 nm

n p* carbonyls 300 nm √
Schematic Diagram of the Ultraviolet Spectrometer

log(I0/I) = A
UV sources I0 I

sample
200 400
l, nm

detector
monochromator/
beam splitter optics I0 I0
reference

Two sources are required to scan the entire UV-VIS band:

• Deuterium lamp – covers the UV – 200-330 nm
• Tungsten lamp – covers 330-700 nm
Detectors
 Detector detects intensity of light transmitted by cuvettes and
sends this data to a meter to record and display the values.
 Electronic detectors calculate and compare the intensities of light
beams.
 The photomultiplier tube is the extensively used detector in UV-Vis
instruments. It includes a photoemissive cathode (electrons are
emitted from the cathode when photons strike it), several dynodes
(a dynode emits multiple electrons when one electron strikes it) and
an anode.
 The incident photon, after entering the tube, strikes the cathode.
The cathode then emits multiple electrons, which are then
accelerated towards the first dynode (whose potential is 90V more
positive than cathode).
 The electrons strike the first dynode, leading to the emission of
several electrons for each incident electron.
 These electrons are then accelerated towards the second dynode, to
produce more electrons which are accelerated towards dynode three
and so on. All the electrons are eventually collected at the anode.
 By this time, each original photon has produced 106 – 107 electrons.
 The resulting current is amplified and measured. Photomultipliers are
highly sensitive towards UV and visible radiations and have fast
response times.
Shifts of Absorption and Intensity
 Shifts of Absorption and Intensity
 Bathochromic Shift (Red Shift)- This type of shift is also known as red
shift. Bathochromic shift is absorption maximum is shifted towards the
longer wavelength (l) or lower energy due to the presence of an
auxochrome or change in solvents.
 Shifts of Absorption and Intensity
 Hypsochromic shift (blue shift)-This effect is also known as blue shift.
Hypsochromic shift is an effect by virtue of which absorption maximum is
shifted towards the shorter wavelength or higher energy. Generally it is
caused due to the removal of conjugation or by changing the polarity of the
solvents.
 Shifts of Absorption and Intensity

 Hyperchromic effect- Hyperchromic shift is an effect by virtue of which

absorption maximum increases. The introduction of an auxochrome in
the compound generally results in the hyperchromic effect.
 Shifts of Absorption and Intensity
 Hypochromic effect- Hypochromic effect is defined as the effect by
virtue of intensity of absorption maximum decreases. Hypochromic
effect occurs due to the distortion of the geometry of the molecule with
an introduction of new group.
 17
https://www.slideshare.net/AMFYH/characterization-of-nanopartical
 Applications of UV spectroscopy

Functional Groups: UV spectroscopy is used to detect the

presence or absence of chromophore in the compound. If the
spectrum of a compound comes out to be transparent above
200 nm, it confirms the absence of a) Conjugation b) a
carbonyl group c) Benzene or aromatic compound d) Bromo
or iodo atoms.
Extent of Conjugation: With the increase in double bonds the
absorption shifts towards the longer wavelength. For instance
if double bond is increased by 8 in the polyenes then the
spectrum appears in the visible region.
Geometrical isomers: cis-alkenes absorb at different
wavelength than the trans-alkenes. The cis-isomer suffers
distortion and absorbs at lower wavelength as compared to
trans-isomer.
 Applications of UV spectroscopy

Estimation of the Purity: UV spectroscopy helps to estimate

the purity of a substance. The intensity of the absorption in
sample compared to the reference can be used for the relative
calculation of the purity of the sample substance.
Identification of an unknown compound: The spectrum of
unknown compound is compared with a reference compound.
If both the spectrums coincide then it confirms the
identification of the unknown substance.
Qualitative & Quantitative Analysis: It can be used to find out
molar concentration of solute under study qualitatively and
quantitatively.
IR spectroscopy
 Principle
 In IR spectroscopy the changes in the vibrational energy
depends upon
i) Mass of the atoms present in a molecule.
ii) Strength of the bonds.
iii)Arrangement of atoms within the molecule.
 Theory

 When a molecule absorb radiation with a frequency less than

100 cm-1, molecular rotation takes place.
 If a molecule absorb more energetic radiation in the region
of 104 to 102 cm-1, molecular vibration takes place.
 A single vibrational energy change is accompanied by a large
number of rotational energy changes & the vibrational
spectra appear as vibrational and rotational bands.
 Theory
 Two criteria must be satisfied by a molecule for the
absorption of IR radiation.
 i) the molecule should possess vibrational and rotational
frequency.
 ii) the molecule must give rise to asymmetrical charge
distribution.
 Three main type of absorption bands occurs in IR spectra.
 i) Fundamental ; ii) Overtone; iii) Combinational
Type of Vibration
Type of Vibration
Instrumentation of IR spectroscopy
Radiation source
The radiation source should emit IR electromagnetic radiations which
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.
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.
Sample
IR spectroscopy is used for the characterization of different samples
like solid, liquid, and gas.
Detector
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. Following other detectors are also
used like
1. Bolometers
2. Thermocouple
3. Thermistors
4. Golay cell
5. Photoconducting cell
6. Semiconductivity cell
7. Pyroelectric detectors
Working of IR-spectrophotometer
We know that compounds that contain functional groups having single
and double bonds always stay in a state of vibration motion. This is
called stretching and wagging. 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.
For Example: Let's say we have a molecule that has stretching and
wagging at 2Hz and 4Hz respectively. If the radiation falls on the
sample suppose has a 1Hz frequency. The sample will transmit 100 %
radiation without any absorption as no stretching and wagging take place
at this frequency.
Now, if the IR radiation of frequency 2Hz falls on the sample then it
coincides with the stretching frequency (2Hz) of the molecule and the
radiation will be absorbed by the sample and there will be a sharp drop
in the value of transmittance and it will be indicated in the graph too.
Now, further 3Hz frequency IR radiations fall on the sample and again
it will show 100 % transmittance as no group in the sample stretch or
wags at 3Hz frequency. Now, the radiation proceeds to 4Hz then again
there will be a sharp drop in the transmittance as it coincides with the
4Hz wagging frequency of the compound. This process goes on till we
get our final graph.
31
FTIR Spectroscopy - Fourier transformation

FT-IR spectroscopy is a modified form of IR spectroscopy, it uses a

complex mathematical equation known as Fourier transformation to plot
a graph. All other principle remains the same that is it depends upon the
vibration of molecules where each functional group has its vibrational
energy that can be used to identify a molecule.
 A typical set-up of FT-IR spectrometer has an IR source, beam
splitter, and two mirrors in which one is fixed at its position and
another is movable, sample, detector, interferogram, computer, and a
recorder. So, when an IR source that may be a Nernst glower or
Globar emits IR radiation then it passes through the beam splitter
then half of the IR radiation go to the fixed mirror and half of them
go to the movable mirror.
 Then, these radiations strike both of the mirrors and return and half
of the radiations again go to the original IR source, and half of them
after combining go to the sample then a specific frequency signal is
absorbed by the sample and the remaining signal is transmitted
towards the detector and it detects the signal.
 Then, an interferogram will be obtained which is further go through
Fourier transformation i.e through a complex mathematical equation
a graph between transmittance and wavenumber is plotted. The
obtained graph will be our final graph for the analysis of the
sample.
 The final graph obtained from the FT-IR spectroscopy will contain
peaks having a constructive peak and destructive peak.
Applications: Compounds Identification
 Applications: H-bonding & Quantative Analysis

Distinction Between 2-Types of H-bonding

 If a compound having intra=molecular H-bonding, it will shows broad
bands in IR spectrum.
 Whereas if a compound having intermolecular H-bonding, it will shows
sharp well defined bands.
 For insatnce o-nitrophenol shows broad band due to the presence of
intra-molecular H-bond, whereas p-nitrophenol shows sharp band due
the intermolecular H-bond.
Quantative Analysis:
It can be done by measuring the intensity of the absorption bands.
i.e. xylene exists as mixture of 3 isomers which shows absorption bands as
 o-xylene bands 740 cm-1
 m-xylene bands 880 cm-1
 p-xylene bands 830 cm-1
Applications: Tautomerism
Applications
 Applications: Compounds Identification

 Increasing ring strain increases the C=O stretching

frequency.
 Increasing ring strain leads to hamper the bond stability.
 This phenomenon and effective IR study helps to identify the
compound.
ELECTRON MICROSCOPY

The limit of
resolution of the
human eye is
about 0.1 mm,
or 100 microns
 Light Microscope Electron Microscope
Illuminating source is the Light. Illuminating source is the beam of
electrons.
Specimen preparation takes usually few Specimen preparation takes
minutes to hours. usually takes few days.
Live or Dead specimen may be seen. Only Dead or Dried specimens are seen.

Condenser, Objective and eye piece All lenses are electromagnetic.

lenses are made up of glasses.
It has low resolving power (0.25µm to It has high resolving power (0.001µm),
0.3µm). about 250 times higher than light
microscope.
It has a magnification of 500X to It has a magnification of 100,000X to
1500X. 300,000X.
The object is 5µm or thicker. The object is 0.1µm or thinner.
Image is Coloured. Image is Black and White.
Vacuum is not required. Vacuum is essential for its operation.
There is no need of high voltage High voltage electric current is
electricity. required (50,000 Volts and above).
41
Light Microscope Electron Microscope
There is no cooling system. It has a cooling system to take out
heat generated by high electric
current.
Filament is not used. Tungsten filament is used to produce
electrons.
Radiation risk is absent. There is risk of radiation leakage.
Specimen is stained by coloured dyes. Specimen is coated with heavy metals
in order to reflect electrons.
Image is seen by eyes through ocular Image is received in Zinc Sulphate
lens. Fluorescent Screen or Photographic
Plate.
It is used for the study of detailed It is used in the study of external
gross internal structure. surface, ultra structure of cell and
very small organisms.

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43
Scanning Electron Microscope (SEM)

44
•A Scanning Electron Microscope (SEM) is a type of electron
microscope that images a sample by scanning it with a high energy beam
of electrons in a raster scan pattern.
•The electrons interact with the atoms that make up the sample
producing signals that contain information about the sample’s surface
topography, composition, and other properties such as electrical
conductivity.

Raster Scan Spiral Scan

•Scanning electron microscopy is used for inspecting topographies of
specimens at very high magnifications.
•SEM magnifications can go to more than 300,000 X but most
semiconductor manufacturing applications require magnifications of less
than 3,000 X only.

Principle
•The basic principle is that a beam of electrons is generated by a
suitable source, typically a tungsten filament or a field emission gun.
•The electron beam is accelerated through a high voltage and pass
through a system of apertures and electromagnetic lenses to produce a
thin beam of electrons.
•Then the beam scans the surface of the specimen, electrons are
emitted from the specimen by the action of the scanning beam and
collected by a suitably positioned detector.
When electrons from the microscope interact with a sample, this can
generate different kinds of other electrons and photons.
 The two types of electrons essential for imaging are backscattered
electrons (BSEs) and secondary electrons (SEs).
 Backscattered electrons are reflected back when the primary
electron beam interacts with the sample object. These are elastic
interactions.
 Secondary electrons are different, because they come from the
atoms of the sample, and are the result of inelastic interactions.
What is the difference between these elastic and inelastic
interactions?
 Elastic interactions occur when there is no loss of energy of the
primary electron, and when this happens, the electrons can change
direction but do not change their wavelength
 Inelastic interactions occur when an interaction causes a loss of
energy of the primary electron.
 BSEs and SEs contain different types of information. BSEs
originate from deeper areas of the sample, whereas SEs come from
surface regions.
 Images from BSEs display high sensitivity to differences in atomic
numbers, which will show up as brighter or darker.
 SE images contain more detailed surface information.
 The scanning electron microscope requires different types of
detectors for backscattered and secondary electrons.
 Typically, for SEs, this will be an Everhart-Thornley detector. This
consists of a scintillator inside a Faraday cage. This detector is
positively charged to attract SEs.
 For detecting BSEs, the microscope will use solid state detectors,
placed above the sample.

Chacteristic X-rays can be measured by Energy-dispersive X-ray

spectroscopy or Wavelength-dispersive X-ray spectroscopy and used to
identify and measure the abundance of elements in the sample and map
their distribution.
 Basic Parts of the SEM

 Electron source
 Condenser lenses
 Objective aperture
 Scan coils
 Chamber (specimen)
 Detectors
 Computer hardware and software.
SEM Instruments
The instrument used for SEM includes these components:
 Electron source
 Anode
 Condenser lens
 Scanning coils
 Objective lens.

 The electron source generates electrons at the top of the

microscope’s column.
 The anode plate has a positive charge, which attracts the electrons
to form a beam.
 The condenser lens controls the size of the beam, and determines
the number of electrons in the beam. The size of the beam will
define the resolution of the image.
 Apertures can also be used to control the size of the beam.
 The scanning coils deflect the beam along x and y axes, to ensure it
scans in a raster fashion over the surface of the sample.
 The objective lens is the last lens in the sequence of lenses that
create the electron beam. As the lens closest to the sample, it
focuses the beam to a very small spot on the sample.
 Electrons cannot pass through glass, so SEM lenses are
electromagnetic. They are made up of a coil of wires inside metal
poles.
 When a current passes through these coils they generate a magnetic
field.
 Electrons are highly sensitive to these magnetic fields, which
therefore enables the lenses in the microscope to control them.
Some SEM Morphology

(
b
)

100 nm
 Applications

 SEMs have wide variety of applications in a number of

scientific and industrial fields.
 It uses to get topographical, morphological and
compositional information.
 SEM can detect and analyze surface fractures.
 SEM provide information in microstructures, examine
surface contaminations.
 It showed the elemental composition and estimations.
 SEMs have practical industrial and technological
applications such as semiconductor inspection, assembly of
microchips for computers.
 SEMs can be essential research tools in fields such as life
science, biology, gemology, medical and forensic science,
and material science.
Advantages of SEM Disadvantages
•It gives detailed 3D and •SEMs are expensive and large.
topographical imaging and the •Special training is required to
versatile information. operate an SEM.
•This works very fast. •SEMs are limited to solid samples.
•Modern SEMs allow for the •SEMs carry a small risk of
generation data in digital radiation exposure associated with
form. the electrons that scatter from
•Most SEM samples require beneath the sample surface.
minimal preparation actions.

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SARS-CoV Cancer Cell
Transmission Electron Microscopy
(TEM)
 Principle

 TEM is a microscopy technique whereby a beam of

electrons is transmitted through an ultra thin specimen,
interacting with the specimen as it passes through.

 An image is formed from the interaction of the electrons

transmitted through the specimen.

 The image is magnified and focused onto an imaging

device, such as fluorescent screen, on a layer of
photographic film, or to be detected by a sensor such as
a CCD (Charge-Coupled Device) camera.
Instrumentation of Transmission electron microscope
Electron source: The electrons used in illumination come from a gun that
uses either thermionic or field emission technology.
Condenser lens: To focus the beam, the first condenser
(Electromagnetic type) lens uses an aperture or slit to restrict the entry
of wide-angle electrons. The beam is concentrated on a small area of the
sample by the second condenser lens.
Objective lens: The objective lens gathers the electrons that pass
through a thin sample. The objective lens’s rear aperture is where the
appropriate operating mode is chosen.
Projective lens: The objective lens creates a focused image, which is
then enlarged by an intermediate lens and a projection lens.
Detector: To capture an image of the sample, the magnified image is
projected onto an electron-sensitive camera.
 Working of TEM

 The electron gun at the top of the microscope produces a stream of

monochromatic electrons.
 By using condenser lenses 1 and 2, this flow is focused into a narrow,
coherent beam. The size of the spot, where the electron beam hits
the sample, is set by the first lens. The second lens condenses the
light to a pinpoint on the sample, transforming it from a broad,
diffused spot.
 The Condenser aperture narrows the beam, blocking electrons at high
angles of incidence (those away from the optic axis, the dotted line
along the center).
 A part of the beam that strikes the specimen is transmitted.
63
 Metal apertures have the ability to confine the beam, with the
objective aperture boosting contrast by excluding high-angle
diffracted electrons and the selected area aperture allowing users
to analyze the periodic diffraction of electrons by organized
arrangements of atoms in the sample.
 Through the intermediate and projector lenses, an image is gradually
magnified as it travels down the column.
 The operator sees the image because light is produced when the
beams strike the phosphor image screen. Areas with low electron
transmission appear darker (thicker or denser) because of this. More
electrons were able to pass through the thinner or less dense parts
of the sample, which are depicted by the brighter parts of the
image.
65
 Application of Transmission Electron Microscope

 Crystallographic, compositional, morphological, and topographic

information can all be obtained from TEM images.
 The images provide a molecular-level perspective, letting scientists
examine sample structure and texture in incredible clarity. Industrial
applications aside, this information is helpful in the study of crystals
and metals.
 Tech businesses rely on TEMs to inspect tiny objects for damage and
defects so that they can improve the product’s quality and longevity.
 TEMs have applications in the analysis and production of
semiconductors, as well as in the production of computer and silicon
chip components.
 Comparison Between TEM and SEM

TEM SEM
 Electron beam passes  Electron beam scan over
through the thin sample. surface of sample.
 Specially prepared thin  Sample can be of any thickness
samples are supported on and is mounted on an
TEM grids. aluminum stub, carbon tape.
 Specimen stage halfway  Specimen stage in the chamber
down column. at the bottom of the column.
 Image shown on fluorescent  Image shown on TV monitor.
screen.  Image is of the surface of the
 Image is a 2D projection of sample.
the sample
Atomic Force Microscope (AFM)
What is AFM?

 The atomic force microscope (AFM) was invented in 1986 by Binnig,

Quate and Gerber.
 Atomic force microscopy (AFM) is one of the foremost tools for
imaging, measuring and manipulating matter at the nanoscale.
 A type of scanning probe microscopy (SPM).
 High-resolution mapping of surface topography, by far the biggest
application of the AFM offers image resolution down to the atomic
scale.
 The AFM raster scans a sharp probe over the surface of a sample and
measures the changes in force between the probe tip and the sample.
Principle
 The AFM consists of a cantilever with a sharp tip (probe) at its end
that is used to scan the specimen surface.
 The cantilever is typically silicon or silicon nitride with a tip radius of
curvature on the order of nanometers.
 When the tip is brought into proximity of a sample surface, laser beam
activates forces between the tip and the sample lead to a deflection
of the cantilever according to Hooke's law.
 Depending on the situation, forces that are measured in AFM include
mechanical contact force, van der Waals forces, capillary
forces, chemical bonding, electrostatic forces.

Working Concept

 The physical parameter probed is a force resulting from different

interactions.
 Thus, an AFM image is generated by recording the force changes as
the probe (or sample) is scanned in the x and y directions.
 The sample is mounted on a piezoelectric scanner, which ensures
three-dimensional positioning with high resolution.
 The force is monitored by attaching the probe to a pliable cantilever,
which acts as a spring, and measuring the bending or "deflection" of
the cantilever.
 Use a laser spot reflected from the top surface of the cantilever
into an array of photodiodes.
Three Modes of operation
Contact mode : hard, stable samples in air or liquid
Non-contact mode : non- invasive sampling
Tapping (Intermittent contact) : No shear and damaging samples
Contact Mode AFM:
 In contact mode AFM, the probe tip is in continuous contact with the
sample surface as it scans across.
 The cantilever maintains a constant force or deflection during
scanning by adjusting the height of the probe tip.
 This mode provides high-resolution topographic images of the sample
surface.
 Contact mode AFM is suitable for imaging relatively flat surfaces and
measuring surface roughness.
Tapping Mode AFM (Intermittent Contact Mode):
 Tapping mode AFM operates by oscillating the cantilever and probe
tip near its resonant frequency.
 As the probe tip oscillates, it lightly taps or intermittently contacts
the sample surface.
 The amplitude of the cantilever oscillation is monitored, and
variations in this amplitude provide information about the sample
surface.
 Tapping mode AFM is less likely to damage delicate samples compared
to contact mode AFM, making it suitable for imaging soft or biological
samples.
 It is also used for studying dynamic processes on the sample surface.
Non-contact Mode AFM:
 Non-contact mode AFM operates with the probe tip hovering very
close to, but not touching, the sample surface.
 It detects interactions such as van der Waals forces between the
probe tip and the sample surface.
 This mode is highly sensitive to weak forces and is often used for
imaging delicate samples or studying surface properties without
physically contacting the surface.
 Non-contact mode AFM can provide high-resolution images of surface
features and properties.
Applications
 The AFM is useful for obtaining three-dimensional topographic
information of insulating and conducting structures with lateral
resolution down to 1.5 nm and vertical resolution down to 0.05 nm.T
 These samples include clusters of atoms and molecules, individual
macromolecules, and biologic al species (cells, DNA, proteins).
 Minimal sample preparation involved for AFM imaging.
 The AFM can operate in gas, ambient, and fluid environments and
can measure physical properties including elasticity, adhesion,
hardness, friction and chemical functionality.
Advantages
 The AFM has several advantages over the scanning electron
microscope (SEM). Unlike the electron microscope which provides a
two-dimensional projection or a two-dimensional image of a sample,
the AFM provides a true three-dimensional surface profile.
 Additionally, samples viewed by AFM do not require any special
treatments (such as metal/carbon coatings) that would irreversibly
change or damage the sample.
 While an electron microscope needs an expensive vacuum
environment for proper operation, most AFM modes can work
perfectly well in ambient air or even a liquid environment.
 This makes it possible to study biological macromolecules and even
living organisms.
 In principle, AFM can provide higher resolution than SEM.
Disadvantages
 The SEM can image an area on the order of millimetres by millimetres
with a depth of field on the order of millimetres.
 The AFM can only image a maximum height on the order of
micrometres and a maximum scanning area of around 150 by 150
micrometres.
 Another inconvenience is that at high resolution, the quality of an
image is limited by the radius of curvature of the probe tip, and an
incorrect choice of tip for the required resolution can lead to image
artifacts
 Slow Imaging Speed, Complexity of Operation, Sample Preparation
Requirements, Probe Tip Wear and Contamination, Limited Imaging in
Liquid Environment, Surface Damage Potential and Limited Depth
Sensitivity
X-ray Powder Diffraction (XRD)
English physicists Sir W.H. Bragg and his son Sir W.L. Bragg developed a
relationship in 1913 to explain why the cleavage faces of crystals appear to
reflect X-ray beams at certain angles of incidence (theta, θ).
 d is the distance between atomic layers in a crystal,
 lambda λ is the wavelength of the incident X-ray beam;
 n is an integer.
This observation is an example of X-ray wave interference (Roentgen strahl
interferenzen), commonly known as X-ray diffraction (XRD), and was direct
evidence for the periodic atomic structure of crystals postulated for several
centuries.

Bragg’s Law
What is X-ray Powder Diffraction (XRD)

X-ray powder diffraction (XRD) is a rapid analytical technique

primarily used for phase identification of a crystalline material and
can provide information on unit cell dimensions. The analyzed material
is finely ground, homogenized, and average bulk composition is
determined.

Fundamental Principles of X-ray Powder Diffraction (XRD)

 Max von Laue, in 1912, discovered that crystalline substances act

as three-dimensional diffraction gratings for X-ray wavelengths
similar to the spacing of planes in a crystal lattice.
 X-ray diffraction is now a common technique for the study of
crystal structures and atomic spacing.
 X-ray diffraction is based on constructive interference of
monochromatic X-rays and a crystalline sample.
 These X-rays are generated by a cathode ray tube, filtered to
produce monochromatic radiation, collimated to concentrate, and
directed toward the sample.
 Conversion of the diffraction peaks to d-spacings allows identification
of the mineral because each mineral has a set of unique d-spacings.
 Typically, this is achieved by comparison of d-spacings with standard
reference patterns.
 The interaction of the incident rays with the sample produces
constructive interference (and a diffracted ray) when conditions
satisfy Bragg’s Law (nλ = 2d sinθ).
 This law relates the wavelength of electromagnetic radiation to the
diffraction angle and the lattice spacing in a crystalline sample.
 These diffracted X-rays are then detected, processed and counted.
 By scanning the sample through a range of 2θ angles, all possible
diffraction directions of the lattice should be attained due to the
random orientation of the powdered material.
X-ray Powder Diffraction (XRD) Instrumentation
 X-ray diffractometers consist of three basic elements: an X-ray
tube, a sample holder, and an X-ray detector.
 X-rays are generated in a cathode ray tube by heating a filament to
produce electrons, accelerating the electrons toward a target by
applying a voltage, and bombarding the target material with
electrons.
 When electrons have sufficient energy to dislodge inner shell
electrons of the target material, characteristic X-ray spectra are
produced.
 These spectra consist of several components, the most common being
Kα and Kβ. Kα consists, in part, of Kα1 and Kα2. Kα1 has a slightly shorter
wavelength and twice the intensity as Kα2.
 The specific wavelengths are characteristic of the target material
(Cu, Fe, Mo, Cr).
 Filtering, by foils or crystal monochrometers, is required to produce
monochromatic X-rays needed for diffraction.
 Kα1 and Kα2 are sufficiently close in wavelength such that a weighted
average of the two is used. Copper is the most common target material
for single-crystal diffraction, with CuKα radiation = 1.5418Å.
 These X-rays are collimated and directed onto the sample. As the
sample and detector are rotated, the intensity of the reflected X-
rays is recorded.
 When the geometry of the incident X-rays impinging the sample
satisfies the Bragg Equation, constructive interference occurs and a
peak in intensity occurs.
 A detector records and processes this X-ray signal and converts the
signal to a count rate which is then output to a device such as a
printer or computer monitor.
 The geometry of an X-ray diffractometer is such that the sample
rotates in the path of the collimated X-ray beam at an angle θ while
the X-ray detector is mounted on an arm to collect the diffracted X-
rays and rotates at an angle of 2θ.
 The instrument used to maintain the angle and rotate the sample is
termed a goniometer. For typical powder patterns, data is collected
at 2θ from ~5° to 70°, angles that are preset in the X-ray scan.
Applications
 X-ray powder diffraction is most widely used for the identification of
unknown crystalline materials (e.g. minerals, inorganic compounds).
 Determination of unknown solids is critical to studies in geology,
environmental science, material science, engineering and biology.
 Characterization of crystalline materials
 Identification of fine-grained minerals such as clays and mixed layer
clays that are difficult to determine optically
 Determination of unit cell dimensions
 Measurement of sample purity
Structure determination of individual crystals and mixed crystals
X-ray pattern of NaCl crystal.
X-ray pattern of KCl crystal..
X-ray pattern of mixture of NaCl and KCl.

XRD spectra of ( a ) amorphous and ( b ) polycrystalline silicon colloids,

( c ) corresponds to single crystal silicon sample powder
Strengths
 Powerful and rapid (< 20 min) technique for identification of an
unknown mineral
 In most cases, it provides an unambiguous mineral determination
 Minimal sample preparation is required
 XRD units are widely available
 Data interpretation is relatively straight forward
Limitations
 Homogeneous and single phase material is best for identification of an
unknown
 Must have access to a standard reference file of inorganic compounds
(d-spacings, hkls)
 Requires tenths of a gram of material which must be ground into a
powder
 For mixed materials, detection limit is ~ 2% of sample
 For unit cell determinations, indexing of patterns for non-isometric
crystal systems is complicated
 Peak overlay may occur and worsens for high angle 'reflections'
Thermogravimetric – Differential Thermal Analysis
 Thermoanalytical Data
What is Thermal Analysis?

 Thermal analysis is a series of techniques that provide physical

property measurement like mass change or enthalpy change, etc as a
function of temperature, time, and other variables.

Thermal Gravimetric Analysis (TGA)

 Measure change in weight during heating or cooling

Differential Thermal analysis (DTA)

 Measure temperature difference between the sample and reference.

Differential Scanning Calorimetry (DSC)

 Measure heat absorbed or liberated during heating or cooling

 Thermal Gravimetric Analysis (TGA)
What is the TGA?

A technique that permits the continuous weighing of a sample as a

function of temperature and/or as a function of time at a desired
temperature.
What Information You Can from TGA ?
1) Thermal Stability of Materials: Explicate decomposition mechanism,
fingerprint materials for identification & quality control.

2) Oxidative Stability of Materials: Oxidation of metals in air,

oxidative decomposition of organic substances in air/O2, Thermal
decomposition in inert atmosphere.
3) Composition of Multi-component Systems: Behaviours sufficiently
different on temperature scale can be identified and reaction
mechanism formulated.

4) Estimated Lifetime of a Product: Related to thermal stability.

5) Decomposition Kinetics of Materials: Rate of reaction, Activation

Energy.

6) The Effect of Reactive or Corrosive Atmospheres on Materials:

Oxidation & Corrosion Studies.

7) Moisture and Volatiles Content of Materials: Loss of moisture,

drying, desorption.
How TGA works
In thermogravimetric analysis (TGA), a sample is continually weighted
while heating, as an inert gas atmosphere is passed over it. Many solids
undergo reactions that evolve gaseous byproducts. In TGA, these
gaseous byproducts are removed and changes in the remaining mass of
the sample are recorded.
In thermogravimetry, null point balances are the most often used form
of the balance system. The null point approach causes the balance beam
to move away from its normal position whenever there is a change in
weight. Hence, a sensor detects this deviation and initiates a force
that will restore the balance to the null position. This restoring force
is proportional to the change in weight.
 TGA: How the balance works
The balance operates on a null-balance principle. At the zero, or
“null” position equal amounts of light shine on the 2 photodiodes.
If the balance moves out of the null position an unequal amount of
light shines on the 2 photodiodes. Current is then applied to the
meter movement to return the balance to the null position.

The amount of current applied is proportional to the weight loss or

gain.
 Thermal Gravimetric Analysis (TGA)
Mechanisms of Weight Change in TGA

Weight Loss:

 Decomposition: The breaking apart of chemical bonds.

 Evaporation: The loss of volatiles with elevated temperature.

 Reduction: Interaction of sample to a reducing atmosphere

(hydrogen, ammonia, etc).

 Desorption.

Weight Gain:

 Oxidation: Interaction of the sample with an oxidizing atmosphere.

 Absorption or Adsorption.
Interpretation of TGA Curves

1. No mass change over entire range of

temperature
2. Desorption / Drying. Mass loss is large
followed by no change in mass.
3. Single stage decomposition
4. Multistage decomposition
5. Similar with 4 but either due to faster
heating rates or due to no intermediates.
6. Atmospheric reaction, increase in mass,
reaction like sulface oxidation.
7. Similar to curve 7, but product decomposes
at higher temperatures.
What does a TGA Thermal Curve look like?

A TGA thermal curve is displayed from left to right. The

descending TGA thermal curve indicates a weight loss occurred
 Differential Thermal analysis (DTA)
What is the DTA?

A technique in which the difference in temperature between the

sample and a reference material is monitored against time or
temperature while the temperature of the sample, in a specified
atmosphere, is programmed.
The sample and the reference are placed symmetrically in the furnace.
The furnace is controlled under a temperature program and the
temperature of the sample and the reference are changed. During this
process, a differential thermocouple is set up to detect the temperature
difference between the sample and the reference.
Also, the sample temperature is detected from the thermocouple on the
sample side.
 When a graph of ∆T Vs temperature
is plotted, the maxima and / or
minima due to exothermic and / or
endothermic process.
 Maxima and minima are called as
peaks.
 Maxima = exothermic process in
which heat is evolved from the
sample causing temperature to
increase.
 Maxima = endothermic process in
which heat is absorbed by the
sample causing temperature to
decrease.
Differential Thermal analysis (DTA)
Information of the Endothermic Peak of the DTA?
Information of the Exothermic Peak of the DTA?
TG-DTA
 TGA and DTA: Calcium Oxalate Monohydrate

H2O

CO

Derivative Weight (%)/min

Weight (%)

CO2
 TGA and DTA: Calcium Oxalate Monohydrate
1st Step, H2O Release
CaC2O4•H2O (s) → CaC2O4 (s) + H2O (g)
2nd Step, CO Release
CaC2O4 (s) → CaCO3 (s) + CO (g)
3rd Step, CO Release
CaCO3 (s) → CaO (s) + CO2 (g)

 Calcium oxalate monohydrate CaC2O4.H2O is stable up to 100 ⁰C.

 Release of water begins at 100 ⁰C and gets completed at 170 ⁰C.
 Then, it shows thermal stability in the range of 170 - 400 ⁰C.
 The decomposition of CaC2O4 to CaCO3 with the elimination of CO
occurs in the temperature range of 400-480 ⁰C.
 Thermal stability of CaCO3 in the temperature range 480-650 ⁰C.
 Above 650 ⁰C CaCO3 decomposes to CaO and CO2.
 The DTA analysis also showed corresponding three endothermic
peaks.
TGA and DTA: CuSO4. 5H2O
 TGA and DTA: CuSO4. 5H2O

1st Step, Release of 2H2O Molecules

CuSO4•5H2O (s) → CuSO4•3H2O + 2H2O ↑
2nd Step, Release of another 2H2O Molecules
CuSO4•3H2O → CuSO4•H2O + 2H2O ↑
3rd Step, Release of rest H2O Molecule
CuSO4•H2O → CuSO4+ H2O ↑
4th Step, Release of SO2 and 1/2O2
CuSO4→ CuO +SO2↑+1/2O2.
5th Step, Release of O2
CuO → Cu2O +1/2O2
 CHEMOFFICE
What is the ChemOffice?

ChemOffice is a scientific tool to efficiently keep track of their work,

gain a deeper understanding of their data and produce scientific reports
professionally and efficiently.

1) ChemDraw

 ChemDraw is used by hundreds of thousands of

scientists around the world to quickly and
effectively to draw molecules and reactions for
use in documents and electronic lab notebooks.

 It is also used to search databases, and to

generate accurate names from structures and to
predict properties and spectra.
2) ChemDraw Excel

 ChemDraw Excel adds chemical intelligence to Excel spreadsheets so that

chemists can use Excel’s analysis, sorting and organization tools to further
manipulate.

 The enrich sets of compounds and data and explore structure-activity

relationships

3) Chem3D

 Chem3D generates 3D models so that chemists can view their compounds

in three dimensions to assess shape and properties to maximize activity or
specificity.

4) ChemFinder

 ChemFinder is a chemically-intelligent personal database system that

scientists use to organize their compounds and to search for and
correlate structures with properties.
 ImageJ
What is ImageJ?
ImageJ is a Java-
based image
processing program.
Applications:
 ImageJ can display, edit,
analyze, process, save, and
print 8-bit color and
grayscale, 16-bit integer,
and 32-bit floating point
images.
 It can read many image file formats,
including TIFF, PNG, GIF, JPEG, BMP, DICOM, and FITS, as
well as raw formats.
 ImageJ supports image stacks, a series of images that share a
single window, and it is multithreaded, so time-consuming
operations can be performed in parallel on multi-CPU hardware.
 ImageJ can calculate area and pixel value statistics of user-defined
selections and intensity-thresholded objects.

 It can measure distances and angles. It can create

density histograms and line profile plots.

 It supports standard image processing functions such as logical and

arithmetical operations between images, contrast
manipulation, convolution, Fourier analysis, sharpening, smoothing,
edge detection, and median filtering.

 It does geometric transformations such as scaling, rotation, and

flips.

 The program supports any number of images simultaneously, limited

only by available memory.
 Origin (Data Analysis Software)

What is Origin ?
 Origin is a proprietary computer program for interactive scientific
graphing and data analysis.

 It is produced by OriginLab Corporation, and runs on Microsoft

Windows.

 Origin is primarily a GUI software with a spreadsheet front end.

 Unlike popular spreadsheets like Excel, Origin's worksheet is column

oriented.

 Each column has associated attributes like name, units and other
user definable labels.

 Instead of cell formula, Origin uses column formula for calculations.

 Recent versions of Origin have introduced and expanded on batch
capabilities, with the goal of eliminating the need to program many
routine operations.

 Instead the user relies on customizable graph templates, analysis

dialog box.

 Themes which save a particular suite of operations, auto recalculation

on changes to data or analysis parameters, and Analysis Templates™
which save a collection of operations within the workbook.

 Origin also has a scripting language (LabTalk) for controlling the

software, which can be extended using a built-in C/C++-based compiled
language (Origin C).

 Other programming options include an embedded Python environment,

and an R Console plus support for Rserve.
 Molecular Docking
 In the field of molecular
modeling, docking is a method which
predicts the preferred orientation of
one molecule to a second when a ligand
and a target are bound to each other
to form a stable complex.

 The associations between biologically relevant molecules such

as proteins, peptides, nucleic acids, carbohydrates, and lipids play a
central role in signal transduction.

 Furthermore, the relative orientation of the two interacting partners

may affect the type of signal produced (e.g., agonism vs antagonism).
Therefore, docking is useful for predicting both the strength and
type of signal produced.
 One can think of molecular docking as a problem of “lock-and-key”,
in which one wants to find the correct relative orientation of
the “key” which will open up the “lock” (where on the surface of the
lock is the key hole, which direction to turn the key after it is
inserted, etc.).

 Here, the protein can be thought of as the “lock” and the ligand can
be thought of as a “key”.

 Molecular docking may be defined as an optimization problem, which

would describe the “best-fit” orientation of a ligand that binds to a
particular protein of interest.