X-_Ray_Crystallography_DHANASHREE KOLHEKAR[1].pptx

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X- Ray Crystallography:
Unlocking the structure of molecules
Presented By
Dhanashree Giridhar Kolhekar
M. Pharm (Pharmaceutical Analysis)
I Semester
Department of Pharmaceutical Sciences,
BBAU Lucknow

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Contents
 Introduction 3
 History 4
 Principle 5
 Bragg’s law 8
 What is x ray and why they are used 10
 Crystals 11
 Types of crystals 12
 Why crystals are used 13
 Different x ray methods 14
 Procedure 24
 Instrumentation 25
 Applications 37
Dhanashree Kolhekar,M.Pharm- I sem(Pharma'ceutical ANALYSIS) BBAU, Lucknow

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Introduction
• X-ray Crystallography is a scientific method used to
determine the arrangement of atoms of a crystalline
solid in three dimensional space.
• A beam of x rays strikes a crystal and causes a beam of
light to diffract into many specific directions.
• The electron density , and the positions of atoms can be
contructed from the diffraction pattern to determine
the mean positions of the atoms in the crystal.

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History
• The English physicist sir William
Henry Bragg pioneered the
determination of crystal structure
by x ray diffraction methods.
• Using X ray crystal data , Dr.
James Watson and Dr. Francis
Crick determined the helix
structure of DNA in 1953.
Original Bragg x ray
spectrometer developed by
William Bragg

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Principle
It uses principle of diffraction into many specific
directions from crystal of the substance
This technique takes advantage of the interatomic
spacing of most crystalline solids by employing
them as a diffraction gradient for x ray light
A crystal can be described with the aid of grid or
lattice , defined by three axis and angles between
them.

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The diffraction pattern obtained helps to
generate the electron density map.
This map reveals where electrons are most
densely located, allowing researchers to build
an atomic model that accurately represents
the 3D arrangement of atoms in the crystal
structure.

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Bragg’s law
• Diffraction of light as well as x ray obeyed by Bragg’s law,
which states that when the x ray is incident onto a crystal
suface , its angle of incidence , θ, will reflect back with a
same angle of scattering , θ. And when the path difference ,
d is equal to whole number, n of wavelength, a constructive
interference will occur . Thus
nλ = 2d sinθ
N is an integer
d is the spacing of the crystal layers
Θ is the incident angle
λ is the wavelength of x-ray

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What is x ray and why they are used?

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Crystals
• A crystal is a solid
whose atoms are
arranged in a highly
ordered repeating
pattern .
• these patterns are
called as crystal
systems .

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Types of crystals

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Why crystals are used?
• When sample is present in liquid form, the
bond angle keep changing, position of atoms
keep changing in every moment so it is very
hard to determine the structure of that
sample.
• In crystalline form the conformtion is fixed in
a single place so it makes easy to determine
the structure of crystal.

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Different x ray methods
x ray absorption method
Auger x ray emission method
X ray fluorescence method
X ray diffraction method
Rotating crystal method
Powder crystal method

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X ray absorption method
• Wavelength at which a
sudden change in
absorption occurs is used
to identify an element
present in a sample, and
the magnitude of the
change determines the
amount of particular
element present.

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Auger x ray emission method
• X-rays eject inner-shell
electrons.
• Instead of emitting a
characteristic X-ray, the
atom releases energy by
ejecting an outer electron,
called an Auger electron.
• The kinetic energy of this
electron provides element-
specific information.

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X ray fluorescence method
• The primary x ray ejects
electrons from inner
energy levels where the
wavelength is equal to
absorption edge.
• But when the
wavelength is shorter
than absorption edge it
emits secondary x ray
when electrons fall into
inner vacant levels.

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X ray diffraction method
• When a beam of monochromatic x radiation is
directed at a crystalline material, one observes
reflection or diffraction of the x rays at various
angle with respect to the primary beam.
• The relationship between the x radiation , angle
of diffraction and distance between each set of
atomic planes of crystal lattice is given by bagg’s
law
nλ = 2d sinθ

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Rotating crystal method
• This method involves rotating a single crystal
within an X-ray beam to obtain diffraction
patterns, which reveal information about the
arrangement of atoms within the crystal
lattice.
• As the crystal rotates, different sets of atomic
planes come into alignment with the incident
X-ray beam at various angles.

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• Each plane will produce a spot on
photographic plate.
• One can take photographs in two ways:
Complete rotation method
 oscillation method

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Powder crystal method
• The powder crystal method, is an X-ray crystallography
technique used to study powdered samples.
• This method is ideal for samples that cannot be prepared as
single crystals.
• The sample is ground into a fine powder, so it contains many
small crystals with random orientations.
• This allows for a range of crystal planes to be exposed to the
X-ray beam at various angles.

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Procedure
Structure determination
Data collection
Protein crystallization
Protein purificataion
It is a multistep process which can be outlined as
below:

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Instrumentation
1.Production of x rays
2.Collimator
3.Monochromator
4.Detectors

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Production of x rays
• X rays are produced inside the x ray tube when high
energy projectile electrons from the filament interact
with the atoms of the anode.
• X ray also called as coolidge tube is a large vaccum
tube containing a heated cathode of tungsten filament
and copper or molybdenum target metal anode.
• Operated at higher voltage upto 60 kV.

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Cont….
• There is cathode which is a filament of tungsten metal
heated by a battery to emit the thermionic electrons.
• This beam of electrons moves towards anode target
and attain the kinetic energy and 99% of energy is
converted into heat via collision and remaining 0.5-1%
is converted to x rays.
• Generally the target gets very hot in use so this
problem has been solved to some extent by cooling
the tube with water.

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Collimator
• X rays produced by the target material are randomly
directed.
• They form a hemisphere with a target at the centre. In
order to get a narrow beam of x rays, the x rays generated
by the target material are allowed to pass through a
collimator which consists of two sets of closely packed
metal plates separated by small gap.
• The collimator absorbs all the x rays except the narrow
beam that passes between the gaps.

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Monochromator
They are of two types:
• Filter monochromator
A filter is a window of material that absorbs undesirable
radiation but allows the radiation of required wavelength
to pass.
• Crystal monochromator
A crystal monochromator is made up of a suitable
crystalline material positioned in the x ray beam so that
the angle of reflecting planes satisfies Braggs equation for
the required wavelength.

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Detectors
1. Photographic method
• In order to record the position and intensity of
x ray beam a plane or cylindrical film is used.
• The film after exposing to x rays is developed.
The blackening of the developed film is
expressed in terms of density units D given by,
• D= Iog Io
/I

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2. Counter methods
• The geiger tube is filled with an inert gas like argon and the
central wire anode is maintained at a potential of 800 to
2500V.
• when an X rays is entering the geiger tube, this ray
undergoes collision with the filling gas , resulting in the
production of an ion pair: the electron produced moves
towards the central anode while the positive ions move
towards outer electrode.

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• The electron is accelerated by the potential
gradient and causes the ionisation of large
number of argon atoms, resulting production
of an avalanche of electrons that are travelling
towards the central anode.
• The geiger tube is in expensive and is
relatively trouble free detector. This tube gives
the highest signal for given x ray intensity.

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Applications of crystallography
• Helps design drugs by revealing the 3D structure of
target proteins.
• used to determine the structure of minerals in geology.
• Helps to understand double-helix structure of DNA and
the organization of RNA.
• Used to study polymorphs of drugs to optimize their
solubility and bioavailability.

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• Differentiation of sugar
• X ray analysis of milk powder

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References
• X-ray Crystallography - Chemistry LibreTexts
• https://www.slideshare.net/slideshow/xray-crystallography-1
18911634/118911634
• www.creativebiomart.net/resource/principle-protocol-x-ray-cr
ystallography-393.htm
• X-ray Crystallography: Definition, Principle, Steps, Data Analysi
s, Applications, and Limitations - The Science Notes
• Instrumental methods of analysis- williards, 7th
edition CBS

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