CONTENT

1. Introduction
2. What is x-ray and x-ray diffraction
3. Review of literature
4. Components and working principle of XRD
5. Types of XRD
6. Use and applications of XRD
7. Advantage and disadvantage of XRD
8. Conclusion
10.Reference
 Introduction

Crystallographers and mineralogists had accumulated the knowledge about crystals

by the measurement of interfacial angles, chemical analysis and determination
ofphysical properties. There was a little knowledge about interior structure,
however, although some shrewd guesses were made, such as crystals were built up
by periodic repetition of some unit, probably an atom or molecule and that these
units were situated some 1 or 2 Ǻ apart. Also, there were indications, that X-rays
might be electromagnetic waves having wavelength of about 1 or 2 Ǻ. In addition to
this, it was known that diffraction occurred when visible light passed through a set
of regularly spaced scattering objects, provided the wavelength of the wave motion
was of the same order of magnitude as the repeat distance between the scattering
centers.

In the year 1913, Sir William Lawrence Bragg and his son Sir William Henry
Bragg put forward a new approach for studying X-ray diffraction by crystals. They
successfully analyzed the Laue experiment and were able to express the necessary
conditions for diffraction in a considerably simpler mathematical form than that used
by Van Laue. The problem of crystal structure with the new tool of X-ray diffraction
solved many structures as that of KC1, KBr, NaCl, and KI, all of which have the
same structure as that of NaCl; these were the first complete crystal-structure
determinations ever made[1].

Further, the two scientists Bragg and Bragg considered an X-ray beam ofwavelength
incident in a direction at an angle to the family of Bragg planes with interplanar
spacing d, and showed that constructive interference takes place between the rays
scattered by the atoms in different Bragg planes when the condition
n= 2d sin (1)

where n is an integer. The above condition is known as Bragg‟s law. The law states
the essential condition which must be met if diffraction is to occur
where n is called the order of diffraction; it may take on any integral value consistent
with sinnot exceeding unity and is equal to the number of wavelengths in the path
difference between rays scattered by adjacent planes . Application of X-ray
diffraction technique offer an excellent tool for the investigation of crystallographic
orientation in polycrystalline materials.

1 in 1912 Max Van Laue2,3 developed . the idea of crystal acting as three
dimensional grating to diffract X-ray since the lattice dimensions are of the
same order of magnitude as the wave-length of X-rays. Since then many X-ray
methods were developed to study crystals.

Amongst them,the most useful and proved to be powerful techniques are

interferrometry and diffractometry as they are of non-destructive type, non-contact,
fast and light sensitive. With the use of these methods, information can be obtained
regarding the deformation of the crystals and electronic energy density in terms of a
function of width.4,5

The principle of this technique is based on scattering of X-rays by a crystal

consisting of well defined array of atoms, ions and molecules. In a crystal, atoms
and molecules are arranged in different planes in a definite manner. These
interplanar spacing (or distances) are of the same magnitude as the wavelength of
X-ray (0.5 to 2 A) and hence, crystal planes act as diffraction gratings. Interaction
of X-rays reflected by a set of parallel planes satisfying Bragg condition leads to
constructive interference.

The Bragg's condition for the occurrence of diffraction is

where, d is the interplanar spacing, 6 is the incidence angle with respect to the
diffraction plane, n is an integer that gives the order of reflection and X is the
wavelength of X-rays.
Diffraction occurs only when Bragg’s Law is satisfied Condition for constructive
interference (X-rays 1 & 2) from planes with spacing d To satisfy Bragg’s Law, q must
change as d changes e.g., q decreases as d increases.

X-RAY & X-RAY DIFFRACTION:

Bragg’s Law:
The Braggs were awarded the Nobel Prize in physics in 1915 for their work in
determining crystal structures beginning with NaCl, ZnS and diamond Although
Bragg's law was used to explain the interference pattern of X-rays scattered by
crystals, diffraction has been developed to study the structure of all states of matter
with any beam, e.g., ions, electrons, neutrons, and protons, with a wavelength similar
to the distance between the atomic or molecular structures of interest.

Deriving Bragg’s Law: Constructive interference occurs only when

Constructive and Destructive Interference of Waves:

Constructive Interference In Phase

Destructive Interference Out of Phase

REVIEW OF LITERATURE

The current research on production and characterization of serratiopeptidase from

Serratia marcescens has been substantiated by many researchers and a few literatures
quoted below have helped in a successful completion of the research and arrival of
a decisive conclusion for a novel production of serratiopeptidase. Steiner et al.,
(1912) used the X-ray analytical methods to discover the atomic architecture of
biopolymers such as proteins and nucleic acids.

The importance of this method of structural analysis can be stated simply by saying
that the structure of DNA was discovered largely due to the availability of X-ray
crystallography data. The wave length of X-rays is of the same order viz 10-8cm as
the distance between individual atoms in a crystal. This similarity led to make a
brilliant suggestion that crystals could act as natural and very fine three dimensional
diffraction gratings of X-rays.

A pattern of this type is generally referred to as the have pattern. X-ray diffraction
method has proved to be unambiguous, fast, sensitive and most reliable tool for
structural analysis of enzymes. It is preferred over chemical methods for its complete
identification of various crystallites present in Enzymes. Energy dispersive
spectroscopy is employed to know the mineral element in enzymes.

Laurence Bragg et al., (1913) determined the structure of NaCl using X-ray analysis.
By use of theory, the three-dimensional structures of nearly 250,000 organic and
biological molecules were determined. Crystallography deals with the study of all
possible type of Crystals and determination of the actual structure of the crystalline
solids by X-rays, neutron beams or electron beams. The goal of crystallization is
usually to produce a well-ordered crystal that is lacking in contamination and large
enough to provide a diffraction pattern when hit with x-ray.

This diffraction pattern can then be analyzed to discern the proteins three-
dimensional structure. Protein crystallization is inherently difficult because of the
fragile nature of protein crystals. Proteins have irregularly shaped surfaces, which
result in the formation of large channels within any protein crystal. Therefore,
noncovalent bonds that hold together the lattice must often be formed through
several layers of solvent molecules.

In addition to overcoming the inherent fragility of protein crystals, the successful

production of x-ray worthy crystals is dependent upon a number of environment
factors because so much variation exists among proteins, with each individual
requiring unique condition for successful crystallization. Therefore, attempting to
crystallize a protein without a proven protocol can be very tedious. Some factors that
require consideration are protein purity, PH, and concentration of protein,
temperature and precipitants.

In order for sufficient homogeneity, the protein should usually be at least 97% pure.
pH conditions are also very important, as different pH can result in different packing
orientations. Buffers, such as Tris-HCl, are often necessary for the maintenance of a
particular pH precipitants, such as ammonium sulfate or polyethylene glycol, are
compounds that cause the protein to precipitate out of solution. X-ray analytical
methods are used to discover the atomic architecture of biopolymers such as proteins
and nucleic acids. protein crystallography on campus was carried out in the 1960s
with a structure determination of g-chymotrypsin, followed by a series of studies on
antibodies and their Fab Fragments, both alone and in complex with antigens
subsequent research has been extended to other proteins.

WORKING :

The XRD can be used to identify single crystals, and to reveal the structure of single
crystals. It can be used to identify crystals which are present in a mixture, e.g.
minerals in a rock. For minerals with variable formulas and structures, such as clays,
XRD is the best method for identifying them and determining their proportion within
a sample. For more background on XRD techniques, see the Wikipedia article on X-
ray crystallography.

A fantastic program on the history of crystallography and X-ray diffraction is

available from the Royal Institution for free on X-ray diffraction has been used to
detect the structure of crystals for over a century, and the basic method has not
changed. A crystalline sample is placed in the path of an X-ray beam. X-rays diffract
through the crystal and into a detector. The beam and detector are rotated through a
range of angles. The angles at which the crystals diffract the beam into the detector
correspond to planes of the crystals.
Each crystal has a characteristic pattern of diffraction angles and corresponding
intensity of the diffracted beam. Most geologists who use XRD do it to identify the
minerals present in a rock sample. The sample is first powdered and packed into a
sample holder, using techniques to insure that the powder is randomly oriented. A
basic scan takes about 20 minutes, and the data usually requires little or no
processing. The peaks in the data are compared to peaks in mineral scans from a
library to identify the minerals present.
This level of analysis can tell you what clay groups are present, (e.g. illite, smectite,
chlorite or kaolinite) but is less reliable for specific information about clay
mineralogy, due to clays' tendancy to orient themselves in the powder. Once the
component minerals have been identified in step 1, a technique called Rietveld
Analysis is used to model the XRD spectrum by weighing the reference spectra the
user has chosen from a library.

There are many sources of uncertainty in this analysis, but with many samples it is
possible to estimate mineral mode to within certainties of <5%. Sources of error
include: using reference spectra which don't actually match the minerals in your
rock, orientation bias in clays and other minerals, and the presence of non-crystalline
material in the sample. Clays present special challenges for XRD for four reasons:
1) they tend to orient themselves due to having one strong cleavage on 001, thus
violating the requirement for random orientation in powder samples, and 2) they
absorb and release interlayer water constantly, which changes the spacing of layers
and thus affects the XRD pattern, 3) different cations may occupy interlayer sites in
the same mineral, also affecting layer spacing, and 4) "different" clay minerals often
form interlayers in the same crystal.

Types of XRD

X-ray Powder Diffraction:

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 K α 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. Kα1and 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.

Laue method:

In the Laue method, a stationary single crystal is bathed in a beam of ‘white’ radiation. Then,
because the specimen is a fixed single crystal, the variable necessary to ensure that the
Bragg’s law is satisfied for all the planes in the crystal has to be provided by the range
of wavelengths in the beam, i.e. each set of crystal planes chooses the appropriate λ from the
‘white’ spectrum to give a Bragg reflection.
Radiation from a target metal having a high atomic number (e.g. tungsten) is often used, but
almost any form of ‘white’ radiation is suitable. In the experimental arrangement shown
in Figure 5.9, either a transmission photograph or a back-reflection photograph may be
taken, and the pattern of spots which are produced lie on ellipses in the transmission case or
hyperbolae in the back-reflection case. All spots on any ellipse or hyperbola are reflections
from planes of a single zone (i.e. where all the lattice planes are parallel to a common
direction, the zone axis) and, consequently, the Laue pattern is able to indicate the symmetry
of the crystal.

For example, if the beam is directed along a [1 1 1] or [1 0 0] direction in the crystal, the
Laue pattern will show three- or fourfold symmetry, respectively. The Laue method is used
extensively for the determination of the orientation of single crystals and, while charts are
available to facilitate this determination, the method consists essentially of plotting the zones
taken from the film onto a stereogram, and comparing the angles between them with a
standard projection of that crystal structure.

In recent years the use of the Laue technique has been extended to the study of imperfections
resulting from crystal growth or deformation, because it is found that the Laue spots from
perfect crystals are sharp, while those from deformed crystals are elongated. This elongated
appearance of the diffraction spots is known as asterism and it arises in an analogous way to
the reflection of light from curved mirrors.
X-ray Crystallography:

Max Von Laue demonstrated in 1912 that X-rays were light waves with wavelengths of a
few tens of the nanometre, i.e. a length corresponding to the spacing between atoms in
crystals. He paved the way to X-ray crystallography (X-ray diffraction, XRD) in 1914, when
he inferred the structure of simple crystals such as NaCl from their diffraction patterns. Father
and son William Henry and William Lawrence Bragg took further the study of diffraction
phenomena and laid the foundations of crystallography and of its application to the
determination of the structure of increasingly large molecules.

The first scientist to investigate the effect of limited particle size on XRD patterns was Paul
Scherrer. He published his results in a paper that included what became known as
the Scherrer equation [59], showing that peak width is inversely proportional
to crystallite size. This led to the use of powder diffraction with X-rays, neutrons and
electrons. XRD was later applied to increasingly complex structures, culminating in 1953 in
the discovery of the double-helix structure of the DNA by Franklin, Watson and Crick.

X-ray crystallography of macromolecular structures relies on growing crystals of sufficient

size (200 nm to several micrometre) to measure their diffraction patterns. For particular
proteins, it is impossible to obtain crystals that are large enough. Moreover, too small crystals
lead to extensive damage of the structure. Modern techniques collect single-crystal XRD
‘snapshots’ from a fully hydrated stream of nanocrystals, using femtosecond pulses from a
hard-X-ray free-electron laser [60].

At present, different integrated approaches are available for the study of increasingly
complex structures and biomolecular processes. These approaches include advanced mass
spectrometry, high-resolutionNMR spectroscopy and X-ray crystallography. Additional
biochemical and molecular techniques are needed to further study the function, localisation
and interactions of biomolecules in intracellular and extracellular environments. These
techniques will not be covered in this work. For a 60-year survey of the determination of

crystallite size, see the study by Langford and Wilson

APPLICATION

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.
Other applications include:

 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
With specialized techniques, XRD can be used to:
 determine crystal structures using Rietveld refinement
 determine of modal amounts of minerals (quantitative analysis)
 characterize thin films samples by:
o determining lattice mismatch between film and substrate and to inferring
stress and strain
o determining dislocation density and quality of the film by rocking curve
measurements
o measuring superlattices in multilayered epitaxial structures
o determining the thickness, roughness and density of the film using glancing
incidence X-ray reflectivity measurements
 make textural measurements, such as the orientation of grains, in a
polycrystalline sample
Advantages and disadvantages

Although it possible to solve crystal structures from powder X-ray data alone, its
single crystal analog is a far more powerful technique for structure determination.
This is directly related to the fact that much information is lost by the collapse of the
3D space onto a 1D axis. Nevertheless powder X-ray diffraction is a powerful and
useful technique in its own right. It is mostly used to characterize and identify phases
rather than solving structures. The great advantages of the technique are:

 Simplicity of sample preparation

 Rapidity of measurement
 The ability to analyze mixed phases, e.g. soil samples

By contrast growth and mounting of large single crystals is notoriously difficult. In

fact there are many materials for which despite many attempts it has not proven
possible to obtain single crystals. Many materials are readily available with
sufficient microcrystalline for powder diffraction, or samples may be easily ground
from larger crystals. In the field of solid-state chemistry that often aims at
synthesizing new materials, single crystals thereof are typically not immediately
available. Powder diffraction is therefore one of the most powerful methods to
identify and characterize new materials in this field.

Particularly for neutron diffraction, which requires larger samples than X-Ray
Diffraction due to a relatively weak scattering cross section, the ability to use large
samples can be critical. In neutron diffraction therefore the powder technique is more
the norm and the single crystal one the exception, although new more brilliant
neutron sources are being built that may change this picture. Structure determination
by neutrons has the great advantage that hydrogen is a strong scattered whereas for
X-rays it is almost invisible.

Since all possible crystal orientations are measured simultaneously, collection times
can be quite short even for small and weakly scattering samples. This is not merely
convenient, but can be essential for samples that are unstable either inherently or
under x-ray or neutron bombardment, or for time-resolved studies. For the latter it is
desirable to have a strong radiation source. The advent of synchrotron radiation has
therefore done much to revitalize the powder diffraction field because it is now
possible to study temperature dependent changes, reaction kinetics and so forth by
means of time dependent powder diffraction.

CONCLUSION & RESULT

X-ray crystallography has been responsible for results leading to fundamental and
important structural concepts. Each crystal structure reveals something about the
molecular function. These structural concepts have contributed significantly to
computer-aided molecular design, rational drug design, etc. 119 '1 20 The thesis
deals with the crystal and molecular structures of some heterocyclic compounds of
biological interest and their complexes. Derivatives of nucleobases and their
analogues are widely used as drugs. For example, acyclovir (2-amino-l,9-dihydro-
9-[(2-hydroxyethoxy)methyl]-6H-purin-6-one] is an antiviral agent. 5-fluorouracil
is an antineoplastic drug. AZT(3'-azido-3'- deoxythymidine) is used in the treatment
of AIDS. Crystallographic studies on a large number of such compounds will enable
the scientists to correlate the structure and drug activity. The conformations of these
drugs can be compared with related structures retrieved from Cambridge Structural
Data Base. For many drugs, their receptor proteins are also known. Crystal structures
of these proteins can also be retrieved from Brookhaven Protein Data Bank(PDB).
Moreover, crystal structures of many protein-drug complexes are also available with
the Brookhaven Protein Data Bank. These structures and their interacting patterns
are very useful for computational studies of drugs and drug-receptor recognition.
The combined results obtained from X-ray studies, NMR studies and computational
techniques contibute towards rational drug design.

 X-ray diffraction is a technique for analyzing structures of biological

molecules.
 X-ray beam hits a crystal, scattering the beam in a manner characterized by
the atomic structure.
 Even complex structures can be analyzed by x-ray diffraction, such as DNA
and proteins.
 This will provide useful in the future for combining knowledge from physics,
chemistry, and biology.
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