X-ray crystallography (JAISMIN) guide hemant yadav.pptx

complete and detailed ppt on x ray of m pharm 1st sem mpat
submitted by jaismin mahlawat under the guidance of hemant yadav m pharm qa
in this you will able to gain complete knowledge of x ray crystallography according to syllabus of m pharm mpat subject all refernces are given and nothing is wrong in this ppt and hemant yadav good guidance makes it easier for me to complete my work i would be thankful to mr hemant yadav registered pharmacist who help me to complete my project work on x ray crystallography

1. X-ray crystallography submitted by : jaismin m. pharm 1 st sem. ( mpat mqa101t) submitted to : ms. anju goyal (assistant professor)

2. Index History Introduction Principle Bragg’s law Production of X-Rays Different X-Rays methods Rotating crystal technique X-Ray powder technique Types of crystals and applications of x-ray diffraction 2

3. History : X-ray crystallography is a powerful technique for visualizing the structure of protein. It is a tool used for identifying the atomic and molecular structure of a crystal. In crystallography the crystalline atoms cause a beam of incident X-rays to diffract into many specific directions. Then crystallographer can produce a three-dimensional picture of the density of electrons within the crystal. From this electron density, the mean positions of the atoms in the crystal can be determined. The English physicist Sir William Henry Bragg determined crystal structure by X-ray diffraction methods. Using X-ray crystal data, Dr. James Watson and Dr. Francis Crick were able to determine the helix structure of DNA in 1953. In 1998 Dr. Peter Kim, a scientist, was able to determine the structure of a key protein responsible for the HIV infection process. 3

4. Intro: • X-ray crystallography is the study of crystal structures through X-ray diffraction techniques. It is not an imaging technique. • When an X-ray beam bombards a crystalline lattice in a given orientation, the beam is scattered in a definite manner characterized by the atomic structure of the lattice. • This phenomenon, known as X-ray diffraction, occurs when the wavelength of X rays and the interatomic distances in the lattice have the same order of magnitude. • Modern X-ray crystallography provides the most powerful and accurate method for determining single-crystal structures. • Structures containing 100-200 atoms now can be analyzed in 1-2 days, whereas before the 1960’s a 20-atom structure required 1-2 years for analysis. 4

5. Through X-ray crystallography the chemical structure of thousands of organic, inorganic, organometallic, and biological compounds are determined every year.
The knowledge of accurate molecular structures is necessary for rational drug design and for structure based functional studies to aid the development of effective therapeutic agents and drugs.
Crystallography can reliably provide the answer to many structure related questions.
X-ray Crystallography is probably the most important technique to determine the structure of any molecule (from small molecules to large assemblies of biological macromolecules) at atomic resolution.
Atomic resolution means that the position of every atom in the molecule is determined within a relatively small range of uncertainty. 5

6. principal 6 Ray diffraction by crystals is a reflection of the periodicity of crystal architecture, so that imperfection in the crystal lattice usually results in poor diffraction properties. Crystals are necessary because it is neither possible to see nor handle single molecules. Molecules in the crystal occur in only one or in very few orientations. This leads to a tremendous amplification of the diffraction image. And not only that, the diffraction image is also much simplified. Since the aim is to acquire information about the position of atoms it is necessary to use radiation of a wavelength close to the distance of atoms in molecules. X-rays have a wavelengths around 10-10 m which is close to the distance between two bonded atoms in a molecule.

7. X-ray crystallography

8. Different x-ray methods : A variety of x-ray technique and methods are in use but we shall classify all methods into three main categories:- X-ray absorption X-ray fluorescence X-ray diffraction ▸ X-ray absorption:- In this method a beam of x-rays is allowed to pass through the sample and the fraction of x-ray photons absorbed is considered to be a measure of the concentration of absorbing substance. ▸ X-ray fluorescence:- In this method x-rays are generated within the sample and by measuring the wavelength and intensity of the generated x-rays one can perform qualitative and quantitative analysis. X-rays fluorescence method is non-destructive and frequently requires very little sample preparation. ▸ X-ray diffraction method:- These methods are based on the scattering of x-rays by crystals. By this methods, one can identify the crystal structure of various solid compounds. This methods are extremely important as compared to other two methods. 8

9. Procedure : First step The process begins by crystallizing a protein of interest. 4 critical steps are taken to achieve protein crystallization: Purify the protein. Determine the purity of the protein and if not pure (usually>99%), then must undergo further purification. Protein must be precipitated by dissolving it in an appropriate solvent(water- buffer soln. w/ organic salt). The solution has to be brought to supersaturation by adding a salt to the concentrated solution of the protein. Let the actual crystals grow. Since nuclei crystals are formed this will lead to obtaining actual crystal growth. 9

10. Production of X-Rays : X-rays are generated when high velocity electrons impinge on a metal target. Approximately 1% of the total energy of the electron beam is converted into x-radiation. The remainder being scattered as heat. Many types of x-ray tubes are available which are used for producing x-rays. Positive voltage in the form of anode having a target. Battery to emit thermos ionic electrons. Cathode-filament of tungsten metal. The electrons are accelerated towards the target a On striking the target the electrons transfer their energy to its metallic surface which gives off x-ray radiation 10

11. X-rAY tube : 11

12. Second Step X-rays are generated and directed toward the crystallized protein. Then, the x-rays are shot at the protein crystal resulting in some of the x-rays going through the crystal and the rest being scattered in various directions. The crystal is rotated so that the x-rays are able to hit the protein from all sides and angles. The pattern on the emulsion due to scattering reveals much information about the structure of the protein. The intensities of the spots and their positions are thus are the basic experimental data of the analysis. 12

13. Third step : An electron density map is created based on the measured intensities of the diffraction pattern on the film. A Fourier Transform can be applied to the intensities on the film to reconstruct the electron density distribution of the crystal. The mapping gives a three-dimensional representation of the electron densities observed through the x-ray crystallography When interpreting the electron density map, resolution needs to be taken into account A resolution of 5Å - 10Å(angstrom) can reveal the structure of polypeptide chains, 3Å - 4Å of groups of atoms, and 1Å - 1.5Å of individual atoms. 13

14. Instrumentation : Source of X- Ray Collimator Monochromator Filter Crystal monochromator Flat crystal monochromator Curved crystal monochromator 14 Detectors Photographic method Counter method Geiger – Muller tube counter Proportional counter Scintillation detector Solid state semi conductor detectors Semi conductor detectors

15. X rays source : The x-ray most common source of x ray is an x ray tube. .The tube is evacuated and contains a copper block with metal target anode , and a tungsten filament cathode with high voltage between them. Collimator : comprises of two closely packed metal plate which are. 0.3mm apart from each other. The x ray beam originate from x ray source passes through this gap and follow single line path. Monochromater : It act as Xray filter which removes unwanted rays ,generally 12-24 Armstrong rays has been in x ray. Name is from greek language mono means single and chroma means colour types of Monochromater : Filter
Crystal Monochromater (a) Flat crystal Monochromater
( b) Curved crystal Monochromater Detector : the captured data has been send to computer for further processing by detector where 3D structure of crystal gets develop. Following detectors are used for detection: Photographic method
Counter method Geiger – Muller tube counter
Proportional counter
Scintillation detector
Solid state semi conductor detectors
Semi conductor detectors 15

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17. These are generally used for investigating the internal structures and crystal structures of various solid compounds.They are Laue's photographic method a) Transmission method b) Back reflection method Bragg's X-ray spectrometer method Rotating crystal method Powder method 17 xray diffraction methods:

18. Xray diffraction method 18 Laue Rotating Crystal Powder Orientation Single crystal Polychromatic beam Fixed angle Lattice constant Single crystal Monochromatic beam Variable angle Lattice parameters Polycrystal(powdered) Monochromatic beam Variable angle

19. Transmission Laue method: In the transmission Laue method, the film is placed behind the crystal to record beams which are transmitted through the crystal.One side of the cone of Laue reflections is defined by the transmitted beam. The film intersects the cone, with the diffraction spots generally lying on an ellipse. Can be used to orient crystals for solid state experiments. Most suitable for the investigation of preferred orientation sheet particularly confined to lower diffraction angles. Also used in determination of symmetry of single crystals. b)Back-reflection Laue Method: In the back-reflection method, the film is placed between the x-ray source and the crystal. The beams which are diffracted in a backward direction are recorded. One side of the cone of Laue reflections is defined by the transmitted beam. The film intersects the cone, with the diffraction spots generally lying on an hyperbola. This method is similar to Transmission method however, black-reflection is the only method for the study of large and thick specimens. Disadvantage: Big crystals are required 19

20. Crystal orientation is determined from the position of the spots. Each spot can be indexed, i.e. attributed to a particular plane, using special charts. The Greninger chart is used for back-reflection patterns and the Leonhardt chart for transmission patterns. The Laue technique can also be used to assess crystal perfection from the size and shape 20

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22. THE BRAGG’S law : Bragg's Law was introduced by Sir W. H. Bragg and his son Sir W. L. Bragg. According to the law, when the x-ray is incident on to a crystal surface, its angle of incidence will reflect back with a same angle of scattering. Diffraction will never occur, if Bragg's equation is not satisfied, in case of infinitely large number of lattice planes . In other words, diffraction may be observed, even if the situation is slightly deviated from the condition, in case of finite number of lattice planes. Bragg's Law means that diffraction can occur only when the following equation is satisfied:

23. Bragg’s equation : 23

24. Rotating crystal method: Photographs can be taken by: Complete rotation method: in this method series of complete revolutions occur Each set of a plane in a crystal diffracts four times during rotation Four diffracted beams are distributed into a rectangular pattern in the central point of photograph Oscillation method: the crystal is oscillated at an angle of 15" or 20° The photographic plate is also moved vack and forth with the crystal The position of the spot on the plate indicates the orientation of the crystal at which the spot wasformed 24

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26. POWDER CRYSTAL METHOD : 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. Fine powder is struck on a hair with a gum it is suspended vertically in the axis of a cylindrical camera. When monochromatic beam is allowed to pass different possibilities may happen. There will be some particles out of random orientation of small crystals in the fine powder. Another fraction of grains will have another set of planes in the correct positions for the reflections to occur. Reflections are possible in different orders for each set. 26

27. 27 If the angle of incidence is e then the angle of reflection will be 2Θ If the radius is r the circumference 2πr corresponds to a scattering angle of 360°. Θ-360*1/πr From the above equation the value of o can be calculated and substituted in bragg's equation to get the value of d.

28. Types of crystal : 28 Crystal Lattice • A crystal lattice is a 3-D arrangement of unit cells. • Unit cell is the smallest unit of a crystal, by stacking identical unit cells, the entire lattice can be constructed. • A crystal’s unit cell dimensions are defined by six numbers, the lengths of the 3 axes, a, b, and c, and the three inter axial angles, α, β and γ

29. 29 Bravais Lattice Bravais Lattice refers to the 14 different 3-dimensional configurations into which atoms can be arranged in crystals.

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32. Applications : 1. Structure of crystals : • X-ray Diffraction Method is non-destructive and gives information on the molecular structure of the sample. • Comparing diffraction patterns from crystal of unknown composition with patterns from crystal of known compounds permits the identification of unknown crystalline compound. • This method can also be used to distinguish between a mixture of crystals. 32

33. 2. Polymer characterization • Determine degree of crystallinity. • Non-crystalline portion scatters x-ray beam to give a continuous background(amorphous materials) • Crystalline portion causes diffraction lines that are not continuous(crystalline materials). 3. State of anneal in metals • A property of metals than can be determined by X-ray diffraction is the state of anneal. • Well-annealed metals are in well-ordered crystal form and give sharp diffraction lines. • It is occasionally necessary to check moving parts for metal fatigue, such as airplane wings. This check can be done by x-ray diffraction without removing the part from its position and without weakening it in the process of testing 33

34. 4.Particle size determination : Spot counting method B. Broadening Of Diffraction Lines C. Low-Angle Scattering 2223. A. Spot counting method This method is used for determining size of particles larger than 5 microns. v=V. δθ . cosθ /2n Where, o V=volume of individual crystallite o V=total volume irradiated o n=no. of spots in diffraction ring at a Bragg angle of θ o δθ =divergence of x-ray beam. Broadening Of Diffraction Lines Crystallites smaller than 120nm create broadening of diffraction peaks. This peak broadening can be used to quantify the average crystallite size of nano particles Using the scherrer’s equation. Low-Angle Scattering From the Bragg relation, nλ =2d sinθ it follows that if one desire to have information about large structural features ( i.e.,large d values),attention should be focussed on small scattering angles,2θ to get better resolution. 34

35. 5. Diffraction methods to complexes Determination of Cis-Trans: Isomerism-X-ray diffraction study has been used to make the distinction between cis and trans isomers of a complex. Determination of Linkage Isomerism: By X- ray studies, it becomes possible to identify linkage isomers of complexes. 35

36. REFERENCES : G. R. Chatwal & Sham K Anand, “Instrumental method of chemical analysis”.(pg. 2.303-2.339). https://www.slideshare.net/slideshow/types-of-crystals-and-application-of-xray-diffraction/248363285 36

37. Thank you Jaismin Modern Pharmaceutical Analytical Techniques (MQA 101T) M.Pharma {quality assurance} 1 st sem