X-RAY DIFFRACTION

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WELCOME 1

X-Ray Diffraction : 

X-Ray Diffraction Presented by- Mr. Sachin.M.Jadhav M.Pharm-I (Pharmaceutics) SVERI’s College of Pharmacy, Pandharpur. 2

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RAY DIFFRACTION 3

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CONTENTS What is light ? What are x-rays ? Why x-rays preferred ? How they are produced ? A) atomic level B) Instrumental level How target metal selected ? Which are useful radiations for analysis ? What is diffraction ? What is BRAGG’s equation ? What is a crystal ? 4

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CONTENTS X-ray diffraction at crystal structure Instrumentation for x-ray diffraction Detectors Different patterns of solid ,liquid & gases Methods of crystal structure determination Various x-ray diffraction patterns with example Fourier transformations Phase problem Phasing methods Ring patterns & sizes achievements 5

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Theory of light 6

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X-ray -0.01nm-10nm Visible light- 400-800 nm 7

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Why do we use X-rays? Visible light- 400-800 nm atomic distances- ~ 1.5 Å X-ray - 0.01nm-10nm (0.1 Å - 100Å) Used x-ray range- 0.7Å - 3Å 8

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Dimensions of life 9 X-rays

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Theory of x-ray at atomic level Inner orbit Outer orbit 10

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Instrumentation for x-ray tube 11

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X-ray production at instrumental level 12

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Coolidge tube  Coolidge tube was invented in the GE company laboratories by W.D. Coolidge. It had a hot cathode in which electrons were liberated through the process of thermionic emission. It also had a metal target, using high atomic number metallic target increased efficiency of x-ray production. 13

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Target metal selection 14

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Intensity Wavelength () Mo Target impacted by electrons accelerated by a 35 kV potential 0.2 0.6 1.0 1.4 White radiation Characteristic radiation → due to energy transitions in the atom K K 15

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Reflection vs Diffraction 16

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BRAGG’s EQUATION Bragg’s equation is a negative law If Bragg’s eq. is NOT satisfied  NO reflection can occur If Bragg’s eq. is satisfied  reflection MAY occur 1915 W.H. Bragg & W.L. Bragg : (Nobel Prize Physics) 17

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BRAGG’s EQUATION d     dSin The path difference between ray 1 and ray 2 = 2d Sin For constructive interference: n = 2d Sin Ray 1 Ray 2  Deviation = 2 18

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Crystal structure 19

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A beam of X-rays directed at a crystal interacts with the electrons of the atoms in the crystal X-Rays and become secondary sources of EM radiation The secondary radiation is in all directions The waves emitted by the electrons have the same frequency as the incoming X-rays  coherent Incoming X-rays Secondary emission 20

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Sets Electron cloud into oscillation Sets nucleus (with protons) into oscillation Small effect  neglected The electrons oscillate under the influence of the incoming X-Rays and become secondary sources of EM radiation 21

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Oscillating charge re-radiates  In phase with the incoming x-rays The emission will undergo constructive or destructive interference with waves scattered from other atoms 22

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Instrumentation for x-ray diffraction 23

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Detectors Scintillators Some materials such as sodium iodide (NaI) can "convert" an X-ray photon to a visible photon; an electronic detector can be built by adding a photomultiplier. These detectors are called "scintillators", filmscreens or "scintillation counters". Geiger-Muller counter most common detection methods were based on the  ionization of gases, as in the : a sealed volume, usually a cylinder, with a mica, polymer or thin metal window contains a gas,acylindrical cathode and a wire anode a high voltage is applied between the cathode and the anode. 26

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It is the new generation of X ray diffraction products from Bruker AXS. The system includes two independent X-ray safety circuits All components like tube housing, X-ray optics, and detectors are mounted on high-precision tracks and can be readily exchanged with reproducible positioning and freely moved along the tracks. The  D8 ADVANCE goniometer is equipped with stepper motors controlled by optical encoders. The Dynamic Scintillation Detector with low background, large dynamic range, and a long life time D8 ADVANCE 28

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Schematic of difference between the diffraction patterns of various phases 29

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Crystal structure determination Monochromatic X-rays Panchromatic X-rays Monochromatic X-rays Many s (orientations) Powder specimen POWDER METHOD Single  LAUETECHNIQUE  Varied by rotation ROTATINGCRYSTALMETHOD 30

Smaller Crystals Produce Broader XRD Peaks : 

Smaller Crystals Produce Broader XRD Peaks 31

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32 Different forms of photographic plate patterns

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a molecule, and its Fourier Transform an atom, and its Fourier Transform 36

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a lattice, and its Fourier Transform a crystal, and its Fourier Transform: 37

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Multiplicity factors 38

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Phase problem 39

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Phase problem 40

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Intensities and phases Phases contain the bulk of the structural information We need both intensities and phases to calculate a realistic Picture Form: 41

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Phasing Methods Heavy atom method (MIR, multiple isomorphous replacement) The basic principle of the MIR method is to collect diffraction data of several crystals of the same protein, that share the same crystal properties (isomorphous), but differ in a small number of heavy atoms. The experimental approach is normally to soak protein crystals with diluted solutions of heavy metal compounds (e.g. mercury or platinum derivatives). These additional atoms cause a slight perturbation of the diffraction intensities. Elements with a high number of electrons (heavy atoms) are used. The differences of the reflection intensities can be used to locate the positions of the heavy atoms within the unit cell, which allows to estimate initial phases 42

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Anomalous scattering (MAD, multiple wavelength anomalous diffraction) The MAD method is based on the capacity of heavy atoms to absorb X-rays of a specific wavelength. Near its characteristic absorption wavelength. This effect is called anomalous scattering. The characteristic absorption wavelengths of typical protein atoms (N,C,O) are not in the range of the X-rays used in protein crystallography and therefore are not contributing to anomalous scattering. However, the use of synchrotron X-ray sources with adjustable wavelengths allows to collect diffraction data under conditions where heavy atoms exhibit strong anomalous scattering. 43

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Molecular replacement (MR) In some cases is structure to be examined is known to be very similar to an other structure, that has already been solved experimentally. This could be e.g. the same protein from an other organism or a mutant of this protein. In these cases the phases computed from of the known protein structure (phasing model) can be used as initial estimates of the phases of the unknown protein. 44

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Crystallite size Size > 10 m  Spotty ring (no. of grains in the irradiated portion insufficient to produce a ring) Size  (10, 0.5)  Smooth continuous ring Size  (0.5, 0.1)   Rings are broadened Size < 0.1   No ring pattern (irradiated volume too small to produce a diffraction ring pattern & diffraction occurs only at low angles) patterns 45

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Ring pattern Broadened Rings Spotty pattern 46

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1895 W.C. Rontgen discovers X-rays (Nobel Prize 1901) 1910 Max von Laue: Diffraction Theory (Nobel Prize: 1912) 1915 W.L. Bragg & W.H. Bragg: NaCl, KCl (Nobel Prize Physics)2 • d • sin Θ = n • λ 1934 D. Bernal & D. Crowfoot examine first Proteins 1950 DNA double helix structure: Watson, Crick, Wilkins (Nobel Prize 1963) 1958 Myoglobin Structure (Nobel Prize 1962 Kendrew, Perutz) 1971 Insulin (Blundell) 1978 First Virus Structure (S.C Harrison) 1988 Nobel Prize: Photosynthetic reaction center (Huber, Michel, Deisenhofer) 1997 Nobel Prize: ATP-synthase structure (Walker) 1997 Nucleosome core particle (T. Richmond) 1999 Ribosome Structures (Steitz, …) 2000 Reovirus core structure (S.C. Harrison) 2000 Rhodopsin structure, GPCR (Palczewski et al.) 2002 ABC-Transporter (D. Rees et al.) 2003 R.MacKinnon: structures of ion channel (Nobel Prize Chemistry 2003) X-Ray diffraction achievements 47

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References- http://imr.chem.binghamton.edu/labs/xray/xray. http://en.wikipedia.org/wiki/xray http://www.imsc.res.in/ Instrumental methods of chemical analysis by G.R.Chatwal and S.K.Anand, Himalaya Publishing House. Page No.2.303-2.330 48 Instrumental methods of analysis by Willard,merrit,dean,settle,cbs publishers,7th edition Page no-372-380

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QUESTIONS ??? 49

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