X-Ray Diffraction

Prof. M.J. Joshi

What is X-Ray?

X-Ray is the Electromagnetic radiation. Wavelength is from 0.01 nanometer to 10 nanometer Frequency is from 30 peta-hertz to 30 exa-hertz (31016 Hz to 31019 Hz) Energies in the range 120 eV to 120 keV. X-rays are shorter than UV rays and longer than gamma rays Wilhelm Conrad Rontgen discovered X-ray in 1895 who got first Nobel Prize for his contribution.

X-Ray Sources
X-rays can be generated by an X ray , a vacuum tube that uses a high voltage to accelerate the electrons released by a hot cathode to a high velocity. The high velocity electrons collide with a metal target, the anode, creating the X-rays. In medical X-ray tubes the target is usually tungsten or a more crack-resistant alloy of rhenium (5%) and tungsten (95%), but sometimes molybdenum for more specialized applications, such as when soft X-rays are needed as in mammography. In crystallography, a copper target is most common. The maximum energy of the produced X-ray photon is limited by the energy of the incident electron, which is equal to the voltage on the tube, so an 80 keV tube cannot create X-rays with an energy greater than 80 keV. When the electrons hit the target, X-rays are created by two different atomic processes:

X-ray fluorescence: If the electron has enough energy it can knock an orbital
electron out of the inner electron shell of a metal atom, and as a result electrons from higher energy levels then fill up the vacancy and X-ray photons are emitted. This process produces an emissions spectrum of X-rays at a few discrete frequencies , sometimes referred to as the spectral lines. The spectral lines generated depend on the target (anode) element used and thus are called characteristic lines. Usually these are transitions from upper shells into K shell (called K lines), into L shell (called L lines) and so on.

Bremsstrahlung: This is radiation given off by the electrons as they are scattered
by the strong electric field near the high-Z (proton number) nuclei. These X-rays have a continuous spectrum. The intensity of the X-rays increases linearly with decreasing frequency, from zero at the energy of the incident electrons, the voltage on the X-ray tube.So the resulting output of a tube consists of a continuous bremsstrahlung spectrum falling off to zero at the tube voltage, plus several spikes at the characteristic lines. The voltages used in diagnostic X-ray tubes, and thus the highest energies of the X-rays, range from roughly 20 to 150 kV.

What is crystal?
In Greek- Kristallos means clear ice. Because of transparency and perfection of crystals a phrase in English is used, as clear as crystal. Crystal has periodic arrangements of its atoms or molecules in three dimensions.

Periodic arrangements in Crystals Periodic arrangements of atoms in crystals Periodic arrangements of soldiers

Braggs Law

Laue Method

The Laue method is mainly used to determine the orientation of large single crystals. White radiation is reflected from, or transmitted through, a fixed crystal. The diffracted beams form arrays of spots, that lie on curves on the film. The Bragg angle is fixed for every set of planes in the crystal. Each set of planes picks out and diffracts the particular wavelength from the white radiation that satisfies the Bragg law for the values of d and involved. Each curve therefore corresponds to a different wavelength. The spots lying on any one curve are reflections from planes belonging to one zone. Laue reflections from planes of the same zone all lie on the surface of an imaginary cone whose axis is the zone axis.

Types of Laue Methods

Transmission mode

Back scatter mode

Powder X-Ray Diffraction Method

Powdered samples are used. Ideally, every possible crystalline orientation is represented very equally in a powdered sample. The resulting orientational averaging causes the three-dimensional reciprocal space that is studied in single crystal diffraction to be projected onto a single dimension. The three-dimensional space can be described with (reciprocal) axes x*, y* and z* or alternatively in spherical coordinates q, *, and *. In powder diffraction, intensity is homogeneous over * and *, and only q remains as an important measurable quantity. In practice, it is sometimes necessary to rotate the sample orientation to eliminate the effects of texturing and achieve true randomness. In accordance with Bragg's law, each ring corresponds to a particular reciprocal lattice vector G in the sample crystal. This leads to the definition of the scattering vector as: G=q =2ksin = (4/) sin Powder diffraction data are usually presented as a diffractogram in which the diffracted intensity I is shown as function either of the scattering angle 2 or as a function of the scattering vector q. The latter variable has the advantage that the diffractogram no longer depends on the value of the wavelength .

Powder X-Ray Diffraction

Powder X-Ray Diffraction Patterns

Patterns are unique for different Crystals. Fingerprint matching can be achieved for rapid identification of unknown substance Various computer soft-wares are available for analysis

Single Crystal X-Ray Diffraction

Single-crystal X-ray Diffraction is a non-destructive analytical technique which provides detailed information about the internal lattice of crystalline substances, including unit cell dimensions, bond-lengths, bond-angles, and details of site-ordering. Directly related is single-crystal refinement, where the data generated from the X-ray analysis is interpreted and refined to obtain the crystal structure.

Instrumentations

X-ray diffractometers consist of three basic elements, an X-ray tube, a sample holder, and an X-ray detector. 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 and K. The specific wavelengths are characteristic of the target material. Filtering, by foils or crystal monochrometers, is required to produce monochromatic X-rays needed for diffraction. Molybdenum is the most common target material for single-crystal diffraction, with MoK radiation = 0.7107. These X-rays are collimated and directed onto the sample. When the geometry of the incident X-rays impinging the sample satisfies the Bragg Equation, constructive interference occurs. A detector records and processes this X-ray signal and convert the signal to a count rate which is then output to a device such as a printer or computer monitor. Details X-rays leave the collimator and are directed at the crystal. Rays are either transmitte Modern single-crystal diffractometers use CCD (charge-coupled device) technology

Single-crystal diffractometers use either 3- or 4-circle goniometers. These circles refer to the four angles (2, , , and ) that define the relationship between the crystal lattice, the incident ray and detector. Samples are mounted on thin glass fibers which are attached to brass pins and mounted onto goniometer heads. Adjustment of the X, Y and Z orthogonal directions allows centering of the crystal within the X-ray beam.

Detectors
X-rays leave the collimator and are directed at the crystal. Rays are either transmitted through the crystal, reflected off the surface, or diffracted by the crystal lattice. A beam stop is located directly opposite the collimator to block transmitted rays and prevent burn-out of the detector. Reflected rays are not picked up by the detector due to the angles involved. Diffracted rays at the correct orientation for the configuration are then collected by the detector. Modern single-crystal diffractometers use CCD (charge-coupled device) technology to transform the X-ray photons into an electrical signal which are then sent to a computer for processing.

Structure Refinements
Once the data have been collected, the phase problem must be solved to find the unique set of phases that can be combined with the structure factors to determine the electron density and, therefore, the crystal structure. A number of different procedures exist for solution of the phase problem, but the most common method currently, due to the prevalence of high-speed computers, is using direct methods and least-squares, initially assigning phases to strong reflections and iterating to produce a refined fit. Structure solution Solution of the phase problem leads to the initial electron density map. Elements can be assigned to intensity centers, with heavier elements associated with higher intensities. Distances and angles between intensity centers can also be used for atom assignment based on likely coordination. If the sample is of a known material, a template may be used for the initial solution. Structure Refinement Once the initial crystal structure is solved, various steps can be done to attain the best possible fit between the observed and calculated crystal structure. The final structure solution will be presented with an R value, which gives the percent variation between the calculated and observed structures.

Phase Problem
The data collected from a diffraction experiment is a reciprocal space representation of the crystal lattice. The position of each diffraction 'spot' is governed by the size and shape of the unit cell, and the inherent symmetry within the crystal. The intensity of each diffraction 'spot' is recorded, and this intensity is proportional to the square of the structure factor amplitude. The structure factor is a complex number containing information relating to both the amplitude and phase of a wave. In order to obtain an interpretable electron density map, both amplitude and phase must be known (an electron density map allows a crystallographer to build a starting model of the molecule). The phase cannot be directly recorded during a diffraction experiment: this is known as the phase problem. Initial phase estimates can be obtained in a variety of ways:

Having obtained initial phases, an initial model can be built. This model can be used to refine the phases, leading to an improved model, and so on. Given a model of some atomic positions, these positions and their respective Debye-Waller factors (or B-factors, accounting for the thermal motion of the atom) can be refined to fit the observed diffraction data, ideally yielding a better set of phases. A new model can then be fit to the new electron density map and a further round of refinement is carried out. This continues until the correlation between the diffraction data and the model is maximized. The agreement is measured by an R-factordefined as

where F is the structure factor. A similar quality criterion is Rfree, which is calculated from a subset (~10%) of reflections that were not included in the structure refinement. Both R factors depend on the resolution of the data. As a rule of thumb, Rfree should be approximately the resolution in angstroms divided by 10; thus, a data-set with 2 resolution should yield a final Rfree ~ 0.2. Chemical bonding features such as stereochemistry, hydrogen bonding and distribution of bond lengths and angles are complementary measures of the model quality.

Fourier Transform
The main goal of X-ray crystallography is to determine the density of electrons f(r) throughout the crystal, where r represents the three-dimensional position vector within the crystal. To do this, X-ray scattering is used to collect data about its Fourier transform F(q), which is inverted mathematically to obtain the density defined in real space, using the formula

F(q)=drf( r )e-iqr
The Fourier transform F(q) is generally a complex number, and therefore has a magnitude |F(q)| and a phase (q) related by the equation

F(q)=|F(q)|e-i(q)
The intensities of the reflections observed in X-ray diffraction give us the magnitudes |F(q)| but not the phases (q). Combining the magnitudes and phases yields the full Fourier transform F(q), which may be inverted to obtain the electron density f(r).

Hemoglobin Structure

Dorothy Hodgkin solved vitamin B12 Structure using X-ray crystallography. She won Nobel Prize in Chemistry

Information Obtained from Single crystal X-Ray Diffraction

Molecular structure

Electron density contours

Packing of Molecules in Unit cell of crystal

Protein Crystallography

NB-protein

Dimeric HIV

Crick and Watson and DNA structure

X-Ray diffracto-gram of DNA