PH-5102 Lecture Notes 10/4/2013. S Raja A, Dept. of Pharmaceutics, IIT (BHU). 1

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1 X-RD CRYSTALLOGRAPHY SUMMARY APPLICATIONS OF SCXRD IN PROTEIN CRYSTALLOGRAPHY THEORY & PRINCIPLE OF SC-XRD INTRODUCTION XRD METHODS THEORY & PRINCIPLE OF XRPD XRPD - INSTRUMENTATION X-RD - INTRODUCTION X-RD is a unique, solid state analytical tool used to study the 3D structure of small or macromolecules by their x-ray diffraction or scattering patterns. X-ray diffraction by a crystal reflects the periodicity of crystal architecture. Any imperfections within the crystal architecture can be easily identified by its poor diffraction pattern. Study of crystals at the atomic or near atomic resolution (0.5 2 angstroms) to reveal the three dimensional structure of the crystals. More than 85 percent of the protein structures (Deposited in global repository, protein data bank have been determined by X-ray diffraction crystallography. GENERAL & PHARMACEUTICAL APPLICATIONS OF XRPD The X-ray diffraction pattern of a pure substance is, like a fingerprint of the substance. The powder diffraction method is thus ideally suited for characterization and identification of polycrystalline phases. 1 2 X-RD - INTRODUCTION The crystal sample is irradiated with x-rays for minutes to several hours and the diffracted radiation is collected by a detector which is controlled by a computer device. Theintensityofeachdiffractedrayisfedintoacomputer,whichusesa mathematical equation called a Fourier transform to calculate the position of every atom in the crystallized molecule. The result the researchers masterpiece is a three-dimensional digital image of the molecule. This image represents the physical and chemical properties of the substance and can be studied in intimate, atom-by-atom detail using sophisticated computer graphics software. X-ray crystallography is the primary method for determining the absolute configuration of a molecule and it provides unambiguous, accurate, and reliable 3-dimensional structural parameters, which are pre-requisites for rational drug design and structure-based functional studies. X-RD - INTRODUCTION Recent advances and developments in X-ray generators, crystallization techniques, detection and hardware and software for solving the structure and refinement have made the technique suitable for the small crystal and large molecule crystallographic studies. Traditional or old X-RD study usually takes a week time, to collect the required crystallographic data. Due to the advent of several new technologies in the area of crystallization and software for solving the structures, the modern X-RD study takes only a few hours to complete the data collection and solving the structure. Thus, X-RD is a unique method of choice for studying macromolecules and their complexes at atomic level as it provides more static description of macromolecular structures than solution/solid state NMR methods. 3 4 S Raja A, Dept. of Pharmaceutics, IIT (BHU). 1

2 X-RD - CLASSIFICATION X-RD - CLASSIFICATION Based on the physical nature of sample Powder/polycrystalline diffraction crystallography (XRPD) FPI Single crystal diffraction crystallography 3D Structure analysis Based on the size of the sample Small molecule or chemical crystallography Macromolecule or biological or protein crystallography Based on the method of data collection Room temperature X-RD Cryogenic X-RD(Especially for protein samples) SMALL MOLECULE X-RD Natural extract / Synthetic compound Pure Crystal 3D Structure PROTEIN X-RD Gene Protein Protein crystal 3D Structure 5 6 Solid matter can be described as : Amorphous - The atoms are arranged in a random way similar to the disorder we find in a liquid. Glasses are amorphous materials. Crystalline-Theatoms arearrangedinaregularpattern, and there is as smallest volume element that by repetition in three dimensions describes the crystal. This smallest volume elementiscalledaunitcell. E.g. we can describe a brick wall by the shape and orientation ofasinglebrick. Almost 90% of solids can be described as crystalline. 7 X-ray diffraction techniques used for characterizing pharmaceutical solids include the analysis of single crystals and powders. The electrons surrounding the atoms diffract X-rays in a manner described by the Bragg* equation, nλ=2dsinθ X-rays will be diffracted at an angle (defined as θ), and this knowledge, together with knowledge of the X-ray wavelength, can be used to calculate the spacing between the planes. *W.H. Bragg and W.L. Bragg, X-rays and Crystal Structure (G. Bell & Sons, London, 1918). 8 S Raja A, Dept. of Pharmaceutics, IIT (BHU). 2

3 Bragg explained the diffraction of x-rays by crystals using a model in which the atoms of a crystal are regularly arranged in space and can be regarded as lying in parallel sheets separatedbyadefiniteanddefineddistanced. Bragg also showed that scattering centers arranged in a plane act like a mirror to x-rays incident on them, so that constructive interference occurs for the direction of specular reflection. Within a given family of planes, defined by a Miller index of (hkl), each plane produces a specular reflectance of the incident beam. The diffraction of x-rays by a crystalline solid leads to constructive interference (such as reflection) at only the critical Bragg angles. When reflection does occur, it is stated that the plane in question is reflecting in the nth order or that one observes nth order diffraction for that particular crystal plane. Therefore, one will observe an x-ray scattering response for every plane definedbyauniquemillerindexof h,k,l. To measure a powder pattern, one would prepare a randomly oriented powdered sample to expose all possible planes of a crystalline powder. *W.H. Bragg and W.L. Bragg, X-rays and Crystal Structure (G. Bell & Sons, London, 1918) One can measure the scattering angle, θ, for each family of crystal planes by slowly rotating the sample (Goniometer) and measuring the angle of diffracted x-rays with respect to the angle of the incident beam. If the incident x-rays are monochromatic having wavelength λ, then for an arbitrary glancing angle of θ, the reflections from successive planes are out of phase with one another. This yields destructive interference in the scattered beams.however,byvarying θ,asetofvaluesfor θ canbefoundsothatthepath difference between x-rays reflected by successive planes will be an integral number n of wavelengths, and constructive interference occurs. One ultimately obtains the following equation, known as Bragg s law, that explains the phenomenon: 2d sinθ = nλ When the angle, wavelength and its order are known then the distance between crystal lattice planes can be deduced 11 Alternatively, the angle between sample and source can be kept fixed while the detector is moved to determine the angles of the scattered radiation. Knowing the wavelength of the incident beam, one can calculate the spacing between the planes (identified as the d- spacings) using Bragg s law. 12 S Raja A, Dept. of Pharmaceutics, IIT (BHU). 3

4 When an X-ray beam hits an atom (in amorphous powders), the electrons around the atom start to oscillate with the same frequency as the incoming beam. In almost all directions we will have destructive interference, that is, the combining waves are out of phase and there is no resultant energy leaving the solid sample. However, the atoms in a crystal are arranged in a regular pattern, and in a very few directions we will have constructive interference. The waves will be in phase and there will be well defined X-ray beams leaving the sample at various directions. The resulting diffracted beam may be described as a beam composed of a large number of scattered rays mutually reinforcing one another. Ideally a crystal can be described as a grid or lattice having three axis and angles between them. Along each axis, a point will be repeated (unit cell constants) and is denoted as a, b and c. The angles between b & c, a & c anda&barerepresentedas α, βand γrespectively. Within the lattice a number of planes (source of diffraction) can be drawn which are represented by h, k and l called Miller indices. These index planes h, k and l are equivalent to the number of parts they cut the a, b and c edges of each cell respectively From the scattering or diffraction pattern of a crystal, the structure factors like amplitudes (F hkl ) and phase (α hkl ) are determined to calculate the electron density at all points to solve the structure. The amplitudes (F hkl ) can be easily determined from the intensitiesof diffractedsignalswhilethephase(α hkl )(PHASE PROBLEM) can not be directly extracted from the intensities. Experimentally the phase problem is addressed by molecular replacement(mr), molecular isomorphous replacement(mir), multi-wavelength anomalous diffraction(mad)& single wavelength anomalous (SAD) diffraction experiments. 15 X-RD CRYSTALLOGRAPHY - INSTRUMENTATION X-RD Sources Rotating anode electrodes(xrpd) Mb(0.71 Angstroms) Cu Kα(1.54 Angstroms) radiation Microfocus rotating anode sources/with advanced optics are capable of generating narrower, more intense and more tightly focused X-ray beams, allowing in-house data collection on more challenging crystals(smaller, weaker diffractors) Synchrotron sources - Synchrotron sources used to produce X- rays of wavelength 0.5 to 2 Angstroms (10-10 m shorter the distance of two atoms in general) to measure the distance between atoms arranged in a identical pattern in the crystal lattice(sc-xrd). 16 S Raja A, Dept. of Pharmaceutics, IIT (BHU). 4

5 X-RD INSTRUMENTATION 2θ- θ GONIOMETER In THETA : 2-THETA Goniometer, the X-ray tube is stationary, the sample moves by the angle THETA and the detector simultaneously moves by the angle 2-THETA. X-RD INSTRUMENTATION θ- θ GONIOMETER In THETA:THETA Goniometer, the sample is stationary in the horizontal position, the X-ray tube and the detector both move simultaneously over the angular range THETA. SCHEMATIC DIAGRAM OF 2THETA-THETA GONIOMETER SETUP SCHEMATIC DIAGRAM OF THETA-THETA GONIOMETER SETUP X-RD INSTRUMENTATION - GONIOMETER X-RD - GONIOMETER The incident angle θ defined as the angle between the incident beam and the sample The 2θ defined as the angle between the incident and diffracted beams. The detector is moved (rotated) at twice the angular rate of the sample to maintain the θ - 2 θ geometry. SCHEMATIC DIAGRAM OF A BRUKKER AXS D5000 GONIOMETER Scintag PAD V Goniometer S Raja A, Dept. of Pharmaceutics, IIT (BHU). 5

6 X-RD SAMPLE PREPARATION TECHNIQUES Powder/Polycrystalline sample preparation In powder or polycrystalline diffraction it is important to have a sample with a smooth plane surface. The powder sample is normally grind down to particles of about0.002mmto0.005mmcrosssection.theidealsampleis homogeneous and the crystallites are randomly distributed. The homogeneous powder sample is pressed into a sample holder to have a smooth flat surface for X-ray irradiation. XRPD APPLICATIONS Identification of single-phase materials minerals, chemical compounds& other other engineered materials. Identification of multiple phases in microcrystalline mixtures Determination of the crystal structure of identified materials Recognition of amorphous materials in partially crystalline mixtures Crystallographic structural analysis and unit-cell calculations for crystalline materials. Quantitative determination of amounts of different phases in multi-phase mixtures by peak-ratio calculations. Determination of crystallite size from analysis of peak broadening. Determine of crystallite shape from study of peak symmetry. Study of thermal expansion in crystal structures using in-situ heating stage equipment XRPD APPLICATIONS 1. Identifications of powder samples It is most common use of powder (polycrystalline) diffraction is chemical analysis which includes phase identification and quantification (1983), high/low temperature phases and determination unit cell parameters of new crystalline materials 2. Polymer Crystallinity The crystallinity parts give sharp narrow diffraction peaks and the amorphous component gives a very broad peak(halo). The ratio between these intensities can be used to calculate the amount of crystallinity in the material. 3. Residual Stress analysis XRPD APPLICATIONS Stress is defined as force per unit area and the residual stress is the stress that remains in the material after the external force that caused the stress have been removed. Positive values indicate tensile (expansion) stress, negative values indicate a compressive state. The principles of stress analysis by the X-ray diffraction is based on measuring angular lattice strain distributions (ALSD). That is, we choose a reflection at high 2-Theta and measure the change in the d-spacing with different orientations of the sample. Using Hooke s law the stress can be calculated from the strain distribution(alsd) S Raja A, Dept. of Pharmaceutics, IIT (BHU). 6

7 4. Texture analysis XRPD APPLICATIONS The determination of the preferred orientation of the crystallites in polycrystalline aggregates is referred to as texture analysis. It tells about the preferred crystallographic orientation in the polycrystalline material, normally a single phase. The preferred crystallographic orientation is usually described in terms of polefigures. By using a four-circle diffractometer, we collect the intensity data for various settings of Chi and Phi angles. Normally we measure all Phi values for a given setting of Chi, then change Chi and repeat the process. X-RPD APPLICATIONS 4. Texture analysis.contd By collecting data for several reflections and combining several polefigures we can arrive at the complete orientation distribution function (ODF) of the crystallites within a single polycrystalline phase that makes up the material. The ODF is a function of three independent angular variables and gives the probability of finding the corresponding unit cell (lattice) orientation. 2D Polefigure display 3D Polefigure display 3D Polefigure display X-ray powder diffraction (XRPD) is the analysis of a powder sample with a typical output being a plot of intensity versus the diffraction angle(2θ). The value 2θ is used based on the configuration of the instrument. Such a plot can be considered a fingerprint of the crystal structure and is useful for determining the crystallographic similarity of samples by pattern comparison (with standard quality pattern in ICCD). A crystalline material will exhibit peaks indicative of reflections from specific atomic planes; these patterns are representative of the structure but do not give positional information about the atoms in the molecule. One peak will be exhibited for all repeating planes with the same spacing. By contrast, an amorphous sample will exhibit a broad hump in the pattern called an amorphous halo. 27 Majority of pharmaceutical products are solids. Most of those solid formulations are meant for oral administration. The solidstate properties like polymorphism, state of solvation and degree of crystallinity are the important parameters used to describe the pharmaceutical performance potential of an API. Eg: Characterization of ampicillin (anhydrous/trihydrate) in ampicillin capsules Every crystalline solid phase has a unique X-RD pattern and in powder mixture, each crystalline phase produces its own diffraction pattern independent of other constituents of the mixture. Thus, X-ray powder diffractometry (XRPD) is a powerful technique for the identification of crystalline solid phases. 28 S Raja A, Dept. of Pharmaceutics, IIT (BHU). 7

8 The solid state parameters (Crystallinity etc) can be very well studied by X-RD powder crystallography (XRPD) which is an unique (Absolutely specific with great degree of accuracy) and powerful solid state analytical tool with tremendous applications in pharmacy and biotechnology. The active pharmaceutical ingredients(s) (API) of powders, Tablets& capsules, Ointments& suppositories, microspheres and many other solid formulations can be directly characterized by X-RD with minimal or no sample pretreatment. In some of the multi-component formulations, there is a pronounced overlap of the powder patterns of ingredients which made identification difficult. This problem was solved by using a pattern subtraction technique, which permitted selective subtraction of the XRD pattern of the constituents of the formulation from the overall XRD pattern. This technique also useful in simultaneous identification of APIs in multi-component pharmaceutical formulations Eg: Trimethoprim & sulphamethoxazole, Paracetamol, aspirin and caffiene combination etc.. Limitations of XRPD TheAPIshouldbeincrystallineform intheformulation The API should constitute to significant weight fraction of the formulation. The crystalline API should constitute at least 5% of the formulation The specific applications of XRPD in Pharmaceutical analysis are Identification of drugs in pure form and in formulation by comparing with the standard XRD pattern (d-spacings and their relative intensity) available with ICCD. Eg. Paracetamol, Paracetamol tablets etc Identification of drugs with their hydration state Most of anhydrate recrystallizes with a different crystal lattice. In this case,thexrdpatternsofthehydrateandtheanhydratewill be different and these differences can be exploited for identification purposes. Eg. Ampicillin, its trihydrate (Both exhibit pronounced differences in their XRD patterns) caffiene, theophylline *Phadnis et al, J Pharm. Biomed analysis, 1997, 15, Identification of polymorphism - α and β form of carbamazepine Identification of drugs in low-dose formulations (<50mg) Benzodiazepines, cardiac glycosides, steroids etc. Identification of drugs in ointment formulations- Zinc oxide ointment etc Simultaneous identification of two or more active drugs Trimethoprim and sulphamethoxazole combination, Acetaminophen, aspirin, caffeine combination, etc. *Phadnis et al, J Pharm. Biomed analysis, 1997, 15, S Raja A, Dept. of Pharmaceutics, IIT (BHU). 8

9 CASE STUDIES OF DRUGS ANALYZED BY XRPD CASE STUDIES OF DRUGS ANALYZED BY XRPD XRD analytical parameters of Benzoic acid X-RD spectrum of Acetaminophen XRPD SPECTRUM OF ACETAMINOPHEN Crystallographic parameters of Roxifiban An oral platelet receptor inhibitor XRPD SPECTRUM OF RANITIDINE HCl X-ray powder diffraction patterns of ranitidine hydrochloride. Form I (thick trace) and Form II (thin trace) S Raja A, Dept. of Pharmaceutics, IIT (BHU). 9

10 XRPD - SUMMARY XRPD represents the methodology of choice for the crystallographic characterization of drug substances produced on a routine, batch-type basis. The resulting powder patterns that contain a scattering peak for each crystal plane or face and therefore constitute an identification test for a given crystalline phase. Since, both polymorphism and solvatomorphism are crystallographic occurrences, XRPD will always be the primary determinant of the existence of such phenomena. Variable-temperature XRPD is a valuable tool to understand thermally induced reactions and to characterize materials during stability studies. 37 S Raja A, Dept. of Pharmaceutics, IIT (BHU). 10