Searching for new API Polymorphs Crystal Structure Determination of Pharmaceutical crystals using Electron Diffraction Tomography Dr. Partha Pratim Das Application Specialist NanoMEGAS SPRL, Belgium
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Searching for API Polymorphs and solving their nanostructure by TEM Electron Diffraction www.nanomegas.com
STRUCTURE ANALYSIS WITH ELECTRON DIFFRACTION Why electrons? 10 4-5 times stronger interaction with matter compared with X-ray single crystal data on powder sample short data collection time - X- Ray peaks broaden with crystals of nm range With Electron microscope we can study nm- and micro-sized crystals
STRUCTURE ANALYSIS WITH TEM Transmission electron microscopy (TEM) Diffraction selected area, nano- and convergent beam electron diffraction Imaging conventional, high resolution (HREM) Chemical analysis EDS and EELS
STRUCTURE ANALYSIS WITH TEM With TEM microscopes we can obtain images of crystalline materials with atomic resolution: Acceleration voltage (kv) Point resolution (Å) TEM grid 3.05 mm size 120 ~3 200 2.0 300 1.7 We can see and analyze nano-crystals Very small quantity of sample!
STRUCTURE ANALYSIS WITH TEM TEM : Electron diffraction advantages TEM goniometer Diameter of grid - 3.05mm Every TEM (electron microscope) may produce ED patterns and HREM from individual single nanocrystals ED information: Cell parameter and symmetry determination Measuring intensity values leads to structure determination
3D Diffraction tomography in TEM : 3D reconstruction of Reciprocal space TEM holder 50 nm 500 nm Kolb et al., Ultramicroscopy 107 (2007) 507 513
TEM - organic samples Cryo Cooling Can be used to Reduce beam damage Keeping Crystal deep Frozen Kolb et al, Polymer Reviews, 50:385 409, 2010
TEM - organic samples What is the allowed maximum (critical) dose? Most biological specimens (proteins) 6 e-/å 2 Glaeser, 1971 Aromatic polymers (such as polystyrene) 36 e-/ Å 2 Anthracence 42 e-/ Å 2 Poly-xylene 120 e-/ Å 2 Kumar & Adams, 1990; Williams & Carter, 2004 Zeolites 100 e-/å 2 Ceramics 600 e-/å 2 Pan & Crozier, 1993
STRATEGY 1: FAST TOMOGRAPHY APPROACH Crystal is continuously rotated during sequential pattern acquisition avoiding intermediate stops for relocation of the crystal The missing wedge of the reciprocal space between the patterns is recorded by longer exposures during the crystal tilt Data Collection data can be recued <15 mint Gemmi et al. J. Appl. Cryst. (2015). 48
STRATEGY 2: FAST TOMOGRAPHY + Direct Electron Detection Camera Ideal detector for low intensity TEM Less radiation damage on sample gives more information For instance: more rotation information Very sharp images (No blurring effects from electron multiplication as in CCD) Medipix-Timepix 1 Electron TimePix = CMOS 1 Count x 100 more 512 x 512 pixels (262k pixels) sensitive than CCD > 120 fps X 10 more sensitive than IP dynamic range is 11810 (13 bit) per frame 55 micron pixel 25 μs exposure Acta Cryst. (2016). A72, 236 242
Low dose with Timepix < 0.0013 e-/ Å 2 /sec High Resolution Diffraction Pattern without Cryo
Data Collection with Fast Tomography + Timepix- CMOS of carbamazepine (CBZ) crystal (Data collected -25 0 to 26 0 ) 55 frames summed / 1 0 NO CRYO USED Resolution: 0.8 Å Collection time < 3 min Acta Cryst. (2016). A72, 236 242
Ab-initio structure solution of carbamazepine (CBZ) crystal from ED Data Experimental a = 7.68 Å b = 11.44 Å c = 13.92 Å β = 91.22 ο NO CRYO USED Literature Reported a = 7.534 Å b = 11.150 Å c = 13.917 Å β = 92.94Å
Data Collection with Timepix-CMOS Nicotinic Acid Crystal Resolution: 0.8 Å (Data collected -10 0 to 26 0 ) NO CRYO USED Collection time < 3 min
Structure was solved ab-initio by DM from ED Data Solved from ED Literature Reported Unit cell from ED a = 7.19 Å b = 11.74 Å c = 7.28 Å β = 112.65 Literature Reported a = 7.19 Å b = 11.69 Å c = 7.23 Å β = 113.55 Å Acta Cryst. (2016). A72, 236 242
Acta A72 (2016) doi:10.1107/s2053273315022500
Acta A72 (2016) doi:10.1107/s2053273315022500 CBZ Nicotinic Acid Nicotinic Acid
API 1 (ED Obtained Unit Cell and SPG) a = 4.99 Å b = 11.59 Å c = 22.95 Å SPG: P2 1 2 1 2 1 (Obtained by X-ray) a = 5.0450 (4) Å b = 12.4178 (11) Å c = 23.187 (2) Å SPG: P2 1 2 1 2 1 Collaboration TeraCrystal
Structure Solution Using ED Data API 1
API 1 Comparison with x-ray structure Blue Structure from SCXRD (Direct Methods) RED- Structure from Electron Diffraction (SA) Collaboration TeraCrystal
API 2 (ED Obtained Unit Cell and SPG) a = 7.56 Å b = 38.07 Å c = 8.61 Å β = 108.86 ο SPG: P2 1 /n (Obtained by X-ray) a = 7.627(1) Å b = 38.007(4) Å c = 8.5629(12) Å β = 108.118(15) ο SPG: P2 1 /n Collaboration TeraCrystal
STRATEGY 3: Random Tomography Collection of 12 random quasi- oriented PED patterns at 300 kv spot size 10 nm scanning at high speed rates > 50 fps CRYSTAL UNIT CELL is calculated from several random patterns (oriented or not) In Collaboration with University of Cambridge, UK
131 ZA ZA 1-1 -3 112 ZA 512 ZA 023 ZA -1-2 1 ZA 1-1 -3 ZA 001 ZA EDIff software : cell parameters are found from 11 random PED patterns a b c alpha beta gamma 6.663 10.68 11.75 90 90 95.0
Validation of Result ASPIRIN form I (: a=11.233(3) Å, b=6.544(1) Å, c=11.231(3) Å, â = 95.89(2)
ZA close to [110] ZA close to [-1-1-5]
Nanoscale Texture Studies Crystalline and Amorphous Phase Mapping Using TEM Electron Diffraction
TEM Orientation imaging /phase mapping characterization at 1 nm scale (EBSD-TEM) from 10-50 nm EBSD-SEM to 1 nm EBSD -like TEM 10 nm www.nanomegas.com
ASTAR : Crystal Orientation Mapping & Phase mapping at nanoscale Scan sample area 1 µm Orientation map Scanning the TEM beam in precession mode Step size 0.1 nm -100 nm Dedicated CCD with > 100 frames /sec Typical area 5 x 5 microns Scanning times (typical) 5-10 min Orientation map www.nanomegas.com
Identification example : nanocrystalline Cu 111 100 110 Diffraction pattern ( nanocrystalline cubic copper) correlation index = 585 Correlation index map For a given ED pattern, the correlation index map is calculated for all possible template orientations and plotted on a map that represents a portion of the stereographic projection (reduced to a double standard triangle). That resulting map reveals the most probable orientation for every experimental spot ED pattern ( in this case ED pattern is found to be close to 110 ZA orientation ) www.nanomegas.com
TEM orientation imaging : Spatial Resolution 1 nm to 500 nm Cu grains of different sizes Courtesy Muriel Veron Grenoble INP
Hydroxyapatite young bone Red: 001 Green:100 Blue: 010 Apatite nanocrystals of different sizes and orientations Courtesy Dr. R. Kroger, Univ of York
TEM : visualize EXACT amorphous / nanocrystalline content in a sample Example : Mg-Cu-Gd partly recrystallized metallic glass with Mg 2 Cu and Cu 2 Gd crystalline precipitates Crystalline or Amorphous? amorphous crystalline www.nanomegas.com
CONCLUSIONS Most important reasons to use Electron Diffraction Nanocrystalline samples that give poor X-Ray patterns Inconclusive cell determination /crystal structure from X-Ray Detailed overview over crystalline vs amorphous content Solve crystal structures ab-intio from 50 nm-micron size crystals How Electron Diffraction can be used? Use any TEM (120, 200, 300 kv LaB6 or FEG) 3D precession diffraction tomography (ADT-3D) can help to find ab-initio the unit cell / crystal structure of any unknown crystal < 50 nm Random precession diffraction tomography (ASTAR) to find unit cell and crystal structure by reconstructing reciprocal space from quasi-oriented PED patterns Reveal detailed local amorphous vs crystalline part in samples
COLLABORATIONS University of Valencia, Spain University of Basel, Switzerland University of Cambridge, UK Center for Nanotechnology Innovation@NEST, Italy SIMAP-INP Grenoble-France, Dr. Edgar Rauch Amsterdam Scientific Instruments/Teracrystal CONTACT US pharma@nanomegas.com/info@nanomegas.com www.nanomegas.com/pharma