x-ray powder diffraction:exposing the bare bones of solid forms
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X-ray Powder Diffraction:exposing the bare bones of solid forms
Dr. Noel Hamill
WelcomeDr Noel Hamill,Team Leader Physical Sciences, ALMAC
noel.hamill@almacgroup.com
• With nearly 10 years experience of working in the pharmaceutical industry, Noel’s current role includes; crystallisation development, solid form screening & characterisation, method development & validation.
• Almac is privately owned, financially stable company with ~ 3,000 employees, global HQ in Northern Ireland with extensive facilities in the UK and US.
• Almac has a comprehensive range of services from research through pharmaceutical & clinical development to commercialisation of product.
www.almacgroup.com
Contents
• Theory of powder diffraction
• X-ray Powder Diffraction– Instrumentation
– Sources of error
– Applications
• Case studies
Theory
• A crystalline powder contains many small crystallites, ideally randomly oriented
• Diffraction occurs when crystallites are oriented such that specific atomic planes are in the correct relationship with the incoming x-rays
Bragg’s law:n=2dsin
Constructive interference is detected when the path-length difference is equal to an integer number of wavelengths
Diffraction by crystal planes
θ ~ 20º
x-rays in x-rays out
top of sample
θ ~ 5ºx-rays in x-rays out
top of sample
crystalline
amorphous
Diffraction from powders
• Rings of x-rays diffracted from a crystalline powder as captured by an area detector
• Not uniform in intensity Particle statistics or preferred
orientation
Need to spin the sample
• Integration of intensity along any radius (in blue) gives a plot of angle 2 vs. intensity
• Small angle = large d spacing
X-ray Powder Diffraction
Information from a powder pattern:– Angular position of diffraction maxima– Peak intensities– Peak shape and width
Reflection mode(Bragg-Brentano geometry)
X-ray Diffractometer
• Traditional set-up
• Prone to errors due to sample preparation
Transmission mode • Reduces preferred orientation
from ‘difficult’ crystallite shapes (tiles, needles)
• Handles small amounts of sample (e.g. ~1mg using glass capillary)
• Samples can be sealed from air or humidity, or kept as a suspension
• Up to 40º 2, focusing mirror gives better data quality vs. reflection mode
Instrument capabilities
• Autosampling• Variable temperature• In situ measurement
– Suspensions– % RH– Radiolabelled– Potent API
• Pattern sorting software
Sources of error
• The powder pattern can be affected by:– Sample height displacement
• Sample is not at the instrument focal plane
– Preferred orientation• Particles (crystallites) are oriented relative to each
other
– Particle statistics• Too few or too large particles (crystallites)
• Sample preparation is very important!
Sample Displacement Error
In reflection mode, if the sample height is different from the focal plane, peak positions will be shifted
This effect is minimised in transmission mode
Incident X-rayspeak shift
Diffracted X-rays
sample
focal plane
Chen et al J. Pharm. Biomed. Analysis 2001, 26, 63
Preferred Orientation• Preferred orientation arises when there is a tendency for
the crystallites in a powder to be oriented in one way
random orientation realistic orientation
• Random orientation of particles can exist only if their shape is spherical
• In real samples, preferred orientation of particles is always present and measured intensities of diffractions are incorrect. This can result in ‘missing peaks’
Preferred Orientation
calculated acetaminophen XRPD pattern (refcode HXACAN01)
XRPD pattern from the (100) face of manually oriented acetaminophen single crystals(Wen et al J. Phys. Chem. B 2004, 108, 11219)
Particle Statistics
• Theory suggests particles < 20 m are best
• Ideally, large particles should be ground– Beware of possible phase
changes upon grinding
• Sieving may help– Beware of separating
different Forms!
• Line broadening occurs for particles <1 m
Reducing measurement errors
• Spinning and oscillation• Reducing particle size to 1-20m (caution!)• Transmission mode (esp. using capillaries)
Applications
• Degree of crystallinity• Phase identification• Unit cell indexing• Crystal structure solution• Mixture analysis
Crystallinity
Phase identification• Diffraction pattern is like a fingerprint for a crystal phase• Rarely, materials will yield matching XRPD patterns
– Other analytical techniques used to distinguish isostructural materials
• One extra peak at low angle is common in isostructural solvates
O
O
O
H3C
OHR
H3C
CH3
CH3
OH
CH3
H3C
H3CO
CH3OHO
OCH3
CH3
O
NH2
CH3
HO
erythromycin A R = OHerythromycin B R = H
Stephenson et al J. Pharm. Sci. 1997, 86, 1239
Polymorph detection
• As instruments become more sensitive, identification of new polymorphs possible
• Detection limits also decrease – caution with term ‘none detected’
05
10152025
3035404550
0.52 0.53 0.54 0.55 0.56 0.57 0.58 0.59 0.6
delta 2theta
sam
ple
ref
A
D
E
Unit cell indexing• Searching for a space group and unit cell
dimensions which match the peak positions in the observed pattern.
• To be successful, the pattern must– Represent one crystalline phase– Have very accurate peak positions
• Indexing a pattern from an unknown sample is compelling evidence that the pattern represents a pure crystalline phase
• Useful for comparison of cell volume (e.g. solvates)
Crystal structure by XRPD
• Rapid advances are being made in techniques to solve a crystal structure from an XRPD pattern alone
• However, currently the structure of crystals containing only relatively rigid molecules have the best chance of being solved in this way
• It is currently difficult or impossible to solve structures of molecules having many degrees of conformational freedom
• Almac/UCC research partnership underway
Mixture analysis
5000
10000
Counts
Position [°2Theta]
15 20 25 30
Position [°2Theta]
10 15 20 25 30
Counts
2000
4000
Crystalline Form A
Crystalline Form B
Amorphous
Resulting XRD pattern
Multi-phase sample
Patterns are additive
Quantitative analysis• Quantitative analysis of mixtures can be carried
out if proper attention is paid to instrument configuration and sample preparation– Be particularly conscious of preferred orientation
• Mixtures of crystalline phases– Ratios of peak areas or intensities– Whole-pattern methods
• Mixtures of crystalline and non-crystalline phases– Integrated intensities of crystalline and non-crystalline
regions– Whole-pattern methods
Method development & validation
• Phase III API with 3 forms• Developed and validated
transmission XRPD method – Quantify Form III in Form I– LOQ: 3% Form III– Limit test Form II (LOD 0.5%)– 22 min run time
• Challenges– Mixing: effect of milling– API potency
Transmission
Method validation issues
• Particle size needs to be controlled• Preferred orientation needs to be eliminated• Standards need to be pure physical forms
(e.g. can they be indexed?)• Robustness of instrument and sample
preparation parameters should be tested routinely
Thank you for your time…
..any questions?
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