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?