EXPERIMENT 5
DETERMINATION OF CRYSTALLINITY USING SOLID STATE
TECHNIQUES. XRPD
The chemicals listed below will be used in this experiment. The likely hazards
associated with each of the chemicals are noted and recommended procedures for
handling are given. You must read this page and the experimental description
carefully before starting the experiment and before coming into the laboratory. Note
any potential hazards and adopt precautions as your safe lab practice. When you are
satisfied that you understand any possible difficulties that might arise and the
recommended procedures for dealing with them, sign the declaration and have it
initialled by a demonstrator. This must be done prior commencing lab work. At the
beginning of the lab session demonstrators will quiz you about the safety information
and experimental procedure in order to identify your ability to work safely and
efficiently. If you fail to prove ability for safe and efficient work you will not be
allowed to start lab practical. Please note, that it is your own responsibility to
complete the lab practical during time that is allocated to you. Be sure to request
information or help if you are in doubt on any point.
| Chemical | Hazard | Precautions |
| Indomethacin | Toxic, very toxic if swallowed |
Do not ingest, avoid skin/eye contact, wear gloves |
AssignmentTutorOnline
Declaration – I have read and understood the contents of the safety information sheet
and the script for the experiment
Signed (student): ……………………………………………………..
Checked (demonstrator): ………………………………………….. Date: ………………………
EXPERIMENT 5
X-RAY POWDER DIFFRACTION (XRPD)
Determination of crystallinity in indomethacin
LEARNING AIMS
To gain familiarity with the use of XRPD equipment
To understand the applications and limitations of XRPD
To gain understanding of the interpretation of X-ray diffractograms
To extend data manipulation and presentational skills
To continue developing GLP skills
LEARNING OUTCOMES
To critically discuss the use of XRPD in pharmaceutical analyses
To manipulate XRPD instrumentation
To enable the differentiation of amorphous from crystalline phases
To learn methods of quantification in XRPD
DIRECTED READING
OMED 0104 – LECTURE NOTES
Cullity, B.D. 1978. Elements of X-ray Diffraction. Addison-Wesley. 555pp.
Whiston, 1987. C. X-ray Methods. Analytical Chemistry by Open Learning. John
Whiley. Chapters 1-3.
Beckett, A.H. & Stenlake, J.B. 1988. Practical Pharmaceutical Chemistry. Part 2.
Athlone Press, London. Chapter 3.
INTRODUCTION
X-Ray Diffraction is used in pharmaceutical analyses particularly in the fields of
crystal structure determination. Areas of interest include phase identification,
determination of degree of crystallinity, estimates of crystallite shape and size,
quantitative analysis of mixtures, determination of polymorphic state.
X-ray Diffractometers consist of an X-ray generator, water-cooling system, an X-ray
tube, a collimation system, slits, sample changer, monochromator, X-ray detector, a
two-circle goniometer. Data are acquired via an interface to pc and are manipulated
using software.
Experimental considerations include selection of: an X-ray tube with suitable anode,
X-ray generator setting (kV and mA), slit widths, step scanning speed, scanning range
in degrees two-theta, suitable sample preparation.
This equipment produces ionizing radiation and as such falls under the remit of the
Ionising Radiations Regulations 1999. All procedures must be carried out in
accordance with the Local Rules which are attached to the equipment.
EXPERIMENTAL
Due to the safety requirements of the Local Rules much of the experimental work in
this practical will be carried out as a laboratory demonstration. Therefore it is
essential that adequate notes are taken while the equipment and methods are being
demonstrated.
Determination of crystallinity in indomethacin
Material: one sample of pure crystalline indomethacin, one sample of pure amorphous
indomethacin, one sample of a mixture of crystalline and amorphous indomethacin.
To achieve good data samples need to be ground to a particle size of 5-10 microns;
there should be an even particle size distribution within the sample. Ground samples
are packed into the plastic sample holder being aware not to introduce any preferred
orientation effects, as this causes incorrect intensity ratios of the diffraction events;
this becomes important when quantification is required. Beware of altering the crystal
structure by grinding, sometimes it may be necessary to use cryogenic grinding.
Diffractograms are collected using suitable scan parameters. Referring to the ICDD
PDF pattern for indomethacin we can see that most of the diffraction events for
indomethacin fall between 10.2 and 37.4O2θ, so for the two end member forms, scan
parameters are chosen to encompass these values i.e. to run from 7 to 40 O2θ. An
intermediate size of exit slit is selected (0.6mm) to give a good compromise between
signal and peak shape resolution. Using a fast position sensitive detector with an
acquisition time of 0.1 second and a step size of 0.02 degrees will take 2 minutes and
45 seconds to collect the data. Better quality data with less noise and a higher signal
can be obtained by increasing the counting time, for example to 0.2 or 0.3 seconds per
step; the data acquisition time will increase accordingly.
For the sample containing a mixture of crystalline and amorphous indomethacin it is
necessary to acquire top quality data for the purposes of Rietveld refinement.
Therefore a smaller exit slit is used (0.2mm) to improve peak shape, and a longer
counting time is used (0.4 seconds per step) to improve signal to noise ratio.
Additionally a longer scan range is selected in order to model the background
effectively.
EVALUATION OF RESULTS
Using the software (Bruker, EVA v.14):
Qualitative assessment:
1) import the data for pure crystalline and pure amorphous forms into EVA.
2) overlay the two diffractograms: crystalline and amorphous, note the
differences.
3) using the crystalline form, perform a peak search and generate a list of dspacings and intensities, export to Excel for output.
4) perform a background subtraction in readiness for the search-match procedure.
5) perform a search-match routine to confirm the identity of the phase: examine
the ICDD card and compare with your data, note any differences and think
about why they may have arisen.
Quantitative assessment:
6) import the mixture data into EVA
7) select an area to measure the total counts under the curve (crystalline +
amorphous)
8) note the measurement of net area (Cps x deg)
9) fit a background curve to the amorphous humps
10) subtract the amorphous background from the total curve to leave crystalline
component only
11) copy the area previously selected to the crystalline curve and note the net area
(Cps x deg) for this curve
12) calculate the percentage crystallinity by ratio. = (net area of the crystalline
component / net area for the total crystalline + amorphous) * 100
data for Qualitative assessment – ICDD data file for indomethacin
triclinic P1, a=9.348Å, b=11.006Å, c=9.764Å, α=69.3, β=110.88, γ=92.76
Angle d value Angle d value
2-Theta ° Angstrom Intensity 2-Theta ° Angstrom Intensity
10.160 8.699 22
11.599 7.623 100
12.734 6.946 15
16.650 5.320 54
17.004 5.210 89
17.281 5.127 25
18.557 4.778 19
19.280 4.600 36
19.599 4.526 60
20.289 4.374 10
20.862 4.255 15
21.795 4.074 89
22.890 3.882 17
23.136 3.841 15
24.001 3.705 22
25.459 3.496 13
26.584 3.350 42
27.437 3.248 15
28.273 3.154 11
28.879 3.089 23
29.335 3.042 31
30.407 2.937 14
33.545 2.669 10
34.112 2.626 11
34.765 2.578 8
37.415 2.402 13
5
QUESTIONS
1. What is the effect on the diffractogram of changing slit sizes?
2. What is the purpose of water-cooling, and why is it necessary?
3. Explain what the effect of a non-random powder sample would be on the diffractogram?
4. What is the effect on peak shape of crystallite size?
5. How is the n-term in the Bragg equation seen in diffractograms?
6. Why do we use monochromatic radiation as a source?
7. How can you explain intensity ratios in your samples which differ from the ICDD?
8. Which terms in the Bragg equation are the instrumental parameters?
9. What would changing the scan speed achieve, and why may you want to do this?
10 why does a diffractogram of an amorphous phase differ from a crystalline phase?