X-Ray Diffraction Analysis

General Description.

X-Ray powder Diffraction analysis is a powerful method by which X-Rays of a known wavelength are passed through a sample to be identified in order to identify the crystal structure. The wave nature of the X-Rays means that they are diffracted by the lattice of the crystal to give a unique pattern of peaks of 'reflections' at differing angles and of different intensity, just as light can be diffracted by a grating of suitably spaced lines. The diffracted beams from atoms in successive planes cancel unless they are in phase, and the condition for this is given by the BRAGG relationship.

nl = 2 d Sin q

l is the wavelength of the X-Rays

d is the distance between different plane of atoms in the crystal lattice.

q is the angle of diffraction.

The X-Ray detector moves around the sample and measures the intensity of these peaks and the position of these peaks [diffraction angle 2q ]. The highest peak is defined as the 100% * peak and the intensity of all the other peaks are measured as a percentage of the 100% peak.

1. J.C.P.D.S. [Joint Committee on Powder Diffraction Standards]

This organisation produced standard diffraction patterns for many of the minerals and inorganic structures suitable for analysis by X-Ray Diffraction Spectroscopy, and published these data as standards. The reference standard for Calcium Hydroxylapatite is 9-432. The procedure was carried out by Hodge.cs. The reference standards for other phosphates and for other possible impurities is given in Table 2. There is no specified method for sample preparation quoted in the J.C.P.D.S. handbook, consequently we have outlined below the sample preparation method to be used.

2. Sample preparation.

The method preferred for the analysis of Hydroxylapatite powders is the method by which all possible crystal orientations are presented simultaneously to the beam of X-Rays. Some crystallites will be correctly orientated to fulfil the Bragg equation for each value of d, and the detector will measure the intensity of the beams of diffracted X-rays at all locations given by the theoretical values of q. Any impurities will give additional lines which can be identified by looking at the pattern expected from likely impurities such as a Tri-Calcium Phosphate or b Tri-Calcium Phosphate etc. The material should be removed from the test plate by bending the mild steel plate in a vice, the flaked material then should be ground by hand in a pestle & mortar sufficiently fine so that it will just pass through a 40 micron sieve. The quantity of powder required to fill the sample holder used is 0.35 grams approx and this should be packed into the holder using a standard back fill method sufficiently firmly such that it will not fall out during the 90° tilt test.

The standard backfill method is as follows:-

Place the sample holder on a glass microscope slide and hold in place with sticky tape. Fill the recess with the ground and sieved sample, tap the holder lightly to ensure the corners are filled, draw another glass microscope slide over the recess to remove excess sample. place the second slide on the sample surface and hold in place with sticky tape. Turn the whole unit over such that the bottom is now the top and remove the first glass slide. The sample is now ready to be inserted into the X-Ray machine.

3. Operation method of the X-Ray diffractometer.

The Xrays used are of the Copper k a wavelength 1.54056 x 10-10m, the scan is taken between 2 theta of 10░ and 2 theta of 45░ at increments of 0.04░ with a count time of 4 seconds for each step. These angles have been selected as this is where the important reflections lie for Hydroxylapatite and other relavent impurities. The count time is selected as 4 seconds to give a good signal to noise ratio and yet to enable the analysis to take place over a reasonable period. The data are collected by computer and stored on floppy discs for later evaluation and hard copy production. See graph 2 enclosed. The intensity of the Xrays are measured on the Y axis, and increasing values of 2 theta are shown on the X axis. The sample is run on the machine and then the Hydroxylapatite standard is run for comparison.

4. Evaluation of data
4a. Relative Crystallinity.

Determination of the relative crystallinity for the Hydroxylapatite sample under test utilises an XRD software package and involves integrating the area under the curve on the graph printout between 2 theta of 30░and 2 theta of 35░ , the result is then divided by a value obtained similarly from a control sample. (standard P120 hydroxylapatite powder supplied by Plasma Biotal and X-rayed on the same day (under the same scan parameters). The ratio expressed in percentage terms gives the relative crystallinity of the sample tested. The Standard P120 hydroxylapatite powder is compared with the international standard for a Alumina [Reference Standard material 674A available from US National Institute of Standards and Commerce. Gaithersburg, MD. 20899.USA.]

N.B. The height of the peaks (intensity) depends upon the number of crystallites diffracting the X-Rays, thus a sample more finely ground will give higher but narrower peaks than the same sample coarsely ground. The area under the graph (as described above) measuring crystallinity will yield the same result in each case whether the sample is finely or coarsely ground.

4b. Quantification of beta Ortho Tricalcium phosphate (Whitlockite).

When equal proportions 50% Hydroxylapatite and 50% beta Whitkockite by weight are mixed then upon analysis the 100% peaks for Hydroxylapatite and for beta Whitlockite are of equal intensity. Graphs have been produced for 10%, 5%, 2%, 1%. mixtures of beta Whitlockite with Hydroxylapatite by weight. these are used to check that the estimate produced for whitlockite by the method below is in the correct range.

The assessment of % by weight of beta Whitlockite is found by integrating the area under the 100% whitlockite peak (2 theta of 31.027) and the 100% Hydroxylapatite peak (2 theta of 31.774). this fraction when multiplied by the overall relative crystallinity give the percentage (weight by weight) of whitlockite present in the sample (to the nearest significant percentage.)

4c. Unit Cell Dimensions.

The Unit cell dimensions are not measured on a regular basis however from many analyses performed (1990-1993) it has become clear that the unit cell dimensions are consistently different in Hydroxylapatite analysed from coating samples, from those obtained by analysing the starting material, this is indicative of minor microstructural changes. These are likely to have occurred during the heating and cooling stages of the process.

4d. Preferred Orientation.

* [When analysing Hydroxylapatite it may be possible under certain circumstances for the `100%' peak, normally at Interplanar spacing 2.814 angstroms units to be reduced height and the `40%' peak at Interplanar spacing 3.440 angstroms units to be of increased height such that the 40% peak is higher than the 100% peak. This is thought to arise due to 'preferred orientation' which is described as follows:-

The crystallites of Hydroxylapatite can be regular 'needle shaped' particles which tend to all lie in a similar direction in the sample holder, this is especially true of the Hydroxylapatite which has been thermally processed by the coating procedure. The X-Ray powder diffraction method requires that the crystallites be randomly packed to give the correct intensity of reflections, thus on occasions the sample may need to be reground and the holder repacked

Table 1 J.C.P.D.S. Standard for Hydroxylapatite 9-432
Interplanar spacings and peak intensity data
Identityd (Andstoms)Intensity/Max Intensity 100hkl

plus other minor lines of less than 1% .

System: Hexagonal

Table 2
MaterialFormulaeJ.C.P.D.S.* reference
Alpha Calcium OrthophosphateCa3(PO4)2
Beta Calcium OrthophosphateCa3(PO4)29-169
Calcium Hydrogen OrthophosphateCa(H2PO4)2.H2O9-390
Calcium PhosphateCaP4O1121-839
Alpha Calcium Di-PhosphateCa2P2O79-345
Beta Calcium Di-PhosphateCa2P2O79-346
Beta Calcium Metaphosphate(CaP2O6)x11-39
Calcium Hydrogen OrthophosphateCaHPO49-77
Calcium OxideCaO4-777
Calcium Carbonate HydrateCaCO3.H2022-147
Silicon DioxideSiO25-0490