Selecting a Specimen Holder for X-ray Powder Diffraction

The specimen-mount procedure is one of the many actions an investigator employs that could dramatically affect Bragg peak positions and intensities in an x-ray powder-diffraction experiment. The Bragg peak positions and, to a lesser extent, the peak intensities are of key interest in pharmaceutical powder-diffraction analysis (1), and it is important to report them as accurately as possible. Of the many systematic errors that affect peak positions and intensities, the most egregious for pharmaceutical compounds is that of preferred orientation (2), which is affected by the shape of the crystallites. In an ideal world, the crystallites in a powder pack together in a random manner, with non-directional orientation, which will allow the incumbent x-rays to illuminate all facets of the crystallites. When crystallites are plate or needle shaped, however, they will pack in an orientation that places the long axis of the crystallite (i.e., short axis of the unit cell) parallel to the packed surface and out of the illumination of incumbent x-rays (3). This phenomenon is common for organic crystals. Although spinning the specimen in the plane of the specimen’s surface does increase the number of crystallites that are illuminated, which is helpful for non-uniformly packed specimens, it will not eliminate the effects of preferred orientation (4), and a different specimen-mounting method is required. Although there are many methods to mount specimens for X-ray powder diffraction to reduce preferred orientation, this work will concentrate on the capillary, side-drifting cavity, back-loading cavity, zero-background holder (ZBH), and an amorphous fill (acacia)/back-load method (4).

To test specimen-mounting techniques for pharmaceutical samples, it is preferable to test a well-known API that displays preferred orientation, such as amiodarone hydrochloride (HCl) (see Figure 1). Amiodarone HCl is an antiarrhythmic agent used for various types of cardiac dysrhythmias, both ventricular and atrial (5). The crystal structure of amiodarone HCl is known, and a predicted powder pattern may be easily generated (6). Comparison of the predicted pattern with patterns generated by various specimen-mounting techniques can aid the investigator in understanding the effects of preferred orientation on the peak positions and intensities.

Materials and methods
Amiodarone HCl (2-butyl-3-benzofuranyl 4-[2-(diethylamino) ethoxy]-3,5-diiodophenyl ketone hydrochloride, C₂₅H₂₉I₂NO₃•HCl, MW 681.8, density 1.71 g/cm³, μ_eff = 2.50 mm-1) and acacia (gum arabic, a mixture of polysaccharides and glycoproteins employed as an amorphous filler, density ~1.4 g/cm^-3, particle size ~140 microns, μ_eff ~0.1 mm-1) were purchased from ALFA AESAR. The amiodarone HCl was sieved (#200, 75 micron) to produce a fine powder.

The predicted powder pattern was determined by employing the program Mercury 3.1 (7) using the metric parameters taken from the room temperature structure of amiodarone HCl (wavelength 1.54056 Å (1.54439 Å), scale 0.5, 4° to 40° 2, step size 0.02°, include hydrogen and full-width half-maximum (FWHM) (2θ) of 0.1°) (5).

A side-drifting cavity mount, back-loading cavity mount,  silicon ZBH, and 0.7-mm capillary were employed for specimen mounting. Powder-diffraction patterns for the flat specimens were collected on a Bruker D8 advanced Bragg-Brentano θ-2θ diffractometer, Cu radiation (40 kV/40 mA), Ni filter, LynxEye PSD detector, and a stationary specimen holder. All flat specimen data collections employed a scan range between 4° and 40° 2θ with 2431, 0.015° scan steps at a constant data-collection speed of 0.7 s/step for a total scan time of 28 min. The capillary data collection was undertaken on a Bruker D8 Discover diffractometer, Cu radiation (40 kV/40 mA), primary beam monochromator (parabolic, Goebel Mirror, 0.6mm slit), LynxEye PSD detector, rotating capillary stage, and two anti-air scatter-beam knives. The specimen was mounted in a 0.7-mm capillary and data was collected with scan range between 4° and 40° 2θ for 1800, 0.02° scan steps at 15 s/step for a total time of 450 min (7.5 h).

The side-drift cavity mount was constructed from a 3-mm thick by 50-mm round stainless-steel disk with 25-mm wide and 40-mm long U-shaped slot (see Figure 2a). The disk was glued to an amorphous quartz disk (10 mm by 50 mm round), to form a cavity approximately 3-mm deep by 25-mm wide. The cavity was filled by clamping a standard glass slide over the closed end of the cavity and turning the holder onto its side (vertical). The specimen was introduced from the top and tapped down until the cavity was filled to the depth of the width of the slide. The holder was then repositioned horizontally and the glass side was carefully removed.

The back-loading cavity mount was constructed from a 50-mm round stainless-steel disk with a 25-mm hole drilled through the center and smooth top and an indented bottom (Bruker-AXS). To fill the holder, the disk was inverted and placed with the top on a clean, smooth, quartz disk (2). The powder was added and gently packed with a 24-mm diameter pestle. A plastic disk was inserted to hold the powder in place. The holder and quartz disk were then quickly inverted so the top of the holder was horizontal, and the quartz disk was removed (see Figure 2a).

The ZBH was constructed with 24.6 mm diameter x 1.0 mm thick, single silicon crystal (MTI Corporation). The silicon crystal was glued in a well on a 3 mm x 50 mm stainless-steel holder in such a manner as to align the surface of the silicon crystal with the surface of the stainless-steel holder. The ZBH was then fastened in a sieve pan with the silicon crystal facing upward. A 200-mesh (75-micron) sieve was attached to the pan and the powder was gently sprinkled onto the sieve above the ZBH holder. After three taps of the sieve assembly, the 200-mesh sieve was removed; the loaded ZBH holder was detached from the sieve pan, and the excess powder was removed from the stainless-steel portion of the ZBH (see Figure 2a).

The capillary mounted sample was loaded by selection of a 0.7 mm (special glass, Charles Supper) thin-walled capillary (8). The capillary was filled by introducing a small amount of the sieved powder into the capillary opening and then transferring the loaded capillary to a long-stemmed funnel (open capillary-end up). The capillary was allowed to drop the length of the stem of the funnel. The entire process was repeated until approximately one-half of the capillary was filled and packed. The capillary was flamed sealed by wrapping the powder-packed portion in a wet tissue, leaving the unfilled capillary uncovered. A small torch was used to heat and pull the uncovered glass to seal the tube. The sealed capillary was glued to a standard brass specimen-mount pin (Charles Supper).

The amorphous filler powder sample was made by mixing equal weight portions (1 g) of acacia and the (1 g) amiodarone HCl into a scintillation vial, which filled the vial to approximately one-quarter full. The vial was then introduced to a long cardboard mailing tube, which was tumbled end-over-end for five minutes. A smaller specimen was removed from the well-mixed sample and pressed into a back-loading cavity mount.

The peak positions for each determined powder pattern and the predicted powder pattern were determined (before background subtraction) with the program package EVA (revision 14.0.0.0, Bruker-AXS) using a second-derivative method with data prepared by a Savitzky-Golay smoothing filter (9). The intensities (%) of each peak position (where 100% pattern line corresponds to the highest intensity point of the scan) were determined with the peak positions. The first 20 (long 20) peak positions were employed for comparison of the peak position and intensities; statistical summaries for these data are summarized in Table I.

FWHM is determined for a dummy peak at 30° 2θ from the full-pattern matching (FPM) subroutine in the program EVA (revision 14.0.0.0, Bruker-AXS) (9) using the metric parameters taken from the room temperature structure of amiodarone HCl.

Each determined pattern was corrected for background by application of a parabolic background-curve algorithm, and their intensities were normalized to share the same maximum intensity of the predicted powder pattern (8). The resulting patterns were offset for clarity and the plot of the combined patterns from 4.5 to 20° 2θ is shown in Figure 3.

Discussion
The values reported in Table I for the standard uncertainties and the average deviations, when comparing the first 20 peaks for amiodarone HCl, are lower than the reference values reported in the US Pharmacopeia (USP) [±0.1° 2θ] (1). As shown, any of the specimen mounting methods described herein would be acceptable in the determination of accurate peak positions. The most accurate method was ZBH, followed by side-drift; capillary and acacia (i.e., amorphous fill/back-load) were approximately equivalent. The back-loading method was the least accurate of the methods studied. The average deviation in the intensities also falls below the 20% reference reported in the USP (1), and the deviations are clearly seen in Figure 3. The most accurate method for intensity was capillary followed by side-drift, acacia and ZBH, and back-load. The capillary method, by nature of the optics employed (parallel), has a slightly larger FWHM compared to the other (Bragg-Brentano) methods. In practice, to differentiate between two reference peak positions the differences in their positions should be two to three times their FWHM (3).

Other errors will also contribute to deviations in accuracies of position and intensities of diffracted x-rays including transparency of the specimen for cavity mounted samples, the breakdown of the constant-volume assumption for the ZBH mounted samples, and micro-absorption effects (10). Also, the rougher surface of the ZBH and side-drift mounted specimens provides voids in the powder material that artificially lower the intensity of the diffracted peaks at lower angles (11). Of the reflection methods, the back-load cavity mount has the smoothest surface with the least voids as is seen in Figure 2b and c. The side-drifted cavity mounted specimen shows the characteristic layering of the material as it is added to the holder and tapped into place (see Figure 2b and c); as expected, the roughest surface is seen for the ZBH mounted specimen, as shown in Figure 2b and c.

The capillary method requires much less sample and provides the most accurate peak intensities; however, the data collection time for the method is much longer (hours, for amiodarone HCl) than the cavity-mounted specimens (minutes). The amorphous-filled method minimizes preferred orientation; however, the filler introduces an amorphous scattering background. The ZBH method requires only micrograms of specimen and is useful in determining accurate peak positions; however, it will not return the most accurate peak intensities. In the end, the best procedure is to measure the x-ray diffraction pattern of the specimen with several different mounting techniques, which in turn will provide more information that can be used in choosing standard peak positions (11).

Acknowledgement
The authors wish to thank Holger Cordes at Bruker AXS, (Madison, WI) for collecting the x-ray powder data from the capillary sample. The x-ray powder diffractometer in the X-ray Diffraction Laboratory at the Department of Chemistry, Texas A & M University was purchased with funds provided by the National Science Foundation (CHE-9807975).

References
1. USP General Chapter <941>, “X-ray Diffraction” (US Pharmacopeial Convention, Rockville, MD, 2007).
2. V.K. Pecharsky and P.Y. Zavalij, Fundamentals of Powder Diffraction and Structural Characterization of Materials (Springer, New York, NY, 2005), pp. 196-202.
3. I. Sunagawa, Crystals Growth, Morphology and Perfection (Cambridge University Press, Cambridge, UK, 2005), pp. 13-17.
4. B. L. Davis, et al., “Specimen Preparation in X-ray Diffraction,” in A Practical Guide for the Preparation of Specimens for X-ray Fluorescence and X-ray Diffraction Analysis, V. Buhrke, R. Jenkins, and D.K. Smith, Eds. (Wiley-VCH, New York, NY, 1998) pp. 130-141.
5. M. B. Rosenbaum, et al., Am. Heart J. 106 (4) 957-964 (1983).
6. V. Cody and J. Luft, Acta Crystallographic, B45, 172-178 (1989).
7. C. F. Macrae, et al., J. Appl. Cryst., 41, 466-470 (2008).
8. J. Reibenspies and N. Bhuvanesh, “Practical Aspects,” in Principles and Applications of Powder Diffraction, A. Clearfield, J. Reibenspies and N. Bhuvanesh Eds. (Wiley, New York, NY, 2008) pp. 149-150.
9. Buker-AXS, EVA V16 Diffracplus Evaluation Manual (Buker-AXS, Madison, WI, 2010) pp. 91-156.
10. P. Whitfield and L. Mitchell, “Phase Identification and Quantitative Methods,” in Principles and Applications of Powder Diffraction, A. Clearfield, J. Reibenspies and N. Bhuvanesh Eds. (Wiley, New York, NY, 2008) pp. 230-234.
11. I. Ivanisevic, R.B. McClurg, and P.J. Schields, “Uses of X-ray Powder Diffraction In the Pharmaceutical Industry,” in Pharmaceutical Sciences Encyclopedia: Drug Discovery, Development, and Manufacturing, S.C. Gad Ed. (John Wiley & Sons, Inc., New York, NY, 2010) pp.1-42.

About the AuthorJoseph H. Reibenspies, PhD, is associate director, j-reibenspies@tamu.edu, and Nattamai Bhuvanesh is manager, both of the X-ray Diffraction Laboratory in the Department of Chemistry at Texas A&M University, College Station, TX.