During different pharmaceutical production processes - including crystallization, granulation, micronization, milling and spray- drying for example - crystallite sizes of the compounds can vary and are dependent on the process parameters employed. X-ray diffraction techniques play an important role in the analysis of crystallite sizes, since traditional methods such as laser granulometry determine only particle size, a parameter that is not directly related to, or correlated with, crystallite size. The SEM (scanning electron microscope) images in Figure 1 show clearly that milling time has only little influence on particle size and a variety of different sizes is always visible. The crystallite sizes cannot be determined from these pictures.
Several X-ray diffraction methods for crystallite size determination are available. These include the widely used, but less accurate, single-peak Scherrer method and the well established Warren-Averbach method for size and strain analysis. Recently the use of the (full-pattern) Rietveld refinement has become popular. This application note describes the use of the Rietveld method for evaluating crystallite sizes in a pharmaceutical drug, milled in a ball mill for different times.
Relevance of crystallite sizes in the pharmaceutical industry
Designing a drug’s formulation is playing an increasingly important role in the pharmaceutical industry. The final dosage form determines the release profile of the drug, and consequently its pharmacokinetics in the body. In particular, the solid state of the API (active pharmaceu- tical ingredient) is crucial to controlling a drug’s performance. Particle design – including such properties as particle shape, crystallite size distribution and degree of agglomeration - begins with crystallization and comprises the entire processing chain leading to formulation. Since the crystallite sizes of a pharmaceutical substance may have a significant influence on the bioavailability of a drug - for example in many poorly water- soluble drugs - it is an important control parameter in the develop- ment and production process.
This note describes the use of the Rietveld method for determining crystallite sizes of pharmaceutical powder samples. In contrast to scanning electron microscopy that could hardly detect changes in the particle size, XRD clearly showed the decrease in crystallite size with increasing milling time of the model compound.
Figure 1. SEM images of the original sample (top) and after a 4 minute milling time
Crystallite size analysis using the Rietveld method
The analysis of crystallite size and/or microstrain by the Rietveld method is based on the change in profile parameters, compared with those of a standard sample. The standard material is used to evaluate the instrument profile function using the chosen optical configuration and therefore should not show microstrain or size broadening; for this purpose the reference material LaB6 (NIST) was used . According to the NIST certificate of this reference material the LaB6 average crystallite size (from SEM analysis) is between 2 and 5 μm.
The Rietveld method uses structure data for the calculation of a full diffraction pattern and compares this with measured data. By least squares fitting, the algorithm calculates (by varying different parameters such as peak shape or lattice constants) the minimum difference between calculated and measured patterns. Small crystallite sizes well below one micrometer show a peak broadening in the diffraction pattern, which results in different profile parameters. These are then used to calculate size and/or lattice strain based on the assumption of a Gaussian size distribution. Calculations were performed using Malvern Panalytical HighScore software with the Plus option for phase identification, crystallographic analysis and Rietveld analysis
Sample preparation and measurement parameters
Azithromycin was used as a model substance. This drug belongs to the class of macrolide antibiotics and prevents bacteria from growing by interfering with their protein synthesis. The azithromycin powder samples were milled in a ball mill with milling times from 1 to 4 minutes. Afterwards they were prepared in back-loading sample holders for analysis in reflection geometry and in 0.7 mm glass capillaries for analysis in transmission geometry. The standard material LaB6 for the analysis of the instrument profile function was measured with the same instrumental setup. For simplicity, in this example it was assumed that peak broadening of the azithromycin samples was related purely to crystallite size effects.
Measurements were performed using a Malvern Panalytical X-ray diffractometer in Bragg-Brentano reflection or transmission geometry. For the transmission geometry the system was equipped with a focusing mirror with an anti-scatter slit as incident beam optic, together with an X´Celerator detector with capillary anti-scatter device on the diffracted beam side. A scan range of 6.5° to 40° 2θ was used. Scan time was 28 minutes with a step size of 0.008°. For best resolution, 0.01 rad Soller slits were used in combination with incident and diffracted beam optics.
Due to the transparency of the investigated organic material Bragg- Brentano reflection measurements were not suitable for this analysis: slight variations in sample preparation cause different sample densities which lead already to uncontrollable peak broadening variations, making crystallite size determination almost impossible.
This problem was overcome using the transmission geometry as described above. Figure 2 shows a graphical comparison of the scans measured in this configuration after different milling times. Clearly visible is peak broadening associated with longer milling times. In our calculation we assume that this is due to the decrease in crystallite size. As can be seen from the plot, beyond a milling time of 3 minutes there is practically no further effect; peak broadening remains constant.
Figure 3 shows as an example a Rietveld refinement of the sample after 1 minute milling time. Using the instrument profile function from the LaB6 analysis, the crystallite size of the milled sample is derived from this refinement. Figure 4 shows the corresponding results derived by Rietveld analysis of the different samples. The graphical representation of crystallite size versus milling time indicates that after 3 minutes milling crystallite size is no longer affected. The calculated crystallite sizes are significantly smaller than the particle sizes visible in the SEM pictures.
Figure 2. Comparison of samples prepared with different milling times scaled to the main maximum (whole range and zoomed into the first part)
Figure 3. Rietveld refinement of the scan performed on a sample milled for 1 minute
Figure 4. Calculated average crystallite size of the azithromycin samples for different milling times
The Rietveld method for determining crystallite size in a powder sample is a powerful tool and can be used easily for pharmaceutical materials. It allows control of this important parameter in the development and production of drugs. Using a Malvern Panalytical diffraction system in transmission geometry and the HighScore software with Plus option, analysis is simple and straightforward, and after proper setup can be used by even less trained operators.