Small Angle X-ray Scattering

Small angle X-ray scattering is a technique that is able to provide a wealth of knowledge about macromolecules on the micro scale. As such it is an ideal platform for the nanostructural analysis of collagen based extracellular matrix materials such as leather.

Figure 3.5. SAXS/WAXS beamline at the Australian Synchrotron.

The Australian Synchrotron is a world class facility that provides scientists and researchers with the opportunity to undertake ground breaking research. Ranging from infrared to hard X-rays the Australian synchrotron is a source of highly intense light that can be used across a broad spectrum of research purposes. A research facility of such a high calibre is of course in high demand and beamtime on every single one of the beamlines at the Australian Synchrotron is highly oversubscribed to. Applications for beamtime to analysis fibrous collagen materials on the SAXS/WAXS beamline (pictured in Figure 3.5) have been successful.

At the SAXS beamline each sample was mounted without tension on a custom made plate with a grid of 64 or 132 holes with a 10 mm diameter. Samples were

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mounted in two orthogonal directions through the leather; edge on and flat on. Figure 3.6 depicts the direction of the beam for each sample. Edge on samples were fixed to the plate so the full thickness of the leather was subjected to the X- ray beam and measurements were made every 0.25 mm from the grain to the corium. Flat on samples were fixed with the uncut face of the leather directed towards the X-ray beam which was perpendicular to the surface of the leather and four measurements were made per sample in a rectangular grid.

Figure 3.6. Direction of beam on the sample for edge on and flat on sample directions.

Each sample was mounted horizontally across one of the holes and attached to the plate using adhesive tape (Fig 3.7a shows edge on mounted samples). When it was critical that a sample did not dry out and kept its moisture content the sample was placed within one of the holes on the plate and sandwiched in place using kapton tape on both sides of the sample (Fig 3.7b). Kapton tape does not interfere with the diffraction process so has no impact on the results and provides protection to stop the sample from drying out. All plate samples were mounted without tension. Once all the samples are taped in place the plate was attached to a sample stage in the

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SAXS beamline. This stage is able to be remotely controlled from outside the beamline hutch and a camera enables the samples to be seen by the beamline user. Static samples only take approximately 10 minutes per sample to collect the diffraction patterns. The remotely controlled sample stage and plate loaded with up to 132 samples is hugely beneficial for maximising the time on the SAXS beamline. If the sample had to be manually changed, the user would have to not only load a new sample into the beamline every time but go through the standard safety procedures required as well, all of which would probably double the allocated time per sample.

Figure 3.7. (a) edge on samples mounted on the plate ready to be inserted into the beam; (b) wet edge on samples sandwiched between kapton tape on the plate and ready to be inserted into the beam.

(a)

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Diffraction patterns were recorded on the Australian Synchrotron SAXS/WAXS beamline, using a high-intensity undulator source. An energy resolution of 10-4

was obtained from a cryo-cooled Si(111) double-crystal monochromator and the beam size (full width at half maximum [FWHM] focused at the sample) was 250 x 80 μm, with a total photon flux of about 2 x 1012 _{photons s}-1_{. Diffraction patterns}

were recorded with an X-ray energy of 8 keV using a Pilatus 1M detector with an active area of 170 x 170 mm and a sample-to-detector distance of 3371 mm. Exposure time for the diffraction patterns was 1 second and data processing was carried out using the SAXS15ID software (Cookson et al., 2006).

Diffraction patterns as shown in Figure 3.8a were obtained for each point analysed. These patterns can be integrated to produce intensity plots that display Bragg peaks (Figure 3.8b).

Figure 3.8. (a) SAXS diffraction pattern; (b) plot of intensity versus q.

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The diffraction patterns obtained from the SAXS beamline at the Australian Synchrotron hold a surprisingly large amount of information on the structure of the material being analysed. This work focuses on the orientation and the D- spacing of the collagen fibrils. Data was processed using scatterBrainAnalysis (Cookson et al., 2006) and Microsoft Excel. Two important structural aspects of collagen, the orientation index and the D-spacing, are able to be extracted from SAXS diffraction patterns and remain at the centre of the work presented in this thesis.

The orientation index, OI, is defined as (90° – OA)/90°, where OA is the minimum azimuthal angle range, centred at 180°, that contains 50% of the microfibrils (Sacks et al., 1997, Basil-Jones et al., 2010). Using the spread in azimuthal angle of one or more D-spacing diffraction peaks. The peak area is measured above a fitted baseline, at each azimuthal angle. OI provides a measure of the spread of microfibril orientation. An OI approaching 1 indicates that the microfibrils are parallel to each other and the leather surface while an OI of 0 indicates the microfibrils are randomly oriented. The OI was calculated from the spread in azimuthal angle of the most intense Bragg peak which occurs at a q value of around 0.059–0.060 Å. Each OI value presented here represents the average of 14– 36 measurements of one sample. For this investigation the sheep and cattle OI averages are derived from 228, 249 and 167 measurements from 15, 14 and 10 samples, respectively and have been reported previously (Basil-Jones et al., 2011). It is not necessary that the samples are highly representative of the particular animal species for general strength-structure relationships to be studied; that there is a range of skins with different strengths is important, although the observed strengths for each species are within industry norms.

The axial periodicity or D-spacing of collagen provides an indication of the nanostructural architecture of collagen microfibrils. The D-spacing is determined for each pattern by taking the central position of a Gaussian curve fitted to one or several of the collagen peaks, dividing these by the peak order (usually from n = 5 to n = 10), and averaging the resulting values. To do this each of the SAXS diffraction patterns is converted to an ASCII file using scatterBrainAnalysis and this is pasted into an Excel template spreadsheet. The Excel template creates a plot

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of intensity versus scattering vector, q (as was shown in Figure 3.8b). The q range to be analysed is manually selected, ensuring the full width of the peak is incorporated. The peak fitting function determines the order of the Bragg peak and the location of it. Standard analysis is based on the 5th _{to the 9}th _{Bragg peaks.}

The scattering vector q can be defined as;

ݍ ൌସగ ୱ୧୬ ఏ_ఒ (equation 1)

Equation 2 can be rearranged to give λ;

ߣ ൌସగ ୱ୧୬ ఏ_௤ (equation 2)

Bragg’s law is:

݊ߣ ൌ ʹ݀ •‹ ߠ (equation 3) Substituting equations 2 into equation 3 gives;

ʹ݀ •‹ ߠ ൌ ݊ସగ ୱ୧୬ ఏ_௤ (equation 4)

Dividing equation 4 by 2 sinθ yields;

݀ ൌʹߨ݊ ݍ

Where n = peak order and d = D-spacing.

The D-spacing was determined for each pattern by taking the central position of several of the collagen peaks, dividing these by the peak order (usually from n = 5 to n = 10) and averaging the resulting values.

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