# A Green Approach for Preparing High-Loaded Sepiolite/Polymer Biocomposites
## Materials and Methods
### Thermal, Spectroscopic and Morphological Characterization of SepX/NR Biocomposites  
The thermogravimetric analysis (TGA) of SepX/NR biocomposite was performed with a Mettler Toledo TGA/DSC1 Star-e System. The variation of weight was analyzed by heating from 30 to 1000 \({}^{\circ}\)C, at 10 \({}^{\circ}\)C min\({}^{-1}\), under an air flux of 50 mL min\({}^{-1}\).  
The crystalline structure of Sep fibers inside the SepX/NR biocomposites was confirmed by X-ray Diffraction (XRD), recorded in X'Pert Pro PXRD (Panalytical) diffractometer (Malvern Panalytical Ltd., Malvern, UK) in Bragg-Brentano geometry, using Cu K-\(\alpha\) source with 1.54 A wavelength.  
The filler-rubber chemical interactions in SepX/NR biocomposites were analyzed through attenuated total reflection9--fourier transform infrared spectroscopy (ATR-FTIR) by a Perkin Elmer Spectrum 100 instrument (Perkin Elmer, Waltham, MA, USA) (4 cm\({}^{-1}\) resolution, 650-4000 cm\({}^{-1}\) range, 8 scans).  
To assess the Sep distribution in Sep/NR biocomposites, SEM-energy dispersive spectroscopy (EDS) analysis was performed by A Vega TS5136 XM Tescan microscope (Tescan Brno s.r.o., Kohoutovice, Czech Republic) operating at 30 kV (25 pA beam current, 12 mm working distance, 38 nm beam spot). Samples were deposited onto a carbon adhesive disc and carbon-sputtered. A map of principal elements of Sep, such as Si, Al and Mg, was detected to visualize the filler dispersion within the rubber matrix.  
The morphological investigation of composites was carried out by TEM using a Zeiss EM 900 microscope (Zeiss, Oberkochen, Germany) operating at 80 kV. Ultrathin sections (about 50 nm thick) of SepX/NR biocomposites previously embedded in an epoxy resin (70 \({}^{\circ}\)C, 24 h) were obtained with a Leica EM FCS cryo-ultramicrome, equipped with a diamond knife, by keeping the samples at \(-130\)\({}^{\circ}\)C.  
Small-angle X-ray scattering (SAXS) was performed on beamline ID2 at the European Synchrotron Radiation Facility (ESRF, Grenoble FR) at a wavelength of 1 A with a sample to detector distance of 2.5 m, yielding a total q-range from 0.001 to 0.15 A\({}^{-1}\). The scattering cross section per unit sample volume d\(\Sigma\)/d\(\Omega\) (in cm\({}^{-1}\)) (scattered intensity I(q)) was obtained by using standard procedures including background subtraction and calibration given by ESRF. The Debye approximation [24] and the Guinier approach were adopted for the analysis of the NR and biocomposite curves, respectively. The scattering intensity I(q) in the Debye model is given by the following equation:  
\[\text{I(q)}=\text{I}_{0}\frac{1}{\left[1+\left(\text{q}\xi\right)^{2}\right]^{2}} \tag{1}\]  
where \(\xi\) is the correlation length of the scattering bodies, in this case associated with the crystalline domains. As particulate systems, the scattering of the Sep rods in the NR can be analyzed using the Guinier approach [25] to get information on the radius of the inorganic fibers and therefore on their dispersion degree. For one dimensional systems the scattering in the intermediate region of the curve can be approximated to:  
\[\text{I(q)}=\frac{\text{I}_{0}}{\text{q}}\exp\left(-\frac{\text{q}^{2}\text{R }_{\text{g}}{}^{2}}{2}\right) \tag{2}\]  
\[\text{R}_{\text{g}}{}^{2}=\text{R}^{2}/2 \tag{3}\]  
where R\({}_{\text{g}}\) and R are the radius of gyration and the radius of the fiber, respectively.  
These parameters are obtained from the linearity dependency of the so called Guinier plot \(\ln(\text{q}\text{I})vs.\) q\({}^{2}\).