# A Green Approach for Preparing High-Loaded Sepiolite/Polymer Biocomposites
## 3 Results and Discussion
### Characterization of SepX and NRL Colloidal Dispersions  
Results of DLS analysis on NRL evidences a bimodal distribution of the hydrodynamic radius, indicating the presence of a fraction of particles with an average size of \(\sim\)300 \(\pm\) 130 nm (65%), and a second population (35%) with an average size of \(\sim\)1200 \(\pm\) 500 nm (Figure S1 of SI) [27]. Generally, NRL particles display high polydispersity with diameters comprised between 0.2 and 1 \(\upmu\)m, due to their natural metabolic origin [27]. As already reported in our previous study [22], SEM micrographs on SepX fibers revealed that both SepS9 and SepB5 appear as rod like particles with similar AR strongly interconnected in bundles-like aggregates (Figure S2 in SI). Table 1 summarizes the average dimensions and AR of SepX fibers. \(\zeta\)-Potential analysis was performed on pristine NRL and SepX dispersion to define the charge of the electric double layer of these colloidal systems. In general, relatively high absolute values of \(\zeta\) (\(\pm\)30 mV) indicate high colloidal stability. In the opposite case (\(\zeta\) values in the range of \(\pm\)5 mV), short-range attractive forces may exceed repulsion and coagulation/flocculation may easily occur. In an aqueous medium, the most common surface charge-determining ions for colloidal systems are H\({}^{+}\) and OH\({}^{-}\). Hence, the pH value readily affects the surface charge and, consequently, \(\zeta\)-potential value. In particular, the \(\zeta\)-potential becomes more positive or negative in magnitude with acidic and basic pH, respectively. Therefore, monitoring \(\zeta\) values, at different pH, allows to determine the isoelectric point (IEP), i.e., the pH at which \(\zeta\)-potential becomes zero and, consequently, colloids lose stability and agglomerate/flocculate (Figure 2). \begin{table}
\begin{tabular}{c c c c} \hline  & **Length (nm)** & **Cross-Section (nm)** & **AR** \\ \hline
**SepB5** & 1398 (\(\pm\)567) & 67 (\(\pm\)15) & 21 (\(\pm\)10) \\
**SepS9** & 1781 (\(\pm\)1004) & 80 (\(\pm\)21) & 22 (\(\pm\)11) \\ \hline \end{tabular} \({}^{\star}\)Calculated as average of the dimensions of 100 fibers in 20 different SEM micrographs.  
\end{table} 
Table 1: Dimensions * and AR of SepX fibers, obtained by scanning electron microscopy (SEM). The \(\zeta\)-potential of pristine NRL-MA at pH 7 is \(-\)28 \(\pm\) 0.05 mV. The negative surface charge is most likely provided by proteins and phospholipids amino and carboxyl functional groups, which can be easily ionized and whose charge balance give rise to a negative electric double layer [28]. Accordingly, decreasing the pH \(\zeta\) of NRL-MA increases (\(\bullet\), - black line in Figure 2), reaching the IEP at pH \(\sim\) 2.9, in agreement with the literature [29]. As concerns on the Sep suspension, Sep bundles, once dispersed in the aqueous medium, form an arrangement of randomly intermeshed elongated fibers, kept together by physical interactions and hydrogen bonding. Water can easily penetrate through these structures, thus increasing the viscosity of the suspension, providing to the colloidal solution a pseudoplastic behaviour [30]. The optimal mixing time to obtain a SepX suspension was \(\sim\)60 min for SepS9 and \(\sim\)20 min for SepB5. At pH 6.5, \(\zeta\) value of the resulting colloidal systems was \(-\)34.8 \(\pm\) 0.05 mV for SepS9, while for SepB5 modified with an alkylbenzyl ammonium salt, a positive \(\zeta\)-potential was measured (+26.9 \(\pm\) 0.05 mV). SepS9 suspension (\(\blacksquare\), - orange line in Figure 2) has slightly positive \(\zeta\) values (+0.38 at pH 2) at low equilibrium pH while negative ones at high equilibrium pH [31]. The IEP lies at pH \(\sim\) 2.4. As reported by [PERSON] et al. [31, 32], at acidic pH SepS9 fibers in water solution display a positive \(\zeta\)-potential due to the protonation of the surface groups, which undergo dehydrodylation or deprotonation at higher pH, leading to significant decrease of the \(\zeta\) value.