# A Green Approach for Preparing High-Loaded Sepiolite/Polymer Biocomposites
## 3 Results and Discussion
### Proposed Flocculation Mechanisms  
Based on the gathered results and taking into account the most common reported flocculation mechanisms, i.e., charge neutralization, polymer bridging and electrostatic patch, two distinct possible flocculation pathways for SepS9/NR and SepB5/NR systems have been proposed (Scheme 1). In the case of SepB5/NR (Scheme 1a), a mechanism simply based on charges neutralization can be inferred. In fact, at the working pH range (9-10), \(\zeta\)-potential of SepB5 and NRL displays an opposite sign, suggesting the occurrence of an attractive potential among the filler and rubber particles, which can collide due to their Brownian motion [47; 48]. The progressive adsorption of polymer segments on SepB5 surface, lowers the free energy of interaction among the fibers, decreasing their distance and leading to flocculation. It must be observed that the proposed mechanism is active only at high SepB5 loading; otherwise a charge reversal may occur resulting in the re-dispersion of the fibers [49]. These considerations are in agreement with the presence, at the early stages of mixing, of homo-aggregated NRL micelles embedding some SepB5 fibers (Figure 4c,d) while, in the final SepB5/NR biocomposite, of large domains constituted by aligned Sep fibers bridged by thin rubber layers (Figure 5b'). In SepS9/NR, both the colloidal systems possess a negative surface charge at the working pH (see Figure 2). Thus, flocculation cannot be explained simply referring to the \(\zeta\)-potential measurements. In fact, when a significant repulsive contribution to the inter-particle potential is present, experimental conditions other than surface charge should affect the flocculation process. In the case of SepS9/NR, flocculation was observed only in the presence of the high loading of Sep fibers, while did not occur for more diluted colloid systems. This suggests that high filler content and a relatively low fraction of NR play a key role in promoting flocculation. It is well known that attractions between colloidal particles can be conveniently induced by the addition of non-adsorbing polymers, due to the so-called depletion attraction [50; 51]. The driving force of this phenomenon lies in the existence of a \"depletion zone\", generated by the conformational entropic restrictions of the polymer coils which are not compensated by adsorption energy. According to these considerations, we can infer that in SepS9/NR system the filler loading is so high that NR is pushed out from the region among the fibers, leading to a higher concentration of the polymer in the colloidal solution (Scheme 1b, top). The difference in the osmotic pressure between these two regions results in the solvent flow out from the gap between Sep particles, inducing attractive forces which drive the flocculation. The hypothesis is supported by the fact that, at low SepS9 content flocculation does not take places, since the binary colloidal systems results sterically stabilized (Scheme 1b, bottom). These observations are in accordance with TEM investigation on Sep9/NR which, at the early stages of mixing, shows (Figure 4a,b) that Sep bundles lies among or in the periphery of the rubber micelles indicating their poor interactions with NRL while, after flocculation reveals (Figure 5a,a') the presence of poorly compact filler aggregates in the biocomposite. Moreover, SAXS analysis also supports the occurrence of weak interactions between the organic and inorganic components in Sep9/NR colloidal system, suggesting that, once in contact with the SepS9 fibers, the polymer chains unfold generating a homogeneous material. Finally, it has to be mentioned that the efficacy of depletion forces is generally enhanced in the presence of colloidal particles with high AR, because they provide extensive and effective confinement regions [52]. This seems to indicate a remarkable influence of Sep anisotropy on the flocculation process of Sep9/NR system.