*3.3. Rheological Properties*

Rheology as reported by Ambrosio et al. [48], is a useful tool to explore the relationships between the mechanical behavior and chemical properties of HA, or other biopolymer solutions. In our study, the flow behavior and viscoelastic measurement were evaluated to understand the difference in the rheological properties of HA and Ch solutions with their complex.

The variation of the viscosity as a function of the shear rate were acquired; in Figure 6a the viscosity curves, with a shear rate (γ) ranging from 0.1 s−<sup>1</sup> to 100 s−<sup>1</sup> , are reported.

<sup>−</sup> γ γ **Figure 6.** (**a**) Viscosity curves of the Ch, HA, and HA/Ch complex. Due to low viscosity, data for Ch were only acquired in the range (1–100) s−<sup>1</sup> (**b**). Frequency sweep test of the Ch (γ = 20%), HA, and the HA/Ch complex (γ = 2%).

HA and Ch (black and purple curves in Figure 6a, respectively) showed different behaviours. Ch viscosity remained constant at a value of 8.8 mPa s over the entire tested shear rate range, thus showing ideal viscous flow behaviour. Such behaviour was mainly related to the sample concentration and substitution degree of chitosan—according to previous results [49]. The pseudoplasticity was inversely proportional to DS and directly correlated to the concentration of sample. For HA, viscosity was shear-dependent, viscosity decreased as γ increased, in agreement with a pseudoplastic behaviour [48]. For the complex, shear-thinning behaviour was also observed. Note that the viscosity values were higher than that for pure HA.

The rheological tests were also performed in the oscillation mode, to evaluate both the storage modulus (G') and the loss modulus (G"). The strain sweep test (data not shown) was initially performed to evaluate the LVE zone, where the intrinsic sample structural properties are independent of the applied strain:2% (HA and Complex) and 20% (Ch) strain value was selected for subsequent frequency sweeps tests. In Figure 6b, values of G' and G" as function of the angular frequency for the Ch, HA, and the complex are reported. In the investigated frequency range (from 0.628 rad/s to 628 rad/s), the mechanical spectrum of Ch showed that G" was greater than G', with both moduli strongly dependent on the frequency, as typically a liquid-like (viscous) behavior dominates over the solid-like (elastic) character. Differently, HA and HA/Ch presented a "weak gel" or viscoelastic behavior with G' and G", which became less dependent on the frequency and the crossover point, when G' = G", was observed. This suggests that at high frequencies, when G' > G", the material show a predominant solid-like behavior.

The angular frequency values, ω**c**, and the corresponding G' = G" values to the crossover point data are reported in Table 4.


**Table 4.** Rheological measurements—evaluation of G' and G" crossover.

<sup>1</sup> value not detected in the frequency range investigated.
