*3.1. Molecular Weight Distribution*

Chromatographic profiles of HA and Ch are shown in Figure 1a,b, respectively. The samples had an elution time between 10 and 15 mL, with a large a-symmetric bell-shape chromatographic peak, caused by a high polydisperse index.

Values of weight-average molecular weight (Mw), number-average molecular weight (Mn), and molecular-weight dispersity (Ð; Mw/Mn) are reported in Table 1. In addition, values of the intrinsic viscosity value (η), hydrodynamic radius (Rh), and Recovery %, derived from TDA, are also reported. To estimate the Recovery %, the OmniSEC software computes actual concentration in each chromatographic slice, based on the *dn*/*dc* value, and a detector constant for the RI. The values of *a* and *log k*, corresponding to the slope and intercept constants of the Mark-Houwink curve, respectively, are also reported. The results refer to the mean values of duplicate injections.

For the HA samples, a recovery value of 90% indicated that no material remained adsorbed in the columns, considering that the water content in the sample was about 10% *w*/*w*; for Ch, the values were close to 100%, due to the low water content in the sample. The Ð value for HA was lower than that for Ch, indicating low molecular weight

dispersion. However, the values were relatively high, indicating the presence of chains of different length.

**Figure 1.** Chromatographic profile (red—refractive index; black—low laser light scattering; green—right angle light scattering; blue—viscometer). (**a**) HA, and (**b**) Ch.



The HA/Ch complex (0.75:1.25 *w*/*w*) molecular weight was also determined by the same technique—results are visualized in Figure 2 As expected, due to very similar molecular weight, the two polysaccharides were eluted together, so it is important to evaluate the *dn*/*dc* of the complex. From analysis on several dilutions of a HA/Ch complex, the refractive index increment (*dn*/*dc*) was determined as 0.118, through a linear regression.

**η** − − Using 0.118 as *dn*/*dc*, the molecular weight analysis results are reported in Table 2. Molecular weight, dispersity, and hydrodynamic radius of the complex (Table 2) remained quite similar to those of the original polymers (Table 1). Generally, through gel permeation chromatography analysis, the average molecular weight of polymers increased due to cross-linked reactions or derivatizations. In the HA/Ch complex, only weak interactions

between the two polysaccharides (hydrogen bonds and Van der Waals forces) are present, which are influenced by high salt concentration of mobile phase.

**Figure 2.** Chromatographic profile (red—refractive index; black—low laser light scattering; green—right angle light scattering; blue—viscometer) of the HA/Ch complex.

**Table 2.** HP-SEC-TDA HA/Ch complex results.

