*2.2. Molecular Weight Distribution by HP-SEC-TDA*

HP-SEC-TDA was used extensively to obtain molecular weight distribution of HA, Ch, and the complex. This method does not require any chromatographic calibration and is considered to be suitable to analyze polysaccharides. Measurements for molecular weight distribution were performed on a Viscotek 305 HPLC system (Malvern Instruments LtD, Malvern, UK). The array exploits simultaneous action of refraction index detector (RI), viscometer, and Right Angle Laser Light-scattering (RALS) detector, using a method adapted from Bertini et al. [35]. To prepare the solutions for HP-SEC-TDA measurements, 10–20 mg was dissolved in a mobile phase volume, to obtain a sample concentration of ~3 mg/mL. The Ch solution was stirred for 3 h and then diluted in a mobile phase to a concentration of ~1 mg/mL. The HA solution was stirred overnight and then diluted in the mobile phase to a concentration of ~0.5 mg/mL, respectively. For the HA/Ch complex, 75 mg of Ch were dissolved in 10 mL of phosphate-buffer saline solution pH 6.9 (PBS), and stirred at room temperature to obtain a completely solubilized solution. Afterwards, 125 mg of HA were added to the solution and the complex was stirred for 12 h. Prior to the analysis of the solution, the HA/Ch complex was diluted in a mobile phase to reach 1 mg/mL sample concentration. The analyses were performed at 40 ◦C,

using 2 x TSKPWXL columns in series (Tosoh Bioscience, 7 mm 7.8 × 30 cm). A total of 0.1 M NaNO<sup>3</sup> for HA, 0.2 M NaNO<sup>3</sup> for Ch, and an HA/Ch complex, both containing 0.05% NaN3, prefiltered using 0.22 mm filter, were used at a flow rate of 0.6 mL/min. Chromatographic profiles were elaborated using the OmniSEC software version 4.6.2. RI increments, referred to as *dn*/*dc*, were determined to enable conversion of RI values into a concentration for Ch and HA/Ch [36]. *dn*/*dc* values equal to 0.129 mL/g and 0.119 mL/g were observed for Ch and HA/Ch, respectively. For HA, the *dn*/*dc* value of 0.155 was taken from the literature [37].
