*3.2. NMR Characterization*

**η /** − Most of the properties of chitosan and its derivates, such as solubility and biodegradability, depend on the proportion between acetylated and non-acetylated glucosamine units, corresponding to degree of acetylation (DA), and eventually substitution degree (DS). NMR spectroscopy is one of the most accurate methods for determining the degree of acetylation for chitosan [38] and DS for Ch compounds [39].

Figure 3 shows the <sup>1</sup>H NMR spectrum of a Ch sample. The characteristic peaks are located at 3.32 and 3.40 ppm (black arrows Figure 3)—such signals represent the H2 of substituted deacetylate units (H2-NHR) and the hydrogens of the CH<sup>2</sup> group involved in the amide bond (-NH-CH2-), respectively. The H2 of unsubstituted deacetylate units (H2-GlcN) appears at about 3.21 ppm. The peaks in the anomeric region, A (5.00 ppm) and B (4.88 ppm), in Figure 3 are attributed to H1 of the substituted and unsubstituted deacetylate units, respectively. These peaks and the CH<sup>3</sup> signal of the acetyl group at 2.06 ppm (C, Figure 3) were used for the DA and DS calculation. In the anomeric region, the two sharp peaks were attributed to the anomeric protons of β-galactose side chains, at 4.57 ppm [17].

By assigning 300 to the integral of the signal related to the methyl group of acetyl (about 2.06 ppm), the percent N-acetylation (%*DA*) and substitution degree (%*DS*) were evaluated, using Equations (1) and (2).

$$\%DA = \frac{\frac{\zeta}{3}}{A + B + \frac{\zeta}{3}} \cdot 100\tag{1}$$

$$\%DS = \frac{A}{A+B} \cdot (100 - \%DA) \tag{2}$$

**Figure 3.** <sup>1</sup>H NMR spectrum of Ch in D2O at 343 K.

From the integral values it was seen that in the Ch sample, *DA* and *DS* were 7.5% and 59%, respectively.

β

 ൌ య ାା య ∙ 100 To confirm the peak attributions and to verify that the integrated signals involved in quantification corresponding to the unique function units, the <sup>1</sup>H-13C HSQC spectrum was acquired (Figure 4). The 2D spectrum confirmed that the anomeric and methyl peaks in the <sup>1</sup>H spectrum corresponded to one peak in the <sup>13</sup>C dimension, and that <sup>13</sup>C chemical shifts were consistent with the attributed signals of product [17].

Pulsed field gradient (PFG) diffusion ordered spectroscopy (DOSY) is the translational diffusion of dissolved molecules. In addition to the overall molecular size and shape, the diffusion coefficient magnitude provides direct information on molecular dynamics, including intermolecular interactions [40], aggregation, conformational changes [41,42], and viscosity [43]. DOSY processing is a particularly suitable technique for complex samples, since it provides a direct correlation of translational diffusion to the chemical shift in the second dimension. Therefore, a prior separation of complex components is not required [44,45]. A DOSY experiment is represented in a 2D spectrum, with chemical shift along one axis and the diffusion coefficients along the other [46,47].

In this study, DOSY was used to evaluate the interaction between HA and Ch in the complex. The outcomes of the DOSY analysis for all samples are visualized in a 2D map in Figure 5a.

**Figure 5.** (**a**) DOSY and (**b**) <sup>1</sup>H spectra of HA (purple), Ch (black), and complex (blue).

For the HA and Ch samples, there is only one population with the same diffusion coefficient, due to a unique molecular weight. For the HA/Ch complex, the results showed different diffusive fronts, due to different hydrodynamic radius of (5a) the HA resonances, and (5b) the resonances due to Ch moiety. The different chemical shift for 1, 2, and 3 signals were related to different small pH of solutions in the presence of HA. Usually, chemical shift of main chain protons, involved in the amino bond (H2) are highly sensitive to the protonation equilibrium [17]. The D values, shown in Table 3, were determined for the peaks corresponding to the components of the complex.

− − − −

− − − − − − − −

− −

− − − − − −

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**Table 3.** DOSY results of HA, Ch, and HA/Ch complex (D = H2O 2.25 × 10−<sup>9</sup> m2/s; D = TSP: 6.50 × 10−<sup>10</sup> m2/s).

The average D value of HA was lower than Ch, despite having similar Mw values. In this case, DOSY was particularly successful in distinguishing among different molar diffusivities, due to the different hydrodynamic radii, confirming the HP-SEC-TDA results. In the HA/Ch complex, the D values of the HA component remained almost constant, meanwhile the Ch diffusion coefficients increased, with values analogous to HA.
