*2.7. NMR Spectroscopy Measurements*

<sup>1</sup>H NMR were performed on Bruker Avance 300 MHz and Bruker Avance 400 MHz instruments. <sup>1</sup>H Diffusion ordered NMR (DOSY) experiments were run on Bruker Avance 700 MHz. Monodimensional NMR spectra were achieved by single-pulse NMR using the solvent suppression only in the case of samples in water solution by using a 90degree pulse and a repetition time as to allow the magnetization to relax completely. The spectra were registered in 8K data point and then transformed in 16K data point. Deuterated solvent for lock purpose was purchased from Sigma Aldrich (Milano, Italy): DMSO-d6, 99.9% D, or D2O 99.9 atom % D. To estimate possible solvent effects on the spectrum of the sample other samples have been prepared in DMSO-d6–D2O mixture (DMSO-d6:D2O = 9:1) or (DMSO-d6:D2O = 8:2) containing all 7.5 mg of sample.

The characteristic resonances of PLA and HA sodium salt were identified: for PLA 3H δ 1.4–1.6 CH3, 1H δ 5.0 CH and for HA sodium salt 3H δ 1.8 CH3(CO-NH); 2H δ 4.5–4.62.

In a diffusion ordered spectroscopy (DOSY) experiment, a series of NMR spectra is normally acquired in a spin or stimulated echo as a function of pulsed-field gradient amplitude, with the amplitude of each signal decaying at a rate determined by the diffusion coefficient. The ideal behavior for unrestricted diffusion is described by the Stejskal–Tanner equation [25].

The diffusion ordered spectroscopy (DOSY) experiments were performed by using the ledbpgppr2s pulse sequence of the Bruker library also in order to suppress the water signal at 4.7 ppm, when necessary. During the DOSY experiment 32 mono dimensional spectra were acquired with 64 scans in a linear increasing gradient varying from 5% to 95% with a pulsed gradient time δ of 70 ms and a diffusion time ∆ of 2 ms. The spectra were then analyzed using the DOSY module implemented in Bruker software TOPSPIN 3.1 (Bruker Italy, Milano, Italy).
