## 3 Results and Discussion
### Characterization of the Phase Transition
\(T_{\text{onset}}\) were generally lower than the cloud point temperatures at 50% transmittance recorded by turbidimetry, especially for pEFS8-46 solutions at lower concentrations, which presented a wider transition (Figure 3b). As for other thermosensitive polymers [49,50], such a difference might be ascribed to the greater sensitivity of the calorimetric technique in detecting the beginning of the phase transition, when the fraction of multichain aggregates is not yet large enough to be detected by turbidimetry. \begin{table}
\begin{tabular}{c c c c c c c} \hline \hline  & \multicolumn{4}{c}{**First Heating Scan**} & \multicolumn{4}{c}{**Second Heating Scan**} \\ \hline  & **Concentration** & \(T_{\text{onset}}\)1  & \(T_{\text{onset}}\)2  \\  & **(g/L)** & **(\({}^{\circ}\)C)** & **(\({}^{\circ}\)C)** & **(KJ/mol)3 ** & **(\({}^{\circ}\)C)** & **(\({}^{\circ}\)C)** \\ \hline pEFS8-26 & 0.5 & 71 & 75.2 & 7.6 & 72.5 & 69.5 & 74.4 \\  & 1 & 70.4 & 73.7 & 7.2 & 71 & 68.4 & 72.2 \\  & 5 & 68.7 & 71.7 & 4.5 & 70 & 65.3 & 70.2 \\ pEFS8-46 & 0.5 & 75.6 & 80.2 & 6.8 & 83 & 75 & 79.8 \\  & 1 & 74.8 & 79.1 & 5.5 & 78 & 73.2 & 78.3 \\  & 5 & 71.8 & 76.8 & / & 73 & 70.7 & 75.2 \\ \hline \hline \end{tabular}
\end{table} 
Table 3: Calorimetric data for pEFS8-x polymers. Figure 4: **Nano-Differential scanning calorimetry** (DSC) thermal profiles of pEFS8-26 aqueous solutions at concentrations of (**a**) 0.5 g/L, (**b**) 1 g/L, and (**c**) 5 g/L. Both \(T_{\text{onset}}\) and \(T_{\text{max}}\) characteristics of the phase transition increased by increasing the length of the polymer (namely, going from pEFS8-26 to pEFS8-46), suggesting that the higher number of repeating units conferred a higher degree of hydrophilicity to the entire polymer system. The dependence of aggregation temperature on the polymer molecular weight was reported in the literature for several thermoresponsive polymers, e.g., PNIPAM [51] or polyoxazolines [52], whereas others, such as POEGMA [53], do not possess such a dependence. At the end of the thermal scans (90 \({}^{\circ}\)C), none of the curves reached a baseline, but the heat capacity tended to lower values with respect to the initial one (\(\Delta\text{C}_{\text{p}}<0\)). The heat capacity change was indicative of the solvent-exposed portions of samples, taking into account their polarity [54]. \(\Delta\text{C}_{\text{p}}\) values lower than 0 were found in literature for the denaturation process of proteins in aqueous solutions and were related to the inter-chain aggregation of proteins in the unfolded form [44; 45; 46; 47]. In analogy with proteins, we attributed the reduction in the heat capacity of pEFS8-x homopolymers to the formation of multichain aggregates, driven by the minimization of the hydrophobic surface in contact with water. An approximate value of the heat quantity exchanged during the process was calculated by measuring the area underlying the peak. The data is reported in Table 3. We observed that the area calculated in this way was in the range of 5-8 kJ per mole of monomeric unit. This value was similar to that reported for PNIPAM (\(\Delta H=5.5\)-7.5 kJ/mol) [22; 24; 50] and slightly higher than that reported for PVCL (4.2-4.8 kJ/mol) [43]. As the enthalpy of an LCST phase transition can be used to estimate the number of hydrogen bonds that are broken per monomeric unit, as well as the amount of water retained by the resulting aggregates [17], it is likely that these phenomena are similar in scale for pEFS8 and PNIPAM. Moreover, it was found that, for both polymers, the peak area decreased by increasing the polymer concentration in water, related to the enhancing of aggregation processes, as previously discussed. The second heating scans had a similar shape as the first ones, but \(T_{\text{onset}}\) and \(T_{\text{max}}\) were 2-3 \({}^{\circ}\)C earlier (Table 3), denoting the non-reversibility of the process in the experimental conditions.