3.1.2. Thermal and Structural Characterization

VT-XRPD (Variable temperature—X-ray powder diffraction) experiment was carried out on **TS3w**. The temperature was slowly increased from to 25 ◦C to 70 ◦C–120 ◦C–155 ◦C–190 ◦C as shown in Figure 3. Once water is removed (120 ◦C) the structure does not collapse and does not lead to the anhydrous form **TSan** either. It dissociates into the starting components. A new diffraction line appears at high temperature (155 ◦C–190 ◦C) belonging to a different theophylline polymorph (elusive form V) [32]. However, at the end of the experiment theophylline can be easily detected and no diffraction lines belonging to **TS3w** pattern is observed.

**Figure 3.** VT-XRPD of Theophylline Squarate trihydrate starting from 25 ◦C to 190 ◦C. Above the reference Theophylline.

Thermogravimetric analysis on **TS3w** (See Supplementary Materials, Figure S2) shows a two steps weight loss while increasing the temperature. The first step might represent the channel-water molecule O3W, and the second step might be interpreted as the other two molecules leaving the system, according to the weight changes (5.7% and 10.6%, respectively). DSC thermogram (See Supplementary Materials, Figure S1) shows two endo peaks at 68.9 ◦C and 89.6 ◦C, associated with the two events observed in the TGA. VT-XRPD experiment confirms that the structure remains intact at 70 ◦C. As described above, the salt structure is disrupted only after all of the water molecules have abandoned the HB network (>70 ◦C), which links Theophylline and Squarate together. The following exothermic event around 138 ◦C could be interpreted as the rearrangement of the molecules into the two pure components, see Figure 3. **TS3w** melting at 227.7 ◦C corresponds to the melting point of the anhydrous form (See DSC thermogram of **TSan** in the Supplementary Materials, Figure S5) and it suggests a previous crystallization of **TSan**, potentially happening where the second exothermic peak (170.5 ◦C) can be detected.

A VH-XRPD (Variable Humidity—X-ray powder diffraction) experiment was also carried out. Theophylline and Squaric Acid (molar ratio 1:1) powders were mixed and exposed to 95% RH for 3 days (Figure 4).

**Figure 4.** VH-XRPD of Theophylline (T) and Squaric Acid (SA). Below the reference Theo-phylline Monohydrate and above the reference Theophylline squarate trihydrate (**TS3w**).

This experiment confirmed the formation of the salts from the two components in the humidity chamber via a solid-state process without the need of dissolving the components in bulk solvents or applying energy from the milling processes. This observation is complementary to what was observed during the VT-XRPD experiment.

Furthermore, along with the formation of the salts, a steep increase in theophylline monohydrate was observed. After a few days though its signal started to drop while the peak of **TS3w** rose. It can thus be tentatively proposed that the formation of the theophylline monohydrate is a necessary step in the salt formation: as observed in VH-XRPD of pure Theophylline (See Supporting Materials, Figure S9) the monohydrate process starts in less than four hours and is completed in 11 h. The experiment never allowed us to see the complete conversion into the pure theophylline salt. That is probably due to the impossibility of mixing the powder during the experiment being the sample laid on a zero-background XRPD sample stage.

The salt formation has been confirmed also by the FTIR analysis showing the protonated nitrogen signal (See Supplementary Materials, Figure S7).

DVS shows that **TS3w** (See Supplementary Materials, Figure S3) might be considered a stable form since after a cycle of increasing and decreasing humidity, XRPD shows again the same pattern without any loss of crystallinity in the powder (See Supplementary Materials, Figure S4). That proved once again that the anhydrous form could be considered a metastable form.

Figure 5 shows the different powder patterns of commercial Theophylline, Squaric acid, Theophylline squarate trihydrate (**TS3w**), and Theophylline Squarate anhydrous (**TSan**). Thermogravimetric analysis on **TSan** does not show any significant weight loss (See Supplementary Materials, Figures S5 and S6).

**Figure 5.** Powder X-ray diffraction of Theophylline (**a**), Squaric acid (**b**), Theophylline Squarate anhydrous (**TSan**, (**c**)) and Theophylline squarate trihydrate (**TS3w**, (**d**)).
