2.2.2. Powder X-ray Diffraction

Powder X-ray diffraction (PXRD) is a very useful tool in identifying the formation of inclusion complexes. It also contributes to investigate the presence of any eventual impurities that may appear, such as crystallites of the host or the guest. PXRD data is presented in Figure 4. Efavirenz presents a diffractogram with several well-resolved peaks that are indicative of its high crystallinity. The most intense reflections occur at 6.3, 10.5, 11.4, 11.9, 12.6, 19.3, 20.0, 20.5, 20.8, 21.2, 21.6, 23.0, 24.7, 25.0 and 27.6 degrees of 2θ.

A first attempt at collecting diffractograms of the two freeze-dried products revealed them to be mostly amorphous (results not shown), which was expected as a result of the preparation method. Restoration of the hydration waters in these samples was performed as a means to increase their crystallinity. The process consisted in placing the bulk materials at ambient temperature in a water-saturated atmosphere during ca. 16 h. PXRD patterns of the rehydrated freeze-dried compounds present overall quasi-similar diffraction patterns and they comprise essentially a new phase, with no traces of crystallites of γ-CD heptahydrate. The diffractogram of the sample with 3:2 stoichiometry exhibits reflections peaking at 5.3, 6.1, 7.5, 8.6, 10.7, 11.5, 12.3, 13.7, 14.2, 15.0, 15.7, 16.1, 16.6, 16.9, 18.9, 20.1, 20.9, 21.6, 22.2 and 22.6 degrees of 2θ. For the compound prepared with a starting stoichiometry of 1:1 (sample FD 1:1 in the Figure 4), the diffractogram is less well resolved, with the main reflections presenting some broadening and peaking at 7.6, 11.5, 11.9, 12.3, 13.7, 15.0, 16.0 and 16.9 degrees of 2θ. It should be noted that the peak centred around 11.9 degrees in the latter sample, albeit poorly resolved, coincides with the most intense reflection of pure efavirenz, thus indicating some degree of contamination with non-included efavirenz (similarly to the observations made from FT-IR data).

Figure 4 also shows the calculated diffractogram of γ-CD·12-crown-4-ether [24], which is herein used as a representative model for the only known isostructural series of γ-CD inclusion complexes [25]. The diffractogram of the 3:2 freeze-dried sample shows an overall diffraction envelope that is similar to that of the model complex, which suggests it belongs to this isostructural series [25]. It is, thus, fair to assume the inclusion complex of γ-CD and EFV should present the host molecules stacked in infinite channels, similarly to the host

organisation reported for the complexes of this isostructural series and herein exemplified for γ-CD·12-crown-4-ether (inset in Figure 4).

**Figure 4.** Experimental powder X-ray diffractograms of γ-CD, efavirenz (EFV), and the freeze-dried samples (FD) with γ-CD:EFV stoichiometries of 1:1 and 3:2 (rehydrated prior to data collection to increase their crystallinity). For comparison it also shows the trace of the inclusion complex γ-CD·12-crown-4-ether [24], calculated from its atomic coordinates using Mercury 3.5.1 (Copyright CCDC 2001–2014). The inset depicts the structure of γ-CD·12-crown-4-ether, as viewed from the top (crystallographic *c* axis) and from the side (*a* axis); the molecules of the crown ether guest are represented in purple for differentiation from those of the γ-CD macrocycle.
