**6.4. Ultraviolet/Visible spectroscopy**

The absorption properties of the host and guest molecules (such as dyes) can be easily altered by functionalization or inclusion complexation. When that happens, ultraviolet-visible spectroscopy confirms the successful complexation or functionalization by monitoring the band broadening or narrowing and/or bathochromic shift [110]. In fact, inclusion complexation can result in hypsochromic or bathochromic shift and/or increase or decrease in the intensity of the absorption maxima. However, this technique does not provide conclusive results on complexation or functionalization [116, 118].

#### **6.5. Fluorescence spectroscopy**

The environment of molecules can greatly influence their fluorescence properties; hence, fluorescence spectroscopy can be used to determine the geometry of complexation. Fluorescence quantum yield is high in complex formation and the maxima emission is often shifted to shorter wavelengths [110]. The enhancement of fluorescence in complexation is a result of shielding caused by quenching and nonradioactive decay processes [116]. This technique can only be used for fluorescent molecules.

### **6.6. Differential scanning calorimetry**

Differential scanning calorimetry (DSC) is an analytical technique based on thermal analysis of compounds. For physical and energetic properties, DSC is one of the most used techniques for CD complexation especially in CD-drug complexes. Endothermal dehydration peaks and decomposition peak are the main characteristics of CDs and are found at 90–130° C and 300°C, respectively. The appearance of a sharp enhanced endothermal peak indicates the formation of a host-guest complex, which is a sum of the individual compound peaks. Since physicochemical properties of guests can be changed during complexation, DSC can show the loss of guest crystallinity by broadening, size reduction and lower temperature shift of guest-melting peaks [116, 119]. However, guest-melting peaks may also indicate the presence of free guest molecules meaning that equilibrium has been reached [120]. In this case, chromatographic techniques can be used to separate the complex and free molecules.
