**6.7. Chromatography**

**6.3. Infrared spectroscopy**

156 Cyclodextrin - A Versatile Ingredient

bonds [116, 117].

**6.4. Ultraviolet/Visible spectroscopy**

plexation or functionalization [116, 118].

only be used for fluorescent molecules.

**6.6. Differential scanning calorimetry**

**6.5. Fluorescence spectroscopy**

Infrared spectroscopic techniques such as the Fourier transform infrared (FTIR) spectroscopy can be used to reveal CD functionalization and inclusion complexation. This can be validated by the appearance of new peaks, shift in peak position or change in peak intensity as a result of changes on pure CD molecules. Noticeable changes may include disappearance of the ─OH peak for functionalization with the appearance of new peaks depending on the type of functional groups introduced. For inclusion complexation, ─CO stretching peaks may be observed [110]. Vibrational modes of the host and guest can be studied using this technique to understand the process of complexation and/or functionalization. Vibrational modes can be restricted to a certain level during complexation and this can result in weak interatomic bonds due to the altered environment around the

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 com-

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

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.

Chromatographic analysis such as thin layer chromatography (TLC) can be very useful for the verification of complexation and modification by monitoring the alterations of the retardation factor Rf values. The complex of modified CDs is found between the Rf values of the CDs and that of the functional group or guest [110, 121]. Another way to study CDs and their complexes using chromatography is by monitoring their volatility using head-space chromatography. This chromatographic technique is specifically for volatile compounds. The increase or decrease in volatility can be observed as influenced by the host-guest interaction and the stability of the complex can be determined [110, 122].

#### **6.8. Microscopic techniques**

Microscopic techniques such as SEM and TEM are used as complementary techniques to analyze the surface morphology, topography and composition of various samples including nanofibers and membranes containing CD species. These two techniques give critical details on the size, size distribution and alignment of fibers as well as the nature of the nanofiber or membrane surfaces. These microscopic techniques are mostly used in the analytic investigation of nanofibers and membranes because of their capability of imaging at high resolutions [11, 43, 49, 69, 73, 77].

#### **6.9. Other characterization techniques**

Other popular techniques often used to study CDs, their derivatives and nanocomposites are thermogravimetric analysis to probe their thermal stability, circular dichroism spectroscopy to study inclusion complexation of ideally sized molecules in the CD cavity, contact angle analysis to understand the hydrophilicity of surfaces, nanosizer instruments for surface charge and Brunauer-Emmett-Teller (BET) to measure the surface area and pore volume.
