**3. Analytical and physicochemical applications**

In the last years, CDs and their derivatives have been used in a variety of fields of analytical chemistry, especially in analytical separations [45, 58–63]. Spectral properties of CD and guest molecules can be altered due to the changes of the electrons distribution in the CD hole. CDs are used as reagent in different analyses such as UV-visible spectrophotometry, fluorescence [64, 65], phosphorescence [66, 67], and nuclear magnetic resonance methods [45, 68, 69].

The complexation of the analyte and/or the colored reagent can effectively change its properties. Among the most notable uses of this effect are: (i) enhancing the solubility of polar or non-polar analyte; (ii) enhancing the stability in polar or non-polar solution of reagents and colored complexes; (iii) increasing UV-visible absorption which improves the sensitivity of the colored reactions; and (iv) enhancing colored reactions selectivity. Luminescence techniques, in terms of fluorimetry and phosphorimetry, have reached a rapid development in routine analysis. However, many compounds luminesce very weakly in aqueous solution and the addition of CDs protects the excited (singlet or triplet) states of the possible dampers present in the solution since the rotation of the molecules is impeded due to the formation of the complex of inclusion with the result of a decrease in vibrational relaxation processes. The formation of inclusion complexes also increases the quantum fluorescence yield and hence the fluorescence intensities of numerous compounds. Sensitivity to certain characteristic reactions also increases.

CDs also increase the emission intensity of the chemiluminescent reactions. This improvement can be attributed to a number of factors, including an increase in the reaction rate and a greater efficiency in the process of excitation and protection of species that emit quenching phenomena. One of the most relevant applications of CDs is to allow the observation of phosphorescence at room temperature [67]. This is because they protect the excited triplet state of the molecules of the shock absorbers present in the solution, and in the case of molecular quenching phosphorescence. They are used as chiral reagents in NMR. In many cases, the formation of inclusion compounds modifies the general characteristics and chemical shifts of two enantiomers. Differences in the chemical shifts of two diastereoisomers can be used for the determination of the isomeric purity of the samples. The formation of inclusion complexes can very significantly modify the redox characteristics [13, 70, 71] of the included molecules. Voltammetric sensors capable of responding to anionic compounds have been developed. The changes produced after the complexation (selective interaction) allow the voltammetry to be used in the study of the complexation between CDs and organic molecules.

CDs increase the selectivity of chromatographic separations [72–74], because the separation process is more selective than that between the eluent and the stationary phase alone. In HPLC, the application of the CDs has achieved a spectacular success. Their incorporation into the mobile phase allows improving the separations, since they are soluble in water and provide reversible and selective complexation. In addition, they are stable and show no absorption in the UV-visible region of the electromagnetic spectrum. These characteristics mean that CDs are generally used in reverse phase separation processes, achieving the separation of isomers, diastereoisomers, and enantiomers [75–78]. The high resolution obtained is due to the differences in the stability constants of the complexes in the mobile phase and the different adsorption of these complexes in the stationary phase. CDs may also be incorporated as support for the stationary phases. Capillary electrophoresis has also found use in chiral analytical separations [79–82].
