Cyclodextrins (CDs) are cyclic oligosaccharides and exhibit a conical molecular structure with a hydrophobic cavity of fixed volume. Thus, a hydrophobic interaction of CDs with different guest molecules in aqueous solution is one of the primary forces involved in the formation of inclusion complexes between them. In addition, the size of the guest relative to that of the CD cavity is also a critical parameter.1-3 As a result of these selectivities afforded by inclusion, CDs have found numerous analytical applications in separation chemistry. Among them, the liquid-chromatographic separation of optical, geometrical and structural isomers using CDs as a stationary or a mobile-phase component has been extensively studied.4-8 When CD is used as a mobile-phase additive in reversed-phase HPLC (RPLC), it has been found that the retention time of a solute usually becomes shorter with increasing concentration of CD in the eluent, and that the change in the capacity factors is closely related to the formation constant of the corresponding inclusion complex. Furthermore, it is known that the capacity factor of a solute is also affected by the type and content of organic modifier in the eluent. Thus, to optimize the eluting conditions and to enhance the selectivity due to the formation of an inclusion complex, it will be essential to obtain a quantitative knowledge concerning the formation reaction of inclusion complexes with special reference to the effect of organic solvents.Flavonols and coumarins have found extensive use as a fluorescent probe for the determination of trace amounts of metal ions in our laboratory. [9][10][11][12][13][14][15][16][17][18][19] Since an understanding of the detailed picture of the inclusion reaction of these fluorophores with CD may contribute to an improvement in the sensitivity and/or selectivity of these probes in the determination of metal ions by the addition of CD, we studied the kinetics and thermodynamics of the inclusion reaction of flavonols with β-CD in a 30%v/v methanolic aqueous solution. 20 In this study, it was found that the fluorescent characteristics of flavonols are largely affected not only by the inclusion complexation with β-CD, but also by the type and content of organic solvents mixed with water.In the present work, we studied the inclusion equilibria of flavonol, 7-methoxyflavonol and 4-phenyl-7-hydroxycoumarin with β-CD to elucidate how the equilibria will be affected by the content of methanol, ethanol, acetonitrile, acetone, dimethyl sulfoxide (DMSO) and N,N-dimethylformamide (DMF). Then, by employing aqueous methanol as a mobile phase and β -CD as a mobile-phase component, we studied the retention behavior of coumarins on a RPLC column to examine how the change in the capacity factors can be described by the inclusion complexation in the mobile phase. Experimental ReagentsThe flavonols and coumarins listed in Tables 2 and 3 were synthesized as described previously [9][10][11][12][13][14][15][16][17][18][19] and were dissolved in a given organic solvent. β -CD wa...