Journal of Chromatography A, 1216 (2009) 5242–5248 Contents lists available at ScienceDirect Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma Determining the stoichiometry and binding constants of inclusion complexes formed between aromatic compounds and -cyclodextrin by solid-phase microextraction coupled to high-performance liquid chromatography Guillaume Chalumot, Cong Yao, Verónica Pino ∗ , Jared L. Anderson ∗∗ Department of Chemistry, The University of Toledo, Toledo, OH 43606, USA article info Article history: Received 23 March 2009 Received in revised form 28 April 2009 Accepted 8 May 2009 Available online 15 May 2009 Keywords: Cyclodextrins Stoichiometry Binding constants Solid-phase microextraction abstract The complexation of native -cyclodextrin (CD) and seven aromatic compounds, namely, phenetole, toluene, m-xylene, naphthalene, biphenyl, fluorene and phenanthrene, has been studied for first time uti- lizing a solid-phase microextraction (SPME)–high-performance liquid chromatography (HPLC) method. The stoichiometries of the analyte:-CD complexes were found to be either 1:1 or 1:2. The formation of 1:2 complexes was confirmed for naphthalene, biphenyl, fluorene, and phenanthrene only when uti- lizing relatively high concentrations of -CD (up to 6.6 mM). The 1:2 stoichiometries were confirmed using the classical modified Benesi–Hildebrand (BH) method. The calculated binding constants for 1:1 stoichiometries (K 1 ) using the SPME method varied from 115.3M -1 for toluene to 3510M -1 for phenan- threne, whereas the corresponding values to the 1:2 stoichiometries (K 3 ) varied from 7.30 × 10 5 M -2 for biphenyl to 9.03 × 10 6 M -2 for naphthalene. © 2009 Elsevier B.V. All rights reserved. 1. Introduction Native cyclodextrins (-, -, or -CDs) are cyclic oligosaccha- rides known to form inclusion complexes in aqueous solutions with a variety of polar and non-polar compounds including monoaro- matic and polyaromatic hydrocarbons via 1:1, 1:2 or even 2:2 stoichiometries [1,2]. The exact nature of the driving force of com- plexation of cyclodextrins with guest molecules is not known. It is a combination of CD-ring strain release upon complexation, geometrical compatibility, van der Waals forces, electrostatic, and hydrophobic interactions and, in some cases, hydrogen bonding between the cyclodextrin and the guest molecule [3]. The formation of these complexes can lead to an increase in the solubility of the solutes in the aqueous phase [4], as well as improved chemical and physical stability [5]. CDs have been widely applied in many areas such as food research [6], environment protection [1,7], and espe- cially pharmacology [8,9]. Due to their usefulness and applications, different studies have been performed to evaluate the stability binding constants and the stoichiometries of the complexes formed by CDs. ∗ Corresponding author. On leave from Department of Analytical Chemistry, Uni- versidad de La Laguna, Spain. Tel.: +34 922318012; fax: +34 922318090. ∗∗ Co-corresponding author. Tel.: +1 4195301508; fax: +1 4195304033. E-mail addresses: veropino@ull.es (V. Pino), Jared.Anderson@UToledo.edu (J.L. Anderson). Many separation-based and non-separation-based methods have been developed to determine binding constants [10], with some of them being recently reviewed [11]. High-performance liquid chromatography [12,13], affinity capillary electrophoresis [8,14,15], and electrospray ionization mass spectrometry [16,17], among others, have been applied to the determination of bind- ing to cyclodextrins, each of which possesses various advantages and shortcomings [11]. In addition to these methods, the modi- fied Benesi–Hildebrand (BH) method is a widely used approach for determining the stoichiometry and equilibrium constants of non- bonded interactions, particularly 1:1 and 1:2 interactions, with CD complexes [18]. Its wide applicability is justified by its facile com- bination with different techniques (UV–vis, fluorescence, infrared, NMR, etc.) [19,20]. Solid-phase microextraction (SPME) is a successful solvent-free extraction technique often used for the determination of a high number of volatile and semivolatile compounds [21–23]. It is a very convenient technique for studying chemical equilibria within a liq- uid matrix due to the small amount of analytes extracted by the SPME coating, which leaves the equilibrium virtually undisturbed [24,25]. In fact, SPME has been applied to the determination of the freely available concentration of different analytes in the presence of complex matrixes [26–29]. SPME combined with GC [30–32] and more recently with HPLC [33] has also been described as a simple and viable method to study the partitioning behavior of different analytes to micelles formed by traditional surfactants or by several ionic liquids. 0021-9673/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2009.05.017