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   Journal of Chromatography A, 1216 (2009) 5242–5248 Contents lists available at ScienceDirect  Journal of Chromatography A  journal homepage: Determining the stoichiometry and binding constants of inclusion complexesformed between aromatic compounds and  -cyclodextrin by solid-phasemicroextraction 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 a r t i c l e i n f o  Article history: Received 23 March 2009Received in revised form 28 April 2009Accepted 8 May 2009Available online 15 May 2009 Keywords: CyclodextrinsStoichiometryBinding constantsSolid-phase microextraction a b s t r a c t The complexation of native   -cyclodextrin (CD) and seven aromatic compounds, namely, phenetole,toluene,m-xylene,naphthalene,biphenyl,fluoreneandphenanthrene,hasbeenstudiedforfirsttimeuti-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 formationof 1:2 complexes was confirmed for naphthalene, biphenyl, fluorene, and phenanthrene only when uti-lizing relatively high concentrations of    -CD (up to 6.6mM). The 1:2 stoichiometries were confirmedusing the classical modified Benesi–Hildebrand (BH) method. The calculated binding constants for 1:1stoichiometries ( 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 forbiphenyl 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-ridesknowntoforminclusioncomplexesinaqueoussolutionswitha variety of polar and non-polar compounds including monoaro-matic and polyaromatic hydrocarbons via 1:1, 1:2 or even 2:2stoichiometries [1,2]. The exact nature of the driving force of com- plexation of cyclodextrins with guest molecules is not known. Itis a combination of CD-ring strain release upon complexation,geometrical compatibility, van der Waals forces, electrostatic, andhydrophobic interactions and, in some cases, hydrogen bondingbetweenthecyclodextrinandtheguestmolecule[3].Theformation of these complexes can lead to an increase in the solubility of thesolutesintheaqueousphase[4],aswellasimprovedchemicaland physical stability [5]. CDs have been widely applied in many areas such as food research [6], environment protection [1,7], and espe- ciallypharmacology[8,9].Duetotheirusefulnessandapplications, different studies have been performed to evaluate the stabilitybindingconstantsandthestoichiometriesofthecomplexesformedby CDs. ∗ Correspondingauthor.OnleavefromDepartmentofAnalyticalChemistry,Uni-versidad de La Laguna, Spain. Tel.: +34 922318012; fax: +34 922318090. ∗∗ Co-corresponding author. Tel.: +1 4195301508; fax: +1 4195304033. E-mail addresses: (V. Pino), (J.L. Anderson). Many separation-based and non-separation-based methodshave 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 advantagesand shortcomings [11]. In addition to these methods, the modi- fiedBenesi–Hildebrand(BH)methodisawidelyusedapproachfordetermining the stoichiometry and equilibrium constants of non-bonded interactions, particularly 1:1 and 1:2 interactions, with CDcomplexes [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-freeextraction technique often used for the determination of a highnumberofvolatileandsemivolatilecompounds[21–23].Itisavery convenienttechniqueforstudyingchemicalequilibriawithinaliq-uid matrix due to the small amount of analytes extracted by theSPME coating, which leaves the equilibrium virtually undisturbed[24,25]. In fact, SPME has been applied to the determination of thefreely available concentration of different analytes in the presenceofcomplexmatrixes[26–29].SPMEcombinedwithGC[30–32]and more recently with HPLC [33] has also been described as a simple and viable method to study the partitioning behavior of differentanalytes to micelles formed by traditional surfactants or by severalionic liquids. 0021-9673/$ – see front matter © 2009 Elsevier B.V. All rights reserved.doi:10.1016/j.chroma.2009.05.017  G. Chalumot et al. / J. Chromatogr. A 1216 (2009) 5242–5248  5243 The purpose of this study is to extend, for the first time,the use of SPME combined with HPLC to the determinationof the stoichiometry and binding constants of the complexesformed with native   -cyclodextrins. The analytes selected inthis study include single aromatic and polyaromatic hydrocar-bons and are evaluated in their possible 1:1 and 1:2 bindingto   -cyclodextrins using the SPME–HPLC approach. The BHmethod was used to confirm the stoichiometry of the complexesformed. 2. Experimental  2.1. Reagents AllexperimentsusedHPLC-gradeacetonitrilesuppliedbyFisherScientific (Fair Lawn, NJ, USA), and deionized water produced bya Milli-Q water purification system (Millipore, Bedford, MA, USA)with a resistivity of 18.2M  cm.The analytes examined in this study were: toluene, sup-plied by Fisher Scientific; m-xylene, supplied by Fluka (St Gallen,Switzerland); phenetole and biphenyl, supplied by Aldrich (Mil-waukee, WI, USA); and naphthalene, fluorene and phenanthrene,supplied by Supelco (Bellefonte, PA, USA). Stock solutions wereprepared by dissolving these compounds in acetonitrile andstored at 4 ◦ C in the dark. The cyclodextrin stock solutions wereprepared by dissolving 1.86g of native   -cyclodextrin suppliedby Fluka in 250mL of deionized water. The stock solutionsof analytes and cyclodextrin were used to prepare workingstandard solutions, maintaining the acetonitrile content at 2%(v/v).  2.2. Instrumentation ThechromatographusedwasaLC-20AliquidchromatographbyShimadzu (Kyoto, Japan) equipped with a DGU-20A 3  degasser, twoLC-20ATpumpsandaSPD-20UV–visdetectoroperatingat254nm.It was connected to a SPME–HPLC interface unit from Supelco andto a C 18  column (250mm × 4.6mm I.D., 5  m particle size) fromAlltech (Deerfield, IL, USA). The separation gradient employed inthis study started with 60% (v/v) acetonitrile, which was linearlyincreased up to 70% (v/v) over 15min, and then kept isocratic for40min, at a flow rate of 1.00mLmin − 1 .The fiber used was a 60  m polydimethylsilox-ane/divinylbenzene (PDMS/DVB) SPME fiber supplied by Supelco.The fiber was conditioned by exposing it in the SPME–HPLCinterface while passing through acetonitrile for 30min (dynamicmode), according to the instructions given by the manufacturer.A fiber holder (Supelco) for manual sampling was used for SPMEanalysis. Extractions were performed in 20mL amber glass vialssupplied by Supelco. The vials were closed by screw caps andPTFE/Butyl septa (Supelco). In all extractions, the vials werecompletely filled with the working solution to leave no headspace.Agitation was achieved by PTFE-coated stir bars and a BarnsteadInternational Super-Nuova stirring plate (Dubuque, IA, USA) at themaximum stirring rate (1200rpm). Due to the fact that sorption of hydrocarbons can take place on PTFE stir bars, they were carefullyrinsedaftereachextractionwithacetone,thenmethanol,thenace-tonitrile and finally with deionized water to avoid memory effects.The stir bars were sonicated daily for 10min with acetonitrile.Fluorescence and UV–vis absorbance measurements wereperformed using a Thermo Electron Corporation AmincoBowman II luminescence spectrometer (Waltham, MA, USA),which uses 1cm quartz cell, and a Hewlett-Packard 8452Adiode array spectrophotometer (Palo Alto, CA, USA), respec-tively.  2.3. Procedures 2.3.1. SPME–HPLC studies In order to determine the stoichiometry and binding constantsof each analyte with  -cyclodextrin, direct-immersion extractionswerecarriedoutin20mLoftheworkingsolutionswithafixedcon-centration of 2.0% (v/v) in acetonitrile, 62.8  gmL  − 1 in phenetole,41.2  gmL  − 1 in toluene, 14.5  gmL  − 1 in m-xylene, 5.70  gmL  − 1 innaphthalene,1.03  gmL  − 1 inbiphenyl,0.74  gmL  − 1 influorene,0.71  gmL  − 1 inphenanthrene,andaconcentrationincyclodextrinvarying from 0 to 6.6mM, depending on the specific experiment.The extraction time of the SPME fiber was fixed at 120min.Following each extraction, the fiber was desorbed for 20min inthe desorption chamber of the SPME interface with the six-portinjection valve in the “load” position. The chamber was previouslyfilled with pure acetonitrile, allowing a static desorption to takeplace.Followingcompletedesorption,thevalvewasswitchedtothe“injection”position,allowingthemobilephasetopassthroughthechamberfor5mintherebytransportingtheanalytestothecolumn.Following each analysis, the fiber was soaked in 20mL of acetoni-trile for 20min under agitation to ensure no obvious carryoverbetween extractions.SPME calibration plots in deionized water were obtainedusing the same extraction conditions but without addition of    -cyclodextrin, and with a varying concentration of analytes.  2.3.2. Fluorescence measurements To perform the fluorescence experiments for the BH method,individual working solutions were prepared for fluorene, naph-thalene and biphenyl using the same concentration of analytes,  -cyclodextrin, and acetonitrile (2%, v/v) as for SPME studies. Allsolutions were allowed to reach equilibrium for over 8h, and pro-tected from light in sealed vials. Fluorescence measurements wereperformed four times from each solution to ensure repeatability,and carried out at room temperature. The instrumental measure-ments were carried out using a slit width and step size of 2nmand 1nm, respectively. The emission wavelengths for the variousanalytes were 314nm for biphenyl, 321nm for naphthalene, and302nm for fluorene, all corresponding to the maximum in theemission spectra. The excitation wavelength was 254nm for allhydrocarbons. The AB2 Luminescence Spectrometer software 5.50by Thermo Electron Corporation was used.  2.3.3. UV–vis absorbance measurements To perform the absorbance measurements for the BH method,working solutions were prepared using the same concentrationof phenanthrene,   -cyclodextrin, and acetonitrile as for the SPMEstudies. All working solutions were allowed to reach equilibriumfor over 8h, and protected from the light. The absorbance of eachsolution was then measured in the diode array spectrophotome-ter at a wavelength of 254nm. All experiments were carried out atroom temperature in quadruplicate. 3. Results and discussion  3.1. Study of the binding behavior using solid-phasemicroextraction with HPLC  The decrease of the free hydrocarbon concentration in an aque-ouscyclodextrinsolutionwhenincreasingthecyclodextrincontentis due to the formation of a hydrocarbon:cyclodextrin complex.When introducing a SPME fiber into an aqueous CD solutioncontaining hydrocarbons, it is assumed that the hydrocarbonsextracted by the SPME coating srcinate from the aqueous phasewhereas the bound hydrocarbon remains as a complex in solution[30,33].  5244  G. Chalumot et al. / J. Chromatogr. A 1216 (2009) 5242–5248 Fig. 1.  Decrease in the extraction efficiencies (expressed as peak-areas) of theSPME–HPLC method for toluene, biphenyl and phenanthrene when increasing the  -cyclodextrin concentration in the experiments. The utilization of external calibration in SPME [34] (standards subjected to the entire SPME procedure) provides the freely dis-solvedfractionofananalyteinthepresenceofcomplexmatricesif (a)thereisanequilibriumbetweenthefreeandmatrixboundfrac-tionoftheanalyte,(b)thefiberextractsonlyanegligibleamountof the free fraction, and (c) the binding matrix does not affect extrac-tion [26–29]. Condition (b) is most important when intending to carry out a negligible depletion SPME (nd-SPME) study [28]. Some studies have indicated possible adsorption of the matrix to theSPMEfibercoating,aconditionalsoknownasfouling.Nevertheless,mostpublicationsondirect-immersionSPMEinmatrix-containingsamples do not report fouling [35] and no visual or quantitative proof has been published for the occurrence of fouling [28]. As statedbyHeringaandHermens[28],iffoulingoccurs,twoopposite effects can take place: the adsorbed matrix can block or decreasethe uptake of the analyte onto the coating; or the adsorbed matrix,with the analyte bound to it, increases the measured amount of compound when the fiber is analyzed. The first effect would resultin a decreased uptake rate of analyte into the fiber coating, whichwould be problematic when measurements are performed in thekinetic phase (when extraction is carried out before equilibrium).Waitingforextractionequilibriumwouldsolvethisproblem,unlessadsorbed matrix components completely inhibit uptake [27].Given these considerations, the SPME extraction time used inall the experiments was 120min in order to ensure equilibration.This extraction time was selected by obtaining profiles in waterandin  -CDsolutionsforalloftheanalytesstudied.Equilibrationisconsideredtobeattainedwhentheamountofananalytesorbedbythe fiber (in terms of chromatographic response) stops increasing.Toevaluatetheeffectofthe  -CDcontentontheextractioneffi-ciency of hydrocarbons by SPME, the chromatographic responsesof the studied analytes were measured at different   -CD con-centrations while keeping constant the remaining experimentalconditions. Fig. 1 shows several examples in which a decrease inthe SPME extraction efficiency is clearly observed. It can be seenthat the extent of the decrease in the SPME extraction efficiencyis also analyte dependent and directly reflects the strength of thehydrocarbon:cyclodextrin inclusion complex.External calibration plots of the SPME–HPLC method wereobtained using an extraction time of 120min in order to quan-tifythebindingbehaviorofdifferenthydrocarbonsinthepresenceof    -cyclodextrin [30,31]. The calibration standards did not con- tain  -cyclodextrin in the sampling matrix resulting in all analytesbeing freely dissolved ( C  analyte free ). The figures of merit for eachcalibration curve are given in Table 1.  3.1.1. Association of   ˇ -cyclodextrin and substituted singlearomatic compounds The studied single aromatic compounds evaluated for a possi-ble 1:1 binding with   -cyclodextrin were phenetole, toluene andm-xylene. In the presence of    -CD in aqueous solution, the rela-tion between the concentration of the free analyte ( C  analyte free ),the total concentration of analyte ( C  analyte total ), and the complexedconcentration of analyte ( C  complex ) is given by Eq. (1): C  analyte total  = C  complex + C  analyte free  (1)If the concentration of the free cyclodextrin in solution isexpressed by  C  CD free , the binding constant of the free analyte (  A )tothe  -cyclodextrinina1:1inclusioncomplex( K  1 )isgivenbyEq.(2):  A + CD free   A − CD( K  1 ) K  1  = C  analyte complex C  analyte free C  CD free (2)If one assumes that (a) the total cyclodextrin concentrationis high (much higher than the analyte’s concentration, therefore, C  CD total ≈ C CD free ), (b) the analyte’s concentration in the headspaceisnegligible(whichissatisfiedbycompletelyfillingtheSPMEvials),(c)theaqueousvolumeissufficientlyhighwithrespecttotheSPMEfiber coating volume (thereby ensuring that the extraction of thehydrocarbonbythe SPMEcoating doesnot disturbthe equilibriumwithinthesamplingvial),and(d)the  -CDisnotstronglyadsorbedontothefiber,Eq.(3)canbederivedwhenintroducingaSPMEfiber in the system [24,25,30]:1 C  analyte free = 1 C  analyte total + K  1 C  CD total C  analyte total (3)Inordertoobtainthebindingconstant, K  1 ,thechromatographicresponse of the studied hydrocarbons was measured at differ-ent  -CDconcentrations(0–6.6mM),whilekeepingtheremainingexperimental SPME extraction conditions constant (i.e., extractiontime and total hydrocarbon concentration). In the case of the threesingle aromatic analytes, the plots of 1/ C  analyte free  (measured bySPME) versusC  CD total  werelinear( R >0.99),whichpermittedcalcu-lation of   K  1 , as shown in Table 2. The obtained  K  1  values are quitesimilar for phenetole ( ∼ 119) and toluene ( ∼ 115), and higher for m-xylene ( ∼ 346), which is in agreement with the hydrophobicity of these analytes. Szaniszlo et al. have reported  K  1  values of 172 ± 4and 100 ± 6M − 1 for toluene and m-xylene, respectively [1]. Val- ues from 140 to 215M − 1 , and from 100 to 160M − 1 have also beenreported for toluene and m-xylene, respectively [1,36]. A  K  1  valueof300M − 1 hasalsobeenreportedforo-xylene[36].Thevariability among binding data in the literature is a common phenomenon,and is attributed to the specific characteristics of each particularmethod employed [16].According to Eq. (3) the obtained intercepts should be close to thetheoreticalvalue,whichistheinverseofthetotalconcentrationof the analyte. For the single aromatic hydrocarbons, the relation-ship between the experimental and the theoretical intercepts wasstatisticallysignificantatthe99%confidencelevelsincethe P  -valuein the ANOVA test was less than 0.1 ( R =0.999).  3.1.2. Association of   ˇ -cyclodextrin and polyaromatic compounds The polyaromatic compounds examined in this study werebiphenyl, naphthalene, fluorene, and phenanthrene. At low   -CD  G. Chalumot et al. / J. Chromatogr. A 1216 (2009) 5242–5248  5245  Table 1 Figures of merit of the calibration curves obtained by SPME–HPLC.Analyte Slope ± SD a Error of the estimate Working range (  gL  − 1 )  R Phenetole 14,060  ±  480 30,643 560–62,000 0.995Toluene 7320  ±  200 9837 36–40,000 0.996m-Xylene 19,660  ±  650 10,286 120–13,800 0.995Naphthalene 422,000  ±  17,000 113,538 52–5710 0.992Biphenyl 9,980,000  ±  530,000 394,842 0.8–690 0.991Fluorene 16,280,000  ±  730,000 452,412 0.6–570 0.992Phenanthrene 47,200,000  ±  1,400,000 853,016 0.6–550 0.996 a SD: error of the slope for  n =10.  Table 2 Binding constants for the 1:1 stoichiometries of the single- and polyaromatic:  -cyclodextrin complexes ( K  1 ) and intercepts obtained by applying Eq. (3).Analyte Theoretical intercept Experimental intercept ± SD a n R K  1  (M − 1 ) ± SD b Range of   -CD (mM) c Phenetole 1946 1965  ±  61 10 0.99 119.3  ±  7.3 0–6.6Toluene 2235 2253  ±  64 10 0.99 115.3  ±  7.4 0–6.6m-Xylene 7320 8220  ±  310 14 0.99 346  ±  16 0–6.6Naphthalene 22,490 22,060  ±  540 6 0.97 870  ±  120 0–0.5Biphenyl 150,000 162,000  ±  11,000 11 0.99 2918  ±  88 0–1.6Fluorene 224,000 356,000  ±  16,000 6 0.98 1670  ±  180 0–0.7Phenanthrene 234,000 236,000  ±  37,000 7 0.98 3510  ±  280 0–1 a SD is the error of the experimental intercept for  n  -CD concentration levels. b SD is the error of the  K  1  for  n  -CD concentration levels (slope error × C  analyte total ). c Studied range of   -CD concentrations for the 1:1 binding. concentrations, these analytes also exhibited a linear relation-ship between 1/ C  analyte free  and  C  CD total  (Fig. 2A as a representative example), which confirms the existence of a 1:1 stoichiometryof the complex and allowed for calculation of   K  1  (Table 2). For naphthalene, biphenyl, fluorene and phenanthrene, the studiedrange of    -cyclodextrin concentration to obtain a 1:1 bind-ing was 0–0.5, 0–1.6, 0–0.7 and 0–1mM, respectively (seeTable 2), and therefore a lower number of    -CD concentrationlevels ( n ) was generally used in the experiments. The obtainedtrend in the  K  1  values for these analytes to   -cyclodextrin wasnaphthalene<fluorene<biphenyl<phenanthrene. Sanemasa et al.have reported  K  1  values around 1500M − 1 for phenanthrene:  -CD ( ∼ 3500M − 1 in this SPME study) and around 630M − 1 fornaphthalene:  -CD( ∼ 870M − 1 inthisSPMEstudy)[37].Hashimoto Fig. 2.  Plots for obtaining biphenyl:  -cyclodextrin binding constant by SPME–HPLC using Eq. (3) (A and B) and Eq. (5) (C).
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