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

a r t i c l e i n f o a b s t r a c t

Article history: The complexation of native ␤-cyclodextrin (CD) and seven aromatic compounds, namely, phenetole,
Received 23 March 2009 toluene, m-xylene, naphthalene, biphenyl, fluorene and phenanthrene, has been studied for first time uti-
Received in revised form 28 April 2009 lizing a solid-phase microextraction (SPME)–high-performance liquid chromatography (HPLC) method.
Accepted 8 May 2009
The stoichiometries of the analyte:␤-CD complexes were found to be either 1:1 or 1:2. The formation
Available online 15 May 2009
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
Keywords:
using the classical modified Benesi–Hildebrand (BH) method. The calculated binding constants for 1:1
Cyclodextrins
Stoichiometry
stoichiometries (K1 ) using the SPME method varied from 115.3 M−1 for toluene to 3510 M−1 for phenan-
Binding constants threne, whereas the corresponding values to the 1:2 stoichiometries (K3 ) varied from 7.30 × 105 M−2 for
Solid-phase microextraction biphenyl to 9.03 × 106 M−2 for naphthalene.
© 2009 Elsevier B.V. All rights reserved.

1. Introduction Many separation-based and non-separation-based methods

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).
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
 G. Chalumot et al. / J. Chromatogr. A 1216 (2009) 5242–5248
The purpose of this study is to extend, for the first time,
the use of SPME combined with HPLC to the determination
of the stoichiometry and binding constants of the complexes
formed with native ␤-cyclodextrins. The analytes selected in
this study include single aromatic and polyaromatic hydrocar-
bons and are evaluated in their possible 1:1 and 1:2 binding
to ␤-cyclodextrins using the SPME–HPLC approach. The BH
method was used to confirm the stoichiometry of the complexes
formed.
2. Experimental
2.1. Reagents
All experiments used HPLC-grade acetonitrile supplied by Fisher
Scientific (Fair Lawn, NJ, USA), and deionized water produced by
a Milli-Q water purification system (Millipore, Bedford, MA, USA)
with a resistivity of 18.2 M 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 were
prepared by dissolving these compounds in acetonitrile and
stored at 4 ◦ C in the dark. The cyclodextrin stock solutions were
prepared by dissolving 1.86 g of native ␤-cyclodextrin supplied
by Fluka in 250 mL of deionized water. The stock solutions
of analytes and cyclodextrin were used to prepare working
standard solutions, maintaining the acetonitrile content at 2%
(v/v).
2.2. Instrumentation
The chromatograph used was a LC-20A liquid chromatograph by
Shimadzu (Kyoto, Japan) equipped with a DGU-20A3 degasser, two
LC-20AT pumps and a SPD-20 UV–vis detector operating at 254 nm.
It was connected to a SPME–HPLC interface unit from Supelco and
to a C18 column (250 mm × 4.6 mm I.D., 5 ␮m particle size) from
Alltech (Deerfield, IL, USA). The separation gradient employed in
this study started with 60% (v/v) acetonitrile, which was linearly
increased up to 70% (v/v) over 15 min, and then kept isocratic for
40 min, at a flow rate of 1.00 mL min−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–HPLC
interface while passing through acetonitrile for 30 min (dynamic
mode), according to the instructions given by the manufacturer.
A fiber holder (Supelco) for manual sampling was used for SPME
analysis. Extractions were performed in 20 mL amber glass vials
supplied by Supelco. The vials were closed by screw caps and
PTFE/Butyl septa (Supelco). In all extractions, the vials were
completely filled with the working solution to leave no headspace.
Agitation was achieved by PTFE-coated stir bars and a Barnstead
International Super-Nuova stirring plate (Dubuque, IA, USA) at the
maximum stirring rate (1200 rpm). Due to the fact that sorption of
hydrocarbons can take place on PTFE stir bars, they were carefully
rinsed after each extraction with acetone, then methanol, then ace-
tonitrile and finally with deionized water to avoid memory effects.
The stir bars were sonicated daily for 10 min with acetonitrile.
Fluorescence and UV–vis absorbance measurements were
performed using a Thermo Electron Corporation Aminco
Bowman II luminescence spectrometer (Waltham, MA, USA),
which uses 1 cm quartz cell, and a Hewlett-Packard 8452A
diode array spectrophotometer (Palo Alto, CA, USA), respec-
tively.
5243
2.3. Procedures
2.3.1. SPME–HPLC studies
In order to determine the stoichiometry and binding constants
of each analyte with ␤-cyclodextrin, direct-immersion extractions
were carried out in 20 mL of the working solutions with a fixed con-
centration of 2.0% (v/v) in acetonitrile, 62.8 ␮g mL−1 in phenetole,
41.2 ␮g mL−1 in toluene, 14.5 ␮g mL−1 in m-xylene, 5.70 ␮g mL−1
in naphthalene, 1.03 ␮g mL−1 in biphenyl, 0.74 ␮g mL−1 in fluorene,
0.71 ␮g mL−1 in phenanthrene, and a concentration in cyclodextrin
varying from 0 to 6.6 mM, depending on the specific experiment.
The extraction time of the SPME fiber was fixed at 120 min.
Following each extraction, the fiber was desorbed for 20 min in
the desorption chamber of the SPME interface with the six-port
injection valve in the “load” position. The chamber was previously
filled with pure acetonitrile, allowing a static desorption to take
place. Following complete desorption, the valve was switched to the
“injection” position, allowing the mobile phase to pass through the
chamber for 5 min thereby transporting the analytes to the column.
Following each analysis, the fiber was soaked in 20 mL of acetoni-
trile for 20 min under agitation to ensure no obvious carryover
between extractions.
SPME calibration plots in deionized water were obtained
using 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. All
solutions were allowed to reach equilibrium for over 8 h, and pro-
tected from light in sealed vials. Fluorescence measurements were
performed 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 2 nm
and 1 nm, respectively. The emission wavelengths for the various
analytes were 314 nm for biphenyl, 321 nm for naphthalene, and
302 nm for fluorene, all corresponding to the maximum in the
emission spectra. The excitation wavelength was 254 nm for all
hydrocarbons. The AB2 Luminescence Spectrometer software 5.50
by 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 concentration
of phenanthrene, ␤-cyclodextrin, and acetonitrile as for the SPME
studies. All working solutions were allowed to reach equilibrium
for over 8 h, and protected from the light. The absorbance of each
solution was then measured in the diode array spectrophotome-
ter at a wavelength of 254 nm. All experiments were carried out at
room temperature in quadruplicate.
3. Results and discussion
3.1. Study of the binding behavior using solid-phase
microextraction with HPLC
The decrease of the free hydrocarbon concentration in an aque-
ous cyclodextrin solution when increasing the cyclodextrin content
is due to the formation of a hydrocarbon:cyclodextrin complex.
When introducing a SPME fiber into an aqueous CD solution
containing hydrocarbons, it is assumed that the hydrocarbons
extracted by the SPME coating originate from the aqueous phase
whereas 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 the
SPME–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-
solved fraction of an analyte in the presence of complex matrices if
(a) there is an equilibrium between the free and matrix bound frac-
tion of the analyte, (b) the fiber extracts only a negligible amount of
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 the
SPME fiber coating, a condition also known as fouling. Nevertheless,
most publications on direct-immersion SPME in matrix-containing
samples do not report fouling [35] and no visual or quantitative
proof has been published for the occurrence of fouling [28]. As
stated by Heringa and Hermens [28], if fouling occurs, two opposite
effects can take place: the adsorbed matrix can block or decrease
the 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 result
in a decreased uptake rate of analyte into the fiber coating, which
would be problematic when measurements are performed in the
kinetic phase (when extraction is carried out before equilibrium).
Waiting for extraction equilibrium would solve this problem, unless
adsorbed matrix components completely inhibit uptake [27].
Given these considerations, the SPME extraction time used in
all the experiments was 120 min in order to ensure equilibration.
This extraction time was selected by obtaining profiles in water
and in ␤-CD solutions for all of the analytes studied. Equilibration is
considered to be attained when the amount of an analyte sorbed by
the fiber (in terms of chromatographic response) stops increasing.
To evaluate the effect of the ␤-CD content on the extraction effi-
ciency of hydrocarbons by SPME, the chromatographic responses
of the studied analytes were measured at different ␤-CD con-
centrations while keeping constant the remaining experimental
conditions. Fig. 1 shows several examples in which a decrease in
the SPME extraction efficiency is clearly observed. It can be seen
that the extent of the decrease in the SPME extraction efficiency
is also analyte dependent and directly reflects the strength of the
hydrocarbon:cyclodextrin inclusion complex.
External calibration plots of the SPME–HPLC method were
obtained using an extraction time of 120 min in order to quan-
tify the binding behavior of different hydrocarbons in the presence
of ␤-cyclodextrin [30,31]. The calibration standards did not con-
tain ␤-cyclodextrin in the sampling matrix resulting in all analytes
being freely dissolved (Canalyte free ). The figures of merit for each
calibration curve are given in Table 1.
3.1.1. Association of ˇ-cyclodextrin and substituted single
aromatic compounds
The studied single aromatic compounds evaluated for a possi-
ble 1:1 binding with ␤-cyclodextrin were phenetole, toluene and
m-xylene. In the presence of ␤-CD in aqueous solution, the rela-
tion between the concentration of the free analyte (Canalyte free ),
the total concentration of analyte (Canalyte total ), and the complexed
concentration of analyte (Ccomplex ) is given by Eq. (1):
Canalyte total = Ccomplex + Canalyte free (1)
If the concentration of the free cyclodextrin in solution is
expressed by CCD free , the binding constant of the free analyte (A)
to the ␤-cyclodextrin in a 1:1 inclusion complex (K1 ) is given by Eq.
(2):
A + CDfree  A − CD(K1 )
Canalyte complex
K1 = (2)
Canalyte free CCD free
If one assumes that (a) the total cyclodextrin concentration
is high (much higher than the analyte’s concentration, therefore,
CCD total ≈ CCD free ), (b) the analyte’s concentration in the headspace
is negligible (which is satisfied by completely filling the SPME vials),
(c) the aqueous volume is sufficiently high with respect to the SPME
fiber coating volume (thereby ensuring that the extraction of the
hydrocarbon by the SPME coating does not disturb the equilibrium
within the sampling vial), and (d) the ␤-CD is not strongly adsorbed
onto the fiber, Eq. (3) can be derived when introducing a SPME fiber
in the system [24,25,30]:
1 1 CCD total
= + K1 (3)
Canalyte free Canalyte total Canalyte total
In order to obtain the binding constant, K1 , the chromatographic
response of the studied hydrocarbons was measured at differ-
ent ␤-CD concentrations (0–6.6 mM), while keeping the remaining
experimental SPME extraction conditions constant (i.e., extraction
time and total hydrocarbon concentration). In the case of the three
single aromatic analytes, the plots of 1/Canalyte free (measured by
SPME) versus CCD total were linear (R > 0.99), which permitted calcu-
lation of K1 , as shown in Table 2. The obtained K1 values are quite
similar 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 K1 values of 172 ± 4
and 100 ± 6 M−1 for toluene and m-xylene, respectively [1]. Val-
ues from 140 to 215 M−1 , and from 100 to 160 M−1 have also been
reported for toluene and m-xylene, respectively [1,36]. A K1 value
of 300 M−1 has also been reported for o-xylene [36]. The variability
among binding data in the literature is a common phenomenon,
and is attributed to the specific characteristics of each particular
method employed [16].
According to Eq. (3) the obtained intercepts should be close to
the theoretical value, which is the inverse of the total concentration
of the analyte. For the single aromatic hydrocarbons, the relation-
ship between the experimental and the theoretical intercepts was
statistically significant at the 99% confidence level since the P-value
in 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 were
biphenyl, 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 ± SDa Error of the estimate Working range (␮g L−1 ) R

Phenetole 14,060 ± 480 30,643 560–62,000 0.995

Toluene 7320 ± 200 9837 36–40,000 0.996
m-Xylene 19,660 ± 650 10,286 120–13,800 0.995
Naphthalene 422,000 ± 17,000 113,538 52–5710 0.992
Biphenyl 9,980,000 ± 530,000 394,842 0.8–690 0.991
Fluorene 16,280,000 ± 730,000 452,412 0.6–570 0.992
Phenanthrene 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 (K1 ) and intercepts obtained by applying Eq. (3).

Analyte Theoretical intercept Experimental intercept ± SDa n R K1 (M−1 ) ± SDb Range of ␤-CD (mM)c

Phenetole 1946 1965 ± 61 10 0.99 119.3 ± 7.3 0–6.6

Toluene 2235 2253 ± 64 10 0.99 115.3 ± 7.4 0–6.6
m-Xylene 7320 8220 ± 310 14 0.99 346 ± 16 0–6.6
Naphthalene 22,490 22,060 ± 540 6 0.97 870 ± 120 0–0.5
Biphenyl 150,000 162,000 ± 11,000 11 0.99 2918 ± 88 0–1.6
Fluorene 224,000 356,000 ± 16,000 6 0.98 1670 ± 180 0–0.7
Phenanthrene 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 K1 for n ␤-CD concentration levels (slope error × Canalyte total ).
c
Studied range of ␤-CD concentrations for the 1:1 binding.

concentrations, these analytes also exhibited a linear relation-
ship between 1/Canalyte free and CCD total (Fig. 2A as a representative
example), which confirms the existence of a 1:1 stoichiometry
of the complex and allowed for calculation of K1 (Table 2). For
naphthalene, biphenyl, fluorene and phenanthrene, the studied
range of ␤-cyclodextrin concentration to obtain a 1:1 bind-
ing was 0–0.5, 0–1.6, 0–0.7 and 0–1 mM, respectively (see
Table 2), and therefore a lower number of ␤-CD concentration
levels (n) was generally used in the experiments. The obtained
trend in the K1 values for these analytes to ␤-cyclodextrin was
naphthalene < fluorene < biphenyl < phenanthrene. Sanemasa et al.
have reported K1 values around 1500 M−1 for phenanthrene:␤-
CD (∼3500 M−1 in this SPME study) and around 630 M−1 for
naphthalene:␤-CD (∼870 M−1 in this SPME study) [37]. Hashimoto

Fig. 2. Plots for obtaining biphenyl:␤-cyclodextrin binding constant by SPME–HPLC using Eq. (3) (A and B) and Eq. (5) (C).
5246 G. Chalumot et al. / J. Chromatogr. A 1216 (2009) 5242–5248

Table 3
Binding constants for the 1:2 stoichiometries of the polyaromatic:␤-cyclodextrin complexes (K3 ) obtained by the SPME–HPLC method and by the BH method.

Analyte SPME–HPLC method BH method

−2
n R K3 (M ) ± SD a
n R K3 (M−2 ) ± SDb

Naphthalene 14 0.996 (9.03 ± 0.23) × 10 6

5 0.993 (5.06 ± 0.39) × 105
Biphenyl 10 0.998 (7.30 ± 0.34) × 105 5 0.997 (1.80 ± 0.09) × 105
Fluorene 15 0.996 (2.27 ± 0.05) × 106 5 0.998 (1.48 ± 0.08) × 105
Phenanthrene 10 0.994 (3.16 ± 0.08) × 106 6 0.995 (1.49 ± 0.16) × 105
a
SD is the error of the K3 for n ␤-CD concentration levels (slope error × Canalyte total ).

2 2
b
SD is the error of the K3 for n ␤-CD concentration levels (K3 × (slope error/slope) + (intercept error/intercept) ).

et al. have reported K1 values for naphthalene:␤-CD of 850 M−1
[38], which is very close to the K1 value obtained by SPME. K1 val-
ues obtained using HPLC with mobile phases modified by ␤-CDs are
much lower, mainly because such studies are normally conducted
with higher content of organic solvents. In these studies, K1 values
of 397 M−1 for fluorene:␤-CD and 602 M−1 for phenanthrene:␤-
CD have been obtained with 35% (v/v) of methanol content [39],
whereas K1 values of 137 M−1 for naphthalene:␤-CD, 181 M−1
for biphenyl:␤-CD, 188 M−1 for fluorene ␤-CD and 72 M−1 for
phenanthrene:␤-CD have been obtained with 20% (v/v) of acetoni-
trile content [12]. The data presented in this SPME study has been
conducted using 2% (v/v) acetonitrile content.
It should be noted that there was not a linear relationship
between 1/Canalyte free and CCD total for all polyaromatic compounds
studied when the experiments were conducted within the entire
range of ␤-CD (i.e., 0–6.6 mM), indicating that at higher ␤-CD con-
tent, the 1:1 stoichiometry is not taking place. Fig. 2B shows a
representative trend for biphenyl.
If one assumes 1:2 binding when working at higher ␤-CD con-
centrations, the following equilibria should be expected to occur:
A + CDfree  A − CD (K1 )
A − CD + CDfree  CD − A − CD(K2 )
Overall : A + 2CDfree  CD − A − CD (K3 )
The binding constant (K3 ) of the analyte to the ␤-cyclodextrin is
given by Eq. (4):
Canalyte complex
K3 = 2
Canalyte free CCD free
Eq. (5) can be therefore obtained to describe 1:2 complex for-
mation following the aforementioned considerations:
2
CCD
1 1 total
= + K3
Canalyte free Canalyte total Canalyte total
The linear relationship obtained when plotting 1/Canalyte free
(measured by SPME) versus CCD 2 (with R > 0.99) for the studied
total
polyaromatic compounds is indicative of a 1:2 hydrocarbon:␤-
cyclodextrin stoichiometry. Fig. 2C shows a representative plot
for biphenyl, whereas all the calculated K3 values are included in
Table 3. The experimental intercepts obtained by applying Eq. (5)
were 17,950 for naphthalene, 456,000 for biphenyl, 397,000 for
fluorene and 44,800 for phenanthrene. The deviation from the the-
oretical intercepts is high for biphenyl, but quite acceptable for the
rest of hydrocarbons (R = 0.998).
In general, 1:1 stoichiometries are observed with ␤-CD, how-
ever, 1:2 and 2:2 stoichiometries have also been reported. A 2:2
stoichiometry has been described for naphthalene:␤-CD under
certain conditions, with K values around 104 M−1 [2]. A 1:2 sto-
ichiometry has also been reported for naphthalene to ␣-CD. To
the best of our knowledge, K3 values for 1:2 stoichiometries
for polyaromatics:␤-CD have not been reported (at least, not for
naphthalene, fluorene, biphenyl and phenanthrene). It should be
commented here that the published studies for naphthalene, fluo-
rene, biphenyl and phenanthrene with ␤-CD do not work with high
␤-CD contents. K3 values for 1:2 stoichiometries (analyte:␤-CD)
have been reported for some other compounds, such as loratadine,
with K3 ∼ 7 × 106 M−2 [40]; carvedilol, with K3 ∼ 9 × 105 M−2 [41];
and epicoccone, with K3 ∼ 2 × 105 M−2 [42], among others. In all
cases, high K3 values on the order of 105 to 106 are obtained, which
is in accordance with the K3 values obtained in this SPME study.
The previously reported studies were conducted using high ␤-CD
concentrations with maximum values of 10 mM [40,42] and 4 mM
[41].
2
It must be added that the plots of 1/Canalyte free versus CCD
total
were non-linear for the single aromatic compounds studied, veri-
fying that a 1:2 binding was not taking place for these analytes.
3.2. Evaluating 1:2 complexation stoichiometry for polyaromatic
compounds using the Benesi–Hildebrand method
The modified Benesi–Hildebrand method [12,43] is the classi-
cal approach to study the complexation of guest molecules with
cyclodextrins and is used in this study to confirm the 1:2 sto-
ichiometry predicted by SPME for the polyaromatic compounds
with ␤-CD. The calculations for the modified BH method are based
in measuring a change in the fluorescence (or UV–vis absorbance)
intensity signals of the analyte in the absence and in the presence
of ␤-CD. The experiments were conducted utilizing fluorescence
measurements for naphthalene, fluorene and biphenyl, and UV–vis
absorbance measurements for phenanthrene.
(4) The modified Benesi–Hildebrand method for a 1:1 stoichiometry
is given by Eq. (6):
1 1 1
= + (6)
I − Io I1 − Io (I1 − Io )K1 CCD free
(5) where I and Io are the fluorescence (or absorbance) intensities
of the aromatic compound in the presence and absence of ␤-CD,
respectively, and I1 is the expected fluorescence (or absorbance)
intensity when all guest molecules are included in a complex. If
the total cyclodextrin concentration is high (much higher than the
concentration of the analyte, therefore, CCD total ≈CCD free ), the ratio
of the intercept to the slope when plotting 1/I versus 1/CCD total
can be taken as an estimation of the K1 formation constant for a 1:1
binding (with I being I − Io ).
The assumption of 1:1 association between polyaromatic com-
pounds and ␤-CD reveals that there is a non-linear relationship
when working in the entire concentration range of ␤-CD (0-6.6 mM)
(see examples in Fig. 3A and C). A wide range of ␤-CD concentra-
tions is used in these experiments as it has been shown that 1:1 BH
plots of 1:2 binding interactions could show a linear behavior due
to use of a narrow concentration range [18].
The modified Benesi–Hildebrand method, assuming a 1:2 stoi-
chiometry and according to the aforementioned considerations, is
 G. Chalumot et al. / J. Chromatogr. A 1216 (2009) 5242–5248
Fig. 3. Benesi–Hildebrand plots for (A) biphenyl, using the whole range of ␤-CD (0–6.6 mM); (B) biphenyl (2–6.6 mM of ␤-CD); (C) fluorene, using the whole range of ␤-CD
(0–6.6 mM), and (D) fluorene (2–6.6 mM of ␤-CD).
given by Eq. (7):
1 1 1 SPME is used, for the first time, to evaluate the binding behav-
= +
I − Io I1 − Io 2
(I1 − Io )K3 CCD free
The experiments were conducted using a ␤-CD concentration
range of 2–6.6 mM, to force the 1:2 binding and to exclude results
originating from the initial 1:1 binding. For each of the polyaromatic
molecules, the plot 1/I versus 1/CCD 2 was linear, with correla-
total
tion coefficient (R) values higher than 0.995. Fig. 3B and D shows
several examples. According to Eq. (7), this proves the formation of
a 1:2 hydrocarbon:␤-CD complex. The obtained K3 values are also
shown in Table 3.
The reliability of the Benesi–Hildebrand method has often been
questioned [20,43]. Thus, several studies have focused on mea-
suring the performance of the Benesi–Hildebrand method when
determining the binding constants of the cyclodextrin complexa-
tion [44,45]. It has been established that a reproducible binding
constant is often difficult to obtain with the BH method if the com-
plexation is too weak or too strong, or if the change in absorption
is too small [44]. The BH method is only recommended when the
complexation is modest (i.e., K ≈ 1000 M−1 for CD complexation)
and the change in absorption is sufficiently large. It has also been
shown with computer simulations that, under certain conditions,
the approach could generate inappropriate stoichiometric conclu-
sions for 1:2 interactions. This problem could occur in the cases
of both weak and strong interactions [18]. This could justify the
lack of agreement between the K3 values obtained by the SPME
method and those obtained by the BH method. In any case, the val-
ues obtained using the SPME–HPLC method for 1:2 stoichiometries
of polyaromatics with high ␤-CD concentrations are in agreement
with those derived from the BH method.
5247
4. Conclusions
(7) ior of several analytes to ␤-cyclodextrins. The SPME–HPLC method
can be used to distinguish among different types of analyte:␤-
cyclodextrin stoichiometries. A 1:1 stoichiometry has been shown
for phenetole, toluene, and m-xylene in the entire range of stud-
ied ␤-CD concentrations (0–6.6 mM), with K1 values ranging from
115.3 to 346 M−1 , which is in agreement with literature values. A 1:1
stoichiometry is also observed for naphthalene, biphenyl, fluorene,
and phenanthrene, if the concentration range of ␤-CD is narrow
(from 0 to 1.6 mM). In this case, the K1 values obtained ranged from
870 to 3510 M−1 , which is also in agreement with literature values.
A 1:2 stoichiometry has been observed for naphthalene, biphenyl,
fluorene, and phenanthrene when working at higher ␤-CD lev-
els (from 2 to 6.6 mM), with K3 values ranging from 7.30 × 105 to
9.3 × 106 M−2 . Due to the absence of literature values, the mod-
ified Benesi–Hildebrand method has been used to confirm such
stoichiometries. SPME has been proven to be a viable and sim-
ple method to study guest–host molecule binding by integrating
sample extraction and loading into a single step.
Acknowledgements
J.L.A. acknowledges funding from the Analytical and Surface
Chemistry Program in the Division of Chemistry and the Separation
and Purification Process Program in the Chemical, Environmental,
Bioengineering, and Transport Systems Division from the National
Science Foundation for a CAREER grant (CHE-0748612). V.P. and
J.L.A. thank the University of Toledo for a visiting faculty grant.
Professor A.M. Afonso is gratefully acknowledged for her valuable
suggestions.
5248 G. Chalumot et al. / J. Chromatogr. A 1216 (2009) 5242–5248

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