Analytica Chimica Acta 504 (2004) 131–135

Bisphenol A-recognition polymers prepared by

covalent molecular imprinting
Takashi Ikegami b , Takashi Mukawa a , Hiroyuki Nariai a , Toshifumi Takeuchi a,c,∗
a Graduate School of Science and Technology, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe 657-8501, Japan
b Graduate School of Information Sciences, Hiroshima City University, Asaminami-ku, Hiroshima 731-3194, Japan
c PRESTO, Japan Science and Technology Corporation (JST), Kawaguchi-shi, Saitama 332-0012, Japan

Received 6 January 2003; received in revised form 25 July 2003; accepted 11 August 2003

Abstract

A specific recognition material for bisphenol A (BPA) was prepared by using a covalent imprinting technique. A chloroform solution
containing bisphenol A dimethacrylate as a template, ethylene glycol dimethacrylate as a cross-linking agent and 2,2 -azobis(isobutyronitrile)
as an initiator was polymerized by UV initiation. When BPA was removed from the resulting polymer by hydrolysis of the ester bonds with
aqueous sodium hydroxide, carboxylic acid residues were generated in the polymer. After the polymer was packed into a stainless steel
column, retention factors of BPA and related compounds were measured. The imprinted polymer adsorbed BPA and structurally related
compounds such as 4,4 -dihydroxybenzophenone, bis(4-hydroxyphenyl)sulfone and 4,4 -dihydroxybiphenyl. A typical association constant
(Ka ) was calculated to be 1.72 × 105 M−1 by Scatchard analysis. Interestingly, 17␣- and 17␤-estradiol were also bound to the imprinted
polymer (Ka = 1.68 × 105 M−1 ), indicating that the polymer could be used as artificial receptors for screening the compounds having
estrogenic action.
© 2003 Elsevier B.V. All rights reserved.

Keywords: Bisphenol A; Endocrine disruptors; Covalent imprinting technique; Artificial receptor

1. Introduction plate. Therefore, the polymers have been utilized in a

Bisphenol A (BPA) has been widely used for the pro-
duction of epoxy resins, polycarbonates and polysulfones,
and is recently suspected as one of endocrine disruptors. It
has been reported that BPA is found to be bound to estro-
gen receptor and causes a weak estrogenic activity in ani-
mal experiments [1]. A slight amount of BPA is eluted from
many plastic packages and tableware, and pollutes foods and
drinks [2]. Since a low dose effect of BPA is not denied
completely [3], the molecular recognition materials, which
can adsorb and remove BPA, are desired to be developed in
the viewpoint of environmental conservation.
Molecular imprinting has been known as a useful tech-
nique for preparation of molecular recognition materials
[4–9]. The polymeric materials synthesized by molecular
imprinting have ‘tailor-made’ binding sites for a target
molecule (template) and strongly interact with the tem-
∗ Corresponding author. Tel./fax: +81-78-803-6158.
E-mail address: takeuchi@scitec.kobe-u.ac.jp (T. Takeuchi).
variety of separation and sensing purposes. Molecularly
imprinted polymers are prepared by co-polymerization of a
cross-linking agent with the complex formed from a tem-
plate and polymerizable monomer(s) that have functional
group(s) specifically interacting with the template through
covalent and/or non-covalent bonds. After the template is
removed from the resulting polymer matrices, the bind-
ing sites having the size and shape complementary to the
template were generated.
The template–functional monomer complex formation
in molecular imprinting is classified into two categories,
i.e., non-covalent [10] and covalent [11–13] approaches.
In the former approach, non-covalent interactions such as
hydrogen-bonding, electrostatic interaction and hydrophobic
interaction, between a template and functional monomer(s)
are used. Attempts have been made for the preparation of
BPA-imprinted polymers by using such non-covalent ap-
proach [14]. Since equilibria between association and dis-
sociation of the template–functional monomer complexes
exist, the binding sites having various affinities would be
formed in the obtained polymers [15].

0003-2670/$ – see front matter © 2003 Elsevier B.V. All rights reserved.
doi:10.1016/j.aca.2003.08.032
132 T. Ikegami et al. / Analytica Chimica Acta 504 (2004) 131–135
Scheme 1. Preparation of BPA-imprinted polymer using bisphenol A dimethacrylate.
Covalent imprinting techniques may produce more ho-
mogeneous recognition sites in polymer matrix, therefore,
in this work, we focused on bisphenol A dimethacrylate
as the template monomer and adopted a covalent imprint-
ing technique for the BPA-imprinted polymer preparation
(Scheme 1). The selectivity of the molecularly imprinted
polymer for BPA, structurally related compounds and estra-
diols was examined chromatographically, and the affinity
was estimated by Scatchard analysis.
2. Experimental
2.1. Materials
Bisphenol A dimethacrylate was purchased from
Aldrich Chemical Co. (Milwaukee, WI). Ethylene gly-
col dimethacrylate (EGDMA), 2,2 -azobis(isobutyronitrile)
(AIBN), BPA, 4,4 -dihydroxybenzophenone, 4,4 -dihydro-
xybiphenyl, hexestrol, 2,4-dihydroxybenzophenone, p-
hydroxybiphenyl, 1-naphthol, 2-naphthol, 17␣-estradiol,
17␤-estradiol, 4,4 -diaminodiphenylmethane, 4,4 -diamino-
diphenyl ether, aniline, methanol and sodium hydrox-
ide were purchased from Wako Pure Chemical In-
dustry (Osaka, Japan). Bis(4-hydroxyphenyl)sulfone,
2,2-bis(4-hydroxyphenyl)butane (bisphenol B), dienestrol,
4,4 -dimethoxybenzophenone, 4,4 -ethylenedianiline and
1-naphthylamine were purchased from Tokyo Chemical
Industry (Tokyo, Japan). Phenol, toluene and hydrochlo-
ric acid were purchased from Katayama Chemical (Osaka,
Japan). Chloroform and acetonitrile were purchased from
Riedel de Haen (Seelze, Germany). EGDMA and chlo-
roform were purified by distillation prior to use. Other
reagents and solvents were used without further purification.
2.2. Preparation of BPA-imprinted polymer
Bisphenol A dimethacrylate (152 mg, 0.418 mmol),
EGDMA (2440 mg, 12.3 mmol) and AIBN (30.0 mg,
0.183 mmol) were dissolved in chloroform (6.25 ml). The
mixture was purged with nitrogen gas for 3 min. The glass
tube was sealed and placed under UV light (XX-15L, UVP,
Upland, CA) for 18 h at 5 ◦ C. After the obtained polymer
was crushed roughly, BPA was removed by refluxing the
polymer (2.00 g) with 1.0 M sodium hydroxide aqueous
solution (400 ml) for 24 h. The polymer was washed with
diluted hydrochloric acid and methanol, then ground by
an automatic mortar (AMN1000, Nittokagaku, Nagoya,
Japan), sieved (32–63 ␮m) in methanol, and packed in a
stainless steel column (150 mm × 4.6 mm ID). BPA was re-
moved by hydrolysis from polymer matrix, and the amount
of BPA recovered was determined by HPLC with a column,
SUPELCOSIL LC-8-DB, 5 ␮m (150 mm × 4.6 mm ID, SU-
PELCO), and a mobile phase of water–acetonitrile (60:40,
v/v) (Scheme 1).
2.3. Chromatographic tests
Chromatographic experiments were carried out using an
HPLC system (Gilson, France), which consisted of two
pumps (models 305 and 306), a degasser (model 503), an
autoinjector (model 234), a dynamic mixer (model 811C)
and a UV/VIS detector (model 119). The mobile phase
was chloroform–acetonitrile (90:10, v/v). The flow rate
was 1.0 ml min−1 . The sample volume was 10 ␮l. The
concentration of all samples was adjusted to 1.0 mM. The
effluent was monitored at 260 nm. Toluene was employed
as a void marker. Retention factor, k , was calculated by
 T. Ikegami et al. / Analytica Chimica Acta 504 (2004) 131–135 133

Fig. 1. Chemical structures of the samples used in this work. (1) BPA; (2) bisphenol B; (3) 4,4 -dihydroxybenzophenone; (4) bis(4-hydroxyphenyl)sulfone;
(5) 4,4 -dihydroxybiphenyl; (6) hexestrol; (7) dienestrol; (8) 4,4 -dimethoxybenzophenone; (9) 2,4-dihydroxybenzophenone; (10) p-hydroxybiphenyl; (11)
phenol; (12) 1-naphthol; (13) 2-naphthol; (14) 17␣-estradiol; (15) 17␤-estradiol; (16) 4,4 -diaminodiphenylmethane; (17) 4,4 -diaminodiphenyl ether; (18)
4,4 -ethylenedianiline; (19) aniline; (20) 1-naphthylamine.

k = (tR − t0 )/t0 , where tR is the retention time of the
sample, t0 is a retention time of the void marker. Tested
samples (Fig. 1) were injected independently in triplicate.
2.4. Scatchard analysis
After the removal of BPA by hydrolysis, the polymer par-
ticles were treated by diluted hydrochloric acid, and were
ground and sieved (32–63 ␮m). The polymers (3.0 mg) were
added to a 1.5 ml chloroform containing BPA (0–1.0 mM)
or 17␤-estradiol (0–1.0 mM). After the suspensions were ro-
tated for 24 h at 25 ◦ C, the polymer particles were removed
by filtration with a 2.5 ml syringe fitted with a disposable
5 ␮m filter cartridge. The filtrate solution (1.0 ml) was col-
lected and the solvent was removed in vacuo. The residue
was redissolved in acetonitrile (1.0 ml), and BPA was an-
alyzed the same HPLC system described above with SU-
PELCOSIL LC-8-DB, 5 ␮m (150 mm × 4.6 mm ID, SU-
PELCO). The mobile phase was water–acetonitrile (60:40,
v/v for BPA and 50:50, v/v for 17␤-estradiol). The flow rate
was 1.0 ml min−1 . The sample volume was 10 ␮l and the
effluent was monitored at 260 nm for BPA and 220 nm for
17␤-estradiol. The determination of free BPA, [BPA] was
carried out in triplicate. The amount of BPA bound to the
polymer, B, was calculated from [BPA] that was determined
by HPLC. Scatchard analysis was provided by the Scatchard
equation, B/[BPA] = (Bmax –B)Ka , where Ka is an associ-
ation constant and Bmax is an apparent maximum number
of binding sites. 17␤-estradiol was evaluated by the same
experimental process and conditions except that the mobile
phase was water–acetonitrile (50:50, v/v).
3. Results and discussion
3.1. Characteristics of the imprinted polymer
The BPA-imprinted polymer was prepared using bisphe-
nol A dimethacrylate. After polymerization, BPA was
cleaved by hydrolysis. Because phenol ester could be
134 T. Ikegami et al. / Analytica Chimica Acta 504 (2004) 131–135

Table 1 carboxyl groups in the binding sites can also interact with
Retention factors (k ) of the samples on BPA-imprinted polymer amino groups through electrostatic interactions. Thus those
Sample k amino derivatives were retained to the imprinted polymer
BPA 2.09
by the simultaneous interactions of two amino groups with
Bisphenol B 1.79 two carboxyl group similar to 4,4 -dihydroxy substituted
4,4 -Dihydroxybenzophenone 5.48 compounds. Despite the imprinted polymer was prepared
Bis(4-hydroxyphenyl)sulfone 5.02 for the recognition of BPA, 4,4 -dihydroxybenzophenone,
4,4 -Dihydroxybiphenyl 3.36 bis(4-hydroxyphenyl)sulfone and 4,4 -dihydroxybiphenyl
Hexestrol 1.26
Dienestrol 1.32
exhibited higher retention factors than BPA. The structures
4,4 -Dimethoxybenzophenone 0.01 of the three compounds are more rigid than BPA, thus those
2,4-Dihydroxybenzophenone 0.54 three compounds may be well retained to the polymer.
p-Hydroxybiphenyl 0.46 Interestingly, 17␤-estradiol that is an estrogen was also
Phenol 0.66 adsorbed on the polymer. It is assumed that the estrogenic
1-Naphthol 0.58
2-Naphthol 0.64
action of BPA is caused by the binding of BPA to es-
17␣-Estradiol 2.06 trogen receptors. The recognition of 17␤-estradiol by the
17␤-Estradiol 1.75 BPA-imprinted polymer indicates that this polymer is ca-
4,4 -Diaminodiphenylmethane 1.98 pable to act as artificial estrogen receptors. The estrogenic
4,4 -Diaminodiphenyl ether 3.06 action of an analyte may be predicted by measuring the re-
4,4 -Ethylenedianiline 1.70
Aniline 0.50
tention factor of the analyte for the BPA-imprinted polymer.
1-Naphthylamine 0.39
3.2. Scatchard analysis

more easily hydrolyzed than aliphatic alcohol-based ester, Batch binding tests were carried out to evaluate the bind-
we adopted a common hydrolysis condition using diluted ing properties of the BPA-imprinted polymer. The amount of
sodium hydroxide as previously reported [16]. After the BPA bound to the polymer, expressed as B, was plotted ver-
hydrolysis, BPA was determined by HPLC. About 40% of sus varied initial BPA concentration and a typical Scatchard
BPA was recovered from the resulting polymer under the plot for BPA was shown in Fig. 2. Two straight lines fitting
condition employed. the Scatchard equation, B/[BPA] = (Bmax –B)Ka , can be
In order to evaluate the BPA-imprinted polymer, BPA drawn, and gave two typical association constants. An asso-
and structurally related compounds were injected to the ciation constant (1.72 × 105 M−1 ) and a maximum number
imprinted polymer-packed column. Retention times of of binding sites (10.7 × 10−6 mol g−1 ) were calculated for
the analytes and a void marker were measured and the high affinity binding sites by fitting the straight line in the
retention factors obtained were listed in Table 1. The poly- range of 0–10 ␮mol g−1 .
mer strongly retained BPA and its analogues having hy- A Scatchard plot for 17␤-estradiol in the BPA-imprinted
droxyl groups at 4- and 4 -positions, namely bisphenol B, polymer was shown in Fig. 3, respectively. The curve fit-
4,4 -dihydroxybenzophenone, bis(4-hydroxyphenyl)sulfone, ting of Scatchard plot gave an association constant of 1.68×
4,4 -dihydroxybiphenyl, hexestrol and dienestrol. The car- 105 M−1 and a maximum number binding site of 10.6 ×
boxyl residues which were produced by hydrolyzing the es-
ter bonds between the polymer matrix and BPA could be ar-
ranged in suitable positions for interacting with two hydroxyl
groups at 4,4 -positions of BPA. The two carboxyl groups in
the binding sites could simultaneously interact with the two
hydroxyl groups of the samples through hydrogen-bonding,
leading to produce strong retention of the samples. There-
fore, 4,4 -dimethoxybenzophenone, of which the hydroxyl
groups were substituted by methoxy groups, was hardly
retained due to the lack of the hydrogen-bonding ability
of the methoxy groups. The compounds having a hydroxyl
group at the 4-position such as 2,4-dihydroxybenzophenone
and p-hydroxybiphenyl are slightly retained due to the for-
mation of only one hydrogen-bonding. The low retention
factors for phenol and phenol derivatives, 1- and 2-naphthol,
are considered in a similar manner. In addition, the polymer
Fig. 2. Scatchard plot for BPA in the BPA-imprinted polymer. [BPA]:
showed high affinity to the compounds having amino groups concentrations of free BPA. The binding constant, Ka , and the maximum
at 4,4 -positions such as 4,4 -diaminodiphenylmethane, number of binding sites, Bmax , were estimated to be 1.72 × 105 M−1 and
4,4 -diaminodiphenyl ether and 4,4 -ethylenedianiline. The 10.7 × 10−6 mol g−1 .
 T. Ikegami et al. / Analytica Chimica Acta 504 (2004) 131–135
Fig. 3. Scatchard plot for 17␤-estradiol in the BPA-imprinted polymer.
[17␤-estradiol]: concentrations of free 17␤-estradiol. The binding constant,
Ka , and the maximum number of binding sites, Bmax , were estimated to
be 1.68 × 105 M−1 and 10.6 × 10−6 mol g−1 .
10−6 mol g−1 in the high affinity binding sites. These values
are comparable to those of BPA. This result was consistent
with the chromatographic tests. Consequently it was shown
that characteristics of this polymer resemble biological es-
trogen receptors.
The imprinted polymers may have organized carboxylic
acids suitable for the specific binding that were induced by
the imprinting effects. In order to confirm the imprinting
effects, people would often use the corresponding blank
polymers that are prepared without the template molecule.
However, in the present case, it is difficult to prepare
the blank polymers that have the same number of active
carboxylic acid residues randomly located in the poly-
mer matrix. Methacrylic acids will form self-associated
dimers in organic solvents by hydrogen-bonding. If you use
methacrylic acid as a functional monomer and polymer-
ized it and a crosslinker without any template molecules,
methacrylic acid will form the dimers and the dimers may
fix the resulting polymer matrix after polymerization. When
the template was bound to the polymer, the stable dimers
should be dissociated, suggesting that such blank polymers
may show less affinity to the tested compounds than ex-
pected. In contrast, the imprinted polymers have no dimer
formation because the template molecule exists between
methacrylic acids. As a result, the imprinting effects may
be overestimated if the both polymers were simply com-
pared. Therefore, we evaluated the imprinted polymers by
the selectivity tests and Scatchard analysis, rather than the
blank polymer experiments.
135
4. Conclusions
Covalently BPA-imprinted polymer using BPA dimethacry-
late as the template monomer was synthesized with a
simple polymerization procedure. The ester bonds of the
obtained polymer were hydrolyzed to generate carboxyl
group-based binding sites. The resulting polymer exhibited
high affinity for BPA, BPA-analogues having two hydroxyl
or amino groups at 4,4 -positions, and estradiols. Thereby,
the BPA-imprinted polymer prepared in this work may ap-
ply to prepare artificial receptors for screening compounds,
which are suspected as endocrine disruptors, instead of
natural receptors.
Acknowledgements
This work has supported by Ministry of Education, Cul-
ture, Sports, Science and Technology (Grants-in-Aid for Sci-
entific Research).
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