Preparation of photoirradiation molecular imprinting polymer for selective separation of branched cyclodextrins

Article abstract of DOI:10.3390/molecules22020288

In the present study, photoirradiation molecularly imprinted polymer (MIP) with azobenzene was used as a functional monomer for the selective separation of the branched cyclodextrins. The functional monomer 4-methacryloyloxy azobenzene (MAA) and the molecular template 6-O-α-D-maltosyl-β-cyclodextrin (G2-β-CD) were implemented for the molecular imprinting. The core-shell structure of photoirradiation MIP was visualized by the transmission electron microscopy (TEM). With Fourier transform infrared spectroscopy (FTIR) and thermogravimetric analysis (TGA), we identified that G2-β-CD was imprinted into the polymer and removed from the MIP. The binding association constant (K_a) and the maximum number of the binding site (Nmax ) were 1.72 × 10⁴ M^-1 and 7.93 μmol·g^-1 MIP, respectively. With alternate irradiation at 365 and 440 nm light, the prepared MIP reversibly released and rebound to the G2-β-CD, which resulted in the nearly zero amount of G2-β-CD in the solution. The HPLC results indicated that the purity of G2-β-CD could reach 90.8% after going through MIP. The main finding of our study was that the photoirradiation of MIP was an easy and effective method for the selective separation of the branched cyclodextrins.

Full text of DOI:10.3390/molecules22020288

molecules
Article
Preparation of Photoirradiation Molecular Imprinting
Polymer for Selective Separation of
Branched Cyclodextrins
Haoran Fan 1 , Jinpeng Wang 1,2,3, Qingran Meng 1,2,3, Xueming Xu 1,2, Tianming Fan and
,2,3
4
Zhengyu Jin 1,2,*
1
The State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Road,
Wuxi 214122, China; fanhaoran0622@163.com (H.F.); jpwang1984@jiangnan.edu.cn (J.W.);
mengqingranxzjscn@163.com (Q.M.); xmxubest@126.com (X.X.)
School of Food Science and Technology, Jiangnan University, 1800 Lihu Road, Wuxi 214122, China
2
3
Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, 1800 Lihu Road,
Wuxi 214122, China
4
Muyang Group Co., Ltd., Muyang Road, Yangzhou 225127, China; wangjinpeng1984@126.com
*
Correspondence: fpcenter@jiangnan.edu.cn
Academic Editor: Bernard Martel
Received: 28 December 2016; Accepted: 8 February 2017; Published: 21 February 2017
Abstract: In the present study, photoirradiation molecularly imprinted polymer (MIP) with azobenzene
was used as a functional monomer for the selective separation of the branched cyclodextrins.
The functional monomer 4-methacryloyloxy azobenzene (MAA) and the molecular template 6-O-
α-
D-maltosyl- -cyclodextrin (G2- -CD) were implemented for the molecular imprinting. The core-shell
β
β
structure of photoirradiation MIP was visualized by the transmission electron microscopy (TEM).
With Fourier transform infrared spectroscopy (FTIR) and thermogravimetric analysis (TGA), we
identified that G2-
β
-CD was imprinted into the polymer and removed from the MIP. The binding
4
−1
association constant (Ka) and the maximum number of the binding site (Nmax) were 1.72 × 10 M
−
1
and 7.93
µmol
·
g
MIP, respectively. With alternate irradiation at 365 and 440 nm light, the prepared
MIP reversibly released and rebound to the G2-
β-CD, which resulted in the nearly zero amount of
G2- -CD in the solution. The HPLC results indicated that the purity of G2-
β
β-CD could reach 90.8%
after going through MIP. The main finding of our study was that the photoirradiation of MIP was
an easy and effective method for the selective separation of the branched cyclodextrins.
Keywords: molecular imprinting polymer; branched cyclodextrins; azobenzene derivatives;
photoirradiation
1
. Introduction
Branched cyclodextrins (CDs), in which one or two primary hydroxyl groups of CD are replaced
by mono- or di-saccharides through the
cyclodextrins ( -CD, -CD, and -CD), such as higher bioadaptability, higher solubility, and lower
hemolytic activity [ ]. Generally, the branched CDs are produced either by the action of cyclodextrin
glycosyl transferase (CGTase) on amylopectin, or by the condensation of the saccharide chains
glucose (G1), maltose (G2), and other saccharides) and CDs with such debranching enzymes as
pullulanase or isoamylase [ ], and branched CDs, unbranched CDs, and acyclic dextrins are formed
in the system [ ]. Special chromatographic methods are used for separating and purifying branched
α-1,6 glycosidic bond, present advantages over the parent
α
β
γ
1–3
(
4
5
CDs from acyclic dextrins yet are impractical for the commercial operations. For instance, Koizumi
et al. isolated three branched CDs by high-performance liquid chromatography (HPLC) from the
mother liquors of a large-scale preparation of unbranched CDs with Bacillus ohbensis cyclomaltodextrin
Molecules 2017, 22, 288; doi:10.3390/molecules22020288
www.mdpi.com/journal/molecules

Molecules 2017, 22, 288
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glucanotransferase [
2
]. Ammeraal et al. developed a method for separating and purifying branched
β-CD from an aqueous solution [5]. In this method, a β-CD complexant which was selected from the
group consisting of p-xylene and toluene was added to a starting solution, the precipitate form was
recovered and then dissolved in water. A second dose of the complexant was added to the solution,
and a branched
β-CD was recovered from the resulting liquor. The separation and purification of
branched CDs from the solution is relatively difficult, complicated, and unsuitable on a large scale [
6].
The easy and effective method for purifying and separating branched CDs from the mother liquors
needs to be developed.
Molecular imprinting is a useful tool for preparing specific molecular recognition materials,
which have tailor-made binding sites for the template molecules [
MIP) syntheses, the functional monomer and the template molecule are assembled through either
covalent or non-covalent interactions (such as ionic interactions, hydrogen bonds, metal-ion chelating
interactions, and hydrophobic interactions) and co-polymerized with a crosslinking monomer [ ].
7]. In molecularly imprinted polymer
(
8,9
After polymerization, the template molecule is removed from the polymer, yielding an MIP with
specific selective binding cavities that are complementary to the size, shape, and functionality
of the template molecule [10
,11]. The MIP has many appealing characteristics such as physical
robustness, thermal stability, and desired selectivity as well as low cost and easy preparation [12
,13].
These physicochemical characteristics make MIP the promising candidates for applications in
separation [14], drug-delivery [15], and chemical sensing [16]. Recently, much research attention
was attracted to the MIP responsivity to stimuli, such as temperature, pH, ionic and solvent
compositions, concentration of specific chemical or biochemical species, electric fields, and light
irradiation [17]. In particular, light is a clean stimulus and its irradiation can be manipulated rapidly
and precisely. Compared with other MIP, a template molecule uptaken by a photoirradiation MIP
could be efficiently and conveniently released under appropriate light irradiation instead of eluting
the template molecule in a solution for a longer time. Gong et al. designed a photoirradiation
functional monomer 4-((4-methacryloyloxy)phenylazo)benzoic acid for preparing photoirradiation
MIP with the aim of uptaking and releasing caffeine [18]. Gong et al. reported that photoirradiation
of molecularly imprinted hydrogels is capable of releasing and uptaking pharmaceuticals in
the aqueous media, where 4-((4-methacryloyloxy)phenylazo)benzenesulfonic acid is used as a
functional monomer [19]. Gomy et al. reported a photoirradiation of MIP containing azo monomer
di (ureidoethylenemethacrylate)azobenzene and their binding properties were investigated [20].
However, there is no report about demonstrating a photoirradiation of MIP for selective separation
of branched CDs. In the present study, the template molecule 6-O-
α-D-maltosyl-β-cyclodextrin
(
G2- -CD) and the functional monomer 4-methacryloyloxy azobenzene (MAA) were used for
β
molecular imprinting.
Among the branched CDs, 6-O-
α
-D-maltosyl-
-CD has much higher solubilizing ability in water and aqueous
methanol solution, higher resistance of the enzymatic degradation, lower local tissue irritancy, and
weaker hemolytic activity on human erythrocytes than its parent -CD [21]. G2- -CD possesses
β-cyclodextrin (G2-β-CD) is generally the most
accessible and useful CD because G2-
β
β
β
a hydrophilic outer surface and hydrophobic internal cavity [22]. This structure is able to form the
inclusion complexes with the azobenzene based on the cis-trans photoisomerization of azobenzene.
Therefore, it is possible that the preparation of the photoirradiation MIP could be implemented for
the selective separation of the branched cyclodextrins. The G2-
maltose through the reverse action with isoamylase or pullulanase in a reaction mixture containing of
-CD and maltose, or by transferring maltosyfluoride to CDs via pullulanase [23]. To our knowledge,
β-CD is synthesized either by grafting
β
no previous study has demonstrated the preparation of the photoirradiation of MIP for the selective
separation of branched CDs by using azobenzene and its derivatives as the functional monomer. In the
current study, we prepared photoirradiation of MIP by using 4-methacryloyloxy azobenzene (MAA) as
the functional monomer to separate G2-β-CD from the maltose. We assumed this approach is a simple
and efficient separation process for G2-β-CD.

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2
. Results and Discussion
2
.1. Characterization of the 4-(Phenyldiazenyl)phenol and MAA
It has been well established that a suitable functional monomer is crucial for the successful
preparation of the MIP for selective separation of the branched cyclodextrins. It is also known that
azobenzene-containing polymer is a widely applied functional monomer in photoirradiation of MIP.
Therefore, ultraviolet–visible (UV-Vis) absorption spectra, fourier transform infrared spectroscopy
1
(
FTIR) spectra, and H-NMR were used to detect the properties of the 4-(Phenyldiazenyl)phenol (PDP)
and MAA.
2
.1.1. Photoisomerization Properties of MAA
Figure 1 showed the spectroscopic responses of the MAA at room temperature upon alternate
irradiation at 365 and 440 nm light. The MAA exhibited one strong absorption peak at ~326 nm and
another very weak peak at ~440 nm, which are typical for the azobenzene chromophore and can
be attributed to the
irradiation with 365 nm light, the peak intensity at ~326 nm decreased rapidly, whereas the peak at
440 nm increased over time (Figure 1a), which was attributed to the trans to cis photoisomerization of
π-π* and n-π* electron transitions of the N=N bond, respectively [19,24]. After
~
the azobenzene. Subsequent irradiation at 440 nm caused cis to trans photoisomerization (Figure 1b).
However, the finally recovered absorbance of trans-MAA was lower than that previous irradiation
at 365 nm light. Similar observations were reported by other studies [25,26]. The rate constants
−
3
−1
−3 −1
for the trans to cis and cis to trans photoisomerization were 2.54
×
10
s
and 11.9
×
10 s ,
respectively. Moreover, the photoisomerization properties became fully reversible upon subsequent
cycles of the 365 nm and 440 nm irradiation (Figure S2 in the Supplementary Materials). No obvious
decline of absorbance was found after five cycles. Similar phenomena were also observed previously
by others [27,28]. It implied that the MAA has good restorability and reversibility in the process of
UV and visible light irradiation. Similar results were also reported by Yang et al. [29], which used
functional monomer 4-((4-methacryloyloxy)-phenylazo)benzenesulfonic acid to polymerize on the
surface of silica microspheres.
0
0
a
10s
b 2.0
10s
20s
30s
3
1
2
4
5
6
8
1
0s
2.0
1.5
1.0
0.5
0.0
min
min
min
min
min
min
0min
1min
2min
4min
6min
8min
10min
15min
20min
30min
40min
50min
1.5
15min
1.0
20min
30min
40min
50min
0min
0.5
6
0.0
200
300
400
500
600
200
300
400
500
600
Wavelength (nm)
Wavelength (nm)
c 1.2
d 1.4
1
1
0
0
.2
.0
.8
.6
1.0
0.8
0.6
0.4
0.2
0.0
0.4
0
.2
.0
0
0
100
200
300
400
500
600
0
100
200
300
400
500
600
t (s)
t (s)
Figure 1. The ultraviolet–visible (UV-Vis) spectra and spectral changes of the 4-Methacryloyloxy
Azobenzene (MAA). ( ) Irradiation at 365 nm; ( ) irradiation at 440 nm; ( ) kinetics of the trans to cis
photoisomerization; (d) kinetics of the cis to trans photoisomerization. a.u.: arbitrary units.
a
b
c

Molecules 2017, 22, 288
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1
2
.1.2. FTIR and H-NMR Spectroscopy of PDP and MAA
The FTIR spectra of the PDP and MAA were presented in Figure 2a. The characteristic peaks of
−
1
−1
azo group) and 1593 cm (υ
PDP were located at 1488 cm
(υ
aromatic). The characteristic
N=N
C=C
−
1
−1
peaks of MAA were located at 2939 cm
644 cm (υC=C methacrylic), and 1593 cm (υC=C aromatic).
(
υ
aliphatic), 1737 cm
(
υ
methacrylic ester),
C=O
CH
−
1
−1
1
1
The H-NMR spectra of the PDP and MAA were shown in Figure 2b,c. The aromatic protons of
PDP appeared between 6.95 and 7.88 ppm. The spectrum of MAA showed similar aromatic protons at
.30–7.99 ppm. The three singlets at 2.09, 5.80, and 6.39 ppm were the methacrylic eater group and
7
their signals were clearly detectable.
a
PDP
MAA
MIP
MIPT
G2-β-CD
1635
1252
1733
4000
3600
3200
2800
2400
2000
1600
1200
800
400
0
-1
Wavenumber
(
cm )
b
d
c
O
N=N
OH
N=N
O
PDP
MAA
e
100
0
.000
80
60
40
20
0
-
0.005
-0.010
MIPT
MIP
2−β-CD
MIP-template
MIP
-0.015
G
G2−β-CD
-0.020
100
200
300
400
500
600
100
200
300
400
500
600
Temperature (
°
C)
Temperature (°C)
Figure 2. Fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA)
1
1
and H-NMR spectrum. (
a
) FTIR spectra; (
b)
H-NMR spectrum of 4-(Phenyldiazenyl)phenol
) derivative thermogravimetry
1
(
(
PDP); (
DTG) thermograms.
c) H-NMR spectrum of MAA; (
d
) TGA thermograms; (e

Molecules 2017, 22, 288
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2
.2. Characterization of MIPT and MIP
2
.2.1. Morphology of Samples
The scanning electron microscopy (SEM) images of MIPT (the Imprinted Polymer Containing
the Template Form) and MIP were presented in Figure 3a,b. SEM revealed that the MIPT and MIP
were uniform with a mean size of ~1.5–2 m. The similar diameters of the MIPT and MIP revealed
µ
that G2-β-CD had less influence on the particle size, which agreed with the previous studies [27].
The size and morphology of the MIPT and MIP were characterized by transmission electron
microcopy (TEM) (Figure 3c,d). TEM images showed that the MIPT and MIP were nearly spherical
with size of ~2
µm. Figure 3d (TEM of MIP) showed a clear and direct visual evidence of the core-shell
structure. More importantly, the TEM micrograph proved that the MIP has a core and a surface
imprinting polymer shell, suggesting that G2-β-CD was removed from the MIP. Similar phenomena
were also observed previously by others [28,30].
a
b
c
d
Figure 3. The scanning electron microscopy (SEM) and transmission electron microcopy (TEM) images.
) SEM of the imprinted polymer containing the template (MIPT); ( ) SEM of molecularly imprinted
(
a
b
polymer (MIP); (c) TEM of MIPT; (d) TEM of MIP.
2
.2.2. FTIR Spectroscopy
The FTIR spectra of MAA, G2-β-CD, MIPT, and MIP were presented in Figure 2a. Three
characteristic peaks of the existence of ethylene glycol dimethacrylate (EGDMA) attributed to the
−
1
−1
C=O stretching (1733 cm ) and C–O–C stretching (1252 and 1144 cm ) in the obtained MIPT
and MIP could be observed. Furthermore, the presence of a well-defined peak of G2- -CD at 1635
-CD was imprinted into the MIPT.
Compared with MIPT, weak absorption bands at 3402 cm and the disappeared characteristic
β
−
1
cm (for H–O–H bending) [31–33] for MIPT suggested that G2-β
−
1
−
1
absorption at 1635 cm could be observed in the spectra of MIP, indicating that G2-
removed from the MIP material.
β-CD was

Molecules 2017, 22, 288
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2
.2.3. Thermogravimetric Analysis
Thermogravimetric Analysis (TGA) and derivative thermogravimetry (DTG) curves of G2-β-CD,
MIPT, and MIP were presented in Figure 2d,e. There were two major weight loss in the TGA curves.
◦
◦
◦
The first weight loss occurred between 25 C and 150 C, and a second one occurred between 300 C
◦
and 400 C. In the first stage, the weight loss of G2-β-CD, MIPT, and MIP was 11.2%, 2.3%, and 0.3%,
respectively. It may be due to the presence of certain compounds at low pyrolysis temperatures and the
vaporization of adsorbed water. In the second stage, the weight of G2-β-CD, MIPT, and MIP reduced
significantly to 9.4%, 30.9%, and 23.0%, respectively. The reduction of weight could be attributed to the
oxidative decomposition of the most organic compounds and some volatile components. Finally,
G2-β-CD, MIPT, and MIP retained ~0.1–8% solid carbonized residue (0.11%, 7.45%, and 4.28%,
respectively). The DTG curves of all three samples showed a roughly consistent trend. However,
there were several different transitions of the samples. Compared to MIP, the weight loss stage
◦
below 150 C of MIPT was observed, which could be ascribed to the template contained in the MIPT.
◦
In DTG curve of G2-
β-CD, similarly, the weight loss between 25 and 150 C was observed. The results
illustrated that G2-β-CD was imprinted into the MIPT and removed from the MIP material.
2
.3. MIP and NIP Binding Properties
The selectivity of MIP toward G2-
β
-CD,
β
-CD, and maltose were presented in Figure 4a. The MIP
-CD than G2- -CD, thus suggesting
-CD, the adsorption capacity was a little higher than
maltose due to its extremely similar structure to G2- -CD, which indicated that the recognition cavity
of MIP was based on the shape, size, and functionality of the template.
had significantly lower binding capacities toward maltose and
a higher selectivity of MIP for G2- -CD. For
β
β
β
β
β
a
c
b
1
1
1
1
6
4
2
0
8
6
4
2
0
0
.20
.16
0.12
.08
.04
.00
MIP
NIP
G2-β-CD (MIP)
β-CD (MIP)
maltose (MIP)
G2-β-CD (NIP)
β-CD (NIP)
50
0
40
0
maltose (NIP)
0
30
20
0
maltose
10
0
0
1
2
3
4
G2-β-CD
β-CD
maltose
MIP/NIP Concentration (mg/mL)
d
-
3
1
.2×10
G2-β-CD (MIP)
β-CD (MIP)
maltose (MIP)
G2-β-CD (NIP)
β-CD (NIP)
5
4
3
2
1
0
0
0
0
0
0
-3
1
.0×10
maltose (NIP)
-
3
0.8×10
Specific binding
-3
0
.6×10
Non-specific binding
-
3
0
.4×10
-8
-8
-8
-8
-7
0
2
4
6
8
10
12
2.0×10
4.0×10
6.0×10
8.0×10
1.0×10
Time (h)
B (mol)
Figure 4. Binding properties of 6-O-
and maltose on MIP and molecularly non-imprinted polymer (NIP). (
(
α
-D-maltosyl-
β
-cyclodextrin (G2-
β
-CD),
β
-cyclodextrin (
) Binding selectivity;
b) equilibrium binding experiments; (c) binding isotherm; and (d) binding kinetics.
β-CD),
a

Molecules 2017, 22, 288
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The equilibrium binding experiments of the G2-β-CD with different amounts of MIP/NIP
(
molecularly non-imprinted polymer) were provided in Figure 4b. The binding amounts of G2-
and -CD increased rapidly with the concentration of MIP. Meanwhile, MIP has a higher binding
capacity for G2- -CD, -CD, and maltose than NIP at the same concentration. Until the MIP
concentration reached to 3 mg/mL, the binding capacity for G2- -CD was achieved to equilibrium.
β-CD
β
β
β
β
However, there were little binding amounts of maltose while the MIP/NIP concentration increased.
The possible reason was that MIP had generated selective recognition sites in the MIP cavities.
Figure 4c shows the binding kinetics of G2-
different time. The binding amounts of G2- -CD rapidly increased within 30 min, and equilibrium was
achieved in 3 h with a binding capacity of ~62.6% (31.3 nmol). For the maltose, the binding capacity of
MIP/NIP was nearly 0. Compared to MIP, NIP has a much lower adsorption capacity for G2- -CD,
-CD, and maltose. The MIP exhibited much higher binding capacity and faster mass transfer rate
for G2- -CD because the existence of more effective binding sites allowed more G2- -CD to bind
rapidly [17].
The Scatchard analysis [34] of MIP with G2-
in the graph indicated that there were two different binding sites for MIP with G2-
β-CD, β-CD, and maltose on the MIP/NIP versus the
β
β
β
β
β
β-CD was shown in Figure 4d. The two straight lines
β
-CD: non-specific
4
−1
binding and specific binding. The K
a
and Nmax values were calculated and they were 1.72
×
10 M
and the 7.93 µmol·g− , respectively.
1
2
.4. Photoregulated Release and Uptake of G2-β-CD and Maltose by the MIP
Figure 5 showed the changes in the binding amount of crude G2-β-CD in the presence of MIP
or NIP under alternate irradiation at 365 and 440 nm light. UV light (365 nm) irradiation caused the
cis-azobenzene to return to trans-azobenzene [35 36], which was unable to form inclusion complexes
with CDs, leading to the release of CDs from MIP into the solution. Then the subsequent irradiation at
40 nm caused MIP rebound CDs. The MIP has significantly higher adsorption capacity for G2- -CD,
-CD, and maltose than NIP. After the suspension was incubated in a dark environment, the adsorption
,
4
β
β
rates of G2-β-CD by MIP and NIP were 56.48% and 10.2%, respectively. The MIP and NIP towards
β
-CD showed a binding of 37.51% and 6.5%, respectively. The subsequent irradiation at 365 nm led to
the release of G2- -CD and -CD from MIP (20.3% and 12.5%, respectively) and NIP (6.3% and 1.5%,
respectively) into solution. The subsequent irradiation at 440 nm caused a rebound in the G2- -CD
and -CD binding. When repeating irradiation cycles, almost all of G2- -CD were rebounded from
the solution into the MIP with four cycles and 50.76% of G2- -CD was released from the MIP into the
solution with four cycles. The obtained G2- -CD was separated and purified by MIP with the purity
of 90.8% (Figure S1e in the supporting information). The change of the amount of uptake and release
of -CD was 44.39% and 27.52% under irradiation of MIP with 365 and 440 nm light with four cycles.
However, both the MIP and NIP showed lower release and uptake ability of maltose.
β
β
β
β
β
β
β
β

Molecules 2017, 22, 288
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60
40
20
0
a 1
00
b
G2-β-CD (MIP)
G2-β-CD (NIP)
β-CD (MIP)
β-CD (NIP)
maltose (MIP)
80
maltose (NIP)
60
40
G2-β-CD (MIP)
G2-β-CD (NIP)
β-CD (MIP)
β-CD (NIP)
maltose (MIP)
20
maltose (NIP)
0
0
1
2
3
4
1
2
3
4
Irradiation cycles (440 nm)
uptake
Irradiation cycles (365nm)
release
Figure 5. Photoregulated uptake and release of G2-
β-CD,
β-CD, and maltose by MIP and NIP.
(
a
) photoregulated uptake of G2- -CD, -CD, and maltose by the MIPand NIP; (b
β
β
) photoregulated
releaseof G2-β-CD, β-CD, and maltose by the azocontaining MIP and NIP.
3
. Materials and Methods
3
.1. Materials
N,N-Dimethylaminopyridine (DMAP), 2,2-Azobisisobutyronitrile (AIBN), Ethylene glycol
dimethacrylate (EGDMA, 98%), tetramethylsilane (TMS), and triethylamine (TEA) were purchased
from Sigma-Aldrich (St. Louis., MO, USA). G2- -CD was purchased from Sinopharm Chemical Regent
Co., Ltd. (Shanghai, China). All other chemicals consumptions were at the analytical level.
β
3
.2. Synthesis of 4-(Phenyldiazenyl)phenol
-(Phenyldiazenyl)phenol was prepared following a previously reported procedure [37] (Scheme S1
4
in the Supplementary Materials). The solution of concentrated HCl (8 mL) and water (8 mL) was
◦
cooled to 0 C and aniline (2.51 g, 27 mmol) was dropwise added. Then, NaNO (2.00 g, 29 mmol) in
2
water (10 mL) was slowly added into the precooled solution. Next, the solution was constantly stirred
in an ice bath for 25 min. After the mixture, separately, phenol (2.54 g, 27 mmol) was dissolved in 25 mL
of 10% NaOH solution. The prepared solution was slowly added into the diazonium salt solution
◦
with stirring while the temperature was maintained at 5 C for 50 min. The yellow-orange pellet was
collected by filtration and washed by water. The crude solid was recrystallized with an ethanol/water
mixture (yield 74.5%).
3
.3. Synthesis of 4-Methacryloyloxy Azobenzene
The functional monomer MAA was synthesized according to a previously reported method [19
]
with some modifications (Scheme 1). DMAP (0.15 g, 1.0 mmol), TEA (4.30 g, 42 mmol), and PDP
3.96 g, 20 mmol) were dissolved in a 150 mL of anhydrous acetonitrile and cooled over an ice bath,
(
resulting in a dark red mixture. After methacrylic chloride (4.50 g, 40 mmol) was added dropwise,
◦
the solution was stirred at 40 C for 24 h and then cooled to room temperature. After saturated brine
solution (50 mL) was added, the formed precipitate was collected by filtration, washed by 2 mol/L
HCl, and dried with freeze-drying. The purified MAA was collected by recrystallization from boiling
glacial acetic acid, yielding 61.7%.

Molecules 2017, 22, 288
9 of 13
HCl/NaNO
2
NH2
+
OH
N=N
PDP
OH
0
-5
o
C
O
TEA/DMAP 40
O
o
C
N=N
O
MIPT
MIP
MAA
Cl
Removal of
G2- -CD
AIBN EGDMA
N2
β
+
G2-β-CD
UV light
In the dark
or visible light
Scheme 1.
Synthetic route for the photoirradiation molecular imprinting polymer. TEA:
triethylamine; DMAP: N,N-Dimethylaminopyridine; AIBN: 2,2-Azobisisobutyronitrile; EGDMA:
ethylene glycol dimethacrylate.
3
.4. Preparation of the MIP
The MIP was prepared according to a previously method [19] with some modifications.
MAA (0.3990 g, 1.5 mmol), G2-β-CD (2.1889 g, 1.5 mmol), and anhydrous acetonitrile (75 mL) were
added to a conical flask (100 mL). The mixture was stirred at room temperature for 4 h in a dark
environment. Then, EGDMA (0.85 mL, 4.5 mmol) and AIBN (0.0086 g, 0.0525 mmol) were added
into the above system. The reaction mixture was bubbled with nitrogen in an ice bath for 30 min,
◦
then the conical flask was sealed and dipped into a 60 C oil bath. After 48 h of polymerization in
a dark environment, the resultant polymer (MIPT, the imprinted polymer containing the template
form) was collected by centrifugation. The G2-
extraction with water at 110 C for 48 h. The resultant material was dried at 35 C under vacuum for
β
-CD was removed from the MIPT through Soxhlet
◦
◦
2
4 h to obtain MIP. As a control, molecularly non-imprinted polymer was prepared in the absence of
G2-β-CD and treated with the same method.
3
.5. Characterization of PDP and MAA
1H-NMR (400 MHz) was measured by a Bruker Advance III instrument (Bruker, Rheinstetten,
Germany) at room temperature using TMS as an internal standard.
The FTIR spectra of the samples were recorded by an FTIR spectrometer (470 FTIR, Nicolet,
Waltham, MA, USA). The dried samples were mixed with KBr, grounded, and pressed into a pellet.
−
1
FTIR spectra were obtained in the wave number range from 400 to 4000 cm
.
3
.6. Photoisomerization Studies
A UV–Vis scanning spectrophotometer (Shimadzu-2600, Osaka, Japan) was used to record the
UV–Vis spectra of MAA. The photoisomerization of the MAA was investigated by first irradiating
with 365 nm light and then with 440 nm light. The photoisomerization kinetics was analyzed by the
following equation:
A − A∞
0
ln
= kt
(1)
At − A∞

Molecules 2017, 22, 288
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where A , A
t
, and A
∞
are the absorbance of the azobenzene chromophores at their correspondence
0
wavelengths at time 0, t, and at the photostationary stage, respectively; k is the rate constant of the
photoisomerization process.
3
.7. Scanning Electron Microscopy and Transmission Electron Microscopy
The morphology of the MIPT and MIP were observed by a SEM microscope (S-4800, Hitachi,
Tokyo, Japan) at an accelerating voltage of 10 kV. The samples were coated with 10-nm-thick platinum
in the aim of conductivity.
MIPT and MIP images were examined using a TEM microscope (HT-7700, Hitachi Instruments
Ltd., Tokyo, Japan). The powdered MIPT and MIP were diluted and spread onto the copper grids
coated with a carbon-supported film. The copper grids were left aside to stand for freeze-drying.
3
.8. Fourier Transform Infrared Spectroscopy
FTIR spectra of G2- -CD, MIPT, and MIP were recorded by an FTIR spectrometer (470 FTIR,
Nicolet, Waltham, MA, USA). The dried samples were mixed with KBr, grounded, and pressed into
β
−
1
a pellet. FTIR spectra were obtained in the wave range from 400 to 4000 cm
.
3
.9. Thermogravimetric Analysis
The thermogravimetric curves of G2-
Mettler, Toledo, USA). The samples (4.0
crucible under air atmosphere at a flow rate of 20 mL/min. The heating rate was 10 C/min.
β
-CD, MIPT, and MIP were performed by a TGA/SDTA851e
◦
(
±
1.0 mg) were heated from 25 to 600 C with a platinum
◦
3
.10. Binding Selectivity
−
5
We dispersed 3.0 mg of MIP/NIP in the 1.0 mL of 5.0
×
10 mol/L G2-β-CD, β-CD and
◦
maltose, respectively. The suspension was shaken at 25 C in a dark environment for 24 h and filtered
using a 0.22 mm porous membrane. The concentration of G2-
was measured by a high-performance liquid chromatograph (Shimadzu, Kyoto, Japan). The HPLC
condition was as follows: Hypersil NH column (4.6 mm diameter 250 mm), K-2301 refractive index
β-CD and maltose in the supernatant
×
2
detector (RID), mobile phase of acetonitrile—water (70:30 v/v), flow rate of 1 mL/min, temperature of
◦
3
0 C, injection of 10 µL.
3
.11. Equilibrium Binding Experiments
−
5
We incubated 5.0
×
10 mol/L of G2-
β-CD or maltose and different amounts of the MIP/NIP at
◦
2
5 C in a dark environment for 6 h. The amounts of G2-
β-CD and maltose which bound to the MIP
were then measured by HPLC.
3
.12. Binding Kinetics
−
5
We incubated 3.0 mg of MIP/NIP and 1.0 mL of 5.0
×
10 mol/L G2-β-CD or maltose in a dark
◦
environment at 25 C [38]. After different intervals, the suspensions were sampled and filtered using
a 0.22 mm porous membrane. The adsorption dynamics were tested by detecting the amounts of
G2-β-CD and maltose remaining in the solution. The concentration of free G2-β-CD and maltose was
measured by HPLC.
3
.13. Binding Isotherm
10 5–1.5
−
×
−4
10 mol/L,
We incubated 3 mg of MIP/NIP and a series of G2-
β
-CD solutions (C = 1.0
×
◦
1
.0 mL) at 25 C for 6 h. The suspensions were filtered using a 0.22 mm porous membrane, and then
the amounts of G2-β-CD bound to the MIP (B) were measured by HPLC. The equilibrium adsorption
capacity of G2-β-CD by the MIP was calculated through the Scatchard equation:

Molecules 2017, 22, 288
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B
F
=
(Nmax − B)Ka
(2)
a
where F is the amount of free G2-β-CD in the solution, K is the binding association constant and Nmax
is the maximum number of the binding site.
3
.14. Photocontrolled Uptake and Release
The G2- -CD was produced through the reverse action with pullulanase in a reaction mixture
containing of -CD and maltose. The reaction mixture was analyzed by HPLC and five peaks were
observed (Figure S1b in the Supplementary Materials). The -CD has a structure similar to G2- -CD,
it has great significance to separate -CD from the obtained crude G2- -CD by 50% methanol owing
to the different solubility of productions [39]. The -CD could be precipitated from the system easily
as precipitation, and the recovery ratio was around 60% (Figure S1c in the Supplementary Materials).
The further purification of G2- -CD was performed with MIP. The photocontrolled release and uptake
studies of the crude G2- -CD by the MIP/NIP were performed by alternate irradiation under 365
β
β
β
β
β
β
β
β
β
and 440 nm light. 3.0 mg MIP/NIP was dispersed in the 1.0 mL of 0.5 mg/mL crude G2-
solution. After the suspension was incubated at 25 C in a dark environment for 6 h, the samples
β
-CD
◦
were centrifuged to determine the amounts of G2-β-CD, β-CD, and maltose in the supernatant by
HPLC. The precipitate obtained by the centrifugation was dispersed in 1.0 mL of distilled water with
stirring and irradiated at 365 nm light for 4 h. Then, stirring was stopped and the centrifugation was
performed again. The amounts of G2-β-CD, β-CD, and maltose in the supernatant were measured by
HPLC. The precipitate of centrifugation and the supernatant of the first centrifugation conducted were
◦
stirred in the visible light (440 nm). After 6 h of visible light irradiation at 25 C, the samples were
centrifuged to determine their binding. The photocontrolled release and uptake studies (i.e., UV light
on for 4 h and visible light on for 6 h, alternately) were conducted similarly as above. The amounts of
samples in the supernatant were determined as well.
3
.15. Statistical Analysis
The data were represented as the averaging of at least three replications. The data were statistically
analyzed using SPSS 17.0 (SPSS Inc., Chicago, IL, USA). The experimental data were analyzed using
analysis of variance (ANOVA) by the Origin Pro 8.0 statistics program (Originlab, Northampton, MA,
USA) and represented by mean
at a significance level of 95% (p < 0.05).
±
standard deviation. The statistically significant differences were set
4
. Conclusions
The innovative usage of molecular imprinting technology, which is an environmentally friendly
method, for the selective separation of the branched CDs resulted in a good performance at
an affordable cost, thus this process is potentially suitable for lab scale. The MIP was prepared
using G2-
The photoirradiation MIP had a core-shell structure for the photocontrolled uptake and release of
G2- -CD. FTIR and TGA showed that G2- -CD was imprinted into the polymer and removed from
MIP. With alternate irradiation at 365 and 440 nm light, MIP reversibly released and rebound to the
G2- -CD, which resulted in the amount of G2- -CD in the solution being nearly 0. The presented
β-CD as the template. The MIP was used to isolate G2-β-CD from the reaction mixture.
β
β
β
β
method was sensitive, effective, and simple. Therefore, it could be used for the selective separation of
the branched CDs. MIP was an effective method of isolation and purification branched CDs for getting
enough G2-β-CD to gain the industrial preparation and application.
Supplementary Materials: Supplementary materials are available online.
Acknowledgments: This project was supported by the National Natural Science Foundation of China (Grant
No. 31230057, 31401524), the Natural Science Foundation of Jiangsu Province (BK20140143), and Jiangsu Province
Science and Technology Support Program (BE2013311).

Molecules 2017, 22, 288
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Author Contributions: Haoran Fan, Jinpeng Wang, and Zhengyu Jin conceived and designed the experiments;
Haoran Fan performed the experiments; Qingran Meng analyzed the data; Xueming Xu and Tianming Fan
conducted critical revision; Haoran Fan wrote the paper. All authors approved the final manuscript.
Conflicts of Interest: Authors declare no conflict of interest.
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©
2017 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).