Materials Sciences and Applications, 2011, 2, 131-140
doi:10.4236/msa.2011.23017 Published Online March 2011 (http://www.SciRP.org/journal/msa)
131
Design of 2,4-Dichlorophenoxyacetic Acid
Imprinted Polymer with High Specificity
and Selectivity
Kizhakekuthiathottil Mathew Annamma1,2, Beena Mathew1
1School of Chemical Sciences, Mahatma Gandhi University, Kottayam, India; 2Department of Chemistry, Devamatha College, Kot-
tayam, India.
Received July 9th, 2010; revised December 7th, 2010; accepted January 28th, 2011.
ABSTRACT
A widely used herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) was imprinted on poly (4-vinylpyridine) (4-VP) using
(40%) ethyleneglycol dimethacrylate (EGDMA) as crosslinking agent. The classical imprinting technolo gy ma kes use o f
a high degree of crosslinking which does not allow the template molecules to move freely. So the binding sites, lo cated
in the central area of the three dimensional polym er matrix are hard to be accessed and the template molecules canno t
be extracted totally. But here we propose a low crosslinked system with high specificity and selectivity. The imprinted
and non-imprinted polymers were characterized by various spectroscopic techniques. The extent of binding was fol-
lowed by batch equilibration method and compared with the respective non-imprinted polymer. Conditions for maxi-
mum specific rebinding were set by altering certain factors like template/monomer ratio, concentration o f template so-
lution, rebinding medium, mass of polymer and time of incubation. The selectivity of the imprinted polymer was inves-
tigated by comparing the binding with structural analogues of 2,4-D like, phenoxyacetic acid (POA), 4-chlorophen-
oxyacetic acid (4-CPOA) and 2,4,5-trichlorophenoxy acetic acid (2,4,5-T). The imprinted polymer exhibited high affinity
towards the template molecule and was selectively rebound to the specific sites. The binding towards the structural
analogues depends on the number of chlorine in the benzene ring.
Keywords: Molecular Imprinting, 2,4-Dichlorophenoxyacetic Acid, Specificity, Selectivity, Sepa ration Factor
1. Introduction
The agricultural efficiency of the world depends on the
effective control of a variety of diseases and pests, espe-
cially weeds. Herbicides are the chemicals that are widely
used in agricultural field for controlling the growth of
herbs, weeds and bushes. Thus the herbicides and their
hydrolysis products are the most abundant pollutants
found in the environment and in agricultural products.
Concern about the health hazards connected with pesti-
cide use has focused on 2,4-D and 2,4,5-T as suspected
cancer-causing agents. Molecular imprinting is a tech-
nique for preparing synthetic polymers possessing recog-
nition sites complementary to a template molecule [1-3].
MIPs can be used for the removal of pesticides, herbi-
cides, endocrine disrupting compounds and heavy metals
from waste and drinking water [4]. The high selectivity
of MIPs for organic compounds, along with other useful
properties opens up wide opportunities for the use of
these materials in analytical chemistry [5-7]. Attempts
were made previously to imprint herbicides in polymers
and to use them in various applications [8-10]. But the
traditional synthesis results in a highly crosslinked rigid
structure, which does not allow the template molecules to
move freely. So the binding sites, located in the central
area of the three dimensional polymer matrix are hard to
be accessed and the template molecules cannot be ex-
tracted totally. Hereafter, many imprinting strategies have
been introduced to solve the problems faced by the tradi-
tional MIPs [11-15]. The reported techniques for herbi-
cide detection using MIPs require high crosslinking, so-
phisticated instruments, skilled technician and longer
analysis time. The most common methods of herbicide
detection are gas chromatography with mass spectrome-
try detection (GC-MS) and HPLC. These methods re-
quire one or more preconcentration steps for trace level

Design of 2,4-Dichlorophenoxyacetic Acid Imprinted Polymer with High Specificity and Selectivity 132
detection and quantification of herbicides due to insuffi-
cient sensitivity of these methods. In the present work
high specific and selective recognition is given by low
crosslinked polymers. Further the UV-visible spectro-
photometric method is technically simple and can per-
form easily. The present paper deals with the synthesis of
low crosslinked (40%) 2,4-dichlorophenoxyacetic acid
(2,4-D) imprinted 4-vinylpyridine (4-VP) polymer, and
the specificity and selectivity studies. The imprinted
polymers were prepared by non-covalent method of im-
printing polymerization in the presence of polar solvents
like methanol and water. Non-imprinted polymers were
also prepared without the template. Factors affecting
specificity and selectivity were also discussed. The re-
binding ability and selectivity performance of the poly-
mer was followed by UV-vis. spectrophotometric method.
The nature of binding sites was followed by Scatchard
analysis.
2. Experimental
2.1. Materials and Methods
4-chlorophenoxyacetic acid (4-CPOA), 2,4-dichlorophen-
oxyacetic acid (2,4-D), 2,4,5-trichlorophenoxyacetic acid
(2,4,5-T), and 4-vinylpyridine (4-VP) were obtained from
Sigma-Aldrich (USA). Ethyleneglycol dimethacrylate
(EGDMA) was from Merck (Germany). Phenoxyacetic
acid (POA) and 2,2’-azobisisobutyronitrile (AIBN) were
purchased from SRL (Mumbai). Methanol, acetonitrile
and chloroform were obtained from Merck (India). 4-
vinylpyridine was distilled under reduced pressure prior
to use. Doubly distilled water was used throughout. The
imprinted polymers were characterized by FT-IR (8400 S,
Shimadzu), UV-vis. (Shimadzu 2450), and 1H (BRUKER
AMX-400) and 13C CP-MAS-NMR (BRUKER DSX-300)
spectrometer.
2.2. Synthesis of EGDMA-Crosslinked 2,4-D
Imprinted and Non-Imprinted Polymers in
Methanol/Water
EGDMA-crosslinked 2,4-D imprinted poly (4-vinylpyri-
dine) with 1:1, 1:2 and 1:4 ratios of 2,4-D and 4-vinyl-
pyridine were prepared (Table 1). The template 2,4-D (1
mmol), functional monomer (4-VP), (4 mmol), required
amount of the crosslinking agent ethyleneglycol dime-
thacrylate and the initiator AIBN (0.32 mmol) were
weighed into glass tubes and dissolved in10 ml solvent
(methanol/water, 4:1, v/v). The resulting mixture was
purged with nitrogen for 15 min. The tubes were then
sealed and inserted in a water bath at 60˚C for 4 h, fol-
lowed by 2 h at 70˚C (Figure 1). The resultant hard bulk
polymers were ground in a mechanical mortar and wet-
sieved in acetone through a 25 m sieve. Non-imprinted
Figure 1. Synthesis of 2,4-D imprinted and non-imprinted
polymers.
polymers were also prepared using the same recipe but
without the addition of the template.
2.3. Extraction of Imprinted Template: General
Procedure
The fine polymer particles were carefully washed by in-
cubation in methanol/acetic acid (7:3, v/v, 2x), acetoni-
trile/acetic acid (9:1, v/v, 2x), acetonitrile (1x), methanol
(2x) for 2 h and Soxhlet extracted with methanol until no
template could be detected under UV (max = 284 nm) in
the wash solution. The particles were then suspended in
acetone and allowed to settle for 4 h. The solvent was
removed by centrifugation and the particles were dried to
constant weight in vacuum.
2.4. Swelling Studies
Fixed amount of the polymer particles were packed into
sintered crucibles, which were filled with different sol-
vents. After 24 h of equilibration, the excess solvent was
removed from the polymer by applying reduced pressure
for 1 min. and the weight of the swollen particles (Ww)
was measured. Subsequently the particles were freeze-
dried for 24 h and were weighed again to obtain the dry
weight (Wd). The swelling ratio (SR) of the polymer in
each solvent was calculated from the following equation:
SR = (Ww − Wd)/Wd.
2.5. Rebinding of Template: General Procedure
The template-desorbed polymers were treated with solu-
tions of the desorbed template and the extent of rebinding
was followed by UV measurements at 284 nm. The poly-
mer particles were put into sample tubes, and the template
solutions of known concentrations were introduced and
equilibrated for a period of time. After this incubation, the

Design of 2,4-Dichlorophenoxyacetic Acid Imprinted Polymer with High Specificity and Selectivity 133
Table 1. Synthesis of 40% EGDMA-crosslinked MIPs and NIPs with varying 2,4-D/4 VP ratio.
Yield (%)
Ratio of 2,4-D:4-VP 2,4-D (1 mmol) 4-VP EGDMA
MIP NIP
1:1 0.221 g 0.11 ml (1 mmol) 0.14 ml (0.75 mmol) 35 45
1:2 0.221 g 0.22 ml (2 mmol) 0.28 ml (1.5 mmol) 65 68
1:4 0.221 g 0.44 ml (4 mmol) 0.56 ml (3 mmol) 72 75
polymer particles were filtered off, and the remaining
concentrations of the template were determined spectro-
photometrically. The amount of template bound to the
polymer Sb (mM/g polymer) was calculated by the equa-
tion, Sb = (Co – Ct)/W. Where Co and Ct are the 2,4-D
molar concentration in the solution at initial and after
interval time ‘t’, respectively. ‘W’ is the weight of the dry
polymer used.
2.6. Optimization of the Conditions of 2,4-D
Rebinding
The optimum conditions for maximum 2,4-D rebinding
were set by changing certain factors like templatemonomer
ratio, rebinding solvent, time, concentration of 2,4-D
solution and mass of polymer.
2.7. Factors Affecting Rebinding
2.7.1. Concentration of 2,4-D Solution
The influence of concentration of template solution on
rebinding was evaluated by batch binding experiments.
From the difference in concentration of template solution
before and after incubation, the amount of template bound
by the polymer was determined.
2.7.2. Rebinding Solvent
The template solutions were prepared in different solvents
and the rebinding in each solvent was determined spec-
trophotometrically.
2.7.3. Time of Rebinding
The time required for the saturation of binding sites was
determined by measuring the absorbance at regular in-
tervals of time till saturation was attained at 30˚C.
2.7.4. Mass of Polymer
Varying amounts of polymers were introduced into equal
volume of template solution for a fixed time and rebinding
was followed.
2.8. Selectivity Studies: General Procedure
The selectivity of imprinted polymers were evaluated by
incubating template desorbed imprinted polymer in solu-
tions of structural analogues for definite time and the
rebinding was followed spectrophotometrically. Separa-
tion and selectivity factors were also determined.
3. Results and Discussion
3.1. Synthesis of EGDMA-Crosslinked 2,4-D
Imprinted and Non-Imprinted Polymers
with Varying Composition of Functional
Monomers and Template
EGDMA-crosslinked 2,4-D imprinted and non-imprinted
polymers with varying template-monomer ratio were
synthesized by free radical polymerization (Figure 1).
3.2. Characterization of 2,4-D Imprinted and
Non-Imprinted Polymers
3.2.1. FT-IR
The incorporation of EGDMA in both imprinted and
non-imprinted polymers was supported by the IR spectra.
The bands at 1396 and 1458 cm−1 correspond to C–H
bend of CH3 and CH2 respectively. The C=O stretch of
the EGDMA crosslinking was observed at 1647 cm−1.
The IR spectrum of 4-vinylpyridine showed an intense
band at 1596 cm−1 due to the C=N stretching. The spec-
tral characteristics of 4-vinylpyridine changed upon addi-
tion of 2,4-D. The 2,4-D/4-vinylpyridine complex re-
vealed a new band at 1546 cm−1 within the C=N mode.
The peaks at 1596 and 1546 cm−1 were assigned to mono-
meric 4-vinylpyridine and H-bonded 4-vinylpyridine
respectively. The C=O and O–H stretching of the car-
boxyl group of 2,4-D at 1735 and 3263 cm−1 were also
shifted and broadened on complexation with 4-vinylpy-
ridine. These changes strongly suggest the formation of
intermolecular H-bonding between the carboxylic group
of 2,4-D and the nitrogen of 4-vinylpyridine.
3.2.2. 1H NMR
The pre-polymerisation complex formation during the
preparation of molecular imprinted polymers can be in-
vestigated by 1H NMR. Due to the low solubility of
2,4-D in chloroform, the 1H NMR titration studies of the
2,4-D/4-VP system were performed in CD3OD. The
study was conducted by adding increasing amounts of
4-VP into 2,4-D solution and the spectra were recorded
after each addition. Because of the rapid exchange be-
tween labile proton of the template and solvent deute-

Design of 2,4-Dichlorophenoxyacetic Acid Imprinted Polymer with High Specificity and Selectivity 134
rium atom, the signal due to acidic proton of the carbox-
ylic acid is not seen in the spectrum. Therefore the
change in chemical shifts of the aromatic and CH2 pro-
tons of 2,4-D as well as aromatic protons of 4-VP were
investigated (Figure 2). As a result of ion-pair formation
between basic nitrogen and acidic proton, the chemical
environment of H2 and H6 (8.8 ppm), H3 and H5 (7.6
ppm) of 4-VP changes and the peaks corresponding to
them were shifted upfield. An upfield shifting of the
peaks corresponding to the aromatic protons of 2,4-D
(H3 - 7.7, H5 - 7.5, H6 - 7.2 ppm) was also observed and
attributed to the participation in - stacking interactions.
The signals due to CH2 protons of 2,4-D (4.9 ppm) was
broadened along with upfield shifting and gradually the
peaks disappeared due to complex formation with 4-VP.
The results confirmed the existence of a 1:3 (2,4-D/4-VP)
complex prior to polymerisation.
3.2.3. 13C CP-MAS-NMR
The incorporation of crosslinking agent and functional
monomer 4-VP in the polymer backbone of imprinted
polymer was confirmed by 13C NMR spectrum (Figure
3). The ester carbonyl of the EGDMA crosslinking was
characterised by the strong peak at 178.8 ppm. The small
peak at 65.0 and the one at 16.7 ppm correspond to the
OCH2 and CH3 carbons of EGDMA. The intense peak at
2,4-D 4-VP
Figure 2. 1H NMR spectra of 2,4-D with increasing concen-
tration of 4-VP.
Figure 3. 13C NMR spectrum of EGDMA-crosslinked im-
printed polymer.
42.8 ppm corresponds to CH2 carbon of the polymer
backbone.
3.3. Determination of Swelling Ratio (SR)
In order to determine the swelling ratios of 2,4-D im-
printed and non-imprinted polymers, the polymers were
immersed in four different solvents such as acetonitrile,
methanol, methanol/water and water. The dispersed poly-
meric particles were recovered by vacuum filtration and
the swollen weight (Ww) of the particles was measured.
Subsequently, the particles were dried and weighed again
to obtain the dry weight (Wd). The swelling ratio [16] of
the polymer was calculated as given below and summa-
rised in the experimental part.
The polymer exhibited higher swelling in methanol-
water, the porogen. Here the swelling ratios of non-im-
printed polymers are higher than that of the imprinted
polymers. This is due to the reluctance of the crosslinked
polymer matrix to expand from the designed geometry
around the template molecule. Whereas there is no such
complementary cavity and framework in the non-im-
printed polymer and so it could undergo extensive swell-
ing. The swelling ratio of both imprinted and non-im-
printed polymer in water is very high compared to other
solvents. This anomalous behaviour in water is due to the
possibility of extensive H-bonding with the nitrogen in
4-vinylpyridine.
3.4. 2,4-D Rebinding Studies
The methodology of molecular imprinting induces the
formation of recognition sites with predetermined selec-
tivity into a synthetic matrix. After removal of the tem-

Design of 2,4-Dichlorophenoxyacetic Acid Imprinted Polymer with High Specificity and Selectivity 135
Table 2. Swelling ratios of 2,4-D imprinted and non-im-
printed polymers.
Swelling Ratio
Solvent
MIP NIP
CH3CN 0.15 0.28
CH3OH 1.72 0.97
CH3OH/H2O 2.25 4.63
H2O 7.09 4.30
plate, complementary binding sites are revealed and a
molecular memory is introduced into the polymer, capa-
ble of rebinding the template with high specificity and
selectivity [3,4,17]. According to this principle of mo-
lecular imprinting, the 2,4-D imprinted polymers must
specifically rebind the print molecule than the non-im-
printed polymers. The imprinted and non-imprinted poly-
mers were investigated for their rebinding capacity by
incubating a fixed amount of each polymer in template
solution of known concentration and equilibrated for a
period of time ‘t’. The concentration of template solution
before and after incubation was determined spectropho-
tometrically. The amount of template bound to the poly-
mer [S]b (mM/g) was calculated as discussed earlier.
Specificity in 2, 4-D Rebinding by Imprinted Polymers
The 2,4-D imprinted polymers showed high specificity
towards the template than the non-imprinted polymer.
The investigation was done by comparing the specific
binding of imprinted polymer (MIP) with a non-im-
printed polymer (NIP) of same crosslinking (40%). Equal
amounts of MIP and NIP were introduced into 0.9 mM
solution of 2, 4-D for a definite time and the amount of
2,4-D bound was determined. From the difference in
binding by the MIP and NIP, it is evident that the im-
printed polymer has a significant affinity towards 2,4-D
than the non-imprinted polymer. Further, the specificity
shown by the imprinted polymer supports the memory of
the polymer towards the print molecule (Figure 4).
3.5. Template-Monomer Ratio on 2,4-D
Rebinding
It has been found that the molar relationship between the
template and functional monomer influences the number
and quality of MIP recognition sites. Hence 2,4-D im-
printed and non-imprinted polymers were prepared in 1:2
and 1:4 template-monomer ratio. To investigate the in-
fluence of this ratio on rebinding, imprinted and non-
imprinted polymers were incubated in equal volume of
2,4-D solutions of definite concentration for a fixed time.
After filtration the 2,4-D binding was followed spectro-
photometrically. The results are summarised in Figure 5.
The 1:4 system offered high specific binding com-
pared to the 1:2 system. In order to accomplish highly
efficient imprinting, the template-monomer adduct must
exist in large excess with respect to free template and
functional monomer. Otherwise non-selective polymeri-
sation would occur concurrently and diminish the effi-
ciency of molecular imprinting. Thus the 1:4 system is
more specific than the 1:2 system. Hence for further
studies the imprinted and non-imprinted polymers in the
1:4 template-monomer ratios were used.
3.6. Optimisation of the Conditions of 2,4-D
Rebinding
There is the need to optimise the conditions for maximum
template rebinding by the imprinted polymers. The total
Figure 4. Specificity in 2,4-D rebinding by 40% EGDMA-
crosslinked MIP and NIP.
Figure 5. Effect of template-monomer ratio on 2,4-D re-
binding by MIP and NIP.

Design of 2,4-Dichlorophenoxyacetic Acid Imprinted Polymer with High Specificity and Selectivity 136
rebinding to a MIP is the sum of specific binding to the
imprinted binding sites and non-specific binding to the
polymer. The non-specific binding to the polymer is
measured as the binding, under identical conditions to a
non-imprinted polymer. If this non-specific binding to the
polymer is dominant, specific binding to the imprinted
sites will be obscured. Thus the ratio of the specific to
non-specific binding should be optimised to observe the
binding at the imprinted sites.
3.6.1. Effect of Concentration of 2,4-D Solution
To evaluate the variation of rebinding with concentration
of 2,4-D solution, batch methods were carried out using
stock solutions of 2,4-D with concentrations ranging
from 0.3 to 1.5 mM. Definite amount of MIPs were in-
cubated in these solutions for a fixed interval of time and
the amount of 2,4-D bound at the saturation point [S]b,
was calculated as described earlier. It was plotted against
initial concentration Co and is given in Figure 6.
The extent of 2,4-D binding increased with concentra-
tion up to 0.9 mM and then remained constant. Thus the
binding attained a saturation point at 0.9 mM. The indi-
vidual cavities in the polymer vary in their affinity and
selectivity towards the template [18]. The binding of the
template molecules to high affinity sites concentrated
inside the polymeric domains led to the increase in spe-
cific binding, which later resulted in shape change of the
polymer network. Binding sites on the surface of the
polymer bound the template without causing much reor-
ganisation of the polymer structure.
3.6.2. Determination of Binding Parameters and
Guest Binding Constants
In the molecular imprinting technique, Scatchard model
was often used to evaluate the binding characteristics of
the imprinted polymers [19,20]. The binding data ob-
tained were treated with Scatchard equation [S]b/[S]f =
(Smax − [S]b)/KD, where KD is an equilibrium dissociation
constant of the binding site, Smax an apparent maximum
number of binding sites and [S]b is the amount of 2,4-D
bound to MIP. [S]f is the fraction of 2,4-D left in solution.
The plot of [S]b/[S]f against [S]b is shown in Figure 7.
The Scatchard plot is not linear and composed of two
straight lines indicating that the binding sites in the
polymer matrix are heterogeneous in respect to the affin-
ity for 2,4-D. Thus two classes of binding sites are
mainly produced in the studied template concentration.
From the slope and intercept of the straight lines obtained,
the values of KD and Smax are determined, where KD1 (50
M/L) and KD2 (201 M/L) are the dissociation constants,
and Smax1 (12.5 mM/g) and Smax2 (19 mM/g) are the ap-
parent maximum number of binding sites at high and low
affinity sites. Since the dissociation constant at high af-
finity site (KD1) is lower than that at low affinity site,
Figure 6. Binding isotherm for the binding of 2,4-D by MIP.
Figure 7. Scatchard plot to determine the nature of binding
sites.
guest binding at high affinity site is stronger.
3.6.3. Effect of Rebinding Medium
Molecular imprinted polymers utilise a solvent in po-
lymerisation to provide porosity within the network poly-
mer. Binding properties of molecular imprinted polymers
are influenced by the type of solvent, or porogen, used in
the polymer synthesis and the solvent used in the par-
ticular application of the MIP [21]. A number of reports in
the literature indicated that the best recognition of im-
printed polymer occurs when the rebinding medium and
the porogen used are the same [22,23]. The ability of the
rebinding medium to recreate the binding site dimensions
formed during the polymerisation determines the binding
performance of the imprinted polymer in that medium. To
determine the effect of binding medium on specific
binding properties, imprinted and non-imprinted polymers
were equilibrated with solutions of 2,4-D in acetonitrile,
methanol, methanol/water, and water. The EGDMA cross-
linked polymer offered maximum specificity in metha-
nol/water, the porogen. The high binding observed in
water is due to a partitioning effect of the lipophilic 2,4-D
into the hydrophobic polymer matrix. The results are given

Design of 2,4-Dichlorophenoxyacetic Acid Imprinted Polymer with High Specificity and Selectivity 137
in Figure 8.
3.6.4. Effect of Time
To optimise the time taken for maximum binding of 2,4-D
by MIP and NIP, definite amounts of the polymer was
equilibrated with 2,4-D solution of known concentration
and the binding was followed spectrophotometrically at
definite intervals of time (Figure 9). The imprinted
polymers took more time for saturation of binding sites
compared to the non-imprinted polymers. The imprinted
polymers possessing shape complementary binding sites
took more time to attain saturation, since the template
molecules have to penetrate through the highly cross-
linked polymer network to access the imprinted cavities
for rebinding. In non-imprinted system, there is no such
specific arrangement of the binding sites, and non-spe-
cific interactions occur only at the surface of the polymer,
the binding of template is rather fast and random. The
MIP attained saturation within 4-6 h. So it is not neces-
sary to equilibrate the polymers for 24 h for getting the
imprinted sites saturated.
Figure 8. Effect of solvent on 2,4-D rebinding by EGDMA-
crosslinked MIP and NIP.
Figure 9. Time dependence on 2,4-D rebinding by MIP and
NIP.
3.6.5. Amount of Polymer
Increasing the amount of polymer is expected to provide
more binding sites for a substrate and as a result the
amount of the substrate bound is also expected to increase.
The effect of the mass of polymer on rebinding was ob-
served by incubating varying amounts of MIP and NIP in
fixed volume of 0.9 mM 2,4-D solution. As the amount of
polymer is increased, the amount of bound substrate also
increased (Figure 10). When higher amount of polymer
was used the substrate bound to the higher affinity binding
sites in preference to the low affinity binding sites. The
increase in the number of binding sites with increase in
mass of the polymer leads to the increase in specificity.
But the substrate bound per gram decreased. This is due to
the possible changes in binding parameters with the dif-
ferent amounts of the polymers used in the batch rebind-
ing studies. The non-imprinted polymers bound signify-
cantly less template than the imprinted polymers.
3.7. Selectivity of 2,4-D Imprinted Polymers
3.7.1. Template Selectivity
The selectivity of MIP is based on the configuration of the
binding sites and the orientation of the functional groups
in the sites. Three structural analogues of 2,4-D, viz.
phenoxyacetic acid (POA), 4-chlorophenoxyacetic acid
(4-CPOA) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T)
were used to evaluate the selectivity of the imprinted
polymer. The molecular structures of these compounds
are given in Figure 11.
The molecules related to 2,4-D in the acidic part and
different in the aromatic part were recognised by the im-
printed polymers, but to a lesser extent than the template
(Figure 12). Since the molecules exactly matching the
template molecule in the carboxylic substructure can be
recognised by the imprinted polymer, it is clear that this
interaction is strongly governed by the ion pair formation.
It is also observed that molecules resembling 2,4-D in the
Figure 10. Effect of the mass of polymer on 2,4-D rebind-
ing by MIP and NIP.

Design of 2,4-Dichlorophenoxyacetic Acid Imprinted Polymer with High Specificity and Selectivity 138
OCOOH
Cl
Cl
Cl
OCOOH
Cl
Cl
OCOOH OCOOH
Cl
POA 4-CPOA 2,4-D 2,4,5-T
Figure 11. Molecular structures of 2,4-D and its structural analogues.
Table 3. Separation factor of MIP and NIP to 2,4-D.
MIP NIP
EGDMA (%) 2,4-D bound
× 10−4 M
2,4-D free
× 10−4 M KMIP 2,4-D bound
× 10−4 M
2,4-D free
× 10−4 MKNIP
Separation factor
α 2,4-D = KMIP/KNIP
40 2.87 6.12 0.468 1.03 7.97 0.128 3.65
Table 4. Selectivity factors of EGDMA-crosslinked MIP.
α 2,4-D α POA α CPOA α 2,4.5-T α 2,4-D/α POA α 2,4-D/α MCPOA α 2,4-D/α 2,4.5-T
3.65 2.00 3.12 3.40 1.82 1.20 1.15
Figure 12. Selectivity between MIP and NIP.
carboxylic substructure, but differ in the substitution on
the aromatic ring are recognised proportionally through
the similarity with 2,4-D. 4-CPOA with only one chlo-
rine atom on the ring is more recognised than POA with
no chlorine atom on the ring. The polymers bound 2,4,5-T
nearly as well as the original template 2,4-D. This may
be due to the accommodation of 2,4,5-T molecule in the
flexible EGDMA-crosslinked networks. The increase in
binding with increasing number of chlorine atoms on the
aromatic ring is reported [24]. The existence of weak
secondary interactions between the ring chlorine and hy-
drogens of 2,4,5-T with 4-VP is suggested by modelling
[25].
3.7.2. Separation and Selectivity Factors of 2,4-D
Imprinted Polymers
A complete secondary screen for binding and selectivity
was performed and binding of the template to the im-
printed and non-imprinted polymers was compared in
terms of separation factor [26]. The high separation fac-
tor (α 2,4-D) of 40% EGDMA-crosslinked polymer proved
the high efficiency of the imprinted system (Table 3).
Separation factor (α 2,4-D) = KMIP/KNIP
Selectivity factor [27] = α 2,4-D/α analogue
The imprinted system has a selectivity factor greater
than unity in all cases and so this system can be used for
the selective recognition of 2,4-D (Table 4).
4. Conclusions
The technique of molecular imprinting leads to highly
stable synthetic polymers possessing selective molecular
recognition properties. In the present work priority is
given to the design of imprinted polymers of the herbi-
cide 2,4-D with maximum specificity and selectivity.
Among the polymers prepared in 1:1, 1:2 and 1:4 tem-
plate-monomer ratios, the 1:4 system exhibited better
specificity and selectivity. The present results proved that
imprinted polymers can very well retain the imprinted
site at moderate extent of crosslinking and can recognise
the template with significant specificity. A crosslinking
of 40% is sufficient for 2,4-D imprinted polymers in
methanol/water. The amount of template binding in-
creased regularly with increasing concentration of the
template solution and attained saturation. From the
Scatchard plots the binding sites are found to be hetero-
geneous in nature with respect to the affinity towards the
template in the studied concentration range. The MIPs
took more time for saturation of binding sites than NIPs

Design of 2,4-Dichlorophenoxyacetic Acid Imprinted Polymer with High Specificity and Selectivity 139
and overnight equilibration is not necessary for getting
the sites saturated. Selectivity studies with structural
analogues revealed that in addition to complementarity in
size and shape, optimal spatial fit also had a role in bind-
ing interactions. The present work in molecular imprint-
ing provided an alternative method against the conven-
tional analytical methods. In the present work high spe-
cific and selective recognition is given by low cross-
linked polymers. Further the UV-visible spectrophoto-
metric method is technically simple and can perform
easily. The attempts made in this work led to the suc-
cessful design of 2,4-D imprinted polymers with high
specificity and selectivity.
5. Acknowledgements
Annamma is grateful to the University Grants Commis-
sion (UGC) for the award of Teacher Fellowship.
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