Anal Bioanal Chem (2008) 390:1101–1109
DOI 10.1007/s00216-007-1765-2
REVIEW
Chromatographic characterization of molecularly
imprinted polymers
Wen-Chien Lee & Chung-Hsien Cheng &
Hsin-Hung Pan & Ting-Hao Chung &
Ching-Chiang Hwang
Received: 10 August 2007 / Revised: 15 October 2007 / Accepted: 22 November 2007 / Published online: 30 December 2007
# Springer-Verlag 2007
Abstract Recent efforts in the investigation of chromato-
graphic characterization of molecularly imprinted polymers
(MIPs) have focused mainly on the nature of heterogeneous
binding sites. More data on the thermodynamics than on the
kinetic features of MIP columns have been published. The
present article addresses the sources of peak broadening
and tailing, which are the main drawbacks often associated
with imprinted polymers in chromatography for practical
applications. With use of the theory of nonlinear chromatog-
raphy, the peak properties of a MIP column, including the
retention and peak broadening and tailing, can be well
interpreted. Efforts to improve chromatographic efficiency
using MIPs prepared by approaches different from the
conventional method, including covalent imprinting and the
format of uniformly sized spherical microbeads, are reviewed
and discussed. This review leads to the conclusion that
nonlinear chromatography theory is useful for characterizing
chromatographic features of MIP columns, since a MIP is
essentially an affinity-based chromatographic stationary
phase. We expect more theoretical and experimental studies
on the kinetic aspects of MIP columns, especially the factors
influencing the apparent rate constant, as well as the analysis
of the influences of mobile-phase composition on the
W.-C. Lee (*) : C.-H. Cheng : H.-H. Pan : T.-H. Chung
Department of Chemical Engineering,
National Chung Cheng University,
Chiayi 621, Taiwan
e-mail: chmwcl@ccu.edu.tw
C.-C. Hwang
Department of Life Science, Mingdao University,
Chang-Hua 523, Taiwan
chromatographic performance. In addition to revealing the
affinity interaction by molecular recognition, slow nonspe-
cific interactions which may be inherited from the imperfect
imprinting and may be involved in the rebinding of the
template to MIPs also need to be characterized.
Keywords Molecularly imprinted polymers .
Liquid chromatography . Nonlinear chromatography .
Band broadening . Peak asymmetry
Introduction
Molecularly imprinted polymers (MIPs) have been widely
studied with regard to highly selective stationary phases in
liquid chromatography, especially for chiral separation. How-
ever, excessive peak broadening and tailing in the chroma-
tography of the more retained compound (normally the
template) are often observed with MIPs at the stationary
phases. The improvement of the chromatographic perfor-
mance in liquid chromatography is thus a challenge for using
MIPs as promising materials [1]. During recent years, many
efforts have been made to apply MIPs to capillary electro-
chromatography rather than liquid chromatography [2, 3].
The chromatographic separation which results in severe
peak broadening and tailing has been often attributed to the
heterogeneity of the binding sites in the MIPs. To address
the problems of imperfect peak profile, this paper gives a
review of the investigation of chromatographic performance
with molecularly imprinted phases through nonlinear chro-
matography theory. Recent papers suggest that nonlinear
chromatography and its associated nonlinear adsorption
isotherm may be a good starting point for understanding
and characterizing MIPs [4–6].
1102
Nonlinear chromatography theory for MIPs
as the stationary phase
The theory of nonlinear chromatography provides models
to describe the complicated thermodynamics and mass-
transfer kinetics involved in chromatographic separation of
solutes in the column. When axial dispersion is negligible,
the chromatographic mass balance and kinetic equations are
written as follows [7]:
@C @q @C
" þ ð1
" Þ þ u0 ¼ 0;
@t @t @z
@q
¼ ka ðqm
qÞC
kd q:
@t
These two equations describe the changes in solute con-
centrations in the mobile phase (C) and stationary phase (q)
with time (t) as well as the axial coordinate along the
column (z). The parameters ɛ and u0 are the total void
rffiffiffiffiffiffiffiffiffiffi h
0 qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffii
k0 * 0 δðτ
1Þ
ðτ
1Þ 1 I 2k d k 0 ð τ
1 Þ þ *
1 kd
C ðt; z ¼ LÞ ¼
K h i
0 *
*
1
T k 0 k d ; k d ð τ
1Þ 1
e
Ru pffiffiffiffiffi
where T ðu; vÞ ¼ e
v 0 e
s I0 ð2 νsÞds, δ is the delta func-
tion, and I0 and I1, respectively, are the first and second
modified Bessel functions. In Eq. 4, K (ka/kd) is the
equilibrium association constant under the elution condi-
0
tions and k0 ¼ ð1
"Þqm K=" is the limiting retention factor,
i.e., the retention factor at zero sample concentration. The
rate constant kd appears in Eq. 4 as a time constant charac-
terizing the adsorption/desorption process. The nondimen-
sional variable and parameter in these equations are defined as
τ=t/t0 and kd* ¼ kd t0 , where t0 (ɛL/u0) is the time required to
pass through the void volume of a column with length L.
Equation 4 describes the dependence of the elution profile on
the loading factor (Lf), and two additional parameters, K and
kd* . The loading factor is equal to the ratio of the amount of
solute injected into the column (C0Vinj) to the saturation
capacity of the column [AcL(1-ɛ)qm], where Ac is the cross-
sectional area of the column [8]. With use of Eq. 4, Baggiani
et al. [4] recently employed different nomenclatures for the
investigation of chromatographic behavior of columns packed
separately with pellicular imprinted beads and with an
imprinted polymer prepared by bulk polymerization.
However, the Langmuir isotherm is somewhat limited
because this isotherm assumes the presence of homogenous
binding sites. MIPs are usually characterized by heteroge-
neous materials containing binding sites with a wide array
Anal Bioanal Chem (2008) 390:1101–1109
fraction and the superficial velocity, respectively. The
chromatographic nonlinearity originates from the kinetics
for the net rate of adsorption according to Eq. 2, describing
the adsorption and desorption processes for the solute onto
the stationary phase. In Eq. 2, qm denotes the maximum
binding capacity of the adsorbents, while ka and kd are the
apparent adsorption and desorption rate constants. The
model consisting of Eqs. 1 and 2 is useful for characterizing
affinity and other adsorption systems, in which the Langmuir
isotherm is followed. That is, in equilibrium conditions, the
ð1Þ following relation is holds:
qm KC
q¼ : ð3Þ
1 þ KC
ð2Þ This nonlinear chromatography model which is based on
the Langmuir isotherm provides an analytic solution for the
elution peak profile. For the elution with an impulse input,
for example, the solution for the model consisting of Eqs. 1
and 2 has been derived by Wade et al. [7] as
!  

k0 kd*
kd* ðτ
1Þ
k0 kd* Lf
0 0
e 1
e
 ; ð4Þ

k0 kd* Lf
0
of binding affinities and selectivities. The affinity (energy)
distribution function, also called the affinity spectrum, is
widely used to describe the heterogeneity of binding sites.
An affinity distribution is a plot of the number of binding
sites against the association (binding) constant (K). With
use of the experimental data of adsorption isotherms, the
affinity distribution describing a continuous distribution of
binding sites in the MIPs prepared by either covalent or
noncovalent imprinting can be theoretically calculated [9].
Continuous heterogeneous binding models such as the
Freundlich and the Langmuir–Freundlich (LF) isotherms
can well characterize MIPs and also be useful for the
calculation of the corresponding binding parameters and
affinity distributions [10]. Although the Freundlich isotherm
provides a good mathematical approximation of the binding
characteristics for noncovalently imprinted polymers [11],
the LF isotherm model is more commonly applied for
adsorption of a print (template) molecule to imprinted sta-
tionary phases, prepared by either covalent or noncovalent
imprinting [12, 13]:
qm aC m
q¼ : ð5Þ
1 þ aC m
Compared with the Langmuir model, the LF model
isotherm offers the advantage that the heterogeneity in
Anal Bioanal Chem (2008) 390:1101–1109
binding sites can be taken account. The parameter a in the
LF model is related to the binding constant, while the
parameter m is the heterogeneity index, varying from 0 to 1.
For a homogeneous material, m=1. When m<1, the material
is heterogeneous. To show the relationship between the LF
and Langmuir models, Eq. 5 can be rewritten as
qm ðaC m
1 ÞC
q¼ : ð6Þ
1 þ ðaC m
1 ÞC
Equation 6 indicates that the apparent association constant
K (aCm−1) is concentration-dependent if the Langmuir
model (Eq. 3) is enforced to represent the adsorption
behavior at every specified solute concentration. Since the
parameter m falls in the range between 0 and 1, a decrease
of the apparent binding constant with increasing solute
concentration is expected for the system when the LF
isotherm is valid. Results from Baggiani et al. [4] show that
the apparent binding constant decreased with increasing
solute concentration whether molecularly imprinted beads
or bulk polymer was used as the stationary phase.
Sajonz et al. [14] and Kim and Guiochon [15] used the
bi-Langmuir model to represent the heterogeneous binding
sites. The bi-Langmuir model with the best isotherm data
on the MIPs is preferably extended to a tri-Langmuir or
tetra-Langmuir model occasionally [16–18]. The bi-Langmuir
model can be expressed as:
qm1 K1 C qm2 K2 C
q¼ þ : ð7Þ
1 þ K1 C 1 þ K2 C
Through the development of the expectation-maximization
method, Stanley et al. [19] demonstrated that the affinity
distributions for a MIP can also be calculated from raw
adsorption isotherm data. The multiple discrete peaks ap-
peared in the affinity distribution for MIPs calculated
numerically from isotherm data, which are fit well to bi-
Langmuir or tri-Langmuir models [16, 18]. Furthermore, it
has been proposed that the bi-Langmuir model (including its
extension) and LF model are equally good isotherm models
to characterize thermodynamic properties of MIPs [12]. The
presence of low classes of binding sites with distinct affinity
in the imprinted polymer is not unusual and can easily be
elucidated by the Scatchard plot analysis [20, 21].
Although Eq. 4 was originally derived to predict the
behavior of a column where the Langmuir isotherm is fol-
lowed, it can be applied to model the MIP chromatography
column as a nonlinear chromatography using the apparent
thermodynamic and kinetic parameters [4]. This means for
every chromatography of a specified sample load, a different
set of association and kinetic constants should be used. Here
we take the chromatography behavior of D-phenylalanine
anilide and L-phenylalanine anilide on an L-phenylalanine
anilide imprinted stationary phase as the example [14]. The
parameters for the isotherm data determined at 40 °C fitting
1103
to the bi-Langmuir model are K1 =0.35 l g−1, qm1K1 =5.62,
K2 =65.3 l g−1, and qm2K2 =10.9. These two sets of
Langmuir parameters, respectively, stand for nonspecific
(low-energy) and specific (high-energy) binding sites in
MIPs. Though the fitting is poor by applying the single
Langmuir isotherm, it results in K=1.03 l g−1 and qmK=
8.31. When these three sets of K and qm are separated and
substituted into Eq. 4, the predicted peak profiles shown in
Fig. 1 are produced. Other parameters for the calculated
peaks using Eq. 4 are based on the published MIP sta-
tionary phase system [14]: ɛ=0.613, t0 =1.019 min, C0 =
0.05 g l−1, and kd =54.12 min−1. The sample volume is 80 μl.
Peaks 1 and 2 in Fig. 1 can be regarded as the chromatography
of the template on the MIP column involving solely specific
and nonspecific binding sites. Peak 3 is less asymmetric
because of the lower K value resulting from the curve-fitting
of the adsorption isotherm based on the single Langmuir
model.
Figure 1 shows the simulated peaks for three sets of
parameters for the Langmuir isotherm. Obviously, the peak
asymmetry is highly dependent on the association constant.
A higher K value leads to a significant peak tailing; that is,
more specific interaction results in more asymmetry in the
elution peak. The locations of the peaks are theoretically
0
determined by the limiting capacity factors k0 , which can be
calculated using the Langmuir parameters for these three
systems. The limiting retention capacity for the Langmuir
0
isotherm model (k0 ¼ 5:25) is located in the middle of the
0 0
nonspecific (k0 ¼ 3:55) and specific (k0 ¼ 6:88) interac-
tions predicted by the bi-Langmuir model. However, this is
Fig. 1 Predicted peak profiles for a molecularly imprinted polymer
(MIP) column based on the theory of nonlinear chromatography using
Langmuir parameters (qm and K) for nonspecific (1) and specific (2)
binding sites in the bi-Langmuir isotherm model, as well as those for
the Langmuir model (3) fit to the data of adsorption of L-
phenylalanine anilide onto L-phenylalanine anilide-imprinted polymer.
The sample injection volume (Vinj) is 80 μl and the solute
concentration (C0) is 0.05 g l−1. The apparent rate constant kd =
54.12 min−1 and all other parameter values were taken from [14]
1104
only true in very dilute conditions. For the retention times
estimated at the peak maximum, the order is obviously not
followed because of the highly asymmetric peak for the
specific binding.
Interpretation of the peak profile from nonlinear
chromatography using MIPs
Retention mechanism
It is suggested by Eq. 4 that the position of the elution peak
0
is dominated by the limiting retention factor k0 as slow
kinetics and overloading effects are all neglected. The ideal
nonlinear model of chromatography describes the elution
curve with a leading shock front appearing at
 
Δq
tR ¼ t0 1 þ F ; ð8Þ
ΔC
where F is the phase ratio [(1-ɛ)/ɛ]. According to Eq. 8, the
0
k0 value used in Eq. 4 is proportional to the slope of the
isotherm evaluated at the dilute conditions (Δq/ΔC at
C→0). Equation 8 also suggests that for the convex
nonlinear isotherms like the Langmuir and LF models an
increase in analyte concentration reduces the retention time.
It is well described by Eq. 4 that the peak position shifts to
shorter times as the sample concentration or the loading
factor increases. That the retention decreases with an
increase in solute concentration under the overloaded
conditions has been interpreted in an analytical form based
on the Langmuir model [22].
Even though the MIPs are well characterized by either
the bi-Langmuir or the LF model, but not the Langmuir
model, Eq. 4 still works when the apparent thermodynamic
parameters (K and qm) are employed, as suggested by
Baggiani et al. [4]. Figure 1 reveals that the peak position is
mainly dependent on the limiting capacity factor, a product
of the phase ratio F and two thermodynamic constants, K
and qm. If the LF mode works well as for common MIP
systems, an increase in analyte concentration leads to a
decrease in the apparent K value and eventually to a decrease
in the limiting capacity factor. In this instance, the decrease
of retention with increasing solute concentration exits even
though the chromatography is not in overloaded conditions.
The compensation for the concentration effect on retention
is the reduction of peak broadening and tailing by the
decrease in the apparent association constant. Since the
concentration effect on retention is much more significant
for a template (higher K value and following the LF isotherm
model) than that for a nontemplate (lower K value), the
0  0
selectively α ¼ kP kNP decreases with increasing sample
concentration. This is why dilution of the sample can
Anal Bioanal Chem (2008) 390:1101–1109
effectively result in an increase in α for the MIP column
systems [6].
The thermodynamics of the chromatography of template
molecules and its related compounds on MIPs has been
extensively studied. The nonlinearity of the adsorption
isotherm results from the unique feature of the affinity-based
retention mechanism. Liquid chromatography on MIPs is
essentially an affinity-based separation owing to its molecular
recognition features. Early works confirmed the experimental
results that multiple additive weak interactions dominate the
recognition of the template molecules in imprinted polymers
[23].
From the computational model of Wu and Li [24], the
interaction energies between the monomer and the template
or its analogues are well correlated with the retention factors
and imprinting factors obtained on high-performance liquid
chromatography (HPLC) columns packed with the corre-
sponding MIP particles.
An investigation of the retention mechanism on nortrip-
tyline MIPs provided evidence that hydrogen bonding
between the pendant amine group on the antidepressants
and a methacrylic acid moiety on the polymer surface was
critical in the molecular recognition process [25]. In addition
to hydrogen bonding, the hydrophobicity of the sample
molecules was also found to play an important role. Yu and
Mosbach [26] discovered that in organic media the degree
of retention on the imprinted polymer was controlled by
hydrogen-bonding interactions between the sample molecule
and the polymer, while in aqueous media it was determined
to a considerable extent by hydrophobic interactions [26].
The study of Baggiani et al. [27] on the binding of herbicide
2,4,5-trichlorophenoxyacetic acid on MIPs revealed that the
molecular recognition mechanism acting on the columns
was dependent on a combination of ion-pair and hydropho-
bic interactions. In summary, in either organic or aqueous
media, the binding and recognition of a template molecule is
attributed to the summation of various interactions, which
result in the affinity nature of the adsorption on MIPs. As
suggested by Toth et al. [6] the experimental adsorption
isotherm should always be reported such that the retention
in the HPLC column of MIPs can be predicted.
Peak broadening and peak asymmetry (tailing)
For chromatographic applications, a large separation factor
is not enough; less peak broadening and skew is very
important. It has been widely cited that heterogeneity of
binding sites is the leading contributor to broad, asymmetric
peaks in chromatographic applications [15]. This hypothesis
needs to be carefully proved. Actually, the peak broadening
and tailing are both thermodynamic and kinetic character-
istics of the chromatography, especially accompanying the
nonlinear adsorption. When axial dispersion is negligible,
Anal Bioanal Chem (2008) 390:1101–1109 1105

the van Deemter equation can be simplified to the following H=L/N and the number of plates is obtained from the peak
equation for the intrinsic plate height contributed by slow profile as N=5.54(tR/W0.5)2, in which W0.5 is the band
kinetics [28]: width estimated at half of the peak height. The peak
0 asymmetry factor As is calculated also from the peak
2u0 1 k0
H¼   ; ð9Þ predicted by Eq. 4. As shown in Fig. 3, As is defined as the
" kd 1 þ k 0 2 ratio (at 10% of the peak height) of the distance between
0
0 the peak apex and the backside of the chromatographic
where kd is the effective desorption rate constant and k0 is
curve to that between the peak apex and the front side of
the limiting capacity factor. kd can be regarded as the
the chromatographic curve. If As >1, the peak shape is
lumped mass transfer rate coefficient taking account of effects
called tailing.
of slow adsorption/desorption, film mass transfer resistance,
The apparent kinetic rate constant kd takes account of both
and intraparticle diffusion; thus, the kd value can be altered by
slow kinetics and the mass-transfer resistances. According to
changes of these resistance sources. Equation 9 is valid for
Miyabe and Guiochon [28], there is a positive concentration
linear chromatography and is exclusive of an axial dispersion
dependence of kd which results from the positive concentra-
effect because of the small contribution of axial dispersion to
tion dependence of surface diffusion. The concentration
overall mass transfer resistance in the MIP column [28].
dependence of the mass transfer rate coefficient following
Whenever the nonlinearity due to the nonlinear isotherm and
the relationship kd =117.3C0.2582 min−1 [14] was used to
sample overloading is taken account, the evaluation of the
0 generate the results shown in Fig. 2. It is noted that in [14,
plate height becomes more complicated. Both kd and k0 are
28] the lump mass transfer coefficient was, respectively,
subject to change with change in the sample concentration.
denoted by kf and km,L. The increase of kd with concentration
Figure 2 shows that the plate height and the asymmetry
can also be found in the chromatography of pyrimethanil on
factor change with association constant according to the
columns packed with pyrimethanil-imprinted polymers [4].
prediction from Eq. 4. The plate height is defined here as
In addition, Fig. 2 shows the plots of plate height (H)
and asymmetry factor (As) versus binding constant K at
different combinations of solute concentration C0 and
injected sample volume Vinj. Both the plate height and the
asymmetry factor have a very similar dependency on K at
every sample load (C0Vinj). At low K value, the asymmetry
factor remains around unity; however, it rises suddenly at
about K=103 M−1 and increases sharply with K. It is
interesting to note that the increase of As occurs slightly
earlier than that of the plate height for the same sample
load. The abrupt increase in As at some threshold K value

Fig. 2 Dependency of asymmetry factor and plate height on the

association constant and loading factor according to the nonlinear
chromatography theory (Eq. 4). The sample load increases in the
following order: 1 (C0 =0.05 g l−1, Vinj =10 μl); 2 (C0 =0.1 g l−1, Vinj =
10 μl); 3 (C0 =0.2 g l−1, Vinj =10 μl); and 4 (C0 =0.05 g l−1, Vinj =
80 μl). The apparent rate constant changes with solute concentration Fig. 3 Predicted peak profiles for a MIP column based on the theory
as kd = 117:3C0 0:2582 min−1. To reveal the behavior at smaller values of of nonlinear chromatography (Eq. 4). The parameters for curve 2 in
the apparent rate constant, the results for C0 =0.05 g l−1, Vinj =80 μl, Fig. 2 were used along with K values of 103, 104, and 2×104 M−1,
and kd =5.412 min−1 are also shown (5). The MIP column in [14] was respectively, for peaks 1, 2, and 3. The definition of the asymmetric
taken as the model column factor As using peak 3 as the example is also shown
1106
suggests that the peak tailing is likely unavoidable at higher
values of the association constant. Besides, it is suggested
by Fig. 2 that the threshold K value decreases with
increasing sample load. As the template load increases
from 0.5 μg (curve 1) to 4 μg (curve 4), the threshold K
value decreases by about 1 order of magnitude. The
dependency of the peak profile on the K value at low sample
load conditions (C0 =0.1 g l−1, Vinj =10 μl) is shown in
Fig. 3. The peak profile remains symmetric up to a K value
of 103 M−1 and it starts to become asymmetric at about K=
104 M−1. Before the peak profile changes to very asymmet-
ric like at K=2×104 M−1, however, the peak position shifts
gradually to shorter time, as suggested by Eq. 8.
Figure 2 indicates that the apparent rate constant kd is
critical in the determination of peak broadening. The plate
height in the low association constant region is inversely
proportional to the value of this rate constant, according to
Eq. 9. When the kd value decreases from 54.12 min−1
(curve 4) to 5.412 min−1 (curve 5) at fixed sample load
(4 μg), the plate height increases sharply from 0.04 to
0.42 cm. In contrast, peak asymmetry is a weak function of
kd. As the rate constant decreases tenfold, As increases only
from 1.1 to 1.3 (at about K=100 M−1).
The results as shown in Fig. 2 agree very well with
experimental findings by Sellergren and Shea [29]. The
strong dependence of the asymmetry factor on sample load
and the weak dependence of it on flow rate suggest that the
nonlinear adsorption isotherm is the main reason for the
broad peaks observed in the MIP column system.
The influence of sample load on band broadening and
peak asymmetry is further discussed as follows. Consider a
system involving two retention mechanisms of close binding
affinity. One is a nonlinear isotherm and the other is a linear
isotherm; the latter exhibits retention that is inversely
dependent on concentration as described by Eq. 8. Increas-
ing the sample load can only shorten the retention time
associated with the nonlinear mechanism. The elution peak
may be broadened owing to the moving of the retention
time caused by the concentration effect in the nonlinear
mechanism. Experimental investigation of the sample load
effect on the peak asymmetry during chromatography of a
template on MIPs shows that the shape of the peak changes
in the abovementioned way until a split is observed [30]. To
explain this phenomenon, Minouraa et al. [30] proposed a
mechanism describing the formation of triple complexes
between a solute molecule (or molecules), an already ad-
sorbed solute molecule, and an adjacent region of the
polymeric stationary phase. These triple complexes could
influence the retention of analytes and contribute to peak
broadening and asymmetry. Therefore, we may hypothesize
that there is a combination of slow linear and nonlinear
kinetics originating from the formation of complexes during
the adsorption/desorption process in the MIP column.
Anal Bioanal Chem (2008) 390:1101–1109
Comparison of MIPs prepared by different approaches
Covalent imprinting has been suggested to improve the
chromatography performance using MIPs. For example,
molecular imprinting strategies suggested by Khasawneh
et al. [31] revealed that there was a great improvement in
the peak asymmetry, i.e., the As value was significantly
reduced from 5.43 or 4.85 to 1.72 by using covalent
imprinting instead of a noncovalent approach. On the other
hand, our previous study indicated that the number of plates
significantly increased when MIPs from covalent imprint-
ing were used [32].
Regarding the chromatographic peak of the template
molecule, the average number of plates in the covalently
imprinted polymer packed column was 4,960 m−1 (Table 1).
This value was about fivefold higher than those obtained
from columns packed with noncovalently imprinted polymers
(880 and 1,040 m−1 for imprinted polymers using meth-
acrylic acid and 4-vinylpyridine as monomers, respectively)
[32]. This increase in the number of plates was a result of a
significant increase in the apparent rate constant. As shown
in Table 1, the calculated kd value increased from 0.54 (or
0.55) to 2.86 when the MIP preparation method was
changed from noncovalent imprinting to covalent imprinting.
In spite of the different preparation methods, the rebinding of
print molecules to the MIP prepared by either covalent or
noncovalent imprinting is in the noncovalent manner, as
represented by Eq. 2. The change in kd value suggests there
are some alterations of the kinetics for the adsorption and
mass-transfer process.
In addition to the increase in the kd value, the increase in
the plate number ratio of print (template) to nonprint
molecules can be an indicator of the improvement in the
chromatography resolution on MIPs. On the basis of Eq. 9,
the plate number at very low sample loading is given by
 
ð1þk0 Þ
0 2
 0
2
NP kd;P k
0
k a 1 þ k 0;P

0
 P ¼ d;P 
¼ 2 ; ð10Þ
NNP kd;NP ð1þk00 Þ2 kd;NP 0
0 a þ k0;P
k0
NP
where α is the separation
. 0 factor, defined by the ratio of two
0
capacity factors k0;P k0;NP . Our previous studies on the
chromatography of cholesterol (print) and β-estradiol
(nonprint) on cholesterol-imprinted polymers prepared by
covalent and noncovalent molecular imprinting approaches
revealed that the plate number ratios were higher. The
values of Np/NNP for columns with methacrylic acid based
and 4-vinylpyridine-based noncovalent imprinting polymers
were 4.2 and 4.4. The plate number ratio increased slightly to
4.6 for a column packed with MIP from covalent imprinting
[32]. The concentrations of cholesterol and β-estradiol were
in total 1 g l−1 in each sample. In this concentration range,
Anal Bioanal Chem (2008) 390:1101–1109 1107

Table 1 Comparison of cholesterol chromatography on cholesterol-imprinted molecularly imprimted polymers (MIPs) prepared by covalent and
noncovalent imprinting by bulk polymerization [32]¶

Imprinting method Capacity factor, k′ Asymmetry factor, Asa Number of plates, N Apparent desorption
rate constant, kd (s−1)

Methacrylic acid based noncovalent imprinting 3.1 0.98 220 0.54

4-Vinylpyridine-based noncovalent imprinting 4.0 0.95 260 0.55
Covalent imprinting 3.5 2.01 1,240 2.86
¶
MIPs prepared by bulk polymerization (25–40-μm ground and sieved particles) were packed in 25-cm columns and the mobile phase was a
mixture of acetonitrile and water (95:5, v/v). The values of the apparent rate constant were calculated using Eq. 9.
a
Calculated based on the chromatograms in Fig. 2 in [32]

the capacity factors for both compounds remained un- ization strategies as reviewed by Turiel and Martin-Esteban
changed. The marginal increase of Np/NNP in the covalent [1] include imprinting on preformed beads, precipitation
imprinting suggests that the apparent rate constant increased polymerization, suspension polymerization, and multistep
for both print and nonprint molecules on the change from swelling and polymerization. One of the attractive methods
noncovalent to covalent imprinting. According to Miyabe to produce MIP packings in the form of uniformly sized
and Guiochon [28], the positive concentration dependence spherical, porous beads is to prepare MIPs inside the pores
of mass-transfer rate is more significant for print than for of silica. The resultant silica–polymer composite MIPs were
nonprint molecules for binding on MIPs. Therefore, we can recently tested for chromatographic characterization [5].
expect NP/NNP to increase with solute concentration. Fur- Comparison of the composite with the conventional sieved
thermore, Eq. 10 suggests that the increase of the separation and ground bulk MIP reveals that the chromatographic peak
factor is not necessary to improve the separation efficiency. for the template (phenytoin) on the silica–MIP composite
Similarly, the decrease in the ratio As,P/As,NP is an column is much sharper, but with tailing, than that on the
indicator to improve the MIP material for enhancing the bulk MIP column. The retention time of the template in the
resolution of print and nonprint molecules. However, since silica–MIP composite system is shorter since the total
the print molecules always have a higher K value than number of binding sites is smaller. On the basis of the data
nonprint molecules, the As,P/As,NP ratio remains greater presented in Fig. 8 in [5], the asymmetry factor and the
unless there are changes in rate constants. The ratio of the number of plates for the bulk MIP column can be calculated
asymmetry factors could be reduced only when the increase as about 5.1 and 28, respectively, while for the column
in the rate constant for the template is much more sig- packed with the silica–MIP composite, As and N are
nificant than that for nonprint molecules. obtained to be about 7.1 and 50. The increase in asymmetry
The second issue is the MIP formats prepared by
different polymerization methods. Traditionally, MIPs are
prepared by bulk polymerization and the resulting polymer
blocks are ground into particles (Fig. 4) prior to use as
packing materials. The ground MIP particles are polydisperse
both in shape and size. Typically, ground MIP particles in a
range of particle sizes, 25–44 μm [32], for example, are
sieved for packing a column. A particle size in this range is
somewhat larger than that for typical HPLC columns. As
indicated in the literature [28], two contributors of the
lumped mass transfer coefficient, the external film mass
transfer and intraparticle diffusion resistances, are strong
dependent on particle diameter. It has also been shown that
intraparticle diffusion contributed much more to the lumped
mass transfer coefficient than the adsorption/desorption
process in the column using MIP as the stationary phase.
Bigger particles thus apparently result in more peak
broadening, which is characterized by a smaller effective
desorption rate constant in Eq. 9. Several methods have
Fig. 4 Scanning electron microscopy image of the MIP particles
been proposed in the literature for the preparation of prepared by the bulk polymerization for 4-vinylpyridine-based non-
micron-sized spherical imprinted particles. Those polymer- covalent imprinting of cholesterol [32]
1108
factor for the column with uniformly sized spherical micro-
beads is due to a greater loading factor. The loading factor is
relatively greater for the same sample size injected into the
silica–MIP composite column, which has a smaller capacity
than the bulk MIP column. On the other hand, the higher
number of plates in the silica–MIP composite column is
obviously due to an increase in the apparent desorption rate
constant. The kinetic dispersion is reduced in the composite
column, since the route for solute traveling for binding sites
in the column is shorter.
However, experimental data suggest that the use of
uniformly sized MIP particles do not necessary result in a
better chromatographic performance. A 10-cm column
packed with D-chlorpheniramine-imprinted spherical particles
prepared by the method of multistep swelling and polymer-
ization, for example, led to 35 plates only at a flow rate of
0.5 ml min−1 [33]. With a longer column (25–cm) packed
with (S)-ibuprofen-imprinted polymers prepared by the
same polymerization method and a higher column temper-
ature, the number of plates increased to 754 [34]. Masci et
al. [35] prepared clenbuterol-imprinted microbeads by the
same two-step swelling and polymerization technique and
used them for the chromatographic separation of clenbuterol
from other β-adrenergic molecules. Although the baseline
separation can be achieved, the peak for clenbuterol is very
disperse in the methacrylic acid based MIP. When the
functional monomer is changed to acryamide, the peak
becomes much less broadened at optimum eluent conditions.
Those results suggest that the chromatographic performance
rather depends on the recipe of monomer and cross-linker,
and the eluent composition but not the MIP formats. If
uniformly sized MIPs are used, the optimization of chro-
matographic conditions such as column temperature and
flow rate is necessary to achieve the baseline separation [36].
Polymer monoliths are the MIP formats prepared by in
situ polymerization in a column and can be directly used as
the stationary phases for liquid chromatography. Basically,
the MIP monoliths are prepared by the method of bulk
polymerization. According to Kim and Guiochon [18], the
monolithic MIPs have fewer nonselective sites than the
conventional bulk MIPs particles. However, the polar porogen
that is needed to prepare the monolithic MIPs can negatively
affect the enantiomeric separation.
Whichever polymerization strategy is used, the degree of
cross-linking can play an important role in determining the
chromatographic performance on MIPs. The mobile-phase
composition and cross-linking density could not only
influence the recognition of print molecules but also the
resolution of print and nonprint molecules [37]. The data of
Yu and Mosbach [37] revealed that the mass transfer in the
low cross-linking density polymer was improved. The
solvent which is used as the porogen during the formation
of the MIP is believed to be of crucial importance in the
Anal Bioanal Chem (2008) 390:1101–1109
determination of morphology and cross-linked structure,
and eventually in determining the binding-site accessibility.
The mass transfer and adsorption/desorption kinetics are
thus controlled by the latter. As suggested by Ansell [38],
the poor column efficiency and peak tailing due to the
limitation of binding-site accessibility may be overcome to
some extent by the optimization of mobile-phase composi-
tion and separation temperature.
Conclusion and future prospects
Chromatographic separation of the template from the mixture
on a MIP column is essentially an affinity chromatography.
The isotherm nonlinearity due to the molecular recognition
onto specific binding sites likely contributes to the band
broadening and tailing in the MIP system. The chromato-
graphic peak broadening and asymmetry depend on the
association constant and sample load, and they suddenly
increase at some threshold values of the association constant.
Numerous studies on the thermodynamics of MIPs indicated
that the binding of the template to imprinted polymers
follows the LF or the bi-Langmuir model, since a combina-
tion of several different weak interactions may be involved in
the adsorption/desorption of the template onto the binding
site. Because of the affinity nature of the chromatography on
MIPs, the isotherm and kinetic nonlinearity is unavoidable
and essential leads to broadening and a tail of the elution
peak. The strong dependence of peak height on the apparent
rate constant and the peak asymmetry as a weak function of
the rate constant can be predicted by the nonlinear chroma-
tography and agreed well with the experimental observation
on MIP columns.
Recent efforts to improve chromatographic efficiency
include the adoption of covalent imprinting to replace the
noncovalent imprinting method and the use of several
strategies for preparing uniformly sized spherical microbeads
instead of the conventional bulk MIP polymers. The covalent
method leads to more specific binding sites and an increase in
the apparent association constant, which results in higher
peak asymmetry. However, the peak asymmetry can be well
compensated by the significant increase in the apparent
kinetic rate constant, which results in a higher number of
plates. The use of uniformly sized spherical microbead MIP
sometimes could also enhance the chromatographic efficien-
cy as reflected by an increase in the rate constant. This
increase in the rate constant may be due to the shorter
diffusion path, and thus the mass-transfer resistance is
reduced.
In order to yield the ideal strategy to improve chromato-
graphic performance, research efforts are still needed to
elucidate how the thermodynamic and kinetic features of
the chromatography of printed molecules on MIPs are
Anal Bioanal Chem (2008) 390:1101–1109
influenced by their preparation methods and polymerization
conditions. Synthetic conditions and mobile-phase compo-
sitions are the most important factors influencing the
chromatographic performance [21]. The accessibility and
structure of binding sites and mobile-phase conditions both
regulate the rebinding of the template and the competitive
adsorption of sample molecules. These two major classes of
factors initiate the proposed salvation mechanism and
higher-order template–template complexation. However,
neither of these mechanisms can well describe the compli-
cated nature of the observed peak shape [30]. In addition to
the specific molecular recognition, it is believed that several
strong, nonspecific interactions which may be involved in
the printing and rebinding of the template need to be clarified.
The good chromatographic separation on MIPs may be
achieved by using an optimal mobile-phase gradient or by
adding mobile-phase modifiers.
Furthermore, more studies on the kinetics of template
binding onto MIPs as well as the mass-transfer rates are
necessary. To our knowledge, the Guiochon group [32] is
the only one addressing the concentration-dependent mass-
transfer kinetics in MIP columns. Both theoretical and
experimental efforts to determine the related aspects are still
required. The adsorption/desorption experiments can be
carried out in the column or the suspension operated in batch
mode [39]. In addition to dealing with elution peaks using
the theory of nonlinear chromatography, theoretical analysis
based on data obtained from the experimental breakthrough
curves is useful for clarifying the characteristics of the mass-
transfer kinetics inside the MIP particles [40].
Also, it should be noted that band broadening and skew
exist even in the linear chromatography. A well-packed
MIP column has higher column efficiency and less peak
tailing owing to the radial heterogeneity of the flow pattern
in the column.
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