Analytica Chimica Acta 528 (2005) 107113

A critical examination of the use of the Freundlich isotherm in

characterizing molecularly imprinted polymers (MIPs)
Gregory T. Rushton, Chelsey L. Karns, Ken D. Shimizu

Department of Chemistry and Biochemistry, University of South Carolina, Columbia, SC 29208, USA
Received 12 April 2004; received in revised form 20 July 2004; accepted 20 July 2004
Available online 17 August 2004
Abstract
The Freundlich isotherm (FI) has previously been shown to be useful in modeling the binding properties of non-covalent molecularly
imprinted polymers (MIPs). The advantage of the FI is that it is a heterogeneous binding model that can accommodate and measure the
heterogeneity inherent in MIPs. However, it is often difcult to verify whether the FI is an appropriate binding model for a particular MIP
because only a narrow portion of the binding isotherm is experimentally measurable. This study takes a critical examination of whether the
FI is an appropriate binding model for MIPs and explores its limitations in characterizing a (+)-cinchonine (CN) imprinted polymer and a
non-imprinted polymer (NIP). A wider portion of the binding isotherm can be examined by systematically measuring a series of isotherms at
different polymer concentrations. This strategy veried that FI can yield an accurate measure of the heterogeneity in the cinchonine MIP and
the NIP. However, in cases of extremely high polymer loading, saturation behavior was observed, and the FI yielded inaccurate measures of
the binding properties even though the experimental isotherm appears to be well modeled by the FI. Further, these studies indicate that the
FI accurately predicts the heterogeneity index for more homogeneous compared to heterogeneous polymers over a wider concentration range
but is subject to considerable error as saturation conditions are approached. These studies demonstrate the importance of correctly applying
the FI to the lowest concentration portion of the binding isotherm that is experimentally measurable.
2004 Elsevier B.V. All rights reserved.
Keywords: Molecular imprinting; Freundlich isotherm; Polymers; Cinchonine; Analytical methods; Adsorption isotherms
1. Introduction
Molecularly imprinted polymers (MIPs) are highly
crosslinked macroporous materials with recognition proper-
ties similar to biological systems such as enzymes and anti-
bodies [14]. However, their use in commercial applications
has been limited by their low overall capacity and selectivity.
MIPs contain a heterogeneous distribution of binding sites
that range in afnity and selectivity. The primary source
of heterogeneity in non-covalent MIPs is the incomplete
monomer template complex formation in the polymerization
mixture (Scheme 1). The monomer template adducts are held
together by relatively weak non-covalent interactions. There-
fore, the functional monomer (M)template (T) equilibrium

Corresponding author. Tel.: +1 803 777 6523; fax: +1 803 777 9521.
E-mail address: shimizu@mail.chem.sc.edu (K.D. Shimizu).
lies far to the left. As a result, most of functional monomer
remains unassociated in the prepolymerization mixture. This
leads to the random integration of the functional monomer
into the polymer matrix and produces a large population
of low-afnity binding sites. Only a small fraction of
functional monomer forms higher order complexes that on
polymerization presumably yield the high-afnity binding
sites. Therefore, the binding properties of MIPs tend to be
dominated by the more predominant low-afnity binding
sites.
Despite the strong inuence of heterogeneity on the bind-
ing properties of MIPs, the breadth of the binding site hetero-
geneity in MIPs has only recently been measured [5,6]. Initial
efforts focused on measuring afnity distributions that yield a
plot of the number of binding sites (N) in an MIPhaving a par-
ticular binding afnity (K) [5]. These studies conrmed that
MIPs contain a broad unimodal distribution of binding sites
0003-2670/$ see front matter 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.aca.2004.07.048
108 G.T. Rushton et al. / Analytica Chimica Acta 528 (2005) 107113
Scheme 1. Functional monomer (M) and template (T) equilibrium in the
prepolymerization mixture. The higher ordered complexes presumably go
on to form high-afnity binding sites that are lined with multiple functional
monomer units. The lower order complexes, in particular the uncomplexed
functional monomer units, form the more numerous low-afnity binding
sites.
that asymptotically tails into the high-afnity region (Fig. 1).
Perhaps the most interesting observation was that imprinted
and non-imprinted polymers share similar unimodal distribu-
tions [7]. The primary difference is that MIPs have a broader
more heterogeneous distribution, which ensures a higher pop-
ulation of high-afnity binding sites. These studies conrmed
that heterogeneity is a key attribute of MIPs and moreover,
that heterogeneity is actually an excellent measure of the im-
printing effect.
The Freundlich isotherm(FI) has been shown to be widely
applicable in measuring the heterogeneity in MIPs [8]. The FI
assumes that the relationship between the bound and free an-
alyte concentrations are dened by a power function (Eq. (1))
with tting parameters a and m. The use of the FI to charac-
terize MIPs has certain advantages. First, the FI is based on
a heterogeneous exponentially decaying distribution, which
ts well to the tailing portion of the heterogeneous distribu-
tion of MIPs. Secondly, the log form of the FI is easily ap-
plied as it can be transformed into a linear function (Eq. (2)).
Isotherms that are well t by the FI will fall on as straight
line when plotted in loglog format (log B versus log F). This
analysis is a useful diagnostic for identifying sources of error
as deviations from linearity are visually evident. Finally, the
slope of the straight line t yields m, which is a measure of the
heterogeneity of a system. Amore homogeneous systemwill
have an mvalue approaching unity and a more heterogeneous
system will have an m value approaching zero.
B = aF
m
(1)
log B = mlog F + log a (2)
Fig. 1. Typical broad unimodal heterogeneous afnity distribution measured
for imprinted (solid line) and non-imprinted (broken line) polymers.
Fig. 2. LangmuirFreundlich isotherm (LFI, solid line) tted with Fre-
undlich isotherms (FI, dashed lines). At low polymer loadings (left side
of graph), the FI accurately models the LFI; at higher loadings (right side of
graph), the FI cannot accommodate the curvature in the data.
Recent studies in our laboratory have utilized the FI to
characterize the heterogeneity in MIPs [7,8]. The majority
of MIP isotherms were well t by the FI. However, in a few
cases, it was observed that the FI measured heterogeneity in-
dexes varied depending upon the concentration window of
the experimental binding isotherm. This was troubling as the
heterogeneity index should be a constant and should be inde-
pendent of the manner in which it was measured. There are
two possible explanations. First, the FI may not be an appro-
priate binding model for these MIP systems. Secondly, these
variations could arise from the limitation of the FI, which
can only accurately model the low concentration subsatura-
tion portion of the binding isotherm (Fig. 2). Application of
the FI to portions of the binding isotherm outside of this win-
dowyields divergent and inaccurate binding parameters. This
study will examine which of these two explanations is a more
plausible description of the underlying physical properties of
MIPs.
A (+)-cinchonine imprinted polymer was chosen because
it displayed varying m values, depending upon the concen-
tration range of the experimental isotherm being modeled
by the FI. The corresponding non-imprinted polymer (NIP)
was also synthesized for comparison. In principle, deviation
of the experimental binding isotherm from the Freundlich
relationship should be easy to verify. This could be accom-
plished by measuring the binding properties of a polymer
over a wide concentration range and plotting the isotherm in
loglog form. Isotherms that are well t by the FI will be
straight over the entire concentration range. Linear regres-
sion analysis will then yield the FI binding parameters m
from the slope and log a from the y-intercept (Eq. (2)). How-
ever, experimental binding isotherms are usually limited to a
narrow portion of the entire binding isotherm, which makes
it difcult to exclude other curved binding isotherm models
[9,10]. This limitation arises due to the solubility limits of
the analyte at high concentrations and the inability to accu-
rately measure analyte concentrations at low concentrations.
For example, the binding isotherms were measured by UV-
spectroscopy in this study and cover only a two orders of
magnitude concentration range (10
1
to 10
3
M).
G.T. Rushton et al. / Analytica Chimica Acta 528 (2005) 107113 109
These practical limitations in measuring the binding
isotherm were circumvented by measuring a series of
isotherms over the same analyte concentration range but us-
ing different masses of polymer. By changing the concentra-
tion of polymer, different portions of the binding isotherm
could be measured while still using the same analytical
method. This is possible because the classes of binding sites
within the polymer matrix that are accessed in rebinding stud-
ies depends on the ratio of polymer to analyte concentrations.
At low polymer loadings, the concentration of binding sites
to analyte concentration is rather large; the analyte will then
presumably access the highest afnity sites in the subsatu-
ration region of the isotherm. At high polymer loadings, the
polymer becomes more saturated with analyte and its bind-
ing properties reveal the predominance of lower afnity sites
as the saturation region of the isotherm is approached. This
strategy allows for a more careful examination of whether the
FI is appropriate to model for a particular system and over
what concentration range. These experimental results are
compared against theoretical binding isotherms generated us-
ing the LangmuirFreundlich isotherm (LFI). Together these
studies revealed that the FI yields accurate binding param-
eters for the MIP and NIP over most conditions. However,
under conditions of high polymer loading, the experimen-
tal binding isotherm shifts into the saturation region where
the FI does not apply and the calculated heterogeneity index
drops well below the value predicted by the LFI. In practice,
it is difcult to determine if the isotherm t to the FI yields
accurate binding parameters because most experiments are
conducted over a sufciently narrow concentration range as
to appear to be well modeled by the FI even in cases when it
is not.
2. Experimental
2.1. General procedure
(+)-Cinchonine or ()-cinchonidine, ethylene glycol
dimethacrylate (EDMA), methacrylic acid (MAA), and 2,2

-
azobis(isobutyronitrile) (AIBN) were obtained fromAldrich.
(+)-cinchonine and all solvents were used as received from
Fisher. EDMA was dissolved in an equal volume of ether,
washed twice with 0.1 M NaOH solution, and washed once
with saturated NaCl solution to remove the inhibitor. Drying
with MgSO
4
and evaporation of the ether afforded the pure
monomer. Methacrylic acid was distilled (5 mmHg, 40

C)
prior to use.
2.2. Polymer preparation (MIP)
To a 40 mL screwcap vial was added methacrylic
acid (MAA, 0.360 mL, 4.26 mmol), ethylene glycol
dimethacrylate (EDMA, 11.7 mL, 62.0 mmol), 2,2

-
azobisisobutryonitrile (AIBN, 118 mg, 0.719 mmol) and
(+)-cinchonine (CN, 213 mg, 0.723 mmol) in toluene
(16.9 mL). The mixture was sonicated under nitrogen for
10 min and then sealed. Polymerization was carried out in
an oil bath at 60

C and allowed to proceed for 18 h. The

resulting monolith was ground with a mortar and pestle
to afford a ne powder. Soxhlet extraction was performed
in two steps to remove the template, rst with a 72:28
(v/v) mixture of toluene:acetic acid, then with a 4:1 (v/v)
acetonitrile:methanol solution. Solid sodium bicarbonate
was added in the second step to encourage the sequestering
of residual acetic acid in the polymer. Drying in vacuo
afforded particles ready for testing. A control polymer was
prepared in an identical manner to the MIP in the absence of
the template.
2.3. Batch rebinding
Cinchonine solutions of varying concentration ranging
from 25 to 500 M in acetonitrile were prepared by serial
dilution of a stock solution and set aside. MIP and NIP sam-
ples with mass 15 mg were weighed out and added to a 5 mL
aliquot of the appropriate solution in a 2 dram screw-cap vial
and shaken for 1 h to ensure equilibration of the template with
the polymer. This procedure was repeated ve times for both
polymers with varying amounts of polymer (25 mg, 40 mg,
50 mg, 75 mg, and 100 mg). A 1.0 mL aliquot of the super-
natant was removed and ltered with a Millex-FG 0.2 M
lter unit to remove ne particles and placed into a HPLC
vial. UV measurements were taken on a Varian ProStar 210
solvent delivery module and Varian ProStar 320 UVvis de-
tector, with a Dynamax C
18
column at 305 nm. Calibration
curves were veried by HPLC analysis (70:30 (v/v), pH 5
acetate buffer/MeCN at 0.8 mL/min).
3. Results and discussion
Two polymers were prepared and tested by batch rebind-
ing studies to generate a series of 12 binding isotherms.
The imprinted polymer (MIP) was prepared in the pres-
ence of a guest molecule, (+)-cinchonine (CN), and a
control polymer (NIP) was prepared under identical con-
ditions in the absence of CN. Binding isotherms were
constructed for each polymer by equilibrating a known
mass of polymer with 5 mL of acetonitrile of varying
CN concentrations. The supernatant was then analyzed for
the remaining free analyte concentration (F). The binding
isotherms were analyzed by plotting the data in log B ver-
sus log F format and applying the FI, using linear regression
analysis.
First, the imprinting effect for the CN MIP was veri-
ed by comparing the binding isotherm for the MIP and
NIP (Fig. 3). The MIP showed higher capacity than the
NIP over the entire concentration range as seen by the
higher vertical displacement of the MIP isotherm. In addi-
tion, the differences in capacity between the MIP and NIP
isotherms systematically increase with decreasing concen-
110 G.T. Rushton et al. / Analytica Chimica Acta 528 (2005) 107113
Fig. 3. Demonstration of the imprinting effect by comparison of the binding
isotherms for the binding of cinchonine to the MIP (lled circles) and the
NIP (open circles) measured using 100 mg of polymer.
tration. This is consistent with the observed higher selec-
tivities of MIPs at lower concentrations [11]. A total of
six isotherms were produced for each polymer by repeat-
ing the rebinding studies with different masses of polymer,
from 15 to 100 mg. In each case, the MIP showed higher
capacity for the template molecule than the corresponding
NIP. The MIP was also more heterogeneous than the NIP
(m = 0.37 and 0.58 for the 100 mg isotherms, respectively),
which is consistent with the previously measured afnity
distributions.
The FI was then applied to the individual isotherms and
a heterogeneity index (m) was calculated. In general, the
FI appears to be a reasonable model as evidenced by the
relatively good t to linear regression analysis (Table 1).
A more quantitative assessment of the validity of applying
the FI to a particular portion of the binding isotherm can
be made from the R
2
of the t and from the stability of the
calculated m values. Theoretically, the binding parameters
measured for a particular polymer should remain constant
regardless of the concentration range or amount of polymer
used to measure the binding isotherm. Variations in m for
isotherms measured at different analyte to polymer ratios
would indicate that the portion of the binding isotherm being
examined falls outside the limits where the FI is accurate
or that the FI is not an appropriate binding for the measured
system.
Table 1
Binding parameters for MIP tested with various amounts of polymer
Isotherm m Range of % bounds R
2
MIP-100 mg 0.37 79.897.6 0.997
MIP-75 mg 0.36 65.197.3 0.992
MIP-50 mg 0.32 67.796.6 0.986
MIP-40 mg 0.34 49.693.4 0.987
MIP-25 mg 0.28 29.786.1 0.905
MIP-15 mg 0.26 13.265.0 0.807
Fig. 4. Loglogisotherms of CNtoMIP. Fromthe lowest data set upward, the
ordering of the isotherms is as follows: 15 mg, 25 mg, 40 mg, 50 mg, 75 mg,
and 100 mg of MIP. All samples were shaken with 5 mL of an acetonitrile
solution containing various concentrations of CN.
3.1. Analysis of the MIP isotherms
The experimental isotherms for the MIP are shown in
Fig. 4inloglogformat andthe bindingparameters inTable 1.
In general, the isotherms were well t by linear regression
analysis. This is conrmed by the comparison of the R
2
val-
ues for the respective isotherms, which are mostly >0.95
(Table 1).
The two isotherms measured using 15 and 25 mg show
slightly poorer ts with R
2
values of 0.904 and 0.807, re-
spectively. This is consistent with the expected deviation of
the experimental binding isotherm from the FI at higher ana-
lyte to polymer ratios as the systemapproaches saturation. An
additional contributor to the lower R
2
values for the 15 mg
and 25 mg isotherms is their higher levels of error as seen
by the greater scatter of the data points about the tted line.
This most likely arises from two sources. First, the low mass
samples are more difcult to weigh out accurately. Second,
the lower amounts of polymer in these studies bind only a
small fraction of the analyte from solution. Under these low
percent bound conditions, the calculated B values are subject
to large errors because the measured F and T concentrations
are of similar magnitude.
The distinction between the four isotherms that t well to
the FI and the two that do not are also evident froma compar-
ison of their m values (Fig. 5). The four isotherms measured
with higher weights of polymer have m values that are rela-
tively constant, from 0.37 to 0.32. In contrast, the isotherms
measured with lower weights of polymer have signicantly
lower m values of 0.28 and 0.26.
3.2. Analysis of the NIP
These discrepancies in the FI measured m values are even
more pronounced for the NIP. The six experimental isotherms
in loglog format for the NIP are shown in Fig. 6. The ability
G.T. Rushton et al. / Analytica Chimica Acta 528 (2005) 107113 111
Fig. 5. Heterogeneity index (m) as a function of polymer loading. The m
values for both the NIP (circles) and MIP (squares) are relatively stable at
low loadings (right side of graph) but decrease at higher loadings due to
deviations from Freundlich behavior and increased experimental error.
Fig. 6. Loglog isotherms of CNto NIP. Fromthe lowest data set upward, the
ordering of the isotherms is as follows: 15 mg, 25 mg, 40 mg, 50 mg, 75 mg,
and 100 mg of NIP. All samples were shaken with 5 mL of an acetonitrile
solution containing various concentrations of CN.
of the FI to t the respective isotherms again decreases with
increasing analyte to polymer ratio. The R
2
values decreases
from0.993to0.859as analyte topolymer mass ratioincreases
(Table 2). This is again indicative of the inability of the FI to
t the subsaturation portion of the binding isotherm.
Table 2
Binding parameters for NIP tested with various amounts of polymer
Isotherm m Range of % bounds R
2
NIP-100 mg 0.58 45.074.2 0.993
NIP-75 mg 0.59 36.162.9 0.964
NIP-50 mg 0.62 32.964.0 0.987
NIP-40 mg 0.66 28.452.2 0.996
NIP-25 mg 0.43 7.636.3 0.890
NIP-15 mg 0.33 3.922.2 0.859
Fig. 7. LangmuirFreundlichisotherms of varyingheterogeneityindex. Cur-
vature is more dramatic as saturation approaches for the most homogeneous
polymer (open squares, m = 0.9) than for the more heterogeneous materials
(open circles, m = 0.6 and lled triangles, m = 0.35).
These differences are even more evident on comparison
of the heterogeneity indexes measured from the respective
isotherms. A dramatic drop is seen for m from an average of
0.61 for the rst four isotherms to 0.33 for the last isotherm
(Fig. 8). This variation in the heterogeneity index again fol-
lows the trend predicted for measurements based on portions
of the binding isotherm approaching the curved saturation
region.
A comparison of the ability of the FI to model the MIP
and NIPreveals some important differences. The more drastic
changes in m for the NIP suggest that the control polymer is
closer to saturation in the concentration range measured. This
is most likely due to the lower capacity and heterogeneity of
the NIP in comparison to the MIP. In summary, the FI derived
m values appear to be accurate for the isotherms measured at
lower analyte to polymer concentration ratios but systematic
deviations are observed as the portion of the binding isotherm
being analyzed approaches saturation. To verify the sources
of the deviations in the FI measured, theoretical models were
studied for comparison.
3.3. Theoretical models
The effects of tting different portions of a hypothetical
heterogeneous binding isotherm with the FI were theoreti-
cally modeled. The LFI (Eq. (3)) was chosen because it can
model both saturation and subsaturation portions of the bind-
ing isotherm for heterogeneous systems. In addition, the LFI
has been shown to be a reasonable heterogeneous binding
model bothphysicallyandmathematicallyfor non-covalently
imprinted polymers [10].
Three hypothetical LFI were generated (Fig. 7). This was
done by selecting arbitrary values of N
t
= 500 M and a =
10 and three different m values (0.9, 0.6, and 0.35). These
were plotted in loglog format over a wide concentration
range (log F = 10 to 5 M). The general features of the
112 G.T. Rushton et al. / Analytica Chimica Acta 528 (2005) 107113
Fig. 8. Heterogeneity index as a function of free analyte concentration for
various m values. From top to bottom, the ordering is as follows: m = 0.90
(lled squares), m = 0.60 (lled circles), and m = 0.35 (open squares).
LFI are evident in loglog format. At lower concentrations,
the isotherm is linear and is well modeled by the FI. At
higher concentrations, signicant curvature is evident as the
system approaches saturation. The isotherms with higher
heterogeneity indexes (lower heterogeneity) show greater
curvature and more rapid transitions from the subsaturation
to the saturation regions.
B =
N
t
aF
m
1 +aF
m
(3)
Two log unit wide portions of the LFI were systemati-
cally t using the FI. At low analyte concentrations, the FI
was able to accurately reproduce the LFI isotherm hetero-
geneity index. The instability of the FI measured m values
were graphically examined (Fig. 8). The FI tting parame-
ter remains accurate and stable at lower concentrations but
at higher concentrations decreases to zero. It is interesting
to note that the more heterogeneous the polymer, the more
gradual the changes are in m. These trends are precisely the
same as those experimentally observed for the CN MIP and
NIP. The inability of the FI to t the curved, higher concen-
tration region of hypothetical binding isotherm can also be
seen in a plot of the R
2
values (Fig. 9). The most dramatic
and precipitous drop was found for the most homogeneous
polymer (m = 0.9).
When the curved portion of the binding isotherm was t
with the FI, two effects were observed: (1) the calculated het-
erogeneity index (m) was lower than the actual value, and (2)
the coefcient of determination (R
2
) dropped signicantly,
reectingthe inabilityof the linear FI functiontot the curved
portion of the isotherm. The magnitude of these trends de-
pends upon the heterogeneity of the system being measured.
Larger changes in the t (R
2
) were apparent for the more ho-
mogeneous polymer upon shifting from the subsaturation to
saturation portions of the binding isotherm. For example, the
value of R
2
drops precipitously from 1.0 to 0.68 for the m
= 0.9 polymer. Much smaller changes are seen for the more
Fig. 9. R
2
vs. log F for polymers with varying heterogeneity indices for a
hypothetical polymer modeled by the Freundlich isotherm. As saturation
(high polymer loading) approaches, the FI is less able to model the data
accurately. From top to bottom, the ordering is as follows: m = 0.35 (lled
squares), m = 0.60 (solid circles), and m = 0.90 (open squares).
heterogeneous polymer (m = 0.35) in which the R
2
drops
from 1.0 only to 0.94. Thus for more heterogeneous systems,
R
2
is an excellent indicator of the ability of the FI to t the
experimental binding isotherm. However, it is a poor indica-
tor for more homogeneous systems. Fortunately, the stability
of m is a good indicator of the appropriateness of the FI to
the concentration window being analyzed for both homoge-
neous and heterogeneous systems (Fig. 8). Agreater absolute
change in m is seen for the more homogeneous polymer (m
= 0.9) on shifting from the subsaturation to the saturation
portion of the isotherm. However, a signicant change is still
evident for the more heterogeneous polymer (m = 0.35).
The theoretical model shows that when the FI isotherm is
appropriately applied, it yields an accurate measure of het-
erogeneity. However, the accuracy of the measurement is de-
pendent on which the portion of the entire binding isotherm
is being analyzed. The FI derived m values accurately repro-
duce the heterogeneity of the hypothetical polymers at low
analyte to polymer concentration ratios (Fig. 8). The accuracy
of the FI based measurement of heterogeneity is also depen-
dent upon the heterogeneity of the system being measured.
The FI analysis yields a more accurate measure of the het-
erogeneity index over a wider concentration range for more
homogeneous adsorbents. For example, for the polymer m =
0.9 the FI derived m values remain stable and consistent with
the absolute value over a wider concentration range (Fig. 8).
In contrast, the more heterogeneous polymer m = 0.35 has a
much narrower concentration region in which m is constant.
This has the practical consequence that more heterogeneous
systems require the FI to be applied to a signicantly lower
concentration window in order to yield an accurate measure
of m.
The trends observed in the theoretical model match those
observed experimentally for CN MIP and NIP. Specically,
the m values measured at lower analyte to polymer ratio are
G.T. Rushton et al. / Analytica Chimica Acta 528 (2005) 107113 113
the highest and are presumably the most accurate. Secondly,
the m values decrease as the FI is applied to higher analyte
to polymer concentration windows. Finally, the more homo-
geneous polymer, which is usually the NIP, shows the more
precipitous drop in m value as FI is applied to higher and
higher concentration regions of the binding isotherm.
4. Conclusions
The results from this study emphasize the importance of
understanding the limitations of the FI. Overall, the FI is
able to accurately measure the heterogeneity of an MIP even
though it is usually restricted to a narrowportion of the entire
bindingisotherm. However, the accuracyof the FI is restricted
to the lower concentration region of the binding isotherm.
Furthermore, when the FI is inappropriately applied to the
saturation region of the binding isotherm, it yields hetero-
geneity indexes that are signicantly lower than the actual
value. Therefore, the FI should be applied to the lowest pos-
sible concentration range. When different values of m are
measured for single polymer, the highest value is most likely
the most accurate. This analysis also conrms the difculty in
verifying that a particular binding model is appropriate for a
particular experimental binding isotherm. Given the narrow
concentration window in which most rebinding studies are
conducted, it is difcult to assess the appropriateness of any
particular model by considering the t of model to the data
alone. For example, in the saturation region of the binding
isotherm, the FI may still appear to yield a good t (as de-
ned by R
2
) even though it is an inappropriate binding model
for this region of the binding isotherm. Amore discriminating
approach is to further consider the underlying assumptions
associated with the application of the model chosen to see if
there are consistent with the systembeing studied. Additional
binding experiments should also be conducted that can mea-
sure different portions of the global isotherm to check for the
consistency of the model over as wide a range as possible. For
our system, the experimentally observed deviations from the
FI were found to be consistent with the LangmuirFreundlich
model. This gives further evidence for the presence of a broad
unimodal distribution of binding sites in MIPs.
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