Focusing Review

Chromatography, Vol.29 No.1 (2008)

Focusing Review

Development and Applications of Fragment Imprinting Technique
Takuya Kubo*
Graduate School of Environmental Studies, Tohoku University, Aoba 6-6-20, Aramaki, Aoba-ku, Sendai 9808579, Japan
Received for review November 20, 2007. Accepted February 4, 2008

Abstract
In analyses of chemicals, selective separation is strongly desired for high accuracy and sensitivity. In order to separate selectively targeting
compounds from complex matrices, molecular imprinting (MI) technique is believed to be one of the most promising techniques. The
polymer-based materials prepared by MI have the selective recognition ability for targeting compounds like as “lock-and-key model”. However, the technique is hard to apply for toxic compounds and rare compounds because the targeting compounds are required directly for thetemplate molecules in the process of preparation of the molecularly imprinted materials. Therefore, we developed alternative technique by thename of Fragment Imprinting Technique (FIT) in which the targeting compounds are not necessary. In FIT, the pseudo-template molecules
are utilized, and the imprinted materials have selective recognition ability for a part of characteristic chemical structure on the targeting compounds. Additionally, FIT was applied for the selective separation of the environmental pollutants and natural toxins.
Keywords: molecular imprinting, fragment imprinting, selective separation

reported such as artificial antibodies, biosensors, and stationary

1. Introduction

solid phases in liquid chromatography (LC) [1-4].

Selective separation technologies are very important for the
purification and/or quantitative analysis of toxins, drugs, biologicalHowever, usual MI technique has some problems for applica-

materials, and chemicals. In general, it is difficult to achieve selec-

tions involving environmental samples and/or biological samples.

tive separation of target compounds from environmental or biologi-

For example, highly toxic or rare compounds cannot be utilized forcal samples due to the complex nature of the sample matrices thattarget as template molecules since the targeting molecule itself isinterfere with the separation and detection of targeting compounds

used in the process of the preparation of MIPs. Another serious

in these samples. Therefore, we urgently require a separation meth-problem is the leakage of template molecules. In the general proce-odology with built-in selectivity for the targeting compounds.

dure, the template molecule cannot be removed completely from

Molecular imprinting (MI) technique is one of the most attrac-

the MIPs even after exhaustive repeated washing with organic sol-tive methods to achieve the selective recognition abilities [1, 2]. In

vents as well as buffered solutions. This is because the imprinted

this technique, the crosslinking agent, polymerization initiator,tem-sites can be formed not only on the surface but also deeply insideplate molecule, and functional monomer which can be interacted

the crosslinked polymer network where it is difficult for the solventwith the template molecule are simultaneously polymerized. Then,reaching [5]. Therefore, contrary to expectations, the template

the template molecules are removed from the prepared polymer bymolecules may leak from the inside of the polymer: this becomes a

washing with organic solvent as well as buffered solution. As a re-serious problem and interferes with the correct quantitative deter-sult, selective recognition based on the imprinting effect can bemination and purification.

achieved. Usually, non-covalent bonding such as hydrogen bond-In this paper, author presents their recent studies focusing on

ing, ionic interactions, and hydrophobic interactions are utilized in

the alternative MI techniques (Fragment Imprinting Technique:the preparation of molecularly imprinted polymers (MIPs). This

FIT) which can overcome the above-mentioned problems. In FIT,

technique is relatively easy so that several approaches have been

we can utilize alternative template molecules which have similar―９―Chromatography, Vol.29 No.1 (2008)

Tetra-Br-BPA. It is very important to know the accurate concentration of these BPAs’derivatives in the environment. Additionally,
the derivatives should be separated by the differences of number,
halogen species and position of substituent because the activity ofeach derivative is much varied.
To achieve the selective separation of these halogenated
BPAs, MI is one of the most attractive techniques. However, general MI method cannot be used for toxic compounds because themethod has above-mentioned problems. Therefore, some alternative molecular imprinting procedures have been reported [13-15]

Figure 1. Basic concept of the Fragment Imprinting Techniqueand we have also previously reported some alternative molecularimprinting procedures that the template molecule has been selectedmoiety of targeting compounds (Figure 1). The separation media

from analogues of the target compound or partially resemble com-

prepared by FIT have selective recognition ability for targeting

pounds of the target compound, but not used the target compoundcompounds and their homologues. We applied this technique toitself [16-18]. In this study, we developed the separation media forseveral type compounds such as halogenated aromatic compounds,

selective separation and concentration of halogenated BPA deriva-

water-soluble compounds, flexible compounds and so on. Also, wetives. FIT (Figure 1) was utilized for preparation of polymer-based

hope to describe each strategy in this paper.

separation media.

Basically, the recognition sites on MIPs lead to decrease the

Uniform-sized MIPs particles prepared by two-step swellinghomogeneousness of separation media and broader peaks on liquidand polymerization method [19, 20] were packed into stainless col-chromatographic evaluations. However, the MIP media has higher

umns and evaluated with liquid chromatography (LC). We investi-

selectivity for targeting compounds than the commonly used sepa-

gated several template molecules for preparation of MIPs and all

ration media. Therefore, the MIPs are well suited to the solid-phase

MIPs were evaluated by LC. As result, BTFB-MIP which was pre-extraction (SPE) and batch separations. In this study, liquid chro-pared with 2,6-bis-(trifluoromethyl)-benzoic acid as template mole-matography was utilized for the evaluation of prepared MIPs tocule indicated the most selective separation ability for a few halo-

clarify the selective adsorption strength. But, we hope to show the-genated BPAs suchas 3,5- and/or 3’,5’- brominated or chlorinatedpossibility of practical application such as separation media of SPE

BPA derivatives. The chromatograms of the brominated BPA using

and others.

BTFB-MIP and Blank (non-imprinted polymer) were shown in
Figure 2. The results showed that 3,3’,5,5’-Tetra-Br-BPA was re-

2. Selective separation of halogenated aromatic compounds

tained selectively on BTFB-MIP whereas the retention strength for2.1. Selective separation of halogenated bisphenol A

3-Br-BPA was almost the same on both the polymers. These results

Endocrine disrupters in the environment have caused an in-suggested that the selective recognition for halogenated BPAs

creasing concern for their possible impact on wildlife and human

could be achieve by the appropriate template based on the molecu-

health. Also, chlorinated (bisphenol A) BPAs are produced during

lar structure and/or structural size [21].

bleaching processes of recycled paper in paper factories [6] and2.2. Selective separation of hydroxy polychlorinated biphenyls

brominated BPAs are primarily used as flame retardants in epoxy
resins used in printed circuit boards [7, 8]. Especially, tetrabromo-(HO-PCBs)

BPA (3,3’,5,5’-Tetra-Br-BPA) has produced the largest size among

Mono hydroxy polychlorinated biphenyls (HO-PCBs) havebrominated flame-retardants in the world. During incomplete com-been shown to inhibit mitochondrial oxidative phosphorylation,bustion such as spontaneous fires at waste disposal sites, polymers

thyroid hormone sulfation, estrogen sulfotranferase and the sulfa-

containing 3,3’,5,5’-Tetra-Br-BPA converted to polybrominatedtion and glucuronidation of 3-hydroxybenzo[a]pyrene, to affectdibenzofurans and polybrominated dibenzo-p-dioxins [9]. Also,

thyroxine (T 4) levels and to exhibit estrogenic or antiestrogenic

3,3’,5,5’-Tetra-Br-BPA has been reported to exhibit some estrogen

activity [22-24]. HO-PCBs are increasing attention as potentially-like properties in vitro [10], and to bind to the thyroid hormone

endocrinologically active metabolites of PCBs. In association withtransporting protein transthyretin in vitro [11] and the thyroid hor-

these risks, the effective separation method has not been developedmone receptor [12]. The chlorinated homologues, tetrachlorinatedand the selective separation is strongly required since targeting

BPA (3,3’,5,5’-Tetra-Cl-BPA) has been also reported to bind tocompounds are extracted together with a lot of biological com-transthyretin in vitro although the affinity was lower than 3,3’,5,5’-

pounds which interfere with the analyses.

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Takuya Kubo

Figure 2. Chromatograms of brominated BPAs on Blank and BTFB-MIP(a): Blank polymer packed column, (b): BTFB-MIP packed column
LC conditions: Mobile phase; 90% aqueous MeOH, Column size; 150 mm×4.6 mm i.d., Flowrate; 1.0 ml min−1, Temperature; 40°
C，Detection; Photo Diode Array (PDA) and MS (selected
ion monitoring)

3. Interval immobilization technique for water-soluble com-

In this study, we developed new separation media by the fragment imprinted polymers having selective recognition ability forpoundsspecific HO-PCBs. The MIPs were also prepared as uniform-sized

In general MI technique, organic solvent is utilized to dissolve

polymer particles. The separation abilities for HO-PCBs on MIPs

the template molecules and construct the porous structure. There-

were evaluated by LC and considered the relationship between re-fore, the water-soluble compounds having ionic groups can betention ability and pKa value dependent on the chemical structure

hardly used for targeting compounds. In order to accomplish theof HO-PCBs. Moreover, the direct separation of a few HO-PCB

selective separation for water-soluble compounds such as alkaloids,

analogues, which may have a possibility of thyroid hormone activ-

we proposed the novel separation media prepared through “interval

ity, from 19 kinds of HO-PCBs was carried out by LC using MIP

immobilization technique”. In this technique, the interval between

packed column.

the two ionic functional groups can be immobilized on crosslinked

As results of the detail LC evaluation, the prepared polymers

polymer using molecular assembly with appropriate pseudo-

had selective separation ability due to pKa value dependent on

template molecule coupled with some ionic functional monomers.

chemical structure of HO-PCBs. Additionally, the pKa and mo-

The concept of this technique for cyanobacterial toxin, cylindros-

lecular volume of template molecules were related on the construc-

permopsin (CYN) is illustrated in Figure 4.

tion of recognition sites [25]. Finally, direct selective separation of

The bulk MIP was crushed and classified at the range of 25

three HO-PCB analogues having a possibility of thyroid hormoneµm ~ 45 µm to be packed into LC column. Results of LC analysis

activity from the mixture sample was completely achieved on BCA

suggested-MIP (template: Biphenyl-4-carboxylic acid) (Figure 3). We antici-

ammonium chloride (TCBA) as pseudo-template had selective rec-pate that the method using in this study will contribute for the se-ognition ability for CYN. Additionally, scatchard analysis [15, 26]

lective separation of thyroid hormone activate compound and thealso supported that the associate constant between CYN and MIP

breakthrough of structure activity relationship of HO-PCBs.

was higher than that of Blank polymer (Figure 5, Table 1) [27].

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that

the

MIP

using

tributyl-(4-carboxybenzyl)-

Chromatography, Vol.29 No.1 (2008)

Figure 3. Chromatograms of HO-PCBs (19 analogues) on ODS and BCA-MIP(a): InertSil ODS-3 (150 mm＊4.6 mm i.d.), (b): BCA-MIP packed column (150 mm＊4.6 mm i.d.)
LC conditions: Mobile phase; A: AN/H2O=8/2, B: AN/0.1% aqueous formic acid＝8/2, 0-10 min; 100% A, 10-20 min; 100% B, stepwise gradient, Flow rate; 1.0 mL min−1, Temperature; 40°
C; Detection; Photo Diode Array

Figure 4. Concept of Interval Immobilization Technique for CYN
SSA: p-styrene sulfonic acid, EDMA: Ethyleneglycol dimethacrylate
According to these results, this technique can be expected as novel

poly (ethylene glycol) diacrylate [28, 29] or acryloyl-cyclodextrins

selective separation media for water-soluble compounds.

with bisacrylamide [30]. These polymers exhibited slight selective

Furthermore, as another problem of MI technique, non-recognition. However, the recognition sites in the polymers wereselective adsorption was observed on MIP-based SPE, originating

mainly controlled through hydrogen bonding, and they appeared tofrom hydrophobic interactions on the surface of the polymer ma-

be heterogeneously dispersed because excess amounts of functional

trix. Consequently, a novel preparation method, which can be per-monomers were used in these cases. Additionally, ionic com-formed in a 100% water system and has much lower hydrophobic

pounds such as guanidyl group-containing alkaloids cannot be util-

characterization, is needed. Recently, several researchers have re-ized in these preparation methods. For these reasons, we developed

ported new MIPs for oligopeptides, that are prepared in water using

a new MIP preparation system using water as the porogenic solvent―１
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Takuya Kubo

Table 1. Binding parameter of polymer and CYN determined byscatchard analysis

Blank

Ka1 (M−1) Ka2 (M−1)

N1 (µ mol/g)

N2 (µ mol/g)

2.7×104 2.3×103

1.4

8.1

0.82

7.6

TCBA-MIP 8.9×10

4

3.2×103

The association constant (Ka) and number of binding sites (N) was
determined from the slope and intercept, respectively, of fitted line
(n/Cf =−Kan+KaN) obtained by least squares regression.

Figure 5. Scatchard plot for CYN on Blank and TCBA-MIP
The association constant (Ka) and number of binding sites (N) was
determined from the slope and intercept, respectively, of fitted line
(n/Cf=−Kan+KaN) obtained by least squares regression.

Figure 6. Chromatograms of the ionic solutes on Blank and MIP
LC conditions: Mobile phase; 0.1 M NaCl aqueous, Flow rate; 0.5 ml min−1, Columnsize; 100 mm＊4.6 mm i.
d., Temperature; 30°
C, Detection; Photo diode array

to apply the FIT to a larger number of compounds, we have to es-

by the interval immobilization technique.

tablish a novel concept to obtain the selective recognition ability

The MIP was prepared by the interval immobilization tech-for compounds having flexible chemical structures.

nique with water and water-soluble crosslinking agent. The MIP
showed improved hydrophobicity and the selective recognition for

In this study, we reported 3 D recognition for the flexible

the template in 100% aqueous solution (Figure 6) [31]. This tech-

structural compound domoic acid, DA (Figure 7) by the fragmentnique should therefore be used in selective SPE of environmental

imprinted polymers. DA is a water-soluble tricarboxylic amino

and/or the biological aqueous samples.

acid with a molecular weight of 311: it acts an analogue of the neurotransmitter glutamate and is a potent glutamate receptor agonist.
Its first identification was reported from the rhodophyta Chondria

4. 3 D recognition for domoic acid having flexible structure
In typically, targeting compounds of MI technique have in-armata as an insecticide [32]. Finally, the cell died and symptoms

flexible and relatively rigid chemical structures. Therefore, in ordersuch as memory loss (which is also a typical feature of ASP) were―１
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Chromatography, Vol.29 No.1 (2008)

observed [33-35]. Additionally, some biological researchers haveand the alteration of the Gibbs free energies calculated by computerreported that DA is assembled with a kainate-type subunit of themodeling (Table 2 and Figure 8). After all, the recognition sites

glutamate receptor. According to that study, the author has de-constructed on 1,3,5-PeTA-MIP could recognize DA by the flex-

scribed that multipoint recognition was worked effectively towardible conformational changes of DA: these conformational changes

selective holding and three COOH groups were selectively recog-

were incorporated into the recognition sites. In other words, these

nized. In our previous study, we have reported “two-point” recog-

results suggested that three-point recognition was achieved by thenition of COOH groups for DA by MIPs [36]. In this study, we in-conformation change of DA onto the recognition site constructedvestigated “three-point” recognition. We prepared MIPs using

by the template molecule (the recognition image is illustrated inmany simple and commercially used template molecules and the

Figure 9) [38]. Additionally, 1,3,5-PeTA-MIP could be used forselectivity of MIPs for DA was evaluated using LC. Additionally,

purification of DA as SPE media from blue mussel extracts (Figure

in order to verify the selective recognition results, we also investi-

10).

gated the conformational alternation of DA by computer modeling.

Here, we proposed a new concept of the selective 3 D recogni-

As results of LC evaluations, simple aliphatic tricarboxylic

tion for the flexible compounds by the FIT. We expect that thisacid (pentane-1,3,5-tricarboxylic acid: 1,3,5-PeTA-MIP) wasconcept will be applied to a greater number of compounds having

worked as a template molecule for selective recognition of DA.

several functional groups and flexible conformation changes.

Moreover, the results of the Gibbs free energies in the chromaConclusion

tographic analysis [37] were compared with the estimated isomers

Author has been studying the modified molecular imprinting
technique. It is expected that the several methodological procedures described in this review will become practical tools for theselective separation of a lot of toxic compoundsand rare naturalcompounds. In the future works, we should develop the novel separation media for the selective separation of biomolecules such asprotein and nucleic acids, and the additional sensor techniques for
detection.
Acknowledgements
Figure 7. Structure of domoic acid
Where (a)-(c) shows each COOH group to compare the distanceson computer modeling.

The author would like to return his grateful acknowledgment
to Professor Kunimitsu Kaya, Professor Ken Hosoya (Tohoku University) and Professor Nobuo Tanaka (Kyoto Institute of Technol-

Table 2. Gibbs free energies of DA onchromatographic evaluation
Polymers

k’of DA∆G-∆G (on BASE) (kJ/mol)∆G-∆G (on BLAMK) (kJ/mol)

BASE

0.10

―――

―――

BLANK

1.10

6.04

―――

OPA-MIP

9.62

11.5

5.46

2,3-NDA-MIP

13.7

12.4

6.35

2,6-NDA-MIP

5.94

10.3

4.25

EDA-MIP

12.6

12.2

6.14

FDA-MIP

11.6

12.0

5.93

KDA-MIP

14.5

12.6

6.51

1,3,5-PeTA-MIP

53.5

15.8

9.79

The Gibbs free energies in the chromatographic analysis were estimated by the equation ∆G=−RTlnK,
where k’=βK (k’ is the retention factor and β is a chemical equilibrium constant). Moreover, ∆∆G between
two stationary phases can also be estimated by the equation ∆∆G=−RTlnα (α is the separation factor).

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Chromatography, Vol.29 No.1 (2008)

Takuya Kubo

Figure 8. Alteration of ∆G by conformation changes in DA
Stable conformation of DA in the mobile phase condition was estimated by RHF/6-31G (d), SCI-PCM in Gaussian 03 W. (1): most stable conformation of DA and (2): conformation of DA after protonation of COOH (b)Chromatographic Science for selecting him as a recipient of the
Encouragement Award in 2007 and giving him an opportunity to
publish this focusing review.
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Figure 10. Chromatograms of blue mussel extracts containing DA(a): passed-through fraction from 1,3,5-PeTA-MIP and (b): collected fraction from 1,3,5-PeTA-MIP
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