Edited by G. Subramanian
Copyright ©2001 Wiley-VCH Verlag GmbH
ISBNs:3-527-29875-4 (Hardcover); 3-527-60036-1 (Electronic)
6Enantiomer Separations using DesignedImprinted Chiral Phases
Börje Sellergren
6.1Introduction
The need for efficient high-throughput techniques in the production of enantiomeri-cally pure compounds is growing in parallel to the increasing structural complexityof new drug compounds [1].
In the absence of synthetic methods allowing the drug to be synthesized in opti-cally pure form,the resolution of racemates using characterized chiral selectors orauxiliaries is the first step in this process. These techniques have the additionaladvantage of providing both enantiomers in preparative amounts,which means thatthe requirements for biological testing of both enantiomers can be met. Convention-ally,preparative optical resolution is performed by fractional crystallization,micro-biological methods,kinetic enzymatic resolution and by chromatography. Ofgrowing importance are methods allowing continuous production of pure enan-tiomers. In chromatography,these can be based on liquid–solid partitioning as insimulated moving bed (SMB) chromatography (see Chapter 10) or liquid–liquid par-titioning as in countercurrent distribution [2,3] or chromatography [4]. In the caseof phases exhibiting particularly high enantioselectivities,batch- [5],membrane-,[6–8] or bubble- based [9] separation techniques may be more attractive.
In chromatography,polysaccharide-based phases (modified amylose or cellulose)are,due to their high site density and broad applicability,the most common phasesused for preparative-scale separations [10]. A problem with these,as well as othercommon CSPs,is the limited predictability of elution orders and separability,mak-ing screening of stationary phase libraries a necessary step in the method develop-ment [10]. Polymers imprinted with chiral templates here promise to alleviate theseproblems offering a new generation of custom-made CSPs with predictable selectiv-ities [11]. In view of the high selectivity often exhibited by these phases,preparativeapplications in the above-mentioned formats are being investigated. This review willsummarize the present state of this research field.
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Fig. 6-1.Approaches to generate imprinted binding sites.6.2Molecular Imprinting Approaches
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6.2Molecular Imprinting Approaches
Molecularly imprinted polymers (MIPs) can be prepared according to a number ofapproaches that are different in the way the template is linked to the functionalmonomer and subsequently to the polymeric binding sites (Fig. 6-1). Thus,the tem-plate can be linked and subsequently recognized by virtually any combination ofcleavable covalent bonds,metal ion co-ordination or noncovalent bonds. The firstexample of molecular imprinting of organic network polymers introduced by Wulffwas based on a covalent attachment strategy i.e. covalent monomer–template,cova-lent polymer–template [12].
Currently,the most widely applied technique to generate molecularly imprintedbinding sites is represented by the noncovalent route developed by the group of Mos-bach [13]. This makes use of noncovalent self-assembly of the template with func-tional monomers prior to polymerization,free radical polymerization with acrosslinking monomer,and then template extraction followed by rebinding by non-covalent interactions. Although the preparation of a MIP by this method is techni-cally simple,it relies on the success of stabilization of the relatively weak interac-tions between the template and the functional monomers. Stable monomer–templateassemblies will in turn lead to a larger concentration of high affinity binding sites inthe resulting polymer. The materials can be synthesized in any standard equippedlaboratory in a relatively short time,and some of the MIPs exhibit binding affinitiesand selectivities in the order of those exhibited by antibodies towards their antigens.Nevertheless,in order to develop a protocol for the recognition of any given target,all of the alternative linkage strategies must be taken into account.
Most MIPs are synthesized by free radical polymerization of functional monoun-saturated (vinylic,acrylic,methacrylic) monomers and an excess of crosslinking di-or tri- unsaturated (vinylic,acrylic,methacrylic) monomers,resulting in porousorganic network materials. These polymerizations have the advantage of being rela-tively robust,allowing polymers to be prepared in high yield using different solvents(aqueous or organic) and at different temperatures [14]. This is necessary in view ofthe varying solubilities of the template molecules.
The most successful noncovalent imprinting systems are based on commodityacrylic or methacrylic monomers,such as methacrylic acid (MAA),crosslinked withethyleneglycol dimethacrylate (EDMA). Initially,derivatives of amino acid enan-tiomers were used as templates for the preparation of imprinted stationary phases forchiral separations (MICSPs),but this system has proven generally applicable to theimprinting of templates allowing hydrogen bonding or electrostatic interactions todevelop with MAA [15,16]. The procedure applied to the imprinting with l-pheny-lalanine anilide (L-PA) is outlined in Fig. 6-2. In the first step,the template (L-PA),the functional monomer (MAA) and the crosslinking monomer (EDMA) are dis-solved in a poorly hydrogen bonding solvent (porogen) of low to medium polarity.The free radical polymerization is then initiated with an azo initiator,commonly azo-N,N’-bis-isobutyronitrile (AIBN) either by photochemical homolysis below roomtemperature [16,17] or thermochemically at 60 °C or higher [15]. Lower thermo-
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chemical initiation at temperatures down to 40 °C or 30 °C is also possible using lessstable azoinitiators [18]. In the final step,the resultant polymer is crushed using amortar and pestle or in a ball mill,extracted using a Soxhlet apparatus,and sieved toa particle size suitable for chromatographic (25–38 µm) or batch (150– 250 µm)applications [16]. The polymers are then evaluated as stationary phases in chro-matography by comparing the retention time or capacity factor (k’) [19] of the tem-plate with that of structurally related analogs (Fig. 6-3). We will refer to the systemshown in Fig. 6-2 as the L-PA-model system.
Fig. 6-2.Preparation of MIPs using L-phenylalanine anilide (L-PA) as template. The L-PA model system.
In the elucidation of retention mechanisms,an advantage of using enantiomers astemplates is that nonspecific binding,which affects both enantiomers equally,can-cels out. Therefore the separation factor (α) uniquely reflects the contribution tobinding from the enantioselectively imprinted sites. As an additional comparison theretention on the imprinted phase is compared with the retention on a nonimprintedreference phase. The efficiency of the separations is routinely characterized by esti-mating a number of theoretical plates (N),a resolution factor (Rs) and a peak asym-metry factor (As) [19]. These quantities are affected by the quality of the packing andmass transfer limitations,as well as of the amount and distribution of the bindingsites.
Some restrictions of this molecular imprinting technique are obvious. The tem-plate must be available in preparative amounts,it must be soluble in the monomermixture,and it must be stable and unreactive under the conditions of the polymer-
6.2Molecular Imprinting Approaches
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Fig. 6-3.Principle of the chromatographic evaluation of the recognition properties of MIPs.ization. The solvent must be chosen considering the stability of the monomer– tem-plate assemblies and whether it results in the porous structure necessary for a rapidkinetics in the interaction of the template with the binding sites. However if thesecriteria are satisfied,a robust material capable of selectively rebinding the templatecan be easily prepared and evaluated in a short time.Table 6-1.Examples of racemates successfully resolved on MIPs.RacemateaSeparationfactorbα1.6ResolutionfactorbRs1.50.980.75ND1.20.50.8ND0.50.9n.d.0.7NotecRefe-renceAmino acidsPhenylalaninePhenylglycineTyrosineAmino acid derivativesPhenylalanine ethyl esterPhenylalanine anilide (PA)Phenylalanine ethyl amidep-Aminophenylalanine ethyl esterArginine ethyl esterTryptophan ethyl esterp-Aminophenylalanine anilidePhenylalanine-N-methyl-anilide (PMA)Leucine-β-naphthylamided,e[104]1.34.92.01.81.51.85.72.03.8[15][105][15][15][105][105][78][25][26]156
Racematea6Enantiomer Separations using Designed Imprinted Chiral Phases
Separationfactorbα3.74.58.42.72.52.22.31.94.34.42.03.21.33.92.35.13.23.63.32.52.82.91.083.41.77.7 / 5.7 f1.53.6high1.9>1.5ResolutionfactorbRs1.41.01.10.42.91.73.1–1.91.91.51.60.82.2ND0.54.54.2> 2NotecRefe-rence[26][26][106][106][27][27][73][107][108][108][108][108][27][108][109][26][110][110][31][111][112][112][113][29][114][113][108][108][96][115][116]N,N’-Dimethyl-phenylalanine anilideProline anilidePyridylmethyl-phenylalanine anilidePyridoxyl-phenylalanine anilideCbz-Glutamic acidCbz-Aspartic acidCbz-PhenylalanineCbz-AlanineCbz-TyrosineBoc-TryptophanBoc-PhenylalanineDansyl-PhenylalanineBoc-Proline-N-hydroxysuccinimide esterAcetyl-Tryptophan methyl esterDiethyl-2-amino-3-phenyl-propylphosphonatePeptidesPhenylalanylglycine anilideCbz-Ala-Ala-OMeCbz-Ala-Gly-Phe-OMeN-Ac-Phe-Trp-OMeCbz-Asp-Phe-OMeCommercial drugsPropranololTimololMetoprololEphedrineNaproxenRopivacaineCarboxylic acidsR-(–)-Mandelic acidR-Phenylsuccinic acid2-Phenylpropionic acidAminesN-(3,5-dinitrobenzoyl)-methylbenzylamine(R)-α-methylbenzylaminea) TRIMVPy-MAAVPy-MAAVPy-MAAVPy-MAATRIMTRIMVPY-MAA1.32.01.21.60.8TRIMdVPYd–2.0high–1.0VPyVPyPYAA/DVBdMAA/DPGLEach racemate was applied on a polymer (ca. 0.1 µmol per gram dry polymer) imprinted with one antipode ofthe racemate. The standard mobile phase,consisting of acetonitrile containing various amounts of acetic acid,was used in most cases. Cbz = Carbobenzyloxy,Boc = t-butyloxycarbonyl.b) αwas calculated as the ratio of the capacity factor (k’) of the template enantiomer to the capacity factor of itsantipode. Rsis the resolution factor.c) The polymers were prepared using MAA as functional monomer and EDMA as crosslinking monomer if nototherwise noted. VPY= 2- or 4-vinylpyridine; TRIM = trimethylolpropane trimethacrylate; DPGL = (R)-N,O-dimethacryloylphenylglycinol; PYAA = 3-(4-pyridinyl)acrylic acid.d) The polymer was evaluated in capillary electrophoresis.e) The polymer was imprinted with L-PA.f) Migration times of the two enantiomers.6.3Structure–Binding Relationships1576.3Structure-Binding RelationshipsA large number of racemates have been successfully resolved on tailor-made MIC-SPs (Table 6-1). Using MAA as functional monomer,good recognition is obtainedfor templates containing Brönsted-basic or hydrogen bonding functional groupsclose to the stereogenic center. On the other hand,templates containing acidic func-tional groups are better imprinted using a basic functional monomer such asvinylpyridine. This emphasizes the importance of functional group complementaritywhen designing the MICSPs. Furthermore,the separation factors are high and higherthan those observed for many of the widely used commercial CSPs [20]. However,the columns are tailor-made and the number of racemates resolved equals nearly thenumber of stationary phases,i.e. each column can resolve only a limited number ofracemates. Although the separation factors are high,the resolution factors are low,but the performance can often be enhanced by running the separations at higher tem-peratures [15] and by switching to an aqueous mobile phase (Fig. 6-3) [21],or byperforming the imprinting in situ in fused silica capillaries for use in capillary elec-trochromatography [22,23]. At low sample loads,the retention on the MICSPs isextremely sensitive to the amount of sample injected,indicating overloading of asmall amount of high energy binding sites. [24] Moreover the peaks correspondingto the template are usually broad and asymmetric. This is ascribed to the mentionedsite heterogeneity together with a slow mass transfer (see Section 6.3.1).Table 6-2.Examples of highly selective recognition by MIPs.Templatek’L(1)6.6α(1)k’L(2)1.05α(2)4.21.071.71.42.12.02.42.00.91.30.41.10.82.3The polymers were prepared by the standard procedure using MAA as functional monomer (see Fig. 6-2) asdescribed elsewhere [25]. a Mobile phase:acetonitrile/acetic acid:90/10 (v/v). Sample:0.2 µmol racemate g–1.b Mobile phase:acetonitrile/water/acetic acid:96.3/1.2/2.5 (v/v).158
6Enantiomer Separations using Designed Imprinted Chiral Phases
6.3.1 High Seletivity
MICSPs are often highly selective for their respective template molecule. This wasthe case for polymer imprinted with L-phenylalanine anilide (L-PA) and L-pheny-lalanine-N-methylanilide respectively (comparing a secondary and tertiary amide astemplate) (Table 6-2) [25]. The racemate corresponding to the template was wellresolved on the corresponding MICSP,whereas the analogue racemate was lessretained and only poorly resolved. Similar results were obtained when comparing apolymer imprinted with L-phenylalanine ethyl ester and one with its phosphonateanalogue (Table 6-2) and have also been observed in comparisons of a primary (1)and a tertiary (2) amine,different in two amino methyl groups,two diacids,N-pro-tected aspartic (3) and glutamic (4) acid,which differed only in one methylene group
in the alkyl chain [26,27]. Pronounced discrimination of minor structural differ-ences have also been reported in the imprinting of N-protected amino acids as (5)and (6) [28],aminoalcohols such as ephedrine (7) and pseudoephedrine (8) [29],monosaccharides [30] and peptides such as 9–11 [31]. Since the polymers imprintedwith templates containing bulky substituents discriminated against those containing
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smaller substituents,the recognition is not purely size exclusion but instead must bedriven by shape complementarity between the site and the substrate,or conforma-tional differences between the derivatives. It was concluded on the basis of 1H-NMRnuclear Overhauser enhancement experiments and molecular mechanics calculationsthat L-PA and the N-methylanilide exhibit large conformational differences. Thus,the torsional angles between the anilide ring plane and the amide plane,as well as inthe E-Z preference over the amide bond (Fig. 6-4) are different [25]. The low energyconformer of the anilide has the phenyl group in a cisconformation to the carbonyloxygen with a torsional angle of about 30 °,whereas in the N-methylanilide thephenyl group is found in a transconformation twisted almost 90 °out of the amideplane. This will result in a different arrangement of the functional groups at the site.In this context it is interesting to note (Table 6-2) that the polymer imprinted withthe N-methylanilide is less selective for its template,i.e. a lower separation factor isseen for the template compared to what is observed using the L-PA-imprinted poly-mer and furthermore,a significant separation of the enantiomers of D,L-PA is alsoobserved. This can be explained considering the smaller space requirements of D,L-PA that thus can be forced into a conformation matching the site of the N-methy-lanilide.Fig. 6-4.Minimum energy conformations of L-PA and L-phenylalanine-N-methyl-anilide (L-PMA)based on molecular mechanics calculations and UV- and NMR-spectroscopic characterizations. (FromLepistö and Sellergren [25].)Table 6-3.Resolution of amino acid derivatives on a MIP imprinted with L-phenylalanine anilide (L-PA).RacematePhenylalanine anilideTyrosine anilideTryptophan anilidePhenylalanine p-nitroanilideLeucine p-nitroanilideAlanine p-nitroanilideData taken from reference [117].k’L3.52.92.43.12.12.0α2.32.22.02.11.61.66.3Structure–Binding Relationships
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6.3.2 Low Seletivity
Numerous examples of MICSPs that are capable of resolving more than the race-mate corresponding to the template have been reported [17,32]. In these cases somestructural variations are tolerated without seriously compromising the efficiency ofthe separation. For instance,a polymer imprinted with L-phenylalanine anilideresolved amino acid derivatives with different side chains or amide substituents [17].Anilides of all aromatic amino acids were here resolved as well as β-naphthylamidesand p-nitroanilides of leucine and alanine (Table 6-3). Furthermore,in aqueousmobile phases,the free amino acid phenylalanine could also be base line resolved onan L-PA-imprinted polymer [32]. Apparently,substitution of groups that are notinvolved in potential binding interactions only leads to a small loss in enantioselec-tivity. Also it was noted that the dipeptide,D,L-phenylalanylglycine anilide wasresolved,while glycyl-D,L-phenylalanine anilide was not. This observation empha-sizes the importance of the spatial relationship between the functional groups at thesites,and indicates that substitutions made at some distance away from the center ofchirality are allowed.
6.3.3 Studies of the Monomer–Template Solution Structures
To what extent do the solution complexes formed between the monomer and thetemplate in solution reflect the architecture of the polymeric binding sites ? Thisquestion is important,since a thorough characterization of the monomer templateassemblies may assist in deducing the structure of the binding sites in the polymerand thus have a predictive value. 1H-NMR spectroscopy and chromatography wereused to study the association between MAA and the template L-PA in solution as a
1H-NMR chemical shifts ofmimic of the pre-polymerization mixture [15]. The
either the template or the monomer versus the amount of added MAA as well as thechromatographic retention of D,L-PA versus the amount of acid in the mobile phase,varied in accordance with the formation of multimolecular complexes between thetemplate and the monomer in the mobile phase. A 1:2 template–monomer complexwas proposed to exist prior to polymerization based on the modeled complex distri-bution curves. Based on these results,hydrogen bond theory,and the assumption thatthe solution structure was essentially fixed by the polymerization,a structure of thetemplate bound to the site was proposed (Fig. 6-5). Since these initial studies,a num-ber of other examples support this model,i.e. the recognition is due to functionalgroup complementarity and a correct positioning of the functional groups in the sitesas well as steric fit in the complementary cavity [33-36]. Rebinding to sites formedof residual nonextracted template have also been proposed as a contributing factor tothe observed recognition [37]. In most imprinted systems however,rebinding selec-tivity or catalytic efficiency increase with increasing recovery of the template [38]and the Langmuir-type adsorption indicates a true receptor behavior [39].
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Fig. 6-5.Model of the binding site for L-PA based on spectroscopic and chromatographic characteri-zation of the prepolymerization monomer–template assemblies.6.4Adsorption Isotherms and Site DistributionAdsorption isotherms can yield important information concerning binding energies,modes of binding and site distributions in the interaction of small molecule ligandswith receptors [40]. In the case of MIPs,a soluble ligand interacts with binding sitesin a solid adsorbent. The adsorption isotherms are then simply plots of equilibriumconcentrations of bound ligand (adsorbate) versus concentration of free ligand. Theisotherms can be fitted using various models where different assumptions are made.The most simple is the Langmuir-type adsorption isotherm (Equation (1)),where theadsorbent is assumed to contain only one type of site,where adsorbate–adsorbateinteractions are assumed not to occur,and where the system is assumed ideal. Thisisotherm depends on two parameters:the saturation capacity (site density),qs,andthe adsorption energy,b [41,42].q=q=a1C1+b1Ca1CaC+21+b1C1+b2C(aandn=numericalparameters)(1)(2)(3)q=aC1/nThe bi-Langmuir model (Equation (2)) or tri-Langmuir model,the sum of two orthree Langmuir isotherms,correspond to models that assume the adsorbent surface6.4Adsorption Isotherms and Site Distribution
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to be heterogeneous and composed of two or three different site classes,and finallythe Freundlich isotherm model (Equation (3)) with no saturation capacity but insteada complete distribution of sites of different binding energies. Depending on the tem-plate-functional monomer system,the type of polymer,the conditions for its prepa-ration and the concentration interval covered in the experiment the adsorptionisotherms of MIPs have been well fitted with all the isotherm models [39,43-45].Thus,most MIPs suffer from a heterogeneous distribution of binding sites. Innoncovalent imprinting,two effects contribute primarily to the binding site hetero-geneity. Due to the amorphous nature of the polymer,the binding sites are not iden-tical,but are somewhat similar to a polyclonal preparation of antibodies. The sitesmay for instance reside in domains with different crosslinking density and accessi-bility [46]. Secondly,this effect is reinforced by the incompleteness of themonomer–template association [15]. In most cases the major part of the functionalmonomer exists in a free or dimerized form,not associated with the template. As aconsequence,only a part of the template added to the monomer mixture gives rise toselective binding sites. This contrasts with the situation in covalent imprinting [33,45,47] or stoichiometric noncovalent imprinting [48,60,90] where theoretically allof the template split from the polymer should be associated with a templated bind-ing site. The poor yield of binding sites results in a strong dependence of selectivityand binding on sample load at least within the low sample load regime.
For determining the adsorption isotherm,the equilibrium concentrations of boundand free template must be reliably measured within a large concentration interval.Since the binding sites are part of a solid,this experiment is relatively simple andcan be carried out in a batch equilibrium rebinding experiment or by frontal analy-sis.
One powerful technique for the study of the interactions between solutes and sta-tionary phases and for the investigation of the parameters of these interactions isfrontal analysis [49]. This method allows accurate determination of adsorption andkinetic data from simple breakthrough experiments,and the technique has proven itsvalidity in a number of previous studies. This has also been used for estimating theadsorption energies and saturation capacities in the binding of templates to MIPs,butoften the data have been modeled only at one temperature and graphically evaluatedusing a simple Langmuir mono site model which in most cases gives a poor fit of thedata [50]. Furthermore,the breakthrough curves are interpreted assuming thermody-namic equilibrium,which is often an invalid assumption in view of the slow masstransfer in these systems. Rather,based on the mass balance equation and by assum-ing kinetic and isotherm values to best-fit isotherms and elution profiles obtained atdifferent temperatures,a more accurate picture of the thermodynamics and masstransfer data can be obtained [49].
The isotherms for the two enantiomers of phenylalanine anilide were measured at40,50,60 and 70 C,and the data fitted to each of the models given in Equations(1–3) [42]. The isotherms obtained by fitting the data to the Langmuir equation wereof a quality inferior to the other two. Fittings of the data to the Freundlich and to thebi-Langmuir equations were both good. A comparison of the residuals revealed thatthe different isotherms of D-PA were best fitted to a bi-Langmuir model,while the
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isotherms for L-PA were slightly better fitted to a Freundlich isotherm model,par-ticularly at low temperatures. However,at concentrations higher than 17 µm (4 ×10–3g L–1),the isotherm data of L-PA were equally well fitted to the Freundlich andto the bi-Langmuir isotherm models,suggesting the existence of binding sites withhigher binding energies (K> 50 000 M–1). At 40 °C for L-PA,the binding constantsand site densities are respectively 84 M–1and ca. 90 µmol g–1for the low-affinitysites,and 16 000 M–1and 1 µmol g–1for the high-affinity sites. For D-PA the respec-tive values are 48 M–1and 136 µmol g–1for the low-affinity sites,and 5520 M–1and0.4 µmol g–1for the high-affinity sites. These values agree well with those deter-mined in previous studies [18]. In view of the small saturation capacities observedfor D-PA on these sites at the other temperatures studied (50,60,70 °C) or after ther-mal annealing of the materials [24],the second site class appears to be specific forL-PA.
For preparative or semipreparative-scale enantiomer separations,the enantiose-lectivity and column saturation capacity are the critical factors determining thethroughput of pure enantiomer that can be achieved. The above-described MICSPsare stable,they can be reproducibly synthesized,and they exhibit high selectivities– all of which are attractive features for such applications. However,most MICSPshave only moderate saturation capacities,and isocratic elution leads to excessivepeak tailing which precludes many preparative applications. Nevertheless,with theL-PA MICSP described above,mobile phases can be chosen leading to acceptableresolution,saturation capacities and relatively short elution times also in the iso-cratic mode (Fig. 6-6).
Fig. 6-6.Overload elution profiles of D,L-PA injected on a column (125 4 mm) packed with the L-PAimprinted stationary phase used in Fig. 6-5. Mobile phase:MeCN:TFA (0.01%):H2O (2.5%). The ten-dency for fronting and the increase in retention with sample load is attributed in part to saturation of themobile phase modifier.
6.5Adsorption–Desorption Kinetics and Chromatographic Band Broadening
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6.5Adsorption-Desorption Kinetics and Chromatographic
Band Broadening
For most applications of specific molecular recognition elements,a rapid associationdissociation kinetics in the ligand receptor binding is important. In chemical sensorsthe response time depends on the association rate between the sensor-bound recep-tor and the target analyte,whereas the dissociation rate determines if,and howquickly,the sensor can be regenerated [51]. The kinetics thus influences the samplethroughput of the analysis,i.e. how many samples that can be analyzed in a certaintime interval. Furthermore,in catalysis the binding kinetics will determine the max-imum rate of the chemical transformation,and in chromatographic separations it willinfluence the spreading of the chromatographic peaks.
When a solute band passes a chromatographic column it is broadened continu-ously due to various dispersion processes [52]. These include processes that showlittle or no flow rate dependence,such as eddy-diffusion or extracolumn effects andflow rate-dependent processes such as axial diffusion,mass transfer processesincluding mobile phase,intraparticle and stationary phase diffusion and slow kineticprocesses upon interaction with the stationary phase. Other factors such as nonlinearbinding isotherms and slow desorption kinetics instead affect the shape of the peak[53]. Altogether,these processes counteract the separation of two compounds andlead to lower resolutions. An understanding of their origin is important in order toimprove the separations as well as to gain insight into the kinetics and mechanismof solute retention.
The dependence of the chromatographic parameters on flow rate and sample loadwas studied in enantiomer separations of d- and l-phenylalanine anilide (D,L-PA) onL-PA-imprinted chiral stationary phases (CSPs) [54].Using a thermally annealed sta-tionary phase,a strong dependence of the asymmetry factor (As) of the l-form onsample load and a weak dependence on flow rate suggested that column overloadingcontributed strongly to the peak asymmetry (Fig. 6-7). This is to be expected in viewof the site heterogeneity discussed in the previous section. However,slow kineticprocesses is another contributing factor to the pronounced band broadening in thechromatography using MIP-based columns. In view of the high binding constantsobserved for MIPs,the desorption rate at the high-energy binding sites should bemuch slower than that at the low-energy sites. The mass transfer rate coefficients,estimated using a MIP prepared in dichloromethane as diluent,were small andstrongly dependent on the temperature and concentration,in particular the rate coef-ficients corresponding to the imprinted L-enantiomer [42]. Recent related studies ofthe retention mechanism of both enantiomers of dansyl-phenylalanine on a dansyl-L-phenylalanine MICSP led to similar conclusions [55],although these processes arestrongly dependent on the system studied,i.e. template-monomer system,crosslink-ing monomer,porogen and method of polymerization.
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Fig. 6-7.Asymmetry factor (As) of the L-enantiomer versus sample load (A) and versus flow rate (B) onL-PA-imprinted polymers. Flow rate:1.0 ml min–1. Mobile phase:MeCN/[potassium phosphate 0.05M,pH 7] (7/3,v/v).
6.6Factors to Consider in the Synthesis of MICSPs
In spite of the fact that molecular imprinting allows materials to be prepared withhigh affinity and selectivity for a given target molecule,a number of limitations ofthe materials prevent their use in real applications. The main limitations are:1Binding site heterogeneity2Extensive nonspecific binding3Slow mass transfer4Bleeding of template
5Low sample load capacity
6Unpractical manufacturing procedure7Poor recognition in aqueous systems
8Swelling–shrinkage:may prevent solvent changes
9Lack of recognition of a number of important compound classes10Preparative amounts of template required
It is clear that improvements aiming at increasing the yield of high-energy bind-ing sites or modifying the site distribution in other ways will have a large impact onthe performance of the materials (affecting limitations 1,2,4 and 5). The strategiesadopted to achieve this have been focusing either on prepolymerization measures,aimed at stabilization of the monomer template assemblies prior to polymerization,or postpolymerization measures aimed at modifying the distribution of binding sitesby either chemical or physical means. The most important of these factors will nowbe discussed,together with techniques allowing their optimization.
6.5Adsorption–Desorption Kinetics and Chromatographic Band Broadening
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6.6.1Factors Related to the Monomer-Template Assemblies
It is of obvious importance that the functional monomers interact strongly with thetemplate prior to polymerization,since the solution structure of the resulting assem-blies presumably defines the subsequently formed binding sites. By stabilizing themonomer–template assemblies,it is possible to achieve a large number of imprintedsites. At the same time,the number of nonspecific binding sites will be minimized,since free functional monomer not associated with the template is likely to be acces-sible for binding. Considering one particular binding site,the following factors havebeen identified that are likely to affect the recognition properties of the site (Fig. 6-8).
Fig. 6-8.Factors affecting the recognition properties of MIPs related to the monomer templateassemblies.
The strength and positioning of the monomer–template interactions are of impor-tance for materials with good molecular recognition properties to be obtained. Thebroad applicability of MAA as a functional monomer is related to the fact that thecarboxylic acid group serves well as a hydrogen bond and proton donor and as ahydrogen bond acceptor [56]. In aprotic solvents such as in acetonitrile carboxylicacids and amine bases form contact hydrogen-bonded assemblies where the associ-
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6Enantiomer Separations using Designed Imprinted Chiral Phases
ation strength for a given acid increases with the basicity of the base [57]. Thus,tem-plates containing Brönsted-basic or hydrogen-bonding functional groups are poten-tially suitable templates for the MAA/EDMA system [15]. Furthermore,more stablecyclic hydrogen bonds can form with templates containing acid [27],amide[26] orfunctionalized nitrogen heterocycles [39,44]. The potential for a given monomertemplate pair to produce templated sites can be predicted by measuring the stabilityconstants,e.g. by spectroscopic techniques,in a homogeneous solution mimickingthe monomer mixture prior to polymerization [15]. This can ultimately be used as a preliminary screening procedure to search for suitable functional monomers. Thus,estimated solution association constants can be correlated with the heterogeneousbinding constants determined for the polymer (Table 6-4). For the prepolymerizationcomplexes discussed thus far,the electrostatic interactions are sensitive to the pres-ence of polar protic solvents. One exception is the complex formed between car-boxylic acids and guanines or amidines [58,59]. Here,cyclic hydrogen-bonded ion-pairs are formed with stability constants that are order of magnitude higher thanthose previously discussed (Table 6-4). This allows amidines such as pentamidine(12) to be imprinted using iso-propanol–water as a porogenic solvent mixture,resulting in polymers that bind pentamidine strongly in aqueous media [60,90].Table 6-4.Association constants for complexes between carboxylic acids and nitrogen bases in aproticsolvents and corresponding association constants and site densities for binding of the base to a molecu-larly imprinted polymer.AcidAcetic acidButyric acid4-Methylbenzoic acidPMAAPMAABaseAtrazine9-Ethyladenine(13)Atrazine9-EthyladenineSolventCCl4CDCl3CDCl3CHCl3CHCl3Ka (m–1)210(1) 114(2) 41>106(1) 8.3 ×104(2) 1.0 ×104(1) 7.7 ×104(2) 2.4 ×103n(µmol g–1)–––20402086Reference[80][79][59][118][39]PMAA refers to polymers imprinted with respective base using MAA as functional monomer.Apart from the successful imprinting discussed above,the recognition for manytemplates is far from that is required for the particular application,even after care-ful optimization of the other factors affecting the molecular recognition properties.Often,a large excess of MAA in the synthesis step is required for recognition to beobserved and then only in solvents of low to medium polarity and hydrogen bond6.5Adsorption–Desorption Kinetics and Chromatographic Band Broadening
169
capacity [61]. In fact,in these cases the optimum rebinding solvent is often the sol-vent used as porogen.[62] Thus,the polymer exhibits memory for the template aswell as the porogen. Moreover,the excess of functional monomer results in a por-tion of the functional monomer not being associated with imprinted sites. These sitesinteract nonselectively with solutes binding to carboxylic acids and limit the degreeof separation that can be achieved. Hence MAA is not a universal monomer. Instead,for the recognition of any given target molecule access to functional monomers tar-geted towards structural features,specific for particular compounds or classes ofcompounds are required.
Based on the structural features of the templates that generate good sites,an inter-esting possibility would be to incorporate these structures in new functionalmonomers for the recognition of carboxylic acids. This concept is somewhat similarto the reciprocity concept in the design of chiral stationary phases [63]. Thus,Wulffet al. synthesized N,N’-substituted p-vinylbenzamidines (13) and showed that thesemonomers could be used to generate high-fidelity sites for the molecular recognitionof chiral carboxylic acids [59]. The binding is here strong enough to provide effi-cient recognition also in aqueous media. Furthermore,due to strong binding thefunctional monomer is quantitatively associated with the template,thus minimizingthe nonspecific binding. Functional group complementarity is thus the basis for thechoice of functional monomer. The search for the optimal structural motif to com-plement the template functionality is preferentially guided by results from the areaof host–guest chemistry and ligand– receptor chemistry. Thus cyclodextrins havebeen used to template binding sites for cholesterol [64] or to enhance the selectivityin the imprinting of enantiomers of amino acids [65]. Based on metal ion co-ordina-tion of amino acids and N-(4-vinylbenzyl)iminodiacetic acid (14),imprinting andsubsequent chiral separation of free amino acids in aqueous solutions has also beenpossible [66].
Based on chiral functional monomers such as (15),MICSPs can be prepared usinga racemic template. Thus,using racemic N-(3,5-dinitrobenzoyl)-a-methylbenzy-lamine (16) as template,a polymer capable of racemic resolution of the template wasobtained [67]. Another chiral monomer based on L-valine (17),was used to prepareMIPs for the separation of dipeptide diastereomers [68]. In these cases the configu-
170
6Enantiomer Separations using Designed Imprinted Chiral Phases
rational chirality inherent in the pendant groups of the polymer are to some extentthemselves chiral selectors,and the effect of imprinting is merely to enhance theselectivity. A good example of this was shown in the imprinting of N-benzyl-L-valineas a bidentate ligand to a styrene-based chiral cobalt complex (18) [69]. The strongenantioselectivity of the imprinted polymer should here be viewed with respect tothe enantioselectivity of the control polymer.
Thus,enhanced separations can be obtained using chiral selectors with configu-rational chirality in combination with molecular imprinting. What about selectorswith conformational chirality ? Can chirality be induced by molecular imprinting ?This concept was elegantly demonstrated by Welch using a brush-type stationaryphases containing a slowly interconverting (in the order of a day) racemic atropiso-mer (19) as imprintable selector (Fig. 6-9) [70]. Leaving the selector in contact withan enantiomerically pure template molecule (20) for more than 2 weeks led to induc-tion of the most stable selector selectand complex. After washing out the selectand,the selector could be used to separate the racemate of 20with similar separation fac-tors as obtained using the reciprocal phase. However due to interconversion,the CSPracemized over a period of 2–3 days,a period that possibly can be extended by stor-ing the CSP at low temperatures. Also mentioned was the interesting possibility ofusing a selection of slowly interconverting selectors to achieve a broadly applicablesystem for atropisomer-based imprinting.
Alternative approaches to imprint peptides via strong monomer template associa-tion have recently been reported,although no results of the chromatographic appli-cation of these phases have been shown. Strong complexation inducing a β-sheetconformation was possible using a designed functional monomer (21) [71]. Peptides
6.5Adsorption–Desorption Kinetics and Chromatographic Band Broadening
171
Fig. 6-9.Imprintable brush-type selectors. (From Welch [70].)
can also be imprinted via a sacrificial spacer approach which potentially will resultin a high yield of templated sites exhibiting pronounced selectivity towards the tar-get peptide (22) [72].
Considering functional group complementarity,other commodity monomers mayalso be used. Thus for templates containing acid groups,basic functional monomersare preferably chosen. The 2- or 4-vinylpyridines (VPY) are particularly well-suitedfor the imprinting of carboxylic acid templates and provide selectivities of the same
172
6Enantiomer Separations using Designed Imprinted Chiral Phases
order as those obtained using MAA for basic templates [73,74]. These polymers are,however,susceptible to oxidative degradation and require special handling.
In the imprinting of carboxylic acids and amides,high selectivities are also seenusing acrylamide (AAM) as functional monomer [28]. Furthermore,combinations oftwo or more functional monomers,giving terpolymers or higher polymers,have in anumber of cases resulted in better recognition ability than the recognition observedfrom the corresponding co-polymers [67,73-75]. These systems are particularlycomplex when the monomers constitute a donor–acceptor pair,sincemonomer–monomer association will compete strongly with template–monomerassociation if neither of the monomers has a particular preference for the template.In a recent series of papers by the group of Liangmo,careful optimization showedthat a combination of acrylamide and 2-vinylpyridine gave significantly higher enan-tioselectivities in the imprinting of N-protected amino acids than the combination of2-vinylpyridine with MAA (23) [76]. Furthermore,better results were obtainedusing acetonitrile as the porogen,in contrast to other systems where solvents oflower polarity (e.g. toluene,CH2Cl2,CHCl3) give the best results. These resultsshow that adequate performance can only be achieved after careful optimizationwhere the related factors are systematically varied.
6.5Adsorption–Desorption Kinetics and Chromatographic Band Broadening
173
Fig. 6-10.Influence of the number of basic interaction sites of the template versus the separation factormeasured in chromatography for the corresponding racemate. The templates were imprinted using MAAas functional monomer by thermochemical initiation at 60/90/120 °C (24 h at each temperature) andusing acetonitrile as porogen. (From Sellergren et al. [15].)
6.6.2Influence of the Number of Template Interaction Sites
Molecular recognition in the biological machinery takes place by the combination ofseveral complementary weak interactions between a biological binding site and themolecule to be bound [77]. A larger number of complementary interactions willincrease the strength and fidelity in the recognition. Thus,templates offering multi-ple site of interaction for the functional monomer are likely to yield binding sites ofhigher specificity and affinity for the template [12]. One example of this effect wasobserved in a study of the molecular imprinting of enantiomers of phenylalaninederivatives (Fig. 6-10) [15,78]. Starting with L-phenylalanine ethyl ester (1) as thetemplate,interactions with carboxylic acids in acetonitrile should consist of theammonium carboxylate ion pair,as well as a weak ester– carboxylic acid hydrogenbond (indicated by arrows). By replacing either the ester group with the strongerhydrogen bonding amide group in (2),or by introducing an aromatic amino group asin (3) – which allows an additional hydrogen bond interaction with another car-boxylic acid group – the enantiomeric selectivity increased. In L-PA (4),where the
174
6Enantiomer Separations using Designed Imprinted Chiral Phases
ethyl amide substituent has been replaced by an anilide group,an additional increasein selectivity is seen. Combining the structural modifications in one molecule,p-amino-phenylalanine-anilide (5),the highest separation factor was obtained. Similarobservations have been made in the imprinting of a number of different classes ofcompounds and thermodynamic evidence for the existence of multiple additive inter-actions in the sites have been provided [35]. In the search for optimal synthetic con-ditions for MIPs,useful start-up information can be obtained from the vast literatureexisting on solution studies of molecular interactions and molecular recognition [Forexample see:79-81].
Fig. 6-11.Stabilization of monomer template assemblies by thermodynamic considerations.
6.6.3Thermodynamic Factors
An important part of the optimization process is the stabilization of themonomer–template assemblies by thermodynamic considerations (Fig. 6-11). Theenthalpic and entropic contributions to the association will determine how the asso-ciation will respond to changes in the polymerization temperature [18]. The changein free volume of interaction will determine how the association will respond tochanges in polymerization pressure [82]. Finally,the solvent’s interaction with themonomer–template assemblies relative to the free species indicates how well it willstabilize the monomer–template assemblies in solution [16]. Here each system mustbe optimized individually. Another option is simply to increase the concentration ofthe monomer or the template. In the former case,a problem is that the crosslinkingas well as the potentially nonselective binding will increase simultaneously. In the
6.5Adsorption–Desorption Kinetics and Chromatographic Band Broadening
175
latter case,the site integrity will be compromised. The above factors have been stud-ied for theL-PA model system. In aprotic media of low polarity,MAA and templatescontaining polar functional groups are only weakly solvated,and the interactionsholding the monomer template assemblies together are mainly electrostatic in nature[77]. In such cases the association of the monomer and template is associated with aloss of one set of rotational and translational degrees of freedom which leads to a netdecrease in entropy [83]. From this follows that the interaction is weakened atincreasing temperature. On the other hand,when the monomer and the template ismore strongly solvated,the association may lead to release of part of the solventshell,leading in turn to a net increase in rotational and translational entropy. In thiscase the interaction will be favored by increasing the temperature.
6.6.4Factors Related to Polymer Structure and Morphology
For the formation of defined recognition sites,the structural integrity of themonomer–template assemblies must be preserved during polymerization to allow thefunctional groups to be confined in space in a stable arrangement complementary to thetemplate. This is achieved by the use of a high level of crosslinking,usually >80% [18].The role of the polymer matrix,however,is to contain the binding sites not only in astable form but also in an accessible form (Fig. 6-12). Porosity is achieved by carryingout the polymerization in presence of a porogen. Most of the crosslinked network poly-mers used for molecular imprinting have a wide distribution of pore sizes associatedwith various degrees of diffusional mass transfer limitations and a different degree ofswelling. Based on the above criteria,i.e. site accessibility,integrity,and stability,thesites can be classified according to different types. The sites associated with meso- andmacro-pores (>20 Å) (sites A and B in Fig. 6-12) are expected to be easily accessiblecompared to sites located in the smaller micropores (<20 Å) (sites C) where the diffu-sion is slow. The number of the latter may be higher since the surface area,for a givenpore volume,of micropores are higher than that of macropores. One undesirable effectof adding an excess of template is the loss of site integrity due to coalescence of thebinding sites,which is related to the extent of template selfassociation. The optimumamount of template is usually about 5% of the total amount of monomer,but can behigher when trivinyl monomers such as TRIM (24) are used as crosslinkers,where alarger fraction of functional monomer is used [84]. In this case higher sample loadcapacities have been observed. The amount of template is of course also limited by thesolubility and availability of the template,although recycling is possible.
176
6Enantiomer Separations using Designed Imprinted Chiral Phases
Fig. 6-12.Different types of binding sites in polymers containing micro- (site B),meso- and macrop-ores (site A); C) Embedded site,D) Site complementary to dimer or multimer,E) Induced binding site,F) Nonselective site,G) Residual template.
Often the materials swell to different extents depending on the type of diluent.The swelling is here normally high in solvents and low in nonsolvents for the poly-mer. Unfortunately,this may lead to large changes in the accessibility and density ofthe binding sites when the solvent is changed [16].
6.7Methods for Combinatorial Synthesis and Screening of
Large Numbers of MIPs
For a complete optimization of all factors,the above-described procedure is notpractical. In order to perform this rapidly,parallel synthesis and screening tech-niques must be developed. These can consist of a scaled-down version of the MIPsin vials that can be handled automatically and analyzed in situ (Fig. 6-13) [85,86].The principle was demonstrated using triazine herbicides as templates and byvarying the type of functional monomer and the monomer composition. With a finalbatch size of ca. 40 mg of monomer,the consumption of monomers and template issignificantly reduced and the synthesis and evaluation can take place in standardhigh-performance liquid chromatography (HPLC) autosample vials. After synthesis,
6.7Methods for Combinatorial Synthesis and Sreening of Large Numbers of MIP
177
Fig. 6-13.Combinatorial imprinting technique suitable for automation.
the primary assessment is based on quantitative HPLC or UV-absorbance analysis ofthe amount of template released from the polymer in the porogenic solvent. Thus inthe case of a rapid and quantitative release the resulting polymer cannot be expectedto rebind a significant amount of the template,and may thus be discarded. After hav-ing established useful functional monomers,a secondary screening for selectivity isperformed. Here,the rebinding of the template to the MIPs was investigated in par-allel to the rebinding to a corresponding control nonimprinted MIP [86]. Alterna-tively,an internal standard,structurally related to the template,may be added and thedifferential binding investigated [85]. An important question is whether the equilib-rium rebinding results reflect the selectivity observed when investigating an up-scaled batch in the chromatographic mode [87]. This was shown in the case of thetriazines,but for other systems suffering from particularly slow mass transfer thismay not be the case. Here,chiral resolution is observed only at low flow rates.
178
6Enantiomer Separations using Designed Imprinted Chiral Phases
6.8New Polymerization Techniques
As indicated above,MIPs have so far been prepared in the form of continuous blocksthat need to be crushed and sieved before use.This results in a low yield of irregu-lar particles,a high consumption of template,and a material exhibiting low chro-matographic efficiency. There is therefore a need for MI-materials that can be pre-pared in high yield in the form of regularly shaped particles with low size dispersityand a controlled porosity. These are expected to be superior in terms of mass trans-fer characteristics and sample load capacity compared to the materials obtained fromthe monolith approach. However,the results obtained so far using alternativeapproaches,although showing some improvements,have been disappointing.
Bead-sized MIPs have been previously prepared through suspension polymeriza-tion techniques either using fluorocarbons (Fig. 6-14) [88] or water [89] as continu-ous phase,dispersion polymerization or precipitation polymerization [90,91]. Thisresulted in spherical particles of a narrow size distribution. These procedures havethe limitation of being sensitive to small changes in the manufacturing conditionsand the type of solvents and polymerization conditions that can be applied,but onceappropriate conditions have been found they should offer an economic alternative forup-scaling. An alternative to this procedure is the coating of preformed supportmaterials [92-94]. MIPs have been prepared as grafted coatings on metal oxide sup-ports [92,93] on organic polymer supports [94] and on the walls of fused silica cap-illaries [95-97]. These techniques however involve many steps and are thus associ-ated with larger batch-to-batch variations. In addition,problems appear in achievinghomogeneous coatings and to suppress secondary interactions with the support sur-face.
Fig. 6-14.Suspension polymerization technique for noncovalent imprinting.
Much effort has been devoted to the development of a multi-step swelling poly-merization technique using water as suspension medium [98]. This has resulted inpolymers showing similar selectivities but slightly improved mass transfer charac-teristics compared with the corresponding monolithic polymers. Of particular rele-
6.9Other Separation Formats
179
vance for bioanalytical applications was the functionalization of the outer surface ofa polymer imprinted with (S)-naproxen with a hydrophilic polymer layer (Fig. 6-15).This led to a slight decrease in the separation efficiency,but allowed on the otherhand direct injection of plasma samples on the columns.
Fig. 6-15.Synthetic scheme of surface-modified MIP for (S)-naproxen. V65 = 2,2’-azobis(2,4-dimethylvaleronitrile); GMMA = glycerolmonomethacrylate; GDMA = glyceroldimethacrylate.
6.9Other Separation Formats
As mentioned in the introduction,due to the high enantioselectivities exhibited bythe imprinted chiral phases,applications in batch-,SMB-,bubble- or membrane-based separation processes may become attractive. The concept of applying MICSPsfor bubble fractionation of enantiomers was demonstrated recently [99]. This sepa-ration principle can be useful for separations of large amounts of material at very lowcosts,and is an important technique for concentrating sulfide ores. For this processto be practical a high enrichment factor is needed and the chiral collector should beeasy to recycle. This is the case of solid collectors such as imprinted polymers whichalso have the benefit of high robustness. Thus L-PA-imprinted polymer particles ofless than 20 µm adhered to air bubbles and were effectively transported to the top ofthe bubble column (Fig. 6-16). The particles were first pre-equilibrated with a solu-tion of the racemate,and then added to the separator. Here,they separated after bub-ble flotation to the top of the column. Enantiomerically enriched compound was thenobtained by washing of the particles that in turn could be recycled. By using fine
180
6Enantiomer Separations using Designed Imprinted Chiral Phases
Fig. 6-16.Bubble fractionation of enantiomers usingimprinted particles (<20 µm) as chiral collectors [99].
floating particles selective for one enantiomer,and large sinking particles selectivefor the opposite enantiomer,the efficiency of this process can most likely beenhanced.
A number of studies have recently been devoted to membrane applications [8,100-102]. Yoshikawa and co-workers developed an imprinting technique by castingmembranes from a mixture of a Merrifield resin containing a grafted tetrapeptideand of linear co-polymers of acrylonitrile and styrene in the presence of amino acidderivatives as templates [103]. The membranes were cast from a tetrahydrofuran(THF) solution and the template,usually N-protected d- or l-tryptophan,removed bywashing in more polar nonsolvents for the polymer (Fig. 6-17). Membrane applica-tions using free amino acids revealed that only the imprinted membranes showeddetectable permeation. Enantioselective electrodialysis with a maximum selectivityfactor of ca. 7 could be reached,although this factor depended inversely on the fluxrate [7]. Also,the transport mechanism in imprinted membranes is still poorly under-stood.
In summary,the present limitations in saturation capacities and selectivity ofimprinted polymers preclude their applications in the above-mentioned preparativeseparation formats.
6.10Conclusions
181
Fig. 6-17.Cast-imprinted membranes. (From Yoshikawa et al. [103].)
6.10Conclusions
A number of conditions will directly influence the development of a new MICSP.The availability of the template in preparative amounts will determine whether it willhave to be recycled,or a template analogue must be used. The latter alternativeshould also be considered in cases where the template is unstable or poorly solublein the monomer mixture. Depending on the format of the separation,the polymermust meet certain requirements. If the material is to be used as a HPLC stationaryphase,then monodisperse spherical particles are desirable and rapid adsorption–des-orption of the template to the sites is necessary for high-performance separations.However,broad and asymmetric band shapes and low saturation capacities due to theheterogeneous distribution of binding sites and slow mass transfer processes areimportant problems that strongly limit the possible applications of these phases inanalytical and preparative chromatography. The use of imprinted polymers in a foamflotation apparatus or in membrane separations have been demonstrated,althoughprobably also here no viable application can be expected in the near future. Never-theless,with designed functional monomers,new polymerization techniques andcombinatorial synthesis and screening techniques,MICSPs that meet the above-mentioned requirements may soon be a reality.
Acknowledgment
The author is grateful to Dr. Francesca Lanza for assistance in preparing themanuscript.
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6Enantiomer Separations using Designed Imprinted Chiral Phases
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