Report
MIPs As Chromatographic Stationary dentists have been working for decades to mimic the exquisite molecular recognition ability of biological molecules such as antibodies, enzymes, and receptors. One approach has been the design and synthesis of "smallmolecule" receptor surrogates such as the crown ethers, cyclodextrins, and cyclophanes. The importance of this work was recognized with the award of the Nobel Prize to Cram, Lehn, and Pedersen in 1987 (1-3). On the other hand, the design and construction of "macromolecular" synthetic receptors has not been as successful. One approach that seems promising is molecular imprinting, a technique for creating three-dimensional networks that have a "memory" of the shape and functional group positions of the template molecule. The resulting molecular imprint polymers (MIPs) can selectively recognize the template molecule used in the imprinting process, even in the presence of compounds with structure and functionality similar to those of the template. Molecular imprinting has become increasingly popular in recent years and several excellent reviews have been published (4-9) MIPs have been applled as artificial antibodies (10) catalysts (11) (9) drug assay tools (12) and chromatographic stationary phases (13) This Report focuses on the application of MIPs as separation media especially as highly selective chiral tionary phases Vincent T. Remcho Z. Jessica Tan Oregon State University 248 A
Molecular imprint polymers recognize specific compounds and show promise as separation media, especially for chiral molecules. There is an increasing demand, especially in the agricultural chemicals and pharmaceutical industries, for better and more efficient means to prepare, purify, and analyze chiral compounds. This demand is driven by the sometimes markedly different biological activities of the enantiomers of a given compound and increasingly stringent regulations regarding optically active molecules. Despite dramatic improvements in asymmetric synthesis, chromatographic methods are still indispensable for analyzing and purifying chiral compounds Commercial chiral stationary phases (CSPs) use immobilized chiral functionalities ranging from small organic compounds to entire proteins For separating a specific enantiomeric pair several and mobile-phase conditions earu
CSp\
may have to be evaluated tain satisfactory results; furthermore the elution order of each enantiomer is often difficult to predict MIPs, in contrast, have predetermined selectivity and can be custom-made. When MIPs are used for chromatographic separations (Figure 1), the isomer used in the preparation of the MIP is always the one that is more strongly retained; therefore,
Analytical Chemistry News & Features, April 1, 1999
the elution order is predictable. This is especially convenient for chiral separations, because further measurement would otherwise be necessary to determine the chirality of the eluted analytes. Many highly effective chiral separations can be achieved using conventional CSPs (14-17). MPP researchers hope to achieve one or more of several specific goals, including exquisite recognition and selectivity, production of low-cost media in bulk for preparative-scale separations, and the fabrication of a highly customizable sorbent. It is unlikely that MIPs will supplant the handful of CSPs that currently dominate the market and accommodate the majority of current chiral separation needs; nonetheless, MIPs offer unique advantages that will keep them at the focus of an expanding research field Basics The concept of molecular imprinting was inspired by Pauling's antibody formation theory, in which an antigen is used as a template to aid in the rearrangement of antibody polypeptide chains so that the antibody has a configuration that complements the antigen molecule (18). Although
Phases For Molecular Recognition Pauling's speculation was proven incorrect for antigen-antibody interactions, chemists found useful the idea of forming a threedimensional structure around a template for preparing synthetic analogs of antibodies that can recognize target molecules with selectivities similar to their biological counterparts. The first experimental attempt at molecular imprinting wss made in 1999 by imprinting a dye on silica gel ({9). However, it wss not until the early 1970s that successful imprints on synthetic organic polymers achieved (4-8). In molecular imprinting, monomers, such as methacrylic acid and styrene, are first assembled around the template molecule as a function of their complementary interactions. This arrangement of monomers is fixed on polymerization. The two general methods for establishing the prearrangement aee covalent (4) and noncovalent (6) imprinting (Figure 2). During covalent imprinting, a monomer is linked to the template molecule via a labile covalent bond, such as boronic ester (4) orketal ((0). The functionalized monomers are then copolymerized with an excess of crosslinker, such as divinylbenzene or ethylene glycol dimethacrylate, in the presence of a porogen (an inert solvent that encourages the formation of a network of pores in the resulting polymer). After polymerization, the polymer is dried, ground, and sieved; the "linker bond" is chemically cleaved, and the templates are freed from the polymer matrix, leaving cavities that are complementary to the template in shape and spatial configuration An excess of crosslinker is ordinarily
used to preserve and stabilize the specific cavities (4). Subsequent interactions between the template and the corresponding
MIP can be in the form of either covalent (19) or noncovalent (21) interactions. During noncovalent imprinting, weak
Figure 1 . Depiction of an HPLC column packed w i t h a MIP stationary phase used for chiral separations. Analytical Chemistry News & &eatures, April 1i 1,19
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Report various imprinted sites gives rise to a nonlinear adsorption isotherm, which, in turn, results in extreme tailing of the more retained peak (23). These peak-broadening and tailing effects can be attenuated to an extent by careful optimization of the synthetic and chromatographic conditions. The mechanism of solute retention on imprinted polymers is, at best, quite poorly understood, although there is compelling evidence to support a cation-exchange mechanism in certain MIPs (23).
Figure 2. Simplified schematics of (a) covalent and (b) noncovalent imprinting procedures.
intermolecular interactions are used to selfassemble the monomer (s) around the template molecule. Typical interactions include metal-ligand complexation; hydrogen bonding; and ionic, 7t-jt, dipole, and hydrophobic interactions. Because there is no covalent bonding between the template and the monomers, the template is readily extracted after polymerization. Care must be taken when choosing the monomers, crosslinkers, and porogens in order to achieve good imprinting. The most commonly used monomers are methacrylates such as methacrylic acid and heteroaromjitic monomers such as vinylDvridine (6) Nonoolar aprotic solvents are generally used although been made to achieve noncovalent imprinting in an aque-
cal reactions that can be used for covalent molecular imprinting also restricts the applicability of this method. Noncovalent imprinting is more flexible and simpler to implement than covalent imprinting; thus, it has become the more popular method for synthesizing MIPs. Functional groups on both the monomer and the template are generally required to provide favorable interactions, and usually more than one interaction site on the template is required to achieve templatespecific recognition. Good imprinting is usually accomplished via relatively strong noncovalent interactions; however such strong interactions also cause extra broadening of the retained peak when the finished MIP is used as a stationary phase
The covalent imprinting approach is claimed to yield more uniform imprinted sites than the noncovalent approach because the monomer and template are held together by a chemical bond during polymerization. However, more quantitative determinations of uniformity are lacking. When the subsequent separation also relies on covalent interactions, the slow kinetics of such an interaction lead to extremely broad peaks and degrade resolution (4) The limited selection of reversible chemi-
In addition, because the assembly of monomelic species around the template is not strictly defined, the resulting binding sites have various affinities toward the template, similar to polyclonal antibodies. A fraction of the imprinted sites may provide the most favorable interactions with the template molecule and exhibit the highest affinity, whereas other imprinted sites may interact with the template in a less favorable or even a nonspecific manner. When such MIPs are used for chromatographic separation, the difference in the the binding strengths of
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News & Features, April 1, 1999
Making MIP stationary phases MIPs can be prepared in several formats for use in chromatography. The conventional approach is to synthesize the MIP in bulk, grind the resulting polymer block into particles, and sieve the particles into the desired size ranges. Particles of
