Adsorption isotherms of a molecular imprinted polymer prepared in the presence of a polymerisable template

Abstract

The current opinion about molecular imprinted polymers (MIPs) is that their molecular recognition properties are due to the presence of nanocavities formed during a polymerization process developed in the presence of a template molecule. According to this principle, the shape of these nanocavities is complementary to that of the template and non-covalent interactions are established between the binding site and a single template molecule. Nevertheless, there are some experimental indications that the real molecular recognition mechanism involves clusters of template molecules being packed into the binding site. Recently, it has been proposed that template molecules covalently linked to the binding site can act as nucleation points, enhancing the formation of these molecular clusters.

We have tested this hypothesis by studying the adsorption isotherms of polymers prepared by imprinting them with 2,4,5-trichlorophenoxyacetic acid (2,4,5-T). Three different polymers were considered: P0, prepared without the template, P1, whose template was represented by 2,4,5-T molecules, and P2, whose template was 1/3 constituted by the polymerisable 2-(2,4,5-trichlorophenoxyacetoxy)-ethylmethacrylate (2,4,5-TEMA) and 2/3 by 2,4,5-T. The polymers were prepared by thermoinduced polymerization of template mixtures, 4-vinylpyridine and ethylene dimethacrylate. The crushed polymers were packed into HPLC columns and frontal chromatographic runs were performed by eluting the columns with a mobile phase containing variable amounts of 2,4,5-T.

The experimental adsorption isotherms were fitted by using several isotherm models, and the Freundlich–Langmuir model was found to give the best fitting in terms of F-test. All the models considered showed a significant difference between the affinity constant values measured for the polymer P1 and P2, with a higher value for the polymer P2 (for Freundlich–Langmuir model: polymer P1, k=(2.00±0.43)×10⁴ M⁻¹; polymer P2, k=(1.93±0.0535)×10⁵ M⁻¹; ratio P2/P1, 9.65±2.09). Such experimental results support the hypothesis that a polymer prepared with a limited amount of template covalently attached to the binding site shows an increased affinity for the template itself.

Introduction

The current opinion about the molecular imprinted polymers (MIPs) is that their peculiar molecular recognition properties are due to the presence of nanocavities formed during a polymerization process developed in the presence of a template molecule and of suitable functional monomers [1], [2].

According to the key-lock principle, the shape of the nanocavities is complementary to that of the template. The non-covalent interactions which govern the molecular recognition mechanism are established between the binding site and a single, isolated template molecule. Moreover, as regards a polymer obtained by the non-covalent imprinting technique, the key-lock principle should be intended as effectively operating not in materials possessing a single, well-defined class of binding site (i.e. ”monoclonal” imprinted polymers), but in a complex environment constituted by a multiplicity of binding sites with a wide and perhaps continuous distribution of affinities for the template (i.e. “polyclonal” imprinted polymers) [3], [4], [5], [6]. It should be remarked that the existence of multiple classes of binding sites could influence the performances of many molecular imprinting application, such as liquid chromatography or solid phase extraction. Thus, a better understanding of the binding behavior could have a significative impact to the field of analytical chemistry.

Some experimental indications show that the molecular recognition behavior of the imprinted binding sites is more complex than that generally considered because it involves not only distinct classes of binding site, but also template–template interactions. The presence of high-order complexes was hypothesized to explain anomalous ${}^1\text{H}$ NMR data obtained from the l-phenylalanine anilide–methacrylic acid pre-polymerization mixtures [7]. In the nicotine-imprinted polymer used as chiral chromatographic stationary phases, increased sample load caused a decrease of the enantioselectivity joined to a marked peak distortion and an increase of the capacity factor. These effects were interpreted by the authors as an indication of the presence of high-order complexes involving template–template interactions, both during the imprinting process and the column elution [8]. Again, interactions between functional monomers and clusters of template molecules were postulated to explain the binding behavior of a cholesterol-imprinted polymer obtained using a polymerizable cholesterol derivative as functional monomer [9].

The concept of a significant influence of residual template molecule, both in the form of high-order complexes and isolated entities, on the molecular recognition properties of silica-based imprinted materials was already the subject of discussions in the 1960s as a possible alternative to the footprint hypothesis [10], [11], [12], but there was not enough experimental evidence that residual template molecules trapped in the imprinted silica effectively played a role in the recognition mechanism. Recently, this hypothesis has again been proposed by Katz and Davis for organic polymers [13]. A polymer imprinted with a mixture of l-phenylalanine anilide and the polymerisable analogue l-phenylalanine-4-vinylanilide showed an increase of the template rebinding compared to the polymer prepared without the analogue. The authors have interpreted those experimental findings as evidence of high-order complexes in the binding sites, due to the presence of anilide molecules covalently bonded to the polymeric structure and acting as nucleating centers for template molecules. Analogously, the bonding should be due to the adsorption of template molecules triggered by residual template molecules already present in the binding site.

Anyway, it should be noted that also in the absence of residual molecules the adsorption of the template by the binding site could be due to the formation of molecular clusters. According to this model, when template structure make it possible, template binding may occur between a binding site and a preformed template cluster, or, more probably, by a progressive clustering of template molecules in the binding site, triggered by increasing template–template molecular interactions with a cooperative-like behavior. In this case, the presence of one or more covalently bonded molecules of template could enhance the overall strength of the binding, thus justifying the experimental findings of Katz and Davis without invoking the hypothesis of residual template molecule.

In this work, we have tested this hypothesis by studying by frontal chromatography the adsorption isotherms of polymers prepared by imprinting them with the herbicide 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) in the presence and in the absence of a polymerisable analogue able to act as a hypothetical nucleating agent. The 2,4,5-T molecule was selected because it has been used as the template in non-covalent imprinting studies previously published by our group, and its binding behavior compared to an imprinted polymer has been well characterized [14], [15].