Leif Schweitz

Capillary electrochromatography with molecular imprint-based selectivity for enantiomer separation of local anaesthetics

Preparation of stationary phases of predetermined selectivity for use in capillary electrochromatography by molecular imprinting was studied. Molecular imprinting of the local anaesthetic (S)-ropivacaine in methacrylate-type polymers was done using an in situ photo-initiated polymerisation process. Such stationary phases could separate the enantiomers of ropivacaine and the enantiomers of the structural analogues mepivacaine and bupivacaine. The influence of several parameters on the ability of the resultant imprinted capillary column to resolve rac-ropivacaine was investigated. These parameters included the type and amount of functional and cross-linking monomers, the molar ratio of the imprint molecule to the monomers and the type of the porogen. The polymer-based, monolithic capillary columns could be rendered super-porous by the use of 1–25% isooctane as a porogenic agent. The best resolution was obtained for polymers made using trimethylolpropane trimethacrylate as the cross-linking monomer. The enantiomer separation increased with increasing molar ratio of the functional monomer methacrylic acid to (S)-ropivacaine, however, at the expense of peak broadening. Initial studies on the optimisation of the electrolyte composition showed that the separation increased for electrolytes as the volume ratio of acetonitrile to buffer increased, the temperature raised and the pH became higher.

Molecular imprint-based stationary phases for capillary electrochromatography

The combination of capillary electrochromatography, which provides a high degree of separation efficiency and short separation times, with molecular imprinting, which provides a means for preparing stationary phases of predetermined selectivity, is discussed in this review. Polymerisation around a templating ligand, using monomers that are selected for their ability to participate in non-covalent interactions, constitute the key step of molecular imprinting. Imprints possessing a permanent memory for the imprint species are formed, enabling the resultant polymer selectively to rebind the imprint molecule from a mixture of closely related compounds. Levels of binding affinity and selectivity typical of antibodies are achievable in the best cases. Several studies have focused on imprinted polymers being used as chiral stationary phases in liquid chromatography (LC) where the enantioselectivity of a given polymer is predetermined by the enantiomeric form of the templating ligand. The use of imprinted LC columns, however, is hampered by their poor chromatographic performance, evidenced by peak broadening and tailing. Being a more efficient chromatography technique, the use of capillary electrochromatography greatly improves the performance of imprinted polymer-based separations. The most successful approach utilises capillary columns filled with a monolithic, superporous imprinted polymer obtained by an in situ photo-initiated polymerisation process. This technique enables imprint-based separation systems to be operational within 3 h from the start of capillary preparation. Enantiomer separations with baseline resolution have been carried out in less than 2 min. Other approaches to capillary electrochromatography (CEC) and capillary electrophoresis (CE) involve immobilisation of imprinted polymer particles inside capillaries using a polyacrylamide gel, the use of imprinted particles as a chiral mobile phase additive and the use of an imprinted polymer prepared by a dispersion polymerisation process.

Molecularly imprinted microparticles for capillary electrochromatographic enantiomer separation of propranolol.

Molecularly imprinted microparticles imprinted against (S)-propranolol were synthesised and studied for use in capillary electrochromatographic separation of propranolol enantiomers. The imprinted microparticles were in the size range of 0.2-0.5 micron as determined by scanning electron microscopy. The microparticles were suspended, in high concentration, in the electrolyte and used to perform enantiomer separation by a partial filling technique.

Molecularly imprinted microparticles for capillary electrochromatography: studies on microparticle synthesis and electrolyte composition.

The use of molecularly imprinted polymer (MIP) microparticles in a partial filling application of capillary electrochromatography (CEC) has previously been shown successful for the enantiomer separation of propranolol. In this investigation, the influence of some important parameters in the preparation protocol, i.e., template to monomer ratio, type of cross‐linker and functional monomers, and the effect of separation condition, i.e., organic modifier content, pH and the temperature of the column, on the electrochromatographic behavior of the MIP microparticles were studied. It was found that ethyleneglycol dimethacrylate (EDMA), having two reactive double bonds, was superior in terms CEC performance to trimethylpropane trimethacrylate (TRIM) and pentaerythritol tetraacrylate (PETEA) having three and four double bonds, respectively. The use of weak functional monomers, i.e., monomers lacking a strong interaction with the template, was shown to increase the separation efficiency. It was found that the template to functional monomer ratio had a pronounced influence on the MIP microparticle partial filling CEC performance as well as the size of the obtained microparticles. The use of a partial filling technique realizes the use of a new MIP phase in every new separation as well as the ability of altering the selectivity of the separation column and length of the MIP without the need for column switching.

Towards molecular-imprint based SPE of local anaesthetics

SummaryThe use of imprinted polymers for selective solid-phase extraction of local anaesthetics from human plasma was investigated. Methacrylic acid-ethylene glycol dimethacrylate based MIPs were developed by the alternative template approach using a structural analogue, pentycaine, as the template. The influence of the type and concentration of detergent in the buffer on selective and non-specific binding was investigated. Tween 20, Triton X-100 and Brij 35 were found to eliminate non-specific adsorption and leave selective imprint-analyte binding essentially unaffected. Efficient pre-concentration of ropivacaine and bupivacaine from plasma samples, both off-line prior to GC and in-line with LC-MS-MS, was demonstrated.

Alternative methods providing enhanced sensitivity and selectivity in capillary electroseparation experiments

The study of alternative and novel techniques for altering selectivity and enhancing sensitivity as well as injection and detection protocols are important in the ongoing development of capillary electroseparation protocols. Some recent research from our laboratory in these fields is presented and discussed in this review. To improve sensitivity an off-line sample enrichment technique utilising solvent evaporation in a levitated drop or an on-line solid-phase extraction protocol was used. The selectivity was tuned by the use of protein gels or molecularly imprinted polymer mediated capillary electrochromatography. Furthermore, a picolitre droplet injection method is described as well as a detection protocol based on laser-induced fluorescence imaging.

Chapter 2 - Solid-phase extraction on molecularly imprinted polymers

This chapter explains the solid-phase extraction on molecularly imprinted polymers (MIPs) and reviews several examples of highly selective pre-concentration of biologic and environment samples. A generic understanding of imprint-analyte binding mechanisms and the various physicochemical binding modes to these extraction materials is essential for a successful development of selective molecular-imprint based solid-phase extraction (MISPE) methods.. Compared with immunoaffinity materials, MIPs have the clear advantage of being highly stable under a large range of buffer pH, solvent, temperature, and pressure conditions, thereby allowing large opportunities for selection of the best experimental conditions for the most efficient use of MIPs. An interesting example is postpolymerization hydrophilization of the external surface of monodisperse MIP beads to render them more compatible with proteins in biosamples, thus allowing direct injection of serum samples into a highly selective MIP pre-column in a coupled-column system. In addition, MISPE with direct detection of the analyte elution is appealing as it eliminates the need for an analytic column, which would simplify the overall separation system and increase the speed of analysis.