## 2 Molecular Imprinting
Template removal should be meticulously performed in order to ensure that the maximum number of imprinted sites are free of the template molecule, especially in analytical applications where template bleeding can affect the results. Complete removal of the template often requires extreme conditions that can impair the imprinted sites. Template removal can be performed by three main approaches, solvent extraction, physically assisted extraction or the use of subcritical/supercritical solvents (subcritical water/supercritical CO\({}_{2}\)). Physically assisted extraction includes ultrasound-assisted, microwave-assisted and pressurized liquid extraction. The most common approach is the use of organic solvents, either by incubating the prepared MIPs in organic solvents or by extraction in a Soxhlet apparatus. MIP incubation is a mild template removal method that requires several hours to be complete but does not impair the imprinted sites. Incubation in organic solvents causes swelling of the MIP structure and favors template removal, while heating and stirring can reduce the duration of template removal. Extraction in a Soxhlet apparatus is a more drastic method where the template is extracted for several hours with heated organic solvent and is more commonly used for MIPs prepared by bulk polymerization. The heated solvent increases template solubility, thus template removal. However, Soxhlet extraction is a time-consuming method (up to 24 h) that requires increased volumes of organic solvent and high temperatures can cause template degradation [41]. There are two main approaches for the preparation of MIPs based on the interactions between the template molecule and the functional monomer that can be either covalent or non-covalent. In the covalent approach, reversible covalent interactions are developed between the functional groups of the template and the recognition unit of the monomer. Covalent imprinting is stoichiometric and functional monomer residues can only be found in the imprinted sites. Template removal is usually achieved by treating the prepared MIPs with organic solvents or Soxhlet extraction, while molecule binding is based on covalent interactions. However, there are limited reversible covalent interactions available and the strong nature of covalent interactions does not favor fast binding and removal on the imprinted sites [38; 40; 42]. In the non-covalent approach, hydrogen bonding, van der Waals forces and ionic or \(\pi\)-\(\pi\) interactions are developed for the formation of the template-monomer complex in a pre-polymerization step, with hydrogen bonding being the most common interaction. Template removal is achieved by washing the prepared MIPs with an organic solvent or a mixture of solvents, while molecule binding is based on non-covalent interactions. Non-covalent imprinting is a simple MIP preparation process that offers fast binding and removal on the imprinted sites and is the most preferred approach. However, the interactions forming the template-monomer are not as stable and prone to disruption [38; 40; 42]. Classic molecular imprinting involves a single template for the preparation of a MIP with imprinted sites that are not able to recognize more than one target molecule. With multi-template imprinting more templates are used for the preparation of MIPs with more than one type of imprinted sites able for simultaneous recognition of multiple target molecules. The multi-template MIPs enable the extraction and determination of multiple analytes; however, they have reduced selectivity compared with one-template MIPs [39]. Like multi-template imprinting, MIPs can be prepared by employing more than one functional monomer. Each functional monomer is able to recognize a different functional group of the template molecule and the resulting in MIPs presents increased selectivity. Multi-functional monomer imprinting can be helpful for the imprinting of macromolecules.