## 2 Synthesis and Characterization of Monoliths
### Monoliths: General Features
In general, to prepare all kinds of monolithic columns, a polymerization mixture with monomers, initiator and porogenic solvent is necessary, and then, this leads to macroporous materials with large through-pores. The polymerization conditions, especially temperature, affect the monolithic structure. Two decades after their introduction, polyacrylamides were used to prepare columns. With the use of these materials, [PERSON] _et al._, 2000, obtained a permanent macroporous structure [15]. However, the use of polymeric materials in HPLC is accompanied by several disadvantages, such as their lower efficiency compared with silica-based columns. The preparation of organic polymer capillary monolithic columns is simple: a fused silica capillary is filled with a polymerization mixture, sealed at both ends, and by heating or by UV, polymerization is initiated [16]. When the polymerization is completed, the seals at the ends of the capillary are removed; the capillary is cut to the required length and washed with an appropriate solvent to remove the porogen and other soluble compounds from the pores of the monolithic column. Generally, organic polymers offer wider variability in chemistries and better biocompatibility than silica [14]. Most of the methods controlling the surface chemistry of porous polymer monoliths described so far rely on copolymerization of functional monomers, chemical modification of reactive groups of the monolith or grafted chains originating from functional monomers [17]. However, it is of great interest to explore new approaches enabling the modification of monolithic supports to obtain materials adapted for specific applications; thus, more dedicated and less common applications of monoliths have recently been found [18]. Moreover, the incorporation of nanostructures in the polymeric scaffold, as well as the preparation of hybrid structures had been described by [PERSON] _et al._, 2012 [19]. There is a variety of nanoparticles that have been successfully used to modify porous polymer monoliths, and this highlights that the field of micro-/nano-material-functionalized polymer monolithic columns is still in its infancy and needs more attention from analysts [20]. Affinity monolith chromatography (AMC) is a type of liquid chromatography that uses a monolithic support and a biologically-related binding agent as a stationary phase. Formats have ranged from traditional columns, to disks, microcolumns and capillaries. Many types of binding agents have been used with monolithic supports in AMC, including antibodies, enzymes, proteins, peptides, lectins, immobilized metal ions and dyes. Thus, a variety of applications have been reported that are based on methods, such as bioaffinity chromatography, immunoaffinity chromatography or immunoextraction, immobilized-metal-ion affinity chromatography (IMAC), dye-ligand affinity chromatography and chiral separations [21]. Nanoparticle-based monoliths are a less popular member of the monolith family. They have emerged as a new class of substrates in sample preparation and separation science, as it has been summarized and highlighted in a recent mini-review of the major advances developed in the last three years [22]. The modification of monolithic supports can also be achieved using \"templating\" approaches, generating new families of porous materials. To synthetized nanoparticle-templated monoliths, nanoparticles are added as a suspension to the polymerization mixture. After polymerization, the nanoparticles are removed by washing the monolith with a strong base [23]. This procedure has been used to increase the ion-exchange capacity of monoliths. Monoliths can also be modified using high internal phase emulsions (HIPEs), achieving the so-called polyHIPEs, reviewed by [PERSON] in 2014 [24].