## 2 Synthesis and Characterization of Monoliths  
### Monoliths: General Features  
Two types of columns have been used as stationary phases for routine HPLC: packed columns and monolithic columns. The particle size, the distribution and the quality of the packing of the particles within the column determine the column quality. In the case of packed columns, silica microspheres are the most used and consist of a tube packed with 3-5-\(\upmu\)m porous silica microparticles. In contrast to HPLC packed columns, monolithic columns are made of a single piece of porous material, which is also called a \"silica rod\". This kind of material entirely fills the column volume without any of the interparticle voids typical of packed columns [7]. One of the main advantages of monolithic columns is that they can work at high flow rates (up to 10 mL min\({}^{-1}\)) in conventional column lengths (4.6 mm I.D.) without generating high back-pressures [8]. According to the size and function, there are two main types of pores in monolithic columns: the flow pores and the mesopores filled with the \"stagnant\" mobile phase, in which the solute molecules migrate to access the active adsorption sites. Large flow pores are responsible for the permeability of the monolith, and they allow LC separations at low pressures. The inner pores of the particles in the packed columns correspond to the mesopores in themonolithic columns. The presence of mesopores increases the total pore surface area and sample capacity of monolithic beds. The structure of monolithic media can be represented as a network of small mesopores, which are responsible for the retention and separation selectivity, interconnected by large flow-through pores [9], and this should lead to higher permeability. Thus, you can get fast separations at the high flow of the mobile phase and moderate back-pressures in comparison to particle-packed columns with similar efficiency [10]. The first generation of commercial monolithic columns has been marketed as \"Chromolith Performance\" columns by Merck since 2000. These columns are made of a C1s chemically-bonded silica monolith. These kinds of materials have some important limitations in their morphology: the broad size distribution, variable geometry and random spatial distribution of the through-pores. The large domain size is in contrast with decreasing diameters of packed bed particles. Furthermore, a radially heterogeneous morphology introduces a mobile phase velocity bias between the local regions of the column cross-section. Additionally, the drawbacks of these columns include the lower column-to-column and batch-to-batch reproducibility. However, the investigations of [PERSON] _et al._ with these HPLC monolithic columns proved that the reproducibilities achieved are similar to those obtained with commercial columns packed with silica-based reversed-phase packing materials [11]. Second generation monolithic columns were also commercialized by Merck, as a welcome addition to the Merck chromatography family of products. The monolithic columns of this second generation can perform fast, high-resolution separations, while keeping the pressure drop to a minimum [12,13]. Rods are prepared by a polymerization process either _in situ_ in a column tube, such as in glass tubes or fused silica capillaries, or in column molds, in which the monolith can later be replaced [1]. The main classification of monolithic materials used in chromatography is according to the nature of their construction materials, organic polymer or silica-based columns. Organic polymers were used for the first monolithic columns, and they were prepared for gas chromatography [3]. Monoliths prepared from organic polymers used as supports for enzyme immobilization and for the preparation of bioreactors are most often formed from either acrylamide derivatives or acrylate/methacrylate-based monomers [14].