# Biomedical Applications of Polymeric Cryogels
## 2 Preparation and Properties of Supermacroporous Cryogels  
Monoliths, also called continuous stationary phases, are important tools for separating biological molecules. Although the studies on monoliths were started in the early 1970s, they gained importance as a big invention in the 1990s. Monolithic materials are characterized by low mass transfer resistance. In all studies involving biological macromolecules, monoliths perform much better than conventional chromatographic systems [6]. Monolithic materials have found wide use in chromatographic applications with the immobilization of different affinity ligands to monoliths [7]. The difference of monoliths from traditional filled columns is due to the one-piece porous structure. The interconnected pores in the structure of the monolith form a cross-channel network. Since the intra-channel mass transfer occurs with the flow, the transfer between the mobile phase and the stationary phase is very rapid. As a result, even very large molecules such as proteins can be separated from monolithic columns in a very short time. In addition, it is desirable to have low pressure during liquid flow in chromatographic systems. Since monoliths have smooth flow channels, high pressure does not occur, thus preventing any back pressure and/or clogging in the column [8]. Cryogel monoliths are gel matrices prepared using partially frozen monomer or polymer solutions. The most important features of the cryogels are their porous structure, non-specific interaction due to their hydrophilic structure, minimum mechanical properties and osmotic stability. The main disadvantage of cryogels is the low surface area. Cryogels characterized by their hydrophilic properties have supermacroopores and are formed by the polymerization process performed at temperatures below zero. The formation of cryogels is given in Figure 1. During cryogel formation, the polymer precursors are first dissolved in a suitable solvent such as water. In the freezing phase, the solvent freezes and forms interconnected ice crystals. The polymer precursors present in the unfrozen portion polymerize to form a network around the ice crystals. After the polymerization is completed, the frozen mixture is brought to room temperature. Then, the ice crystals melt and the porous polymer network, called \"cryogel\", is obtained. Solvent crystals act as pore forming agents. The melting of the ice crystals due to the surface tension of the solvent around the pore wall allows the pores in the cryogels to have a circular shape. The shape and size of the pores depend on many factors such as monomer concentration and temperature at which polymerization takes place are the most important factors. Micropore formations are also observed in the polymeric phase of the cryogels. Thus, in addition to supermacroopores, there are also micropores in the cryogel structure [9]. The supermacroopores in the structure of cryogels vary in size from 10 to 200 \(\upmu\)m.  
The pore size, the thickness and the density of the pore walls are the parameters that affect the performance of cryogels. The pore size affects the flow resistance. The thickness and density of the pore walls determine the macroscopic mechanical properties of the cryogel. The pore volume of a chromatographic material is very important. An average of 3-4% of the total weight of a cryogel that is completely swollen by water is the dry polymer weight. 4-5% of the weight consists of polymer bound water. Thus, 90% of the remaining weight of the cryogel constitutes of water inside pores. Due to the elasticity of the cryogels, 70% of the water in the large pores can be removed by mechanical compression. This feature indicates that a large part of the cryogel volume consists of interconnected pores [10]. Many advantages of cryogels make them useful in the field of biochromatography. The large pores of the cryogels interconnected with each other offer advantages when working with viscose biological fluids such as blood, plasma, plant or animal tissue extracts.