Size exclusion chromatography (SEC) is a separation technique in which molecules are separated primarily according to their size or, more precisely, their hydrodynamic volume in solution. It is also known as gel filtration chromatography when aqueous solvents are used and gel permeation chromatography when organic solvents are used, particularly for polymers. Unlike adsorption, partition, or ion-exchange chromatography, SEC does not depend mainly on chemical interactions between the solute and the stationary phase. This makes it a particularly useful and gentle method for separating proteins, polymers, polysaccharides, and other molecules according to molecular size. The most distinctive feature of SEC is the nature of its stationary phase. The column is packed with porous particles containing pores of controlled dimensions. Common stationary materials include cross-linked dextran, agarose, polyacrylamide, and porous silica. When a mixture passes through the column, molecules of different sizes have different degrees of access to these pores. Large molecules cannot enter many of the smaller pores and therefore travel mainly through the spaces between the stationary-phase particles. They consequently move through the column more rapidly. Smaller molecules can enter a greater number of pores, so they follow a longer effective path and are eluted later. Thus, in SEC, larger molecules generally emerge before smaller molecules. The separation mechanism is mainly based on molecular size and does not require strong chemical interactions. This is one of the most important unique characteristics of the method. In an ideal SEC system, the stationary phase should not adsorb the sample molecules, bind them chemically, or cause ion-exchange interactions. The separation is therefore governed by the physical accessibility of the molecules to the pores. Because of this, SEC is often considered a relatively simple and reproducible separation technique. Another important feature is that SEC can be performed under mild conditions. This is especially valuable for biological substances such as proteins and enzymes, which may lose their activity under harsh chemical or thermal conditions. In aqueous SEC, biological molecules can be separated using suitable buffers that maintain their natural structure. Since there is normally little chemical interaction between the sample and the stationary phase, the technique can often be used without causing major changes in the chemical nature of the separated substances. SEC has a characteristic separation range determined by the pore size and structure of the stationary phase. Each packing material is suitable for molecules within a particular molecular-size range. Molecules that are too large to enter the pores are excluded from them and are therefore eluted together in or near the void volume of the column. At the other extreme, molecules that are sufficiently small to enter nearly all accessible pores are able to explore almost the entire pore volume and tend to elute near the total permeation volume. The most effective separation occurs between these two extremes, where molecules have different degrees of pore accessibility. The concepts of void volume and total column volume are therefore particularly important in SEC. The void volume is the volume outside the porous particles through which completely excluded molecules travel. Very large molecules that cannot enter the pores elute close to this volume. The total permeation volume represents