Principle: The principle of the chromatography technique is illustrated in Figure 32.2. The column is packed with the beads containing pores to allow entry of molecules based on their sizes. Smallest size in the inner part of pore followed by gradual increasing size and largest molecule excluded from entering into the gel. The separation between molecules occur due to the time they travel to come out from the pores. When the mobile phase pass through the column, it takes protein along with it. The small molecules present in the inner part of the gel takes longer flow of liquid (or time) and travel longer path to come out where as larger molecules travel less distance to come out. As a result, the large molecule and small molecule get separated from each other. A schematic gel filtration chromatogram is given in Figure 32.3.

Figure 32.2: Principle of Gel Filtration Chromatography.

Suppose the total column volume of a gel is V_t and then it is given by-

V_g is the volume of gel matrix, V_i is the pore volume and V_o is the void volume. The volume of mobile phase flow to elute a column from a column is known as elution volume (V_e). The elution volum is related to the void volume and the distribution coefficient Kd as given below

Kd is the ratio of inner volume available for an analyte and it is independent to the column geometry or length.