Proteins elute from a gel filtration column in order of their size, with the largest molecules eluting first and the smallest molecules eluting last.

In gel filtration chromatography, beads with tiny tunnels are used to separate proteins. The size of the tunnel is called the ‘exclusion limit’. Only proteins with molecular sizes smaller than the exclusion limit can enter the tunnels. On entering the tunnels, they have a longer path to pass through the column and hence take longer to elute. Proteins with molecular sizes larger than the exclusion limit are unable to enter the tunnel. Instead, they make their way between the beads easily and pass through the column quickly, eluting first from the gel filtration column.