There are two extremes in the separation profile of a gel filtration column. There is a critical molecular mass (large mass) which will be completely excluded from the gel filtration beads. All solutes in the sample which are equal to, or larger, than this critical size will behave identically: they will all eluted in the excluded volume of the column. There is a critical molecular mass (small mass) which will be completely included within the pores of the gel filtration beads. All solutes in the sample which are equal to, or smaller, than this critical size will behave identically: they will all eluted in the included volume of the column Solutes between these two ranges of molecular mass will elute between the excluded and included volumes (Fig. 2). Thus, while deciding a size exclusion matrix for protein purification, included and excluded range should be considered. For example: Sephadex G 75 matrix has fractionation range 3-80 kDa. This tells that the matrix has included volume range 3kDa and excluded volume range 80 kDa. If protein of interest and impurities both are close to 80kDa or above they are likely to co-elute in excluded volume. Thus purification will not work. Now you can think what is the use of a size exclusion matrix Sephadax G25 (range 1-5 kDa)? This is generally used for desalting as all proteins are above 5kDa and comes in excluded volume and salts are eluted late in included volume.