In our case, the solvent will be a buffer. The porous nature of the gel matrix forms the basis for the separation method. The matrix that is packed into the column is often referred to as the column bed . Protein in the mobile phase will interact with the matrix as it moves through the column. The degree of interaction will depend on the properties of the protein.
Proteins interact with the matrix based on their molecular weight .
A sample containing a mixture of proteins of different sizes flows into the matrix. As the mobile phase exits the bottom of the column, it is collected as aliquots of constant volume. The exiting mobile phase is called the column eluate , and each successive portion of the eluate is called a column fraction . A linear relationship is obtained if the logarithms of the molecular weights of standard proteins are plotted against their respective elution volumes. The molecular weight of the unknown protein can be estimated by extrapolating from the standard graph.