Gel-Filtration Chromatography

Gel filtration chromatography is a method for separating proteins and peptides based on their size (1), The chromatographic matrix consists of porous beads, and the size of the bead pores defines the size of macro-molecules that may be fractionated. Those proteins or peptides that are too large to enter the bead pores are “excluded,” and thus elute from the column first (Fig. 1). Since large molecules do not enter the beads, they have less volume to pass through, which is why they are the first to elute from the column. Smaller macromolecules that enter some, but not all of the pores are retained slightly longer in the matrix and emerge from the column next. Finally, small molecules filter through most of the pores, and they elute from the column with an even larger elution volume. This method is also called gel permeation, molecular sieve, gel-exclusion, and size-exclusion chromatography. Since no binding is required and harsh elution conditions can be avoided, gel-filtration chromatography rarely inactivates enzymes, and often is used as an important step in peptide or protein purification (see Note 1).