In ion-exchange chromatography, the beads in the column are coated with charged substances so
that they attract or bind compounds that have an opposite charge. For instance, a positively
charged compound will attract and hold a negatively charged backbone of DNA or protein as it
passes through the column, either increasing its retention time or retaining it completely. After all
other compounds have moved through the column, a salt gradient is used to elute the charged
molecules that have stuck to the column.
Size-Exclusion Chromatography
In size-exclusion chromatography, the beads used in the column contain tiny pores of varying
sizes. These tiny pores allow small compounds to enter the beads, thus slowing them down. Large
compounds can’t fit into the pores, so they will move around them and travel through the column
faster. It is important to remember that in this type of chromatography, the small compounds are
slowed down and retained longer—which may be counterintuitive. The size of the pores may be
varied so that molecules with different molecular weights can be fractionated. A common approach
in protein purification is to use an ion-exchange column followed by a size-exclusion column.
Affinity Chromatography
In affinity chromatography, a protein of interest is bound by creating a column with high affinity
for that protein. This can be accomplished by coating beads with a receptor that binds the protein
or a specific antibody to the protein; in either case, the protein is retained in the column. Common
stationary phase molecules include nickel, which is used in separation of genetically engineered
proteins with histidine tags, antibodies or antigens, and enzyme substrate analogues, which mimic
the natural substrate for an enzyme of interest. Once the protein is retained in the column, it can be
eluted by washing the column with a free receptor (or target or antibody), which will compete with
the bead-bound receptor and ultimately free the protein from the column. Eluents can also be
created with a varying pH or salinity level that disrupts the bonds between the ligand and the
protein of interest. The only drawback of the elution step is that the recovered substance can be
bound to the eluent. If, for example, the eluent was an inhibitor of an enzyme, it could be difficult to
remove.