Column Chromatography

Ion Exchange Chromatography

• Ion exchange chromatography is commonly used to separate charged

biological molecules such as proteins, peptides, amino acids, or nucleotides.

• The amino acids that make up proteins are zwitterionic compounds that contain
both positively and negatively charged chemical groups.

• Depending on the pH of their environment, proteins may carry a net positive

charge, a net negative charge, or no charge. The pH at which a molecule has
no net charge is called its isoelectric point, or pI.
• In a buffer with a pH GREATER than the
pI of the protein of interest, the protein
will carry a net negative charge;
therefore, a positively charged ANION
EXCHANGE resin is chosen to capture
this protein.
• In a buffer with a pH LOWER than the pI of
the protein of interest, the protein will carry a
positive net charge; thus a negatively-
charged CATION EXCHANGE resin is chosen.
Ion exchange chromatography

• After loading an impure protein sample

onto an ion exchange chromatography
column, the column is washed to
remove undesired proteins and other
impurities, and then the protein(s) of
interest is eluted using either a salt
gradient or a change in pH.

• To elute proteins from an anion exchange

resin, a decreasing pH gradient is chosen,
while an increasing pH gradient is chosen
for elution from cation exchangers.
 Affinity Chromatography

• Affinity chromatography is a separation method based on a specific

binding interaction between an immobilized ligand and its binding partner.

• Affinity chromatography offers high selectivity, resolution, and capacity in most protein
purification schemes. It has the advantage of utilizing a protein's biological structure or
function for purification. As a result, purifications that would otherwise be time consuming
and complicated, can often be easily achieved with affinity chromatography.

• A commonly used metaphor to illustrate affinity binding is the lock and key analogy. A
unique structure present on the surface of a protein is the key that will only bind to the
corresponding lock, a specific ligand on a chromatographic support.
ELUTION

• pH elution
A change oh pH alters the degree of ionization of charged groups on the ligand and/or bound
protein. This change may affect the binding sites directly, reducing their affinity , or cause indirect
changes in affinity by alterations in conformation
• Chaotropic eluents
Chaotropic agents such as guanidine hydrochloride or urea can be used to alter the structure of
proteins, but these eluents may cause permanent or temporary damage to the ligand.

• Competitive elution
Selective eluents are used to separate substances on group specific or when the binding affinity of
the ligand/target protein interaction is relative high. The eluting agents competes either for binding
to the target of protein or for binding to the ligand. The concentration of competing compound
should be similar to the concentration of coupled ligand.
 Size exclusion chromatography

• There are great diversity of proteins in the body and they all vary in size. One common
purification method that separate proteins based on their size is gel filtration or size exclusion
chromatography.

• The setup consists of long column that contains special gel beads. These beads are insoluble
but porous. They typically consist of a hydrabed polymer such as dextran.

• SEC only works if there is relatively large differences size protein in the mixture that we want to
separate it.
Size exclusion chromatography

A porous column acts

as a “molecular
sieve”, in which
smaller molecules get
stuck in pores and
take longer to travel
through the column ,
but the larger
molecules pass quickly
to the bottom.
Size exclusion chromatography

• The small proteins enter

the porous beads while
the larger proteins
cannot fit into the
internal volume of the
beads

• therefore the larger

protein reach the
bottom first while the
others emerge last.