Ion exchange chromatography

Principle of Ion Exchange Chromatography: Ion exchange chromatography separates
proteins or other molecules based on differences in their accessible surface charges. In ion
exchange chromatography the analyte molecules are retained on the column based on
coulombic (ionic) interactions. The stationary phase surface contains ionic functional groups
of opposite charge that interact with analyte ions. The elution is done by increasing salt
gradient. Most commonly used salt is NaCl, exists in equilibrium with Na+
(cation) and Cl-
(anion) in aqueous solution. As the concentration of salt increases concentration of Na+
(cation) and Cl-
(anion) also increases. The basic principle of ion exchange chromatography
is the reversible exchange of analyte ions bound to solid support with similar ions generated
from salt in liquid phase. Many biological molecules such as proteins, amino acids,
nucleotides and other ions have ionisable groups which carries a net charge (positive or
negative) dependent on their pKa and on the pH of the solution, which can be utilised in
separating mixture of such molecules as explained in box. Ion exchange chromatography
experiments are carried out mainly in columns packed with ion exchangers. On the basis of
type of exchanger used for separation this chromatography is further subdivided into cation
exchange chromatography and anion exchange chromatography.
Many biological molecules, especially proteins, are stable within a narrow pH range so the
type of exchanger selected must operate within this range. Suppose if protein is most stable
below its isolecteic point (pI), there will be net positive charge on the protein surface, so for
separation of this protein cation exchanger should be used (experimental pH value should be
between lowest pH wehere protein is stable and pI). If protein is most stable above its pI,
there will be net negative charge on the protein surface and anion exchanger should be used
(experimental pH value should be between highest pH where protein is stable and pI value).
If protein is stable over a wide range of pH, it can be separated by either type of ion
exchanger (experimental pH value may be decided considering lowest and highest pH value
stability of the protein). Weak electrolyte requires very high or very low pH for ionisation so
it can only be separated on strong exchanger, as they only operate over a wide pH range,
whereas in case of strong electrolytes, weak exchangers are preferred.
Proteomics & Genomics Dr. Vikash Kumar Dubey
IIT Guwahati Page 3 of 5
Cation Exchange Chromatography: Commonly used cation exchange resins functional
groups are following:
Carboxymethyl (CM) -O-CH2-COOSulphopropyl
(SP) -O-CH2-CH2-CH2SO3
-
Methyl sulphonate (S) -O-CH2SO3
-
Solid support with these functional groups can be prepared with various beads. They differ in
few properties like flow rate, stability, binding capacity (linked with porocity) etc. Cation
exchangers based on dextran (Sephadex), agarose (Sepharose) and cross-linked cellulose
(Sephacel) are the ion exchange matrices with high porosity, leading to improved flow
properties and high capacities for macromolecules. Typically, cation exchange
chromatography is performed using buffers at pH s between 4 and 7 and running a gradient
from a solution containing just this buffer to a solution containing this buffer with 1M NaCl.
The surface charge on the molecules (proteins, nucleic acids etc), which binds to cation
exchanger should be net positive. Thus, to get binding of a specific protein pH should be
below the pI of that protein. Once salt concentration increases, it results in increase in Na+
(cation) and Cl-
(anion). Beyond a certain point, positively charged protein is exchanged with
cation. Thus, this type of chromatography is called cation exchange chromatography