Ion exchange chromatography (IEC) is a type of column chromatography that separates ions based on their charge. The stationary phase contains fixed charged groups that can attract either positively charged cations or negatively charged anions. Proteins and nucleic acids can be positively or negatively charged depending on pH for separation. The procedure involves preparing the stationary phase, exchanging ions to activate it, applying the sample, eluting fractions, and regenerating the column. IEC has applications in amino acid analysis, nucleic acid analysis, water purification, and separation of biological compounds.

1. Ion Exchange
Chromatography
(IEC)
Amandeep Singh
Assistant Professor,
Department of Biotechnology,
GSSDGS Khalsa College,
Patiala.

2. Principle
It is a type of column chromatography. It works on the principle that opposite charged
particles attract each other. Stationary phase consist of fixed charges on a solid support
of the column. These fixed charges can be either positive or negative. If the fixed charge
is negative, then it can be used for exchange of positive ions (cations), therefore the
technique become cation exchanger. On the other hand, if the fixed charge on the
stationary phase is positive, then it can be used for exchange of negative ions (anions),
therefore the technique become anion exchanger.
-ve +ve
Cation exchanger Anion exchanger
Macromolecules (proteins, nucleic acids) can be made negative (by increasing the pH) or
positive (by decreasing the pH) for their separation in IEC.
Fixed Charges

3. Different macromolecules (e.g. proteins) with different ionic strength will leave the column at
different rate as they separate in IEC.

4. Procedure
1. Preparation of stationary phase
2. Preparation of exchange medium
a) Swelling
b) Removal of fines
c) Equilibration
3. Sample application
4. Elution
5. Regeneration

5. 1. Preparation of stationary phase
Resin is used to make stationary phase.
2 Main groups of resins are used commonly:
1. Polystyrene 2. Cellulose
Polystyrene Resins
Prepared by polymerization of styrene and divinyl benzene. Divinyl benzene
produce cross linkages. Higher the cross linkage, higher the polymerization.
Effects of increasing cross linkage:
1. Increase rigidity
2. Reduce swelling
3. Reduce porosity
4. Reduce solubility of polymeric structure

6. Cellulose Resins
• Have much greater permeability
• Much lower charge density
Examples of cellulose resins for making stationary phase:
Strong Cation Exchanger Weak cation exchanger
Sulphoethly cellulose Carboxymethyl Cellulose or
CM-Cellulose
Strong anion exchanger Weak anion exchanger
Guanidoethyl cellulose DEAE Cellulose or Diethyl
aminoethyl cellulose
The exchanger made by resin of these types, need to be activated before
using, by the technique known as preparation of exchange medium.

7. 2. Preparation of Exchange medium
Conversion of exchanger from the form in which it is supplied, to the form in which it is
to be used is known as preparation of exchange medium. OR Activation of stationary
phase is called preparation of exchange medium in IEC. Because it is the medium
which exchange opposite charged ions in the column. Following steps are required for
the preparation of exchange medium:
3 Steps:
1. Swelling of medium
2. Removal of fines
3. Equilibration
1. Swelling of medium
Dry exchanger contains densely packed polymers, have buried functional groups
which are unavailable for ion exchange. To expose the functional groups out, the dry
exchanger is treated with acids or bases first and this process is called swelling.
Swelling
Anion exchanger Cation exchanger
1st with acid, then with base 1st with base, then with acid
Finally if impurities are present, matrix treated with a chelator such as EDTA

8. 2. Removal of fines
Removal of very small particles of exchanger (fines) is done after swelling
Because these small particles (called Fines) decrease flow rate and results
in low resolution. Therefore they must be removed from the column.
3. Equilibration
Equilibration is known as attachment of counter ions with the stationary
phase packed inside the column. Ions bound electrostatistically to the
exchanger are called counter ions.
After removal of fines, Exchanger is finally equilibrated with suitable
counter ion.
Chemical used for equilibration Counter ion to be introduced
NaOH Na+
HCl H+
NaNO3 NO3
-
Formic acid Formate

9. 3. Sample application
Sample to be separated is dissolved in the
appropriate buffer.
Choice of Buffer is dictated by compounds to
be separated.
For anion exchanger = cationic buffer is used
For cation exchanger = anionic buffer is used

10. E- = Charged cation exchanger
Y+= counter ion
X+= charged molecule in sample to be separated
With increasing concentration of Y+ With increasing pH of solvent
Greater the charge by X+, higher the
concentration of Y+ required to elute it
Higher the pK of X+, higher will be
the pH required to elute it.
E- Y+ + X+ E- X+ + Y+
Elution of X+
4. Elution
Therefore, elution of sample ions form the column can be done by two methods:
1. By increasing the concentration of counter ions
2. By increasing the pH of the solvent used as mobile phase

11. 5. Regeneration
• Regeneration is a process that takes ion exchange resin beads that
are exhausted (fully loaded), and removes ions that have been
picked up during the in-service cycle so the resin can continue to be
used.
• Regeneration of an ion exchange resin bed involves multiple
processes, including:
1. Backwash (by running water to remove dirt, debris, air pockets in
the column)
2. Chemical injection (by brine solution or other regenerant, which
drives off the hardness or other ions and restores the resin back to
the required starting form for beginning a new service cycle)
3. Slow rinse (with water, for removal of brine solution or
regenerant)
4. Fast rinse (with raw water to ensure that water quality is being
met after regeneration)

12. Applications
1. Amino acid analysis
2. Base composition of nucleic acids
3. Water purification
4. Ultrapure, metal free reagent preparation
5. Low level metal detection in biological samples
6. Separation of vitamins, biological amines, organic
acids & bases.