Ion exchange chromatography separates ions and polar molecules based on their affinity for an ion exchange resin. It works through the reversible electrostatic interaction between ions in solution and ions attached to the resin. There are four main types of resins: strong cation, weak cation, strong anion, and weak anion. Organic resins like polystyrene with divinylbenzene crosslinking are commonly used. The process involves equilibrating, applying the sample, eluting components at different rates depending on their affinity, and regenerating the resin. Ion exchange chromatography has applications like water softening, enzyme purification, and separation of ions, sugars, amino acids and proteins.

1. ION EXCHANGE CHROMATOGRAPHY
By
M.Vharshini
B.Sc. Bio Medical Science
Sri Ramachandra University

2.  Ion-exchange chromatography is a process that
allows the separation of ions and polar molecules
based on their affinity to the ion exchanger.
 It can be used for almost any kind of charged
molecule including large proteins, small nucleotides
and amino acids.
 Cations or Anions can be separated using this
method.
PRINCIPLE
 It is based on the reversible electrostatic
interaction of ions with the separation matrix (i.e.)
The separation occurs by reversible exchange of
ions between the ions present in the solution and
those present in the ion exchange resin.

3. CLASSIFICATION OF RESINS
According to the chemical nature they classified as-
1. Strong cation exchange resin
2. Weak cation exchange resin
3. Strong anion exchange resin
4. Weak anion exchange resin

4.  Natural resins : Cation - Zeolytes, Clay
Anion - Dolomite
 Synthetic resins: Inorganic & Organic resins
◘Organic resins are polymeric resin matrix.
The resin composed of –
 Polystyrene (sites for exchangeable functional
groups)
 Divinyl benzene(Cross linking agent)-offers
stability.

5. »It must be chemically stable.
»It should be insoluble in common solvents.
» It should have a sufficient degree of cross
linking.
»The swollen resin must be denser than water.
»It must contain sufficient no. of ion exchange
groups.

6. Cross linking:
It affects swelling & strength & solubility
Swelling:
When resin swells, polymer chain spreads apart
Polar solvents → swelling
Non-polar solvents → contraction
Swelling also affected electrolyte concentration.

7. Particle size and porosity
Increase in surface area & decrease in particle
size will increase the rate of ion exchange.
Regeneration
 Cation exchange resin are regenerated by
treatment with acid, then washing with water.
 Anion exchange resin are regenerated by
treatment with NaOH, then washing with water
until neutral.

12. 1.Column
» glass, stainless steel or polymers
2.Packing the column
» Wet packing method:
A slurry is prepared of the eluent with the stationary
phase powder and then carefully poured into the
column. Care must be taken to avoid air bubbles.
3.Application of the sample
 After packing, sample is added to the top of the
stationary phase, use syringe or pipette.
 This layer is usually topped with a small layer of sand
or with cotton or glass wool to protect the shape of
the organic layer from the velocity of newly added
eluent.

13. 4.Mobile phase
Acids, alkalis, buffers…
6.Stationary phase
The ionic compound consisting of the cationic species
(M+) and the anionic species (B-)
7.Elution
 Components of mixture separate & move down the
column at different rates depending upon the affinity of
the ion for ion exchanger.
 The eluates are collected at different stages
8. Analysis of the eluate
Spectrophotometric, flame photometry
polarographic, conductometric…

16. a. Nature & properties of ion exchange resins:
 Cross linking & swelling is important.
 If more cross linking , they are more rigid
but swelling is less.
 swells less → separation of ions of different
sizes is difficult.
b. Nature of exchanging ions:
1. valency of ions.
2.Size of ions.

17. 3.Polarizability
4.Concentration of solution.
5.Concentration & charge of ions.
c. pH of the mobile phase
d. Ionic strength
e. Mobile phase modifiers
f. Temperature

18.  The basic process of chromatography
using ion exchange can be represented in
4 steps:
1. Equilibration
2. Sample application and wash
3. Elution
4. Regeneration

25. APPLICATIONS
◘ softening and demineralisation of
water.
◘ For extraction of enzymes from
tissues.
◘ purification of solutions free from
ionic impurities.
◘ separation of inorganic ions.
◘ separation of sugars, amino acids
and proteins.
◘ ion exchange column in HPLC.

26.  Himmelhoch, SR (1971) Chromatography of
proteins on ion-exchange adsorbents. Meth.
Enzymol. 22:273-286.
 Scopes, RK (1982) Ion exchangers-principles,
properties and uses. In “Protein
 www.harvardapparatus.com/ion exchange
chromatography
 http://machias.edu/basic-principles-of-ion-
chromatography.html