ION EXCHANGE CHROMATOGRAPHY

ByM.Vharshini

B.Sc. Bio Medical Science

Sri Ramachandra University

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.

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

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.

»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.

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.

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.

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.

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…

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.

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

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

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.

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