Ion exchange chromatography

ION EXCHANGE CHROMATOGRAPHY

By�M.Vharshini
B.Sc. Bio Medical Science
Sri Ramachandra University
ION EXCHANGE CHROMATOGRAPHY
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
According to the Source they can -
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.
Ion exchange resin should have following requirements
»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.
Physical properties of ion exchange resins�
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.


EXPERIMENTAL SETUP OF ION EXCHANGE CHROMATOGRAPHY
Metrohm 850 Ion chromatography system
Instrumentation of ion exchange chromatography

PRACTICAL REQUIREMENTS
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

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

2. ION EXCHANGE CHROMATOGRAPHY 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. According to the Source they can - 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. Ion exchange resin should have following requirements »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. Physical properties of ion exchange resins 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.

8. EXPERIMENTAL SETUP OF ION EXCHANGE CHROMATOGRAPHY

9. Metrohm 850 Ion chromatography system

10. Instrumentation of ion exchange chromatography

11. PRACTICAL REQUIREMENTS 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. T his 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.

12. 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…

13. Factors affecting ion exchange separations 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. Nature of exchanging ions: 1. valency of ions. 2.Size of ions.

14. 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

15. Working principle 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

16. APPLICATIONS ◘ softening and demineralisation of water. ◘ F or 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.

17. References 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