Ion exchange chromatography is a separation technique based on charge that can be used to separate a wide range of charged molecules like proteins, nucleotides, and amino acids. It works by exploiting ionic interactions between oppositely charged solute ions and the stationary phase. The stationary phase is typically a resin with covalently attached anions or cations. Cation exchange chromatography retains positively charged molecules, while anion exchange chromatography retains negatively charged molecules. Separation is achieved as molecules are differentially retained on and eluted off the column based on their affinity for the stationary phase. Ion exchange chromatography is widely used for applications like water softening, demineralization, and separation of molecules like amino acids, sugars, and lan

1. Ion Exchange Chromatography
Guided by- Presented by-
Dr.A.Suneetha M.pharm,Ph.D. G.Parimala Devi
Professor and HOD Y15MPh226
Dept. of Pharm.Analysis

2. DEFINITION
Ion exchange chromatography may be
defined as a reversible reaction in which free
mobile ions of a solids called ion exchange
are exchanged for different ions of similar
charge present in solution
EX:- insoluble organic polymer in to
which charged group is introduced.

3.  Ion exchange chromatography -- is a separation based
on charge
 Used for almost any kind of charged molecules ---
large proteins, small nucleotides and amino acids
 Ion-exchange chromatography preserves analyte
molecules on the column based on ionic interactions
 Mobile phase – buffer, pH and salt concentration---
opposite charged solute ions attracted to the
stationary phage by electrostatic force
 Stationary phase– resin is used to covalently attach
anions or cations onto it

4. Common properties of all ion exchangers
 They are almost insoluble in water and inorganic
solvents such as benzene, carbon tetrachloride, ether
etc.,
 They are complex in nature, i.e., infact they are
polymeric.
 They have active or counter ions.

5. Ion-exchange resin

6. Principle
 The principle involved in the chromatography is the
attraction between oppositely charged particles.
 Many biological materials such as amino acids and
proteins, have ionisable groups and they carry a net
positive or negative charge can be utilized in separating
mixtures of such compounds.
 The net charge of such compounds is dependent on
their pKa and pH of the solution.

8. Cation exchange chromatography retains
positively charged cations because the stationary
phase displays a negatively charged functional
group:
Anion exchange chromatography retains anions
using positively charged functional group:
.

10. ION EXCHANGE MECHANISM:-
 Diffusion of the ion to the exchanger surface.
 Diffusion of the ion through the matrix surface of the
exchanger to the exchange site.
 Exchange of the ions at the exchange site.
Cation exchanger
Anion exchanger

11.  Diffusion of the exchanged ion through the
exchanger to the surface.
 Selective desorption by the eluant and diffusion of
the molecule in to the external solution.
CATION EXCHANGERS
 A cation exchanger is a high molecular weight, cross
linked polymer having sulphonic, carboxylic,
phenolic etc., groups as an integral part of the resin
and an equivalent amount of cations.

12.  Thus, a cation exchanger is nothing but a polymeric anion
to which active cations are attached .
 In these cation exchangers the hydrogen ions are mobile
and exchangeable with other cations
 When cation exchanger is kept in a solution of salt, some
of the H+ ions of the resin enter the solution and in
equivalent amount of the cations of the salt get attached to
resin.

13.  The resin having sodium ions produced in the above
reaction, can exchange these ions with other cations.
 Some commercially available cation exchangers:

14. ANION EXCHANGERS:-
 An anion exchanger is a polymer having amine or
quaternary ammonium groups as integral parts of the
resin and an equivalent amount of anions such as Cl-,
OH- ions etc.
 These ions are mobile and exchangeable.

15. Commercially available anion exchangers:

16. Regeneration
 anion exchanges are generally supplied in the form of
 Salts ,amines, in particular ,are stable only in this form.
 Cation exchanges can be converted in to the H+ form by
treatment with aqueous acid followed by washing the
ammonium base having hydroxyl group from the strongly
basic anion exchanger on treatment with sodium
hydroxide while the weakly basic anionic exchanges are
converted in to free amines

17. ION EXCHANGE COLUMN USED IN
CHROMATOGRAPHIC SEPERATIONS:-
The principle is based on different cations or anions
which have different capacities to undergo exchange
reaction.
This should depend upon the charge and the size of
hydrated ion in solution.
Therefore ionic charge plays an important role in
determination of exchange reaction.
The decrease in the order of capacity is as follows:
Thorium>Aluminium>Calcium>Sodium
Similarly, the decrease in the capacity among anions is as
follows
Phosphate ion>sulfate ion> iodine

18. Similarly the decrease in the capacity of univalant cations is
H⁺>Cs⁺>Rb⁺>K⁺>NH₄⁺>Na⁺>Li⁺
The decrease in the capacity of doubly charged cations is :-
Ba²⁺>Pb²⁺>Sr²⁺>Ca²⁺>Ni²⁺>Cu²⁺>Co²⁺>Zn²⁺>Mg²⁺
Similarly for univalent anions is :-
I⁻>NO₃⁻>Br⁻>CN⁻>Cl⁻>OH⁻>F⁻

19. SELECTION OF SUITABLE SYSTEMS:-
SPECIFICATION OF ION
EXCHANGER
IONIC GROUPS SUITABLE
CHROMATOGRAPHIC
MEDIUM
Strong acid -SO₃⁻ Acidic and alkaline
Weak acid -COO⁻ Alkaline
Strong base -N⁺R₃ Acidic and alkaline
Weak base -NHR2 Acidic

20. ION EXCHANGE TECHNIQUES
There are two methods:
Batch method
Column method
BATCH METHOD:-
It involves single step equilibrium processes
 Resin+ Solution are mixed in a vessel.
 Filter the solution.
 The extent to which the ions from the solution are exchanged for
those on the resin depends on the selectivity coefficient.

21. The batch method is used for softening of water and production of
deionized water.
E.g. Exchange of calcium and magnesium ions which
causes hardness.
2RSO ₃⁻ Na ⁺ + Ca⁺ (RSO ₃⁻)2 Ca²⁺+ 2 Na ⁺
When resin is exhausted, a 15% of NaCl is added to the resin for
removal of calcium ions.
From deionized water all cations and anions of electrolytes have
been removed and these cations are replaced by the hydrogen
ions.
E.g. RSO ₃⁻ H⁺ + M⁺ RSO ₃⁻M⁺+ H ⁺

23. COLUMN METHOD:-
It involves in separation of components of a mixture by selecting
different coefficient of resin.
The difference in selectivity coefficient leads to different migration
rates on ion exchange column.
This involves frontal analysis, elution analysis, displacement
development.

26. APPLICATIONS
1. Separation of similar ion from one another.
2. Removal of interfering radicals.
3. Softening of hard water.
4. Complete demineralization of water.
5. Separation of lanthanides.
6. Separation of actinides.
7. Separation of amino acids.
8. Separation of sugars.

27. REFERENCE
https://www.harvardapparatus.com/download
s/Ion%20Exchange%20Chroma%20SpinColu
mn%20Guide.pdf.
Instrumental analysis by Gurdeep R. Chatwal
Instrumental analysis by Skoog.

28. Thank u all