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
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.
Ion exchange chromatography -- is a separation based
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
Common properties of all ion exchangers
They are almost insoluble in water and inorganic
solvents such as benzene, carbon tetrachloride, ether
They are complex in nature, i.e., infact they are
They have active or counter ions.
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.
Cation exchange chromatography retains
positively charged cations because the stationary
phase displays a negatively charged functional
Anion exchange chromatography retains anions
using positively charged functional group:
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.
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.
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.
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
The resin having sodium ions produced in the above
reaction, can exchange these ions with other cations.
Some commercially available cation 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.
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
ION EXCHANGE COLUMN USED IN
The principle is based on different cations or anions
which have different capacities to undergo exchange
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:
Similarly, the decrease in the capacity among anions is as
Phosphate ion>sulfate ion> iodine
Similarly the decrease in the capacity of univalant cations is
The decrease in the capacity of doubly charged cations is :-
Similarly for univalent anions is :-
SELECTION OF SUITABLE SYSTEMS:-
SPECIFICATION OF ION
IONIC GROUPS SUITABLE
Strong acid -SO₃⁻ Acidic and alkaline
Weak acid -COO⁻ Alkaline
Strong base -N⁺R₃ Acidic and alkaline
Weak base -NHR2 Acidic
ION EXCHANGE TECHNIQUES
There are two methods:
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.
The batch method is used for softening of water and production of
E.g. Exchange of calcium and magnesium ions which
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
E.g. RSO ₃⁻ H⁺ + M⁺ RSO ₃⁻M⁺+ H ⁺
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
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.
Instrumental analysis by Gurdeep R. Chatwal
Instrumental analysis by Skoog.