Summary on Ion Exchange Chromatography

1. Ion exchange chromatography
Anil Shrestha
Msc Medical microbiology

2. Principle
• The principle feature is the attraction between
oppositely charged particles.
• Many biological materials have ionisable
group, they may carry a net +ve or –ve charge.
• The net charge exhibited by such compounds
is dependent on the pH of the solution and
isoionic point of the compound.
• Carried out in column mode.
• Packed with ion exchange resins.

3. Types of ion exchanger
• Cation exchanger which posses negative
charge and attract positively charged
molecules.
• Anion exchanger which posses positive charge
and attract negatively charged particle.

4. Ion exchange mechanism
It is thought to be composed of five distinct steps:
1)Diffusion of the ion through the matrix surface. This
occurs very quickly in homogeneous solutions.
2)Diffusion of the ion through the matrix structure of
the exchanger to the exchanger site. This is dependent
upon the degree of cross-linkage of the exchanger and
the concentration of the solution. This process is
thought to be the feature that controls the rate of the
whole ion-exchange process.

5. 3)Exchange of ions at the exchanger site. This is thought
to occur instantaneously and to be an equilibrium
process.
Cation exchanger:
RSO3
-………..Na+ + R’-NH3
+ RSO3
-…….NH3
+-R’ + Na+
(Exchanger) (Counter ion) (Charged Bound molecular ion Exchanged ion
Molecule to be
Separated)
Anion exchanger:
(R4)N+………….Cl - + R’-COO_ (R4)N+………_OOC-R’ + Cl-
(Exchanger) (Counter ion) (Charged Bound molecular ion Exchanged ion
Molecule to be
Separated)

6. The more highly charged the molecule to be exchanged, the tighter it
binds to the exchanger and less readily it is displaced by other ions.
4)Diffusion of the exchanged ion through the exchanger to the surface.
5)Selective desorption by the eluent and diffusion of the molecule to
the external eluent. The selective desorption of the bound ion is
achieved by changes in pH and /or ionic concentration or by affinity
elution. In affinity elution the ion having greater affinity for the
exchangers than for the bound molecule is introduced into the system.

7. Ion exchange material
• Most are polymeric resins. The resins incorporate
various functional group, strong or weak acids or
bases, to provide the ion exchange capacities. All
exchangers are generally supplied with an
appropriate counter ion, normally Na+ or Cl-.
Type Polymer Functional group
Cation exchange (Weakly
acidic)
Agarose
cellulose
-COO-
-CH2CCOO-
Cation exchange (Strongly
acidic)
Cellulose
Dextran
-SO3
-
-CH2SO3
-
Anion exchanger (Weakly
basic)
Agarose
Cellulose
-CH2CH2NH3
+
-CH2CH2NH+(CH2CH3)2
Anion exchanger (Strongly
basic)
Polystyrene
Callulose
`-CH2N+(CH3)3
-CH2CH2-N+(CH2CH3)2
CH2CH(OH)CH3
Table: List of ion-exchange resin

8. • Low pressure ion-exchange chromatography can be carried
out using a variety of matrices and ionic group. Matrices
used include polystyrene, cellulose and agarose. Functional
ionic groups include sulphonate (-SO3
-) and quaternary
ammonium(-NR3), both of which are strong exchangers
because they are totally ionized at all normal working pH
values. Carboxylate (-COO-) and diethylammonium
(HN(CH2CH3)2),both of which are termed weak exchangers
because they are ionized over only a narrow range of pH
values.
• All exchangers are characterized by a total exchange
capacity, which is defined as the number of milliequivalents
of exchangeable ions available, either per gram of dried
exchanger or per unit volume of hydrated resins. Thus the
exchange capacity of Bio-rex 70 is 3.3 meq.cm-3, DEAE-
Sephadex A-25 is 0.5 meq.cm-3.

9. Exchanger Selection
• The choice of ion exchanger depends upon the stability
of the sample components, their relative molecular
mass and the specific requirements of the separation.
• Many biological compounds, especially proteins, are
stable within only a fairly narrow pH range so the
exchanger selected must operate with in this range.
Generally, if the sample is most stable below its
isotonic point, giving its net positive charge, a cation
exchanger should be used, whereas if it is most stable
above its isotonic point, giving it a net negative charge,
an anion exchanger should be used. Compounds that
are stable over a wide range of pH may be separated
by anion or cation exchanger.

10. • Although the degree of cross-linking af an exchanger does
not influence the ion-exchange mechanism, it does
influence its capacity. Resins with a low degree of cross
linking are more permeable to high molecular weight
compounds. The meshsize of polystyrene resins determines
the flow rate.
• The pH of the buffer used should be at least one pH unit
above or below isoionic point of the compounds being
separated. In general, cationic buffers, eg, TRIS, pyridine,
alkyl amines are used in conjugation with anion exchangers;
anionic buffers, eg, acetate, barbiturate and phosphate are
used in conjugation with cation exchangers. Initially the
buffer pH and ionic strength should be such as to just allow
the binding of the sample components to the exchanger. A
buffer of the lowest ionic strength should initially be used
for the subsequent elution of the components.
• The amount of the sample that can be applied to a column
is dependent upon the size of the column and the capacity
of the exchanger.

11. APPLICATION
• Water demineralization
• Recovery of antibiotics from fermentation
broth
• Recovery of vitamins from fermentation broth
• Separation of amino acids
• Water softening
• Recovery of uranium from acid leach solution

12. Application in amino acid separation
• The separation of amino acids (in a protein
hydrolysate) is usually achieved using a strong acid
cation exchanger. The sample is introduced on top the
column at a pH 1-2 thus ensuring complete binding.
Gradient elution using increasing pH and ionic
concentration results in the sequential elution of the
amino acids. The acidic amino acids( aspartic and
glutamic ) are eluted first, followed by the neutral
amino acids(glycine and valine). The basic amino acids(
lysine and arginine) retain their net positive charge
upto pH 9-11 and are eluted last. Automatic aminoacid
analyzers use these principles.

13. • The gist of ion-exchange chromatography is
that ions of one types will be attracted to the
charges on the resins beads more or less
strongly than ions of a different type. Upon
elution, least attracted ion will emerge from
the bottom of the column before the others.
So solutions of different ions can be collected
in different flasks.