This document provides an overview of ion exchange chromatography. It describes the principle, components, instrumentation, technique, limitations, advantages and applications of ion exchange chromatography. The principle involves separation of charged molecules based on their affinity for oppositely charged functional groups on a stationary resin phase. Key components are the stationary resin phase (e.g. cation or anion exchangers), mobile buffer phase, and column. The technique involves sample loading, washing, and eluting molecules using changes in buffer pH, salt concentration or both. Advantages include low cost, reusability, and efficient separation of charged biomolecules like proteins. Applications include water softening and purification processes.

2. CONTENTS
Introduction
Principle
Components
Instrumentation
Technique
Limitations
Advantages and Applications
References

3. • Laboratory technique for the separation of a
mixture, which is dissolved in a fluid (mobile
phase) and carries it through a system
(stationary phase).

4. – The liquid entering the column.
– The liquid leaving the column.
– The process by which the adsorbed
ions are removed from the column.
– The solution used for elution.
– The solution obtained as a result of
elution.

5. HISTORY OF ION-EXCHANGE
CHROMATOGRAPHY
• In 1850 H.Thompson and J.T Way, who proved that soil
can remove potassium or ammonium salts from water
with release of equivalent amount of calcium salt.
• Modern ion exchange resin were first used in 1935 by
Adams and Holms.
• In 1940 Modern Ion-exchange Chromatography was
developed during the war to separate and concentrate
the radioactive elements needed to make an atomic
bomb.

6. PRINCIPLE
• Ion-exchange chromatography is commonly used
to separate charged biological molecules such as
proteins, peptides, amino acids or nucleotides.
• The amino acids that make up proteins are
zwitter ionic compounds that contain both
positively and negatively charged chemical
groups.
• Depending on the pH of their environment,
proteins may carry a net positive charge, a net
negative charge, or no charge.

7. PRINCIPLE
• Ion exchange chromatography involves separation of ionic and polar
analytes using chromatography supports derivatized with ionic
functional groups that have charges opposite that of the analyte
ions.
• The analyte ions and similarly charged ions of the matrix compete
to bind to the oppositely charged ionic functional group on the
surface of the stationary phase.
• The pH at which a molecule has no net charge is called it’s
isoelectric point or pI.
• The choice of buffer pH then determines the net charge of the
protein of interest.
• In a buffer with a pH greater than the pI of the protein of interest,
the protein will carry a net negative charge; therefore, a positively
charged anion exchange resin (cation resin) is chosen to capture
this protein.

8. • In a buffer with a pH lower than the pI of the
protein of interest, the protein will carry a net
positive charge; thus a negatively- charged cation
exchange resin is chosen.

9. • Choosing a buffer pH:
a) Anion exchanger- 0.5 – 1.5 pH units greater than the pI of the
protein of interest.
b) Cation exchanger- 0.5 – 1.5 pH units less than the pI of the
protein of interest.

10. COMPONENTS OF IEC
I. Stationary phase or Ion exchange resin:
a) Natural: Cation - zeolytes, clay etc.
Anion – dolomite
b) Synthetic resin: In-organic and organic resins
• are polymeric resin matrix
• Polystyrene (sites of exchangeable functional groups)
• Divinyl benzene (cross linking agent) offers stability
1. Anion exchangers
2. Cation exchangers

11. 1. Anion Exchangers:-
• The anion exchangers have positively
charged exchanger with negatively
charged mobile counter ion available
for exchange.
• If the basic functional groups are
introduced (via buffer), the resin
becomes anion exchanger.
• Functional groups on anion exchange
resins:
Quaternary amines Strong anion
(-N+R3) exchangers
Tertiary amines weak anion
(-NR2) exchangers

12. 1. Anion exchangers
• In anion exchange chromatography, the
exchanging ions are anions and the equation is represented
as follows;
e.g. anion exchanger may be tertiary or quaternary
ammonium form:
X-N+R3OH-
X = Matrix (resin)
-N+R3 = Fixed charge (cationic),
Non-exchangeable
-OH- or -Cl- = Counter ion (anion), Exchangeable
[They are supplied as the chloride rather than hydroxide as
the chloride form is a more stable.]
X-N+R3Cl- + B-- X-N+R3B-- + Cl-
• B-- = anionic protein in sample

13. 2. Cation exchangers:
• The cation exchangers have negatively
charged exchangers with positively
charged mobile counter ion available
for exchange.
• If acidic functional groups are
introduced, then the resin becomes
cation exchangers.
• Functional groups on resins:-
Sulphonic acid strong cation
(SO3H) exchangers
Carboxylic acid Weak cation
(COOH) exchangers

14. 2. Cation exchangers
• Assuming that analytes are cations, the competition
can be explained using the following equation;
X_COO-H+
X = Matrix (resin)
-COO- = Fixed charge (anion), Non-exchangeable
H+ = Counter ion (cation), Exchangeable
They are usually (but not always) supplied in the Na+
form:
X-COO-Na+
Na+ will exchange with anion (A++) of sample
X-COO-Na+ + A++ X-COO-A++ + Na+

15. COMPONENTS OF IEC
II. MOBILE PHASE:
BUFFER
1. In ion exchange chromatography, pH values is an important
parameter for separation and can be controlled by means of
buffer substances.
2. The charged species in buffers used for ion exchange
chromatography should thus generally have the same sign as the
charged species of the ion exchange resin.
3. For example, although phosphate buffers are commonly used for
protein purification, they’re not appropriate for anion exchange
chromatography because the phosphate ion interacts strongly
with positively charged anion exchange resins.

16. II. MOBILE PHASE
5. For cation exchange chromatography,
Buffer Buffering range
Acetic acid 4.8 – 5.2
Citric acid 4.2 – 5.2
Lactic acid 3.6 – 4.3
Phosphate 6.7 – 7.6
6. For anion exchange chromatography,
Buffer Buffering range
Diethanolamine 8.4 – 8.8
Diethylamine 9.5 – 11.5
Tricine 7.4 – 8.8
Tris 7.5 – 8.0
7. Slow flow-rates during column loading and elution increases the
interaction time between the protein and the exchange resin,
promoting specific binding interaction during loading.

17. COLUMN PREPARATION
1. The apparatus used in this method consist of a glass column fitted
with a glass wool plug at a lower end.
2. A slurry of resin is made in distilled water and any fine particles
are removed by decantation.
3. The slurry is then slowly poured into vertically fixed column.
4. To ensure that no air bubbles remain in the column and that the
resin is uniformly distributed, the column is backwashed with
distilled water.
5. The flow of water is then stopped and the resin is allowed to
settle.
6. The excess water is then drained off.
7. The level of water must never be allowed to fall below that of
surface of the resin as otherwise the resin may dry up and
channels may be formed in the resin bed.

18. Column Preparation
Distilled
water
Resin
Flow rate
controler

19. ION CHROMATOGRAPHY

20. 1.INJECTOR
3.DETECTOR 4.COMPUT
ER SYSTEM
2.COLUMN
IEC INSTRUMENTATION

21. THE TECHNIQUE
STEP I - Equilibration
• Equilibration buffer is run on the column in a particular
flow rate.
• Elution of buffer is detected by detector and a straight
line (base line) will form on the graph.
• Straight line shows that the column is properly packed
and is ready for purification.

22. THE TECHNIQUE
STEP II – LOADING
1. An impure protein sample is loaded into the ion exchange
chromatography column at a particular pH and allowed to flow slowly in
the column.
2. Charged proteins will bind to the oppositely charged functional groups in
the resin.
STEP III – WASHING
1. The remaining neutral, oppositely charged protein (other than sample)
and counter ions will get eluted by running buffer.
2. This unwanted or unbounded molecules with resin will give a peak
during washing.

25. THE TECHNIQUE
STEP IV – ELUTION
1. Bounded interest of ion (X+
) can now be eluted by either of the two ways,
a) by adding salt – by adding a component M+
having magnitude of charge
more than that of X+ so that M+ will replace X+ and X+ will be eluting out.
Proteins with few charged groups will elute at low salt conc., whereas
proteins with many charged groups will have greater retention times and
elute at high salt conc.
b) by changing pH of the solvent (mobile phase) so that X+ have no charge
and is then unbounded from the matrix and can be eluted out.
2. For anion exchange resin, elution is followed by decreasing the pH
gradient (mobile phase), will cause the molecule to become more
protonated (less negatively charged) allowing it’s elution.
3. For cation exchange resin, increasing the buffer pH of the mobile phase
causes the protein to become less protonated (less positively charged ) so
it cannot form an ionic interaction with the negatively charged resin,
allowing it’s elution.
4. A new peak of the interested protein formed on the system.

26. After calculating the area of the
peak, conc. of the interested
protein in a mixture can be
known

28. REGENERATION OF RESIN
• After separation of components, the resin is
not useful for another separation.
• It loses it’s exchangeable functional groups.
• Replacing the exchangeable functional groups
does the regeneration.
• Using strong alkali like NaOH and KOH usually
does regeneration of anion-exchange resins.
• For cation exchange resins strong acids like
HCl can be used

29. FACTORS AFFECTING
CHROMATOGRAPHIC SEPARATION
1. Ion – Exchange Resin:
• The swelling factor and cross – linking is
important for the effective separation.
• The cross – linking and swelling must be proper/
controlled as it helps in proper exposure of
charged functional groups for exchange of ions.
2. Sample:
The conc. and charge of ions...
3. Rate of flow of eluent
4. Buffer

30. ADVANTAGES
• Cost effective
• Re-usable
• Low maintenance cost
• Efficient technique
• Quick separation
APPLICATIONS
1. Softening of hard water
2. Demineralization of water
3. To analyze base composition
of nucleic acid
4. To separate protein mixtures
5. To detect any additives in
food and drugs
• Fermentation - Cation
exchange resins are used to
monitor the fermentation
process during ß-galactosidase
production.

31. REFERENCES
1. https://www.bio-rad.com/en-in/applications-technologies/ion-
exchange-chromatography?ID=MWHAY9ESH
2. https://www.news-medical.net/life-sciences/How-Does-Ion-Exchange-
Chromatography-Work.aspx
3. https://bitesizebio.com/31744/basics-ion-exchange-chromatography/
4. https://www.intechopen.com/books/column-chromatography/ion-
exchange-chromatography-and-its-applications
5. https://chemistry.stackexchange.com/questions/115874/retention-time-
and-dead-time
6. https://www.slideshare.net/MahendraMahi28/ion-exchange-
chromatography-142600420
7. https://www.slideshare.net/kskuldeep1995/ion-exchange-
chromatography-46831790
8. https://www.slideshare.net/AlexaJacob1/ion-exchange-chromatography-
ppt-86978892