1. ION EXCHANGE CROMATOGRAPHY AND
AFFINITY CHROMATOGRAPHY
By
KAUSHAL KUMAR SAHU
Assistant Professor (Ad Hoc)
Department of Biotechnology
Govt. Digvijay Autonomous P. G. College
Raj-Nandgaon ( C. G. )

2. CONTENTS
• Introduction.
• Bygone days.
• Basic terms related to chromatography.
• Different type of chromatography techniques.
– Ion exchange chromatography:
• Principle of ion exchange chromatography.
• Resin selection in ion exchange chromatography.
• Commonly used ion exchangers.
• The applications of ion exchange chromatography.
• Merits and demerits of ion exchange chromatography.
– Affinity chromatography:
• Why use affinity chromatography?
• Steps involved.
• An example illustrating about the technique.
• Choice of ligand.
• The applications of affinity chromatography.
• Merits and demerits of affinity chromatography.
• Conclusion.
• Bibliography.

3. INTRODUCTION
• The word ‘Chromatography’ comes from the two
Greek words ‘Chroma = color’ and ‘Graphein = to
write’.
• ‘Chromatography’ is a physical method
commonly used for separating a mixture of
chemical substances into its individual
components.
• WHY SO??
– Its main aim behind separation of mixtures
into their components is in order to analyze,
identify, purify, and/or quantify the mixture or
components.

4. LETS HAVE A LOOK AT BYGONE DAYS
• 1900

5. BASIC TERMS RELATED
TO CHROMATOGRAPHY

6. TERM DEFINITION
Mobile phase or carrier solvent moving through the column
Stationary phase or adsorbent substance that stays fixed inside the column
Eluent fluid entering the column
Eluate fluid exiting the column (that is collected in flasks)
Elution the process of washing out a compound through a column using a
suitable solvent
Analyte mixture whose individual components have to be separated and
analyzed
Chromatograph the assembly of apparatus for carrying out chromatographic
separation.
Solute Referring to the sample components in partition chromatography.
Solvent Referring to the liquid stationary phase in partition chromatography.
Elution time The time between the start of the separation (The time at which the
solute enters the column) and the time at which the solute elutes.
Retention Time And Retention
Factor
It is a measurement of the time taken for a solute to pass through
a chromatography column.
It is calculated as the time from injection to detection.

7. CATEGORIZATION

8. PREPARATIVE
CHROMATOGRAPHY
More sample
required
AIM- To remove
impuritiesfor
commercial
product
ANALYTICAL
CHROMATOGRAPHY
Less sample
required
AIM- to separate
compound

9. CHROMATOGRAPHY
ADSORPTION
PAPER
CHROMATOGRAPHY
THIN LAYER
CHROMATOGRAPHY
PARTITION
NORMAL
REVERSE
GAS
CHROMATOGRAPHY
SIZE-EXCLUSION
ION- EXCHANGE
CATAION
ANION
AFFINITY
HPLC

10. ION EXCHANGE
CHROMATOGRAPHY

11. Ion exchange chromatography is applicable for the separation
or purification of mixture of proteins on the basis of their net
charge.
• Faciliated by-
a) Manipulated by changing buffer pH.
b) By altering salt concentration.
• In this chromatographic technique, the stationary solid
phase commonly consists of an insoluble matrix with
covalently attached anions or cations (called ion
exchanger).
• Solute ions of the opposite charge in the mobile liquid
phase are attracted to the ion exchanger by electrostatic
forces.

13. COMMONLY USED ION EXCHANGERS
NAME TYPE FUNCTIONAL GROUP
Anion exchanger
DEAE-cellulose Weakly basic Diethylaminoethyl (DEAE)
QAE-Sephadex Strongly basic Quaternary aminoethyl (QAE)
Q-Sepharose Strongly basic Quaternary ammonium (Q)
Cation exchanger
CM-cellulose Weakly acidic Carboxymethyl (CM)
SP-Sepharose Strongly acidic Sulfopropyl (SP)
SOURCE S Strongly acidic Methylsulphate (S)

14. CATEGORIZATION OF EXCHANGER
ACCORDING TO THE STRUCTURE:
NAME PARTICLE SIZE ION EXCHANGE
EFFICIENCY
STRUCTURE
(a)Pellicular type with ion
exchange film
30-40 μ 0.01-0.1 meq/g
(b)Porous resin coated with
exchange beads
5-10 μ 0.5-2 meq/g
(c) Macroreticular resin bead Not highly
efficient
Very low exchange
capacity
(d) Surface sulfonated and
bonded electrostatically with
anion exchange
Less efficient 0.02meq/g.

15. SELECTION OF THE ION EXCHANGER
• The choice of the ion exchanger whether
cationic or anionic for the purification of a
biomolecule largely depends on the isoelectric
point, pI of the biomolecule.
pH>pI net negative charge, binds to anion
exchanger.
pH =pI no net vharge, no binding to ion exchanger.
pH<pI net positive charge, binds to cation
exchanger.

17. THE APPLICATIONS OF ION EXCHANGE
CHROMATOGRAPHY
 Separation and Purification of blood components such as
albumin,recombinant growth factors and enzymes.
 Biotechnology - Analytical applications such as quality control and
process monitoring.
 Food and clinical research - to study wheat varieties and the
correlation of proteinuria with different renal diseases.
 Fermentation - Cation exchange resins are used to monitor the
fermentation process during ß-galactosidase production.

18. MERITS AND DEMERITS
MERITS DEMERITS
Long life of resins. Inextensibility of properties of
resins
Cheap maintenance
Environmental friendly Some inorganic factors may cause
resins foul

19. AFFINITY
CHROMATOGRAPHY

20. • It’s a technique enabling purification of biomolecules
with respect to biological function or individual chemical
structure.
• The substance to be purified is specifically and reversibly
absorbed to a ligand (binding substance),immobilized by
a covalent bond to a chromatographic bed material(i.e.
matrix).
• Recovery of the substance can be achieved by changing
experimental conditions to favour desorption.
• In 1910, the German scientist, Emil Starkenstein came
up with the concept.
• The term affinity chromatography introduced in 1968 by
Pedro Cuatecasas, Chris Anfinsen and Meir Wilchek.

21. WHY USE AFFINITY
CHROMATOGRAPHY?
Affinity chromatography offers:
• high selectivity,
• resolution,
• and capacity.
It has the advantage of utilizing a protein's biological
structure or function for purification. As a result,
purifications that would otherwise be time consuming
and complicated, can often be easily achieved with
affinity chromatography.

23. TYPES OF LIGANDS USED
TYPES OF LIGAND TARGET MOLECULES OF INTEREST
Enzyme Substrate analog, inhibitor, cofactor
Antibody Antigen
Lectin Polysaccharide, glycoprotein, cell surface
receptor, cell
Nucleic acid Complementary base sequences, nucleic acid
binding protein
Hormone Receptor
Avidin Biotin
Calmodulin Calmodulin-binding molecule
Poly(A) RNA containing poly(U) sequences
Glutathione Glutathione-S-transferase or GST fusion
protein
Proteins A and G Immunoglobulins
Metal ions Poly(His) fusion proteins, native proteins with

24. AN EXAMPLE

25. APPLICATIONS
1. Purify and concentrate a substance from a mixture into a
buffering solution.
2. Reduce the amount of a substance in a mixture.
3. Purify and concentrate an enzyme solution used in Genetic
Engineering - nucleic acid purification.
4. Production of Vaccines - antibody purification from blood
serum.
5. And Basic Metabolic Research - protein or enzyme purification
from cell free extracts.

26. MERITS AND DEMERITS OF AFFINITY
CHROMATOGRAPHY
ADVANTAGES
 Extremely high
specificity.
 High degrees of purity
can be obtained.
 The process is very
reproducible.
 The binding sites of
biological molecules can
be simply investigated.
DISADVANTAGES
 Expensive ligands.
 Leakage of ligand.
 Degradation of the solid
support.
 Limited lifetime.
 Non-specific
adsorption.
 Relatively low
productivity.

27. CONCLUSION
Chromatography is one of
several separation techniques defined
as differential migration from a
narrow initial zone. Chromatography
has numerous applications
in biological and chemical fields. It is
widely used in biochemical research
for the separation and identification
of chemical compounds of biological
origin.

28. BIBLIOGRAPHY
BOOKS:
Leninger-Principles of biochemistry, Fifth edition by David L. Nelson and Michael
M.Cox
Fundamentals and applications of biophysics and molecular biology, Path Finder by
Pranav Kumar
INTERNET SOURCES:
https://www.britannica.com/science/chromatography
https://www.khanacademy.org/test-prep/mcat/chemical-processes/separations-
purifications/a/principles-of-chromatography
https://study.com/academy/lesson/what-is-chromatography-definition-types-
uses.html
YOUTUBE CHANNELS:
Ak lectures
Shomu’s biology
Agilent technologies.