Introduction Chromatography terms History Protein purification Purpose Chromatographic methods – a) Size exclusion b) Ion exchange c) Affinity d) HPLC Conclusion Reference

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PROTEIN SEPARATION BY 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
 Chromatography terms
 History
 Protein purification
 Purpose
 Chromatographic methods – a) Size exclusion
b) Ion exchange
c) Affinity
d) HPLC
 Conclusion
 Reference
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3. INTRODUCTION:
 The term chromatography is derived from Greek meaning “
coloured writing” .
 It is a technique which separates molecules on the basis of
difference of size, shape, mass, charge, solubility & adsorption
properties.
 THEORY: It is based on the principle of partition coefficient of
molecules i.e. a separation method that exploits the differences in
partitioning behavior between a mobile phase and a stationary
phase to separate the components in a mixture.
Types
Plane chromatography Column chromatography
(e.g; paper, TLC) (e.g; SEC, ion exchange,
affinity, HPLC etc)
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4.  CHROMATOGRAPHY TERMS
 The analyte is the substance which is to be purified or isolated
during chromatography
 A chromatogram is the visual output of the chromatograph.
Different peaks or patterns on the chromatogram correspond to
different components of the separated mixture
 The mobile phase is the analyte and solvent mixture which
travels through the stationary phase
 The retention time is the characteristic time it takes for a
particular molecule to pass through the system
 The stationary phase is the substance which is fixed in place for
the chromatography procedure and is the phase to
which solvents and the analyte travels through or binds to.
Examples include the silica plate in thin layer chromatography.
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5. HISTORY:
 Chromatography was invented by the Russian botanist Mikhail
Tswett (1872-1919) in 1906.
Mikhail Tswett
 Affinity chromatography was discovered in 1968 by Pedro
Cuatrecasas and Meir Wilcheck
 Meir Wilcheck Pedro Cuatrecasas
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6. Protein Purification:
 Protein purification is a series of processes intended to isolate a
single type of protein from a complex mixture.
 The starting material is usually a biological tissue or a microbial
culture.
 Mostly various column chromatographic techniques are employed
for protein separation.
 Separation steps may exploit differences in (for example) protein
size, physico-chemical properties, binding affinity and biological
activity.
 PURPOSE:
 Purification may be preparative or analytical. Preparative
purifications aim to produce a relatively large quantity of purified
proteins for subsequent use.
 Analytical purification produces a relatively small amount of a
protein for a variety of research or analytical purposes, including
identification, quantification, and studies of the protein's
structure, post-translational modifications and function.
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7. Chromatographic methods for protein purification
 Usually a protein purification protocol contains one or more
chromatographic steps.
 The basic procedure in chromatography is to flow the solution
containing the protein through a column packed with various
materials.
 Different proteins interact differently with the column material,
and can thus be separated by the time required to pass the column,
or the conditions required to elute the protein from the column.
A] Gel Filtration Chromatography ( Molecular Sieve or Size
Exclusion Chromatography):
 Separation by: SIZE and SHAPE
 Stationary phase: porous beaded gels composed up of Agarose or
Polyacrylamide & Sephadex ( carbohydrate dextran).
 Elution: with continuous buffer. Larger molecules elutes out first
followed by smaller once as they get trapped in the pores.
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8. Fig: SEC for
protein separation
 Advantage: It can also be used for determining the tertiary
structure and quaternary structure of purified proteins, especially
since it can be carried out under native solution conditions.
 Disadvantage: It is generally a low-resolution chromatography
technique . 8

9. B] Ion Exchange Chromatography (anion exchange or cation
exchange):
 Separation by: NET CHARGE
 Stationary phase: Hydrated beads of carbohydrate polymer (e.g.
cellulose or agarose) or other polymer (e.g. polystyrene), with
covalently-attached charged functional group (e.g. phosphate (–)
or a carboxymethyl group (–) or diethylaminoethyl group (DEAE,
+).
 Elution: usually increasing salt concentration in buffer is method
of elution. can also elute (wash proteins off column) by changing
pH .
 Types: a) Cation exchange: matrix having +vely charged groups.
b) Anion exchange: matrix having –vely charged groups.
 In both cases molecules having similar charge elutes first followed
by opposite charged molecules.
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10. 
 Fig: Anion exchange chromatography
 Advantage: Ion exchange chromatography is a very powerful tool
for use in protein purification and is frequently used in both
analytical and preparative separations.
 Disadvantage: Cannot be used for the separation of uncharged
species.
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11. C] Affinity Chromatography:
 Separation by: binding specificity of protein to particular ligand.
 Stationary phase: Agarose or Polyacrylamide bead derivatives
(Sepharose or bio-gel) which is activated by “Cyanogen bromide”
for ligand attachment (50-300 mg/ml of gel prepared).
 Table: Specific ligands used to bind particular protein
Ligands Proteins
a) Enzyme cofactor, inhibitor (analogue)
b) Lectin glycoprotein, cell surface receptor
c) Antibody antigen (protein)
d) Hormone, Vitamins receptor, carrier protein
e) Glutathione glutathione-S-transferase or GST
fusion proteins
f) Metal ions ( Ni/Co) Poly (His) fusion proteins, native proteins
with histidine, cysteine and tryptophan
residues on their surfaces 11

12.  Elution: competitive binding, salt conc., pH alteration, temp.
 Fig: Affinity chromatography
 (glucose-protein binding)
Advantages:
 Rapid separation is achieved while avoiding contamination.
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13.  Affinity chromatography is a fairly achievable technique because
of the great selectivity of the glucose residues and the target
protein, giving purified product with a high yield of recovery.
 It can be a one step process in many cases.
 Technique can be used for substances of low concentration.
 Unlike Gel filtration chromatography and ion-exchange
chromatography, affinity chromatography would be able to isolate
one specific protein at a time, where other techniques will isolate
proteins with similar characteristics.
Disadvantages:
 The interaction of proteins of interest and ligand has to be
determined carefully. This process required expensive materials,
time, and small amount of protein that can be processed at once.
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14. D] HPLC (High performance liquid chromatography):
 Separation by: Hydrophobicity.
 Stationary phase: Hydrated beads of carbohydrate polymer (e.g.
cellulose or agarose) or other polymer (e.g. polystyrene),.
 Fig: RPLC
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15. Elution: The proteins are eluted by a gradient of increasing amounts
of an organic solvent, such as acetonitrile
Advantage:
 The diffusion is limited and the resolution is improved.
 Faster.
Disadvantage:
 HPLC purification frequently results in denaturation of the
purified proteins and is thus not applicable to proteins that do not
spontaneously refold.
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17.  REFERENCES:
 Principles of Biochemistry, 4th edition- Nelson & Cox.
 Principles & techniques of Biotechnology & Molecular
Biotechnology- K. Wilson & J. Walker.
 Biochemistry, 6th edition- Berg
 Biochemistry- Stryer
 Internet sources
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