Application of Ion Exchange

and
Affinity Chromatography in
protein purification
Group - 3
Name

Riddhi Halder 30008222004

Arunima Samanta 30008222002
Ashwini Shivani 30008222017
Riyana Sinha Roy 30008222019
Debashmita Das 30008222020
Introduction

What is Ion Exchange Chromatography?

Ion exchange chromatography (also known as ion chromatography) is a
method of separating ions and polar molecules based on their affinity for ion
exchangers.

What is Affinity Chromatography?

Affinity chromatography is a type of liquid chromatography for the separation,
purification or specific analysis of sample components.
ION EXCHANGE CHROMATOGRAPHY PRINCIPLE

Ion-exchange chromatography is used to separate charged biomolecules. The

charged analyte samples will be used as a liquid phase. When the analyte passes
through the chromatography column in the stationary phase, it is opposite the
charged sites. The analytes that have been isolated based on their charge are
eluted using a solution of varying ionic strength. The analytes are very selectively
separated according to their different charges by moving such a mobile phase
through the chromatography column.
Ion Exchange Chromatography Procedure
The Ion Exchange Chromatography procedure is as follows

❖ Ion exchange separations are carried out mainly in columns packed with
an ion-exchanger.
❖ These ionic exchangers are commercially available. They are made up of
styrene and divinylbenzene. Example. DEAE-cellulose is an anionic
exchanger, CM-cellulose is a cationic exchanger.
❖ The choice of the exchanger depends upon the charge of particle to be separated.
To separate anions “Anionic exchanger” is used, to separate cations “Cationic
exchanger” is used.
❖ First the column is filled with ion exchanger then the sample is applied followed
by the buffer. The tris-buffer, pyridine buffer, acetate buffer, citrate and phosphate
buffers are widely used.
❖ The particles which have high affinity for ion exchanger will come down the
column along with buffers.
❖ In next step using corresponding buffer separates the tightly bound particles.
❖ Then these particles are analyzed spectroscopically.
 ION EXCHANGE CHROMATOGRAPHY
ADVANTAGES
High Efficiency: IEC excels at separating
charged particles, including large proteins,
small amino acids, nucleotides, and complex
samples.
Versatility: A wide range of charged
compounds can be purified using IEC, from
small ions to large proteins.
Specificity: IEC can target specific ions,
minimizing interference from unwanted
components and enhancing the accuracy of
results.
DISADVANTAGES
Limited Detection: A common detection
method, conductivity, while sensitive to ions,
doesn't provide structural information about
the separated molecules. Coupling IEC with
other detectors like mass spectrometry or
UV-Vis spectrophotometry can help address
this limitation.
Minimal Sample Prep: Often, minimal preparation
Non-Ionic Applicability: IEC is
is required for samples before running IEC,
ineffective for separating non-ionic
reducing the risk of contamination or analyte
species or molecules that don't interact
loss.
well with the ion exchange resins.
Ease of Use: The technique is relatively simple,
making it accessible to researchers of varying
experience levels.
Regenerability: In many cases, the resins used in
IEC can be regenerated and reused for multiple
separations.
Quantitative Separations: IEC allows for precise
quantification of separated components, which is
suitable for analytical and preparative purposes.
Application of ION Exchange
❖ An important use of ion-exchange chromatography is in the routine analysis of amino
acid mixtures.
❖ The 20 principal amino acids from blood serum or from the hydrolysis of proteins are
separated and used in clinical diagnosis.
❖ This is most effective method for water purification. Complete deionization of water
(or) a non-electrolyte solution is performed by exchanging solute cations for hydrogen
ions and solute anions for hydroxyl ions. This is usually achieved by method is used for
softening of drinking water.
❖ In the analysis of products of hydrolysis of nucleic acids. In this way,
information is gained about the structure of these molecules and how
it relates to their biological function as carriers of hereditary
information.
❖ Chelating resins are used to collect trace metals from seawater.
❖ To analyze lunar rocks and rare trace elements on Earth.
AFFINITY CHROMATOGRAPHY PRINCIPLE
The principle of this method is:
The stationary phase consists of a support medium, on which the substrate (ligand)
is bound covalently, in such a way that the reactive groups that are essential for
binding of the target molecule are exposed.
As the crude mixture of the substances is passed through the chromatography
column, substances with binding site for the immobilized substrate bind to the
stationary phase, while all other substances is eluted in the void volume of the
column.
Once the other substances are eluted, the bound target molecules can be eluted by
methods such as including a competing ligand in the mobile phase or changing the
pH, ionic strength or polarity conditions.
Affinity Chromatography Procedure

1. Preparation of Column

❖ The column is loaded with solid support such as sepharose, agarose,

cellulose etc.
❖ Ligand is selected according to the desired isolate.
❖ Spacer arm is attached between the ligand and solid support.
 Affinity Chromatography Procedure
1. Preparation of Column

❖ The column is loaded with solid support such as sepharose, agarose, cellulose etc.
❖ Ligand is selected according to the desired isolate.
❖ Spacer arm is attached between the ligand and solid support.
2. Loading of Sample
❖ Solution containing a mixture of substances is poured into the
elution column and allowed to run at a controlled rate.

3. Elution of Ligand-Molecule Complex

❖ Target substance is recovered by changing conditions to favor
elution of the bound molecules.
 AFFINITY CHROMATOGRAPHY
ADVANTAGES
High Specificity: It utilizes highly specific
interactions between a ligand (attached to a solid
matrix) and the target molecule, resulting in
exceptional purity of the isolated biomolecule.
Single-Step Purification: In many cases, affinity
chromatography can achieve high purification in a
single step.
DISADVANTAGES
Costly Ligands: Developing and obtaining highly
specific ligands can be expensive, particularly for
complex biomolecules. Additionally, some ligands
may have limited stability, requiring careful storage
and handling.
Optimization Challenges: Optimizing elution
conditions to ensure target molecule recovery while
minimizing non-specific binding can be
time-consuming and require careful
experimentation.
Minimal Sample Disruption: Often, the technique
Limited Applicability: Affinity
requires minimal sample manipulation, reducing
chromatography is most effective for
the risk of denaturation or degradation of the
well-characterized biomolecules with
target biomolecule.
known binding partners. It may not be
suitable for isolating novel or poorly
Study of Binding Interactions: The technique can
understood targets.
be used to investigate the binding properties of
molecules, providing valuable insights into
Potential for Denaturation: Harsh
protein-protein or protein-ligand interactions.
elution conditions used to recover the
target molecule can sometimes lead to
denaturation or loss of its activity.
Applications
Its major application includes:

❖ Separation of mixture of compounds.

❖ Removal of impurities or in purification process.
❖ In enzyme assays.
❖ Detection of substrates.
❖ Investigation of binding sites of enzymes.
❖ In in vitro antigen-antibody reactions.
❖ Detection of Single Nuceotide polymorphisms and mutations in nucleic
acids.
Comparison
COMBINING THE TWO TECHNIQUES
Combining ion exchange chromatography (IEC) and affinity chromatography is a powerful
strategy for protein purification. It leverages the strengths of both techniques to achieve high
purity and yield of the target protein.

Examples of Applications:

Antibody Purification: IEC can be used to isolate antibodies based on their charge, followed by
affinity chromatography with protein A or G for high-purity antibody preparations.

Enzyme Purification: An initial IEC step can separate enzymes based on charge, followed by
affinity chromatography using a ligand specific to the enzyme's active site.
Protein Complex Isolation: A combination of IEC and affinity
chromatography can be used to isolate specific protein complexes from
cell lysates.

The complementary nature of these techniques provides a robust and

efficient approach for various protein purification applications.
CONCLUSION
Ion exchange chromatography (IEC) and affinity chromatography are powerful tools for protein
purification, each offering distinct advantages. IEC excels at separating proteins based on their net
charge, making it a versatile technique for initial purification steps or separating proteins with similar
sizes but different charges. Its ease of use, cost-effectiveness, and scalability make it a popular choice
for diverse applications.

Affinity chromatography, on the other hand, provides unmatched specificity by utilizing targeted
interactions between a ligand and the protein of interest. This allows for high-purity isolation in
potentially fewer steps, making it ideal for downstream purification or isolating specific proteins from
complex mixtures. However, developing suitable ligands can be expensive, and the technique may not be
ideal for novel or poorly understood proteins.

Therefore, the most effective approach often involves a combination of these techniques. IEC can be
used for initial enrichment, followed by affinity chromatography for final purification with high specificity.
This combined approach leverages the strengths of each method to achieve the desired level of purity
and yield for various research and bioprocessing applications.
References:
Application, Advantages & Disadvantages of Ion exchange chromatography [refer to] Firsthope
(https://www.firsthope.co.in/ion-exchange-chromatography-introduction-principle-components-applications-ad
vantages)

Advantages and disadvantages of affinity chromatography [refer to] Science Info

(https://www.pharmastuff4u.com/2023/04/advantages-and-disadvantages-of.html)

Affinity Chromatography and Importance in Drug Discovery [refer to] IntechOpen

(https://www.intechopen.com/books/1490)

Ion Exchange Chromatography and Affinity Chromatography in Protein Purification ([refer to] Journal of
Chromatography B, Volume 848, Issue 2, 15 February 2007, Pages 141-147) by JE Porter et al.