Affinity Chromatography

“Basic Note – 2020”

affinity chromatography

"affinity chromatography principle, procedure and Basic Note - 2020".

Affinity Chromatography Advance Detailed Note 2020

Today In this post I’m am going to teach you about Affinity chromatography which is also known as a liquid chromatographic technique. There are so many other blog available on google but not specific and detailed. Here I’m trying to solve this problem and give a complete and detailed note on affinity chromatography.

In this in-depth post you’ll learn:

    • What is Affinity chromatography?
    • Who discovered affinity chromatography?
    • What is the principle of affinity chromatography?
    • How does affinity chromatography work?
    • What are advantages and disadvantages of affinity chromatography?
    • What are the applications of affinity chromatography?

So, if you are ready to go “all in” with this post, this post is for you.

Let’s dive right in

Affinity Chromatography

What is Affinity Chromatography?

In this section, I’ll help you get a handle on the fundamentals.

In this section, we will cover the introduction of affinity chromatography in detail.

What is the Principle of Affinity Chromatography [Basic Note] 2020

Affinity chromatography is a type of chromatography which is a separation technique. It is also called as a sample purification technique.


This technique is basically used for the biological molecules. Ex: Various Proteins.

By means of specific interaction of molecules, this method separates our desired molecules from the mixture.

This specific interaction is done with the component known as a ligand.

In this technique, the ligand is immobilized on a support.

To understand the basic working process of this technique, suppose we are passing a solution of a mixture of proteins through a column, the separation will be done on the basis of affinity.

If the proteins have a higher affinity with the ligand on the column, it will stay there for longer period of time and other molecules will elute faster.

Likewise, the separation takes time on the basis of the affinity. Here, ligand and molecules interact just like a lock and key model.


Affinity chromatography is a separation method that involves the separation of biochemical mixtures based on the highly specific interaction between enzyme and substrate, receptor and ligand, or protein and nucleic acid, etc.

Who discovered Affinity Chromatography?

In 1987 Pedro Cuatrecasas and Meir Wilchek discovered and developed affinity chromatography and its applications to biomedical sciences.

What is the principle of Affinity Chromatography?

What is the principle of affinity chromatography?

These interactions are reversible.

You can easily understand this concept if you stay tuned with this article.

If you don’t understand the concept, just write in the comment below, I’ll try my best to give you an answer.

Let’s move on to the next section of this post,

How does affinity chromatography work?

Actually, there are various kinds of procedures seen in USP (United States Pharmacopoeia), BP (British Pharmacopoeia) or IP (Indian Pharmacopoeia), but all follow the same concept in the background.

Indian Pharmacopoeia.
british pharmacopoeia for affinity chromatography.
British Pharmacopoeia.
US pharmacopoeia for affinity chromatography.
US Pharmacopoeia.

You can follow this procedure for your lab work,

Here, the matrix which is on the column is in bead form to which a specific ligand is bound by a covalent bond. In order to be effective for a matrix, it must have certain parameters:

1. It must be insoluble in buffers and solvents employed in the process

2. It must be mechanically and chemically stable.

3. It must be easily bound to a ligand or spacer arm onto which the ligand can be attached.

4. It must show good flow properties and must have a relatively large surface area for the attachment.

If an enzyme is to be purified, a substrate analog, inhibitor, cofactor, or effector may be used as an immobilized ligand.

Attachment of ligand to the matrix

There are so many procedures for the attachment of the ligand to the stationary phase by a covalent bond. Steps for the elution process:

1. Preparation of Column

Initially, the column is loaded with solid support such as cellulose, sepharose, agarose, etc.

Ligand is selected based on the desired isolate.

Spacer arm is attached between the ligand and solid support.

2. Sample Loading

A Solution having a mixture of substances is poured into the elution column and allowed to run at a (considerable) controlled rate.

3. Ligand-Molecule Complex Elution

The target substance is recovered by changing the conditions to favor the elution of the bound molecules.

Now, how you can perform this procedure:

protein purification (Affinity Chromatography)

Firstly, prepare your column part.

For the column part, first take the inner material of the column which is called a stationary phase which is cellulose, sepharose, agarose.

These all are basically used as a stationary phase because they are easily available and comparatively cheaper also.

Now you have prepared the column by putting the stationary phase inside the column and then you should fix it on the stand.

Then the ligand and spacer arm is added to make a solid matrix. Which will hold our solution mixture for a certain period of time for the effective interaction with the ligand and stationary phase?

Then the sample loading is to be done in which we have to load our sample from the above side of the column and then the solution mixture will react with the stationary phase for some period of time.

After the completion of the interaction, our desired material is eluted from the column which indicates the completion of the separation process.

Now, you can use your sample for further analysis. Here, the process of separation is completed and now we are going to discuss commercially used gels that are widely used and easily available.

List of Commercially used Gels






This gel is used widely because it is easily available and comparatively it is compatible with almost all materials.

1. CYANOGEN BROMIDE – ACTIVATED AGAROSE used in affinity chromatography

All ligands which are containing primary amino groups are easily attached to the agarose that is why this gel is especially versatile

Very gentle conditions may be used to couple the ligand Since the gel is extremely reactive.


Between ligand and matrix, stearic interference problems are seen.

These activated gels help overcome the steric interference problem by placing a six-carbon spacer arm between ligand and matrix.

Affinity chromatography gels

Ligands containing free primary amino group couple covalently with CH-Agarose.

Ligands containing free carboxyl group couple covalently with AH-Agarose.


Epoxy-Activated Agarose gel which is used for the attachment of ligands containing thiol, amino or hydroxyl groups.


These are the group-specific adsorbents that contain ligands that have an affinity for a specific class of biochemically related substances.

For example,

vibration blue-agarose specifically reacts with the enzymes which are having nucleotide cofactors like DNA polymerase, serum albumin, and Kinase.

Components of Affinity Chromatography

1. Matrix

2. Spacer  Arm

3. Ligand

1. Matrix

In order to be effective, it must have certain characters:

It should be insoluble in buffers & solvents employed in the process

The matrix is actually inert support to which a ligand can be indirectly or directly joined.

The matrix should be inert physically as well as chemically and must be easily coupled to a spacer arm or ligand onto which the ligand can be attached.

It must show good flow properties and should have a considerably large surface area for effective attachment.

It must be chemically and mechanically stable.

Mostly used matrix materials are polyacrylamide & agarose.

2. Spacer arm

Spacer arm is used to improve the binding between the target molecule and ligand by removing any effects of steric hindrances.

3. Ligand

The molecule that binds reversibly to a specific target molecule is called as a Ligand.

When we know the nature of the macromolecule to be isolated, only then we can select the ligand of our choice or requirement.

The hormone itself is a good or an ideal candidate for the ligand when a hormone receptor protein is to be purified by using this chromatography method.

For the isolation of antibodies, a hapten or antigen may be used as ligand.


Firstly it’s consumes a large amount of time.

When the solution mixture comes at the bottom of the column, it needs some time to react with the support or stationary phase for the better separation and that is why it takes time.

As the column contains affinity resins and other stuff in a compacted form that’s why the solution mixture takes time to come at the bottom from the top.

It is expensive because more amounts of solvents are required.

Non-specific adsorption cannot be removed; it can just be minimized.

In this chromatography method, there is limited availability of immobilized ligands along with their high cost.

If the required pH is not adjusted, proteins get denatured.

You know very well that everything which has its application is preferred first in the market.

Application of Affinity Chromatography

1. It is one of the most useful methods for the separation and purification of specific products from the mixture.

2. It is a sample purification technique and it is used basically for biological molecules. Ex: proteins, nucleic acid, and enzymes, etc.

3. It has its application for concentrating and purifying the compound from a mixture.

Applications of Affinity Chromatography.

Its major application includes:

1. For the separation of a mixture of various compounds.

2. In the purification process or removal of impurities.

3. In the assays of enzyme

4. Substrates detection

5. For the investigation of enzymes binding sites

6. In in-vitro antigen-antibody reactions

7. For the detection of Single Nucleotide polymorphisms and knowing the mutations in nucleic acids

What are the Advantages and Disadvantages of Affinity Chromatography?

Advantages of Affinity Chromatography:

1. Mostly, It’s used to study enzymes and proteins in the pharmaceutical field.

2. In the pharmacy world, affinity chromatography is used to check the quality of the proteins and enzymes which is present in the formulation.

3. It’s is also used in the genetic engineering field for the gene therapy and identification of genes.

4. It is also used during the production of vaccines.

5. This chromatography is mostly used in a pharmaceutical company, in the production department of the vaccines for maintaining the quality of the product.

6. In pharmacy and other research work where they require high purity, it can be achieved by affinity chromatography.

7. High specificity is achieved with affinity chromatography.

8. For increasing the Solubility.

9. It prevents proteolysis.

10. The elution volume is related to molecular weight.

11. In Affinity Chromatography, there is less zonal spreading than in other techniques.

12. It does not depend on the temperature, pH, Ionic Strength, and buffers composition. Therefore, separation can be carried out under any condition.

13. High degrees of purity can be obtained.

14. The binding sites of biological molecules can be simply investigated.

15. In affinity chromatography the process is reproducible it is a major advantage.

16. Highly sensitive for the detection of the compound like halogen, peroxides, quinines, nitrites, etc.

17. More sensitive than TCD & FID.

Disadvantages of Affinity Chromatography:

1. Compared to another chromatography method the affinity chromatography is non-specific for adsorptions.

2. Ligands used in this method are too much expensive that’s why it is used in specific work only.

3. Low productivity.

4. Sometimes Ligands leakage problems are also seen.

5. The presence of metal-ions contaminates the purified protein solution, because they may destabilize or stabilize the protein.

6. Metal-ion transfer and metal ion leakage lead to protein loss.

7. Degradation of the solid support.

8. Expensive Ligands.

9. Interfere with structure.

10. Limited sample volume.

11. For using it, the person must have skills to handle it.

12. Considerable optimization is required.