Without a doubt, protein purification has been an important process in protein research, particularly for investigating the function and structure of a protein. To help you prepare for protein purification, our article will guide you through the significance of this process, some common methods of protein purification, and materials to perform this method.

In this article:

Why is Protein Purification Important?

What is Chromatography?

What is Column Chromatography?

Common Terms used in Column Chromatography

The Main Phases of Column Chromatography

How Does Column Chromatography Work?

Two Common Approaches used in Column Chromatography

1.Size Exclusion Chromatography

2.Affinity Chromatography

Related Products


Why is Protein Purification Important?

The reason why you need to purify your protein of interest before performing downstream experiments is your protein may come from a mixture of many molecules and other proteins. It means your protein could be only a tiny fraction of the starting material—as little as 1%.

illustration of how protein purification works using column chromatography. Protein purification separates out a mixture of molecules to isolate your protein of interestProtein purification, purified protein

Your sample or starting material may contain many proteins with different amino acid sequences, sizes, charges, structures, and binding sites. Many of these proteins can alter the findings of your study when you use your unpurified protein.

To purify your protein from unwanted molecules, you can use a chromatography method.

What is Chromatography?

Chromatography is a technique to separate, identify, and purify molecules for quantitative and qualitative analysis. In chromatography, molecules in a mixture of substances separate with the use of adsorbents.

There are different types of chromatography techniques based on the type of equipment, the type of materials used, or the interactions between material and the target molecules.

One common chromatography approach used to purify a protein is a column chromatography technique.

What is Column Chromatography?

Column chromatography is a chromatography technique to separate a mixture of unpurified molecules into a single molecule by using an adsorbent, taking place in a plastic column or a glass tube.

Common Terms used in Column Chromatography

Affinity chromatography

A chromatography technique to separate molecules based on their interaction with a ligand cross-linked to a matrix.

Agarose resin/Agarose bead

A common matrix used in column chromatography.


A substance to be separated during chromatography.


A tube used in the protein purification.

Column chromatography

A chromatography technique involving the use of a column containing a matrix (such as agarose beads).


A solution containing the target protein after elution.


A solution entering the column.


The process of dissolving the target protein.


The process of collecting the target molecule in a collection tube by using an elution buffer.

Elution buffer

A buffer for dissolving the target protein.

Gas chromatography

A chromatography technique to separate molecules by using a gas in the mobile phase.


A molecule with a specific affinity that forms a complex with another molecule to perform a biological function.


A solid material used as a stationary phase in the column.

Mobile phase

A mixture of analyte (substance being separated) and solvent flowing through the column.

Separated molecules

Molecules or proteins of interest.

Size exclusion chromatography

A chromatography technique to separate molecules based on size.


A buffer to dissolve the substance being separated.

Stationary phase

A solid and immobile phase in the column.

Target protein

Protein of interest or purified protein. This can also be the separated molecules.


The removal of unbound molecules or nonspecific proteins by using a buffer.

The Main Phases of Column Chromatography

There are two distinct phases during chromatography. When performing chromatography, think of the phases as different stages when performing this technique.

two phases of column chromatography illustration: mobile phase and stationary phase. Column chromatography, stationary phase, mobile phase

For example, in column chromatography, agarose beads acts as the stationary phase, immobile within the column.

The addition of analyte and solvent and the action of them running through the column is considered the mobile phase.

Stationary Phase

The stationary phase is an adsorbent or immobile surface inside the column. The analyte and solvent flow through the stationary phase.

Examples of a stationary phase are a solid surface, such as a thin layer or paper, or a porous matrix in a column. The matrix is highly porous, so the analyte and solvent can migrate through the pores.

Some examples of porous matrices used for chromatography are cross-linked polyacrylamide, agarose (such as Sepharose™), and dextran (such as Sephadex™). Typically, these matrices are in bead form.

Mobile Phase

The mobile phase is a mixture of analyte and solvent, which moves (mobile) together during chromatography. As this phase travels in the column, the components of the analyte begin to separate and continue moving slowly toward the end of the column.

In most cases, including column chromatography, the mobile phase is liquid; however, in the case of gas chromatography, the mobile phase can be gaseous.

How Does Column Chromatography Work?

A mixture of solvent is loaded in the column with a porous matrix as a stationary phase. In the process, molecules in the mixture migrate at different rates, inhibiting some of them to leave the column. After the elution with a buffer, the target molecules finally flow through the column.

Two Common Approaches used in Column Chromatography

1.Size Exclusion Chromatography

Size exclusion chromatography is a method of separating proteins and other molecules based on their size.

Small molecules flow through the pore of the matrix and fit better than large molecules. They can easily get trapped in the pore. After entering the pore of matrix, they can still flow through and travel to the other end of the column, but these small molecules migrate slow through the column.

size exclusion chromatography separates molecules based on size as they flow through agarose beads within a column, column chromatography, agarose beads

Conversely, large molecules go through the spaces between the beads and travel faster, because they are too big to enter the pore. Consequently, the large molecules migrate faster than the small molecules.

2.Affinity Chromatography

This chromatography uses a molecule binding specifically to a protein, or a ligand. The ligand is cross-linked directly to a matrix. After your protein of interest binds to the ligand, this complex then stays immobilized inside the column. Whereas, the unbound proteins flow through the column.

Affinity chromatography uses molecular binding to purify proteins. In affinity chromatography the protein of interest binds to a ligand

After the unbound proteins leave the column, you use elution buffer to release your target protein from the ligand and collect the eluate in a tube.

To purify your protein by performing a particular column chromatography method, you must choose the right type of agarose beads or resins as a matrix by considering the characteristics of your protein and matching them with the agarose resin.

To learn more about how to pack your column with the agarose resins for protein purification, watch this video below:

Related Products

Chromatography Columns

Plastic Spin Columns (Cat. No: P-300)


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