Column Chromatography

In this column chromatography definition and types post we have briefly explained about principle, different types of column chromatography methodology and its applications.

What is Column Chromatography?

The term chromatography is derived from the Greek chroma, which means “colour,” and graphein, which means “to write”; the column chromatography methodology was first used around the turn of the twentieth century to separate plant pigments with easily visible colours. Column Chromatography Methodology is based on the fact that different compounds can distribute to varying degrees between different phases, or separable portions of matter. The stationary phase is one, and the mobile phase is the other.

The mobile phase flows over the stationary material, carrying with it the sample to be separated. To varying degrees, the sample’s components interact with the stationary phase. Some components interact with the stationary phase relatively strongly and are thus carried along more slowly by the mobile phase than those that interact less strongly. The separation is based on the components’ different mobilities. Many column chromatography methodology used in protein research are variations on column chromatography, in which the stationary phase material is packed in a column. 

The sample is a small volume of concentrated solution applied to the column’s top; the mobile phase, known as the eluent, is passed through the column. The eluent dilutes the sample, and the separation process increases the volume occupied by the sample. A successful experiment results in the removal of the entire sample from the column.

Column Chromatography

Column Chromatography Principle and Instrumentation

Types of Column Chromatography

1. Gel-filtration chromatography

Size-exclusion column chromatography methodology, also known as gel-filtration chromatography, separates molecules based on size, making it a useful method for sorting proteins with different molecular weights. It’s a type of column chromatography methodology where the stationary phase is made up of cross-linked gel particles. Gel particles are typically bead-shaped and made of one of two types of polymers.

The first is a carbohydrate polymer, such as dextran or agarose; these two polymers are often referred to by the trade names Sephadex and Sepharose, respectively. The second is based on polyacrylamide, which is sold under the trade name Bio-Gel.

The cross-linked structure of these polymers produces pores in the material. The extent of cross-linking can be controlled to select a desired pore size. When a sample is applied to the column, smaller molecules, which are able to enter the pores, tend to be delayed in their progress down the column, unlike the larger molecules.

As a result, the larger molecules are eluted first, followed later by the smaller ones, after escaping from the pores. The advantages of this type of column chromatography methodology are (1) its convenience as a way to separate molecules on the basis of size and (2) the fact that it can be used to estimate molecular weight by comparing the sample with a set of standards.

Column Chromatography

Gel-filtration chromatography

2. Affinity Chromatography Method

Affinity column chromatography methodology uses the specific binding properties of many proteins. It is another form of column chromatography with a polymeric material used as the stationary phase. The distinguishing feature of affinity column chromatography methodology is that the polymer is covalently linked to some compound, called a ligand that binds specifically to the desired protein.

The other proteins in the sample do not bind to the column and can easily be eluted with buffer, while the bound protein remains on the column. The bound protein can then be eluted from the column by adding high concentrations of the ligand in soluble form, thus competing for the binding of the protein with the stationary phase.

The protein binds to the ligand in the mobile phase and is recovered from the column. This protein–ligand interaction can also be disrupted with a change in pH or ionic strength. Affinity column chromatography methodology is a convenient separation method and has the advantage of producing very pure proteins.

Column Chromatography

Affinity chromatography

3. Ion-Exchange Chromatography

Ion-exchange column chromatography methodology is logistically similar to affinity chromatography. Both use a column resin that binds the protein of interest. With ion-exchange chromatography, however, the interaction is less specific and is based on net charge. An ion-exchange resin has a ligand with a positive charge or a negative charge. A negatively charged resin is a cation exchanger, and a positively charged one is an anion exchanger.

The column is initially equilibrated with a buffer of suitable pH and ionic strength. The exchange resin is bound to counterions. A cation-exchange resin is usually bound to Na+ or K+ ions, and an anion exchanger is usually bound to Cl– ions. A mixture of proteins is loaded on the column and allowed to flow through it. Proteins that have a net charge opposite to that of the exchanger stick to the column, exchanging places with the bound counterions.

Proteins that have no net charge or have the same charge as the exchanger elute. After all the nonbinding proteins are eluted, the eluent is changed either to a buffer that has a pH that removes the charge on the bound proteins or to one with a higher salt concentration. The latter outcompetes the bound proteins for the limited binding space on the column. The once-bound molecules then elute, having been separated from many of the contaminating ones.

Column Chromatography

Ion-Exchange Chromatography

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