What is chromatography?
Chromatography is an essential instrumentation technique. It is a powerful separation tool that is utilized by almost all branches of science. It is a vital technique as it allows the separation of components of complex mixtures.
Chromatography enables the separation of substances based on size, structure, nature, and other properties. There are many types of chromatography techniques. Based on the mode, they can be classified into Planar Chromatography and Column Chromatography.
Similarly, based on the property of the sample, they can be classified into affinity chromatography, ion-exchange chromatography, flash chromatography, gas chromatography, gel filtration chromatography, etc. In this article, the focus is on column chromatography.
What is it?
It is a technique used to separate a single chemical compound from a fluid mixture. Generally, chromatography is used for separation, identification, or purification. Column chromatography is a separation technique.
It can be used on a small scale as well as on large-scale materials to purify. This technique is utilized both in Biology as well as Chemistry. It is a solid-liquid chromatography process in which the stationary phase is a solid & the mobile phase is a liquid or gas.
To understand the working principle, one has to understand what is adsorption.
Adsorption is when solids attract molecules of solutions or gases that they may be in contact with. Adsorption is different from absorption. Adsorption happens only at the surface level, whereas absorption happens both at the surface and core levels.
Column chromatography is the separation technique where the components of a complex mixture are separated through differential adsorption with respect to the stationary phase.
For example, if there are three components in a solvent, with an absorption coefficient of 5(A), 10(B), and 15(C), C is going to get adsorbed first, then B, and then A. Hence, understanding adsorption is key to understanding this type of chromatography.
There are two essential components of all chromatography techniques. They are the Mobile Phase and the Stationary Phase.
The mobile phase is the solvent, and it carries the substances(or components) of a complex mixture down the stationary phase. It is known as mobile because it moves through the chromatographic system. In this type of chromatography, this movement is under the influence of gravity.
It is selected based on the component to be separated and the type of chromatography performed. In this chromatography, the stationary phase is called the adsorbent. It is a solid substance, generally silica, alumina, or cellulose.
Procedure for the experiment
1. Preparation of the column
- The column is prepared by taking a glass tube, which is cleaned and dried and then coated with a thin layer of the Stationary Phase.
- The column’s bottom is packed with glass wool or cotton wool. Then the top of the column is covered with paper so that the Stationary Phase is not disturbed.
- There are two types of packing: Wet packing and Dry packing; in the former, the selected adsorbent is added as a slurry, while in the latter, the selected adsorbent is added as a dry powder.
2. Sample Introduction
- The solvent or the Mobile Phase introduced in the column. The components get separated as bands due to the phenomenon of relative adsorption, as discussed earlier.
3. Collecting Fractions
- Once the separation is completed, the samples are collected as fractions. This process is called Elution. The separated molecules can further be analyzed for various purposes.
- Pushing compressed air from the top of the column can speed up the process.
- One can further analyze the fractions by Thin Layer Chromatography for impurities and get the desired end product.
The different types
- Adsorption Chromatography: If the stationary phase is adsorbent material like silica or alumina.
- Ion-exchange Chromatography: If the stationary phase is an ion-exchange resin.
- Affinity Chromatography: If the stationary phase is attached with a Ligand that binds with specific molecules like protein or enzyme. This chromatography is used in Biology.
- Gel Permeation Chromatography: If the stationary phase is a porous gel or beads.
- Partition Chromatography: The Stationary Phase and the Mobile Phase are liquid in partition chromatography.
- To discover drug estimation from drug solutions.
- Used for the isolation of metabolic fluid from biological fluids.
- To teach students simple principles of chemistry.
Any reaction will have unwanted side products, unreacted starting material, and other impurities in the product mixture. In such a situation, we need a way to separate everything to isolate the desired product, and often, that way is column chromatography.
This process involves low cost, simple understanding, and disposability of the stationary phase used in the process. Hence, it is always a go-to technique for the basic requirements of a chemistry experiment.