What is Chromatography and it's types? Do you know the applications of Chromatography?

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CHROMATOGRAPHY is the technique for the separation, purification, and testing of compounds.

The term "chromatography" is derived from Greek, chroma meaning, "color", and graphein meaning " to write".

In this process, we use the mixture to be separated on a stationary phase (solid or liquid) and a pure solvent such as water or any gas is allowed to move slowly over the stationary phase, carrying the components separately as per their solubility in the pure solvent. 

Principles of Chromatography


chromatography is a separation method where the analyte is combined within a liquid or gaseous mobile phase, which is pumped through a stationary phase. Usually, one phase is a hydrophilic and the other lipophilic. The component of the analyte interacts differently with these two phases. Depending on their polarity they spend more or less time interacting with the stationary and are thus retarded to a greater or lesser extent. This leads to a separation of the different components present in the sample. Each sample component elutes from the stationary phase at a specific time, it's retention time. As the components pass through the detector their signal is recorded and plotted in the form of a chromatogram.

Types of Chromatography

The four main types of chromatography are   

1. Adsorption Chromatography 


Source: Internet

In this process, different compounds are adsorbed on the adsorbent to different degrees based on the absorptivity of the component. Here also, a mobile phase is made to move over a stationary phase, thus carrying the components with higher absorptivity to a lower distance than that with lower absorptivity.

 

2. Thin Layer Chromatography

Source: Internet


In the process of TLC, The mixture of substances is separated into its components with the help of a glass plate coated with a very thin layer of adsorbent, such as silica gel and alumina.

The plate used for this process is known as a chrome plate. The solution of the mixture to be separated s applied as a small spot at a distance of 2 cm above one end of the plate. The plate is then placed in a closed jar containing a fluid term as an eluant, which then rises up the plates carrying different components of the mixture to different heights.

3. Column Chromatography

Source: Internet

Column chromatography is a technique used to separate the component of the mixture using a column of suitable adsorbent packed in a glass tube. The mixture is placed on the top of the column, and an appropriate eluant is made to flow down the column only.

Depending upon the degree of adsorption of the components on the wall adsorbent column, the separation of components takes place. The component with the highest absorptivity is remained at the top, while the other flow down the different heights accordingly.

4. Partition Chromatography 

Source: Internet


In this process, a continuous differential partitioning of components of a mixture into a stationary phase and mobile phase takes place. The example of partition chromatography can be seen in paper chromatography. In this process, chromatography paper is used as a stationary phase which is suspended in a mixture of solvents that act as a mobile phase.

Her, we put a spot at the base of the chromatographic paper with the mixture to be separated and as the solvent rises up this paper, the components are carried to different degrees depending upon their retention on the paper. The components are thus separated at different heights.

Application of Chromatography  

In bioanalytical chemistry, chromatography is mainly used for the separation, isolation, and purification of proteins from complex sample matrices. In cells, for example, proteins occur alongside numerous other compounds such as lipids and nucleic acids. In order to be analyzed, these proteins must be separated from all the other cell components. Then the proteins of interest might have to be isolated from other proteins and purified further.

A number of different chromatographic techniques are used for purification and analysis of proteins. Typical examples include reversal phase chromatography, ion-exchange chromatography, affinity chromatography, and size exclusion chromatography.

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