All About Chromatography

Chromatography is a technique used for the separation, purification, and testing of different types of substances.

The term “chromatography” is derived from the Greek words chroma, which means “color,” and graphein, which means “to write,” as in “to write in color.”

We apply the mixture to be separated on a stationary phase (solid or liquid), and a pure solvent such as water or any other gas is allowed to move slowly over the stationary phase, carrying the components separately based on their solubility in the pure solvent, as shown in the diagram.

Chromatographic Principles and Procedures

Chromatography is a method of separation in which the analyte and a mobile phase are combined in a liquid or gaseous medium that is pumped through a stationary phase. A typical two-phase system has one phase that is hydrophilic and another that is lipophilic. The components of the analyte interact with two phases in a different way than one another. In accordance with the polarity of their interactions with the stationary phase, they spend more or less time interacting with the stationary phase and are thus retarded to a greater or lesser extent. As a result, the different components present in the sample are separated from one another. Each sample component elutes from the stationary phase at a specific time, which is referred to as its retention time in the laboratory. During the course of a component’s passage through the detector, the signal from the component is recorded and plotted on a chromatogram.

Types of Chromatography

The four primary types of chromatography are as follows:

1) Adsorption Chromatography

Adsorption chromatography is a process in which different compounds are adsorbed on the adsorbent to varying degrees depending on the absorptivity of the component. As with the preceding example, in this case the mobile phase is made to move over the stationary phase, thereby transporting the components with higher absorptivity to a lower distance than those with lower absorptivity. The following are the most common types of chromatographic techniques that are used in industrial settings.

2)Thin-Layer Chromatography (TLC)

Thin-layer chromatography (TLC) is a technique in which a mixture of substances is separated into its constituents with the help of a glass plate coated with a very thin layer of adsorbent, such as silica gel or alumina.

Chrome plate is the name given to the plate that is used in this process. The solution of the mixture to be separated is applied as a small spot 2 cm above one end of the plate, at a distance of 2 cm from the other end. The plate is then placed in a closed jar containing a fluid known as an eluant, which then rises up the plate, carrying different components of the mixture to different heights as it rises up the plate.

3. Column Chromatography 

Column chromatography is a technique for separating the constituents of a mixture that uses a column of suitable adsorbent packed in a glass tube, as illustrated in the figure below. Afterwards, the mixture is placed on the top of the column, and an appropriate eluent is made to slowly flow downward through the column.

The separation of the components is dependent on the degree of adsorption of the components on the wall adsorbent column during the separation procedure. The component with the highest absorptivity is retained at the top, while the other components flow down to different heights in accordance with their absorptivities.

4. Partition chromatography 

The components of a mixture are continuously partitioned differentially into two phases, which are referred to as the stationary phase and the mobile phase, in this process. Using the example of paper chromatography, we can see how partition chromatography is used. In this procedure, chromatography paper is used as a stationary phase, and it is suspended in a mixture of solvents that serves as the mobile phase in the process.

In this case, we place a spot at the base of the chromatographic paper with the mixture to be separated, and as the solvent rises up the paper, the components are carried to different degrees depending on how long they have been retained on it. Because of this, the components are separated at different heights.

Differential Extraction

In aqueous solutions, differential extraction is a technique for separating any organic component present from the rest of the solution. The solubility of the desired compound in the organic solvent used in this process is greater than that of the compound in water, which is why it is used in this process. In addition, the organic solvent is chosen in such a way that it is immiscible with the aqueous solution that it can form layers and be easily separated using a separating funnel, which is not always the case.

A process known as distillation or evaporation is used to recover the organic compound after it has been extracted from the solution. Continuous extraction is used in situations where the solubility of the compound in the organic solvent is less than 50% of its initial solubility.

Chromatography has a variety of applications

Chromatography is a technique used in bioanalytical chemistry that is primarily used for the separation, isolation, and purification of proteins from complex sample matrices. Proteins, for example, coexist with a plethora of other compounds in cells, including lipids and nucleic acids, among others. All of the other cell components must be separated from these proteins before they can be examined further. Afterwards, it is possible that the proteins of interest will need to be isolated from other proteins and purified even further.

When it comes to protein purification, chromatography is a critical component of almost any strategy. Protein purification and analysis are accomplished through the use of a variety of different chromatographic techniques. They can be divided into groups based on the physical principle that is involved in the separation procedure. Reversed phase chromatography, ion exchange chromatography, affinity chromatography, and size exclusion chromatography are all examples of chromatographic techniques.

Conclusion

Chromatography is a technique used for the separation, purification, and testing of different types of substances.Chromatography is a method of separation in which the analyte and a mobile phase are combined in a liquid or gaseous medium that is pumped through a stationary phase. A typical two-phase system has one phase that is hydrophilic and another that is lipophilic.Adsorption chromatography is a process in which different compounds are adsorbed on the adsorbent to varying degrees depending on the absorptivity of the component.Thin-layer chromatography (TLC) is a technique in which a mixture of substances is separated into its constituents with the help of a glass plate coated with a very thin layer of adsorbent. Differential extraction is a technique for separating any organic component present from the rest of the solution. When it comes to protein purification, chromatography is a critical component of almost any strategy.