Chromatography: Principle, Types & Applications

Chromatography is a technique used for separating a mixture of chemical substances into their individual components.

• It is used for the separation, purification, and testing of compounds.
• The term ‘Chromatography’ was coined by Russian botanist Mikhail Tswett in 1906.
• There are two principles of Chromatography namely Adsorption Chromatography and Partition Chromatography. 
• In Chromatography, The mixture is dissolved in a fluid solvent called the mobile phase, which carries it through a stationary phase.
• In the stationary phase, different components of the mixture travel at different speeds, causing them to separate.
• These phases determine which substances travel more quickly or slowly, and this is how they are separated.
• The different travel times are referred to as retention time.

There are many types of chromatography such as Liquid Chromatography, Gas Chromatography, and Ion-exchange Chromatography, however, all of these types employ the same basic principles. 

Key Terms: Chromatography, Adsorption Chromatography, Partition Chromatography, Paper Chromatography, Thin Layer Chromatography, Mobile Phase

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Chromatography Definition

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Chromatography is a laboratory technique used to separate components or solutes of a mixture. The components are separated on the basis of the relative amounts of each solute distributed between a moving fluid stream, called the mobile phase and a contiguous stationary phase.

• The mobile phase in Chromatography can be either a liquid or a gas. 
• The stationary phase is either a solid or a liquid.
• The solute molecules are continuously exchanged by the Kinetic molecular motion between the two phases. 
• Different types of Chromatography can be categorized depending on the type of mobile phase used.
• If the mobile phase is liquid, the technique would fall under the category of liquid chromatography.
• If the mobile phase is gas, the technique will fall under the category of gas chromatography.

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Principles of Chromatography

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Chromatography is based on the principle where molecules are transferred between the mobile phase and the stationary phase due to the absorbance or partition of molecules present in the mixture which we want to separate.

In order to understand the principle of chromatography, one must be thoroughly clear with the following terms:

Chromatography is a separation technique where the analyte is combined within a mobile phase which is pumped through a stationary phase.

• Generally, one phase is hydrophilic and the other phase is lipophilic.
• The components of the analyte interact differently with both of these two phases.
• Depending on their polarity, they interact more or less with the stationary phase, retarding to a greater or lesser extent.
• Sample components separate as they travel through the stationary phase at different speeds.
• Each sample component elutes from the stationary phase at a specific time which is called retention time.
• As the components of the sample pass through the detector, their signal is recorded and plotted in the form of a chromatogram.

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Types of Chromatography

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There are four main types of chromatography. These are

Adsorption Chromatography

Based on the absorptivity property of the component, different components are adsorbed on the adsorbent to different extents.

• The most common adsorbents are silica gel and alumina.
• When the mobile phase is allowed to pass through the stationary phase (adsorbent phase) the compounds of the mixture are moved to different distances over the stationary phase.

Thin Layer Chromatography (TLC)

This is another type of chromatography which is used to separate the mixture over a thin layer of adsorbent coated on a plate made of glass. The adsorbent used is silica gel or alumina.

• An adsorbent layer is spread across a glass plate.
• The mixture to be separated is dissolved in the suitable solvent.
• With the help of a fine capillary at a distance of 2 cm, the mixture solution is placed on the glass plate.
• A glass jar is filled with a suitable solvent and then a glass plate is placed in the jar at a vertical position.
• It is important to note that the mixture placed on the adsorbent-coated plate is not dipped in the solvent.
• With the help of capillary action, the solvent will automatically increase as the compartment is closed this process is called elution.
• As the elution moves up, depending on the degree of adsorbent and separation the mixture is separated. 
• The separated spots can be coloured and will be visible to the naked eye and the transparent spots can be viewed with the help of iodine or UV light.
• The relative adsorbent of each component of the mixture is expressed in terms of retardation factor (R_f value).
• R_f is the distance moved by the substance from the baseline Distance moved by the solvent from the baseline
• The R_f value of the substance depends on the nature of the substance, solvent, adsorbent and temperature.

Thin Layer Chromatography

Column Chromatography

Separating a mixture over a column of absorbent (Stationary Phase) is called Column Chromatography.

• The stationary phase (adsorbent) is filled in the glassed tube.
• The adsorbents like Alumina, silica gel, activated charcoal, manganese, etc can be used.
• The mixture which is to be separated is mixed with the suitable solvent and is allowed to run through an adsorbent.
• When the mixture is run through the adsorbent it slowly gets separated and forms a layer at different heights.
• Due to the difference in properties, the mixture is separated when run through the stationary phase.
• The component with higher absorptivity remains at the top and the remaining components flow down.
• The components are separated and removed by dissolving into suitable solvents and this process is called elution.
• Elution acts as a moving phase. As it is passed from the adsorbent phase the component is slightly dissolved in elution depending on the component's nature. 
• Thus the components get separated.

Column Chromatography

Partition Chromatography

Chromatography that is based on the continuous differential partitioning of components of a mixture is known as Partition Chromatography. The most common type of partition chromatography is Paper Chromatography.

Paper Chromatography

The below diagram shows the process of Paper Chromatography:

Paper Chromatography

In Paper Chromatography, a special type of paper is used which is called Chromatography paper. The paper contains water stored in it, which acts as a Stationary Phase.

• The mobile phase in this process is called the irrigant or developing solvent.
• The solvent travels through chromatography paper by the capillary action.
• The drop of mixed solution is dropped on chromatography paper and one end of the paper is dipped into the solvent.
• The mixture undergoes partly partition on the water molecules present in the paper and partly adsorption on the paper.
• A mixture is separated and displaced on the chromatography paper based on partition and adsorption rates.
• Components may be visible to the naked eye because they are of different colours, or they can be viewed under ultraviolet light or by spraying the reagents.

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What is Differential Extraction?

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Differential extraction refers to the method of separation of any organic component present in a given aqueous solution.

• An organic solvent is used for which the solubility of the desired compound is more than compared to that of water.
• Using a separating funnel, organic solvents are chosen so that they are immiscible with the aqueous solution and can form layers easily.
• It is later recovered by the process of distillation or evaporation.
• The process of continuous extraction is brought into use when the solubility of the compound is less in the organic solvent.

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Applications of Chromatography

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There are various uses of Chromatography. The most common uses are mentioned below-

• Chromatography is used for the separation, isolation and purification of proteins from complex sample matrices.
• It is an essential part of almost any protein purification strategy. 
• Organic compounds like amino acids, alcohol, acids, antibiotics, etc are used for characterising and isolating with the help of Chromatography.
• It is used to determine chemicals in pharmaceutical products.
• Chromatography is used to determine the alcohol level present in blood samples at hospitals.
• Clinical tests like urine analysis and antibiotic analysis can be done with the help of chromatography.
• It is used to determine components present in samples collected from the crime scene or from the suspects.
• The nutritive value of food additives in food industries can be determined by using chromatography. 
• It can be used to determine the level of pollution present in the water.

Also Read:

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Chromatography Handwritten Notes

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Here are the handwritten notes on Chromatography and related topics:

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Things to Remember

• Chromatography is the technique used to separate the components of a mixture.
• Chromatography is used for the separation, purification, and testing of compounds in laboratories.
• The three major Chromatography techniques are Column Chromatography, Thin layer Chromatography and Paper chromatography.
• The principle of Chromatography states that the molecules are transferred between the mobile phase and the stationary phase due to the absorbance or partition of molecules present in the mixture.
• Chromatography has a wide range of applications in different sectors and industries such as pharmaceuticals, chemical, and food industries.

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Previous Years’ Questions 

1. Paper chromatography is a type of partition chromatography….
2. Those substances can be separated by steam distillation which are…
3. The phenomenon in which adsorption and absorption takes place…
4. If dichloromethane (DCM) and water (H2O) are used for differential extraction… [JEE Main 2019]
5. Which of the following is not correct… [UP CPMT 2007]
6. The Lassaigne's extract is boiled with concentrated…. [NEET 2011]
7. For which of the following compounds Kjeldahl method… [JEE Main 2013]
8. In Dumas method one gram of carbon compound gives… [AP EAMCET 2019]
9. When metal M is treated with NaOH, a white gelatinous precipitate… [JEE Main 2018]
10. Liquation process is used for the purification of… [COMEDK UGET 2005]