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CHROMATOGRAPHY - Types, Applications, Column Chromatography, Thin layer Chromatography, Partition Chromatography, Paper chromatography, Practice Problems & FAQs 

Do you ever think of a technique that keeps you safe and healthy every day?

The technique is chromatography, which helps us in many ways in our everyday life.

It aids in the production of various antibodies that aid in the fight against diseases and viruses. It played an important role in the fight against the Ebola virus.

The contaminants, and hence the quality of the food and beverages we are consuming, are tested routinely by chromatography.

It helps to identify and catch criminals by analyzing blood and clothing samples.

Table of content:

Chromatography

Chromatography is an important technique extensively used to separate mixtures into their components, to purify compounds and to test the purity of compounds.

Terms used in Chromatography

  1. An adsorbent is a solid substance that is used to bind solute molecules from a liquid or a gas phase.
  2. Adsorbate: A substance that binds with an adsorbent during adsorption is an adsorbate.
  3. Mobile phase and stationary phase: The mobile phase is the solvent that moves through the column, while the stationary phase is the material that remains fixed inside the column.

Types of chromatography

Chromatography is classified into two categories based on the principles involved.

  1. Adsorption chromatography
  2. Partition chromatography

Adsorption chromatography

It is based on the principle that various components (adsorbates) in a mixture bind to a suitable adsorbent (silica gel, alumina) to a different extent. Some components are more strongly adsorbed than others.

Adsorbates dissolved in a solvent hence travel through the column at different rates and thus get separated with respect to time.

On the principle of differential adsorption, there are two main types of adsorption chromatography.

  1. Column chromatography
  2. Thin layer chromatography

Column chromatography

The adsorbent is filled into a cylindrical column at regular intervals. The mixture is dissolved in a suitable solvent and added slowly at the top of the column to flow under gravity. Because of the differential adsorption of substances on the adsorbent, the compounds move along the column at various rates, which allows them to be separated into fractions in column chromatography.

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Thin layer chromatography

A thin-layer chromatography plate is used to separate substances in a mixture. In thin layer chromatography, the adsorbent is coated as a thin paste on an inert plate and placed in a glass chamber containing solvent at the bottom. Components of a mixture move to various distances with the solvent (eluent) due to capillary action depending on their degree of adsorption.

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Partition chromatography

The components of a mixture are continuously differentially partitioned between the stationary and mobile phases in partition chromatography. It involves paper chromatography.

Paper chromatography

Partition chromatography is the principle of paper chromatography in which the components are spread or partitioned between the liquid and stationary phases. Synge and Martin first discovered paper chromatography in 1943. Paper chromatography is a chromatography technique that uses paper sheets or strips as the adsorbent and the stationary phase through which a solution is made to flow.

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Types of Paper Chromatography

  1. Ascending Paper Chromatography: Soluble travels in an upward direction in this technique.
  2. Descending Paper Chromatography: In this technique, due to gravity and capillary action, the flow of solvent moves downward.
  3. Ascending: Descending Paper Chromatography:- After a certain point, the solvent moves in two directions in this kind of paper chromatography. The solvent moves upwards on the paper that has been folded over a rod at first, then crosses the rod and continues its downward journey.
  4. Radial or Circular Paper Chromatography: The sample is placed in the circular filter paper's center. The filter paper is tied horizontally on a Petri plate containing the solvent once the spot has dried.
  5. Two Dimensional Paper Chromatography: Two-dimensional paper chromatography can be used to separate substances with similar rf values.

Application of chromatography

  1. Water samples are tested for purity.
  2. Vitamins and preservatives are separated and analyzed in the food industry.
  3. Used in forensic science to detect small amounts of substances in the stomach.

Practice Problems

Q 1. Separation of organic compounds by column chromatography is based on:

A. Selective absorption

B. Selective adsorption

C. Different solubilities

D. Both A and B

Solution: B)

The process of column chromatography is used to isolate a single chemical ingredient from a mixture dissolved in a fluid. It separates substances based on differences in compound adsorption to the adsorbent as the compounds pass through the column at different rates, allowing them to be separated into fractions.

Q 2. Adsorption chromatography does not include which of the following?

A. Paper chromatography

B. Thin-layer chromatography

C. Column chromatography

D. All of the above

Solution: A)

Partition chromatography is exemplified by paper chromatography. Adsorption chromatography includes thin layer chromatography and column chromatography.

Q 3.  In adsorption chromatography, which of the following can be used as an adsorbent?

A. Alumina

B. Silica

C. Both A and B

D. None of the above

Solution: C)

The differential adsorption of distinct components of a mixture on a suitable adsorbent such as silica gel or alumina is the basis of the adsorption chromatography method.

Q 4. Chromatography is based on which principle?

A. The rate of movement of components in a column is different

B. The rate of movement of components in the mixture is different

C. The capture of one solute on the adsorbent allows it to be separated from other components

D. None of the above

Solution: B)

The interaction of solute molecules with active sites on the stationary phase is the basis of chromatography. The polarity of the solutes determines this attachment or interaction. This technique demonstrates the axiom "polar like polar." Because the stationary phase is more polar than the mobile phase, the mixture's high polar compounds will be strongly attached to the stationary phase, while less polar compounds will be loosely bound. The mobile phase will elute less tightly bound compounds earlier than tightly bound compounds.

Frequently asked questions

Q 1. Can the chromatography technique be used to separate coloured substances?

Answer: Yes, Thin layer chromatography is used to separate or identify coloured substances in mixtures.

Q 2. What types of solvent are used in chromatography? 

Answer: In most cases, low viscosity solvents are used in chromatography. This is because the rate of flow of a solvent is inversely proportional to its viscosity.

Q 3.  Why are developing reagents used in chromatography?

Answer: When the colourless components are separated by chromatography, the separated components on the chromatogram are not visible. To make the components visible in an iodine chamber some suitable reagents(ninhydrin) are sprayed called developing reagents. 

Q 4.  How does a solvent rise against gravity in paper chromatography?

Answer: A solvent rises through a filter paper by capillary action in paper chromatography.

Q 5.  Why is the chromatography technique preferred over other purification techniques?

Answer: It detects even a very small amount of component is present in a mixture and also detects any number of components present in a mixture.

Related Topics 

Distillation Sublimation
Crystallisation Filtration and Decantation
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