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Q8.18 (c):
Give a brief description of the principles of the following techniques taking an example in each case. (c) Chromatography

Solution :

Theoretical Foundation of Chromatography

Chromatography is a physical method of separation in which the components to be separated are distributed between two phases: a stationary phase and a mobile phase. The stationary phase can be a solid or a liquid supported on a solid, while the mobile phase can be a liquid or a gas [Per IUPAC definition of analytical separation techniques].

Step 1: The Underlying Principle

The separation is governed by the differential migration of individual components of a mixture. As the mobile phase moves over the stationary phase, the components move at different rates based on their adsorption affinity (for Adsorption Chromatography) or solubility (for Partition Chromatography) toward the stationary phase. Those components with a higher affinity for the stationary phase move more slowly, while those with a higher affinity for the mobile phase move more rapidly [By the Principle of Differential Distribution].

Step 2: Graphical Representation of Paper Chromatography

Component A Component B Solvent Front

Step 3: Example - Separation of Plant Pigments

Consider the separation of chlorophyll and carotenoids from a plant extract using Paper Chromatography:

  • Stationary Phase: A strip of chromatographic paper, which contains water trapped in the cellulose fibers.
  • Mobile Phase: A suitable solvent mixture (e.g., petroleum ether/acetone).
  • Process: The plant extract is spotted on the base of the paper. As the solvent rises through capillary action [By Capillary Action Principle], the pigments partition between the stationary water phase and the mobile organic phase based on their polarity.

Step 4: Analytical Quantification ($R_f$ Value)

The efficiency and identification of components are defined by the Retardation Factor ($R_f$), calculated as:

$R_f = \frac{\text{Distance travelled by the substance from the baseline}}{\text{Distance travelled by the solvent front from the baseline}}$

Since the distance travelled by any substance is always less than or equal to the distance travelled by the solvent, $0 \le R_f \le 1$.

Feature Description
Stationary Phase Fixed solid or liquid layer.
Mobile Phase Flowing fluid (liquid/gas).
Separation Criterion Differential affinity/solubility.

Final Solution: Chromatography is an analytical technique relying on the differential partitioning of mixture components between a stationary and a mobile phase, allowing for high-resolution separation based on specific molecular interactions.


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