Chromatography and it’s
Applications
Learning Objectives
Understand the basic principles of chromatography
Differentiate between various types of chromatography
Learn the working of Thin Layer Chromatography (TLC)
Explore applications of TLC
Introduction to Chromatography
Origin: Greek words 'chroma' (color) and 'graphein' (to write)
A separation technique used to separate individual components in a mixture
Compounds separate based on their differing affinities for the stationary and mobile phases
The stationary phase, which stays fixed in place
like the silica coating on a TLC plate.
The mobile phase, which is the solvent
moving through the stationary phase.
Classification of Chromatography
Based on the physical state of mobile and stationary phase:
Gas Chromatography (GC), Liquid Chromatography (LC)
Based on mechanism of separation:
Ion-exchange chromatography, partition, and adsorption
Based on chromatographic techniques:
Paper Chromatography, Column Chromatography
Thin Layer Chromatography (TLC)
• Stationary phase: silica/alumina on glass/plastic
• Mobile phase: solvent or solvent mixture
• Solvent carries components upward by capillary action
Empty TLC plate
• The mechanism of separation in TLC is called adsorption
chromatography.
TLC – Procedure Overview
1. Prepare TLC plate
2. Spot the sample
3. Preparation of TLC chamber
4. Dry the plate and visualize spots
5. Calculate Rf values
Thin Layer Chromatography (TLC)
Figures a) Prepared samples, b) TLC chamber made with a beaker and watch glass, c)
Adding eluent to a TLC chamber, d) Prepared plate.
Thin Layer Chromatography (TLC)
Figures a) Tipping the vial to withdraw liquid into the spotter, b) Spotting samples onto a TLC p
c) Excess sample drained on a paper towel
Thin Layer Chromatography (TLC)
TLC Process: Visual Demonstration of Separation
Thin Layer Chromatography (TLC)
UV Chamber Visual under UV light
Thin Layer Chromatography (TLC)
a) An iodine chamber using silica gel b-d) Inserting a plate into the chamber and jostling
e) Developed TLC plate with iodine
Retention Factor (Rf Value)
• Formula:
Rf = Distance traveled by compound /
Distance traveled by solvent front
• Each component have different Rf
values
Applications of TLC
• Purity Check
• Monitoring Reaction Progress
• Identification of Compounds
• Detection of Functional Groups
Advantages and Limitations of TLC
Advantages: Limitations:
• Not suitable for volatile compounds
• Simple and quick
• Less precise than GC/HPLC
• Cost-effective
• Requires small sample size
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