Chromatography
Content
Chromatography
Paper chromatography
Objective
Paper Chromatography
• At the end of this
lecture, student will be able to
Discuss the principle involved in paper chromatography
Explain the procedure involved in paper chromatography
Explain the various types of paper chromatography
Chromatography
– Paper Chromatography
• Principle – Partition chromatography
• Stationary – liquid – usually water, held in the fibres of
paper
• Moving liquid – mobile phase
• Components of mixture to be separated – migrate at
different rates and appear as spots
• A drop of test solution applied as a small spot, spot
dried
• Paper kept in closed chamber
• Edge of the filter paper dipped into the solvent
• Paper gets the solvent through capillary action
• Solvent reaches the spot (mixture)
• Various substances are moved by the solvent at various
speeds
• After the solvent reaches a suitable height, paper is
dried
• Visualised by suitable reagents – visualizing reagents
• Movement of substances relative to the solvent is
expressed as Rf
• Rf = distance travelled by the solute from the origin
line/distance travelled by the solvent from the origin line
Rf depends on
• Solvent
• Medium used for separation
• Nature of the mixture
• Temperature
• Size of the vessel
Paper
Chromatography – types
Descending
chromatography
• Development of paper is done by allowing the solvent to
travel down the paper
• Well sealed glass tank of suitable size and shape,
provided with a trough for the mobile phase in the upper portion
• Paper with sample spotted is inserted
Advantages
• Development can be continued indefinitely even though the
solvent runs off the paper
Ascending
chromatography
Ascending descending
chromatography
• Upper part of ascending chromatography is folded over a
glass rod
• Ascending development changes over into descending after
crossing the glass rod
Radial or circular
paper chromatography
• Radial development
Two dimensional
chromatography
• Square or rectangular paper is used
• Sample applied in one corner
• Development is done
• Second development is performed at right angle to the
direction of the first run
• Identical solvent systems or two different solvent systems
Paper
Chromatography – Qualitative Analysis
Choice of proper chromatographic
technique
Depends on the nature of the substance
Choice of filter
paper
– Qualitative or quantitative, Analytical or preparative
– Hydrophilic or lipophilic, Neutral or charged species
Various type of
Whatmann filter paper available
– Whatmann paper – 99% α cellulose and rest is mineral
– For polar substances – exchange capacity increased by
carboxyl content (1.4 %) by partial oxidation
– Capillarity of the paper can be increased by partial
hydrolysis – soaking filter paper for 24 hrs in 7% hydrochloric acid and
washing with water and ethanol
Modified papers
• Carboxyl papers – cationic separation
• Acetylated papers – RP chromatography of lipophilic
substances
• Kieselghur papers, Alumina papers, Zirconia papers, Silica
papers – Separation of low polarity substances
• Ion exchange papers – ion exchange paper chromatography
Developing solvents
• Rf – 0.05 – 0.85
• Difference 0.05
• Distribution ratio of the components should be independent
of concentration
• Should not undergo chemical reaction with any components
or paper
• Should not interfere with the detection of spots
• Composition should not alter any time
Solvents of
increasing polarity
• n-hexane, cylcohexane, carbon tetrachloride, Benzene,
Toluene,
Trichloroethylene, Diethylether, Cholrofrom, Ethylacetate, N
butanol, n propanol, Acetone, Ethanol, Methanol, Water
Multiple developments
• Spot resolution increased
• Two solvent systems are used
• Useful in separating closely related compounds
Sample preparation
• Depends on the sample
Summary
Paper chromatography
Principle
involved is partition
Types of paper chromatography
Ascending
Descending
Ascending-Descending
Radial/Circular
2 Dimensional
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