Paper Chromatography - Pharmacognosy and Phytochemistry II B. Pharma 5th semester PDF Notes

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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