Paper Chromatography – Principle, Instrumentation and Application – Pharmacognosy and Phytochemistry II B. Pharma 5th semester PDF Notes




 Paper chromatography


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

– Paper Chromatography

• Principle – Partition chromatography

• Stationary – liquid – usually water, held in the fibres of

• 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

• 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

• After the solvent reaches a suitable height, paper is

• 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

Chromatography – types


• 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


• Development can be continued indefinitely even though the
solvent runs off the paper


Ascending descending

• 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

• 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

Chromatography – Qualitative Analysis

Choice of proper chromatographic

 Depends on the nature of the substance

Choice of filter

– 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

• 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,

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


 Paper chromatography

     Principle
involved is partition

 Types of paper chromatography

    Ascending

    Descending


    Radial/Circular

    2 Dimensional

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