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

Paper Chromatography

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


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

Principles of Paper Chromatography:

Paper chromatography is a separation technique based on the differential migration of solutes in a mixture through a porous paper medium. It relies on the principles of selective partitioning and capillary action. The main principles involved in paper chromatography are as follows:

  1. Selective Partitioning: In paper chromatography, a stationary phase (the paper) and a mobile phase (the solvent) are used. The solutes in the mixture have varying affinities for the stationary and mobile phases. Some solutes have a stronger affinity for the paper (stationary phase), while others have a stronger affinity for the solvent (mobile phase).
  2. Capillary Action: Capillary action is the ability of a liquid to flow in narrow spaces without the assistance of external forces. The paper’s porous nature allows the solvent to move upward through the paper due to capillary action. As the solvent moves, it carries the solutes along with it.
  3. Separation by Partitioning: As the solvent travels up the paper, it carries the solutes with it. Different solutes move at different rates based on their partition coefficients, which determine their affinity for the paper relative to the solvent. This leads to the separation of the components of the mixture.

• 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

Types of Paper Chromatography


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


• 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


 Paper chromatography

     Principle involved is partition

 Types of paper chromatography

    Ascending

    Descending

    Ascending-Descending

    Radial/Circular

    2 Dimensional

Use in Pharmacognosy:

Paper chromatography is extensively used in pharmacognosy for the separation and identification of plant constituents, such as alkaloids, flavonoids, and essential oils. It is particularly useful in determining the purity of natural extracts and identifying specific compounds present in medicinal plants. This information is valuable in quality control, formulation, and the standardization of herbal medicines.

FAQ (Frequently Asked Questions):

  1. Q1: What is the most critical factor affecting the separation in paper chromatography? A1: The choice of solvent system is a critical factor. The solvent’s polarity and its interaction with the stationary phase have a significant impact on separation.
  2. Q2: Can paper chromatography be used for quantitative analysis? A2: While paper chromatography is excellent for qualitative analysis and separation, it is less precise for quantitative analysis due to factors like irregular sample application and variable solvent flow rates.
  3. Q3: How is the Rf value useful in paper chromatography? A3: The Rf value helps in identifying and comparing the migration of different compounds on the paper. It’s a useful tool for the qualitative assessment of the separated components.
  4. Q4: Are there any safety precautions to consider when performing paper chromatography? A4: Yes, safety precautions include working in a well-ventilated area, avoiding exposure to harmful solvents, and handling chemicals with care. Always follow safety guidelines and use appropriate protective equipment.
  5. Q5: What is the purpose of the sample application line in paper chromatography? A5: The sample application line is where the mixture is applied. It ensures that the sample components start at a common point, allowing for accurate separation and comparison.

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