Paper Chromatography – Principle, procedure, Applications

Paper Chromatography - Principle, procedure, Applications

What is Paper Chromatography?

Paper chromatography definition explains that is an inexpensive and powerful analytical technique, which requires a piece of paper or strips serving as an adsorbent in the stationary phase across which a particular solution is allowed to pass.

For the separation of dissolved chemical substances and lipid samples (in particular), paper chromatography is found to be very trustable. This analytical tool employs very few quantities of material.

Principle of Paper Chromatography

Paper chromatography is a form of liquid chromatography where the basic principle involved can be either partition chromatography or adsorption chromatography.

In paper chromatography separation of component is distributed between phases of liquid. Here, one phase of liquid is water that is held amidst the pores of filter paper and the other liquid is the mobile phase that travels along with the filter paper. Separation of the mixture is the result that is obtained from the differences in the affinities towards the water and mobile phase when travelling under capillary action between the pores of the filter paper.

Though in a majority of paper chromatography applications, the principle is based on partition chromatography but sometimes, adsorption chromatography can take place where the stationary phase is the solid surface of the paper and the mobile phase is the liquid phase.

Paper Chromatography procedure

  1. Selection of the ideal type of development: Based on factors such as the complexity of the solvent, mixture, paper, etc. the development type is chosen. Mostly either Radial or Ascending type of paper chromatography is employed because of the easiness they offer while handling and performing which ultimately leads to obtaining the chromatogram faster within a shorter duration of time.
  1. Selection of Filter paper: As per the pores’ size and the sample quality.
  2. Sample preparation: This involves the dissolution of the sample in an ideal solvent that is being utilized in developing the mobile phase.
  3. Sample loading or spotting on the paper: With the help of a capillary tube, micropipette, the sample is spotted on the paper at an accurate position. This promotes the interpretation of the chromatogram more quickly and easily.
  4. Chromatogram development: This is carried by the paper immersion in the mobile phase. The mobile phase crosses over the sample on the paper because of the capillary action of the paper.
  5. Drying of paper and detection of the compound: With the aid of air drier, the paper is dried as soon as the chromatogram is developed. On the chromatogram developed paper, the detecting solution is sprayed and dried thoroughly for the identification of the sample chromatogram spots.

Types of Paper Chromatography

Ascending Paper Chromatography
As per the name, the developing solvent is found to be moving in an upward direction. Here, a sufficient quantity of mobile phase is poured into the development chamber. Sample and reference are spotted on the line drawn a few centimetres from the bottom edge of the paper suspended from a hook or clip at the top.

Descending Paper Chromatography
Here, the solvent front travels down the length of paper suspended from the top inside the developing chamber. The mobile phase is kept in a trough in the upper chamber. The paper with spotting on the line drawn a few centimetres from the top is clamped to the top. Before elution, the jar is covered and equilibrated with the mobile phase vapour.

Ascending – Descending Chromatography
It is a mixed type of chromatography where the solvent first travels upwards on the paper that is folded over a rod and after crossing the rod it moves downwards. 

Horizontal or Circular Paper Chromatography
This allows the separation of sample components in the form of concentric circular zones through the radial movement of the liquid phase. 

Two-Dimensional Chromatography
This helps in resolving substances that have similar Rf values.

Where, Retardation factor (Rf) = The distance travelled by the solute/ distance travelled by the solvent front

Applications of paper chromatography 

In the analysis of different classes of compounds namely:

  • Amino acids and organic acids
  • Alkaloids
  • Polysaccharides
  • Proteins and peptides
  • Natural and artificial pigments
  • Inorganic cations
  • Plant extracts

Applications of paper chromatography in different key areas

Paper chromatography uses are not confined to any particular field. A number of the necessary areas include:

  • Reaction monitoring – The progress of the reaction can be estimated by developing the chromatogram over different time intervals by spotting the reactants.
  • Isolation & Purification – This technique is useful in the purification and isolation of components of mixtures. Here, the separated components on the paper are cut, dissolved in suitable solvents and using spectroscopic methods, their absorption is characterised at specific wavelengths.
  • Foods – Analysis of food colours in synthetic drinks and beverages, ice creams, sweets, etc. Only edible colours are permitted for use, this is why identification and quantification are of utmost importance.
  • Forensics – Provides a basis for identification and comparison against reference standards for drugs and their metabolites. Paper chromatography offers a vital role in the viable analysis of samples that are available in milligrams or microlitre quantities. 
  • Pharmaceuticals – Provides information related to the development of new drugs molecules, reaction completion and progress of manufacturing processes. This process is cost-effective and hence used as an alternative method in monitoring the active ingredients present in the drug forms. Paper chromatography is also applicable in colour identifications of pharmaceutical formulations.

The technique of Paper Chromatography is being extensively used for the last several years and still have preserved their ground associated with the separation of different classes of compounds.


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