Paper Chromatography Principle and Application

Paper chromatography is a type of liquid chromatography in which the basic principle can be partition or adsorption chromatography.

Paper chromatography is a low-cost and powerful analytical technique that uses a piece of paper or strips as an adsorbent in the stationary phase through which a specific solution is allowed to pass. Paper chromatography method has been found to be very reliable for the separation of dissolved chemical substances and lipid samples (in particular). Paper chromatography method makes use of a small amount of material.

Paper Chromatography Principle

Paper chromatography method is a type of liquid chromatography in which the basic principle can be partition or adsorption chromatography.

Separation of components is distributed between liquid phases in paper chromatography. In this case, one liquid phase is water, which is held within the pores of the filter paper, and the other liquid phase is the mobile phase, which travels with the filter paper. The separation of the mixture is the result of differences in the affinities of the mixture towards the water and mobile phase when it travels under capillary action between the pores of the filter paper.

Though partition chromatography is used in the majority of paper chromatography method applications, adsorption chromatography can occur when the stationary phase is the solid surface of the paper and the mobile phase is the liquid phase.

Types of Paper Chromatography

Paper Chromatography Principle and Application

Paper Chromatography Principle and Application

  1. Ascending Paper Chromatography: According to the name, the developing solvent is found to be moving upward. A sufficient amount of mobile phase is poured into the development chamber at this point. The sample and reference are positioned on a line drawn a few centimetres from the bottom edge of the paper, which is suspended from a hook or clip at the top.
  2. Descending Paper Chromatography: The solvent front descends the length of paper suspended from the top of the developing chamber. The mobile phase is kept in an upper chamber trough. The paper is clamped to the top with spotting on the line drawn a few centimetres from the top. The jar is covered and equilibrated with the mobile phase vapour prior to elution.
  3. Ascending Descending Chromatography: It is a mixed type of chromatography in which the solvent first travels upwards on the paper that is folded over a rod before moving downwards after crossing the rod.
  4. Radial mode Paper Chromatography: The solvent moves from the centre (mid-point) to the periphery of the circular chromatography paper in this case. For the development of the chromatogram, the entire system is kept in a covered Petri dish. The wick in the centre of the paper dips into the mobile phase in a petri dish, causing the solvent to drain onto the paper and move the sample radially, forming concentric rings of different compounds.
  5. Two-dimensional chromatography: The chromatogram develops in two directions at right angles in this case. In this mode, samples are spotted to one corner of rectangular paper and allowed to develop for the first time. For the second chromatogram, the paper is immersed in the mobile phase at a right angle to the previous development.

Paper Chromatography Instrumentation

i) Stationary phase

The most common type of paper is made of highly purified cellulose. Cellulose, a glucose homopolysaccharide, contains thousands of anhydro-glucose units linked by oxygen atoms. However, many of the hydroxyl groups in glucose are partially oxidised during the manufacturing process. Typically, the oxidation products are aldchyde, ketone, or carboxyl functional groups.

ii) Mobile phase

The mobile phase, which is less polar, flows over the polar stationary phase. The mobile phase may not be necessarily immiscible with water if water is being used as the stationary phase. This is because the stationary phase water is very tightly held by cellulose and will not mix with the mobile phase on this account. The mobile phase is usually a mixture of various solvents such as alcohols, acids, esters, ketones, phenols, amines, and hydrocarbons etc. The solvents are selected in such a way that the resolution of sample components is satisfactory.

Partition coefficient of substances to be analysed should range from 1-100 in favour of the aqueous phase. The solvent should be removable from the paper and to this effect its boiling point should ideally be less than 200oC. The solvent should be stable. It should not become oxidized when spread over the paper. Some solvents, for example phenolic solvents, react with small quantities of copper present in the paper and get oxidized to produce brown or black tarry material.

iii) Developing Chamber

The chromatographic chambers are made of a variety of materials, including glass, plastic, and stainless steel. Glass tanks are the most popular. They are available in a variety of dimensional sizes based on the length of the paper and the type of development. The atmosphere in the chamber should be saturated with solvent vapour.

iv) Detecting agents

There are several detection methods available. If the sample components are coloured, the analysis is simplified because the component’s distinctive colour identifies it. When the components are colourless, they can be coloured by spraying the paper with color-producing reagents. A good example is the detection of amino acids. Ninhydrin reagent, when applied to paper, reacts with amines and amino acids to produce a blue or purple colour.

Steps in Paper Chromatography

Paper chromatography method involves applying a sample mixture to a piece of filter paper, immersing the paper’s edge in a solvent, and allowing the solvent to move up the paper via capillary action.

  1. Solid Support Selection: Fine cellulose paper with defined porosity, high resolution, negligible sample diffusion, and a good solvent movement rate.
  2. Mobile Phase Selection: Depending on the analyte, different combinations of organic and inorganic solvents may be used. Example. Butanol: Acetic acid is a type of acid that is found in nature. Water (12:3:5) is an appropriate solvent for separating amino acids.
  3. Tank Saturation: To improve resolution, the inner wall of the tank is wrapped in filter paper before the solvent is added.
  4. Loading Samples: When using a solid sample, it is dissolved in a suitable solvent. To prevent diffusion, a spot of sample (2-20ul) is added to the base line with a micropipette and air dried.
  5. Development: The sample loaded filter paper is carefully dipped into the solvent at a height of no more than 1 cm and waited until the solvent front reaches near the edge of the paper.
  6. Chromatogram Drying: Following development, the solvent front is marked and dried in a dry cabinet, oven or air dryer.
  7. Detection: Colorless analytes are detected through staining with reagents such as iodine vapour, ninhydrin, and others. Analytes that have been radiolabeled or fluorescently labelled are detected by measuring radioactivity and fluorescence, respectively.

Paper Chromatography Rf Value

For a given compound, the distance travelled relative to the solvent is constant as long as other parameters such as the type of paper and the exact composition of the solvent are constant. 

Paper chromatography Rf value is the distance travelled relative to the solvent. Thus, in order to obtain a measure of the extent of movement of a component in a paper chromatography method, the “paper chromatography Rf value” for each separated component in the developed chromatogram is calculated. 

Paper chromatography Rf value is a number that represents the component’s distance from the application point.

Application of Paper Chromatography

1) The technique of paper chromatography method has revolutionized biochemistry where difficult analyses with vanishingly small sample volumes are legion. 

2) The control of purity of pharmaceuticals, the detection of adulterants and contaminants in foods and drinks, the study of ripening and fermentation, the detection of drugs and dopes in animals and humans, the analyses of cosmetics, and to top it all, the analyses of the reaction mixtures in biochemical labs are all performed routinely with paper chromatography method.

Advantages of Paper Chromatography

1) It requires fever quantitative material. 

2) Separation of compounds in a short time. 

3) Analysis requires a low amount of sample. 

4) Easy to handle and setup. 

5) The less sample quantity required for the analysis. 

6) Cost-effective method. 

Disadvantages of Paper Chromatography

1) Volatile substances cannot be separated using paper chromatography method. 

2) Paper chromatography method not compatible with large amounts of sample. 

3) Quantitative analysis is not useful in paper chromatography method. 

4) Paper chromatography method cannot be separated complex mixture. 

5) Compared to the HPLC, HPTLC, paper chromatography method has less accuracy. Data cannot be saved for long periods