Over the last century, the procedure of chromatography has given important participation in the field of molecular characterization in addition to the purification method. The quantitative chromatographic examination is precious in the pharmaceutical industry for research as well as for quality control. Likewise, biotechnology industries widely use chromatographic procedures and in many cases, alternative techniques are not possible. As an example, chromatographic techniques have been applied for the separation of stereoisomers that are very similar in structure and properties. The growth of biotherapeutics would have been impossible without chromatography-based purification strategies. The reasons for the popularity of these techniques lie in their unique sensitivity, flexibility, and scalability. Moreover, chromatographic separations are comparatively fast and simple and afford important ease of operation compared with the other instrumental techniques.
A method of partition chromatography using filter paper strips as the carrier or inert support.
The factor governing the separation of compounds of solutes on filter paper is the partition between two immiscible phases.
One is normally water adsorbed on cellulose fibers in the paper (stationary phase).
The second is the organic solvent flows past the sample on the paper (stationary phase)
Partition occurs between the mobile phase and the stationary aqueous phase bound by cellulose. The isolation depends on the separation coefficient of the solute.
In paper chromatography, substances are distributed between the paper as a stationary phase and a suitable mobile phase. The chromatography paper has an extremely consistent composition and thickness. It is made from cotton cellulose and has a low content of additives. For the separation of the compound, a discreet spot is applied to the paper. The components of the mixture partition between the two phases: aqueous phase consisting of water adsorbed in the pores of the chromatography paper (stationary phase) and solvent (mobile phase) that moves over the paper due to capillary action. The mixtures in the compound get separated due to variations in their affinity towards the two phases. An additional factor could be the adsorption of the elements of the mixture by the filter paper. Paper chromatography is a useful technique for the separation and analysis of molecules because it is quick and requires small quantities of material.
The material for paper chromatography consists of a vapor-tight glass chamber with inlets for the addition of solvents in addition to supports for solvent troughs and chromatography paper.
In Paper Chromatography, a volatile solvent is used to carry the elements of the sample along with the paper. This is called the developing solvent. The choice of solvent depends on the chemical nature of the materials to be analyzed and needs to be optimized for every type of mixture. Usually, solvents such as ethanol, methanol, isopropanol, acetone, diethyl ether, chloroform, methylene chloride, formamide, acetic acid, ammonia, cyclohexane, benzene can be used alone or as a mixture as the mobile phase.
The common procedure in paper chromatography is as follows:
• Preparation of Chromatographic Chamber:
The procedure for paper chromatography starts with equilibration of the chamber with the solvent vapors. The pre-saturation of the chromatography chamber is carried out well in advance before the development. The saturation of the chamber with the solvent may be facilitated by lining the chamber with filter papers wetted with the developing solvent.
• Preparation of Paper:
The paper may be used as such, where water adsorbed in the fibers acts as the stationary phase. Alternatively, the paper may be impregnated with a polar solvent other than water, such as buffer, salt solution, methanol, or glycol. Paper may also be modified with silica, diatomaceous earth, and alumina. Such types of modification papers would illustrate the adsorbing capacity of the respective modifiers. Paper may be made hydrophobic by creating with silicone or other non-polar solvents. Paper may also be acetylated so that it becomes more hydrophobic and holds a similar hydrophobic element.
• Application of Samples:
The compound to be examined is dissolved in a suitable solvent. A fine line is drawn on the filter paper so that it is a few centimeters away from the level of developing solvent. The dissolved samples are applied to the spots (diameter: 6-10mm) that lie at least 3 cm apart on the line drawn on the chromatography paper. The sample application is usually performed using micropipettes. The sample application may be repeated after the solvent volatilizes from the previous spot, to concentrate the sample.
After preparing the chamber and spotting the samples, the paper is ready for development.
• Development of the Chromatogram
Depending upon the type of development, paper chromatography can be classified as:
• Ascending Paper Chromatography
Here the developing solvent is poured at the bottom of the chromatography chamber. The solvent moves in an upward direction due to capillary action.
• Descending Paper Chromatography
Here the solvent is allowed to travel down the paper. The mobile phase is kept in a trough placed at the top of the chromatography chamber and it is permitted to descend over the chromatography paper.
• Ascending-Descending Paper Chromatography
It is the combination of both the above technique. The upper portion of the ascending chromatographic sheet is folded on a rod while allowing the descent of paper after crossing the rod.
• Radial Paper Chromatography
It is also called Circular Chromatography. Here a circular filter paper is taken and the sample is applied at the center of the paper. After drying the spot the filter paper is tied horizontally on a shallow chamber containing solvent. A wick of the paper is dipped inside the solvent. The solvent rises through the wick and the elements get separated in the form of a concentric circular zone.
• Two-Dimensional Paper Chromatography
In this technique, square or rectangular paper is used. Here the sample is applied at one corner and development is made at a right angle to the direction of the first run, using a different solvent. This helps better the separation of the elements of the mixture.
Examination of results
After the development, any strongly retained compound can be visualized as colored spots on the chromatogram. Colorless molecules can be visualized by heating the paper in a hot air oven so that the material in the spots char and can be seen as black spots.
The paper may be sprinkled with a solution of sulphuric acid before heating for better charring. Fluorescent materials can be visualized with ultraviolet light. Reagents specific for certain components may be sprayed on to make spots colored. Radioactive spots can be located with a detector, or the X-ray film may be exposed to the chromatogram.
Applications of Paper Chromatography
• Paper chromatography has been used for the separation of mixtures having polar and non-polar compounds such as amino acids, hormones, drugs, and other biochemicals.
• It has also been used for the evaluation of inorganic compounds.
• Although paper chromatography has become very popular due to its simplicity.
• It suffers from various limitations such as long development time, poor separation, low accuracy in quantitative determinations, and low reproducibility.