Paper Chromatography DefinitionBack to Top
Paper chromatography is the type of chromatography where it take place upon the stationary phase of cellulose filter paper when the mobile phase of solvent along with the mixture components run across and separate out due to difference in affinity with water.
Principle of Paper ChromatographyBack to Top
The principle of paper chromatography is about partition chromatography where the components of the mixture are distributed and duly partitioned within the mobile solvent phase. The solvent phase, usually water, is trapped within the pores of the stationary phase of filter paper whereas the mobile phase mixture moves over the paper. This is an adsorption type of chromatography which exist between the solid and liquid phase. The constituents of the coloured mixture get separated because of the difference in affinity existing for each of these mixture components.
Paper Chromatography MethodBack to Top
The paper chromatography applies the difference in polarity of the substances and the limit of affinity with water.
The paper chromatography is usually carried out with the help of different colour ink mixture where the different ink mixture separate out as per the affinity with the water or solvent which works as the mobile phase. The ink mixture is marked a little above from one end of the cellulose filter paper. A line is drawn with the help of a pencil just below the ink mixture mark. A beaker with water is kept ready for the filter paper to be dipped in till the pencil mark.
A stand with clips to hold the paper strip is fixed above the beaker. Once the paper is dipped in it is allowed to soak water. The water absorbs through the paper strip and rises along the ink mixture mark. The ink mixture rises along the mobile water phase and slowly splits into its individual colours. The splitting of these colour components continues till the last of the colours spread along the mobile phase.
Once the separation of colours is complete the paper is unclipped and kept out for drying. The distance of each component and the mobile phase solvent is measured from the pencil mark and used for calculations. The calculation is carried out on the basis of Rf or retention or retardation factor.
The experiment method overall involves the following steps:
- Selecting the right kind of development as the method depends upon the mixture complex, the mobile phase and the stationary phase.
- Selecting the right kind of cellulose filter paper as the size of pore of these filter papers is very essential as the adhesion depends upon the pore size of the stationary phase
- Preparing the right kind of sample mixture as the mobile phase should not react with any of the mixture components
- The sample mixture needs to be spotted at right places on the stationary phase filter paper (usually with a capillary tube)
- The chromatogram is prepared with caution as the mobile phase moves due to capillary action of the cellulose filter paper
- After the chromatogram is ready it is allowed to dry
During the development of chromatogram should be carried out in a room with no wind movement or fan running as it might affect the capillary action. Care should be taken to hold the filter paper after the chromatogram is ready as any smudging of the spots could alter the reading.
Types of Paper ChromatographyBack to Top
There are five types of paper chromatography based mainly on the development of chromatogram.
1. Radial paper chromatography
In this type of paper chromatography the solvent moves from the centre towards the peripheral regions of the filter paper. The radiating mixture component is allowed to spread till all the components have separated out. For precaution the entire system is covered with the help of a Petri dish.
The centre of the paper is allowed to be dipped into the solvent and the coloured components radiates out in concentric circles.
2. Ascending paper chromatography
The chromatogram of this paper chromatography ascends slowly due to the mobile phase movement in a upward direction. The solvent is kept at the bottom of the filter paper or stationary phase with the end of the filter paper dipped in.
The component mixture spot is kept well above the solvent level and is not allowed to touch the spot.
3. Descending paper chromatography
The mobile phase in this type is kept at the top of the chromatogram and the components of the mixture separates out downward due to gravity and capillary action of the filter paper.
4. Two dimensional paper chromatography
The chromatogram in this type of paper chromatography develops at right angle to each other and the filter paper is dipped at right angle once the first chromatogram is complete.
The second chromatogram then develops at right angle to the first one.
5. Ascending and descending paper chromatography
The chromatogram in this type first develops in upward direction and then is reversed and allowed to move again in reverse direction which overall shows a bi-directional movement of the mixture components.
Advantages of Paper ChromatographyBack to Top
- This type of chromatography helps us identifying the affinity of molecules especially when a mixture of polar and non-polar components are taken for separation.
- The separation of various types of amino acids is carried out using this method.
- Paper chromatography very essential to segregate and determine the molecules present in urine sample.
- The forensic science and pharmaceutical industries make use of this method to determine the level of hormones and drug levels in samples.
- The paper chromatography is very useful in evaluating the various salts and metal complexes present in sample compounds.
Paper chromatography, in analytical chemistry, technique for separating dissolved chemical substances by taking advantage of their different rates of migration across sheets of paper. It is an inexpensive but powerful analytical tool that requires very small quantities of material.
The method consists of applying the test solution or sample as a spot near one corner of a sheet of filter paper. The paper is initially impregnated with some suitable solvent to create a stationary liquid phase. An edge of the paper close to the spot is then immersed in another solvent in which the components of the mixture are soluble in varying degrees. The solvent penetrates the paper by capillary action and, in passing over the sample spot, carries along with it the various components of the sample. The components move with the flowing solvent at velocities that are dependent on their solubilities in the stationary and flowing solvents. Separation of the components is brought about if there are differences in their relative solubilities in the two solvents. Before the flowing solvent reaches the farther edge of the paper, both solvents are evaporated, and the location of the separated components is identified, usually by application of reagents that form coloured compounds with the separated substances. The separated components appear as individual spots on the path of the solvent. If the solvent flowing in one direction is not able to separate all the components satisfactorily, the paper may be turned 90° and the process repeated using another solvent. Paper chromatography has become standard practice for the separation of complex mixtures of amino acids, peptides, carbohydrates, steroids, purines, and a long list of simple organic compounds. Inorganic ions can also readily be separated on paper. Comparethin-layer chromatography.