In analytical chemistry, paper chromatography is defined as a technique for separating the dissolved chemical substances by taking advantage of their varied rates of migration across sheets of paper. It is an inexpensive method but a powerful analytical tool that needs very small quantities of the material.
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About Paper Chromatography Method
This method contains applying the sample or test solution as a spot near one corner of a sheet of the filter paper. Initially, this paper is impregnated with some quantity of suitable solvent to create a stationary liquid phase. An edge of the paper, which is close to the test spot, is then immersed in the other solvent, where the components of the mixture become soluble in differential degrees. The solvent also penetrates the paper by capillary action and, in passing over the sample spot, carries along with it the different components of the sample. These components move along with the flowing solvent at velocities, which are dependent on their solubilities present in the flowing and stationary solvents.
Separation of these components is brought if there are differences in the relative solubilities present in the two solvents. Prior to the flowing solvent reaches the farther paper edge, both the solvents are evaporated, and the location of the separated component can be identified, generally by the application of reagents that produce colored compounds with the separated substances. Then, the separated components appear as individual spots on the solvent’s path. If the solvent that is flowing in one direction is not able to separate all the components in a satisfactory manner, the paper can be turned 90°, and the process will be repeated using the other solvent.
The paper chromatography method has become a standard practice for the separation of complex mixtures of peptides, amino acids, steroids, carbohydrates, purines, and a long list of simple organic compounds. Also, the inorganic ions can readily be separated on paper.
Pigments and Polarity
Paper chromatography is a method that is used for testing the purity of compounds and the identification of substances. The paper chromatography method is a useful technique due to the reason it is relatively quick and needs only small quantities of material. Separations in the paper chromatography method involve the partition principle. In the method of paper chromatography, the substances are distributed between a mobile phase and a stationary phase.
The stationary phase is defined as the water trapped between the paper’s cellulose fibers. The mobile phase is defined as a developing solution, which travels up the stationary phase by carrying the samples with it. The sample components will readily separate as per how strongly they adsorb onto the stationary phase vs. how readily they dissolve in the mobile phase.
When a sample of colored chemical is placed on a filter paper, the colors get separated from the sample simply by placing one end of the paper in a solvent. Then, the solvent diffuses up the paper by dissolving the different molecules in the sample as per the polarities of the molecules and the solvent. If the sample has more than one color, it means it must contain more than one kind of molecule. Because of the various chemical structures of every kind of molecule, the chances are high that each molecule will have at least a slightly different polarity by giving each molecule a variable solubility in the solvent.
The unequal solubility causes the different color molecules to leave the solution at various places as the solvent continues to move the paper up. The more soluble a molecule, the higher it will migrate up the paper. The chemical will not dissolve in a very polar solvent if it is very non-polar. This is similar to a very polar chemical solvent and a very non-polar solvent.
It is also important to make a note that when using water (which is a more polar substance) as a solvent, when the color is more polar, it will rise higher on the papers.
Types of Paper Chromatography
Let us discuss the types of paper chromatography given below.
Chromatogram development is done by allowing the solvent to travel down the paper. In this case, the mobile phase is placed in the solvent holder at the top. And, the spot is kept at the top, and solvent flows from top to bottom of the paper.
Ascending Paper Chromatography
In the ascending paper chromatography case, the solvent travels up the chromatographic paper. Both the ascending and descending paper chromatography are used for the separation of both organic and inorganic substances. The solvent and sample move upward.
This is the hybrid type of both of the techniques given above. The upper part of the ascending chromatography is folded over a rod to allow the paper to become descending after the rod crossing.
In radial chromatography or circular chromatography, a circular filter paper can be taken, and the sample is deposited at the paper’s center. After the spot gets dried, the filter paper can be tied horizontally on a Petri dish that contains a solvent so that the paper’s wick is dipped in the solvent. The solvent then rises through the wick, and the components get separated into concentric rings.
In this particular technique, rectangular or square paper can be used. In this case, the sample is applied to one of the corners, and the development can be performed at a right angle to the direction of the first run.