Paper chromatography is one of the easiest methods of
chromatography. It is a method of planar chromatography (stationary
phase is in form of a plane). Paper chromatography follows the basic
principle of chromatography, which states that substances or components
are distributed in between the stationary phase and the mobile phase. It
is an analytical technique, where only a small amount of a sample is
used for separating and identifying its components. Like any other
method of chromatography, paper chromatography has two phases - the
stationary phase and the mobile phase. In paper chromatography, porous
paper serves as the stationary phase, and a solvent, either water or
ethanol serves as the mobile phase. Paper can be a filter paper or any
special paper. However, it is better to use Whatman No.1 filter paper or
chromatography paper, since they are pure and have uniform fibers.
The Requirements
The materials and equipment required for performing paper chromatography are easily available. One can even carry out a simple paper chromatography experiment at home for understanding. Let's look into the requirements for a simple method.
Sample: Sample is a mixture, in which components are to be separated and studied. Sample should be in liquid form for paper chromatography. In schools and colleges, various ink types are used as sample for paper chromatography practical. Sample can be a complex mixture such as blood, polluted water, plant pigments, amino acids and so on.
Developing Chamber: Developing chamber is a container or glassware that can be sealed properly. Sealing is important in order to create an atmosphere saturated with solvent vapor inside the chamber. Shape and size of a chamber differs with reference to a particular experiment.
Chromatography Paper and Solvent: The chromatography paper or the stationary phase is cut based on the shape and size of the developing chamber. Usually, the paper shape is in the form of a rectangle, for example, 10-15 cm length and 4-5 cm width. The paper size should be such that it can be suspended without touching the sides of the developing chamber. The solvent or the mobile phase can be water, alcohol or a mixture of solvents. It should be added into the developing chamber up to a certain level in the bottom.
Other requirements include pencil, ruler, tape, scissors, capillary tube, calculator, notepad, etc.
The Procedure
The procedure for paper chromatography method is quite simple as compared to other methods of chromatography. In order to have a good separation, one has to follow the following procedure carefully.
Cut chromatography paper into rectangular strips and mark a line on the paper at about 2-3 cm from the bottom. Marking should always be done with pencil, otherwise the solvent will carry away the markings and there will be unnecessary contamination. Label the paper with its corresponding sample to avoid confusion. Clean your hands before handling chromatography paper.
With the help of capillary tube, take sample and place a spot on the starting line (line drawn in first step). While loading the sample, care should always be taken to avoid spilling.
Now, place the chromatography paper in the developing chamber, which contains the solvent or the mobile phase. While placing the paper, it is important that the solvent level doesn't reach the starting line or the sample spots. Make sure that the paper is suspended without touching the sides of the chamber; otherwise it will lead to poor separation. Seal the chamber in a proper manner.
The solvent rises up the paper or the stationary phase by capillary action and dissolves the sample. The components of the sample move along with the solvent in upward direction. The speed of movement depends on two factors, the attraction of the solvent molecules to the paper and the differential absorption of the solute components in the solvent. The more attraction or affinity a component has, the slower it moves up and vice-versa. Thus, different components cover different distances within the same time.
Check if the solvent has reached near the top level of chromatography paper. Remove, the paper when it reaches the top and mark the level with pencil. This level or height is called the "solvent front". Examine the different spots of varied colors. Each spot represents a specific component of the sample. Sometimes, spots are not distinct to locate. In such condition, the paper should be viewed using UV light, ninhydrin or iodine vapors. Carefully circle the spots with pencil.
Retention Factor (Rƒ): How to Calculate it?
Retention Factor (Rƒ) is the ratio of the distance traveled by the substance to the distance traveled by the solvent. Rƒ value is always between 0 and 1 and has no unit. The Rƒ value of unknown compounds is compared with the Rƒ value table of known compounds for identification.
Measure the distances of the solvent front and also the distances traveled by the components (take the center point of the spots). Calculate the retention factors of the components by using the relation, Rƒ = distance traveled by the substance/distance traveled by the solvent. Since the distance covered by the components varies, the resulted Rƒ values will also vary. Compare and match the Rƒ values of the unknown components with Rƒ value table and identify the substances present in the particular sample.
As we have seen, this type of chromatography is easy to carry out. It can be an ascending chromatography or a descending chromatography, based on the direction of running a chromatogram. Time required for running a chromatogram varies from one hour to several hours. In general, it is used in clinical research, hospitals, manufacturing industries and forensic science studies. The disadvantage of paper chromatography is that it is not a reliable method for separating complex mixtures
Read more at Buzzle: http://www.buzzle.com/articles/paper-chromatography.html
The Requirements
The materials and equipment required for performing paper chromatography are easily available. One can even carry out a simple paper chromatography experiment at home for understanding. Let's look into the requirements for a simple method.
Sample: Sample is a mixture, in which components are to be separated and studied. Sample should be in liquid form for paper chromatography. In schools and colleges, various ink types are used as sample for paper chromatography practical. Sample can be a complex mixture such as blood, polluted water, plant pigments, amino acids and so on.
Developing Chamber: Developing chamber is a container or glassware that can be sealed properly. Sealing is important in order to create an atmosphere saturated with solvent vapor inside the chamber. Shape and size of a chamber differs with reference to a particular experiment.
Chromatography Paper and Solvent: The chromatography paper or the stationary phase is cut based on the shape and size of the developing chamber. Usually, the paper shape is in the form of a rectangle, for example, 10-15 cm length and 4-5 cm width. The paper size should be such that it can be suspended without touching the sides of the developing chamber. The solvent or the mobile phase can be water, alcohol or a mixture of solvents. It should be added into the developing chamber up to a certain level in the bottom.
Other requirements include pencil, ruler, tape, scissors, capillary tube, calculator, notepad, etc.
The Procedure
The procedure for paper chromatography method is quite simple as compared to other methods of chromatography. In order to have a good separation, one has to follow the following procedure carefully.
Cut chromatography paper into rectangular strips and mark a line on the paper at about 2-3 cm from the bottom. Marking should always be done with pencil, otherwise the solvent will carry away the markings and there will be unnecessary contamination. Label the paper with its corresponding sample to avoid confusion. Clean your hands before handling chromatography paper.
With the help of capillary tube, take sample and place a spot on the starting line (line drawn in first step). While loading the sample, care should always be taken to avoid spilling.
Now, place the chromatography paper in the developing chamber, which contains the solvent or the mobile phase. While placing the paper, it is important that the solvent level doesn't reach the starting line or the sample spots. Make sure that the paper is suspended without touching the sides of the chamber; otherwise it will lead to poor separation. Seal the chamber in a proper manner.
The solvent rises up the paper or the stationary phase by capillary action and dissolves the sample. The components of the sample move along with the solvent in upward direction. The speed of movement depends on two factors, the attraction of the solvent molecules to the paper and the differential absorption of the solute components in the solvent. The more attraction or affinity a component has, the slower it moves up and vice-versa. Thus, different components cover different distances within the same time.
Check if the solvent has reached near the top level of chromatography paper. Remove, the paper when it reaches the top and mark the level with pencil. This level or height is called the "solvent front". Examine the different spots of varied colors. Each spot represents a specific component of the sample. Sometimes, spots are not distinct to locate. In such condition, the paper should be viewed using UV light, ninhydrin or iodine vapors. Carefully circle the spots with pencil.
Retention Factor (Rƒ): How to Calculate it?
Retention Factor (Rƒ) is the ratio of the distance traveled by the substance to the distance traveled by the solvent. Rƒ value is always between 0 and 1 and has no unit. The Rƒ value of unknown compounds is compared with the Rƒ value table of known compounds for identification.
Measure the distances of the solvent front and also the distances traveled by the components (take the center point of the spots). Calculate the retention factors of the components by using the relation, Rƒ = distance traveled by the substance/distance traveled by the solvent. Since the distance covered by the components varies, the resulted Rƒ values will also vary. Compare and match the Rƒ values of the unknown components with Rƒ value table and identify the substances present in the particular sample.
As we have seen, this type of chromatography is easy to carry out. It can be an ascending chromatography or a descending chromatography, based on the direction of running a chromatogram. Time required for running a chromatogram varies from one hour to several hours. In general, it is used in clinical research, hospitals, manufacturing industries and forensic science studies. The disadvantage of paper chromatography is that it is not a reliable method for separating complex mixtures
Read more at Buzzle: http://www.buzzle.com/articles/paper-chromatography.html
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