Paper chromatography is a method of planar chromatography (stationary phase is in form of a plane). It follows the basic principle of this technique, which states that substances or components are distributed in between the stationary and the mobile phase. It is an analytical method, wherein only a small amount of a sample is used for separating and identifying its components. In this process, a porous paper (filter or any special paper) serves as the stationary phase, and a solvent - either water or ethanol serves as the mobile phase. However, it is better to use Whatman No.1 filter paper or chromatography paper, since they have uniform fibers.
The materials and equipment required for performing this technique are easily available. One can even carry out a simple paper chromatography experiment at home.
Sample: It is a mixture, in which components are to be separated and studied. Sample should be in liquid form. In schools and colleges, various ink types are used as samples for paper chromatography practical. It also can be a complex mixture such as blood, polluted water, plant pigments, amino acids, etc.
Developing Chamber: It is a container or glassware, which 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 stationary phase (chromatography paper) 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 in length and 4-5 cm in width. Its 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.
- Cut the paper into rectangular strips, and mark a line on it 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. Always clean your hands before it.
- With the help of capillary tube, take a 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 a poor separation. Seal the chamber in a proper manner.
- The solvent rises upwards on the stationary phase by capillary action, and dissolves the sample. The sample components move along with the solvent in the 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 the 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, the spots are not distinct to locate. In such cases, the paper should be viewed using UV light, ninhydrin, or iodine vapors. Carefully circle the spots with a pencil.
Retention Factor (Rƒ): Calculations
Retention Factor (Rƒ) is the ratio of the distance traveled by the substance, to the distance traveled by the solvent. This 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 values of the unknown components with the table, and identify the substances present in the particular sample.
This technique can be an ascending or a descending one, 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.