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A small concentrated spot of solution that contains the sample of the solute is applied to a strip of chromatography paper about two centimeters away from the base of the plate, usually using a capillary tube for maximum precision. This sample is absorbed onto the paper and may form interactions with it. Any substance that reacts or bonds with the paper cannot be measured using this technique. The paper is then dipped in to a suitable solvent, such as ethanol or water, taking care that the spot is above the surface of the solvent, and placed in a sealed container. The solvent moves up the paper by capillary action, which occurs as a result of the attraction of the solvent molecules to the paper,also this can be explained as differential adsorption of the solute components into the solvent. As the solvent rises through the paper it meets and dissolves the sample mixture, which will then travel up the paper with the solvent solute sample. Different compounds in the sample mixture travel at different rates due to differences in solubility in the solvent, and due to differences in their attraction to the fibers in the paper. Paper chromatography takes anywhere from several minutes to several hours. In some cases, paper chromatography does not separate pigments completely; this occurs when two substances appear to have the same values in a particular solvent. In these cases, two-way chromatography is used to separate the multiple-pigment spots.

Ascending Chromatography

In this method, the solvent is in pool at the bottom of the vessel in which the paper is supported.It rises up the paper by capillary action against the force of gravity.

Descending Chromatography

In this method, the solvent is kept in a trough at the top of the chamber and is allowed to flow down the paper. The liquid moves down by capillary action as well as by the gravitational force. In this case, the flow is more rapid as compared to the ascending method. Because of this rapid speed, the chromatography is completed in a comparatively shorter time. The apparatus needed for this case is more sophisticated. The developing solvent is placed in a trough at the top which is usually made up of an inert material. The paper is then suspended in the solvent. Substances that cannot be separated by ascending method, can be separated by the above descending method.

Analysis

After development, the spots corresponding to different compounds may be located by their color, ultraviolet light, ninhydrin (Triketohydrindane hydrate) or by treatment with iodine vapors. The paper remaining after the experiment is known as the Chromatogram.The components which have been separated differ in their retention factor i.e Ratio of distance traveled from the spot or origin by the solute component to that of the distance traveled from the spot or origin by the solvent. Retention Factor can never be greater than one. To calculate Rf, use the following: Distance traveled by sample/ distance traveled by solvent.

The final chromatogram can be compared with other known mixture chromatograms to identify sample mixes using the Rf value in an experiment. The retention values found can be compared to known values, and from that conclusions can be drawn.

Composition of Filter Paper

The original work in paper chromatography was carried on whatman no.1 filter paper. At first, it was necessary to use the ordinary grades of papers but the makers now apply products specially made for chromatography in which the chemical and physical properties of the material are closely controlled in manufacture to the papers of high purity and uniformity. Paper for chromatography is made from cotton cellulose. The paper is generally made up of α-cellulose with other substances e.g. β-cellulose, ether soluble matter, NH3, N2 and minerals. Cellulose is a highly hydroxylated poly saccaride which consists of a polymer of glucose with a molecular wt. about 50000.

Rƒ value

Rƒ value may be defined as the ratio of the distance travelled by the substance to the distance travelled by the solvent. Rƒ values are usually expressed as a fraction of two decimal places but it was suggested by Smith that a percentage figure should be used instead. If Rƒ value of a solution is zero, the solute remains in the stationary phase and thus it is immobile. If Rƒ value = 1 then the solute has no affinity for the stationary phase and travels with the solvent front.