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INTRODUCTION

Paper chromatography is an analytical chemistry technique for separating and identifying mixtures that can be colored, especially pigments. This can also be used in secondary or primary colors in ink experiments. This method has been largely replaced by thin layer chromatography, however it is still a powerful teaching tool. Two-way paper chromatography, also called two-dimensional chromatography , involves using two solvents and rotating the paper 90° in between. This is useful for separating complex mixtures of similar compounds, for example, amino acids.

PRINCIPLE OF PAPER CHROMATOGRAPHY

In paper chromatography, chromatography paper is used. This paper contains water trapped in it, which acts as the stationary phase. On the base of this chromatography paper, the solution of the mixture is spotted. The paper strip is then suspended in a suitable solvent, which acts as the mobile phase. This solvent rises up the chromatography paper by capillary action and in the procedure, it flows over the spot. The components are selectively retained on the paper (according to their differing partition in these two phases). The spots of different components travel with the mobile phase to different heights. The paper so obtained is known as a chromatogram.

INTRODUCTION Paper chromatography is an <a href=analytical chemistry technique for se p aratin g and identifying mixtures that can be colored, especially pigments . This can also be used in secondary or primary colors in ink ex p eriments. This method has been largely replaced by thin layer chromatography , however it is still a powerful teachin g tool. Two-wa y p a p er chromatography , also called two-dimensional chromatography , involves using two solvents and rotating the paper 90° in between. This is useful for separatin g com p lex mixtures of similar compounds, for example, amino acids . PRINCIPLE OF PAPER CHROMATOGRAPHY In paper chromatography, chromatography paper is used. This paper contains water trapped in it, which acts as the stationary phase. On the base of this chromatography paper, the solution of the mixture is spotted. The paper strip is then suspended in a suitable solvent, which acts as the mobile phase. This solvent rises up the chromatography paper by capillary action and in the procedure, it flows over the spot. The components are selectively retained on the paper (according to their differing partition in these two phases). The spots of different components travel with the mobile phase to different heights. The paper so obtained is known as a chromatogram. Paper Chromatography: Procedure ∑ Cut chromatography paper into rectangular strips and mark a line on the paper at about 2-3 cm from the bottom. Marking " id="pdf-obj-0-43" src="pdf-obj-0-43.jpg">

Paper Chromatography: Procedure

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 it undergoes contamination. Label the paper. Clean your hands before handling chromatography paper. With the help of capillary tube, take sample and place a spot on the starting line and avoid spilling. Now, place the chromatography paper in the developing chamber, which contains the solvent or the mobile phase. 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. 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 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.

Ascending chromatography

In this method, the solvent is in pool at the bottom of the vessel in which the paper is supported. The ascending chromatogram is folded over the glass rod whose other half become the descending chromatogram. This technique gives as quick separation as that of the individual techniques.

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. Though the liquid

moves down by capillary action as well as by the gravitational force, this method is also known as the gravitational method. In this case, the flow is more rapid as compared to the ascending method, and the chromatography is completed more quickly. 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 sometimes be separated by the descending method.

Retention Factor (Rƒ): Calculation

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. 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.

Paper chromatography 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 .The disadvantage of paper chromatography is that many a times complex mixtures can’t be separated by using this method.

The essential structure of paper

Paper is made of cellulose fibres, and cellulose is a polymer of the simple sugar, glucose.

Paper is made of cellulose fibres, and cellulose is a polymer of the simple sugar, glucose.

application of paper chromatography

The oldest of all chromatographic techniques, paper chromatography (PC) is still being used today in several industries to either separate and identify, or to purify a chemical. In agriculture, the metabolism of herbicides by bacteria is analyzed by PC. In microbiology, plant extracts' bacteria and proteins (amino acids) are separated out by this technique as well. The classic use for this technique is for separation/ identification of dyes and pigments (colored species- hence the origin of the term "chroma") in ink and paint samples. By far, one of the more common applications is in forensic science: amounts of narcotics, hallucinogens, even aspirin are detected by PC in both urine and blood samples.