Paper Chromatography Experiment 1 PDF

EXPERIMENT 1 PAPER CHROMATOGRAPHY THEORY Paper chromatography is a simple and widely used technique in biochemistry. It is useful not only for testing the purity of compounds and identifying substances but also in the separation of closely related compounds from a...

EXPERIMENT 1 PAPER CHROMATOGRAPHY THEORY Paper chromatography is a simple and widely used technique in biochemistry. It is useful not only for testing the purity of compounds and identifying substances but also in the separation of closely related compounds from a mixture. It is based on the principle of partition of compounds to be separated between two phases. In paper chromatography, substances are distributed between a stationary phase and a mobile phase. The stationary phase is usually a piece of high quality filter paper. Water held back by the paper is called the stationary phase. The mobile phase is a developing solution that travels up the stationary phase, carrying the samples with it. 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. The paper is then dipped into a suitable solvent, such as ethanol 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; this can also 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. The more soluble the component the further it goes. Rƒ value may be defined as the ratio of the distance travelled by the substance to the distance travelled by the solvent. 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. An application of chromatography in medicine is the detection of the presence of drugs such as narcotics in urine and in blood samples. OBJECTIVES 1. To practice paper chromatography in determining the solubility of a solute in various solvents. 2. To apply the principles involved in paper chromatography. MATERIALS beaker (50 ml) oslo paper aspirator capillary tubes 3 hard glass test tubes with cork 3 iron clamps Mortar and pestle iron stand 1  Pipette (10 ml) pencil Scissors ruler masking tape REAGENTS Ethyl alcohol Diethyl ether Acetone PROCEDURE 1. Cut 3 pieces of oslo paper with the dimension shown in the figure below. -2 cm- 22 cm -1 cm- 2. Grind in a mortar and pestle small pieces of green leaves. 3. Get the leaf extract (sample) by adding enough amount of ethyl alcohol in a small beaker. Set aside. Prepare the solvent system using 3 hard glass test tubes. Mix 3 ml ethyl alcohol with 3 ml water in the first test tube; 3 ml acetone with 3 ml water in the second test tube and 3 ml diethyl ether with 3 ml water in the third test tube. Cover each test tube containing the solvent system with a cork. (Caution: Ethyl alcohol, acetone and diethyl ether are flammable liquids. Keep away from flame, instead place in a fume hood.) 4. By means of a capillary tube, spot the leaf extract into each of the 3 previously cut oslo papers. The spot should be 2 cm from the edge of the narrow tip of the oslo paper. Let it dry. 5. Prepare the set up as shown below: cork ethyl alcohol and H2O masking tape Diethyl ether and H2O oslo paper acetone and H2O leaf extract spot 2 1 cm solvent system Note: The narrow tip of the oslo paper must touch the solvent system by dipping 1 cm of it into the solvent. The wider end of the oslo paper may be suspended into the cork using a masking tape. The oslo paper should hang straight and should not touch the sides of the test tube. 6. Allow the solvent to reach the maximum height. Mark the height reached by the solvent and the height reached by the leaf extract sample. Let the oslo paper dry. 7. Solve for the Rf value after measuring the height reached by the solvent and the leaf extract sample using the formula: Maximum height reached by the leaf extract Rf = Maximum height reached by the solvent Precaution: Diethyl ether can cause irritation of the eyes and skin when inhaled excessively. 3 Name ______________________________ Rating ________ Course/year & section ________________ Date __________ EXPERIMENT 1 PAPER CHROMATOGRAPHY REPORT / ANSWER SHEET DATA AND RESULTS Solvent Maximum height Height of leaf Rf value of solvent extract Ethyl alcohol acetone Diethyl ether CALCULATIONS QUESTIONS 1. Which solvent yields the highest Rf value? What does it indicate? 2. Can Rf value be used to identify a substance in the mixture? Why? 3. How are the components of a sample mixture separated and identified in paper chromatography? GENERALIZATION 4