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SEMESTER 3 Practical Science 2 Experiment 8 TOPIC: Separation Process-Chromatography PURPOSE: To separate and to identify the colors found in a type

of commercial food dye. THEORY: In chemistry , a separation process is used to transform a mixture of substances into two or more distinct products. There are many types of separation process, some of them are chromatography, cyclonic separation (separation based on density differences), crystallization, demister (removation of liquid droplets from gas streams), electrophoresis and many others. Chromatography is a technique used to separate the different components in a mixture, which are present in small trace quantities. There are several types of chromatography, one of which is paper chromatography. It is extensively used in research and industry, to analyze and identify different dyes. All chromatography techniques consist of two major components; the stationary phase and the mobile phase. The method consists of dissolving the mixture in a suitable solvent and which is then carried over the surface of the stationary phase. Separation is possible because different substances have different degrees of absorption solubility between the stationary phase and the mobile phase. The components will move along the stationary phase at different speeds and are thus separated. In paper chromatography, the stationary phase is the strip of chromatography paper and the mobile phase is suitable solvent. The principle on which chromatography works is very simple. The pulp in the filter paper used for chromatography, is composed of thin threads acting as capillaries.

When this paper is suspended in a beaker of water, touching the water surface, water ascends through these capillaries. Different soluble dyes move upwards at different speeds and to different levels, by capillary action. In research and industry a special kind of filter paper is used for chromatography. In the laboratory, a fine quality of ordinary filter paper can also be used. The Rf defines the movement of the substance relative to the solvent front in a given chromatographic system. Rf value comes under thin layer chromatography and paper chromatography. Thin layer chromatography is used extensively for the quantitative determination of high molecular weight compounds. Quantitative

identification can be based on the rate at which a compound flows along the plate. Here, we can identify the compounds in the sample by measuring the Rf value of the sample.

By comparing the Rf values of the components with Rf values of known substances, the components in the mixture can be identified. The value of Rf for each of the component is calculated using the formula: Rf =
h h

MATERIALS AND APPARATUS: i. ii. iii. iv. v. food dye ethanol container chromatography paper pointer

PROCEDURE: 1. 100 cm3 of ethanol and water solvent in the ratio 3:1 is prepared. This solvent is poured into a 1dm3 tall container. The mouth of the container is covered with plastic and it is left to stand for 30 minutes. 2. On a strip of filter paper measuring about 4 cm wide and 38 cm long, a line is marked lightly with a pencil about 2 cm from one end. Using a fine glass capillary pipette, the solution of food dye is spotted onto a marked spot in the middle of the line as shown in the diagram below. The spot id dried with a hair dryer. 3. This paper is suspended in the tall container with its lower end touching the solvent. The pencil line is ensured to be above the solvent level and the sides of the filter paper do not touch the walls of the high container as shown in the diagram below. 4. The apparatus is left to stand for 1 to 2 hours. Then, this filter paper is removed and the position of solvent front is marked immediately. The paper is dried with a hair dryer. 5. The Rf values for each of the spots separated is obtained. The distance moved by the solvent front is measured and the distance moved by the colour components from the starting line.

RESULTS: Substance Component Colour of component Distance of movement of component/cm Distance of solvent front/cm Rf

Food dye




= 0.17

DISCUSSIONS: 1. Using your results from the table above and information given by your lecturer identify the color components in the sample of food dye provided. Blue

2. In this paper chromatography experiment, state the substance that functions as the 1) Stationary phase = Chromatography paper 2) Mobile phase = Ethanol mixture

3. Explain how you would ensure that the drop of food dye at the starting line is concentrated and small in size. By using a sharp pointer, take a drop of dye and placed on the stationary line. Let it dry first and placed another drop of dye. This process is repeated until get a small and concentrated drop.

4. Why must the starting line containing the drops of food dye solution be placed above the solvent level at the beginning of the experiment? Because the ethanol solvent can move slowly and evenly towards the food dye.

5. State the steps taken to ensure effective separation. i. ii. iii. The measuring cylinder used needs to be large enough that the chromatography paper will fit easily inside without touching the sides of the cylinder. Make sure the origin line is at the bottom. Cover the measuring cylinder with a cellophane tape. Do not disturb the beaker while the ethanol rises up the paper. iv. Make sure the drop is at the middle of the stationary line.

v. vi. vii.

Make sure the drop is concentrated. Make sure the food dye drop dry first before it placed in ethanol solvent. Do not fold the chromatography paper. The paper must be perpendicular to the ethanol solvent. So those, the ethanol solvent can moves evenly.


Do not immerse the chromatography paper into the ethanol till exceed the food dye drop.

CONCLUSION: From the experiment, the Rf value of blue component in the food dye is 0.17.