BIOLOGICAL INSTRUMENTATION (BOE 201) PAPER CHROMATOGRAPHY REPORT SEMESTER I ACADEMIC SESSION 2020/2021 DATE: 25/11/2020 Topic : Principle and technique of paper chromatography Introduction: Chromatography is a technique that is used to separate and to identify components of a mixture. Chromatography uses a solvent or gas stream to move the components of a mixture from a narrow starting point into a specific medium (tissue paper in this experiment) in different ways. It is used to purify and separate different substances. Paper chromatography is an inexpensive method of separating dissolved chemical substances by their different migration rates across the sheets of paper. In Paper Chromatography, it consists of stationary phase (a solid, or a liquid supported on a solid) and a mobile phase (a liquid or a gas). Stationary phase is usually a piece of filter paper (tissue paper in this experiment) while mobile phase is the solvent that carried the solutes through the stationary phase. When the mobile phase moves, the separation of the mixture takes place. The compounds in the mixture are separated according to their different affinities to the stationary phase and how readily they dissolve in the mobile phase solvents. The different components in the sample will travel along the tissue paper at different rates. Objectives: To observe how chromatography can be used to separate mixtures of chemical substances. To separate different colour markers/highlighters (inks) into their components. To obtain a paper chromatography of various colour inks. To identify components of colour inks by calculating Rf values. Materials: Tissue paper Container filled with shallow water Ruler Pencil Samples: water-based colour markers or any water-based colouring agent and oil A4 paper Methods: 1.Using a pencil and ruler, draw a straight line across the paper that above the bottom edge. 2. Put a small dot-spot at that line using different colours of highlighters or water-based colour markers. 3. Then, as a reference for hydrophobic agent, drop a small spot of oil at that line. 4. After the spots are dry, place the tissue paper on the water in the container and let the solvent move up until the solvent front about 80% of chromatography tissue paper. Notes: Make sure to carefully place the tissue paper on the water slightly below the line. 5. Mark and measure how far each colour(samples) and solvent travelled in cm. 6. Calculate the retention factor (Rf) for each spot and record the values. The distance travelled relative to the solvent is called the Rf value. Results: I)Observations No. Colour inks Distance travelled component(cm) by Distance travelled by Rf value solvent(cm) 1 Purple 10.9 14.4 0.76 2 Pink 10.2 14.4 0.71 3 Blue 11.4 14.4 0.79 Blue colour ink has the highest Rf value=0.79. It travelled the farthest which means, the more soluble a component is, the more readily it will dissolve in the mobile phase and the farther it will usually travel as it is carried by the water through the paper. While purple colour ink has the second higher Rf value=0.76 and travelled farther than pink colour ink but smaller distance than blue colour ink. Lastly, the pink colour ink with the lowest R f value=0.71and travelled the smallest distance among these components. Discussions: a. state the principle for paper chromatography. The principle of separation is usually partition rather than adsorption. The substance is distributed between the stationary phase and the mobile phase. The stationary phase is the tissue paper in this experiment while the mobile phase is the water(solvent) that carries the components(solutes) through the tissue paper. Generally, the higher the solubility of a solute in a solvent, the greater the solute mobility in that solvent. If a solute dissolve more readily in the mobile phase, then it will travel with the solvent, hence the partition occurs between two phases. Different solutes travel at different rates up the paper due to their different solubility in two phases. Components of the sample will separate readily according to how strongly they adsorb onto the stationary phase versus how readily they dissolve in the mobile phase. If a component is more dissolved to the solvent than to the paper, it will move a large distance up the paper. If a component is more adsorbed to the paper than to the solvent, it will travel a small distance up the paper. The movements of substances relative to the solvent are expressed in terms of Rf values (retention factor). The principle can also be adsorption chromatography between solid and liquid phases, where the stationary phase is the solid surface of the tissue paper and the liquid phase is the mobile phase. But most applications of paper chromatography are based on the principle of distribution chromatography. b. identify mobile phase and stationary phase. The mobile phase is the water which acts as solvent that carried up the colour inks(solute). While the stationary phase is the tissue paper. c. differentiate between tested samples Blue ink has the highest Rf value=0.79. It travelled the farthest which means, the more soluble a component is, the more readily it will dissolve in the mobile phase and the farther it will usually travel as it is carried by the water through the paper. While purple ink has the second highest Rf value=0.76 and travelled farther than pink ink but smaller distance than blue ink. Lastly, the pink ink with the lowest R f value=0.71and travelled the smallest distance among these components. d. troubleshooting/ problem(s) encountered for this experiment The solvent accidentally covers the pencil line on the tissue paper because did not carefully place the tissue paper into the solvent. The solvent wash away the colour inks, and not get to see the colours travel up the paper. So have to conduct the experiment again to get the better result. Error analysis occurred. There could have inaccurate measurement of the distances travelled by the colour inks and the mistakes or careless when calculating the retention factor which is the distance travelled by the ink(solute) and the water(solvent). When spotting, there is not enough space between the spots, which cause the spots to be crowded, and cause the analytes to overlap when moving along the stationary phase. e. differentiate between this chromatography with other chromatography Chromatography can be defined as laboratory technique for the separation of the mixture. The mixture is dissolved in a substance known as mobile phase (liquid or gas), which carries it through a system that is column, a capillary tube, a plate, or a sheet on which it is fixed the material called the stationary phase. In paper chromatography the stationary phase is the adsorbent paper that is placed in the solvent. It is an analytical method used to separate colored chemicals or substances. Paper chromatography is a type of solid-liquid partition method with the stationary phase is the cellulose filter paper and the mobile phase is liquid. In thin-layer chromatography, the stationary phase is the adsorbent that is a relatively thin, uniform layer of dry applied to a glass, plastic, or metal sheet or plate, glass plates being most commonly employed. It is a solid-liquid adsorption chromatography technique used to isolate non-volatile mixtures. It can be defined as a convenient way to check the purity of organic compounds. The distinguishing features of gas chromatography are a gaseous mobile phase and a solid or immobilized liquid stationary phase. Liquid stationary phases are available in packed or capillary columns. Gas chromatographs used at airports to detect bombs and use them in forensics in many different ways. It can be used to analyze the fiber on the human body, and also analyze the blood found at the crime scene. 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