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Download presentation Paper Chromatography is a method of separating and identifying various components in a mixture, which are present in small trace quantities. There are several types of chromatography, one of which is paper chromatography. This uses a stationary phase (paper) and a mobile phase (solvent). As the solvent wicks up the paper by capillary action, an equilibrium is established for each chemical in the mixture. Part of the time the particular pigment is moving in the solvent, while at other times it is adsorbed into the paper. The pigments separate in the sample due to polarity; the paper is more polar than the solvent so nonpolar substances will spend more time in the nonpolar mobile phase and will travel further up the paper Spinach Chromatography Lab Objective 1. Separate pigments from spinach using paper chromatography 2. Calculate Rf values of separated pigments *Rf = distance substance travels distance solvent front travels Materials • Chromatography paper strips • Petroleum Ether: Acetone Solution • 1 vial spinach leaf extract • Capillary pipet • Parafilm squares • MSDS • Transparent Tape • Metric rulers • Graduated Cylinder (100 m. L) • Graphite pencil Pre-lab Questions • What is chromatography? • Calculate the Rf of a substance that traveled 5. 2 cm on chromatography paper. The solvent front traveled 8. 1 cm. • Does each spot on the developed chromatography paper represent one substance? Procedures 1. 2. 3. 4. 5. Prepare a piece of chromatography paper by measuring 2 cm from bottom of paper and drawing a line with pencil At the other end, attach a piece of tape long enough to drop the paper to the bottom of the graduated cylinder and attach the top of the tape to the cylinder mouth At center station, touch the capillary pipet with pigment to center of line; allow the spot to dry Pour 5 m. L petroleum ether: acetone solution into 100 m. L graduated cylinder Cover the top of cylinder with Parafilm square 6. Place chromatography paper inside cylinder so that the paper extends into the solvent 7. Wait 10 minutes, then remove the paper from the cylinder, and mark the location of the solvent front with pencil. Trace pigment outlines with pencil after solvent evaporates 8. In your notes, draw a life-size picture of the developed chromatography paper, labeling the locations and colors of the spots. 9. Measure the distance each spot and the solvent front traveled from the pencil line; calculate the Rf values for each pigment. Follow-up Questions • Find the distance traveled by: – – – Solvent front _______cm Carotene ____cm Xanthophyll _______cm Chlorophyll a ______cm Chlorophyll b_______cm • Calculate Rf for each pigment: Carotene ____ Xanthophyll _____ Chlorophyll a ____ Chlorophyll b ____ • Which pigment is most polar? Which is least?
The following is adapted from from: Paper chromatography is a procedure used to separate substances in a mixture. In a biology lab, this is usually a solution of liquid plant pigments : these contain different kinds of chlorophylls and other colored photosynthetic pigments.* A small, concentrated sample of a mixture is placed on the chromatography paper – above the line of a solvent mixture.* Paper is then placed in contact with a solvent solution, at its bottom.* Solvent is drawn up through the paper due to capillary action.* As solvent hits the spot, it dissolves many of the pigments.* Some pigments have a greater attraction for the chromatography paper, so they move a lesser distance.* Some have a lesser attraction, so they move a greater distance.Experimental setupExample of a resultAnother example, showing how the different pigments each moved a different distance.The sample will separate into consistent patterns – as long as the solvent, paper, and time it runs, stay the same.(Time matters because the longer the amount of time that the experiment runs, the further the pigments will move.) Different solvents will change the separation pattern of the mixture.The retardation factor, Rf value, of a pigment is a statistic used to identify how a pigment behaves.Each pigment has it’s own specific Rf (for the same solvent, same time)Retardation factor (Rf) is defined as the ratio of the distance traveled by the center of a spot, to the distance traveled by the solvent frontfrom Bio C2005/F2401x 2010 Lec.5 Lawrence Chasin, September 221, 2010 How can we separate a solution of amino acids? Paper chromatography does this based on the differential solubility of the different amino acids in organic (non-polar) solvents.This solubility is determined by the nature of the amino acid’s side group.The amino acid mixture is spotted onto a filter paper; one edge of the paper is immersed in a mixture of aqueous and non-aqueous solvents.The liquid will be drawn up the paper by capillary action.As it rises, the water in the liquid mixture is bound by the paper (cellulose, with its many OH groups), forming a stationary water layer, or stationary phase.The organic solvent (e.g., propanol) moves up without as much interaction with the solid cellulose; it is considered the mobile phase.The amino acids will be constantly equilibrating between being in the mobile organic phase or the stationary water phase.The more polar the side chain, the more time the amino acid will spend in the stationary phase.The more hydrophobic the side chain, the more time it spend in the mobile organic phase.By using a series of different solvents, all 20 amino acids can be separated in this way. It works for many other organic molecules as well.The distance that an organic molecule moves in a particular chromatographic system is called the Rf, which stands for mobility Relative to the Front:that is, the distance the organic molecule in question migrated divided by the distance that the front of liquid has risen on the paper at that time.Rf’s in a particular solvent and at a given temperature are reproducible and are published for many organic compounds, including all the amino acids*text |
Paper chromatography is a method used in regents
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