Lab_Report_-_Photosynthesis

LAB REPORT – CHROMATOGRAPHY Chamodi Basnayake PROCEDURE: 1. Cut out a piece of chromatography paper and draw a parallel line about 2 cm from the bottom of the paper using a pencil. 2. Use a coin to extract the pigments form the spinach leaf by, placing the leaf on the line drawn previously and using the edge of the coin to squeeze out the juice onto the paper leaving a band of about 3mm wide. 3. Use a 50ml beaker; pour acetone into the beaker, until it reaches about 1 cm from the bottom of the beaker. 4. Place the chromatography paper in the beaker making sure the line drawn does not touch the solvent. 5. Let the liquid move up the paper by osmosis, carrying the cell pigment upward with it. 6. Let it be there for 20 minutes or so and put an end to the experiment when acetone doesn’t seem to move any further along the paper. 7. Mark the maximum distance acetone travelled and the other pigment bands that had different colors. Band Number | Distance Travelled(cm) | Band Color | Identity | 1 | 2.2 | Orange | Carotenoid | 2 | 2.3 | Dark Green | Chlorophyll A | 3 | 2.4 | Light Yellow | Xanthophyll | 4 | 2.6 | Light Green | Chlorophyll B | Maximum Distance the Solvent (Acetone) moved = 2.8 cm | PROCESSING THE DATA Calculating the Rf Values Rf= Distance traveled by PigmentDistance traveled by Solvent MOLECULE | Rf | 1. Carotenoid | 0.79 | 2. Chlorophyll A | 0.82 | 3. Xanthophyll | 0.86 | 4. Chlorophyll B | 0.93 | Example Calculations: Rf=yx ; y = Distance pigment travelled; x = Distance Solvent travelled Chlorophyll B: Y = 2.6; x = 2.8 Rf=2.62.8 * 0.93 Chlorophyll A: Y = 2.3; x = 2.8 Rf=2.32.8 * 0.82 Xanthophyll: Y = 2.4; x = 2.8 Rf=2.42.8 * 0.86 Carotenoid: Y = 2.2; x = 2.8 Rf=2.22.8 * 0.79 CONCLUSION: Paper chromatography is a technique used to separate substances in a mixture based on movement of the different substances up a piece of chromatography paper by osmosis. Pigments extracted from plant cells contain a variety of molecules that can be separated into different bands of colors according to which we can name each pigment’s color and compare its size to the other pigments. Given that the orange stripe is Carotenoid, Dark green – Chlorophyll A, Light yellow- Xanthophyll and Light green – Chlorophyll B, from the smallest molecule to the largest molecule: Chlorophyll B, Xanthophyll, Chlorophyll A and Carotenoid. The size of the pigment can be decided by how far each pigment traveled on the chromatography paper. The cellulose fibers in chromatography paper are arranged in grids. Therefore, the molecules that will be able to move fast through the paper will be the small molecules that are able to quickly make it through the tiny holes in the grids. The largest molecules travel the least amount of distance as they are simply too big to move through the holes without struggling. The factor that’s involved in separation of the pigments is the size. Rf value is the retardation factor or the sometimes called the retention factor. That’s the ratio of the distance traveled by the pigment to the distance travelled by the solvent front. The bigger the Rf value, the future it traveled down the paper and the closer it is to the distance the solvent front traveled.