Identifying Plant Pigments by Chromatography

Karol Mnich Mrs. Farrell SBI 4U April 14, 2010 Holy Cross Catholic Secondary School

Introduction Chromatography can be used in police work, to be more specific it is used in a forensics laboratory. An example of how it could be used is when there was an incident and there was lipstick left on someones cup they were drinking out of left at the scene. Chromatography would help identify which persons lips it was by the lip stick. The chemist would dissolve a small piece of each of the two samples of lip stick found on the cup. They would then use chromatography to compare each of the chromatograms. This will show a distinct difference between the two lips if one was exactly the same as the chromatogram produced from the cup stain then we have a positive identification. This can also be used with finger prints when the police are trying to identify people in murder cases. Colours everywhere appear to be one solid gradient. What appears as a bright red maple leaf on our Canadian flag is actually made up of many blue, red and green specs. The maple leaf appears to be red because the number of red dots overpowers the amount of blue and green dots. In this lab we were to investigate the colour green on a leaf. This lab helps understand if the leave contains a single pigment that reflects green light only, or are there other colours that are not seen because of amount of green pigments. This investigation specifically helps us create a visual for each pigment colour by separating them.(Nelson 2003) RF values are important in the lab being done. RF is a measurement that is based on relative distance traveled by a sample of a substance (answers.com, 2010).It is important to have accurate Rf values in chromatography because accuracy will make it easier to compare results. In this lab, our actual Rf value in the end is compared to the theoretical value of each pigment seen. The formula for RF is the distance of pigment sample over distance of solvent front times 100. Chromatography is a method used to separate and or to analyze complex mixtures, by separating them into the chemicals from which they are made. Molecules in pigments and other mixtures have different characteristics, such as size and solubility; they travel at different speeds when pulled along the piece of chromatography paper. There are two phases that chromatography paper can be in; stationary phase and mobile phase. The stationary phase is an absorbent paper. The mobile phase is a suitable liquid solvent or mixture of solvents. (Clark, 2007 ) Pigments are chemical compounds that reflect specific wavelengths of visible light. Autotrophs, organisms that produce their own food, utilize certain pigments to carry out photosynthesis. These autotrophs have pigments because of the chlorophyll that is used in photosynthesis. Due to the wide range of the visible light spectrum, several different classes of pigments are needed in order to maximize the energy taken from the sun. Because of the porphyrin ring structure of chlorophyll, electrons can move easily, allowing chlorophyll to capture the sun's energy. (Aqua Media, 2009) Purpose The purpose of this lab is to separate a green leaf extract to determine what types of pigments are in the leaves of spinach.

Hypothesis It was predicted that the spinach pigments should include xanthophylls, carotene, chlorophyll A and chlorophyll B. Materials Safety goggles Leaves of Spinach Medicine dropper Beaker Funnel Mortar and pestle 20mL of acetone Pencil Two Test tubes Test tube rack Cork stopper Chromatography paper Scissors Apron

Safety Precautions 1. Acetone and chromatography solvent was handled with extreme care due to the hazard of being highly flammable and to avoid spilling and contact with skin 2. Vapours of acetone and chromatography solvent were avoided when breathing near the substances. 3. Goggles were worn to avoid chemical contact with eyes Procedure 1. Safety goggles and lab apron were put on 2. One leaf of spinach was grinded up in the mortar and pestle along with 3mL of acetone. The mortar and pestle were cleaned with 1 mL of acetone. This was done to prevent contamination and also to prevent cross contaminate the specimens in anyway. The spinach leaf was not over grinded to avoid a pulpy mess that absorbs all of the solvent. A second spinach leaf was grinded up with a mortar and pestle without the presence of acetone. 3. 3.0 mL of chromatography solvent was put in two separate test tubes for each leaf extract. 4. Two pieces of chromatography paper was cut precisely to fit the test tube 5. A pigment extract of the juice from each leaf was transferred 3 cm from the bottom of each chromatography paper. This pigment extract was placed on the paper with the help of a dropper.

6. The papers were than waved in the air to make it as dry as possible. 7. The chromatography paper containing the pigment extract with acetone was placed in the test-tube without the extract touching the solution. The chromatography paper containing the pigment extract without the acetone was also placed in a test-tube without the extract touching the solution. 8. After a period of time the apparatus was observed until the chromatography solvent reached the top of the chromatography paper. 9. The distance from the middle of the original extract spot to each pigment band line and from the extract spot of the solvent line. The Rf values of each pigment according to the equation. 10. The chromatography solution was discarded, the unused spinach was put back into the bag and the station was cleaned up. Hands were washed with soap and water. Observations Leaf Pigment Colours, Theoretical Rf value, and Actual Rf Value Pigment Theoretical Rf Value Actual Rf Value Colour Carotene 0.98 0.10 Orange Chlorophyll a 0.59 0.96 Blue-Green Chlorophyll b 0.42 0.90 Yellow-Green Pheophytin 0.81 -----Olive Green Xanthophyll 1 0.28 -----Yellow Xanthophyll 2 0.15 -----Yellow

Discussion The results were that Carotene with the orange colour has the highest Rf value with 0.100. Chlorophyll A followed with an Rf value of 0.96 which contained a bluegreen colour. Chlorophyll B finished with an Rf value of 0.90 which contained a yellowGreen colour. Pigments such as pheophytin, xanthophyll 1 and xanthophyll 2 were not present on our strip of chromatography paper. When using chromatography in this lab there were a few ways that our results could have been inaccurate and pigments not being seen. Errors like the solvents used came in contact with foreign substances, the chromatography paper also could have been contaminated, and the dropper did not obtain the same amount of spinach juice for each paper. When the chromatography solvent was being transferred from the bottle to the test tube there was contact with the air. This caused vapours to escape and this also caused a competing reaction. The elements in the air might have contaminated with the solvent and caused a flaw in the reaction. The stopper was placed over the test tube to prevent vapours from escaping and to avoid vapours to come in contact with air. The amount of space left between the test tube and the stopper could have caused the vapours to evaporate rapidly and dry out the chromatography paper quicker. The chromatography paper given was either touched by a human hand or by scissors. Handling the chromatography paper with our hands could have resulted in a contamination on the paper. Having a foreign substance involved with the reaction could have altered any results shown. The scissors were also used to cut the end of the chromatography paper to the right shape. The scissors also could have contained many substances on each blade which would result in the reaction to compete with other unwanted matter. The dropper that picked up the solvent and the grinded up spinach did not pick up the same amount all the time. There was no precise way to get the exact same amount repetitively. This also affected the amount of pigment that was placed on the chromatography paper because the same amount was not placed on each piece of chromatography paper. This would cause the solvent to have a harder time separating the pigments because there would be too much in one experiment and if there was too little the solvent would separate the pigment quickly, and this would cause the reaction to occur faster. Conclusion In conclusion, it was found that there was more than one pigment within the spinach. These pigments were carotene, chlorophyll a, chlorophyll b, and xantophyll.

References Clark, Jim. "Paper chromatography." Chemguide: helping you to understand Chemistry - Main Menu. 2007. Web. 29 April 12, 2010. <http://www.chemguide.co.uk/analysis/chromatography/paper.html>. Hardy, James K. "Analytical Chemistry." HordeNet at The University of Akron. 2000. Web. 29 April 11, 2010. <http://ull.chemistry.uakron.edu/analytical/index.html>. Simms, John. "Paper Chromatography". All sands. September 9, 2006. April 12, 2010 <http://www.allsands.com/science/paperchromatogr_xqw_gn.htm>. Smith, S.E. What is chromatraphy. Wise Geek. March 30, 2010. April 13, 2010. <http://www.wisegeek.com/what-is-chromatography.htm> What are pigments?. Aquamedia. May 18, 2009. April 13, 2010. <http://www.feap.info/home/FAQ/Answers/ans5_en.asp>