Chromatography of Plant Pigments

CHROMATOGRAPHY OF PLANT PIGMENTS Marquez, Ma. Rica Paulene, Moises, Patrisha Kate, Policarpio, Jairus Paolo, Rolda, Zylene Joy Department of Biology, College of Science, University of the Philippines Baguio April 23, 2013 ABSTRACT The objective of this experiment was to apply the technique of paper chromatography as a method for separating individual plant pigments contained in plant tissue extracts containing pigment blends. The process of chromatography separates molecules because of the different solubilities of the molecules in a selected solvent.In paper chromatography, paper marked with an unknown, such as plant extract, is placed in a beaker covered with a foil containing a specified solvents. The solvent carried the dissolved pigments as it moved up the paper. The pigments were carried at different rates because they were not equally soluble. The most soluble pigment traveled the longest distance while the others traveled in a shorter length. The distance of the pigment trave led was unique for that pigment in set conditions and was used to identify the pigment.The ratio was then used to measure the Rf (retention factor) value. | INTRODUCTION As primary producers in the food chain with some bacteria and algae, plants produce their own food by using the sun’s energy to transform carbon dioxide and water into glucose. In this process of photosynthesis, plants convert the sun’s energy into chemical energy that is stored in the bonds of the glucose molecule. Glucose is a simple carbohydrate that provides immediate fuel to cells but it is also a building block for more complex carbohydrates stored by living organisms for future use.For photosynthesis to transform light energy from the sun into chemical energy (bond energy) in plants, the pigment molecules absorb light to power the chemical reactions. Plant pigments are macromolecules produced by the plant, and these pigments absorb specified wavelengths of visible light to provide the energy req uired for photosynthesis. Chlorophyll is necessary for photosynthesis, but accessory pigments collect and transfer energy to chlorophyll. Although pigments absorb light, the wavelengths of light that are not absorbed by the plant pigments are reflected back to the eye.The reflected wavelengths are the colors we see in observing the plant. Plants contain different pigments, and some of the pigments observed include: ? chlorophylls (greens) ? carotenoids (yellow, orange red) ? anthocyanins (red to blue, depending on pH) ? betalains (red or yellow) (Plant Traveling Lab. TTU/HHMI at CISER. 2010) As you may know from the popular media, there is currently a substantial research effort in place to explore the potential health benefits of plant pigments to humans. In popular literature, these plant-based compounds are often collectively referred to as â€Å"phytochemicals†; most are also pigments.Flavonoids, anthocyanins, and carotenoids are just some of the categories of plant pigme nts known to have antioxidant properties. â€Å"Antioxidant† is a general term used to describe any substance that has the ability to neutralize â€Å"free radicals† which cause cellular damage by removing electrons from surrounding molecules. Many lines of research suggest that consuming a diet rich in plant pigments may slow the process of cellular aging and reduce the risks of some types of disease, such as cancer, heart disease, and stroke.The point of this experiment is to look at the polarity of some of the common pigments in plant leaves and how that polarity affects their interactions with the cellulose fibers in paper and a few solvents and to apply the technique of paper chromatography as a method for separating individual plant pigments contained in plant tissue extracts containing pigment blends. (https://www. msu. edu) RESULT Table 1 shows the Rf values of Kangkong (Ipomoea aquatic), Bloodleaf (Iresine herbstii), and Golden bush (Duranta repens) plant| Rf value| | 50% acetone50% pet. ther| 40% acetone, 10% isopropanol, 50% pet. ether| 40% acetone, 10% pet. ether, 50% isopropanol| 30% acetone70% pet. ether| 30% pet. ether 70% acetone| Kangkong| Yellow: 0. 5Brown: 0. 01| Yellow: 0. 33Brown: 0. 23| Yellow: 0. 79Brown: 0. 08| Yellow: 0. 04Brown: 0. 96| Yellow: 0. 31Brown: 0. 76| Bloodleaf (purple leaf)| Purple:0. 05Green:0. 11Yellow:0. 11| Purple:0. 06Green:0. 13| Purple:0. 13Green:0. 58| Purple:0. 03Green:0. 58Yellow:0. 38| Purple:0. 097Green:0. 86| Golden bush| Green:0. 5Brown:0. 5| Green:0. 47Brown:0. 09| Green:0. 6Brown:0. 067| Green:0. 3Brown:0. 96| Green:0. 094Brown:0. 96| DISCUSSION The extracts of kangkong, golden bush, and purple leaves were applied to a horizontal line about ? of an inch from the bottom of a filter paper using a capillary tube. The filter paper then was soaked one by one on a beaker with a mixture of 40% acetone, 10% petroleum ether and 50% isopropanol. These solvents are used because they are capable of separa ting mixtures that contain both polar and non-polar compounds, or to increase separation of mixtures of compounds that have similar behavior with a single solvent.The beaker was covered with aluminum foil to make sure that the atmosphere in the beaker is saturated with the solvent’s vapor. Saturating the atmosphere in the beaker with the solvent’s vapor stops the solvent from evaporating as it rises up the filter paper. As the solvent slowly travels up the paper, the different components of the extract travel at different rates and the extracts are separated into different colors. After 3-5 minutes, the distance traveled by each pigment and solvent were measured. Plants extracted| Color/ Pigment| Rf values| Kangkong| Yellow (solvent)| 0. 9 mm| | Brown (solute)| 0. 08 mm| Purple leaves| Green (solvent)| 0. 58 mm| | Purple (solute)| 0. 13 mm| Golden bush| Green (solvent)| 0. 6 mm| | Brown (solute)| 0. 067 mm| Table 1: Pigments and Rf values for each plant extract The dis tance traveled relative to the solvent is called the Rf value, or the Retardation value. It can be computed with the formula: Rf = distance travelled by the solute Distance travelled by solvent We had the following computations: A. For Kangkong: Rf= 0. 08 mm 0. 79 mm = 0. 1012 B. For purple leaves: Rf= 0. 13 mm 0. 58 mm = 0. 241 C. For Golden bush: Rf= 0. 067 mm 0. 6 mm = 0. 1117 These values imply that the larger Rf value a compound has, the larger the distance it travels. It also means that it is less polar because it interacts less strongly with the polar absorbent on the filter paper. So similarly, the smaller the Rf value a compound has, the shorter the distance it travelled. It also means that is is more polar because it interacts more strongly with the polar absorbent on the filter paper. Comparing to the other groups’ results, there were similar colors that sprung up.The distances travelled by the pigments were significantly different than ours because they used diffe rent percentages of solvents. CONCLUSION Paper chromatography proved to be an accurate method of separating and observing the various colors of plant pigments. The pigments dissolved in the solvent and migrated upward. The colors were observed and their migration distances measured & recorded. The  Rf  value  of each pigment was determined by dividing its migration by the migration of the solvent. We have always understood chlorophyll, a pigment that is very important in photosynthesis, to be green.However, through this experiment we have discovered that many other pigments are also present in the leaves. For example the kangkong leaf also contains different pigments even though the leaf is dominated by the color green. We have observed that the kangkong leaf not only carries a green pigment, but that it also carries yellow and brown pigment through the chromatography LITERATURE CITED * Mcmurry, John. 2010. Foundations of Organic Chemistry. Pasig City, Philippines: Cengage Lea rning Asia Pte Ltd. * Thompson, R. 2008. Illustrated guide to Home Chemistry experiments. Canada: O’ Reilly media.Page 109 * Clark, J. 2007. http://chemguide. co. uk/analysis/chromatography/paper. html. Retrieved April 22, 2013 * Unknown. PlantTravelingLab. TTU/HHMI at CISER. 2010 . http://www. ciser. ttu. edu. * 2010. 04-20-13. * Unknown. separation of pigments by paper chromatography. http://www. hsu. edu/pictures. aspx? id=1653, n/a. 04-20-13 * Unknown. plant pigment analysis. https://www. msu. edu. n/a. 04-20-13 * http://apnewtechbiology. wordpress. com/lab-6-plant-pigments/ * http://www. biologyjunction. com/chromatography_of_simulated_plan. htm ANSWERS TO QUESTIONS