LAB REPORT Exercise 13 PHOTOSYNTHESIS: Pigment Separation, Starch Production, and CO2 Uptake Jim Goetz Lab Section

12 March 20, 2012

Introduction The most important chemical reaction in the biosphere is photosynthesis. It is the pre-requisite for all life on earth. In this process, light from the sun is converted into chemical energy, which is used directly as nutrition by the photosynthetic organisms. Energy is then transferred by animals, which eat such organisms (e.g. cows eating grass), to other living beings (e.g. humans) eating these animals, and so on through the nutritional chain. The energy necessary for life processes is liberated in the combustion of carbohydrate and fats by the oxygen of the air in cellular respiration. This process may continue indefinitely because the consumed nutritional substances are continuously produced in the process of photosynthesis of green plants. With the aid of solar energy, plants build up complicated organic compounds from two simple inorganic molecules, carbon dioxide and water, with the concomitant liberation of oxygen. Thus photosynthesis and respiration result from the sun driving a continuous cyclic process in the biosphere:

A simpler form of photosynthesis, which leads to the formation of organic material without liberation of oxygen, is found in certain bacteria. Photosynthesis and respiration comprise electron transfer between proteins. These often contain metal ions, e.g. iron, in specific electron-transport chains. The electron-transport proteins in photosynthesis as well as in respiration are organized as complicated molecular aggregates bound to membrane systems of two specific cell organelles, chloroplasts and mitochondria. The energy liberated during the electron transport is used to pump

protons across the membranes, so that a difference in pH and electrical potential between the two sides is created. This electrochemical potential is then used to drive the synthesis of adenosine triphosphate (ATP), the universal energy storage molecule in living cells (Mitchell 1961). The summary equation for photosynthesis is: CO2 + 12H20  C6 H12 O6 + 6H2O + 6O2 Carbon dioxide and water, with the addition of light, yield sugar with water and oxygen as byproducts. The oxygen is released into the environment, sugar us used to fuel growth or it is stored as starch. In the light reactions, one molecule of the pigment chlorophyll absorbs one molecule of the pigment chlorophyll absorbs one photon and loses one electron.This electron is passed to a modified form of chlorophyll called pheophytin, which passes the electron to a quinone molecule, allowing the start of a flow of electrons down and electron transport chain that leads to the ultimate reduction of NADP to NADPH. In addition, this creates a proton gradient across the chloroplast membrane; its dissipation is used by ATP synthase for the concomitant synthesis of ATP. The chlorophyll molecule regains the lost electron from a water molecule through a process called photolysis, which releases a dioxygen molecule. The overall equation for the lightdependent reactions under the conditions of non-cyclic electron flow in green plants is:

2 H2O + 2 NADP+ + 3 ADP + 3 Pi + light → 2 NADPH + 2 H+ + 3 ATP + O2

In the light- independent or dark reactions, the enzyme RuBisCO captures CO2 from the atomosphere and in a process that requires the newly formed NADPH, called the Calvin- Benson Cycle, releases three-carbon sugars, which are later combined to form sucrose and starch. The overall equation for the light-independent reactions in green plants is: 3 CO2 + 9 ATP + 6 NADPH + 6 H+ → C3H6O3-phosphate + 9 ADP + 8 Pi + 6 NADP+ + 3 H2O

Both of these reactions occur in chloroplasts. Chloroplasts are plastids that contain chlorophyll and in which photosynthesis takes place. The photochemical reactions convert light energy to chemical energy captured in ATP and NADPH. The biochemical reactions use the ATP and NADPH produced by the photochemical reactions to reduce CO2 to sugars. The photochemical reactions occur on the thykaloid membrane and the biochemical reactions occur in the stroma. This is depicted in the diagram below.

Entropy is disorder. A chemical reaction, which proceeds with a positive entropy change is one which produces a more disordered system than was present before the reaction occurred. Negative entropy refers to reactions, which produce less disorder. The less disorder the greater amount of energy produced. In the process of photosynthesis, multiple molecules of carbon dioxide are combined into single molecules of sugars, which are in turn connected to form larger molecules of carbohydrates. Since this process involves taking several units and combining them into one, it proceeds with a loss of disorder or, in other words, negative entropy (increased energy). The purpose of this lab was to use paper chromatography to separate dissolved compounds such as chlorophyll, carotene and xanthophyll. When a solution of these pigments (substances that absorb light) is applied to strips of paper, the pigments absorb onto the fibers of that paper. When the tip of

the paper is immersed in a solvent, the solvent is absorbed and moves up through the paper. As the solvent moves through the spot of applied pigments, the pigments dissolve in the moving solvent. The pigments to not always keep up with the moving solvent. Some pigments move almost as fast as the solvent, where others move more slowly. This differential movement of pigments results from each pigment solubility and characteristic tendency to be absorbed by the cellulose fibers of the paper. A pigments molecular size, polarity and solubility determine the strength of this tendency. Pigments absorbed strongly move slowly whereas those absorbed weakly move fastest. Thus each pigment has a characteristic rate of movement and the pigments can be separated from each other. The solvent used is composed of a 9:1 mixture of petroleum ether and acetone. This is a non- polar solvent. Non-polar molecules in the mixture has little attraction for the water molecules attached to the cellulose. It will spend most of its time dissolved in the moving solvent. Molecules like this will therefore travel a long way up the paper carried by the solvent. They will have relatively high Rf values. Polar molecules will have a high attraction for the water molecules and much less for the non-polar solvent. They will therefore tend to dissolve in the thin layer of water around the cellulose fibers much more than in the moving solvent. The spinach extract and chromatography paper are polar and thus have an attraction to one another. This is what enables the differentiation of pigments along the chromatography paper. The relationship of the distance moved by a pigment to the distance moved by the solvent front is specific for a given set of conditions. This relationship can be defined by the equation: Rf= Distance moved by pigment/ Distance from pigment origin to solvent front Rf is the given constant for a given pigment in a particular solvent matrix system. In this experiment, the test tube is covered in order to make sure that the atmosphere in the beaker is saturated with solvent vapor. Saturating the atmosphere in the beaker with vapor stops the solvent from evaporating as it rises up the paper and thus proper results can be seen. The hypothesis using paper chromatography, the pigments that give a leaf its color can be separated and observed to determine the Rf value of each pigment and their function during

photosynthesis Materials and Methods Used in this experiment is chromatography strip, acetone, petroleum ether, pencil, Pastuer pipet, test tube, plastic film, liquid spinach extract Procedure 13.1 Separate plant pigments by paper chromatography 1. Observe the contents of the provided container labeled “Plant Extract” 2. Obtain a strip of chromatography paper from lab instructor. Handle the paper by its edges so oil on your fingers do not contaminate the paper 3. Use a pencil to mark a faint line across the paper, approximately 2 cm from the tip of the paper. Use a Pastuer pipet to apply a stripe of plan extract over the pencil mark. Blow the stripe dry and repeat this application 20 times. 4. Place the chromatography strip in a test tube containing 2 m: of chromatography solvent (9 parts petroleum ether: 1 part acetone). Position the chromatography strip so that the tip of the strip (not the stripe of the plant extract) is submerged in the solvent. 5. Place the tube in a test tube rack and watch as the solvent moves up the paper. Keep the tube capped and undisturbed during the solvent movement 6. Remove the chromatography strip before the solvent reaches the tip of the strip. Mark the position of the solvent front with a pencil and set the strip aside to dry. Observe the bands of color, then draw your results on figure 13.5. 7. Measure the distance from the pigment origin to the solvent front and from the origin to each pigment band. Calculate the Rf number for each pigment. Record data in table 13.1

Table 13.1 Rf Numbers For four Plant Pigments Pigment Carotene Xanthophyll Chlorophyll a Chlorophyllb Rf 0.96 0.70 0.61 0.45

Carotene: a = 8.1 cm Rf = 8.1 / 8.4 = 0.96 Xanthophyll: a = 5.9 cm Rf = 5.9 / 8.4 = 0.70 Chlorophyll a: a = 5.1 cm Rf = 5.1 / 8.4 = 0.61 Chlorophyll b: a = 3.8 cm Rf = 3.8 / 8.4 = 0.45 The result of this experiment was there was a proper breakdown of the pigments (carotene, chlorophyll a, chlorophyll b, and xantophyll) located within the spinach extract. As noted in the table above, the pigments of carotene, xanthoplyll, chlorophyll a and chloroplyll b were all properly identified. The pigments were identified by their unique colors. The colors were: Carotene- yellow orange Xanthopyll- yellow Chlorophyll a- bright green Chlorophyll b- olive green These were able to be identified by their separation on the chromatography paper. Please see figure 13.5 attached to lab report Discussion My hypothesis was confirmed as the pigments that give a leaf its color were separated and observed. The Rf value of each pigment was determined. Based upon this, their function during photosynthesis was possible to be ascertained. When green is seen in a plant, there are many pigments in that “green” plant. The pigment that

reflects green light in leaves is chlorophyll. There are two forms of chlorophyll: chlorophyll A and chlorophyll B. There are also gold and yellow pigments (the carotenes and xanthophylls) in the leaf that are "masked" by the green. These pigments were separated the different pigments found in the spinach solution by using paper chromatography. Chromatography in this experiment worked the different pigment molecules, when dissolved in the solvent, migrated through the chromatography paper at different rates. The rate at which each specific substance moved on the chromatography paper was determined how soluble the substance is in the solvent being used and how well the substance adhered to the chromatography paper. Once the pigment molecules were separated by chromatography they were identified by calculating Rf values. The experimental Rf values were compared to standard Rf values, which was found in the book, Heme, Chlorophyll, and Bilins. Two substances that have the same Rf for a given solvent are probably the same molecule. The probability that the tested molecules are the same substance increases if they continue to have the same Rf value in a variety of solvents. Rf values can be used to identify unknown substances. In this case, the substances were known by the color comparison given in the lab manual but the Rf values were found and had to be looked up.

Any errors in this experiment would be most likely due to the oil from fingers on either the chromatography paper or in the test tube. The covering for the test tube may also have not been covered the entire time, thus allowing outside atmospheric conditions to settle within the test tube and effect the evaporation of the solvent.

References -Press Release: The 1988 Nobel Prize in Chemistry". 31 Mar 2012 -Peter Mitchell (1961). "Coupling of phosphorylation to electron and hydrogen transfer by a chemi-osmotic type of mechanism". Nature 191 (4784): 144–148. -Smith et. Al. (2001) Heme, Chlorophyll, and Bilins

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