Lab - 4 Photosynthesis-Pigment-Revised(1).pdf

PHOTOSYNTHESIS  Photosynthesis An anabolic, endergonic, carbon dioxide (CO2) requiring process that uses light energy (photons) and water (H2O) to produce organic macromolecules (glucose). SUN photons 6CO2 + 6H2O ® C6H12O6 + 6O2...

PHOTOSYNTHESIS  Photosynthesis An anabolic, endergonic, carbon dioxide (CO2) requiring process that uses light energy (photons) and water (H2O) to produce organic macromolecules (glucose). SUN photons 6CO2 + 6H2O ® C6H12O6 + 6O2 glucose Photosynthesis is the process that converts solar energy into chemical energy that is used by biological systems (that means us). Photosynthesis has 3 major events: 1. Sunlight is converted into chemical energy 2. Water (H2O) is split into oxygen (O2) 3. Carbon dioxide (CO2) is fixed into sugars (C6H12O6) The photosynthesis reaction: 6 CO2 + 12 H2O + sunlight à C6H12O6 + 6 H2O + 6 O2 6 Carbon 12 Water 1 Sugar 6 Water 6 Oxygen dioxide molecules (glucose) molecules molecules molecules molecule  Plant Chef Glucose in Plants Why do plants make glucose? What is it plants do with glucose? “My masterpiece!” Glucose Glucose molecules can be broken apart for energy to power reactions. Plants can also make glucose into carbohydrate chains called polysaccharides. poly = many saccharide = carbohydrate Glucose is a simple sugar Glucose is a monosaccharide. Glucose because it is one of the smallest units of carbohydrates. mono = one saccharide There are 2 polysaccharide chains in plants: Cellulose Starch Cellulose is the structural Starch is a long term energy store that component of cell walls. the plant can use later. O O O O O O O O Question: Where does photosynthesis take place? Plants Autotrophs: self-producers. Location: 1. Leaves a. stoma b. mesophyll cells Mesophyll Chloroplast Cell Stomata  Stomata (stoma) Pores in a plant’s cuticle through which water and gases are exchanged between the plant and the atmosphere. Oxygen (O2) Carbon Dioxide (CO2) Guard Cell Guard Cell  Mesophyll Cell Nucleus Cell Wall Chloroplast Central Vacuole  Chloroplast Organelle where photosynthesis takes place. Stroma Outer Membrane Thylakoid Granum Inner Membrane  Thylakoid Thylakoid Membrane Thylakoid Space Granum Question: Why are plants green? Chlorophyll Molecules Located in the thylakoid membranes. Chlorophyll have Mg+ in the center. Chlorophyll pigments harvest energy (photons) by absorbing certain wavelengths (blue-420 nm and red-660 nm are most important). Plants are green because the green wavelength is reflected, not absorbed.  Redox Reaction The transfer of one or more electrons from one reactant to another. Two types: 1. Oxidation 2. Reduction  Oxidation Reaction The loss of electrons from a substance. Or the gain of oxygen. Oxidation 6CO2 + 6H2O ® C6H12O6 + 6O2 glucose  Reduction Reaction The gain of electrons to a substance. Or the loss of oxygen. Reduction 6CO2 + 6H2O ® C6H12O6 + 6O2 glucose  Factor affects - Photosynthesis What affects photosynthesis? Light intensity: as light increases, rate of photosynthesis increases  PHOTOSYNTHESIS What affects photosynthesis? Carbon Dioxide: As CO2 increases, rate of photosynthesis increases  PHOTOSYNTHESIS What affects photosynthesis? Temperature: Temperature Low = Rate of photosynthesis low Temperature Increases = Rate of photosynthesis increases If temperature too hot, rate drops Experimental Video – Light is necessary for photosynthesis Move the cursor in the middle of slide Experimental Video – Effect of Light on photosynthesis Move the cursor in the middle of slide  PIGMENTS IN PLANT AND ITS EXTRACTION BY LIQUID PARTITIONING  INTRODUCTION: WHAT ARE PIGMENTS v Pigments are “molecules that absorb specific wavelength (energies) of light and reflect all others.” v Pigments are coloured. v The colour we see is the net effect of all the light reflecting back at us…! v They are the substances produced by living organisms that have a colour resulting from selective colour absorption.  PIGMENTS IN PLANTS The Principal pigments in plants are 1. Chlorophyll 2. Carotenoids 3. Xanthophylls 4. Anthocyanins 5. Betalins  PRIMARY FUNCTIONS OF PIGMENT IN PLANTS ¢ Primary Function : Photosynthesis ¢ Uses green pigment chlorophyll along with several red and yellow pigments. ¢ Help to capture as much light as possible. ¢ Other functions include attracting insects to flowers to encourage pollination.  CHLOROPHYLL ü Primary pigment in plant ü Chlorophyll absorbs red and blue wavelengths of visible light ü Reflects green and yellow ü All land plants and green algae have two types of chlorophyll - Chlorophyll a and Chlorophyll b ü Kelps and photosynthetic heterokonts (Dinoflagellates) contain Chlorophyll c. ü Chlorophyll serves as fuel for photosynthesis  CAROTENOIDS q Red, orange or yellow pigments. q Function as accessory pigments in plants. q Absorb wavelength not readily absorbed by chlorophyll. q Ex: Carotene (Found in Carrots) q Lutein (Yellow pigment found in fruits and vegetables) q Lycopene (Red pigment in tomatoes)  ROLE OF CAROTENOIDS ¢ Attract pollinators and seed dispersers ¢ Accessory photosynthetic Pigment in periods of low light, ¢ Absorbs excess light energy, ¢ Antioxidant roles, ¢ Substrate for hormones  CAROTENOIDS Carotenoids are Present in - ¢ Carrot, ¢ Sweet potato, ¢ Winter squash, ¢ Pumpkin, ¢ Green leafy vegetables, ¢ Cantaloupe, ¢ Apricot  ANTHOCYANINS ¢ Literally “Flower blue” ¢ Water soluble flavanoid pigments ¢ Colour appear as red to blue, acc to pH. ¢ Occur in all tissues of higher plants but colour not noticeable. ¢ Have purple colour and are present in: ¢ Vegetables, (onions, cabbage, potatoes), red, blue & purple berries, black beans  ROLE OF ANTHOCYANINS ¢ Attractpollinators and seed dispersers ¢ Repel predators, protect cells from damage by excess light, ¢ Improve plant tolerance to stress such as drought, UV-B, ¢ Improve night vision and other vision ¢ Disorders, protect against heart disease,  XANTHOPHYLLS ¢ Fourth common class of pigments ¢ Essentially oxidized carotenes ¢ Usually red and yellow ¢ Do not absorb energy  BETALINS ü Red or yellow pigments. ü Water soluble. ü Synthesized from tyrosine. ü Never co-occur in plants having anthocyanins. Occur in: ü Beets (red and yellow ), spinach, fruit of prickly-pear cactus.  ROLE OF BETALINS ¢ Antioxidant, ¢ May protect against heart disease, ¢ Various cancers, ¢ Ulcers, ¢ Liver damage  CONCLUSIONS q Life would be nothing without Plants… q And Plants would be nothing without colors… q Green plants having chlorophyll have a vital role in photosynthesis, a process neccessary for life on earth. q Red and yellow plants and flowers having carotenoids or Betalins provide fresh fruits, and help in pollination. q Blue plants and flowers having Anthocyanins are strong antioxidants. OBJECTIVES To distinguish and study the various pigments present in plants through the process of paper chromatography.  What is Chromatography? nThe term chromatography is derived from Greek words Chroma- colour and Graphe-write. nThere are many types of chromatography: paper chromatography, column chromatography, thin layer chromatography and partition chromatography nChromatography is a technique used to separate molecules on the basis of differences in size, shape, mass, charge, solubility and adsorption properties. These techniques involve the interaction between three n components: the mixture to be separated, a solid phase and a solvent. 35  How does paper chromatography work? n In paper chromatography, the mixture is spotted onto the paper, dried and the solvent is allowed to flow along the sheet by capillary attraction. n As the solvent slowly moves through the paper, the different compounds of the mixture separate into different colored spots n The paper is dried and the position of different compounds is visualized n The principle behind the paper chromatography is that the most soluble substances move further on the filter paper than the least soluble substances n Different plant pigments can be separated by using the technique of paper chromatography 37  What is Retention Factor or Rf value? Retention factor or Rf value is applied in chromatography to make the technique more scientific than a mere analysis. The retention factor or Rf is defined as the distance travelled by the compound divided by the distance traveled by the solvent. Rf=(Distance travelled by the compound)/(Distance travelled by the solvent) 38  Theory n The chloroplast pigments can be separated and identified by paper chromatography. n Absorptive paper containing a concentrated spot of chloroplast extract is dipped into a suitable solvent. n The various pigments have different sized molecules, with the result that as the solvent ascends the absorptive paper it carries the pigments with it at different rates, the smaller molecules being more mobile than the larger ones. n In this way they can become separated from each other and can be identified by their different colours and positions. 39 Materials Required Spinach leaves, chromatography chamber, Mortar and Pestle, Ether- acetone solvent, scissor, acetone, pencil, capillary tube, spatula and scale, filter paper strips, watch glasses, thread, stapler Procedures Ø Take a few freshly plucked green spinach leaves. Ø Using scissors, cut the spinach leaves into small pieces and weigh it for 2 grams. Ø Take a measuring cylinder that contains 3 ml of acetone and pour it into the mortar. Ø Grind the spinach leaves using the mortar and pestle. Ø Place the extract into a watch glass using a spatula. Ø Take a strip of filter paper having a narrow notch at one end of the strip. Ø Take a pencil and a scale and draw a horizontal line with a pencil about 1.5 cm away from the tip of the notch. Ø Put a drop of the pigment extract in the middle of the line with the help of a capillary tube. Ø Allow the drop to dry and repeat till four or five drops are placed on the paper. Ø Take the chromatographic chamber and pour ether acetone solvent in it. Ø Fold one end of the filter paper strip and staple it. Ø Using a thread, hang the filter paper strip in the chromatographic chamber. Ø The loading spot should remain about 1.5 cm above the solvent level. Ø Leave the chromatographic chamber undisturbed for some time. Ø We can observe, as the solvent moves through the paper, it spreads the different pigments of the mixture to various distances. Ø When the solvent rises about 3/4th up the strip, remove the strip carefully and let it dry. PREPARING THE CHROMATOGRAPH CHAMBER 1. Cut a length of chromatography paper/filter paper of sufficient length to almost reach the bottom of chromatography chamber. 2. Rule a pencil line across the strip of paper 30 mm from one end. Make a cut from each side of the line to the center bottom to form an arrow head. 3. The bottom tip of the strip should almost reach the bottom of the tube when the cork is inserted. 41 Setup of chamber and appearance of pigments 42 Cut to form an arrow head 43 PREPARATION OF LEAF PIGMENTS 1. Quickly immerse leaves in water and wash it to remove any insects and dust particles. 2. Blot the leaves with towel tissue and weigh 2 gram of leaves and then cut into small pieces. 3. Transfer small pieces of leaves in mortar and pestle and grind these up with a small amount of absolute acetone (3 ml). 4. With help of capillary tube make the spot on chromatography paper with dark green paste and allow it to dry. 5. Repeat this step 4 for 4-5 times. 44 Add chloroplast extract 45 Finished pigment dot 46 SEPARATING THE PIGMENTS 1. Add the solvent to a depth of 15 mm in the chromatography paper. 2. Carefully place the filter paper/chromatography paper into the chromatography chamber. 3. Do not let the spot of extract touch the solvent. 4. Do not shake or move the tube for at least 15 minutes. 5. Remove the chromatograph from the tube when the solvent has almost reached the top of the paper. 47  Solvent front Do not let the spot of extract touch the solvent. 48 Leave the tube for 15+ minutes Solvent front 49 Remove the chromatograph 50 Look for different colours n Mark and measure furthest distance travelled by each different pigment n Record n Draw the outline of each of the pigment spots n These fade very quickly 51  Rf values n The ratio of the rate of movement of the pigment to solvent (the Rf value) can then be calculated for each pigment n Rf = distance moved by the substance from the original position distance moved by solvent from the same position n Use table to identify leaf pigments n Measure the width and depth of each of the pigment bands n Estimate the relative quantities of each pigment present 52 Identify the leaf pigments NAME COLOUR Rf Carotene Orange Xanthophyll Yellow Chlorophyll a dark-green Chlorophyll b Yellowish-green 53 Experimental Video – Separation of pigment Move the cursor in the middle of slide 54