1. Experiment No 4

2. 5 Experiment Material and Chemicals Overview Introduction Procedure Objective 1 2 3 4

3. Introduction Introduction

4.  Chlorophyll is vital for photosynthesis, which allows plants to absorb energy from light.  Good and bad absorbance from the electromagnetic spectrum.  First isolated by Joseph Bienaime Caventou and Pierre Joseph Pelletier in 1817.  Chlorophyll molecules are embedded in the thylakoid membranes of chloroplasts. Chlorophyll

5.  Two primary functions of Chlorophyll  Absorbance of light.  Transfer of that light energy by resonance energy transfer.  Photo system units and their reaction centers.  Why green and not black?  Chlorophyll is a chlorin pigment, which is structurally similar to a pigment named as Heme.  The general structure of chlorophyll a was elucidated by Hans Fischer in 1940.

6. Structure of Chlorophyll a and Chlorophyll b

7.  In plants chlorophyll is synthesized from succinyl- CoA and glycine.  Chlorosis is a condition in which leaves produce insufficient chlorophyll, turning them yellow.  Chlorosis can be caused by a nutrient deficiency of iron, called iron chlorosis or by a shortage of magnesium or nitrogen. Source of Chlorophyll and Chlorosis

8. Experiment

9.  ice bath  Mortar  Pestle  Beaker  Graduated cylinder  Funnel  Cheesecloth  Centrifuge  Centrifuge Tubes  Funnel  Sucrose Apparatus and Chemicals

10. Layout and protocol of the Experiment Layout and protocol of the Experiment

11. Preparation of an ice bath Maintaining of chilled condition throughout chloroplast isolation Removal of petiole and midrib

12. Measuring Weight and cutting of leaves Addition of 50 ml of cold, 0.5M sucrose and Grinding

13. Use of Cheesecloth in extraction process

14. Process of Suspension and re suspension

15.  Prepare an ice bath to keep mortar and pestle, 100mL beaker and graduated cylinders chilled throughout chloroplast isolation.  Remove the petiole and midrib from several spinach leaves, then weigh out 10g of leaf material.  Cut the leaf fragments into small pieces in the mortar then add 50 ml of cold 0.5M sucrose. Grind the leaves for 2 minutes to prepare a homogenate.  Line a funnel with four layers of cheesecloth. Pour the homogenate through the cheesecloth layers and collect the filtrate in a chilled 100 ml beaker. Protocol

16.  Transfer the green filtrate to two chilled 15 ml centrifuge tubes. Balance the tubes.  Centrifuge the filtrate at 500 rpm for 5 min.  Transfer the supernatant to two chilled centrifuge tubes and balance the tubes (use a Pasteur pipette to transfer the supernatant to ensure the pellet is not disturbed).  Centrifuge the samples at 1500 rpm for 7 min. While the samples are being centrifuged, take time to clean your work area and any glassware that is no longer needed.  Carefully discard the supernatant using a Pasteur pipette and gently resuspend the chloroplast pellet in 10 ml of cold 0.5M sucrose. Keep the chloroplast suspension on ice.

17. Chlorophyll content meter measuring chlorophyll content by absorption

18.  Chlorophyll is registered as a food additive (colorant), and its E number is E140.  Chefs use chlorophyll to color a variety of foods and beverages green, such as pasta.  Healing properties of chlorophyll Advantages of Chlorophyll in food industry

19. Thank You