Photosynthesis Experiments Anglo European School, Ingatestone

Chemistry of Photosynthesis In the process of photosynthesis, energy from sunlight is trapped by chlorophyll and used in the manufacture of carbohydrate from carbon dioxide and water.
light chlorophyll

2 #34 1 2 *43

#2*$432 1 2 34

This equation is a useful summary of the process, but it is a gross over simplification in its suggestion that photosynthesis takes place in one step. Photosynthesis comprises two main steps, each containing multiple complicated reactions. Photosynthesis as a Two-Stage Process a) The light dependent stage requiring light) b) The light independent stage capable of occurring in the dark but can also proceed in the light). The light dependent stage results in the formation of products which are then fed into the light independent stage. ! " light dependent stage) " # light independent stage) In the light dependent stage, chlorophyll traps the light energy of sunlight and converts it into chemical energy. !t the same time, water molecules are split into hydrogen and oxygen. The oxygen is evolved as oxygen gas and the hydrogen atoms are used to reduce carbon dioxide, with the formation of carbohydrate. The Light Dependent Stage In the late $%&'(s, )obert *ill of the +niversity of #ambridge found that when isolated chloroplasts were illuminated, they produced oxygen and acquired reducing properties. #arbon dioxide was not involved. *ill concluded that water had been split into hydrogen and oxygen. This is now called the *ill reaction, and is one of the main events in the light dependent stage of photosynthesis.
#34 4*4 4*43 34

chlorophyll

#*43

*43

,hen light strikes chlorophyll, electrons are emitted from the chlorophyll. -ome of these excited electrons are used, together with hydrogen ions from the dissociation of water, to reduce a hydrogen acceptor. nicotinamide adenine dinucleotide phosphate /!0P1) is reduced to /!0P*. The /!0P* then enters the light independent stage of photosynthesis, handing on the hydrogen, which is then used in the reduction of carbon dioxide. see over)

GHB 2004 1

Photosynthesis Experiments Anglo European School, Ingatestone

6ight 0ependent -tage

-ite. Thylakoid membranes in the chloroplasts

6ight Independent -tage

-ite. -troma of the chloroplasts

34 *43)

chlorophyll

3*

!TP
electron carrier system

splitting

*43

of water

!0P
chlorophyll

carbohydrate

1 1

/!0P

The #alvin #ycle

/!0P*

GHB 2004 2

Photosynthesis Experiments Anglo European School, Ingatestone

The Effect of Chloroplast Concentration on the Rate of the Light Dependent Reaction 0#PIP is an indicator which is red in acid and blue in neutral and alkali conditions. It is also readily reduced to form a colourless compound. ,e can used 0#PIP to test the rate of the light dependent reaction in a leaf extract containing chloroplasts. 7aterials fresh cabbage leaves $$$ ml chilled sucrose8phosphate buffer blender crushed ice $'' ml beaker Preparation of Leaf Extract $. !voiding the midrib, roughly tear 4:g of fresh cabbage leaves 4. Place the leaves in the blender and add $''ml of chilled sucrose8phosphate buffer. "lend the mixture for a minute or so, until the green tissue is pulped. *old the lid on;) &. Place the beaker in the tub and surround the beaker with crushed ice. Place two layers of muslin over the top of the beaker. Pour the mixture through the muslin to obtain the leaf extract. <eep your beaker of leaf extract in the ice and maintain it at a low temperature. Place a drop of the extract on a microscope slide, put a cover slip on top and examine under the microscope. )ecord your observations. Making Dilutions of the Leaf Extract $. Take 9 clean universal bottles and label them , , = . 4. !dd sucrose8phosphate buffer to the bottles, in the amounts indicated in the diagram opposite. &. Top the level of the liquid in each bottle up to : ml with leaf extract. #ap the bottles and place them on ice until you are ready to use them.
6eaf extract
reduced 0#PIP colourless)

4,2 dichlorophenolindophenol 0#PIP > red in acid? blue in neutral and alkali solutions)

tub muslin microscope ,clean slides and cover slips pipettes 9x universal bottles

0#PIP solution 49ml chloride buffer 2x clean test tubes $:', halogen lamp stop clock

sucrose8phosphate buffer

9 ml

& ml

4 ml

$ ml

GHB 2004

Photosynthesis Experiments Anglo European School, Ingatestone

Testing the Leaf Extract $. 6abel 2 test tubes ', $, 4, &, 9 = :. Place them in a rack and add 9.' ml of chloride buffer solution and $ .' ml of 0#PIP solution to each test tube. 4. Place the rack approximately $' cm in front of the halogen lamp, but do not turn on the lamp. 3. !dd '.: ml of sucrose8phosphate buffer to tube '. !dd '.: ml of the appropriate dilution of leaf extract to each of the other tubes. e.g. add '.: ml of diluted leaf extract to tube $) The table below shows the final contents of each tube.
T+"@
9.' ml chloride buffer solution $.' ml 0#PIP solution '.: ml leaf extract8buffer

'
sucrose8 phosphate buffer

$
diluted extract

4
diluted extract

&
diluted extract

9
diluted extract

:
neat extract

9. -hake the tubes gently to mix the contents and replace them back into the rack. :. Turn on the lamp and start the timer. )ecord the time taken for the colour of the 0#PIP to disappear from each tube. riting !p In your write5up, you should attempt to answer the following questions. ,hat conclusion can you draw about the reaction you observed from your result in tube 'A Biven your biological knowledge, how do you explain the effects of the leaf extract on the 0#PIP solution. The buffer solution used to make the leaf extract has a p* of 2.: and contains sucrose. ,hy are these properties a requirement for this bufferA ,hat problems did you encounter in determining the end5point of the reaction seen in the test tubesA *ow did this affect the accuracy and8or reliability of your investigation. *ow could you make improvements to your method to counteract these problemsA

GHB 2004 4

Photosynthesis Experiments Anglo European School, Ingatestone

"uffer )ecipes $. Stock Phosphate "uffer. 0issolve 4.Cg /a4*P39 anhydrous 7.,. $9$.%D) and 2.9g <*4P39 anhydrous 7.,. $&2.'%) in $ dm& of cold, distilled water. 4. Sucrose#Phosphate "uffer. 0issolve :$.9g of sucrose in :'' cm& of the stock phosphate buffer. #hill this in a refrigerator for several hours. &. Chloride "uffer. !dd '.&Cg potassium chloride to :'' cm& of the stock phosphate buffer. 9. DCP$P. we found that a $ in $' dilution of the solution used for vitamin # titrations gave results within a reasonable amount of time

GHB 2004 !