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LABORATORY 7 -- PHOTOSYNTHESIS

In the first exercise, we will look at how plants obtain the water and oxygen needed for photosynthesis. We will then examine florescence and absorption by chlorophyll. We will then examine in oxygen production and of the light-dependent reactions and carbon dioxide fixation of the synthesis reactions.

LEAF ANATOMY

Background: For photosynthesis to occur, water is necessary as it provides the needed electrons for the light reaction. Also, carbon dioxide is needed for the Calvin cycle. Water is brought to photosynthetic cells via the xylem and leaf veins. Carbon dioxide is obtained via stomata.

Procedure:

1. Observe a leaf cross section slide with a microscope and identify the following structures: vascular bundle, bundle sheath, xylem, phloem, and mesophyll cells.

1. Remove the epidermis from a leaf and place it on a slide with a drop of water and a coverslip and observe the stomata and guard cells.

CHLOROPHYLL PREPARATION

Background: We will extract chlorophyll extracted from spinach. We will first blend spinach leaves and filter, then extract pigments and other compounds from the chloroplast with acetone, and then will remove some non-chlorophyll components from the acetone extract using ether.

Procedure:

1. Homogenize 20 g of frozen spinach leaves in 100-ml cold water containing 0.2 g MgO.

1. Filter through eight layers of cheesecloth.

2. Add 95-ml acetone.

3. Add the acetone extract to a separatory funnel with 200 ml petroleum ether. (FLAMMABLE!)

4. Add 50 ml 10% (w/v) NaCl.

5. Drain and discard lower aqueous layer. (It may take up to 15 min for the solvent to partition.)

6. Add 120 ml of 80% (v/v) methanol to the funnel and shake.

7. Add just enough 10% NaCl to form two layers, then drain and discard lower layer.

8. Add 20 ml 10% NaCl., drain aqueous phase, and repeat.

9. Add 3 g anhydrous Na2SO4.

FLUORESCENCE OF CHLOROPHYLL

Background: When an atom absorbs a photon, the photon's energy is used to excite one of the atom's electrons to a higher orbital. This is an unstable state, and the electron usually returns to ground level, releasing its energy as heat or light (fluorescence). In photosynthesis, the electron is captured by the primary acceptor, and the electron's energy is given to the electron transport chain or NAD to form NADH. In this experiment, we will excite the chlorophyll preparation sample with white light. The reaction center has most likely be removed from the chlorophyll in this preparation. What do you think will happen to the electrons of chlorophyll when they excited by the light?

Procedure:

1. Place a tube of chlorophyll in front of a bright light.

1. View the tube from the side and record observations.

VISIBLE LIGHT SPECTRUM AND CHLOROPHYLL ABSORPTION

Background: White light is actually composed of many wavelengths of light that make up the visible spectrum. Chlorophyll will absorb certain colors of the spectrum and use that energy for photosynthesis, while letting other colors pass through. We will examine these phenomena a spectroscope (separates white light into its component colors) and a spectrophotometer (measures the intensity of light at varying wavelengths).

Procedure 1:

1. View white light through a spectroscope and record your observations.

2. Now insert a chlorophyll sample between the spectroscope and the light and record observations.

Procedure 2:

1. Place a piece of white paper in a cuvette.

2. Insert the cuvette into the spectrophotometer and adjust to 400 nm.

3. Looking down into the spectrophotometer, record the color of light at 400 nm.

4. Repeat at 15 nm intervals to 700 nm.

Procedure 3:

1. Adjust the wavelength of the spectrophotometer to 400 nm

1. Adjust a spectrophotometer to zero absorbance using ether as a blank.

10. Add 4 ml of your chlorophyll preparation to a cuvette.

2. Record the absorbance.

3. Increase the wavelength of the spectrophotometer by 15 nm.

11. Repeat steps 2 through five, until 700 nm.

OXYGEN RELEASE BY PLANTS

Background: During the light reaction of photosynthesis, electrons from chlorophyll that are lost when excited by light are replaced by electrons from water. The waste product of this reaction is oxygen gas, which is released from the plant. In this exercise, we will examine the oxygen output of plants using a volumeter. To negate carbon dioxide uptake by plants, we will add a substitute carbon soluble source, sodium bicarbonate (NaHCO3). Remember, O2 is also used for respiration.

Procedure:

1. Place some plant leaves into a test tube and fill with a 3% NaHCO3 solution.

2. Place a stopper containing a bent glass rod on top, making sure the solution enters the glass rod. If the solution does not go into the glass tube, and some more to the test tube.

4. Place next to a light source and note the position of fluid in the glass rod. (Do not bump or readjust the apparatus, as this will move the position of the fluid).

5. After 15 minutes, measure the distance the fluid has moved. Record this as net oxygen release.

6. Wrap tube in foil and wait 10 minutes, then record position of fluid in the glass rod.

7. After 15 minutes, measure the distance the fluid has moved. Record this as net O2 consumption.

8. Gross photosynthetic O2 production = net O2 release + net O2 consumption.

CARBON DIOXIDE UPTAKE BY PLANTS

Background: Animals give off carbon dioxide as a waste product. Plants take up CO2 for fixation in the Calvin cycle. Phenol red is a pH indicator that is red above pH 7 and yellow below pH 7.

Procedure:

1. Obtain a beaker of slightly basic water containing phenol red, and using a straw, blow bubbles into the water until it turns yellow.

3. Add plant leaves and place in front of a light source, and observe the color of the water every 10 min or so, until it turns red.

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