flourescence of chlorophyll€¦ · web viewthis feat is accomplished because of the ability of...

Wilkes University Science in Motion

Fluorescence of Chlorophyll

PA S. T. & E. Standards:

3.1.10.A2. Explain cell processes in terms of chemical reactions and energy changes.

3.2.C.B3. Explain the differences between an endothermic process and an exothermic process.

3.2.10.B5. Describe the components of the electromagnetic spectrum.3.2.10.B6. Explain how the behavior of matter and energy follow predictable

patterns and are defined by laws.

Introduction and Background:

Photosynthesis:

Basic Equation: 6 CO2 + 6 H2O → C6H12O6 + 6 O2

In the world of life, it is the green plants, the producers, which are able to produce the sugars that then become the major food source for animal life. This feat is accomplished because of the ability of plants to absorb light energy and convert it into chemical energy in the form of bonds within ATP in a process called photosynthesis. In fact, the word photosynthesis comes from the Greek words photo, meaning “light” and synthesis, meaning “putting it all together.” Usually plant cell organelles called chloroplasts are involved; within the chloroplast membranes plant cells have developed thousands of structures called photosystems, and each chloroplast contains thousands of pigment molecules, the main type is chlorophyll. Chlorophyll and other pigments are responsible for absorbing certain photons (violet, blue, and red wavelengths) of light from the visible spectrum of sunlight or artificial light.

Photosynthesis occurs in two stages. These stages are called the light reaction and the dark reaction. As expected, the light reactions take place in the presence of light. The dark reactions do not require direct light, however dark reactions in most plants occur during the day. Light reactions occur mostly in the thylakoid stacks of the grana. Here, sunlight is converted to chemical energy. Since light energy cannot be destroyed, it is converted to another state. In this case electrons in the pigment molecules are “energized” by moving to higher molecular energy levels. The “energized” electrons fall back to a lower energy state (original molecular energy levels) almost immediately and the energy is given off. Some of the energy is trapped by special chlorophyll molecules within the chloroplast’s photosystems; this energy is used to initiate a series of electron



transfers which eventually lead to the production of two high energy molecules in the form of ATP and NADPH, and oxygen gas (through the splitting of water). Oxygen is released through the stomata.

Both ATP and NADPH are used in the dark reactions to produce sugar. Dark reactions occur in the stroma. Carbon dioxide is converted to sugar using ATP and NADPH. This process is known as carbon fixation or the Calvin cycle. Carbon dioxide is combined with a 5-carbon sugar creating a 6-carbon sugar. The 6-carbon sugar is eventually broken-down into two molecules, glucose and fructose. These two molecules make sucrose or sugar, which is the primary energy food source for nearly all plants and animals in the world. The remainder of the unused energy is given off as heat and/or light.

Light and the Electromagnetic Spectrum:

White light is separated into the different colors (= wavelengths) of light by passing it through a prism. Wavelength is defined as the distance from peak to peak (or trough to trough). The energy is inversely proportional to the wavelength: longer wavelengths have less energy than do shorter ones.

Wavelength and other aspects of the wave nature of light. Image from Purves et al., Life: The Science of Biology, 4th Edition, by Sinauer Associates (www.sinauer.com) and WH Freeman (www.whfreeman.com).

The order of colors is determined by the wavelength of light. Visible light is one small part of the electromagnetic spectrum. Longer wavelengths of visible light are towards the red side of the spectrum, and shorter wavelengths are towards the violet side. Wavelengths longer than red are referred to as infrared, while those shorter than violet are ultraviolet.


http://www.whfreeman.com/

http://www.sinauer.com/

http://www.botany.hawaii.edu/faculty/webb/BOT311/BOT311-00/PSyn/PsynDark1.htm


The electromagnetic spectrum. Image from Purves et al., Life: The Science of Biology, 4th Edition, by Sinauer Associates (www.sinauer.com) and WH Freeman (www.whfreeman.com).

A pigment is any substance that absorbs light. The color of the pigment comes from the wavelengths of light reflected (in other words, those not absorbed). Chlorophyll, the green pigment common to all photosynthetic cells, absorbs all wavelengths of visible light except green, which it reflects to be detected by our eyes. Black pigments absorb all of the wavelengths that strike them. White pigments/lighter colors reflect all or almost all of the energy striking them. Pigments have their own characteristic absorption spectra, the absorption pattern of a given pigment.

Chlorophyll is a complex molecule. Several modifications of chlorophyll occur among plants and other photosynthetic organisms. All photosynthetic organisms have chlorophyll a. Accessory pigments absorb energy that chlorophyll a does not absorb. Accessory pigments include chlorophyll b (also c, d, and e in algae and protists), xanthophylls, and carotenoids (such as beta-carotene). Chlorophyll a absorbs its energy from the Violet-Blue and Reddish Orange-Red wavelengths, and little from the intermediate (Green-Yellow-Orange) wavelengths. Chlorophyll b absorbs its energy from the Indigo-Blue and reddish orange-Red wavelengths. In green leaves the quantities of xanthophylls and carotenoids are much lower proportionately to the chlorophyll pigments.


http://www.emc.maricopa.edu/faculty/farabee/biobk/BioBookglossC.html#carotenoids

http://www.emc.maricopa.edu/faculty/farabee/biobk/BioBookglossC.html#chlorophyll%20b

http://www.emc.maricopa.edu/faculty/farabee/biobk/BioBookglossC.html#chlorophyll%20a

http://www.emc.maricopa.edu/faculty/farabee/biobk/BioBookglossC.html#chlorophyll




In this lab, you will be working with chlorophyll extracts taken from the chloroplasts in green plant leaf cells. When illuminated, the chlorophyll molecules will absorb certain wavelengths of light and become “excited.” Electrons in the excited molecules will immediately fall back to the original molecular energy state since this state is unstable. Since there are no photosystems in the extract, all the absorbed energy will be released as heat and low energy light (dull red). This is referred to as fluorescence and the sum of the heat and light given off will equal the amount of energy absorbed from the light source originally. So if a solution of chlorophyll isolated from chloroplasts is illuminated, it will fluoresce in the red part of the spectrum and also give off heat.

Guiding Questions:

1. Why is photosynthesis such an important process?2. How is light energy from the sun related to future sugar production by

green plants?3. What function do chloroplasts serve in this process?4. Is photosynthesis an endergonic or exergonic reaction?5. Describe the location and major events of the light reaction of

photosynthesis.6. Describe the location and major events of the dark reaction of

photosynthesis.7. Describe the main colors of the visible part of electromagnetic spectrum-

which color(s) have longer wavelengths? Shorter?8. What is the relationship between wavelength and energy?



9. What color(s) would you expect to produce the highest absorbencies when the chlorophyll extract is placed in the spectrophotometer? Lowest?

10.Explain why “fluorescence” occurs in this experiment.

Vocabulary:

1. Absorption is the process by which light energy is retained without reflection or transmission when passing through a medium.

2. Carotenoids are accessory pigments in plants that have a yellow-orange color.

3. Chlorophyll is a green pigment which constitutes much of the color of green leaves. The two main forms are chlorophyll a and b.

4. A Chloroplast is a plant organelle that contains photosystems and chlorophyll and is important in the photosynthetic process.

5. An Endergonic reaction results in a net absorption of energy from the surroundings; this energy can be stored in chemical bonds.

6. An Endothermic reaction absorbs heat from the surroundings (gets colder). 7. An Exergonic reaction results in a net release of energy into the surroundings; this can occur

when chemical bonds are broken.8. An Exothermic reaction release heat to the surroundings (gets hotter). 9. Fluorescence occurs when chloroplasts have been ground in acetone

and removed from living systems after being exposed to a light source. Absorbed energy is released as heat and low energy light (dull red) is observed. This reaction is exergonic and exothermic.

10. The Grana are structures in the chloroplast where the light reactions occur.

11. Photosynthesis is the process by which plants absorb light energy and convert it into chemical energy in the form of bonds within ATP which is then used to produce sugars to provide energy for living organisms.

12. Photosystems are functional and structural units of protein complexes involved in photosynthesis that together carry out the primary photochemistry of photosynthesis: the absorption of light and the transfer of energy and electrons.

13. A Spectrophotometer is an instrument that passes a beam of light through a liquid to be studied. The amount of light absorbed by the liquid (or transmitted) at different wavelengths can be measured.

14. The Stroma is the part of the chloroplast where the dark reactions occur.

15. Transmittance is the process by which light passes through a medium.

16. Wavelength is the distance from peak to peak (or trough to trough) in a light wave.

17. Xanthophylls are accessory pigments in plants that have a dull yellow color.

Materials: (per group) 8-10 green spinach leaves 2 gauze squares flashlight

2 small test tubes2 test tubes with caps mortar & pestle filter paper acetone



Funnel graduated cylinder plastic spoongloves

safety goggles test tube rack green food coloringspectrophotometer

Safety Notes:1. Safety glasses must be worn during this lab as acetone can irritate

eyes.2. Gloves should also be worn to protect hands. 3. Although no flames will be used it should be noted that acetone is

flammable. 4. Chlorophyll can stain your clothing, so be careful of spills. Wear a

lab coat or apron if available. 5. Quickly replace caps on acetone bottles and test tubes as you work

to minimize the fumes. Also keep acetone on ice when not using to minimize fumes.

6. Acetone/spinach test tube products should be placed in a special container and returned to Wilkes for disposal.

7. Place the filter paper/gauze/spinach debris into the garbage or a bag provided.

8. Clean your lab table and wash your hands before leaving the room.

Procedure:1. Grind 8-10 spinach leaves using a mortar and pestle.2. Add 20 mls of acetone to the ground leaves. This should produce

about 10 mls of a deep green liquid after filtering. You may add an additional 5 mls of acetone to the leaves and mix if amount of liquid is less than 10 mls.

3. Fold the filter paper and place it into the funnel on top of a test tube placed in one slot of the rack.

4. Add two 2- layered gauze squares, which you will fold in half on top of the filter paper.

5. Guiding the liquid with a spoon, filter the chlorophyll extract into a test tube. You may gently push to allow more liquid to filter through, but be careful not to force leaf debris or make a hole in the filter paper. If this happens you will need to re-filter as the chlorophyll extract should be dark but clear for the fluorescence to be seen.

6. Place the cap on the tube to decrease acetone fumes. If possible, dim room lights for this step and shine the flashlight at a 90 degree angle to the test tube.

7. Observe the fluorescence- you should see a dull red circle on the outside of the test tube glass. Feel with your hand to see if any heat is evident.



8. If you have time, place 10 mls of tap water and one drop of green food coloring into a second test tube. Add the cap and if possible, dim room lights for this step and shine the flashlight at a 90 degree angle to the test tube.

9. Answer the questions on the Student Data Sheet.10. Continue the lab with the Absorbance of Spinach Chlorophyll

Extract using a Spectrophotometer.



Fluorescence of ChlorophyllStudent Data Sheet

Name: Date:

Observations:

1. What color does the chlorophyll fluoresce?

2. What color did the green food coloring show?

Discussion:

1. Why did you have to grind the spinach leaves using the mortar and pestle?

2. Why were the leaves mixed with acetone?

3. Explain in scientific terms why the fluorescence occurred.

4. Discuss why the results with the green food coloring were different from the observations with the chlorophyll.

5. Explain how the results would be different in a living plant and what chemical processes would be occurring.

6. Is photosynthesis an exergonic or endogonic reaction? Is the fluorescence of chlorophyll exergonic or endogonic? Would fluorescence be exothermic or endothermic? Defend your choices for each question.



Absorbance of Spinach Chlorophyll Extract

If time and equipment allow, the absorption spectrum of spinach pigments can be determined and plotted by using your pigment extract and a spectrophotometer. This instrument passes a beam of light through a liquid to be studied. The amount of light absorbed by the liquid (or transmitted) at different wavelengths can be measured. Students should measure and plot the absorbance of the pigment extract from 400 to 700 nanometers at 25 nanometer wavelength intervals. Every time you change the wavelength, you must reset the spectrophotometer to “0 ABS 100 % T” with the “blank” cuvette (containing acetone) before reading the absorbance of the sample cuvette. Always wipe cuvettes before placing into the spectrophotometer chamber.

Procedure:

1. Fill one cuvette 3/4 full with chlorophyll solution.

2. Fill the second cuvette with acetone to be used as a blank.

3. Set the spectrophotometer to 400 nm by pushing nm button up or down.

4. Wipe the blank cuvette with a Kimwipe. Place into chamber and set to zero absorbance (100% transmittance) using the “0 ABS 100% T” button.

5. Remove the blank, wipe the sample cuvette with a Kimwipe, and place the solution into the spectrophotometer. Do not push any buttons and record the absorbance on the Data Table.

6. Reset the wavelength to 425 nm, and repeat steps 4 and 5.

7. Repeat steps 4 and 5, recording absorbencies at every 25 nm, using this technique until you reach 700 nm.

8. Graph the data to determine the wavelength(s) of maximum absorbance and answer the questions on Student Data Sheet.



Name: Date:

Color of solution:

Data Table

Wavelength (nm)

400 425 450 475 500 525 550 575 600 625 650 675 700

Absorbance (A)

Using a computer program or graph paper, graph your results. Be sure give a Title to your graph and correctly label x and y Axes with Terms and Units.

Questions:

1. What is the independent variable on your graph? What is the dependent variable?

2. What is (are) the wavelength(s) of maximum absorbance for the chlorophyll solution? (There may be more than one peak).

3. What color(s) of light corresponds to the wavelength(s) of maximum absorbance for the chlorophyll solution?

4. What is(are) the wavelength(s) of minimum absorbance for the chlorophyll solution?

5. What color(s) of light correspond(s)to the wavelength(s) of minimum absorbance for the chlorophyll solution?

6. Why is it necessary to wipe the cuvettes?



OPTIONAL EXCEL GRAPHING ACTIVITY Fluorescence of Chlorophyll*

1. Open Excel workbook.2. Type WAVELENGTH (nm) in second column and record units from

400-700.3. Type ABSORBANCE (A) in fourth column and numbers obtained.4. Highlight entire typed numbers area.5. Click on Insert tab at top. 6. Click on graph type- Scatter –choose second picture. 7. Click on Chart Tools-layout-Chart Title-Above chart-Type Title:

Spinach Absorbance vs. Wavelength.8. Click on Axes Titles- Horizontal(x-axis) (Title below)-

WAVELENGTH (nm); 9. Axes Titles- Vertical (y-axis)- Rotate Title- Type ABSORBANCE (A).10. Click on Series on actual graph- click delete. (This is if you

have two different lines on same graph). 11. Save your work (just in case); you may keep graph in view.12. Go to Google Images; Type any

Spinach/Plant/Popeye/Salad photo/drawing you would like. 13. Choose a picture by left clicking. You will see a small photo

at top left of screen. (No not enlarge). Right click and then left click on Save Picture As and save as (Spinach photo, etc.) to desktop or documents.

14. Go back to your graph and click on the background.15. On toolbar at top click on Format then Shape Fill-Click on

Picture.16. Choose your photo, click on it then Insert.17. You will get your spinach picture behind the graph; save or

print your work.

*This procedure may also be used any other graphing lab such as the Catalase Enzyme Lab- just change x & y axes to Hydrogen peroxide % (x) and Time (Sec.) (y) and find pictures of potatoes, Mr. Potato, etc. This is an older Excel version; adjust for your present computer.

References:

Bailey, R. 2011. Photosynthesis. {Retrieved 26 Feb. 2011}. http://biology.about.com/od/plantbiology/a/aa050605a.htm.Fluorescence of chlorophyll. 2008. Juniata College: Huntingdon (PA): www.scienceiinmotion.org. Miller, K., Levine, J. Biology. Boston (MA): Pearson Prentice Hall; 2006.


http://www.scienceiinmotion.org/

http://biology.about.com/od/plantbiology/a/aa050605a.htm


Terzaghi, W. 1 Mar 2011. Wilkes University (PA): Interview on endogonic and exergonic reactions.The electromagnetic spectrum. Image from Purves et al., Life: The Science of Biology, 4th Edition, by Sinauer Associates (www.sinauer.com) and WH Freeman (www.whfreeman.com).Wavelength and other aspects of the wave nature of light. Image from Purves et al., Life: The Science of Biology, 4th Edition, by Sinauer Associates (www.sinauer.com) and WH Freeman (www.whfreeman.com).






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