250-7053s ap lab 4 plant pigments - wikispacesap...ap biology lab #4 plant pigment and ... drop of...

For many centuries, people believed that the increase in the size of plants was caused by the intake of material from the soil. It was not until a Belgian physician, Jan Baptista van Helmont (circa 1577–1644), performed an experiment that demonstrated conclusively what we accept today: the increase in the size of a plant is not due simply to the plant obtaining a mystery substance from the soil; plants gain what they require through the process of photosynthesis. Photosynthesis uses energy from light captured by photosynthetic pigments. Water molecules are split in the process; the plants fix car-bon from carbon dioxide into glucose and fructose chains and oxy-gen, a byproduct, is released. In many plants the sugars then combine to form long chains known as starches. Many plants store their photo-synthetic products this way. Most plants produce their own organic molecules and do not need to obtain them from another organism; through photosynthesis these plants are autotrophs. However, not all plants are able to carry out photosynthesis. Many parasitic plants, without photosynthetic pig-ments, rely entirely on other, host species for nourishment; these plants are heterotrophs. Examples of photosynthetic pigments are chlorophyll a, chlorophyll b, and carotene. Green plants usually have high chlorophyll content; in a typical plant, approximately three-quarters of the chlorophyll is chlorophyll a and the rest is chlorophyll b. The presence of other pig-ments becomes apparent in some plants in the fall when the chloro-phyll no longer masks their presence. Some plants are high in pig-ments that mask the chlorophylls during the whole growing season (e.g., red cabbage remains red because of the presence of anthocya-nin). Each pigment absorbs light of a specific range of wavelengths. Chlorophyll a, the primary pigment used in photosynthesis, absorbs blue and red light. Chlorophyll b absorbs light in the blue-green and orange-red portions of the spectrum. Carotenoids absorb light in the blue and blue-green regions.

BACKGROUND

Copymaster. Permission granted to make unlimited copies for use in any one school building. For educational use only. Not for commercial use or resale. 1

AP Biology Lab #4 Plant Pigment and

Photosynthesis Lab Activity

Student Study Guide

TM

DID YOU KNOW? Chlorophyll a not only pro-duces all oxygen for respira-tion on Earth, but also pro-duces oxygen for the forma-tion of ozone, which protects the planet from dangerous ultraviolet light.

© 2002 WARD’S Natural Science Establishment, Inc. All Rights Reserved

Plants lose water in two ways: from inside the leaves through transpi-ration, and from the surface of the leaves or from the soil through evaporation. The total loss of water from both sources is called evapotranspiration. To reduce the water lost through transpiration, leaves can close their pores, or stomata, using special cells called guard cells. However, this also limits the exchange of air and other gases, which enter and exit the leaves through the stomata, so photo-synthesis might also be limited. Plants may adapt to this by capturing sunlight’s energy during the day, storing the energy until night, and exchanging gases at that time when water loss is lower due to the lower temperatures. This is known as the “light phase” of photosynthesis. The “dark phase” of photosynthesis can occur during the light or the dark, while the light phase can occur only when wavelengths of light that will stimulate photosynthetic pigments are present. The first step in the conversion of light to chemical energy is the ab-sorption of light by a pigment system. In all photosynthetic cells, ex-cept photosynthetic bacteria, the pigment system includes chloro-phyll a. Chlorophyll a occurs in all photosynthetic eukaryotes and in prokaryotic blue-green algae. In vascular plants, bryophytes, green algae, and euglenoid algae, chlorophyll b, an accessory pigment, is also found. In the leaves of green plants, chlorophyll b generally con-stitutes about one-fourth the total chlorophyll content. Chlorophyll b absorbs light wavelengths different from chlorophyll a, extending the range of light that can be used for photosynthesis. It shares with chlo-rophyll a the ability to absorb light energy and produce an excited state in the molecule. The excited molecule of chlorophyll b transfers its energy to a molecule of chlorophyll a, which then transforms it into chemical energy. Chlorophyll c or chlorophyll d takes the place of chlorophyll b in other groups of plants. Carotenoids are also accessory pigments involved in the capture of light energy in photosynthesis. Carotenoids are red, orange, or yellow fat-soluble pigments found in all chloroplasts and also, in association with chlorophyll a, in the prokaryotic blue-green algae. There are two classes of carotenoids: those that do not contain oxygen are called carotenes, and those that do contain oxygen are called xanthophylls. In green leaves, the color of the carotenoids is masked by the much more abundant chlorophylls; in some tissues, such as those of a ripe tomato or the petals of an orange flower, the carotenoids predomi-nate. During autumn, chlorophyll begins to break down as the leaf begins to senesce, allowing the carotenes and xanthophylls to display the brilliant colors we associate with fall. Carotenoids, which are not water soluble, are not found free in the cytoplasm, but like the chlorophylls are bound to proteins within the plastids. Only certain carotenoids serve as accessory pigments, but these are important for the overall process of photosynthesis in the green plant.

2 © 2002 WARD’S Natural Science Establishment, Inc. All Rights Reserved Copymaster. Permission granted to make unlimited copies for use in any one

school building. For educational use only. Not for commercial use or resale.

DID YOU KNOW? Pliny the Elder (23-79AD) first reported the separation of dyes on papyrus.

DID YOU KNOW? The word chromatography comes from the Greek: chromatos, meaning color and graphe, meaning writing.

To measure the percentage of each wavelength of light absorbed by a pigment (the absorption spectrum), a spectrophotometer is used. A spectrophotometer directs a beam of light of a specific wavelength at the object to be analyzed, and records what percentage of the light of each wavelength is absorbed by the pigment or pigment system. The absorption spectrum is different from the action spectrum, which graphs the efficiency of different wavelengths of light in promoting a given photoresponse, as in photosynthesis or phototropism. A spectrophotometer can also be employed to measure the rate of photosynthesis. In the dye-reduction technique, the compound DPIP (2,6-dichlorophenol-indophenol) is substituted for NADP (nicotinamide adenine dinucleotide phosphate), the primary electron-accepting compound of photosynthesis. As DPIP is reduced by chloroplasts in the presence of light, it changes from blue to colorless. The spectrophotometer measures the increase in light transmittance over time, and thus indicates the rate of photosynthesis. Chromatography is a technique for analyzing or separating mixtures of gases, liquids, or dissolved substances such as chlorophyll pig-ments. There are many types of chromatography, including column, paper, thin-layer, gas–liquid, ion exchange, and gel filtration. In gen-eral, all types of chromatography involve two distinct phases: the sta-tionary phase and the mobile phase. The separation depends on com-petition for molecules of sample between the mobile phase and the stationary phase. Column, paper, and thin-layer chromatography can be used to sepa-rate extracted plant and algal pigments. In paper chromatography, the separation takes place through absorption and capillary action. A drop of the mixture to be separated is placed at the bottom of a strip of chromatography paper, which holds the substance by absorption. The chromatography paper and developer are then placed in a cham-ber. The paper acts as a wick, drawing the developer upward by cap-illary action and dissolving the mixture as it passes over it. The com-ponents of the spotted mixture move upward at differing rates, deter-mined by both the solubility of the pigments in the solvent and their relative attractions to the cellulose of the chromatography paper, re-sulting in the different pigments in the mixture showing up as col-ored streaks or bands. The pattern formed on the paper is called a chromatogram. To establish the relative rate of migration for each pigment, the Rƒ value of each pigment is calculated. The Rƒ value represents the ratio of the distance a pigment moved on the chromatogram relative to the distance the solvent front moved. It is calculated using the following formula: Rƒ = Distance Substances Traveled Distance Solvent Traveled Any molecule in a given solvent matrix has a uniquely consistent Rƒ . The Rƒ value is used by scientists to identify molecules.



DID YOU KNOW? Forensic scientists use certain chromatography techniques to analyze drugs and the dyes in cloth fibers.

• Separate plant pigments using chromatography • Calculate Rƒ values for four different plant pigments • Measure the rate of photosynthesis using a spectrophotometer MATERIALS NEEDED PER GROUP 1 Vial 1 Chromatography paper strip 4 Cuvettes 15 Pipets Chromatography solvent, 10 ml Quarter Spinach Indophenol solution, 3 ml Phosphate buffer, 4 ml Prepared boiled chloroplast Prepared unboiled chloroplast Parafilm Test tube rack Spectrophotometer Floodlight, 100 W Aluminum foil PART A: CHROMATOGRAPHY OF PLANT PIGMENTS

1. Obtain a chromatography vial from your teacher and label it with your initials using a permanent marker or wax pencil.



OBJECTIVES

MATERIALS

PROCEDURE

FUME HAZARD

Protective gloves, goggles, and an apron should be worn throughout this activity.

When working with the chromatography solvent, use a chemical hood or proper ventilation. Refer to the en-closed MSDS for disposal instructions for the solvent.

DID YOU KNOW? A typical plant cell may con-tain as many as fifty chloroplasts.

2. Go to a fume hood or a well ventilated area and remove the cap

from a chromatography vial. Using a disposable pipet, add 1 ml of chromatography solvent to the vial. Replace the cap and allow the chamber to sit undisturbed until needed in Step 7. This will ensure that the atmosphere within the vial is saturated with sol-vent vapors (equilibration).

3. Obtain a chromatography strip from your instructor. Be sure to

handle the chromatography strip by the edges. Do not touch the surface of the strip. The oils from your fingers can interfere with the chromatogram.

4. Measure 1.5 cm from one end of the chromatography strip and

draw a pencil line across the width of the strip. 5. Use a pair of scissors to cut two small pieces below the pencil line

to form a pointed end (Figure 1). The pointed end will be referred to as the bottom end of the chromatogram.

6. Obtain a fresh piece of spinach and place it over the line on the

chromatography strip. Rub the ribbed edge of a coin (dime or quarter) over the spinach leaf to extract the pigments. Repeat 5 to 10 times with different portions of the spinach leaf, making sure you are rubbing the coin over the pencil line (Figure 2).

7. Remove the cap from your chamber and carefully place the chro-

matography strip into the vial so that the pointed end is barely immersed in the solvent. Make sure not to immerse the pigments in the solvent (Figure 3).

8. Cap the vial and leave it undisturbed. Observe as the solvent is

drawn up the chromatography strip by capillary action. You will be able to see the plant pigments separating along the strip. No-tice the different colors that you see during this process.

9. When the solvent reaches approximately 1 cm from the top of the

strip, remove the cap from the vial. Using forceps, remove the strip from the vial. This is a chromatogram.



Step 2 should be performed under a chemical fume hood or with proper ventilation. !

CAUTION

Steps 7-11 should be performed under a chemical fume hood or with proper ventilation. !

CAUTION

Figure 1

Figure 2

Figure 3

10. Immediately mark the location of the solvent front. The solvent will evaporate quickly.

11. In Table 2 in the Analysis section, list the pigment colors that you

observe. Once the strip has dried, mark the middle of each pig-ment band on your chromatography strip with a pencil.

12. Using a metric ruler, measure the distance from the original pencil

line with the spinach extract to the solvent front and each mark you have made for each pigment band (Figure 4). Record these distances in millimeters in Table 1 in the Analysis section.

13. Calculate the Rf value for each pigment on your chromatogram

using the following formula and record your answers in Table 1. Rf = Distance pigment traveled Distance solvent traveled 14. Follow your teacher’s instructions for proper disposal of all

materials. Part B: Photosynthesis / The Light Reaction 1. Turn on the spectrophotometer to warm it up. Refer to the in-

struction manual for your spectrophotometer to determine how long it will take to warm up. Adjust the wavelength control knob to 605 nm.

2. Set up an incubation area with a floodlight, a flask of water for a

heat sink, and a test tube rack or similar sample holder (Figure 5).

6 © 2002 WARD’S Natural Science Establishment, Inc. All Rights Reserved

WASTE � DISPOSAL

Refer to the MSDS for proper disposal of chromatography solvent.

Figure 4

Copymaster. Permission granted to make unlimited copies for use in any one school building. For educational use only. Not for commercial use or resale.

Figure 5

3. With a glass-marking pen, label four cuvettes at the very top rim

1, 2, 3, and 4. Wipe down the outside of each cuvette with lens paper to remove any fingerprints and oils.

4. Wrap the outside of cuvette 2 with foil and make a foil cap for the

top to keep the chloroplast solution in complete darkness. It will be used as the experiment control.

5. Add the following: 6. Zero the spectrophotometer by adjusting the amplifier control

knob until the meter reads 0% transmittance. 7. Obtain 2 ml of boiled chloroplast and 2 ml of unboiled chloro-

plast. Transfer three drops of unboiled chloroplasts to cuvette 1. Cover the top of the cuvette with parafilm and invert to mix.

8. Place cuvette 1 in the sample holder of the spectrophotometer. Be

sure the cuvette is wiped clean and is inserted into the sample holder in the same direction every time to ensure consistent read-ings. Adjust the light control knob until the meter reads 100% transmittance.

Cuvette 1 will be used to recalibrate the spectrophotometer between readings.

9. Transfer three drops of unboiled chloroplasts into cuvette 2 with

a pipette. Immediately cover the cuvette with parafilm and invert to mix.

10. Remove the foil sleeve and foil top, and place the cuvette in the

sample holder. Read the percent transmittance and record it as Time 0 in Table 2 in the Analysis section of the lab.

11. Place the cuvette back into its foil sleeve and cover it with the foil

top. Place the cuvette in the test tube rack between percent trans-mittance readings.

12. Repeat the readings at 5, 10, and 15 minutes. Be sure to cover and

mix the cuvette before each reading. Read the percent transmit-tance and record the data in Table 2.


NOTE

DID YOU KNOW? Many scientists believe that chloroplasts and mitochon-dria have evolved from prokaryotic “trespassers” that invaded other, larger cells, a speculation known as the endosymbiotic theory.


1 2 3 4

Phosphate Buffer 1 ml 1 ml 1 ml 1 ml

Distilled Water 4 ml 3 ml 3 ml 3 ml

DPIP — 1 ml 1 ml 1 ml

Cuvette

Be sure to use cuvette 1 to check and recalibrate the spec-trophotometer between each reading to ensure consistent results.

13. Transfer three drops of the unboiled chloroplasts into cuvette 3. Immediately cover the cuvette with parafilm and invert to mix.

14. Place the cuvette in the sample holder. Read the percent transmit-

tance and record it as Time 0 in Table 2. 15. Place the cuvette in the test tube rack between percent transmit-

tance readings. 16. Repeat the readings at 5, 10, and 15 minutes. Be sure to cover and



17. Transfer three drops of the boiled chloroplasts into cuvette 4. Im-

mediately cover the cuvette with parafilm and invert to mix. 18. Place the cuvette in the sample holder. Read the percent transmit-

tance and record it as Time 0 in Table 2. 19. Place the cuvette in the test tube rack between percent transmit-

tance readings. 20. Repeat the readings at 5, 10, and 15 minutes. Be sure to cover and




NOTE

NOTE

DID YOU KNOW? The cartoon character “Popeye” attributes his amaz-ing strength to a daily diet of spinach. In fact, spinach is an excellent source of both vita-min A and folic acid, as well as iron and potassium.


NOTE

ANALYSIS



Band Number Pigment Migration Distance

(mm) Rf Value

1 (top)

2

3

4

—

Table 1 Chromatography of Plant Pigments

Table 2 % Transmittance

Cuvette 0 min. 5 min. 10 min. 15 min.

2 (Dark)

3 (Unboiled)

4 (Boiled)

Time

WARD’S Name: AP Biology Lab #4 Group: Plant Pigments and Photosynthesis Date: Lab Activity

WARD’S Name: AP Biology Lab #4 Group: Plant Pigments and Photosynthesis Date: Lab Activity



1. Which pigment migrated the farthest? Why? 2. During summer, leaves are generally bright green. What would you hypothesize that this indicates

about the role of green light wavelengths, chlorophyll, and the photosynthetic process? 3. Design an experiment to test your hypothesis from the question above. Describe your experiment or

draw a picture of your experimental setup. If you draw your setup, be sure to label each component and its purpose.

4. Why do leaves change color in autumn? 5. What is the function of the chlorophylls in photosynthesis?

ASSESSMENT



6. What are the accessory pigments and what are their functions? 7. In your experiment you used paper chromatography to separate various pigment molecules. There

are several other chromatographic techniques employed to separate a variety of molecules. Research another form of chromatography and describe it below.

8. What does the Rƒ value represent? If you were to perform your experiment on a chromatography

strip that was twice the length of the one you used, would your Rƒ values still be the same? 9. Shown below is a strip of chromatography paper and a list of five molecules and their various Rƒ

values. Assuming the solvent front traveled 54 mm, place each molecule where it would be found on the finished chromatogram.

Molecule Rƒ value

N,N-fictionol .41

cis-2,4-pretendium .20

dl-made-upelene .72

d(+)-tetra-imaginase .91

polysynthetic acid .78

Solvent front



10. What is the absorption spectrum? 11. In what way is the spectrophotometer used to measure the rate of photosynthesis? 12. Below is graphic representing the steps in the process of photosynthesis. Place the components from

the list into their proper place on the graphic.

H2O O2

CO2 ATP Light

NADPH Sugar

Calvin Cycle Light Reactions

250-7053s ap lab 4 plant pigments - wikispacesap...ap biology lab #4 plant pigment and ... drop of...

Documents