Discussion: Connecting Your Learning

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Discussion: Connecting Your Learning

Discussion: Connecting Your Learning


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Photosynthesis is a biological process that occurs in plants, some bacteria, and some protists. This process relies on pigments, most importantly chlorophyll, to capture light energy and drive the chemical reaction of photosynthesis. This lab involves the extraction of several plant pigments that convert light energy into glucose. Discussion: Connecting Your Learning

Resources and Assignments

Multimedia Resources


Required Assignments

Lesson 5 Lab 5

Required Materials

From the Lab Kit:

· 1test tube

· 4 strips of chromatography paper

· 10 mL syringe

· 2 Phenol red tablets

· Test tube stopper

· 2 Micropipettes

· 2 paper clips

· Rubber band

· 100 mL graduated cylinder

· Mortar and pestle

· Bag of sand (about 2 teaspoons)

· Goggles

· Metric ruler

· Hole punch

· Straw

· Forceps

Student Supplied:

· Acetone (either nail polish remover or acetone from paint section of a hardware or home improvement store)(Acetone is flammable so be certain to keep it away from open flames.) Discussion: Connecting Your Learning

· Light source (lamp with 75W or greater light bulb)

· Scissors

· 1/4 teaspoon baking soda

· 2 cups plus 50 mL distilled water

· Liquid dish soap

· 10 Spinach leaves (large, standard-sized leaves; do not use small, baby spinach leaves) Discussion: Connecting Your Learning

· 1 large glass jar with lid

· Paper towel

· 2 Glass bowls

· 2 Cups or Glasses (4 oz.)

· Hairdryer

· Pencil

· Stopwatch or clock

· Tape

· Marker or Pen

Focusing Your Learning

Lab Objectives

By the end of this lab, you should be able to: Discussion: Connecting Your Learning

1. Study the nature of light and its effect on biological systems.

2. Provide the chemical reaction for photosynthesis.

3. State the importance of pigments to photosynthesis.

4. List variables which affect the rate of photosynthesis.

5. Describe the separation of photosynthetic pigments by chromatography.

6. State the purpose of determining Rf values. Discussion: Connecting Your Learning

7. Calculate Rf values.

Background Information

Energy from the sun travels in waves, similar to the way that waves move across the ocean. All waves share characteristics, such as a crest, the highest part of a wave, and a trough, the lowest part of a wave. The distance measured between either the crests or troughs of two successive waves is knows as wavelength. Discussion: Connecting Your Learning

Waves of energy from the sun are comprised of photons. Low energy photons travel in longer wavelengths while high energy photons travel in shorter wavelengths. Wavelength is measured in nanometers (1 nm = 1 billionth of a meter). Photosynthetic organisms absorb light of wavelengths between approximately 380 and 750 nanometers. Discussion: Connecting Your Learning

Waves of radiant energy are organized according to the electromagnetic spectrum. The portion of the electromagnetic spectrum that is visible to the human eye is called the visible spectrum and ranges in wavelength from 400 nm to 700 nm. Different wavelengths of light are viewed as different colors. Wavelengths of 700 nm are seen as red light, while wavelengths of 400 nm are seen as violet light. From highest wavelengths to lowest, the visible colors are red, orange, yellow, green, blue, indigo, and violet. This order is often abbreviated as ROYGBIV, from the first letter of each color. Beyond the visible spectrum are other forms of energy, such as infrared and gamma rays, which are undetectable without specialized equipment. Discussion: Connecting Your Learning

Pigments are molecules that absorb only photons of specific wavelengths of light. Photons that are not absorbed are reflected back as a color.Chlorophyll a and chlorophyll b reflect wavelengths of approximately 510 nm, which appear green in color. Carotenoids reflect wavelengths of 650 nm (red), 590 nm (orange), and 570 nm (yellow). Xanthophylls reflect wavelengths of 650 nm (red) and 570 nm (yellow).

Click on image to enlarge.

Plants absorb sunlight and convert the solar energy into chemical energy in the form of glucose as a food source through a process called photosynthesis.  Photosynthesis  is the process of combining water and carbon dioxide in the presence of light energy to produce glucose and oxygen. The chemical equation for photosynthesis is:

Click on image to enlarge.

The process of photosynthesis is carried out in the  chloroplasts  of plant cells, which are concentrated in the interior of the leaves. Gas exchange of carbon dioxide and oxygen occurs through small openings in the leaf called  stomata . The figure below details the location of the stomata in comparison to the chloroplasts in a leaf.

Click on image to enlarge.

All life on earth is either directly or indirectly dependent on photosynthesis. Each chloroplast consists of a double membrane. The inner membrane houses a chamber filled with a fluid called  stroma . Suspended in the stroma are membranous sacs called  thylakoids , which are stacked into structures called  grana . Below is an image that details the structure of a chloroplast.

Click on image to enlarge.

Chloroplasts appear green in color due to the presence of a light-absorbing pigment called  chlorophyll , which is found in the thylakoid membranes. The process of photosynthesis mainly employs two types of chlorophyll ( chlorophyll a  and  chlorophyll b ), each activated by a different wavelength of light. In order for the process to proceed, the plant cell must absorb (take in) the particular color (wavelength) of light needed to activate chlorophyll. Chlorophyll a and b absorb blue and red light while they reflect green light; giving leaves their green appearance.

While chlorophyll is required for photosynthesis, it is not the only pigment that plants contain. Many plants contain accessory pigments, including carotene  (orange) and  xanthophyll  (yellow). Even though these pigments do not directly participate in photosynthetic reactions, they do transfer the energy they receive to chlorophyll, which, in turn, uses the energy for photosynthesis. Because these pigments absorb light at different wavelengths, the presence of accessory pigments allows a plant to maximize the amount of sunlight captured. Another pigment is  anthocyanin  (red or purple), which helps protect the plant from ultraviolet damage. These accessory pigments contribute to the colors found in fall foliage. As chlorophyll levels decline in the fall, the accessory pigments are able to be seen, producing the vivid orange, yellow, and red colors seen in leaves. Discussion: Connecting Your Learning

The first part of this lab will involve a demonstration of CO2 use in photosynthesis. To test if plants require CO2 for photosynthesis, an experiment will be conducted using phenol red. Phenol red is an indicator that turns yellow in the presence of CO2. If the CO2 is removed, phenol red returns to its original color (red). Discussion: Connecting Your Learning

In the second experiment in this lab, four pigments found in plant leaves will be identified through the process of  chromatography . The four pigments to be observed include carotene (yellow-orange color), xanthophyll (yellow color), chlorophyll a (blue-green color), and chlorophyll b(yellow-green color). The pigments will be extracted using one-way paper chromatography. To conduct this type of chromatography, an extract of the compound must first be obtained. The chromatography paper must be marked with a pencil, as ink will dissolve in the solvent used in this experiment and will also travel up the paper. At the conclusion of the experiment, the Rf factor will be calculated for each pigment.

In chromatography, chemicals can be compared to one another based on their Rf values. Rf stands for “ratio of fronts” and is characteristic for any given chemical. Rf values are calculated using the following equation:

Rf =

Distance the pigment traveled

Distance the solvent traveled    (solvent front will be near the paper clip)

For example, if the solvent travels 10 cm, and the pigment travels 3 cm, the Rf value for that pigment would be:

3 cm/10 cm = 0.3000

Note that the Rf value should be calculated to four decimal places. For example, if the result is 0.345678, the Rf value should be documented as 0.3457.

The higher the Rf factor, the more soluble that pigment is in the particular solvent.

As will be demonstrated in this experiment, pigments have different Rf values. This occurs because pigments travel at different rates depending on their solubility in the solvent, molecular mass, and affinity for bonding with the paper (or their chemical charge). In general, the less chemically charged and lighter pigments will travel further up the paper. Those that are heavier (have a higher molecular weight) and are chemically charged travel a shorter distance. If the pigment does not travel, it is NOT soluble in the particular solvent.

The final experiment will determine the influence of light intensity and carbon dioxide concentration on the rate of photosynthesis in plant leaves. Plant leaves contain extracellular spaces that are filled with gases that enter and exit the leaves. Recall that the overall reaction for photosynthesis includes an input of carbon dioxide gas and an output of oxygen gas. As described earlier in the lab, these gases are exchanged through small openings in plant leaves called stomata. Air that is found in the extracellular spaces of leaves gives them buoyancy, causing them to float on water. In the experiment, a solution of baking soda and water will be used to supply a source of carbon for photosynthesis. Circular disks will be cut from spinach leaves for the experiment. The air will be extracted from the extracellular space of the spinach leaves and replaced with water, which will cause the leaves to sink in a sodium bicarbonate solution. If cells in the leaves are performing photosynthesis, oxygen gas will be generated as a by-product, causing the leaves to float in the solution.

The amount of time it takes for the disk to float will be used as a measure of photosynthetic activity. In order to account for variability in photosynthetic rates between the disks, the time required for 50% of the disks to float (five disks) will be determined. This rate can be determined by constructing a graph that plots the number of disks floating as a function of time, and then determining the time at which 50% of the disks floated. In other words, the number of disks will be on the y axis and the time will be on the x axis, as seen below. The more time it takes for 50% of the disks to float, the slower the rate of photosynthesis and vice versa. This lab will investigate the effects that different concentrations of carbon and different light intensities have on the rate of photosynthesis in the disks.

Click on image to enlarge.


1. Demonstrate the use of CO2 in plants for photosynthesis.

A. Pour distilled water into the graduated cylinder to the 50 mL level.

B. Add one phenol red tablet to the graduated cylinder and stir until dissolved.

C. Place a straw into the graduated cylinder and blow slowly and steadily, until the phenol red changes to a yellow color.  BE CAREFUL NOT TO BLOW TOO HARD OR THE SOLUTION MAY SPLASH INTO THE FACE.

D. Place a small piece of spinach leaf into the test tube.

E. Pour the phenol red solution from the graduated cylinder into the test tube to the top of the tube.

F. Carefully put the tube cork into the test tube, leaving no air bubbles.

G. Place the test tube in front of a light source (light or window) for 30 minutes. Record results of any observed color change.

2. Chromatography

Remember, acetone is flammable so be certain to keep it away from open flames.

A. Wrap a rubber band around the jar lengthwise so that the mouth of the jar has a stretch of rubber band around it. The chromatography paper strip will be attached to this rubber band so be certain that it is centered across the opening of the jar.

B. Attach two paper clips to the rubber band so that they hang loosely in the opening of the jar.

C. Using the forceps to minimize handling, remove one chromatography strip and while holding it at the terminal end (straight edge), attach the chromatography strip to the paper clip. Note: Only handle the chromatography paper by the extreme edges (as opposed to the flat surfaces) or at the terminal portion of the end that will not go into the acetone/water as the oils present on skin will inhibit the absorption of the pigments and can potentially skew the results.

D. The strip should not touch the bottom of the inside of the glass jar. If it does, it may be necessary to fold over the top of the strip. The strip should hang no more than one cm from the bottom of the jar because the solvent (acetone) needs to be poured into the jar. Only the very tip of the chromatography strip should touch the solvent. Once the strip is positioned, remove it from the paper clip and place it on a paper towel. Use this strip to make any adjustments to the second strip. The remaining strip may be cut to size, or the top may be folded over so that it is the same size as the test strip. Place both strips on a clean paper towel and set it aside.

E. Place a small piece of tape on each of the two bowls. Using a marker or pen, label the first bowl “acetone” and the second bowl “water.”

F. Remove two, large spinach leaves. Use the scissors to cut up the leaves, or use clean hands to tear the spinach into small bits and place the pieces into the mortar. Be sure to use only the leaves of the spinach and not the stems.

G. Cover the leaf tissue with 10 mL of acetone and add 1/2 teaspoon of fine sand. Using the pestle and sand, grind the spinach leaves until the entire mixture becomes a slurry of a dark liquid (about five to seven minutes). Add additional acetone (no more than 5 mL) and grind for another minute. Let the solution stand for a few minutes (about five) to be sure the pigments are extracted. A very dark pigment solution should be formed.

H. Carefully pour the liquid into the bowl labeled “acetone.”

I. Repeat steps E-G, this time using water to cover the plant leaves and pouring the liquid into the bowl labeled “water.”

J. Using a  pencil  (ink cannot be substituted as it will migrate along with the solvent) and the metric ruler, draw a horizontal line across each strip about 1 to 1 1/2 centimeters from the tip (bottom). Label one strip “A” for acetone and the second strip “W” for water to identify the solvent used to extract the pigment.

K. Using a micropipette, draw up a small volume of the spinach/acetone slurry and add a small drop to the center of the pencil line on the chromatography paper labeled with the “A.” Attempt to keep the spot as small as possible to prevent the pigment from spreading across the paper.

L. Repeat step J a second time, using the second chromatography strip (labeled with the “W”) and the spinach/water slurry.

M. Allow the spots to dry (use of a hairdryer on a low setting may facilitate the drying process). Repeat steps J and K several more times until a deep green spot is achieved.

N. Pour acetone into the jar to a height of about 1 to 1 1/2 cm.

O. Place the jar in a location where it will not be subject to shaking or vibration. If the jar is bumped, or disturbed in any way, the solvent will quickly migrate up the paper and the pigments will not be removed.

P. Attach the prepared chromatography strips to the paper clips. The strips should hang so that only the extreme end of the chromatography strip touches the solvent (acetone). DO NOT ALLOW THE PIGMENT LINE TO TOUCH THE SOLVENT.

Q. Cover the jar with the lid. The solvents will begin to migrate up the strip, carrying the pigments along with them.

R. Watch the strips carefully as the process can proceed rapidly. Check the strips every minute or so and remove the strips as soon as all four pigments are visible.  Be very careful not to let the pigments or solvent reach the paper clip.

S. Remove the paper strips and place them on the paper towel. Using a pencil, immediately draw a freehand line across the paper to identify the solvent front (end location of the acetone or water), as well as the location where each pigment was extracted (the top of the solute line).

T. Measure and record the distance for the solvent front (end distance of acetone), as well as the distance for each pigment from the starting point to the top of the respective pigment line.

U. Calculate the Rf value for each pigment by dividing the distance from the origin (slurry spot) to the top of the pigment line, by the distance from the origin (slurry spot) to the solvent front.

V. Record the values obtained.

3. Factors That Affect the Rate of Photosynthesis

A. Prepare a 0.8% bicarbonate solution. Take one plastic cup and label it 0.8%. Using the 100 mL graduated cylinder, pour a total of 150 mL of distilled water into the labeled cup. Add ¼ teaspoon of baking soda and stir until dissolved.

Click on image to enlarge.

Click on image to enlarge.

B. Take a second plastic cup and label it 0.2%. Using the 100 mL graduated cylinder, measure out 50 mL of the 0.8 mL solution and pour it into the cup labeled 0.2%. Using the 100 mL graduated cylinder, add an additional 100 mL of distilled water to the cup.

Click on image to enlarge.

C. Add a few drops of dish soap to each cup and stir.

D. Using a hole punch, cut 30 leaf disks from fresh spinach leaves, trying to avoid any major veins.

Click on image to enlarge.

E. Remove the plunger from a 10 mL syringe. Place 10 disks into the body of the syringe and then gently re-insert the plunger. Take care not to damage the leaf disks when inserting the plunger.

Click on image to enlarge.

F. Insert the syringe into the 0.2% bicarbonate solution and draw up about 8 mL. The disks should be floating at this time.

Click on image to enlarge.

G. Hold the syringe upward (tip up) and slowly depress the plunger to remove any excess air.

H. Cover the tip of the syringe with the thumb and with the opposite hand, pull back on the plunger to create a partial vacuum and move air out of the disks. Hold for 10 seconds.

Click on image to enlarge.

I. Simultaneously release the thumb and the plunger. Tap the side of the tube to dislodge any bubbles. The disks should start to sink.

Click on image to enlarge.

J. Repeat steps H and I until all of the disks sink to the bottom of the syringe (Note: Be patient as this may take several attempts).

K. Place the syringe upright under a light source 10 cm from the syringe and start a stopwatch or record the time.

Click on image to enlarge.

L. After the end of one minute, invert the syringe to agitate the disks and then immediately return the syringe to its position under the light source. Record the number of disks that are floating in the syringe.

Click on image to enlarge.

M. Repeat step L after each one minute interval, until all of the disks are floating.

Click on image to enlarge.

N. Empty the contents of the syringe, including the solution and the disks, down the drain of a sink. Flush the syringe with tap water.

O. Repeat the process two more times, beginning with step F. The first time, modify the experiment by using the 0.8% solution. For the last trial, modify the experiment by using the initial 0.2% solution, but place the syringe 15 cm away from the light source.

P. Calculate the rate of photosynthesis (as indicated by 50% of the disks floated for each trial) by graphing the number of disks that floated as a function of time and extrapolating the point of time where 50% of the disks were floating.

Assessing Your Learning

Compose answers to the questions below in Microsoft Word and save the file as a backup copy in the event that a technical problem is encountered while attempting to submit the assignment. Copy the answers from Microsoft Word by simultaneously holding down the Ctrl and A keys to select the text, and then simultaneously holding down the Ctrl and C keys to copy it. Then, click on the link below to open up the online submit form for the laboratory. Paste the answers into the online dialog box by inserting the cursor in the submit box and simultaneously holding down the Ctrl and V keys. The answers should now appear in the box. Repeat this process for each question. Review all work to make sure that all questions have been answered and then click on the Submit button at the bottom of the page.



a. What is the name of the pigment that captures light? (2 points)

b. Why does the pigment appear green? (2 points)

2. List two variables besides the wavelength (color) of light which might affect the rate of food production in plants. (4 points)



3. Why is chlorophyll important for all biological life? (5 points)


a. In Part I of the procedure, what is the name of the indicator used to identify the presence of CO2? (2 points)

b. What color did the indicator turn after blowing air into the water through the straw? (2 points)


a. What color did the indicator turn after the tube was placed under a light source for 30 minutes? (2 points)

b. Why did this occur? (3 points)

6. List the four common pigments found in plants and their functions. (4 points)





7. If the Rf factor of a pigment is .8400 and the distance that the solvent traveled is 12 cm, how far did the pigment travel? (5 points)

8. List the pigments extracted from the spinach leaves and their corresponding Rf values, from lowest to highest Rf value (4 points).

a. pigment, Rf value

b. pigment, Rf value

c. pigment, Rf value

d. pigment, Rf value

9. Based on the results, which pigment has the highest molecular weight? (5 points)

10. From the chromatography lab, which pigments were soluble in the acetone? (5 points)

11. The earth’s early atmosphere did not contain oxygen. This changed dramatically once the early cells underwent photosynthesis. Explain why photosynthesis could have occurred in such an atmosphere and how it eventually affected the evolution of other organisms. (10 points)


a. In reviewing the data from the floating disk experiment, which factor had a greater impact on the rate of photosynthesis (light intensity or concentration of carbon dioxide)? (5 points)

b. How did the student come to this conclusion? (5 points)


Independent Variable: This is the cause.

Dependent Variable: This is the response or effect.

One hundred samples of several different plants were placed in each of six sealed containers with water in them. At the end of two days the amount of oxygen produced was measured. Results are shown in the table below.



Height of Plant

Light Intensity

Source of Light

Distance from Light

mL O2 Produced











































13. Based on the data presented in the table, which two containers could be correctly used to compare the rate of photosynthesis at two different light intensities? (5 points)

a. 1 and 2

b. 2 and 3

c. 1 and 5

d. 5 and 6

e. 4 and 5

14. Compare Containers 1 and 2. What independent variable is tested by this comparison? (5 points) Discussion: Connecting Your Learning

a. Kind of plant

b. Height of plant

c. Light intensity

d. Distance from light source

e. Light source

15. Which container had the slowest rate of photosynthesis? (5 points) Discussion: Connecting Your Learning

a. 1

b. 2

c. 3

d. 4

e. 5

f. 6

16. (Application) How might the information gained from this lab pertaining to photosynthesis and pigments be useful to the student or how can the student apply this knowledge to everyday life as a non-scientist? The application paragraph will be graded according to the rubric below. (20 points)











Below 12

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