Materials Included In Kit

Additional Materials Required

Prelab Preparation

Column Chromatography of Plant Pigments Demonstration

1. Place the tip on the bottom of the chromatography column. Pour about 2.5 mL of hexane into the column so that the liquid fills the narrow portion of the column.
2. Slowly pour 1 g of aluminum oxide into the column to fill the narrow portion of the column until it is about ⅔ full. Tap the tip of the column on the lab bench while pouring the aluminum oxide to eliminate spaces in the column.
3. Carefully pour a small amount of sand into the column so it forms a 2-mm layer on top of the aluminum oxide. Place the blue cap on the column to prevent evaporation of solvents. The column will look like the one in Figure 3.
   {11411_Preparation_Figure_3}
4. Secure the column to a support stand with the clamp around the wide portion of the column.
5. Place an empty 250-mL beaker underneath the column.
6. Pour 10 mL each of hexanes and acetone into separate, labelled 50-mL beakers or flasks.
7. Pour 5 mL of hexanes and 5 mL of acetone into another labelled 50-mL beaker or flask to make a 50/50 hexane–acetone mix.
8. Cover all three beakers with Parafilm^® to prevent evaporation of the solvents.
9. Weigh 0.5 g of spinach powder and place it in a 13 x 100 mm test tube.
10. Add 1 mL of the 50/50 hexane–acetone mixture. Insert a stopper into the test tube and shake vigorously. Continue shaking until the liquid (extract) begins to turn a dark green. Place the test tube in a test tube rack with the stopper in place.
11. Label three clean test tubes pigment 1, pigment 2 and pigment 3 and place them in a test tube rack with stoppers.

Part 3. Photosynthesis with Sodium Alginate Spheres Guided-Inquiry Activity

Part A. Culturing Chlorella

1. To ensure an adequate population, begin culturing at least two weeks before the lab. The culture should be dark green before beginning.
2. Add one liter of distilled water to a clear container, such as a large, glass jar or two-liter bottle with the top cut off. A volumetric or round bottom flask is also good for this purpose.
3. Add ten mL of Bristol’s culture media concentrate and stir well.
4. Add chlorella culture.
5. Place the jar near a diffused light source or use grow lights or wide spectrum bulbs placed 12–18" from the container.
6. Maintain the temperature near 21° C by adding small quantities of water or by adjusting the light source if it becomes too warm. Temperatures higher than 27° C can be fatal to the culture.

Part B. Preparing Sodium Alginate with Algae

1. Add two grams of sodium alginate to a 250-mL beaker containing 60 mL of distilled water and stir, using a stir plate on a low setting, until all the sodium alginate is incorporated and the solution is thick and uniform. This may take more than one hour. This step may be done ahead of time. Cover the beaker and store in a laboratory refrigerator for up to two weeks.
2. Collect 300 mL of algae from near the bottom of the algae culture in a 400-mL beaker.
3. Allow the algae to settle into the bottom of the beaker for about an hour, then pipet off the top 150 mL that is not as concentrated, leaving 150 mL of the most concentrated algae.
4. Just before lab, combine the concentrated algae solution and the sodium alginate solution in the 400-mL beaker, stirring slowly to ensure the mixture is homogenous. You may choose to divide this stock sample into 15-mL quantities for each lab group or each lab group may use their syringe to remove a 10-mL portion from the stock.

Part C. Other Preparation

1. Cut eight 20 cm x 20 cm squares from the cheesecloth.
2. Add 25 mL of 0.04% bromthymol blue to 175 mL distilled water to make 0.005% bromthymol blue indicator solution. Adjust the pH using a dilute acid or base to achieve a green starting color. This corresponds to a pH of about 7.

Safety Precautions

Acetone and hexanes are flammable liquids and dangerous fire risks. Acetone is also slightly toxic by ingestion and inhalation. Hexanes are a respiratory irritant. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. Remind students to wash their hands thoroughly with soap and water before leaving the laboratory. Please review current Safety Data Sheets for additional safety, handling and disposal information.

Disposal

Please consult your current Flinn Scientific Catalog/Reference Manual for general guidelines and specific procedures, and review all federal, state and local regulations that may apply, before proceeding. Dispose of remaining acetone or hexane by evaporating in a fume hood. After evaporating solvents from the column, the dry spinach powder, sand, aluminum oxide, and the chromatography column may be placed in the trash according to Flinn Suggested Disposal Method #26a.The leftover calcium chloride solution may be reused or rinsed down the drain with excess water according to Flinn Suggested Disposal Method #26b. The sodium alginate spheres and excess sodium alginate may be placed in the trash according to Flinn Suggested Disposal Method #26a. The bromthymol blue solution may be neutralized with sodium hydroxide according to Flinn Suggested Disposal Method #24b. Excess algae culture can be sterilized and handled according to Flinn Suggested Biological Waste Disposal Type 1.

Lab Hints

• Enough materials are provided in this kit for 8 groups of students to complete the guided-inquiry lab activity and for the demonstration to be completed four times. There are enough materials for students to complete the introductory activity and additional trials as part of the guided-inquiry activity. Each group can complete three trials at a time using the included vials.
• This module can reasonably be completed in five, 50-minute class periods. Complete the POGIL^® activity on day one, the demonstration on day two, the introductory activity on day three, and designing and carrying out the guided-inquiry activity on days four and five.
• The rate of photosynthesis may be accelerated by placing the algae in the dark for 12 hours before the experiment.
• A number of factors will affect the pH rate of change. A noticeable change in pH may take place in as little as thirty min- utes or as great as several hours. Light quality, algae density and metabolic condition of the algae all may vary.
• Rather than using bromthymol blue, a pH meter can be used to measure the pH at the beginning and end of the experiment.
• For information regarding proper algae care, see Culturing Algae, Publication No. 10583, available on the Flinn Scientific website.

Teacher Tips

• Students should have some background knowledge on polarity and intermolecular forces before starting this activity.
• The POGIL^® activity is designed to be completed in class using the POGIL teaching method. This includes students working in groups with assigned roles to construct their own learning using modeling. For more information, visit www.pogil.org.
• The separation of pigments during the demonstration takes time. Students can read the overview and complete the pre-demonstration questions while the demonstration is being prepared and started. Alternately, the demonstration could be recorded and replayed at a faster speed.
• Using a document camera during the demonstration will help students see the column chromatography separation with more detail.
• Models are expressed as diagrams on paper, however using manipulatives to create three dimensional models is a great extension. Student must be able to explain their models and how they fit with the observations they made during the demonstration.
• The following student laboratory kits can be used to further explore photosynthesis: Plants in the Spotlight—A Photosynthesis Investigation Student Laboratory Kit (Flinn Catalog No. FB1780) and Plant Pigment Chromatography Student Laboratory Kit (Flinn Catalog No. FB0586).
• This learning module incorporates the following kits: Basic Column Chromatography Student Laboratory Kit (Flinn Catalog No. AP7392) and Sodium Alginate Photosynthesis Student Laboratory Kit (Flinn Catalog No. FB2125).

Answers to Prelab Questions

1. Examine Figure 1. Of the three pigments, which absorbs wavelengths associated with the color red?

   Chlorophyll a and chlorophyll b

2. What colors are not absorbed by chlorophyll a and b?

   Green and yellow are not absorbed by chlorophyll a and b.

3. When a color is not absorbed, it is transmitted. What colors are transmitted by carotenoids?

   Green, yellow, orange and red are transmitted.

4. What determines the color of an object?

   The color of an object is determined by the wavelengths of light in the visible spectrum that are transmitted, or not absorbed.

5. Explain the role of the adsorbent in column chromatography.

   The adsorbent attracts polar molecules, which slows their movement through the column. This separates molecules with different polarities.

6. Explain the role of the eluent in column chromatography.

   The eluent is a liquid with a polarity that is less than that of the adsorbent. It carries molecules through the column that have a similar polarity to the eluent. Several different eluents are used to carry molecules of differing polarities.

Sample Data

Part 2. Column Chromatography of Plant Pigments Demonstration

Observations

Models Part 3. Photosynthesis with Sodium Alginate Spheres Guided-Inquiry Activity
Change in pH

Answers to Questions

Part 3. Photosynthesis with Sodium Alginate Spheres Guided-Inquiry Activity

1. What was the change in pH of the sample?

   Answers will vary based on data collected. The pH changed from 7.7 to 7.5 for a total change of 0.2 in 45 minutes.

2. What does this change in pH indicate about the productivity of the sample?

   Answers will vary based on data collected. The decrease in the pH indicates that respiration occurred at a faster rate than photosynthesis, however the small change indicates that photosynthesis is also occurring in the sample.

3. Explain the role of light in photosynthesis.

   Light provides the energy needed to make glucose during photosynthesis. The energy from the light is transformed into chemical potential energy that is stored in the bonds of the glucose molecules. The pigments in chlorophyll absorb the energy from specific wavelengths of light in the blue and red portions of the visible spectrum. Light sources that have a higher amount of these wavelengths will encourage growth by increasing the rate of photosynthesis.

Guided-Inquiry Design
In your group, design an investigation that uses this method to measure photosynthetic activity and tests a different variable.

1. Record the research question.

   The research questions may include manipulating light, temperature, algae type, sphere size or other reasonable questions that test one variable.

2. Define the limitations of the testing methods.

   Student answers will vary. The use of a colored indicator impacts the wavelengths of light that can interact with the algae. Using a very dilute indicator solution helps with this. The color indicator does not give precise pH readings. Temperature can be difficult to control and impacts the solubility of carbon dioxide in water. The time allowed may limit the ability to get meaningful results.

3. Identify the control, independent variable, dependent variable and critical constants.

   Student answers will vary.

4. Record the working procedure. If changes must be made to the working procedure, record those changes here.

   Student answers will vary.

5. Explain the safety procedures needed to carry out the investigation safely.

   Student answers will vary. Students should always wear goggles, gloves and a chemical-resistant apron or lab coat. Students must follow appropriate disposal guidelines.

6. Carry out the investigation and record relevant data.

   Student answers will vary.

7. Display relevant data in a meaningful way to help communicate the results of the investigation.

   Student answers will vary.

Post-Lab Analysis

1. Using a claims, evidence and reasoning model, explain the results the experiment.
   1. Propose a claim based in scientific understanding.

      Student answers will vary. A claim is a short statement that connects the observed evidence to scientific understanding of a concept. In this case, the claim would connect the observed change in pH to productivity.

   2. Discuss specific evidence from the experiment.

      Student answers will vary. The evidence must match the observations the student made during data collection. For example, if the student claims algae in the dark did not perform photosynthesis, the pH data should show a decline in pH.

   3. Discuss the reasoning for the claim based on connections to the POGIL^® activity, the demonstration and the introductory lab activity.

      Student answers will vary. The reasoning must include a connection to the role of pigments to photosynthesis. It must also connect the variable they chose to the rate of photosynthesis.

References

Special thanks to Pam Bryer, Bowdoin College, Brunswick, ME, for sharing the sodium alginate photosynthesis activity with us.

What’s in a Leaf—Photosynthesis. POGIL^™ Activities for High School Biology. Trout, L., Editor; Flinn Scientific: Batavia, IL (2012).

The demonstration activity was adapted from Flinn ChemTopic^™ Labs, Volume 2, Elements, Compounds and Mixtures; Cesa, I., Editor; Flinn Scientific: Batavia IL (2005). 

Introduction

Photosynthesis is a set of biochemical reactions that transfers light energy from the sun to chemical energy in glucose. The light energy is captured by pigments in chlorophyll, a component of chloroplasts. Carbon dioxide and water are used during photosynthesis and oxygen and glucose are produced. By measuring the reactants or the products, scientists can determine the rate of photosynthesis.

Concepts

• Chromatography
• Photosynthesis
• Polarity of pigments

Background

Part 2. Column Chromatography of Plant Pigments Demonstration
A pigment is a molecule or compound that absorbs certain wavelengths of visible light and transmits or reflects the remaining wavelengths of visible light. Different pigments have different molecular structures, and thus reflect different specific wavelengths of visible light. The color of an organism is due to the reflection of specific wavelengths of light by pigments in the organism. The major pigments of photosynthetic organisms are the chlorophylls and accessory pigments. There are two types of green chlorophyll, called chlorophyll a and chlorophyll b. Chlorophyll a and chlorophyll b are found in all plants, most algae and some bacteria. The accessory pigments in plants are a class called carotenoids. The absorbance of these three classes of pigments are shown in Figure 1.

Each of these pigments is a different color due to differences in their chemical structure. Just as they have different colors, they also have differences in polarity, caused by the unequal sharing of electrons in the covalent bonds of the atoms. This affects the intermolecular forces between the pigment molecules and molecules they interact with.

Using column chromatography, chlorophyll and other pigments can be separated and the pigments characterized using a spectrophotometer or colorimeter (see Figure 1). Column chromatography is a type of chromatography called adsorption chromatography. The column contains a solid, which acts as the adsorbent. A thin layer of the plant material is placed on top of the adsorbent. A flow of a liquid eluent or solvent is washed through the column, carrying the components of the plant material down the solid column. The intermolecular forces either make the pigment molecules stick to the adsorbent or the eluent, which have differences in polarity.

Successful separation of substances via column chromatography is based on two properties of the substance being separated—its adsorptivity on the solid and its solubility in the eluent. Adsorptivity is the adhesion of the molecules in the substance being separated to the molecules on the surface of the adsorbent. The adsorbent is a relatively polar material and the eluent is rather nonpolar. Therefore, the more polar the compound in the mixture being separated, the higher the adsorptivity on the solid, which results in the compound moving more slowly through the column.

Part 3. Sodium Alginate Photosynthesis Guided-Inquiry Activity
A novel method of controlling the amount of photosynthetic organisms during laboratory testing is by encapsulating algae in sodium alginate. Sodium alginate is a polysaccharide extracted from brown seaweed. When it contacts a solution containing calcium ions, it forms a gel. By dropping a mixture of sodium alginate and algae into the calcium solution using a syringe, small spheres are made in a process called spherification. These spheres are permeable to small molecules, including the reactants and products of photosynthesis. The algae remain alive throughout this process and can continue with photosynthesis and respiration. Photosynthesis is the process of using light energy to convert water and carbon dioxide to glucose molecules with high chemical potential energy.


During photosynthesis, autotrophs, such as algae, remove carbon dioxide from the atmosphere and from aquatic environments. The removal of carbon dioxide from aquatic environments results in an overall increase in pH because aqueous carbon dioxide reacts with water to form carbonic acid (H₂CO₃) as shown in the following reaction. When carbon dioxide is removed, a proportional amount of carbonic acid reacts to reform carbon dioxide and water to maintain equilibrium.

Because of this, pH is a good indicator for measuring the relative rate of photosynthesis or respiration. When both are happening simultaneously, an increase in pH indicates that photosynthesis is occurring faster than respiration and a decrease in pH indicates that respiration is occurring faster than photosynthesis.

Experiment Overview

The purpose of this learning module is to build understanding of the process of photosynthesis, particularly the light reactions involving the chloroplasts, through collaborative modeling and experimentation. First, use models in the POGIL^® activity to discover how the structure of leaves enable them to carry out photosynthesis. Then, observe as pigments from chlorophyll are isolating using column chromatography. Collaborate with your team to make a model to show how pigments are separated. Finally, carry out an investigation to learn how photosynthesis, light and pH are connected in an aquatic environment with photosynthetic algae spheres.

Part 1. Establishing Background Knowledge 

In groups, complete the Photosynthesis: What’s in a Leaf POGIL activity. 

Part 2. Column Chromatography of Plant Pigments Demonstration

The purpose of the introductory activity is to practice a method of measuring the amount of photosynthesis in an aquatic environment using sodium alginate to encapsulate algae.

Demonstration 
Before watching the demonstration, complete the Pre-Demonstration Questions from Part 2 of the Photosynthesis Worksheet with your group. While watching the demonstration, complete the table of observations. Next, complete the Modeling section on the Photosynthesis Worksheet with your group. Produce models in the form of drawings that show how each of the three pigments interact with the adsorbent and each eluent. 

Part 3. Sodium Alginate Photosynthesis Guided-Inquiry Activity

The purpose of the introductory activity is to practice a method of measuring the amount of photosynthesis in an aquatic environment using sodium alginate to encapsulate algae.

Materials

Safety Precautions

All materials are nonhazardous. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. Wash hands thoroughly with soap and water before leaving the laboratory. Please follow all laboratory safety guidelines.

Procedure

Part 3. Sodium Alginate Photosynthesis Guided-Inquiry Activity

Part A. Preparing Sodium Alginate Solution

1. Add 30 mL of calcium chloride solution to a 50-mL beaker.
2. Draw up 10 mL of sodium alginate and algae mixture into the syringe.
3. Secure the syringe with a clamp attached to a support stand and place the calcium chloride solution beneath the syringe as shown in the Figure 2.
   {11411_Procedure_Figure_2}
4. Allow the sodium alginate and algae mixture to drip into the beaker until approximately 30 spheres of equal size have been made. You may need to apply a slight amount of pressure to the plunger.
5. Fit the cheesecloth in the funnel or use a strainer with a fine mesh.
6. Pour the algae spheres into the funnel and strain the calcium chloride solution into a clean, empty beaker. This may be reused for the guided-inquiry portion.

Part B. Introductory Activity

7. Add 20–30 algae spheres to the vial, filling it most of the way to the top.
8. Using a pipet, fill the vial with bromthymol blue solution so there is minimal air space at the top. Screw on the cap.
9. Record the color and corresponding pH of the solution in Part 3 of the Photosynthesis Worksheet.
10. Place the vial 15 cm from the light source.
11. Place a 400-mL beaker of water between the light source and the vial to act as a heat sink.
12. Turn on the light.
13. After 30 minutes, check for color change. If time allows, check for color change every 30 minutes. It may take several hours to see a change in color.
14. Once a color change is detected, record the total time elapsed, the color of the solution and the pH of each vial on the worksheet.
15. Calculate the change in pH and record on the worksheet.
16. Answer all analysis and calculation questions on the worksheet.

Guided-Inquiry Design

1. With your group, discuss the variable that impact the rate of photosynthesis. Choose one variable that can be tested using the experimental design used above.
2. Research the limitations to experimental design above and make changes as approved by your instructor.
3. Use the guide on the Photosynthesis Worksheet to plan and write appropriate procedures with safety considerations.
4. Once you have obtained permission from your instructor, carry out your investigation.
5. Consult your instructor for appropriate disposal procedures. The calcium chloride solution can be reused many times.

Column Chromatography of Plant Pigments Demonstration

1. Remove the tip from the chromatography column and allow the hexane in the column to drain until the liquid level is just above the sand layer. Replace the tip on the column.
2. Shake the test tube containing the spinach extract and allow the solid to settle. Place a clean pipet into the test tube and fill the pipet with only the liquid portion of the extract.
3. Remove the tip from the column and add 5 drops of the spinach extract to the top of the column by running it down the inside of the column in a circular fashion so as not to disturb the sand layer.
4. As soon as the extract is absorbed into the sand layer, carefully add a pipet-full of hexane to the column by running it down the inside of the column in a circular fashion. Continue adding hexane in this manner until the entire column is full.
5. As the solvent moves through the column, it will begin to carry one of the pigments with it. This will take several minutes as the pigments interact with the adsorbent phase. Colored bands should become visible in the column. As this is taking place, review with the students the prelab information concerning the separation of the pigments.
6. As the first pigment band begins to exit the tip of the column, place the appropriate test tube underneath the column to collect the solvent containing this pigment. As soon as the band has completely exited the column, remove the test tube, stopper it and set it out so students are able to see it and record observations.
7. The same general procedure will be used to collect additional pigments as they pass through the column. Different solvents, first 50/50 hexane–acetone and then acetone, will be needed to collect pigments 2 and 3, respectively. The same general procedure should be followed in each case. Never allow the top of the column to run completely dry. Collect each pigment band solution in a different test tube. Replace the test tube with a beaker to collect eluent that does not contain a pigment.

Student Worksheet PDF

11411_Student1.pdf