Laboratory 9: Photosynthesis

Objectives

After completing this lab, you should be able to:

• Identify the reactants and products of photosynthesis.

• Explain the importance of photopigments in photosynthesis.

• Apply and interpret results from paper chromatography experiments.

Key Terms

Introduction

Life on Earth can be divided into two categories:

• Autotrophs – organisms that make their own food.

• Heterotrophs – organisms that consume others for food.

Photoautotrophs—such as plants, algae, and cyanobacteria—use light energy to convert carbon dioxide and water into glucose through photosynthesis, storing energy in chemical bonds.

Overall Equation for Photosynthesis

$$\text{Light Energy} + 6CO₂ + 6H₂O → C₆H₁₂O₆ + 6O₂$$

Light Energy+6CO2​+6H2​O→C6​H12​O6​+6O2​

The products, glucose and oxygen, sustain nearly all life on Earth.

Photopigments and Light Absorption

Photosynthesis occurs in chloroplasts, which contain photopigments capable of capturing light energy.
The primary pigment is chlorophyll a (blue-green), aided by accessory pigments such as:

• Chlorophyll b – yellow-green pigment that expands absorption range.

• Carotenes – orange-red pigments (like β-carotene in carrots).

• Xanthophylls – yellow pigments that absorb blue light.

These pigments enable plants to utilize a broader portion of the visible spectrum.

Visible Light Spectrum

Visible light consists of multiple colors, each representing a specific wavelength.
When light strikes an object, it can be absorbed, reflected, or transmitted.
An object’s color corresponds to the wavelength that is reflected—the others are absorbed.

Example: A red surface appears red because red wavelengths are reflected while others are absorbed.

Paper Chromatography

Chromatography is a laboratory method used to separate components of a mixture.
In this lab, we use a polar paper (stationary phase) and a non-polar solvent (mobile phase) to separate the pigments in spinach extract.

• Polar pigments adhere more strongly to the paper and remain lower.

• Non-polar pigments move farther with the solvent front.

Rf (Retention factor) values indicate pigment mobility:

$$R_f = \frac{\text{Distance pigment traveled}}{\text{Distance solvent traveled}}$$

Rf​=Distance solvent traveled/Distance pigment traveled​

Materials

• Spinach leaves

• Mortar and pestle

• Acetone

• Petroleum ether/acetone solvent (9:1)

• Chromatography jar and lid

• Chromatography paper (15 × 5 cm)

• Pencil and ruler

• Paintbrush

• Scissors

• Graduated cylinder

• Spectrophotometer and pigment samples

Procedure

Experiment – Separation of Spinach Pigments via Paper Chromatography

1. Prepare three chromatography samples per group.

2. Cut chromatography paper to 15 cm × 5 cm. Handle only by the edges.

3. Draw a pencil line 2 cm from the bottom (origin line).

4. Grind 5–6 spinach leaves with 5 mL acetone in a mortar and pestle.

5. Using a paintbrush, apply 10–15 thin lines of pigment along the origin line.

   • Allow the paper to dry for 10–15 minutes.

6. Place the paper (pigment side down) into the chromatography jar with 9:1 petroleum ether/acetone solvent.

   • Ensure the solvent level is below the pigment line.

7. Cover the jar and allow it to sit undisturbed for 15–30 minutes.

8. When pigment bands separate, remove the paper and mark the solvent front immediately.

9. Label each pigment band (see Figure 2 placeholder).

10. View your chromatogram under UV light — some pigments will fluoresce pink.

11. Measure distances (origin → pigment and origin → solvent front).

12. Calculate Rf values for each pigment and record them in Table 1.

13. Safely dispose of used solvent in the fume hood waste container.

Data

Table 1. Rf Value Analysis of Spinach Photopigments

Review Questions

1. Write the overall equation for photosynthesis.

2. Compare and contrast the equations for photosynthesis and cellular respiration.

3. What is the main photopigment of photosynthesis?

   • How do accessory pigments enhance the efficiency of photosynthesis?

4. Why do leaves change color in the fall?

5. How do your Rf values compare with the established ones?

   • Which pigment is most polar and which is least polar? Explain your reasoning.

Exercise Sheet

Experiment Analysis

1. Chromatogram Figure

Prepare a figure of your chromatogram.

• Label: solvent front, all pigments (by name), and any visible UV bands.

2. Rf Comparison

Create a table comparing observed and established Rf values.

• Identify the most polar and least polar pigments.

• Explain how your data supports this conclusion.

Licenses and Attribution

CC Licensed Content, Original:

Other Licensed Content Included:

• Adapted with permission from BioScience I Laboratory Manual by Jamie Colson-Moon and Denise Bristol. Per request from these original authors, this content is now licensed (CC BY-NC-SA).