The Arizona STEM Acceleration Project
Analysis of Chlorophyll Concentrations using Spectrophotometry
Measuring the Absorbance of Chlorophyll Solutions at Different Concentrations
Grades 9-12 Biology and Chemistry
Reny Mathew
Date: 11/28/23
Notes for teachers
Leaf Selection: Ensure fresh and healthy leaves are available for students to extract pigments. Different plant species can be used to showcase variations in pigment concentrations.
Standard Solutions: Prepare standard solutions with known pigment concentrations to calibrate the colorimeter. This establishes a reference for interpreting absorbance values.
Vernier Colorimeter or Spectrophotometer calibration: Instruct students on the proper calibration of the colorimeter using a blank solution (solvent only) before measuring the pigment samples. This step ensures accurate absorbance readings.
Wavelength Selection: Discuss the importance of selecting the appropriate wavelength for measurement, typically corresponding to the absorption peaks of chlorophyll (around 430 nm). Explain why this specific wavelength is relevant to the pigments under investigation.
Pigment Extraction: Guide students in choosing a suitable solvent (e.g., acetone or ethanol) for extracting pigments from leaves. Emphasize the importance of using the same solvent consistently across all samples.
Crushing and Extraction: Provide clear instructions on the proper technique for crushing leaves and extracting pigments. Remind students to be consistent in their methods to maintain the integrity of the experiment.
List of Materials
Standards
HS.L2U1.21 Obtain, evaluate, and communicate data showing the relationship of photosynthesis and cellular respiration; flow of energy and cycling of matter.
HS.P4U1.10 Construct an explanation about the relationships among the frequency, wavelength, and speed of waves traveling in various media, and their applications to modern technology.
HS+B.L2U1.8 Develop and use models to develop a scientific explanation that illustrates how photosynthesis transforms light energy into stored chemical energy and how cellular respiration breaks down macromolecules for use in metabolic processes.
Standards
Cross Cutting Concepts
Science and Engineering Practices
Objectives:
Agenda (90 minutes)
What are the pigments seen in plants?
Green plants contain several pigments responsible for the various colors observed in different parts of the plant. The primary pigments in green plants are chlorophylls, carotenoids, and anthocyanins.
Chlorophylls: Chlorophylls are the most important pigments in green plants, responsible for their green color. The two main types of chlorophyll found in plants are chlorophyll-a and chlorophyll-b. These pigments are crucial for photosynthesis, the process by which plants convert light energy into chemical energy.
Carotenoids: Carotenoids are pigments that contribute to the yellow, orange, and red colors in plants. They are accessory pigments that assist in capturing light energy during photosynthesis and protecting the plant from damage caused by excess light.
Anthocyanins: Anthocyanins are water-soluble pigments responsible for red, purple, and blue colors in plants. While not always present in green plants, they can be found in certain parts such as fruits, flowers, and leaves. Anthocyanins serve various roles, including protection against UV radiation and attraction of pollinators.
Xanthophylls: Xanthophylls are yellow pigments that, like carotenoids, play a role in capturing light energy during photosynthesis. They are often found in chloroplasts alongside chlorophylls.
The combination and relative abundance of these pigments contribute to the overall coloration of plants. Chlorophylls dominate the green color, but other pigments can become more apparent when chlorophyll levels decrease, such as during the fall when chlorophyll breaks down and other pigments become more visible.
How does the variation in chlorophyll concentration correlate with changes in absorbance values?
Spectrophotometry is a technique used in analytical chemistry and biochemistry to measure how much a substance absorbs light at different wavelengths. It involves the use of a spectrophotometer, an instrument that measures the intensity of light as it passes through a sample solution.
The basic principle of spectrophotometry is based on Beer's Law, which states that the absorbance of a substance is directly proportional to its concentration in a solution.
Spectrophotometer: An instrument that emits light at different wavelengths and measures the amount of light absorbed by a sample.
Wavelengths: Spectrophotometers typically operate across a range of wavelengths, allowing the analysis of a variety of compounds with different absorption characteristics.
Sample Solution: The substance of interest (spinach leaves) is dissolved in a solvent, and the absorbance of light passing through the solution is measured.
Beer's Law describes the relationship between absorbance (A), concentration (C), l is the path length of the cuvette (the distance the light travels through the solution and the molar absorptivity (ε) of a substance: A=ε⋅C⋅l
Spectrophotometer
Colorimeter
Activity:
Steps 1-3
Step 5
Assessment
Students will record and graph the data and analyze the results in their groups.
Students will identify the wavelength of maximum absorption
Students will compare absorbance and concentration using Beer’s law formula
Post-lab questions:
1. What are the two peak absorbance readings and what wavelengths do they occur?
2. What colors on the spectrum do these readings represent?
3. Draw a graph to represent your findings for each sample.
DATA TABLE:
Concentration | wavelength | Absorbance | wavelength | Absorbance | wavelength | Absorbance |
100% | 450 nm | | 550 nm | | 660 nm | |
50% | 450 nm | | 550 nm | | 660 nm | |
25% | 450 nm | | 550 nm | | 660 nm | |
Differentiation
Provide extra support by working with struggling learners in small groups or one-on-one sessions.
For students who have struggle understanding about the wavelengths they can be referred to the concept of absorbance spectrum in the visible light range.
Demonstrate each step and allow for guided practice before independent work. Struggling learners will understand the reasoning behind the three wavelengths, 450 nm(blue), 550 nm (green) and 660nm(red).That means, chlorophyll absorbs blue and red light well and transmits or reflects green light. This is why we perceive chlorophyll and the plant leaves that contain it to be various shades of green. Plants tend to absorb blue and red light well – green light, not so well.
Consider using a simple checklist or rubric that highlights key procedural steps rather than complex scientific concepts.
Remediation
Extension/Enrichment
As an extension to the lesson, students can make connection to Beer’s law in Chemistry.
According to Beer's Law: A=ε⋅l⋅C where A is the absorbance, ε is the molar absorptivity (a constant for a given substance and wavelength), l is the path length of the cuvette (the distance the light travels through the solution), C is the concentration of the solution.
Given this relationship, when concentration increases, absorbance tends to increase, and when concentration decreases, absorbance tends to decrease.
For example,if students are comparing a concentration of 100% to a dilution of 12.5%, the 100% concentration is likely to have a higher absorbance compared to the 12.5% dilution, assuming a linear relationship between concentration and absorbance.