In this lesson students will utilize spectrophotometry to identify the wavelength of maximum absorbance for a food dye. They will also generate a Beer's Law Standard Curve, and utilize their skills to identify the different dyes and their concentrations in an unknown mixture. The lesson culminates with an extension to utilizing a similar method in color matching paint.
This activity will help prepare your students to meet the performance expectations in the following standards:
- HS-PS4-3: Evaluate the claims, evidence, and reasoning behind the idea that electromagnetic radiation can be described either by a wave model or a particle model, and that for some situations one model is more useful than the other.
- HS-PS4-4: Evaluate the validity and reliability of claims in published materials of the effects that different frequencies of electromagnetic radiation have when absorbed by matter.
- HS-ETS1-2: Design a solution to a complex real-world problem by breaking it down into small, more manageable problems that can be solved through engineering.
- Scientific and Engineering Practices:
- Using Mathematics and Computational Thinking
- Developing and Using Models
- Analyzing and Interpreting Data
AP Chemistry Curriculum Framework
- Big Idea 1: The chemical elements are fundamental building materials of matter, and all matter can be understood in terms of arrangements of atoms. These atoms retain their identity in chemical reactions.
- 1.16 The student can design and/or interpret the results of an experiment regarding the absorption of light to determine the concentration of an absorbing species in a solution.
By the end of this lesson, students should be able to
- Determine the wavelength of maximum absorbance of a water soluble dye using a spectrophotometer.
- Use a Beer’s Law Curve to determine the concentration of an unknown sample of a dye.
- Identify the components of a mixture using absorbance spectra of the dyes present.
- Design a procedure to identify components in a paint mixture using concepts learned in this activity.
This lesson supports students’ understanding of
- Beer’s Law
- Electromagnetic spectrum
Teacher Preparation: 45 minutes
- Engage: 15-30 minutes
- Explore: 50-90 minutes
- Explain: 10-20 minutes
- Elaborate: 15-20 minutes
- Evaluate: 45-60 minutes
- Colored flashlights or flashlights with colored saran wrap
- Paint chips from hardware store/paint store
- FD&C food dyes (Blue #1, Yellow #5, Red #40) stock solution of 0.050% was used to generate 1-L solutions of each dye.
- Spectrophotometer (this lab was written for the use of Vernier Labquests & Vernier Spectrophotometer). Spec20 can be used but more time may need to be allotted.
- Distilled or Deionized Water
- Beakers, 50-mL (4 per lab group)
- Graduated pipets (4 per lab group)
- Pipet bulbs or fillers (1 per lab group)
- Cuvettes (6 per lab group)
- Kimwipes or lens tissue
- Graph Paper (if not using graphing technology)
- Always wear safety goggles when handling chemicals in the lab.
- Students should wash their hands thoroughly before leaving the lab.
- When students complete the lab, instruct them how to clean up their materials and dispose of any chemicals.
- Engage: Begin by shining colored light on different colored paint chips. For example, using a red light, shine the light on a red paint chip. Do the same with blue and green light. Then shine the different colored lights on different papers—red on blue, etc. Have students compare the different lights on the different papers and discuss what they find.
- Hold up colored solutions and ask the students to discuss the difference between the colored solutions and the opaque paint chips. Use the opportunity to discuss the transmittance of light through a solution and the reflection of light from the opaque paint chips.
- Discuss with the students how this is occurring and/or show this 7 minute video, Color: White light, Reflection & Absorption, that does a great job explaining the basics about how color is reflected from an object.
- Using a paint chip, ask students to discuss the following questions. Allow students to generate conversation regarding the topic.
- How one might be able to go to the paint/hardware store and get a can of the exact same color?
- How does the technician know what paints to mix?
- How does the technician know the paint will end up reflecting color the same way the unknown color does?
- Does the technician have a means to measure this or does it simply get “eyeballed”?
- Tell the students the technology used to measure reflection on an opaque object is similar to the technology used to measure transmittance. In both situations, a spectrophotometer is used. In this lab, students will identify an unknown in a water-soluble system. Students will then be asked to make an analogy to how this can be done when the unknown is opaque, like with the paint chips. Video tutorial are provided on the student handout—teachers may want to make the links easily available for students.
Part One: Prepare dye solutions for this portion of the lab. Each dye solution can be made using the 0.050% stock solution. Add 25 mL of the dye to a volumetric flask. Dilute to 1-L.
- In order to determine the initial concentration of the dye, find the wavelength of maximum absorbance for each dye (approximately 630 nm for FD&C Blue dye #1, approximately 427 nm for FD&C Yellow #5, and approximately 503 nm for FD&C Red #40). Use Beer's Law to calculate the concentration of the new stock solution:
- A = abc
- A is absorbance measured
- a = molar absorptivity of the dye (see below for values)
- b = path length of cuvette (1 cm)
- c = concentration
- a for FD&C Blue Dye #1 is 130,000 M-1cm-1
- a for FD&C Yellow Dye #5 is 27,300 M-1cm-1
- a for FD&C Red Dye #40 is 25,900 M-1cm-1
- Concentrations are reported in microMolar units (µM). Students may need to be reminded of the conversion of Molar to microMolar.
- In this portion of the lab, the students will get comfortable with the spectrophotometer while evaluating a food dye to determine the wavelength best absorbed by the dye. The teacher may want to delegate each color to a specific group if computer interfaces are not used for time sake. Each lab group is to find the absorbance spectrum for the given food dye, graph their findings, pinpoint the wavelength of maximum absorbance and then collaborate as a class to provide all groups the data. If the student is using a computer interface (as in this procedure), the teacher may choose to have each group find an absorbance spectrum for each dye. The students will use these spectra in part two of the lab.
- Part Two: Students will complete dilutions on each of the dyes to develop a Beer's Law Standard Curve. The teacher may choose here to delegate a specific dye to each group for time's sake. Students will be told the initial concentration of the dye as determined earlier by the teacher. Students will use M1V1=M2V2 to perform the remaining concentration calculations. Students will need to complete these calculations before generating the Beer's Law Standard Curve.
- The teacher will provide an unknown solution. This unknown solution can be made with blue and yellow. If red and yellow are mixed, students will not be able to get conclusive results due to the additive properties of the two dyes’ absorbances. This is referenced in Analysis Question #1.
- Each group should get a different unknown mixture—for example, the unknown used in the “Predicted Results” available in the Answer Key Document was made with 2 mL yellow, 2 mL blue, 6 mL water mixture. Mixtures of blue and red work well also.
- The students are to match the peaks in the absorbance spectrums of the unknown with the peaks of their known substances. The peak for the blue dye should be approximately 630 nm and the peak for the yellow dye should be approximately 427 nm. Students should then be able to use the absorbance value (of the unknown) at each of the peaks to determine the concentration of each dye in the unknown by comparing to the Beer's Law Standard Curve.
- Explain: The students will identify the concentration of each dye in the unknown, providing supporting evidence for their reasoning.
- Elaborate: Students are asked to extend their thinking to determine how paints are matched at a hardware store. If provided with a specific color, how can a recipe be determined to match that color? Point out to students, that paints are not water soluble dyes, but are colored by insoluble particles called pigments (this is also pointed out in the lab).
- Evaluate: The teacher may choose to request students to create a lab report to relay the group’s findings. Or use a tool such as EduCreations or ShowMe to make a short video clip highlighting the important discoveries and concepts learned, answers to the analysis questions found at the end of the lab activity, and pictures of graphs generated.
- References: Flinn Scientific, Inc. “Analysis of Food Dyes in Beverages”