1. How can we use spectroscopy to quantify amount of substance?
Lab Session 7
This is an experiment in which they will explore how to use absorption spectroscopy to quantify the amount of dye in a commercial product.
How can we use spectroscopy to quantify amount of substance?

2. How much dye is added per gram of food product?
Your Challenge
Imagine that you work for the FDA and you have been asked to monitor the amount of coloring added to different food products.
Your task is to use absorption spectroscopy to quantify the mass of dye in commercial food products.
Present the challenge to be faced in this week’s experiment.
How much dye is added per gram of food product?

3. Absorption Spectroscopy
Chemical substances absorb “electromagnetic radiation” (EM) of specific wavelengths.
Dyes absorb EM in the visible portion of the spectrum.
Remind students how absorption spectroscopy works.

4. How would you compare the concentration of the different solutions?
Quantitation
How would you expect the absorbance (A) change with the concentration (C)?
Spectra can also be used to quantify the concentration of different substances in a system C
lmax
Discuss the meaning of C (as # of particles per unit volume, not mass per unit volume)
Discuss the idea of using the absorbance at the maxima as a way to compare relative concentrations (this helps to minimize noise).
A α C
How would you compare the concentration of the different solutions?
Compare A at given l

5. Measuring Concentrations
For many materials, the absorbance (A) at a given l is proportional to the concentration of the absorbing species (number of moles or molecules per unit volume; C).
A α C
How would you expect A to change with b?
A α b
How can we convert this proportionalities into a math relationship?
b (path length)

6. Beer’s Law
Putting this all together we come up with the Beer – Lambert Law:
For any particular wavelength,
Concentration
Mol/L
The answer: Introducing a proportionality constant (epsilon)
Discuss the meaning and implications of the relationship. Stress that epsilon is specific for a particular substance at an specific wavelength.
Molar absorptivity  L/(mol cm)
Path length  cm
Absorbance

7. We need to determine e for the dye. How?
Quantitation
Given
How can we use this type of data to determine the actual concentration of dye in a solution?
Students need to realize that somehow we need to determine the molar absorptivity of the substance using reference solutions of known concentration.
We need to determine e for the dye. How?

8. Why do we need many data points? Can we use the graph to determine C?
References
We need to build a “reference system” using standard solutions of known concentrations. ?
Slope = eb
Why do we need many data points?
Discuss the fact that e must be experimentally determined with known solutions. The value is too dependent on the conditions under which it is measured to be accurately reproduced from lab to lab. Thus, it is not normally possible to look up accurate e values.
Unknown
Can we use the graph to determine C?

9. Skill Building Tests
Based on your food product, select a dye to analyze.
Determine its lmax;
II. Build a reference graph and determine e for the selected dye.
Use this slide to introduce their first challenge, DON’T SEND THEM INTO THE LAB YET. Go over the next five slides to guide students in the design of a good plan to build the reference graph.
Available resources:
Dye solutions of known C,
Spectrometer.
But before you proceed, let’s build a plan

10. Food Coloring What do you have?
In the USA, the following artificial colorings are permitted in food as of 2007:
Blue No. 1 – (Blue shade)
Blue No. 2 – (Dark Blue shade)
Red No. 3 –(Pink shade)
Red No. 40 – (Red shade)
Yellow No. 5 – (Yellow shade)
Yellow No. 6 – (Orange shade)
Make them check what food coloring is in their product by reviewing the product label.
It is best if each group selects products to analyze with the same type of dye, or two at most. So that they can
Prepare the corresponding reference graphs. Yellow 5 should be avoided as its lambda max may be too low for our spectrometers.
What do you have?

11. lmax
As you collect data, reflect on the measured value for lmax and make sure it is reasonable given the color of your dye.
color
wavelength interval
red
~ 630–700 nm
orange
~ 590–630 nm
yellow
~ 560–590 nm
green
~ 490–560 nm
blue
~ 450–490 nm
violet
~ 400–450 nm
For each dye consider two things. (1) are the measured lambamax values consistent with the color of the dye (its complement)? This will require knowing the wavelengths of the various colors. (2) For multiple values, are the all acceptable or should one or more be discarded?

12. Is this the best approach?
Dilutions
You will need to prepare solutions of different concentrations. For that purpose, you can use serial or parallel dilutions.
2.0 mol/L  0.2 mol/L  0.02 mol/L 
serial
Is this the best approach?
Discuss how to make dilutions with the students. Make them think about the problems of using serial dilution to generate a reference graph A C ?

13. Is this the best approach? Parallel dilution will work best for us
Dilutions
Parallel
Is this the best approach? A C
Make them think what happens when you do parallel dilutions. Discuss why this is the best approach for this experiment.
Parallel dilution will work best for us

14. Your Plan
Design a plan to generate reference graph with points well distributed in the absorbance range from 0.1 to 1.
Make sure all groups have a clear plan before they move into the lab.

15. Skill Building Tests
Based on your food product, select a dye to analyze.
Determine its lmax;
II. Build a reference graph and determine e for the selected dye.
Available resources:
Dye solutions of known C,
Spectrometer.
Design and implement your plan!
The purposes is for them to use the equipment to generate the reference graph for their food dye, and determine the molar absorptivity. Make sure they know how to use the spectrometer in absorbance mode.
You have 60 minutes

16. Share Results and Reflect
Present your results to the class. Decide as a group which data should be shared to facilitate the analysis.
Dye
lmax e
Blue 1
630
Blue 2
610
Red 3
527
Red 40
502
Yellow 5
428
Yellow 6
484
Does your dye follow Beer’s law?
Identify any potential sources of error in your data.
Discuss the results with the class. Analyze the validity of the data.
For each molar absorptivity consider three things. (1) Does the value make sense in the equation given that the stock solutions should have absorbances near to 1.0? (2) for multiple values for the same dye are they measured at the same lambdamax? and (3) For multiple values, are they all acceptable or should one or more be discarded?

17. How much dye is added per gram of food product?
Your Challenge
Imagine that you work for the FDA and you have been asked to monitor the amount of coloring added to different food products.
Your task is to use absorption spectroscopy to quantify the mass of dye added to commercial food products.
Present the challenge to be faced in this week’s experiment.
How much dye is added per gram of food product?

18. Your Plan
Discuss in your group how you plan to determine the amount of dye in your food product (you need to take into account whether your sample is liquid or solid).
Discuss how you will calculate the amount of dye in grams per gram of food product.
Have the groups discuss how they are planning to face the challenge. Discuss and evaluate their plan of action and their proposal to go from concentration to amount of dye in grams. Once you approve it, they may go into the lab.
You may need to have them contrast preparation of a solid sample and a liquid sample.

19. Your Real Challenge
Determine the amount of dye per gram of your selected food product.
Design and implement an experimental procedure!
Available resources:
Food product;
Spectrometer;
Glassware;.
I would suggest to use an unknown mixture to guide students on how to analyze the data for a mixture. Ask them to focus their attention on the 4 more intense peaks for the reference spectra, and use these peaks as a guide in the detection. It is likely that the emissions from sodium will be seen in different salts. Make sure students adjust intensities to the net more intense peak.
You have 60 minutes

20. Share Results
Present your results to the class. Decide as a group which data should be shared to facilitate the analysis.
Have the different students summarize their results on the whiteboard.

21. Claims and Evidence
Based on the results of your experiments, present your major claims and the evidence that you have to support the answer to the challenge and your own research question.
Is the group answering the central question for this experiment?
Are their claims clear?
Is the evidence reliable?
Is the evidence appropriate to support the claims?
How do you explain the results?
Ask each of the groups to report their results on the board. Have the rest of the groups question the claims and the evidence using the questions on the slide as a guide.

22. Final Reflections What did you learn from doing your experiment?
How would you improve what you did?
How have your ideas changed as a result of this lab?
What do you not completely understand?
What new questions do you have?
Use these questions to motivate a discussion and emphasize major points you want students to understand.

23. Your Report
Beginning questions (2 p): What questions guided your explorations?
Safety Considerations (2 p): What did you do to stay safe in the lab?
Procedures and Tests (2 p): What experiments did you do to answer your questions?
Data, calculations, and representations (6 p): What observations did you make? What data did you collect? What calculations and representations helped you make sense of the data?
You will now go over the basic components of the report. Students should work together in groups, but insist on having an individual report from each student. Talk to your students about your expectation for the report. Have them share ideas on what to write in the different sections.

24. Your Report Claims (2 p): What can you claim to answer your questions?
Evidence and Analysis (6 p): How did you interpret your results to support your claims?
Reflections and additional questions (10 p): What did you learn? What do you not completely understand? How have your ideas changed as a result of this lab? What new questions do you have? How would you improve what you did?
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25. Midterm Exam A two part exam:
Part 1 A written test. Short answer questions. You will have one hour to complete.
Part 2 A practical. You will perform the experiment described in a handout posted in D2L. You will collect data and write a report, all in class. Unlike all experiments so far, you will perform this experiment by yourself.
Announcement. The practical is conducted like a normal experiment with two major variations. 1) once in lab, they work alone and (2) they submit the report before leaving the lab.