1. GenChem Week 3 Absorption Spectroscopy Today’s Agenda:  Review understanding of chemical toxicity.  Develop understanding of absorption spectroscopy.  Develop the day’s problem.  Perform the first challenge: identify λ max for your dye.  Perform the second challenge: determine molar absorptivity for the dye.  Perform the third challenge: prepare samples for analysis  Perform the fourth challenge: determine dye content of sample.

2. GenChem Chemicals and the environment.

3. GenChem Let′s think! In your groups:  Based on what you have heard from the media, what are the dangers posed by chemicals.  Name some chemicals you think are toxic.

4. GenChem Consider a chemical with the following properties:  It is a major component of acid rain.  It is a green house gas which significantly contributes to “global warming".  As a gas it can cause severe burns.  It is a major cause of farmland erosion.  It accelerates the corrosion and rusting of many metals.  It can cause electrical failures and decrease the effectiveness of automobile brakes.  In 2000, more than 409,000 people died due to lethal exposure to this chemical. What is it? What is it? Dihydrogen Monoxide

5. GenChem What about chemicals makes them dangerous? “All things are poison, and nothing is without poison: only the dose permits something not to be poisonous.” - Paracelsus

6. GenChem The hazardous properties of chemicals are best summarized by the NFPA chemical safety placard required on all shipments of hazardous chemicals.

7. GenChem The importance of amounts is reflected in the use of the term “lethal dose”. The most commonly used measure of lethality is the LD 50

8. GenChem MaterialLD50 (mg/kg)subject, route sucrose 29700rat, oral (cane sugar) sodium bicarbonate 4220rat, oral (baking soda) sodium chloride 3000rat, oral (table salt) ethanol 2080rat, oral (grain alcohol) caffeine192rat, oral sodium cyanide6.4rat, oral sarin 24human, skin contact (nerve agent) VX 0.14human, skin contact (nerve agent)

9. GenChem For most chemicals, the most critical question is “how much”? The question posed to you with this experiment is “how much would you have to consume to achieve an LD 50 exposure to a common food dye?”

10. GenChem Common Food Dyes

11. GenChem Foods are a mixture of many chemicals. Only a small fraction are dye molecules. To answer our question we will need a method for counting the dye molecules in a food sample. There are not many techniques for doing this directly. One of the quickest, cheapest and easiest is absorption spectroscopy.

12. GenChem Lets look at the relationship between concentration and absorbance. Absorbance is directly proportional to the concentration. Absorbance  concentration Time for a Demonstration!

13. GenChem What do we mean by concentration? What are some ways we can express concentration? How do chemists measure concentration? Molarity = moles solute / liter solution

14. GenChem Why did chemists invent this? It allows chemists to measure moles (amounts) by measuring volumes of solutions.

15. GenChem Putting this all together we come up with the Beer – Lambert Law aka Beer’s Law for any particular wavelength,

16. GenChem In your groups:  Discuss how you can use Beer’s Law to determine the concentration of something in solution. Given values for  and b, A can be measured and C calculated. Let′s think!

17. GenChem In your groups:  How can b be determined?  How can  be determined? Let′s think! b – measure cuvet.  – from the slope.

18. GenChem In your groups:  What wavelength should be used?  Why? Wavelength of maximal absorbance, symbolized as max. Maximum signal minimizes effect of noise. Let′s think!

19. GenChem Finding max Share with the class your response to the pre-lab question. This experiment requires you to provide your own sample of a food containing a food dye. Describe the sample you intend to use. Indicate which food dye you will be evaluating. Form a group with 2 or 3 others who will be evaluating the same food dye. In this group come up with a consensus answer to the following question. Outline a procedure to display a spectrum of your food dye on the spectrophotometer. Let′s think!

20. GenChem Your First Challenge Determine max for your selected food dye. Available resources:  Food dye solution of known concentration.  Spectrometer. Just do it! Let′s explore! You have 10 minutes

21. GenChem max Consider the reported values for max Are they reasonable? Should any be discarded? colorwavelength 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

22. GenChem In your groups:  What will you need to determine the molar absorptivity of your food dye?  Outline how you expect to achieve this. Let′s think!  a number of solutions of known concentration.  perform a serial or parallel dilution of the stock solution.

23. GenChem Standard solutions In your groups review your responses to the following pre-lab question and come up with a consensus answer. Create a procedure you can use to prepare a series of solutions to determine the molar absorptivity of your food dye. Let′s think!

24. GenChem Your Second Challenge Determine the molar absorptivity (ε) for your selected food dye at your previously determined max. Available resources:  Food dye solution of known concentration.  graduated pipet and bulb  volumetric flasks  Spectrometer. Implement your experimental procedure! Let′s explore! You have 45 minutes

25. GenChem Consider the reported values for ε at λ max Are they reasonable? Should any be discarded?

26. GenChem You how have enough information to use Beer’s Law. How are you going to use it to determine the dye in your food sample? Measure the absorbance of the sample, its volume and calculate its concentration.

27. GenChem Dissolve a known amount of the sample in water until absorbance is less than 1.0. Measure everything. As a group review each other’s responses to the following pre-lab question. Make necessary corrections. Create a procedure for preparing your sample for analysis. Describe how you will have to treat it to be able to put it in the cuvet, measure its absorbance and obtain a good value.

28. GenChem Volumetric Measurements The following volumetric devices are available for your use. Make sure you know how to properly use them. 10 mL graduated pipet Pipet bulb (blue) 50 mL volumetric flask 100 mL volumetric flask

29. GenChem Your Final Challenge Once you have a procedure approved by your instructor, perform it. When you have a value for the mass of food dye in your original sample, report it to your instructor. Let′s explore! You have 60 minutes Available resources:  glassware.  Spectrometer.

30. GenChem The Report With this report you will learn how to compose a scientific discussion. From your Technique Book: Discussion. Analysis of the data and presentation of conclusions regarding original hypothesis. This should include a defense of the conclusions citing the results and a discussion of the reliability of the data used.

31. GenChem Discussion Outline What were the major goals of today’s lab? Determine the λ max and molar absorptivity of a selected food dye. Determine the amount of food dye present in a sample. Determine the amount of the food stuff required to reach the LD50 value.

32. GenChem Discussion Outline For each goal, what were the results? Determine the λ max and molar absorptivity of a selected food dye. Determine the amount of food dye present in a sample. Determine the amount of the food stuff required to reach the LD50 value. λ max = 630. ε at 630 = 1.7 x 10 5 Abs mol –1 cm –1 Mass of dye present in 10 g of peas = 4.5 x 10 -3 g. Mass of peas to reach LD50 = 4,400 g per kg of body mass.

33. GenChem Discussion: Goal 1 For Goal 1: Interpret/analyze result. What is the result? (Cite data/give examples). What does it mean? Does it make sense? Explain the chemistry. Is it what was expected? Is it consistent with your observations? Comparisons to literature or peers. If something went wrong: What were you expecting and why? What went wrong? How do you know? How could you fix it next time?

34. GenChem Discussion: Goal 1 The dye being studied was Fast Green FCF. The absorbance spectrum of a 6.10 x 10 -6 M solution is shown in figure 1. The λ max was determined to be at 630 nm. A parallel dilution of this solution produced the solutions given in table 1. A plot of absorbance vs concentration is given in figure 2. Excel was used to generate a trend line through these points and produced a value of 1.7 x 10 5. The diameter of the cuvet was determined to be 1.0 cm yielding a value for the molar absorptivity at 630 nm of 1.7 x 10 5 Abs mol –1 cm –1 The data points are all a reasonable close fit to the trend line. The r 2 value of 0.992 supports this conclusion. The fact that the trend line does not go through the origin introduces some uncertainty as the origin should be a point on the graph, but the difference is small. The value is also consistent with observations. Food dyes in general are strongly colored and expected to have large molar absorptivities. The values obtained by classmates for the other food dyes all were on the order of 10 5 and the stock solutions all appeared to have similar color intensities.

35. GenChem Additional Goals If the experiment has more than one goal, you will discuss the results of each like we did for Goal 1. In addition to discussing the results of each goal, you also need to discuss data quality…

36. GenChem Discussion: Data quality In a paragraph, discuss the quality and reliability of your data: Discuss pros/cons/accuracy/precision of tools/techniques. Use technique book! Be specific and give examples. Suggest fixes for the problems you identify and additional experiments to improve findings. Avoid using generic “human error”. Any discussion of human error should focus on specific, known errors and their effect on the results.

37. GenChem Discussion: Data quality The data presented in table 1 and figure 2 fit nicely the Beer’s Law equation. This indicates that Beer’s Law is valid for the concentration ranges studied and provides confidence that this calibration plot can be reliably used for other determinations. It also implies that the spectrometer is suitable over this concentration range and that the parallel dilution was reliably performed. The fact that the absorbance value for the sample solution tested falls within the range of standard solutions strongly suggests that the overall outcome of the experiment is valid to at least two significant figures.

38. GenChem Discussion See Technique book pp. 68-69 for more help with writing a Discussion.

39. GenChem The Report Report Content:  Raw data. A copy of all measurements and observations you collected in lab.  Results. An organized presentation of the results obtained.  Calculations. Examples of the various calculations performed.  Discussion. A discussion of the results obtained. You will now write your report for this experiment. It is a limited report. Only the following sections are required. We’ll add the “introduction” next week. As an additional pre-lab assignment for next week, construct what you think will be an appropriate introduction for the aspirin synthesis.