1. Experiment 6 Amount of Dye in a Sports Drink

2. Goal To make a Beer’s Law standard curve To use the standard curve (and spectrophotometry) to determine the concentration of red dye that is in a sports drink

3. The Intro Should Include… What is a Spectrophotometer? What does it measure? What fundamental Chemistry principle governs the use of this method? Explain what the principle is and how it applies to this experiment What type of analysis is done using this method and how does it allow you to determine the [red dye]? What is the relationship between a particular substance and light?

4. Color and Light For a colored molecule, the higher the concentration the darker the color Every color represents a different wavelength, λ What is happening when we see a color? Because each color has a specific λ, each colored molecule has an optimum λ where it will absorb the greatest amount of light, λ max

5. Determining Which Wavelength Should be Used Absorbance Wavelength λ λ max You should include a graph like this one in your notebook and indicate on the graph what your λ max value is

6. A=εbc Note: ε is a constant for a specific molecule and is dependent on wavelength b is constant for our experiments because we use the same cuvet throughout the experiment A=εbc is linear y=mx+b, our b is zero Beer’s Law only applies to ______________ Beer’s Law only works for _______________ Beer-Lambert Law (AKA Beer’s Law)

7. The Spectrophotometer LIGHT SOURCE MANY λs MONOCHROMATOR 1 λ I0I0 Sample Cell I DETECTOR

8. Spectrophotometer continued What does it directly measure? It can then convert %T to absorbance This is the absorbance value that relates back to Beer’s Law

9. How Does This Allow Us to Determine the Concentration of an Unknown? We already said that Beer’s Law is linear for dilute solutions How can we use that to our advantage? A=εbc, ε and b are both constant Make solutions (> two) of known conc. Measure Absorbance (at same λ) for each What will we get when we plot A vs C? Note: You will not get linear data for solutions that are too concentration. Therefore, it is critical that you have a standard curve that is within the range of your measurement, or that your measurement is within the range of your standards.

10. Standard Curve

11. Standard Curve continued This will be our __________ plot. We now have the equation for the line We can measure Abs for the unknown and plug into our equation and solve for conc. X is the concentration of the solution that was analyzed in the spectrophotometer. You may have to do additional calculations to take into account any dilutions that you made to the unknown sample.

12. Applications of This Method Can be used any time that you have a solution and want to find the concentration of a colored molecule in that solution Quantitative analysis of: - Trace chemicals - Environmental agents (heavy metals in H 2 O samples) - Biochemicals ([DNA] or [protein]) - Metabolites - Degradation products (to test for oxidation of, for example Fe +2 to Fe +3 ) Kinetic studies (ie enzyme kinetics) Spectroscopy also used in: Blood sugar monitors (use near IR wavelengths) Blood oxygen level monitors (uses red and IR wavelengths)

13. Critical Steps/Measurements

14. Experimental Design Variables