Introduction to SpectroscopyVisible spectroscopy is the study of the interaction of radiation from the visible part of the electromagnetic spectrum with a chemical species. Understanding visible spectroscopy requires understanding visible light. Light travels in packets of energy called photons. Each photon has a specific energy related to a certain frequency or wavelength (E = hν = hc/λ). Visible light consists of wavelengths ranging from roughly 400 nm (blue violet) to 700 nm (red). Absorption When all wavelengths of visible light are present, the light appears "white" to our eyes. If any wavelength is removed (absorbed), we perceive the remaining combination of wavelengths of light as the "complementary" color, the color across the color wheel (Figure 1).Figure 1: Color Wheel - Numbers are in λ (nm)Colored compounds, chromophores, are colored because of the absorption of visible radiation. The color is often a result of the compound absorbing a certain color of light, leading to the perception of the compound being the complementary color. For example, if white light passes through a test tube containing a solution of copper (II) sulfate (CuSO4), the solution will be blue because the Cu2+ ions strongly absorb “orange” photons of light (photons of light with λ ~ 600 nm).When a photon of colored light is absorbed by a chromophore (a colored compound) an electron transitions from lower energy orbital to higher energy orbital. The energy of absorbed radiation is equal to the energy difference (ΔE) between the highest energy electronic occupied orbital (OO) and an unoccupied orbital (UO). Many transition metal complexes and large conjugated organic molecules are brightly colored because this energy difference is equal to an energy within the visible region of the electromagnetic spectrum. Mathematically, this relationship is expressed by equation 1:Elight = hνlight = hc/λlight = ΔE = EUO – EOO Before the absorption of a photon, the electrons within the compound are in the lowest energy orbitals possible. Such an electron configuration is called the ground state. The absorption of a photon of visible radiation by a compound promotes an electron from an occupied orbital to an unoccupied orbital. The result is a higher energy compound in an excited state. (Figure 2)Figure 2. Absorption of light resulting in the excitation of an electronThe wavelength (i.e., frequency, energy, or color) of light required to promote an electron from the ground to the excited state is specific to each chemical, just as the energy difference between EOO and EUO is dependent on chemical identity. The goal of this experiment is to determine the concentration of FD&C Red Dye #40, a red chromophore, in red bottled sports drinks. Here is the structure of FD&C Red Dye #40 (molecular formula = C18H14N2Na2O8S2). You may need to refer to it during your experiment.The visible spectrum of FD&C Red Dye #40 is shown below. This type of spectrum is a plot of absorbance on the y-axis versus wavelength on the x-axis. The key thing to look for in a molecular absorption spectrum is the wavelength that best absorbs light, typically termed “λmax”, which is short for “wavelength of maximum absorbance”. It is at this wavelength that the instrument can best detect changes in concentration. Notice that the spectrum indicates that the λmax for FD&C Red Dye #40 is at ~506 nm. Notice that 506 nm is in the "green" region of the visible light spectrum and is the complement to "red", which is the color of the dye. Figure 3: Absorbance spectrum of FD&C Red-Dye #40Spectrophotometer Absorption and fluorescence are quantitatively measured by an instrument called a spectrometer. The spectrometer used in our lab contains absorption and excitation light sources, a cuvette holder, a diffraction grating to split light into different wavelengths, and a detector connected to a display (Figure 4). The solution to be analyzed is poured into a special vessel called a cuvette. A cuvette is a rectangular box with clear walls. The cuvette either holds the blank (typically, the solvent only) or the sample dissolved in the same solvent. Figure 4. From Vernier’s Website, Go Direct SpectroVis Plus Spectrophotometer To prevent spilling and insure a good measurement, fill a cuvette with a solution to about 75% of its total volume. Wipe the outside of a cuvette with a tissue before placing it in the sample holder. Beads of water, fingerprints, or bubbles in the solution interfere with the transmission of light through the sample. Gently insert a cuvette into the sample holder so the light passes through its clear walls. Mark a side of the cuvette at the very top and always insert the cuvette into the spectrometer the same way to avoid random fluctuations in the readings due to variations within the cuvette. Take care not to spill solutions into the spectrometer and replace scratched cuvettes. Figure 5: Plastic reusable cuvette with 1 cm pathlength.Click on the following link and watch how to insert the cuvette and read absorbance of samples in the Vernier Spectral Vis:https://www.youtube.com/watch?v=CEUa1JgKcp0#action=shareJulia Schafer-Mar 01, 2021, 1:38 PM PSTProcedureIan Ball-Mar 01, 2021, 1:38 PM PSTSAFETYFD&C Red Dye #40 is on the GRAS (“generally recognized as safe” by the FDA) list so it poses no safety threat.WASTEAll solutions may be poured down the drains. It is just food coloring. PROCEDUREEquipment:Spectral Analysis (or other data-collection and analysis program)Vernier SpectroVis Plus (or compatible)Cuvette25 mL, 50 mL & 100 mL volumetric flasks5 mL, 10 mL, 15 mL & 20 mL volumetric pipets and bulbsDistilled waterPipet pump or pipet bulbTissues (preferably lint-free)Work individually to measure your own unknown for this experiment. Note: Your instructor may have you work in groups to generate a rough standard curve of the FD&C Red Dye #40.Preparation of a Stock and Standard Solutions1. Prepare a primary standard solution of FD&C Red Dye #40 by weighing out precisely 0.105 g of solid solute and quantitatively transferring it to a 500 mL volumetric flask. Next, a stock solution is prepared by making a 1:10 dilution of the primary standard solution – this is Standard #1. 2. From the stock solution, "Standard #1" of FD&C Red Dye #40, prepare four dilutions using water as the solvent: a) 5 to 25 mL; b) 10 to 25 mL; c) 15 to 25 mL, and d) 20 to 25 mL. These four solutions plus Standard #1 constitute your five standard solutions. Note: In the Prelab assignment you will have already calculated the concentration of FD&C Red Dye #40 in each of the standard solutions.Preparation of Unknown Sports Drink Sample3. Obtain 25.00 mL of a sports drink sample to be compared to your standard curve. Your instructor will assign you a sports drink. Be sure to record the sample in your lab notebook. The goal is to use the absorbance of the unknown sample and the generated data from the standards to determine the amount of FD&C Red Dye #40 in the sample. Depending on the initial absorbance value of the sample, it may require dilution. Analysis of the Standards and Unknown on the Vernier SpectroVis Plus4. Prepare a blank by filling an empty cuvette 3/4 full with distilled water. To correctly use a cuvette, remember:All cuvettes should be wiped clean and dry on the outside with a tissue.Handle cuvettes only by the top edge or the ribbed sides.All solutions should be free of bubbles.Always position the cuvette so the light passes through the clear sides.5. Launch Spectral Analysis. Connect the SpectroVis Plus to your Chromebook or computer. Click or tap Absorbance vs. Concentration.6. To calibrate the spectrometer, place the blank cuvette in the spectrometer and select Finish Calibration. Note: If necessary, wait for the spectrometer to warm up before selecting Finish Calibration.7. Determine the optimal wavelength for creating the standard curve.a. Remove the blank cuvette, and place the highest concentration standard into the cuvette slot.b. The live graph will update with the spectrum of the sample. Click or tap the desired wavelength or enter the Wavelength. Click or tap Done.8. You are now ready to collect absorbance-concentration data for the five standard solutions. Prepare a cuvette for each standard solution. To prepare a cuvette, rinse it twice with the standard solution and then fill it about 3/4 full. Repeat this with all five standards until you have the five cuvettes complete. To correctly use a cuvette, remember:All cuvettes should be wiped clean and dry on the outside with a tissue.Handle cuvettes only by the top edge or the ribbed sides.All solutions should be free of bubbles.Always position the cuvette so the light passes through the clear sides.You are now ready to collect absorbance-concentration data for the five standard solutions.a. Click or tap Collect to start data collection.b. Empty the stock solution from the cuvette. Using the 5:25 standard dilution, rinse the cuvette twice with ~1 mL amounts and then fill it 3/4 full. Wipe the outside with a tissue and place it in the device.c. When the value has stabilized, click or tap Keep and enter the concentration of the standard in units of Molarity (mol/L). Click or tap Keep Point. The absorbance and concentration values have now been saved for the first solution.d. Discard the cuvette contents as directed by the safety guidelines. Using the 10:25 standard solution, rinse the cuvette twice with ~1 mL amounts, and then fill it 3/4 full. Place the cuvette in the device, wait for the value displayed on the screen to stabilize, and click or tap Keep. Enter the concentration of the standard in units of Molarity (mol/L). Click or tap Keep Point.e. Repeat the procedure for the 15:25 and 20:20 dilutions standards. Note: Wait until Step 10 to test the unknown.f. Click or tap Stop to stop data collection. Preparation of Standard Curve9. Display a graph of absorbance vs. concentration with a linear regression curve.a. Download and open the data file (.smbl file) prepared for the class in Vernier Spectral Analysis.b. Edit the concentration column to reflect the actual molarities used in the experiment. c. Click or tap Graph Tools, , and choose Edit Graph Options.d. Enter 0 as the value for both the Left value for the x-axis and the Bottom value for the y-axis. Dismiss the Graph Options box.e. Click or tap Graph Tools, , and choose Apply Curve Fit. f. Select Linear as the curve fit and Dismiss the Curve Fit box. The linear-regression statistics for these two data columns are displayed for the equation in the form:y = mx + bwhere x is concentration, y is absorbance, m is the slope, and b is the y-intercept. Note: One indicator of the quality of your data is the size of b. It is a very small value if the regression line passes through or near the origin. The correlation coefficient, r, indicates how closely the data points match up with (or fit) the regression line. A value of 1.00 indicates a nearly perfect fit. The graph should indicate a direct relationship between absorbance and concentration, a relationship known as Beer’s law. The regression line should closely fit the five data points and pass-through (or near) the origin of the graph.Measure the Absorbance of the Unknown10. Determine the absorbance value of the unknown sports drink solution.a. Obtain a new cuvette. Rinse the cuvette twice with the unknown solution and fill it about 3/4 full. Wipe the outside of the cuvette and place it into the device.b. Monitor the absorbance value. When this value has stabilized, record it.c. If the sample is outside the range of the calibration standards, you will need to quantitatively dilute the sample and re-analyze it.CalculationsUsing the absorbance of your unknown sample, determine the concentration of FD&C Red Dye #40 in the sample measured. Taking into account any dilutions and using the serving size on the container, calculate how many grams of FD&C Red Dye #40 per serving of your unknown sports drink. POST-EXPERIMENT DISCUSSION QUESTIONS – Answer these questions in your Conclusion as directed by your instructor1. If we had blue dye, red dye, and yellow dye in the samples. How could we focus on just the red dye’s concentration?2. What is the wavelength setting on the spectrophotometer for this experiment? Why does the instrument need to be set at this wavelength?3. Why did we need to prepare and analyze a set of standards?4. What is Beer’s Law? Was Beer’s Law used in this experiment? If so, how? If so, why could we use it?5. Of what, exactly, are we measuring the absorbance? Be specific. 6. Discuss any error(s) that may have been introduced in the analysis of the red dye.