Materials Included In Kit

Additional Materials Required

Prelab Preparation

1. Turn on the spectrophotometer. Allow to warm up for 15–20 minutes. Set wavelength to 630 nm.
2. Add 25 mL of FD&C Blue 1 concentrated stock solution to a 1000-mL volumetric flask and dilute to the mark with distilled or deionized water. Provide this diluted solution to the students as their “stock” solution. Dilute dyes Red 40 and Yellow 5 following this step.
3. Blank the spectrophotometer with distilled or deionized water. Be sure to clean the outside walls of the sample cell.
4. Using the same sample cell, rinse with the diluted stock solution from step 2 twice before measuring the absorbance. Collect an absorbance reading of the FD&C Blue 1 solution at 630 nm. Absorbance (A) should read between 0.7 and 1.0.
5. Use Beer’s law, A = abc to calculate the concentration of the new stock solution.

   A is absorbance measured.
   a is molar absorptivity of FD&C blue 1 dye (130,000 M^–1cm^–1).
   b is path length of the sample cell or cuvet, 1 cm.
   c is concentration.
   
   Absorbance measured, A = 0.938
   0.938 = (130,000 M^–1cm^–1)(1 cm)(c)
   c = 0.938/(130,000 M^–1cm^–1)(1 cm)
   c = 7.22 x 10^–6 M or 7.22 μM

Safety Precautions

The FD&C dyes are slightly hazardous by eye and skin contact. The dyes have been stored with other, nonfood-grade chemicals and are not for consumption. Avoid contact with eyes, skin and clothing. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. Remind students to wash their hands thoroughly with soap and water before leaving the laboratory. Please review current Safety Data Sheets for additional safety, handling and disposal information.

Disposal

Please consult your current Flinn Scientific Catalog/Reference Manual for general guidelines and specific procedures, and review all federal, state and local regulations that may apply, before proceeding. Any remaining or excess dye solutions and sports drinks may be rinsed down the drain with plenty of water according to Flinn Suggested Disposal Method #26b.

Lab Hints

• For a cooperative study, each group should make at least 2–3 dilutions from their stock solution; there should be a range of dilutions and section data in order to construct an accurate calibration curve.
• Inform your students that the goal is to identify a linear graph with the data collected by the section.
• Students may collect data in % transmittance.
• Students should collect at least one spectroscopic measurement of their dilutions and one reading for their blue sports drink sample. We suggest using Gatorade^® as the consumer beverage samples. An example of a sports drink that contains FD&C Blue 1 is Gatorade Glacier Freeze. The label ingredients should include Blue 1 and no other food dyes. Fruit Punch Gatorade is an excellent sample for a red drink. Lemon-Lime Gatorade contains yellow food dye.
• Absorbance measurements for the Beer’s law plot will be most accurate if the same sample cell is used for all readings. This may not be practical, but it should be discussed as a source of error.
• Spectrophotometer measurements are typically in percent transmittance or absorbance. Remember that transmittance (T) is the decimal fraction of % T. Thus, if % T = 56.1, T = 0.561. You may assign different graphs to the groups and have students determine which graph provides linear data that has a positive slope and goes through zero. They may plot on an Excel program.
• Colorimeters may be used if spectrophotometers are not available. Set the colorimeter to 635 nm to analyze Blue 1, 470 nm for Red 40 and 430 nm for Yellow 5.
• Students will report that –log T vs. [dye] plot provides a linear relationship between the concentration and the transmittance. The graph contains a positive slope and goes through zero.
• Discuss the relationship between transmittance (T) and absorbance (A) and the best-fit line that may be used to determine the concentration of FD&C Blue 1 in the blue sports drink sample.

  –log T = slope [dye]
  A = –log (T)

• The best-fit line of the linear graph should be used to calculate the concentration of food dyes in the sports drink samples. Each group will determine the concentration of their sports drink sample based on the classes’ calibration curve. See Sample Data.

  y = mx + b
  A = m[concentration] + 0

• Based on the relationship A = –log T, the higher the absorbance, the lower % transmittance of light beamed through the sample. Measurements greater than 1.5 A should not be accepted. The sample should be diluted and a new absorbance reading collected.
• The literature values of a (the molar absorptivity coefficient) are 25,900 M^–1cm^–1 for FD&C Red 40 (λ_max = 503 nm) and 27,300 M^–1cm^–1 for FD&C Yellow 5 (λ_max = 427 nm).

  Note: When using the colorimeter, the molar absorptivity coefficient needs to be adjusted for your lab data. Since the colorimeter reads at 635 nm, not 630 nm, the absorbance will be different. For the blue dye, using a colorimeter, the stock solution has an absorbance of approximately 0.51. The new molar absorptivity coefficient, for a wavelength of 635 nm, can be found by multiplying a ratio of stock solution absorbances to the molar absorptivity constant at 630 nm.

  {13833_Hints_Equation_4}

  In our lab, we found the colorimeter to measure 0.51 for the stock solution and 0.915 with the spectrophotometer. Therefore, the constant for the colorimeter at 635 nm would be (0.51/0.915) • 130,000 M^–1cm^–1 or 72,000 M^–1cm^–1. You can perform the same calculations for the molar absorptivity constants of the yellow and red dyes (using the absorbance of the stock solution performed in your colorimeter and our sample spectrophotometer absorbance data for the stock solutions).

Further Extensions

Opportunities for Undergraduate Research
FD&C dyes are organic molecules with chemical structures containing multiple carbon rings with double bonds (see Figure 3 in the Background). When molecules have a series of double bonds separated by single bonds, the bonding pattern is called conjugation. This pattern of bonding results in a reduced separation between the ground state and the excited state of the electrons. The energy difference corresponds to the energy of photons in the visible region. As the amount of conjugation increases, the energy of the absorbed photon decreases. FD&C Blue 1 dye should absorb light that is least energetic of the three, followed by Red 40 and Yellow 5 with higher absorbed energies. Measure the visible spectra of Red 40 and Yellow 5 and identify the wavelength of light that results in the maximum absorbance value, λ_max, for each dye. Plot the visible spectra of all the dyes on one graph to create an overlay. Compare the dyes and make observations. Prepare solutions and generate Beer’s law plots to analyze the concentrations of these dyes in various beverages. The structures and molar masses of FD&C Red 40 and Yellow 5 are shown in Figure 7.

Answers to Prelab Questions

1. What would be an optimum wavelength for measuring the absorbance versus concentration of a series of FD&C Blue 1 dye solutions? Explain your answer. Absorbance measurements are most accurate and sensitive in the range 0.2–1.0.

   The wavelength of maximum absorbance for FD&C Blue 1 is 620–630 nm. The maximum absorbance is approximately 0.9 for this solution. This is the accurate range.

2. 2. To construct a calibration curve, a series of known concentration standards is prepared. Using the estimated concentration of the FD&C Blue 1 stock solution, determine the concentration of each of the following dilutions. Hint: M₁V₁ = M₂V₂.
   {13833_PreLabAnswers_Table_2}
3. Using the information provided in Question 1, predict the absorbance of each solution A–H at the optimum wavelength. Refer to Equation 3 in the Background section. The value of a is 130,000 M^–1 cm^–1 and b = 1 cm.

   Predicted absorbance values are shown in the table. Sample calculation for c = 2.8 μM:
   A = (130,000 M^–1cm^–1) (1 cm) (2.8 x 10^–6 M) = 0.36

Sample Data

Spectroscopic Data and Analysis for FD&C Blue 1 in Beverages

Sports Drink Tested: Glacier Freeze Gatorade^®
%T = 58.5 and A = 0.234

Best-fit line calculation:
y = 0.134x + 0
0.234 = 0.134x
x = 1.75 μM

Mass of FD&C Blue 1 in a 1-L sample of Glacier Freeze Gatorade:
Molar mass of FD&C Blue 1 is 793 g/mol.
1.75 x 10^–6 M = moles/1 L or 1.75 μM
1.75 x 10^–6 moles = x g/793 g/mole
x = 0.00139 g (1.4 mg) FD&C Blue 1 in a 1-L bottle of Glacier Freeze Gatorade

Spectroscopic Data and Analysis for FD&C Yellow 5 in Beverages Sports Drink Tested: Lemon-Lime Gatorade^®
A = 0.31

Best-fit line calculation:
y = 0.0270x + 0
0.31 = 0.0270x
x = 11.5 μM

Mass of FD&C Yellow 5 in a 1-L sample of Lemon-Lime Gatorade:
Molar mass of FD&C Yellow 5 is 534 g/mol.
1.15 x 10^–5 M = moles/1 L or 11.5 μM
1.15 x 10^–5 moles = x g/534 g/mole
x = 0.00609 g (6 mg) of FD&C Yellow 5 dye in a 1-L bottle of Lemon-Lime Gatorade

Spectroscopic Data and Analysis for FD&C Red 40 in Beverages Sports Drink Tested: Fruit Punch Gatorade^®
A = 0.40

Best-fit line calculation:
y = 0.0259x + 0
0.40 = 0.0259x
x = 15.4 μM

Mass of FD&C Red 40 in a 1-L sample of Fruit Punch Gatorade:
Fruit Punch sample was diluted by adding 1 mL to 5 mL of distilled or deionized water in order to obtain an absorbance within calibration limits.

Molar mass of FD&C Red 40 is 496 g/mol.
M₁(0.001 L) = (1.54 x 10^–5 M)(0.006 L)
M₁ = 9.24 x 10^–5 M = moles/1 L
9.24 x 10^–5 moles = x g/496 g/mole
x = 0.0458 g (46 mg) of FD&C Red 40 in a 1-L bottle of Fruit Punch Gatorade

Answers to Questions

1. Calculate the value of %T for an absorbance value A = 1.5. Using this result, explain why absorbance measurements > 1 may not be accurate.

   A = 1.5 = – log T
   T = 10^–1.5 = 0.032
   %T = 0.032 x 100 = 3.2%
   At an absorbance reading A = 1.5, only 3% of the incident light is transmitted. Since not much light is being transmitted, the readings are likely to have greater error or be less precise.

2. Spectrophotometric studies can be conducted on any colored compound. The transition metal group of the periodic table exhibits a wide array of different colored compounds. The complex ion tetraamminecopper( II ) contains four ammonia molecules covalently bonded to a copper( II ) ion. In aqueous solutions, Cu²⁺ ions will bond to four water molecules in a square planar geometry. The ion is a light blue color. The water molecules can be displaced by ammonia molecules, which are stronger Lewis bases than water. The appearance of the intense dark blue-violet color of the [Cu(NH₃)₄]²⁺ ion is often used as a positive test to verify the presence of Cu²⁺ ions.
   1. Write a balanced chemical equation for the reaction of copper(II) sulfate and concentrated ammonia to produce tetraamminecopper(Ii) sulfate.

      CuSO₄ + 4NH₃ → [Cu(NH₃)₄]SO₄

   2. [Cu(NH₃)₄]²⁺ solutions exhibit a deep blue-violet color. How can you use spectrophotometry to confirm that this reaction has occurred and that the product formed is in fact tetraaminecopper(II) sulfate? Would you expect the wavelength of maximum absorbance (λ_max) for Cu(NH₃)₄²⁺ to be greater than or less than λ_max for Cu(H₂O)₆²⁺? Explain.

      λ_max for Cu(NH₃)₄ will be less than the value of λ_max for Cu(H₂O)₆²⁺ because absorbance will be shifted from red–yellow toward yellow–green.

3. The electron transitions responsible for the colors of transition metal ions involve d → d transitions. Why are zinc ions colorless in aqueous solution?

   Zinc ions have completely filled d-subshells. There are no empty d orbitals available for d → d transition. The ions do not absorb visible light.

References

Pavia, D. L. et al. Introduction to Spectroscopy, 4th ed; California: Brooks/Cole, 2009. Print.

Skoog, D. A. et al. Principles of Instrumental Analysis, 6th ed; California: Thomson Higher Education, 2007. Print.

Introduction

Assume an investigative role and design a valid procedure using spectroscopy and graphical analysis to determine the concentration of FD&C food dyes in sports drinks. The investigation will develop—and test—your skills in preparing accurate serial dilutions, understanding spectroscopic measurements, and extrapolating from graphical data.

Concepts

• Spectroscopy
• Wavelength
• Beer’s law
• Absorbance vs. transmittance
• Consumer science
• Solution concentration

Background

The color of a solution is an important tool used by scientists to gain information about the composition of the solution. Color is a physical property that is useful for both qualitative and quantitative analysis. A qualitative method yields information about the nature or type of compound in a sample, whereas a quantitative method provides numerical data for the amount of a compound in a sample.

Spectroscopy is the study of the interaction of light and matter. A spectrophotometer is an instrument that uses electromagnetic radiation from a selected region of the electromagnetic spectrum, such as ultraviolet, visible or infrared light, to analyze the absorption or transmission of radiation by a sample. The basic function of a spectrophotometer is shown in Figure 1.

The electromagnetic spectrum (see Figure 2) is the entire range of possible wavelengths or frequencies of electromagnetic radiation. In this investigation a visible spectrophotometer will be used—it scans the visible region of the electromagnetic spectrum, from 380 nm to 750 nm. Typical light sources for visible spectrophotometers include xenon and tungsten lamps. Glass cuvettes or test tubes may be used as sample cells for visible spectrophotometers. More specialized spectrophotometers require quartz cells, which are “invisible” to and do not absorb ultraviolet radiation. In addition to the energy source used in spectrophotometers, a diffraction grating called a monochromator is also incorporated. The monochromator spreads the beam of light into the light’s component wavelengths. The desired wavelength is then focused onto the sample cell to detect any absorption or emission of light by a substance in a sample.

Spectrophotometry is an analytical procedure that uses electromagnetic radiation to measure the concentration of a substance. The success of a spectrophotometric technique requires that the absorption of light by the substance being analyzed must be distinct or different from that of other chemical species in solution. How do scientists select the desired wavelength for spectrophotometry?

The absorption of visible light by a substance results from electron transitions, that is, the promotion of a ground state electron to a higher energy atomic or molecular orbital. Both light energy and electron energy levels are quantized, so that the specific wavelength of light absorbed by a substance depends on the energy difference between two electron energy levels. The optimum wavelength for spectrophotometric analysis of a substance is selected by measuring the visible spectrum of the substance, corresponding to a plot of absorbance (A) versus wavelength (λ, “lambda”).

Just seven unique dyes are approved by the Food and Drug Administration for use in foods, drugs and cosmetics. These seven FD&C dyes give rise to the entire palette of artificial food colors. Three FD&C dyes, FD&C Blue 1, FD&C Red 40 and FD&C Yellow 5, are discussed in this advanced inquiry lab for the analysis of sports drinks and other beverages. The structure of FD&C Blue 1 is shown in Figure 3. Notice the extensive series of alternating single and double bonds (also called conjugated double bonds) in the center of the structure. This feature is characteristic of intensely colored organic dyes and pigments. Every double bond added to the system reduces the energy difference between the bonding and nonbonding molecular orbitals so that the resulting energy gap corresponds to visible light.

A solution containing FD&C Blue 1 appears blue under normal white light—blue is the color of light transmitted by the solution. The colors or wavelengths of light that are absorbed by this solution are complementary to the transmitted color. A color wheel (see Figure 4) provides a useful tool for identifying the colors or wavelengths of light absorbed by a substance. The blue solution absorbs orange light and we would expect the visible spectrum of FD&C Blue 1 to contain a peak in the 600−640 nm region. The optimum wavelength for spectrophotometric analysis of a dye solution is generally determined from the wavelength of maximum absorbance (abbreviated λ_max, or “lambda max”). The value of lambda max for FD&C Blue 1 is 630 nm.

The wavelength of light absorbed by a substance is characteristic of its molecular or electronic structure. The intensity of light absorbed depends on the amount of the substance in solution. Generally, the more concentrated the solution, the more intense the color will be, and the greater the intensity of light the solution absorbs. A digital spectrophotometer measures both the percent transmittance of light and the absorbance. When light is absorbed, the radiant power (P) of the light beam decreases. Transmittance (T) is the fraction of incident light (P/Po) that passes through the sample (see Figure 5). The relationships between transmittance and percent transmittance (%T) and between transmittance and absorbance (A) are given in Equations 1 and 2, respectively. The amount of light absorbed by a solution depends on its concentration (c) as well as the path length of the sample cell (b) through which the light must travel. (Equation 3), which is known as Beer’s law. The constant a in the equation is a characteristic of a substance and is known as the molar absorptivity coefficient.

Experiment Overview

The purpose of this inquiry lab is to use spectroscopy and graphical analysis to determine the concentration of dye in a sports drink. The investigation begins with a an introductory activity for preparing a series of standard dilutions of an FD&C Blue 1 stock solution and measuring the percent transmittance of each. The results will be analyzed graphically to identify an optimum linear relationship among various functions (T, %T, log T and A) for a Beer’s law calibration curve. The procedure provides a model for guided-inquiry analysis of the concentration of food dye(s) in sports drinks and other consumer beverages. Additional dyes, FD&C Yellow 5 and FD&C Red 40, are also available for optional extension or cooperative class studies.

Materials

Prelab Questions

The visible absorption spectrum for FD&C Blue 1 is shown in the graph below. The estimated concentration of the dye was 7.0 μM (7.0 x 10^–6 M ).

1. What would be an optimum wavelength for measuring the absorbance versus concentration of a series of FD&C Blue 1 dye solutions? Explain your answer. Absorbance measurements are most accurate and sensitive in the range 0.2–1.0.
2. To construct a calibration curve, a series of known concentration standards is prepared. Using the estimated concentration of the FD&C Blue 1 stock solution, determine the concentration of each of the following dilutions. Hint: M₁V₁ = M₂V₂.
   {13833_PreLab_Table_1}
3. Using the information provided in Question 1, predict the absorbance of each solution A–H at the optimum wavelength. Refer to Equation 3 in the Background. The value of a is 130,000 M^–1cm^–1 and b = 1 cm.

Safety Precautions

The FD&C dyes are slightly hazardous by eye and skin contact. The dyes have been stored with other, nonfood-grade chemicals and are not for consumption. Avoid contact with eyes, skin and clothing. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. Wash hands thoroughly with soap and water before leaving the laboratory. Please follow all laboratory safety guidelines.

Procedure

Introductory Activity

1. Turn on the spectrophotometer and allow to warm up for 15–20 minutes.
2. Based on the maximum absorbance of the dye being tested, select the appropriate wavelength on the spectrophotometer.
3. Read the entire Procedure. Construct an appropriate data table to record measurements and the results of calculations. Note: As part of a cooperative lab activity, your instructor may assign different groups to prepare and analyze different solutions, and to graph the results. Each group will need to transcribe data and analyze the results for all solutions in order to complete the guided-inquiry activity.
4. Obtain approximately 50 mL of stock solution containing FD&C Blue 1 dye.
5. Using a serological pipet for accurate volume measurements, dilute the stock solution as indicated in the following table to prepare 10 mL each of a series of standard solutions, B–H. Carefully mix each solution. Hint: To avoid contaminating the stock solution, first use the pipet to add the required amount of distilled water to each test tube. Rinse the pipet three times with the stock solution. Then measure and add the required amount of stock solution to each test tube.
   {13833_Procedure_Table_2}
6. Use the “blank” test tube (H) to set the 100% transmittance value for the spectrophotometer at the desired, optimum wavelength for this study.
7. Measure and record the percent transmittance (% T) of the stock solution and each standard solution (B–G) at the optimum wavelength. Remember to handle the test tubes only at the top and to polish the test tube with lens tissue. To avoid spills, do not place test tubes in the spectrophotometer if they are more than 75% full. Remove some solution if needed from each test tube before inserting it in the instrument.
8. Convert %T to transmittance (T) for each measurement, and calculate the appropriate values of both log T and −log T. Record all results.
9. Use Beer’s law to calculate the precise concentration of FD&C Blue 1 in the stock solution. The molar absorptivity (a) of FD&C Blue 1 is 130,000 M⁻¹m⁻¹ at 630 nm and the path length (b) is 1 cm. Record the micromolar (μM) concentration (1 μM = 1 x 10^–6 M) in your data table.
10. Prepare separate graphs of (a) %T, (b) T, (c) 1/T, (d) log T and (e) –log T (on the y-axis) versus dye concentration (on the x-axis) for each solution. Note: Dye concentrations were calculated in the Prelaboratory Assignment using the estimated concentration of the stock solution. Recalculate the concentrations of solutions B–G, if needed, based on your answer to Question 9. Use the dilution equation.

Guided-Inquiry Design and Procedure

Concentration of FD&C Blue 1 in Beverages

1. Based on the graphs obtained in the Introductory Activity, identify the optimum linear relationship or calibration curve for quantitative analysis of the concentration of an “unknown” solution containing FD&C Blue 1 food dye.
2. Which graph would provide the most accurate means to determine the concentration of an “unknown” solution whose transmittance has been measured spectroscopically? Explain in terms of Beer’s law and give an example of how the analysis would be carried out.
3. Consult the ingredients label for a blue-colored sports drink or other consumer beverage. Identify the dyes that are present and explain whether the beverage can be analyzed using the calibration curve described.
4. Obtain the necessary spectroscopic data for the beverage containing FD&C Blue 1 food dye. Recall that absorbance measurements are most accurate in the range of A values from 0.2 to 1.0. Treat the beverage sample if needed to make sure the data is in the acceptable range.
5. Determine the concentration (micromolar, μM) of the dye in the beverage and calculate the amount (mass) of dye in milligrams per liter of the beverage. The molar mass of FD&C Blue 1 is 793 g/mole.

Student Worksheet PDF

13833_Student1.pdf