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 503 nm, λ_max of FD&C Red 40.
2. Add 25 mL of FD&C Red 40 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 Yellow 5 following this step (optional). Half of the class may analyze yellow 5 in a yellow sports drink.
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.
5. Collect an absorbance reading of the FD&C Red 40 solution at 503 nm. Absorbance (A) should read between 0.7 and 1.0.
6. 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 Red 40 dye (25,900 M^–1 cm^–1)
   b is path length of the sample cell or cuvet, 1 cm
   c is concentration
   Absorbance measured, A = 0.999
   0.999 = (25,900 M^–1 cm^–1)(1 cm)(c)
   c = 0.999/(25,900 M^–1 cm^–1)(1 cm)
   c = 3.85 x 10^–5 M or 38.6 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

• This laboratory activity was specifically written, per teacher request, to be completed in one 50-minute, wet lab class period. It is important to allow time between the Prelab Homework Assignment and the Lab Activity. Prior to beginning the homework, show the students the materials they will be using for the analysis, including the spectrophotometer—this will get the procedure thought process rolling. Once students turn in the homework answers, graphs and figures and their procedure, check it for safety and accuracy before implementation in the lab.
• Students may work on the Prelab Homework Assignment and procedure in pairs outside of class.
• Prior to assigning the homework, show the students the lab materials and equipment. Display the glassware, sample cells, pipets, pipet bulbs or fillers, beakers and spectrophotometer on the bench top. Give a brief introduction into how to operate the spectrophotometer, such as turning it on and other available options on the unit.
• Stock solutions should be prepared for the students before lab day.
• Turn the spectrophotometer(s) on for the students before lab to allow a 15–20 minute warm-up of the lamp.
• We suggest using Gatorade^® as the consumer beverage samples. An example of a sports drink that contains FD&C Red 40 is Gatorade Fruit Punch. Lemon-Lime Gatorade contains yellow food dye, sample data is included for both in this activity.
• 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.

Teacher Tips

• 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.

• The literature value or molar absorptivity coefficients are 25,900 M^–1 cm^–1 for FD&C Red 40 (λ_max = 503 nm) and 27,300 M^–1 cm^–1 for FD&C Yellow 5 (λ_max = 427 nm).
• 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 Answers to the Prelab Homework Assignment.

  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.
• Another option is to assign half of the class FD&C Red 40 and the other half FD&C Yellow 5.

• 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

Alignment to the Curriculum Framework for AP^® Chemistry 

Enduring Understanding and Essential Knowledge
Atoms are so small that they are difficult to study directly; atomic models are constructed to explain experimental data on collections of atoms. (1D)
1D3: The interaction of electromagnetic waves or light with matter is a powerful means to probe the structure of atoms and molecules, and to measure their concentration.

Learning Objectives
1.15 The student can justify the selection of a particular type of spectroscopy to measure properties associated with vibrational or electronic motions of molecules.
1.16 The student can design and/or interpret the results of an experiment regarding the absorption of light to determine the concentration of an absorbing species in a solution.

Science Practices
2.2 The student can apply mathematical routines to quantities that describe natural phenomena.
4.1 The student can justify the selection of a kind of data needed to answer a particular scientific question.
4.2 The student can design a plan for collecting data to answer a particular scientific question.
5.1 The student can analyze data to identify patterns or relationships.
6.4 The student can make claims and predictions about natural phenomena based on scientific theories and models.

Answers to Prelab Questions

1. A visible absorption spectrum for a 7.0 μM (7.0 x 10^–6 M) FD&C Blue 1 solution was collected on a spectrophotometer. The resulting data are shown in Table 1. Graph absorbance vs. wavelength (nm) and report the wavelength value of maximum absorbance (lambda max, λ_max) in nm for FD&C Blue 1. Make sure to label the axis, include units and title the graph. Hint: Look at the tallest peak.
   {14108_PreLabAnswers_Figure_7}
   1. Report the lambda max for FD&C Blue 1: λ_max ___630___nm
   2. Look at the color wheel (see Figure 4 in the Background section). Does the wavelength of maximum absorbance for FD&C Blue 1 you selected make sense? Explain.

      Yes. Since the solution appears blue under normal white light, we would expect it to absorb orange light (its complimentary color), in the range of 600 nm to 640 nm.

   3. What would be an optimum wavelength for measuring the absorbance versus concentration of a series of FD&C Blue 1 dye solutions? Explain. Hint: 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 630 nm. The maximum absorbance is approximately 0.83 for this solution. This is the accurate range.

2. In lab, a student turns on the spectrophotometer and allows a 15–20 minute warm-up, sets the optimum wavelength for analysis on the unit, and prepares a series of FD&C Blue 1 dye solutions from a 7.0 μM (7.0 x 10^–6 M) FD&C Blue 1 stock solution using a stock solution rinsed serological pipette (for accurate volume measurements). See Figure 6 for the student setup of serial dilutions.

   Example student data table for answers to 2a, 2b and 2c.

   {14108_PreLabAnswers_Table_3}
   1. Each sample has a final volume of 10-mL and was prepared separately in small beakers and mixed. The gray shaded area (Figure 6) represents the FD&C Blue 1 stock solution. How much water should the student transfer to each?

      See data table.

   2. Determine the concentration of each dilution. Hint: M₁V₁ = M₂V₂.

      Example calculation for Sample B:
      7.0 μM)(8 mL) = (M₂)(10 mL)
      56.24/10 mL = M₂
      5.60 μM = M₂

   3. If each sample, A–H, was transferred to a sample cell (test tube or cuvet) and analyzed in the spectrophotometer, predict the absorbance of each solution, A–H, at the optimum wavelength. Refer to Equation 3 in the Background section. The molar absorptivity (a) of FD&C Blue 1 is 130,000 M^–1 cm^–1 and the test tube’s path length (b) is 1 cm.

      Example calculation for Sample B:
      A = abc
      A = (130,000 M^–1 cm^–1)(1 cm)(5.60 x 10^–6 M)
      A = 0.728

3. After making the serial dilutions, the student “blanked” the spectrophotometer with sample H and analyzed samples A–H using the spectrophotometer. Table 2 shows the student’s spectrophotometric data for FD&C Blue 1.
   {14108_PreLabAnswers_Table_4}
   1. Use Beer’s law to calculate the precise concentration of test tubes B–H and fill in the blanks in Table 2. Hint: Refer to Equations 1, 2 and 3 in the Background section. Watch the concentration units; convert μM to M. Show your work.

      Example calculation for B
      A = –log(T)
      Convert % T to A: –log(T)
      18.7/100 = 0.187
      –log(0.187) = 0.728
      c = 0.728/130,000 M^–1 cm^–1
      c = 5.60 μM

   2. Use the data in Table 2 to 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).
      {14108_PreLabAnswers_Figure_8}
   3. Identify the optimum linear relationship or calibration curve for quantitative analysis of the concentration of an “unknown” sports drink containing FD&C Blue 1 food dye. Make observations of the data points on the calibration curve. List possible lab techniques for data set improvement (i.e., solution prep, spectrophotometer set-up).

      Student answers may vary.
      
      The optimum linear relationship is (e). Graphing the data by absorption (–log T) vs. dye concentration gives the optimum straight line with the equation y = mx + b, where m is the slope of the line (rise over run), and b is the y intercept, which goes through zero.
      
      Ideally, when the optimum line is forced through zero, a correlation coefficient, R², should be close to “1.” The reported R² in graph (e) is 0.992.
      
      Students report if any errors occurred (e.g., solution spill, incorrect serial dilutions were made, glassware was not clean or dry, student accidentally used tap water instead of DI water).

      • When preparing serial dilutions, practice great care to ensure accurate measurements of dye and water.
      • Allow plenty of lamp warm-up time on the spectrophotomer.
      • Positioning the sample cell (test tube) the same direction in the spectrophotometer sample cell is ideal. It would also be ideal to use the same sample cell, but was not practicle in the alotted time.
4. Finally, the student obtained a blue sports drink (after consulting the ingredient’s label for FD&C Blue 1 dye), transferred 10-mL to the sample cell, and collected the necessary spectroscopic data, A = 0.234. Calculate the concentration of FD&C Blue 1 in mg/L.

   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
   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

5. Write a step-by-step procedure to determine the concentration of FD&C Red 40 in a sports drink, such as fruit punch flavored. The procedure should be written in the lab notebook to be used on the day of lab.

   Student procedures will vary; helpful tips were provided. Students set the wavelength on the spectrophotometer to 503 nm. Provided with the stock solution, students conduct the necessary serial dilutions, analyze on the warmed-up spectrophotometer, and finally run the red sports drink sample. The red sports drink will need dilution to collect an accurate absorbance reading. After the lab, students build the calibration curve and calculate the amount of red dye in the sports drink in mg/L.

Sample Data

Spectroscopic Data and Analysis for FD&C Red 40 in Beverages

Sports Drink Tested: Fruit Punch Gatorade^®
A = 0.400
Best-fit line calculation:
y = 0.0259x + 0
0.400 = 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

Spectroscopic Data and Analysis for FD&C Yellow 5 in Beverages Sports Drink Tested: Lemon-Lime Gatorade^®
A = 0.310
Best-fit line calculation:
y = 0.0270x + 0
0.310 = 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

References

AP^® Chemistry Guided-Inquiry Experiments: Applying the Science Practices; The College Board: New York, NY, 2013.

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

Experience and learn the concepts you need to help you succeed in the AP^® Chemistry exam with this guided-inquiry lab! Assume an investigative role and design a valid procedure using spectroscopy and graphical analysis to determine the concentration of FD&C Red 40 in a sports drink. A thorough practice homework set will guide you through this investigation to develop your skills in preparing accurate serial dilutions, understanding spectroscopic measurements and extrapolating from graphical data.

Concepts

• Spectroscopy
• Wavelength
• Beer’s law
• Consumer science
• Absorbance vs. transmittance
• 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 cuvets 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”).

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 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/P_o) 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. See 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 advanced inquiry lab is to use spectroscopy and graphical analysis to determine the concentration of FD&C Red 40 dye in a sports drink. The investigation begins with an introductory homework assignment, where you will be asked to answer, observe and form conclusions on the spectrophotometric analysis of FD&C Blue 1 dye:

• Prior to completing the homework, observe the equipment you will be using for analysis, this may include glassware, electronic balances, a spectrophotometer, etc.
• The homework begins with providing the visible region spectrophotometric data for FD&C Blue 1 dye to be graphed and analyzed.
• A serial dilution model guides you through the dilution equation.
• Use Beer’s Law to answer questions from given spectrophotometric transmittance dye data, select the optimum linear relationship or calibration curve among various functions (T, %T, 1/T, log T, –log T), and calculate the amount of FD&C Blue 1 dye, in mg/L (ppm), of a sports drink.
• Finally, write a detailed, step-by-step procedure, including necessary data tables, to analyze and determine the amount of FD&C Red 40, in mg/L (ppm), in a red sports drink.

Completion of the homework assignment will promote success in the lab!

Prelab Questions

Complete the following homework set and write a lab procedure to be approved by your instructor prior to performing the lab. Along with your procedure, you will turn in any graphs or figures you were asked to create in this homework set and answers to the questions.

1. A visible absorption spectrum for a 7.0 μM (7.0 x 10^–6 M) FD&C Blue 1 solution was collected on a spectrophotometer. The resulting data are shown in Table 1. Graph absorbance vs. wavelength (nm) and report the wavelength value of maximum absorbance (lambda max, λ_max) in nm for FD&C Blue 1. Make sure to label the axes, include units and title the graph. Hint: Look at the tallest peak.

   Wavelength and Absorbance Data for FD&C Blue 1

   {14108_PreLab_Table_1}
   1. Report the lambda max for FD&C Blue 1: λ_max ________nm
   2. Look at the color wheel (see Figure 4 in the Background section). Does the wavelength of maximum absorbance for FD&C Blue 1 you selected make sense? Explain.
   3. What would be an optimum wavelength for measuring the absorbance versus concentration of a series of FD&C Blue 1 dye solutions? Explain. Hint: Absorbance measurements are most accurate and sensitive in the range 0.2–1.0.
2. In lab, a student turns on the spectrophotometer and allows a 15–20 minute warm-up, sets the optimum wavelength for analysis on the unit, and prepares a series of FD&C Blue 1 dye solutions from a 7.0 μM (7.0 x 10^–6 M) FD&C Blue 1 stock solution using a serological pipette, rinsed with stock solution, for accurate volume measurements. See Figure 6 for the student’s setup of serial dilutions.
   {14108_PreLab_Figure_6}

   Create a data table to answer Question 2.

   1. Each sample has a final volume of 10-mL and was prepared separately in small beakers and mixed. The gray shaded area (Figure 6) represents the FD&C Blue 1 stock solution. How much water should the student transfer to each?
   2. Determine the concentration of each dilution. Hint: M₁V₁ = M₂V₂.
   3. If each sample, A–H, was transferred to a sample cell (test tube or cuvette) and analyzed in the spectrophotometer, predict the absorbance of each solution, A–H, at the optimum wavelength. Refer to Equation 3 in the Background section. The molar absorptivity (a) of FD&C Blue 1 is 130,000 M^–1 cm^–1 and the test tube’s path length (b) is 1 cm.
3. After making the serial dilutions, the student “blanked” the spectrophotometer with sample H and analyzed samples A–H using the spectrophotometer. Table 2 shows the student’s spectrophotometric data for FD&C Blue 1.

   Spectrophotometric Data for FD&C Blue 1

   {14108_PreLab_Table_2}
   1. Use Beer’s law to calculate the precise concentration of test tubes B–H and fill in the blanks in Table 2. Hint: Refer to Equations 1, 2 and 3 in the Background section. Watch the concentration units; convert μM to M. Show your work.
   2. Use the data in Table 2 to 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).
   3. Identify the optimum linear relationship or calibration curve for quantitative analysis of the concentration of an “unknown” sports drink containing FD&C Blue 1 food dye. Make observations of the data points on the calibration curve. List possible lab techniques for data set improvements (i.e., solution prep, spectrophotometer setup).
4. Finally, the student obtained a blue sports drink (after consulting the ingredient’s label for FD&C Blue 1 dye), transferred 10-mL to the sample cell, and collected the necessary spectroscopic data, A = 0.234. Calculate the concentration of FD&C Blue 1 in mg/L.
5. Write a step-by-step procedure to determine the concentration of FD&C Red 40 in a sports drink, such as fruit punch flavored. The procedure should be written in the lab notebook to be used on the day of lab.

   Helpful tips:

   1. Think safety first. Make sure you have the proper PPE available to perform this lab (i.e., goggles, apron and gloves).
   2. Make a list of the equipment and glassware needed for this lab.
   3. Number the steps in your procedure; remember to be as detailed as possible, from set-up to clean-up.
   4. Draw necessary data tables in your notebook for data collection during the lab.
   5. Find the ingredients label for a red-colored sports drink or other consumer beverage. Make sure FD&C Red 40 is part of the ingredient list.
   6. Remember, absorbance measurements are most accurate and sensitive in the range 0.2–1.0. What should you do if your sports drink data is not within the acceptable absorbance range?
   7. When setting-up the spectrophotometer, think about the optimum settings. Spectrophotometers come with options to collect absorbance and transmittance data and the wavelength can easily be adjusted within the visible spectrum. Hint: Research the visible spectra of FD&C Red 40 to identify the wavelength of light that results in the maximum absorbance value.

Safety Precautions

The FD&C dyes are slightly hazardous by eye and skin contact. The dyes have been stored with other non-food-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.