Amounts of Artificial Food Colors in Commonly Consumed Beverages Report

provided Raw Data for Result Section

-Red Dye #40 dilution preparation, calculate the V1 for each dilution and complete the table. Include this in your result section of the lab report.

Concentration (M2)

V2 (mL)

M1 (stock ,M)























-Data for calibration plot :Red Dye # 40. Lambda max at 490 nm.

Concentration (M)













-Data for unknown drink. Use the calibration plot and equation you created to solve for the concentration of Red due #40 in the two unknown drinks: 

Sample containing red dye#40


Mystery red drink #1


Mystery red drink #2


TableDescription automatically generated





Spectroscopy. n fascinating field of chemistry and physics, has bccn used in an array of diverse applications from the analysis of chemicals in foods and pharmaceuticals to the detertnination of’ age nnd composition of distant stars and entire galaxies. The fundamental idea behind spectroscopy involves the interaction bctwccn matter (molcculcs and atoms) and radiation

(e.g. infrared radiation). For exatnple. thc dctailcd images of thc human body obtained from MRI (magnetic resonance inmging) scans arc the product of the interactions of hydrogen atoms with radio waves (under certain controlled conditions). We use these interactions of matter with radiation to obtain information about substances, specifically, the structure and behavior of molecules.

Not all molecules interact with radiation in the same way. The nature of the interaction and the region of the electromagnetic spectrum where these interactions occur depend on the structure

Figure I: diluted solution from a

color (Figure 1). You or someone you know may have

commercial drink oxytops. wordp

used this technique, called colorimetry (measure of the

word image 3094 of the molecules (or atoms). Color results from the interaction of substances with a specific portion of the electromagnetic field that we call “visible”. As expected, the amount of substance influences the extent of that interaction. You can easily tell which of the solutions is more concentrated just by looking at the intensity of their

intensity of color). For instance, you can find fast, cheap and easy to use medical assays to assess blood glucose levels that use colorimetric principles. They use strips of paper impregnated with chemicals that change color with the amount of glucose present in a small drop of blood. The final pattern of colors on the strip is the “reading” that indicates the glucose levels.

word image 3095

In this project, your team will apply the principles of spectrophotometry for chemical analysis and you will use your chemistry knowledge and understanding to explain the interaction of UV-Visible radiation and molecules in commercial products.



Figure 2: Spectrophototneter from Ocean Optics Company

molecules and how these interactions can be used determine their concentration in solutions. Food dyes

word image 3096 Food dyes, widely used in t11C food industry as additives, may possess potential negative health efTccts related to their use and have recently sparkcd somc controversy. To help the ptiblic make inli)rtned decisions. n non-profit orgnni?lltion called Clean Eating Advocates (CEA) is creating an online datnbnse With the food dye content in various commercial drinks. They propose using spectrophotomctry for the analyscg. measuring the absorption of tJV-Visible radiation by a solution and using it to find the concentration of dyes in thc drinks. Ijnaginc that you have been asked to assist CEA in this projcct. Sincc their project rcccntly started, the database iB “till small. Ilowcvcr, the CEA provided us with n list or target dyes, Your team will investigate the interaction of WV-visible radiation with food dye to

absorb electromagnetic radiation in the visible light spectrum, and the absorption of light and the concentration of dyes are related. Thus, your team will use a spectrophotometer (Figure 2) to determine the unknown dye concentrations in solutions.

Standard Curve vith In•nginary Data You will determine the concentration of color dyes in commercial products. Your team will have to identify the wavelength at which the absorbance is greatest (lambda maxima, Imax) per color. Then, create a calibration curve per color which must comply with industrial standards (linearity of R2 = 0.95 and a minimum offive data points). Each calibration curve can be used to calculate the unknown concentration of that

word image 3097


CorEentration per cent (VIV)

Figure 3: This is an example of a word image 3098 word image 3099 standard curve. A best fit line has been generated and the resulting equation

specific dye in the commercial product (Figure 3).

It is important to use the linear equation from the calibration curve and correlate it with Beer’s law (A =

and r-squared value are shown below tlc) to calculate the unknown concentration. The word image 3100 the X-axis label word image 3101

spectrophotometer that you will be using gives more

word image 3102

accurate readings if the absorbance is between the range of 0.1 and l, then you will have to adjust


the concentrations of the dye solutions to obtain values within this range. To start, your TA will assign three of the dyes available in the stockroom to your team. Specific instructions for the use of the spectrophotometer will be provided to you during class. Create a calibration curve for each assigned color (same as mini-project 2) and get the linear equation (Beer’s law). Use Microsoft excel or similar software to create your calibration curve (absorbance vs concentration). Create the calibration curve during class as you perform the readings on the spectrometer. Both tasks should be completed at the stitne tinte. Discord nnd repeat samples that deviate from the linearity of the graph so your team can have n five point graph with and R2 of 0.95 or higher. Answer the following questions before you stnrt your experiment:

t. What is the goul Cor the entire project?

  1. What are the chemical formulas and the maximum absorbance of the dyes assigned to your team?
  2. How are color and wavelength of maximum absorbance related?
  3. What will the concentration of your initial stock solution be?
  4. word image 3103 word image 3104 word image 3105 word image 3106 word image 3107 What measurements will you collect and how will you organize them in your notebook?

Topics you may need to review depending on your experimental decisions are: electromagnetic spectrum, light wavelength and color, UV-Visible absorption spectroscopy, spectrophotometer, concentration of solutions, dilution calculations, Beer’s law, calibration curves, lambda maxima (Xmxx), accuracy and precision of measurements, interpretation of data using Excel. Reviewing the following techniques before coming to the lab will make your work easier and more productive: measuring volumes at different levels of accuracy (use of burets and pipets), serial and parallel dilution, and graphing using Excel.


word image 3108

These links provide information about: concepts, research connection and laboratory techniques. Do not limit your search of information to these resources only.

    • USF Laboratory Toolbox: Canvas Course Homepage


    • Principle of Spectrophotometry:

http://www.chm.davidson.edWvcc Spectrophotomctry Spectrophotometry.html

word image 3109 Absorbance Spectrum:

http://www.chm.davidson.cdu/vcc Spectrophotometry/Absorbance.Spectrum.html word image 3110 Beer’s Law: word image 3111BeersLaw.htrnl

  • Spectroscopy information (Michigan State University, Department of Chemistry):

http://www2.chetnistry.tnstl.edt1/ract11ty/rcusgh/VirC11xtJrnl/SpectrPY=-IYV—t——!J! word image 3112 Spectrophotometer, how does it work? reusch/Virt•rxtJlnl/Spectrpy/UVVis/uvspec.httnl/uv I

  • Preparation of standard solutions: http:/ word image 3113 How to dilute a solution? word image 3114


You may be asked to leave the laboratory if you do not abide by safety norms:

  • Laboratory coat, splash proof goggles and gloves MUST be worn during this experiment.
  • Wash your hands thoroughly at the end of the experiment.
  • Water is not a chemical hazard; however, other chemicals you may propose to use in your experiments may present hazards. Consult the SDS (Safety Data Sheet) for each chemical; some chemicals may irritate/burn eyes and skin.
  • Do not pour any of your solutions or samples down the drain. Use the proper waste containers (solid, liquid waste).


The following will be available in the laboratory. You will need to ask your TA for authorization before using any substance or equipment not listed here:

*All dyes are in solid form unless otherwise stated by the TA or the stockroom staff

  • General laboratory glassware (volumetric flask, beaker)
  • Red Dye word image 3115 word image 3116 Red Dye word image 3117


  • Yellow Dye #5,6
  • Green Dye #3
  • Blue Dye #2
  • Other dye colors may be available
  • Graduated pipettes
  • Pipette pumps
  • Cuvettes
  • Ocean Optics Spectrophotometer Instrument (the “operation instructions” for this instrument will bc shared with you before the experiment)


  1. What safety protocols need to be followed for this project? What are the specific safety concerns for each chemical used? Consult the SDS for each.
  2. Mix two of your dyes, predict what the spectrum will look like.
  3. Why is finding the wavelength of maximum absorbance necessary?
  4. What glassware will you need to use? What equipment will you need?
  5. Suggest the concentration of food dyes in a few specific products.
  6. You will need to prepare a calibration curve. What is a calibration curve? What is your independent variable? What is your dependent variable? What are you measuring? How are you going to measure it? How do all these components fit together to allow you to obtain useful data from a calibration curve?
  7. When performing serial and parallel dilutions, accuracy is extremely important. A small error in an initial dilution propagates throughout the experiment becoming a large error by the end. How will you minimize error when measuring solutions? word image 3118
  8. The absorbance values for your solutions must be in the range of 0.1-1.0. If the absorbance value is lower than 0.1 or higher than 1.0 what will you have to do to get a value between those numbers?
  9. How many different concentrations are needed to produce a calibration curve? word image 3119
  10. You will need to find a line of best fit. What is the point of finding the line of best fit? What does the variance show? Do you want a high or low R2 value? word image 3120
  11. Analyze your data and prepare a graph of absorbance vs. concentration using Excel.

Determine the “line of best fit” or “trend line” from the graph. How can you relate this line of best fit to the Beer-Lambert Law?

  1. How can you determine if the correlation for the line-of-best-fit works?
  2. Use your graph to calculate the molar absorptivity (c) for the food dye. What is the physical meaning of the molar absorptivity?
  3. Does your molar absorptivity match the reported value in the literature? What error sources could affect the accuracy of your measurement?
  4. What type ol’ sample preparations arc necessary? For example, how would you prepare a solid sample? What happens if your sample is too concentrated? Too dilute?
  5. What other factors are ilnportant to consider when analyzing commercial products?
  6. If a samples absorbance value falls outside the range of the calibration curve? How would you address this?

Check your planning with your TA. If there is enough time left, your TA may allow you to start working on the analysis of one or more samples. This will allow you to advance some work or simply get acquainted with the glassware and the preparation of your sample. If any new concepts or techniques came up during your planning, you need to review them before coming to the lab next week.


I. What is the goal of the entire project? Relate your results to the goal of the overall project.

  1. Prepare a table of wavelength of maximum absorbance vs. solution color using your data and observations from the initial activity. word image 3121
  2. Compare the spectra for three dyes. Does the wavelength of maximum absorbance for word image 3122 each color solution match color of the solution?
  3. How are your spectra similar? How are your spectra different? What causes these similarities and differences? You have an idea of how the absorbance spectrum for a single substance looks. How do you think the absorbance spectrum will look if instead of a single dye in a solution, you have multiple food dyes? What do you notice? What does


this tell you about the relationship between and the dyes? Can you differentiate between multiple dyes?

  1. Analyze your data and determine the concentration of food dye in your food products. Label concentrations with proper units.

word image 3123

  1. What is the concentration of food dye reported in the commercial product you studied?
  2. word image 3124 List possible sources of error in this analysis. How did the errors affect your measurements? Label them as random or systematic.
  3. Several dyes have been banned. Why have they been banned?
  4. Are dye concentrations safe in the products you studied?
  5. Fill out the table below and compare your results to the results of other teams using the same color dyes. Is there any difference? Why?

word image 3125 Table 1: Data collection from color samples.


Imax (cm-I)

word image 3126



(M) sample 1


(M) sample 2







Make sure you follow the lab report or oral/poster presentation guidelines provided by your instructor. Present your experimental data in an easy-to-read format. Sometimes it is better to use graphs instead of tables for specific purposes; this helps the reader understand your findings and your claims. Always use and show the appropriate units for measurements and significant figures. Consider describing general aspects and findings from your study of color and wavelength. Was there anything that initially seemed counterintuitive? Do not overemphasize procedures that you know other teams performed as well. Stress steps that you think are new to other teams, for example details of food sample preparation and analysis.

Remember that we want you to connect your experimental work to research. What kind of similarities do you find between your lab work and what you understand for research?



Many researchers in the natural and medical sciences at USF routinely use spectroscopic techniques. Dr. Dean F. Martin, Distinguished University Professor Emeritus at the University of South Florida researches aspects of environmental chemistry motivated by his interest in fast and cheap removal of toxic chemicals from the environment. One method he is interested in uses substances that act as ”molecule traps”. They encapsulate the toxic molecules within their structure, something like a fishing net catching fish of specific sizes and letting others free. One of these molecule-trapping substances, Ortogil@, has been widely studied by undergraduate and graduate students working under the supervision of Dr, Martin. Ortogil@) shows particular attraction to certain food dyes and pharmaceutics present in watcr samples. Professor Martin uses spectrophotometry to determine how clean the water sample is after treatment with Ortogil@.

word image 3127 The picture shown illustrates an experimental setup used by Dr. Martin’s students. The yellow solution in the bottle is a sample contaminated by the organic molecule 4-nitrophenol. Dr. Martin and his students quantify the concentration of 4-nitrophenol using the spectrophotometer on the right. The solution is pushed through a glass column filled with a packing material covered with Ortogil@. As you can see, the liquid eluting from the column in the graduated cylinder is clear. Dr. Martin’s students quantify the content of 4-nitrophenol by subtracting the initial concentration from the final concentration measured by the spectrophotometer and calculate Photo taken in Dr. Martin’s Laboratory at USF how much contaminant was removed.

Medical research findings suggest that the presence of food dyes is correlated with development of attention deficit hyperactivity disorder (ADHD) in children. You can imagine the impact of Dr. Martin’s development of cheap and easy ways to remove harmful materials from our

drinking water sources.


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