Food dye lab report 2nd submission

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Identifying and Replicating the Concentration of Food Dyes in Purple Powerade Through the Use of Spectroscopy, Beer’s Law, and the Dilution Formula Chemistry 1065 Fall 2023 Alex Wu, , , Lucas Harrison Anna Dittrich Katie Reimer

Abstract This experiment aimed to determine the concentration of various food dyes in a purple Powerade. In this experiment, FD&C Blue #1, FD&C Blue #2, FD&C Red #3, and FD&C Red #40 at different dilutions, along with the purple Powerade, were tested with an Ocean Optics spectrophotometer in order to compare and contrast the peak wavelength and absorbance levels to those found in the purple Powerade. Through the use of a spectrophotometer, it was determined that Blue #1(628nm) and Red #40(498.3nm) were used in the drink as the peak wavelengths matched closely with those of the purple Powerade, which had red and blue wavelengths 628nm and 494nm respectively. Calibration curves were then created to find the concentration using Beer’s Law. The resulting calculations found a concentration of 8.75*10^-6M of Red #40 and 3.37*10^-6 concentration of Blue #1 in the drink. Using the dilution formula, trivially, 4.86ml of Red#40, 8.43ml of Blue #1 and 36.7ml of distilled water are needed to recreate the Powerade. The recreated Powerade was then retested and compared to the original to verify accuracy. Introduction Color spectroscopy and spectrophotometry have become essential tools in many scientific fields due to their versatility and ability to accurately determine the composition of the atmosphere and other tissues and objects. A 2020 meta-analysis published in the journal “Progress of Biomedical Engineering,” showed the applications of optical spectroscopy in diagnosing the in vivo disease states of certain tissues without the need for invasive biopsies. When light is delivered in a highly localized state on tissues, certain combinations of optical processes occur 1 . Analysis of these optical processes can rapidly provide in-depth insight into the current pathological state of the tissue 1 , leading to faster, less invasive diagnosis and more

favorable patient outcomes. The tools and applications in this meta-analysis are much more advanced than those used in this paper; however, the fundamental theory and purpose are similar. Color spectroscopy is particularly useful in astronomy and searching for extraterrestrial life in the universe. Every chemical element or molecule produces a specific light spectrum; color spectroscopy equipment allows for the identification and analysis of these wavelengths from distant celestial bodies. NASA’s exoplanet exploration department uses a type of color spectroscopy called transmission spectroscopy in order to find life on other planets. The molecules, methane, oxygen, and water, are all signs of potential life on other planets 2 ; each of these molecules has its own unique color signature, giving astronomers an unmistakable sign of potential life 2 . Color spectroscopy's versatile and easy application provides a fundamental basis for discovering life on other planets and propels the understanding of the nature of the universe forward. The purpose of this experiment is to identify and replicate the concentration of food dyes in an artificially colored beverage using a spectrophotometer, Beer’s Law, and the dilution formula. Due to the nature of this experiment, a hypothesis cannot be provided; however, if the procedure is pertinently followed, the concentration of dyes will be found, and the color of Powerade will be replicated. The contents of this report will elucidate the the procedures followed in the experiment, the calculations done to find the concentration of food dyes in the beverage, and the final result of the experiment. Experimental An artificially colored beverage with 2 different food dyes was obtained. The beverage used in this experiment was a purple Powerade. The Powerade contained red #40 and blue #1 dyes in order to create its light purple color. An Ocean Optics spectrophotometer was used in

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order to determine the wavelength and absorbance levels of the dyes in the Powerade. The spectrophotometer was connected to a laptop with LoggerPro software installed. The spectrophotometer was calibrated using a cuvette filled with DI water. After calibration, the cuvette was cleaned, dried, and filled with the Powerade. The software was then run with the Powerade until two peaks occurred on the screen. The data was recorded in a spreadsheet. After calibration and running the drink through the spectrophotometer, 4 dyes, Red #40, Blue #1, Blue #2, and Red #3 at a pre-dilution concentration of 9*10 -5 M were tested. 5 different concentrations of each dye were created. For each dye, a 25ml volumetric flask was filled with water and poured into a beaker, then 25ml of the dye was poured into the beaker in order to create a 50% concentration. This concentration was then measured by filling a cuvette and running it through the spectrophotometer and LoggerPro until a peak occurred, with the data recorded into a spreadsheet. Then, 25ml 50% concentration was disposed of into the sink and replaced with 25ml of DI water to create a 25% concentration. The steps were repeated until the concentration got down to 3.125%. After measuring the wavelength and absorbance values of each concentration, the =max() function in google docs was used in order to find the peak wavelength and absorbance values of each dilution. The peak values of the Red #40 and the Blue #1 dilutions were then plotted to form a linear regression. A trivial manipulation of Beer’s Law leads to the concentration value of the two dyes. A simple bijection of the dilution formula leads to the amount needed for each dye in order to replicate the color of the beverage. The beverage was replicated using 4.86ml of Red #40, 8.43ml of Blue #1, and 36.7ml of water. The replicated color was then tested in the spectrometer and compared to the original color. Data

Table 1: Peak wavelength and absorbance values of Powerade and food dyes Sample Tested Peak Absorbance(AU) Wavelength (nm) Purple Drink(Red peak) 0.39 628. Purple Drink(Blue peak) 0.18 494. Blue #1 1.1 628. Blue #2 0.39 608. Red #40 0.32 498. Red #3 0.04 528. The Powerade was tested to show its peak red and blue wavelength and absorbance levels. All dyes were then tested at 5 different concentrations in order to determine the peak absorbance and wavelength. Graph #1: Absorbance vs Wavelength in Powerade

The figure above shows two peaks in the Powerade, indicating the presence of different dyes in the Powerade. Graph #2: Concentration Curve of Red #40 The table above shows the linear regression of Red #40’s absorbance at its average wavelength of all the dilutions of Red #40. The linear regression equation for this curve was Y=2223X+0.09928.

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Graph #3: Concentration curve of Blue #1 The table above shows the linear regression of Blue #1’s absorbance at its average wavelength of all the dilutions of Blue #1. The equation for this graph was Y= 110000364X. Equation 1: Beer’s Law A = (εl)(C) Where: In this equation, A is equal to absorbance, εl is equal to calibration curve slope, and C is equal to concentration. Trivially, C = A/εl. This equation allows for the calculation of the concentration of the red and blue dyes in the Powerade. Equation 2: Dilution formula C 1 V 1 =C 2 V 2 Where: In this equation, C 1 is equal to the concentration of dye in Powerade, V 1 is equal to the volume of Powerade, C 2 is equal to the concentration of dye used in replication Powerade V 2 is equal to the volume of replicated Powerade. From Beer’s law, C 1 = A/εl. V 1 , V 2 are given. Trivally, C 2 = ((A/εl)(V 1 ))/V 2 . This equation allows for the calculation of the concentration needed to recreate the solution.

Discussion A UV/vis spectrophotometer was used on a cuvette filled with Powerade to determine the Powerade's wavelength and absorbance peaks. This testing showed that the Powerade had a red peak at 628nm and a blue wavelength of 494nm, leading to the conclusion that Blue #1 and Red #40 were the dyes used in the drink as their wavelengths(628nm and 498nm respectively) were nearly identical to those in the Powerade. The peak values in each graph are able to be measured as each wavelength has a specific energy level associated with it, meaning that when light hits molecules, a wavelength with enough energy will be absorbed by the electrons of a molecule, causing electrons to be promoted to a higher energy level. The graphs show this as the Y axis shows the absorbance value, meaning that when wavelengths with enough energy to promote an electron are absorbed, the graph spikes up, thereby showing the peak absorbance and wavelength values. The accuracy of these peak points is of high importance in this lab as they ensure the accuracy of the concentration values. The concentration curves were created by plotting and running a linear regression on each concentration's peak absorbance and wavelength values for the two dyes. The specific molar concentrations of these two dyes were 4.5E^-5mol/L, 2.3E^-5mol/L, 1.1E^-5mol/L, 6.25E^-6 mol/L, and 2.8E^-6mol/L. The slope of these graphs allows for the concentration of the two dyes to be calculated through Beer’s Law. In this experiment, the extinction coefficient is the slope of the concentration curves. The value of the extinction coefficient in this experiment for Red #40 was 22230molcm, and the extinction coefficient for Blue #1 was 110000364molcm. The absorbance values for the dyes were determined by finding the corresponding absorbance values. These values were 0.32AU for Red #40 and 1.1AU for Blue #1. Knowing these values, an elementary application of algebra leads us to concentration values of 3.37E-6mol/L of Blue #1 in the drink and a concentration of 8.75E-6mol/L of Red #40. After finding the concentration values through Beer’s law, 4.86ml of Red #40, 8.43ml of Blue #1 and 36.7ml of DI water were used in order to recreate the Powerade. While the calculations that were made were all accurate, the visual appearance of the recreated drink was visually a lot bluer than the original drink. This error can be attributed to the other chemicals present in the Powerade; these chemicals could change the drink's color by clouding up the sample, making it seem lighter or darker, affecting the spectra of the Powerade. Other errors during this experiment can be attributed to the spectrophotometer's heat or the cuvettes' quality and handling. In this experiment, the spectrophotometers were run for very long periods of time, potentially causing the system to heat up, so when cuvettes were inserted into the system, they could have absorbed this heat, subtly changing the properties of the molecules in the solution, leading to an inaccurate reading. Improper handling or poor quality of the cuvettes could skew results by potentially clouding up the outside of the cuvettes, preventing the light from passing through easily and increasing the absorption reading as it would prevent light from easily arriving at the sensor. This could lead to the spectrophotometer reading this as absorption 3 , skewing the results. Conclusion

This experiment aimed to determine the concentration of different dyes in an artificially colored drink and replicate it. This was done using a spectrometer, Beer’s Law, and the dilution formula. The spectrophotometer determined that the Powerade solution contained the dyes Blue #1(498nm) and Red #40(628nm), as their wavelengths matched those seen in the Powerade(494nm, 628nm) nearly perfectly. After plotting the concentration curves and applying Beer’s Law, the concentration of the dyes in the Powerade was found to be 8.75E^-6M for Red #40 and 3.37E^-6M for Blue #1. This information created a solution with 4.86ml of Red #40, 8.43ml of Blue #1 and 36.7ml of water. The sample solution was similar but not completely identical to the original Powerade. This discrepancy could be caused by the other chemicals present in the Powerade, heating up of the spectrophotometer, improper handling or the poor quality of the cuvettes used. This experiment can be expanded upon by using a different type of spectroscopy, such as infrared spectroscopy. UV/Vis spectroscopy uses much higher energy and smaller wavelengths in order to cause electron transitions in orbitals 3 , whereas infrared spectroscopy uses much lower energy and longer wavelengths to affect protons within a molecule. The difference in the use of these two techniques is that UV/Vis spectroscopy is often used for measuring concentrations of known compounds in a solution, whereas infrared spectroscopy is useful in providing information on the unknown compounds in the solution 4 . While UV/Vis spectroscopy is enough for the scope of this lab, infrared spectroscopy can expand upon the scope by allowing for both quantitative and qualitative understanding of solutions with unknown compounds. This lab is important as it provides the fundamental basis for how UV/Vis spectroscopy determines the various concentrations of compounds in a solution. UV/Vis spectroscopy is extremely important in modern pharmaceutical quality control. With the mass production of drugs such as ibuprofen, it is an efficient way to ensure that these drugs have the advertised dosages 5 .

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References (1) Kim, J. A.; Wales, D. J.; Yang, G.-Z. Optical Spectroscopy for in Vivo Medical Diagnosis—a Review of the State of the Art and Future Perspectives. Prog. Biomed. Eng. 2020 , 2 (4), 042001. https://doi.org/10.1088/2516-1091/abaaa3. (2) Why We Search | The Search For Life . Exoplanet Exploration: Planets Beyond our Solar System. https://exoplanets.nasa.gov/search-for-life/why-we-search (accessed 2023-11-28). (3) 4.4: UV-Visible Spectroscopy . Chemistry LibreTexts. https://chem.libretexts.org/Bookshelves/Analytical_Chemistry/Physical_Methods_in_Chemi stry_and_Nano_Science_(Barron)/04%3A_Chemical_Speciation/4.04%3A_UV-Visible_Spe ctroscopy (accessed 2023-11-29). (4) Infrared Spectroscopy . Chemistry LibreTexts. https://chem.libretexts.org/Bookshelves/Physical_and_Theoretical_Chemistry_Textbook_Ma ps/Supplemental_Modules_(Physical_and_Theoretical_Chemistry)/Spectroscopy/Vibrational _Spectroscopy/Infrared_Spectroscopy/Infrared_Spectroscopy (accessed 2023-11-29). (5) Al Ktash, M.; Stefanakis, M.; Boldrini, B.; Ostertag, E.; Brecht, M. Characterization of Pharmaceutical Tablets Using UV Hyperspectral Imaging as a Rapid In-Line Analysis Tool. Sensors (Basel) 2021 , 21 (13), 4436. https://doi.org/10.3390/s21134436.