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Absorption Spectroscopy: Beer-Lambert Law

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Absorption spectroscopy is a practical way to find what the light absorption of a substance is, and whit this data combining the Beer’s law equation you can determine the concentration of different things in a solution, in this case using a orange Gatorade looking the concentration of its dye which is yellow dye #6 and what amount of this Gatorade sample is needed to kill someone. The outcomes in this experiment were that the yellow dye #6 concentration is 5.36 x10-6 M and that the LD50 (lethal dose) is 22,515 L / KG.

In this experiment, we are going to use the Absorption Spectroscopies to determine the level of risk posed to the consumer by the amount of dye present in a commercial food product and how much is needed to get a LD50 of this dye. The determination will be accomplished using a Beer’s Law analysis. In this case using an Orange Gatorade with the yellow dye #6 Absorption Spectrometry is the study light absorbed by molecules. In it white light is cause to pass through a sample and then through a device that breaks the light up into a spectrum. When such light is passed through a sample, under right conditions, the electrons of the sample will absorb certain wavelengths of light, and the device will read the others and compare them to the one in the beginning and that’s how the absorption is calculated. With the data obtained from absorption spectroscopy is most often evaluated using the Beer-Lambert Law. This is how absorption measurements are converted into concentration values. The expected outcomes are that we get the concentration of the dye in the sample in this case the orange Gatorade and determine the LD50 of this sample.


For this of the experiment we used:

Lab Equipment:

Stock Dye (Yellow dye #6)
Orange Gatorade Sample
4 test tubes
Test tube rack

First, with the stock dye in this case Yellow #6 also known as Sunset Yellow, we start making 4 dilutions 1/10, 2/10, 3/10 and 4/10 in a dye/water ratio in ml with the pipette we add each of the amounts of dye to the already filled up test tubes with water.

Next, we place each of the samples into cuvettes and then into the spectrometer, start collecting data and then localize the highest intensity with the respective wavelength and converting this information to concentration.

Then by plugging this data into excel and making a chart and treating the chart as a function we can get the slope which in this case is the molar absorptivity which is part of the Beer’s law.
Following, placing the Gatorade sample into a cuvet and place it in the spectrometer and look for the wavelength that was recorded in the stock dye samples

Finally with the Molar absorptivity and the absorption of the Gatorade sample we can get the concentration of the dye in the sample and then we can get the LD50 of this sample.

With all the process and data collected we got that:

Dye/Water RatioWavelength (nm)AbsorptionConcentrationAbsorption 1/104800.0703.73×10-6 M0.070

2/104800.1506.83×10-6 M0.150
3/104800.2019.46×10-6 M0.201
4/104800.2591.17×10-5 M0.259

Molar Absorptivity = 21657
After we get the Molar Absorptivity we can get the concentration of the Gatorade Sample Sample/water ratioWavelength (nm)Absorption

By plugging the Absorption of the Sample and the Molar Absorptivity to the Beer’s Law we get that the concentration of the yellow dye #6 is 5.36 x10-6 M and that the LD50 is 22,515 L / KG

As we can see on the results, we can determine that in the Gatorade sample, the concentration of yellow die is not dangerous unless you drink 22,515 L / KG which is almost impossible to one person to retain that amount of liquids, first the person would die of drinking excessive amounts of water called overhydration.

The calculations done in this experiment were to get the concentration of the stock dye dilutions, the concentration of the dye in the Gatorade using Beer’s law sample and the LD50 of the Gatorade.

Concentration = (Dye amount)/(Total amount of Solution)
C = (1.0 ml of yellow dye #6)/(11ml of water+dye)
C = 3.7×10-6 M
Absorption = εbC
C =Absorption/εb
C = 0.116/(21657(1))
C = 5.356×10-6 M
LD50= C x MM=g/L then with LD50 of the stock dye x L/g to get the Liters per kilogram needed to kill someone. 5.356×10-6 M x 4.10×10-5MM = 0.0024226259 g/L
6g/Kg x 1L/0.0024226259g=2476.651471 L/kg
And then because the solution used was diluted to 1/10 ratio we need to multiply it by 9.09 which is the percentage of dye in the solution. And the result was 22,515 L / Kg.

Wrap Up Discussion

What is the dye content of your sample? How much of the food must you consume to reach an LD50 exposure.. R: Yellow dye #6, 22,515 L / KG

Discuss your confidence in your answer Here is where you will address any problems you encountered during the experiment. Are there other possible answers? If so, what should you do next? R: yes because the sample was not made that same day, it was made some months ago, that’s probably why the results may vary between the same Gatorades but with other dates which they were made. Discuss what you learned about the suitability of Absorption spectroscopy to determine molar concentrations. R: with Absorption spectroscopy it is easy to determine the molar concentration, but as the past spectroscopy, it is always some experiments needed to be done before identifying and determining what you are looking for.

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