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Determining the Concentration of Red Food Dye #40 in a Solution
Objectives:
 Prepare 5 solutions of red # 40 of known concentration and measure %T.
 Create a standard curve of absorbance vs concentration.
 Determine concentration of a solution of unknown concentration.
Introduction:
The objective of this experiment is to determine the concentration of a food dye, Red #40 in a food sample. You will prepare 5 standard solutions of red # 40 by dilution from a stock solution and calculate the new concentration of each solution.
Example calculation A student was provided a 0.0009384 M stock solution of red #40. The student diluted 3.00 mL of the stock solution with water to a total volume of 25.00 mL. What is the new concentration of the red # 40 solution? C_{1}= 0.0009384 M V_{1}= 3.00 mL C_{2}= ? V_{2}= 25.00 C_{1}V_{1}=C_{2}V_{2} Solve for C_{2} 
Each of the 5 solutions will have a different concentration, the higher the concentration the deeper the red color of the solution. We will use a spectrophotometer to relate the concentration of red # 40 to the “color intensity” of the solutions.
A spectrophotometer is an instrument that can be used to measure the amount of light that can pass through a solution, a clear colorless solution will allow more light to pass through a than a more pigmented sample.
We will use a spectrophotometer to measure percent transmission. If the sample absorbs no light the percent transmittance = 100%. If the sample absorbs the light completely percent transmittance = 0%. So, the greater the amount of light absorbed by the sample, the smaller the percent transmittance.
Percent transmission 
Percent transmittance can be used to calculate absorbance of the solution. When you plot absorbance vs concentration you will observe a direct relationship, this is known as Beer’s Law. The BeerLambert law describes absorbance of light through a solution of a substance is dependent on the path length, molar absorptivity and the concentration of the absorbing substance by the relationship shown in equation 1
A= bc Equation 1
Where:
A – absorbance
– molar absorptivity with units of Lmol^{1}cm^{1} (this is a measure of the amount of light absorbed per unit concentration or a measure of how well the substance absorbs light)
b – path length, this is the width of the cuvette in which the sample is contained, with units of cm (the cuvettes we will be using have path length = 1 cm)
c – the concentration of the compound in solution, expressed in mol/L
we will measure % transmittance in this experiment and from % transmittance you will calculate the absorbance of each solution.
Equation 2
Absorbance: A = 2 – log %T
Equation 2 will be used to calculate absorbance from % transmittance data which we will be measuring in this experiment using a spectrophotometer. Now let us look at the BeerLambert law and explore it’s significance. For dilute solutions, the Beer Lambert law states that the absorbance of the solution will be directly proportional to its concentration. As the concentration increases, the absorbance also increases. If you plot Absorbance vs Concentration the slope of the best fit line is the molar absorptivity, .
Example Data:
 Example standard curve of absorbance vs concentration Best fit Line from sample graph 1 y=21.577X + 0.0339 slope= For this data set the slope= =21.557 
You can use Beer’s law to determine the concentration of a sample of food dye once you calculate the absorbance of the solution and obtain from the best fit line seen described by equation 1.
Example calculation solving for concentration given an absorbance measurement A= b C Rearranges to
Given: A= 0.107 =21.557/ M cm b= 1.0 cm 
In part 1 of this experiment you will prepare 5 different samples of known concentrations, measure % transmittance, calculate the absorbance of each solution, and then plot absorbance vs concentration. The slope of the bestfit line is equivalent to the molar absorptivity. In Part 2 you will measure the % transmittance of a food sample containing red food dye #40. You will calculate the absorbance, then use Beers law to determine the concentration red # 40 in your food sample.
Safety:
Wear safety glasses at all times. Red # 40 solutions are nonhazardous and can be disposed of down the drain with water.
Equipment:
 Cuvettes (2)
 Pasteur pipette
 Pipette bulb
 Volumetric pipette one of each:
 (1 mL, 2 mL, 3 mL , 5 mL,10 mL
 25 mL Volumetric flask (5)
 Red Food dye #40
 Unknown Red food dye #40 sample
 Distilled water
 50 mL beaker
 A/B pipette bulb
 Spectrophotometer (shared)
 Marker
 250 mL beaker
Procedure:
Part A: Preparation and % transmittance measurements of known solutions
 Obtain ~ 30 mL of the sock solution of red food dye # 40, record the exact concentration written on the bottle on your datasheet.
 Obtain five, 25 mL volumetric flasks, number the flasks 15.
 Condition the 1 mL pipette with the stock solution and discard the conditioning liquid to a waste beaker.
 Using the 1 mL volumetric pipette carefully deliver 1.00 mL of red food dye #40 that you obtained in step 1 into the volumetric flask labeled #1, then add distilled water to the calibration line.
 Cap the volumetric flask and mix the solution well by inverting 20 30 times.
 Repeat steps 35 for the remaining solutions as described in table 1.
Table 1
Volumetric flask  Volume of Stock solution of Red #40 (mL) (mL)  Total volume of solution 
1  1.00  25.00 
2  2.00  25.00 
3  3.00  25.00 
4  5.00  25.00 
5  10.0  25.00 
 Obtain 2 cuvettes
 Fill the first cuvette with ¾ full with distilled water.
 Fill the second plastic cuvette with the solution from volumetric flask #1, about ¾ full
 Setting the spectrophotometer to 100% T “Blank”.
Genesys 20 spectrophotometer
 Insert the cuvette containing distilled water into the sample holder inside the spectrophotometer, and then close the shutter.
 The wavelength of the spectrophotometer should be set to 504 nm. Ask your instructor to help you adjust the settings if the wavelength is not set to 504 nm.
 Press the A/T/C button until the spectrophotometer is displaying % Transmittance.
 Press the 0 ABS/100% transmittance button to set the percent transmittance to 100%.
 Remove the cuvette containing water from the spectrophotometer.
 Measuring the % Transmittance of your solutions
 Insert the cuvette containing the red food dye solution from volumetric flask #1 into the spectrophotometer sample holder, then close the shutter.
 The wavelength of the spectrophotometer should be set to 504 nm. Go back to step 8 if the wavelength is not set to 504 nm.
 Record the %T on your datasheet in Table 2.
 Remove the cuvette from the spectrophotometer
 Repeat steps 67 for each food dye sample. You may think performing the “blank” for each sample is unnecessary, trust me you want to reset to 100% Transmittance in between each measurement when using the Genseys 20 Spectrophotometer.
Part 2 Measuring percent transmittance of unknown
 Measure the %T of a food sample containing red dye #40 of unknown concentration.
 Obtain a sample of the red food dye #40 of unknown concentration, record the unknown number on your datasheet. Fill a cuvette ¾ full with the unknown sample.
 Repeat step 8 setting the spectrophotometer to 100% T “Blank”.
 Insert the cuvette containing your unknown red #40 sample into the spectrophotometer sample holder, then close the shutter.
 Record the %T on your datasheet.
 Remove the cuvette from the spectrophotometer.
Clean up: All red #40 solutions and unknown can be disposed of down the drain as they are nonhazardous, rinse the cuvettes, volumetric pipettes and volumetric flasks with distilled water 35 times and return them to the proper location.
Date_________________ Name__________________________________________
Datasheet
 Stock concentration of Red food Dye # 40 ___________________________
(written on the bottle of stock solution)
 Fill in table 2 by completing the following:
 Calculate the concentration of each diluted solution of Red food dye # 40 using the equation C_{1}V_{1}=C_{2}V_{2}
_{Show example calculation for #1 here}
 Record the % transmittance for each sample
 Calculate the absorbance using : A = 2 log(%T ) for each sample
Show example calculation for #1 here
Table 2:
Volumetric flask #  Volume of Stock solution of Red #40 (mL)  Total Volume of solution (mL)  Concentration of diluted solution of Red food dye # 40  % Transmittance  Absorbance

1  1.00  
2  2.00  
3  3.00  
4  5.00  
5  10.00  
Unknown Red #40  Unknown # 
 Calibration curve
 Using Excel plot Absorbance vs Concentration of the 5 Red #40 dye solutions. (if you do not know how to use excel see this website for help https://chem.libretexts.org/Bookshelves/Ancillary_Materials/Laboratory_Experiments/Wet_Lab_Experiments/General_Chemistry_Labs/Online_Chemistry_Lab_Manual/Chem_11_Experiments/01%3A_Using_Excel_for_Graphical_Analysis_of_Data_(Experiment))
 Generate the best fit line, record the best fit line equation on Table 3.
 Insert the equation of the line and the R^{2} value on the graph.
 Give your graph a title.
 Label the X and Y axis.
 Attach a printed copy of your excel graph to your datasheet.
 Record the molar absorptivity in table 3.
Table 3
 
 

Date_________________ Name__________________________________________
Postlab
 Your unknown food red # 40 sample was from an undiluted sample of diet Gatorade. Using the concentration, you determined from the experiment calculate the mass of Red # 40 in a 11.6 oz can of the Gatorade, the molecular formula of red # 40 is: C_{18}H_{14}N_{2}Na_{2}O_{8}S_{2}.
 Use Beer’s Law and the Absorbance and final concentration of volumetric flask 3 to calculate the molar absorptivity. Compare the two molar absorptivity values (from Beer’s Law and from the trendline). Which do you think is more accurate?
 According to the FDA, the acceptable daily intake for Red # 40 is 420 mg for an adult weighing 60 kg. How much would an 80.5 kg person need to drink to reach this daily acceptable intake for red # 40 in the Gatorade you analyzed?
 What is the purpose of using a blank? What is the purpose of zeroing the spectrometer?
Date_________________ Name__________________________________________
Prelab
 Fill in table 4 by completing the following:
 Calculate the concentration of each diluted solution of Red food dye # 40 using the equation C_{1}V_{1}=C_{2}V_{2}
_{Show example calculation for flask #1 here}
 Calculate the absorbance using : A = 2 log %T for each sample
Show example calculation for flask #1 here
Flask #  Initial concentration of Colored solution (M) C_{1}  Volume of Stock solution of colored solution (mL) V_{1}  Total volume V_{2}  Concentration of diluted solution (M) C_{2}  % Transmittance of colored solution  Absorbance A = 2 log %T 
1  0.00145 M  1.00  10.0  74.2  
2  0.00145 M  2.00  10.0  62.5  
3  0.00145 M  3.00  10.0  54.2  
4  0.00145 M  4.00  10.0  42.3  
5  0.00145 M  5.00  10.0  32.5 
 Calibration curve
 Using Excel plot Absorbance vs Concentration of solutions(if you do not know how to use excel see this website for help: https://chem.libretexts.org/Bookshelves/Ancillary_Materials/Laboratory_Experiments/Wet_Lab_Experiments/General_Chemistry_Labs/Online_Chemistry_Lab_Manual/Chem_11_Experiments/01%3A_Using_Excel_for_Graphical_Analysis_of_Data_(Experiment))
 Generate the best fit line, record the best fit line equation on Table 5
 Insert the equation of the line and the R^{2} value on the graph
 Give your graph a title
 Label the X and Y axis
 Attach a printed copy of your excel graph to your prelab.
 Record the molar absorptivity in table 5
Table 5
 

 The % Transmittance of an unknown sample was measured to be 48.3% Calculate the absorbance of the sample. Use the molar absorptivity and Beers law to calculate the concentration of the unknown sample.