Chem 1A
Dr. White
1 Fall 2013
UV-Vis Absorption Spectroscopy of Dyes in Beverages
Introduction
The intensity of the color in solutions can be quantified by measuring the absorbance of light by the solution.
Absorption spectroscopy involves measuring, via a spectrometer, the intensity of light after it passes through a
colored solution. The deeper the color, the more light will be absorbed.
The UV-Vis Spectrometer we will use consists of a white light source, a sample compartment and a detector.
Incident light from the source is focused on the sample. Depending on the color of the sample, some wavelengths
of light are absorbed and some are transmitted into the detector. The color of the light absorbed is directly related
to the color of the light transmitted; i.e., the color we see. The color absorbed is the complement of the color we
see. A color wheel, which shows the colors of the rainbow, is shown to the right. On a color wheel, the
complementary color is the color found directly opposite on the wheel. For example, the complementary color to
red would be green.
The table below summarizes the relationship between the color
observed and the color absorbed as well as the wavelength of
the absorbed light.
Table 1: Approximate Relationship of Wavelength of Visible
Light Absorbed to Color Observed.
Color Observed
Color Absorbed
Wavelength of
Absorbed Light (nm)
Violet
Dark Blue
Blue
Green
Yellow
Red
Yellow-Green
Yellow
Orange
Red
Blue
Blue-Green
570
580
600
650
450
490
To determine the wavelength of absorbed light, an absorption spectrum is taken. The process of obtaining an
absorption spectrum can be thought of as occurring in three steps. (1) A substance is exposed to a source of
incident electromagnetic radiation at various wavelengths. (2) The substance absorbs the incident radiation at
those wavelengths that coincide with transitions of its electrons from lower to higher energy levels. The substance
transmits – that is, it does not absorb – the energy of the wavelengths that do not correspond to its characteristic
electron transitions. (3) The wavelengths of transmitted radiation are recorded by a detector.
A simple picture of the concept is shown below. White incident light is focused on the colored sample. The sample
absorbs some wavelengths of light and transmits the complementary wavelengths of light.
Incident Light (I0)
Transmitted Light (I)
b
Chem 1A
Dr. White
Fall 2013
2 The transmittance of light (T) is defined as:
(1)
The absorbance of light (A) is related to the transmittance by:
(2)
Besides the concentration of the absorbing species (c), two other factors determine the absorbance of a sample
solution. The further light must travel through a solution, the greater the absorbance. This distance is referred to as
the pathlength and is denoted with the symbol b. The ability of the molecule to absorb light at the given
wavelength, known as the extinction coefficient (ε) a quantum mechanical effect, will also determine the
absorbance. The relationship between these influences and the absorbance is given by the Beer-Lambert Law:
(3)
The Beer-Lambert Law states that there is a linear relationship between concentration and absorbance. Generally,
dilute solutions follow the Beer-Lambert Law quite well. The absorbance of a compound is usually measured at the
maximum point (λmax) in its absorption spectrum. By choosing λmax, we choose the point where the detection of the
molecule in the solution is the most sensitive, since ε is the largest.
If we prepare a series of solutions of the same molecule in known concentrations and plot the linear relationship
between absorbance and concentration for these solutions according to the Beer-Lambert law, the result should be
a straight line. This graph can allow us to determine the concentration of an unknown solution by correlating the
absorbance of that solution to its corresponding concentration.
In part 1 of the lab you will investigate the absorbance spectra of 4 food dyes in order to see how they differ. In part
2 of the lab, you will create a standard curve for Red Dye #40 and use it to determine the concentration of Red Dye
TM
#40 in Fruit Punch Gatorade . Then, you determine the number of servings of Red Dye #40 you would have to
ingest to reach the accepted daily intake (ADI), the safe daily intake level for a healthy adult of normal weight. ADI
values are based on current research, with long-term studies on animals and observations of humans and are
expressed by body mass, usually in milligrams (of the substance) per kilograms of body mass per day. For Red
Dye #40, the ADI is 7 mg/kg (from the World Health Organization Website).
Procedure
Part 1: UV-Vis Absorption Spectra of Food Dyes
1. If it is not already open, start the Logger Pro program on your computer.
2. Calibrate the spectrometer by filling a clean, dry cuvette ¾ full with deionized water. Mark one side of the
cuvette with a grease pencil so that you can place the cuvette in the spectrometer with the same orientation
throughout the experiment. Wipe the cuvette with a KimWipe and place it in the spectrometer (make sure
the smooth side of the cuvette faces the light source). Select Calibrate ► Spectrometer from the
Experiment menu. The calibration dialog box will display the message: “Waiting . . . seconds for lamp to
warm up.” The minimum warm-up time is one minute. Follow the instructions in the dialog box to complete
the calibration. Click
.
Chem 1A
Dr. White
3 Fall 2013
3. Empty the cuvette and rinse it twice with small amounts of the blue dye solution. Fill the cuvette ¾ full with
the blue dye solution and place it in the spectrometer. Click
solution will be displayed. Click
. A full spectrum graph of the
to complete the analysis. Note that one area of the graph
contains a peak absorbance. Record the wavelength of this peak absorbance (λmax) and the absorbance at
the λmax.
4. Repeat step 3 for the yellow, red and green dyes. When you click
, a dialog box will appear
that will ask you if you want to erase the data. Select “store latest run” so that you can plot all 4 spectra on
one plot.
5. Save the spectra to a flash drive so that it can be printed in the computer lab or by your instructor at the
instructor’s desk.
6. Once the data is saved, go to the Data menu and select “Clear All Data.”
Part 2: Determination of the Amount of Red Dye #40 in Fruit Punch Gatorade using a Standard Curve
1.
Make solutions of the Red Dye #40 as described by Table 2 below using a graduated 10-mL pipette (Use
the stock red dye solution, NOT the Gatorade to make these solutions). Cover the top of each tube with
parafilm and mix the solutions in each tube completely.
Table 2: Volumes and Concentrations for Red Dye #40 solutions.
2. Calibrate the spectrometer again by repeating
Solution
Volume of Stock
Red Dye Solution
(mL)
Volume of DI
Water (mL)
1
10.00
0.00
Concentration
(M)
3.0 x 10
-5
step 2 from Part 1.
3. Empty the cuvette and rinse it twice with small
amounts of the solution in test tube 1. Fill the
cuvette ¾ full with the test tube 1 solution,
2
8.00
2.00
2.4 x 10
-5
3
6.00
4.00
1.8 x 10
-5
4
5.00
5.00
1.5 x 10
-5
spectrum graph of the solution will be
5
4.00
6.00
1.2 x 10
-5
displayed. Click
6
2.00
8.00
6.0 x 10
-6
wipe it with a KimWipe and place it in the
spectrometer. Click
. A full
to complete the
analysis. Click the Configure Spectrometer
Data Collection icon,
, on the toolbar. A
dialog box will appear. Select Abs vs. Concentration under Set Collection Mode. The wavelength of peak
absorbance (λmax) will be automatically selected. Record this wavelength on your data sheet. Click
to proceed.
Chem 1A
Dr. White
4. Leave the cuvette in the spectrometer. Click
4 Fall 2013
. When the absorbance reading stabilizes, click
. Enter the concentration of the red dye solution in tube 1 as found in Table 2 and click
-5
. (Note: to enter 3.0 x 10 , type “3.0E-5”). Record the absorbance value in your notebook.
Discard the cuvette contents down the drain. Using the solution in test tube 2, rinse and fill the cuvette ¾
full. Wipe the cuvette with a KimWipe and place it in the spectrometer. When the absorbance reading
stabilizes, click
. Enter the concentration. Repeat step 4 for test tubes 3, 4, 5 and 6. (DO NOT
click on stop until you have analyzed all 6 solutions!)
5. When you have finished testing the standard solutions, click
.
6. To determine the best-fit line equation for the standard solutions, click the linear fit button,
, on the
toolbar. An equation for the line will appear as something like this:
Linear Fit for: Latest ⎢Absorbance at 502 nm
Abs-502 = mx+b
m(Slope): 2.340E+004
b(Y-intercept): 0.00570
Correlation: 1.000
RMSE: 0.001725
4
This would be written in equation form as: y = 2.340 x 10 x + 0.00570 (where y is the Abs at 502 nm and x
is the concentration in M)
Write down the equation for the standard solutions in your data lab notebook. Save your graph by
selecting “Save As” from the file menu. Save it as a different file name as before to a flash drive so that it
can be printed.
7. Now prepare your Gatorade
TM
solution for analysis by measuring 2.00 mL of it with a graduated pipette and
diluting it by adding 8.00 mL of water. Make sure to mix the solution completely.
8. Click the Configure Spectrometer Data Collection icon,
, on the toolbar. A dialog box will appear.
Select Abs vs. Wavelength under Set Collection Mode. Empty the cuvette and rinse it twice with small
amounts of the Gatorade
spectrometer. Click
TM
solution. Fill the cuvette ¾ full with the Gatorade
TM
solution and place it in the
. A full spectrum graph of the solution will be displayed. Click
to complete the analysis. Record the Absorbance of the solution at the same wavelength used for the red
dye standard solutions. Save your spectrum by selecting “Save As” from the file menu. Save it as a
different file name as before to a flash drive so that it can be printed.
Chem 1A
Dr. White
Fall 2013
Name______________________________
Data/Results:
Part 1: UV-Vis Absorption Spectra of Food Dyes
Table 1: Concentration and absorption data and results for blue, red, yellow, and green food
dye solutions.
Dye
λ max (nm)
Absorbance at λ max
Color of Absorbed Light
at λ max
Blue
Red
Yellow
Green*
*Green has 2 peaks, record data at each peak.
In terms of peaks, discuss how the spectrum from the green dye compares to the spectra from
the blue and yellow dyes.
Attach a copy of the combination graph containing the spectra from the four food dyes. Make
sure to label the dye associated with each spectrum.
5 Chem 1A
Dr. White
Fall 2013
Part 2: Determination of the Amount of Red Dye #40 in Fruit Punch GatoradeTM using a
Standard Curve
Table 2: Concentrations and Absorption Data for Red Dye #40 solutions.
Solution
1
2
3
4
5
6
Concentration (M)
Absorption at
________nm
3.0 x 10-5
2.4 x 10-5
1.8 x 10-5
1.5 x 10-5
1.2 x 10-5
6.0 x 10-6
Best Fit Equation of the Line in the Standard Curve:_______________________________
Absorbance of diluted GatoradeTM Solution______________
Concentration of the diluted Red Dye #40 in Gatorade (use the best fit equation and the
Absorbance of the diluted Gatorade)
______________M
Concentration (molarity) of the undiluted Red Dye #40 in Gatorade
M undiluted =
M diluted ×10.00 mL
2.00 mL
€
______________M
6 Chem 1A
Dr. White
Fall 2013
Use dimensional analysis to solve the following:
According to the bottle, a serving size of Gatorade is 240 mL. The molar mass of Red Dye
#40 is 496.43 g/mol. Use this information along with the concentration of Red Dye #40 in
Gatorade to calculate the mass (in mg) of Red Dye #40 ingested per serving of Gatorade.
__________mg/serving
The ADI for Red Dye #40 is 7 mg/kg. Use this information along with the mass of Red Dye
#40 per serving determined above to calculate the number of servings an 80.kg person would
need to ingest to receive the ADI of Red Dye #40.
____________servings
Follow-up Question:
Red Dye #2 was banned in 1976. It has an ADI of 0.8 mg/kg. If the concentration of Red Dye
#2 in Fruit Punch Gatorade is the same as the Red Dye #40 determined in the experiment,
how many servings of Fruit Punch Gatorade would an 80. kg human need to consume to
reach the ADI? (the molar mass for Red Dye #2 is 604 g/mole)
____________servings
7