Experiment 37B-1
FV 3-15-16
SPECTROSCOPIC ANALYSIS OF DYES – MORE THAN PRETTY COLORS1
PART 1
MATERIALS:
FD&C food stock solutions (Red, Blue, Yellow), 4 50 mL beakers, 250 mL beaker, 3 10 mL
volumetric pipets, 2 5 mL volumetric pipets, 4 100 mL volumetric flasks, plastic dropper,
Spectronic 200, cuvettes.
PURPOSE:
The purpose of this experiment is to understand how light interacts with a dye solution and
to use this knowledge to determine the amount of dye in an unknown.
LEARNING OBJECTIVES:
1.
2.
3.
4.
5.
6.
By the end of this experiment, the student should be able to demonstrate the
following proficiencies:
Prepare diluted solutions and calculate their concentrations.
Understand what an absorbance spectrum is and select max, the wavelength of maximum absorbance.
Relate the color observed of a solution to its max value (and color absorbed).
Understand Beer’s Law and what factors affect it.
Create a calibration curve using standard solutions.
Determine a concentration of an unknown using a calibration curve.
PRE-LAB:
Complete the pre-lab questions on pages E37B1-7 and E37B1-8 before lab.
DISCUSSION:
Mars
When light of a particular wavelength () is absorbed by a sample, the intensity of light that initially strikes
the sample (Io) can be compared to the intensity of light that passes through the sample (I).
Io
I
The transmittance (T) is defined as the fraction of the light striking the sample that passes through the sample:
T = I/Io
%T = (I/Io) x 100
at a given 
Absorbance (A) is related to transmittance logarithmically:
A = log10(1/T) = log10(Io/I) =  log(%T/100)
at a given 
Thus, when no light is absorbed by a sample, I = Io, T = 1, %T = 100, and A = 0. Conversely, when all of the light
that strikes a sample is absorbed by it, I = 0, T = 0, %T = 0, and A is infinite.
1
This lab is based on “Spectroscopic Analysis of Food Dyes” by Barbara A. Reisner, Joycette Santos-Santori, Dawn
Rickey, and Melonie Teichert.
E37B1-1
In this lab, you will be using an instrument called a spectrophotometer, which measures %T and A at various
wavelengths to investigate food colorings in the visible region of the electromagnetic spectrum.
What are the FD&C dyes?
FD&C (Food, Drug, & Cosmetics) dyes are additives approved by the Food & Drug Administration (FDA) to
enhance the color of consumer products. While there are many natural dyes derived from natural products (e.g., beet
juice, tumeric, saffron, paprika), the manmade FD&C dyes are considered safe for human consumption. A list of
dyes that are currently approved by the FDA is provided below. The molecular structures for some of these dyes can
be found below:
 FD&C Blue No. 1 - Brilliant Blue FCF, E133 (blue shade)
 FD&C Blue No. 2 - Indigotine, E132 (dark blue shade)
 FD&C Green No. 3 - Fast Green FCF, E143 (bluish green shade)
 FD&C Red No. 40 - Allura Red AC, E129 (red shade)
 FD&C Red No. 3 - Erythrosine, E127 (pink shade)
 FD&C Yellow No. 5 - Tartrazine, E102 (yellow shade)
 FD&C Yellow No. 6 - Sunset Yellow FCF, E110 (orange shade)
The color choices provided by the FD&C dyes are somewhat limited. However, it is possible to produce a great
number of colors by mixing these dyes. If you read the ingredient lists on consumer products, you may see the
presence of FD&C dyes or other natural ingredients used to produce the colors that we associate with these products.
BLUE No. 2
O
H
C
-
O3S
C
H
C
H
N
C
C
HC
C
C
H
CH
C
C
C
C
C
N
H
SO3-
C
H
O
YELLOW No. 6
HC
HO
RED No. 3
I
CH
C
C
-
CH
O
C
C
C
HC
N
HC
CH
I
C
HC
CH
C
C
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CH
C
-
C
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H
I
COO-
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HC
O
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SO3-
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O3 S
Electromagnetic Spectrum
E37B1-2
(this is better viewed in color)
PROCEDURE:
You will work with a lab partner. Each lab partner must record the data and answer the questions individually, after
discussing them.
Demo: View the instructor demo and record your observations on page E37B1-4.
Part A. Solution Preparation
1.
2.
3.
4.
5.
6.
Into separate dry, 50 mL beakers obtain the following: ~20 mL stock Blue, ~20 mL stock Yellow, and ~20
mL stock Red solutions.
Dividing the work among the lab partners, prepare a 1:10 diluted solution of each of the stock dye solutions
(Blue, Yellow, Red) by using these steps:
a. Pipet 10 mL of the stock solution into a 100 mL volumetric flask. (If the pipet is wet, pre-rinse it
with a little of the stock solution and discard the rinse.)
b. Using distilled water, dilute the solution to the line of the volumetric flask. Use a dropper to add
the last drops of water.
c. Cap the flask securely and invert the flask a couple of times to mix the contents.
d. You should have 3 diluted (1:10) solutions of Blue, Yellow, and Red.
Once the diluted Blue and Yellow solutions are prepared (from step 2), mix equal volumes together in a
small beaker. For example, pipet 5 mL of the diluted Blue solution into the beaker, then with a clean pipet,
pipet 5 mL of the diluted Yellow solution into the beaker. (Make sure not to contaminate your solutions by
using dirty pipets.) Mix the solution with a glass stirring rod. Was the color what you expected?
Once the diluted Red solution is prepared (from step 2), prepare a further diluted solution of it by pipetting
20 mL of the diluted Red solution into a 100 mL volumetric flask. You will need to use the 10 mL pipet
twice. Fill to the line with distilled water and mix. How does this further diluted Red solution compare to
the original one?
Record your observations of each solution.
Once all of your solutions are prepared, inform your instructor that you are ready to obtain the absorbance
spectrum of each solution.
Part B. Obtaining Absorbance Spectra
1.
2.
3.
4.
5.
6.
Bring your 5 solutions to the designated spectrophotometer.
Follow the directions for using the Spectronic 200. You will begin by calibrating the instrument, then
collecting the absorbance spectrum. This instrument sends all visible wavelengths of light through the
sample and records the absorbance at each wavelength. The output will be a plot of absorbance vs.
wavelength (), which is known as an Absorbance Spectrum.
For each solution, record the wavelength of maximum absorbance (max) in the data section as well as the
absorbance value at max. Note that some solutions may have more than one max value (record them all).
Also collect a printout of each absorbance spectrum by using the shared printer (print a copy for each lab
partner). Make sure to label which solution gave which spectrum. Submit your absorbance spectra with
your lab.
Rinse your cuvettes well with distilled water.
Clean up all of your glassware. The dye solutions can be disposed down the drain. Make sure to remove
any remnants of dye from glassware as they can stain if left to dry.
Start working on the post-lab questions and calculations.
Example Absorbance Spectrum:
max (nm) = wavelength of maximum absorbance
Absorbance
Wavelength (nm)
E37B1-3
Name ____________________________________________
Section ___________________________
DATA AND ANALYSIS
Experiment 37B-1
Demo:
Observations: ___________________________________________________________________________
What did you learn from the demo? __________________________________________________________
_______________________________________________________________________________________
Give units
Prepared Solutions
Observations (including color)
max (
)
Absorbance at max
1:10 diluted Blue
1:10 diluted Yellow
1:10 diluted Red
1:50 diluted Red
Mixture of diluted Blue
and diluted Yellow
1. a. Explain why the dye solutions (B, Y, R) were listed as “1:10 diluted”. Where do the numbers 1:10 come
from?
b. Based on the lab procedure (i.e., what was mixed), explain why the further diluted Red solution was listed as
“1:50”. Show a calculation which shows where the numbers “1:50” come from.
c. Which solution, 1:10 diluted Red or 1:50 diluted Red, contained more Red dye molecules in it (in a 100 mL
volume)?
Based on your absorbance data, which solution, 1:10 diluted Red or 1:50 diluted Red, absorbed more light at
max?
2. What are the similarities and differences between the absorbance spectra of the 1:10 diluted Red and 1:50 diluted
Red solutions (note the scale on the y-axis)?
Similarities:
Differences:
What are the origins of these similarities and differences? How does this evidence relate to what is
happening on the molecular level?
E37B1-4
3. Explain how the absorbance spectrum of the Mixed Blue/Yellow solution compared to the individual Blue and
Yellow solution spectra.
What is the origin of the Mixed Blue/Yellow spectrum being lower in absorbance (note scale on the y-axis)?
4. As seen in the pre-lab, a Red laser pointer emits red light at a wavelength of about 650-670 nm. Did your Red
dye solution have a max value close to this range? (Close would be within 10 nm.)
How did the observed color of each dye solution relate to the max value you found?
Refer back to your data.
Is this surprising to you? Why?
Color wheel
In general, a color wheel can be used to predict wavelength regions absorbed for a
particular color. Speculate how this is done using the color wheel. 
5. Suppose you had a solution that was violet in color. Sketch the predicted absorbance spectrum for this solution
(between 400 and 750 nm) and give an approximate max value (label on plot). Label the axes on your plot.
For this violet solution, what color is
observed?
________________
absorbance
spectrum
What color is
absorbed?
6. An orange solution should have a max of approximately what wavelength?
E37B1-5
________________
max  _______________
7. a. Based on what you learned from this lab, refine your Initial Model (from the pre-lab) of concentrated and
dilute Red dye solutions. Be sure to address the questions: “What happens when you shine light on
concentrated and dilute Red dye solutions?” and “Why do we see it as Red?”. Make sure to include
Refined
the macroscopic and molecular-level drawings, labeling and explaining your drawings and symbols.
model
Clearly communicate what you know.
Macroscopic (concentrated and dilute red solns)
Molecular-level (conc’d and dilute red solns; why do your see Red?)
What happens when you shine light on concentrated and dilute Red dye solutions? Why is the Red dye solution Red? Why do we
see it as Red?
b. Explain what has changed and/or stayed the same from your initial model. Explain why (using what you have
learned in the lab, including experimental evidence) you made changes and why you did not. Be specific.
Stayed the same
Changed from initial to refined model
Explain why (using the lab or data) you didn’t change
your thinking on some aspects. What lab results
confirmed your thinking in the initial model?
Explain why (using the lab or data) your thinking changed. What data or
evidence from the lab made you change your thinking?
E37B1-6
Name ______________________________
Section ___________________________
PRE-LAB QUESTIONS
Experiment 37B-1
Complete these questions before lab.
1. a. In this lab, you will be studying how light interacts with dye molecules in a solution. What similarities
can you find in the structures of the FD&C dyes shown on page E37B1-2?
b. FD&C dyes are found in many drinks and foods. Find an example of one of these dyes (check food
and drink labels).
FD&C dye = ______________________ found in __________________________________________
(which dye)
(give name of drink or food)
2.
A laser (which stands for Light Amplification by Stimulated Emission of Radiation) emits essentially a
single wavelength of light (i.e., it is monochromatic light). This wavelength of light emitted correlates to
the observed color of the laser beam.
Roughly, at what wavelength () does a Red laser pointer emit? _____________________ nm
Roughly, at what wavelength () does a Green laser pointer emit? _____________________ nm
Are these wavelengths in the visible region of the electromagnetic spectrum? ______________
Which has higher energy photons, Red or Green light? __________________
Which has higher wavelength, Red or Green light? __________________
Which has higher frequency, Red or Green light? __________________
Emission is somewhat similar to transmittance (light leaving a sample). Explain how Absorbance (A) and
Transmittance (T) are related.
Assume a red laser is directed separately into a red and a green solution. Draw what you think is
transmitted through each solution. Does the red laser light go through unaffected? Is it absorbed (partially
or completely)? What happens to that red laser light in each case?
Red laser light

Red laser light
Red
Solution

E37B1-7
Green
Solution
Initial
model
3. Suppose you are looking at 2 Red dye solutions, one concentrated and one dilute. Construct an initial
model of your understanding of these 2 solutions. Focus on the appearance of the solutions for your
macroscopic model, and then develop a molecular-level model that explains the appearance of the
solutions. Consider the questions: What happens when you shine light on concentrated and dilute Red
dye solutions?” and “Why do you see the solution as Red?” in your model. Include both macroscopic
and molecular-level drawings and use words to clarify your drawings. If you use symbols, be sure to
include a key or explanation.
Macroscopic (concentrated and dilute red solns)
Molecular-level (conc’d and dilute red solns; why do your see Red?)
Conc’d:
Conc’d:
Diluted:
Diluted:
Explain what happens when you shine light on concentrated and dilute Red dye solutions. Why do you see the solution as Red?
E37B1-8