Experiment 9: Determination of Allura Red

Concentration in Mouthwash
GOAL AND OVERVIEW
The spectral profile of allura red, a dye in some commercial products, will be
measured using a Spec 20 spectrometer. From the spectral profile, the
wavelength of light absorbed most strongly by allura red will be determined. Using
the absorbance values obtained for a series of volumetric dilutions made from a
stock solution of known allura red concentration, a Beer's law plot of absorbance
versus concentration will be constructed. The Beer's law plot will be used to
determine the concentration of allura red in a sample of commercial mouthwash.

Objectives of the data analysis

 perform volumetric dilutions and understand the effect of dilution on number
of moles
 calculate absorbance from measured transmittance values
 understand how and why Beer's law is used
 graphically represent data and interpret the plots

SUGGESTED REVIEW AND EXTERNAL READING

 reference material on spectroscopy and dilution; textbook information on
spectroscopy

BACKGROUND
The study of the interaction of light with matter is called spectroscopy.
Spectroscopy had been a fundamental part in the development of chemical
molecular theory, and it can be used for both qualitative and quantitative analysis
of matter. Most modern chemistry, biology, geology, astronomy, and physics labs
use some type of spectroscopy. Light provides the foundation for spectroscopic
measurements; a few properties of light are reviewed below.

Recall that visible photons correspond

to a very small part of the electromagnetic
spectrum between 400 (violet) and 700
(red) nanometers (1 nm = 10-9 m). This lab
uses absorption of visible light to
Energy

measure the amount of a molecule present

in solution.
A
Absorption and emission strictly follow E
the law of conservation of energy. The
photon’s energy is transferred directly to
or from the molecule:
Ephoton = ∆ Emolecule Molecular Absorption
and Emission

1
 Molecular absorption and emission spectra are called band spectra because
they appear as broad peaks (“bands”). Molecules can absorb or emit photons
corresponding to the energy differences between vibrational states as well as
electronic states.

Molecular
Absorption Emission

ty
si
n
te
In

Atomic Emission

40 50 60 70 λ
0 0 0 0 (nm)

PHOTON ABSORPTION and BEER'S LAW

As it turns out, few molecules undergo emission processes that can be
measured spectroscopically. Absorption measurements are much more common.
Every molecule or atom absorbs some frequencies of light over a broad range of
the electromagnetic spectrum. Beer's law describes the relationship between the
number of photons absorbed and the number of absorbing molecules in a sample.

In a typical absorption experiment, a cell (transparent container) is filled with a

sample containing a certain amount of the molecule of interest. The cell is placed
into a spectrometer, which provides light of known wavelengths. The
spectrometer can be used in several ways, but the most common uses in this
course are:

i) to scan over a range of wavelengths to identify which wavelength is most

strongly absorbed by the molecule of interest (to find λ max); or,

ii) to quantify the number of absorbing molecules in the sample by measuring

how many photons are absorbed at a specific wavelength (usually λ max)
using Beer’s Law.

The following section describes the mathematical basis for Beer’s Law, with a
pigment acting as the absorbing molecule. The pigment is dissolved in a solvent,

2
and the solution is placed into the cell. The cell is placed in the sample holder in
the spectrometer.

pathlength In the spectrometer, incident light of intensity I0

is directed through the cell. As the light passes
through the cell, each pigment molecule has an
I0 I1 equal probability of absorbing a photon from the
light.
The light emerging from the far side of the cell
concentrati has a reduced intensity, I1, as shown in the figure.
on The fraction of the incident light that is transmitted
cell through the cell is I1/I0. This ratio is called the
transmittance and given the symbol T.
A given cell absorbs the same fraction of the incident light regardless of
incident light intensity. This means that the cell transmits the same fraction of the
incident light.

Suppose the light of intensity I1 emerges from the first cell and enters another
cell that is identical to the first one, as shown. The light that emerges from the
second cell has intensity I2.

I0 I1 I2 I1 I 2
(1a) T1 = = = T2
I 0 I1
2
I I I I 
(1b) TTotal = 2 = 1 ⋅ 2 =  1 
I1 I2 I 0 I 0 I1  I 0 

I0 I1

Beer's law asserts that the second cell transmits the same fraction of I1 as the
first cell did of I0 (Eq. 1a). The overall transmittance Ttotal is the product of the two
individual transmittances (Eq. 1b).
The same effect would be noted if a third (or fourth, fifth, …) cell were inserted:
the total transmittance is the product of the individual transmittances.
If a cell with pathlength b is used, its transmittance can be referenced to that of
a 1 cm pathlength cell with the same concentration of absorbing molecules. The
“cell number” is just the pathlength b in cm. In this more general case:
b
I I I I 
Ttotal = b = 1 ⋅  ⋅ b =  1 cm  = (T1 cm ) b
I0 I0 I b-1  I 0  (2)

Note that the pathlength b appears as a power. Think of it this way: if one, 2-cm
cell were used, it would be the same as using two, 1-cm cells.
This idea also explains the observed concentration dependence. If
concentration were doubled, it would be equivalent to having two identical cells in
the path of the incident light. Tripling the concentration would be equivalent to
three identical cells, and the transmittance would be cubed.
For a concentration c, as referenced to a 1 M solution:

3
 c
I I 
Ttotal = conc =  1 M  = (T1 M ) c
I0  I0  (3)

Note that the concentration c appears as a power.

This brings up a central ideal in Beer’s Law: transmittance depends on the
number of absorbing molecules in the path of the light. Transmittance will
decrease (more light absorbed) as the number of absorbing molecules in the path
of the light increases; it makes no difference whether those molecules are there
because the pathlength is longer or because the concentration is increased.

Combining Eqs. 2 and 3, using a 1-cm reference cell and a 1 M solution, total
transmittance becomes:
bc
I 
TTotal = (T1M, 1cm ) =  1M, 1cm 
bc
(4)
 I0 
Eq. 4 asserts that whatever fraction of light is transmitted in a 1 cm light path,
the square of that fraction will be transmitted in a 2 cm path, the cube in a 3 cm
path, etc. Also, whatever fraction of light is transmitted by a 1 molar solution, the
square of that fraction will be transmitted by a 2 molar solution, the cube by a 3
molar solution, etc.

The discussion of transmittance has only been defined on the number of

absorbing molecules, not their identity. To take into account how strongly a
particular molecule absorbs light of a given wavelength, the molar extinction
coefficient, ε , has been defined in terms of the fraction of light transmitted by a 1
ε
cm path containing a 1 molar solution of the molecule of interest, where T = 10- .

ε is specific to a molecule and to the wavelength where measurements are

made. A larger value for ε means that a molecule absorbs “better” at that
wavelength (less transmitted); ε = 0 means the molecule does not absorb that
wavelength. ε is usually determined experimentally by making a Beer’s Law plot
(discussed in later sections), but it can also be calculated using quantum
mechanics.
When the above description of the extinction coefficient is included in the
general description of transmittance (Eq.4), the following relationship between
measured transmittance, pathlength and solute concentration can be written as:
bc
I 
T =  1 cm, 1M (
 = 10 -ε ) bc
= 10 -εbc (5)
 I0 
where b is pathlength of the light through the cell in cm and c is the concentration
of the absorbing molecule in molarity (M = mol/L). Eq. 5 demonstrates the
exponential dependence observed in transmittance on the extinction coefficient ε ,
pathlength b, and concentration c, of the sample. Values of ε have been
measured for many substances and are listed in reference tables.

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 Values for ε are usually given at the particular wavelength of maximum
absorbance, λ max, for the substance. The molecule is most efficient at absorbing
at λ max, so the spectrophotometer is most sensitive for quantitative analysis at
λ max. Also, λ max represents the peak of the absorption curve, which is usually flat
right around its maximum. This means that if the wavelength of the light
generated by the spectrometer happens to fluctuate slightly during the
experiment, the absorption should not be greatly affected (and precision is not
compromised).

Spectrometers can often be read in either transmittance, T (or percent

transmittance, %T, which is 100% × T) or the absorbance, A. The relationship
between A and T is Beer’s Law:

A = − log ( T ) = − log (10 −εbc ) = εbc (6)

Absorbance, A, is the preferred unit for discussing the amount of light absorbed
by a sample because A changes linearly with concentration. If concentration of a
compound doubles, then its absorption doubles. By measuring changes in
absorption, you can qualitatively infer that concentration is changing. If you know
ε b, you can use absorption measurements to quantitatively calculate
concentration.
You will determine ε for two different molecules by preparing Beer's Law plots
for each substance. A Beer’s Law plot shows absorbance graphed versus
concentration (y vs. x). This should be a straight line with slope = ε b and y-
intercept = 0. Finding ε requires two steps:

i) the spectral profile (absorbance over a range of wavelengths) is collected to

determine λ max (the best wavelength to study the molecule; see above);
and,
ii) absorbance measurements are taken at λ max using several solutions of
known concentration to create a Beer’s Law plot. The slope divided by the
cell pathlength (measured in cm) is the experimentally-determined value for
ε .

You will use a Spec 20 UV-Visible Absorption spectrometer for three of your
experiments. Please read the general description of a spectrometer and the
specific description of the Spec 20's in the instrument usage section of the section
3B in the reference section.

Photon Observation:
When our eyes see color, it is the result of two scenarios:
i) the light comes from the emitting source directly; or,
ii) the photons have reflected from some molecules (an object).

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 The color of light that the brain interprets is
determined by the light’s wavelength. White R
light is a mixture of all visible wavelengths O RV
(400-700 nm). When white light shines on an
object that appears to be colored, the object is V
Y
absorbing some wavelengths and is
transmitting (or reflecting) the rest; it is this
“rest” that is observed. YG BV

If one color of light is mixed with light of its

"complementary color," the resulting light will G B
look white. A color wheel for light is used to BG
determine a color’s complement, where: R =
red, O = orange, Y = yellow, G = green, B = blue, V = violet.

For an object to be colored under white light, it must absorb the light of its
complementary color. For example, green leaves absorb red and violet, reflecting
green to our eyes.
This is slightly different from the color wheel for pigments used in art classes.

ALLURA RED, ABSORPTION SPECTROSCOPY, AND CHEMICAL ANALYSIS

Many food dyes that formerly were considered safe are today known to be
either toxic, carcinogenic (cause cancer), teratogenic (cause birth defects), or
cause heart trouble. In 1960, the Food and Drug Administration started requiring
all new dyes submitted for approval to be tested extensively.

Allura red, red dye #40, was approved for use in food in 1971 after Allied
Chemical Corporation, its manufacturer, subjected it to the most expensive and
thorough testing program that had ever been given to a food dye. The molar mass
of allura red is 496.42 g/mol.

PRELAB HOMEWORK (to be filled out in your bound lab notebook before you
perform the experiment)
Title and date
Define: (1) incident intensity, (2) transmittance, (3) molar extinction coefficient, (4)
λ max, (5) absorbance, (6) spectral profile, (7) aliquot
Answer:
1. What colors are absorbed by dark blue glass?
2. What colors are transmitted by dark blue glass?
3. In what range of wavelengths would you expect a red dye to be most effective
at absorbing radiation?
4. What is the mathematical relationship between transmittance and absorbance?
5. Give a verbal description of Beer's law.
6. What equation governs volumetric dilutions?
Procedure (Experimental Plan)
Data Tables

6
PROCEDURE
Part 1. Spectral Profile for Allura Red – Find λ max
1) Take about 40 mL of the stock solution of allura red
2) Record the concentration of the stock solution.
3) Put stock solution in the spectrometer cell.
4) Take its absorption spectrum by recording the percent transmittance (%T) over
the entire visible range (400 – 700 nm).
a. Near the absorbance maximum (transmittance minimum), use 10 nm
intervals.
b. Away from the minimum T range, use 20 nm intervals.

Part 2. Transmittance (and Absorbance) for Various Known Concentrations of Allura

Red
In this part, you will make a number of volumetric dilutions. Dilution equation :
To do this: n1 = n2
c1 ⋅ V1 = c 2 ⋅V2
i) Take a known volume, V1, of a solution of concentration
c1 ⋅ V1
c1 . c2 =
ii) Add pure solvent until the final total volume is V2. V2
iii) The concentration of the diluted solution is given by the
dilution equation, which is based on the fact that
the number of moles of solute does not change during the dilution.
iv) The ratio of volumes is the "dilution factor" which multiplies the original
concentration to determine the final concentration.
v) The number of moles of solute is constant during the dilution, because you
are adding pure solvent that has no solute in it.
vi) Initial and final volumes appear in the numerator and denominator of the
dilution equation, so their units will cancel out as these units are the same.
vii) The dilution equation works for concentrations in any units (most
importantly, mol/L), and the volumes can be measured in any consistent unit
(most importantly, mL).
viii) Use a volumetric pipet for measuring V1. This amount is called an aliquot
ix) Run the aliquot into a volumetric flask of the proper volume V 2 to yield the
final desired concentration, c2. Do not use graduated glassware such as a
graduated pipet or cylinder. They are not precise enough.
x) Use DI water for dilutions. Use a disposable pipet to make the volume come
precisely to the mark on the volumetric flask.

You will make a Beer's law plot of absorbance against concentration for a range
of concentrations. The concentration range needs to include the unknown
concentrations you plan to measure (the mouthwash). Four dilutions of a stock
solution of allura red, each of ½ the concentration of the one before it, will be
necessary. Each is made by a process called “diluting by a factor of two”. The
volume goes up by a factor of 2, so the concentration goes down to half of what it
was.

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1) Record the concentration of the stock solution of allura red. It is approximately
0.002% by mass:
(gallura red/gsolvent)×100%.
2) Use a 25 mL volumetric pipet and a 50 mL volumetric flask.
3) Make a set of four dilutions using the procedure outlined above.
4) The four dilutions, the stock solution, and a blank consisting of pure DI water
make six different known concentrations of allura red.
5) For each solution, measure the %T at λ max
6) Calculate the absorbance for each. Be sure you know why you make
measurements at λ max.
%T
A = −log ( T ) = εbc T= = 10 −εbc = 10 − A
100 %
Part 3. Determination of Allura Red (Red Dye #40) Concentration in Mouthwash
The concentration of allura red in the commercial mouth wash is too high to read
directly, so you will have to dilute it.
1) Obtain about 7 mL of the mouthwash. This is enough for three trials.
2) Make a 1 to 25 dilution using a 2 mL volumetric pipet and an appropriate
volumetric flask.
3) Gently transfer the solutions to minimize creating bubbles that can make it
difficult to observe the meniscus.
4) Measure the %T of the diluted sample.
5) Be sure to use the same Spec 20 throughout the entire experiment.

DATA ANALYSIS
Part 1. Determining λ max
1) Calculate the values of absorbance at each wavelength using transmittance
values between zero and one (%T between zero and a hundred); A = -log T.
Notice the relationship between absorbance and transmittance, how the one
changes with the other.
2) Make a plot of A vs. λ ; this is called the spectral profile
3) Determine the value of λ max. Does it make sense λ max would have this value,
given that you are looking at a red dye?

Part 2. Beer's Law Plot – determining ε Allura Red at λ max

1) The stock solution of allura red is approximately 0.002% by mass: (gallura
red/gsolvent)×100%.
2) Convert this from percent by mass to mol/L.
a. Convert percent by mass to mass fraction = mass of dye over mass of water
(divide by 100).
b. To convert to molarity, convert the numerator (mass of dye) to moles of dye
using molar mass. Convert the denominator (mass of water) to liters of
water using density of water and a unit conversion. This should give you
moles of allura red per liter of water.
3) Plot absorbance against concentration
4) Fit the best straight line to the data points.
With this Beer's law plot:

8
 i) given concentration, you should be able to find absorbance; or,
ii) given absorbance, you should be able to find concentration

An example plot is shown.

Absorbance vs. Concentration - Allura Red
1.200
5) Express your best-fit line
mathematically in the form y = 24943x
1.000
A = slope × concentration. Recall 2
R = 0.9995
A=(ε b)c. 0.800

Absorbance
6) Use your ruler to measure b, the
inner diameter (pathlength) of 0.600
your cuvette.
7) Calculate your experimental value 0.400

of ε , the extinction coefficient, for

0.200
allura red.
0.000
A = −log ( T ) = εbc 0.E+00 1.E-05 2.E-05 3.E-05 4.E-05

Molarity (mol/ L)
8) With this value, use the
absorbance equation (instead of
your Beer’s Law plot) to convert between A and c.

Part 3. Concentration of Allura Red in Mouthwash

1) Calculate the concentration of dye in the mouthwash. Take into account the
dilution and report the concentration of the original mouthwash (undiluted).
2) Average the three values obtained from your three trials.
3) Calculate the standard deviation.
4) Also calculate the average number of allura red molecules in one milliliter of
mouthwash.

REPORTING RESULTS
If a report is required in place of a lab summary
Abstract
Results
%T measurements for known concentrations
λ max and ε of allura red
%T of mouthwash dilution
Concentration of allura red in undiluted mouthwash
Sample Calculations:
Absorbance from transmittance
Dilution
Slope and extinction coefficient
[Allura red]cuvette and [Allura red]mouthwash
No. allura red molecules in a mL of mouthwash
Discussion/Conclusion

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 What you did, how you did it and what you determined.
Does λ max agree with observed color?
How was Beer's law used?
What were the final results?
Review Questions

REVIEW QUESTIONS
1. Around what wavelength would yellow-appearing dye have its maximum
absorption? What is the energy difference between the initial and final state of
the dye molecule when it absorbs light of this color?
2. If the extinction coefficient of a dye is 4.86×106 L/cm.mol, what is the
concentration of a solution of this dye if 78.4% of the light at the dye's λ max is
absorbed though a one-cm path of the solution?
3. Suppose you start with a solution of red dye #40 that is 0.0035% by mass. If
you do three successive volumetric dilutions pipetting 2 mL of solution and
diluting with water in a 25 mL volumetric flask, what is the molarity of the final
dilutions?
4. Why is it important to use water as a blank when setting 100%T?
5. Why is absorbance a preferred unit over transmittance?
6. What information is given by an extinction coefficient?
7. Does a molecule have the same extinction coefficient at all wavelengths?
Explain.
8. If you need to make a 1:5 dilution of something because your absorption
measurement was about 5 times too high, how would you do it? You have a
100 ml volumetric flask and the ability to transfer any volume of solution
between 1 to 50 ml very precisely.

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