Absorption Spectroscopy

“It would be nice if the Food and Drug Administration stopped
issuing warnings about toxic substances and just gave me the
names of one or two things still safe to eat.”
—
Robert Martin Fuoss
Author and Editor
Food safety
is a continuing public concern. In the United States, the food nearly everyone eats has been
processed to some degree. During the processing, chemicals are often added to improve food safety by
retarding spoilage and preventing the growth of unwanted organ
isms. Chemicals also make our food more
appetizing by adding color and aroma. But some of these chemicals may also have a dark side, and this is
the primary source of our concern.
The danger posed by any chemical depends on the amount. Chemicals that are
dangerous in large
amounts can be quite beneficial in small doses, something also true of most drugs. Measuring the
amounts of chemicals present in food and drugs is of critical importance. And the most common way this
is done is using absorption spectros
copy.
Educational Objectives:
A student who has successfully completed this experiment will be able to
collect absorbance measurements from a simple spectrophotometer,
perform a serial or parallel dilution to prepare standards,
perform a Beer’s law analys
is to determine a solution’s concentration, and
determine a percent composition.
Experimental Objectives
:
A student who performs this experiment is asked to
prepare a series of standards from a known solution using a serial or parallel dilution,
prepare a
n unknown sample,
measure absorbance of all samples,
determine molar absorptivity of a food dye, and
determine percent composition of the food dye in a food sample.
Background
Chemical reactions are governed by the number of molecules involved in the reac
tion. Chemical
equations are written to reflect the number of molecules involved. But atoms and molecules are so small,
they can’t be counted directly
—
other means must be employed. A critical aspect of chemical
experimentation is the determination of the n
umbers of molecules involved in a chemical reaction.
The concepts of the mole, atomic mass and molar mass were developed to address this problem. These
concepts can be employed to convert mass measurements into numbers of molecules. Combined with
density,
it is possible to use both volume and mass measurements to “count” molecules.
But mass and volume are indirect measures of the number of atoms and molecules. Spectroscopy provides
a way to make direct measurements. This is because the absorption of light
is a molecular phenomenon.
The amount of light absorbed is directly proportional to the number of atoms or molecules, making
spectroscopy a chemist’s most valuable tool. It is by far the quickest, easiest and cheapest method
available for the direct deter
mination of numbers of atoms and molecules. As a result, it is widely
employed in labs in many disciplines.
Absorption spectroscopy is discussed in more detail in chapter 8 of the technique book. This material
should be reviewed before continuing. Pay par
ticular attention to the material on absorption spectroscopy
in chapter 8
–
6
.
The Beer
–
Lambert Law
The data obtained from absorption spectroscopy is most often evaluated using the Beer
–
Lambert Law.
This is how absorption measurements are converted into co
ncentration values.
You will need to be
familiar with the law and its application. This is discussed in detail in section 10
–
7 of the lab book. Pay
particular attention to the material on
molar absorptivity
.
Chemicals
In common usage, the term “chemica
l” tends to have a negative connotation. This is because of the
dangers posed by certain chemicals. The reality is that every chemical can be dangerous under the right
conditions. Oxygen, which is essential for life, is the demon chemical that antioxidants
combat. Excess
exposure to water causes more deaths than any other chemical. We even have a name for death from
overexposure to water: drowning.
When considering the dangers of chemicals, we must consider the amounts. One common way to express
the dange
r of any chemical is with its LD50: the amount of that chemical that will cause death in 50% of
a population. The higher the LD50 value the safer the chemical is. Extremely large LD50 values indicate
a chemical that is safer than breathing. It is our desir
e as consumers that food additives fall into this
category.
Food Dyes
The coloring of food to make it more appetizing has been going on
for at least 3500 years. Yellow pigments were being added to butter
as early as 1300 A.D. because people expect butter
to be yellow.
Originally, all food colorants were natural products. For example,
Romans were known to use saffron to impart a yellow color.
In the 19th century, with the dawn of the chemical industry, many
new synthetic compounds became available, some o
f which were
used to color foods. These compounds included many things we
now know to be toxic. For example, licorice and hard candy were
commonly colored with arsenic compounds.
By 1900, some 80 compounds were being used to color foods. Concerns about t
heir safety led Congress
to create the Food and Drug Administration (FDA), which was charged with assessing and certifying the
safety of chemicals added to foods and used as drugs. Today only nine chemical dyes are certified by the
FDA as being safe for us
e in foods and only seven are currently used in processed foods.
Compounds that have been thoroughly tested by the FDA’s Department of Food, Drugs & Cosmetics
(FD&C) are given a number, which has become the common way to identify these dyes. The seven
com
mon food dyes are all relatively small organic molecules that strongly absorb light in the visible
region and have pure colors. These are given in Table 1. It is interesting to note that, of these seven, only
three (allura red, tartrazine and brilliant blu
e) account for most of the colorings used in food products.
Figure 1.
Nutritional content label.
The Problem
The problem posed for this experiment is to
determine the level of risk posed to the consumer by the
amount of
dye present in a commercial food
product
. The determination will be a
ccomplished using a
Beer’s Law analysis. A serial or parallel dilution of a solution of the pure dye will produce a series of
solutions of known concentration. The absorbance of these solutions will be measured and a calibration
plot generated. This plot c
an then be used to convert absorbance measurement of sample solutions into
molar concentrations. This in turn can be converted into grams, which will ultimately be related back to
the original weight of the sample and the dye’s LD50.
Procedure
1.
Your Ass
ignment
You are to determine the amount of
one
dye present in
one
food sample. Consultation is
encouraged, but ultimately you are responsible for your sample and dye. If your sample has more
than one dye, then you may work with a partner: you evaluate one
dye and your partner evaluates
the other. Begin by identifying the dye, the sample and your partner, if appropriate.
2.
Sample Selection
You will need to provide your own sample to evaluate.
Make sure you bring one with you to class.
When
selecting a samp
le keep the following in mind.
•
The sample must contain one or
more of the dyes
listed in table 1. This can be determined by
referencing the nutritional content label on the
package (figure
1
).
•
The dyes must NOT be identified as “lakes.” Lake dyes
do no
t dissolve in water.
•
If a solid, the sample must freely dissolve in water to
produce a transparent solution.
•
Carbonated beverages need to “go flat” (be de
–
carbonated) before they can be used. This must be
done prior to class.
Suitable samples are any
transparent drink, powdered
drink mixes and certain medications. Solid samples such
as hard candies are also suitable if the dye can be easily
dissolved.
Table 1.
Known food dyes available for analysis.
Common Name
FD&C Name
Formula
Molecular
Weight
LD50*
Erythrosin B
Red Dye #3
Na
2
C
20
H
6
O
5
I
4
879.42 g/mol
6
–
10
Allura Red
Red Dye #40
Na
2
C
18
H
14
N
2
O
8
S
2
496.42 g/mol
6
–
10
Tartrazine
Yellow Dye #5
Na
3
C
16
H
9
N
4
O
9
S
2
534.39 g/mol
6
–
10
Sunset Yellow
Yellow Dye #6
Na
2
C
16
H
10
N
2
O
7
S
2
452.37 g/mol
6
–
10
Fast Green FC
F
Green Dye #3
Na
2
C
37
H
34
N
2
O
10
S
3
808.85 g/mol
2
Brilliant Blue FCF
Blue Dye #1
Na
2
C
37
H
34
N
2
O
9
S
3
792.86 g/mol
6
–
10
Indigo Carmine
Blue Dye #2
Na
2
C
16
H
8
N
4
O
9
S
2
510.38 g/mol
6
–
10
* Numbers are grams/kg body weight.
3.
Stock Dye Solution
You will need a dye sol
ution of known concentration for your Beer’s Law analysis. A stock solution
having an absorbance value of about 1.0 at the wavelength of maximal absorbance (
?
max
) is
provided. Plan to o
btain about 20 mL of the stock solution of your dye in a labeled container
to use
during the course of the experiment
.
4
.
Preparing Standards
For each dye, a series of solutions of known concentrations must be prepared for the B
eer’s Law
analysis. You can prepare them from the stock solution by performing either a serial or a parallel
dilution. This is discussed in chapter
11
of the technique book.
The number and concentrations of solutions to be made is to be decided by you in c
ollaboration
with your classmates. You are to come up with a plan as part of your pre
–
lab assignment. The plan
will be refined during the pre
–
lab activities the day of the lab. Since you only need enough of each
solution to make an absorbance measurement,
10 mL will be plenty. 6? test tubes are very
convenient for holding these solutions.
For measuring volumes you will be allowed to check out a 10.00 mL graduated pipet and pipet bulb.
If you are not familiar with this device here is a short video demonstr
ating the operation of a
volumetric pipet, a close relative of the pipet available for your use.

5
.
Preparing Your Sample
In order to determine how much of each dye is in your food sa
mple, a solution of the sample must
be made up quantitatively. This means that you will need to know exactly how much of the sample
(the weight or volume) was used and what the final solution volume is. One problem to avoid is a
solution that is too concen
trated. If the color is too dark, the absorbance will be difficult to read
NOTE: Using Cuvets
To avoid dilution
error, make sure you
rinse the cuvet with
so
me of the solution
before filling it. Cuvets
should be filled to
between 2/3 and 3/4.
Note
If the absorbance of
your sample solution
is greater than 1.0, it
is too concentrated.
You will need to
dilute it.
accurately. The ideal solution should have a lower color intensity than the stock dye solution.
How you prepare the solution will depend on the nature of the samples.
For liquids,
some dilution of the sample will probably be required. Remember that this will need to
be quantitative. To determine how much to dilute the sample, take 1 mL and compare the color
intensity to 1 mL of the stock dye solution. (The comparison is best done in
test tubes.) If the
sample is too intense, dilute with water until the intensity is less than the stock dye solution.
Measure the new sample volume. You will now know how much to dilute your sample.
For solids, such as Kool
–
Aid, massing by difference is t
he preferred technique.
a.
Put exactly 10.00 mL of distilled water into a graduated cylinder.
b.
Accurately mass a small amount (0.1 g should suffice) of the powdered sample on weighing
paper.
c.
Quantitatively transfer part of this massed sample into
the graduated cylinder. Mix and
compare the color intensity to the stock dye solution. If the color is more intense, add more
water. If it is weak, add some more of the massed sample. Continue until the color is intense, but
not as intense as the stock dye
solution. When you are satisfied with your sample, re
–
mass the
remaining powdered sample and then determine the mass of sample added to the graduated
cylinder. Also measure the final volume of the solution. Make sure to record these values.
d.
Store this
solution in a properly labeled container.
6
.
Initial Measurements
Prepare the spectrophotometer for absorbance measur
ements as described in chapter 8
–
6
of the
technique book.
A
detailed set of instructions can be found here.
A short vi
deo presentation
can be found here.

Rinse and then fill the sa
mple cuvet with the stock dye solution.
Put it in the cell holder and adjust the spectrophotometer to
produce a good spectrum.
Identify
the
?
max
. Use the cursor to determine the wavelength.
Record this value in your notebook. You may also want to save
the spectrum data file.
7
.
Confirming the Presence of the Food Dye
in your Sample
Empty the sample cuvet and then refill it with your sample
s
olution (remember to rinse the cuvet with a small amount).
Locate all the ?
max
positions and confirm that one corresponds to your known food dye. Consult
your instructor if there are any problems.
8
.
Estimating [Dye]
sample
Before making any measurement
s, compare the color
intensity of your sample solution to the standard solutions
you have prepared. Identify which two solutions “bracket”
your sample and which appears to be closest in appearance
to your sample. Record this information in your notebook.
W
hen you have completed calculating the concentration of
dye in your samples, you can compare the results to these visual observations. If they are not
consistent, you should look for problems in your calculations.
Figure 2
.
Estimating [sample].
9
.
Beer’s
Law Plot
If your measurements do not produce a linear plot, then the Beer’s Law analysis will not be valid. It
is useful to plot absorbance vs. concentration for the known dye solutions as the measurements are
made. Any deviations from linearity should be
questioned and addressed while you are still in lab.
Set up a blank graph of concentration vs. absorbance in your notebook.
Measure the absorbance of all your known dye standards at
?
max
. As you go, plot the absorbance
values against concentration. Any deviations from linearity should be investigated. It may be
necessary to remake questionable solutions. It may also be necessary to make more solutions to
ensure enough data points on
the plot.
Measure the absorbance values for your sample at the same ?
max
value.
Data Analysis
and the Worksheet
The objective of this analysis requires you to determine the weight percent of the original sample that is
dye molecules. The following is an outline of the ope
rations you’ll need to perform to obtain your answer.
You will be reporting your results in a worksheet you will complete in lab. Here
’
s the link to the
worksheet
.

http://slb.faculty.arizona.edu/sites/slb.faculty.arizona.edu/files/151/Absorption%20Spectroscopy%20Lab

%20Worksheet.pdf
1.
Using
the graph paper on the worksheet
, draw the best straight line yo
u can through the data points.
2.
Interpolate the [dye] from this plot as described above
3.
Use the total sample volume and its concentration to determine the moles of dye present in your
sample.
4.
Use the dye’s molecular weight to convert moles to g
rams.
5.
Compare the grams of dye in the original solid sample to the total weight of the sample to get a
percent composition.
An Alternative Way to Determine
the [Dye] in Yo
ur Sample
If you use Excel® or some other spreadsheet program to construct your plot,
you will not be able to interpolate your [dye] from the plot. In this case, you will
have to have the program calculate the slope for you. If the data is plotted
correctl
y, the slope will be equal to
?
l in the Beer’s Law equation. The slope and
the absorbance of your sample solution can then be used to calculate [dye].

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