Visible Spectrophotometry Analysis of a Food Dye in a Commercial

Visible Spectrophotometry
Analysis of a Food Dye in a Commercial
Beverage: Skill Building Lab
 2011, Sharmaine S. Cady
East Stroudsburg University
Skills to build:
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Using a volumetric flask
Using a micropipettor
Using a Vernier Spectrometer
Solving dilution problems
Preparing calibration curves
Visible Light and Spectrophotometry
When white light passes through a prism, it forms a continuous spectrum of
colors found in the visible region of the electromagnetic spectrum (Figure 1).
chemical species absorbs photons of visible light when the photon energies match
energy required to move the valence electrons to higher energy levels. The color of
compound is a blend of the wavelengths of visible light that are transmitted and
absorbed by the sample.
Figure 1. Continuous spectrum of visible light
the
A
the
the
not
Food Dye in Beverages
There are two ways by which color arises from absorption of visible light.
A
color wheel will aid in the explanation (Figure 2).
First, a species may transmit one
color and absorb all the rest. For example, if a chemical species absorbs all
wavelengths of visible light except red, the observed color of the solution is red.
Second, the compound may absorb several wavelengths of light and transmit the rest.
The color perceived will be a blend of the transmitted wavelengths. For example, if a
chemical species absorbs blue light, then the remaining red, orange, yellow, green,
indigo, and violet will recombine to give an orange color since orange lies opposite of
blue on the color wheel. This color is known as the complementary color. Copper(II)




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

Violet: 400 - 424 nm
Indigo: 424 - 430 nm
Blue: 430 - 491 nm
Green: 491 - 575 nm
Yellow: 575 - 585 nm
Orange: 585 - 647 nm
Red: 647 - 700 nm
Figure 2. Color wheel and
wavelengths of visible light
complexed with ammonia, [Cu(NH3)4]2+appears blue in solution (Figure 3) because it
absorbs red and orange light and transmits the remaining wavelengths that produce the
complementary blue-green color.
Figure 3. Visible absorption of [Cu(NH3)4]2+ in solution
(Source: http://www.chem.purdue.edu/gchelp/cchem/color.html)
Usually a species has a maximum absorption of light at a particular wavelength.
This maximum wavelength of absorption allows for measurement of the concentration of
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Food Dye in Beverages
a colored species in dilute solutions. Table 1 gives the observed complementary color
for the range in which a maximum wavelength falls.
For example, -carotene is a
carotenoid found in plant pigments. It absorbs light at 453 and 483 nm. From the table,
the expected complementary color is yellow-orange.
Table 1. Maximum Wavelength and Their Complementary Colors
Beer-Lambert Law
To determine the concentration of a colored species in solution, the BeerLambert Law is applied to the spectral data obtained. The Beer-Lambert Law states that
the concentration of a colored species is directly proportional to its absorbance at a
given wavelength. This can be seen from the mathematical representation of the law
given below, where A is the absorbance, is a constant known as the molar absorptivity
or extinction coefficient, b is the cell path length in cm, and c is the concentration of the
colored species. This equation gives a straight-line graphical plot (y = mx + b) at
concentrations below 0.10 M,
A   bc
where y = A, the slope m = b, and x = c. In a Beer-Lambert plot, the y-intercept b = 0.
Figure 3 shows a typical Beer-Lambert plot with the equation for the straight line shown.
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Food Dye in Beverages
Absorbance
Absorbance vs. Fe(II) Concentration
0.900
0.800
0.700
0.600
0.500
0.400
0.300
0.200
0.100
0.000
0.00E+00
y = 11006x
2.00E-05
4.00E-05
6.00E-05
8.00E-05
Concentration (M)
Figure 3. Graphical representation of Beer-Lambert Law
The above plot represents a calibration curve used to determine an unknown
concentration. A calibration curve is prepared by plotting the absorbance of standard
solutions of known concentration vs. concentration as shown in Figure 3. The equation
for the straight line is shown. The absorbance value for an unknown is substituted for y
in the equation, and the value of x is determined. For example, an unknown solution
with an absorbance of 0.680 has a concentration of 6.18 x 10-5 M.
Food Dyes
The Food and Drug Administration (FDA) began its regulatory functions in 1906
with the passage of the Federal Food and Drugs Act. The FDA is responsible for the
regulation of all color additives used in the United States since the passage of the
Federal Food, Drug, & Cosmetic (FD&C) Act of 1938, which made food color certification
mandatory. There are nine certified food color additives that have received FDA
approval at the present time, seven of which have no restrictions as to use (Table 1).
Color additives are dyes, pigments, or other substances that impart color when
added or applied to food, drugs, cosmetics, or the human body. Color additives may be
classified as a dye or lake. Dyes are water-soluble and are manufactured as powders,
granules, liquids, or other special purpose forms. Dyes are used in beverages, dairy
products, pet foods, and other various consumer products. Lakes are water-insoluble
and more stable than dyes. They are often used to color products that have high fat or
oil content or lack sufficient moisture to dissolve dyes, such as coated drug tablets, cake
and donut mixes, hard candies, and chewing gum.
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Food Dye in Beverages
Table 1. Certified Food Color Additives
Name
Color
Common Food Use
FD&C Blue No. 1
Brilliant Blue FCF
Bright blue
FD&C Blue No. 2
Indigotine
Royal blue
FD&C Green No. 3
Fast Green FCF
Sea green
FD&C Red No. 40
Allura Red AC
Orange-red
FD&C Red No. 3
Erythrosine
Cherry-red
FD&C Yellow No. 5
Tartrazine
Lemon yellow
FD&C Yellow No. 6
Sunset Yellow
Orange
Beverages, icings, jellies,
condiments, extracts,
confections
Ice cream, snack foods,
confections, cereals, baked
goods
Beverages, pudding, ice
cream, confections, baked
goods
Beverages, gelatins,
pudding, confections,
condiments
Fruit cocktail cherries,
baked goods, snack foods,
confections
Beverages, ice cream,
custard, cereals,
confection, preserves
Beverages, snack foods,
cereals, ice cream, baked
goods, confections
Note the number of conjugated double bonds in the molecular structures (FD&C
Blue No. 1 (1), FD&C Blue No. 2 (2), FD&C Green No. 3 (3), FD&C Red No. 40 (4),
FD&C Red No. 3 (5), FD&CYellow No. 5 (6), and FD&C Yellow No. 6 (7)) that can
establish a set of bonding and anti-bonding molecular orbitals that allow electronic
transitions from the ground to the excited state through the absorption of visible light.
_
SO3 Na
+
O
+
H
N
_
Na SO3
N
N
H
_
SO3 Na
+
O
2
_
SO3 Na
_
SO3
+
+
N
+_
Na O3S
N
HO
+
_
Na SO3
1
+
N
3
_
SO3
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Food Dye in Beverages
I
OCH3
I
+_
O
Na O
O
HO
+_
Na O3S
N
I
N
I
_
+
COO Na
H3C
_
+
SO3 Na
4
5
+_
HO
Na OOC
+_
Na O3S
+_
N
Na O3S
N
N
N
N
N
H3C
HO
_
+
SO3 Na
6
_
+
SO3 Na
7
Determination of Food Dye Concentration in a Beverage
To determine the dye concentration, standard solutions of known dye concentration are
prepared to obtain data to plot a calibration curve. A stock solution of dye is used to
prepare five standard solutions whose concentrations are determined using dilution
calculations. (See supplement under the experiment listing at the web site.) Then the
absorbances of the five standard solutions are determined, and a Beer-Lambert plot is
obtained as a calibration curve. This calibration curve is used to determine the
concentration of the dye in the beverage.
In this experiment, you will determine the concentration of FD&C Blue No. 1 or
FD&C Red No. 40 in a beverage. FD&C Blue No. 1 and FD&C Red No. 40 have
maximum absorbances at 629 nm and 503 nm, respectively. The measured absorbance
for the beverage will be used to determine the dye concentration.
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Food Dye in Beverages
Experimental Methods and Materials
Safety considerations
Wear suitable protective clothing, gloves, and eye/face protection!
You should read the online MSDS for:
FD&C Blue No.1
Preparation of Standard Solutions
With a micropipettor, place 125 L of the stock FD&C Blue No. 1 solution or
FD&C Red No. 40 solution into a 100.00-mL volumetric flask. Record the
concentration of the dye in the stock solution. Add distilled water to the mark on the
flask. Invert 10 times to insure thorough mixing. In a similar fashion, prepare four more
standard solutions with the values given in the table below. Determine the concentration
of dye in each solution.
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Food Dye in Beverages
Table 2. Data for Standard Dye Solutions
Standard Solution
Volume Stock Solution, L
1
120.00
2
250.00
3
500.00
4
750.00
5
1000.00
Concentration, M
Calibrating the Vernier Spectrometer
Click on Logger Pro 3.5 from the Programs list on the computer. Click on
Experiment  Calibrate  Spectrometer on the menu bar. Allow the spectrometer
to warm up for 3 minutes. Place a cuvette ¾ full with distilled water in the cuvette
holder. Click Finish Calibration. Once the OK button is ready, click it
Collecting Data for the Calibration Curve
Fill a cuvette ¾ full with Solution 5. Click on the Configure Spectrometer Data
Collection icon
on the right-hand side of the menu bar.
Click Abs. vs.
Concentration under Set Collection Mode. Click the OK button to close the dialog
box.Click Experiment Start Collection and then Keep. Enter the concentration of
the solution into the dialog box. Check that the wavelength for absorbance readings is
correct for the color dye you are sampling. If so, repeat Start Collection and then Keep
with Solutions 4, 3, 2, and 1. If not, repeat the calibrationuntil the correct wavelength is
showing.
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Food Dye in Beverages
Beer-Lambert Plot
Click the Linear Fit icon on the menu bar. Is the correlation factor acceptable
(close to one)? If not, prepare a new set of standard solutions. Record the wavelength
for which the data set was collected.
Determination of Dye Concentration in Beverage
Record the name of the beverage and the dye present based on the label
information. Fill a cuvette ¾ full with the beverage. Click on AnalyzeInterpolation
Calculator on the menu bar. Click the OK button. Record the absorbance and
concentration. Print the screen.
Laboratory Report
Answer the following questions as part of your conclusions.
1. Did the standard concentrations and absorbances provide an acceptable linear
calibration curve? Discuss the correlation value for your curve. The closer the
correlation value is to 1, the more reliable the experimental data and the more
the line representsan accurate relationship between absorbance and the
concentration. When the value = 1, all experimental data points are on the line.
See Figure 3.
2. What is the class average concentration for the beverage dye? Do any values
seem unreasonable?
3. Calculate the m/v% dye in the solution based on the class average. Is the
concentration of the dye within the OSHA standard (see MSDS) for a
nonhazardous solution?
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Food Dye in Beverages
References
Reusch, W. Visible and Ultraviolet Spectroscopy.
http://www.cem.msu.edu/~reusch/VirtualText/Spectrpy/UV-Vis/spectrum.htm (accessed
September 2005)
U. S. Food and Drug Administration. Food Color
Facts.http://www.cfsan.fda.gov/~Ird/colorfac.html (accessed January 24, 2007)
Sigmann, S. B.; Wheeler, D. E. The Quantitative Determination of Food Dyes in
Powdered Drink Mixes.J. Chem. Ed.2004, 81, 1475-1478.
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