# CHEM 111 Suffolk University Chemistry Lab Report

Lab 5: Spectrophotometry of Food ColoringCite this procedure as:
Pavlac, M. M. Suffolk University, Boston, MA. Spectrophotometry of Food Coloring, 2020.
Introduction:
One of the most common tasks for a chemist is quantifying how much of a particular
substance is in a sample. For example, an environmental chemist might want to know the
concentration of phosphate in a lake. A forensic chemist might investigate how much arsenic is
in wine. Or a clinical biochemist could determine the amount of aspirin in blood. The
experimental techniques used by these chemists vary. However, they all involve experimental
measurements that indicate the concentration of the analyte, the substance being investigated.
In this experiment, you will be playing the role of a food chemist. Your goal is to
determine the concentration of food coloring in an unknown solution. The units for
concentration will be in parts per million (ppm). This unit compares the amount of solute
(substance being dissolved) to the amount of solution (the entire mixture). For example, lake
water that is 1 ppm phosphate contains 1 gram of phosphate for every million (106) grams of lake
water. The same relationship can also be used for volumes. A beverage that is 1 ppm red food
coloring contains 1 mL of red food coloring for every million mL of beverage.1
The food coloring concentration will be determined by measuring the absorbance of the
sample using spectrophotometry (as described below). In order to evaluate an unknown, you
will first need to investigate the mathematical relationship between absorbance and
concentration. This involves creating a standard curve, a graph that features the detector
response (in this case absorbance) on the y-axis and concentration of the analyte on the x-axis.2
A standard curve is constructed by analyzing a series of standards with known concentrations.
You will be making four standard solutions for your assigned food coloring. This will
involve diluting a provided stock solution. The amount of stock necessary to make each standard
can be calculated using the dilution equation:
C1V1 = C2V2
[1]
In Equation 1, “C1” is the concentration of the stock, and “V1” is the volume of the stock that
needs to be diluted. On the other side, “C2” is the desired concentration, and “V2” is the total
volume of the standard solution. Once you have made your standard solutions, you will measure
their absorbance using a spectrophotometer and construct your standard curve.
Method Details:
Spectrophotometry encompasses any method that uses light to determine the chemical
concentration of a sample. All spectrophotomers have the same basic set-up. A light source
inside the instrument provides the radiation that is necessary for the analysis. This radiation
travels through a grating device called a monochromator, which allows for the selection of
wavelength. A sample (inside a transparent cuvette) is inserted into the spectrophotometer and
illuminated with the incident light. A detector on the opposite side then measures how much of
the light is transmitted through the sample.2
The absorbance of a sample is defined with the equation below:
A = -log (I/I0)
[2]
In Equation 2, “I0” represents the intensity of the light illuminating the sample. This value is
determined by calibrating the instrument with a blank. The “I” is the intensity of the light that
travels through the sample and hits the detector.1 The spectrophotometer evaluates the
relationship between the two intensities and then digitally converts the results into an absorbance
measurement. Because of the ratio, these absorbance measurements are unitless.
The absorbance of a sample is linked to its concentration through Beer’s Law:
A = 𝜀bc
[3]
In Equation 3, “A” is the absorbance of the sample, and “c” is the concentration. The other two
variables include the molar absorptivity (𝜀), which indicates how much light is absorbed by a
substance at a certain wavelength. The pathlength (b) is determined by the size of the cuvette.
In most applications, both the molar absorptivity and the pathlength can be assumed to be
constant. Beer’s Law therefore illustrates how absorbance and concentration are directly
proportional.2 This relationship will form the basis of the linear standard curve used in this
experiment.
Procedure3
Formation of Groups and Planning the Experiment
You will work in groups of three. Each group needs to analyze three food colors in total
(red, blue, and yellow). Determine which group member will be responsible for which primary
color. You will then come together at the end of the experiment to analyze secondary color
mixtures. The group will be provided with one SpectroVis Plus spectrophotometer. The
SpectroVis Plus is attached to a LabQuest sensor. Read through the provided directions on how
to set up the spectrophotometer and measure the absorbance of samples using the LabQuest.
Follow the directions to calibrate the spectrophotometer using a cuvette filled with deionized
water.
Part 1: Making the Standard Dilutions for a Primary Color (Individual)
A stock solution (2000 ppm) is provided for each primary color. This stock will need to
be diluted to construct a series of standard solutions. You want to make four solutions with the
following concentrations: 200 ppm, 400 ppm, 600 ppm, and 800 ppm. Use the dilution equation
[1] to determine what volume (mL) of stock is necessary to make 10 mL of each of these
Obtain four 10-mL volumetric flasks, and label them with the standard concentrations.
Make the first standard by adding a small amount of deionized water to the first flask. Then add
in the correct volume of stock using an appropriate volumetric instrument. Swirl to mix. Fill the
volumetric flask to the line with deionized water, cap the flask, and invert several times to mix.
Repeat this dilution process to make the remaining standard solutions. Pipet a small amount of
each standard into its own cuvette.
Part 2: Constructing a Standard Curve for a Primary Color (Individual)
Set the spectrophotometer to read a “Full Spectrum.” Insert the cuvette with your most
concentrated standard (800 ppm) into the cuvette holder. Measure a complete spectrum for the
sample, and then determine the wavelength of maximum absorbance. Record this wavelength in
nm. Then change the mode of the spectrophotometer to “Events with Entry.” Set the
wavelength to the wavelength of maximum absorbance for your primary color. At that
wavelength, measure the absorbance of all your standard samples, as well as the blank.
Construct a standard curve that compares absorbance on the y-axis to sample concentration in
ppm on the x-axis.
Part 3: Analysis of a Primary Color Unknown (Individual)
Obtain the unknown sample for your primary color. Pipet a small amount of the
unknown into a cuvette. Make sure the spectrophotometer is set to “Events with Entry” mode
and the appropriate wavelength for your color. Measure the absorbance of the unknown sample.
Then use the linear trendline equation from your standard curve to calculate the concentration of
the unknown in ppm.
Part 4: Analysis of Color Mixtures (Group)
Create the three secondary colors by mixing together the primary colors. The solution for
each secondary color should be made by combining equal volumes of two primary color
standards (800 ppm). The total volume of each mixture should be at least 3 mL in order to have
enough to fill a cuvette. Set the spectrophotometer to read a “Full Spectrum.” Read the
spectrum for each secondary color solution. Note down all wavelength peaks and the associated
absorbance values.
References
1. Kenkel, J. Analytical Chemistry for Technicians; CRC Press: Boca Raton, 2003; pp. 193.
2. Harris, D. Quantitative Chemical Analysis; W. H. Freeman: New York, 2016.
3. Wink, D.; Fetzer-Gislason, S.; Kuehn, J. Working with Chemistry: A Laboratory Inquiry
Program; W. H. Freeman: New York, 2005; pp. D3-D7.
LAB 5: SPECTROPHOTOMETRY
OF FOOD COLORING
EDUCATIONAL OBJECTIVES
• Become familiar with the operation of a
spectrophotometer.
• Investigate Beer’s Law and the relationship
between absorbance and concentration.
• Learn how to construct and apply a standard
curve.
VISIBLE LIGHT
Wavelength
(nanometers)
Color
Absorbed
Color
Observed
< 400 nm Ultraviolet Colorless 400–450 nm Violet Yellow 450–500 nm Blue Orange 500–550 nm Green Red 550–580 nm Yellow Purple 580–650 nm Orange Blue 650–700 nm Red Green Infrared Colorless > 700 nm
Range:
400 – 750 nm
The colors are
differentiated
according to
wavelength.
OBSERVED COLOR
• Complementary colors:
opposites on the color wheel
• Colored substances
absorb complementary
color
• Example: Chlorophyll
(green) absorbs red light
SPECTROPHOTOMETRY
• Measurement technique based on interaction of
radiation with a sample
• Options:
• Absorbance
• Transmittance
• Reflection
• Different applications:
• Qualitative – detecting presence of a substance
• Quantitative – determining amount of a substance
HOW SPECTROPHOTOMETERS WORK
Comparison of:
• Intensity of light passing through
sample (It)
• Intensity of light passing through
“blank” (I0)
TRANSMITTANCE AND
ABSORBANCE
• % Transmittance: Amount of
light passing through a sample
%T = (It / I0) x 100
• Absorbance: Amount of light
NOT passing through a sample
• Calculated from %Transmittance
A = -log(%T/100)
BEER’S LAW
Absorbance proportional to concentration:
A = ebc
A = Absorbance
e = molar absorptivity of a substance in L/(mol•cm)
Note: This is a constant at a given wavelength.
b = light path length of spectrophotometer
Note: Pathlength is 1 cm for standard cuvettes.
c = concentration of sample
STANDARD CURVE
• Created using a series of standard solutions with
known concentrations
Absorbance
• Graph of absorbance (y) vs. concentration (x)
y= mx + b
R2 = #
Concentration (appropriate units)
EXPERIMENT SET-UP
• Working in groups of 3
• Each assigned a dye color
• Individually:

Make standard solutions
Determine maximum wavelength
Create standard curve
Calculate concentration of unknown solution
• As a Group:
• Make color mixtures and read on spectrophotometer.
PART 1: STANDARD SOLUTIONS
• Want four standard solutions
• Total volume for each: 10 mL
• Standard Concentrations: 200, 400, 600, 800 ppm
• Prepared from stock solution
• Stock Concentration: 2000 ppm
• Need to calculate how much stock for each
• Dilution equation!
DILUTION EQUATION
• Involves:
• Concentration of solutions (in any consistent units)
• Volume of solutions (in any consistent units)
• The Equation:
C1V1 = C2V2
• Where:
C1 = concentration of stock solution
V1 = dispensed volume of stock solution
C2 = concentration of dilution
V2 = total volume of dilution
DILUTION EXAMPLE
A student wants to make 250 mL of solution with a
concentration of 800 ppm. He has a stock solution
with a concentration of 4000 ppm. How much stock
must the student dilute to 250 mL to get the desired
concentration?
C1 = 4000 ppm
V1 = ?
C2 = 800 ppm
V2 = 250 mL
C1V1 = C2V2
(4000 ppm)(V1) = (800 ppm)(250 mL)
V1 = 50 mL
MAKING THE DILUTIONS
• Use 10-mL volumetric flasks

Pipet in correct volume of stock
Swirl to mix
Fill to line with DI water
Stopper and invert carefully to mix
• Pipet each into a cuvette
DETERMINING λmax
• Where substance has maximum absorbance
• Found by reading absorbance over range
• Aka collecting a spectrum
• Absorbance measurements at λmax more
sensitive to changes in concentration
USING THE SPEC
• Calibrate at beginning
• With deionized water
• Set to correct mode:
• “Full spectrum” to read at multiple λ
• “Events with entry” to read samples at specific λ
• General Notes:
• Fill cuvettes 3/4 full (to line)
• Wipe with Kimwipe
• Insert with arrow facing arrow
PART 2: MAKING THE STANDARD
CURVE
• Determine wavelength of maximum absorbance
• With “Full Spectrum”
• Darkest sample (800 ppm)
• Measure standards at maximum wavelength
• With ”Events with Entry”
• Set spec to read at specific wavelength (from spectrum)
• Insert samples and record absorbance
• Also read blank sample
GRAPHING DATA
Concentration
(ppm)
Absorbance
0
0.000
200
0.118
400
0.244
600
0.363
800
0.486
Independent
Variable:
Concentration
x axis
Dependent
Variable:
Absorbance
y axis
Absorbance should have a
LINEAR dependence on
concentration according to
Beer’s law.
PART 3: THE UNKNOWN
SAMPLE
• Obtain “unknown” solution
• Measure A at own max wavelength
• Use trendline equation:
• Plug in Absorbance for y
• Solve for x
Example:
Unknown red solution with an
absorbance of 0.200.
(0.200 − −1.200 x 10)* )
6.085x 10)/
x = 331 ppm
EVALUATION OF ACCURACY
• Compare results to actual concentration
• Calculate the %Error:
(experimental value − actual value)
% Error =
x 100
actual value
Note: %Error less than 10% is reasonably accurate!
PART 4: MIXTURES
• How do mixtures compare to three primary colors?
• Prepare 3 mixed solutions:
• red (800 ppm) and blue (800 ppm)
• red (800 ppm) and yellow (800 ppm)
• blue (800 ppm) and yellow (800 ppm)
• Read spectra and record max wavelengths
• NOTE: two max wavelengths/absorbances
2020/2/24
Take Test: Prelab: Spectrophotometry of Dyes – …
Welcome
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Course Content
Lab 5 Spectrophotometry of Dyes
Chaojie Lu
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Take Test: Prelab: Spectrophotometry of Dyes
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Take Test: Prelab: Spectrophotometry
of Dyes
Test Information
Description
Instructions
Multiple Attempts This test allows 2 attempts. This is attempt number 1.
Force
Completion
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test can be saved and resumed later.
Question
Completion
QUESTION 1
10 points
This experiment involves making solutions of food
dyes and water. Since the reagents used are all
nontoxic, you don’t need to wear goggles while
working in the laboratory this week.
True
False
QUESTION 2
10 points
“If 2.00 mL of a stock solution is diluted to 25.00 mL,
what is the concentration of the final solution relative
to the concentration of the stock?”
QUESTION 3
10 points
Scientists study light and color quantitatively using
spectrophotometry- the quantitative determination of
light intensity by wavelength. This means that when a
spectrophotometer is used to generate the spectrum
of a solution, absorbance is the
Click Save and Submit to save and submit. Click Save All Answers to save all an
variable and wavelength is the
variable.
You will use the spectrophotometric data to generate a
lib ti
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i
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calibration curve in this experiment. You will use the
absorbance data recorded for solutions of varying
concentrations to generate the calibration curve. In
this case,
and
is the dependent variable
is the independent variable.
*By convention, when data is plotted on a graph, the
independent variable is the
axis, and
the dependent variable is the
axis.
QUESTION 4
10 points
“If the absorbance of a solution made by diluting 4.00
mL of stock to 10.00 mL is 0.2200, what would you
predict the absorbance of a solution made by diluting
8.00 mL of the same stock solution to 10.00 mL to
be?”
Question Completion Status:
QUESTION 5
10 points
Color arises when an object absorbs some wavelengths but reflects others. An
observer perceives the complement of the light that is absorbed. Using the
color wheel, indicate what color would be observed if a solution absorbed light
at 470 nm.
Selected Coordinates
Clear
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