Part 1 - Get a Lab Appointment and Install Software:
Set up an Account on the Scheduler (FIRST TIME USING NANSLO):
Find the email from your instructor with the URL (link) to sign up at the scheduler.
Set up your scheduling system account and schedule your lab appointment.
NOTE: You cannot make an appointment until two weeks prior to the start date of this lab assignment.
You can get your username and password from your email to schedule within this time frame.
Install the Citrix software: – go to http://receiver.citrix.com and click
download > accept > run > install (FIRST TIME USING NANSLO).
You only have to do this ONCE. Do NOT open it after installing. It will work automatically when you go
to your lab. (more info at
http://www.wiche.edu/info/nanslo/creative_science/Installing_Citrix_Receiver_Program.pdf)
Scheduling Additional Lab Appointments:
Get your scheduler account username and password from your email.
Go to the URL (link) given to you by your instructor and set up your appointment.
(more info at http://www.wiche.edu/nanslo/creative-science-solutions/students-scheduling-labs)
Changing Your Scheduled Lab Appointment:
Get your scheduler account username and password from your email. Go to http://scheduler.nanslo.org
and select the “I am a student” button. Log in to go to the student dashboard and modify your
appointment time. (more info at http://www.wiche.edu/nanslo/creative-science-solutions/studentsscheduling-labs)
Part 2 – Before Lab Day:
Read your lab experiment background and procedure below, pages 1-12.
Submit your completed Pre-Lab Questions (page 9) per your faculty’s instructions.
Watch the Spectrophotometer Control Panel Video Tutorial
http://www.wiche.edu/nanslo/lab-tutorials#beerlambert
Part 3 – Lab Day
Log in to your lab session – 2 options:
1)Retrieve your email from the scheduler with your appointment info or
2) Log in to the student dashboard and join your session by going to http://scheduler.nanslo.org
NOTE: You cannot log in to your session before the date and start time of your appointment. Use
Internet Explorer or Firefox.
Click on the yellow button on the bottom of the screen and follow the instructions to talk to your lab
partners and the lab tech.
Remote Lab Activity
SUBJECT SEMESTER: ____________
TITLE OF LAB: Beer-Lambert Law – Food Dye Concentrations
in Sports Drinks
Lab format: This lab is a remote lab activity.
Relationship to theory (if appropriate): This activity is designed to be done after a basic
introduction to the Beer-Lambert Law. It assumes that students will have been introduced to
the concepts of chemical bonds and covalent and ionic compounds. It introduces the idea of
solving simultaneous equations for multiple unknowns.
Instructions for Instructors: This protocol is written under an open source CC BY license. You
may use the procedure as is or modify as necessary for your class. Be sure to let your students
know if they should complete optional exercises in this lab procedure as lab technicians will not
know if you want your students to complete optional exercises.
Instructions for Students: Read the complete laboratory procedure before coming to lab.
Under the experimental sections, complete all pre-lab materials before logging on to the
remote lab. Complete data collection sections during your online period, and answer questions
in analysis sections after your online period. Your instructor will let you know if you are
required to complete any optional exercises in this lab.
Remote Resources: Primary – UV/Vis Spectrometer; Secondary – Cuvette Holder.
CONTENTS FOR THIS NANSLO LAB ACTIVITY:
Learning Objectives............................................................................................................. 2
Background Information .................................................................................................... 2-6
Estimating Error ................................................................................................................. 7-9
Pre-lab Questions ............................................................................................................... 9
Equipment .......................................................................................................................... 10
Preparing for this NANSLO Lab Activity ............................................................................. 10
Experimental Procedure .................................................................................................... 10
Exercise 1: Calculating the Absorbance and Concentration of Blue #1 and
Yellow #5 in Solutions ..................................................................................................... 11-12
Creative Commons Licensing ............................................................................................. 12
U.S. Department of Labor Information .............................................................................. 12
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LEARNING OBJECTIVES:
After completing this laboratory experiment, you should be able to do the following things:
1. Measure and analyze the visible light absorbance spectrum of a standard solution to
determine the maximum wavelength of absorbance (λmax).
2. Create multiple solutions of differing concentrations of a stock solution.
3. Solve simultaneous equations for multiple unknown concentrations.
4. Determine and report the concentrations of two food dyes in a sports drink, along with
associated experimental uncertainty (error).
BACKGROUND INFORMATION:
Food dyes are used in many products to enhance or provide colors that make foods and drinks
more appealing or unique in some way. Sports drinks come in a wide variety of colors in an
attempt to appeal to different consumer groups. Food colorings are used in very small
quantities and provide no nutritional value at all. Because these molecules contain lightabsorbing features, which absorb visible light very strongly, even a small amount of food
coloring can result in very strong colors.
Anything that absorbs light can be quantified by using the Beer-Lambert Law, also sometimes
just called “Beer’s Law”:
A=abc
Where A=Absorbance (unitless); a = molar absorptivity (molarity-1∙cm-1), which is a constant for
the absorbing species, b = path length, or thickness of the absorbing layer of a solution (cm), and
c = concentration of the solution (molarity).
Beer’s law tells us that the absorbance of a particular species is directly proportional to the
concentration of the absorbing species. The measurement of a reference sample (one with
everything except the substance being analyzed) allows us to factor out the absorbance of light
by the solvent, and by the cuvette itself.
So A = abc. And if a and b are constant for any given species and path-length, we can see that
the absorbance of a solution is directly proportional to the concentration of the absorbing
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species. Because the absorbance of a solution is easy to measure, this technique is frequently
used to measure concentrations of unknown solutions, and this is what you will be doing in this
experiment.
Food dyes absorb light because of conjugated systems of double bonds, which means double
bonds are alternating with single bonds in either a long chain or one or more rings. For
example, a food dye called FD&C Blue #1 (Food, Drug and Cosmetics) looks like this:
Figure 1: FD&C Blue #1 (Public Domain)
Notice the alternating single and double bonds in the rings? See Figure 1. Having a lot of these
alternating bonds is what allows this molecule to absorb light very strongly.
The other dye we will be working with in this activity is called FD&C Yellow #5. It looks like
Figure 2.
Figure 2: FD&C Yellow #5 (Public Domain)
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It has a very different system of conjugated bonds, so it absorbs different wavelengths of light
than Blue #1. In fact, if you remember your color wheel (or look one up), you can probably
predict what color and wavelength or frequency of visible light each of these dyes absorbs
based on their names. Of course, if only one of these two molecules is present, it is pretty easy
to apply Beer’s Law and determine its concentration. But what if both of them are present?
While each molecule has a wavelength at which it absorbs the most strongly (λmax), they also
absorb light at many other wavelengths. For example, here is the absorbance spectrum of Blue
#1. See Figure 3.
Figure 3: Absorbance Spectrum of Blue #1
And here is the absorbance spectrum of Yellow #5. See Figure 4.
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Figure 4: Absorbance Spectrum of Yellow #5
There are several features to take note of here. The first is the absorbance features below 400
nm. We aren’t interested in these peaks because they are in the ultraviolet region of the
electromagnetic spectrum and therefore don’t affect the perceived color of the substance since
our eyes don’t register in that range of wavelengths. Also, notice how the absorbance of each
dye is NOT zero at the λmax value of the other dye.
Therefore, each molecule will affect the absorbance of light at the λmax of the other molecule,
making it impossible to just solve for the concentration of each one at its respective λmax. In
that case, we have two unknowns so we must have two equations, and we do! The absorbance
at the λmax for each molecule is composed of the absorbance of Yellow #5 and the absorbance
of Blue #1. What we need to know is how much light each of the two dyes absorbs relative to
the other one. Luckily, some folks at the Chemistry Department of Appalachian State University
in Boone, NC have done that work for us (Sigmann and Wheeler).
So we can write an equation for each one like this (where B1 = Blue #1 and Y5 = Yellow #5):
A631 = AB1 + 0.047(AY5)
And
A427 = AY5 + 0.003(AB1)
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Doing a little algebra, we get the following equations:
𝐴𝐵1 =
𝐴631 − (0.047 ∙ 𝐴427 )
0.9998
And
𝐴𝑌5 =
𝐴427 − (0.003 ∙ 𝐴629 )
0.9998
Where A631 is the measured absorbance at 631 nm and A427 is the measured absorbance at
427 nm.
To calculate concentrations, we will also need to know the molar absorptivity of each molecule.
This is the factor “a” in the Beer’s Law equation, and it tells us how much light a 1.00 Molar
solution of it will absorb in a 1.00 cm path-length. This is a property of the molecule, just like its
mass, and it doesn’t change. For these two molecules, the molar absorptivity values are
(Sigmann and Wheeler):
Dye
Blue #1
Yellow #5
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Molar Absorptivity (M-1cm-1)
1.30 x 105 at 631 nm
2.73 x 104 at 427 nm
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ESTIMATING ERROR:
There are many sources of noise, or uncertainty,
in any experimental measurement that you will
ever make. The goal of any scientific data
collection is to gather data with as little noise as
possible, although it can never be totally
eliminated. For example, using a spectrometer
with fiber optic light transfer, as you will do in
this lab activity, has noise in its measurements
due to the inconsistencies in the fiber optics,
interference between the light and the sample
being measured, inefficiencies in the detector,
etc. Most of this noise is random, meaning that
it has the same probability of resulting in a
measurement that is too high as it does resulting
in a measurement that is too low. When noise is
random, it can be minimized by averaging
multiple measurements. In a spectrometer such
as this one, there are two methods of averaging
measurements. One is through the use of
“boxcar averaging” and the other method is to
average multiple complete spectra.
Boxcar averaging is a smoothing method by
which multiple points on a curve are averaged
into one point. This reduces the overall number of points in the curve, but it averages out some
of the random noise in those points. One must be careful not to average too many points on
the curve together, because the more points you average in each “boxcar”, the less information
there is in that curve. For example, in the extreme case where you averaged ALL the points on
the curve, you would end up with just one point, which isn’t very useful. Essentially, you
specify how many points you want in the averaging function. Let’s say that you specify 2 points
for the boxcar average, and let’s further assume that the curve you are smoothing has 1000
points in it. The process works like this:




Points #1 and #2 are averaged to become a single point. We’ll call it NewPoint #1.
Then, the ORIGINAL point #2 is averaged with the ORIGINAL point #3 to become the
second point in the new curve which we’ll call NewPoint #2.
The process continues with the boxcar moving ahead one point at a time until all the
original points are averaged to become NewPoints in the smoothed curve.
The original 1000 points are turned into 999 NewPoints, and you have a slightly
smoother curve.
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Figure 5 illustrates how this process would proceed for a small curve of 50 original points. The
“y” curve is the 50 original points and the “y-box” curve is the 49 “new” points.
The other type of averaging is to average the entire curve. The spectrometer will collect one
entire spectrum about once per second. The spectrum consists of many thousands of points,
and each time a spectrum is collected, these points are slightly different (due to random noise).
If you average two spectra together, you reduce the noise by a factor of √2. If you average
three spectra, you reduce the noise by a factor of √3, etc. Of course, the more spectra you
average, the longer it takes to get a result, so there is a balance between decreasing noise and
collecting data in a reasonable amount of time.
In addition, you can always estimate the error in any measurement by taking several
measurements of it and calculating the standard deviation of the set of results. In other words,
if you measure something several times, you are likely to end up with a set of results that are
not all exactly the same number. The standard error of that set of numbers is an estimate of
how much error there is in your results, and it is equal to the standard deviation divided by the
square root of the number of samples in the data set. Here’s an example. Let’s say we
measured the absorbance of a sample at 500 nm five times, and the result was:
Trial #
Absorbance
1
0.324
2
0.332
3
0.325
4
0.328
5
0.322
We would report that the absorbance at 500 nm was: average +/- standard error, which in this
case is: 0.326 +/- 0.002. This means that the uncertainty in your result (0.326) starts in the
third decimal place, because that’s the first digit in your standard error. Since the uncertainty
starts in the thousandths place, we have to limit the average value to the thousandths place as
well. When reporting uncertainty in this way, you always report only one digit in the standard
error.
The standard deviation can be calculated manually (you’ve probably done this in a math
course), or you can perform the calculation using a spreadsheet program like Excel. If you have
fewer than 100 samples to work with, use the “stdev.s” function, which is more accurate for
small samples.
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Sources:
Sigmann, S. B., and Wheeler, D. E. The Quantitative Determination of Food Dyes W in Powdered
Drink Mixes. J Chemical Education 2004, 81, 10, pp 1475-1478.
PRE-LAB QUESTIONS:
1. How are the dyes called Blue #1 and Yellow #5 similar to each other? How are they
different?
2. What ionic group does Yellow #5 have that Blue #1 does not?
3. Describe in your own words what the “molar absorptivity” of a molecule is.
4. If you have a mixture of solutions containing Blue #1 and Yellow #5, what data will you
need to determine the concentration of each dye using Beer’s Law?
5. Create a table to record the data you will need for question #4.
6. Complete the following sentences:
a. When solutions containing Blue #1 and Yellow #5 are mixed together, the value
of the molar absorptivity for each dye will _____________________. (increase,
stay the same, decrease)
b. When solutions containing Blue #1 and Yellow #5 are mixed together, the color
of each dye will ________________________. (get lighter, stay the same, get
darker)
7. Explain your answers to question 6.
a.
b.
8. What information does the standard deviation convey about a set of numbers?
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EQUIPMENT:



Paper
Pencil/pen
Computer with Internet access
PREPARING FOR THIS NANSLO LAB ACTIVITY:
Read and understand the information below before you proceed with the lab!
Scheduling an Appointment Using the NANSLO Scheduling System
Your instructor has reserved a block of time through the NANSLO Scheduling System for you to
complete this activity. For more information on how to set up a time to access this NANSLO lab
activity, see
www.wiche.edu/nanslo/scheduling-software.
Students Accessing a NANSLO Lab Activity for the First Time
For those accessing a NANSLO laboratory for the first time, you may need to install software on
your computer to access the NANSLO lab activity. Use this link for detailed instructions on
steps to complete prior to accessing your assigned NANSLO lab activity –
www.wiche.edu/nanslo/lab-tutorials.
Video Tutorial for RWSL: A short video demonstrating how to use the Remote Web-based
Science Lab (RWSL) control panel for the Spectrometer can be viewed at
http://www.wiche.edu/nanslo/lab-tutorials#beerlambert.
NOTE: Disregard the conference number in this video tutorial.
AS SOON AS YOU CONNECT TO THE RWSL CONTROL PANEL: Click on the yellow button at the
bottom of the screen (you may need to scroll down to see it). Follow the directions on the pop
up window to join the voice conference and talk to your group and the Lab Technician.
EXPERIMENTAL PROCEDURE:
Read and understand these instructions BEFORE starting the actual lab procedure and collecting
data. Feel free to “play around” a little bit and explore the capabilities of the equipment before
you start the actual procedure.
Once you have logged on to the Remote Lab, you will perform the following Laboratory
procedures.
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EXERCISE 1: Calculating the Absorbance and Concentration of Blue #1 and
Yellow #5 in Solutions
Set-up Spectrometer
1. Turn on temperature controller. Ensure the temperature of the system is adjusted to
25.0 degrees C.
2. Ensure that the stirring control is turned on and ensure that cuvette 0 (the reference
sample) is selected.
3. Turn on the spectrometer light, and you will see the spectrum of the light source.
4. Play around with the Boxcar Width and # Spectra to Average to get the least noisy
(“jumpy”) spectrum that you can. Make sure that you don’t smooth it out TOO much,
because then you lose valuable information in the signal. Boxcar width should be
somewhere in the range of 2 to 6.
5. Turn off the light and store the Dark Spectrum.
6. Turn the light back on and store the Reference spectrum.
7. Click on the Show Absorbance Spectrum button to view the Absorbance Spectrum.
Determine Concentrations for Experiment
1. Select cuvette 1 in the Qpod. This cuvette will be empty.
2. Inject 4.0 mL of stock solution (a mixture of solutions containing Blue #1 and Yellow #5)
from Pump #1.
3. Measure the absorbance at the two values of λmax for the two dyes. If each
absorbance value is less than 0.8, you can proceed.
4. Assuming that the absorbance is linear with respect to concentration, figure out how
much you need to dilute the stock solution to bring the absorbance values into the
appropriate range.
Collect Data
1. Advance the carousel to select the next cuvette.
2. Use the pumps to deliver appropriate volumes of water and stock solution.
3. After allowing the mixture to stir in the cuvette for a couple minutes, record the
absorbance values at both λmax locations, unless the absorbance values are still too
high. In that case, you will need to come up with a different dilution to use for the next
one.
4. Another student should take control of the interface.
5. Repeat steps 1 through 5 to collect at least three measurements where both
absorbance readings at each λmax is lower than 0.8.
6. After each student has collected some data (and everyone has recorded the full data
set), you can log out of the lab and work on the data analysis portion. If you have time
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left in your scheduled lab period, you can continue working with your lab partners to
analyze the data.
Data Analysis (to be done offline if necessary)
1. Using the data you collected, along with information in the Background section above,
calculate the absorbance of Blue #1 and Yellow #5 in each solution that you worked
with.
2. Use Beer’s Law to calculate the concentration of Blue #1 and Yellow #5 in each of the
solutions you worked with.
3. Taking into account the dilutions you made, calculate the concentration of Blue #1 and
Yellow #5 in the stock solution. You should now have at least three measurements of
Blue #1 and Yellow #5 concentrations (one for each solution you made).
4. Calculate the average and standard deviation of your set of measurements for Blue #1
from step 3. The concentration of Blue #1 in the stock solution is: _______ M ± _______
5. Calculate the average and standard deviation of your set of measurements for Yellow #5
from step 3. The concentration of Yellow #5 in the stock solution is: _______ M ±
_______
6. Look up the molar masses of Blue #1 and Yellow #5 and properly reference your source.
7. Based on your results in steps 4, 5, and 6 calculate how many grams, each, of Blue #1
and Yellow #5 are present in 946 mL of sport drink with the same composition as the
stock solution you worked with in this lab activity.
For more information about NANSLO, visit www.wiche.edu/nanslo.
All material produced subject to:
Creative Commons Attribution 3.0 United States License 3
This product was funded by a grant awarded by the U.S.
Department of Labor’s Employment and Training Administration.
The product was created by the grantee and does not necessarily
reflect the official position of the U.S. Department of Labor. The
Department of Labor makes no guarantees, warranties, or
assurances of any kind, express or implied, with respect to such
information, including any information on linked sites and
including, but not limited to, accuracy of the information or its
completeness, timeliness, usefulness, adequacy, continued
availability, or ownership.
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