Part 1 - Get a Lab Appointment and Install Software:
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 1-27.
Submit your completed Pre-Lab Questions (page 6) 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
Lab format: This lab is a remote lab activity.
Relationship to theory (if appropriate): This activity quantitatively relates the concentration
of a light-absorbing substance to the absorbance of light.
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 ....................................................................................................... 2-5
Pre-lab Questions................................................................................................................... 6
Equipment ............................................................................................................................. 6
Preparing for this NANSLO Lab Activity ................................................................................ 6
Experimental Procedure ....................................................................................................... 7
Exercise 1: Determining the Absorbance and Concentration
of Solutions ....................................................................................................................... 7-9
NANSLO Control Panel Instructions for Absorbance Apparatus ..........................................10-26
Creative Commons Licensing ................................................................................................ 27
U.S. Department of Labor Information ................................................................................. 27
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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 NiSO 4 solution
to determine the maximum wavelength of absorbance (λmax).
2. Measures the absorbance of several standard NiSO4 solutions.
o Calculate the concentrations of these standard solutions using information
provided by the Laboratory Technician.
3. Create tables to display observations.
4. Construct a standard curve for the standard solutions. Find the relationship between
absorbance and concentration for NiSO4.
5. Measure the absorbance of an unknown concentration of the NiSO4 solution.
6. Calculate the concentration of the unknown NiSO4 solution using the standard curve
that you derived.
BACKGROUND INFORMATION:
Visible light represents only a very small part of the electromagnetic spectrum. Visible light
consists of light having wavelengths from about 3.8 x 10-7m to 7.8 x 10-7 m (380 nm to 780 nm).
Many substances interact with electromagnetic radiation in the visible and ultraviolet regions of
the spectrum. Substances that have color absorb some wavelengths from the visible region of
the spectrum and reflect others. The energies associated with photons of visible and ultraviolet
light are in the same range as energies required to promote outer level (valence shell) electrons
to higher energy level in many substances.
𝐸 = ℎ𝜈 is the difference in energy between the ground state and the excited state. When light
of the appropriate wavelength impinges on a substance, it may be absorbed by promoting an
electron to a higher energy level. This happens in the visible and ultraviolet regions of the
spectrum.
The energy of a photon of electromagnetic radiation is given by the
relationship:
𝐸 = ℎ𝜈 where 𝐸 = energy in joules, 𝜈 = frequency in cycles per second, and ℎ
= Planck’s constant = 6.62607 𝑥 10−34 J • s
The relationship between wavelength and frequency of electromagnetic
radiation is: 𝛌𝛎 = 𝐜 where 𝜆 = wavelength in meter and 𝑐 =
2.996 𝑥 108 𝑚⁄𝑠, the speed of radiant energy in a vacuum.
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In making measurements of the amount of radiant energy absorbed or transmitted by a
sample, we use a blank so that the change in absorbance of the sample holder and the solvent
can be factored out. That is, a blank containing all substances that will be in the sample, except
the one under investigation, is placed in the spectrophotometer, and a measurement is taken
so that we know how much light is absorbed by everything except the substance we are trying
to investigate.
The absorbance of a solution can be related to the concentration of the absorbing species in
the solution. This relationship is called the Beer-Lambert law, after Augustus Beer (a German
physicist) and Johann Lambert (a Swiss physicist), but is commonly referred to as Beer’s Law,
although it has nothing to do with beer. The Beer-Lambert Law can be expressed as:
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 blank, as described above,
allows us to factor out the effect of the solvent, cell walls and cell length.
So 𝑨 = 𝑨𝒃𝒄, and if 𝐴 and 𝑏 are constant for any given species and cell length, we can see
that the absorbance of a solution is directly proportional to the concentration of the absorbing
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.
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 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 method is 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
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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 is not very useful. Essentially, you
specify how many points you want in the averaging function. Let’s say that you specify two
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 which we will call 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.
Figure 1 below 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.
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Figure 1: Example of Boxcar averaging.
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.
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PRE-LAB Questions:
1.
2.
3.
4.
5.
Why are we measuring the absorbance of various samples in this lab activity?
What are the sources of noise in the measurements you will be making?
How can you reduce the noise in your measurements?
What two factors must you balance when deciding how to collect your data?
Read the procedure below and ask your Instructor if you need any clarification on any of
the steps.
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. The instructions found on pages 924 provide a written description of the RWSL control panel features.
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.
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EXPERIMENTAL PROCEDURE:
See NANSLO Control Panel Instructions for Absorbance Apparatus starting on Page 10 for
more information on how to perform the steps noted here.
Once you have logged on to the Remote Lab, you will perform the following Laboratory
procedures.
EXERCISE 1: Determining the Absorbance and Concentration of Solutions
1. Turn on temperature controller. Ensure the temperature of the system is adjusted to
25.0 degrees C.
2. Ensure the spectrometer’s light source is turned off.
3. Store a Dark Spectrum.
4. Ensure that cuvette 0 (the reference sample) is selected.
5. Turn on the light and you will see the spectrum of the light source.
6. Play around with the Boxcar Width and # Spectra to Average to get the least noisy
spectrum that you can.
7. Store the Reference spectrum.
8. Ask the Lab Tech for information about the standard NiSO4 solutions. You will use this
information to calculate the concentration of each standard solution (during data the
analysis portion of the activity).
9. Select one of the 𝑁𝑖𝑆O4 standards (cuvettes 1 through 4) in the Qpod.
10. View the Absorbance Spectrum.
11. Determine the location of 𝜆𝑚𝑎𝑥.
12. Record the Absorbance of the 𝑁𝑖𝑆O4 sample at 𝜆𝑚𝑎𝑥. Each student in the group
must write the measurement down for later use.
13. Repeat step 9 for all remaining samples, including cuvette #5 which contains the
unknown concentration of 𝑁𝑖𝑆O4.
14. Another student should take control of the interface and repeat the process starting at
step 2.
15. After each student has collected a complete set of data (and everyone has recorded
each data set), you can log out of the lab and work on the data analysis portion. If you
have time left in your scheduled lab period, you can continue working with your lab
partners to analyze the data.
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Data Analysis (to be done offline if necessary):
Plot a standard graph using the concentration and Absorbance values for the standard
solutions. Plot the concentration on the X-axis and Absorbance values on the Y-axis. Draw a
best-fit line going through the origin. From the Absorbance of the unknown solution, you can
calculate the concentration of the unknown solution using the line equation of the standard
curve.
In Excel, the best-fit line and its equation can be determined by this method:
1. Insert a scatter plot of the data, making sure that absorbance is on the y axis and
concentration is on the x axis. If they are switched, then delete the graph, change the
positions of the absorbance and concentration columns and insert the scatter plot graph
again.
2. Right-click one of the data points on the graph and select Add Trendline.
3. Make sure “Linear” is selected and check the box to set the intercept to zero and also
the one to display the equation on the chart.
4. You will now have the best-fit line and the equation for that line. You can use this line
equation to calculate the concentration of the unknown NiSO4 solution
Questions (show all necessary calculations):
A. Why do you have to first take an absorbance measurement of a cuvette filled with
distilled water? Why does this measurement have to be subtracted from the
measurements of the 𝑁𝑖𝑆O4 samples?
B. Why didn't we just measure one or two samples with known concentrations of 𝑁𝑖𝑆O4?
C. How many significant digits can you report in the concentration of the unknown
sample? What limits the number of significant digits in this result?
D. What is the energy, in Joules, of one photon of light at 𝜆𝑚𝑎𝑥?
E. Use Figure 2 to determine what color light is being absorbed for 𝜆𝑚𝑎𝑥.
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Figure 2: Visible portion of EM Spectrum
F. Figure 3 demonstrates the relationship between absorbed and reflected colors of light.
Absorbed is opposite of reflected on the wheel. For example, if a substance absorbs
orange light, it will reflect blue light, and therefore appear blue. Compare the color of
the NiSO4 solution to the color of the light it absorbs. Does it agree with the color
wheel? What can you deduce from this?
Figure 3: By Sakurambo at English Wikipedia [GFDL
http://www.gnu.org/copyleft/fdl.html) or CC-BY-SA-3.0
(http://creativecommons.org/licenses/by-sa/3.0/)] via
Wikimedia Commons
G. If a chemical solution was primarily orange in color, approximately what wavelength
would you expect λmax of the absorbed light to be? Why?
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NANSLO CONTROL PANEL INSTRUCTIONS FOR ABSORBANCE APPARATUS
The Remote Web-based Science Lab (RWSL) spectrometer is controlled remotely by using a
web interface as shown below. This NANSLO control panel allows you to control every function
of the spectrometer just as if you were sitting in front of it.
Figure 4: Spectrometer lab interface for emission spectroscopy lab.
Communicating with your Lab Partners
As soon as you have accessed this lab interface, click on the “Voice Conference” yellow button
(you may need to scroll down to see it) to view instructions for communicating with your lab
partners and with the Lab Technicians. Only one person can be in control of the equipment at
any one time so talking together on a conference line helps to coordinate control of the
equipment and creates a more collaborative environment for you and your lab partners.
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Gaining Control of the Absorbance Apparatus
Right click anywhere in the gray area of the lab interface and choose “Request Control of VI”
from the dialogue box that appears when multiple students are using the spectrometer at the
same time. After you request control, you may have to wait a short time before you actually
receive control and are able to use the features on this lab interface.
Figure 5: Take control of the lab interface by right clicking
and selecting “Request Control of VI.”
Releasing Control of the Absorbance Apparatus
To release control of the spectrometer so that another student can use it, right click anywhere
in the gray area of the lab interface and choose "Release Control of VI" from the dialogue box
that appears.
Figure 6: Release control of the lab interface by right clicking
and selecting "Release Control of VI."
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How to use the Spectrometer, Qpod, and Qpod Temperature Controller using the NANSLO
Control Panel
The NANSLO Control Panel provides controls for three pieces of equipment. More detailed
instructions are provided below on using the controls for each piece of equipment.
1. Spectrometer analyzes the light absorbed in a sample and visually displays the absorbance
spectrum of the sample, the absorbance value, and the wavelength at λmax. Spectrometer
controls are available when the “Spectrometer” tab of the lab interface is selected.
2. Qpod is a carousel that holds six cuvettes. A selected cuvette is rotated into place so that light
can be shined through it in order to measure absorbance. Qpod controls are available when the
“Cuvette Holder (Temp Control)” tab is selected and then the “Cuvette Selection” tab is
selected.
3. Qpod temperature controller raises the temperature of the sample in the cuvette placed inside
of it based on the temperature selected. The temperature controller is available when the
“Cuvette Holder (Temp Control)” tab is selected and then the “Temp Control Display” tab is
selected.
Figure 7 shows the equipment configuration for this lab. The light path is indicated with yellow
arrows. Some of the fiber optic cabling that the light flows through is not visible in the photo.
The light is produced by a Xenon strobe inside the spectrometer. The light passes through a
fiber optic cable into one side of the Qpod where it then passes through whatever sample is
inside the Qpod. The light next enters a fiber optic cable on the other side of the Qpod and is
returned to the sensing unit in the spectrometer. The spectrometer measures how much light
is passed and how much light is returned, thus determining how much light is absorbed in the
sample.
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Figure 7:
Figure 7: Experimental apparatus (Spectrometer, Qpod and Cuvettes)
for Beer-Lambert law NANSLO lab activity.
Spectrometer Controls
When the “Spectrometer” tab is selected, controls are available for the spectrometer.
To activate the spectrometer to start the data feed, click the "Start" button on the far left
portion of the lab interface. The button will now turn yellow and say “Pause”.
Figure 8: After clicking on the "Start" button, it turns to a "Pause" button.
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The Image View Window on the upper right-hand side of the screen displays the real-time video
feed from a digital camera focused on the equipment used for this NANSLO lab activity based
on the Camera Preset selected.
The controls in the lower left hand corner of this screen allow you to capture lab data
generated by the spectrometer. See Figure 9.
Figure 9: Controls for the spectrometer.
Zooming In and Out on the Spectrum Graph on the Left-hand Side of the Spectrometer Screen
You will need to “zoom out” on the spectrum window to view the entire spectrum properly.
Here’s how to zoom out and in on the spectrum.
Click on the center button at the lower right of the graph, shown below in Figure 10.
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Figure 10: Click button to access other options.
This brings up a small sub-menu containing other buttons. The only two buttons that are useful
to you are the left-most buttons in the top and bottom rows. Select the left-most button in the
bottom row to view the entire spectrum.
Figure 11: Spectrum Zoom Out Button
Select the left-most button in the top row to select specific parts of the spectrum to “zoom in”
on and view more closely (see Figure 12). After clicking this button, your mouse turns to a
magnifying glass. Use your mouse (hour glass) to select a portion of the spectrum to inspect
more closely by holding down your mouse button and drawing a box around the area of
interest. Be sure you draw the box so that it includes some area past the top of the peak you
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are interested in or else it will chop off the top of it in the viewing window. The enlarged image
of that area will appear in the spectrum window. If you accidentally zoom in too far or on the
wrong part of the spectrum, use the zoom out button shown in Figure 11 and start over again.
Figure 12: Spectrum Zoom In Button
Qpod Controls
To access the Qpod Controls, select the “Cuvette Holder (Temp Control)” tab and then the
“Cuvette Selection” tab. The Qpod controls are used to select a sample to analyze and collect
data on. The sample contained in each cuvette is determined based on the lab being
performed. A cuvette is a small laboratory vessel used to hold the sample. All of our cuvettes
have a path length (distance that the light travels through them) of 1.00 cm. (Photo from
http://cuvette.net/).
Using Camera Preset 4, zoom in to actually see the Qpod carousel rotate.
To select the Reference Sample (if not already selected,) you would use the back and forth
arrows until “0” is displayed in the “Selected Cuvette” field or click the “Re-set to Reference”
button.
For all other selections, you would use the back and forth arrows until the sample of interest is
displayed in the “Selected Cuvette” field, e.g. “3.” Your lab procedure will provide more detail
on the samples to select.
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Figure 13: Accessing the Qpod Carousel Controls
Qpod Temperature Controller Controls
To access the Qpod Temperature Controller, select the “Cuvette Holder (Temp Control)” tab
and then the “Temp Control Display” tab.
1. First select the “Temperature Controller” button to turn on the heater.
2. In the “Set Temperature *C” field, enter the temperature indicated in your lab
procedure (typically this is 25o C.)
3. View the current temperature in the “Current Temperature *C” area and wait until it
reaches the set temperature before proceeding to college data.
Using Camera Preset 2, zoom in to actually see the temperature on the Qpod Temperature
Controller.
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Figure 14: Accessing the Qpod Temperature Controller Controls
Camera Presets and Pan-Tile-Zoom Controls Available for Spectrometer, Qpod, and Qpod
Temperature Controller
Several camera preset positions have been programmed for use with this lab interface.
Hovering over the gray area where the buttons are will give you a pop-up menu that describes
where each preset is assigned to. Each Camera Preset is set to focus in on a specific area.
Preset 1, for example, zooms in on a portion of experimental setup, Preset 2 on the
temperature control, Preset 3 on the sample in the cuvettes; and Preset 4 on the carousel
holding the cuvettes.
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Figure 15: Absorbance NANSLO lab activity camera presets.
Figure 16 shows the Pan-Tilt-Zoom Controls available. The four arrows are used to pan and tilt
allowing you to move the camera right to left and up and down. The two zoom buttons allow
you to zoom in to see a closer look at the equipment or zoom out to view more of the room.
Figure 16: Pan, tilt and zoom capabilities.
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STEPS IN PERFORMING YOUR LAB ACTIVITY
Storing the Dark Spectrum Before Turning on the Spectrometer Light
To establish a level of baseline “noise” in the instrument, which will automatically be
subtracted out later in the process, you need to store the “dark spectrum” which is merely a
measurement of what the spectrometer is measuring when there is no light present. With the
“Spectrometer” tab is selected, click on the “Store Dark” button. There will be no indication
that anything happened, so if you’re not sure you clicked this button, just click it again – you
won’t hurt anything by storing another dark spectrum. See Figure 17.
Figure 17: Storing the Dark Spectrum.
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Turning on the Spectrometer Light
Now turn on the spectrometer’s light source by clicking the “Light” button. The button turns
green. You should now see a spectrum (left-hand window of this lab interface) that looks
similar to the one shown in Figure 18.
Figure 18: Turning on the spectrometer light.
Turning on the Qpod’s Temperature Control System
Turn on the Qpod Temperature Controller heating unit. Several steps must be performed to
complete this step.
1. Select the “Cuvette Holder (Temp Control)” tab.
2. Select the “Temp Control Display” tab if not selected by default.
3. Select the “Temperature Control” button. It will turn green and display “On”
when selected.
4. In the “Set Temperature *C” field, ensure that it is set to 25.00 °C, which is the
standard temperature for most Absorbance measurements, or whatever
temperature is indicated in your lab procedure.
5. Watch the temperature curve for a few minutes to ensure that the temperature
of the Qpod is adjusted to 25.00 °C +/- 0.05 °C.
Note that if you have selected Camera Preset 2 you can see the temperature on the Qpod.
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Figure 19: Setting the Qpod temperature.
Selecting the Reference Sample
The “Cuvette Holder (Temp Control) tab is currently selected. Select the “Cuvette Selection”
tab. As mentioned above, the Cuvette Selection tab allows you to rotate the carousel that
holds the six cuvettes. They are numbered 0 through 5. You will use this tab to select each
sample that will be used for this lab activity.
For now, make sure that the “reference sample” is selected: “Cuvette 0 = Reference Sample.”
The reference sample is a cuvette full of distilled water. If the reference sample is not selected,
either use the arrows to navigate to that cuvette or select the “Reset to Reference” to return to
Cuvette 0.
Note that if Camera Preset 4 has been selected, you can see the carousel on the Qpod move.
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Figure 20: Selecting the Reference Sample.
Storing the Spectrum for the Reference Sample
Next, select the “Spectrometer” tab. Click the “Store Ref” button to collect and store the
spectrum of the “reference sample”. A spectrum where light is being absorbed by the cuvette
and by water is stored and allows it to be subtracted out from your later sample
measurements, thus allowing you to measure the Absorption of light that is only due to the
material you are interested in (𝑁𝑖𝑆𝑂4 in this experiment).
Figure 21: Storing the spectrum for the Reference Sample.
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Measuring the Absorbance of Nickel (II) Sulfate
Background: There are several standard 𝑁𝑖𝑆𝑂4 solutions that you will measure the absorbance
of. This range of concentrations was chosen because the Absorbance is directly proportional to
the concentration (obeys Beer’s law) in this concentration range. By plotting Absorbance on the
y-axis and concentration of 𝑁𝑖𝑆𝑂4 on the x-axis, you will draw a best-fit straight line (which is
called the “standard curve”) passing through the origin. When you measure the absorbance of
the sample, you must do so at a single wavelength. This is called the 𝜆𝑚𝑎𝑥, and corresponds to
the tallest peak in the absorbance spectrum. It is important that this wavelength be the one at
which the sample absorbs light the most strongly, because this results in the most favorable
signal-to-noise ratio and gives an absorbance measurement with the least amount of
uncertainty.
Selecting the Standard: Select the “Cuvette Holder (Temp Control)” tab and then the “Cuvette
Selection” tab. Select one of the standard solutions (cuvettes 1 – 4). Tell the lab tech which
cuvette you have selected, and you will be provided information that will allow you to calculate
the concentration of the standard solution selected. You will do this for each standard solution.
Viewing the Spectrum of the Selected Standard: Select the “Spectrometer” tab. With the
cuvette containing 𝑁𝑖𝑆𝑂4 in the Qpod, select the “Show Absorbance Spectrum” button. Notice
that the line on the absorbance spectrum may look flat. If so, you need to adjust the scale so
you can see it.
Figure 22: Selecting the “Show Absorbance Spectrum” button.
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Zoom out to view the whole spectrum (see Figure 11 on zooming out.) You will see a peak that
is the absorbance peak and noise on either side of it where we have run out of light intensity.
Use spectrum “zoom in” tool to look at the tallest peak more closely.
Identifying 𝝀𝒎𝒂𝒙: 𝜆𝑚𝑎𝑥 is the tallest peak in the Absorbance Spectrum. You only need to
identify λmax once for a chemical. No matter how many different concentrations of that
chemical you measure, λmax is always the same. So, you don’t need to adjust the cursor
location after you have it set.
You can ignore the noisy parts of the spectrum on either end. There may be one peak or there
may be more than one. Always use the tallest absorbance peak. Once you have identified the
tallest peak, you can read the wavelength and absorbance in the “Cursor Location Information”
area. In Figure 23, the tallest peak is at 395.6 nm.
To select the peak:
1. Select the “Enable Cursor” button. The button will turn a bright green when selected.
2. Select the Cursor Control button from the menu that appears on the lower right hand
side of the spectrum graph.
3. Move your cursor into the spectrum graph area, hold down your left mouse button, and
drag the green line displayed in the spectrum to the tallest peak. The location of the
cursor will correspond to the wavelength and the intensity (“Absorbance”) shown in the
“Cursor Location Information” area.
If you lose your cursor, disable and re-enable it by clicking on the Cursor Control button
twice. It will re-appear in the center of the screen.
Figure 23: Identifying the 𝜆𝑚𝑎𝑥 which is the tallest peak in the Absorbance Spectrum.
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Exporting to Your Clipboard: To capture an image of the spectrum graph, do the following:
1. Select “Graph Image” if not already selected from the drop-down menu to
capture the spectrum graph image.
2. Select the “Export to Clipboard” button.
Figure 24: Capturing an image
Open a document, e.g. Word, and paste the graphic into the document.
Documenting 𝝀𝒎𝒂𝒙: Once you have recorded 𝜆𝑚𝑎𝑥, select the next standard and record the
𝜆𝑚𝑎𝑥 for that standard. Notice that because you are looking at the same chemical but just a
different concentration the 𝜆𝑚𝑎𝑥 does not change. However, the absorbance has changed
since it is either more or less concentrated than the solution(s) previously selected. Continue
until you gather data for all standards and the unknown, cuvette #5. When you have finished
recording all data, reset the carousel to sample 0 and turn off the temperature control.
Analyze data with partners if you have time left.
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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
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availability, or ownership.
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