CHEMISTRY SEMESTER ONE
SPECTROSCOPY LAB
BEER-LAMBERT LAW
Lab format: this lab is a remote lab activity
Relationship to theory: 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 exercise.
Remote Resources: Primary - UV/Vis Spectrometer; Secondary - Cuvette Holder
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 on-line period, and answer questions in analysis
sections after your on-line period. Your instructor will let you know if you are required to complete
any optional exercises in this lab.
Contents
BEER-LAMBERT LAW ...................................................................................................... 1
Learning Objectives ..................................................................................................... 2
Background Information............................................................................................... 2
Equipment ................................................................................................................... 3
Preparing to Use the Remote Web-based Science Lab (RWSL) ................................. 3
Introduction to the Remote Equipment and Control Panel ........................................... 4
Experimental Procedure .............................................................................................. 4
Appendix: Introduction to the Remote Equipment and Interface ................................. 7
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LEARNING OBJECTIVES

Measure and analyze the visible light absorbance spectrum of a standard NiSO 4 solution
to determine the maximum wavelength of absorbance (λmax).

Measure the absorbance of several standard NiSO4 solutions.
o Calculate the concentrations of these standard solutions using information
provided by the Laboratory Technicians.

Create Tables to display observations.

Construct a standard curve for the standard solutions. Find the relationship between
absorbance and concentration for NiSO4.

Measure the absorbance of an unknown concentration of the NiSO4 solution.

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
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
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SPECTROSCOPY LAB
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.
EQUIPMENT

Paper

Pencil/pen

Computer with Internet access
PREPARING TO USE THE REMOTE WEB-BASED SCIENCE LAB (RWSL)
Click on this link to access the InstallGuide for the RWSL: http://denverlabinfo.nanslo.org
Follow all the directions on this webpage to get your computer ready for connecting to the
remote lab.
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SPECTROSCOPY LAB
INTRODUCTION TO THE REMOTE EQUIPMENT AND CONTROL PANEL
Watch this short tutorial video to see how to use the RWSL control panel:
http://denverlabinfo.nanslo.org/video/absorbance_spectroscopy.html
There are appendices at the end of this document that you can refer to during your lab if you
need to remind yourself how to accomplish some of the tasks using the RWSL control panel.
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:
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. Store a Dark Spectrum.
3. Ensure that Sample 0 is selected.
4. Turn on the light and you will see the spectrum of the light source
5. Play around with the Integration Time, Boxcar Width, and # Spectra to Average to get
the least noisy spectrum that you can.
6. Store the Reference spectrum.
7. 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).
8. Select one of the NiSO4 standards in the Qpod. View the Absorbance Spectrum.
9. Determine the location of λmax.
10. Record the Absorbance of the NiSO4 sample at λmax. Each student in the group must
write the measurement down for later use.
11. Repeat step 10 for all remaining samples, including cuvette #5, which contains the
unknown concentration of NiSO4.
12. Another student should take control of the interface and repeat the process starting at
step 2.
13. 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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SPECTROSCOPY LAB
Data Analysis (to be done offline if necessary):
Plot a standard graph using the concentration and Absorbance values for the standard
solutions. Plot Concentration on the X-axis and Absorbance values on the Y-axis. Draw a bestfit 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:
a. 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.
b. Right-click one of the data points on the graph and select Add Trendline.
c. 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.
d. 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 NiSO 4?
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 1 to determine what color light is being absorbed at λmax.
750 nm
620 nm
600 nm 580 nm
500 nm
450 nm
380 nm
Figure 1 - Visible portion of EM Spectrum
F. Figure 2 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
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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 2 - By Sakurambo at English Wikipedia [GFDL (www.gnu.org/copyleft/fdl.html) or CC-BYSA-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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APPENDIX: INTRODUCTION TO THE REMOTE EQUIPMENT AND INTERFACE
When you access the RWSL through the course website, you will see an interface that looks like
this (Figure 3). You can gain control of the interface by right-clicking anywhere on the screen
and selecting “Request Control of VI”. If someone else has control, you will get a message
telling you that you need to wait. You will be placed in queue and will get control of the
interface when the other person releases it.
Figure 3 - RWSL Interface
The controls on the right side of the screen are for controlling the camera. The preset positions
allow you to quickly zoom in to a different part of the setup, but you can also pan, tilt and zoom
the camera using the keypad controls on the screen.
On the left side of the screen, you can see the controls for one of the pieces of equipment that
is used in this experiment. It is called a Qpod, and it is a device into which a cuvette containing
sample is placed so that light can be shined through it in order to measure absorbance. All of
our cuvettes have a path length (distance that the light travels through them) of 1.00 cm.
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SPECTROSCOPY LAB
Here is a cuvette:
(photo from http://cuvette.net/ , really, that’s the name of the site)
The Qpod is also capable of controlling the temperature of the sample inside of it. For clarity,
here is a labeled picture of the equipment:
Bucket of water that allows
the Qpod to control the
cuvette temperature
Cuvettes in carousel
Qpod (the black
unit) and
temperature
controller
(underneath).
Spectrometer
Figure 4 - Equipment for this lab
The light path is also indicated with yellow arrows. Some of the fiber optic cabling that the light
flows through is not visible in the photo, but you probably get the idea. The light is produced
by a Xenon strobe inside the spectrometer, and then passed through a fiber optic cable into
one side of the Qpod. The light passes through whatever sample is inside the Qpod, and then
enters a fiber optic cable on the other side of the Qpod and is returned to the sensing unit in
the spectrometer.
Controlling the Qpod:
The first thing to do is to turn on the Qpod’s temperature control system and ensure that it is
set to 25.00 °C, which is the standard temperature for most Absorbance measurements. You
do this by gaining control of the interface and clicking the button labeled “Temperature
Controller” (see Figure 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.
The Cuvette Selection tab allows you to rotate the carousel that holds the six cuvettes.
They are numbered 0 through 5 (see Figure 5).
There is also a tab for “Ramping Controls”, but they will not be used for this experiment,
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so you can ignore them.
Figure 5 - Temperature and Cuvette Selection Controls
Basic Functions of the Spectrometer:
After the temperature of the Qpod has been set properly, you are ready to proceed with taking
Absorbance measurements. Click the “Spectrometer” tab to proceed.
The first thing to do is to take a “dark spectrum”, which is merely a measurement of what the
spectrometer is measuring when there is no light present. This establishes a level of baseline
“noise” in the instrument, which will be automatically subtracted out later in the process.
First, on the Spectrometer tab of the interface, click the green button labeled “Start”. This
enables the spectrometer to operate, and will change the button to a yellow “Pause” button.
You take and store the dark spectrum by ensuring that the “Light” is not on, and then clicking
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 6).
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Click Here to store Dark spectrum
Figure 6 - Storing a Dark Spectrum
At this point, turn on the spectrometer’s light source by clicking the “Light” button, which will
then turn green. You should now see a spectrum on the screen that looks like this (Figure 7):
Figure 7 - Light Spectrum
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Now you need to collect and store the spectrum of the “reference sample”. The reference
sample is just a cuvette full of distilled water. Selecting this cuvette into the Qpod and clicking
the “Store Ref” button will store a spectrum where light is being absorbed by the cuvette and
by water. Having this spectrum stored 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).
Click Here to store Reference Spectrum
Figure 8 - Storing the Reference Spectrum
Now you are ready to measure the absorbance of nickel (II) sulfate. 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.
Zooming in and out on the Spectrum:
With a cuvette containing NiSO4 solution in the Qpod, make sure the light is on and that the
spectrum graph is zoomed all the way out. Here are the steps for doing this:
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a. Click on the button at the lower right of the graph, shown below in Figure 7.
b. This brings up a small sub-menu of other buttons. The only two that are useful
to you are the left-most buttons in the top and bottom rows (See Figure 8),
although you can play around with the others if you want to. Select the leftmost button in the bottom row to view the entire spectrum.
c. Select the left-most in the top row to select specific parts of the spectrum to
“zoom in” on and view more closely. After clicking this button, you use the
mouse to draw a box around the area that you want to zoom in to. Be sure you
draw the box so that it includes some area past the top of the peak you are
interested in, or else it will chop off the top of it in the viewing window.
d. If you accidentally zoom in too far or on the wrong part of the spectrum, just
zoom out and start over again.
Zoom In
Zoom Out
Figure 9 – Spectrum Manipulation
Button
Figure 10 - These two buttons are
most useful
Finding λmax:
This is merely identifying the tallest peak in the Absorbance spectrum, and you only need to do
this once for a chemical, no matter how many different solution concentrations you measure.
Use the Cuvette Selection controls to select a cuvette containing NiSO4 solution, and with the
light turned on, and the cursor enabled, click the “Show Absorbance Spectrum” button and
then zoom all the way out on the spectrum. You will now see the absorbance spectrum of the
sample, as in Figure 11. Click the Cursor Control button and move the cursor on top of the
tallest peak using the mouse.
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Cursor Control button
Enable Cursor
button
Absorbance
button
Absorbance Value
at cursor location
Figure 11 - Absorbance Spectrum
You can ignore the noisy parts of the spectrum on either end. There may be one peak as shown
below, or there may be more than one. Always use the tallest absorbance peak. Now, you can
read the wavelength of the tallest peak on the “Cursor Location Information” line. In this case,
the tallest peak is at 395.6 nm. This is λmax, and the absorbance at λmax is shown in the
Absorbance at Wavelength box.
Remember, λmax does not change as long as you are measuring the absorbance of the same
chemical (NiSO4 in this experiment), so you do not need to adjust the cursor location after you
have it set.
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Smoothing out the Absorbance spectrum:
Do you notice how much the Absorbance reading is jumping around? This is due to noise in the
data. Look back at the Spectrometer Tab (Figure 10). See the fields called “Integration Time”,
“Boxcar Width” and “# Spectra to Average”? These are variables that you can adjust to “clean
up” the noise in the spectrum. The integration time is how many milliseconds the spectrometer
will wait before it stores a spectrum. The Boxcar Width is
how many sequential points in the spectrum will be
averaged to produce one point on the curve. The
“Average” variable tells the spectrometer how many
spectra to average before it reports a result. Just like any
other measurement that contains random error (“noise”),
averaging several measurements can average out the noise
and “clean up” the signal. Play around with these settings
to see what effect they have on the spectrum. Once you
find a setting that gives you results that you think are good,
stick with them for the rest of the experiment.
Figure 12 - Variables
Spectrometer Tab
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