Science in Motion
INTRODUCTION TO SPECTROPHOTOMETRY
PA Standards:
3.4.10.C
3.4.12.C
3.7.10.B
3.7.12.B
Distinguish among the principles of force and motion – Describe light effects.
Apply the principles of motion and force – Evaluate wave properties of motion and force.
Apply appropriate instruments and apparatus to examine a variety of objects and processes
Evaluate appropriate instruments and apparatus to accurately measure materials and processes.
Background:
Spectrophotometry is a method of analyzing that involves how light interacts with the atoms (or
molecules) in a sample of matter. This can be done quantitatively, meaning the properties of that
interaction are actually measured and compared. Visible light is only a small portion of the entire
electromagnetic spectrum and it includes the colors commonly observed (red, orange, yellow, green,
blue and violet). The visible spectrum consists of electromagnetic radiation whose wavelengths range
from 400 nm to nearly 800 nm.
When white light is observed, what is actually seen is a mixture of all the colors of light. When this
light passes through a substance, certain energies (or colors) of the light are absorbed while other
color(s) are allowed to pass through or are reflected by the substance. The Energy of light is related to
its frequency by the equation [E=hν]. This is why some substances appear colored. The color that we
see is the combination of energies of visible light that are not absorbed by the sample. If the substance
does not absorb any light, it appears white (all light is reflected) or colorless (all light is transmitted). A
solution appears a certain color due to the absorbance and transmittance of visible light. For example, a
blue solution appears blue because it is absorbing all of the colors except blue. A sample may also
appear blue if all colors of light except yellow are transmitted. This is because blue and yellow are
complementary colors. (See the color wheel below.)
Consider the following experiment with three different students: one is wearing a white shirt, one red
and the other green. Based on the previous paragraph, you should understand why they appear those
particular colors. We will put these students in a closet with no windows, shut the door and turn off the
lights. What color is each of the shirts at this time? The answer is rather obvious. What will happen if
we only allow red light to fill the room? What color is the white shirt? How about the red shirt? What
about the green shirt? Not sure, watch the following demonstration carefully. We will produce a
spectrum on the white movie screen with the overhead projector and a diffraction grating. Pass a white
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Lab #531, Introduction To Spectrophotometry, (5/21/07) Advancing Science Program, Gettysburg College, Gettysburg, PA 17325,
www.advancingscience.org, Science in Motion
Science in Motion
piece of paper through each color and what do you observe? Pass a bottle of red solution
through the white light, the red light and then the green light. What color does the solution
appear in each case? Are you surprised?
The wavelength associated with the complementary color is known as the wavelength of maximum
absorbance. The wavelength of maximum absorbance is used when determining the concentration of a
colored solution since at this wavelength, a slight change in concentration allows for a significant
change in the absorbance of light.
The amount of light absorbed by a solution is dependent on the ability of the compound to absorb light
(molar absorptivity), the distance through which the light must pass through the sample (path length)
and the molar concentration of the compound in the solution. This relationship is known as Beer’s Law
and is represented by the equation:
A = εℓC
where A is absorbance, ε is molar absorptivity, ℓ is path length, and C is molar concentration. If the
same compound is being used and the path length is kept constant, then the absorbance is directly
proportional to the concentration of the sample [A α C].
A spectrophotometer is used to provide a source of light of certain energy (wavelength) and to measure
the quantity of the light that is absorbed by the sample. The basic operation of the spectrophotometer
includes a white light radiation source that passes through a monochromator. (See below). The
monochromator is either a prism or a diffraction grating that separates the white light into all colors of
the visible spectrum. (An example of a grating is a CD ROM surface. The reflective surface has tiny
grooves etched onto it which separate white light the same way a prism does.) After the light is
separated, it passes through a filter (to block out unwanted light, sometimes light of a different color)
and a slit (to narrow the beam of light--making it form a rectangle). Next the beam of light passes
through the sample that is in the sample holder. The light passes through the sample and the unabsorbed
portion strikes a photodetector that produces an electrical signal which is proportional to the intensity of
the light. The signal is then converted to a readable output that is used in the analysis of the sample. The
spectrophotometer displays this quantity in one of two ways: (1) Absorbance -- a number between 0 and
2 and (2) Transmittance -- a number between 0 and 100%. The entire range of wavelengths can be
measured for a particular sample which if graphed produces a characteristic spectrum for that sample.
Light Bulb
Prism
Filter
Slit
Sample
Detector
The sample for a spectral analysis is prepared by pouring it into a cuvette which looks similar to a small
test tube. A cuvette is made using a special optical quality glass that will itself absorb a minimal amount
of the light. It is also marked with an indexing line so that it can be positioned in the light beam the
same way each time to avoid variation due to the differences in the composition of the glass.
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Lab #531, Introduction To Spectrophotometry, (5/21/07) Advancing Science Program, Gettysburg College, Gettysburg, PA 17325,
www.advancingscience.org, Science in Motion
Science in Motion
Viewing the Visible Spectrum
Introduction:
The spectrophotometer is designed to detect absorbances of light at different wavelengths when the light
passes through a solution of some given concentration. Some compounds absorb more light at one
wavelength than another, so the wavelength must be changed every time a different colored compound
is being analyzed to achieve optimum results from a spectrophotometer. The wavelength of light is
selected by adjusting the wavelength dial and read on the wavelength display. In this lab, the color of
light associated with each wavelength will be observed with the eye. The visible range of light is
approximately 400 to 700 nm. The very ends of the visible spectrum will also be determined in this
experiment. Please note that the accepted symbol for wavelength is the Greek letter lambda ().
Wavelength is related to frequency (ν) through the equation C=λν, where C is the speed of light.
Guiding Question: What is the relationship between wavelength and the color of visible light?
Student Prediction: _________________________________________________________________
Vocabulary:
wavelength, frequency, spectrum, nanometer
Materials Needed:
A piece of white chalk 1-2 cm long
Spectrophotometer
A cuvette/cuvette rack
Safety: Always wear safety glasses.
Procedure : (Best results are obtained by doing this experiment in a dimly lit room)
1. Place the piece of chalk in a cuvette with the angle end directed up.
2. Set the wavelength of the spectrophotometer to 425 nm. Be sure the filter switch is set to the left.
3. Place the cuvette in the spectrophotometer so the angle of the chalk faces to the right of the
spectrophotometer.
4. Open the light slit by turning the transmittance adjustment knob (right knob) clockwise.
5. Look into the sample compartment. Record on the data sheet the color of the light striking the chalk.
6. Repeat Step 5 increasing the wavelength by 25 nm each time. Continue the process until reaching
675 nm. At 600 nm, move the filter lever (#11 in the diagram) to the right.
7. While looking at the piece of chalk, slowly increase the wavelength to the point where the color is
no longer seen. This is one end of the visible spectrum. Record this wavelength value.
8. Adjust the wavelength back to 425 nm. While looking at the piece of chalk, slowly decrease the
wavelength to the point where the color is no longer visible. This is the other end of the visible
spectrum. Record this wavelength value.
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Lab #531, Introduction To Spectrophotometry, (5/21/07) Advancing Science Program, Gettysburg College, Gettysburg, PA 17325,
www.advancingscience.org, Science in Motion
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DATA SHEET
Period ____________________________
Name _____________________________
Date ______________________________
Lab Partner _______________________
Frequency
(Hertz)
Wavelength
(nm)
7.1 x 1014
425
6.7 x 1014
450
6.3 x 1014
475
6.0 x 1014
500
5.7 x 1014
525
5.5 x 1014
550
5.2 x 1014
575
5.0 x 1014
600
4.8 x 1014
625
4.6 x 1014
650
4.4 x 1014
675
Observed
Color
End of
Spectrum
Notes (anything unusual)
End of
Spectrum
Questions:
1. Why was a white piece of chalk used for this lab?
2.
a) At what wavelength do you no longer see the red color in the spectrum?
2.
b) At what wavelength do you no longer see the violet color in the spectrum?
3.
a) What would be an approximate wavelength of infrared light?
b) What would be an approximate wavelength of ultraviolet light?
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Lab #531, Introduction To Spectrophotometry, (5/21/07) Advancing Science Program, Gettysburg College, Gettysburg, PA 17325,
www.advancingscience.org, Science in Motion
Science in Motion
Viewing the Visible Spectrum
Teacher Notes
Lab Time:
Grade Level:
Objective:
10 – 15 minutes
8 – 12, physical science, chemistry, and physics
To observe the color of light emitted by the spectrophotometer at various wavelengths.
Preparations:
 Teachers should prepare chalk for students.

Turn the spectrophotometers on to warm up at least 20 minutes prior to the start of the lab. If the
spectrophotometers are not warmed up, you will notice some fluctuation of color when the
instrument is set at one wavelength.

Encourage the students to turn the wavelength knob and watch the colors change. Encourage
them to be as descriptive as possible. A good description of a color is for them to compare the
color to an object, for example if someone tells you that a shirt was pink like Pepto Bismol, you
KNOW the color!
Depending on individual perception of color, here is what the data should look like.
Wavelength (nm)
Color Observed
425
violet
450
violet/blue (lighter violet)
475
blue (aquamarine)
500
green with hints of blue
525
green, pure
550
green with yellow
575
yellow
600
orange
625
red with orange
650
red
Answers to Questions
1. Why was a white piece of chalk used for this lab? White chalk was used because white reflects all
colors of light and allows the entire visible region of the spectrum to be seen.
2.
a)
At what wavelength do you no longer see the red color in the spectrum? This will vary from
student to student, but should be between 700-710 nm.
2.
b)
At what wavelength do you no longer see the violet color in the spectrum? This will also vary
amongst students, but should be between 405-415 nm.
3.
a)
What would be an approximate wavelength of infrared light? Above 700 nm
3.
b)
What would be an approximate wavelength of violet light? Below 400 nm
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Lab #531, Introduction To Spectrophotometry, (5/21/07) Advancing Science Program, Gettysburg College, Gettysburg, PA 17325,
www.advancingscience.org, Science in Motion
Science in Motion
Considerations:
This lab is very quick, easy and creates no mess. It gives the students the experience of
seeing exactly how a spectrophotometer operates inside. It is recommended that this lab be used to
introduce the spectrophotometer before other spectrophotometer labs are used. By performing this lab,
students will begin to associate a wavelength with the particular color of light transmitted. This is useful
in understanding why certain wavelengths of light are chosen for various colored solution analyses.
More Fun Facts (Use as you see fit!)
The units for wavelength of light are nanometers, abbreviated nm. Most small things we deal with are
measured using the unit called “centimeter”. “Cent-” means “one hundred times smaller than”. “Nano” means “one billion times smaller than.”
The wavelength of a wave on a pond or in the ocean is measured using meters or centimeters. We can
easily pick out the “wave” on a pond. For light, however, the wavelength is SOOOO small, we cannot
even see that light is a wave!!
Wavelength
Did you know? The human eye can distinguish somewhere between 200 and 270 different colors.
Websites of Further Interest;
http://science.hq.nasa.gov/kids/imagers/ems/visible.html
http://en.wikipedia.org/wiki/Visible_spectrum
http://en.wikipedia.org/wiki/Electromagnetic_spectrum
http://www.glenbrook.k12.il.us/GBSSCI/PHYS/CLASS/light/u12l2a.html
Introduction to Spectrophotometry, Used with permission from the Chemistry in Motion program, Juniata College, Huntington, PA. Edited
and adapted by Jack Sipe, Jr., Jeanne Suehr, and Gregory J. Anderson for Advancing Science, Gettysburg College, Gettysburg, PA 17325.
http://www.advancingscience.org
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Lab #531, Introduction To Spectrophotometry, (5/21/07) Advancing Science Program, Gettysburg College, Gettysburg, PA 17325,
www.advancingscience.org, Science in Motion
← Circle pre or post lab →
Pre Lab
Post Lab
Advancing Science/SIM
Pre/Post Lab Quiz – Visible Spectrophotometry
1. A visible spectrophotometer (like a Spec 20)
a) produces its own visible light
b) absorbs light from the sun
c) analyzes samples that give off their own light
d) causes samples to glow in the dark
2. Samples that are analyzed on a Spec 20 must:
a) be colored
c) be dilute enough for light to pass through
b) be in solution form
d) all of the above
3. A “blank” must be used:
a) before every measurement
c) whenever the wavelength is changed
b) to set at 0% Transmittance
d) none of the above
4. A spectral curve showing wavelength of maximum absorbance (λmax) would look
like which of the following graphs?
a) A
b) A
c) A
d) A
5. The Beer-Lambert equations, A = c•ε•ℓ is best represented by which of the following
graphs?
a)
b)
c)
d)
Gettysburg College Advancing Science Pre/Post Quiz for activities including #531, 532,
533, 534, 536 and 537.