S5: Generating an Absorption Spectrum with the OPN Spec
Chris Stewart and John Giannini*
St. Olaf College, Biology Department, 1520 St. Olaf Avenue, Northfield, MN 55057
* Email: [email protected]
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To generate a simple Absorbance spectrum using the OPN Spec, we followed several
of the steps that are set forth in the Supporting Information for our OPN Colorimeter
paper.1 As a result, we summarize our protocols here. We also briefly describe the
major hazards associated with this exercise, and we offer some helpful hints for
conducting this activity as part of a teaching lab at the conclusion of this supplement.
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Generating a Simple Absorbance Spectrum for Methylene Blue
Before beginning the assay, we prepared a 100 mg/L solution of Methylene Blue
(Certified Biological Stain) (Fisher Scientific; Hampton, NH) using deionized water (DI
H2O). We then set up the 3D-printed and wooden versions of the OPN Spec as
described in the prior supplements (S1 and S4), using removable mounting putty (i.e.,
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poster tack) to hold the various components in place on our table top. In each set-up,
we positioned the cuvette holder, so that its face was roughly 15 cm away from the
center of the DVD attached to the rotating post. For our light source, we used a Coast
G20 inspection light, and we used a PDV-P8103 photocell (i.e., light dependent resistor
or LDR) connected to a Craftsman digital multimeter (No. 82170) as our detector.
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Next, we turned on the Coast G20 light, rotated the DVD, and adjusted the cuvette
holder for the OPN Spec so that a narrow band from the middle of the violet spectrum
fell on the LDR. For both versions of the OPN Spec, we covered the empty cuvette
chamber with a lid and waited roughly 15 minutes for the flashlight, LDR, and
multimeter to warm up.
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During the warm-up period, we recorded the resistance values displayed on the
multimeter every two minutes to track the percentage change in these values. We also
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organized the materials needed for the activity: 200- and 1,000-μL commercial
pipettes, disposable pipette tips, plastic transfer pipettes, a large (e.g., 250 mL) beaker
and a separate squirt bottle both containing DI H 2O, a small (e.g., 2 mL) sample of the
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Methylene Blue solution (100 mg/L), and another large (e.g., 1,000 mL) empty beaker
for waste.
At the conclusion of the warm-up period, we recorded the resistance value
associated with an empty chamber in the violet spectrum, which generally ranged
between 47 and 293 kOhms for the 3D-printed version of the OPN Spec and between 16
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and 24 kOhms for the wooden version. However, these values could differ given the
strength of the batteries used, the distance between the DVD and the cuvette holder,
the specific wavelength striking the LDR, or other factors (e.g., depending upon the
color of the light hitting the LDR, the resistance for an empty chamber ranged between
5 and 293 kOhms in our tests).
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We used the resistance value associated with an empty chamber as the intensity of
the incident light (I ) striking the cuvette. We then filled a standard plastic cuvette with
3 mL of DI H2O, placed it into the OPN Spec, and recorded the corresponding resistance
value (for our “zero” reading). Next, we added 40 µL of the Methylene Blue solution to
the cuvette using a commercial pipette and gently suspended the mixture (5 times)
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using a disposable transfer pipette to ensure to thorough mixing. We then recorded the
corresponding resistance value, which we used as the intensity of the transmitted light
(T ) passing through the solution. Once finished, we emptied the contents of the cuvette
into a “waste” beaker and thoroughly rinsed out the cuvette into the beaker several
times to ensure that it was clean.
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Next, we rotated the DVD, so that a narrow band from the middle of the indigo
spectrum fell on the LDR. Then, we repeated the steps above, taking a reading for (i) an
empty chamber, (ii) the cuvette containing 3 mL of DI H2O, and (iii) the cuvette
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containing 40 µL of the Methylene Blue solution gently suspended in the 3 mL of DI
H2O. After emptying out and rinsing the cuvette into the waste beaker, we repeated the
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above steps for wavelengths in the middle of the blue, green, yellow, orange, and red
spectrums, respectively.
We then used the resistance values to calculate the Absorbance readings for each
cuvette at the different red wavelengths. Specifically, A = log10( T / I ), omitting the
negative sign from the traditional Beer-Lambert formula since the resistance of our LDR
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is inversely proportional to the amount of light striking it. In this equation, T denotes
the resistance associated with the transmitted light passing through the cuvette and I
denotes the resistance of the incident light striking the cuvette (i.e., the resistance of an
empty chamber). In addition, to account for the Absorbance of the DI H2O and its
container, we subtracted the Absorbance value associated with 3 mL of DI H2O in the
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cuvette for each color wavelength (i.e., our “zero” readings) from the corresponding
Absorbance values for the Methylene Blue solution. We then displayed our results as a
column graph to show how the Absorbance of our Methylene Blue sample varied with
wavelength.
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Hazards
As explained in the Supporting Information for the OPN Colorimeter, 1 Methylene
Blue is a hazardous chemical, and readers should exercise great care when working
with it. The compound can cause serious skin, eye, and respiratory irritation upon
contact, and it can also prove harmful if swallowed or inhaled. Methylene Blue has also
caused mutagenic effects and adverse reproductive effects in laboratory animals, and
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exposure to the chemical in large doses or over long periods of time can cause tissue or
organ damage. In addition, the substance can ignite if exposed to an open flame,
sparks, a hot surface, or other sources of heat or ignition. As a result, teachers and
students should exercise the proper level of care when working with the compound,
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including wearing the appropriate protective equipment (e.g., gloves, goggles, lab coats,
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masks or respirators, etc.). Teachers and students should also avoid the formation of
any vapors or mists when handling the substance, and readers should further work in a
well ventilated room away from any possible sources of ignition when using the
chemical.
For these reasons, we recommend that instructors prepare any dilute solutions of
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Methylene Blue if the compound is used as part of a laboratory exercise. In the
process, instructors should make sure to wear an appropriate dust mask or respirator
to protect against inhaling any Methylene Blue particles. Teachers and students should
also employ the proper lab etiquette when handling the substance, such as not eating
or drinking around the compound and thoroughly washing their hands after working
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with the chemical. Finally, readers should make sure to review the Material Safety Data
Sheet for Methylene Blue before working with the chemical, and any solutions
containing the compound should be disposed of as chemical waste (and not poured
down any drains) at the conclusion of any lab activity or classroom demonstration
involving the substance.
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Helpful Hints
In addition to the helpful hints provided in the Supporting Information for the 3Dprinted (S1) and wooden (S4) versions of the OPN Spec, we include a few additional
suggestions here, which might be useful in the context of a classroom demonstration,
teaching lab, or educational exercise.
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First, although a typical Absorbance spectrum would cover every wavelength in the
visible spectrum (and possibly even the ultra-violent and infra-red spectrums,
depending upon the instrument used), for simplicity and in the interests of time, we use
our OPN Specs to create a simple Absorbance spectrum for each one of the colors in the
rainbow (i.e., red, orange, yellow, green, blue, indigo, and violet). In our experience, this
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assay takes about 15 to 20 minutes to complete (after the warm-up period has
finished). Of course, depending upon the level of the class, instructors could have their
students take more readings (e.g., by recording two resistance values per color) or less
(e.g., by recording one resistance value in the blue, green, and red wavelengths only),
which of course would also change the amount of time needed for the exercise.
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Second, instructors could instead use “matched” cuvettes for the exercise, which
would expedite the process since students could use one cuvette as their “zero” sample
(i.e., by filling it with 3 mL of DI H2O only) and the other cuvette as their “sample” (by
suspending 40 µL of the Methylene Blue solution in the 3 mL of DI H2O that is already
there). However, matched cuvettes can be rather expensive (e.g., they cost between $40
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to $300 online for a single pair at October 2016 prices, depending upon the source).
While some readers might consider using two different plastic cuvettes from the same
batch instead, we do not recommend this approach because even small variations in
the cuvettes can lead to significant differences in the subsequent Absorbance
calculations. For example, in testing two different plastic cuvettes from the same batch
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from our stockroom (each filled with 3 mL of DI H2O), we found that their corresponding
resistance values varied by roughly 0.85% on average with a standard deviation of
0.96% (n = 23). However, the corresponding Absorbance readings for different
wavelengths in the visible spectrum varied by approximately 39.8% on average with a
standard deviation of 71.4% (n = 23). This large discrepancy is likely due to the
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logarithmic nature of the Absorbance function (A = -log10[ T / I ] ) because, depending
upon the value of the incident light I, small differences in the amount of transmitted
light T can lead to substantial differences in the calculated Absorbance value.
Finally, depending upon whether students use the 3D-printed or wooden version of
the OPN Spec, they may obtain slightly different Absorbance readings in their
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experiments (especially if generating a simple Absorption spectrum for one or more
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chemicals in solution). For example, if using a wooden version of the OPN Spec without
a cuvette lid and using only an index card for the face of the cuvette holder, then a
greater amount of ambient light will likely strike the sample (S4). As a result, “higher
Absorbance” wavelengths contained in that light may artificially inflate the readings of
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“lower Absorbance” colors (Fig. S5-1). Depending upon the sophistication of the class,
however, instructors could use the opportunity to help students think about the
reasons for the different results and possibly even design experiments to test their
hypotheses.
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Figure S5-1. Difference in Absorbance readings from a simple Methylene Blue spectrum generated
using the 3D-printed and wooden versions of the OPN Spec.
References
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(1) Stewart, C.; Giannini, J. Inexpensive, open source colorimeters that are easy to
make and use. http://pages.stolaf.edu/opn-lab/equipment/ (accessed Oct. 2016).
(2) Mahmood, T.; Anwer, F.; Mahmood, I.; Kishwar, F.; Wahab, A., Solvatochromic effect
of methylene blue in different solvents with different polarity. European Academic
Research 2013, 1, (6), 1100-1109.
(3) Prahl, S. Optical absorption of methylene blue. http://omlc.org/spectra/mb/
(accessed Aug. 2016).
(4) Whang, T. J.; Huang, H. Y.; Hsieh, M. T.; Chen, J. J., Laser-induced silver
nanoparticles on titanium oxide for photocatalytic degradation of methylene blue. Int. J.
Mol. Sci. 2009, 10, (11), 4707-18.
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