Alexis Musso*, Thomas Bateson
Using Spectrophotometry to Determine the More Effective Sequestering Agent of Procion Dye
Chem 112 – 553
April 10-17, 2013
Abstract
In the lab, spectrophotometry was used to determine whether magnetized Bentonite clay or
charcoal was the more effective sequestering agent of Procion Dye, a model PAH. By making a
calibration curve from known concentrations of dye solutions, the concentration of dye remaining after
adding clay and charcoal was determined from the plot. The major result was that charcoal is a better
sequestering agent because the charcoal removes more moles of dye per gram than the magnetic clay.
Introduction
PAHs, polycyclic aromatic hydrocarbons, are compounds that result largely from incomplete
burning of carbon-containing materials and pollute both the environment and our food 1. Because PAHs
include a large group of chemical compounds known to be cancerous, is it becoming increasingly
important to find ways to remove these compounds from the environment 2. Often, charcoal is used in
industries to adsorb PAHs from polluted water sources, but alternative adsorbents, such as clay, are being
investigated due to different requirements in economics, scale, and more 3. Therefore, in the lab,
previously learned methods of spectrophotometry were used to study the comparable effectiveness of
charcoal and magnetic clay as sequestering agents for PAHs, modeled by Procion, a red dye.
Materials and Methods
First, the Bentonite clay was synthesized. 250 mL of distilled water in a 600 mL beaker was
heated to 70°C on a hot plate using a thermometer. Then, 40 mL of distilled water, 0.7852 g of FeCl3, and
0.3928 g FeSO4·7H2O was added to a 250 mL Erlenmeyer flask, swirled to dissolve the iron compounds,
and then added to the water bath. After about 5-10 minutes in which the iron color darkened, the flask
was removed and 0.2977 g Bentonite was added and swirled to disperse the clay. Next, 20 mL of 2.5 M
NaOH was added to the solution drop wise, which formed a black, magnetic precipitate. Once the
solution was cooled, it was transferred to two centrifuge tubes and centrifuged for 5-10 minutes at 4000
rpm. The supernatant was disposed of in a waste container and the remainder of the solution was added to
the tubes, which were centrifuged a second time for 5-10 minutes at 4000 rpm. Once again, the
supernatant was disposed of. About 10 mL distilled water was added to the tubes, the tubes were shaken
to suspend the precipitate back into solution, and then they were centrifuged a final time. The supernatant
was disposed of one last time and the magnetic clay was scrapped into a beaker using a small spatula.
* Primary Author
Second, the wavelength of maximum absorbance for Procion Red dye was determined by
referencing Appendix 2 in the lab manual, which states the λmax for Procion Red Dye is 538 +/-3 nm 3.
After the Spec 20 spectrophotometer was allowed to warm up for 15 minutes, the wavelength knob was
set to 538 nm. Next, a standard curve was created. Using pipets, a 10 mL volumetric flask, and cuvettes, a
series of three dilutions of Procion Red dye with concentrations equal to 100%, 50%, 25%, and 12.5%
were prepared. The 100% standard was the 0.05 mM dye stock solution. The 50% standard was 5 mL of
0.05 mM of dye diluted with 5 mL of distilled water to create a concentration of 0.025 mM of dye.
Repeating the dilution process, the 25% standard was 0.0125 mM of dye and the 12.5% standard was
0.00625 mM of dye. Then, the Spec 20 was blanked with distilled water by turning the knob to 100%
transmittance. Finally, the % transmittance of the stock solution and its 3 dilutions were measured. In an
excel spreadsheet, these measurements were recorded, converted to absorbance, and plotted against
concentration to create the standard curve.
Finally, two centrifuge tubes were obtained and each filled with 15 mL of the 0.05 mM Procion
Red dye. About 0.3134 g of Bentonite clay and 0.3106 g of charcoal were weighed out by an analytical
balance, mildly grinded using a mortar and pestle, and added to the two separate centrifuge tubes. (Note:
Charcoal was redone a second time using 0.1007 g) The tubes were swirled to mix in the clay and
charcoal and allowed about 5 minutes to adsorb the red dye. Next, the centrifuge tubes were filtered with
filter paper into a 10 mL graduated cylinder. The filtered solutions were put into two different cuvettes.
The spectrophotometer was set to the wavelength of maximum absorbance λmax = 538 nm, blanked, and
the % transmittance of each cuvette measured. In the excel spreadsheet, the transmittances were recorded,
converted to absorbance, and read off the standard curve in order to determine the concentration of
Procion Red dye remaining.
Results and Discussion
In order to identify whether charcoal or magnetic clay was better at removing Procion dye (the
model PAH) from water, a calibration curve was first made using λmax = 538 nm and the samples from the
serial dilutions.
Chart 1: Serial Dilutions of Procion Red Dye to Make a Calibration Curve
Standard
Solutions
100%
50%
25%
12.50%
Concentration
(mM)
0.05
0.025
0.0125
0.00625
%T at 538 nm
18
43.2
63.4
79.4
T (%T/100)
0.18
0.432
0.634
0.794
A (-log(T))
0.7447275
0.3645163
0.1979107
0.1001795
Figure 1: Calibration Curve for Procion Red Dye at λmax = 538 nm
Absorbance vs. Concentration
0.8
0.7
y = 14.893x
R² = 0.999
Absorbance
0.6
0.5
λmax = 538 nm
0.4
Absorbance vs.
Concentration
0.3
0.2
0.1
0
0
0.01
0.02
0.03
0.04
0.05
0.06
Concentration (mM)
According to Beer’s Law, A = Ɛbc. For this lab, A is absorbance, Ɛ is molar absorptivity in units
mM-1·cm-1, b is the path length, and c is concentration in mM. From the graph, A = 14.893c, so the slope
14.893 = bƐ. Since b = 1 cm, Ɛ = 14.893 mM-1·cm-1 or 14893 M-1·cm-1. After the charcoal and clay were
added to remove the dye, the % transmittance of each filtered solution was measured and recorded from
the spectrophotometer into Chart 2. The remaining dye concentration was then calculated in Chart 3.
Chart 2: Charcoal and Clay Transmittance and Absorbance Results
Chart 3: Determining Concentration of Dye Remaining
Chart 4: Determining Moles of Dye Removed Per Gram of Substance
According to the results, charcoal removed more dye as seen by the higher % transmittance and
lower absorbance yielded from its filtered solution. Decreased absorbance means less dye was in the
solution to absorb light so more light was transmitted. Using the calibration curve equation, it was found
that the filtered charcoal solution had a lower concentration of dye remaining than the filtered magnetic
clay solution. Finally, in Chart 4, by analyzing the moles remaining by using molarity times concentration
to find the initial and final moles of dye, the amount of moles removed per gram of substance was found.
Charcoal is more effective because it removed 6.7 x 10-6 moles dye removed /g charcoal which is greater
than 1.1 x 10-6 moles dye removed / g clay.
To compare the usefulness of the magnetic clay to the charcoal, one parameter is higher effective
absorbance, which in the lab was determined as charcoal. A second parameter is the availability and cost
of each. Since the US is the largest producer of Bentonite clay, it is widely available and relatively cheap.
A third parameter important to industries is the ability to filter one substance over the other. Bentonite
clay is a very fine powder and thus difficult to filter. Making the clay magnetic to more easily remove it
though may have reduced its ability to adsorb PAH since charcoal was found to be more effective than the
magnetic clay. Furthermore, Procion Red Dye was a suitable model for a PAH because of its similarly
thin structure. Therefore, both are relatively flat molecules and can be adsorbed by Bentonite clay through
intercalation. Intercalation is due to the clay’s alternating layers of tetrahedral and octahedral atoms that
allows it to adhere to relatively flat molecules, such as PAHs, in the gaps between the layers 3.
Errors may have occurred. One error includes not filtering out all the charcoal and clay particles
from the solution, which would cause the absorption to increase and the transmittance to decrease, the
opposite of the results desired. That is the reason a second trial was done for charcoal because in the first
trial too many charcoal particles got into the filtered solution. Another error is not cleaning the cuvettes,
which means the data may have been slightly inaccurate due to dirty smudges or fingerprints.
Conclusion
The purpose of the lab was to design an experiment that tested a modified adsorbent material and
evaluated its efficacy compared to known standards. The results showed that charcoal, the known
standard, was the more effective sequestering agent as compared to magnetized clay because charcoal’s
filtered solution yielded a higher transmittance, lower absorbance, and lower concentration of dye as
compared to that of the magnetic clay. Charcoal removed 6.7 x 10-6 moles dye removed /g charcoal which is
greater than 1.1 x 10-6 moles dye removed / g clay. Overall, the experiment was successful because both
charcoal and magnetic clay were found to remove the model PAH from water, but as expected charcoal
removed more since magnetizing the clay may have reduced its effectiveness.
References
1. Srogi, K. "Monitoring of Environmental Exposure to PAHs: A Review." 5(2007): 169-195. Web.
15 Apr. 2013.
2. Wenzl, Thomas, Rupert Simon, Juliane Kleiner, and Elke Anklam. " Analytical methods for
polycyclic aromatic hydrocarbons (PAHs) in food and the environment ..." Trends in Analytical
Chemistry. 25.7 (2006): 716-25. Web. 15 Apr. 2013. <http://www.pfigueiredo.org/met_8.pdf>.
3. Leung, Tom, and Amber Schaefer, eds. "Appendix 2 Procion Red MX-5B." General Chemistry of
the Texas Environment. 3rd ed. Plymouth, MI: Hayden-McNeil, LLC, 2013. 315. Print.