Chemistry 315

Experiment 01:
Fluorescence Measurements

Victoria Wisniewski

School of Chemical Sciences, 505 S. Mathews Ave., Urbana, IL 61801

vjw2@ilinois.edu

Lab partner: Sam Smith & Kean Zhu

Lab section: AB2, Tuesday PM

Major: Chemical Engineering

Date of lab work: Sept. 10th 2019

Due date of report: Sept. 17th 2019

This experiment utilized the RF-35301 Spectrofluorimeter. There were two parts to this
experiment. In the first part, the quenching rate constant of carbon tetrachloride was
determined using fluorescence spectroscopy and both Stern-Volmer expressions and
plots. The rate constant, kq, was determined to be 2.54 ns -1. The second part of this
experiment also utilized fluorescence spectroscopy, but also involved standard addition
methods. The purpose of this was was to determine the concentration on quinine in tonic
water B. The concentration was found to be 73.36 ppm quinine.

Keywords: FLUORESCENCE SPECTROSCOPY, QUENCHING, STERN-VOLMER

PLOT, QUININE, STANDARD ADDITION

1
Introduction
A quantum of light energy is called a photon, and when photons hit molecules, the
newly given energy pushes the molecule into the next excited state. The molecule returns
this energy once it reaches back to ground state, and the is considered fluorescence,
which is what this experiment is based on. For a molecule to go through the radiative
process that is fluorescence, it will jump from the first excited state to ground state.

The addition of a quencher will reduce the impact of fluorescence, and this
technique is used in multiple disciplines. For example, in an experiment done by South
China University of Technology, fluorescence quenching was used to better understand
the electron transfer mechanisms of biomass carbon quantum dots. This will allow a
better understanding of the role of quantum mechanics in nanotechnology, and further let
technology evolve to better imaging and sensing.2

In the experiment done in lab, carbon tetrachloride (CCl4) was the quencher for
anthracene, meaning that it reduced the intensity of the fluorescence of anthracene. The
molecules can be seen below.

Figure 1: Anthracene

Cl

Cl Cl
Cl

Figure 2: Carbon Tetrachloride

The Stern-Volmer expression is one that is heavily used with fluorescence. It is as

follows:

2
 Φ0 I 0
= =1+ k q∨Q∨ ¿ ¿ (1)
Φ I k f + k nr

Φ0
The variables explain relative quantum yield. Quantum yield is a fraction of how
Φ
much light energy was absorded that appears as emitted light using fluorescence.1 The

I0 Intensity without quencher

variables are basically . The concentration of the quencher
I Internsity with quencher
is expressed as Q. The rate constants in the Stern-Volmer expressions are as follows: kq is
the quenching rate constant and kf and knr are first-order rate constants. Something to note

I0
about the denominator (kf+knr) is that when is plotted against [Q] (otherwise known as
I
a Stern-Volmer plot), the trendline is:

ln [ A∗( t ) ]=ln [ A∗ ( 0 ) ] −( k f + k nr ) t (2)

This means that the absolute value of the slope in the trendline is (kf + knr), and
this was used in the prelab to determine that (kf + knr) = 0.28 ns-1. The quenching rate
constast can then be found using the Stern Volmer equation. A Stern Volmer plot can be
created with expiremental data points, and the Stern Volmer equation can be solved for
kq. Because of the ease of these calculations, using fluorescence for determining kq is a
very good choice.

The second part of the experiment is using fluorescence to determine the

concentration of quinine in tonic water Brand B.

HO N
O

Figure 3: Quinine

3
 In addition to using fluorescence, this part also uses standard addition. Standard
addition involves small additions to dilute a sample of initial volume V0. Because of the
dilution, the intensities that the instrument will give out are smaller than what they
actually are, so they need to be corrected using:

' V 0 +V
I= xI (3)
V0

Initial volume is noted as V0, while the total volume is V. The initial intensity is I, while
the corrected version is I’. 1

This method of determining concetration is very well-known and efficient. In a

study done at San Francisco State University, researchers were able to determine the
concentration in calcium in powdered milk by trying both external standard based
calibration and standard addition, along with fluorescence. With this study, they
determined that the method of standard addition was very accurate, only being 0.01% off
of the concentration that the manufacturer gave.3 With experiments like this in mind, it’s
clear that the use of fluorescence and standard addition can give an accurate value of
concentration of quinine in tonic water.

4
Experimental Details
This lab has two parts associated to it. Before either part was started, the RF-
35301 Spectrofluorimeter was turned on and calibrated properly. A slight deviation from
the lab manual guidelines is that the instrument’s software, RFPC, was recently updated
so the instructions given for the calibration weren’t accurate More specifically, settings
such as Confgiure -> Instrument were not found in the software. Additionally, the
recording range could not be found on the newly updated software. The parameters were
as follows: EX Wavelength: 366 nm, EM Wavelength Range (nm): Start: 350, End: 500,
Slit Width (nm): EX: 3, EM: 3.

In the first part of the expirement, a solution of 0.40M CCl 4 was made. Six 10 mL
were filled with 5.0 mL 4.0 x 10 -5 M anthracene, and then incriments of the CCl4 solution
as follows: 0.00 mL, 1.00 mL, 2.00 mL, 3.00 mL, 4.00 mL, and 5.00 mL. A sample was
pipetted into a quartz cuvette, and then inserted into the instrument. The Start button was
pressed and the instrument read the sample. Intensities were recorded at peaks around
382, 402, and 425 nm. This was repeated with the other samples.

In the second part of the experiment, the instrument was re-configured. The
parameters were as follows: EX Wavelength: 337 nm, EM Wavelength Range (nm):
Start: 350, End: 500, Slit Width (nm): EX: 1.5, EM: 3.A solution of 2.00 mL tonic water
Brand B (this is another deviation - the lab manual specified Bramd B but that wasn’t
found in lab), 1.00 mL 3M H2SO4, and 97.0 mL of DI water. Exactly 3.00 mL of the
solution was pipetted into a quartz cuvette and placed into the instrument. The instrument
read the sample, and the intensity of a peak around 450 nm was recorded. Then, in
increments of 0.10 mL, the solution was added to the cuvette and the sample was read
again. This was repeated until there were six total samples read. All waste was properly
disposed, and the instrument was turned off. 1

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Data and Results
In this section, both parts will be analyzed separately. The first part’s purpose was
to figure out the quenching rate constant of CCl 4. Three Stern-Volmer plots were created,
each showing the data from the three different peaks: 378 nm, 400 nm, and 425 nm. The
x-axis shows the concentration of the quencher, while the y-axis shows an intensity ratio:

I 0 Intensity without quencher

= (#)
I Intensity with quencher

[CCl4] vs Intensity at 378 nm

3
f(x) = 9.29 x + 1.1
2.5 R² = 0.92

1.5
I0/I

0.5

0
0 0.05 0.1 0.15 0.2 0.25
[CCl4] (M)

Figure 1: Graph of [CCl4] and intensity values associated at wavelength 378 nm

6
 [CCl4] vs Intensity at 400 nm
3
f(x) = 8.74 x + 1.06
2.5 R² = 0.89

1.5
I0/I

0.5

0
0 0.05 0.1 0.15 0.2 0.25
[CCl4] (M)

Figure 2: Graph of [CCl4] and intensity values associated at wavelength 400 nm

[CCl4] vs Intensity at 425 nm

3.5

3
f(x) = 9.19 x + 1.1
2.5 R² = 0.88

2
i0/I

1.5

0.5

0
0 0.05 0.1 0.15 0.2 0.25
[CCl4] (M)

Figure 3: Graph of [CCl4] and intensity values associated at wavelength 425 nm

To determine the quenching rate constant, the Stern-Volmer expression is used:

Φ0 I 0
= =1+ k q∨Q∨ ¿ ¿ (1)
Φ I k f + k nr

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The variables of this equation are elaborated upon in the Introduction section of the lab
report. It was determined that the slope of Figures 1-3 are equal to:

kq
slope= (#)
k f +k nr

It was pre-determined that

k f +k nr=0.28 n s−1 (#)

With (#) and (#), and using the slope from the slope intercept equation of each figure, the
kq can be determined. An example calculation is seen bellow for Figure 1, revolving
around the peak at 378 nm.

y=9.2913 x +1.0988

kq
9.2913=
0.28 n s−1

k q=2.60 n s−1

For figure 2 with wavelength peak at 400 nm, the quenching rate constant was
determined to be kq = 2.45 ns-1. For figure 3 with wavelength pea at 425 nm, the
quenching rate constant was determined to be kq = 2.57 ns-1.

Table 1: Summary of kq of quencher CCl4 in part one

Quenching rate constant (ns-1)

Peak of 378 nm 2.60

Peak of 400 nm 2.45

Peak of 425 nm 2.57

Average 2.54 ± 0.082

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 In the second part of this experiment, the concentration of quinine in tonic water
brand B was determined using the following steps.

First, the date collected by the instrument needed to be corrected using the below
equation. This is due to the standard addition method that was used to dilute the sample.
Using this method signifies that the intensity recordings are smaller than what they
actually are.

' V 0 +V
I= xI (#)
V0

An example calculation can be seen below:

3+ 0
I '= x 191.4=191.4
3

The values for all six samples can be seen in table 3 in the supplementary information
section. These values were plotted against the amount of quinine sulfate added, as seen in
Figure 4.

Volume of quinine sulfate vs intensity

1400

1200
f(x) = 1959.67 x + 203.92
1000 R² = 1

800
Intensity

600

400

200

0
0 0.1 0.2 0.3 0.4 0.5 0.6
Volume of quinine sulfate added (mL)

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Figure 4: Volume of quinine sulfate versus intesity at peak with wavelength by 450 nm

To find the initial amount of quinne sulfate in tonic water brand B, the slope intercept
line was used:

y=1959.7 x +203.92

0=1959.7 x+203.92

x=0.104 mL

Next, the following equation is used to determine the concentration of quinine sulfate in
the tonic water:

μL 1 100 mL
volum e0 ( mL ) x 50 x x =[ quinine sulfate ] ∈tonic water (ppm )
mL 3 mL 2 mL quinine sulfate

μL 1 100 mL
0.104 mL x 50 x x =86.71 ppm
mL 3 mL 2 mL

As determined in the prelab, quinine sulfate is 84.6% quinine, inducing the following
calculate:

( 86.71 )∗.846=73.36 ppm quinine

Discussion
This experiment can be considered successful. According to a journal publication
written by Rhode Island College, the quenching rate constant of CCl 4 is 2.9 ns-1.4 This is
close to the expiremental values found, with a percent error of 9.29%. A large portion of
this error is that the six samples of CCl 4 were too diluted with CCl4 due to human error.
This explanation fits well with the data. The samples had too much of the quencher,
therefore decreasing the intensities more than what was needed.

Quenchers absorb certain wavelengths that are emitting a photon due to a

fluorescent dye, which makes the light no longer visible and unreadable to the
instrument. This is how the CCl4 works – it does not allow light to come through, and

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therefore, the intensities are lowered. This is also why diluting the sample too much with
the CCl4 caused the intesnties to be even lower. The R2 values of all three figures were
large, rangin from 0.88-0.92. This means that the trendlines on the Stern Volmer plots
were accurate and consistent. There is, however, variation in the last data point in all
three figures, meaning that it didn’t follow the trend well. This most likely has to do with
the dilution of the last sample. It is reasonable to assume that the last sample, at 0.2M
CCl4, was the sample that was diluted too much as it has a lower intensity than expected.

In the second part of this expiment, tonic water was found to have 73.36 ppm of
quinine in tonic water brand B. The R 2 value is incredibly high, at 0.99, meaning that the
trendline is very consistent with all the data points, and this gives confidence that the data
is, at the very least, consistent with itself. To further prove that the concentration of
quinine is 73.36, it falls under the FDA’s regulations of quinine in tonic water. Quinine
content must be under 83 ppm, which the value calculated falls under. 1 These two points
prove that the experiment was run smoothly, and the results are accurate. The reason
standard addition was used in constast to other methods is that the sample is being used
for all experiments, and the samples only get more dilutes. This allows the sample to be
consistent since there is no variance in how the sample was made and is the reason why
the R2 value is so high.

Both parts in the experiment are considered successful, with results that have
good statistical accuracy and have values that were expected.

Chemical Engineering Question

If I were the CEO of the company, I would not be using mercury in CFLs.
Mercury is a very dangerous chemical, especially just being dispoed of in a landfill. This
loose chemical can now either evaporate into the air or leaching can occur, which is very
dangerous for the environment.5 In our current political state, many consumers are now
very influenced by the environmental impacts of a company, and therefor will be wary of
buying a product that contains such a damaging chemical. Additionally, if a company
knows that their products are damaging and is waiting for the government to ban it, I do

11
not think that is an ethical company. I would begin production in light-emitting diode
bulbs (LEDs). These bulbs last 2.5 times longer than CFLs, have an average $0.20
deduction in energy cost when compared to CFLs, and while are a much more expensive
investment, they are overall a much cheaper and more efficient option. They also do not
contain any mercury, therefore eliminating the unnecessary environmental impact that
CFLs have.6 Approximately 40 tons of mercury is released into the air each year,
meaning in 5 years, there will be an accumulation of 200 tons of mercury.7

References
1. Baker, C.J.; Dowd, S.E.; Gibson, B.; Hisao, G.S.; Lanni, E.J.; McCurry, D.A.;
Rabe, P.C.; Schilter, D.; Schmitt, K.G. Experiment 1. CHEM 315 Laboratory
Notes [Online] 2012. http://compass.illinois.edu (accessed Sep 13 2019).
2. Liang, Z.; Kang, M.; Payne, G. F.; Wang, X.; Sun, R. Probing Energy and
Electron Transfer Mechanisms in Fluorescence Quenching of Biomass Carbon
Quantum Dots. ACS Applied Materials & Interfaces 2016, 8 (27), 17478–17488.
3. Chan, J. C.; Palmer, P. T. Determination of Calcium in Powdered Milk via X-Ray
Fluorescence Using External Standard and Standard Addition Based
Methods. Journal of Chemical Education 2013, 90 (9), 1218–1221.
4. Chan, J. C.; Palmer, P. T. Determination of Calcium in Powdered Milk via X-Ray
Fluorescence Using External Standard and Standard Addition Based
Methods. Journal of Chemical Education 2013, 90 (9), 1218–1221.
5. Summary of Research on Mercury Emissions from Municipal Landfills.
http://www.newmoa.org/prevention/mercury/landfillfactsheet.cfm (accessed Sep
14, 2019).
6. Office of Pollution Prevention and Techincal Assistance. Lighting Alternatives.
https://www.in.gov/idem/files/factsheet_lighting_alternatives.pdf (accessed Sep
14, 2019).
7. Bolate, Y. Inventory of U.S. sources of mercury emissions to the atmosphere.
Columbia University, New York, NY, 2017.

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Supplementary Material
Table 2: All recorded intensities for CCl4 data for part one

Sample Wavelength (nm) Intensity

378 972.8

399 1015.9
1
423 547.6

379 677

299 764
2
423 393.9

378 530.9

399 584.1
3
423 300.3

378 401.8

399 433
4
423 221.1

378 344

399 369.5
5
423 191.6

379 367.2

399 410.2
6
423 213.8

Table 3: Recorded and corrected intensities for quinine sulfate for part two

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Sample Volume Wavelength Intensity Corrected
Quinine Added (nm) Intensity
(mL)

1 0 450 191.4 191.4

2 0.10 450 392 405.1

3 0.20 453 568 605.9

4 0.30 452 727.5 800.3

5 0.40 450 867.2 982.8

6 0.50 453 1009.4 117.6

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