Time Resolved Pulsed Laser Photolysis Study of Pyrene Fluorescence Quenching by I- Anion
Michael Buchanan
Partners: Derek Rowe, Alex Schneider, and Justin Miller
Chem 457 Sec. 006
February 27, 2015
Abstract
This experiment focuses on fluorescence which is the process of exciting an electron by
absorbing a high energy photon to an excited state and then emitting that photon to drop back
down to ground state, this can then be seen in the visible light spectrum. The time it takes for an
electron in an excited state to drop to ground state can be changed by altering the variables of the
system. This experiment shows the rate of emission to the ground state of an electron from
pyrene was changed by adding I- anions to the pyrene solution. The excited state of pyrene is
able to accept electrons from I- causing it to form Py- and I. In this experiment the rate of
emission to the ground state of pyrene and the change in rate emission to the ground state with
the addition of I- were measured. These two measurements were then taken to find the rate
constants of the quenching of pyrene and the pyrene fluorescence.
Introduction
Fluorescence spectroscopy is process used to analyze the fluorescence of a given
sample using ultraviolet light. The laser is used to excite the electrons of the molecules to cause
them to go into an excited state and then emit photons as they go back to their ground state. From
the emitted photons, the excited state of a specific compound can be examined and determined.
Using the properties of the compound, the concentration of the sample can then be determined if
need be. Using Fluorescence spectroscopy is important to be careful because it uses an extremely
sensitive detector as a way to determine different structures and solution states. Being able to
determine the structure of a molecule is extremely important and useful in modern day
application like determining the difference in malignant tumors from benign tumors.1
The laser has to give off a certain wavelength to excite the compound so then once the
compound is excited, the compound goes to the excited state and then emits photons as it goes
back to its ground state. The photons produce a charge on the photodiode in the laser and then
the photodiode uses the charge to find the intensity of the fluorescence in the compound. The
process of quenching is used to help the transition back to the ground state for the compound and
it decreases the photon intensity given off during the process.
Experimental Method
Five mixtures were created for the experiment. These mixtures consisted of pyrene,
potassium iodide, and 50/50 ethanol/water in a 10 mL flask. Each flask contained 10 μM of
pyrene and some concentration of KI (0mM, 10mM, 20mM, 30mM, and 40mM). One by one,
the flasks were put into a cuvette and were purged under pure nitrogen for five minutes to avoid
quenching by oxygen from the air. The cuvettes were then placed in the photolysis apparatus
(Figure 1). 2
Figure 1: Experimental Apparatus
Three data points were taken for each of the flasks and then were saved onto an USB drive to
later analyze and use for our data.
Results
The oscilloscope measures the fluorescence intensity over time. It then displayed a graph
of Intensity vs time and using an exponential curve for the different graphs, we were about to
find kobs.
Figure 2: Fluorescence Intensity vs Time
These data points are the average of the three runs for each flask to give an average kobs
for each of the flasks. By taking the equations above for each flask and applying it to the
equation below, kobs was able to be found for each of the flasks. Example, for the 10mM
solution, the 𝑘𝑜𝑏𝑠 = 4 × 106
𝑚𝑀
𝑠
.
𝐼 = 𝐼𝑜 𝑒 −𝑘𝑜𝑏𝑠 𝑡
Equation 1: Equation if Intensity I is treated as proportional to excited pyrene concentration.
Once the kobs was found for each of the flasks, it was then plotted against the
concentration of iodide anions to determine k0 and kq by using the graph below.
1.E+07
9.E+06
Kobs
8.E+06
7.E+06
y = 1.385E+8x + 3.484E+6
R² = 9.001E-1
6.E+06
5.E+06
4.E+06
3.E+06
0
0.005
0.01
0.015
0.02
0.025
0.03
0.035
0.04
[I-]
Figure 3: kobs vs [I-]
Plotting the kobs against the concentration of the iodide anion gave the values for the rate
constants, k0 and kq. Both of these rate constants can be determined by the slope of the line (𝑘𝑞 =
1.385 × 108
𝑚𝑀
𝑠
) and by the y-intercept (𝑘0 = 3.484 × 106
𝑚𝑀
𝑠
).
Discussion
The rate of emission to the ground state of pyrene with iodide both depends on the
amount of pyrene and iodide anions within the solution. Using the graphs and equations for the
data, we can see that as there are more iodide anions than pyrene the rate is a pseudo first order
reaction. With this information, the rate of emission to the ground state is proportional to the
amount of pyrene in the solution while the rate constant is proportional to the amount of iodide
anions in the solution. If the equations are rearranged, then the rate constant for quenching can
be found while changing the amount of iodide anions. This can be seen in the emission rate
constants kobs that were calculated for each of the five flasks that contained pyrene and different
concentrations of potassium iodide. The relative intensity of fluorescence vs. time was plotted to
for kobs by using linear regression analysis. Then after calculating the kobs for each of the flask,
kobs vs [I-] was plotted to find the emission rate of pyrene and the quenching rate (k0 and kq). In
the graph of kobs and [I-], k0 is the y-intercept and kq is the slope of the line.
As seen in the two graphs, the residual value for the Figure 3 is much smaller than those
that are in Figure 2. This is likely due to the way the experiment way done. As the mixtures
were made, the potassoium iodide had to measured out and put in the 40mM flask and was
mixed with the 50/50 ethanol/water solution. Each of the other flasks would have some sort of
amount put in them from this original flask. The amount take from the original flask and put into
the pther flasks may not have been the exact concentration as need. Another aspect is the purging
under pure oxygen to prevent quenching from oxygen in the air. As the purging concluded for
each sample, the extraction point of the purging needle being taken out of the cuvette might have
let air in causing the samples to quench a bit.
Conclusion
To calculate the emission rate constant, the reaction of adding photons to pyrene to an
excited state had to be treated as a first order reaction. Using this knowledge, he fluorescence
intensity of pyrene was plotted against time, which gave us the observed rate constants. Once we
had kobs, it was plotted against the concentration of iodide anions to find k0, the emission rate
with no quencher present, and kq, the quenching rate. Using these rate constants and assume it to
be a pseudo-first order reaction, the rate constants for quenching of pyrene by iodide were
calculated. This experiment demonstrated the importance of fluorescence spectroscopy in all
types of chemical systems, making it a very useful analytical tool that can be used to determine
unique properties of molecules.
Acknowledgements
Chemical and Mechanical support from Dr. Milosavljevic and the whole Department of
Chemistry. Assistance with experiment and accurately assessing questions from Codey
Henderson.
Literature Cited
1. Choi, J; Gupta, S; Park, I; Lee, D; Ye, J.C. Breast cancer diagnosis from fluorescence
spectroscopy using support vector machine. Optical Tomography and Spectroscopy of Tissue
2007, 6434.
2. Milosavljevic, B. Lab Packet for CHEM 457 Experimental Physical Chemistry. The
Pennsylvania State University. Spring 2015 pp. 5-6.
Appendix
Sample Calculations
Mass of KI = (M ∗ V) ∗ MW
(0.1 M ∗ 0.01L) ∗ (166
g
)
mol
= 0.1676 g KI
6𝑡
𝑘𝑜𝑏𝑠 : 𝐼 = 𝐼𝑜 𝑒 −𝑘𝑜𝑏𝑠𝑡 For example, 10mM 𝑦 = 0.2901𝑒 −4×10
𝑘𝑞 = 1.385 × 108
𝑚𝑀
𝑠
& 𝑘0 = 3.484 × 106
𝑚𝑀
𝑠
→ 𝑘𝑜𝑏𝑠 = 4 × 106
𝑚𝑀
𝑠
, both are taken from the equation of kobs vs [I-].
Uncertainty analysis/error propagation
Uncertainty of the slope and y-intercept of the kobs versus [I-] (observed rate constant vs
concentration of the iodide anions).
Slope: ∆95% 𝑚 =
𝑠𝑚 ×𝑡95%
√𝑁
Y-Intercept: ∆95% 𝑏 =
Other Data
=
(2.753×107 )×2.78
𝑠𝑏 ×𝑡95%
√𝑁
√5
=
= 3.423 × 107 (
(6.74×105 )×2.78
√5
𝑚𝑀
𝑠
= 8.38 × 105 (
)
𝑚𝑀
𝑠
)
SUMMARY OUTPUT
Regression Statistics
Multiple R
0.953462617
R Square
0.909090962
Adjusted R Square
0.878787949
Standard Error
870535.1601
Observations
5
ANOVA
df
Regression
Residual
Total
Intercept
X Variable 1
1
3
4
SS
MS
F Significance F
2.2735E+13 2.27E+13 30.00002 0.011967
2.27349E+12 7.58E+11
2.50085E+13
Coefficients Standard Error
t Stat
P-value Lower 95%Upper 95%
Lower 95.0%
Upper 95.0%
3115655.593
674313.6354 4.620484 0.019082 969688.7 5261623 969688.7 5261623
150781160.8
27528738.89 5.477227 0.011967 63172427 2.38E+08 63172427 2.38E+08
0mM KI
K1
3,476,372.13
K2
3,485,766.36
K3
3,489,507.44
Average
3483881.979
30mM KI
K1
8,219,674.26
K2
8,214,372.25
K3
8,103,426.32
Average
8179157.61
10mM KI
K1
3,523,941.39
K2
3,511,544.31
K3
3,523,172.16
Average
3519552.622
40mM KI
K1
8,671,045.79
K2
8,712,155.41
K3
8,696,211.37
Average
8693137.523
20mM KI
K1
6,706,591.03
K2
6,760,705.24
K3
6,874,696.66
Average
6780664.31