Time Resolved Pulsed Laser Photolysis Study of Pyrene Fluorescence Quenching by IAnion
CHEM 457, Fall 2013
Submitted: September 10, 2013
Undergraduate Chemistry Department, The Pennsylvania State University
Kristen Woznick
Lab Partners: Tim Haggerty, Kelly Helfrich, Tim Haggerty, and Arjun Plakkat
Abstract
In this experiment, fluorescence decay using a laser photolysis technique was used to measure
the rate constants for unimolecular decay of the pyrene’s first singlet state. This experiment
allowed the relaxation rate of excited Pyrene to be observed due to both fluorescence and
quenching. The quenching rate constant, kq, following a pseudo-first order reaction system had a
value of (1.0 ± 0.4) x108 M-1s-1 due to the Iodine quencher. The rate constant for inherent
unimolecular decay of pyrene was (3.9 ± 0.9) x106 s-1 due to spontaneous fluorescence
decay.
1.0 Introduction
Molecules differ in molecular properties in ground state versus excited state.3 When a
molecule is in excited state, the excited electron is paired with an electron in the ground state
orbital by opposite spin.4
Py + hν1*Py
When the electron returns to the ground state, this is completed either through fluorescence, a
highly sensitive technique, or radiationless decay; photons are emitted and undergo small
vibrations thereby lowering the energy.3
*PyPy + hν2 (fluorescing)
*PyPy + heat (radiationless decay)
The intensity of the fluorescence is decreased through the quenching by another molecule, in this
case Iodine.1 Through quenching, the relaxation time will be increased for the electron to
transition from the excited to ground state.
Pyrene is an alternant aromatic compound comprised of four benzene rings seen in
Figure 1.5 Pyrene is a polycyclic aromatic hydrocarbon that is a product of the burning coal, oil,
gas, or garbage.5
Figure 1. Pyrene
When excited, Pyrene undergoes redox only in the company of a compound with lower reduction
potential.3 Excited Pyrene reluctantly accepts electrons from and Iodine anion in order to reach
the ground state and therefore forms a Pyrene anion and an Iodine radical through photo induced
electron transfer reaction, an energetically favorable reaction.3
*PyPy- + I*
This Iodine is our quenching concentration.
In looking more specifically at the kinetics of this reaction, it is noticed that concentration
of Iodine is greater than the concentration of excited Pyrene allowing the assumption to be made
of pseudo first order reaction. Consequently, the concern in this experiment is the Pyrene of
4.79*10-7 M concentration. From this assumption, it is then possible to determine the rate
constants for the decay of the excited Pyrene molecules, the fluorescence rate constant, and the
quenching rate constant.
2.0 Experimental Method
In reference to the CHEM 457 Experimental Physical Chemistry Lab Packet, Fall 2013,
the following 0 mM, 10 mM, 20 mM, 30 mM, and 40 mM samples were prepared in 10 mL
volumetric flasks through serial dilution. The stock solution, the 40 mM [KI] concentration, was
made by transferring .1659 g ± 0.0004 g KI using the AE 100 Mettler Balance into the 10 mL ±
.05 mL volumetric flask type B. Then the remaining volume in the volumetric flask was filled
with 50% ethanol-water solution to dissolve the KI particles and then different amount were
taken from the 40 mM and transferred to the 0 mM, 10 mM, 20 mM, and 30 mM solutions with a
1 mL ± .01 mL pipette. All samples were added with 2 mL of 100 μM pyrene in ethanol solution
and then varying volumes of 50% water-ethanol solution to give the total 10 mL. These
appropriate volumes can be seen in Table 1.
Table 1. Sample Preparation with appropriate volumes of components
Volume of 0.1 M
Volume of 100 μM
Volume of 50%
pyrene in ethanol
water-ethanol
solution
solution
solution of KI + 50%
Sample Number
KI Concentration
water-ethanol
solution
1
0 mM
0 mL
2 ± .01 mL
8 ± .01 mL
2
10 mM
1 ± .01 mL
2 ± .01 mL
7 ± .01 mL
3
20 mM
2 ± .01 mL
2 ± .01 mL
6 ± .01 mL
4
30 mM
3 ± .01 mL
2 ± .01 mL
5 ± .01 mL
5
40 mM
4 ± .01 mL
2 ± .01 mL
4 ± .01 mL
After sample preparation, the oxygen was removed from the samples through nitrogen purging
for five minutes by inserting a needle into the sample containing cuvet with cap in order for
bubbling to occur. The needle was cleaned with ethanol between the purging of the different
samples due to contamination precaution due to the high sensitivity of the oscilloscope. Oxygen
was required to be removed because this can also act as a quencher within the defined system of
aromatic hydrocarbons. However, there was no precise manner to measure the oxygen content
that could have leaked into the system. A suggestion for this is offered in the discussion.
Figure 2. Laser Photolysis Setup
In Figure 2, the first piece of equipment, the nitrogen pulsed laser, excited photons by emitting
Ultraviolet light at a constant wavelength, 337.1 nm, emitting 2.883*1014 photons. This laser was
emitted 5 different instances, one for each concentration of the samples. The samples in a cuvet
were clamped in front of the laser. The pulse rate did not need to be adjusted since the cartridge
was recently changed. The photons were collected using the semiconductor photodiode while the
light below 375 nm was absorbed by the optical filter in order to prevent interference. This
photovoltaic characteristic produced a voltage on the oscilloscope. Voltage is then proportional
to the number of photons that underwent fluorescence. This experiment was only repeated once
due to time limitations within the lab.
Results/ Discussion
Using the laser photolysis setup, the raw data contained the fluorescence intensity
versus time data. In order to better see the graph, the time was converted to nanoseconds
from the given seconds. This data was then best-fit with an exponential best-fit trend line
using Microsoft Office Excel. In order to plot the exponential fit, the initial baseline and
increase in the fluorescence curve values were deleted. This can be seen in Figure 3.
Figure 3. Plot of fluorescence intensity (arb units) versus time (ns) from the raw experimental data taken from
the oscilloscope best fitted with an exponential trend line.
The k(observed values) can be seen as 0.003, 0.006, 0.007, 0.009, 0.008 ns-1 are seen for the 0,
10, 20, 30, 40, and 50 mM concentrations respectively. The 30 mM concentration [KI] sample
has a steeper and faster exponential decay than compared to the 40 mM [KI] sample. This is in
question and needs verified with possible error. The more concentrated [KI] solution should
decay faster due to more quencher. In order to gain a better perspective, the natural logarithm of
the intensity was plotted due to the greater accuracy within excel to fit data towards linear
regression instead of exponential decay. Figure 4 was produced. In looking at this data, the
natural logarithm of intensity versus time yields has a linear regression line with a slope that is
equal to –ko. This data was truncated to reduce the noise in the data that occurred after some time
as it can be seen that as the concentration increased the lifetime was shorter than the previous.
Figure 4. Plot of the natural logarithm of intensity (arb units) versus time (ns) yields a line with a slope equal to –
Ko(s-1)
As can be seen, approximately 99% of the data correlates with the best-fit equations and
linear regressions analysis located in the appendix. The appropriate ko values were seen
and increased with the increased concentration of Iodine quencher.
[I-] mM
0
10
20
30
40
kobs (s-1)
337.1x104
511.0x104
70.1x105
90.6x105
77.2x105
Standard error (s-1)
0.3x104
0.7x104
0.1x105
0.2x105
0.1x105
R2
0.9975
0.9973
0.9960
0.9966
0.9963
Table 2. Ko values correlated with appropriate [KI] concentrations.
In using these observed K values, these were plotted against the [I-] concentration (mM) to
determine the quenching rate constant, which is the slope of the best-fit line and yielding a
y intercept of the ko value.
Figure 5. K(observed) (ns-1) versus Iodine concentration (mM) to find the best-fit equation to determine value of
quenching constant.
The quenching rate constant is (1.0 ± 0.4) x108 M-1s-1 and the ko value is (3.9 ± 0.9) x106 s-1
for no Iodine quencher concentration. The ko value only accounts for fluorescence. This
large quenching constant indicates that Iodine is a strong quenching agent. However, in
looking at this best-fit line the experimental ko value should be 3.4x106 s-1 offering a
disagreement since only approximately 79% of the data is within correlation. Therefore,
possible error can be seen in doing the serial dilution in preparing the samples. The
calculated uncertainty of the [KI] was determined.
Table 3. The Determined and reported uncertainties for [KI] for each serial dilution with calculations located in
the appendix.
[KI] (mM)
Calculated Concentration of [KI] (mol/L)
-3
Standard Deviation (mol/L)
±0.7x10-3
40
99.9x10
30
30x10-3
±1x10-3
20
2.0x10-3
±0.5x10-3
10
10.0x10-3
±0.1x10-3
0
0
-
This uncertainty explains why the intensity of the 30 mM is greater than the 40 mM Iodine
concentration as time advances. In doing the serial titration to make the 30 mM sample, 3 mL of
solution was removed from the stock solution, 40 mM [I-]. Taking into account relative error of
the pipetting and the volumetric flask, the [KI] concentration could have been 31 mM and the 40
mM [I-] sample could have been 99.2 mM. Yet, the 30 mM solution could have been more
concentrated than the diluted 40 mM sample thereby having less [KI] concentration. Human is
an error source in the pipetting technique by not adding enough or too much of the intended
volume of the solution during the dilution technique.
3.0 Conclusion
Overall, the inherent spontaneous decay of Pyrene provided a good interpretation of the
unimolecular fluorescence decay showing the expected exponential best-fit data. Although the
quenching rate constant and rate constant without quenching were calculated, difference in error
between the 30 mM and 40 mM Iodine concentration samples resulted. This was explained due
to the serial dilution technique in which the 40 mM [I-] acted as the stock solution. Repeat of
experiments could have provided better statistical data and a more precise calculation for the
needed rate constants. In order to improve the experiment, diverse quenchers could have been
engaged in the experiment to further support the theory that was observed in the experiment in
order to see the effects of quencher on the rate constants given different molecular properties.
4.0 Acknowledgements
I would like to acknowledge my other laboratory group members: Tim Haggerty, Kelly
Helfrich, and Arjun Plakkat for their effort and problem solving expertise through the experiment
as well as the availability of Jennifer Tan to provide guidance in the data analysis.
5.0 References
(1) “Fluorescence Quenching by Electron Transfer.”
http://www.d.umn.edu/~psiders/courses/chem4643/labinstructions/etQuench.pdf
(accessed September 7, 2013).
(2) Lakowicz, Joseph R. 2006. Principles of Fluorescence Spectroscopy (3rd edition), pp 1-24.
(3) Milosavljevic, B.H. Lab Packet for CHEM 457: Experimental Physical Chemistry, Time
Resolved Pulsed Laser Photolysis Study of Pyrene Fluorescence Quenching by I- Anion.
University Press: University Park, 2013.
(4) “Photochemistry of Aromatic Molecules.”
http://turroserver.chem.columbia.edu/courses/MMP_Chapter_Updates/MMP+Ch%2012
%20050704.pdf (September 7, 2013).
(5) “Pyrene.”
http://www.epa.gov/osw/hazard/wastemin/minimize/factshts/pyrene.pdf (September 7,
2013).
6.0 Appendix
Photons in the laser pulse of 170 μJ:
𝑚
−34
𝐽 ∗ 𝑠)(3.00 × 108 𝑠 )
ℎ𝑐 (6.626 × 10
𝐸=
=
= 5.9 × 10−19 𝐽
𝜆
337.1 × 10−9 𝑚
170 × 10−6 𝐽 ×
1 𝑝ℎ𝑜𝑡𝑜𝑛
= 2.9 × 1014 𝑝ℎ𝑜𝑡𝑜𝑛𝑠
5.9 × 10−19 𝐽
Concentration of excited photons:
𝐶=
𝑛 2.883 × 1014 𝑝ℎ𝑜𝑡𝑜𝑛𝑠
1 𝑚𝑜𝑙𝑒
=
×
= 4.79 × 10−7 𝑀
𝑉
0.001 𝐿𝑖𝑡𝑒𝑟𝑠
6.02 × 1023 𝑝ℎ𝑜𝑡𝑜𝑛𝑠
Calculation to determine amount of KI needed to prepare 10 mL of 0.1 M solution
. 1 𝑀𝑜𝑙𝑒𝑠
1 𝐿𝑖𝑡𝑒𝑟
166.0277𝑔 𝐾𝐼
× 10 𝑚𝐿 ×
×
= .1660 𝑔 𝐾𝐼
𝐿𝑖𝑡𝑒𝑟
1000 𝑚𝐿
𝑚𝑜𝑙
Uncertainty for [KI] for 40 mM solution:
Concentration:
1 𝑚𝑜𝑙
𝑀 (0.1659 𝑔)(166.0277 𝑔 )
𝑀𝑜𝑙
𝐶= =
= 9.992 × 10−2
= 99.9 × 10−3 𝑀 = 99.9 𝑚𝑀
1
𝐿
𝑉
𝐿
(10 𝑚𝐿)(
)
1000 𝑚𝐿
Relative error:
(𝑏𝑎𝑙𝑎𝑛𝑐𝑒) + (𝑣𝑜𝑙𝑢𝑚𝑒𝑡𝑟𝑖𝑐 𝑓𝑙𝑎𝑠𝑘) =
0.0004 𝑔 0.00005𝐿
+
= (7.411 × 10−3 )(𝐶)
. 1659 𝑔
0.01 𝐿
= 0.7 × 10−3 𝑀 = 0.7 𝑚𝑀
Uncertainty:
99.9 𝑚𝑀 ± 0.7 𝑚𝑀
Uncertainty for [KI] for 30 mM solution:
Concentration:
1𝐿
−2
𝑀 (3 𝑚𝐿 × 1000 𝑚𝐿)(9.99 ∗ 10
𝐶= =
1𝐿
𝑉
(10 𝑚𝐿)(
)
𝑚𝑜𝑙
𝐿
)
= 30.0 × 10−3 𝑀 = 30.0 𝑚𝑀
1000 𝑚𝐿
Relative error:
(3 × 𝑝𝑖𝑝𝑒𝑡𝑡𝑒) + (𝑣𝑜𝑙𝑢𝑚𝑒𝑡𝑟𝑖𝑐 𝑓𝑙𝑎𝑠𝑘) =
. 00001 𝐿 . 00001 𝐿 . 00001 𝐿 0.00005 𝐿
+
+
+
0.001 𝐿
0.001 𝐿
0.001 𝐿
0.01 𝐿
= (3.500 × 10−2 )(𝐶) = 1 × 10−3 𝑀 = 1 𝑚𝑀
Uncertainty:
30 𝑚𝑀 ± 1 𝑚𝑀
Uncertainty for [KI] for 20 mM solution:
Concentration:
1𝐿
−2
𝑀 (2 𝑚𝐿 × 1000 𝑚𝐿)(9.99 ∗ 10
𝐶= =
1𝐿
𝑉
(10 𝑚𝐿)(
)
𝑚𝑜𝑙
𝐿
)
= 2.0 × 10−2 𝑀 = 2.0 𝑚𝑀
1000 𝑚𝐿
Relative error:
(2 × 𝑝𝑖𝑝𝑒𝑡𝑡𝑒) + (𝑣𝑜𝑙𝑢𝑚𝑒𝑡𝑟𝑖𝑐 𝑓𝑙𝑎𝑠𝑘) =
. 00001 𝐿 . 00001 𝐿 0.00005 𝐿
+
+
0.001 𝐿
0.001 𝐿
0.01 𝐿
= (2.500 × 10−2 )(𝐶) = 0.5 × 10−3 𝑀 = 0.5 𝑚𝑀
Uncertainty:
2.0 𝑚𝑀 ± 0.5 𝑚𝑀
Uncertainty for [KI] for 10 mM solution:
Concentration:
1𝐿
−2
𝑀 (1 𝑚𝐿 × 1000 𝑚𝐿)(9.99 ∗ 10
𝐶= =
1𝐿
𝑉
(10 𝑚𝐿)(
)
𝑚𝑜𝑙
𝐿
)
= 10.0 × 10−3 𝑀 = 10.0 𝑚𝑀
1000 𝑚𝐿
Relative error:
(1 × 𝑝𝑖𝑝𝑒𝑡𝑡𝑒) + (𝑣𝑜𝑙𝑢𝑚𝑒𝑡𝑟𝑖𝑐 𝑓𝑙𝑎𝑠𝑘) =
. 00001 𝐿 0.00005 𝐿
+
= (1.500 × 10−2 )(𝐶)
0.001 𝐿
0.01 𝐿
= .1 × 10−3 𝑀 = 0.1 𝑚𝑀
Uncertainty:
10.0 𝑚𝑀 ± 0.1 𝑚𝑀
Linear regression analysis of kq for 0 mM [I-]:
Linear regression analysis of kq for 10 mM [I-]:
Linear regression analysis of kq for 20 mM [I-]:
Linear regression analysis of kq for 30 mM [I-]:
Linear regressions analysis of kq for 40 mM [I-]:
Linear Regressions analysis for kq and ko: