Fluorometric Measurement of the Rate Constant and Reaction
Mechanism for Ru(bpy)32+ Photophorescence Quenching by O2
Matt Beaner
Ahmed Rebh
Matt Micklin
David Belias
Penn State University
Chem457
Section 4
03/19/15
Abstract:
The rate constant for relaxation of *Ru(bpy)32+ by oxygen quenching was found to be
4.1584x109 ± 1.074x108. The Kd, or collision rate of Ru(bpy)32+ was found to be 6.483x109. A UV
spectrum was used to determine the wavelength that Ru(bpy)32+ would become excited from
and a fluorimeter provided quantum yield data. The rate constants were determined by firing
337.1nm laser photons at samples of Ru(bpy)32+ containing nitrogen, air, and oxygen.
Fluorescence lifetimes and quenching constants are important in the field of biology where
fluorescence lifetime imaging microscopy can be used to observe samples.
Introduction:
Ru(bpy)32+ can be excited via 337.1nM light. This moves thee molecule into a higher
energy state. The excitation takes place via equation 1;
Ru(bpy)32++ hv * Ru(bpy)32+ – excitation mechanism(1)1
The excited molecule wants to return to its original energy state, or to at least to a lower
energy state. It can relax by either oxygen quenching, fluorescence, or non-radiative decay.
Within each of these mechanisms there are other possibilities such as: intersystem crossing,
internal conversion, and vibrational relaxation to name a few.2
Figure 1 – Generalized Jablonski diagram for
different possibilities2
Figure 1 is a generalized Jablonski diagram which shows the different possible ways or
an excited molecule to move to a lower energy state. The initial adsorption, internal
conversion, fluorescence, intersystem crossing, and phosphorescence are all shown. This shows
why the rate constants even matter and why they are being studied. A molecule wants to reach
a lower energy state as quickly as possible, this is limited by its rate constant for each individual
method and therefore it moves to a lower energy state through different ways.
Oxygen quenching occurs when molecular oxygen reacts with the excited state molecule
which leads to a lower energy state. There are a few different mechanisms for quenching but
they all involve excited Ru(bpy)32+ moving to a lower energy state.
*Ru(bpy)32+ +O2  Ru(bpy)32+ - oxygen quenching mechanism (2)1
Fluorescence occurs when the excited molecule gives off a photon of light causing it to
move to a lower energy state.
*Ru(bpy)32+  Ru(bpy)32+ + hv – fluorescence relaxation mechanism (3)1
Finally non-radiative decay occurs when energy is given off through heat loss to the
surrounding solution.
*Ru(bpy)32+  Ru(bpy)32+ + heat – non-radiative relaxation mechanism (4)1
The quenching rate constant Kq, collision rate constant Kd, and fluorescence rate
constant Kf are all based on their respective method of relaxation. The rate constants can all be
related through a rate constant ratio called quantum yield (Φ). This quantum yield is
determined through fluorimeter measurements.
Φ=
𝛷°
𝛷
𝑟𝑎𝑡𝑒 𝑜𝑓 𝑓𝑜𝑟𝑚𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑝𝑟𝑜𝑑𝑢𝑐𝑡
𝑠𝑢𝑚 𝑜𝑓 𝑎𝑙𝑙 𝑟𝑎𝑡𝑒𝑠
– quantum yield (5)1
𝐾𝑞
= 1 + 𝐾𝑓+𝐾𝑛𝑟 [𝑂2] - phi°/phi relation to rate constants (6)1
8𝑅𝑇
Kd= 3𝑛 – equation for collision rate constant (7)1
One use for all of these rate constants is in fluorescence lifetime imaging microscopy
(FLIM). FLIM is useful when looking at thick samples of tissue because the fluorescence is
independent of original excitation wavelength and is instead only dependent upon the lifetime
of the fluorescence.3
Experimental Method:
The first test run on the Ru(bpy)32+ sample was UV spectroscopy. A Varian 4000 UV-Vis
Spectrometer and cuvette were used to scan the sample between 250 and 650nm to determine
the excitation wavelength. This wavelength was later used for exciting the molecules in laser
photolysis.
Next a Horiba Jobinyvon Fluorolog ihr320 and cuvette were used to scan the air,
nitrogen, and oxygen saturated samples. This measurement would then be used to determine
the area°/area and phi°/phi values for each sample and Kq/(Kf+Knr) as it gives the actual
intensity of the fluorescence peaks.
Finally a Tektronix TDS2022B oscilloscope, SRS NL100 laser, and Electro-Optics
Technology ET2030A photodiode were used to run a laser photolysis analysis on each of the
samples. This provided intensity vs time data and allowed for the calculation of Kf+Knr.
Results:
The results of the UV spectrum showed that Ru(bpy)32+ absorbs light at 337.1nm. This
was then used as the wavelength of laser fired at the samples in the latter procedures.
The fluorimeter data was used to determine values of
𝛷°
𝛷
for each sample and
corresponding O2 concentration. This table was then used to make a graph of area°/area vs.
concentration of O2 in molarity, this is also known as a Stern-Volmer plot. The slope of this line,
𝐾𝑞
2079.2 ± 53.23, is equal to 𝐾𝑓+𝐾𝑛𝑟.
Figure 2 – Table of O2 concentration and
Sample
Air
Nitrogen
Oxygen
[O2] %
21
0
100
𝑎𝑟𝑒𝑎°
𝑎𝑟𝑒𝑎
∅°
or ( ∅ )
[O2] M
φ0/φ
0.000269 1.488607
0
1
0.00128 3.639277
Figure 3 - area°/area vs. concentration and respective slope
Area°/area vs. Concentration O2
Area°/Area
4
3
2
y = 2079.2x + 0.9689
1
0
0
0.0002
0.0004
0.0006
0.0008
Concentration of O2(%)
0.001
0.0012
0.0014
Next the laser photolysis data was used to make a plot of ln(intensity) vs time. All three
samples were plotted on the graph and they are in the order they are due to the concentration
of O2. Because Ru(bpy)32+ is not quenched by nitrogen, it has the smallest slope. Air has 21%
oxygen in it and therefore the intensity dies off slightly faster and the slope is slightly larger.
Finally in 100% oxygen the Ru(bpy)32+ is quenched quickly and therefore the decrease in
intensity of fluorescence, and consequently the slope, is much larger than the other two
samples. The slope of the nitrogen saturated line, 2x106 ± 6790.24 is equal to Knr + Kf.
Figure 4 – ln(intensity) vs time for all three samples and their respective slopes
Ln intensity vs time
0
Ln intensity
-1
0
0.0000005
0.000001
0.0000015
0.000002
0.0000025
-2
-3
y = -2E+06x - 1.257
y = -2E+06x - 1.1646
-4
y = -6E+06x - 1.2276
-5
Nitrogen
Air
O2
-6
-7
Time
Finally by using the above two rate constants, the value for the quenching rate constant,
Kq, could be determined. It was found to be 4.1584x109 ± 1.074x108. The inverse of this
quenching rate constant is related to the time it takes for oxygen to quench the excited
Ru(bpy)32+.
The value of Kd, the collision rate constant, was determined using equation 7 and found
to be 6.483x109. This is related to the frequency of excited Ru(bpy)32+ coming into contact with
an oxygen molecule.
Discussion:
Quenching occurs between the quenching molecule, in this case O2, and the excited
molecule Ru(bpy)32+. This can occur in one of three ways. The first being collisional deactivation,
where the two molecules go back to their original states.
S* + Q  S + Q – collisional deactivation mechanism (8)4
The second mechanism for bimolecular quenching is energy transfer, where the energy from
the excited molecule is transferred to the quenching molecule.
S* + Q  S + Q* - energy transfer mechanism (9)4
The third mechanism for bimolecular quenching is electron transfer, where an electron is
transferred to or from the excited molecule to the quenching molecule.
S* + Q  S+ + Q- or S- + Q+ - electron transfer mechanism (10)4
The quenching constant has quite a large error compared to the value. Some of this
error may have come from the propagation of error from the Fluorolog which was later used in
the calculation of the quenching constant.
The collision rate constant Kd is directly connected to the quenching rate constant Kq.
The inverse of the quenching rate constant is the average time it takes for a given Ru(bpy) 32+
molecule to be quenched by O2. The collision rate constant is how often oxygen and Ru(bpy)32+
come together. This means that oxygen does not quench Ru(bpy)32+ every time they come
together.
Conclusion:
Ru(bpy)32+ was studied using a UV spectrum, fluorimeter, and laser photolysis setup to
determine rate constants of relaxation of Ru(bpy)32+. The UV spectrum showed that Ru(bpy)32+
is excited by 337.1nm light while the Fluorolog and laser photolysis were used to measure the
relaxation rates. The rate constant for relaxation of * Ru(bpy)32+ by oxygen quenching was
found to be 4.1584x109 ± 1.074x108 and the collision rate constant was found to be 6.483x109.
Acknowledgments:
Thank you to Ahmed Rebh, Matt Micklin, and David Belias for their assistance in the
recording, analyzing, and interpretation of this data and experiment. Also thank you to Jovan
Livada and Phil Donahue for their assistance in data gathering and questions related to the
experiment. Finally thank you to Dr. M and the Pennsylvania State University Chemistry
Department for allowing us access to their facilities and knowledge.
References:
1. Milosvljevic, Bratoljub H. "Chapter 4 - Real Gas Behavior." Lab Packet for Chem 457
Experimental Physical Chemistry. N.p.: n.p., n.d. 10-1--11. Print. Spring 2015.
2. Jablonski Diagram. Digital image. US National Library of Medicine. National Center for
Biotechnology Infrmation, 12 May 2011. Web. 17 Mar. 2015.
3. Berezin, Mikhail Y., and Samuel Achilefu. "Fluorescence Lifetime Measurements and
Biological Imaging." Chemical Reviews. U.S. National Library of Medicine, n.d. Web. 17
Mar. 2015.
4. Atkins. "Kinetics of Complex Reactions." Instructors Solution Manual 23 (n.d.):
446. Chem.uci.edu. University of California Irvine. Web. 18 Mar. 2015.