ADH 1/28/2015

Laser Induced Fluorescence of Quinine Sulfate and the Kinetics of Cl- Induced Quenching
of the Fluorescence
adapted from Jonathan Gutow (1/95, last revised 12/03) by Audrey Dell Hammerich

Prelab Reading Assignment: Notes on Electronic Spectroscopy; for experimental -

Oscilloscope and Function Generator Instructions and Oscilloscope, Laser, PMT Instructions

Introduction

In this experiment you will be concerned with the decay kinetics of the fluorescence of
quinine sulfate in water solutions and the physical processes that effect these. Quinine is used as
a fluorescence standard because the fluorescence is not quenched (decreased) by oxygen but can
be quenched in a controlled manner by adding atomic anions (F-, Cl-, I- and Br-) to the solution .1
Many molecules re-emit some of the light energy they have absorbed as light, but
delayed in time and at a different energy than the absorbed light. This is usually referred to as
fluorescence or phosphorescence. As shown in Figure 1, fluorescence is the emission of light by
a molecule in the same electronic state it was initially photoexcited into (usually S1), whereas
phosphorescence is the emission of light from a molecule in a different electronic state (usually
T1). Phosphorescence usually has a longer average time between photon absorption and
emission than fluorescence. For a more detailed discussion of these phenomena see any standard
physical chemistry text.2,3 The Notes on Electronic Spectroscopy should be consulted for this
experiment.

Figure 1: Some electronic transitions that can accompany fluorescence and phosphorescence.

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 In this experiment an N2 gas laser system will be used to excite the quinine molecules.
An advantage of lasers over typical arc lamp /monochromator combinations is that lasers produce
much more intense light, allowing excitation of more molecules thus increasing the fluorescence
intensity and making it easier to detect. Also the N2 laser produces very short pulses of light,
<10-9 sec in duration, which means the excitation light is on for a short time compared to the
fluorescence lifetime of many molecules, ~20.0 × 10-9 sec for quinine in water solutions at room
temperature. Because of these advantages lasers have made it possible to use fluorescence for
remote analysis of gases. One system, called LIDAR (light detection and ranging), focuses a
powerful pulsed laser through a telescope at a remote point, such as the top of a smoke stack, and
monitors the concentrations of gas at that point through the telescope by measuring the laser
induced fluorescence (LIF) spectrum.4

Kinetics of Fluorescence Quenching

BASIC KINETICS OBSERVED

The sample consists of a solution of quinine sulfate dihydrate (C40H54N4O10S) in water,
with a little acid and salt impurities.

The acid keeps the quinine protonated so that it fluoresces more strongly. Your primary task is
to decide whether the Cl- ions from the salt quench the quinine fluorescence by collisionally
deactivating the excited fluorophore (commonly referred to as “dynamic” quenching) or by
forming a nonfluorescing complex with the ground state quinine (commonly referred to as
“static” quenching). This is not a question that can be answered using simple fluorescence
intensity measurements.5 Thus it is necessary to collect the time resolved decays of the
fluorescence.
Because they are adequate to describe the observed results only the simplest kinetic
models possible for dynamic and static quenching will be considered. The unexcited quinine, the
fluorophore, will be represented by the capital letter ‘F’, the excited quinine by ‘F*’, the
quenching anion by a capital ‘Q’, the exciting light photon by ‘hυ’, and the fluorescence photon
by ‘hυf’. Their concentrations will be represented by the symbol in square brackets.
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 Dynamic Quenching. In the case of dynamic quenching, the quinine is excited at some
rate by the light and then either relaxes by giving off a photon or by colliding with the quencher
and relaxing without fluorescing. This simple kinetic mechanism is shown below:
ka
F + hυ → F* (1a)
kf
F* → F + hυf (1b)
kq
F* + Q → F + Q (1c)

The nonradiative decay rate (transition to the ground state So without emitting a photon) is
missing from this mechanism. It has been absorbed into kf because the experiment cannot
differentiate between the two. Another thing ignored in this simple scheme is stimulated
emission. This becomes important when the light intensity is so high that the rate of excitation,
ka[hυ], is large compared to kf. Then the process of stimulated emission becomes an important
route for returning F to its ground state. This is represented in equation 1d below, but will be
assumed unimportant for the analysis that follows.

F* + hυ → F + 2hυf (1d)

.
You should be able to show that at high light intensity the combination of equations 1a and 1d,
leads to the conclusion that the transition can be saturated. What is the maximum fraction of F
that can be in the excited state if the light intensity was high? For more details look up Einstein
coefficients in any physical chemistry2,3 or spectroscopy text.
From these equations the differential rate laws can be written. Although the rate law for
equation (1a) is often written by absorbing the number of photons available into the rate constant
ka, here this is not done to keep the intensity dependence explicit. By so doing the photons can
be treated as just another reagent. To avoid confusion with the use of “I” representing intensity
(power in W/m2) in Beer’s law, [hυ] will be used to represent the concentration of photons in
moles/L. This convention is commonly employed when studying laser induced chemistry.6 At
low light intensity and high light intensity the appropriate value for [hυ] is directly proportional
to the mean average of the light intensity across the sample. In the intermediate range where the
intensity is only high enough to cause saturation of the transition part way across the sample the
relationship is more complicated.
At any instant one can calculate an effective photon concentration by dividing the
number of photons in the cell by the volume of the cell filled with light (often called the
excitation volume). In this representation the rate constant ka = cα(υ), where c = speed of light,
and α(υ) = the absorptivity (loge) for absorption at the particular frequency. Notice that ka has
proper second order rate constant units of m3 mol-1 s-1 (or L mol-1 s-1 or M-1 s-1). This treatment is
exactly equivalent to using Beer’s law to calculate the number of photons absorbed per second,
but allows us to treat the light like any other reagent. The differential rate laws using this
notation are:
d[F*] − d[F]
= = k a [F][hυ ] − (k f + k q [Q]) [F*] (2a)
dt dt

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 d [hυ] Q
d[hυ]
= f (t ) and =0 (2b)
dt dt

where f(t) varies rapidly during a light pulse. If a constant intensity source was used instead f(t)
= 0.
If a light pulse was used that is fast compared to the sum of the quenching plus
fluorescence decay rates (kf + kq[Q]), once the light pulse ends some excited molecules will
remain. Their concentration will be denoted by [F*]i. With the light off, the first term in the rate
equation (2a) is zero, giving us:
= − (k f + k q [Q]) [F*]
d[F*]
(3)
dt
.
This is just a first order rate equation, and can easily be integrated for the time after the light is
off to get:

{
[F*] = [F*]i exp − (k f + k q [Q]) t } (4)

Thus, in the case of dynamic quenching, the fluorescence is expected to decay as a single
exponential but with a decay rate that is linearly proportional to [Q].
Static Quenching. In the case of the static quenching mechanism, the quencher forms an
equilibrium concentration of a complex with the unexcited fluorophore, F. One of two things
can happen: either the complex will not absorb an excitation photon, or the excited complex will
relax by some route other than fluorescence. The reaction scheme is:

F+Q ← →
K
 FQ
eq
(5a)
ka
F + hυ → F * (5b)
k
F * f → F + hυf (5c)

In this mechanism assuming the equilibrium is reached the differential rate laws are:
[FQ]
K eq =
[F][Q]
(6a)
d[F*] − d[F]
= = k a [F][hυ ] − k f [F*]
dt dt
(6b)
d [hυ] d [Q]
= f (t ) and =0
dt dt
. (6c)
Equation 6a implies that the concentration of uncomplexed F available when the light
turns on will depend on Keq[Q]. Recognizing that [FQ] + [F] = [F]o is a constant one can solve
for the concentration of [F] at equilibrium:

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 [F]o
[F] =
K eq [Q] + 1 . (7)

Substituting for [F] in equation 6b shows us that when the light is on the rate of production of
[F*] decreases with increased [Q]:
d[F*] k a [hυ][F] o
= − k f [F*] (8)
dt K eq [Q] + 1

So as the quencher concentration increases fewer molecules reach the excited state during the
light pulse. Just as with the dynamic mechanism the first term in this equation is zero when the
light is off. The resulting equation is easily integrated to find the time dependence of the excited
fluorophore concentration after the light goes off:

[F*] = [F*] i exp{− k f t} (9)

where [F*]i decreases with increasing [Q], but the decay rate is independent of the quencher
concentration.
Thus to determine if the quenching was static or dynamic, plot the observed fluorescence
decay rate versus the quencher concentration. The dynamic case will yield a sloped line.
Consider how the slope and intercept are related to kq and kf. In the static case the decay rate is
constant.

RELATIONSHIP OF QUENCHING RATE TO COLLISION RATE IN LIQUIDS

If the quenching was dynamic kq can be extracted from the data. It is interesting to
compare kq with the theoretical diffusion limited collisional rate constant (kD). Since the
hydrodynamic radii of both the quencher and the fluorophore are not known, an approximate
form of kD is used where the hydrodynamic radii are assumed to be equal. In this way they
cancel out and an expression for kD is obtained which depends only upon temperature and the
solvent viscosity:
8RT
kD =
3η (T ) , (10)

where kD is the rate constant for diffusion, R is the gas constant, T is the temperature in kelvin
and η(T) is the solvent viscosity which in general drops as temperature increases.2,3 Since the
temperature in the laboratory varies between 20˚C and 25˚C, η(T) for the 0.5 M H2SO4 (about
5% by weight) solution used is between 1.12 × 10-3 kg m-1 s-1 and 1.01 × 10-3 kg m-1 s-1.7,8 So, if
all collisions lead to quenching, kq will be equal to kD and will fall in the range 5.8 × 109 M-1 s-1
to 6.5 × 109 M-1 s-1. If some collisions do not lead to quenching, kq should be somewhat less
than the calculated kD.

WHAT YOU SEE IF YOU DO NOT HAVE TIME RESOLUTION (STERN-VOLMER ANALYSIS)
For continuous excitation the situation is different. Experimentally, the intensity of the
fluorescence, which is proportional to [hυf] ∝ [F*] while the light is left on, is measured. The
5
differential rate equations are still those of equations 2a and b. Since the light is on all the time
the first term from equation 2a cannot be dropped for analysis of the observed fluorescence. By
substituting [F]o - [F*] for [F], equation 2a can be integrated to find the time dependence of the
excited state concentration, [F*]. This shows that there is an initial period after turning on the
light where the [F*] grows and then it reaches a steady state. The initial induction period is very
short and it is easier to analyze the mechanism by making measurements after the initial
induction period has finished and assuming steady-state conditions. The steady state equilibrium
is reached when [F*] gets large enough, because the first and second terms in equation 2a will
cancel. Substituting for [F] and assuming steady-state conditions in equation 2a gives:
ka [ hυ][ F ]o
[ F *] =
k f + k q [Q] + ka [hυ ]
(11)
Mathematically the inverse of this expression is easier to work with:

1 k + k q [Q] + ka [hυ ]  1 kf  kq
= f = + + [Q] (12)
[ F *] ka [ hυ][ F ]o  [ F]o ka [hυ ][ F ]o  k a [hυ ][ F ]o

Note that this is the equation for a straight line versus the concentration of Q, since [F]o, [hυ], kf,
kq, and ka are all constants. The terms in brackets are the y-intercept and the fraction before [Q]
is the slope of the line. Plotting 1/(fluorescence intensity) versus [Q] yields a straight line with a
positive slope, if Q quenches the fluorescence. This type of procedure is called a Stern-Volmer
analysis and is commonly used to analyze concentration dependent kinetics.3,9 This analysis is
often carried one step further to generate what is called a reduced Stern-Volmer plot. If the
fluorescence intensity (equation 11) is divided by the fluorescence intensity with [Q] = 0, and
then the inverse taken, an equation for a line with a y-intercept of 1 is obtained:

[ F*][Q]=0 kq
=1 + [Q] (13)
[ F*] ka [hυ] + k f

The slope now depends only upon ka, the rate of photon absorption, [hυ], the photon
density, kf, the rate of fluorescence, and kq, the rate constant for quenching. [hυ] can be
calculated from the light intensity, ka from the Beer’s law absorption coefficients at the excitation
frequency, and kq is assumed to equal kD, which can be estimated based on the diffusion
coefficients, as shown above.2,3 Thus kf can be determined. In most formulations of this
equation the intensity of the light is assumed to be so small that the ka[hυ] term in the
denominator can be ignored, making the analysis even easier. Often this equation is written in
terms of the quantum yield, φ ∝ [F*], and the factor in front of [Q] is referred to as KSV, the
Stern-Volmer constant:
φo
= 1 + KSV[Q]
φ . (14)
The problem is that the same functional form is obtained if static quenching is occurring.
If it is assumed that the equilibrium in equation 5a is fast enough that it can adjust to any change
6
in [F] caused by absorption or fluorescence, you should be able to derive the following reduced
Stern-Volmer equation:
[ F*] [ Q ] 0 k f Keq
=
=1 + [Q] (15)
[ F*] k f + ka [hυ ]

Notice that the slope of this line is a different function of the constants than in the dynamic case,
but you will not be able to tell the difference, since only the slope can be measured.

Overview of the Experiment and Analysis

You will use a laser, a monochromator, a photomultiplier tube (PMT) and a digital
storage oscilloscope to collect the time resolved laser-induced fluorescence of quinine sulfate at
approximately 10-5 M and 0.5 M H2SO4 in water. You will observe the effects of Cl- as a
quencher by varying its concentration in the solution.

USE OF THE N2 LASER

Read the instructions about using the laser in the handouts provided: Oscilloscope and
Function Generator Instructions and Oscilloscope, Laser, PMT Instructions. Be careful: the UV
pulse produced by the laser can easily damage your eyes. Wear plastic glasses that do not transmit

signal
PMT oscilloscope
S2
M3
M4 photodiode
G

M2 M1
trigger
S1
F1
F2
monochromator
cuvette B

L
sample chamber

laser

Figure 2: Block diagram of experimental setup; solid lines represent bnc cables (for bayonet Neill–Concelman)

7
the laser light. The output from the laser is split by beamsplitter B. Part of the laser beam is
focused by lens L onto the sample cuvet while the other part passes through a neutral density
filter F1 to reduce its intensity and strikes a photodiode, triggering data acquisition by the digital
oscilloscople.

USE OF THE MONOCHROMATOR

The laser beam travels through the sample. The fluorescence is collected at right angles
to the incoming laser beam, passes through a neutral density filter F2, and then into the
monochromator. The monochromator is an Ebert optical mount. The light passes through the
entrance slit S1, striking a 45o mirror M1, and is reflected to a large collimating mirror M2.
From this mirror it is it is reflected to the grating G and dispersed back to the collimating mirror
M3. The light beam, returning to focus, is reflected by a 45o mirror M4 out through exit slit S2
and into the photomultiplier housing. The monochromator should be set to detect 480 nm
fluorescence. This is far from the laser frequency of 337.1 nm and is the frequency at which the
fluorescence exhibits simple exponential decay. At other wavelengths the emission rate is
affected by a number of other processes that have not been considered, and requires a sum of two
or more exponential functions to fit the decay.1,10,11 If there is time, you might want to collect
fluorescence at about 390 – 400 nm from a sample without quencher to see if the biexponential
decay can be observed.

USE OF THE PMT

The light that gets through the monochromator is detected by a photomultiplier tube.
PMTs are based on the photoelectric effect. After a light photon ejects an electron from the
photocathode the electron is accelerated by a potential voltage into another electrode. The
accelerated electron knocks many electrons out of the electrode, which are then accelerated to
another electrode. This process is repeated 9 times here (9 stage PMT) giving a current gain of
about 106 – 107! It is possible to see the signal from a single photon; if you have a 350 MHz or
greater oscilloscope and the fluorescence intensity is low enough you may be able to see
individual spikes (photons) in the decay signal from the PMT. Typically PMTs operate at a
negative voltage of between 500 and 1250 V. Be very careful: high voltages are dangerous and
a PMT can be easily damaged by exposing it to too much light while voltages are applied. To
avoid damaging the PMT do not apply voltage to the tube until the room lights are off and start
looking for signal at a low voltage increasing the voltage only enough to get a signal of a few
hundred millivolts at its peak. DO NOT TURN THE VOLTAGE ABOVE 800 V! If you have
any questions, consult with the professor.

USE OF THE DIGITAL OSCILLOSCOPE

Explicit instructions for the particular scope being used are contained in the handouts:
Oscilloscope and Function Generator Instructions and Oscilloscope, Laser, PMT Instructions. A
digital oscilloscope is different from a standard analog scope. Digital scopes take an input
voltage versus time and at regular intervals convert the voltage level to a digital number using
standard (usually successive approximation) analog-to-digital conversion techniques. This
8
means that if something happens between samplings one will not see any sign of it (see Figure
3).

A c t ual Slow Signal

Fast Signal

Observed on
Digit al Scope

Observed on
Analog Scope

Figure 3: Comparison of a digital scope missing a fast signal and the response of an analog
scope. The black dots represent the values actually measured by the digital scope and the dashed
lines the time the samples are taken.

Using an analog scope, even if something happens faster than the scope can respond, it
will react in some way; thus the signal looks like a smoothed (averaged) version of the real
signal. The advantage of a digital scope for the kind of experiment done here is that one can
conveniently save and average together multiple traces of a repeated event. There is no way to
save and average on a normal scope. Sometimes the term transient digitizer is used. This is just
a digital oscilloscope without some of the features usually found in an oscilloscope.
The oscilloscope only displays data when told to do so by the occurrence of a trigger. In
this experiment a small amount of the light pulse out of the laser is used to trigger the beginning
of data collection via the photodiode. This is done since the signal of interest only occurs over a
very brief period of time right after the laser pulse. Mounds of useless data would be collected
were the signal from the PMT collected at other times.

Trigger ( volt age signal f rom a phot odiode)

PMT Signal

t =0 ( st art collect ing dat a)

Figure 4: Timing diagram for data collection with a trigger.

9
SAMPLES
You should make 5 samples with different concentrations of Cl- ions. The quinine
sulphate dihydrate should be about 10-5 M in the samples and the same for each sample. The
H2SO4 in the solution should be 0.5 M. A good range for the quencher concentrations is 0.00 –
0.04 M. Make sure the cell is very clean before filling it with each sample and use the same
cuvette for all of your samples. Average enough time scans together on each sample that the
signal to noise level is > 20:1. 50 scans per sample should be used (per laser shot). Do not
forget to collect a background, so that any radio-frequency noise can be subtracted out. Collect
the background by averaging the signal with the fluorescence blocked by a piece of cardboard.
For each sample take a UV/VIS spectrum to verify that the absorbance does not change
with quencher concentration. This will allow verification, independent of the time-dependent
experiment, that the quenching occurs in the excited state. Presented in Figure 5 are an
absorption spectrum and an emission spectrum for quinine. If time permits absorption and
fluorescence spectra of the quinine sulfate will be taken.

Figure 5: Absorption and emission spectra of quinine.

ANALYSIS
You will be able to measure the dependence of the fluorescence lifetimes on quencher
concentration and also perform a Stern-Volmer analysis. The data should be saved in a format
appropriate to be transferred to a spreadsheet or a data analysis program for processing.
Before doing any analysis subtract out the background, which consists mostly of radio-
frequency interference from the spark gaps in the N2 laser. To get the observed rate constant for
fluorescence decay, (kf + kq[Q]) or kf, fit the decaying part of the signal to a single exponential.
To find kq plot the observed decay rate versus [Q] and fit it to a straight line. To perform the
Stern-Volmer analysis you must get the data into a form that is equivalent to the cw (continuous
wave) fluorescence intensity. This can only be accomplished if the conditions were exactly the
10
same for all the samples. This means the laser power must stay the same, the slits must not be
adjusted, etc. Just integrate the total area under the decaying portion of each decay curve to
measure the total fluorescence.

Details of the Experimental Procedure and Analysis

On the first day you will learn how to use the digital oscilloscope and make
measurements on various waveforms. There should be sufficient time for you to also prepare
your samples. The following lab periods will be devoted to working with the laser. You will
learn how to measure the width in time of the laser and obtain the laser induced fluorescence
decay curves for your samples. Finally you will obtain the absorption and fluorescence spectra
on the samples that you have analyzed. Be sure to work up your data early to see whether you
need to return to lab the second week to collect more data or prepare fresh new solutions.

FUNCTION GENERATOR
Experiment. Follow the procedure in the Oscilloscope and Function Generator Operation
Instructions. For each of the sine, square, and triangular waveforms obtain the waveform and the
derivative of the waveform. For each waveform and its derivative measure the maximum,
minimum, amplitude, period, and frequency of the wave obtained by using the 1) markers, 2)
quick measurements, and 3) function generator settings. Put in your notebook a copy of all
waveforms, data collected, answer the two questions on the derivative waveform, and all values
that were requested to be recorded in the Oscilloscope and Function Generator Operation
Instructions handout
Analysis. For the sine wave, show your calculation of its frequency. For each waveform
and its derivative overlay their waveforms on the same plot. Make sure that both can be clearly
seen and differentiated. Tabulate the measurements of the maximum, minimum, amplitude,
period, and frequency of the waveforms and derivatives obtained by using the 1) markers, 2)
quick measurements, and 3) function generator settings.

SAMPLES
A total volume of 10 mL is sufficient to fill the 1 cm cuvettes for the LIF measurements
and the UV/VIS and fluorescence spectra. 10 mL volumetric flasks are provided. You will need
to quantitatively dilute the following two stock solutions in order to prepare 5 quinine samples
with different quencher concentrations in the range of 0.00 – 0.04 M KCl:
~ 2.00 × 10-5 M quinine sulfate dihydrate in ~ 1.0 M H2SO4
~ 0.150 M KCl
The actual concentrations are to be found on the stock solution bottles. Be sure to determine
how much of each stock solution you need and pour out this small amount into a beaker for your
use. Do not return excess solution to the stock bottles! In each of the five samples the
concentration of quinine sulfate dihydrate should be near 1.00 × 10-5 M and needs to be the same
for all samples. The concentration of H2SO4 should be ~ 0.5 M. The exact concentration of
quencher (0.00 – 0.04 M Cl-) in your 5 samples is unimportant but its value should be known to
11
three places. Include all calculations for your sample preparations in your laboratory notebook.
FLUORESCENCE EXPERIMENT
Experiment – Laser Pulse. Follow the procedure in the Oscilloscope, Laser, and PMT
Operation Instructions. You will begin by obtaining the waveform for the pulsed laser used in
this experiment and determining its pulse width. The pulse width is the duration in time of the
optical pulse. Measure the full width at half maximum (FWHM) (width of the wave, measured
at half the maximum height) of your pulse with the markers and record in your notebook along
with a copy of the laser pulse waveform.
Experiment – Fluorescence Decays. Start with the sample containing no quencher and
obtain its fluorescence decay curve. Adjust the voltage on the power supply and the vertical
offset until you obtain a peak that almost fills the screen vertically when the vertical setting of
channel 3 is set to 10 mV/div. Allow an empty region of ~ 10 mV above and below the peak.
Adjust the horizontal offset so that you can observe the decay going to zero. Use the markers to
find the time interval between the peak maximum and 1/e of the maximum (Δ row on marker
measurement screen). This is an approximate measure of the fluorescence lifetime. Proceed to
collect the fluorescence decays and measure the lifetimes of the remaining samples in the order
of increasing concentration of quencher using the same cuvette for all samples. You have just
completed trial 1. Obtain two more trials of your 5 samples in the same manner. Put in your
notebook your lifetime measurements and all values that were requested to be recorded in the
Oscilloscope, Laser, and PMT Operation Instructions handout.
Experiment – Absorption and Fluorescence Spectra. For your best trial obtain a
UV/VIS spectrum of each sample. Obtain a fluorescence spectrum of a sample without any
quencher.
Cleanup. All solutions can be poured down the drain with plenty of water. The plastic
cuvettes should only be rinsed out several times with distilled water. Avoid the use of any
solvents. They will attack the plastic.
Analysis – Laser Pulse. The width of a laser pulse is commonly measured by fitting a
plot of voltage versus time to a Gaussian function. The pulse width is defined as the full width at
half maximum (FWHM) of this plot. Even when the laser waveform is not a “perfect” Gaussian
function, the FWHM serves as a measure of the pulse width. Fit your voltage versus time data
on the laser pulse to a Gaussian function. Put the laser pulse and its Gaussian fit on the same
plot. Be sure that both can be seen and are distinguishable. Place Origin’s dialogue box with the
regression parameters and their errors on your plot without obscuring anything. Determine the
FWHM from the functional form defining the Gaussian. Report the pulse width of the nitrogen
laser from its FWHM as given by the 1) markers, 2) equation defining the Gaussian, and 3)
Origin’s Gaussian fitting program.
Analysis – Fluorescence Decays. For each trial plot the fluorescence decays (entire
“peak”) for all 5 samples on one graph. For each sample plot the fluorescence decay (entire
“peak”) and its exponential fit of the decaying portion from a nonlinear regression analysis on
the same graph. Appropriately adjust the thickness of the plots so that the original data and the
fit can each be seen. Include Origin’s dialogue box with the regression parameters and their
errors for the fit on your plot, again without obscuring anything. You have three runs for each
12
quencher concentration. Tabulate the value of the observed fluorescence decay rate (use the
definition for decay rate given on pp. 3-4) for each sample and the average value for each
quencher concentration. Using error bars, plot the average of the observed fluorescence decay
rate versus Cl- concentration. Perform a linear regression on this data, obtain the regression
parameters and their errors, and include Origin’s dialogue box on the plot. You should now be
able to identify and give the values and errors for the fluorescence rate constant kf and quenching
rate constant kq.
Analysis – Stern-Volmer. To obtain the reduced Stern-Volmer plot you will need to
integrate the area under just the decaying portion of each fluorescence decay curve. You have
three runs for each quencher concentration. Tabulate the value of the integral under the
fluorescence decay for each sample and the average value for each quencher concentration.
Using error bars produce the reduced Stern-Volmer plot. Perform a linear regression on this
data, obtain the regression parameters and their errors, and include Origin’s dialogue box on the
plot. You are now able to compare your previously determined kf and kq rate constants with their
Stern-Volmer analogue.
Analysis – Absorption and Fluorescence Spectra. Determine whether the UV/VIS
absorbance changes as the concentration of Cl- changes. Based upon your conclusion, determine
whether the UV/VIS spectra do or do not verify that the quenching of quinine occurs in the
excited state. Can you observe and identify a biexponential decay in the fluorescence spectrum
of your sample without any quencher? Note: in the LIF experiment you were observing the
decay of the fluorescence as a function of time. Here the spectra that you are collecting are a
function of wavelength.

Questions

1) Show all of your work and derive equation 7 from equation 6a. (Hint: [F]o = [FQ] + [F])
2) Show all of your work and derive equation 11 from equation 2a using a steady-state
approximation on [F*]. (Hint: [F]o = [F] + [F*])
3) Show all of your work and derive equation 13 from equation 11.
4) Use the absorption and fluorescence spectra that you took or, if they were not obtained, use
Figure 5 and explain why the experiment was performed with a 337.1 nm laser and why the
fluorescence was detected at 480 nm.
5) In Figure 5 there is a slight shoulder at approximately 310 nm in the absorption spectrum of
quinine. Give your reasoning and suggest its origin.
6) The rise time of the PMT is about 2.2 ns (the rise time is the time for the PMT signal to go
from 10 to 90% of its final value), the laser pulse width is < 3 ns and the sampling rate of the
oscilloscope is 4 gigasamples/sec. What is the shortest fluorescence decay time you will be
able to measure directly? Show how you calculate or estimate this. Suggest ways you can
improve upon this.
7) Is the quinine fluorescence quenched dynamically, by complex formation, or by a
combination of both? Explain why using your experimental observations.
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8) Report your values for kq and kf. Compare these values from your time-resolved experiment
to your Stern-Volmer analysis. Also compare kq with kD. If they are different explain the
significance
9) The literature value for the lifetime (1/kf) = 19.3 ± 0.6 ns (95% confidence limits).1,10,12
Compare this with your results (show how you convert to 95% confidence limits) and
discuss any discrepancies.
10) The best available literature value of kq = 6.2 × 109 M-1s-1 determined from lifetime
measurements by Barrow and Lentz.10 This was determined in 0.05 M H2SO4 whereas you
obtained your data in 0.5 M H2SO4. The viscosity of 0.05 M H2SO4 is very close to that of
pure water (not the same as 0.5 M). Use equation 10 and convert the above Barrow and
Lentz kq value to what it would be in 0.5 M H2SO4. Then quantitatively compare your
results with this and discuss any discrepancies.

References

(1) Pant, D.; Tripathi, U. C.; Joshi, G. C.; Tripathi, H. B.; Pant, D. D. J. Photochem. Photobio.
A 1990, 51, 313-325.
(2) Atkins, P. W.; de Paula J. Physical Chemistry, 10th ed.; W. H. Freeman and Company:
New York, 2014.
(3) Barrow, G. M. Physical Chemistry, 6th ed.; McGraw-Hill: New York, 1996.
(4) Steinfeld, J. I.; Francisco, J. S.; Hase, W. L. Chemical kinetics and dynamics, 1st ed.;
Prentice-Hall, Inc.: Englewood Cliffs, NJ, 1989.
(5) Fraiji, L. K.; Hayes, D. M.; Werner, T. C. Measurement of Fluorescence Intensity and
Lifetime. In Physical Chemistry: Developing a Dynamic Curriculum; Moore, R. B.,
Schwenz, R. W., Eds.; American Chemical Society: Washington, D.C., 1993; pp 269-278.
(6) Gutow, J. H.; Zare, R. N. J. Phys. Chem. 1992, 96, 2534-2543.
(7) CRC Handbook of Chemistry and Physics; 95th ed.; Haynes, W. M.., Ed.; CRC Press, Inc.:
Boca Raton, Fl, 2014 (Website: http://www.hbcpnetbase.com).
(8) Viscosity Data for Sulfuric Acid (Website:
http://204.151.174.101/htm/businesses/ecoserv/brochures/sa/graphics/fig07.gif); Rhodia,
Inc.: 1999.
(9) Stern, O.; Volmer, M. Z. Phys. 1919, 20, 183.
(10) Barrow, D. A.; Lentz, B. R. Chem. Phys. Let. 1984, 104, 163-167.
(11) Pant, D.; Tripathi, H. B.; Pant, D. D. J. Lum. 1992, 51, 223-230.
(12) Chen, R. F. Anal. Biochem. 1974, 57, 593-604.

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