Franziska Hofmann, Stephan Steinmann

February 17, 2007

Fluorescence and Fluorescence Quenching

1 Introduction
Fluorescence is one of the two phenomena described as "luminscence", which is the emission of light by relax-
ing from excited states. The second phenomenum is phosphorescence. The difference is, that phosphorescence
involves spin-forbidden transitions, whereas fluorescence does not. (see also section 4.7)
Typical fluorescent molecueles are shown in Figure 1. Atoms are nonfluorescent, with the exception of the
lanthanoides.

Figure 1: Quinine, Fluorescein and 7-Hydroxy-coumarin (or Umbelliferone)

Fluorescence is not only a scientific method for the determination of properties of excited molecules such
as the pKa -value of phenol in the excited state (which is significantly lower), but a powefull analytical method,
too. Fluorescence is used in environmental monitoring, clinical chemistry, DNA sequencing and genetic analysis.
There are special methods using fluorescence: fluorescence microscopy, enzyme-linked immunoassay and fluores-
cence polarisation immunoassay, the two latter-ones used instead of radio-immunoassays, which use inconvenient
radioactive substances. [1]
Fluorescence is influenced by the polarity of the solvent, since the dipole-moment is different in the excited
state and a polar solvent can lower the energy of the excited state, causing a more pronounced Stokes shift. By
Stokes shift the shift in the wavelength between absorption and fluorescence is meant: Fluorescence occures at
higher wavelength (lower energy) than the absorption primarly because fluorescence occures from the lowest
vibrational level of the electronically excited state. (see also p. 5)
The Stokes shift occurs, because the excited state lives long enough for interaction with the solvent: Collisions
with the solvent results in a fast nonradidative energy loss down to the lowest vibrational level of the electronically
excited state. Because the energy difference between the two electronic states may be too high to be accepted by
the solvent, this excited state lives until it experiences spontaneous relaxation by emission: Fluorescence.
There can be a process called "solvent relaxation", too: Often, the dipole moment is lager in the excited state than
in the ground state, due to the increase in bondlengths. The rotation of solvent molecules is fast in comparision
with the lifetime of the excited state, hence the solvent reorientates around the molecule, causing a lowering of
energy and a shift to longer wavelengths of the emission.[2]

2 Experimental Part
A solution of quinine (3.3 · 10−5 M) in sulfuric acid (0.05 M) was diluted according to Table 1. In the case of NaF
and NaCl a concentration of 5 · 10−3 M was used, for NaBr and NaI solutions of 2 · 10−3 M were given.
The solutions were measured relative to the reference solution (no quencher) at an irradiation wavelength of 322
nm and a emission wavelength of about 458 nm. The reference solution was measured before every sample and
from time to time a new reference sample was made, since quinine is destroyed during the measurements.

1
 Quinine/µl Quencher/µl Water/µl
500 0 1500
500 1500 0
500 1200 300
500 900 600
500 600 900
500 300 1200

Table 1: Prepared solutions.

3 Results
Table 2 shows an overview about the measurements made, the intensities are relative to 100 for the reference
sample.

Quencher 1500µl 1200µl 900µl 600µl 300µl1

NaCl 73 / 70.5 / 70 79 / 79 / 81 87.5 / 84.75 / 85 99.5 / 95.5 / 81 43.5 / 46 / 44
NaBr 90 / 89 / 89.5 94 / 94.5 / 95 98.5 / 98.5 / 99
NaI 81 / 81 / 83 90 / 94.5 / 90 95 / 94.5 / 95
NaF 99 / 105 / 98

Table 2: Measured solutions and the relative fluorescence intensities.

Table 3 shows the data used for the Stern-Volmer analysis.

I0
Quencher cNaX /mM I
NaCl
3.75 1.41±0.04
3.0 1.26±0.03
2.25 1.17±0.03
1.5 1.03±0.032
4.95 2.25±0.08
NaBr
1.5 1.117±0.007
1.2 1.058±0.006
0.9 1.014±0.004
NaI
1.5 1.22±0.02
1.2 1.09±0.04
0.9 1.054±0.004
NaF
3.75 0.99±0.05

Table 3: Results of the measurements. The values in italic are not used for the Stern-Volmer analysis.

Table 4 shows the results of the Stern-Volmer plot: a linear regression, weightened according to the error was done.
The results, which can be calculated from this data are given in section 4.14 to 4.16
1
A 0.033 M NaCl solution was used in this case!
2
This value was calculated without the value of 81, since this seems not to fit.

2
 Quencher Slope 95% CI(slope)
NaCl 162 75 / 249
NaBr 168 4 / 331
NaI 268 -153 / 688

Table 4: Slopes and 95% confidence levels for Stern-Volmer analysis. The final results for KSV,NaX are given in
section 4.14.

4 Discussion
It is obvious, that the experiment didn’t work as it should, but we are unaware of the cause (or causes). We
estimate, that even imprecise pipetting could not produce such results. Sometimes, it looked like not all the
cuvettes responded in the same way, but since fused silica cells should be UV/Vis permeable this seems highly
unlikely. The cells were cleaned after every measurement, first with water, then with acetone and the acetone was
removed by a steam of nitrogen, so obvious dirt-sources are thought to be avoided.
If we wouldn’t know, that the experiment used to work properly, we would propose some modifications in the
proceedure: With beginning at higher quencher concentrations, a wider range of the scale could be used, which
could enhance the accurancy but of course the variability would increase, since the random error is higher for the
lower intensities. To cope with the varibility, one could run more measurements, as [3] advises anyway. In order
to minimise effects due to variabilities in ionic strenght and viscosity of the solutions it could be considered, if
instead with water the solutions should be complemented with a solution of an inert salt, such as NaF. In addition
to this, it could be wise, if the reference sample would be renewed regularly: It seemed to us, as if the loss of
quinine during the measurements was quite considerable.

4.1 Why are the quantitative measurements best made at the absorption maximum?

The absorption maximum is chosen for the excitation wavelength since a

higher absorbance means more excited states. More the excited states, more
fluorescence can be observed and it doesn’t matter, which particular excited
state is reached, because fluorescence does occure from the lowest vibrational
level of the electronically excited state anyway. (see also Figure 5)
The fluorescence is best measured at the maximum of the fluorescent spectrum,
since there the sensitivity is highest.

4.2 The Frank-Condon Principle

The Frank-Condon principle states:
Because the nuclei are so much more massive than the electrons,
an electronic transition takes place very much faster than the nuclei can
respond.

This results in the most likely transition being the one, where the internu-
Figure 2: The vertical transi-
clear distance at the turning point of the excited state has the same value as the
tion. [2]
equilibrium position in the ground state, therefore the most likely transition is
often called the vertical transition. Quantum mechanically the vertical transi-
tion is determined by computing the overlap integral of the ground states and the excited states. The higher the
value of this integral, the more intense the transition between these two states and the highest value indicates the
vertical transition.[2] For illustration, see Figure 2.

4.3 How do the absorption and fluorescence bands look like?

Since we didn’t take an absorption and a fluorescence spectrum, we have to relay on the literature. The spectra for
quinine are not very interessting in this behalf, as can be seen from Figure 3. Clearly, the case of quinine does not
follow the mirror rule (see for example Figure 4). This is explaind by the fact, that the first peak in the absorption

3
spectrum (at about 320 nm) is the transition from the ground state to the second excited state; fluorescence however
occures only from the first excited state to the ground state.

Figure 3: The absorption and fluorescence spectrum of quinine sulfate in 1 M sulfuric acid. [1]

4.4 How can the intensities be connected to the Frank-Condon Principle?

As allready mentioned above (4.2), the intensity of a transition is proportional to the overlap integral of the wave-
function for the two states.
In general, the intensity is proportional to the transition dipole moment µfi = τ Ψf µ̂Ψi dτ With Ψ f refering to the
R

wavefunction in the final and Ψi to the initial state.

4.5 Why is the fluorescence spectrum the mirror image of the absorption spectrum?

Since in most cases, in the excited state the geometry of the molecule is
only slightly changed (bonds are usually longer, but the symmetry as a whole
remains more or less the same), the spacing of vibrational levels of the excited
state is similar to the spacing of these levels in the ground state. Therefore, the
corresponding transitions are of a similar intensity, because the overlap integral
is similar: The overlap of S0 (v = 0) with S1 (v = 2) is more or less the same as
Figure 4: Illustration to the mir-
the overlap between S1 (v = 0) with S0 (v = 2).
ror rule. [1]
Absorption starts always from the ground state, hence, the higher the final vi-
bronic state, the shorter the wavelength of the absorbed photon. However, fluorescence is the transition from the
vibronic ground state of the electronic first excited state to some of the vibronic excited states of the electronic
ground state, therefore the higher the final vibronic state, the longer the wavelength of the photon emitted, since
less energy is set free.
Therefore, the relative intensities of comparable transitions are similar in the fluorescence spectrum to the ones in
the absorption spectrum, but their ordering is reversed, which results in the phenomenum sometimes called "mir-
ror rule": The fluorescence spectrum is often the mirror image of the absorption spectrum, an example is given in
Figure 4.

4.6 Jablonski Diagram

A Jablonski Diagram gives an overview over the processes that can happen to a molecule irradiated with light of
an appropriate3 wavelength. An example is given in Figure 5.
The internal conversion is responsible for a part of the Stokes shift mentioned above. Intersystem crossings occur,
if there is a state of different multiplicity but with similar energy. Emission from a state of the same multiplicity is
called fluorescence (hνF ), whereas emission from a state of different multiplicity is refered to as phosphorescence
(hνP ).

3
If the energy of the radiation does not correspond to the energy difference for an allowed transition, only scattering occurs.

4
 Figure 5: An example for a Jablonski Diagram. [1]

4.7 Which transitions are spin allowed and which are spin forbidden and how does this influence
the lifetime (τ)?
Transitions which happen without a spin conversion are spin allowed, i.e. singlet-singlet or triplet-triplet transition
are spin allowed, but singlet-triplett transition are spin forbidden, since the spin of an electron has to be altered.
The allowedness has a great impact on the lifetime: Whereas the lifetime of a singlet excited state is about 10 ns,4
the lifetime of a triplet excited state is in the order of ms to s! Therefore fluorescence is observed within a far
shorter period after absorption than phosphorescence.

4.8 Fluorescence Quenching and its Reaction Sequence

Fluorescence quenching is a process of relaxation without emission of a photon. The excess energy can for example
be converted in translational, rotational or vibrational energy of a different molecule, atom or ion, which then
undergoes relaxation itself or, in the case of a gain in translational energy, it’s temperature raises. Quenching is a
second order reaction.
The quantum yield in this case will be given by the general Equation 2.
For a system, where quenching occurs, the ongoing processes are summarised in Table 5.

Description Process
Absorption M + hν → M∗
Fluorescence M∗ → M + hν’
Nonradidative decay M∗ → M
Quenching5 M∗ + Q → M + Q

Table 5: Possible processes in a fluorescing system with a quencher.[4]

4.9 Definition of the Fluorescence Quantum Yield and Derivation of it’s Value for Continous
Measurements
The fluorescence quantum yield is the ratio between the absorbed photons and the emitted photons.

] Molecules fluorescing per unit of time an volume

Φf = (1)
] Photons absorbed per unit of time and volume
4
more general 10−12 to 10−6 s
5
An other possibility would be: M∗ + Q → M + Q∗ followed by Q∗ → Q

5
The molecules fluorescing are given by Γ[M∗ ], the photons absorbed can be expressed as the molecules, which
absorb a photon, this would be kabs [M]. But these experssions are not very usefull, since neither the actual con-
centration of the excited state, nor the one of the ground state molecules is easily determined. For continuous
irradiation, the steady state approximation is sensible:

d[M∗ ]
− = Γ[M∗ ] + knr [M∗ ] + kq [Q][M∗ ] − kabs [M] = 0
dt
This can be rearranged to:
kabs [M]
[M∗ ] =
Γ + knr + kq [Q]
Now, the quantum yield Φ f is conveniently expressed through the rate constants for emission Γ, nonradidative
decay knr and quenching kq :

Γ · [M∗ ] Γ · kabs [M] Γ

Φf = = = (2)
kabs [M] kabs [M] · (Γ + knr + kq [Q]) Γ + knr + kq [Q]

4.10 When does Φ f become unity?

According to Equation 2 the quantum yield becomes unity, if there is no quencher in the system or the rate of
emission is much higher than the rate of nonradidative decay.

4.11 How is the lifetime defined?

M∗ → M + hν (3)
The lifetime (τ) of the excited state is the time, the average molecule stays in the excited state before returning to
the ground state. Since process 3 has first order characteristic, the concentration of excited state molecules can be
expressed by:
[M∗ ] = [M∗ ]0 e−k·t
In order to get the lifetime, one has to apply the definition:
∞ ∞ #∞
e−kt
Z Z "
1 1
τ= [M ] dt =
∗
e −kt
dt = − = (4)
[M∗ ]0 0 0 k 0 k

The "natural" lifetime, that means, the lifetime for an ideal fluorescing system is the inverse of the rate constant of
emission, since k = knr + Γ and in the ideal case, knr = 0
1
τn =
Γ

4.12 Composition of the (real) Lifetime for Process (3)

The real lifetime is, of course, not the same as for the ideal system. There is nonradidative decay (knr ) and there
can be quenching (kq ), too. The nonradidative decay includes for example intersystem crossing or (irreversible)
first order reactions of the excited state.
Generally the lifetime is given by:
1
τ= (5)
Γ + knr + kq [Q]

4.13 Derivation of the Stern-Volmer Equation and the Stern-Volmer Diagram

If one forms the ratio of the quantum yield in absence of a quencher (Φ0 ) to the quantum yield if there is a quencher
(Φ) it can be written:
Φ0 Γ Γ + knr + kq [Q]
= ·
Φ Γ + knr Γ

6
After canceling Γ this can be rearranged into:

Φ0 Γ + knr kq [Q]
= +
Φ Γ + knr Γ + knr

According to Equation 5 if [Q]=0 the lifetime is given by τf = 1

Γ+knr . Now the upper equation can be written in the
form of the Stern-Volmer equation:
Φ0
= 1 + τf · kq [Q] = 1 + KSV · [Q] (6)
Φ
Stern-Volmer plots are given in Figure 6.
1.14 1.24

1.22
1.45 1.12
1.20
1.40
1.10 1.18
1.35
1.16
1.30 1.08
1.14
1.25
1.06 1.12
1.20
1.15 1.04 1.10

1.10 1.08
1.02
1.05 1.06

1.00 1.00 1.04

0.0015 0.0020 0.0025 0.0030 0.0035 0.0040 0.0010 0.0012 0.0014 0.0010 0.0012 0.0014

Figure 6: The Stern-Volmer plots. The data of Table 3 is used.

4.14 The Stern-Volmer constants KSV,NaX

According to Equation 6 the Stern-Volmer constant is equivalent to the slope of the Stern-Volmer plot. With the
data of Table 3 the Stern-Volmer constants are 160±90 M−1 , 200±200 M−1 and 300±500 M−1 for KSV,NaCl ,
KSV,NaBr and KSV,NaI . Obviously, the data is not worth anything else than a weak hint.

4.15 Rate Constant for the Quenching Process, if τ f equals 19.1 ns

From Equation 6 it follows, that:
KSV
kq =
τf
∆KSV
∆kq =
τf
The quenching constants then are: (8±5) 109 M−1 s−1 , (9±9) 109 M−1 s−1 and (10±30) 109 M−1 s−1 for NaCl,
NaBr and NaI.
In [3] only the value of kq,NaCl is given: 6.2 10−9 M −1 s−1 . This means, that at least our value has the correct
order of magnitude.

4.16 Trend in Quenching Ability of the Halogenides

Unfortunatly, the data does not allow a description of a trend, not only because of the really high uncertainty (the
confidence intervals are wide) but the mean values do not really depict a trend. However, if the trend is known, it
can be guessed.
In the literature, (e. g. [5]) the quenching ability increases from chloride to iodide. This is connected to the
screening power of water: Iodide has a lower charge density because of it’s higher radius, hence in a polar solvent,
the solvent shell of iodide is smaller and therefore it is more likely to collide directly with the excited molecule,
which is necessary for the quenching process.

7
4.17 Error Calculation
A gaussian error calculation is to be done for the error in kq if τf has an error of 1%,6 the balance one of 0.1 mg
and the intensities of I and I0 one of 1 (if I0 = 100).
From Equation 6 one can write: !
I0 1
kq = −1 ·
I τf [Q]
Gauss Error Calculation is done by noting that:

∂kq 2 ∂kq 2 ∂kq 2 ∂kq 2

! ! ! !
∆(kq ) =
2
(∆τf ) +
2
(∆[Q]) +2
(∆I) +
2
(∆I0 )2
∂τf [Q],I,I0 =const ∂[Q] τf ,I,I0 =const ∂I τf ,[Q],I0 =const ∂I0 τf ,[Q],I=const
(7)
!2 !2 2
1 I 0 1 1 I 0 1 1 1 I 1
∆(kq )2 = 2 −1 (∆τf )2 + −1 (∆[Q])2 + 2 (∆I0 )2 + 04 (∆I)2
τf I (τf [Q])2 [Q]2 I (τf [Q])2 I (τf [Q])2 I (τf [Q])2
 2 (I0 −I)2
Division by kq2 = II0 − 1 (τ [Q]) 1
2 =
1
I2 (τ [Q])2
leads to:
f f

∆(kq )2 1 1 1 I20
= 2 (∆τf ) +
2
(∆[Q]) +
2
(∆I0 ) + 2
2
(∆I)2
kq2 τf [Q] 2 (I0 − I)2 I (I0 − I) 2

∆[Q] can be related to the error of the balance, if we notice, that

c · V1
[Q] = (8)
V2
with c the concentration of the salt in the prepared solution, V1 the pipetted volume and V2 the volume of the
sample. Then ∆[Q] = V V2 ∆c. Since c = V·M with m the used amount of salt, V the volume of the volumetric flask
1 m
∆m
an M the molecular weight of the salt, ∆c = V·M , therefore:
V1 ∆m
∆[Q] = · (9)
V2 V · M
With equation 8 and 9 it can be wirtten:
!2
1 ∆m
(∆[Q])2 =
[Q]2 c·V·M
Now, the relative error in kq can be wirtten as:
s
∆kq ∆τ f 2
!2 !2 !2
∆m ∆I0 I0 ∆I
!
= + + + (10)
kq τf c·V ·M I0 − I I(I0 − I)
The relative errors in kq are summarised in Table 6. V is taken to be 100 ml.
∆kq ∆kq
Salt c/M M/(g mol−1 ) Imin kq min Imax kq max
NaCl 5 · 10−3 58.44256 70 5.9% 99.5 26.5%
NaBr 2 · 10−3 102.8939 89 13.7% 99 142%
NaI 2 · 10−3 149.894242 81 8.4% 95 284%

Table 6: Relative errors in kq

For I the highest an the lowest value measured was taken, since Equation 10 shows a strong dependence of the
error on the measured intensity I: For moderate values of I, the last two terms in Equation 10 are relatively small,
too. But for higher values of I, the error gets realy high, as can be seen in Table 10. This whole error calculation
is based on the assumption, that kq is calculated out of the single measurements, not with the linear regression
according to the Stern-Volmer equation.
6
According to [3] an error in the lifetime of about 3% would seem more realistic.

8
4.18 How does the error vary at the end of the experiment (low intensities)?
In theory: The lower the intensities, the higher the error, because the uncertainty of measurement becomes more
and more important (relativly).
In our experiment there were no really low intensities with one exeption and it seems to us, that the error show no
significant pattern.

4.19 Where was made the biggest error in measurement?

Generaly, the probably biggest error in measurement is made by reading the intensity on the analogous device, if
one is not used to it. Where our really big errors come from, we don’t know.

4.20 Why is a reference cell needed?

A reference intensity has to be measured for the use of the data according to the Stern-Volmer equation. The use
of the reference before every measurement is considered necessary in order to be sure no instrumental drift does
interfere with the fluorescence quenching: Without a reference cell, one can not be certain, the different intensities
are really due to the added quencher.
Strictly, for every measurement a new reference sample should be used. Like this a systematic error due to the
reference sample could be avoided and one has to be aware of the fact, that UV radiation damages irreversible the
analyte.

References
[1] Lakowicz, J. R. Priciples of Fluorescnce Spectroscopy; 1999.

[2] Atkins, P.; de Paula, J. Physical Chemistry; 7th ed.; 2002.

[3] Gutow, J. Journal of Chemical Education 2005, 82, 302.

[4] Bagott, G. Essentials of Molecular Photochemistry; 1991.

[5] Eisenbrand, J. Fresenius’ Zeitschrift für analytische Chemie 1961, 179, 406.