V

Stern-Volmer Quenching of Conjugated Polymers:

A Study of Fluorophore Concentration

An Undergraduate Thesis by
Christopher Vaughns
Faculty Advisor:
Dr. Uwe Bunz
Georgia Institute of Technology
School of Chemistry & Biochemistry

In Partial Fulfillment of
Bachelors of Science Degree in Chemistry

Georgia Instutite of Technology

School of Chemistry & Biochemistry
Fall 2007

Vaughns 2
Abstract
The purpose of this thesis is to re-examine the independence of Stern Volmer constants
on the fluorophore concentration. Conventional theory suggests that Stern-Volmer
constants are independent of fluorophore concentration. Using PPEs as fluorophore, the
Stern-Volmer constants were obtained for different PPE concentrations quenched with
TbCl3. Stern-Volmer constants increased with decreasing PPE concentration.

Keywords:
Stern-Volmer quenching
Conjugated polymers
poly(para-phenylenethynylenes)

Vaughns 3
Introduction
The objective of this thesis is to investigate the dependence of fluorophore
concentration with regard to Stern-Volmer quenching of conjugated polymers. This
research will provide new insight on the quenching that takes place in conjugated
polymers. Conjugated polymers are important as sensing materials for heavy metal ions
in water, organic vapors, and explosives through fluorescence quenching via SternVolmer behavior.1 The classical sensing methods are atomic absorption spectrometry,
anodic stripping voltametry and X-ray fluorescence spectrometry, which all are
expensive methods of sensory determination. Fluorescence quenching of conjugated
polymers in the presence of analyte may provide quick inexpensive methods for the
sensing of dangerous substances.
Fluorescence quenching involves two chemical species, the fluorophore and the
quencher. A fluorophore is a chemical species that, when excited, emits fluorescence,
and a quencher is a chemical species that reduces the fluorescence of a fluorophore.
Fluorescence quenching is measured quantitatively with the Stern-Volmer equation. In
equation 1, the quencher concentration is [Q], the Stern-Volmer constant is Ksv, Fo is
the measured fluorescence intensity without quencher present, and F[Q] is the measured
fluorescence intensity with [Q] present. After plotting (Fo / F[Q]) against [Q], the slope
can be determined to give the value of Ksv, the Stern-Volmer constant. Generally, a
more sensitive system will have a steeper slope and, as a result, a higher Ksv value. The
Stern-Volmer equation does not contain a variable for the fluorophore concentration and
suggests that fluorescence quenching is independent of the fluorophore concentration.2

Vaughns 4

Fo/F[Q] = 1 +Ksv[Q] or Ksv =((Fo/F[Q]) -1)/[Q]

Equation 1: The Stern-Volmer equation does not have a variable for fluorophore concentration, suggesting
fluorophore independence

Fluorescence quenching typically occurs through the mechanisms of either static

or dynamic quenching. In dynamic quenching, a collision between the quencher and
fluorophore takes place during the excitation lifetime of the fluorophore. The efficiency
of dynamic quenching is limited by the lifetime of the excited state of the fluorophore
and the quencher concentration. In static quenching, complexation occurs in the ground
state between the quenching species and the fluorophore. In this mechanism, quenching
of the excited fluorophore by the complex quencher occurs upon irradiation. Due to the
short lifetimes (300 ps) of PPEs, they only exhibit static quenching.
Poly(aryleneethynylene)s (PAEs) are a category of conjugated polymers by which
arene groups are bonded together by alkyne linkers. Generally, PAEs present distinct
chromogenic and fluorescent behavior. Though PAEs have similar structural
characteristic to other conjugated systems, PAEs exhibit exceptional fluorescence
capabilities when compared to other conjugated systems. Poly(paraphenylenethynylene)s (PPE) fall within this class of polymers, and demonstrate
extraordinary fluorescence in solution and in the solid state. 1

Vaughns 5

Figure 1: PPEs are conjugated molecules where the arene groups are in conjugation with the alkyne linkers.
Diverse functionality can be introduced to PPEs by simply altering the R groups. n is the number of
subunits.

Many quenching studies involving PPEs as a fluorophore have been conducted to

evaluate the substituent effects, fluorophore arrangement, and the solvent effects on
Stern-Volmer quenching. These studies have been vital in the advancements of
conjugated polymeric fluorescence quenching because they have given insight on
creating ideal chemo sensors. The investigation of fluorophore concentration effects on
Stern-Volmer quenching behavior is at the forefront of current research, and there have
not been any publications on these effects.
Research groups have placed a great emphasis of the methods in which PPE
experience fluorescence quenching. There have been significant strides in the research of
the molecular wire effect with regard to fluorescence quenching. The main premise is that
chemical sensors attached together on a molecular backbone may act similarly to an
electrical circuit wired in series. Swager demonstrated increased sensitivity to
conjugated polymers when comparing quenching of PPE wired in series with PPE endcapped with anthracence units.3 While using methyl viologen as a quencher, results
showed a 67-fold increase when comparing the PPE wired in series to a non-wired PPE.

Vaughns 6
The conclusion from Swagers work is that augmented quenching occurs when the
fluorophore is in a molecular wire arrangement. Like an electrical circuit, the rest of the
molecular chain ceases to fluoresce when a quencher complexes to one part of the chain.
As figure 2 demonstrates, isolated chemo sensors can continue to emit fluorescence after
one sensor has been quenched with quencher where as fluorescent sensors wired in series
cannot fluoresce after one sensor has been quenched. Swagers work shows that
fluorescent chemosensory systems wired in series are advantageous because of the
increased sensitivity, which is vital for sensing dangerous materials.

Figure 2: Illustration of the emission difference with isolated and wired PPE.

In studies in which sugar-substituted PPEs were quenched with mercury and lead
ions, Kim has observed a phenomenon known as cooperative quenching. The
experiments concluded that two or more sugar substituents could participate in the
complexation of a single mercury ion, which would give significantly higher Ksv values
than a PPE not experiencing cooperative quenching. 4

Vaughns 7

Figure 3: Cooperative quenching is illustrated as two sugar-PPE sensors bind to mercury

Un-aggregated PPEs in solution exhibit a short fluorescence lifetime of

approximately .3 to .5 ns.3 These fluorescence lifetimes do change upon the addition of
quencher. As a result, static quenching is the established quenching mechanism in PPE
fluorescence quenching. In these cases of static quenching, the Stern-Volmer constant is
equal to the binding constant of the quencher to the fluorophore (PPE).
Schanze reports anionic PPEs that are significantly quenched by methyl viologen.
In addition to demonstrating anionic PPE quenching, this study also reflects the
differences of quenching when immersed into different solvents. In methanol, sulfonated
7
4
-1
PPE and its analog expressed Ksv values of 1.4 x10 and 2.2 x 10 M , respectively. In

water, the Ksv values were 2.7 x107 M-1 and 7.0 x103 M-1, respectively. This polymer was
more aggregated in water but not in methanol because of the polymers more polar
character. This aggregation may result in an increased self-quenching.
Quenching studies have focused primarily on the mechanism of PPE quenching as
well as the effects of aggregation in chemical sensing.

Vaughns 8
Procedure & Methods
The methods for re-evaluating fluorophore concentration dependence on SternVolmer quenching involves synthesizing un-aggregatated PPEs, preparing stock and
sample solutions, and conducting quenching experiments.
Synthesis of PPE
Before any quenching studies can take place, the polymers must first be
synthesized. One of the polymers used for the concentration-dependent quenching studies
was also used in the sensing of lead ions. As indicated in scheme 1, synthesis of the
polymer takes place by starting from 2,5 diiodohydroquinone in the presence of
potassium carbonate, which yields compound 1. Trimethylsilylacetylene and a
Pd(PPh3)2Cl2 / CuI catalyst are placed with triethylamine to produce compound 2.
Coupling of 1 and 2 at 50 C for 72 hours produced polymer 3 (carboxylate- substituted
PPE) with a 93% yield and molecular weight of 12 x 103 g/mole. A solution of polymer 3
in methanol was treated with NaOH to yield the water-soluble carboxylate-substituted
PPE.

Vaughns 9

OH
I

Br
I

TMS
(Ph3P)2PdCl2
CuI/NEt3

OEt
I

K2CO3

HO

O
(Ph3P)2PdCl2

I
then Bu4N+F-

O
O

O
O

n*

CuI/NEt3
Ch2Cl2
O

Na -O

Na OH
CH3OH

O
*

*
n
O

(Ph3P)2PdCl2
CuI/NEt3

OMe

then NaOH/MeOH

+ Na -O
+ Na -O

O
Polymer Carboxylate-Substitued PPE

O
MeO

OMe
O
+

Na -O

5
Model Carboxylate-Substitued PPE

Scheme 1: Synthetic route for the synthesis of water-soluble carboxylated PPEs

Preparation of Stock and Sample Solutions:

After obtaining synthesized PPE, a stock solution of concentration 5 x 10-4 M was
prepared by dissolving the appropriate amount of PPE into 20 mL of deionized water.
The stock solution would serve as the principal source of PPE for the preparation of the
varying concentrations of PPE sample solutions. Using a pre-determined volume of the
PPE stock solution, a series of sample solutions were prepared via serial dilution as seen
below in figure 4.

Vaughns 10

PPE Stock Solution

PPE Sample Solution

Quenching Vials

Figure 4: A concentrated PPE stock solution is prepared and used to make up all diluted PPE samples.
Schematic also indicates that quenching experiments take place for each PPE sample solution.

Vial #
0
1
2
3
4
5
6
7
8
9
10

Volume of Sample
PPE solution
(mL)
5
5
5
5
5
5
5
5
5
5
5

Volume of
TbCl3 quencher
(L)
0
10
20
30
40
50
60
70
80
90
100

Table 1: The fluorescence quenching of each vial is measured using fluorimetry and a quenching curve is

generated for each vial. The concentration of quencher added depends on PPE sensitivity.

Vaughns 11
Quenching Experiments
For these quenching experiments, TbCl3 was used as the quencher. As described
earlier, PPE substituents can affect the sensitivity to a particular quencher so a method of
guess and check was applied to determine the appropriate quencher concentration range
for the experiments. Before data collection occurs, a volume of 10 L of TbCl3 of
either .01M, .001 M or .0001 M was added to three different vials containing 5mL of PPE
solution. The different quenching effects were observed and an appropriate range was
determined. After determining the appropriate quencher concentration range, a
quenching experiment was conducted for each concentration of PPE sample solution. A
series of 11 vials were prepared that all contained the same volume and concentration of
PPE, but also contained different volumes of TbCl3. The contents of each vial were
transferred into a cuvette and measured by spectrofluorimetry. PPEs typically absorb at
430 nm, and an emission spectra was obtained for each vial with excitation at 405 nm.
The quenching effects were examined for each concentration of PPE so that the
differences in Stern-Volmer activity could be observed. Both PPEs identified as IBK 85
and IBK 146 were used for the quenching experiments.
RO

+Na -O

O
*

Na -O

*
n

O
RO
O

O
N

OR

O
+

O
N

O
OR

O
OR

Scheme 2: Chemical Structure for IBK 85 (on the left); Chemical Structure for IBK 146 (on the right)

Vaughns 12
Results
250
0 M
4 M
6 M

150

50
40

8 M
Fo/F

Intensity (a.u)

60

2 M

200

10 M
12 M

100

30
20

16 M
20 M

10

50

0
0

0
425

475

525
Wavelength
(nm)

2.5

10

15

20

25

[Q] * 10-6 (M)

625

y = 0.1582x + 1
R2 = 0.9101

Fo/F

575

1.5
1
0.5
0
0

[Q] * 10-6 (M)

Figure 5: The quenching curves, and Stern-Volmer plots for PPE IBK 85 at 5 x 10 M
-5

700

80
70

0 M

500

2 M

60

4 M

400

6 M

50
Fo/F

Intensity (a.u)

600

8 M

300

10 M

30

12 M

200

20

16 M

10

20 M

100

40

0
0

0
425

475

525

575

625

10

15
[Q] * 10-6M

Wavelength (nm)
y = 0.7639x + 1
R2 = 0.9061

7
6

Fo/F

5
4
3
2
1
0
0

[Q] * 10-6M

Figure 6: The quenching curves, and Stern-Volmer plots for PPE IBK 85 at 1 x 10 M
-5

20

25

Vaughns 13

250

150

50
40
Fo/F

200
Intensity (a.u)

60

0 M
2 M
4 M
6 M
8 M
10 M
12 M
16 M
20 M

100

30
20
10

50

0
0

0
425

475

525
Wavelength (nm)

575

10

15

20

25

[Q] * 10-6 (M)

625

y = 1.0741x + 1
R2 = 0.9636

9
8
7

Fo/F

6
5
4
3
2
1
0
0

-6

[Q] * 10 (M)

Figure 7: The quenching curves, and Stern-Volmer plots for PPE IBK 85 at 5 x 10 M
-6

200
0 M

180

2 M
4 M

140

6 M

80

120

8 M

70

100

10 M

60

12 M

80

50

16 M

60

Fo/F

Intensity (a.u)

160

20 M

40
30

40

20

20

10

0
425

475

525

575

625

Wavelength (nm)

0
0

10

15

[Q] * 10-6 (M)

12
10

y = 1.3949x + 1
R2 = 0.893

Fo/F

8
6
4
2
0
0

[Q] * 10-6 (M)

Figure 8: The quenching curves, and Stern-Volmer plots for PPE IBK 85 at 1 x 10 M
-6

20

25

Vaughns 14

1x10-6M

30

5x10-6M
1X10-5M

25

5x10-5M

Fo/F

20
15
10
5
0
0

10

-6

[Q] * 10 (M)

Figure 9: Summary of Stern-Volmer plots for PPE IBK 85

PPE Concentration
(M)
5 x 10-5
1 x 10-5
5 x 10-6
1 x 10-6

Ksv
(M-1)
1.6 x 105
7.6 x 105
1.0 x 106
1.4 x 106

Linear Regression
Correlation Coefficient
.91
.91
.96
.89

Table 2: Stern-Volmer constants in quenching curve are generated for each vial. The concentration of
quencher added depends on PPE sensitivity.

Vaughns 15

400

0.0 M
0.5 M

350

1 M

16

1.5 M
2.0 M

14

2.5 M

12

200

3.0 M

10

150

3.5 M

100

4.5 M

50

5.0 M

250

4.0 M

Fo/F

Intensity (a.u)

300

8
6
4
2

0
425

475

525

575

625

0.0

1.0

2.0

2.5

4.0

5.0

6.0

[Q] * 10 (M)

y = 0.604x + 1
R2 = 0.9457

Fo/F

3.0
6

Wavelength (nm)

1.5
1
0.5
0
0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

-6

[Q] * 10 (M)

Figure 10: The quenching curves, and Stern-Volmer plots for PPE IBK 146 at 5 x 10 M
-6

250

0.0 M

18

0.5 M

16

200

1.0 M

14

1.5 M

12

2.0 M

10

150

2.5 M

100
50
0
425

475

525

3.0 M

3.5 M

4.0 M

4.5 M

6255.0 M

575

Fo/F

Intensity (a.u)

300

Wavelength (nm)

0.0

1.0

2.0

3.0

4.0

[Q] * 10-6 (M)

3
y = 1.0431x + 1
R2 = 0.8885

2.5

Fo/F

2
1.5
1
0.5
0
0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

[Q] * 10-6 (M)

Figure 11: The quenching curves, and Stern-Volmer plots for PPE IBK 146 at 1 x 10 M
-6

5.0

6.0

Vaughns 16

350

0.0 M
0.5 M

35
30

1.0 M

250

1.5 M

25

200

2.0 M
2.5 M

20

150

3.0 M

Fo/F

Intensity (a.u)

300

3.5 M

100

10

4.0 M
4.5 M

50

5.0 M

0
425

475

525

575

15

0
0.0

625

1.0

2.0

3.0

4.0

5.0

[Q] * 10 (M)

Wavelength (nm)

3
2.5

y = 0.9071x + 1
R2 = 0.9184

Fo/F

2
1.5
1
0.5
0
0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

[Q] * 106 (M)

Figure 12: The quenching curves, and Stern-Volmer plots for PPE IBK 146 at 5 x 10 M
-7

250
0.0 M

25

0.5 M
1.0 M

150

20

1.5 M
2.0 M

100

2.5 M
3.0 M

Fo/F

Intensity (a.u)

200

15
10

3.5 M

50

4.0 M

5
0

425

475

525

575

625

3.5

0.0

1.0

2.0

3.0

[Q] * 10-6 (M)

Wavelength (nm)

y = 1.1822x + 1
R2 = 0.9681

Fo/F

2.5
2
1.5
1
0.5
0
0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

[Q] * 10-6 (M)

Figure 13: The quenching curves, and Stern-Volmer plots for PPE IBK 146 at 1 x 10 M
-7

4.0

5.0

Vaughns 17

3.5

5x10-6M

1x10-6M
5x10-7M

Fo/F

2.5

1x10-7M

2
1.5
1
0.5
0
0

0.5

1.5

-6

[Q] * 10 (M)

Figure 14: Summary of Stern-Volmer plots for PPE IBK 146

PPE Concentration
(M)
5 x 10-6
1 x 10-6
5 x 10-7
1 x 10-7

Ksv
(M-1)
6.0 x 105
1.0 x 106
9.1 x 105
1.2 x 106

Table 3: Summary of Stern Volmer constants for PPE IBK 146

Linear Regression
Correlation Coefficient
.95
.89
.92
.89

Vaughns 18
Discussion
Data was obtained for PPE IBK 85 quenching with terbium (III) chloride at PPE
concentrations of 5 x 10-5 M, 1 x 10-5 M, 5 x 10-6 M, and 1 x 10-6 M. An appropriate
quenching range was determined to be between 0 and 20 M of TbCl3 through the
procedure described in the methods section. Quenching experiments were conducted for
each concentration at the specified quenching range, and the fluorescence of each
solution containing quencher was measured. The maximum intensity for the solutions
without quencher was seen at 461nm, and as a result, the intensities at this wavelength
were compared to intensity without quencher. This procedure gave the ratio of Fo/F[Q],
which was plotted against the quencher concentration range. A fluorescence spectrum,
quenching curve, and Stern-Volmer plot are present for each concentration quenching
experiment in figures 5 -8. The quenching curve demonstrates the quenching behavior
over the entire pre-determined quenching range, while the other plot zooms in on the
more classical linear Stern-Volmer activity.
As seen in figure 5, the quenching experiment of concentration of 5 x 10-5 M PPE
-1
IBK 85 had an apparent Ksv constant of 1.6 x 105 M . Figure 6 demonstrates an apparent

Ksv constant of 7.6 x 105 M-1 at a PPE IBK 85 concentration of 1 x 10-5 M. PPE IBK 85
-1
of concentration 5 x 10-6 M yielded an apparent Ksv constant 1.0 x 106 M , as seen in

figure7. Figure 8 illustrates that an apparent Ksv constant of 1.4 x 106 M-1 was observed
for PPE IBK 85 concentration of 1 x 10-6 M.
Figure 9 shows the quenching curves for all the experimented PPE concentration
are one plot, and demonstrates that sensitivity for the terbium quencher increases with
decreasing PPE concentration. PPE sensitivity to the terbium quencher was at its lowest

Vaughns 19
at 5 x 10-5 M and sensitivity was at its highest at the concentration of 1 x 10-6 M. As seen
in table 2, all the linear regression coefficients were approximately .9, which suggests a
high linear correlation. This high linear correlation between the quencher concentration
and Fo/F[Q] is in accordance with the Stern-Volmer equation 1.
The results suggest there is a fluorophore concentration effect on Stern-Volmer
quenching. Generally, there is a higher sensitivity observed with lower concentration of
PPE than with higher concentrations. More dilute concentrations of PPE have high SternVolmer constant in quenching with terbium (III) chloride. As figure 5- 8 demonstrate,
concentrations varying from 5 x 10-5 M to 1 x 10-7 M had increasing Stern-Volmer
constants, which suggest that there is a greater sensitivity to the terbium quencher with
decreased PPE concentration.
Though this data agrees with the prediction of increased sensitivity with
decreased PPE concentration, this data comes with a cautionary note because there are
some problems with the fluorescence spectrum for each of the PPE IBK 85
concentrations. Blue shift is a phenomenon by which the maximum emission of the PPE
shifts towards the blue region of the electromagnetic spectrum. The major suggestion for
this phenomenon among this data set is that longer chains of PPE are efficiently
quenched first, and that unquenched shorter chains are attributing to a lower maximum
emission wavelength. This phenomenon is similar to Swagers work in comparing the
molecular wire chemo sensor to an isolated chemo sensor, where longer chains of PPE
are completely quenched before the shorter chains. Despite this cautionary note, it is
important to acknowledge that the Stern-Volmer constants changed with fluorophore PPE
concentration and seem in accordance with predicted behavior.

Vaughns 20

Since blueshift activity was observed in the PPE IBK 85 solutions, quenching
studies were conducted with another PPE. As seen below in figure 15, PPE IBK 146 has
a lesser degree of blueshift than PPE IBK 85.

400

0.0 M
1 M

300

0 M
2 M

200

1.5 M

4 M
6 M

2.0 M

250

2.5 M

200

3.0 M
3.5 M

150

4.0 M

100

In te n s ity (a .u )

Intensity (a.u)

250

0.5 M

350

8 M
10 M

150

12 M
16 M

100

4.5 M
5.0 M

50

20 M
50

0
425

475

525

575

625

0
425

Wavelength (nm)

475

525
575
Wavelength (nm)

625

-6

Figure 15: On the left PPE IBK 146 at 5x 10 M does not have any blueshift activity

On the right PPE IBK 85 shows blueshift activity at 5x 10-6 M

Data was obtained for PPE IBK 146 quenching with terbium (III) chloride at PPE
concentrations of 5 x 10-6 M, 1 x 10-6 M, 5 x 10-7 M, and 1 x 10-7 M. An appropriate
quenching range was determined to be between 0 and 5 M of TbCl3 through the
procedure described in the methods section. Quenching experiments were conducted for
each concentration at the specified quenching range, and the fluorescence of each
solution containing quencher was measured using the same procedure for PPE IBK 85.
Using the same technique to obtain Stern-Volmer plot for IBK 85, the ratio of Fo/F[Q]
was plotted against the quencher concentration range. A fluorescence spectrum,
quenching curve, and Stern-Volmer plot are present for each concentration quenching
experiment in figures 10-13.
As figure 10 illustrates, the quenching experiment for the concentration of

Vaughns 21
5 x 10-6 M PPE IBK 146 had an apparent Ksv constant of 6.0 x 105 M-1. Figure 11 shows
an apparent Ksv constant of 1.0 x 106 M-1 at a PPE IBK 146 concentration of 1 x 10-6 M.
-1
PPE IBK 146 of concentration 5 x 10-7 M yielded an apparent Ksv constant 9.1 x 105 M ,

as seen in figure 12. Figure 13 points to an apparent Ksv constant of 1.2 x 106 M-1 was
observed for PPE IBK 146 concentration of 1 x 10-7 M.
When considering the elimination of blue shift activity as seen in PPE IBK 146,
the data agrees with the prediction of increased sensitivity with decreased PPE
concentration. As figure 14 indicates, PPE sensitivity was minimized at concentration of
5 x 10-6 M and maximized at 1 x 10-7 M. Though the exact dependence of fluorophore
cannot be equated to a variable in the Stern-Volmer equation, the data leads to the
conclusion that there is some dependence on the fluorophore concentration in SternVolmer behavior. This research has grave implications on chemo sensing and suggests
that lesser concentrated chemo sensors will provide greater sensitivity. Future research
needs to involve different PPEs with different substituents to allow for greater insight in
the general fluorophore concentration effect of Stern-Volmer quenching on conjugated
polymers.

Vaughns 22
References:
(1)

Bunz, U. H. Poly(arylene ethynylene)s 2005, 1-52.

(2)

Lackowicz, J. R. Principles of Fluorescence Spectroscopy 1983, 262268.

(3)
Zhou, Q.; Swager, T. M. Journal of the American Chemical Society 1995,
117, 12593-12602.
(4)
Kim, I.-B.; Erdogan, B.; Wilson, J.; Bunz, U. Chemistry European
Journal 2004, 10, 6247-6254.