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1/98 WWI-M14

FLUORESCENCE BASED SENSING FOR CRYSTALLIZATION AND FOR
SOLUTION CHARACTERIZATION
By

Qiuxia Wang

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTORS OF PHILOSOPHY

Departments of Chemical Engineering and Chemistry
1998

ABSTRACT

FLUORESCENCE BASED SENSING FOR CRYSTALLIZATION AND FOR
SOLUTION CHARACTERIZATION
By

Qiuxia Wang

Production of a specified crystal size distribution (CSD) is one of the primary goals
of a crystallization process because CSD affects the cost of operation. Filtration and drying
are often the limiting steps in chemical manufacturing processes and significant cost
reductions can be realized by creating CSDs that have favorable filtration and drying
properties. The CSD is determined by supersaturation of solution. Control of the CSD is
realized by control of supersaturation which requires the accurate measurement of the
supersaturation. No technique is available for the measurement of supersaturation of low
solubility systems with solubility around 2wt%.

In this research, pyranine, 1-pyrene butyric acid and carminic acid were
investigated as potential fluorescence probes to measure the supersaturation of low
solubility systems, such as tryptOphan. nicotinic acid, phenylalanine and benzoic acid,
using steady state fluorescence. Pyranine was shown to be a very promising probe to be
used as the sensor to measure supersaturation in solutions due to the smooth and nonlinear
calibration curves which show a higher signal response at supersaturated region. In
addition pyranine will not enter the crystal lattice. Pyranine is not listed as a food additive
by FDA, so it can not be added directly into food and pharmaceutical production processes.
Carminic acid (CA), a FDA approved food additive, was shown to be a good sensor for

phenylalanine solutions by following the fluorescence intensity change with the
concentration of phenylalanine. With pyranine immobilized on a membrane, the PIR (Peak
Intensity Ratio) changes with the concentration of solutes linearly. The fluorescence probe
technique is a very promising technique to be used to monitor crystallization in situ.

The formation of nuclei is the beginning point of the crystallization. The nucleation
of the solute must somehow depend upon the solute molecules in a supersaturated solution
and also depend upon the various molecular interactions. Understanding the structure of
supersaturated solutions can help us better design and control crystallization. Although lots
of work has focused on this subject, no generally accepted theories are available, especially
for low solubility systems, and there are no reports on supersaturated solution structure at
all.

Fluorescence quenching requires the contact of fluorophores and quenchers, so the
interaction of fluorophore and quenchers can provide information about the organization of
solutes in solutions. Quenching curves show different trends of quenching in unsaturated
and supersaturating regions which was taken as the evidence of the formation of aggregates
of the solutes in supersaturated solutions. Different molecules show totally different
quenching behaviors to pyranine in solution. The different quenching behaviors are the
results of the different interactions of pyranine with solute molecules. The contact complex
was suggested as the mechanism of the interaction between pyranine and solute molecules.
The stronger quenching abilities of tryptophan and nicotinic acid are the results of
formation of hydrogen bonding complex with pyranine duo to the amine in the rings. The
different steady state quenching and lifetime quenching behavior of phenylalanine support
strongly the contact complex assumption. The associates were suggested in tryptophan
solutions and phenylalanine due to the downward quenching curves. It was shown only
73% of phenylalanine and tryptophan are accessible to fluorophores to quench the
fluorescence. The excitation wavelength dependence of tryptophan maximum emission

wavelength supports the exists of the associates in tryptophan solutions.

ACKNOWLEDGEMENTS

First, I would like to thank my mentor Kris A. Berglund for his encouragement,
guidance and support. He helped me a lot personally and professionally. A body of work
such as this would not be possible without his assistance. It has been a privilege to have
had him as my preceptor. What I have learned from him is far beyond the appreciation of
chemistry and chemical engineering, and what I learned will continue to influence me in my
future.

I would like also to thank my committee members, Drs. Gary Blanchard, Greg
Baker and Alec Scranton for their time and assistance.

I would also like to thank my colleagues in the research group for their caring and
help. Everyone I have had the opportunity to interact with has meant something to me and
helped in many different ways. Special thanks to Sanjay Yedur who I can always turn to
wheneverI need help in research and in my personal life. Also, special thanks to Dale
LeCaptain who opened opportunities for me to experience American culture by inviting me
to numerous social activities.

Many thanks are also due to my family for their love and my friends here in the
USA and in China for their caring and support. They shared my frustrations and the
happiness. All those unforgettable long hour talks on the phone helped me through the
tough times and made me a happier person (of course the big phone bill annoyed my
landlady sometimes). Out of many, I in gpin Jia, one of my great friends, helped me get
settled here at MSU. I knew that whatever happened, I could count on her to be right there
with her caring and support, that means a lot to me.

To all these people, thank you from the bottom of my heart.

iv

TABLE OF CONTENTS

LISTS OF TABLES ................................................................................ viii
LIST OF FIGURES .................................................................................. x
CHAPTER 1
INTRODUCTION ................................................................................... 1
1.1 Control of crystallization by control of supersaturation ........................ 1
1.2 The fluorescent probe technique for measurement of supersaturation ........ 5
1.3 The structure study of supersaturated solutions by fluorescence
quenching ............................................................................ 8
1.4 Introduction of the systems for study ........................................... 10
1.5 Overview of this work ............................................................ 13
1.6 References ......................................................................... 16
CHAPTER 2
SUPERSATURATION MEASUREMENTS OF LOW SOLUBILITY SYSTEMS
USING A STEADY STATE FLUORESCENCE PROBE TECHNIQUE ................... 19
2.1 Introduction ....................................................................... 19
2.2 Materials and methods ........................................................... 21
2.3 Results and discussions .......................................................... 21
2.3.1 Steady state fluorescence ............................................... 21
2.3.2 Measurement of concentration ......................................... 28
2.4 Conclusions ...................................................................... 33
2.5 References .......................................................................... 33
CHAPTER 3
SUPERSATURATION MEASUREMENTS BY USING THE STEADY STATE
FLUORESCENCE OF IMMOBILIZED PYRANINE ......................................... 35
3.1 Introduction ....................................................................... 35
3.2 Materials and methods ........................................................... 36
3.3 Results and discussions ........................................................ 37
3.3.1 Fluorescence spectra of immobilized pyranine ...................... 37
3.3.2 Measurement of supersaturation ....................................... 42
3.3.3 Measurement of concentration .......................................... 44
3.4 Conclusion ......................................................................... 44
3.5 References ......................................................................... 46
CHAPER 4
THE EXPLORATION OF OTHER POSSIBLE PROBES--PBA AND CA ................. 47
4.1 Introduction ....................................................................... 47
4.2 Materials and methods ........................................................... 49
4.3 Results and discussions .......................................................... 51
4.3.1 Steady state fluorescence of PBA as probe ............................ 51
4.3.2 Calibration curve of PBA as probe ................................... 51

V

4.3.3 Steady state fluorescence of carminic acid as probe .................. 54

4.3.4 Quenching behaviors of tryptophan and phenylalanine .............. 59
4.3.5 Detection of PBA and CA in crystalline products ................... 62
4.4 Conclusion ........................................................................ 64
4.5 References .......................................................................... 64
CHAPTER 5
MECHANISM OF PYRANINE FLUORESCENCE QUENCHING AND THE
APPLICATION IN THE STUDY OF SUPERSATURATED SOLUTION ................ 66
5.1 Introduction ........................................................................ 66
5.2 Theory .............................................................................. 68
5.3 Materials and methods ............................................................ 70
5.4 Results .............................................................................. 71
5.4.1 Tryptophan quenching of pyranine .................................... 71
5.4.2 Phenylalanine quenching of pyranine .................................. 73
5.4.3 Nicotinic acid quenching of pyranine fluorescence .................. 77
5.4.4 The absorption spectra of pyranine in water and in solutions ....... 82
5.5 Discussion ......................................................................... 82
5.6 Conclusions ........................................................................ 91
5.7 References .......................................................................... 91
CHAPTER 6
QUENCHING STUDY BY MEASUREMENT OF LIFETIME OF PYRANINE IN
DIFFERENT SOLUTIONS ....................................................................... 94
6.1 Introduction ....................................................................... 94
6.2 Materials and methods ........................................................... 96
6.3 Results and discussions .......................................................... 97
6.3.1 Fluorescence lifetime quenching of pyranine at 510nm
in different solutions .................................................... 97
6.3.2 Fluorescence lifetime quenching of pyranine at 382nm
in different solutions .................................................... 102
6.4 Conclusion ........................................................................ 105
6.5 References ....................................................................... 107
CHAPTER 7
THE DEPENDENCE OF EXCITATION WAVELENGTH OF TRYPTOPHAN
FLUORESCENCE IN TRYPTOPHAN AQUEOUS SOLUTION .......................... 108
7.1 Introduction ....................................................................... 108
7.2 Materials and experiments ...................................................... 110
7.3 Results ............................................................................ 110
7.3.1 The fluorescence spectra of tryptophan in aqueous solution ....... 110
7.3.2 Excitation spectra versus concentration of tryptophan .............. 114
7.3.3 Excitation wavelength dependence of maximum emission
wavelength .............................................................. 114
7.3.4 Lifetime of tryptophan in different solvents at different
concentration ............................................................ 1 17
7.3.5 Comparison of indole fluorescence and tryptophan
fluorescence ............................................................. 119
7.4 Discussion ....................................................................... 123
7.5 Conclusion ........................................................................ 126
7.6 References ....................................................................... 126

CHAPER 8
EXCITATION SPECTRA STUDY OF PYRANINE IN WATER AND DIFFERENT

SOLUTIONS WITH DIFFERENT CONCENTRATIONS .................................. 128
8.1 Introduction ...................................................................... 128
8.2 Materials and methods .......................................................... 129
8.3 Results and discussions ......................................................... 130
8.3.1 Excitation spectra at 510nm emission wavelength ................ 130
8.3.1.1 Excitation spectra of pyranine in water and low
concentration solutions at 510nm .................. 130
8.3.1.2 Excitation spectra Of pyranine in different
concentration of solutions ............................ 138
8.3.2 Excitation spectra at 382nm emission wavelength ................. 139
8.3.2.1 Excitation spectra of pyranine in water and low
concentration solutions ............................... 139
8.3.2.2 Excitation spectra of pyranine in different
concentration of solutions ............................ 141
8.4 Conclusion ........................................................................ 145
8.5 References ....................................................................... 145
CHAPTER 9
SUMMARY OF THE WORK AND PROPOSED FUTURE WORK ...................... 147
9.1 Summary of the work .......................................................... 147
9.2 Future work ...................................................................... 149
9.2.1 Structure study of supersaturated solutions .......................... 149
9.2.2 Flow Injection Analysis (FIA) ........................................ 151
9.3 References ....................................................................... 154
APPENDIX ........................................................................................ l 55

vii

Table A1
Table A2
Table A3
Table A4
Table A5
Table A6
Table A7
Table A8
Table A9
Table A 10
Table A1 1
Table A l 2
Table A 1 3
Table A 14
Table A 15
Table A16
Table A17
Table A 1 8
Table A19
Table A20
Table A21
Table A22

Raw
Raw
Raw
Raw
Raw
Raw
Raw
Raw
Raw
Raw
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Raw
Raw
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Raw
Raw
Raw
Raw
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data
data
data
data
data
data
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data
data
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data
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for

for

LIST OF TABLES

Figure
Figure
Figure
Figure
Figure
Figure
Figure
Figure
Figure
Figure
Figure
Figure
Figure
Figure
Figure
Figure
Figure
Figure
Figure
Figure
Figure

Figure

2.4 ........................................................ 155
2.5 ........................................................ 156
2.6 ........................................................ 156
3.3 ........................................................ 157
3 4 ........................................................ 158
4.3 ........................................................ 159
4.4 ........................................................ 160
4.6 ........................................................ 161
4.7 ........................................................ 161
4.8 ........................................................ 162
4.9 ........................................................ 163
4.10 ...................................................... 163
5.1 ........................................................ 164
5.2 ........................................................ 165
5.3 ........................................................ 166
5.4 ........................................................ 167
5.5 ........................................................ 167
5.6 ........................................................ 168
5.7 ........................................................ 168
5.8 ........................................................ 169
6.1 ........................................................ 169
6.2 ........................................................ 170

viii

Table A23
Table A24
Table A25
Table A26
Table A27
Table A28
Table A29
Table A30
Table A31

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Raw
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Raw
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Figure 6.3 ........................................................ 171

Figure 6.4 ........................................................ 172
Figure 6.5 ........................................................ 173
Figure 6.6 ........................................................ 173
Figure 7.2 ........................................................ 174
Figure 7.3 ........................................................ 174
Figure 7.6 ........................................................ 175
Figure 7.7 ........................................................ 177
Figure 8.4 ........................................................ 177

ix

Figure 1.1

Figure 1.2

Figure 2.1
Figure 2.2

Figure 2.3

Figure 2.4

Figure 2.5

Figure 2.6

Figure 2.7

Figure 3.1

Figure 3.2

LIST OF FIGURES

The supersaturation versus corresponding time profiles for
different cooling rates (3) ......................................................... 3

Figure 1.2. Schematic diagram of rate of processes in exciplex formation,
dissociation and quenching. Solid lines, radiative processed; broken lines,
radiationless processes. (i) formation of exciplex between fluorophore M
and quencher Q, (ii) fluorescence of exciplex, (iii) internal conversion of
exciplex, (iv) desociation of exciplex (v) energy transfer, (vi) electron
transfer, (vii) intersystem crossing of exciplex ............................... 11

Molecular structure of 8-hydroxyl-1,3,6—pyrenetrisulfonate (pyranine) ...22

Scheme for acid-base equilibria in the ground state and excited state of
pyranine. Deprotonated form of pyranine (PyO‘) will be excited

at 440nm and emit at 510nm. Protonated form of pyranine (PyOH)

will be excited at 405nm and emit at 450nm. pKa=7.5 and pKa*=0.5....24

Fluorescence of pyranine in water (0) and in aqueous tryptophan
solutions: 1 (0.0793g). 6 (0.51G), 11 (1.02g) and 18 (1.73g)
tryptophan in 100ml H20 at room temperature ................................ 25

PIR (peak intensity ratio) of pyranine(1x105M) versus concentration
of aqueous tryptophan solutions at room temperature. Lines are
polynomial fitting, and arrow indicates the solubility of tryptophan ........ 29

PIR (peak intensity ratio) of pyranine (4x10'5M) versus concentration
of aqueous nicotinic acid solutions at room temperature. The arrow
indicates the solubility of nicotinic acid at 25°C ............................... 30

PIR (peak intensity ratio) of pyranine(10’5M) versus concentration
of aqueous benzoic acid solutions at room temperature. The arrow
indicates the solubility of benzoic acid .......................................... 31

Fluorescence spectra of tryptophan excited at 342nm before and after
recrystallization from aqueous solution containing pyranine at a
M ............................................................

concentration of 10' 32
Fluorescence spectra of immobilized pyranine and free pyranine
in water at room temperature ..................................................... 38

Fluorescence spectra of the immobilized pyranine saturated with tryptophan,
phenylalanine and nicotinic acid in pure water at room temperature ......... 40

Figure 3.3

Figure 3.4

Figure 4.1

Figure 4.2

Figure 4.3

Figure 4.4

Figure 4.5

Figure 4.6

Figure 4.7

Figure 4.8

Figure 4.9

Figure 4.10

Figure 5.1

Figure 5.2

Peak intensity ratio (PIR) of pyranine versus concentration of tryptophan,
nicotinic acid and phenylalanine aqueous solutions at room temperature.
The values in parentheses represent the emission peaks for PIR ............ 43

Reproducibility of peak intensity ratio (PIR) of pyranine versus
concentrations for both byptophan and nicotinic acid aqueous
solutions at room temperature .................................................. 45

Molecular structures of l-pyrene butyric acid (PBA) and
carminic acid (CA) ............................................................... 48

Fluorescence spectra of PBA in aqueous tryptophan solutions:
1(0.0118M), 4(0.0410M) and 9(0. 100M) ..................................... 52

Fluorescence intensity of PBA versus the concentration of
tryptophan in aqueous solution at room temperature.
The lines are smooth fit of data .................................................. 53

Peak intensity ratio (PIR) of PBA versus the concentrations of
tryptophan in aqueous solutions at room temperature ......................... 55

Fluorescence spectra of carminic acid in aqueous tryptophan and
phenylalanine solutions at room temperature. Phe l: 0.0128M,
Phe 2: 0.0138M, Trp 1: 0.0119M and Trp 2: 0.0702M ..................... 56

Fluorescence intensity of CA at 595nm versus concentration of
phenylalanine in aqueous solutions at room temperature. The plot is
polynomial fitting .................................................................. 57

Fluorescence intensity of canninic acid at 595nm versus concentration
of aqueous tryptophan at room temperature. The arrow indicates the
solubility of tryptophan at 25°C. The line is smooth fitting of data ......... 58

Quenching of PBA by tryptophan in aqueous solutions at 375nm and
395nm fit to Stern-Volmer law at room temperature. The arrow
indicates the solubility of tryptophan at 25°C .................................. 60

Quenching of carminic acid at 595nm by tryptophan in aqueous
solutions fit to 1/I=1/Io+(K/Io)[Q] at room temperature ....................... 61

The quenching curve of tryptophan in aqueous solutions to CA
at 595nm fit to Io/(Io-I)=(1/[Q])(l/fK")+1/f at room temperature .......... 63

Quenching of pyranine(10'5M) fluorescence at 382nm by tryptophan
in aqueous solutions at room temperature fit to 10/1- 1=KW[Q].
The arrow shows the solubility of tryptophan at 25°C ...................... 72

Quenching of pyranine fluorescence at 510nm by tryptophan in aqueous

solutions at room temperature fit to 1/I=1/Io+(K o)[Q]. The arrow
indicates the solubility of tryptophan at 25° ................................ 74

xi

Figure 5.3

Figure 5.4

Figure 5.5

Figure 5.6

Figure 5.7

Figure 5.8

Figure 5.9

Figure 5.10

Figure 5.11

Figure 6.1

Figure 6.2

Figure 6.3

Figure 6.4

Quenching of pyranine fluorescence (10'5M) at 382nm by phenylalanine
in aqueous solutions at room temperature. The arrow shows the
solubility of phenylalanine. The curve is fit to loll-1=K"[Q] .............. 75

Quenching of pyranine fluorescence at 382nm by phenylalanine in aqueous
solutions at room temperature fit to IO/(IO-I)=(1/[Q])(1/fK")+1/f ......... 76

Quenching of pyranine fluorescence at 382nm by nicotinic acid
in aqueous solutions at room temperature fit to Io/I-1=K,,[Q5].
The arrow indicates the solubility of nicotinic acid at 25 C ................. 78

Quenching of pyranine fluorescence at 382nm by nicotinic acid in
aqueous solutions at room temperature fit to (Io/I-l)/[Q)]=K,+K2[Q].
Arrow indicates the solubility of nicotinic acid at 25 C ...................... 79
Quenching of pyranine fluorescence at 382nm by nicotinic acid in

aqueous solutions at room temperature fit to (L/I)exp(-V[Q])=1+K"[Q]
Arrow indicates the solubility of nicotinic acid at 25°C ......................
Quenching of pyranine fluorescence at 510nm by nicotinic acid at room
temperature at different excitation wavelengths of 342nm and 360nm

fit to loll-1=K"[Q] . Arrow shows solubility of nicotinic acid .............. 81

Absorption spectra of pyranine in water with water as reference and in
tryptophan, phenylalanine and nicotinic acid aqueous solutions with the
same concentration of solution as reference. The concentrations of all
solutions are at solubility ......................................................... 83

The molecular structures of nicotinic acid; tryptophan and
phenylalanine. Solubility in water at 25 C is given in parentheses ......... 84
Scheme of quenching mechanism of pyranine by amine in tryptophan and
nicotinic acid ring through hydrogen bonding complex at ground and
excited state ....................................................................... 87

Lifetime Stem-Volmer quenching of pyranine fluorescence at 510nm by
tryptophan in aqueous solutions at room temperature.
The arrow indicates the solubility of tryptophan at 25°C ..................... 98

Lifetime Stern-Volmer quenching of pyranine fluorescence at 510nm
by nicotinic acid in aqueous solutions at room temperature.
The equation shown is linear fitting ............................................. 99

Lifetime and steady state quenching of pyranine fluorescence
at 510nm by phenylalanine in aqueous solutions at room
temperature fit to Stern-Volmer Law .......................................... 100

Lifetime and steady state quenching of pyranine fluorescence at 382nm

by tryptophan in aqueous solutions at room temperature fit to Stem-
Volmer Law. The arrow shows the solubility of tryptophan at 25°C ..... 103

xii

Figure 6.5

Figure 6.6

Figure 7.1

Figure 7.2

Figure 7.3

Figure 7.4

Figure 7.5

Figure 7.6

Figure 7.7

Figure 7.8

Figure 7.9

Figure 8.1

Figure 8.2

Figure 8.3

Figure 8.4

Lifetime and steady state quenching of pyranine fluorescence at 382nm
by nicotinic acid in aqueous solutions at room temperature fit to
Stern-Volmer law. The arrow shows solubility of nicotinic acid .......... 104

Lifetime and steady state quenching of pyranine fluorescence at 382nm
by phenylalanine in aqueous solutions at room temperature fit to
Stern-Volmer law ................................................................ 106

Fluorescence spectrum of tryptophan in aqueous solution excited
at 308nm at room temperature .................................................. 111

Maximum emission intensity dependence on the concentration of
tryptophan at different excitation wavelengths which are smaller
than 310nm at room temperature ............................................... 112

Maximum emission intensity dependence on the concentration
of tryptophan at different excitation wavelengths which are
higher than 320nm at room temperature ....................................... 113

Excitation spectra of tryptophan at different tryptophan concentrations
monitoring at 450nm emission wavelength ................................... 115

Fluorescence spectra of supersaturated tryptophan solution
at different excitation wavelength at room temperature ...................... 116

The dependence of the maximum emission wavelength on the excitation
wavelength at different concentration of tryptophan at room temperature.118

Lifetime of tryptophan in the mixture of water+ethenol+propyl acetate
(2:2:1) at different concentrations of tryptophan at room temperature ..... 120

Excitation spectra of indole and tryptophan aqueous solutions at room
temperature monitoring at 500nm emission wavelength .................... 121

Fluorescence spectra of tryptophan and indole in water
at room temperature .............................................................. 122

Excitation spectra of pyranine in water and in

tryptophan(0.266g/100ml H20), phenylalanine(0.33g/100ml H20)

and nicotinic acid(0.302g/100ml H20) aqueous solutions at 510nm
emission wavelength at room temperature .................................... 131

Emission spectrum of pyranine in water excited at 342nm and excitation
spectrum of pyranine in water with emission wavelength 510nm
at room temperature .............................................................. 133

Scheme of solvated Structure of PyO- and the contact complex between
pyranine and tryptophan, phenylalanine and nicotinic acid ................ 135

The concentration ratio of PyO' and PyOH calculated based on pH and
fluorescence intensity ratio of 467nm/402nm versus pH of solutions in
tryptophan and phenylalanine solutions at room temperature ............... 137

Figure 8.5 Excitation spectra of pyranine in water and in
tryptophan(0.266g/100ml H20). Phenylalanine(0.33g/100ml H20)
and nicotinic acid(0.302g/100m1 H20) aqueous solutions at 382nm
emission wavelength at room temperature .................................... 140

Figure 8.6 Emission spectrum of pyranine(10'5M) in water excited at 342nm and
excitation spectrum of pyranine(10‘5M) in water with emission
wavelength at 382nm at room temperature .................................... 142

Figure 8.7 Excitation spectra of pyranine in different concentration of
tryptophan aqueous solutions at 382nm errrission wavelength at room
temperature. Unsaturated: 0.266g, saturated: 1.112g,
supersaturated: 1.547 g in 100ml H20 ......................................... 143

Figure 8.8 Excitation spectra of pyranine in different concentration of
phenylalanine aqueous solutions at 382nm emission wavelength
at room temperature. Undersaturated: 0.33 g, saturated: 2.648 g,
supersaturated: 3.2g in 100ml H20 ............................................ 144

Figure 9.1 Suggested molecules can be used to study fluorescence quenching
systematically as compared to tryptophan , phenylalanine and
nicotinic acid ...................................................................... 150

Figure 9.2 A schematic diagram of basic flow injection analysis (FIA) system

and reverse flow injection analysis (rFIA)
showing the various components .............................................. 153

xiv

Chapter 1

INTRODUCTION

1 . l . Control of crystallization by control of supersaturation

Crystallization from solution is one of the most widely used unit operations in the
food, pharmaceutical and chemical industries. It ranks second behind distillation in use as
a separation process (1). Most bulk pharmaceutical and organic fine chemicals are
marketed as crystalline products. That crystallization becomes such an important separation
technique in industrial production is not a historical accident, but the consequence of some
important factors. First, crystals are usually of extreme purity. Crystallization is cited as
one of the best and least expensive method to produce pure solids from impure solutions.
The purity of product is always an important aspect in industrial production, especially in
food and pharmaceuticals. Second, crystallization improves the product’s appearance, an
important aspect of consumer acceptance. This is one sale factor. Third, one of the most
important factors, the production of uniform crystals results in good flow, handling and
packaging characteristics which facilitates subsequent finishing steps, such as filtering or
drying (1, 16). When we talk about crystals making the finishing steps easier, we assume
we have bigger crystals. If crystals are very tiny the finishing steps are harder. So
production of a specified crystal size distribution (CSD) is one of the primary goals of a
crystallization process. The CSD affects the cost of operation of down-stream units such
as filtration equipment and dryers. These subsequent steps are often the limiting steps in
chemical manufacturing processes and significant cost reductions can be realized by

creating CSDs that have favorable filtration and drying properties.

1

2
Supersaturation, defined as the amount of solute in excess of the equilibrium

solubility at a given temperature, has great influence in the resulting CSD (1,2). The
effects of supersaturation on CSD reveal the influence of nucleation rate and growth rate.
Supersaturation of the solution is the driving force for both crystal nucleation and growth
but in different orders (1). In general, when supersaturation is high, nucleation rates will
be high, resulting in formation of many nuclei and many tiny crystals. When the
supersaturation is not very high, for example just a little bit over the solubility limit, the
nucleation rate will not be very high, and the growth rate will become more dominant.
Fewer nuclei formed in the solution will grow to become larger crystals, which is often the
desired result in industry. Therefore, there is an industrial need to control the
supersaturation of solution during crystallization processes which requires the measurement
of supersaturation accurately.

Supersaturation is essential for any crystallization, so reaching supersaturation is
always the first step of crystallization. Many crystallization processes start with a hot,
saturated solution that is cooled and/or evaporated to create supersaturation. Different
cooling processes of the solution will have an impact on the CSD because different cooling
will give us different supersaturation profiles. Natural cooling will produce a
supersaturation peak leading to high rates of nucleation and excessive fine crystals (1,3).
Linear cooling results in larger crystals as compared with natural cooling. The largest
crystals can be obtained by controlling the cooling rate (1,3), where the crystallizin g system
responds to a programmed cooling profile. The supersaturation versus corresponding
time profiles for different cooling rates are given in Figure 1.1 (3). Programming cooling
means we control the cooling curve just a little bit over the solubility curve which will
achieve a low supersaturation. Low supersaturation results in low nucleation rates and the
growth rate becomes a more dominant rate, thus producing larger crystals. The control of
supersaturation is realized by controlling the cooling rate in a cooling crystallization.

Evaporation rates and anti-solvent addition rates should be similarly controlled to produce

Supersaturation

 

 

 

 

 

 

 

0.016 t i i E F
— - - - - Linear cooling
1- — - - - - Programmed cooling
0.014-1- Natural cooling _
L
1.
0.012% ‘"
0.01 ~~ "
0.008% T
0.006“ T
. J
0.004-— J”
Time (min)

Figure 1.1. The supersaturation versus corresponding
time profiles for different cooling rates (3).

4
the supersaturation profile as prescribed for controlling cooling to achieve larger CSDs in

these processes.

The control of supersaturation of solution is critical to industrial crystallization.
Any attempt to control an industrial crystallization process requires the in situ measurement
and control of supersaturation. Therefore, the in situ measurement of supersaturation has
been cited as one of the most important needs of the industry (1,15). One reason is that
many industry crystallization processes are usually done in a batch mode. Control of batch
industrial crystallization processes is more difficult than continuous processes due to the
complexity and difficulty of the crystallization itself and also the different retention time
needs for the required growth of crystals. Another consideration is batch to batch
fluctuations, which can cause considerable variation in the crystallization resulting in final
product divergence. Such variation is often unacceptable to the Food and Drug
Administration (FDA). Reworking a batch that does not meet specifications incurs
additional costs and opens the opportunity for additional contamination. All of these
reasons show us that there is a need to implement control strategies that provide a
quantitative output that can be used either by an operator or implemented in an automatic
control scheme. Supersaturation has been shown to be a very sensitive factor in batch
crystallization. Minor changes in operation sometimes result in the random fluctuations of
supersaturation. What is desired in industry is to find an in situ technique which can be
used to monitor supersaturation and to control the supersaturation just in the metastable
zone by adjusting the rate of cooling and/or the rate of evaporation to maintain optimal
conditions during the operation. An in situ monitoring system can eliminate the need to
draw off sample aliquots, thus reducing the possibility of experimental error due to changes
that occur in the solution due to its metastablity. Clearly, there is a need for the

development of in situ measurements to follow the crystallization processes.

5
1.2. The fluorescent probe technique for measurement of supersaturation

In order to control supersaturation in the solution, we must first be able to measure
the supersaturation accurately. If the concentration of a solution can be measured and the
solubility is known, the supersaturation can be calculated. The measurement of
supersaturation turns out to be dependent on the measurement of concentration of the
solutions. Any techniques which can be used to measure the concentration can be used to
measure supersaturation. The measurement of concentration can be realized by measuring
properties of the system which are sensitive to solute concentration. Numerous analytical
techniques for the measurement of supersaturation of solutes in solutions have been
proposed such as conductomeuy, polarimetry, refractometry, viscometry, density
calibrations, etc.(1,4). Solution density and refractive index are probably the two
properties most commonly used to measure the supersaturation. In all cases, it always
starts with making up a series of solutions of known concentration and measuring the
physical properties of solutions to get the calibration curve. The primary difficulty in using
these conventional analytical techniques is that the separation of the saturated or the
supersaturated solution from the crystals is required prior to analysis. Supersaturated
solutions are unstable and changes may occur during the separation and subsequent
handling procedures. Special care must be taken to maintain process conditions, and to
avoid adsorption of solute on any of the sampling and analytical equipment. This special
care poses some difficulties for routine measurements in industrial practice. Therefore, we
can not be sure the properties we measured are the properties of the system. For example,
refractometry is one of the most common techniques to be used to measure supersaturation,
the change of refractive index is very small with changes of concentration of the solution,
which usually happens in the fourth or fifth decimal place. The sensitivity and accuracy of
the measurements are not reliable. For low solubility systems with the concentration
changes around 1%, the physical properties change, such as density and refractive index,

will not be large. It will be very difficult or impossible to measure accurately physical

6
properties changes with the concentration of solutions by using these conventional

techniques. To date there are no suitable techniques to measure the supersaturation in low
solubility systems. These low solubility systems are very common in industry, so there are
needs to find some suitable technique for the measurement of supersaturation of low
solubility systems. Fluorescence spectroscopy, due to its high sensitivity, can be a
potential technique to be used in study of low solubility systems.

Fluorescence spectroscopy is of extreme sensitivity and high specificity and has
played a major role in the field of analysis, particularly in the determination of trace analysis
in our environment, industries and bodies (13, 17-19). High sensitivity results from the
difference in wavelengths between the exciting and fluorescence emission, which results in
a signal contrasted with essentially zero background. A small signal can be measured
directly .

It is always easier to measure a small signal than a small difference between two
large signals as is done in absorption spectrOphotometry. High specificity results from two
separated spectra, excitation and emission spectra, and the possibility of measuring the
lifetime of the fluorophore. Two compounds that are excited at the same wavelength, but
emit at different wavelengths are readily differentiated without the use of chemical
separation techniques. The same two compounds may fluoresce at the same wavelength
but require different excitation wavelength (17).

Fluorescence spectroscopy is one of the most established techniques. It continues
to be one of the most active research fields in science. This is evidenced by the
exponentially increasing number of papers, monographs and reviews published each year
and by the increasing sales of those companies selling fluorescence instruments.
Fluorescence has some other advantages which other techniques can not compete with. In
addition to the extreme sensitivity, fluorescence spectroscopy is experimentally simple,
easy to control and generally inexpensive for operation. Very useful information can be

obtained from this simple technique (13,17- 19).

7
The basis for the use of fluorescence probes is that certain molecules display a

selective affinity for a unique site on the molecule and the structure and dynamic properties
of the host system are reflected in the fluorescence properties of the probe. Changes in the
emission of the extrinsic probe molecule can be conelated to corresponding changes in the
local solution environment. In a photochemical investigation, changes in emission of the
fluorescin g species gives information about the electronic state of fluorescing species (5).
Fluorescence probes can be covalently linked or physically bound to specific sites in
molecules such as proteins. The magnitude of the observed fluorescence signal provides
information about energy transfer processes, the distances between reactive sites.

The fluorescence probe technique can be used in nonfluorescent systems. Although
a host system does not fluoresce, a change in the host system will change the fluorescence
of a probe. The change of probe fluorescence then reveals this change and gives us
information about the host system. The concentration of the probe can be low enough, as
low as 10'5M, to preclude any effects other than that arising from the interaction of the
probe molecule with its local environment. Additionally, this low concentration can
minimize any potential effects on crystallization. Often the sensitivity of a probe to the
solvent environment is enhanced in its excited state. Another advantage of using the
fluorescence probe technique is the application of remote sensing. Remote sensing is often
required in industrial production and the previously discussed convention measuring
techniques are not easy to be used in remote sensing. The fluorescence probe technique
can be made remote sensing by applying fiber optic technology.

The fluorescence probe technique has been shown to be effective for monitoring the
supersaturation of high solubility systems (6, 7). This approach has been used to monitor
the degree of supersaturation utilizing a peak intensity ratio (PIR) of pyranine in aqueous
solutions of sucrose, glucose, fructose, and lactose (6). It has been demonstrated that

pyranine can also be used as a fluorescent probe to measure supersaturation in citric acid

8
solutions and that the probe responds to the solvent microenvironment as well as changes

in the pH of the solution (8).

l . 3 . The study of structure in supersaturated solutions by fluorescence
quenching

It is well known that crystallization takes place with the formation of a stable
crystal nucleus from a homogeneous supersaturated solution first, then these nuclei will
grow to form crystals (1). The formation of nuclei is the beginning point of the
crystallization. The nucleation of solute must somehow depend upon the structure of solute
molecules in a supersaturated solution and also depend upon the various molecular
interactions, such as solute-solute and solute-solvent interactions (1). Understanding the
structure of a supersaturated solution can help us better design and control crystallization.

In aqueous solution, each ion is surrounded by dipole oriented water molecules due
to the coulombic forces between ions and water molecules, which are called solvated ions.
Water molecules who are closer to the ions are bonded to the ions much firmer than those
water molecules which are far away from ions. Those far away molecules are bonded to
ions non oriented and loose. The hydrated ions can be very big or small depending on the
properties of the ions (1). The structure of the supersaturated solutions probably are more
complex than undersaturated solutions.

Many techniques have been used to study the structure of supersaturated
solutions, including Raman, FI'IR, NMR, and X-ray diffraction (1). Researchers are
trying to find the distinguishing features of supersaturated solutions by investigating the
dependence of various physical properties on concentration. The discontinuity of any
property versus concentration at the equilibrium saturation point has rarely been observed
(1). Some theories, such as the cluster theory, have been provided about the su'ucture of
supersaturated solution (1,9) Solute cluster theory was reported by several researchers

through observing the concentration gradients or different diffusion rate at high

9
concentration in high solubility systems (20-22). Previous work was focused on high

solubility systems and theory was based on the study of high solubility systems whose
solubility can be as high as 50wt%. No information about the organization of low
solubility systems at supersaturated region has been given. A concentration gradient,
which is the evidence for the formation of solute clusters, will not be noticeable in low
solubility systems. Therefore, there is a need for suitable techniques to study low
solubility systems.

Steady-state fluorescence spectroscopy represents a powerful optical spectroscopic
technique for monitoring the dynamics of macromolecular interactions in solution (10—12).
Steady state fluorescence spectroscopy using extrinsic trace fluorescent probe molecules is
solvent sensitive. Specific information about the immediate molecular environment of the
probe molecule, either free in solution or coupled to a macromolecule, can be obtained from
changes in the peak intensities and wavelengths.

The fluorescence quenching technique is a very powerful tool for studying the
dynamics of micellar system (13,18, 23). Quenching follows two mechanisms where one
is viscosity-independent static quenching and the other is viscosity-dependent dynamic
quenching. Dynamic quenching is a diffusion controlled process. Both of the quenching
processes require the contact of the fluorophore and quenchers. Quenching is always
interpreted as the contact complex formation between fluorophore and quenchers to some
degree. Contact complexes are the results of Van der Waals molecular forces between
probe molecule and quencher which are not very strong. Sometimes a stronger bonding
such as hydrogen bonding may form between probe and quencher which will show
stronger interaction and quenching ability. Static quenching is due to the ground state
contact complex between fluorophore and quencher, which absorbs the incident excitation
light and yield excited complex which is called as exciplex. In many cases ground state
fluorophore and quencher can not form a su'ong contact complex, but when fluorophore is

excited the change of dipole moment will make the formation of complex much easier.

10
Dynamic quenching involves the diffusion-controlled encounters of fluorophores and

quenchers. The possible physical process of quenching can be shown by the schematic
diagram in Figure 1.2 (13). M represents the fluorophore and Q represents quencher.
(MQ)* represents the exciplex between M and Q. The triplet is described by 3. The
quenching of M* can occur by processes ii(exciplex fluorescence), iii(exciplex internal
conversion to ground state), v(energy transfer from 1M"‘ to ‘Q*), vi(exciplex dissociation
in ions or direct electron transfer) and vii(exciplex intersystem crossing to the triplet
exciplex state). Process iv does not quench the fluorescence of M*. The fluorescence of
the exciplex is not very common in the quenching process and the same exciplex internal
conversion (process iii) to the ground state is not very common either due to the big large
energy gap. With heavy atom quenchers, such as xenon, bromobenzene and various
bromide and iodide heavy atom quenchers, the exciplex intersystem crossing to the triplet
exciplex state is a very important mechanism of quenching. Direct electron transfer
between 1M* and ‘Q* is another common quenching mechanism which can be proved by
transient absorption spectra (13). Quenching mechanisms can be fitted to different models.
By interpreting quenching data we can deduce the behavior of quenchers in the solution,
thus the information about solution. The investigation of the microscopic interactions
between the solvent and solute by fluorescence quenching will result in a more directed and

rational approach for understanding and control of crystallization.

l . 4 Introduction of the systems for study

A significant amount of research has been done in our group by using the
fluorescence probe method (6,8). It has been used to monitor the degree of supersaturation
with a ratio of peak intensity of pyranine in aqueous solutions of sucrose, glucose,
fructose, and lactose (6). It has been demonstrated that pyranine can be used as a
fluorescent probe to measure supersaturation in citric acid solutions and that the probe

responds to the solvent microenvironment as well as changes in the pH of the solution (8).

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12
Previous studies have been done with very highly concentrated solutions, e. g. 50 wt %.

For these studies the PIR is a measure of the ratio of water associated with the solute to that
not associated with the solute (6,8). Pyranine responds to the available water in its
immediate environment, but for dilute solution the behavior of solute should be different.
In the current project tryptophan, nicotinic acid and phenylalanine will be studied. The
solubility of tryptophan, nicotinic acid and phenylalanine are 1.136g, 1.67 g and 2.965g in
100ml water at 25°, respectively (Merck Index). Such low concentrations will probably
not greatly affect the solvent behavior in the solution. The physical properties which are
used to measure the supersaturation of high solubility systems will not change as much as
in high solubility systems. This poses difficulty in measurement of supersaturation of low
solubility systems by conventional methods. Another question is whether the structure of
such dilute solution are the same as the highly concentrated solutions. Will the
fluorescence probe method work for such dilute solution? This research is to understand
the crystallization of low solubility systems by studying the supersaturation solution
structure using the fluorescence quenching technique, and measuring supersaturation of
solution in low solubility systems by fluorescence probe technique.

The reason that we choose L-tryptophan as our research system is that L-tryptophan
is one of the essential amino acids which is not synthesized in the body. L- tryptophan is a
major article of commerce. Additionally, the combination of aromatic and ionic groups
makes L-tryptophan a good model system for complicated molecules. Tryptophan is an
antidepressant drug used to improve the quality of people’s sleep. It is a precursor for
serotonin, an important neurotransmitter which can be produced during the deep sleep state
(restorative phase of sleep during which the sleeping brain can restore the neurotransmitters
and also muscles truly relax) (14). Serotonin may also reduce certain reactions and have a
tranquilizing effect. L-tryptophan appears to be useful in the treatment of some depressive
and anxiety disorders. It has been said that tryptophan reduces cravings for fatty foods,

alcohol, among others.

13
Nicotinic acid is needed for proper circulation; healthy skin; functioning of the

nerve and digestive system; Nicotinic acid helps to eliminate canker sores, reduces
cholesterol and triglycerides, and improves the unpleasant symptoms of vertigo in
Meniere's syndrome and is effective in the treatment of mental disorders (Information from
intemet).

Most bulk pharmaceutical and organic fine chemical are marketed as crystalline
products, including tryptophan and nicotinic acid. But the crystallization of tryptophan is
very hard to handle in industry because only fluffy crystals result. As we already know,
this will add some problems to the filtration and/or drying processes and so will increase
the cost of operation.

There is no suitable technique for measurement of the supersaturation of low
solubility systems such as tryptophan and nicotinic acid solutions, which are very common
in industrial crystallization. Clearly, there is a need for the development of a measurement
technique to follow the crystallization process of low solubility systems and a suitable
method to study the structure of supersaturation of solution of low solubility systems. It is
the objective of this research project to develop sensors capable of measuring the
supersaturation of tryptophan and nicotinic acid solutions then extend to general low
solubility systems and study the structure of supersaturated solutions by using the

fluorescence probe method.

1.5. Overview of this work

In chapter 2, pyranine in solution is used as a probe for measurement of
supersaturation in tryptophan solution and nicotinic acid solutions. Results show that the
PIR (Peak Intensity Ratio) of pyranine versus the concentration of tryptophan and nicotinic
acid are smooth and nonlinear with a greater slope in the higher concentration region. PIR
curves can be used as the calibration curve for measurement of the supersaturation of

tryptophan and nicotinic acid solutions. The fluorescence of tryptophan crystallized in the

14
presence of pyranine from solution exhibited no pyranine, which indicates pyranine does

not go into the crystal lattice. We can conclude that pyranine is a good probe and the
fluorescence probe method is a promising technique for measurement of the supersaturation
of low solubility systems.

In Chapter 3, pyranine is immobilized on a membrane as the probe to measure the
supersaturation. Pyranine is not listed as a food additive by FDA and can not be added
directly into food or pharmaceutical processes. Therefore it is necessary to immobilize
pyranine onto a surface for use as a sensor. Pyranine is immobilized onto a membrane for
this purpose. Results Show that PIR versus concentration of solute is linear and can serve
as the calibration curve for measurement of supersaturation. The fluorescence of the
solution without probe membrane exhibit no pyranine emission, which confirms the
stability of the sensor.

Chapter 4 is an investigation of other potential probes for supersaturation .
measurement. l-Pyrenebutyric acid (PBA) and carbonic acid (CA) are studied. Carminic
acid is an approved food additive by the FDA. If carminic acid can serve as a probe in this
fluorescence probe technique, we can add carminic acid to of food and pharmaceutical
processes. The fluorescence intensity of CA versus concentration of tryptophan and
phenylalanine are smooth curves which can be used as calibration curves to measure
supersaturation. Especially in phenylalanine solutions, intensity increases with the increase
of phenylalanine concentration. Although the PIR of PBA versus the concentration of
tryptophan and phenylalanine are smooth, PBA will enter the crystal lattice. So PBA can
be used as the calibration curves to measure supersaturation. Tryptophan shows quenching
to both CA and PBA, but not phenylalanine. Different quenching behaviors of tryptophan
and phenylalanine to PBA and CA can give us information about different interactions of
probe molecules with tryptophan and phenylalanine in solution.

The PIR versus solute concentration is the result of fluorescence quenching of

pyranine by solutes. Different wavelengths have different quenching constants. The level

15
of supersaturation is an essential requirement for all crystallization operations. The

formation of nuclei from supersaturation solutions is the beginning point of the
crystallization. The nucleation of solute must somehow depend upon the solute molecules
in a supersaturated solution and also depend upon the various molecular interactions, such
as solute-solute and solute-solvent interactions. The interpretation of quenching behaviors
can reveal information about the accessibility of solutes to probes then information about
the organization of solutes in solution can be deduced from the interactions of solutes and
probes. The fluorescence quenching studies of pyranine by tryptophan, nicotinic acid and
phenylalanine are presented in detail in Chapter 5.

Chapter 6 deals with the fluorescence quenching study of pyranine by lifetime
measurement The emission lifetime of a probe molecule is highly sensitive to its
environment. The measurement of lifetime can determine static and dynamic
conformational information about macromolecules, make state assignments, and provide
information on the diffusive processes such as collision quenching and proton transport.
By using the lifetime Stem-Volmer quenching fitting, dynamic quenching and static
quenching are very easily distinguished. Different quenching behaviors of tryptophan,
nicotinic acid and phenylalanine to pyranine result from the organization in the various
solutions.

Fluorescence of tryptophan shows excitation wavelength dependence at all
excitation wavelengths which is uncommon. Fluorescence occurs from the lowest
vibration state and the energy difference between the excited state and ground state will not
change with the excitation. In general, the emission is not excitation dependent. Only
when a fluorophore is in a polar and viscous environment where its mobility is restricted,
the wavelength of maximum fluorescence will depend on the excitation wavelength. The
maximum fluorescence emission will show red—shift when excited with extreme red edge
wavelength. This phenomenon is well known as red edge excitation shift (REES). The

fluorescence of tryptophan is studied in detail in chapter 7.

16
Excitation spectra of pyranine in water and in aqueous solutions of tryptophan,

phenylalanine and nicotinic acid are presented in chapter 8. The excitation spectrum of
pyranine shows fine vibronic structure around 460nm which is the result of the high dipole
moment of solvated deprotonated form of pyranine which is represented by PyO'. The
introduction of even trace amount of solutes into solution change the excitation spectra
dramatically. The introduction of solutes shifts the equilibrium between the protonated
form of pyranine (PyOH) and the deprotonated form of pyranine (PyO') to the protonated
form especially with nicotinic acid. The contact complex between pyranine and solute
molecules is another reason for the change of the excitation spectra which matches the
results we obtained from quenching data.

Chapter 9 gives a summary of the work and an overview of future work. The
fluorescence quenching technique deserves more attention for solution study. Some
molecules which have similar molecular structures as tryptophan, phenylalanine are
suggested as target molecules to conduct the systematic study in order to understand the
interactions better. New methods which can yield information about interactions may be
developed to investigate solution structure. Time resolved fluorescence can be applied as a
useful investigation tool. Fluorescence has been shown to be a very useful tool to measure
supersaturation. How we apply this technique to the real measurement of the
supersaturation in industrial crystallization is a consequence of industrial need. Flow

injection analysis (FIA) is discussed as an possible application in industrial crystallization.

1.6. Reference
1. J. W. Mullin, Crystallization, Butterworth-Heinemann, Oxford, England (1993)
2. A. D. Randolph, and M. A. Larson, Theory of Particulate processes,
Academic Press, New York, USA (1988)
3. A. G. Jones, and J. W. Mullin, Chem. Eng. Sci., 29,105 (1974)
4. H. K. Zimmerman, Chemical Review, 51,25 (I952)

10.
11.

12.

13.

14.

15

16.

17.
18.

19.

20.
21.

17
V. Ramamurthy, Photochemistry in Organized & Constrained Media,

VCH (1991)

R. Chakraborty, and K. A. Berglund, J. Crystal. Growth, 125, 81-96 (1992)
R. Chakraborty, and K. A. Berglund, AICHE Symposium Series, 284-7,
114-123 (1991)

S. K. Yedur, and K. A. Berglund, Applied Spectroscopy, 50(7),

866-870 (1996)

J. W. Mullin, and C. Leci, AICHE Symp. Ser., 68, 8 (1972)

H. Kondo, I. Miwa, and J. Dunamoto, J. Phys. Chem, 86, 4826-31 (1982)
E. Bardez, B. Goguillon, E. Keh, and B. Valeur, J. Phys. Chem, 88,
1909-13 (1984)

G. D. Correll, R. N. Cheeser III, F. Mome, and J. H. Fendler,

J. Am. Chem. Soc., 100:4, 1254-1262 (1978)

J. B. Birks, Photophysics of Aromatic Molecules, New York,
Wiley-Interscience (1970)

A. S. Gissen, Vitamin Research Products Newsletter, May/June (1993)

G. A. Hussman, M. A. Larson, and K. A. Berglund, Industrial Crystallization ‘84
S. J. Jancic, and E. J. DeJong, Eds. Elsevier, Amstrdam (1984)

P. A. Belter, E. L. Cussler and Wei-shou Hu, Bioseperations Downstream
Processing for Biotechnology, Johnm Wiley & Sons, , New York (1988)

G. G. Guilbault, Practical Fluorescence, M. Dekker, New York (1990)

J. R. Lakowicz, Principles of Fluorescence Spectroscopy, Plenum Press,
New York and London (1983)

J. R. Lakowicz, Topics in Fluorescence Spectroscopy, Plenum Press,

New York and London (1992)

J. W. Mullin, and C. L. Leci, Phil. Mag., 19 (1969)

E. L. Cussler, AIChE J. 26(1), 43 (1980)

18
22. L. S. Sorrel, and A. S. Myerson, AIChE J., 28(5), 772 (1982)

23. M. R. Eftink and C. A. Ghiron, Biochemistry, 15(3), 672 (1976)

Chapter 2

SUPERSATURATION MEASUREMENTS OF LOW SOLUBILITY
SYSTEMS USING A STEADY STATE FLUORESCENCE PROBE
TECHNIQUE

2.1. Introduction

Supersaturation, which is defined as the amount of solute in excess of the
equilibrium solubility, is the driving force for both crystal nucleation and growth. Different
nucleation rates and growth rates will result in different crystal size distributions (CSDs),
one of the most important parameters in industrial crystallization. Uniform CSD can make
the finishing steps, such as filtration and drying faster and easier (1). Filtration and drying
are usually limiting steps in chemical manufacturing processes and the determining factor
for operation costs. Reduction of cost can be realized by a desired CSD. Since
supersaturation is a detenninin g factor of the final CSD of crystallization systems, the
control of supersaturation is critical to industrial crystallization. Any attempt to control an
industrial crystallization process requires the real time measurement and control of
supersaturation. Therefore, the in situ measurement of supersaturation has been cited as
one of the most important needs of the industry ( 1).

Numerous analytical techniques for the measurement of supersaturation of solutes
in liquids have been proposed, including refractometry, interfertometry, viscometry and
density calibrations (2). These techniques require the separation of the saturated or the
supersaturated solution from the crystals prior to analysis. This separation from solution is

difficult and the solution properties may change. All these techniques involve the

19

20
measurement of the physical properties which change with concentration. For low

solubility systems, such as tryptophan and nicotinic acid, the concentration change is just
around 1%, so the physical properties do not change very much with the concentration.
This low concentration change will make conventional measurements inaccurate. Low
solubility systems are common in industrial production; therefore, techniques for
measurement of supersaturation in low solubility systems is needed.

Many studies have attempted to develop new techniques for in situ control of
crystallization by measurement of supersaturation. A method using the steady state
fluorescence of extrinsic trace fluorescent probe molecules as sensors for monitoring the
supersaturation of high solubility solutions was developed by Chakraborty and Berglund
(3). This approach has been used to monitor the degree of supersaturation utilizing a peak
intensity ratio (PIR) of pyranine in aqueous solutions of sucrose, glucose, fructose, and
lactose (3). It has also been demonstrated that pyranine can be used as a fluorescent probe
to measure supersaturation in citric acid solutions and that the probe responds to the solvent
microenvironment as well as changes in the pH of the solution [4]. These studies were
done with very highly concentrated solutions, often higher than 50 wt %. In such cases the
PIR is a measure of the ratio of water associated with the solute to that not associated with
the solute. Pyranine responds to the available water in its immediate environment. This
behavior, in turn, is related to the amount of citric acid and sugar present in solution.

Thus, the change in emission properties of pyranine can be related to the concentration of
aqueous solution of citric acid. In other words the peak intensity ratio (PIR) can be used as
an indicator of the degree of supersaturation. However, a technique that is suitable for in
situ measurements for low concentration solutions, such as a few weight percent, has not
been developed.

The current study utilizes pyranine as fluorescent probe by measuring the peak
intensity ratio (PIR) versus solute concentration for solutions of tryptophan, nicotinic acid

and benzoic acid with the aim of supersaturation measurement.

21

2 . 2 . Materials and methods

Pyranine (8-hydroxy-1,3,6-pyrenetrisulfonate), also known as FDA D&C Green
No. 8,was obtained from Lancaster, Windham, NH and used as received. Aqueous
solutions of pyranine were prepared by dissolution of 0.026 g pyranine in 5m] HPLC
grade water. Different concentrations of solutions were prepared by dissolving the
appropriate amount of solute in 5m] HPLC water. Supersaturated solutions were prepared
by heating the solution in the water bath until all the solute was dissolved and subsequent
cooling to room temperature. The solutions were used as prepared without any degassing.

Probe molecules were introduced by addition of five microliters of the pyranine
solution (10'2M) to the aqueous tryptophan and benzoic acid solutions resulting in a probe
concentration of 10 5M, and 20 microliters of pyranine (10'2M) for nicotinic acid resulting
in a probe concentration of 4x10‘5M. Fluorescence spectra of the pyranine in the aqueous
solutions were collected within several hours of the preparation the solutions. All spectra
were taken at room temperature with a Spex 1681 Fluorolog spectrometer equipped with a
Xenon lamp source and a PMT detector. A quartz sample holder was used in all cases.
Spectra was recorded at 1.00mm increment and an integration time of 1.0 sec. An
excitation wavelength of 342nm for pyranine was used. The emission spectra were
recorded over a range from 350-600nm for pyranine. Monochromator slits were 0.5 mm
for spectra.

All experiments are conducted more than twice with a newly prepared solution

series.

2.3. Results and discussions
2.3.1. Steady state fluorescence
Pyranine (8-hydroxy-1,3,6-pyrenetrisulfonate), whose chemical structure is shown

in Figure 2.1, was used as a probe for all studies. Pyranine is water soluble and is known

22

NaO3S OH

Figure 2.1 Molecular structure of 8-hydroxy-1,3,6-pyrenetrisulfonate (pyranine)

23
to have a highly sensitive, polarity dependent equilibrium between two excited states that

fluoresce (5,6).

The photochemistry of pyranine has been well characterized (5) (Figure 2.2). In
aqueous solution, pyranine exists with the sulfonate groups entirely dissociated. It has two
excited fluorescent states: one in which the hydroxyl proton is associated with the molecule
and one in which it is dissociated. Therefore, pyranine can be used to investigate the
proton-accepting character of the solution microenvironment. In aqueous solution when
pH is low, the absorption spectrum of pyranine is characteristic of the acidic form PyOH
alone (max. 405nm) and the emission band at 440nm is the fluorescence of PyOH*. At
high pH values, the absorption band at 445nm appears, revealing the presence of the basic
form PyO' in the ground state. The band at 510nm is the fluorescence of PyO’*. When
pH is between this high and low value, depending on the rate of proton transfer, emission
can be observed from the protonated species, the ionized species, or both (Figure 2.2) (6).

Fluorescence spectra of pyranine in aqueous solution of tryptophan are shown in
Figure 2.3. The emission spectrum of pyranine exhibits two bands: one is at 510nm which
is assigned to deprotonated pyranine fluorescence and the other is a set of peaks 380nm to
440nm, which consists of three fine vibronic structure peaks. The spectra of pyranine in
tryptophan solutions are different from the previous results in citric acid solutions in which
the 445nm emission peak is missing in tryptophan solutions. In the previous work the
concentration of citric acid was very high which is around 50wt%. The pH of the solution
was low in such high concentrations so pyranine was in the form of PyOH* and the
emission of 445nm is observed. In our systems the solubility of tryptophan is
1.136g/100mlI-120 and the solubility of nicotinic acid is 1.67g/100ml I120, and benzoic
acid 0.34g/100m1H20 at 25°C. The pH of the solutions was pretty high (around 6). At
this high pH pyranine exists in the form of PyOH mainly in the ground state and when it is
excited it becomes PyOH*. We can not see the emission of PyOH in tryptophan solution,

because of the fast excited-state proton transfer reaction (7). The high value of pK,* (0.5)

24

 

pKa* .
PyOH* + H20 ‘___*: PyO'* + H3O
Em(PyOH*) Em(PyO‘*)
pKa

 

PyOH + H20 PyO‘ + 1130*

 

Figure 2.2 Scheme for acid-base equilibria in the ground state and excited
state of pyranine. Deprotonated form of pyranine (PyO') will be excited
at 440nm and emit at 510nm. Protonated form of pyranine (PyOH)
will be excited at 405nm and emit at 450nm. pKa=7.5 and pKa*=0.5.

25

233.352 88. a of .52: 2 5:85? 33: M: ea 88. : : .338 e $88.8 _
”meow—:8 5.3399 3833 E can 2: 853 E 05523 mo cocoomoeoaa MN Semi

«Osmond Nc+wom.m

r p
A

 

3559.226;
mo+woo.m ~o+wom.v

 

~o+moo.v ~o+mom.m
oo+mood

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mo+m5mé
a mo+MooN

.. mo+momN

 

 

: mo+moo.m

I, mo+mom.m

 

 

mo+moo.v

(saomsuum

26
in the excited state often differs greatly from the ground state value (pK,=7.5), leading to

deprontonation of the excited species. Although at ground state pyranine is in the form of
PyOH, when it is excited the equilibrium between PyOH* and PyO‘* will favor PyO‘* due
to the low concentration of H,O" in the solution and the proton transfer from PyOH*

to base is within the lifetime of fluorescence, so the fluorescence occurs from PyO'* which
results in that the 510nm peak The absence of the 445nm peak in our spectra suggest that
fast proton transfer occurred before the fluorescence of pyranine. Peak 445nm will show
up in highly concentrated sugar solutions. From the equilibrium consideration, PyO‘* will
be dominant form. But because of the high viscosity of sugar solutions, the proton transfer
can not be done within the lifetime of fluorescence. So 445nm emission was observed.
This supports the conclusion of fast proton transfer reaction in low solubility systems.

The 382nm, 401nm and 430nm emissions peaks can be attributed to typical
emission spectra of the polycyclic aromatic heterocycle (PAH). The typical emission
spectrum of the PAH monomer will consist of five major vibronic bands which are
generally labeled as I-V in progressive order, such as the 0-0 band is labeled 1, etc.(8,9).
The possibility of a pyrene impurity was considered and discounted because pyrene is very
hydIOphobic and its solubility in water is very low (~5-8 x 10‘7 M). Pure pyrene was
purchased from Aldrich Chemical Co. (99+%). The emission spectrum of a saturated
pyrene aqueous solution possessed two peaks at 371nm and at 390nm, but the intensity
measured was much lower than corresponding intensities for pyranine emission.
Therefore, we can conclude that the peaks around 400nm in pyranine are not the
consequence of a pyrene impurity.

The fluorescence spectra show no differences in shape and maxima in the presence
and absence of tryptophan at low tryptophan concentration. No new fluorescence peak is
observed at longer wavelengths which indicates that no strong ground state complex
formed. The intensity of the 510nm peak of pyranine increased upon addition of tryptophan

in the solution as compared to the intensity of pyranine in pure water. If we compare the

27
intensity of pyranine emission at 510nm in different tryptophan concentrations, we see the

peak intensity decreases with the increase of the tryptophan concentration.

The peak intensity of pyranine at 380—430nm decreases with the addition of
tryptophan and keeps decreasing with the increased tryptophan concentration before a
definite concentration. When the tryptophan concentration is very high, the peak intensity
of pyranine at 380—430nm increases with the increase of tryptophan concentration, and high
concentration of solute leads to the loss of fine vibronic structures of the emission spectra.

The shape and maximum of the fluorescence spectra of pyranine in nicotinic acid
solution and in benzoic acid solution are the same as the fluorescence spectra of pyranine in
tryptophan solutions. The effects of solute concentration on the peak intensity are
different. The peak intensity of pyranine fluorescence at both 510nm and 380nm-430nm
decreases with the addition of the benzoic acid in benzoic acid aqueous solution. The only
difference between the fluorescence spectra of pyranine in benzoic acid solution and in
nicotinic acid is that the intensity of fluorescence of pyranine decreases dramatically with
the addition of niCOtinic acid. The intensity keeps decreasing rapidly with the increase of
the concentration of nicotinic acid. Here we should notice that the concentration of
pyranine in nicotinic acid solution is four times higher than the concentration of pyranine in
benzoic acid and in tryptophan solutions.

The decrease of the intensity with the increase of the solute concentration is due to
the solute quenching of pyranine fluorescence. Tryptophan, benzoic acid and nicotinic acid
have different quenching abilities to pyranine. N icotinic acid can effectively quench
pyranine fluorescence. Tryptophan and benzoic acid show more moderate quenching of
pyranine fluorescence. The different pyranine fluorescence quenching behaviors of

tryptophan, nicotinic acid and benzoic acid will be discussed in chapter 5.

28
2.3.2. Measurement of concentration

The peak intensity ratio (PIR), defined as the ratio of the peak intensity at 401nm,
382nm or 430nm to the intensity at 510nm is used to measure the concentration of solution.
The use of a ratio as compared to simple intensity can provides an internal standard for the
spectrophotometer. Graphs of the PIR versus concentration of tryptophan, nicotinic acid
and benzoic acid are shown in Figures 2.4, 2.5 and 2.6, respectively. In order to
determine the supersaturation of the solution it is necessary to develOp a calibration curve to
monitor signal versus concentration of solute. Changes in emission properties of pyranine
can be related to the concentration of aqueous solutions. The smooth nature of the PIR
curve indicates that it can be used to determine the concentration, hence the supersaturation,
so it can serve as calibration curve to follow the concentration changes during the
crystallization process. The measurement of supersaturation of solutions is realized by
measuring PIR of the probe sensor fluorescence. The concentration of solutions can be
read from the calibration curve. The supersaturation can then be calculated by the
difference of concentration and the solubility. The presence of an upward curvature
indicates that the response is nonlinear. It shows larger gain at high concentration region
which is really desired in industrial practice because bigger signal response means higher
sensitivity of the technique. It has been shown elsewhere (3,10) that the supersaturation
measurement using the PIR is more sensitive than other techniques. We can tell the high
sensitivity of fluorescence probe technique from our results since the concentration change
in our research is from 0.5%-1.8% in tryptophan solutions, but the signal PIR changes
from 0.29 to 0.72. So a remarkable signal can be realized by using fluorescence probe
sensor technique.

A potential problem for using external probe is that the probe molecule may go into
the crystal lattice. The cocrystallization of the probe in the crystal is not acceptable in

industrial production because it will affect the purity of products. To test whether the probe

PIR

29

 

    

 

 

 

 

 

0.8 rrrrrrrrr i . v . . . r f v v } ........ f
F 4
" 1
Oo7__ -n—
- 4
i- 4
0.6“ J_
0.5“ -c
i A ‘
401nm/510nm .
.1
J
%
0.1 rrrrrrrrr % AAAAAAAAA : rrrrrrrrr
0 5 1 1 5 2

Concentration of tryptophan ( g/ 100ml H20 )

Figure 2.4 PIR (peak intensity ratio) of pyranine(10'5M) versus
concentration of aqueous tryptophan solutions at room temperature.
Lines are polynomial fitting, and arrow indicates the solubility of tryptophan.

PIR

30

 

0.9

0.4"

0.3“

0.2-

-¢-

1 l
r T I 1 T— I Y T ‘Y T

428nm/509nm

 

0.1 ‘ I ‘ ‘ ‘ ‘ I 4 I ‘
0 0.5 1 1.5 2 2.5 3

 

 

A J L l L A A A l l A A A A 1 E A L l L A 1

Concentration of nicotinic acid (g/100ml H20)

Figure 2.5 PIR (peak intensity ratio) of pyranine (4x10‘5M) versus

concentration of aqueous nicotinic acid solutions at room temperature.

The arrow indicates the solubility of nicotinic acid at 25°C.

PIR

0.85

0.8-
0.75a
0.7-

0.65-

0.55-~ -_

0.45

31

 

TfiT‘Y

‘1—
-—
—o—
-n—

382nm/509nm

I T j r T Y I
G

I
l
I

L A A 4 1 A J A A

 

A Q A l A A A L A A A i l A A A

A A J

 

 

0 0:1 0:2 0:3 0:4 0:5 0.6
Concentration of Benzoic Acid (g/ 100ml H20)
Figure 2.6 PIR (peak intensity ratio) of pyranine(10'5 M) versus

concentration of aqueous benzoic acid solutions at room temperature.
The arrow indicates the solubility of benzoic acid.

32
molecules will go into the crystal lattices. Recrystallized tryptophan with the presence of

pyranine is dissolved into pure water and the fluorescence is measured by using 342nm
excitation wavelength and scanning the emission spectrum from 350nm to 600nm. For
comparison, a pure aqueous tryptophan emission spectrum is recorded by using 342nm
excitation wavelength. The fluorescence spectra of both solutions are shown in Figure 2.7.
Only one peak will show up for both case which is fluorescence of tryptophan. There are
no peaks from the fluorescence of pyranine. Mass spectra also show no traces of pyranine
in the recrystallized tryptophan. Therefore, we can conclude that pyranine does not go into

the crystal lattice.

2 . 4. Conclusions

The emission properties of a fluorescence probe molecule like pyranine in trace
amounts can be used to obtain an estimation of the supersaturation of low concentration
solutions. The PIR is strongly correlated to the solute concentration and the relation
between the PIR and concentration is highly non-linear and has an increasing signal in the
supersaturated region of concentration. This technique is highly sensitive and

experimentally simple which makes it promising for industrial use.

2.5. References

l. J. W. Mullin, Crystallization , Butterworth-Heinemann, Oxford, England (1993)
H. K. Zimmerman, Chemical Review, 51, 25 (1952)

R. Chakraborty, and K. A. Berglund, J. Crystal Growth, 125, 81-96 (1992)

S. K. Yedur, and K. A. Berglund, Applied Spectroscopy, 50(7), 866-870 (1996)
H. Kondo, I. Miwa, and J. Dunamoto, J. Phys. Chem, 86, 4826-31 (1982)

9995”!"

V. Ramamurthy, Photochemistry in Organized & Constrained Media, VCH
Verlagsgesellschaft mbH and VCH publishers (U K) Ltd. (1991)

33

 

30000.00
Tryptophan recrystallized from aqueous solution
containing pyranine as a probe (106M)
25000.00 .
l
20000.00 ~ '

Intensity (CPS)
8

Tryptophan as received
dissolved in water

10000.00 4 /

/ x
...- ”m.

 

'f‘l

 

01m 7 Y I I I V T r l 1
350.00 370.00 300.00 410.00 4:30.00 60.0) 4701!) 400.00 510.00 530.00 560.00

Wavelength (nm)

 

Figure 2.7 Fluorescence spectra of tryptophan excited at 342nm
before and after recrystallization from aqueous solution containing

pyranine at a concentration of 10'5 M

10

34
Q. Chen, W. J. Walczak, and M. D. Barkley, J. Am. Chem. Soc.,

117, 556-7 (1995)

S. A. Tucker, W. E. Acree, JR., and M. J. Tanga, S. Tokita, K. Hiruta,

and H. Langhals, Applied Spectroscopy, 46(2), 229 (1992)

M. U. Kumke, H. G. Lohmannsroben and Th. Roch, Analyst, 119, 997 (1994)
R. Chakraborty, and K. A. Berglund, AICHE Symposium Series,

284-7, 114-123 (1991)

Chapter 3

SUPERSATURATION MEASUREMENTS BY USING THE STEADY
STATE FLUORESCENCE OF IMMOBILIZED PYRANINE

3 . 1 Introduction

Steady state fluorescence of extrinsic trace fluorescent probe molecules as sensors
as introduced by Chakraborty and Berglund (1) has been shown to be a very useful method
to monitor the supersaturation of high solubility solutions. It has been used to monitor the
degree of supersaturation with the peak intensity ratio (PIR) of pyranine in aqueous
solutions of sucrose, glucose, fructose, lactose and citric acid (1, 2). In high solubility
systems, the PIR is related to the available water in its immediate environment. Pyranine in
solution can be used to monitor the crystallization of low solubility systems as well by
explaining the pyranine quenching behavior the solutes as described in chapter 2. The PIR
of pyranine was shown to change smoothly and nonlinearly with the concentration of
tryptophan, nicotinic acid and benzoic acid, and had higher signal response in
supersaturation region.

For previous studies pyranine (1x10‘5M) was introduced into the crystallization
solution. A potential problem is that pyranine is not approved by the FDA as a food
additive. Phannaceutical applications of crystallization demand a high degree of purity
which precludes the tolerance of even trace amounts of the probe incorporated in the
crystal. The control of crystallization of food and pharmaceutical production processes is
an important industrial need.

35

36
Previous studies have attempted to develop suitable methods to measure

supersaturation of food and pharmaceutical crystallization. ATR-FI‘IR was shown to be a
useful in-situ crystallization monitor for high solubility systems (3). ATR-FTIR can not be
used to monitor low solubility systems, such as tryptophan and nicotinic acid solutions,
due to low concentration changes which will produce not detectable signal. Pyranine is still
the best probe to measure the supersaturation of low solubility systems due to its high
signal response, sensitivity, stability and high quantum efficiency. Probe immobilization is
an approach for avoiding contamination, however, the probe must response to the changes
of the solution concentration. An immobilized pyranine probe is the target of current study.
In this research we have immobilized pyranine on an ion-exchange membrane and
have determined the calibration curve of PIR (Peak Intensity Ratio) versus the
concentration of solutions. The solutes studied are tryptophan, phenylalanine, and

nicotinic acid all of which have low solubility around 2wt%.

3 . 2 Materials and Methods

Pyranine, also known by the FDA as D&C Green No. 8,was obtained from
Lancaster, Windham, NH and used as received. Aqueous solutions of pyranine were
prepared by dissolution of 0.026 g in 5ml HPLC grade water to get the pyranine at the
concentration of 10'2M.

Different concentrations of solutions were prepared by dissolving the appropriate
amount of solutes in 5ml HPLC water. Supersaturated solutions were prepared by heating
the solution in the water bath until all the solute was dissolved and subsequent cooling to
room temperature. The solutions were used as prepared without any degassing.

Immobilized pyranine was prepared by immersing a membrane for 2.5mins in the
pyranine solution (4x10‘5M) obtained by addition of 20ul pyranine at 10 2M to 5ml HPLC
water. The membrane was DEAE cellulose acetate NA45 anion exchange membrane with

pore size 0.45 micro meter obtained from Schleicher & Schuell, Keene, NH. The

37
immobilized pyranine membrane was immersed in the solutions of tryptophan, nicotinic

acid or phenylalanine for over 24 hours to saturate the membrane with the solutes before
use in an experiment. Fluorescence spectra of the immobilized pyranine in the aqueous
solutions were collected within a couple of hours of the preparation of the solutions. All
spectra were collected at room temperature with a Spex 1681 Fluorolog spectrometer
equipped with a Xenon lamp source and a PMT detector and a front angle set up. A quartz
sample holder was used in all cases. Spectra were recorded at a 1.00mm increment and an
integration time of 1.0 sec. An excitation wavelength of 342nm for pyranine was used.
The emission spectra were recorded over a range from 350-600nm for pyranine.

Monochromator slits were at 0.5 mm for all spectra.

3 . 3 Results and Discussion
3.3. 1. Fluorescence spectra of immobilized pyranine

The spectra of immobilized pyranine and free pyranine in water are shown in Figure
3.1. There is a broad peak at 510nm for both immobilized and free pyranine. There is a
set of peaks around 400nm which are different for immobilized pyranine and free pyranine.
With free pyranine, three fine vibronic peaks will show up around 400nm. In immobilized
pyranine, only two peaks will show up and the peak symmetry is not as good as free
pyranine. Another remarkable difference is the relative intensity at 510nm and at 400nm.
There is a big increase of peak intensity at 510nm and a big decrease of peak intensity at
400nm when pyranine is immobilized on membrane. The peak at 510nm is assigned as
fluorescence of deprotonated pyranine and the other bands from 380nm to 420nm are
characterized peaks of polycyclic aromatic hydrocarbon (PAH) (4,5).

As we know the amount of pyranine on the membrane is very low although we can
not tell how much it is. We can tell that is low from the preparation of immobilized
pyranine. Four piece membranes were immersed in pyranine solution (4x10'5M) for

2.5mins at the same time. Even thought we can assume the ion exchange is very fast,

Intensity (CPS)

4.55%

38

 

4UE+£B J

3.$E+(5 1

SIDEKB J

a
i

E
a

1.50545 .

1.00905

5.(DE+O4 4

 

O.CDE+CD -“

 

 

 

immobilized pyranine

 

Free
free pyranine
im
400 450 500 550
Wavelength (nm)

Figure 3.! Fluorescence spectra of immobilized pyranine and
free pyranine in water at room temperature

 

39

there is limitation of 1x10’5M. Actually we know it is impossible that all pyranine will be
exchanged onto the membrane, but free pyranine concentration in water is 10'5M. So the
concentration difference is not hard to tell.

It is commonly known that the fluorescence intensity of an excited probe molecule
can be drastically enhanced when bound to a micelle assembly (6,7,8). The enhancement
of fluorescence in micelle system stem from altered micropolarity, restricted motion,
effective shielding of the excited single state from quenchers present in solution (8). The
shielding of the probe from vibrational quenching by the hydrogen-bond structure of water

is also the reason for the enhanced fluorescence (6). Hoshino and Irnamure also reported

the increase of fluorescence of benzene derivatives by forming inclusion complexes with B-

cyclodextrin in aqueous solution (10). The enhancement is due to the decrease of the
rotational freedom of florescence molecules and the elimination surrounding fluorescent
molecules in a aqueous solutions. It is obvious when pyranine is immobilized on
membrane, the rotational freedom of pyranine molecule is restrained which will result in the
increase of the fluorescence. Effective shielding of the excited single state from quenchers
present in solution with immobilized pyranine and also the shielding of the probe from
vibrational quenching by the hydrogen-bond structure of water are the results of the
enhancement of immobilizes pyranine fluorescence too.

The fluorescence spectra of immobilized pyranine saturated with three different
solutes, tryptophan, phenylalanine and nicotinic acid are shown in Figure 3.2.
Immobilized pyranine saturated with phenylalanine has the same fluorescence spectrum as
immobilized pyranine which is not saturated with solutes. There are no big change for the
peak at 510nm either. But peaks around 400nm are different when immobilized pyranine
saturated with tryptophan and nicotinic acid. Tryptophan blurred this set of peaks, and
nicotinic acid enhanced the peak at 430nm. The differences of spectra saturated with

different solutes can be interpreted as the different interaction of pyranine with tryptophan,

Intensity (CPS)

611E“

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AGE“ .

3006-05 -

laid-(5 -

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40

 

iv,

 

\ / Phe

0‘.

NA

Phe

Trp

/\

."
I
t
1'
t
I

I

 

fir

350 400

a. 560 500
Wavelength (nm)

Figure 3.2 Fluorescence spectra of immobilized pyranine saturated with
tryptophan, phenylalanine and nicotinic acid in pure water at room

temperature

 

41
nicotinic acid and phenylalanine which are the results of different structures of solute

molecules. As we know fluorescence occurs from the lowest vibronic level at fist excited
state, but electrons will relax to different vibrational level of ground state. So fluorescence
reflects the vibrational levels of ground states. The change of fluorescence at 400nm when
immobilized pyranine saturated with tryptophan and nicotinic acid can be taken as the
evidence of the stronger interaction with pyranine compared to phenylalanine which show
no interference to immobilized pyranine. N icotinic acid shows the strongest interaction
with pyranine which changes the ground state energy profile of pymaine.

The fluorescence spectra of immobilized pyranine in tryptophan, phenylalanine and
nicotinic acid solutions show no differences in shape and maxima in the presence and
absence of tryptophan, phenylalanine and nicotinic acid. The immobilized pyranine
fluorescence intensity changed with the concentration of solutes. But so many factors can
result in the change of intensity so intensity change is unpredictable.

The fluorescence intensity of pyranine will increase with the addition of the solutes
into the solution. When we immersed immobilized pyranine which did not contact with
any solutes into tryptophan solution less than an hour, a very large fluorescence intensity
enhancement at all bands can be realized. As we know nicotinic acid quenches pyranine
strongly when pyranine is free in the water because the hydrogen bonding complex
between pyranine and nicotinic acid (refer to chapter 5). Tryptophan will quench
fluorescence of pyranine in solution too and phenylalanine will quench pyranine
fluorescence in solution slightly. The different quenching ability of solutes results from the
different interaction of pyranine with different solutes. When immobilized pyranine
contacts with solutes, solute molecules will enter the membrane. Because the similar ring
structure of pyranine as tryptophan, nicotinic acid and phenylalanine, the dipole moment
interaction of pyranine with tryptophan, nicotinic acid and phenylalanine will be stronger
than the interaction with water. This interaction is interpreted as the contact complex

referred to chapter 5. The increase of the fluorescence intensity can be attributed to the

42
protection of quenching by water because of the formation of the contact complex between

pyranine and solute molecules. Water quenching fluorescence by proton transfer has been
proven by isotope effect (11,12). As we know the most important quenching mechanism
is dynamic quenching which involves the collisional contact between quencher and
fluorophore. When pyranine is immobilized in the membrane, the solute molecules can not
diffuse into the membrane to quench the fluorescence of pyranine due to the bigger size of
solute molecules. Water is much smaller molecules which are easier to diffuse to pyranine
to quench the fluorescence. But the contact complex between pyranine and solute
molecules provide the shielding of the probe from formation of hydrogen bonding with
water molecules than from vibrational quenching by the hydrogen-bond structure of water.

Thus the fluorescence intensity increases.

3 . 3.2. Measurement of supersaturation

The peak intensity ratio (PIR), defined as the ratio of the peak intensity at 401nm or
382nm to the intensity at 510nm for tryptophan and phenylalanine and 429nm to 510nm for
nicotinic acid, is used to measure the concentration of solution. The use of this ratio can
provide an internal correction in the spectrophotometer setup. The fluorescence intensity of
immobilized pyranine is really unpredictable. So many different factors can affect the
intensity, especially the membrane surface. Even different light shooting spot will alert the
intensity. By using the PIR, these random change can really be corrected which is such a
good news for us. Figure 3.3 is the calibration curves of immobilized pyranine in
tryptophan, phenylalanine and nicotinic acid solutions. We can see the PIR changes with
the concentration of solutes linearly. By measuring PIR, concentration of solutes can be
read out from calibration curves and the supersaturation can be calculated by the difference
of solubility and concentration. So we can control the supersaturation by adjusting the
outer conditions, such as the temperature of environment. It has been shown that the

supersaturation measurement using the PIR is more sensitive than other techniques,

PIR

43

 

 

 

 

 

 

0.7 i .
r
06-- -_
r
0.5-- ~-
. O O
i Tryptophan (384/510)
04-» --
F 'i
0.3+ .-
’ i
J
Phenylalanine (382/510) 4
b n O O C o 03 0‘9
0.2“” O—_ 3 V _-
f
l
1.
0.1 n A I i r A . n I A L L n % L 44; n I . I n n % n n n m % L a n n I A I A
0 0.5 1 1.5 2 2.5 3 3.5 4

Concentration of solutes (g/100ml H20)

Figure 3.3 Peak intensity ratio (PIR) of pyranine versus concentration of
tryptophan, nicotinic acid and phenylalanine aqueous solutions at room
temperature. The values in parentheses represent the emission peaks for PIR.

44
we can see this in our research since the concentration change in our research is from

0.06%-1.8% of Tryptophan, PIR changes from 0.3 to 0.5. The concentration is 0.5% to
2.5% for nicotinic acid and the gain of PIR is from 0.5 to 0.7. This is a big signal
response compared to traditional methods which changes occurs at fourth or even fifth
decimal places.

Reproducibility of PIR versus the concentration was checked for both tryptophan
and nicotinic acid solutions. It turned out different immobilized pyranine will give us
different linear curves, but the same immobilized pyranine will have the same PIR versus
concentration plots. Figure 3.4 shows two sets of the PIR vs. concentration of tryptophan
and nicotinic acid with the same immobilized pyranine at different experiment dates. For
tryptophan, two experiments were conducted by 6 days difference and nicotinic acid is 8
days difference. Two sets data for both tryptophan and nicotinic acid are overlap each
other. Another experiment using the same immobilized pyranine and conducted in two
different days in tryptophan solutions showed an overlap PIR versus concentration curves
too which is not shown here. So we can conclude the relation between PIR and the
concentration of solutes is reproducible.

Pyranine has very high solubility in water, so we have to consider whether
immobilized pyranine will dissolve in water. If even trace dissolution happens, it will
make this technique not good enough for monitoring food and pharmaceutical processing.
So we immerse immobilized pyranine membrane into water, then detect the fluorescence of
pyranine after the membrane was taken away. The spectra shows no fluorescence of
pyranine. So we can conclude pyranine will not dissolve into water once it is immobilized

in the membrane. It is safe for it to be used in food processing.

3 . 4 . Conclusion
The fluorescence of immobilized pyranine was enhance due to the restriction of

movements of pyranine molecule and also due to the protection of quenching by water

PIR

45

 

 

0.l~— —

 

 

 

L-
1.
r-
0 AllA%AAAA%1_LJL¥%ALLA%AAAA%AALL
0 0.5 1 1.5 2 2.5 3

Concentration of tryptophan and nicotinic acid (g/100ml H20)

Figure 3.4 Reproducibility of peak intensity ratio (PIR) of pyranine
versus concentrations for both tryptophan and
nicotinic acid aqueous solutions at room temperature.

46

molecules. The introduction of solutes to immobilized pyranine on membranes will

enhance the fluorescence intensity by formation of contact complex which will protect the

quenching from water. The PIR of immobilized pyranine is strongly correlated to the

solute concentration and the relation between the PIR of irnmcbilized pyranine and

concentration is linear. This technique is highly sensitivity which gives it more promise.

3.5.

10.

ll.

12.

References

R. Chakraborty and K. A. Berglund, J. Crystal Growth, 125, 81(1992)
Sanjay K. Yedur and Kris A. Berglund, Applied Spectroscopy, 50(7),
866(1996)5.

Dunuwila, D. D., PhD Thesis, Michigan State University(1996)

S. A. Tucker, W. E. Acree, JR., and M. J. Tanga, S. Tokita, K. Hiruta,
and H. Langhals, Applied Spectroscopy, 46(2), 229, 1992

M. U. Kumke, H. G. Lohmannsroben and Th. Roch, Analyst, 119, 997(1994)
W. L. Hinze, H. N. Singh, Yoshirrri Baba and Noel G. Harvey,

Trends in Anal. Chem, 3(8), 193(1984)

H. N. Singh and W. L. Hinze, Analyst, 107, 1073(1982)

H. N. Singh and W. L. Hinze, Anal. Lett. 15, 221(1982)

M. Hoshino, M. Imamura, K. Ikehara and Y. Hama,

J. phys. Chem. 85, 1820(1981)

J. D. Ingle, Jr.., and S. R. Crouch, Spectrochemical Analysis,
Prentice-Hall, Inc., 1988

L. P. McMahon, W. J. Colucci, M. L. McLaughlin and M. D. Barkley
J. Am. Chem. Soc., 114, 8442-8448(1992)

Yu Chen, Bo Liu, Hong-Tao Yu and Mary D. Barkley,

J. Am. Chem. Soc., 118, 9271-9278(1996)

Chapter 4.

THE EXPLORATION OF OTHER POSSIBLE PROBES--PBA AND CA

4 . 1 . Introduction

It was shown in chapter 2 that pyranine is a good probe for measurement of
supersaturation in low solubility systems. The PIR (peak intensity ratio) curves of
pyranine with the concentration of tryptophan, nicotinic acid are smooth, nonlinear curves
which have a larger gain at higher concentrations.

Our goal is to evaluate other probes and compare them to pyranine. PBA (1-
Pyrenebutyric acid) is another useful probe which is used widely to study rrricellar
properties and fluorophores’ location in membranes (1, 2). The molecular structure of
PBA (l-Pyrenebutyric acid) is shown in Figure 4.1. PBA has strong fluorescence in
aqueous solution. There are three emission peaks for PBA in MeOH originating from the
first excited singlet state and shows fine structure as is common for many PAH compounds
(3). Pharmaceutical and food production applications of crystallization demand a high
degree of purity which precludes the tolerance of even trace amounts of the probe
incorporated in the crystal. Pyranine and PBA, which are not approved by the FDA as
food additives, can’t be added directly in food and pharmaceutical processing to monitor
the process if there is a possibility of the probe contamination in the product. Although we
already have shown pyranine will not go into crystal lattice, the addition of pyranine is not
allowed by FDA in food processing. We need to explore other possible probes approved
by FDA. Carminic acid (CA) is a choice.

47

48

 

l-pyrene butyric acid (PBA)

H0 CHZOH
H O 0 0H 0 CH3
COOH
"' 999
H O I O H
O H O
Canninic acid (CA)

Figure 4.1. Molecular structures of l-pyrene butyric acid (PBA)
and carminic acid (CA)

49
Carminic acid (CA), shown in Figure 4.1, is a natural food pigment making it a

potentially good probe for food processing and pharmaceutical processing. Carminic acid
is the major coloring substance of cochineal used in food and beverages, and can be added
directly into pharmaceutical and food processing.

CA is weakly fluorescent in aqueous solution. The photochemistry of CA has been
studied and results show that the fluorescence of carminic acid is moderately dependent on
pH in acidic and neutral aqueous solution. There are four species of CA in solution---
LH3(CA), LHZ”, LHZ",and L3“ (4,5). Each species has a different emission spectrum.

The state of supersaturation is an essential requirement for all crystallization
operations. The formation of nuclei is the beginning point of the crystallization. The
nucleation of solute must somehow depend upon the solute molecules in a supersaturated
solution and also depend upon the various molecular interactions, such as solute-solute and
solute-solvent interactions (6). Understanding the structure of supersaturated solutions can
help us better design and control the crystallization.

Fluorescence quenching requires the contact of the quenchers and fluorophores so
fluorescence quenching technique is a very useful tool to study the interaction of
fluorophore and quencher then information about the organization of solution can be
deducted from quenching data. Different quenching behaviors are the results of different
interaction of fluorophors and quenchers.

In this chapter we measure the fluorescence spectra of PBA and CA in aqueous
solutions to evaluate the feasibility of PBA sand CA for measurement of supersaturation in
low solubility systems. The quenching behavior of tryptophan and phenylalanine to PBA

and CA are also studied to probe the organization of solute molecules in solutions.

4.2. Material and Methods
PBA (l-Pyrenebutyric acid) was obtained from Eastman Kodak, Rochester NY and
used as received. PBA solutions were prepared by dissolution of 0.0134g PBA in 20ml

50
ethanol because the solubility of PBA in water is pretty low. Different concentrations of

solutions were prepared by dissolving the appropriate amount of solute in 5ml HPLC
water. Supersaturated solutions were prepared by heating the solution in the water bath
until all the solute was dissolved and subsequent cooling to room temperature. The
solutions were used as prepared without any degassing.

Probe molecules were inuoduced by addition of five microliters of the PBA
solution (4x10'3M) to the aqueous tryptophan and phenylalanine solutions resulting in a
probe concentration of 4x10‘M. Fluorescence spectra of PBA in the aqueous solutions
were collected within two hours of the preparation of the solutions. All spectra were taken
at room temperature with a Spex 1681 Fluorolog spectrometer equipped with a Xenon lamp
source and a PMT detector. A quartz sample holder was used in all cases. Spectra were
recorded at a 1.00mm increment and an integration time of 1.0 sec. An excitation
wavelength of 326nm for PBA was used. The emission spectra were recorded over a
range from 340—4500nm for PBA. Monochromator slits were at 0.5 mm for spectra.

Carminic acid was obtained from Aldrich Chemical Company, Inc. Milwaukee, WI
and used as received. Carminic acid solutions were prepared by suspending 0.01 g CA in
20ml 1120. The suspension was centrifuged in a Eppendorf Centrifuge 5415C at the speed
of 14x1000rnin‘1 for 5 mins to obtain a saturated aqueous solution of CA. The appropriate
concentration of CA was introduced by addition of 5 microliters of the saturated CA
solution to the aqueous tryptophan and phenylalanine solutions which were prepared by
dissolving the appropriate amount of solute in 5ml HPLC water. Fluorescence spectra of
CA in the aqueous solutions were collected within two hours of the preparation the
solutions. All spectra were taken at room temperature with a Spex 1681 Fluorolog
spectrometer equipped with a Xenon lamp source and a PMT detector. A quartz sample
holder was used in all cases. Spectra were recorded at a 1.00mm increment and an

integration time of 1.0 sec. An excitation wavelength of 500nm was used. The emission

5 1
spectra were recorded over a range from 530nm to 700nm. Monochromator slits were set

at4mm.

4.3. Results and Discussions
4.3.1. Steady state fluorescence of PBA as probe

There are two peaks in the PBA emission spectra in tryptophan solution shown in
Figure 4.2, one peak is at 375nm and the other is at 395nm. This is a typical emission
spectrum of a polycyclic aromatic hydrocarbon (PAH). The typical emission spectrum of
the PAH monomer will consist of five major vibronic bands which are generally labeled as
I-V in progressive order, such as the 0-0 band is labeled I, etc.(3,7,8). The fluorescence
intensity versus the concentration of PBA (Figure 4.3) shows that the intensity of
fluorescence decreases with the concentration of tryptophan at both wavelengths. The
intensity shows a larger decrease at 375nm. The intensity differences at 375nm and 395nm
are smaller with the increase of the concentration of tryptophan. When the concentration of
tryptophan is high, the fluorescence intensity increases very little with the concentration of
tryptophan, especially the intensity at 395nm. The intensity of fluorescence at 375nm and
395nm approach the same value at high uyptophan concentrations. The intensity decrease
with the concentration of tryptophan showed the quenching of tryptophan to PBA.

The emission spectra of PBA in aqueous phenylalanine solution has the same shape
and peak position as in tryptophan solutions. There are two peaks, one is at 375nm and the
other is at 395nm. The intensity of both peaks changes little with the concentration of

phenylalanine which means phenylalanine does not quench the fluorescence of PBA.

4.3.2. Calibration curve of PBA as probe
The peak intensity ratio (PIR) is defined as the ratio of intensity at 395nm and

375nm in tryptophan solutions. The PIR increases with the increase of concentration of

Intensity (CPS)

SIDEKB

52

 

4.505% 1

4.00905 <

3.50905 i

3.CDE+(B

2.3)E+% 4

2.(DE+(5 1

1 50905

roots-«05 J

5.00904 i

 

OWE-0(1)

 

 

\

\

 

 

Y— _ f

50 am 370 380 390 «D 410 420 433 44) 450

Wavelength (nm)

Figure 4.2 Fluorescence spectra of PBA in aqueous
tryptophan solutions:
1(0.0l 18M). 4(0.04lOM) and 9(0. 100M)

Intensity (CPS)

53

 

 

 

 

 

5105....i..e-i...r.-
i'
4105i—
_ 375nm
..
310%
i
F O
2105-— ‘
.. A
° 0
o 0 g o, - - _ /’
3951111] . . ; ; ? ‘3":/
1105- , .
G . . 1 1 I . . r . lr . r n I 1 I n
0 0.5 1 1.5

Concentration of tryptophan (g/100ml H20)

Figure 4.3 Fluorescence intensity of PBA versuses the
concentration of tryptophan in aqueous solution
at room temperature. The lines are smooth fit of data.

54
tryptophan shown in Figure 4.4. The increase is smooth, but there is not a larger gain at

higher concentration as shown with pyranine as the probe in chapter 3. In contrast the PIR
plot curves downward at higher tryptophan concentration; However, the PIR curve is
changing with the concentration of tryptophan smoothly and the signal is big enough to be
used to measure the supersaturation of tryptophan solutions.

The intensity of PBA did not change with the concentration of phenylalanine, so the
PIR of 395nm/375nm is constant at all concentrations of phenylalanine. Therefore, PBA

can not be used as probe to measure the supersaturation of phenylalanine.

4.3.3. Steady state fluorescence of carminic acid as a probe

There are four emission peaks in the emission spectra of carminic acid because there
are fun species in the solution (5). They are difficult to deconvolute because they overlap
each other. In general, we obtain one broad peak, or one wide peak with a shoulder when
one species is dominant. From the emission spectra (Figure 4.5) we can see that in
tryptophan and phenylalanine solutions there are two different dominant species, which
position at 580nm in phenylalanine and 595nm in tryptophan. The emission intensity of
carminic acid increases with the concentration of phenylalanine (Figure 4.6), but decreases
with the concentration of tryptophan (Figure 4.7). The PIR is defined as the peak intensity
ration of 580nm/595nm. No changes of the PIR occurs with the concentration changes of
phenylalanine. Therefore peak intensity will be used as the signal to measure the
supersaturation for CA as probe. From Figure 4.6 the fluorescence intensity of CA will
increase with the concentration of phenylalanine smoothly and can be used as the
calibration curve to measure the supersaturation of the phenylalanine solutions. From
previous results we know phenylalanine does not quench the fluorescence of PBA and
quenches the fluorescence of pyranine weakly shown in chapter 2, therefore it is hard to

get strong signal to measure the concentration of phenylalanine using PBA and pyranine as

PIR

 

0 K A A L 4 l L A I A 1 A A A A l A J

55

1.1 . . . ' i
395nm/375nm

 

—n—

 

 

I

0 0:5 1 1:5 2
Concentration of tryptophan (g/100ml H20)

 

Figure 4.4 Peak intensity ratio (PIR) of PBA versus the
concentrations of tryptophan in aqueous solutions at room temperature.

 

 

 

 

4.50905
Phet
4.009054 Phe 2
/ I. Trp 1
3.509054
3.00905.
2 2.50905.
0
V
.E‘ .
(I)
5 200905
E ' Trp 2
1.50909
1.00905 ~
5.00904.
0.00900
530 550 570 500 610 6:30 660 570 090
Wavelength (nm)

Figure 4.5 Fluorescence spectra of carminic acid in aqueous tryptophan
solutions and phenylalanine solutions at room temperature. Phe l: 0.0128M,
Phe 2: 0.0138M. Trp I: 0.0] l9M and Trp 2: 0.0702M

Intensity (CPS)

2 106

57

 

1.5 106--

I

1 10‘-

I

5105-

 

 

 

 

1 2 i 4 5
Concentration of phenylalanine (g/100rnl H20)

Figure 4.6 Fluorescence intensity of CA at 595nm versus
concentration of phenylalanine in aqueous solutions
at room temperature. The plot is polynomial fitting.

58

4 105

—iy—-

 

—tr—
.l
.4

T

3.5 105-

I

3 105—

Intensity (CPS)

2.5 105-

I

2 105.-

 

1.5 105 . . L I i . . . . i . . . 4 i I I . .
0 0.5 1 1.5 2
Concentration of tryptophan (g/100ml H20)

 

 

Figure 4.7 Fluorescence intensity of carminic acid at 595nm
versus concentration of aqueous tryptophan at room temperature.

The arrow indicates the solubility of tryptophan at 25°C.
The line is smooth fitting of data.

59
probes. Here we show CA will work as a probe for phenylalanine. However, CA can not

be used to measure the supersaturation of tryptophan solutions because the PIR curve
doesn’t change with the concentration of tryptophan and fluorescence intensity of CA

decreased with the concentration of tryptophan.

4.3.4 Quenching behavior of tryptophan and phenylalanine

The decrease of the fluorescence intensity of PBA in tryptOphan solutions is the
result of quenching by tryptophan. The Stem—Volmer quenching curve of PBA by
tryptophan is shown in Figure 4.8. Quenching of PBA by tryptophan follows a straight
line at low concentration of tryptophan at both wavelengths. When the concentration of
tryptophan increases, the plot curves downward. When the concentration of tryptophan
increases, aggregates of tryptophan probably form in the solution reducing the quenching
efficiency, so the plot curves down. Tire fluorescence intensity of PBA does not change
with the concentration of phenylalanine which indicates that phenylalanine does not quench
the fluorescence of PBA.

The fluorescence intensity of CA in tryptophan solutions decreases with the
concentration of tryptophan, but increases with the concentration of phenylalanine
solutions. So we can conclude tryptophan quenches the fluorescence of CA but
phenylalanine does not quench the fluorescence of CA. The quenching curve of CA by
tryptophan in solutions is shown in Figure 4.9. The graph shows a downward quenching
curve. The downward curvatures can be the result of the heterogeneity of the system or
selective quenching. Different binding sites in large molecules make some fluorophores
easier to be quenched than others (9). The negative deviation from the Stem-Volmer law

can be described by modified Stem-Volmer Law III (10).

1111

0

= —+
1‘1 fKlel f

O

 

 

(4.1)

f is the fraction of the fluorescence accessible to quenching.

Io/I-l

 

 

 

 

 

10 .L i - v i
84- . . o . —4>-
375nm
o ’ o o
o
6—-— . ~-
x X
x x
x x
x 395nm
. " i
L.
2-- ' .._
0 L L A I § 1 J A L E A A A A : A A A J
0 0.5 1 1.5 2

Concentration of tryptophan (g/100ml H20)

Figure 4.8 Quenching of PBA by tryptophan in aqueous solutions
at 375nm and 395nm fit to Stern-Volmer law at room temperature.

The arrow indicates the solubility of tryptophan at 25°C.

61

 

‘—
IAI-
-i-
«r-
-b

6 10'6

5.5 106—

5 10'6-

 

 

 

 

A l A A l A A l A J A
I

3.510“
0.4 0.6

A l A A A
I

0.8 1 1'2 {.4 116 1.8 2
Concentration of tryptophan (g/100ml H20)

Figure 4.9 Quenching of carminic acid at 595nm by tryptophan
in aqueous solutions fit to 1/I=1/Io+(K/Io)[Q] at room temperature.

62
By using this modified Stem-Volmer curve to fit the quenching data, Figure 4.10 is

obtained which is a straight line. The intercept 1.372 results in the fraction of the
fluorescence accessible to the quenching as 73% and the quenching constant is 1.2 (g
tryptophan/100ml HZOI)". Some of tryptophan molecules can not access CA to quench the
fluorescence because of the formation of multiple molecular associates among tryptophan
molecules. Detailed discussion regarding the structure of tryptophan in solution will be
presented in chapter 5 and chapter 7.

The different quenching behaviors of tryptophan and phenylalanine stem from the
different host-probe interaction of probe molecules with solute molecules resulting from the
different structures of solute molecules. We know the structural difference of tryptophan
and phenylalanine is only in the ring, tryptophan has an indole ring and phenylalanine has a
benzene ring. If the dynamic quenching were the quenching mechanism, phenylalanine
should have a larger quenching ability due to its relative smaller size compared to
tryptophan. However, phenylalanine does not show any quenching ability at all; therefore
we interpret the quenching from tryptophan is static domain quenching due to the amine in

the ring.

4.3.5. Detection of PBA and CA in crystalline products

In order to test if PBA and CA will enter the crystals lattice of tryptophan, an
experiment is conducted as follows: dissolving tryptophan in solution, adding PBA and
CA into the solution, then recrystalizing tryptophan from solutions with PBA and CA in the
solutions. Recrystalized tryptophan is dissolved in water and the fluorescence of PBA and
CA is recorded. For tryptophan recrystallized from PBA solution, the solution was excited
at 326nm, spectra were collected from 340nm-450nm. For tryptophan recrystallized from
CA solution, the solution was excited at 500nm. The spectra for material recrystallized

from PBA exhibits the peaks from PBA indicating that PBA is incorporated into the crystal

63

fit—
i

 

4,5....:....:4......
y = 1.372 + 1.1417x R2: 0.99874 .

 

 

r

1.5 A A A I % A I I I I A I l I I I I L L
0.5 1 1.5 2 2.5

The reciprocal of concentration of tryptophan (g/100ml H20)"l

 

 

 

Figure 4.10 The quenching curve of tryptophan in aqueous solutions to
CA at 595nm fit to Io/(Io-I)=( l/[Q])(1/stv)+1/f at room temperature.

64
of tryptophan. Therefore, PBA can not be used to measure the supersaturation because it

will contaminate the crystal product Conversely, no fluorescence of CA is detected which

tells us that CA does not go into the crystal of tryptophan.

4 . 4 . Conclusion

Tryptophan will quench the fluorescence of PBA, but phenylalanine will not. This
can be interpreted as the static quenching domain in the tryptOphan quenching behavior.
The formation of aggregates in high concentration solution reduce the quenching efficiency
resulting in the downward curvature of the quenching plot.

However, PBA can incorporate into the lattice of the crystal of tryptophan which
will contaminate the product; therefore, PBA can not be used as the probe to monitor the
crystallization.

Carminic acid is an excellent potential probe for the food processing because it is a
food additive. Tire fluorescence intensity of carminic acid at 595nm changes smoothly with
the concentration of solutes. Monitoring the change of the intensity we can determine the
concentration of the solution. It is shown that carminic acid will not go into the lattice of
the tryptophan crystal. In conclusion, carminic acid can be used as in situ monitors to
follow the crystallization of phenylalanine.

Tryptophan shows a quenching ability for CA and phenylalanine will enhance the
fluorescence of CA by protecting CA from quenching by water. Only 73% of tryptophan
can access CA which confirms that multiple molecule associates form among tryptophan

molecules in tryptophan solution.

4.5. References

1 F. H. Quina and V. G. Toscano, J. Phys. Chem. 81(18), 1750(1977)

2. M. Deumie, M. El Baraka and E. Quinones, J. Photochem. Photobio,
A: Chemistry, 87, 105-113(1995)

\IO‘iUI-h

10.

65
T. L. Netzel, Min Zhao, K. Nafisi, J. Headrick, M. S. Sigman and B. E. Eaton,

J. Am. Chem. Soc., 117, 9119-9128(1995)

H. Stapelfeldt, J. Huang, and L. H. Skibsted, Food Chemistry, 48, 1-11(1993)
M. J. Schwing-Weill, Analusis, 14(6), 290-295(1986)

J. W. Mullin, Crystallization, Butterworth-Heinemann, Oxford, England(l993)
S. A. Tucker, W. E. Acree, JR., and M. J. Tanga, S. Tokita, K. Hiruta,

and H. Langhals, Applied Spectroscopy, 46(2), 229(1992)

M. U. Kumke, H. G. Lohmannsroben and Th. Roch, Analyst, 119, 997(1994)
M. R. Eftink and C. A. Ghiron, Biochemistry, 15(3), 672(1976)

J. R. Lakowicz, Principles of Fluorescence Spectroscopy, Plenum Press,

New York and London(1983)

Chapter 5

MECHANISM OF PYRANINE FLUORESCENCE QUENCHING AND THE
APPLICATION IN THE STUDY OF SUPERSATURATED SOLUTION

5 . 1 . Introduction

Fluorescence quenching is a very useful method used to study both physical and
biochemical phenomena (1-5). Fluorescence quenching experiments can yield valuable
information about fluorophore and quencher interactions. The diffusion-controlled process
of fluorescence quenching has been used to study various dynamic properties of aggregated
systems of micelles and lipid vesicles (1). Quenching processes require the contact of
quencher and fluorophore, so quenching measurements can reveal the accessibility of
fluorophores to quenchers. By studying the interaction between the probe molecules and
solute molecules, we can get insight about the organization of solute molecules. Therefore,
fluorescence quenching is a potential technique to allow us to study the structure of
supersaturation solution.

Crystallization takes place by the formation of a stable crystal nucleus from a
homogeneous supersaturated solution fnst, then those nuclei will grow to form crystals.
The formation of nuclei is the beginning point of the crystallization. The nucleation of
solute must somehow depend upon the solute molecules in a supersaturated solution and
also depend upon the various molecular interactions, such as solute-solute and solute-
solvent interactions. Supersaturated solutions definitely have different structures from
unsaturated solutions. Understanding of the structure of supersaturated solutions can help

us in the control of industrial crystallization. Several studies have focused on the structure

66

67
of supersaturated solutions and many techniques have been applied, such as NMR, X-ray

diffraction and Raman spectroscopy, and many theories about the structure of
supersaturated solution have been proposed (6-10). Mullin and Leci suggested that solute
clusters form in a supersaturated solution of citric acid by observing an isothermal column
of a supersaturated solution of citric acid which generated a concentration gradient over
period of several days. The concentration gradient developed as a result of the density
difference between the clusters and the solution (6). Cussler observed different rates
between cluster diffusion and molecular diffusion in many binary systems, which
supported the cluster assumption (7). Myerson and co-workers observed the decrease in
the diffusivities of urea and glycine solutions, which provided evidence for molecular
aggregation (8). Hussman et a1. and McMahon et al. also confirmed cluster formation on
alkali nitrate solutions by using Raman spectroscopy (9,10). Although significant work
has been done on structure study, there are still no generally accepted theories.

Previous research focused on studying high solubility systems with solubility
exceeding 50wt%. No information about the organization of supersaturated solutions of
lower solubility systems has been reported. A suitable method is needed to study lower
solubility systems to get information about the organization of supersaturated solutions.
The interaction between s solute and a probe can be studied by fluorescence quenching
combined with the sensitivity of fluorescence spectroscopy which makes fluorescence
quenching a possible technique for low solubility systems. Fluorescence quenching will be
used to study the organization of supersaturated solutions in this research.

In this chapter fluorescence quenching of pyranine by tryptophan, phenylalanine,
and nicotinic acid in aqueous solutions are studied to probe the organization of solutions.
Tryptophan, phenylalanine and nicotinic acid showed different quenching behaviors which
are the results of the different interactions between the pyranine and solutes. By
interpreting the different quenching behaviors, insight into the organization of solution can

be gained.

68
5.2. Theory

Fluorescence quenching involves a variety of processes, such as excited state
reactions, excited state proton transfer, excited state electron transfer, energy transfer,
complex formation and collision quenching (11). The quenching from diffusion-controlled
encounters, the most common quenching process, is called dynamic quenching.

Dynamic quenching Of fluorescence can be described by the Stem-Volmer Law (11)

Lo
I

-1=K.v[Ql (5.1)
I0 is initial intensity without the quencher, I is emission intensity of sin gle-emission
wavelength with the quencher. K" is the Stem-Volmer quenching constant. Q is the
concentration of quencher. If the mechanism of quenching is by dynamic quenching, a
straight plot of loll versus concentration of quencher will be obtained and the slope of the
line is the Stem-Volmer quenching constant.

Static quenching is a frequent complicating factor in the analysis of dynamic
quenching. Static quenching can be described by a ground state complex model or a sphere
of action static quenching model. Ground state complex static quenching occurs as a result
of the formation of a nonfluorescent ground state complex between the fluorophore and
quencher. When this complex absorbs light it immediately returns to the ground state
without emission of a photon (11). The sphere of action static quenching model for static
quenching is described as “active sphere” which is the volume around a quencher molecule.
The fluorophore is quenched without the need for a diffusion-controlled collision
interaction if it is in the active interaction volume (12). In many instances the fluorophore
can be quenched both by collisions and also by static quenching. The characteristic feature
of the Stem-Volrner plots in such circumstances is an upward curvature, concave towards

the y axis. In the ground state complex model the modified form of Stem-Volmer law I
(11) can be expressed as

69

 

I
—I°— : (1+ Klel)(1+ Kdel) (5.2a)
I0 _.

{2 1 = K, + Kd + K8Kd[Q] (5-2b)

K, and Kd are the quenching constants of static and dynamic quenching respectively. When
K,>>Kd static quenching is dominant or when Kd>>K, dynamic quenching is dominant,
the Stem-Volmer quenching plot will follow a straight line. When there is no dominant
form of quenching, which means K, is very close to Kd, the Stem-Volmer plot will be
upward, but the plot of (loll-1)/[Q] versus [Q] (equation 5.2b) should be a straight line.
From the slope and intercept we can get static and dynamic quenching constants.

Birks (12) described the active sphere model using the modified Stern-Volmer Law
II

I
fCXIX-VIQD = 1+ KWIQ] (5.3)

K,v is the collision quenching constant, and V is the interaction volume or is called the static
quenching constant which is related to the probability of finding a quencher molecule close
enough to a newly formed excited state. A plot of (Io/I)exp.(V[Q]) versus Q by adjusting V
should result in a straight line.

Downward curvature of the S-V plot was reported by Lakowicz (11), Eftink and
Ghiron (l3), Kikuchi and Sato (14) and by Pan and Cherry (15). Negative deviations
from the Stem-Volmer Law are the results of multiple emitters with different quenching
constants or inefficient quenching (11,13). Pan and Cherry pointed out that the downward
curvature is the characteristic of the particular quenching effect. Also, downward
curvatures can be the results of the heterogeneity of the system or selective quenching,
such as different binding sites in large molecules which make some fluorophores easier to
be quenched than others (16). The negative deviation from the Stem-Volmer law can be

described by modified Stem-Volmer Law III (27).

70

1° = 1 1 +1 (5.4)
Io-I fl("[Q] f

 

f is the fraction of the fluorescence accessible to the quenching.
Different fits of the Stern-Volmer plot indicates the different quenching
mechanism. By interpretation of the different quenching mechanisms we can infer the

interaction of the probe and the solute and the organization of solution.

5 . 3 . Materials and methods

Pyranine, also known as FDA D&C Green No. 8, was obtained from Lancaster,
Windham, NH and used as received. Aqueous solutions of pyranine were prepared by
dissolution of 0.026 g in 5ml HPLC grade water. Different concentrations of solutions
were prepared by dissolving the appropriate amount of solutes in 5ml HPLC water.
Supersaturated solutions were prepared by heating the solution in the water bath until all the
solute was dissolved and subsequent cooling to room temperature. The solutions were
used as prepared without any degassing.

Probe molecules were introduced by addition of five microliters of the pyranine
solution to 5ml aqueous tryptophan and phenylalanine solutions resulting in a probe
concentration of 10’5M and 20 microliters of the pyranine to 5ml nicotinic acid aqueous
solutions resulting in a probe concentration of 4x10‘5M. Fluorescence spectra of the
pyranine in the aqueous solutions were collected within two hours of the preparation the
solutions. All spectra were collected at room temperature with a Spex 1681 Fluorolog
spectrometer equipped with a Xenon lamp source and a PMT detector. A quartz sample
holder was used in all cases. Spectra were recorded at a 1.00mm increment and an
integration time of 1.0 sec. An excitation wavelength of 342nm for pyranine was used.
The emission spectra were recorded over a range from 350-600nm for pyranine.

Monochromator slits were at 0.5 mm.

7 1
Absorption spectra were collected by using a Perkin-Elmer Lambda Array 3840

UV/V is Spectrophotometer. The absorption spectra of pyranine in water were recorded
with HPLC water as the reference and the absorption spectra of pyranine in solution of
tryptophan, phenylalanine and nicotinic acid were taken by using the same concentration of

tryptophan, phenylalanine and nicotinic acid solutions as references.

5 . 4 . Results
5.4.1. Tryptophan quenching of pyranine

The fluorescence spectra of pyranine in aqueous tryptophan solutions (Figure 2.3)
exhibit no differences in shape and maxima in the presence and absence of tryptophan
when the concentration of tryptophan is not very high although there is appreciable
quenching, i.e. reduction in the intensity in the presence of uyptophan. When the
concentration of tryptophan is very high, peaks around 400nm started losing their vibronic
fine structure. No new fluorescence peak is observed at longer wavelengths indicating that
no excimers or exciplex are formed.

The Stem-Volmer quenching curve of pyranine by tryptophan at 382nm is shown
in Figure 5.1 and shown that when the concentration of tryptophan is low, the quenching
response of tryptophan to pyranine is linear. With the increase of the concentration a
maximum is reached and then the quenching plot curves downward.

With the addition of tryptophan in water, the fluorescence intensity at 510nm will
increase compared with the intensity of pyranine in pure water. But with the increase of the
concentration of tryptophan the fluorescence intensity of pyranine will decrease.
Tryptophan quenches the fluorescence of pyranine but we can not fit the data by using a
normal Stem-Volmer law because of the lower of Io than I. We just rearrange it as follows:

1 1 K“
I- 'I—+ —Io—[Q] (5-5)

0

Io/I-l

72

 

 

l A A A A A A A A L l L

 

 

I

0 0: 5 1 1:5
Concentration of tryptophan ( g/100ml H20 )

Figure 5.1. Quenching of pyranine(10'5M) fluorescence at 382nm
by tryptophan in aqueous solutions at room temperature fit to

Io/I-1=KW[Q]. The arrow shows the solubility of tryptophan at 25°C.

73
In this case the intercept will be the reciprocal of initial intensity. The different initial

intensity Io will not change the shape of the quenching curve. The only thing can be
changed by initial intensity is the intercept which will not affect the interpretation of our
results. The quenching of pyranine fluorescence at 510nm by tryptophan in aqueous
solution can be fitted by using equation 5.5 and is shown in Figure 5.2. The upward curve
shows up which tells us the quenching of pyranine by tryptophan at 510nm is coexisting of

static and dynamic quenching.

5 .4.2. Phenylalanine quenching of pyranine fluorescence

The fluorescence spectra of pyranine in phenylalanine aqueous solutions have the
same shape and maximum as in tryptophan aqueous solutions and show no differences in
shape and maxima in the presence and absence of phenylalanine at low phenylalanine
concentration. No new fluorescence peak is observed at longer wavelengths indicating that
no excirrrers or exciplex are formed.

The Stem-Volmer quenching curve of pyranine fluorescence at 382nm by
phenylalanine is shown in Figure 5.3. The quenching curve deviated downward smoothly
with the concentration of phenylalanine. The difference means that the quenching
mechanism of pyranine by phenylalanine and by tryptophan was different. The quenching
data were fitted to the modified Stem-Volmer Law III (Equation 5.4), and a straight line
was obtained with intercept 1.37 and slope 1.30 (Figure 5.4). The intercept is the
reciprocal of the fraction of the fluorescence accessible to the quenching. The fraction of
phenylalanine accessible to pyranine is calculated as 72.8%. This value suggests there are
only 73% of phenylalanine molecules accessible to pyranine and 27% of phenylalanine can
not access pyranine. The quenching constant from the slope is 1.05. If we fit the
quenching curve at 382nm by normal Stern-Volmer Law, the quenching constants of
pyranine by phenylalanine can be obtained from the initial slope of the quenching curve

which is 0.9.

1/1

74

 

1.4 10'5

1.2 10"-

1 10:5—

810%

6 105-

4 10“—

r

I

I

I

I

l l l
I I I

T I fi I V r T T I T fi

Y = 2.37x10‘6 + 2.13x10’°*x +1.69x10'6 *x2

 

l A A

 

2 10'6

 

I

0i5 1 1T5 2
Concentration of tryptophan (g/100ml H20)

Figure 5.2 Quenching of pyranine fluorescence at 510nm by tryptophan
in aqueous solutions at room temperature fit to m=mo+(st/Io)[QJ°

The arrow indicates the solubility of tryptophan at 25°C.

I.”

75

 

2.2

—>—
"P-
—>—
.11—

«i

at
.1
—U—

1.8-— .._

1.6‘“‘ +-

1.4-~ -—

1.2-- __

J A A A A A A A A

11"; A A 1 Ir; A r A I *1 A r r
0

0.5 i {.5 2 2.5

 

 

 

3.5

p

Concentration of phenylalanine (g/100ml H20)

Figure 5.3 Quenching of pyranine fluorescence ( 10-5 M) at 382nm
by phenylalanine in aqueouse solutions at room temperature. The arrow
shows the solubility of phenylalanine. The curve is fit to 10/1- 1=KW[Q].

(I -I)

I/

76

l l l
T V I I

 

3.5 “r

2.5 ‘—

 

r 1 l T V

y = 1.3664 + 1.7372x R: 0.99914

 

A A A A A l A A A A A A

 

 

 

0:5 i 1i5 2
The reciprocal of concentration of phenylalanine (100ml HZO/g)
Figure 5.4. Quenching of pyranine fluorescence at 382nm

by phenylalanine in aqueous solutions at room temperature
fit to Io/(Io-I)=(1/[Q])(1/stv)+1/f .

77
5.4.3. Nicotinic acid quenching of pyranine fluorescence

The spectra of pyranine in nicotinic acid aqueous solutions have the same shape and
maximum as in aqueous tryptophan solutions and show no differences in shape and
maxima in the presence and absence of nicotinic acid although there is strong quenching in
the presence of nicotinic acid. With the addition of nicotinic acid into pure water, the
fluorescence intensity of pyranine decreased dramatically. The spectra were not excitation
wavelength dependence. No new fluorescence peak is observed at longer wavelengths
indicating that no excimers or exciplex are formed.

The Stern-Volmer quenching plot of pyranine fluorescence at 328nm by nicotinic
acid curved upward (Figure 5.5) which was the evidence of coexistence of dynamic
quenching and static quenching. The initial slope of Stem-Volmer plot was 11.6-10.8.

Fitting the quenching data to the modified Stem-Volmer law I (equation 5.2) yields
the plot of (loll-1)/[Q] versus [Q] shown in Figure 5.6. The quenching curve is a straight
line at lower concentrations. Quenching constants obtained by using the straight part of the
plot are 8.6109 and 1.6i0.3. At high concentrations, the plot curves downward.

The quenching data were fitted to a modified Stem-Volmer law 11 (Io/Dexp(-V[Q])
versus [Q] (Figure 5.7). When the concentration of nicotinic acid was low we obtain a
straight line with the slope 14.5 :08, the dynamic quenching constant and V=0.45, the
static quenching constant.

The quenching plot of pyranine fluorescence at 510nm shows upward curvature
(Figure 5.8). From the curve we can see two sets of data at different excitation
wavelengths 342nm and 360nm overlapped each other which tells us the excitation
wavelength does not affect the quenching behavior at 510nm.

Fitting data to the modified Stem-Volmer law I (Equation 5.2b) we can obtain plots
which would still curve a little bit upward for both excitation wavelengths. Fitting data to

the modified Stem-Volmer law 11 (Equation 5 3), we can see when the concentration of

Io/Il

78

 

80

40"

20-

10'

70*

50~

30~

I
I
I

I
l
I

I
l
I

I
A I
I

A A A A

 

 

l A J

 

6 035 i 1T5 2 2.5

Concentration of nicotinic acid (g/100ml H20)

Figure 5.5 Quenching of pyranine fluorescence at 382nm by nicotinic

acid in aqueous solutions at room temperature fit to Iofl'1=st[Q]'

The arrow indicates the solubility of nicotinic acid at 25°C.

79

 

 

 

 

1 A 1 l A
40-- -—
O
'i
35 " -_
b
30 “ __
Q
A
H
I
:3
25 -_ ~-
.
t
20* -r
t '1
.- 4
i
)-
10 A A A A ll A A A A % A A A A J]; A A A A % A AA. A A. % A A. A A
0 0.5 1 1.5 2 2.5 3

Concentration of nicotinic acid(g/ 100 ml H20)

Figure 5.6. Quenching of pyranine fluorescence at 382nm by nicotinic acid
in aqueous solutions at room temperature fit to (Io/I-l)/[Q]=K1+K2[Q].

Arrow indicates the solubility of nicotinic acid at 25°C.

(Io/I)exp(-0.45*[Q])

30

80

 

 

 

 

 

 

0'5 '1 1‘.5 '2 2.5
Concentration of nicotinic acid(g/ 100ml H20)
Figure 5.7 Quenching of pyranine fluorescence at 382nm by

nicotinic acid in aqueous solutions at room temperature

fit to (lo/I)exp(—V[Q])=1+K,,[Ql

Arrow indicates the solubility of nicotinic acid at 25°C.

10/11

81

 

 

 

 

 

15()....§....JI 4, ,1,,,
—e— 360nm Excitation i
i D 342nm Exciation
r i
lOO-F d~
50* T
.- 4
a
O I I l I I I I % I I I L I I I I I 1 I I I I
0 0.5 l 1.5 2 2.5

Concentration of nicotinic acid(g/ 100ml H20)

Figure 5.8 Quenching of pyranine fluorescence at 510nm by nicotinic acid
at room temperature at different excitation wavelengths of 342nm and 360nm
fit to Io/I-1=K“[Q]. Arrow shows solubility of nicotinic acid.

82
nicotinic acid is low the quenching curves are straight lines with the slope 7.9 i0.2, the

dynamic quenching constant, and V=0.9, the static quenching constant. When
the concentration is high the plots curve downward. Quenching curves showed different

behavior of unsaturated and supersaturated solutions.

5.4.4. The absorption spectra of pyranine in water and in solutions

The absorption spectra shown in Figure 5.9 showed that low wavelength bands
236nm and 246nm were missing by introducing tryptophan, nicotinic acid and
phenylalanine and the broad band around 454nm was missing in the presence of
tryptophan, phenylalanine and nicotinic acid too. Absorption bands at 282nm and 291nm

were missing with the addition of tryptophan and nicotinic acid into solution.

5 . 5 . Discussion

The structures of tryptophan, nicotinic acid and phenylalanine are shown in Figure
5.10 . Tryptophan and phenylalanine are different only in the ring, one is benzene and the
other is the indole ring which has a nitrogen heteroatom in a five member ring. Nicotinic
acid is a six member ring with one nitrogen heteroatom in the ring. Both nicotinic acid and
tryptophan have strong quenching ability for pyranine, which implies that the ring nitrogen
contributed to the quenching of pyranine or a ground and/or excited state complex form
through nitrogen in the ring.

The formation of a transient excited state complex or exciplex as an intermediate in
the fluorescence quenching process was proposed by many workers (17-26). Such as
exciplex can be emissive which will present broad and structureless emission spectra at
longer wavelength (18). In many cases there were non-emissive exciplexes as involved in
the excited state quenching. An exciplex is defined as a complex of simple integer
stoichiometry formed between an excited molecule and one or more non-excited

molecules(19). The formation of an exciplex is the result of intermolecular forces or

83

 

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84

N
0
/
COOH

Nicotinic acid (NA) (1.67g/100ml P120)

N/H
@
CHZCHCOOH

NH2
Tryptophan (Trp) (1.14g/100ml H20)

/NH2

CH2— CH

e w

Phenylalanine (Phe) (2.96g/100ml H20)

Figure 5.10 The molecular structures of nicotinic acid, tryptophan and
phenylalanine. Solubility 1n water at 25 °C 18 given in parentheses

85
Van der Waals interactions which have an electromagnetic origin and arise in a variety of

ways such as dipole—dipole, polarizability-dipole, polarizability-polarizability and
dispersion interactions. Hydrogen bonding interaction is often cited as an efficient
quenching mechanism by charge transfer or electron transfer via the hydrogen bond or by
proton transfer (20). The orbital overlap interactions play an important role in the correct
exciplex geometry and also help to stabilize the exciplex but they can not become a
dominant factor for the formation of exciplex (19).

Pyridine which has the same structure as nicotinic acid except the carboxyl group in
nicotinic acid was reported to be a strong fluorescence quencher to many fluorophores
such as l-pyrenol, 2-naphthol, l-aminopyrene and 13H— and 7H.dibenzocarbazole which
was suggested as hydrogen bonding interaction between pyranine and fluorophore in the
hydrogen bonded complex (20).

The strong quenching ability of nicotinic acid and tryptophan for pyranine comes
from the hydrogen bonding with pyranine. Amines are shown to be very efficient
quenchers of aromatic molecules because they are organic elecu'on donors (21). Amines
can form a hydrogen bonding complex with pyranine in the ground state. But the
fluorophore-quencher interaction did not change the absorption and fluorescence spectral
properties, because the ground-state complex formed between pyranine and tryptophan or
nicotinic acid were not sufficiently stable owing to weak interaction and the exciplexes are
non emissive species. We are able to tell there are ground state complexes because the
absorption spectra bands shown in Figure 5.7 show a small red shift for u'yptophan, and a
larger red shift for nicotinic acid, which tell us the stabilization of the excited state will be
improved due to the hydrogen bonding interaction. With the addition of nicotinic acid and
tryptophan all peaks at lower wavelengths disappear. Those lower peaks can be assigned
to some structure of pyranine with water molecules. The absence of those peaks is due to
the formation of a complex of pyranine with nicotinic acid and tryptophan which will

destroy the structure of pyranine with water. At our experimental conditions (pH~7 and

86
low concentration of acid) pyranine (pK.=7.5) will exist as a protonated species. This

protonated species will form a hydrogen bonding complex with tryptophan and nicotinic
acid. When pyranine is excited, the proton transfer will take place between pyranine and
amine due to the low pK. (0.5) in the excited state. The excited energy will be lost by
proton transfer then will quench the fluorescence of pyranine. The mechanism of
quenching, including both the ground state complex and exciplex, is summarized in the
scheme shown in Figure 5.11.

The Stem-Volmer plot of tryptophan-pyranine is a straight line when the
concentration of tryptophan is low. With the increase of the concentration of tryptophan
the Stern-Volmer plot curves downward Negative deviations from the Stem-Volmer Law
are the results of multiple emitters with different quenching constants or inefficient
quenching (11,13) or many possible quenching mechanisms existing at the same time.
Kikuchi and Sato (14) showed in their paper that the quenching curve of oxygen sometimes
would deviate downward from a straight line when the concentration of oxygen was high
which was reported as the evidence for exciplex formation. Negative curvatures of the
plots in the pyranine-tryptophan system is the result of the two or three dimensional
structure among tryptophan molecules which can be proved by the excitation dependence of
tryptophan fluorescence (see chapter 7 for details). In pH range 4-8 the predominate
species of tryptophan is the zwitterion +l-IaNRCOO‘ , so the Coulombic interaction and/or
hydrogen bonding between two or more zwitterions can form chain structures or a three

dimensional structure such as

+H3NRcoo- ---------- +1.13NRCOQ' ---------- +l'i3NRCOO"

+H3NRcoo- ---------- +113NRcoo- ---------- +H3NRcoo-

We refer these two or three dimensional structure as associates.
The effective concentration of quenchers will not be directly proportional to true

concentrations because the molecules in the center of the associates could hardly contact

87

 

 

 

_ 1 ,,,
9 Light :
8999 > g
-0 O— H _____ O'- "' ' " 'H— N
Ground 3011:078th N\Exc:tcd state complex
N| NH3 I
(31-12:... (in .....C00 COOH
H

Tryptophan Nicotinic acid

Figure 5.11 Scheme of quenching mechcnism of pyranine
by amine in tryptophan and nicotinic acid ring
through hydrogen bonding complex at ground and excited state

88
with pyranine, making the quenching by dynamic encounter inefficient. Quenching by

diffusion was also inhibited due to the larger size of associates. The Stem-Volmer plot
should curve upward with the coexistence of static quenching and dynamic quenching.

The coexistence of dynamic quenching and static quenching appear in the pyranine-
tryptophan system which can be seen from the quenching plot of fluorescence at 510nm
shown in Figure 5.2. The associates causes the downward curvature and coexistence of
dynamic and static quenching causes an upward curve. These two effects cancel each other
to result in a straight quenching plot of pyranine fluorescence at 382nm at low
concenuation of tryptophan. We notice the difference of quenching curves between 510nm
and 382nm. The emission at 510nm is from the deprotonated form PyO'. The hydrogen
bonding occurs at the hydroxyl group, so the hydrogen bonding complex will affect the
emission of PyO' much more. The emission at 382nm is the result of general it <—> 1t *
polycyclic aromatic hydrocarbon (PAH) transmission. The hydrogen bonding complex
will not affect the 382nm emission as much as the 510nm emission resulting in more static
quenching effects in the quenching of pyranine at 510nm. Also the hydrogen bonding
between tryptophan and pyranine is not as strong as the bonding between pyranine and
nicotinic acid, which explains why the quenching plots show the similar upward curves at
382nm and 510nm in nicotinic acid solutions.

When the concentration of tryptophan is high, a different situation exists.
Aggregates will form in the solution at high concentration of tryptophan. As we know at
high concentration in high solubility systems, the observation of a concentration gradient
was taken as the evidence of the formation of aggregates in solution (6). In low solubility
systems, the concentration gradient will not be noticeable. The much lower quenching
ability occurs at very high concentration which implies the formation of aggregates in a low
solubility system such as tryptophan solutions. The aggregates will have much less ability
to quench fluorescence of pyranine due to their relatively large size which reduces

diffusion. Some of the molecules in the middle of aggregates can not contact with pyranine

89
which is the requirement for the quenching, therefore, quenching plots will curve

downward.

The similar associates should appear in the phenylalanine solutions. But
phenylalanine does not have a cyclic amine in its structure, so there is no hydrogen bonding
complex between pyranine and phenylalanine. It is reasonable for us to infer the formation
of a contact complex between pyranine and phenylalanine because they both have benzene
rings. Phenylalanine will like pyranine better than phenylalanine likes water. The missing
of two lower peaks in adsorption spectra of pyranine with the addition of phenylalanine
shows that phenylalanine destroys the structure of pyranine with water. The interaction is
not as men g as tryptophan so only two peaks disappear. The contact complex between
pyranine and phenylalanine is not strong enough to quench fluorescence of pyranine
effectively. Quenching requires the contact of fluorophores and quenchers. Some
molecules in the middle of associates can not contact with pyranine even they can diffuse to
pyranine during lifetime. The data fit to the modified Stem-Volmer law III (Equation 5.4)
shown in Figure 5.4 shows only 73% of phenylalanine were accessible to pyranine. These
27% of phenylalanine that could not reach pyranine may be in the middle of associates or
may be too big to effectively diffuse to pyranine.

The higher concentration of tryptophan and phenylalanine, the more the associates
present, and the more downward the quenching plot would curve. When the concentration
was very high almost all molecules are involvec in the formation of associates or aggregates
which makes the quenching much more inefficient and the curve would go down instead of
curving down.

The higher quenching ability of nicotinic acid to pyranine is because nicotinic acid
can not form associates among molecules. When the concentration of nicotinic acid was
low all the single molecules were good quenchers. Some of them which were far from
fluorophores would diffuse to pyranine, meet pyranine and quench fluorescence. Other

molecules would form a hydrogen bonding complex with pyranine and they would quench

9O
pyranine by proton transfer from excited pyranine to nicotinic acid. Both dynamic

quenching and static quenching exist at the same time which result in the upward Stem-
Volmer plot. At the 510nm emission fitting to the modified Stern-Volmer I (equation 5.2)
still results in an upward curve. So we can tell how strong the static quenching is. The red
shift of adsorption spectra of pyranine in nicotinic acid solutions provides the evidence of
the formation of ground state complex between pyranine and nicotinic acid. The ground
state complex model can better explain the quenching behavior of pyranine fluorescence by
nicotinic acid also.

When the concentration of nicotinic acid was high the quenching was not as
effective as for lower concentrations. Thus, the quenching curve would not follow the
same trend. We can interpret this result as aggregate formation which makes the diffusion
of molecules more difficult, then the dynamic quenching was restrained. Also the
formation of aggregates inhibited the formation of the complex between pyranine and
nicotinic acid, so the static quenching was restrained too.

The higher quenching ability of nicotinic acid can be because it does not have a
benzene ring beside the amine, so less spatial hindrance occurs for the formation of the
complex between pyranine and nicotinic acid as compared to tryptophan. The complex
between pyranine and nicotinic acid is more stable than the complex between pyranine and
tryptophan. The proof comes from the absorption spectra which show a larger red shift in
nicotinic acid solutions as compared to the red shift in tryptophan solutions.

In all three systems the discontinuity occurs in the quenching plots at high
concentration. All quenching curves will curve downward at high concentration of solutes.
We can interpret this result as the different structure of supersaturated solutions and
unsaturated solutions. The structure of supersaturation solutions is always an interesting
topic since the supersaturation is the starting point for crystallization. But in very few cases
the discontinuity of physical properties on concentration have been observed In our

quenching study, the quenching plot changes at high concentration in all cases. The change

91
of quenching shows the different quenching behavior of low and high concentration.

High concentration solutions show weaker quenching ability. This phenomenon is taken
as a evidence of the formation of aggregates at high concentration. So we can conclude
aggregates will form in the low solubility system as in the high solubility system at high
concentration. Especially in tryptophan solutions, the zwitterion form will help the
formation of aggregates, so the change of the quenching plots is really dramatic. At high
concentration the plot will go down with the increase of concentration (Figure 1). In
nicotinic acid the quenching plot will just curve downward at high concentration to indicate
the formation of aggregates.

The fluorescence quenching technique is a very easy and simple method.
However, the quenching studies can be performed with a great degree of precision and get

very important information about the organization of solutes.

5 . 6 . Conclusion

Fluorescence quenching is a useful tool for studying solute behavior. Different
quenching behaviors of tryptophan, phenylalanine and nicotinic acid to pyranine indicate
the differences of the host-probe interaction. The differences come from different structures
of tryptophan, phenylalanine and nicotinic acid. Nicotinic acid and tryptophan will form a
hydrogen bonding complex with pyranine for both ground and excited state due to the
electron deficiency of amine. Aggregates are pmposed in the supersaturated solution which

are the reason for the discontinuity of the quenching curves.

5 . 7 . References
1. H. G. Marcelo, and F. C. De Schryver, Chem. Rev., 93, 199(1993)
2. F. S. Abrams, and E. London, Biochemistry, 31, 5312(1992)
3. K. Kachel, E. Asuncion-Punzalan, and E. London,
Biochemistry, 34, 154750995)

10.

11.

12.

13.
14.

15.
16.
17.
18.

19.
20.

21.
22.

92
C. Peviani, W. Hillen, N. Ettner, H. Lami, S. M. Doglia, E. Piemont, C. Ellouze,

and M. Chabbert, Biochemistry, 34, 130070995)

M. R. Eftink, and C. A. Ghiron, Biochemistry, 15(3), 672-680(1976)

J. W. Mullin, and C. L. Leci, Phil. Mag., 19(1969)

E. L. Cussler, AIChE J. 26(1), 43 (1980)

L. S. Sorrel, and A. S. Myerson, AIChE J., 28(5), 772(1982)

G. A. Hussman, M. A. Larson, and K. A. Berglund, Industrial Crystallization ‘84
S. J. Jancic, and E. J. DeJong, Eds. Elsevier, Amstrdam (1984)

P. M. McMahon, K. A. Berglund, and M. A. Larson, Industrial Crystallization
‘84, Jancic, S. J. and DeJong, E. J. Eds. Elsevier, Amstrdam (1984)

J. R. Lakowicz, Principles of Fluorescence Spectroscopy, Plenum Press,
New York and London(1983)

J. B. Birks, Photophysics of Aromatic Molecules, New York,
Wiley-Interscience(1970)

M. R. Eftink and C. A. Ghiron, Anal. Biochem. 114, 199(1981)

K. Kikuchi, C. Sato, M. Watabe, H. Ikeda, Y. Takahashi and T. Miyashi,

J. Am. Chem. Soc.., 115, 5180(1993)

Rui-jun Pan and Richard J. Cherry, Biochemistry, 34, 4880(1995)

M. R. Eftink and C. A. Ghiron, Biochemistry, 15(3), 672(1976)

D. Goswarrri, R. S. Sarpal and S. K. Dogra, Bull. Chem. Jpn., 64, 3137(1991)
H. Pal, D. K. Palit, T. Mukherjee and J. P. Mittal, Chem. Phys. Letts.

173, 354(1990)

Photochem. Photobio, 27, 819(1978)

H. Miyasaka, A. Tabata, S. Ojima, N. Ikeda and N. Mataga, J. Phys. Chem.
97, 8222(1993)

M. Mac, J. Najbar and J. Wirz, Chem. Phys. Letts., 235, 187(1995)

A. Sharma, 0. S. Wolfbels and M. K. Machwe, Anal. Chim. Acta,

23.
24.
25.

26.

27

93
230, 213(1990)

St. L. Logunov and M. A. Rodgers, J. Phys. Chem, 96, 2915(1992)

U. Graf, H. Niikura, and S. Hirayama, J. Phys. Chem. A, 101, 1292(1997)
E. M. Talavera, B. Quintero and J. M. Alvarez,

J. photochem. Photobiol. A: Chem., 66, 171(1992)

H. Pal, D. K. Palit, T. Mukherjee and J. P. Mittal,

J. Chem. Soc. Faraday Trans., 89(4), 683(1993)

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J. of Luminescence, 46, 291-299(1990)

Chapter 6

QUENCHING STUDY BY MEASUREMENT OF LIFETIME OF
PYRANINE IN DIFFERENT SOLUTIONS

6 . 1 Introduction

Time-resolved fluorescence is a powerful optical spectroscopic technique for
monitoring the dynamics of macromolecular interactions in solution and a useful tool to
study the interactions of solute and solvent (1-4). A wealth of information about the
organization and dynamics of solvent—solute interactions in the microenviomment of a
probe molecule can be obtained by studying the relaxation processes on a variety of time
scales. Time-resolved techniques are able to directly separate and quantify the relevant
fluorescence parameters that contribute to the steady-state emission parameters. The
emission lifetime of a probe molecule is highly sensitive to the solvation. The measurement
of lifetime can determine static and dynamic conformational information about
macromolecules, make state assignments, and provide information on the solvent polarity
as well as diffusive processes such as collision quenching and proton transport. The use of
time resolved fluorescence methods to investigate the microscopic interactions between the
solvent and solute and the crystallization behavior should result in a more directed and
rational approach to understand and control the crystallization.

Frequently, materials having very similar spectra can have greatly different lifetimes
which can be used to identify different materials. The lifetime is defined as the time

required for the population of the excited state to decrease to 1/e of its original value after

94

95
the excitation source is turned off. Lifetime measurements have become an indispensable

tool in the physical and biological sciences (4).

Fluorescence quenching is another very useful method used to study both physical
and biochemical phenomena (5-9). Fluorescence quenching experiments can yield valuable
information about fluorophore and quencher interactions. The quenching from diffusion-
controlled encounters is called dynamic quenching. Dynamic quenching is very common in
the bimolecular systems. Fluorescence of fluorophore can be quenched by collisional
processes between fluorophore and quencher. The dynamic quenching can be expressed as
Stem-Volmer Law

10/1-1=K..[Q] (6.1)

Static quenching is a frequent complicating factor in the analysis of dynamic
quenching. When the fluorophore and quencher are close enough, the fluorescence can be
quenched immediately on excitation. Static quenching decreases the emission intensity but
has no effect on the lifetime of the unassociated excited fluorophores. In order to
distinguish dynamic quenching and static quenching we use the lifetime form of the Stem-

Volmer Law to describe dynamic quenching
130/13-1=KW[Q] (6.2)
“I:0 is the lifetime of fluorophore without quencher, and “t is the lifetime with quencher.

The two plots should be indistinguishable if the simple diffusional quenching model

holds. The discrepancies between the I and 1: Stem-Volmer plots can supply useful

chemical information. There are, however, times when only 1: data can be used

conveniently. These include situations in which the quencher absorbs intensely in the
region of excitation and/or emission. Under these conditions, trivial absorption can
produce unacceptable distortions of intensity quenching data unless complicated, and

frequently unreliable, corrections are applied.

96
The coexistence of static quenching and dynamic quenching will result in upward,

nonlinear Stem-Volmer plots. It can be described as

 

I
0 —1
/IQ = K, + Kd + K,Kd [Q] (6.3)

Where KI and Kd are the static and dynamic quenching constants respectively.

Static quenching does not affect the lifetime Stem-Volmer plot Therefore from lifetime
quenching data we can get the dynamic quenching constant, then the static quenching
constant can be obtained readily.

Chapter 5 focused on steady state quenching of pyranine fluorescence by solutes in
aqueous solutions. In this chapter more attention will be given to the dynamic quenching
study by lifetime measurements with pyranine as probe in solutions of tryptophan,
phenylalanine and nicotinic acid to investigate the interaction between the host system and
probe. Some steady state fluorescence quenching will be shown as a comparison to the

time resolved results.

6 . 2 Materials and methods

Pyranine, also known as FDA D&C Green No. 8, was obtained from Lancaster,
Windham, NH and used as received. Aqueous solutions of pyranine were prepared by
dissolution of 0.026 g in 5ml HPLC grade water. Different concentration of solutions
were prepared by dissolving the appropriate amount of solutes in 5ml HPLC water.
Supersaturated solutions were prepared by heating the solution in the water bath until all the
solute was dissolved and subsequent cooling to room temperature. The solutions were
used as prepared without any degassing.

Probe molecules were introduced by addition of five microliters of the pyranine
solution to 5ml aqueous tryptophan and phenylalanine solutions resulting in a probe
concentration of lO'SM and 20 microliters of the pyranine to 5ml nicotinic acid aqueous

solutions resulting in a probe concentration of 4x10’5M for the lifetime measurement at

97
510nm. For the lifetime measurement at 382nm, 25 microliters of pyranine solutions were

added into tryptophan solutions and phenylalanine solutions and 150 microliters of
pyranine were added into nicotinic acid solutions to compensate for the weaker
fluorescence at 382nm. Lifetime measurements of pyranine in the aqueous solutions were
collected within several hours of the preparation the solutions. All lifetimes were measured
by using the single photon counting time correlated method with PMT as the detector and a
YAG laser with LDS 698 dye as excitation light source. The excitation wavelength was
342nm and emission wavelengths were 382nm and 510nm. The other conditions were

high voltage 3200v, TAC range 100ns, power 120mw/cavity dump.

6 . 3 Results and Discussion
6.3.1. Fluorescence lifetime quenching of pyranine in different solutions at 510nm

The decay curve of pyranine is fit to a first order kinetic processes. The lifetimes of
pyranine in different concentrations of tryptophan, phenylalanine and nicotinic acid were
measured by using the single photon counting technique. The results show that the
fluorescence lifetime of pyranine in tryptophan solutions will decrease with the increase in
the concentration of tryptophan. The same decrease of lifetime was observed in nicotinic
acid solutions. Conversely, the lifetime of pyranine in phenylalanine solutions increased
with increased concentrations of phenylalanine. The decrease of the lifetime with the
increase of concentrations is due to the fluorescence quenching of pyranine by tryptophan
and nicotinic acid. The increase of lifetime of pyranine with the increase of concentration
of phenylalanine is due to the protection of pyranine from quenching by water. Water
quenching fluorescence by proton transfer has been proven by the isotope effect (10,11).

Lifetime Stem-Volmer quenching curves are shown in Figures 6.1 and 6.2 for
tryptophan and nicotinic acid, respectively. The quenching of pyranine by phenylalanine at
510nm for steady state and lifetime Stem-Volmer Law are shown in Figure 6.3. In

tryptophan solutions the plot is linear at lower concentration and then curved downward at

 

- m ___.__ _ .

98

 

1,7....:.-..:...-t.-f.

TO/T

 

 

 

 

0.9 4 1 1 1 I 1 1 1 1 l 1 1 1 1 l 1 1 1 1
0 0.5 1 1.5 2

Concentration of tryptophan(g/100ml H20)

Figure 6.1 Lifetime Stem-Volmer quenching of pyranine fluorescence
at 510nm by tryptophan in aqueous solutions at room temperature.

The arrow indicates the solubility of tryptophan at 25°C.

to/t

 

99

 

y = 3.3554X + 1 o
R2 = 0.995

 

 

 

0 0.5 1 1.5
Concentration of nicotinic acid(g/ 100ml H20)
Figure 6.2 Lifetime Stern-Volmer quenching of pyranine fluorescence at

510nm by nicotinic acid in aqueouse solutions at room temperature.
The equation shown is linear fitting.

 

100

 

   

Lifetime

 

 

fib—

0.95 1 1 1 1_% 1 1 1 1 1 1 l 1 1 1 l 1 1
0 0.5 1 1.5 2 2.5 3
Concentration of phenylalanine (g/ 100ml H20»

Figure 6.3 Lifetime and steady state quenching of
pyranine fluorescence at 510nm by phenylalanine in

3.5

 

aqueous solutions at room temperature fit to Stem—Volmer Law.

101
higher concentrations. In nicotinic acid the plot is linear. In phenylalanine solutions, the

lifetime plot goes downward with the increase in concentration of phenylalanine which is
seldom observed and the steady state plot is going up with the increase of the
concentration.

The linear Stern-Volmer plot for nicotinic acid shows that the lifetime Stem-Volmer
law holds for nicotinic acid. The slope is the dynamic quenching constant which is 3.4
(100g HZO/g nicotinic acid). At low concentration of tryptophan, the dynamic quenching
Stern-Volmer law holds. As previously discussed, tryptophan tends to form associates
among themselves. With the increase of concenu'ation, the population of associates will
increase. At very high concentration, aggregates will form in the solution. Those
associates and aggregates can not quench fluorescence of pyranine efficiently due to the
larger size and also the lack of contact of molecules in the middle of associates, thus the
lifetime decrease will not follow the same trend. The plot curves downward. In
phenylalanine solutions, the lifetime quenching plot goes down with the concentration of
phenylalanine which can be interpreted that phenylalanine will not quench the fluorescence
of pyranine at 510nm dynamically. Steady state phenylalanine shows quenching behavior
to pyranine with an increasing quenching curve. We can conclude phenylalanine will
quench pyranine fluorescence statically but not dynamically. Alternatively we can say
phenylalanine is poorer quencher as compared to H20 because water can form a hydrogen
bonding complex with pyranine which was shown a very effective quenching mechanism.
When phenylalanine is added to water, a contact complex will form between pyranine and
phenylalanine. This contact complex will protect pyranine from quenching by water. This
contact complex will quench fluorescence of pyranine statically by formation of an

exciplex.

102
6.3.2. Fluorescence lifetime quenching of pyranine at 382nm in different solutions

The fluorescence decay of pyranine at 382nm is a first order kinetic process with
life time around 4ns. The fluorescence lifetime of pyranine at 382nm was measured in
tryptophan, phenylalanine and nicotinic acid at different concentrations. The results show
that lifetime decreases with the increase of the concentration of solutions. In tryptophan
and nicotinic acid solutions the increase is greater. The decrease in the lifetime of pyranine
with the increase of concentration of solutes results from the fluorescence quenching of
pyranine by the solutes.

The quenching plots of pyranine in tryptophan solutions for steady state and time
resolved measurements are shown in Figure 6.4. The lifetime quenching plot is linear at
low concentration of tryptophan and curves downward at higher concentration which is
taken as the evidence of the formation of aggregates at the higher concenuations. From the
linear part, the dynamic quenching constant is shown to be 4.3 (100g HzO/g tryptophan).
The steady state quenching curve is distinguishable from the lifetime quenching curve,
which tells us the steady state quenching at low concentration is not just dynamic
quenching although it is straight line. As discussed in chapter 5, a straight line at low
concentration of u'yptophan is result of the double functions of static quenching and
associates of tryptophan in solutions. The steady state quenching curve has a larger slope
which means higher quenching constants. The higher quenching constant is dynamic
quenching plus static quenching.

The quenching plots of pyranine in nicotinic acid for steady state and time resolved
measurements are shown in Figure 6.5. Because nicotinic acid has a strong quenching
capability for pyranine, the peak intensity is low which makes the measurement of the
lifetime difficult. We can measure some lifetimes by increasing the concentration of
pyranine. The concentration of pyranine can not be too high in the solution because of the
sclfquenching of pyranine. Therefore, the fluorescence lifetime of pyranine can be

measm'ed only at low concentrations of nicotinic acid. The lifetime quenching plot shows

103

 

0' ' 0 0 Steady state.
s Q .

d

  
   

Steady state

10/1-1

ro/r—l

Lifetime

 

L L A l

O .f 1 . 1 1 1 1 I 1 1 1 1 1 1 1
0 0.5 l 1.5 2
Concentration of tryptophan (g/ 100ml H20)

A J

 

Figure 6.4 Lifetime and steady state quenching of pyranine fluorescence
at 382nm by tryptophan in aqueous solutions at room temperature

fit to Stern-Volmer Law. The arrow shows the solubility of tryptophan at 25°C.

104

 

 

 

 

30 . . . l . . . l l l . . i
r-
. 1
25* *
1 Steady stat
20* r-
l i
1. O
15* *
l . 1
l.
10* *
L 1
5* *
i 1
i , a lifetime <
0.: v1 r;1 1 1 1 1 1 1 1 1 4 1 i 1 1
0 0.2 0.4 0.6 0.8 1.2

1
Concentration of nicotinic acid (g/ 100ml H20)

Figure 6.5 Lifetime and steady state quenching of pyranine fluorescence
at 382nm by nicotinic acid in aqueous solutions at room temperature fit to
Stern-Volmer law. The arrow shows solubility of nicotinic acid.

 

 

 

 

105
linear relationship with the concentration of nicotinic acid with the dynamic quenching

constant 4.1 (100g HZO/g nicotinic acid). Steady state quenching curves upward which
shows the coexistence of dynamic quenching and static quenching.

The lifetime quenching plot of pyranine in phenylalanine solutions exhibits an
uncommon shape (Figure 6.6). As previously discussed for quenching at 510nm,
phenylalanine will only quench pyranine fluorescence statically by formation of a contact
complex. The formation of a contact complex can protect pyranine from quenching by
water. When the concentration of phenylalanine is not high, the protection is not quite
complete. Water will quench the fluorescence of pyranine dynamically which will decrease
the lifetime of pyranine. When the concentration of phenylalanine is higher, the quenching
from water is stopped. The protection of quenching from water is much more effective at
510nm which the lifetime increase with the concentration of phenylalanine all the time. The
reason is that the quenching of pyranine from water is by hydrogen transfer through a
hydrogen bonding complex. The quenching of PyO' at 510nm will be more effective than

quenching at 382nm which is the emission of the PAH structure.

6.4 Conclusion

Time resolved fluorescence decay can help us understand steady state quenching
better. Lifetime Stem-Volmer quenching plots of pyranine by tryptophan curve downward
suggesting the formation of associates and aggregates at higher concentrations of
tryptophan. Phenylalanine does not quench the fluorescence of pyranine dynamically at
510nm due to the increase of the lifetime with the increase of the phenylalanine
concentration. Steady state studies show that phenylalanine will quench the fluorescence of
pyranine. This behavior can be interpreted as the evidence of the formation of contact
complex between phenylalanine and pyranine and this complex will protect the quenching

from water by proton transfer through the hydrogen bond. Nicotinic acid will quench

106

 

In

steady state 0

’1

 

 

 

 

 

 

1.6*
L’
PO A
O
1.4*
1 O
1 . 1.x— 1‘ A 0 4‘6 lifetime
. 0'; o .
1.2+o
/
I
o
11411111111111111111111111¥1111
1 I l I l I l
O 0.5 1 1.5 2 2.5 3

Concentration of phenylalanine (g/lOOml H20)

Figure 6.6 Lifetime and steady state quenching of pyranine
fluorescence at 382nm by phenylalanine in aqueous solutions
at room temperature fit to Stem-Volmer law

107

fluorescence of pyranine strongly dynamically and statically due to the small size, non-

associates and strong hydrogen bonding with pyranine.

6.5
1.

10.

11.

References

H. Kondo, I. Miwa, and J. Dunamoto, J. Phys. Chem, 86, 4826-310982)
E. Bardez, B. Goguillon, E. Keh, and B. Valeur, J. Phys. Chem, 88,
1909(1984)

G. D. Correll, R. N. Cheeser III, F. Mome, and J.H. Fendler,

J. Am. Chem. Soc., 100:4, 1254—12620978)

J. N. Demas, Excited State Lifetime Measurements, Academic Press,

New York (1983)

H. G. Marcelo, and F. C. De Schryver, Chem. Rev., 93, 199(1993)

F. S. Abrams, and E. London, Biochemistry, 31, 5312(1992)

K. Kachel, E. Asuncion-Punzalan, and E. London, Biochemistry,

34, 154750995)

C. Peviani, W. Hillen, N. Ettner, H. Lami, S. M. Doglia, E. Piemont, C. Ellouze,
and M. Chabbert, Biochemistry, 34, 130070995)

M. R. Eftink, and C. A. Ghiron, Biochemistry, 15(3), 672-6800976)

L. P. McMahon, W. J. Colucci, M. L. McLaughlin and M. D. Barkley
J. Am. Chem. Soc., 114, 8442—84480992)

Yu Chen, Bo Liu, Hon g-Tao Yu and Mary D. Barkley,
J. Am. Chem. Soc., 118, 9271-9278( 1996)

Chapter 7

THE DEPENDENCE OF EXCITATION WAVELENGTH OF TRYPTOPHAN
FLUORESCENCE IN TRYPTOPHAN AQUEOUS SOLUTION

7 . 1 Introduction

The fluorescence of tryptophan has been investigated extensively due to the
importance of the photophysics of tryptOphan and the use of its fluorescence in the study of
the structure and function of proteins and enzymes0-6). Tryptophan is a very important
intrinsic probe for the study of proteins and enzymes. The quenching study of tryptophan
in proteins can provide us information about the folding of proteins and many other
important information.

It had been reported that tryptophan fluorescence is very sensitive to environment.
The emission maximum of uyptophan in protein changes in response to the different
polarity and also maybe rigidity of the surrounding matrix from 308nm to 360nm (4).
There are two excited states, 1L. and 1Lu, for indole ring which have different dipoles
where 1L. is more polar than the ‘11,, state (2,4). The fluorescence decay of tryptophan
shows a double exponential decay at pH values where the zwitterion predominates with
emission maxima at 350nm and 335nm. The two species were assigned to the non-
interconverting relaxed 1L. and 1Lb states (2). Szobo and Rayner interpreted the dual
exponential decay of tryptophan fluorescence in terms of the presence of more than one
tryptophan conformer in solution (1). Many people suggest the emission of tryptophan is

comprised of two bands. However despite the extensive work regarding to fluorescence of

108

109
tryptophan, there are no reported studies on the excitation wavelength dependence of

tryptophan fluorescence.

Fluorescence emission occurs from the zero-point vibrational level of the first
excited singlet state, so a fluorescence emission spectrum is generally not dependent on the
excitation wavelength. The fluorescence spectrum yields data about the vibrational level of
the ground electronic state. When a fluorophore is in a polar and viscous environment
where its mobility is restricted, the wavelength of maximum fluorescence will depend on
the excitation wavelength. The maximum fluorescence emission will show a red-shift
when excited with extreme red edge wavelength. This phenomenon is well known as red
edge excitation shift (REES) (7,8). The origin of the REES phenomenon result from the
reorientation process of solvent molecules around fluorophore when it is excited. The
fluorophore in the solution will exist in the solvated state with solvent molecules around it.
When the fluorophore is excited then the dipole of the fluorophore will change then the
solvent molecules around fluorophore will reorient themselves to meet the minimum energy
requirement This reorientation process or so-called solvent relaxation process is dependent
on the viscosity of the solvent. If the viscosity is low the reorientation will be very fast
around 10128 which is within the excited state lifetime 10 95. So when the viscosity is not
very high the emission is not excitation wavelength dependence because emissions all occur
from the solvent-relaxed state. That is the general situation for water, a very common
solvent, which has no very high viscosity. But when the viscosity of the medium is very
high, the solvent relaxation process will take longer time because the restriction of moving.
When the solvent relaxation time is the same as or longer than the lifetime of fluorophore,
the relaxation can not be done within the lifetime of fluorescence. So fluorescence will not
happen from the lowest vibration state. In this case each excitation wavelength will excite
selectively some special state fluorophores which have the similar solvent orientation in the
excited state as solvent relaxed state. Fluorescence will occur from the special excited

states and will be red shifted.

 

 

 

1 10
REES is rarely observed in aqueous solution. In this work we find that the

emission spectra of tryptophan is excitation wavelength dependence in aqueous solution at
all wavelengths which is not very common. The fluorescence of tryptophan is studied in

this research. For comparison, the fluorescence of indole is studied at the same time.

7 . 2 Materials and Experiments

Tryptophan was purchased from Aldrich Chemical Company, Inc., Milwaukee, WI
and used as received. Different concentrations of tryptophan solutions were prepared by
dissolving the appropriate amount of tryptophan in 5ml HPLC water. Supersaturated
solutions were prepared by heating the solution in the water bath until all the solute was
dissolved and subsequent cooling to room temperature. The solutions were used as
prepared without any degassing.

Fluorescence spectra of tryptophan in the aqueous solutions were collected within a
couple hours of the preparation the solutions. All spectra were taken at room temperature
with a Spex 1681 Fluorolog spectrometer equipped with a Xenon lamp source and a PMT
detector. A quartz sample holder was used in all cases. Spectra were recorded at a 1.00mm
increment and an integration time of 1.0 sec. Monochromator slits were at 0.5 mm for both

excitation and emission spectra.

7 . 3 Results
7.3.1. The fluorescence spectra of tryptophan in aqueous solution

Fluorescence spectra of tryptophan in aqueous solution shown in Figure 7.1 show
a broad peak with the maximum emission around 356nm when excited at 308nm. Figure
7.2 and Figure 7.3 show the maximum fluorescence intensity dependence on the
concentration of tryptophan at different excitation wavelengths. When the excitation
wavelength is lower than 310nm, the maximum emission intensity shows a parabolic plot

regarding to the concentration of tryptophan (Figure 7.2). That is, when the concentration

Intensity (CPS)

111

300000

 

250000 4

200000 4

150000 4

100000 ~

50000 1

 

 

 

O I I I l r I
320 340 360 380 400 420 440

Wavelength (nm)

Figure 7.1 Fluorescence spectrum of tryptophan in aqueous
solution excited at 308nm at room temperature

Maximum Emission Intensity (CPS)

112

 

c-l—

3105....1....§.-+
.

2510*

ll

1
D
1

Excitation
wavelengths

1

210$
LSIOL

1101

 

l 1 1 l 1 l A 1 J J J k 4 A 4

 

 

 

0 0.5 1 1.5 2
Concentration of Tryptophan (g/ 100ml H20)

Figure 7.2 Maximum emission intensity dependence on
the concentration of tryptophan at different excitation wavelengths
which are smaller than 310nm at room temperature

Maximum Emission Intensity(CPS)

113

—i—
-F—
I-O-
q!—

 

1 105

T

8 10“

O‘
t—I
O
A
J
I

Excitation
a l ths
w ve eng X 320nm

 

 

 

 

4 104* 4-

2 10‘-- __
O 1 l 1 1 D I 1 1 1 l 1 1 1 l 1

o 0.2 0.4 0.6 0.8 1

Concentration of tryptophan (g/100ml H20)

Figure 7.3 Maximum emission intensity dependence on the
concentration of tryptophan at different excitation wavelengths
which are higher than 320nm at room temperature.

l 14
of uyptophan is low, the intensity of tryptophan fluorescence will increase with the

increase of the concentration of tryptophan. When the concentration of tryptophan is high,
the fluorescence intensity of tryptophan will decrease with the increase of the concentration
of tryptophan. Intensity changes with the concentration are sharper as shown in Figure
7.2. At different excitation wavelengths, the parabolic maximum occurs at different
concentrations. When the excitation wavelength is greater than 320nm, the intensity of the
maximum emission increased with the increase of concentration of tryptophan shown in

Figure 7.3.

7.3.2. Excitation spectra versus concentration of tryptophan

The excitation spectra at different concentrations are shown in Figure 7.4. The
emission wavelength is 420nm and the spectra were collected from 300nm to 400nm. The
maximum of excitation wavelength shifts to red with the increase of concentration of
tryptophan and the intensity of excitation spectra increased with the concentration. The
excitation spectrum becomes broader with the increase of the concentration of tryptOphan.
With the increase of concentration of u'yptophan the shoulder at longer wavelength

becomes higher and higher.

7 .3.3. Excitation wavelength dependence of maximum emission wavelength

Figure 7.5 shows the fluorescence spectra of tryptophan with the concentration of
1.632g tryptophan in 100ml I-l20 which is supersaturated solution at different excitation
wavelengths. The same solution excited with different excitation wavelengths shows
different maximum emission and different emission intensity.

The maximum emission wavelength will depend on the excitation wavelength at all
concentrations of tryptophan. The different tryptophan concentration is 0. 37 g/ 100ml H20
which is much unsaturated, 1.108g/100m1 H20 which is saturated solution and

1.632g/100ml H20 which is supersaturated solution. The plot of the maximum emission

 

 

115

LOOE+05

 

900E+04~

800E+041
700E+04~
<300E+04«
- ‘ Concentration increasing
400EH04- :
300E+041 f

Intensity (CPS)
8
111
§

2.00E+04 ~

 

 

L00E+044

 

 

 

0.00E+00 I I I
300 320 340 360 380 400

Wavelength (nm)

Figure 7 .4 Excitation spectra of tryptophan at different tryptophan
concentrations monitoring at 450nm emission wavelength

116

11138-5

 

8.U)E+O4 1

7.CDE+O4 .

6.mE+O4 i

Intensity (CPS)
§
é

§
§

30090“ ‘ 350nm
308nm

2.00504 4

370nm
1.00904 4

 

DRIED

 

 

320 340 360 360 400 420 440 460 480 500 520
Wavelength (nm)

Figure 7.5 Fluorescence spectra of supersaturated tryptophan
solution at difi‘erent excitation wavelengths at room temperature

 

1 17
as a function of excitation wavelength for different concentrations is shown in Figure 7.6.

From the plot of wavelength of excitation versus wavelength of maximum emission we can
see the wavelength of maximum emission will increase with the increase of the wavelength
of excitation for almost all excitation wavelengths. At lower excitation wavelengths, lower
than 310nm, the emission wavelength does not change with the excitation wavelength and
when the excitation wavelength is bigger than 310nm and lower than 330nm, the maximum
emission wavelength increases sharply and nonlinearly with the excitation wavelength.
When the excitation wavelength is larger than 350nm, the increase of the emission
wavelength is linear with the increase of the excitation wavelength. When the excitation
wavelength is between 330nm-350nm the relation between maximum emission and
excitation is pretty complicated. It shows different changes with different concentrations.
At low concentration, such as 0.37 g tryptophan in 100111] H20, a sudden drop of
fluorescence intensity occurs at 335nm, and at higher concentration, such as 1.108 g
u'yptophan in 100ml H20, a sudden intensity drop occurs at 340nm. For a supersaturated
solution, there is a curve down at wavelength 340nm but no sudden intensity drops. Three
different solution concentrations have almost the same maximum emission versus excitation

wavelength plot.

7.3.4 Lifetime of tryptophan in different solvents at different concentration
According to the literature tryptophan has a double-exponential decay at neutral pH.
The major component has a life time of 3.1ns with emission max. at 350nm, and the minor

component has a lifetime of 0.5ns with emission maximum at 335nm.

The decay curve of tryptophan also can be fitted by two exponentials with t, around

2ns and 1:2 around 5ns at different solvents. The shorter lifetime will not change with the

solvents, but the longer lifetime will change with the solvents. It almost becomes double

Emission maximum emission wavelength (nm)

118

 

 

 

 

 

 

500nwnwmvinwfiei rt 1 .
- o 0.37g/100mlH20
* o 1.108g/100mlH20 O
A 1.632g/100ml H20
0
O
450—— é -_
' J
6
O
a
2
9 J
8 .
e
. 8 1
400» -_
. 0
£800
a“ 8
r 0 0
. A 4
0 4
9 1
+ new?
350111111+,%J:%l1
280 300 320 340 360 380 400 420 440

Excitation wavelength (nm)

Figure 7.6 The dependence of the maximum emission
wavelength on the excitation wavelength at different
concentration of tryptophan solutions at room temperature

1 19
when the solvent is changed from water to the mixture of water and alcohol (1:1). The data

is shown next:

H20 t,=2.03ns 12:3.168ns x2=0.658
EtOH+HZO( l : l) I,=2.34ns 12:6.12ns x2=0.638

EtOH+I-I,O+PropylAcetate(2:2:l) r,=l.920ns 12:3.770ns x2=0.637

The lifetime of tryptophan will change with the concentration of u'yptophan. In the
mixture of water, ethanol and propyl acetate (2:2: 1), the shorter lifetime of tryptophan just
decreased slightly with the concentration of tryptophan and larger lifetime will have a large
decrease with the tryptophan concentration increase (Figure 7.7). The lifetime of
tryptophan in water will decrease with the concentration of tryptophan too. But the change

is not as regularly as in mixture.

7.3.5 Comparison of indole fluorescence and tryptophan fluorescence

The fluorescence of tryptophan results from the indole ring. In order to support the
hypothesis of the formation of associates due to the side chain of tryptophan in tryptophan
solutions, the fluoresce of indole is studied as comparison. The excitation wavelength
dependence of indole fluorescence is checked first. It turned out that the fluorescence of
indole is not excitation wavelength dependence. The excitation spectra of tryptophan
(0.116g/100ml water) and indole (0.051 g/ 100ml water) are shown in Figure 7.8. Indole
has a sharp peak at 299nm. The excitation spectrum of u'yptophan is a sharp peak at
305nm but there is a tail followed this peak. The emission spectra of tryptophan and indole
in water excited using the highest excitation intensity wavelength are shown in Figure 7.9.
Tryptophan and indole have very similar emission spectra Both are broad peaks. The

maximum emission wavelength of indole will not change with the excitation wavelengths.

Lifetime of tryptophan (us)

120

 

 

 

 

 

 

7 . i i . . I l i l .
i
. . i
_ Tryptophan 1n mixture-4:2 *
6 * 1
I
.
5 2_ -1.
.
4 -- .—
l- 1
3 * *
, . .
Tryptophan 1n mrxtur "151
i N l
2 --<.— V v \ C ...—
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1r 1 1 1 l 1 1
0.2 0.4 0.6 0.8 l 1.2 1.4 1.6

Concentration of tryptophan (g/ 100ml H20)

Figure 7.7 Lifetime of tryptophan in the mixture of
water+ethenol+propyl acetate (2:2: 1) at different
concentrations of tryptophan at room temperature

121

6.00E+O3

 

Tryptophan
5.00E+03 «

Indole

4.00E+03 i

3.00E+03 1

Intensity (CPS)

2.00E+03 4

1.0013403 1

‘~ J”

0.00E+00

 

 

 

T T 7

280 290 300 3 10 320 330
Wavelengh (nm)

340 350

Figure 7.8 Excitation spectra of indole and tryptophan aqueous solutions
at room temperature monitoring at 500nm emission wavelength

Intensity (CPS)

4150E+05

‘LOOE+05 -

350E+05

.300E+05

.2505+05

200E+05

150E+05

LOOE+05

500E+04

(100E+00

122

 

 

 

Indole
i Tryptophan
J .
320 340 360 380 400 420 440

Wavelength (nm)

Figure 7 .9 Fluorescence spectra of tryptophan and indole
in water at room temperature

 

123
7.4 Discussion

Tryptophan in solution at a pH around 6.5 is a zwitterion. 80 two, three or more
tryptophans may interact with each other to form dimers, trimmers or more complicated
complexes, they can be two dimensional or three dimensional. These two or three
dimensional structures produce different species of uyptophan in the solution which can be
called associates. These associates will have different numbers of tryptophan molecules.
Due to the co-bondin g among rings or atoms, they will have different electronic structures,
and different energy levels. The broad peak of tryptophan suggests many energy levels
exist in the tryptophan solution. These different energy levels are close to each other, and
they can not be distinguished so a broad emission band results.

In general, the larger the associate is, the lower the energy will be. Because many
molecules together will share electrons which make the electricity cloud more spread, then
lower energy level can be realized. Each associate will have a specific excitation which
means each excitation wavelength will favor one specific associate which the energy
difference between excited state and ground state is the same as the excitation energy.
Different excitation wavelengths will excite selectively specific tryptophan associates in the
solution. Different concentrations will result in different distributions of associate species
and also different dominant associates. This explains why we get different excitation
spectra at different concentrations. The red shift of the excitation wavelengths with the
increase of the concentrations is the result of larger associates in higher concentrated
solutions. The more molecules in the associate, the lower energy it will be. Then lower
energy will be needed to excite the larger associate. The intensity of excitation spectra
increase with the concentration is much easier to understand. More species will accumulate
in the solution with the increase of concentration, so higher concentration solution will have
higher intensity, and the same, broader peak at high concentration solutions will occur too.

The maximum emission intensity dependence on the concentration of tryptophan at

different excitation wavelength is the result of the different population of species and each

1 24
species has specified excitation wavelength (Figure 7.2 and 7.3). When the excitation

wavelength is lower than 320nm, a parabolic curve will appear regarding to the relation of
maximum emission wavelength to concentration of tryptophan. Different excitation
wavelengths will have different favor associates to excite which results in the different
position of parabolic peak. Different concentrations of solution will have different
populations of different species. This population difference results in the fluorescence
changing with the concentration. When the excitation is lower than 310nm, due to the
higher energy, many species can be excited resulting in the intensity dependence on the
population of the species. The higher population, the higher the intensity. The larger the
associates are, the lower the energy is. Longer excitation wavelength will tend to excite
larger associates. So with the increase of the excitation wavelength, the parabolic peak will
move toward higher concentration. When the excitation wavelength is longer than 320nm,
those small species can not be excited at all due to the lower excitation energy. Only those
larger associates can be excited and those large associates tend to exist only in high
concentrated solutions and the population of those larger associates will increase with the
increase concentration of tryptophan. So when the excitation wavelength is larger than
320nm, the intensity of maximum emission will increase with the concentration of
tryptophan all the way.

The increase of emission wavelength with the excitation wavelength at longer
wavelengths such as over 360nm can be interpreted as the REED phenomena detailed in the
introduction. Due to the larger size of tryptophan associates, the relaxation process will not
be very fast. Or we can say the larger size of uyptophan associates makes the energy of an
associate much lower than a single solvated molecule. Low excitation energy will only
excite selectively those larger associates. The size of the molecule makes the relaxation
difficult, so the emission will not happen from lowest vibration level. The lower the
excited energy is, the larger associates will be excited and the lower the emission energy

will be, so the red shift will occur.

125
The same reason can be used to interpret the dependence of emission wavelength on

the excitation wavelength at shorter wavelength range. We suppose there are many
different tryptophan associates in the solution and each species will have definite excitation
wavelength. When a definite excitation wavelength is used only specific species will be
excited to its biggest degree and those excited species will emit at different wavelengths
with the maximum emission intensity.

That the relation between maximum emission and excitation wavelength is not
tryptophan concentration dependent supports specific species exist in the solution of
tryptophan. At different concentrations the population of different species will be different,
but not the types of species. Each type of species will have a definite emission maximum.
A definite excitation wavelength will excite a definite species, then result in the dependence
of maximum emission wavelength on excitation wavelength.

Exponential decay behavior of tryptophan fluorescence is dependent on excitation
wavelength (1,9) The dependence is due to the associates in the solutions. As we know
different excitation wavelengths will excite different species. It is very easy to understand
different species will have different decay behaviors so dependence of decay on the
excitation is the result. The decay of tryptophan is concentration dependent and solvent
dependent. The different concentration will have different distribution of associates, in
other words different concentration of excitation favor species.

It has been demonstrated that the fluorescence properties of some probes are
sensitive to the hydrogen-bondin g character of the solvent (10). The clearest manifestation
of this effect is the drastic increase in the fluorescence lifetime in going from protic to polar
approtic media. So the increase of the lifetime in the mixture of ethanol and water is due to
the decrease of hydrogen bonding in the mixture than in pure water.

The fluorescence of tryptophan is due to the indole ring. The indole fluorescence is
not excitation wavelength dependent. The only difference in indole and tryptophan is that

tryptophan has side chain. The side chain of tryptophan is the reason for the formation of

126
associates. So the nondependence of indole emission on the excitation wavelength

supports strongly our proposal of associates of tryptophan in the solution.

7 . 5 Conclusion

The maximum emission of tryptophan fluorescence in solution is excitation
wavelength dependent. The dependence is the result of multiple molecule associates of
tryptophan. These multiple molecule associates will have specific electronic structure and
specific excitation energy. Different excitation wavelength will favor different specific
species, resulting in different emission wavelengths.

The nondependence of emission on the excitation wavelength of indole fluorescence

support strongly the assumption of associates in tryptophan solutions.

7 . 6 References
1. A. G. Szabo and D. M. Rayner, J. Am. Chem. Soc., 102, 554-563(1980)
2. David Creed, Photochem. Photobio., 39, 5370984)
3. L. P. McMahon, W. J. Colucci, M. L. McLaughlin and M. D. Barkley
J. Am. Chem. Soc., 114, 8442-84480992)
4. Hong-Tao Yu, W. J. Colucci, M. L. Mclaughlin and M. D. Barkley,
J. Am. Chem. Soc., 114, 84490992)
5. Hong-Tao Yu, M. A. Vela, F. R. Fronczek, M. L. McLaughlin and
M. D. Barkley, JAm. Chem. Soc., 117, 348-3570995)
6. Yu Chen, Bo Liu, Hong-Tao Yu and Mary D. Barkley,
J. Am. Chem. Soc., 118, 9271-92780996)
7. S. Guha, S. S. Rawat, A. Chattopadhyay and B. Bhattacharyya, Biochem.,
35,134260996)
8. J. R. Lakowicz, Principles of Fluorescence Spectroscopy, Plenum Press,

New York and London(1983)

 

 

127
9. W. X. Balcavage and T. Alvager, Mol. Photochem., 7, 309 (1976)

10. Ramamurthy, V., Photochemistry in Organized & Constrained Media,
VCH(1991)

 

Chapter 8

EXCITATION SPECTRA STUDY OF PYRANINE IN WATER AND
DIFFERENT SOLUSITIONS WITH DIFFERENT CONCENTRATIONS

8 . 1 Introduction

The excitation spectrum is the dependence of emission intensity at a single
wavelength upon the excitation wavelength (1). The excitation spectrum represents the
relative quantum yield at each excitation wavelength. The excitation specu'um of a
fluorophore is typically superimposable on its absorption spectrum, but excitation has
advantage over adsorption on the sensitivity. Much higher sensitivity can be realized in the
excitation spectra which means more information can be obtained Excitation spectra reflect
the vibrational levels of excited states. In general, the excited states and ground states will
have the same vibrational structures so we can obtain the information of ground state
vibrational structure by interpreting the data we obtained from excitation spectra.

The intensity of the excitation spectrum can be much higher than emission intensity
for the same concentration solution. Therefore, the excitation spectrum can provide us
information which would not show up in the emission spectra, such as the fine vibronic
structure. It is a good supplement to the emission spectrum.

The majority of chemical or biological reactions occur in solutions. The interaction
and behavior of solvent and solute in solutions has remained an interesting topic to many

researchers for many years. It is clear that the properties of solute molecules will be

128

129
modified by solvents. Understanding the interaction is essential for the intrinsic properties

of solutes to be understood better (2). Many crystallization processes occur from solutions;
therefore, understanding the structure of solution, especially supersaturated solution
structures, can help us to design and control crystallization better. Although such research
has been conducted for decades, it is still not clear about what happens in solutions.

Excitation is seldom used as research method although Tucker (3) showed it a more
convenient, fast and reliable for study of fluorescence quenching phenomena and less
tedious and time consuming for the examination of spectra than simple emission (3).

In this research the excitation spectra of pyranine in water and in different
concentrations of tryptophan, phenylalanine and nicotinic acid solutions at 510nm and
382nm emission wavelengths will be examined The information about the interaction of

solute molecules with pyranine will be described.

8 . 2 Materials and methods

Pyranine, also known as FDA D&C Green No. 8, was obtained from Lancaster,
Windham, NH and used as received. Aqueous solutions of pyranine were prepared by
dissolution of 0.026 g in 5ml HPLC grade water. Three different concentrations of
solutions were prepared by dissolving the appropriate amount of solutes in 5ml HPLC
water. They are under saturated , saturated and supersaturated solutions. Supersaturated
solutions were prepared by heating the solution in the water bath until all the solute was
dissolved and subsequent cooling to room temperature. The solutions were used as
prepared without any degassing.

Probe molecules were introduced by addition of five microliters of the pyranine
solution to 5ml aqueous tryptophan, nicotinic acid and phenylalanine solutions resulting in
a probe concenuation of 105M. All excitation spectra were collected at room temperature
with a Spex 1681 Fluorolog spectrometer equipped with a Xenon lamp source and a PMT

detector. A quartz sample holder was used in all cases. Spectra were recorded at 1.00mm

1 30
increment and an integration time of 1.0 sec. Emission wavelengths of 382nm and 510nm

were used. The excitation spectra were recorded over a range from 260nm to 370nm for
the 382nm emission wavelength and 280nm to 480nm for the 510nm emission wavelength.

Monochromator slits were set at 0.5 mm for all spectra.

8 . 3 Results and discussions
8.3.1 Excitation spectra at 510nm emission wavelength
8.3.1.1. Excitation spectra at 510nm of pyranine in water and low concentration solutions
Excitation spectra of pyranine in water and low concentration of tryptophan,
phenylalanine and nicotinic acid solutions at 510nm are shown in Figure 8.1. There are
large differences between the excitation spectrum of pyranine is in water and excitation
spectra of pyranine in uyptophan, phenylalanine and nicotinic acid solutions. There is a set
of peaks around 460nm which are pretty strong when pyranine in water. Fine vibrational
su'ucture shows up in this set of peaks. There are some broad peaks from 360nm to
440nm. There is also a broad band which has two peaks at 290nm and 298nm
respectively. The addition of solutes into the water alters the excitation spectrum of
pyranine greatly even in trace amounts. With even trace amounts of phenylalanine and
nicotinic acid in solution, the intensity of the band around 460nm decreases dramatically.
The band at 402nm and 380nm shows up with the addition of solutes into water. The band
around 460nm decreases a lot with the addition of tryptophan and phenylalanine and
disappears with the addition of nicotinic acid. With the addition of phenylalanine the peak
at 298nm disappears and the peak at 290nm increases. With the addition of uyptophan and
nicotinic acid in solution, the band at lower wavelengths disappears. The band at lower
wavelength can be assigned to the interaction of pyranine with water. The disappearance of
the lower wavelength peaks can be interpreted as that the interaction between pyranine and

solutes. The interaction between pyranine-nicotinic acid and pyranine-tryptophan are

 

131

2.506%

 

Pyranine
2.mE+08 j

Phe

15E“ ‘ H
1mE+06+ K

5me <

 

Intensity (CPS)

 

NA

 

o-“m ‘ I Y I I Y 1 T r T
25) an 320 30 w 3&3 400 420 44) M 4m

Wavelength (nm)

 

 

Figure 8.1 Excitation spectra of pyranine in water and in tryptophan(0.266g/100ml
H20), phenylalarrine(0.33g/100ml H20) and nicotinic acid(0.302g/100ml H20) aqueous
sohrtions at 510nm emission wavelength at room temperature.

 

l 32
stronger which destroys the interaction between pyranine and water. The interaction of

phenylalanine with pyranine is not as strong as nicotinic acid with pyranine but it is
stronger than pyranine with water. Only one peak disappears. This results match quite
well with the quenching results which is shown in chapter 5.

The excitation spectrum at emission wavelength 510nm and the emission spectrum
with peak at 510nm of pyranine in water is shown in Figure 8.2. The spectra were
obtained from the same solution at the same time. The intensity of the excitation is much
higher than the emission spectra. In order to make them in the same scale, we decrease the
slits to 0.25mm for the excitation spectra and the emission spectrum is at 0.5mm slit open.
The emission spectrum shows a broad and single band with the peak at 510nm. The
excitation spectrum shows the fine vibrational structure with five peaks around 460nm.
They are at 450nm, 452nm, 457nm, 462nm, and 467nm respectively. The shape of this
band is quite similar as the mirror of typical emission spectra of the polycyclic aromatic
hydrocarbon (PAH). The typical emission spectrum of the PAH monomer will consist of
five major vibronic bands which are generally labeled as I-V in progressive order, such as
the 0-0 band is labeled 1, etc.(4-9). If we ignore the fine structure in the excitation spectra,
the emission spectra can be considered as a mirror image of the excitation image. The
fluorescence spectrum of 1-aeabenz[a]anthracene in trifluoroethanol and in HClO4-
trifluoroethanol is reported to move from 360nm-405nm to 460nm-560nm with the
protonation of the nitrogen lone electron pair by a hydrogen ion. At the same time the a
broad, single band will replace the original fine vibrational structure bands (7). When
pyranine is in high concentrations of weak acid solutions, two excited fluorescent states
will exist and one in which the hydroxyl proton is associated with the molecule (PyOH)
and one in which it is dissociated in pyranine (PyO') (10). The emission from PyOH* is at
440nm and the absorption at 405nm. PyO‘* will absorb at 445nm and emit at 510nm.

The fluorescence of pyranine in water exhibits two bands one is from 370 to 430nm which

contains three fine vibrational structure peaks and another is a broad band centered at

133

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1 34
510nm. The excitation from 370nm to 430nm will be discussed later. From the excitation

spectrum we see a peak around 405nm which is assigned as the excitation of PyOH. The
band with the fine vibrational structure around 460nm will be assigned to the excitation of
PyO‘. The emission spectra will reflect the vibrational levels of the ground state and the
excitation specu'um will reflect the vibrational levels of the excited states. The excited state
has a higher dipole moment than ground state. PyO'* will have a greater dipole moment
than PyOH* because of the lack of the hydrogen ion which results in much larger energy
spacing between the various vibrational levels. The fine vibronic structure can be detected
in the excitation of PyO‘. In water, pyranine is surrounded by dipole orientated water “ _
molecules. In the inner layer water molecules will be bonded firmly with entirely
dissociated sulfonate groups with a scheme shown in Figure 8.3. This is the reason that
pyranine has such a high solubility in water. Peaks around 405nm and 460nm are the
excitation of solvated PyOH (4.15x10'6M) and PyO‘ (5.85x10‘M), respectively. The
firmly bounded solvated pyranine PyO' will show fine vibronic structures due to a higher
dipole moment which is not hard to tell from the su'ucture. We see almost continuous
increasing in intensity of excitation spectrum from 340nm to 440nm which can be
interpreted as at outer layer some non-orientated water molecules will bound with pyranine
loosely. These loosely bounded water molecules at the outer layer will alter the energy of
pyranine in some way. Because the bonding is really loose, and those loosely bonded
water molecules move around in some region, the energy change is really small. The much
higher intensity of the excitation than the emission is the result of quenching from water.
Water will quench the fluorescence of pyranine but the excitation intensity will not be
affected by quenchers that effectively.

The larger change in excitation spectra at 460nm with the addition of solutes into
water is because all the solutes are acids. The addition of acid will favor PyOH in the

equilibrium in solutions. The peak at 405nm, which is assigned as the excitation of PyOH,

 

135

H20 H20
H20 H2O

02H H20 H20 H20
02H
02H '03 803- H2O

Omsk.— 02H 9 H20
cc
02H 9 H20 ___> Inner
- layer
02H 038 0‘

02H H20 H20

H20

Solvated pyranine molecule in water

H o
H o 2
O H 02H 2 H20 H20
2 '03 so, /NH2 H20
02H 9 CH; CH\
COOH
cc ------ c \
H20
02H 9 ” ’ N H2 H20
02H ’03 0'— H’ H20
02H H20
H20

Solvated of complex between pyranine and tryptophan

Figure 8.3 Scheme of solvated structure of PyO- and the contact complex
betweeen pyranine and tryptophan, phenylalanine and nicotinic acid

 

 

1 36
increases and the peak around 460nm, which is assigned as the excitation of PyOH' ,

decreases. Especially, with the addition of nicotinic acid, the acidity of the solution has a
larger increase, all pyranine will exist in the form of PyOH according to the calculation.
The peak at 460nm disappears with the addition of nicotinic acid. However, the emission
of pyranine in tryptophan, nicotinic acid and phenylalanine are all from PyO'* due to the
fast proton transfer reaction at excited states (refer to chapter 2 for details).

Even trace amounts of phenylalanine in solution, which does not change the pH of
solution much, will decrease the intensity of the peak at 467nm significantly. We may
conclude that the shift between PyOH and PyO- equilibrium may not be the only reason for
the intensity change of two peaks. If the shift of the acid-base equilibrium between PyOH
and PyO’ is the only reason for the peak change at 402nm and 467nm, the ratio of [PyO-
]/[PyOH] should be equal to the ratio 146va at different pHs. Figure 8.4 shows the
ratio of [PyO-l/[Pyofl] and Immflm at different pH values for phenylalanine and
tryptophan solutions. The two ratios do not change the same way with the pH. The acidity
of the solutions increases with the addition of phenylalanine and tryptophan to solution.
The equilibrium between PyOH and PyO' shifts from PyO‘ to PyOH. The concentration of
PyOH increases, so does the intensity at 402nm. The intensity increase is larger than the
concentration increase which means some other factors will affect the intensity at 402nm.
The Other factor is the interaction of phenylalanine and tryptophan with pyranine. As we
know from chapter 5, phenylalanine will quench fluorescence of pyranine slightly. If
phenylalanine is far away from pyranine, the fluorescence of pyranine will be much more
easily quenched by water around pyranine which is much smaller and will form hydrogen
bonding complex with pyranine. The hydrogen bonding complex is the most effective
quenching mechanism as evidenced by the stronger quenching of nicotinic acid and
tryptophan. Pyranine can form a hydrogen bonding complex with water, therefore, water

will quench the fluorescence of pyranine. Phenylalanine has a benzene ring su'ucture. It is

 

137

 

 

 

 

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13
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' 1‘.
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467nm/402nm (Phe u
1
l» ’
G c 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
6 6.5 7 7.5

pH of solutions with pyranine

 

 

 

 

Figure 8.4 The concentration ratio of PyO' and PyOH calculated based
on pH and fluorescence intensity ratio of 467nm/402nm versus pH of
solutions in tryptophan and phenylalanine solutions at room temperature.

1 38
very possible for pyranine and phenylalanine to be close to each other to form a kind of

contact complex. It is feasible because phenylalanine will like pyranine better than water.
When phenylalanine is added into water, it will u'y to find pyranine and to stay close to
pyranine. When pyranine is excited, the distance of pyranine and phenylalanine becomes
closer which is an exciplex. By formation of the exciplex, the fluorescence of pyranine is
quenched. This contact complex will stabilize PyOH better because of the lower dipole
moment of PyOH as compared to PyO’ and this contact complex can protect pyranine from
quenching by water.

Tryptophan has two interactions with pyranine. Tryptophan will form a contact
complex with pyranine similar to phenylalanine. The amine in the ring of tryptophan can
form hydrogen bonding with the hydroxyl group in pyranine as shown in Figure 8.3. This
function of tryptophan will pull the hydrogen ion from pyranine so it favors PyO'. Trace
amounts of tryptophan will not eliminate the peak at 467nm at all because the acid-base
equilibrium will favor PyOH. These double functions of tryptophan results in the
downward curve for the change with pH in tryptophan solution (Figure 8.4).

With the addition of nicotinic acid, the acidity of the solution will change
significantly and the equilibrium between PyO’ and PyOH will all shift to PyOH. The shift
will be the dominant factor.

The formation of a contact complex between pyranine-uyptophan and pyranine-
phenylalanine will hinder the water accessibility of pyranine and limit the rotation of
pyranine especially around hydroxyl group side (Figure 8.3). The addition of tryptophan
and phenylalanine will increase the fluorescence intensity of pyranine at 510nm. N icotinic
acid has a strong quenching ability for pyranine and even trace amounts of nicotinic acid

decrease the fluorescence intensity significantly.

1 39
8.3.1.2. Excitation spectra of pyranine in different concentration of solutions

The shape and maximum of the excitation spectra of pyranine does not change
much with the increase of solute concentration except that the peak around 460nm
disappears at higher concentration of phenylalanine. The intensity of the excitation spectra
of pyranine decreases with the decrease of the concentration of tryptophan. The intensity
changes a little with the concentration of phenylalanine. In nicotinic acid the intensity
decreases dramatically with the increase of the concentration of nicotinic acid, but the shape
and maximum remains unchanged. The intensity decrease results from the quenching of

pyranine by solutes.

8.3.2 Excitation spectra at 382nm emission wavelength
8.3.2.1. Excitation spectra of pyranine in water and low concentration solutions

Figure 8.5 shows the excitation spectra of pyranine in water and in low
concentrations of tryptophan, phenylalanine and nicotinic acid. There are four peaks in the
excitation spectrum of pyranine in water and they are 270nm, 281nm, 448nm and 366nm
and also a shoulder at 334nm. There is a peak at 308nm which is the excitation peak of
tryptophan. With the addition of solutes in the water, the excitation intensity will drop.
Nicotinic acid has the highest drop in intensity and tryptophan has an intermediate intensity
drop and phenylalanine drops the least The different decreases of the excitation intensity
matches the different quenching abilities of the solute molecules to pyranine. As shown in
chapter 5, nicotinic acid has the strongest quenching ability to pyranine and phenylalanine
quench the fluorescence of pyranine only slightly.

With the addition of tryptophan and nicotinic acid into the water, the excitation
peaks of pyranine at lower wavelengths disappears. Those peaks at low wavelengths can
be assigned as the interaction between water and pyranine. The addition of tryptophan and

nicotinic acid into water destroys the interaction between pyranine and water molecules

140

 

 

 

 

 

 

 

 

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Wavelength (nm)

Figure 8. 5 Excitation spectra of pyranine in water and in tryptophan(0.266g/100ml
H20), phenylalanine(0.33 g/ 1001111 H20) and nicotinic acid(0.302g/ 100ml H20)
aqueous solutions at 382nm emission wavelength at room temperature.

 

141
because of the stronger interaction of pyranine with tryptophan and nicotinic acid.

Phenylalanine will have weaker interactions with pyranine compared with tryptophan and
nicotinic acid, so the lower wavelength peak at 281nm remains in phenylalanine solutions,
but the peak at 270nm is diminished.

The emission spectrum and excitation spectrum with 382nm emission wavelength

of pyranine in water are shown in Figure 8.6. A good mirror image is observed.

8.3.2.2. Excitation spectra of pyranine in different concentration of solutions

The excitation spectra of pyranine in different concentrations of tryptophan are
shown in Figure 8.7. The peak around between 300nm to 320nm is the excitation peak of
tryptophan. As shown in chapter 7, the excitation spectra of uyptophan will shift to longer
wavelengths with the increase of the concentration and the intensity will increase at the
same time without pyranine in solution. With pyranine in solution, the red shift is the
same, but the intensity decreases with the increase of the concentration. The pyranine
excitation intensity at peak 348nm and 366nm decreases a lot with the increase of the
concentration of uyptophan. Once again it matches with the quenching of tryptophan to
pyranine.

Excitation spectra of pyranine in different concentrations of phenylalanine solutions
are shown in Figure 8.8. The positions of the peaks are almost the same as pyranine in
water. The peak intensity will decrease with the increase of concentration of phenylalanine.
Because the unstability of supersaturated solutions of phenylalanine, we can not go to high
supersaturations. As discussed in Figure 8.5, with the addition of phenylalanine, the peak
at 270nm is eliminated, but with the increase of phenylalanine concentration, the peak at
270nm disappears. The disappearance of the peak can be interpreted as that the interaction
of phenylalanine with pyranine is stronger than the interaction of water with pyranine

although there is no hydrogen bonding between phenylalanine and pyranine. The contact

 

 

Intensity (CPS)

142

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Excitation
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5.00904 - r ‘v\
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'1
0.00900 1 . 4 . . .

Wavelength (nm)

 

 

 

 

Figure 8.6 Emission spectrum of pyranine00‘5 M) in water excited at

342nm and excitation spectrum of pyranine00'5 M) in water with emission
wavelength at 382nm at room temperature

143

 

2.00915
1HE+CB 4

1.60905 <
Unsaturated

Saturated
1.0915 ~ .5

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Intensity (CPS)

8.G)E+O4 <

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4.(I)E+O4 .

 

 

 

 

 

 

 

 

 

sic 111 111
Wavelength (nm)

340

:50 3a) 370

Figure 8.7 Excitation spectra of pyranine in difl‘erent concentration of tryptophan
aqueous solutions at 382nm emission wavelength at room temperature. Unsaturated:
0.266g, saturated: 1.112g, supersaturated: 1.574g in 100111] H20

 

 

 

144

4.505%

 

4.CDE+% 4

3.505% .

Unsaturated
3.00905 ~

 

a
a

 

Intensity (CPS)

§
1

 

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1.50905 1 Saturated and supersaturated

1.00905 1

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0.00900 .

 

ado 111 T
Wavelength (nm)
Figure 8.8 Excitation spectra of pyranine in different concentration of phenylalanine

aqueous solutions at 382nm emission wavelength at room temperature.
Undersaturated: 0.33g, saturated: 2.648g, supersaturated: 3.2g in 100ml H20

 

145
complex forms between pyranine and phenylalanine; therefore, with the addition of

phenylalanine, the interaction between water and pyranine which has the peak at 270nm is
destroyed by phenylalanine. With the increase of the concentration, the replacement of
water by phenylalanine increases, thus the decrease of the peak at 270nm, and finally
disappearance of peak 270nm.

Excitation spectra of pyranine in different concentration of nicotinic acid solutions
show a large decrease in the excitation intensity with the increase of the nicotinic acid
concentration This sharp decrease is the result of the strong quenching ability of nicotinic

acid.

8 . 4 Conclusion

The excitation spectra of pyranine change dramatically with the addition of solutes
in even trace amounts. For the excitation spectra at 510nm, a large decrease at 460nm and
increase at 402nm is the results of the acid-base shift between PyO‘ and PyOH and contact
complex formation between pyranine and solute molecules. The disappearance of peaks at
lower wavelengths of excitation with the addition of solutes into water was taken as the
evidence of the formation of the complex which destroys the interaction of pyranine with

water.

8.5 Reference

1. J. R. Lakowicz, Principles of Fluorescence Spectroscopy, Plenum Press,
New York and London(1983)

2. J. F. Coetzee, C. D. Ritchie, Solute-solvent interactions, Marcel Dekker,
New York and London(l969)

3. S. A. Tucker, W. E. Acree, JR., Applied Spectroscopy, 46(9), 13880992)
4 Jan Stahlberg and Mats Almgren, Anal. Chem, 57, 817-821(1995)
5 W. E. Acree, JR., 8. A. Tucker, A. I. Zvaigzne, Hans-FR. Grutzmacher,

 

10.

146
Applied Spectroscopy, 44(3), 4770990)

S. A. Tucker, W. E. Acree, JR., and M. J. Tanga,

Applied SpeCtroscopy, 45(1), 570991)

8. A. Tucker, W. E. Acree, JR., and M. J. Tanga, M. Zander, J. C. Fetzer, S.
Tokita, K. Hiruta, K. Kitahara and H. Nishi,

Applied Spectroscopy, 45(7), 11880991)

S. A. Tucker, W. E. Acree, JR., and M. J. Tanga, S. Tokita, K. Hiruta,

and H. Langhals, Applied Spectroscopy, 46(2), 229(1992)

M. U. Kumke, H. G. Lohmannsroben and Th. Roch, Analyst, 119, 997(1194)
H. Kondo, I. Miwa, and J. Dunamoto, J. Phys. Chem, 86, 4826-310982)

 

 

Chapter 9

SUMMARY OF THE WORK AND PROPOSED FUTURE WORK

9 . l . Summary of the work

In this research pyranine, PBA and CA were investigated as potential fluorescence
probes to measure the supersaturation of low solubility aqueous systems, such as
tryptophan. nicotinic acid, phenylalanine and benzoic acid. The measurement of
supersaturation is realized by measuring the concentration of solution. Any physical
properties which change with the concentration can serve as the calibration curves to
measure the supersaturation. Fluorescence was shown to be a very useful technique to
measure the supersaturation of low solubility systems due to the change of PIR (Peak
Intensity Ratio) of fluorescence with concentration. Monitoring the PIR during the
crystallization, we can know the supersaturation. By changing the outside conditions, we
can control the supersaturation resulting in control of the crystallization for desired crystal
size distributions (CSD).

Pyranine was shown as a very promising probe for use as the sensor to monitor
crystallization in the solution due to the smooth and nonlinear calibration curves which
show a higher signal response at supersaturated region. Additionally, pyranine will not
enter the crystal lattice of the crystalline products. Pyranine is not listed as a food additive
by FDA, so it can not be added directly into food and pharmaceutical production process.
Carminic acid (CA), one of approved food additives by FDA, was investigated as a
potential probe to measure the supersaturation. CA was shown to be a good sensor for

phenylalanine by following the intensity change with the concentration of phenylalanine.

147

148
Another way to avoid adding the probe into the system directly is to immobilize the probe

molecule. Due to the good behavior of pyranine in water solution as a probe sensor,
pyranine was immobilized onto an ion-exchange membrane to prevent it from possibly
contaminating the crystal products. The PIR (Peak Intensity Ratio) changes linearly with
the concentration of solutes for immobilized probe.

Fluorescence quenching requires the contact of fluorophores and quenchers, so the
interaction of fluorophore and quenchers can provide information about the organization of
solutes in solutions. All quenching curves show discontinuities in supersaturated region
which indicates the different solution structure of supersaturated solutions and unsaturated
solution. This discontinuity was taken as the evidence of the formation of aggregates of the
solutes in supersaturated solutions. Different molecules such as tryptophan, nicotinic acid
and phenylalanine show totally different quenching behaviors relate to pyranine in solution
or to immobilized pyranine. The different quenching behaviors are the results of the
different interactions of pyranine with solute molecules. The contact complex was
suggested as the mechanism of the interactions between pyranine and solute molecules.
The stronger quenching abilities of tryptophan and nicotinic acid are the results of
formation of hydrogen bonding complex with pyranine due to the presence of the amine in
the rings. The different steady state quenching and lifetime quenching behavior of
phenylalanine support strongly the contact complex assumption. The excitation spectra of
pyranine changes significantly even with the addition of trace amount of solutes into water
and the different dependence of concentration ratio and intensity ratio on pH of solutions
also support the formation of contact complex assumptions. The associates were suggested
in tryptophan solutions and phenylalanine due to the downward curvatures of quenching
plots of phenylalanine for pyranine and tryptophan for carminic acid. It was shown only
73% of phenylalanine and tryptophan are accessible to the fluorophore to quench the
fluorescence. The excitation wavelength dependence of the tryptophan maximum emission

wavelength support the exists of the associates in tryptophan solutions.

 

 

149

9 . 2 Future work
9.2.1 Structure study of supersaturated solutions

Due to the importance of the supersaturation to crystallization , the structure of
supersaturated solutions is always an important subject. The fluorescence quenching
technique is shown as a powerful tool for system study. So much useful information can
be obtained from such a simple technique. The fluorescence quenching technique should
be paid much attention due to its high sensitivity to the interaction. Some systematic
research can be conducted to investigate the reason for such different quenching behaviors
by different molecules. By choosing different structures of molecules, such as some
molecules with the similar structure as tryptophan. nicotinic acid and phenylalanine, we can
tell what is the main reason for the different quenching behavior, then the different
interactions will be interpreted. Suggested molecules are shown in Figure 9.1 which have
similar structure as tryptophan or phenylalanine. Using these molecules as target molecules
to study the different quenching then to obtain the information about interactions.

The interaction of molecules will change the properties of the molecule somehow.
So any techniques which can detect the change due to the interaction can be employed as
tools to study the structure of solutions. Due to the improvement of the sensitivity of old
techniques, such as Raman, FITR and UV-Vis, they may be utilized as the analytical
techniques to detect the interactions in solutions because the interaction may change the
vibration frequency. The change of the vibration frequency can be used as the evidence to
interpret the interaction of molecules. The same material in solid and solution phase will
have different Raman shifts which may be provide some information about the structure.
New method may be developed for this purpose.

Time resolved fluorescence is always a very powerful technique to study the

interaction of molecules. Measurements of the rotational transport properties of the

 

150
: NH NH
CHZCOOH CH2CCH3
indoleacetic acid 3-indolylacetone
NH HOCHCHZNHZ
@ ‘
N
CHZTHCOOH
NH2
tryptazan phenylethanolarnine
N
I \
/
CHZOH
nicotinyl alcohol

Figure 9.1. Suggested molecules can be used to study fluorescence
quenching systemically as compared to tryptophan, phenylalanine and nicotinic acid

 

 

1 5 1
fluorophores, obtained from fluorescence depolarization techniques, are highly sensitive to

molecular scale interactions (1,2). The time dependent anisotropy measurements result in
the direct quantization of rotational correlation times (1). The use of time resolved
fluorescence methods to investigate the microscopic interactions between the solvent and
solute and the supersaturated solution behavior should result in a more directed and rational
approach to understand and control the crystallization. So more researches should be
conducted by using time resolved fluorescence technique. The contact complex model can
be invested by anisotropy measurements because the contact complex will slow the rotation

rate of probe.

9.2.2. Flow Injection Analysis (FIA)

The automation of chemical methods is necessary for cost. safety and reliability.
Conventional batch operations are not cost effective. In general, batch methods require
highly skilled chemists to carry out the individual tasks and the reproducibility for batch
Operation is low (3). Batch analysis always involve multiple stages of careful operation,
with frequent transfers of solutions from one vessel to another, and thus exposing the
sample to serious contamination risks. Also during the transfer it is very easy to cause
some changes, which makes the result different from the true situation (4).

Despite the obvious drawbacks of the manual batch procedure, little has been
attempted for its automation, presumably owing to difficulties in designing efficient
automated procedures. The difficulties in on-line continuous manipulation of a
heterogeneous system come from potentially serious blockage problems.

The inception of FIA (flow injection analysis) is the result of a long search for
better laboratory techniques in solution manipulation which could match the efficiency of
the computer age. FIA was found to be powerful technique, not only for performing serial
analysis, but also for separation operations. FIA is a non-chromatographic flow analysis

technique for quantitative analysis, performed by reproducibly manipulating sample and

 

“-1

 

152
reagent zones in a flow under thermodynamically non-equilibrated conditions. Two basic

features of FIA are: 1) reproducible manipulation of sample and reagent zones through
precise timing; 2) quantitative evaluation of analyte concentration under thermodynamically
nonequilibrated conditions (4). FIA methods exhibit extremely favorable features over
batch analysis. They are high sample throughput, high enrichment efficiencies for
preconcentration systems, low sample consumption, low reagent consumption, low
contamination risks owing to closed and inert separation system, and high reproducibility.
The most important is that simple automated operation which allows implementation with
continues monitoring systems and use in process control (4)

A schematic diagram of basic FIA system showing the various components are
shown in Figure 9.2. In this traditional FIA system the sample is added by pulses and
reagent is continuously pumped. In our system the amount of sample available is large, so
the design of such a system is a little different from the traditional way (3). In our system
the reverse flow injection analysis (rFIA) is utilized. The main difference between FIA and
rFIA is that in rFIA the samples are continuously pumped and the reagent which is probe in
our case is injected(The schematic diagram of our system is shown next). The other
differences of our system from conventional FIA:

1) Size of sample--sizes of sample are large in our system

2) Not filtered-mo filtration is needed for our system

4) Recycle--after the analysis the solution can go back to crystallizer so there is no waste.
5) Slurry scattering-may be useful for measuring the amount of crystals

The advantage of using FIA for fluorescence based measurements is that the
performance of fluorimetric detectors can be enhanced significantly. FIA stabilizes the
solution conditions thereby increasing precision. The reproducibility of fluorescence
measurements are reportedly improved by better conuol of the reaction conditions in the
FIA systems, and selectivity may be enhanced by on-line removal of potential quenching

interference using FIA separation techniques (3,4).

 

153

 

: T #5/ H_.._,w

—

 

 

 

P: pump; C: carrier; R: reagent; S: sample injector; F: Filter
M: mixing(reaction) coil; D: flow through detector; W: Waste.

 

 

 

 

 

   

 

C: crystallizer; P: pump; S: crystallization slurry; R: probe solution;
M: mixing coil; D: fluorometer; A: remove probe;

Figure 9.2 A schematic diagram of basic flow injection analysis (FTA) system and
reverse flow injection analysis (rFIA) showing the various components

 

 

 

154
In experiments crystallization solution is pumped through the tube and mixes with

probe solution, the mixture flows through fluorolog spectrometer to measure the PIR,
therefore supersaturation can be obtained. A crystallizer condition, such as temperature,
can be changed to control the supersaturation as low as possible in order to obtain the
desired CSD. The process is flowing all the time, and the supersaturation can be measured

in-situ in real time. Using FIA, in-situ monitoring the crystallization can be realized.

9.3. References

1. V. R. Kaufman, D. Anvir, D. Pines-Rojanski, and D. Huppert,
J. Non-crystalline Solids, 99, 379-386 (1988)
2. J. C. Pouxviel, B. Dunn, and J. I. link, J. Phys Chem, 93, 2134-2139 (1989)
3. B. Karlberg, and G. E. Pacey, Flow Injecrion Analysis-a Practical Guide,
Elsevier0989)
4. Z. L. Fang, Flow Injection Separation and Preconcentration, VCH0993)

Appendix

Appendix

Table A1 Raw data for Figure 2.4

Concentration of Peak Intensity Ratio Peak Intensity Ratio

tryptophan (g/100ml (PIR) of 382/510 (PIR) of 401/510

 

11,0)
0.510 0.290 0.194
0.588 0.285 0.200
0.692 0.286 0.208
0.812 0.302 0.233
0.934 0.320 0.254
1.02 0.342 0.283
1.10 0.369 0.308
1.20 0.388 0.337
1.32 0.432 0.388
1.43 0.474 0.433
1.53 0.530 0.491
1.65 0.575 0.543
1.73 0.630 0.595
1.89 0.716 0.696

155

 

 

156

Table A2 Raw data for Figure 2.5

Concentration of nicotinic acid (g/100ml 1120) Peak Intensity Ratio (PIR) (428/510)

0.00 0.101
0.320 0.145
0.836 0.232

1.04 0.274

1.38 0.358

1.74 0.465

1.91 0.561

2.24 0.653

2.46 0.705

2.71 0.839

Table A3 Raw data for Figure 2.6

Concentration of bezoic acid (g/ 100ml H20) Peak Intensity Ratio (PIR) (382/509)

 

0.000 0.612
0.056 0.454
0. 121 0.499
0.248 0.553
0.320 0.601
0.412 0.659
0.462 0.732
0.556 0.803

157

 

Table A4 Raw data for Figure 3.3
Trp PIR Phe PIR NA PIR

0.634 0.307 0.766 0.196 0.460 0.501
0.820 0.332 1.088 0.201 0.694 0.547
0.986 0.348 1.636 0.204 0.844 0.556
1.129 0.369 2.156 0.214 1.058 0.563
1.368 0.411 2.452 0.217 1.322 0.594
1.624 0.476 2.704 0.212 1.520 0.621
1.784 0.471 2.874 0.207 1.790 0.625
1.880 0.513 3.168 0.207 2.052 0.636

3.260 0.214 2.234 0.658

3.456 0.217 2.652 0.683

 

3.690

0.219

 

 

158

Table A5 Raw data for Figure 3.4

Reproducibility of immobilized pyranine in tryptophan solutions

Concentration of PIR Conecntration of PIR
tryptophan (1 1/ 12) tryptophan (1 1/ 18)

0.00 0.1840 0.634 0.3074
0.214 0.2295 0.820 0.3321
0.394 0.2526 0.986 0.3479
0.640 0.2636 1.128 0.3696
0.832 0.2957 1.368 0.41 13
1.004 0.3717 1.624 0.4757
1.128 0.3522 1.784 0.4706
1.308 0.3899 1.880 0.5131
1.400 0.4185
1.892 0.5259

Reproducibility of immobilized pyranine in nicotinic acid solutions

Concentration of
nicotinic acid (01/20)

0.00
0.460
0.694
0.844
1.058
1.322
1.520
1.790
2.052
2.234
2.652

PIR

0.2228
0.5012
0.5472
0.5556
0.5632
0.5933
0.62 10
0.6249
0.6365
0.6576
0.683 1

Concentration of
nicotinic acid (01/28)

0.000
0.666
0.910
1.124
1.286
1.524
1.774
1.938
2.142
2.348

PIR

0.2016
0.531 1
0.5686
0.5748
0.6003
0.6102
0.6413
0.6554
0.6773
0.6699

 

159

Table A6 Raw data for Figure 4.3

Concentration of tryptophan Intensity at 375nm Intensity at 395nm
(g/IOOmI H20)

0.068 4.7900e+05 3.0300e+05
0. 137 3.2000e+05 2.0600e+05
0.216 2.2700e+05 1.5000e+05
0.296 1.8300e+05 1.2500e+05
0.390 1.4800e+05 l.0700e+05
0.522 l.2600e+05 97027

0.588 1.2500e+05 98512

0.710 1.1600e+05 96381

0.818 1.1400e+05 98811

0.906 1.1200e+05 1.0000e+05
1.020 1. 1400e+05 1.0500e+05
1.112 l.1300e+05 1.0800e+05
1.222 1. 1600e+05 1.1200e+05
1.428 1.2400e+05 1.2000e+05
1.528 1.2400e+05 1.2300e+05
1.612 1.2600e+05 1.2800e+05
1.700 1.2400e+05 1.2700e+05
1.828 1.3100e+05 1.3400e+05

0.068

0.1367

0.216
0.296
0.390
0.522
0.588
0.710
0.818
0.906
1.020
1.1 12
1.222
1.428
1.528
1.612
1.700
1.828

Table A7

Concentration of tryptophan
( g/ 100ml HzO)

160

Raw data for Figure 4.4

PIR (395/375)

0.631
0.645
0.661
0.685
0.724
0.768
0.787
0.829
0.867
0.895
0.922
0.950
0.966
0.991
1.017
1.024
1.024
1.039

161

Table A8 Raw data of Figure 4.6

Concentration of phenylalanine Fluorescence intensity at 595nm

<g/100m1 H20)
0.212 3.253le+05
0.228 3. 5235e+05
0.276 4. 2897e+05
0.652 6. 4569e+05
0.940 6. 593 le+05
1.420 9.03 12e+05
1.967 1.2616e-t-06
2.400 1.3453e+06
2.950 1.4850e+06
3.585 1.698364%
4.217 1.97856+06

Table A9 Raw data of Figure 4.7

Concentration of tryptophan Fluorescence intensity at 595nm
(g/ 100ml HZO)
0.243 3.5910e+05
0.420 2.5895e+05
0.616 2.3754e+05
0.852 2. 1484e+05
1. 166 1.9538e+05
1.266 1.9151e+05
1.434 1.8342e+05
1.660 1.7880e+05
1.890 1.7131e+05

162

Table A 10 Raw data for Figure 4.8

Concentration of tryptophan S-V at 375nm S-V at 395nm
(g/100ml P120)
0.000 0.000 0.000
0.068 1.128 1.116
0.137 2.190 2.104
0.216 3.489 3.264
0.296 4.577 4. 108
0.390 5.911 4.993
0.522 7.070 5.596
0.588 7.147 5.497
0.710 7.770 5.640
0.818 7.951 5.477
0.906 8.115 5.394
1.020 7.971 5.107
1.112 8.011 4.954
1.222 7.778 4.699
1.340 7.199 4.325
1 .428 7. 252 4.225
1.528 7.128 4.016
1.612 7.246 4.051
1.700 6.774 3.766
1 .828 6.522 3.542

 

163

Table All Raw data for Figure 4.9
Concentration of tryptophan Reciprocal intensity at 595nm
(g/IOOmI P120)
0.243 2.785e-06
0.420 3.862e-06
0.616 4.210e-06
0.852 4.655e-06
1.166 5.1 18e-06
1.266 5.222e-06
1.434 5.452e-06
1.660 5.593e-06
1.890 5.837e-06
Table A12 Raw data for Figure 4.10
Reciprocal concentration of tryptophan S-V-II at 595nm
2.380 4.089
1.624 3.257
1.174 2.679
0.858 2.325
0.790 2.266
0.698 2.151
0.602 2.090
0.529 1.999

0.000
0.079
0.180
0.220
0.290
0.422
0.510
0.588
0.692
0.812
0.934
1.020
1.096
1.196
1.316
1.426
1.532
1.648
1.730

Table A 13

Concentration of tryptophan
( g/ 100ml 1-120)

164

Raw data for Figure 5.1

S-V at 382nm

0.000
0.448
0.839
1.316
1.807
2.558
3.034
3.470
3.863
4.243
4.552
4.664
4.640
4.717
4.638
4.637
4.424
4.41 1
4.277

 

 

165

Table A 14 Raw data for Figure 5.2
Concentration of tryptophan Reciprocal intensity at 510nm
(g/ 100ml H20)
0.079 2.688e-06
0.180 2.703e-06
0.220 2.890e-06
0.290 3.086e-06
0.422 3.584e-06
0.510 3.876e-06
0.588 4.219e-06
0.692 4.608e-06
0.812 5.236e-06
0.934 5.882e-06
1.020 6.410e-06
1.096 6.897e-06
1. 196 7.353e-06
1.316 8.065e-06
1.426 8.850e-06
1.532 9.524e-06
1.648 1.031e-05
1.730 1.101e-05
1.888 1.264e-05

 

0.000
0.224
0.306
0.420
0.522
0.632
0.732
0.860
1.008
1.388
1.410
1.622
1.852
2.024
2.216
2.424
2.598
2.842
3.036
3.240
3.440

Table A 15

Concentration of phenylalanine
(g/ 100ml H20)

166

Raw data for Figure 5.3

S-V at 382nm

1.000
1.128
1.084
1.296
1.275
1.316
1.355
1.431
1.475
1.622
1.624
1.697
1.745
1.831
1.873
1.913
1.962
2.013
2.084
2.127
2.162

1.916
1.582
1.366
1.163
0.992
0.720
0.709
0.617
0.540
0.494
0.451
0.413
0.385
0.352
0.329
0.309
0.291

0.000
0.214
0.322
0.464
0.698
0.848
1.080
1.334
1.494
1.796
1.960
2.272
2.420

Table A16

Reciprocal Concentration of phenylalanine

Table A17

Concentration of nicotinic acid

167
Raw data for Figure 5.4

Raw data for Figure 5.5

S-V at 382nm

S-V III at 382nm

4.631
4.166
3.817
3.320
3.104
2.609
2.601
2.435
2.343
2.203
2.145
2.095
2.040
1.987
1.923
1.887
1.861

0.000
2.584
4.409
6.995
12.87
16.99
25.62
33.97
41.06
51.58
58.48
67.35
75.73

 

 

0.320
0.836
1.040
1.384
1.744
1.910
2.244
2.456
2.708

0.000
0.214
0.322
0.464
0.698
0.848
1.080
1.334
1.494
1.796
1.960
2.272
2.420

Table A 18

Concentration of Nicotinic acid

Table A19

Concentration of nicotinic acid

168

Raw data for Figure 5.6

S-V

Raw data for Figure 5.7

S-V II at 382nm

I at 382nm

14.09
22.78
26.00
31.19
35.67
38.52
39.55
39.81
39.54

1.000
3.255
4.679
6.488
10.13
12.28
16.38
19.18
21.47
23.43
24.62
24.59
25.82

 

 

169

Table A20 Raw data for Figure 5.8
Concentration of nicotinic S-V at 510nm 342nmEx S-V at 510nm 360nmEx
acid (g/100ml 1120)
0.000 0.000 0.000
0.214 1.430 1.421
0.322 2.815 2.735
0.464 5.110 4.974
0.698 10.87 10.39
0.848 16.37 15.52
1.080 26.06 25.48
1.334 40.69 39.49
1.494 50.91 49.62
1.796 78. 19 74.52
1.960 93.64 91.23
2.160 105.6 112.3
2.272 117.1 125.0
2.420 140.4 142.0
Table A21 Raw data for Figure 6.2

Concentration of tryptophan Lifetime S-V at 510nm

0.000 1.000

0.151 1.085

0.232 1.132

0.454 1.238

0.638 1.295

0.820 1.365

0.956 1.418

1.030 1.429

1.268 1.502

1.396 1.518

1.592 1.564

1.810 1.602

 

 

0.000
0.210
0.354
0.616
0.786
0.964
1.102
1.318
1.632
1.894

Table A22

Concentration of nicotinic acid

170

Raw data for Figure 6.2

Lifetime S-V at 510nm

1.000
1.836
2.253
2.977
3.463
4.083
4.517
5.428
6.441
7.638

.T

171

Table A 23 Raw data of Figure 6.3

Concentration of S-V at 510nm Concentration of Lifetime S—V at
phenylalanine phenylalanine 510nm
(g/IOOmI Hp) (g/100m1 H20)

0.000 1.000 0.000 1.000
0.462 1.033 0. 290 0.998
0.758 1.040 0.492 0.999
1.132 1.010 0.720 0.984
1.592 1.066 0.944 0.984
1.946 1.079 1.144 0.980
2.234 1.062 1.300 0.974
2.404 1.132 1.550 0.980
2.524 1.105 1.694 0.976
2.622 1.125 1.956 0.971
2.720 1.113 2.196 0.975
2.806 1.122 2.414 0.962
2.896 1.125 2.596 0.970
3.022 1.141 3.060 0.963
3.142 1.125 3.234 0.964
3.400 1.185
3.602 1.146

 

172

Table A 24 Raw data of Figure 6.4

Concentration of S—V at 382nm Concentration of Lifetime S-V at
tryptophan tryptophan 382nm
(g/100m1 Hp) <g/100m1 H20)

0.000 0.000 0.000 0.000
0.079 0.448 0.206 0.927
0.180 0.839 0.308 1.332
0.220 1.316 0.460 1.879
0.290 1.807 0.660 2.784
0.422 2.558 0.904 3.885
0.510 3.034 1.096 4.751
0.588 3 .470 1.224 5.176
0.692 3.863 1.422 5.682
0.812 4.243
0.934 4.552
1.020 4.664
1.096 4.640
1.196 4.717
1.316 4.638
1.426 4.637
1.532 4.424
1.648 4.41 1
1.730 4.277
1.888 4.334

173

Table A 25 Raw data of Figure 6.5

Concentration of S—V at 382nm Concentration of Lifetime S-V at

nicotinic acid nicotinic acid 382nm
(g/ 100ml 1120) (g/ 100ml 1-120)
0.000 0.000 0.000 0.000
0.214 2.584
0.322 4.409 0.087 0.261
0.464 6.995 0. 145 0.522
0.698 12 87
0.848 16.99 0.216 0.904
1.080 25.62 0.352 1.450
1 334 33.97
1.494 41.06 0.462 1.929
1.796 51.58 0.686 2.749
1 960 58.48
2 160 60.09
2 272 67.35
2 420 75.73
Table A 26 Raw data of Figure 6.6
Concentration of S-V at 382nm Concentration of Lifetime at
phenylalanine phenylalanine 382nm
(g/100m1 HzO) (g/100ml 11,0)
0.000 0.000 0.000 0.000
0.310 ’ 0.293
0.462 0.312 0228 0' 198
0.758 0.496 0.544 0.252
1.132 0.664
1. 592 0.827 0.718 0.264
1.946 0.941 0.912 0.295
2.234 1.010
2.404 1.159 ”’96 0309
2.524 1.139 1.248 0.330
2.622 1.183
2.720 1.142 1.574 0.326
2.806 1.192 1.854 0.323
2.896 1.174
3.022 1.269 2'024 “328
3.142 1.225 2.316 0.310
3.400 1.382
3.602 1.346

174

Table A27 Raw data of Figure 7.2
Concentration 300nm Ex 305nm Ex 308nm Ex 310nm Ex
of tryptophan
0.062 1.8886e+05 2.0247e+05 1.2050e+05 76216
0.0787 1.6750e+05 2.3890e+05 1.5689e+05 1.0960e+05
0. 124 99470 2.6750e+05 2. 1390e+05 1.5880e+05
0.236 32924 2. l293e+05 2.6068e+05 2. 1527e+05
0.322 23159 1.5571e+05 2.5484e+05 2.3180e+05
0.504 77477 2. 1596e+05 2.4894e+05
0.704 21253 42269 1.6130e+05 2.2780e+05
0.882 20464 28521 1. 1661e+05 2.0090e+05
1.054 19050 22627 88747 l.7453e+05
1.270 16909 17316 61175 1.4330e-1—05
1.550 17741 16885 41082 1.0720e+05
1.780 16444 17500 28469 83971
Table A28 Raw data of Figure 7.3
Concentration of 320nm Ex 330nm Ex 340nm Ex 350nm Ex
tryptophan
0.062 13067 201 11 19430 15639
0.0787 32244 29733 16479 16226
0.124 41225 35099 31437 29154
0.236 47020 34134 27937 23398
0.322 51897 38014 30003 24844
0.504 65829 45257 351 13 27058
0.704 81749 56484 39646 29363
0.882 91910 63039 43695 29459
1.054 1.0122e+05 69543 46903 32522
1.270 1. 1370e+05 79225 52504 32417
1 .550 1.2582e+05 88773 57678 363 15
1.780 1.3195e+05 94932 62964 36918

 

175

Table A29 Raw data of Figure 7.6

Excitation Emission Excitation Emission
wavelength at wavelength at wavelength at wavelength at
unsaturated unsaturated supersaturateconce supersaturated
concentration of concentration of ntration of concentration of

tryptophan tryptophan tryptophan tryptophan
300.00 357.00 308.00 357.00
302.00 357.00 310.00 358.00
305.00 356.00 312.00 360.00
307.00 358.00 315.00 366.00
309.00 357.00 318.00 376.00
312.00 360.00 320.00 380.00
315.00 363.00 325.00 386.00
318.00 373.00 330.00 387.00
320.00 379.00 335.00 391.00
325.00 384.00 340.00 393.00
330.00 386.00 350.00 398.00
333.00 388.00 355.00 404.00
334.00 390.00 360.00 411.00
335.00 380.00 365.00 416.00
340.00 384.00 370.00 422.00
345.00 390.00 375.00 429.00
350.00 397.00 380.00 434.00
355.00 404.00 385.00 444.00
360.00 411.00 390.00 452.00
365.00 417.00
370.00 422.00
375.00 429.00
380.00 435.00

176

Continue of Table A 29

Excitation wavelength at saturated Emission wavelength at saturated

concentration of tryptophan concentration of tryptoplmn
300.00 357.00
305.00 358.00
308.00 357.00
310.00 358.00
312.00 361.00
315.00 365.50
317.00 372.50
320.00 380.00
325.00 386.00
330.00 387 .00
335.00 389.00
337.00 392.00
339.00 390.00
340.00 385.00
341.00 387 .00
342.00 390.00
345.00 390.50
350.00 396.00
355.00 401.00
360.00 410.00
365.00 417 .00
370.00 424.00
375.00 431.00
380.00 439.00
385.00 442.00
390.00 451.00
400.00 464.00
410.00 477.00
420.00 490.00

177

Table A30 Raw data of Figure 7.7

Concentration of tryptophan Lifetime in mixture Lifetime in mixture
(g/100ml 1-120) solvent (ns) solvent (ns)
0.356 2.12 5.78
0.506 2.04 6.12
0.724 2.01 5.76
1.048 1.90 4.20
1.490 1.92 3.77

Table A31 Raw data of Figure 8.4

pH of PyO‘ 467nm/402 pH of PyO’leOH in 467nm/402nm

tryptophan IPyOH in run in phenylalanine phenylalanine phenylalanine
solutions tryptophan tryptophan solutions solutions solutions
solutions solutions

7.13 0.427 0.391 6.06 0.126 0.0337

7.15 0.447 0.571 6.73 0.170 0.1934

7.38 0.757 1.852 7.54 1.10 1.368

7.43 0.852 2.069 7.65 1.41 2.033

7.65 1.41 2.517

   

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