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LIBRAML "
Mkhigan State
University

 

 

This is to certify that the

dissertation entitled

 

nuclear Aromatic Hydrocarbon Compounds in
Organized Media

1
Room-temperature Phosphorescence Study of Poly— \
presented by i

l

Hai-Dong Kim

has been accepted towards fulfillment
of the requirements for

Ph.D. degree in Chemistry

 

fl/M/éwa

Major! professor

Date September 12, 1989

 

MS U is an Affirmative Action/Equal Opportunity Institution 0-12771

 

 

 

 

 

 

 

 

 

PLACE IN RETURN BOX to remove this checkout from your record.
TO AVOID FINES return on or before date due.

DATE DUE DATE DUE DATE DUE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

MSU Is An Affirmative Action/Equal Opportunity Institution

 

 

"/1—

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ROOM-TEMPERATURE PHOSPHORESCENCE STUDY OF POLYNUCLEAR
AROMATIC HYDROCARBON COMPOUNDS IN ORGANIZED MEDIA

By

Hai-Dong Kim

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

Department of Chemistry

1989

 

 

 

 

 

(064'013%

 

ABSTRACT

ROOM-TEMPERATURE PHOSPHORESCENCE STUDY OF POLYNUCLEAR

AROMATIC HYDROCARBON COMPOUNDS IN ORGANIZED MEDIA

By

Hai-Dong Kim

Room-temperature phosphorescence spectrometry (RTP) is a
very convenient method compared to conventional low-
temperature phosphorimetry. Although the importance of RTP
has been realized in the analytical world.over the last two
decades, it still needs a number of improvements to be used
in routine chemical analysis. This research was intended to
develop a new RTP methodology using a synthetic enzyme model
compound, N,N,N’,N’,N",N",N’",N’"-octamethy1-2,11,20,29-
tetraaza[3.3.3.3]paracyclophanetetraammonium tetrafluoro-
borate (methyl-APO).

In the first part of the research, a computer-controlled
versatile luminescence spectrometer was develOped with
completely menu-driven software for the control of the
instrument, data acquisition, and data manipulation on a
personal computer. The use of a disk-type chopper gave a

number of advantages over the conventional rotating-can type

 

 

 

 

phosphoriscope. Results are presented to show the
versatility of the developed instrument.

A new decay kinetic model of micellar stabilized RTP (MS-
RTP) is proposed based on triplet quenching on the micellar
surface. The dependence of MS-RTP lifetimes on environmental
conditions was investigated. It was found that heavy atoms
were the major contributor in MS-RTP lifetimes. Also,
solution temperature and the method of deoxygenation affect
observed lifetimes significantly.

The potential analytical applicability of methyl-APO in
RTP was examined. Methyl-APO showed a very strong binding
ability toward anionic and neutral compounds, but did not
induce RTP when used alone. The addition of premicellar
concentrations of surfactant effectively covered both open
ends of the AFC-guest inclusion complex and, thereby,
induced RTP. Methyl-APO also showed more selectivity and
sensitivity than cyclodextrins due to the hydrophobic and

electrostatic interactions involved.

 

 

 

 

ACKNOWLEDGEMENTS

It took a long time to realize the true meaning of "That’s

"

the way of science mentally and physically. Whenever I
sank down hopelessly during my research, other fellows in
the Lab tried to persuade me how science is difficult by
saying "That’s the way of science.". At that time I had no
answer for that, but now, I have an answer for that. "But
it’s worth to try."

I would like to thank sincerely Dr. Stanley R. Crouch and
Dr. Matthew J. Zabik for their warm advice and guidance
throughout my research. Both of you really inspired me to
complete my work. When I joined the Crouch group, there were
already eighteen people hanging around to make me dizzy. I
was really surprised to see Stan’s ability to manage each
kid including me. Dr. Zabik’s Lab was more spacious compared
to: the 'Lab in chemistryu Thanks to Dr. Zabik’s special
attention, I could complete my research in his Lab.

I would like to thank to all group members of both groups.
These include, Peter, Jimmy, Mayda, Steve, Larry, Pavlos,
Kris, Susan, Mary Ann, Gamal, Glen, Ines, Salah, and
everyone else who shared the same adviser. I also thank Dr.
Leavitt and his group members who helped me in many ways
during my research.

Dr. Nelson Herron and Dr. Paul Kraus were the old fellows

Who deserve my deep acknowledgement. We were the same funny

iv

 

 

 

 

guys who tried to catch the tough fruit, RTP. Also, we were
the same guys who shared the same bitterness and fragrance
of that fruit. I still remember a loud shouting session when
Nelson exchanged punches with the famous, old instrument.

A special thanks is reserved for my family: my wife, Sock;
our pretty daughter, Suyoun; our baby son, Junyoung; and my

parents who prayed and waited patiently for my success.

 

 

 

 

 

 

TABLE OF CONTENTS

List Of Tables 0.0.000.000...00.00.00.000...0.0.0..0000.

List Of Figures 0.0.000.00.00.00000....00000.00000000.00

I. IntrOduction.00.0...00.000.000.0000000000000.00.0.0.

References 000.......0.00...000..0.....O0.0000...00..

II. Molecular Luminescence .............................

A. The Absorption and Emission of Light by Molecules .

B. The Effect of Molecular Structure .................

C. Medium Effect 0.00.0.00.000.00.00000000

1. Effect of Temperature and Solvent .

2. Effect of pH and Dissolved Oxygen ...

3. Effect of Solute Concentration ......

4. Effect of Coordination by Metal Ions

D. Quenching of Photoluminescence ........

References .............................
III. Room-Temperature Phosphorescence ....
A. Conditions for RTP Observation ......
B. Methods for RTP Measurement .........
1. Solid State RTP ...................

2. Sensitized RTP ....................

3. Micellar Stabilized RTP ...........

4. Cyclodextrin Enhanced RTP .........

5. Other Methods of RTP ..............

C. Deoxygenation Methods ...............

vi

0....000...

0.00

0...

ix

X

12
14
15
15
16
17
18
21
23
25
27
23
so
33
38
41
43

 

 

 

 

1. Physical Methods of Deoxygenation ..............
2. Chemical Methods of Deoxygenation ..............

D. HeavyAtom EffeCts 0.00.00.0000.0000.000.000.00...

References

0.00.00.00.00000.0......00.00.00.000000000

 

IV. Applications of Macrocyclic Compounds in

ChemicalAnaIYSis 00.000.000.000.00.00.00.000000000
A. Host-Guest Molecular Inclusion ...................
B. Synthetic Enzyme Model Compound: Cyclophanes ....

C. Other Macrocyclic Compounds in Chemical Analysis

References

......0...0.0....0..00.......000000000000

V. Instrumentation .............................

A. Luminescence Spectrometer .................

1. Principle of Operation of the Instrument

2. Hardware Construction ...................

a. Computer Interfacing to Spectrometer ..

b. Light Source Modulation and Detection .

c. RTP and RTP Lifetime Measurement ......

3. Software Development

4. Results and Discussion

B. Modification of IBM-DACA board ............

1. Hardware Modification ...................

a. Hardware Triggered ADC Operation ......

b. Modification of Counter/timer Circuit .

2. Software Development ....................

3. Results and Discussion

C. Conclusions

References

0....00...0.0..000

vii

00.0...000.0000.....0

ooooooooooooo00000000000009.0000...

43
44
45
49

53
55
58
64
70
74
75
76
78
78
82
86
88
90
98
99
99
102
104
107
108

110

 

 

VI. Room-temperature Phosphorescence Lifetimes ......... 112
A. Methods for Determining Decay Lifetimes ........... 114
B. RTP Decay Lifetimes of Organic Compounds in

Micellar Solutions ................................ 121
1. Decay Kinetics Model for MS-RTP ................. 121

2. Experimental Section. ........................... 124

 

3. Results and Discussion .......................... 126
C. Conclusions ....................................... 143
References ........................................... 144
VII. RTP of Organic Compounds in Mixed Organized
Media: Azaparacyclophane—Surfactant System ........ 146
A. Experimental Section

1. Materials and Methods ........................... 148'

 

‘ 2. Synthesis of Azaparacyclophane .................. 149
B. Results and Discussion ............................ 156
C. Conclusions ....................................... 173
References ........................................... 170
VIII. Conclusions and Future Prospects ................. 176
A. Luminescence Instrumentation ...................... 176
B. RTP Decay Lifetimes of PAHs in Organized Media .... 178
C. Application of Synthetic Macrocyclic Compounds..... 180
References ........................................... 182
Appendix
. A. Source Code of TPLIB .............................. 183
B. Source Code of EMISPEC-II ......................... 196
C. Source Code of IBMDAC ............................. 215

Do source cede Of LS-DATAooooooooo09.000000000000000. 224

 

viii

 

 

LIST OF TABLES

Table Title Rage
6.1 MS-RTP Spectral Characteristics of PAHs.......... 133
6.2. MS-RTP Lifetimes of Selected Arenes.............. 135
6.3. The Effect of Temperature on RTP of Pyrene....... 138

6.4. The Effect of Heavy Atom on RTP Lifetime......... 139

ix

 

 

 

301'.

3.2.

4.4.
4.5.
5.1.
5.2.
5.3.

5.4

5.5.

506.

LIST OF FIGURES

1111: 2:82

Jablonskii diagram of a molecule upon excitation.

(A, absorption; F, fluorescence; 10, internal
conversion; 180, inter-system crossing; P, phos-
phorescence; VR, vibrational relaxation).......... 10

Schematic representation of sensitized phos-
phorescence. The analyte (acting as the energy

donor) absorbs light, and the acceptor molecule

emits phosphorescence. (10, internal conversion;

180, inter-system crossing; Sn, singlet state; Tn,
triplet state; Q, quenching)...................... 31

Simplified model representation of the ionic

micelle. The electric double layer is composed of

a Gouy-Chapman layer outside and a Stern layer

inside the shear surface.......................... 35

Simplified representation of B -cyclodextrin
StrUCtureoooooooo00000000000000.0000...on...oooooo 39

Diederich’s water-soluble cyclophanes............. 60
Water-soluble heterocyclophanes................... 61

Schematic representations of modified water-

soluble heterocyclophanes......................... 63
Some representative crown ethers......§........... 65
Lehn’s cryptands.................................. 68
Block diagram of the luminescence spectrometer.... 77

Schematic representation of the Lab Master board.. 79

Interfacing of the control signals from Lab Master
to the Aminco-Bowman spectrometer (SPF-500)....... 81

Circuits built for (A) photodiode light sensor,
(B) current-to-voltage convertor(lower)........... 83

Timing diagram of the chopper phase detector.

The opto-interrupter output is connected to the
multivibrator and the pulse width of the multivib-
rator is adjustable with a variable register...... 85

Timing diagram of the counters for RTP and RTP
lifetime measurement.............................. 87

X

 

 

 

5.7.
5.8.A

5.9

50100A

5.10.3

5.11.

5.12.
5.13.

6.5.

6.6.

Block diagram of the EMISPEC program.............. 89

Fourier smoothing of the luminescence spectrum of
naphthalene in micelle (SDS). Degree of smoothing,
9; dot, raw data; line, smoothed data............. 93

Fourier smoothing of the luminescence spectrum of
naphthalene in micelle (SDS). Degree of smoothing:
25. dot: raw data; line: smoothed data............ 93

Fourier smoothed luminescence spectrum of
naphthalene (1.5x10'5 M). Degree of smoothing: 15.
The "end-effect" was corrected at both ends....... 95

Excitation-emission spectrum of pyrene (1.5x10'6 M)
in micelle (SDS 0.1 M). Excitation scan range:
280-380 nm; emission scan range: 300-500 nm....... 97

Excitation-emission spectrum of naphthalene at
room temperature. Excitation scan range: 180-360 nm;
emission scan range: 280-480 nm................... 97

Modification of the timer/counter circuit of
the IBM-DACA000000.00000.000.00.00.00.00000000000 101

Jumper board pin layout........................... 103

Sine wave (6250.us/Cycle) captured in mode 0

(A,line), in mode 3 (A,dotted line), in mode 9

with trigger pulse interval of 27.ns (B,dotted

line) and 62 us (B,line).......................... 106

Schemetic representation of typical RTP decay
curve and data acquisition process................ 115

RTP development of 2-bromonaphthalene with time... 127

The effect of sodium sulfite and SDS on the RTP
intensity of naphthalene.......................... 129

Excitation and emission spectra of 2-bromo-
naphthalene (A,C) and pyrene (B,D). A and B are
excitation spectra and C and D are emission
spectra........................................... 131

Excitation and emission spectra of naphthalene
(A,C) and biphenyl (B,D). A and B are excitation
spectra and C and D are emission spectra.......... 132

MS-RTP decay of 5x10'5 M naphthalene (upper) and
pyrene (lower). Solutions were deoxygenated with
0.01 M sodium sulfite, and the concentrations of
SDS were 0.05 M for both, the concentrations of T1’

 

 

 

 

6.7

7.1

7.5

7.1

70!

 

 

7.1.

7.2

7.3

7.4.

7.5.

7.6.

7.7.

7.8.

7.9.

7.10.

were 0.04 M for naphthalene and 7.5x10'3 M for
Pyrene.‘O.......OOCOOCCO......OOOCOOOOOOOOOCOOO0.0 137

Plot of observed decay rate constant of pyrene
versus heavy atom (Tl‘) concentration in SDS
micelle....O...00...............OOOOOOOOOOOOOCOOO. 141

Synthesis of N,N,N’,N,’,N",N”,N’",N’"-octamethyl-
2,11,20,29-tetraaza[3,3,3,3]paracyclophanetetra-.
ammoniumtetrafluoroborate......................... 151

NMR spectrum N,N’,N",N’"-tetramethyl-Z,11,20,29-
tetraaza[3,3,3,3]para cyclophane.................. 154

Mass Spectrum N,N’,N",N’"-tetramethyl-2,11,20,29-
tetraaza[3,3,3,3]para cyclophane.................. 155

Simplified structural representation of the host
molecule, methyl-APC (upper), and anionic guest
meleculeSOOOO0..0..0.00.00........OOOOOOOOOOOOOOOO 157

Fluorescence enhancement of ANS (2.5)(10‘5 M,
excitation=375nm, upper spectrum) and TNS (2.5X10‘5

M, excitation=337nm, lower spectrum) on addition of
1X10'4 M of 8 -cyclodextrin (dotted line) and methyl-
APC (upper solid line)............................ 160

Double reciprocal plot for methyl-APC-ANS complex..162

RTP spectrum of TNS (2.5x10‘5 M, upper spectrum)

and 2-bromonaphtha1ene (2.5x10‘5 M, lower spectrum)
upon addition of 0.1 mM of B-CD(dotted line) and
methyl-AFC (upper solid line). The lower solid line
in both spectra were obtained with SDS'only.
Excitation: TNS=337 nm; ANS=293 nm. .............. 164

The effect of host (upper graph) and SDS (lower
graph) on the RTP intensity of TNS (2.5x10'5 M)... 167

RTP development of 2-bromonaphthalene (5X10"5 M)

with 0.02 M sodium bromide (lower line) and 0.02 M
thallium nitrate (upper line). [NazSOa]=0.04 M;
[SDS]=5 mM; [APC]=0.1 mM. Excitation=293 nm;
emission=525 nm................................... 169

RTP decay of TNS (51(10'5 M) in 5 mM SDS and 0.02 M
TlNOa; dotted line, no host; bottom line, on

addition of 0.2 mM BeCD;upper line, on addition of
002 mM methYI-APCOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO 172

xii

 

 

 

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CHAPTER I
INTRODUCTION

Luminescence spectrometric methods are among the most
popular analytical methods. Luminescence spectrometric
methods allow a greater sensitivity and selectivity in
chemical analysis than absorption spectrometry. Also,
luminescence spectrometry provides a lower limit of
detection and a larger linear dynamic range compared to the
absorption spectrometry.

Room-temperature phOSphorimetry (RTP) is quite different
from the classic low temperature phosphorescence technique
which is typically performed in glass matrices at liquid
nitrogen temperature (1). Since the discovery of RTP,
numerous techniques have been developed to induce RTP from
various molecules. These include solid-state RTP (2),
micellar-stabilized RTP (3): sensitized RTP (4),
yclodextrin enhanced RTP (5), and colloidal or
icrocrystalline RTP (6). The probability for observing RTP
(especially in solutions) is enhanced in a rigid molecular
nvironment due to reduced quenching by oxygen or other
'mpurities and in the presence of a heavy atom due to an
ncrease in the rate of intersystem crossing.

Photophysical and photochemical properties of organic

olecules included in the cavity of cyclodextrins (CDs) have

 

 

 

 

 

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been of considerable interest in the past decade (7).
Cyclodextrins form complexes with hydrophobic organic and
organometallic molecules in aqueous solution. Although there
is no direct proof for a fixation of the guest molecules
within the void space of the cyclodextrin, the complexes are
usually regarded as inclusion compounds, host-guest
compounds, in which hydrogen bonding, van der Waals forces,
and hydrophobic interactions are the main binding forces
(8). When lumiphores are included inside the cyclodextrin
molecules, the resulting RTP shows enhanced intensity and
lifetime because the cyclodextrins protect the lumiphores
from quenchers (9).

The synthetic macrocyclic enzyme model compounds,
azaparacyclophanes (APCs) can act as inclusion hosts capable
of molecular organization by forming complexes ,with a
variety of hydrophobic molecules (10). Water-soluble
azaparacyclophane, N,N,N’,N’,N",N",N’",N’"-octamethyl-2,11,
20,29-tetraaza[3.3.3.31paracyclophanetetraammonium tetra-
fluoro-borate (mehtyl-APC), is an excellent inclusion host
toward certain organic substrates. A unique substrate
specificity was observed due to its cavity size and
functionality (11). The macrocyclic cavity is surrounded by

he wall which is formed by four benzene rings and four

uarternary ammonium residues around the macrocyclic ring.
The objectives of this research are divided into three

arts. The first objective of this research was to develop a

ersatile computer-controlled luminescence instrument for

 

 

 

measuring fluorescence, RTP, and RTP lifetimes. The second
objective was to study the decay kinetics for micellar
stabilized RTP in order to understand the mechanism of
triplet quenching in micellar solution. The last objective
of this research was to develop a new RTP method using a
synthet ic enzyme model compound , methyl-APO . This
dissertation presents work performed mainly on these three
projects.

The entire dissertation is divided into eight chapters
plus appendix. Following introductory remarks in this
chapter , the fundamental background 0 f molecular
luminescence is described in chapter II. This chapter is
divided into four main categories: the absorption and
emission of light; the effect of molecular structure; the
effect of medium condition; and, quenching of luminescence.
This background is very important for understanding the RTP
phenomenon.

In chapter III, recent developments in RTP .are reviewed.
Various conditions necessary for the observation of RTP are
discussed first. A brief historical survey of RTP and
various methods for RTP measurements develOped so far are
described in the following part. The last two parts discuss
the essential requirements to observe RTP in solution,
deoxygenation methods and the heavy atom effect.

Applications of macrocyclic compounds in chemical analysis
are discussed in chapter IV. The host-guest molecular

inCIUSion phenomenon is discussed first. The general

 

 

 

 

overview of synthetic enzyme model compounds,
azaparacyclOphanes, in host-guest chemistry and their
applications are presented in the second part. The last part
reviews various macrocyclic compounds currently used in
chemical analysis.

The deve10pment of a computerized RTP instrument for this
research is described in chapter V. This chapter is divided
into two broad parts: the luminescence spectrometer and a
modification of the IBM-PC data acquisition board. The first
part describes the general principles of the new instrument,
computer interfacing, excitation source modulation and
detection for time-resolved spectrometry, the use of the
instrument for RTP and RTP lifetime measurements, and the
software developed. for instrument control and data
acquisition are also discussed. The second part describes a
modification of the IBM-PC data acquisition board used later
in this research with the Perkin-Elmer LS-5B spectrometer.

The RTP lifetime measurements and the study of decay
kinetics in micellar-stabilized RTP are presented in chapter
VI. Various methods for determining RTP lifetimes are
discussed first. And then, a new decay kinetics model in
micellar stabilized RTP is pmoposed. In the last part, RTP
lifetime data for polyaromatic hydrocarbon molecules,
pyrene, naphthalene, biphenyl, and 2-bromonaphthalene are
presented. Also various factors affecting the observed RTP

lifetime are discussed with the experimental data.

 

 

 

 

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Chapter VII presents RTP data of organic compounds in
mixed organized media: the azaparacyclophane-surfactant
system. The strength of molecular association of
azaparacyclophane with anionic and neutral compounds in
solution is compared to cyclodextrin. The factors affecting
RTP in this system as well as an analytical figure of merit
are discuSsed along with the experimental data.

Conclusions and future prospects on instrumentation,
application of synthetic macrocyclic compounds, and RTP
decay lifetimes in chemical analysis are described in the
final chapter. Source codes for the software developed

during this research are presented in the appendix.

 

 

 

 

 

CHAPTER I REFERENCES

(1) Hurtubise, R. J. Anal. Chem. 1983, 55, 669A.

(2) Ward, J. L.; Walden, G. L.; Winefordner, J. D. Tulanta
1981, 28, 201.

(3) Cline Love, L. J.; Skrilec, M.; Habarta, J. G. Anal.
Chem. 1980, 52, 754.

(4) Donkerbroek, J. J.; Elzas, J. J.; Goojier, C.; Frei, R.
W.; Velthorst, N. Thlanta 1981, 28, 717.

(5) Scypinski, S.; Cline Love, L. J. Anal. Chem. 1984, 56,
322.

(6) Weinberger, R.; Cline Love, L. J. Appl. Spectrosc. 1985,
39, 516.

(7) Ramamurthy, V.; Eaton, D. F. Acct. Chem. Res. 1988, 21,
300.

(8) Cramer, F.; Mackensen, G. Angew. Chem. Intern. Ed. Engl.
1966, 5, 601.

(9) Turro, N. J.; Bolt, J. D.; Kuroda, Y.; Tabushi, I.
Photochem. Phatobiol. 1982, 35, 69.

(10) Tabushi, I.; Kuroda, Y}; Kimura, Y. 7btrahedron Lett.
1976, 37, 3327.

(11) Tabushi, I.; Kimura, Y.; Yamamura, K. J} Am. Chem. Soc.

981 , 103, 6486 .

 

 

 

 

 

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CHAPTER II
MOLECULAR LUMINESCENCB

The emission of light by electronically excited molecules
is the basis of molecular luminescence spectroscopy.
Photoluminescence originates from atoms and molecules that
are excited by an external light source, as opposed to
bioluminescence, chemiluminescence, and electroluminescence,
which are excited by a biological process, by a chemical
reaction, and by electrical energy, respectively.
Fluorescence and phosphorescence are the two major types of
luminescence which occur in molecules as a. result of
photophysical process after absorption of light.
Fluorescence is a radiative transition from the lowest
excited singlet state in: the ground state. Phosphorescence
is the result of a radiative transition from the lowest
excited triplet state to the ground state.

This chapter describes the nature of the photophysical
processes of photoluminescence. The theoretical aspect of
Photoluminescence is discussedyfirst. The next two parts
deal with the dependence of photoluminescence upon molecular
structure and. the molecular environments. The last part
briefly discusses quenching effects for the two major types

of photoluminescence, fluorescence and phosphorescence.

 

 

 

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A. Absorption and Emission of Light by Molecules.

A light wave may be thought of as electromagnetic
radiation traveling with a speed of 3.0 x 1010 cm/s. When a
molecule is exposed to electromagnetic radiation, it can
absorb a photon of that radiation. The absorption or
emission process entails the interaction of the electric
field associated with the exciting light with the loosely
bound p or nonbonded electrons of the absorbing molecule.
This interaction distorts the electronic distribution of the
absorbing molecule and causes energy to be absorbed from the
electric field of the exciting light wave.

Electronic absorption entails the promotion of an
electron, by the absorption of energy, from an originally

occupied bonding or nonbonding orbital to an originally

  
  
   
  
  
  
   
  
  

unoccupied molecular orbital. Because 6 electrons are
usually bound very tightly by the molecule, a great deal of
energy (which is beyond the conventional luminescence

spectroscopic region) is required to promote these electrons

 

to vacant molecular orbitals. 0n the other hand, delocalized
I electrons are not as tightly bound as J electrons.
Hence, their' electronic 'promotion do not 'require higher
energy than do the d electrons. The nonbonding electrons are
nly slightly lower in energy than atomic valance shell
lectrons. As a result, the energy gap between. the n-
rbitals and vacant ‘l’ orbitals is very small (1). In most

rganic molecules of spectroscopic interest it is only the J

 

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electrons and n electrons which are involved in electronic
transition to vacant 1 molecular orbitals.

The amount of light absorbed by the molecules is given by

the Beer-Lambert law:

Po
A=log(-——)=&bc (2.1)
P

where A is the absorbance, Po and P are the incident and
transmitted light intensity, respectively, t is the molar
absorptivity of the molecule, and c is the molar
concentration of the absorber in light path length b. To
utilize the above equation properly there are several

requirements: the solution must be sufficiently dilute so

 

that changes in the refractive index are negligible, the
radiation must be monochromatic, and stray light must be
negligible. Quantum mechanically the electronic excitation
of particular molecular species occurs only if the exciting
radiation energy corresponds to the difference in energy

between the electronically excited state and the ground

 

state of the absorber.

There are several vibrational levels within each
electronic state in molecules. In the ground electronic
state, almost all molecules occupy the lowest vibrational
level at room temperature. When a molecule absorbs a
particular frequency of radiation it can be excited to one
of several vibrationally, as well as electronically excited

energy levels (Figure 2.1). If the absorbed radiant power by

 

 

 

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Figure 2.1. Jablonskii diagram of a molecule upon excitation.

(A, absorption; F, fluorescence; 10, internal conversion;ISC,

inter-system crossing; P, phosphorescence; VR, vibrational

relaxation).

the
the

chi

11

the molecules is measured as a function of the wavelength of
the radiation, a molecular absorption spectrum can be
obtained (2).

The excess vibrational and electronic energy is then
dissipated by rapid (10'13 - 10'12 's) radiationless
processes, vibrational relaxation and internal conversion.
Vibrational relaxation occurs when ther excited molecule
loses vibrational energy within a given electronic state,
and internal conversion occurs when the molecule undergoes a
radiationless transition from various excited energy levels
to the lowest vibrational level of the lowest excited
electronic singlet state. The excited molecules may return
to the ground electronic state with. emission of radiant
energy whose frequency is governed by the gap between the
lowest excited singlet state and the ground electronic
state. This radiative transition between excited and ground
states of the same spin multiplicity occurs in a time frame
of 10'11 - 10‘7 s after excitation, and is called
fluorescence.

Some portion of the molecules in the lowest excited
singlet state may deactivate by crossover' to the lowest
excited triplet state, a process called intersystem
crossing. This crossover entails a change in spin angular
momentum. This is, of course, forbidden in absorption
quantum mechanically. In the excited singlet state, the
Spins of the promoted electrons are still paired with the

round state electrons; however, in the triplet state the

 

sp:

1m
rat
or!

ph'

wi

De
in
me

Dr

ex

12

spins of the electrons have become unpaired and are thus
parallel with the ground state electron. Molecules in the
lowest triplet state can return to the ground state without
radiation being emitted by triplet - singlet intersystem
crossing, or with radiation being emitted, which is called
phosphorescence. Because phosphorescence is spin forbidden
process, the lifetime of the triplet state is very long
(10" - 10 s) as compared with an average lifetime of 10‘5 -
10'8 s for an excited singlet state (3).

Molecules in the excited states which do not deactivate
with radiation through either fluorescence or
phosphorescence usually deactivate without radiation.
Deactivation of an excited electronic state may involve

interaction and energy transfer between the excited
‘molecules and the solvent or other solutes (quenchers). This
process is called external conversion. The details of
external conversion processes are not well understood.
External conversion competes so successfully with
phosphorescence that phosphorescence could not be observed
without reducing the effectiveness of the external

conversion.
B. The Effect of Molecular Structrue on Photoluminescence.
Molecular structure can have a profound effect on the

photoluminescence. Both fluorescence and phosphorescence are

most often observed in highly conjugated organic molecules

 

 

wit
mo]
fr:
bet
tr:
1m
in‘

qu.

su
to
su
1a

fr

lc

ti.

 

 

 

13

with rigid molecular skeletons. Those molecules whose
molecular structure have less vibrational and rotational
freedom may have high probability that the energy gap
between the ground state and the lowest excited singlet or
triplet state will be large and require deactivation by
luminescence. Typically, aromatic hydrocarbon molecules show
intense fluorescence and can give high phosphorescence
quantum yield in certain situations.

Aromatic hydrocarbon molecules containing freely rotating
substituents, or lengthy aliphatic side chains, usually tend
to luminescence less intensely than those without those
substituents (4). This results from the introduction of a
large number of rotational and vibrational degrees of
freedom by the exocyclic substituents.

The energy difference between the ground state and the
lowest excited singlet or triplet states becomes smaller in
those molecules which have a more extended conjugated
system. Therefore, benzene, naphthalene, and anthracence
fluoresce maximally at 262 nm, 320 nm, and 379 nm,
respectively.

Certain substituents strongly affect fluorescence (5). A
substituent that delocalizes the 1 electrons, such as -NH2,
‘OH, -F, -OCH3, and -NHCH3 groups, often enhances
fluorescence. These electron-donating substituents tend to
increase the transition probability between the lowest

excited singlet state and the ground state.

 

 

 

 

 

NHCO
fluo
stro
incr
the

stat
This

chap

non:
rota

of t

fern
met:

and

Pro

80m

 

 

?7—i

14

 

Electron-withdrawing groups containing -Cl, -Br, -I, -
NHCOCHa, -N02, and -COOH decrease or quench the
fluorescence. The influence of halogen substituents is
strong. As the atomic number of the halogen substituent
increases, the fluorescence of the molecule decreases, and
the probability for intersystem crossing to the triplet
state increases at the expense of the excited singlet state.
This heavy atom effect will be discussed more in detail in
chapter III.

Molecular rigidity lessens the probability of competing
nonradiative transitions by restricting the vibrational and
rotational degrees of freedom of the molecule. The influence

of the molecular rigidity results in a decreased probability

 

 

of collisional deactivation and intersystem crossing. The
formation of chelates of certain organic molecules with
metal ions also promotes fluorescence by promoting rigidity

and minimizing internal vibrations.

C. Medium Effects.

The chemical environment affects the photophysical

processes of the molecules to a large extent. The effects of

 

some of these environmental variables are considered briefly

in this section.

 

 

 

1. E1

mole:
inert
imprt

COIN!

elect
eleC'

exci'

V and

in d

such
effe
of 1
m°le
sing
P0pu
lowe
flue

Pho:

 

 

15

1. Effect of Temperature and Solvent.

The quantum efficiency of photoluminescence by most
molecules decreases with increasing temperature because the
increased frequency of collisions at elevated temperatures
improves the probability for deactivation by external
conversion.

Solvent interactions with solute molecules are largely
electrostatic. It is usually the differences between the
electrostatic stabilization energies of the ground and
excited states that contribute to the relative intensities,
and spectral positions of fluorescence and phosphorescence
in different solvents.

Solvents containing heavy atoms, or other solvents with
such atoms in their structure, also have a substantial
effect on the photoluminescence of solute molecules. Atoms
of high atomic number in the solvent cage of the solute
molecule enhance spin-orbital coupling in the lowest excited
singlet state of the solute (6). This increases the
population of the lowest triplet state at the expense of the
lowest excited singlet state. Thus, the intensity of
fluorescence becomes less intense while that of

Phosphorescence becomes more intense in heavy atom solvents.

2. Effect of pH and Dissolved Oxygen.
Many aromatic compounds containing acidic or basic ring
substituents show a dependence on the pH of the medium (7).

0th ionized and nonionized forms of the compound are likely

 

 

 

 

to
in
Pr:
3e]

moi

gr

in
mo
Tr
hi

hi

 

 

16

to show different wavelength and intensity dwe to a change
in the nature and rates of the photoluminescence.
Protonation and dissociation can alter the relative
separation of the ground and excited states of the reacting
molecules. The protonation of electron-withdrawing groups,
such as carbonyl and nitrogen, results in a shift of the
luminescence spectra to lower wavelengths, while the
protonation of electron-donating groups, such as the amino
groups, produces spectral shift to shorter wavelengths.

The presence of dissolved oxygen reduces the emission
intensity of the photoluminescent molecules. This effect is
more severe in phosphorescence than in fluorescence spectra.
Triplet state oxygen. and other' paramagnetic species are
highly effective in deactivating excited triplet states (8).
Micromolar amount of oxygen can completely quench the
phosphorescence of most aromatic compounds in solution. Due
to the long intrinsic lifetime of the triplet state
engendered by the spin-forbidden nature of phosphorescence,
there is a comparatively long period for oxygen to interact

with excited triplet molecules.

3. Effect of Solute Concentration.

At lower solute concentrations, a plot of the
photoluminescence of the solution versus concentration of
the emitting species is normally .linear. But at higher
solute concentrations, there is a tendency for molecules to

form aggregates in both ground and excited states. Molecular

 

 

 

 

8!!

of

qut
f0:
sol
f r
so

CO

ov
of
th
ab

8!

17

aggregation can substantially affect the photoluminescence
of molecules (9).

Self—quenching and self-absorption are the two well known
quenching effects due to the high solute concentration. The
former is the result of collisions between excited state
solute molecules. Radiationless transfer of energy occurs
from the excited state of the solute molecules to the
solvent molecules. Self-quenching increases with solute
concentration.

Self-absorption occurs when the wavelength of emission
overlaps an absorption peak. This is due to the absorption
of emitted radiation (secondary absorption) by analyte. But,
there is also absorption of the incident radiation (primary
absorption) which can cause nonlinearity. These two effects

are often called the inner filter effect (3).

4. Effect of Coordination by Metal Ions.

Photoluminescence of aromatic ligands can be affected by
coordination with metal ions. Nontransition metals will
shift the wavelength of fluorescence and phosphorescence of
luminescing ligands to which they are coordinated. This
results from the positive polarization, caused by the metal
ion, at the sites of coordination on the ligand (10). The
luminescence of the ligand may be somewhat enhanced or
quenched by coordination, depending on the influence of the
metal ion has on the nonradiative processes competing with

luminescence.

 

 

 

Th1
ions
much

some

arom
tran
tran
One

heav
orbi

then

M

0013

 

 

18

The coordination of aromatic ligands with transition metal
ions usually produces electronic spectral shifts which are
much greater than the shifts produced by complexation of the
same ligands with nontransition metal ions.

The fluorescence and phosphorescence of '1uminescing
aromatic ligands are usually quenched by complexation with
transition metal ions. The reasons for this quenching of
transition metal complexes are not completely understood.
One of the proposed theories is that the paramagnetic and
heavy atqm effects of the transition metal ion cause spin-
orbital coupling which populates low lying states which are

then deactivated by internal conversion (11).

D. Quenching of Photoluminescence.

Quenching of luminescence is the result of the interaction
of the chromophore, either in ground or excited state, with
the various other species present in the system. Quenching
processes may be divided into two broad categories depending
on the state of the chromophore when it actually interacts
to give radiationless deactivation.

Static quenching occurs when an interaction takes place
between the chromophore and the quencher in the ground state
forming a nonluminescent complex. The efficiency of
quenching is governed. by the formation constant of the

complex as well as the concentration of the quencher.

 

 

 

 

 

pot
lif
res
que
lit
anc
eqi

fol

wh.
thc
pm
It

qu

Yi
th
qu

st

 

19

In dynamic quenching, the quenching species and the
potentially luminescent molecules interact during the
lifetime of the excited state of the chromophore with no
resulting radiation. As a result, the efficiency of dynamic
quenching depends on the viscosity of the solution, the
lifetime of the excited state of the luminescent species,
and the concentration of the quencher. The Stern-Volmer
equation describes this dynamic quenching process as
follows:

’P/Po = --------------- (2.2)
1 + kqf [Q]
where kq is the bimolecular quenching constant, Po and P are
the quantum yields ‘of the luminescence in the abscence and
prescence of the quencher [Q], respectively, and 1 is the
lifetime of the luminescent molecule in the absence of the
quencher.

It should be noted that in dynamic quenching, the quantum
yields of fluorescence and phosphorescence are governed by
the kinetics of the photoreaction. However, in static
quenching, they are generally governed exclusively by the
strength of ground state complexation.

Static and dynamic quenching can be distinguished by
arious methods. In static quenching, the observed lifetime
f the luminescence is unaffected by the quencher, while it
ecreases in dynamic quenching due to a quenching process
hich occured during the lifetime of the luminescent

pecies. Also, absorption spectra of the possible

 

 

 

 

lumine
that f
format

state.

 

 

 

 

 

20

luminescent species are different in static quenching from

that. in the absence of the quencher due to a complex

formation between lumiphore and quencher in the ground

state.

 

 

 

 

 

1.
Met.
Yor
2.
bum

Int

SPe

Lot

21.

 

21

CHAPTER II REFERENCES

1. Schulman, S.G., Mblecular Luminescence Spectroscopy:
Methods and Applications; Part 1, Wiley-Interscience, New
York, 1985.

2. Winefordner, J. D.; Schulman, S. G.; O’Haver, T. C.,
Luminescence Spectrometry in Analytical Chemistry, Wiley-
Interscience, New York, 1972.

3. Ingle, J. D.; Crouch, S. R., Spectrochemical Analysis,
Prentice-Hall, Englewood Cliffs, N.J., 1988.

4. Willard, H. W.; Merrit, L. L.; Dean, J. A.; Settle, F.
A., Instrumental Methods of .Analysis, Sixth Ed., D. Van
Nostrand Company, New York, 1981.

5. West, W., Chemical Application of Spectroscopy
(Techniques of' Organic Chemistry, Vol. IX), Interscience,
New York, 1956.

6- Zander, M, Phosphorimetry, Academic Press, N.Y., 1968.

7- Schulman, S. G.; Winefordner, J. D., Talanta 1970, 17,
607.

3. Lower, S. K.; El-Sayed, M. A., Chem. Rev., 1966, 66, 199.
9- Schulman, S. 6., Fluorescence and Phosphorescence
Spectroscopy: Physical Principles and .Practice, Pergamon,
London, 1977.

10. Bhatnagar, D. C.; Forsta, L. S., Spectrochim. Acta 1965,

21, 1803.

 

 

 

 

11. Mo
Spectr

Cliffs

 

22

11. McGlynn, S. P.; Azumi, T.; Kinoshita, M., Molecular
Spectroscopy of the Triplet State, Prentice-Hall, Englewood

Cliffs, N.J., 1969.

 

 

 

 

Ph
(1)
trans
molec
phos1
Most
fluo:
life
Wher
San
anal
from
With
Pesc
the
len,
dea<
QUE]
Pho:

“it:

 

 

23

CHAPTER III

ROOM TEMPERATURE PHOSPHORESCENCE

Phosphorescence was identified first by Lewis and Kasha
(1) in 1944 as the emission of light as a result of a
transition from the lowest excited triplet state of a
molecule to the ground state. .A number of features of
phosphorescence distinguish it clearly from fluorescence.
Most importantly, phosphorescence is distinguished from
fluorescence by its much longer lifetime. Fluorescence
lifetimes are typically in the range of 10'8 - 10'6 3,
whereas phosphorescence lifetimes normally lie between 10"
s and 10 8. These long lifetimes are potentially valuable in
analysis, as they enable phosphorescence to be distinguished
from fluorescence and scattered light. Also phosphorescence
with different lifetimes may be distinguished using time-
resolved methodology. Unfortunately, long lifetimes are also
the principle disadvantage of phosPhorimetry: in these
lengthy periods, the excited molecules are normally
deactivated by collisions with solvent molecules and other
quenchers such as oxygen molecules. As a result, traditional
phosphorimetry is performed in rigid media at liquid
nitrogen temperature (77 K) to minimize the radiationless

deactivation by various quenching effects.

 

 

 

 

 

inv<
fla:
the
the
wil

sel

bac
the
lim
phc
brc
Hm
of

tel

881

We

t!

l.

24

Sample preparation in low temperature phosphorimetry
involves lowering a long capillary cell into a quartz Dewar
Elask filled with liquid nitrogen. The rate of cooling of
the sample cell, the chemical nature, and the composition of
the solvent system will determine whether the cooled matrix
will be a clean glass, a cracked glass, or a snow. Thus, the
selection of the proper solvent is critical. It should form
a clear glass at 77 K and have a low phosphorescence
background. Also, the analyte should be readily soluble in
the solvent at 77 K. Experimental difficulties and
limitations associated with traditional methods in
phosphorimetry, have been the primary factors in preventing

broader application of low temperature phosphorimetry.

 

However, recent developments have shown that a wide variety
‘of organic compounds exhibit strong phosphorescence at room
itemperature when certain conditions are applied to the
fsample system (2).

t

iWalling (4) generalized room temperature phosphorescence

Although the studies by Roth (3) and by Schulman and

.(RTP) first in an analytical application, perhaps the first
fRTP phenomenon was observed by Wiedemann and Schmidt (5) in
\

‘u896. Phosphorescence spectra obtained at room temperature

.5

'fire generally similar to those observed at 77 K, but they
:Lhow less vibrational fine structure due to increased
. \

’yibrational freedom of the molecule at the higher

.temperature. RTP emission peaks are also shifted to slightly

Zionger wavelengths at room ‘temperature compared. with low

 

 

temp:
typic
inva1
and 1
also

RTP

MOIGI

temp
RTP

desc
nece

heav

A. C

fluj
teml
Stat
brii
flu:
5001

Pho

the

tem

 

 

25

temperature phosphorescence spectra, although shifts are
typically less than 10 nm (6). RTP lifetimes are almost
invariably shorter than phosphorescence lifetimes at 77 K
and rarely exceed a few hundred ms (7). RTP intensities are
also generally lower than those obtained at 77 K; however,
RTP is still capable of low detection limits for certain
molecules.

This chapter describes several features of room
temperature phosphorescence. The conditions necessary for
RTP are discussed first, and then various RTP techniques are
described. The last two parts cover the most important and
necessary conditions in RTP: deoxygenation methods and the

heavy atom effect.

A. Conditions for the Observation of RTP.

Most organic compounds which emit strong fluorescence in
fluids, commonly do not give any phosphorescence at room
temperature. Because of the long lifetime of the triplet
state, collisional deactivation is highly effective in
bringing about radiationless decay of triplet states in
fluid media. Quenching of the triplet state by a small
amount of dissolved oxygen is very efficient in preventing

phosphorescence in a liquid solution. As a result, some

   

technique must be used to reduce or to prevent such
uenching in order to observe phosphorescence at room

emperature. The ‘essential requirements for RTP are;

 

 

 

 

deo:

mic:

3am]
sail]
the
mic
red
it

and
dis

sol

gen
rec‘
co]
anc
tri
trJ'
th
Pil
or,
mi.
in
cy
Co

No

26

deoxygenation of the sample solution, a rigid molecular
microenvironment, and a heavy atom source.

Deoxygenation can be done physically or chemically during
sample preparation and emission measurement. By bubbling the
sample solution with an inert gas such as nitrogen or argon,
the concentration of dissolved oxygen can be reduced below
micromolar amounts. Chemical deoxygenation is another way to
reduce molecular oxygen in a sample solution by converting
it into other species. Sodium sulfite (8), chromium(II) (9),
and zinc. (10) have been used successfully to convert
dissolved molecular oxygen to other inactive species in
solution.

It has been observed that, a rigid microenvironment

generally increases the phosphorescence intensity by

 

ireducing collisional quenching of the triplet state. Because
collisional quenching is very efficient at room temperature
and is often considered as the main pathway for loss of
triplet state energy, a rigid molecular environment for the
triplet state is very important for the observation of the
phosphorescence at room temperature. In solid state RTP, the
rigid matrix is formed by the hydrogen bonding of ionic
organic molecules to hydroxyl groups of the filter paper. In
micellar stabilized RTP, sample molecules are confined
inside micelles which are semi-rigid organized media. In
cVelodextrin RTP, molecules form trimolecular inclusion
complex with cyclodextrin and a heavy atom. Because sample

molecules are included inside the cyclodextrin cavity, there

 

 

 

27

is less probability for collisional quenching of the triplet
state.

Although RTP can be observed in deoxygenated rigid media
at room temperature, the observed intensity of RTP is
generally too low to be useful in analytical work. However,
by adding a 'heavy atom such as Tl’, I', or Ag‘, into the
sample solution, the intensity of’ RTP can ‘be increased
significantly. This is due to an increase in the intersystem
crossing rate from excited singlet state to triplet state
through spin-orbital coupling induced by the heavy atom.
These effects are discussed in more detail later in this

chapter.
B. Methods for RTP Measurement.

Since the introduction of analytical RTP by Roth (3) in
1967, numerous methods for RTP measurement have been
developed. These are solid surface RTP, micellar-stabilized
RTP, sensitized RTP, cyclodextrin RTP, and collidal or
microcrystalline RTP. Though all these different methods of
RTP measurement look different from each other, they have
many common factors. Each different RTP technique tries to
minimize collisional quenching which is believed to be the
main pathway for radiationless deactivation of the triplet
state, by employing different methods to fulfill the three
requirements discussed ix: the above section. This section

discusses the major RTP methods developed thus far.

 

 

 

 

 

1.

on

ad:

po.
am
so
no
so
to
of
ad
11

 

28

1. Solid Surface RTP.

In 1967, Roth (3) reported a new analytical method based
on the RTP emission from a variety of organic compounds
adsorbed on the filter paper. Schulman and Walling (11)
observed strong RTP from salts of a wide variety of
polynuclear carboxylic or sulfonic acids, phenols, and
amines adsorbed on paper, silica, alumina, and other
supports. The observed phosphorescence lifetimes of several
molecules ‘were in the range of 100-700 ms, 'which were
somewhat shorter than those observed. at .liquid nitrogen
temperature. In 1974, Paynter et al (12) reported the range
of linearity and detection limits for several compounds
adsorbed on filter paper. Calibration curves had a wide
linear range, detection limits were in the nanogram region,
and precision ranges from about 3% to 10% depending on the
solid surface and experimental conditions.

In most cases of solid surface RTP, drying of the samples
was essential to enhance the RTP signal. It was noted that
moisture on the solid substrate caused radiational
quenching. It seemed that moisture acts to disrupt any
binding of analyte on the solid surface so that the rigid
molecular environment becomes loose and aids in the
transport of oxygen into the sample matrix.

Although no general model has been developed to explain
the interactions involved in the production of RTP from

compounds adsorbed (n1 filter paper, it seems that hydrogen

 

 

 

 

bond
soli

rigi

imp:
intt
sol‘
non;
fr01
ion
rig

rad

Pap
Mos
Pho
Wav
bac
8t

frc
Wit
he:
ba<
in]
him
be

29

bonding of ionic organic molecules to hydroxyl groups of the
solid surface is the primary mechanism for providing the
rigid matrix for RTP.

The ionic nature of the analyte molecules plays an
important role in producing RTP. While ionic molecules show
intense RTP signals when spotted on solid surfaces from
solvents containing a large excess of a strong acid or base,
nonionic compounds exhibit extremely weak or no RTP even
from acidic or basic solutions (11). It is believed that the
ionic state of the molecule results in great molecular
rigidity via adsorption to the substrate, which reduces
radiationless decay due to collisional deactivation (12).

The major disadvantage of solid surface RTP using filter
paper, is the strong background from the solid substrate.
Most grades of paper exhibit substantial background
phosphorescence, which is difficult to remove in the
wavelength range 400-600 nm. Attempts to reduce the
background emission from paper have been described by Ward
et al (13). Experiments designed to wash the contaminants
from the paper have met with little success (14). Beteh and
Winefordner (15) tried to photobleach the lignins or
hemicelluloses believed. to be a constituent of the
background. They concluded that such a pretreatement
improved the absorption characteristics of the filter paper,
but did not significantly reduce the phosphorescence

background.

 

 

 

0t
acet
mixt
obse
acet
the

cart

(I)

an.

a 8

upo

sen
ana
Sui
eff
Pat
Pr.
moi
so

do

Ph

 

 

30

Other substrates used for solid surface RTP include sodium
acetate, starch, inorganic substances, polymer-salt
mixtures, and silica gel. Wandruszka and Hurtubise (16)
observed RTP from a number of compounds adsorbed on sodium
acetate. The main interaction proposed with this system was
the formation of hydrogen bonds of the analyte with the

carbonyl group of sodium acetate.
2. Sensitized RTP.

Sensitized phosphorescence refers to the process whereby
an acceptor (or emitter) having no appreciable absorption in
a given region of the spectrum, is made to emit radiation
upon excitation as a result of triplet energy transfer from
a donor (or sensitizer) molecule. The overall process of
sensitized phosphorescence is shown in Figure 3.1. .The
analyte is excited by means of light. absorption.
Subsequently, these non-fluorescent compounds with a high
efficiency of intersystem crossing, deactivate without
radiation through intersystem crossing. However, in the
presence of an acceptor, the triplet energy of the donor
molecule can be transferred to the triplet state of the
acceptor molecule, and the excited acceptor molecule
deactivates with radiation.

There are several requirements for successful sensitized
Phosphorescence. In general, a suitable acceptor should have

a triplet energy lower than that of the donor, a low molar

 

 

 

 

 

 

82 .
Sl

FiEur.
Phospj
abso,‘
PhOsp
cross

(inane

 

 

31

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

32 +_ [C '
31 V“ ISC 82
F 31
it La _-..
I, “"I ’ T1
: ' I'
[C
: Q Q P
ISC
SO ' J L 30
DONOR MOLECULE ACCEPT OR MOLECUIE

Figure 3 . 1 . Schematic representation of sensitized

phosphorescence . The analyte (acting as the energy donor)
absorbs light , and the. acceptor molecule emits
Phosphorescence. (IC, internal conversion; IS inter-system

crossing; Sn, singlet state; Tn, triplet state; Q,

quenching).

 

 

 

abs:
str<
88p
suf‘
mecl
con
wid
con
if

max

RTE
chi
. ab:
ph<
on
as
Dr
de

8.0

bi

 

 

32

absorption in the excitation region of the donor, and a
strong phosphorescence yield at room temperature. The energy
gap between the donor and acceptor triplet state should be
sufficiently high so that it prevents the reverse transfer
mechanism (2). Biacetyl has favorable properties in this
context: its molar absorptivity is exceedingly small over a
wide wavelength range (17). For a given analyte
concentration, the maximum sensitized RTP signal is reached
if the excitation wavelength chosen corresponds to the
maximum in the excitation spectrum of the analyte.
Donkerbroek et al (18) introduced sensitized and quenched
RTP detection for flow injection analysis and liquid
chromatography. In sensitized RTP, the analyte, after
absorption of l ight , induces biacetyl to emit
phosphorescence. The quenched RTP detection method is based
on a dynamic quenching process in which the analyte acting
as a quencher, reacts with excited biacetyl, thus
prohibiting phosphorescence emission. The limits of
detection for several substituted aromatic compounds in
acetonitrile:water (1:1) were in the range of 10"8 M (19).
DeLuccia and Cline Love (20) demonstrated that sensitized
biacetyl RTP can be enhanced via molecular organization for
many aromatic compounds. Micelles composed of sodium dodecyl
sulfate (SDS) and B-cyclodextrin were used to enhance the
energy ‘transfer reaction ‘by organizing the reactants in
close proximity to one another. Both organized media

Provided more favorable environments for induction of

 

 

 

88115

in:

3.]

molt
pol
can
hyd
car
sul

pol

sol
cor
mor
mi<
Spr
dil
as:
by.

in

Dr

 

 

33.

sensitized RTP than homogeneous solutions of the reactants

in acetonitrile.
3. Micellar Stabilized RTP.

Surfactants (Surface Active Agents) are amphiphilic
molecules composed of a hydrophobic portion and a charged or
polar portion. The hydrophobic backbone of the surfactant
can vary in length from eight to twenty carbons and the
hydrophilic portion can be a partially' dissociable
carboxylate (soap), a fully ionized moiety (such as anionic
sulfate or cationic trimethyl ammonium), or an uncharged
polar species (such as alcohol).

A fascinating feature inherent in. aqueous surfactant
solutions is the phenomenon of self-organization. At low
concentrations in solution, the surfactants exist mostly as
monomers. Above a certain concentration, .the critical
micelle concentration (CMC), surfactant molecules associate
spontaneously to build up structural entities of collidal
dimensions called micelles. The architecture of these
agglomerates is such that the interior contains the
hYdrophobic alkyl chain of the amphiphile, while the
hYdrophilic head groups are located at the surface and are
in contact with bulk water.

Micelle formation is believed to be the result of three
Primary forces: hydrophobic repulsion between the

hYdrocarbon chains and the aqueous environment; charge

 

 

 

 

repulsi
attract
roughly
100 nor

Most
measure
The en
solutie

the CM(

Where
incorp
The
Packin
micell
solve,
toget}
Phase,
head
Illicaali

s°1Ve1

 

 

34

repulsion. of ionic head. groups; and, the van. der' Waals
attraction between the alkyl chains (21). Micelles are
roughly spherical in shape and they consist of typically 60-
100 monomers.

Most micelles are Optically clear and they do not cause
measurable light scattering in conventional spectrometry.
The concentration of micelle at various bulk surfactant
solution can be calculated from the aggregation number and
the CMC

[surfactant] - CMC

[micelle] = --------------------- (3.1)
aggregation number

where the aggregation. number is ‘the number' of monomers
incorporated into a micelle (22,23).

The shape of micelles depends upon the solvent and the
packing parameters of the surfactant molecule in the
micellar assembly. When surfactants are dissolved in polar
solvents, the polar head groups and counterions orient
together in an outward fashion in contact with the water
phase. However, in apolar solvents the orientation of the
head groups and the aliphatic tail is reversed (reversed

micelle) such that the hydrophobic tails contact the apolar

solvent.

 

 

 

 

 

 

Fisu:
mice]
Chap:

Surf;

 

35

o a o .
”0‘. . . 0 .
”a. . a @ e

 

lo@... .. . 0
Range ol-O‘ f. ‘ ‘ °
shoot Con

 

 

 

 

 

«doc. 20 - 30 x .
-o Inc-Stern layer
up to a fur
—oe t0- Gouy . Chapman
' layer up to

several hundred 7.

Figure 3.2._ Simplified model representation of the ionic

micelle. The electric double layer is composed of a Gouy-
Chapman layer outside, and a Stern layer inside the shear

surface.

 

 

 

 

 

The
discus:
rough
double
layer
shear
surfac
outsid
contai

Mi
applic
solubi
°r8anz
as 81
Phase
enZYm
usefu
stabi
the a
Phase

°f ma

miCe]
deox;
The

attr

 

 

36

The most advanced theoretical model of an ionic micelle
discussed so far is a combination of a Stern layer with a
rough rather than a smooth sphere and a Gouy-Chapman diffuse
double layer (24) as shown in Figure 3.2. The aqueous Stern
layer is between the smooth surface of the core and the
shear surface, which contains ionic heads of the micellized
surfactant ions. The Gouy-Chapman region is immediately
outside of the Stern layer and is a diffuse double layer
containing unbound counterions.

Micelles are responsible for many of the practical
applications of detergents, such as enhancement of the
solubilities of organic compounds in water, and catalysis of
organic reactions. Furthermore, micelles have been proposed
as simple model systems for a variety of important "two-
phase" systems, such as monolayers, colloids, proteins,
enzymes, and membranes. Among the primary reasons for the
usefulness of micelles as models are: (a) the thermodynamic
stability and reproducibility of many micellar systems; (b)
the simplicity of the structure of the micelle; (c) the two—
phase nature of the micelle; and (d) the ionic composition
of many micellar surfaces (25).

RTP of aromatic hydrocarbons was first observed in
micelles by Kalyansundaram et a1 (26). The solutions were
deoxygenated by bubbling them with nitrogen gas for 30 min.
The ready observation of RTP in micellar solution was
attributed to the protective screening of the triplet probe

from external quenchers by the micelle assembly. Cline Love

 

 

 

 

and
(MS
deo
enh
giv
sur
que
We

had

ti
fo

th

 

 

37

and coworkers (27) further developed micellar-stabilized RTP
(MS-RTP) for analytical applications. In addition to
deoxygenation by an inert gas, heavy atoms were required to
enhance the sensitivity. Thallium counterions were found to
give higher signals than silver~ counterions' in 0.15 M
surfactant solutions, and about 90 X of the fluorescence was
quenched by heavy atoms. The limits of detection were
typically in the nanomolar range, and the calibration curves
had a wide linear dynamic range for several hydrocarbons.
The incorporation of the analyte molecule into the core of
a micelle imparts certain advantages (28):
(i) The structural conformation of the micelles protects the
triplet state of the analytes from external quenchers.
(ii) The orientational constraint decreases vibrational
deactivation.
(iii) The altered. microenvironment can jprovide favorable
polarity and acid/base equlibrium for enhanced
phosphorescence quantum efficiency.
(iv) The micellar solutions can improve the detection limits
for hydrophobic species in aqueous solution by increasing
their solubility.
(v) The proximity’ of interacting species (phosphors and
heavy atoms) is increased, and can result in a more

effective spin-orbital coupling.

 

 

 

 

 

 

 

 

 

4. Cycle:

Cyc]
oligosacu
starch.
glucose
8 ,and 1
moieties
hollow i
This wat
can cut.
c°ll|I>osed
Elicosic‘
hidrOphc
cavities
reSpecti
FiSure 1

The h
trap CO]
molecul.
inclusi.
comi’lex
intermo
Waals
macmcy
their

 

 

38

4. Cyclodextrin Enhanced RTP

Cyclodextrins (CDs) are a series of macrocyclic
oligosaccharides produced by the bacterial degradation of
starch. The most widely used CDs consist of 6, 7, and 8
glucose monomers arranged in torus shapes and denoted as a ,
B ,and T CD, respectively (29). The coupling of the glucose
moieties gives a rigid, torus molecular structure with a
hollow interior that contains one or more water molecules.
This water can easily. be displaced by other species which
can enter the CD cage. The interior of the cavities are
composed of two rings of C-H groups with a ring of
glycosidic oxygen in between, allowing them to be
hydrophobic in nature. The internal diameters of these
cavities are approximately 5.7, 7.8, and 9.5 A ,
respectively, and the depths are roughly 7.8 A as shown in
Figure 3.3.

The hydrophobic nature of the cavities enables the CDs to
trap compounds, such as aromatic and alkyl halides as guest
molecules in their interior, resulting in the formation of
inclusion complexes (30). The stability of an inclusion
complex depends on the size of the CD cavity and on
intermolecular forces such as hydrogen bonding, van der
Waals attraction, and hydrophobic interactions. These
macrocyclic carbohydrate molecules can be discriminating in

their inclusion complexing tendencies toward different

   

structural, positional, or stereomeric molecules. A more

 

 

 

 

 

 

 

“Sure

Struct,

 

 

Figure 3.3.

structure.

 

39

 

 

 

 

 

 

 

 

Simplified

 

AP-

 

representation

 

of

B -cyclodextrin

 

 

 

detailed
next cha
The CI
luminesc
enhanced
several
which 0
revealee
inclusie
complex
They ce
aqueous
c°mPlet
quenche
Fol]
coworke
a“film
demonst
9°1Ynue
heavy
deoxyg,
sensit
trimol
moleCu
Phosph
baged

cuPVes

 

 

40

detailed discussion of macrocyclic compounds is given in the
next chapter.

The CD molecule provides a favorable microenvironment for
luminescent probes. Turro et al (31) reported cyclodextrin-
enhanced RTP in 1982. They investigated the emission of
several 1,3-bichromophoric systems in aqueous solutions
which contained various cyclodextrins. In all cases, CDs
revealed structural (size and shape) selectivities toward
inclusion complex guest molecules. The tight CD-lumiphor
complex also showed partial immunity to oxygen quenching.
They' could observe RTP of 4-bromo-1-naphthoyl groups in
aqueous solution even under 1 atm of oxygen (32). Oxygen
completely quenched the fast decay, but only partially
quenched the slow decay.

Following the work of Turro’s group, Cline Love and
coworkers further developed cyclodextrin enhanced RTP for
analytical applications. Scypinski and Cline Love (33)
demonstrated cyclodextrin enhanced RTP for the analysis of
P01ynuclear aromatic hydrocarbons (PAHs). They found that a
heavy' atom. source, 1,2-dibromoethane, was :necessary, and
deoxygenation with nitrogen gas was required to enhance the
sensitivity. The .proposed inclusion complex is a
trimolecular complex of CD-lumiphor-heavy atom. Only
molecules that can physically enter the CD cavity are
Phosphorescent, which provides considerable selectivity
based on lumiphor size. Shapes of analytical calibration

curves are similar to those obtained for micellar-stabilized

 

 

 

 

RTP. '
range
Tl
utili
RTP o
bindi
Hurtb
on a
size
betwe

level

50 0

A:
cond
(ii)
meth
diff

mic:

PAH:
PAH
inh
the
0f

"83

 

41

RTP. Typical detection limits are in the 10'11 to 10‘13 M
range.

The unique inclusion. capability of cyclodextrins was
utilized in other areas of RTP. Vo-Dinh et a1 (34) reported
RTP of anthracene on cyclodextrin treated filter paper. The
binding constant of anthracene with BeCD was 32. Bello and
Hurtbuise (35) observed RTP of several aromatic hydrocarbons
on a solid surface of CD-sodium chloride. By utilizing the
size requirements of the CD molecule, they could identify
between seven to nine PAHs in the mixtures at nanogram

levels.

5. Other Methods of RTP.

As mentioned earlier, in order to observe RTP, certain
conditions are required: (i) a rigid molecular environment;
(ii) deoxygenation; and (iii) a heavy atom source. Other
methods of RTP try to fulfill these requirements with
different methods. These methods include RTP of colloidal or
microcrystalline suspensions and silicalite RTP.

Weinberger and Cline Love (36) reported RTP observation of

 

PAHs in colloidal suspensions in water. In this method, the
PAH molecule, which is insoluble in the aqueous. media, is
injected rapidly into the aqueous solution. This results in
the formation of a microcrystalline or colloidal suspension
of the sample in the aqueous media. The observed spectrum

was very similar in resolution, shape and symmetry to

 

 

 

spectra at:
to quenchi
manner ana
pathways
minimized.
and provic
is present
oxygen thl
immune to
Casal e
ketones
thmphob
Phenylpro
five 0rd
cavities.
restricti
Channels
part of
topc’lilflie
°f this
which are
The diam.
“088-38.
limiting
temPErat,
Zeolite

Effect d

 

42

spectra observed at 77 K. Also, this system is insensitive
to quenching by dissolved oxygen. The microcrystals act in a
manner analogous to solid-surface RTP, in that the quenching
pathways that normally operate at room temperature are
minimized. The microcrystals serve as their own substrate
and provide stability to the triplet state. Since the solute
is present as a solid in suspension, diffusion of molecular
oxygen through the solute is not possible, thus rendering it
immune to oxygen quenching (37).

Casal and Scaiano (38) observed RTP of several aromatic
ketones included in the channels of silicalite, a
hydrophobic zeolite. They found that the lifetime of B -
phenylpropiophenone at room temperature is enhanced by over
five orders of magnitude by inclusion “in silicalite
cavities. This enhancement ‘was attributed. to 'the steric
restrictions imposed on the included guest molecules in the
channels of the silicalite. Silicalite ( 99 2 SiOz) forms
part of the class of zeolite molecular sieves with a new
topologic type of tetrahedral framework. The channel system
of this zeolite consists of near circular zig-zag channels

which are cross-linked by elliptical straight channels (38).

The diameter of the circular channels is 5.4 A and the free

 

cross-section of the elliptical ones is 6A . Therefore, the
limiting size for adsorption is around 6A at room
temperature. One of the remarkable properties of this

zeolite is its hydrophobicity, which ensures no deactivation

effect due to water.

 

 

 

 

 

 

C. Deoxyge

Oxygen
RTP requix
solution
this sect

for solut:
1. Physic;

The mos
is bubbli
as, nitro
min with
dissolved
amounts,
°°n3uming
in 8 mice

Reim (4
0"men. :
high ox:
0xYflen t
differem
Membrane

dissolve:

 

 

43

C. Deoxygenation Methods.

Oxygen quenches RTP so efficiently that every method of
RTP requires protection of the triplet state from oxygen. In
solution RTP, deoxygenation is a necessary condition. In

this section, the deoxygenation methods developed to date

for solution RTP are discussed.
1. Physical Methods of Deoxygenation.

The most common method for deoxygenation in solution RTP,
is bubbling of the sample solution with an inert gas, such
as, nitrogen and argon. Ifthe solution is bubbled for 30
min with oxygen-free inert gas, the concentration of
dissolved oxygen can be reduced to less than micromolar
amounts. The disadvantages of this method are the time
consuming process and the fact that it generates excess foam
in a micellar solution.

Reim (40) used semipermeable membranes to remove dissolved
oxygen. Silicon rubber was used as membrane because of its
high oxygen permeability and chemical inertness. Since
oxygen transport is directly proportional to the pressure
difference across the membrane, evacuation around the
membrane was necessary. With this method about 95 X of the

dissolved oxygen could be removed in 25 min.

 

 

 

 

 

Other 111
thaw cycle
the diffi

degassing

2. Chenict

In most
into inaC‘
Rollie
dissolved
formed by
in contac

membrane.

Zn(H

4Cr2

Although
time rem
in RTP.
MacCr.
disSOlve.
column p:

(”Ween a

 

 

44

Other methods are vacuum degassing and repeated freeze-
thaw cycles. However, these methods are not popular due to
the difficult and time consuming process involved for

degassing of sample solution.

2. Chemical Methods of Deoxygenation.

In most chemical methods the dissolved oxygen is converted
into inactive other species by a chemical reaction.

Rollie and coworkers (41) used chromium(II) to convert
dissolved molecular oxygen into water. Chromium(II) is
formed by the reduction of chromium(III) with Zn(Hg), and is
in contact with the sample solution through a semipermeable

membrane. The chemical reaction is as follows:

 

Zn(Hg) + 2Cr3* : Zn2* + 2Cr3* + Hg (3.2)

4Cr2* + 02 + 4H30*-————~r 4Cr3* + 6H20 (3.3)

Although this method is effective, the long equilibration
time required for the reaction prohibits further application
in RTP.

MacCrehan and May (42) used a zinc column to remove
dissolved oxygen. A sample solution is passed through a

column packed with zinc particles and allowed to react with

oxygen as follows:

 

 

 

Zn+

Zn+

Since zinc
a finite 1

Recentl
deoxygenat

reaction c

2!

The react:
on the

concentra
a °°mPlet
by the mj

and effec

D. The He

It ha

affects
heavy on
°°lnmon p:
of which
triDlet

as the e

 

 

45
Zn + 02 + ZH’ ; Zn“ + H202 (3.4)
Zn + H202 + 2 H‘ -————4. Zn2* + 2HzO (3.5)

Since zinc is oxydized by the reaction, the zinc column has
a finite lifetime.

Recently, Diaz Garcia and Sanz-Medel (43) reported a
deoxygenation method based on the following chemical

reaction of sulfite with oxygen:

28032' + 02 -————+ 28042' (3.6)

The reaction time for oxygen consumption showed a dependence
on the surfactant concentrat ion in MS-RTP . As the
concentration of surfactant increases, it took more time for
a complete reaction due to the restriction of ionic movement
by the micelles. This method has been shown to be an easy

and effective deoxygenation method.

D. The Heavy Atom Effect.

It has been observed that the presence of heavy atoms
affects luminescence significantly.. The use of external
heavy atoms to increase phosphorescence yields has become a
common practice in RTP. By mixing two chemical species, one
of which contains a heavy atom, the probability of singlet-
triplet crossover increases. This phenomenon is referred to

as the external heavy atom effect (44), in contrast to the

 

 

 

 

 

internal h
chemically
intercombi
interactic
may freque
quantum
fluoresce]
lifetimes
without t
The heat
orbital c
The exter
one, by c
by a 1
distribut
ph“Whore
rate con:
increase:
lifetime:
"hich de.
increase
the T1 .
and the
due to J
(49),
while

orbital

 

46

internal heavy atom effect, observed when the heavy atom is
chemically affixed to the molecular skeleton whose
intercombinational transition it perturbs (45). These
interactions between the heavy atom and the sample molecule
may frequently result in an increase in the phosphorescence
quantum yield, and a corresponding decrease in the
fluorescence yield. Also, the observed phosphorescence
lifetimes with the heavy atom are usually shorter than
without the heavy atom.

The heavy atom effect has been ascribed to increased spin-
orbital coupling induced by the heavy atom perturber (46).
The external heavy atom effect may take place in two ways:
one, by complex formation with the heavy atom, and another,
by a long range interaction through a statistical
distribution of the heavy atom molecules around the
phosphorescing molecule (47). It has been shown that the
rate constants of both 81 -> T1 and T1 -> SO .processes are
increased (48). This is evident from the observed RTP
lifetimes of probe molecules in micelles with heavy atoms
which decrease substantially as the heavy atom concentration
increases. Furthermore, it has been also demonstrated that
the T1 -> SO radiationless transition is not much enhanced,
and the observed reduction of triplet lifetimes is mainly
due to an increase in the radiative transition probability
(49).

While various mechanisms to explain the enhanced spin-

orbital interaction and the reduced triplet lifetime of

 

 

 

 

 

aromatic 1
still not
been previ
singlet at
phosphores
the emitti
other. Th
molecule 1
of the moi
Bower
several h
Paper, an.
> pb2+
formation
Providing
cases.
White
effect of
RTP of ac
enhanceme
Hence, H
In this
affect, 1
nonradia1
The ex
RTP (56)

SOlutiOn

 

r——*-—

aromatic hydrocarbon molecules have been proposed, it is
still not well understood. The various schemes which have
been previously proposed share a common factor in that the
singlet states are ultimately responsible for the reduced
phosphorescence lifetime (50). That is, the triplet state of
the emitting molecule is mixed with the singlet state of the
other. The sources of the singlet state can be the same
molecule (51), the perturber (52), or charge transfer state
of the molecule-perturber complex (53).

Bower and Winefordner (54) investigated the effects of
several heavy metal ions on the RTP of PAHs adsorbed on
paper, and found the following enhancement trend: Tl’ > Ag’
> Pb2+ > ngt. It has been suggested that 1 -complex

formation between the heavy metal ions and the PAH might be

 

providing an internal heavy atom effect in some of these
cases.

White and Seybold (55) have investigated in detail the
effect of the addition of various sodium halide salts on the
RTP of sodium Z-naphthalenesulfonate adsorbed on paper. The
enhancement in RTP follows the trend of I' > Br‘ > 01' > F‘.
Hence, the heavier ions induce the greater RTP enhancement.
In this study (55), the heavy atom perturber was found to

affect the radiative RTP emission rate more than the

 

nonradiative rate.
The external heavy atom effect is extensively used in MS-
RTP (56). The RTP of many PAHs is not detectable in a 0.1 M

solution. of NaLS. However, when thallium(I) is ‘used to

 

 

replace 30

RTP signal

 

 

48

replace 30 X of the sodium counterions in NaLS, an intense

RTP signal can be detected.

 

 

 

 

 

 

1. Lewis,
L Vo-Dinl
Analysis,
3. Roth, 1
4.Schulm
5. Wieden
1896, 58,
5. Miller
7- Cline

Chem., 15
3. Diaz (
103.

9. Rolli

1983, 55

10. MacC
625.

 

 

49

CHAPTER III REFERENCES

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2. Vo-Dinh, T. , Room Temperature Phosphorimetry for Chemical
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3. Roth, M., J. Chromatogr., 1967, 30, 276.

4. Schulman, E. M.; Walling, 0., Science, 1972, 178, 53.

5. Wiedemann, E.; Schmidt, G. 0., Ann. Phys. (Leipzig),
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6. Miller, J. N., Trends Anal. Chem., 1981, 1, 31.

7. Cline Love, L. J.; Habarta, J. G.; Skrilec, M., Anal.
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8. Diaz Garcia, M. E.; Sanz-Medel, A, Anal. Chem., 1986, 58,
103.

9. Rollie, M. E.; Ho, C. N.; Warner, I. M., Anal. Chem.,
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10. MacCrehan, W. A.; May, W. 151., Anal. Chem., 1984, 56,
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12. Paynter, R. A.; Wellons, S. C.; Winefordner, J. D.,
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13. Ward, J. L.; Bateh, R. P.; Winefordner, J. D., Analyst,
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14. Bateh, R. P.; Winefordner, J. D., Talanta, 1982, 29,
713.

 

 

 

 

 

15. Ward,
1981, 28:
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49, 2164.
17. Gooije
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R. W., An
20. DeLuc
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21. Cline
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22- Kalya
23. Hoff:
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24. Stig1
25. Turn.
Int. Ed.
26' Kaly
Ph’s- Le
27' Clin
Chem” 1
28. Ing]

Prenti“

50

15. Ward, J. L.; Bower, E.L.Y.; Winefordner, J. D., Talanta,
1981, 28, 119.

16. Wandraszka, R.M.A.; Hurtubise, R. J., Anal. Chem., 1977,
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17. Gooijer, C.; Velthorst, N. H.; Frei, R. W., Trends Anal.
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18. Donkerbroek, J. J.; Veltkamp, A. C.; Gooijer, C.;
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19. Donkerbroek, J. J.; Gooijer, C.; Velthorst, N. H.; Frei,
R. W., Anal. Chem., 1982, 54, 891.

20. DeLuccia, F. J.; Cline Love, L. J., Anal. Chem., 56,
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21. Cline Love, L. J.; Habarta, J. G.; Dorsey, J. G., Anal.
Chem., 1984, 56, 1133A.

22. Kalyanasundaram, K., Chem. Soc. Rev., 1978, 7, 453.

23. Hoffmann, H.; Ebert, G., Angew. Chem. Int. Ed. Ehgl.,
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24. Stigter, D., J. Phys. Chem., 1964, 68, 3603.

25. Turro, N. J.; Gratzel, M.; Brawn, A. M., Angew. Chem.
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26. Kalyanasundaram, K.; Grieser, F.; Thomas, J. K., Chem.
Phys. Lett., 1977, 51, 501.

27. Cline Love, L. J.; Skrilec, M.; Habarta, J. G., Anal.
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28° Ingle, J. D.; Crouch, S. R., Spectrochemical Analysis,

Prentice Hall, Englewood Cliffs, N.J, 1988.

 

 

 

29. Bende:
Spring-Vex
30. Saenge
31. Turrc
Photobiol
32. Turro
1983, 2,
33. Scypi
322.

34. Vo-Di
35. Bellc
1285.

36~ Weinl
40A, 49.
37. Weinl
516.

33. Case
628.

39' Case
1308.

40' Rein
41' Roll
1983, 5,
42' Mac
625.
43. m.

58: 143

 

 

I

51

:9. Bender, M. L.; Komiyama, M., Cyclodextrin Chemistry,
Spring-Verlag, Berlin, 1978.

30. Saenger, W., Angew. Chem. Int. Ed. Engl., 1980, 19, 344.
31. Turro, N. J.; Okubo, T.; Weed, G. C., .Photochem.

Photobiol, 1982, 35, 325.

32. Turro, N. J.; Cox, G. S.; Li, X., Photochem. Photobiol.
1983, 2, 149.

33. Scypinski, S.; Cline Love, L. J., Anal. Chem., 1984, 56,
322.

34. Vo-Dinh, T.; Alak, A., Appl. Spectrosc., 1987, 41, 963.
35. Bello, J. M.; Hurtubise, R. J., Anal. Chem., 1988, 60,

1285.

36. Weinberger, R; Cline Love, L. J., Spectroch. Acta, 1984,
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37. Weinberger, R; Cline Love, L. J., Appl. Spec., 1985, 39,
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38. Casal, H. L.; Scaiano, J. 0., Can. J. Chem., 1984, 62,
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39. Casal, H. L.; Scaiano, J. 0., Can. J. Chem., 1985, 63,
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41. Rollie, R. E.; Ho, C. N.; Warner, I. M, Anal. Chem.,
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42. MacCrehan, W. A.; M87: W. 16., Anal. Chem., 1934, 55.
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43. Diaz Garcia, M. E.; Sanz-Medel, A, Anal. Chem., 1986,
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44. Kasha,
45. M00111]
46. 81-38:
47. Ebara
Japan, 19
48. Baker
1978, 58,
49. Eisen
42, 794.

50. Chauc
42, 1947.
51. Isubc
32. 5966

52. 8013'
53. Murr-

54- Bowe

1978, 16

55' Tim

2035.

56. 0111

Chem. :

 

52

14. Kasha, M, J} Chem. Phys., 1952, 20, 71.
45. McClure, D. S., J. Chem. Phys., 1949, 17, 905.
46. El-Sayed, M. A., Acct. Chem. Res., 1968, 1, 8.

47. Ebara, N.; Yajima, Y.; Watanabe, 11.; Bull. Chem. Soc.
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48. Baker, R. H.; Moroi, Y., Gratzel, M., Chem. Phys. Lett.,
1978, 58, 207.

49. Eisenthal, K. R.; El-Sayed, M. A., J. Chem. Phys., 1965,
42, 794.

50. Chaudhurl, N. K.; El-Sayed, M. A., J. Chem. Phys., 1965,
42, 1947.

51. Isubomura, H.; Mulliken, R. S., J. Am. Chem. Soc., 1966,
82, 5966.

52. Hoijtink, G. J., Moll. Phys., 1960, 3, 67.

53. Murrel, J. N., Mbll. Phys., 1960, 3, 319.

54. Bower, E. L. Y.; Winefordner, J. D., Anal. Chim. Acta.,

1978, 102, 1.

55. White, W.; Seybold, P. G., J. Phys. Chem., 1977, 81,
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56. Cline Love, L. J.; Skrilet, M.; Haberta, J. G., Anal.
Chem., 1980, 52, 754.

 

 

 

 

APPLICAT

There h
in the 31
compounds
cavities
macrocycl
interesti
ions and
the macrt
great an
been don
cOmpared

Althou
°°mpound
been knc
deCades
°°mP0unc
been I

macr°cy

Polythi

numerou

natural

p°rph91

53

CHAPTER IV
APPLICATION OF MACROCYCLIC COMPOUNDS IN CHEMICAL ANALYSIS

Theme has been an increasing amount of research interest
in the general properties and applications of macrocyclic
compounds. Macrocyclic compounds typically contain central
cavities ’ringed with long frameworks. Many of these
macrocyclic compounds have been shown to possess very
interesting and unusual binding properties. Various metal
ions and molecules can be encapsulated inside the cavity of
the macrocyclic compounds. The complexes thus formed are of
great analytical interest. But relatively few studies have
been done on the analytical applications of these compounds
compared to the fundamental studies performed.

Although metal complexes of naturally occuring macrocyclic
compounds, such as, porphyrins and certain antibiotics have
been known for over 50 years, it is only during the past two
decades that a large number of synthetic macrocyclic
compounds capable of binding metal ions or molecules have
been prepared and investigated (1). These synthetic
macrocyclic compounds include cyclic polyethers,
polythioethers, polyamines, and cyclophanes. There are
numerous publications and patents on applications of
naturally occuring macrocyclic compounds, cyclodextrins and

Porphyrins (2). The important properties and usefulness of

 

 

 

 

 

these corn
decade (3'
Inclusi
having a '
and chemi
These ul
inclusion
organic 1
those per
Since the
hiSh deg:
ions ca:
sPecific
designin
aPPr0pri
m01ecula
Water
°°mPOunc
“acrocm
quaterm
Were m.
functio
have 6.
Certain
was Obs

Th

 

 

54

these compounds have become well understood during the past
decade (3).

Inclusion complexes are formed with macrocyclic compounds
having a variety of selectivities, depending on the physical
and chemical property of the host macrocyclic compounds.
These unique properties of macrocyclic compounds in
inclusion complex formation stimulated chemists to devise
organic compounds which will perform functions similar to
those performed by naturally occuring macrocyclic compounds.
Since these macrocyclic compounds are synthetic in nature a
high degree of selectivity in binding of guest molecules or
ions can be obtained by designing host molecules for
specific 'purposes (4). A wide variation. is ‘possible in
designing host structures which will have the most
appropriate size, shape, and functionality for specific
molecular recognition.

Water soluble heterocyclophanes are synthetic macrocyclic
compounds which have been proposed as enzyme models (5). The
macrocyclic ring is composed of a wall of benzene rings and
quaternary nitrogens. Several variations of the structure
were made to provide host cyclophanes with different
functionality and substrate specificity. Heterocyclophanes
have been shown to be excellent inclusion hosts toward
certain organic substrates. A unique substrate specificity
was observed due to their cavity size and functionality.

This chapter discusses analytical applications of

macrocyclic compounds. In the first part» a brief

 

 

 

 

 

 

 

introduct
chemical .
of the 3
molecular
the curr

compounds

A. Host-(

A b;
catalyti
driving
guest cc
Suest mo
usually
formatic
Molecule
mOlecul.
that c
Postula
the sa
°°mpati
SimPle
for mu
m°iecu1

Vari

years I

55

introduction to host-guest chemistry and its usage in
chemical analysis is discussed. Next, potential applications
of the synthetic enzyme model compounds, cyclophanes, in
molecular luminescence are addressed. The last part reviews
the current status of applications of other macrocyclic

compounds in chemical analysis.

A. Host-Guest Molecular Inclusion.

A basic understanding of the specific binding and
catalytic behavior of enzymes is one of the significant
driving forces for studying host-guest chemistry. In a host-
guest complex, the host molecule is the larger, and the
guest molecule is the smaller of the two. The host molecule
usually contains one or several binding sites for,complex
formation (inclusion complex) with guest molecules. The host
molecule must recognize and complex best_ those guest
molecules that contain binding sites and steric features
that complement those of the host. The self-evident
postulate that two objects cannot occupy the same space at
the same time indicates that host and guest must be
compatible with respect to shape if they are to complex. The
simple electrostatic attraction of opposite charges accounts
for' much of the binding forces between host and guest
molecules (4).

Various investigations have been carried out in recent

years on some new synthetic catalysts which show enzyme-like

 

 

 

 

 

behavior
polymers
phenomena
Micellar
site thrc
between 5
intra-con
effect
effectiv:
these ca
and sub:
Thus, t
substrat
°°mPouné
due to 1
(i) T
due to
nature.
(ii)
the Sec
maCroc;
36bstra

(iii

 

56

behavior (6). Micellar surfactants and water soluble
polymers bearing various functional groups have shown
phenomenological similarities with enzyme systems (7).
Micellar surfactants have ‘been known to form a hydrophobic
site through micelle formation. The hydrophobic interaction
between substrates and micelles results in the formation of
intra-complexes. Additionally, micelles. provide proximity
effect by raising local concentrations of substrates
effectively. The formation of hydrophobic binding sites in
these cases is in dynamic equilibrium with the bulk phase,
and subject to the external medium effects, consequently.
Thus, these mobile structures may provide a limited
substrate specificity. On the other hand, macrocyclic
compounds may exhibit several novel and unique characters
due to the following effects (8).

(i) The macrocyclic cavity provides a stable binding site
due to a characteristic ring conformation of hydrophobic
nature.

(ii) A high substrate specificity can be brought about by
the geometric requirements for binding substrates into these
macrocyclic cavities, as well as by spatial geometries of
substrate molecules incorporated into these cavities.

(iii) A high possibility of introducing a charge relay or
an electrostatic antenna system, which is provided by
spatial arrangements of appropriate functional groups.

Consequently, a well-designed macrocyclic compound may

 

 

 

 

 

 

exhibit :
certain ‘
Subst:
specific
binding-
atoms re
for the
the spe:
guest i1
complex1
hydrogel
interac‘
The
affecte
discrim
affecti
complex
the ri:
(iii) .
°avity:
(V) st
chaYSe
In
triPle
e8Peci
the in

COuld

 

 

57

exhibit a unique behavior in inclusion complex formation for
certain types of substrates.

Substrate specificity is. understood to indicate that a
specific substrate has a "best fit" to a unique array of
binding-site residues and that the spatial arrangement of
atoms relevant to the catalysis is particularly favorable
for the stabilization of the transition state (9). Although
the specific mechanism and requirements for a stable host-
guest inclusion complex formation may be different for each
complex, it is known that the main binding forces are
hydrogen bonding, van der Waals forces, hydrophobic
interaction, and steric and electrostatic interactions (10).

The stability of host-guest inclusion complexes can be
affected by various forces which are at the core of the
discriminative nature of substrate binding. The factors
affecting the formation and stability of host-guest
complexes include: (i) the type or types of binding sites in
the ring; (ii) the number of binding sites in the'ring;
(iii) the relative sizes of the guest and the macrocyclic
cavity; (iv) the physical placement of the binding sites;
(v) steric hindrance in the ring; and (vi) the external
charge of the guest (11).

In. room-temperature phosphorescence, quenching of the
triplet state of a molecule is the most annoying problem
especially in solution. Judging from the above discussion,
the inclusion behavior of macrocyclic enzyme model compounds

could provide an ideal environment for observing room-

 

 

 

 

temperatu
reduced 1
cavity of
possible
requireme
Also, si
specific
guest in

of this

3. Synth

Since
Years 8
selectis
developE
melecul:
the enz;
Followi1
innovat
supremo
(13,14,
°°mpoun
f°r the

In
inclusj

“Prom

 

58

temperature phosphorescence. Triplet quenching should be
reduced by inclusion of the triplet molecule inside the
cavity of the host molecule. At the same time, it might be
possible to observe a high degree of selectivity due to the
requirements for the host-guest inclusion complex formation.
Also, since the host molecule is synthetic in nature,
specific host molecules could be tailored for a specific
guest in chemical analysis. These are the underlying ideas

of this research.

B. Synthetic Enzyme Model Compounds: Cyclophanes.

Since the discovery of crown ethers by Pedersen twenty
years ago, the chemistry of synthetic hosts for the
selective complexation of organic and inorganic guests has
developed rapidly. Soon after the fundamental studies on
molecular complexation by cyclodextrins, Cramer investigated
the enzyme-like catalytic properties of these systems (12).
Following this, a large variety of stimulating and
innovative studies on the catalytic pr0perties of the
supramolecules have been conducted by many research groups
(13,14,15). Of the fascinating synthetic enzyme model
compounds, water-soluble cyclophanes seem quite interesting
for the examining possible RTP applications

In aqueous solution, cyclOphanes form stoichiometric
inclusion complexes with aromatic guest molecules; these can

aPproach.«enzyme-substrate complexes in their stabilities

 

 

 

 

 

 

(16). D11
soluble c
hosts wit
residues,
introduce
showed
molecule:
Urushi
azaparac:
Tabushi
Of the s
N’"-octa
tetraamm
°°mPlexe
and st!
molecule
in wate
quarter,
The m
cavity
benzene
Pseudo
312% of
NMR Stt
molem

are per

 

 

59

(16). Diederich. and. his coworkers (17) introduced water
soluble cyclophanes. For the construction of water-soluble
hosts with apolar cavity binding sites shaped by hydrocarbon
residues, ionic groups remote from the cavity were
introduced as 'shown in Figure 4.1. Complexation studies
showed stable 1:1 complexes with various hydrocarbon
molecules in solutions of a wide polarity range (18).

Urushigawa et al (19) reported the synthesis of various
azaparacyclophanes (APCs) containing four benzene rings.
Tabushi and his coworkers (20) studied substrate specificity
of the water-soluble heterocyclophane, N,N,N’,N’,N",N",N’",
N’"-octamethyl-2,11,20,29-tetraaza[3,3,3,3]paracyclophane-
tetraammonium tetraborate (methyl-APC). Strong inclusion
complexes were formed through hydrophobic, electrostatic,
and steric interactions between the host and guest
molecules. Solubilization of the host methyl-APO molecules
in water was achieved by the introduction .of the four
quarternary nitrogens around the macrocyclic ring.

The methyl-APO molecule in Figure 4.2-A has a hydrophobic
cavity resembling a square box surrounded by a wall of
benzene rings that form angles of 60. with respect to a
pseudo molecular plane defined by the four N atoms (21). The
size of macrocyclic cavity is 5.5-7 A wide and 6 A deep. An
NMR study showed that the benzene rings of the methyl-APO
molecule favor a "face" conformation, in which benzene rings

are perpendicular to the hypothetical molecular plane (22).

 

 

 

 

 

15C

FiBUre

 

60

 

 

Figure 4.1. Diederich’s water-soluble cyclophanes.

 

 

 

 

FiSUre

 

61

.. o
+ /
\
cu3 3c
o
/

s
/N
cu, H3c\
"3
H

u\_._/N

I

A

3

O - 484',”

3

O

cu3
+
+
\

on,

on
s S"
s s"

\
CH

\

/
C

C

C
“a

Figure 4.2. Water—soluble heterocyclophanes.

 

 

 

 

Variati
to impro*
Nitrogen
replaced
original
wide pH 1
in aqueOI
its elec

The b:
hydropho
'shallow
With 10!
been 1

Specific

Cationic

favors ,

having

binding

Skeleto

hYdroca

Synthes

Thea.

Promisi

chemic:

11) n.

Site t

(ii)T

 

62

Variations of the basic structure of methyl-APO were made
to improve the enzyme-like action of the host molecule.
Nitrogen atoms in the original structure of methyl-AFC were
replaced with sulfur atoms as shown in Figure 4.2-B. The
original methyl-APO was more stable in an aqueous media of a
wide pH range, while sulfonium heterocyclophane was unstable
in aqueous solution above pH 9, where a remarkable change in
its electronic spectrum was observed (20).

The basic methyl-APO molecule without modifications with
hydrophobic substituents, containing a relatively small and
.shallow cavity for host-guest interactions. APC molecules
with long alkyl chains of different functional groups have
been introduced (Figure 4.3) to improve substrate
specificity further. Anionic APC (Figure 4.3-A) favors
cationic guests (23), while cationic: APC (Figure 4.3-B)
favors anionic guests (24). Octopus-like APC (Figure 4.3-C)
having eight long alkyl chains gives extra hydrophobic
binding efficiency (25). By using“ two “rigid macrocyclic
skeletons, a capped APC (Figure 4.3-D) with four flexible
hydrocarbon chains connecting the macrocycles was
synthesized (26).

These synthetic enzyme model compounds (APCs) are highly
Promising macrocyclic compounds for various applications in
chemical analysis. Their most prominent characteristics are:
(i) The macrocyclic skeleton may provide a stable binding
site through hydrophobic interaction.

(ii) The stable binding site may show a high substrate

 

 

FiSUre

Solubl

 

63

 

/‘..——-cyc:eumo¢m mg

         
  
   

-
an

I

Announce m.

 

 

 

 

 

 

 

/ z"""Guoss melted:

 

 

. '10.! J ”anemone"
"‘9

 

Figure 4.3. Schematic representations of modified. water-

soluble heterocyclophanes.

 

 

 

specifici
requirem
(iii) A
wall of ‘
Thus,
for trij
inside
quenchin
Also, a
and char

using A}

C. Othe:

In 19
class c
ethers
Structu
couple,
can 31,
and no]
sTnthe:
iOtal
attach

The
p°1Tet

Genera

 

64

specificity due to electrostatic and geometrical
requirements for host-guest interaction.
(iii) A rigid microenvironment may be obtained due to the

wall of benzene rings.

Thus, the APCs may provide a favorable microenvironment
for triplet state molecules. Triplet molecules included
inside the macrocyclic cavity may be protected from
quenching by dissolved oxygen or impurities in the solution.
Also, a high selectivity in RTP is expected due to the size
and charges of the APC. In this research, the possibility of

using APC molecules in RTP was examined.

C. Other Macrocyclic Compounds in Chemical Analysis.

 

In 1967, Charles Perdesen reported the synthesis of a new
class of macrocyclic compounds called crowns (27). Crown
ethers are cyclic polyethers having various sizes and
structures. These compounds have the ability to selectively
complex metal ions, and in particular, the alkalies. They
can also solubilize inorganic and organic solutes in polar
and nonpolar solvents. Over 60 cyclic polyethers have been
synthesized with ring structures containing from 9 to 60
total atoms, from 3 to 20 oxygen atoms, and from 1 to 4
attached hydrocarbon rings (28).

The nomenclature recommended by Perdesen for his cyclic
Polyether compounds does not follow complicated IUPAC rules.

Generally, the groups attached to the crown are mentioned

 

 

 

 

Figure

 

65

J
J

[U
B
U

32'9"“ moneys-12mm

Figure 4.4. Some representative crown ethers.

 

 

 

 

 

 

first, t
"crown" .
Examples
been fou
with a I
indicati
the met
formed.
of the I
0n the
structul
cYclic
sandwic]
P01yeth.
Crown
membran
related
Prepare
ionic a
In 19
ligands
metal
central
cation
°°ntai1
becOme

Smalle

 

66

first, then the number of atoms in the ring, followed by
"crown" and then by the number of oxygen atoms in the crown.
Examples are given in Figure 4.4. The cyclic polyethers have
been found to form primarily 1:1 polyether:metal complexes
with a large array of metal ions. However, there are some
indications that, depending on the ratio of the cavity to
the metal ion. diameter» 2:1 and 3:2 complexes are also
formed. The 1:1 complexes are generally assumed to consist
of the metal ion bound in the cavity of the polyether ring.
On the other hand, 2:1 complexes are "sandwich" type
structures, in which the metal ion is located between two
cyclic polyether' molecules. The 3:2 complexes are "club
sandwich" types, in which two metal ions are located between
polyethers.

Crown ethers have been used in extraction studies (29,30),
membrane transport (31,32), ion-selective electrodes and
related electrochemical procedures (33), ion-exchange resin
preparation (34), and liquid chromatographic separation of
ionic and neutral species (35,36).

In 1967, Lehn and his coworkers introduced the macrocyclic
ligands called cyrptands (37). Cryptands form complexes with
metal ions, in which the metal ion is located within the
central cavity (crypt). In the cryptates, the complexed
cation is completely enclosed in three dimensions by ligands
containing 0 or N which bind to the cation. Thus, the ion
becomes isolated from external species if it fits, or is

smaller than the diameter of the cavity. The cavity is

 

 

 

 

 

 

 

hydrophi]
outside 1
solvents
which he
hydrocar
The
importan
earth i
macrocyc
2.1.1.,
imports:
The u
crown e
metal
determi
using c
ion-exc
Rec:
Chemica
applic;
Chapte;
f°rmat
(44) ,
Cycl
ooluum

SePhad

adVant

 

 

67

hydrophillic, and hydrophobic hydrocarbon groups form the
outside envelope to make cryptands quite soluble in organic
solvents. Thus, the cryptand is a three-dimensional ether,
which has a hydrophillic cavity and hydrophobic exterior
hydrocarbon groups (38,39).

The cryptands promise to become of great analytical
importance, as their' complexes with. alkali and alkaline
earth ions are much more stable than those with other
macrocyclic compounds, such as crown ethers. The cryptands,
2.1.1., 2.2.1, and 2.2.2. (Figure» 4.5), .are .analytically
important and readily available.

The usage of cryptands in chemical analysis is similar to
crown ethers. Kirch and Lehn (40) used cryptands in alkali
metal ion extraction. Czerwenka and Scheubeck (41)
determined sodium and potassium by potentiometric titration
using cryptands. Cryptands were used for the preparation of
ion-exchange resins (42).

Recently, cyclodextrins have been used extensively in
chemical, analysis. The structure, properties, and their
application in luminescence are discussed in the previous
chapter. Mueller and Rodin (43) studied inclusion complex
formation of cyclodextrins with metal ions. Cramer et al
(44) studied cyclodextrins as enzyme model system.

Cyclodextrin polymers are used as filling materials for
column chromatography (45). When compared with customary
Sephadex, it is apparent that cyclodextrin polymers have the

advantage in that they strongly retard molecules of an

 

 

 

 

 

 

Fizur

 

 

Cryptand 2.2.2

 

Figure 4.5. Lehn’s cryptands.

 

 

 

 

appropri
even isc
446,47)
chromatc
difficul
and pea
accompl
Recen
of cycl
solubil
increas
(50,51)
and s1
certail

m0dlfil

 

 

69

appropriate size. Molecules with similar molecular weight or
even isomers can be separated. Armstrong and his coworkers
(46,47) used cyclodextrins extensively in liquid
chromatographic separations. They demonstrated that many
difficult separations, such as enantiomers, diastereomers,
and positional, geometric, and structural isomers can be
accomplished (48).

Recently, an attempt was made to increase the solubility
of cyclodextrins in ‘the aqueous 'phase. Using ‘urea, the
solubility of cyclodextrins in aqueous solution was
increased up to 10 times (49). Tabushi and his coworkers
(50,51) modified cyclodextrins to provide more functionality
and structural rigidity. Considerable enhancement of a
certain elementary recognition interaction was observed with

modified cyclodextrins.

 

 

 

 

 

 

1. Keith
2. Saeng
3. Tabu:
1439.
4. Cram1
5. Tabu:
6. Jenc
McGraw-E
7. Fend
Macrame
3. Murz
Bull. (
9. Tab!
1981, ,
10. Ch:
1971,
11. Cr
1966,
12. Cr
13~ Mu
144 B:
15' K:
16. u.

70

CHAPTER IV REFERENCES

1. Kolthoff, I. M., Anal. Chem., 1979, 51, 1R.

2. Saenger, W., Angew. Chem. Int. Ed. Engl., 1980, 19, 344.
3. Tabushi, I. M.; Mizutani, T., Tetrahedron, 1987, 43,
1439.

4. Cram, D. J.; Cram, J. M., Science, 1974, 183, 803.

5. Tabushi, I, Tetrahedron, 1984, 40, 269.

6. Jencks, W. P., Catalysis in Chemistry' and .Enzymology,
McGraw-Hill, New York, 1969.

7. Fendler, J. E.; Fendler, E. J., Catalysis in Micellar and
Macromolecular Systems, Academic Press, New York, 1975.

8. Murakami, Y.; Sunamoto, J.; Okamoto, H.; Kawanami, K.,
Bull. Chem. Soc. Japan, 1975, 48, 1537.

9. Tabushi, I; Kimura, Y; Yamamura, K, J. Am. Chem. Soc.,
1981, 103, 6486.

10. Christensen, J. J.; Hill, J. 0.; Izatt, R. M., Science,
1971, 174, 459.

Ed. Ehgl.,

11. Cramer, F; Mackensen, G., Angew. Chem. Int.

1956, 5, 601.

12. Cramer, F., Angew. Chem., 1961, 73, 49.

13. Murakami, Y., Top. Curr. Chem., 1983, 115, 107.

14. Breslow, R., Science, 1982, 218, 532.
15. Kellogg, R. M., Angew. Chem., 1984, 96, 769.
16. Murakami, Y., J. Inclusion Phenom. 1984, 2, 35.

 

 

 

17. Die:
362.
18. Diet
8024.
19. Uru:
Japan,
20. Tel
Soc., 1
21. Tai
Higuchi
22. Ta
1975, .
23. Mu
Chem.
24- Mu
Chem.
25. Mu
Lett.,
26. M
Chem.
27. p.
28. c‘
Rev.,
29. v
Micrc

30, r]

71

17. Diederich, F., angew. CHem. Int. Ed. Ehgl., 1988, 27,
362.

18. Diederich, F.; Dick, K., J. Am. Chem. Soc., 1984, 106,
8024.

19. Urushigawa, Y.; Inazu, T.; Yoshino, T., Bull. Chem. Soc.
Japan, 1971, 44, 2546.

20. Tabushi, I.; Kimura, Y.; Yamamura, K., J2 Am. Chem.
Soc., 1981, 103, 6486.

21. Tabushi, I.; Yamamura, K.; Nonoguchi, H.; Hirotsu, K.;
Higuchi, T., J. Am. Chem. Soc., 1984, 106, 2621.

22. Tabushi, I.; Yamada, H.; Kuroda, Y., J) Org. Chem.,
1975, 40, 1946.

23. Murakami, Y.; Nakano, A.; Miyata, R.; Matuda, Y., J.
Chem. Soc. Perkin I, 1979, 1669.

24. Murakami, Y.; Nakano, A.; Akiyoshi, K., Fukuya, K., J.
Chem. Soc. Perkin I, 1981, 2800.

25. Murakami, Y.; Kikuchi, J.; Suzuki, M.; Takaki, T., Chem.
Lett., 1984, 2139.

26. Murakami, Y.; Kikuchi, J.; Tenma, H., J. Chem. Soc.
Chem. Commun., 1985, 753.

27. Perdesen, C. J., J. Am. Chem. Soc., 1967, 89, 7017.

28. Christensen, J. J.; Eatough, D. J.; Izatt, R. M., Chem.
Rev., 1974, 74, 351.

29. Vasilikiotie, G. S.; Papadoyannis, I. N.; Kovimtzis, A.,
Microchem. J., 1984, 29, 556.

30. Tsay, L.; Shih, J.; Wu, S. 0., Analyst, 1983, 108, 1108.

 

 

31. Lam
Christen
32. Tent
33. Meye
54, 278.
34. Fer]
1980, 5.
35. Kim‘
36. 818
163.

37. Die
Lett.,
38. Let
39. Lel
40. Ki
1975,
41. C2
34.

42. St
43. M
COMmut
44- CI
45' H<
46- A:
22: 4‘.
47. A

1985,

 

 

72

)1 . Lamb, J. D. ; Izatt, R. M. ; Robertson, P. A. ;
3hristensen, J. J., J. Am. Chem. Soc., 1980, 102, 2452.
32. Tsukube, H., Tetrahedron Lett., 1982, 23, 2109.

33. Meyerhoff, M. E.; Franticelli, Y. M., Anal. Chem., 1982,
54, 27R.

34. Fernando, L. A.; Miles, M. L.; Bowen, L. H., Anal. Chem.
1980, 52, 1115.

35. Kimura, K.; Shono, T.; J. Liq. Chromatogr. 1982, 5, 223.
36. Blasius, E.; Janzen, K. P., Top. Curr. Chem., 1981, 98,
163.

37. Dietrich, B.; Lehn, J. M.; Sawvage, J. P., Tetrahedron.
Lett., 1969, 2885,2889.

38. Lehn, J. M., Pure & Appl. Chem., 1977, 49, 857.

39. Lehn, J. M., Acct. Chem. Res., 1978, 11, 49.

40. Kirch, M.; Lehn, J. M., Angew. Chem. Int. Ed. Engl.,

1975, 14, 555.

41. Czerwenka, G.; Scheubeck, L., Z. Anal. Chem., 1975, 276,
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42. Stefanak, 2.; Simon, W., Chimica, 1966, 20, 436.

43. Mueller, P.; Rodin, D. 0., Biochem. .Biophys. Res.

Commun., 1967, 26, 398.
44. Cramer, F., Angew. Chem. Int. Ed. Engl., 1966, 5, 601.
45. Hoffman, J. L., Anal. Chem., 1970, 33, 209.

46. Armstrong, D. W., DeMond, W., J. Chromatogr. Sci., 1984,
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47, Armstrong, D. w.; DeMond, W.; Czech, B. P., Anal. Chem.,

1985, 57, 481.

 

 

48. Arms
49. Phat
Anal. Ch
50. Tabu
Fujita,

51. Tab

Soc., 1!

 

73

.8. Armstrong, D. W., Anal. Chem., 1987, 59, 84A.

19. Pharr, D. Y.; Fu, Z. S.; Smith, T. K.; Hinze, W.
4nal. Chem., 1989, 61, 275.

50. Tabushi, I.; Shimokawa, K.; Shimizu, N.; Shirakata,

Fujita, K., J. Am. Chem. Soc., 1976, 98, 7855.

51. Tabushi, I.; Yamamura, K.; Nabeshima, T., J} Am. Chem.

Soc., 1984, 106, 5267.

 

 

Altho
over tl
instrum:
instrum
needs d
(1) des
RTP wit
(2) con
c°mPone
Nithipz
for det

The
acquis
labora
acquis
other
acquis
cons“n
finely:

°f ti
exPer:

than .

 

 

74

CHAPTER V

INSTRUMENTATION

Although RTP has grown as an important analytical.method
over the last two decades, there are few commercial
instruments available for RTP. Modifications to commercial
instruments for RTP studies have been made to aid individual
needs depending on the RTP technique employed. Vo-Dinh et a1
(1) designed an automatic phosphorimeter for solid-surface
RTP with a continuous filter paper device. Cline Love et al
(2) constructed a phosphorimeter from commercially available
components and used it in micellar—stabilized RTP studies.
Nithipatikom and his coworkers (3) designed a spectrometer
for determining single and multiple RTP lifetimes.

The use of microcomputers for instrument control and data
acquisition has become a common tool in scientific
laboratories. In many cases, experimental control and data
acquisition require complex timing and synchronization with
other experimental conditions. Instrument control, data
acquisition, complex timing and synchronization, time
consuming event monitoring, tedious calculations, and data
analysis can be done with a personal computer. This ability
of the personal computer has enabled scientists to do

experiments easier with more sophisticated instrumentation

than previously possible.

 

 

 

In thi
controll
fluoresc
acquisit
modificz
adaptor

Perkin-I

A. Lumi

In t
500) w:
Person:
and da
lumine:
Comput

The
Xenon
monoch
mm fo
emissi
°Ptim4
°°ntr4
allow.

monoC
sPect

the m

 

 

 

 

75

In this chapter, the construction of a versatile computer
controlled luminescence spectrometer for measuring
fluorescence, RTP, and RTP lifetimes with comprehensive data
acquisition and analysis software is described. Also, the
modification of an IBM-PC data acquisition and control
adaptor board is presented. This board was used for the

Perkin-Elmer LS-5B spectrometer later in this research.

A. Luminescence Spectrometer.

In this work, an Aminco-Bowman spectrofluorometer (SPF-
500) was modified and interfaced to the IBM-KT compatible
personal computer for keyboard control of the instrument,
and data acquisition. This instrument allows semiautomatic
luminescence data acquisition and analysis on a personal
computer.

The Aminco-Bowman spectrofluorometer (SPF-500) contains a
Xenon lamp which produces an intense 250 W Xenon arc. The
monochromators are single-pass Czerny-Turner tYPe With a 250
mm focal length and f/4 aperature. Both excitation and
emission monochromators use 600 line/mm gratings blazed for
optimal wavelength response. These monochromators are
controlled by precision 500 step/rev stepping motors. This
allows precise control of position and scanning rate of both
monochromators. The interface connector at the back of the

spectrometer contains all the necessary control lines for

the monochromator control.

 

 

 

 

 

 

 

1. Princ

In th
monochrc
sample
without
shown i
used as
modulat
allows
has dec

For
acQuisi
Ohio) s
°UIPuts
monoch;
Turbo
contro

A :
excite
beam,
adiust
adjust
°06nt4

Th

ConVe.

 

 

 

 

1. Principle of Operation of the Instrument.

In this work, the Optical unit, excitation and emission
monochromators, PMT, xenon lamp with power supply, and
sample compartment unit of the spectrometer, were used
without modification. A block diagram of the instrument is
shown in Figure 5.1. A continuous Xe-arc lamp (250W) was
used as an excitation source. The exciting radiation was
modulated by a rotating single-disk type chopper. This
allows phosphorescence to be measured after any fluorescence
has decayed.

For data acquisition and monochromator control, a data
acquisition board (Lab Master, Scientific Solution, Solon,
Ohio) was used with an IBM-XT compatible computer. Digital
outputs from the Lab Master board controlled both
monochromators for scanning. Menu driven software written in
Turbo Pascal and Macro Assembler was used for instrument
control, data acquisition, and data manipulation.

A photodiode was placed at the opposite side of the
excitation beam to sense the modulation of the excitation
beam. The pulsed signal from the Optointerrupter was
adjusted to be in-phase with photodiode signal. This
adjusted chopper signal ‘was used to trigger' the AM9513
counter/timer for time-resolved data collection.

The current signal from the PMT (Hamamatsu R928) was

converted into a voltage signal, and a variable gain

 

 

 

 

 

 

77

 

 

 

 

 

 

 

 

 

 

 

 

 

 

i Opts-Memphi-
1m chopper l
_; = ——> Scope
[ fin-llono ...... I Sample
4 u ..
Hester

 

 

 

 

E Photo Diode

Variable Gain
C” ——> ““3

Personal j
Computer
--' _ I l '

[Mime l

 

 

‘r—— 1"

 

 

 

 

 

 

 

 

 

Figure 5.1. Block diagram of the luminescence spectrometer.

 

 

 

 

amplifie
input V4
converts
filter w
between
amplifie

decay c4

2. Hard

(a) Com

The
digital
multipj
two 12
Channe
5.2, .
counte
insta]
Scannj
signa:

The
1/0 m
chann
range

Was U

78

amplifier (AD507, Analog Device) was used to get the desired
input voltage signal (0-10 V) for the analog-to-digital
converter (ADC). To reduce high frequency noise, a low-pass
filter was used for fluorescence and RTP measurements placed
between the current-to-voltage converter and the main
amplifier. .It was turned off by a manual switch when RTP

decay curves were to be measured.
2. Hardware Construction.
(a) Computer Interfacing to Spectrometer.

The Lab Master board (4) contains a 12 bit analog-to-
digital converter (ADC) with a built-in 16 channel
multiplexer (MP 6812), a 24 bit parallel port (Intel 8255),
two 12 bit digital-to-analog converters (DACs), and a five
channel 16 bit counter/timer, AM9513, as shown in Figure
5.2. The AM9513 LSI chip is one of the most powerful
counter/timers (5). The Lab Master data acquisition board
installed on the IBM-XT compatible computer controlled the
scanning module of the spectrometer, and digitized analog
signals from the spectrometer.

The Lab Master board address was set to 1808 (decimal) and
I/O mapped. The last channel of the multiplexer was set to
channel 15. Input mode was set to pseudo-differential 0-10 V
range, and no interrupts were used. Channel 15 of the ADC

was used for luminescence data read, and channel 13 and 14

 

 

 

 

FiEUI

bOQrd

79

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

24 bit
Parallel Port ta] l/O
.4 .. l+—* 4
5 channel
,___. —[(“9 Counter )tlmer 4____, Counter l/O
IBII
Board
BUS h- control late 1 Int: 1
... mp ml
logic control t p
_—+
i 12 hit an in l
+ <— 0: 9n
.___. (I? 6612)
DAC 7 12 Mt Anal tp l
0; on u
(ch so) ’

 

 

 

Figure 5.2. Schematic representation of the Lab Master

board.

 

 

 

were u:
Pin 24
interf:
ADC cl
wavele:
l
monoch
latch
turns
contro
approp
output
monoch
scanni
excite
select
An 1
Progre
Port
monocl
latch
modull
°°nneq
In
COUnt.
after

 

80

were used for reading positions of the both monochromators.
Pin 24 (analog output of excitation wavelength) of the
interface connector of the spectrometer must be connected to
ADC channel 13, and pin 23 (analog output of emission
wavelength) must be connected to ADC channel 14.

For control of both excitation and emission
monochromators, a parallel output was used along with a
latch (74LS75). The direction of rotation and the number of
turns of the monochromator to scan wavelength were
controlled by the software. This was done by sending the
appropriate digital outputs to the latch inputs, whose
outputs were connected to the control lines of both
monochromators. This allowed total computer control of the
scanning modes (emission , excitation , synchronous ,
excitation-emission) and automatic initial wavelength
selection of both monochromators.

An Intel 8255-5 parallel I/O port on Lab Master board was
programmed to work in mode 0, and digital data bits 0-5 of
port A were used to send control pulses for both
monochromators. Data bits 1-4 were latched by a 74LS75 quad
latch in order to keep each control signal of the scanning
module until next operation. Each digital signal and latch
connection is shown in Figure 5.3.

In the phosphorescence mode, counter 5 of the AM9513
counter/timer was used to trigger the ADC start conversion
after a given delay time. In the RTP lifetime mode, counter

4 was used to control the delay time after lamp excitation,

 

 

F18n:

Mast

81

 

ADC channel 11255-5 m am Mt
[ 1a 14 o 1 z a 4 51 Computer ride
‘ I r

 

 

 

 

 

 

 

Q 16 15 10

 

 

 

 

 

 

 

 

 

Spectrometer
[ 22 23 43 43 44 so I interface connector
I l l . W l l l
E! El Scan El 8! Scan lamp
Invelength Pd“ Iotor clutch dlrectlon lgnltlon
analog output)

‘ To control so ennlneufumodul ebdlrectly loom
pin 83 (motor 111 hold and pint auto lmtllrectlon enable)
must be connected to pln tnrn.)(

Figure 5.3. Interfacing of the control signals from Lab

Master to the Aminco-Bowman spectrometer (SPF-500).

 

 

and con
ADC. 0U
GATE4 a
order t
of the
extern:
order

betwee
operat

sectic

(b). 1

Se‘
Aminc
excit
rota)
freq!
dete
most
solu
give
11v:
Sen:
or
ter

and

 

 

 

 

82

and counter 3 was used to control the sampling rate of the
ADC. OUT4 of the counter/timer must be connected to GATE3.
GATE4 and GATE5 must be connected to the chopper signal in
order to count the delay time in microseconds. 0UT3 and OUTS
of the counter were connected to the control pin of the
external start conversion of ADC through a rotary switch in
order to select external start conversion signals of ADC
between 0UT3 and OUTS. More detailed explanations about the
operation of the counter/timer will be shown later in this

section.

(b). Light source Modulation and Detection.

Several commercial spectrophosphorimeters, including the
Aminco-Bowman (SPF-500), use a rotating can to modulate the
exciting light source. The major disadvantage of the
rotating-can type phosphoriscope is that the modulation
frequency of the excitation source, and the luminescence
detection time window are limited. But the RTP lifetimes of
most organic phosphors are very short, especially in
solution. As a result, the rotating-can type phosphoriscope
gives very low or negligible luminescence signals for short-
lived phosphors. The short delay time will increase the RTP
sensitivity of the short lived phosphors. A pulsed source,
or a single-disk type chopper, gives better performance in
terms of flexibility for selecting appropriate delay times

and gate times for the experimental requirements.

 

 

 

 

Fieu
(B)

 

 

 

 

 

 

 

10H

 

 

 

 

 

 

 

 

 

 

 

Figure 5.4. Circiuts built for (A) photodiode light sensor,

(B) current-to-voltage converter.

 

 

 

In
replace
disk wa
diamete
was d1
control
types .
were u
lifeti
static
beam,
choppe

The
accomj
inter
trigg
chop;
{GEHE
Plan:

was :

Phot
diSp

out;

int:

Sim

 

 

 

84

In this design, the Xe-arc lamp was modulated by a

  

replaceable rotating single-disk type chopper. The chopper
disk was constructed with a stiff, thin plastic blade (10 cm
diameter) and coated with non-glare black paint. The chopper
was driven by a synchronous motor, and its speed was
controlled by a variable, precision DC power supply. Two
types of disks, which have a different number of open slots,
were used depending on the type of experiment (RTP or RTP
lifetime). When fluorescence ‘was measured, the disk was
stationary with its open slot facing the incoming excitation
beam, so that all the exciting light could pass through the
chopper disk.

The phase detection of the rotating chopper was
accomplished through the use of a photodiode and an opto-
interrupter. A photodiode (EG&G UV-lOOBQ) with a Schmitt
trigger (74LSl4) was placed on the opposite side of the
chopper across the sample cell, and an opto-interrupter
(GEHZlBl) with monostable multivibrator (74L8121N) was
placed under the chopper blade. The phase detection signal
was adjusted in the visible range as described below.

At the beginning of the experiment, signals from
photodiode circuit and opto-interrupter circuit ,were
displayed concurrently on a dual-trace oscilloscope. The
output of the photodiode circuit is the true chopped signal
of the excitation source, whereas the output from the opto-
interrupter circuit is not the true source chopping signal,

since its signal depends on the mounting position of the

 

 

 

 

mm

Photo
Mode

lull-
Vibrator

Fig‘.
The
null

adj-

 

85

 

 

 

 

 

 

 

 

 

 

luliivlhrntor
.3.
‘ Q
.4. 7412111 H— -
.—L— Q
-.5.
14! 7l1oLc 511
' , um
I :AM 3(203)

 

 

 

 

1.1__l ' |
mp1. (1) 1 l l l—

 

 

 

 

 

 

iiulti- ‘ . . .
Vibrator

 

 

 

 

 

 

 

Figure 5.5. Timing diagram of the chopper phase detector.
The opto-interrupter output is connected to the
multivibrator and the pulse width of the multivibrator is

adjustable with a variable register.

 

 

 

opto-in
pulse c
by a ‘
falling

opto-ii

(c). R

For
AM9515
‘ choppe
500 H
signa.
conne
PPOSI‘
board
outpt

Conve

exci
For

simu
rOte
Cour

Out]

Out

86

opto-interrupter under the chopper blade. The low-going
pulse output of the opto-interrupter was adjusted manually
by a variable register of the multivibrator, until the
falling edges of both signals from the photodiode and the

opto-interrupter were exactly matched (Figure 5.5).

(c). RTP and RTP Lifetime Measurement.

For RTP and RTP lifetime measurements, a timer/counter,
AM9513, on the Lab Master board was used. A single-disk type
chopper with four opening slots was rotated to obtain a 200-
500 Hz modulated source for RTP measurements. The chopper
signal from the one-shot of the phase detector circuit was
connected to gate 1 of the counter 1, and counter 1 was
Programmed to generate a pulse at the terminal count. The on
board 1.000 MHz clock was used as the clock source. The
output of counter 1 was connected to the ADC external start
conversion pin. By varying the count number of the counter
1, data can be acquired at different time delays after
excitation for time-resolved phosphorimetry (Figure 5.6).
For RTP lifetime measurement, two counters were used
simultaneously. A chopper with one or two open slots was
rotated to obtain a 5-10 Hz modulated source. Gate 1 of the
counter 1 was connected to the chopper signal, and the
output of the counter 1 was connected to gate 2 of counter
2. The analog-to-digital conversion was triggered by the

output of counter 2. At the falling edge of the chopper

 

 

t

Counter
Gilt

Counter

Count 1

Count:

count

Fig}
111.

 

87

 

Counter F—

Gate _.

 

 

Counter 1

 

 

'i'enninal
Count 1

 

 

Counter 2

 

 

Terminal
Count 2

 

 

 

 

Figure 5.6. Timing diagram of the counters for RTP and RTP-

lifetime measurement.

 

hahle
Counter

(ho
11.22”

Delay

Gate

liietime

 

signal,
time a1
edge 1
initia
repeti
At eac
read 1
delay
starti
minim:
read j
At
the
wavel
CVCIE
POini
The

C0111

3. s

PAS(
ASS]
lum
XT

EMI

 

7;—

88

time and generates a pulse at the terminal count. The rising

edge of the terminal count signal of counter 1 then

 

initiates counter 2 to count down for a given gate time,
repetitively, to generate pulses at each gate time interval.
At each terminal count pulse of counter 2, an RTP signal was
read to obtain a decay signal. The delay time is the time
delay from the falling edge of the chopper signal to the
starting point of the data reading. The gate time (33 .113
minimum) is the time interval between the successive data
read points.

At the beginning of each experiment, the user can specify
the number of data points to read at each emission
wavelength for RTP measurement, or the number of decay
cycles to repeat per experiment, and the number of data
points per each decay cycle for RTP lifetime measurement.
The final data. are obtained. by signal averaging of ‘the

collected raw data in real time.

3. Software Development.

A menu driven program, EMISPEC,.was developed using TURBO
PASCAL (Version 4.0, Borland International) and MACRO
ASSEMBLER (Version 4.0, MicroSoft) for instrument control,
luminescence data acquisition, and manipulation on the IBM-

XT compatible computer. A block diagram of the program,

 

EMISPEC, is shown in Figure 5.7. There are six items in the

 

signal, counter 1 starts the count down for a given delay.

File

. load
. Save
.. list

Lies!

 

Fig'

 

89

 

' MSPEC-II

I

I_ i | t I 1

File | Data ] Set-up J Graph J Lifetime’J ToolsJ

 

 

 

 

 

 

 

 

 

 

 

 

 

 

.. load _ Display _ Set-up _ Screen Plot .. Calc LTil .. Help

 

 

 

 

 

 

1.. Save .. Print _ lode .. Printer Plot _ Simulate LTii _ Change Par-m
.. List _ Smoothing .. Plotter Plot L. Semilog Plot l. Test Data Read
_ Dos Shell _ Start Read .. Plotter File

_ llulPlot

Figure 5.7. Block diagram of the EMISPEC program.

 

 

 

main 1
For n1
transi
devel<
node
scan
wavel
scan
emiss
spect
nonoc
ways
The :
an E
rout

soft

4-i

inf:
the
Inca

Var

00C

(7:

 

90

main menu: File, Data, Set-up, Plot, Tools, and Lifetime.
For noisy data, smoothing can be employed using a Fourier
transformation technique, according to the unique algorithm
developed by Eric E. Aubanel (6). Set-up parameters include
mode (Fluorescence, RTP, RTP Lifetime), initial setting and
scan ranges for both monochromators, number of data per
wavelength or number of decay cycles for signal averaging,
scan mode (emission, excitation, synchronous, excitation-
emission), delay time, and gate time. Fluorescence and RTP
spectrum data are plotted on the screen in real time as the
monochromator scans. The plot routine plots data in five
ways: screen plot, printer plot, plotter plot, plotter file.
The spectrum drawn on the screen can be printed with either
an EPSON printer or an HP plotter. Data plot can also be
routed into a file in HPGL format for use with other

software.

4. Results and Discussion.

The primary output of any experiment in which chemical
information is to be extracted, is the signal which measures
the phenomenon under observation. In most cases the signal
measured has some superimposed noise which arises from
various sources. Noise can be distinguished from the signal
by its frequency characteristics and by the time of
occurrence or phase coherence of their frequency component

(7). The removal of noise can be performed by hardware and

 

 

software.
The thrl
moving-aver
In the mo
replaced by
on either 1
it is very
first and ‘
degree as '
n neighbor
Signal nor
The less
you Specif
distances
The advan
Statistics
d°es not 1
intervals,
Senerates
bad data
Spread th
F°uPier
best meth
and elimi
usually j
c°"‘P°nent

 

91

software.

The three most common methods for smoothing data are
moving-average, least-squares, and Fourier transformation.
In the moving-average method (8), each data point is
replaced by the average of itself and n neighboring points
on either side of it. The advantage of this method is that
it is very easy to program. The disadvantage is that the
first and the last data points are not smoothed to the same
degree as the rest of the data set, because they don’t have
n neighbors on each side of them. This method flattens the
signal more than the other methods.

The least-squares method identifies the line of the order
you specify which minimizes the sum of the squares of the
distances between the data points and the calculated line.
The advantages of this method are that it generates
statistical information on the goodness of the fit, and it
does not require that the data be collected at regular time
intervals. The disadvantage of this method is that it
generates disappropriately biased data by one or two very
bad data points, because it will twist the line of fit to
spread the error over the entire data set.

Fourier transformation and inversion (9) is probably the
best method, since it lends itself naturally to identifying
and eliminating noise. The reason for this is that noise is
usually present at high frequencies, whereas, the signal
components are usually present at low frequencies. Fourier

transformation produces the frequency spectrum. By

 

 

 

 

 

eliminating

performing a
the original
In this s
smoothing me
frequency 10
converter an
fluorescence
long time
decay signa
lifetime w
averaging s
enhance the
If the noiSI
the noise '1:
signal is r
improved by
Also, by 1

reduce nois

of Fourier

Transforms

iii. In t

smoothing)

 

92

eliminating the high frequency portion of the spectrum and
performing an inverse Fourier transformation, one can obtain
the original data without much of—the noise.

In this system, we employed both hardware and software
smoothing methods. A simple passive low-pass filter (cut-off
frequency 10 Hz) was placed between the current-to-voltage
converter and the variable gain amplifier (10) for RTP and
fluorescence measurements. Because the low-pass filter has a
long time constant which can affect the phosphorescence
decay signal, it was turned off by a manual switch when RTP
lifetime was being measured. Two other methods, signal
averaging and Fourier smoothing, were used to further
enhance the signal to noise ratio (S/N) of the measurement.
If the noise is random and the number of measurements is N,
the noise is proportional to the square root of N, while the
signal is proportional to N. As a result, the S/N will be
improved by the square root of N with signal averaging (11).
Also, by performing Fourier smoothing, one can further
reduce noise components from the measured data. The method
of Fourier smoothing used is a modified Discrete Fourier
Transformation (DFT), which runs much faster than regular
DFT. In this method, the user can control the degree of
smoothing, and the data size is not limited to a multiple of
2, which is the limiting factor of the Fast Fourier
Transformation (FFT).

The effect of the degree of smoothing of the data is well-

demonstrated in Figure 5.8. A typical luminescence data set

 

 

 

 

natal

 

m m m -M

t“ agate-ea

 

 

93

 

 

 

 

I000
.00
.0.
, I
o: . .
. g :3 ..s..
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3 m . 0. ° . ......
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o L I I I I I I I I
300 325 350 375 400 425 450 475 500 525

immanent 000

5,3, A. .Fourior ssoothinn of the lusinsscencs spectrum of Naphthajgng in
missusiSOSI . Degree of smoothing: 9. new data: dot. smoothed data: line.

 

j

V

 

E

3....

E

:3

tune»

a“.
goo.
zoo-'
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o -...

soc

 

 

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o o.
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s .... s I
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o ' o o
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0‘... o. I, 0...... . 0. ....‘~. I ..
s s g s
0....... .. ... .0 ~ { 0.0
O O
_I I L I I _I I I I
325 350 375 400 4a 450 475 500 525

“V3.85?" M

3.3. Fourier- ssoothing of the Inslnescsncs spectrum of Naphthalene in

sicsnslSOSl .Deares of smoothing: 25. new dots: dot. smoothed data:

line.

 

 

 

 

 

in Figure 5.

naphthalene
solution. Pea
above 400 nm
dots in F
smoothing, a
Fourier smoo
Figure 5.
smoothing o
undersmoothe
Choosing the
and error.
smoothed (18‘
Figure 5.8,
which the f i
This end ei
beginning a1

data as per

points are

Figure 5.9,

line method

The gene

phosphoriu

Fisher and

94

in Figure 5.8 was obtained with an aqueous solution of
naphthalene in a sodium dodecyl sulfate (SDS) micellar
solution. Peaks below 400 nm are fluorescence, and the peaks
above 400 nm are phosphorescence at room temperature. The
dots in Figure 5.8 are the raw data 'without signal
smoothing, and the solid line is the smoothed data after
Fourier smoothing.

Figure 5.8.A shows oversmoothed data with a degree of
smoothing of 9, while with a degree of smoothing of 25,
undersmoothed data were obtained as shown in Figure 5.8.3.
Choosing the right degree of smoothing is a matter of trial
and error. With a degree of a smoothing of 15, properly
smoothed data were obtained as shown in Figure 5.9. In
Figure 5.8, both smoothed data sets show "end effects" in
which the first and last part of the data set are distorted.
This end effect is due to the discontinuity between the
beginning and the end of the data set. The DFT treats the
data as periodic; that is, it assumes that the last data
points are followed by replicas of the initial points. In
Figure 5.9, this "end effect" was corrected by the straight
line method (6).

The general principles and applications of time-resolved
phosphorimetry were well demonstrated by Winefordner (12).
Fisher and Winefordner (13) showed a detailed comparison of
the pulsed source and continuous mechanical phosphoriscope
system in. phosphorimetric instrumentation. The major

advantage of pulsed source phosphorimetry over the rotating

 

 

 

 

 

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coo“

 

 

 

 

 

 

can type me

selecting ex
gate time,

under optim
RTP, the (r
inadequate

minimum) to
after excit
lived RTP

detection

hand, if 1:
type blade
phosphoresc
phosphoresc
choosing ti

resolved s:

the measur
type phosp)
Accurate

source Pu

ueasureme

is used

adjustuen

its out)“

 

 

96

can type mechanical phosphoriscope, is the possibility of
selecting experimental conditions, such as, delay time, and
gate time, so that the species of interest is measured
under optimum conditions. For measurement of short-lived
RTP, the rotating can phosphoriscope was found to be
inadequate since it takes a relatively long time (1.5 ms
minimum) to open the emission window by rotating 90 degrees
after excitation. As a result, the intensity of a short-
lived, RTP signal has decreased significantly' before the
detection of actual RTP signal is started. On the other
hand, if the exciting light source is modulated by a disk
type blade or pulse, the detector is always open to the
phosphorescence signal. The measurement of the
phosphorescence signal can be initiated at any time by
choosing the proper delay time after excitation for time-
resolved spectrometry. This system gives more versatility in
the measurement of short-lived RTP over the.rotating can
tYPe phosphoriscope.

Accurate detection of the end of the modulated excitation
source pulse is very important in RTP and RTP lifetime
measurements. Typically, an apto-interrupter or photodiode
is used to detect the chopper phase. However, manual
adjustment of the position of the opto-interrupter so that
its output matches exactly with the true source chopping, is
very difficult. Also, most photodiodes (even UV sensitive)
are not very sensitive to light below 300 nm; however, many

organic compounds require excitation under 300 nm. In

 

 

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Excitation acan range: 280-380. aaiaaion acan range: 300-500 nu.

 

 

 

ute 5.10.3. Excitation-Salaaion apactrua of Naphthalene at real temperature.
Excitation acan range: sea-350. aaiaaion acan range: 280-48‘0.

 

 

addition, typ
high concentr
difficult to
signal from
difficulties,
and an opto-
system gives
which is ind
the chopper,
the sample s
Figure 5.
Pyrene and 1
emission s.
wavelength
Spectrum is
the Sample
Wavelength
different 1

different a
3- Modific:

Recently
have becon
One °f the
IBM-q)“ A (

Adapter).

 

98

addition, typical RTP sample solutions are turbid due to the
high concentration of heavy atoms. These conditions make it
difficult to obtain a reliable and accurate phase detection
signal from the rotating chopper. To overcome these
difficulties, we used a combined system using a photodiode
and an opto-interrupter for chopper phase detection. This
system gives a very reliable and accurate chopper signal,
which is independent of any variation of the motor speed of
the chopper, excitation source wavelength, and condition of
the sample solution.

Figure 5.10 shows the excitation-emission spectrum of
pyrene and naphthalene in SDS micellar solution. The entire
emission spectrum was obtained by changing excitation
wavelength. at certain intervals. The excitation-emission
spectrum is useful for examining the spectral response of
the sample molecules over certain excitation and emission
wavelength ranges. Also, any two similar. spectra of
different samples at a particular excitation will show a

different excitation-emission spectrum.
B. Modification of IBM-DACA.

Recently, personal computers and data acquisition adapters
have become commercially available at much lower prices.
One of the big bargains at the time of this writing is the
IBM-DACA (IBM Personal Computer Data Acquisition and Control

Adaptor). The IBM-DACA provides both analog and digital I/O

 

 

 

 

capabilities
general purP<
does not req
throughput, f
acquisition 1
functions: a
digital conv:
timer/counte
This sec1
board for g
SYstem cont:
this modif:
collection <
unmodified
trigger, by
Small jumpe
board timer
timing and

this reseaz
1‘ Hardware

( a) Hardwa:

The anal
which are

c("W'El‘ter

 

99

capabilities as well as an on-board counter/timer (14). For
general purpose data acquisition and system control which
does not require complex timing, process control, or high
throughput, it is satisfactory. But for more complex data
acquisition and system control, it lacks two very important
functions: a hardware trigger capability for the analog-to-
digital conversion; and total control of the three on board
timer/counters.

This section describes a modification of the IBM-DACA
board for general purpose laboratory data acquisition and
system control, along with a software package developed for
this modified IBM-DACAJ IBy this modification, data
collection can be initiated by a software trigger as in the
unmodified original IBM-DACA, as well as by a hardware
trigger, by simply switching the jumpers installed on a
small jumper board. Also all three channels of the on-
board timer/counter can be programmed and used for complex
timing and counting operations. This board was used later in

this research with Perkin-Elmer LS5 spectrometer.
1. Hardware Modification.

(a) Hardware Triggered Analog-to-digital Conversion.

The analog input device of the IBM-DACA has four channels
which are multiplexed into a single analog-to-digital

converter (ADC), AD574K (15). The ADC converts the analog.

 

 

signal in cm
of 0 to 4095
to most pop‘
controllers .
control the
63: are both
or a conver
(CE) or chi
analog-to-di
DACA, both
design, whi.
to-digital
conversion,
CE Signal .
its inverte
ADC CE sign
analog-tow-
can not 3,
analOS't.o-¢
triBSered
the oriSin.
In this
trigger of
the line
conversi0n
board' L:

disconnect

 

 

100

signal in one of three ranges to digital values in the range
of 0 to 4095 (12 bit). The AD574K is designed to interface
to most popular microprocessors with peripheral interface
controllers. The control signals CE, CS and R/C of AD574K,
control the operation of ADC. The state of R/C, when CE and
.3 are both asserted, determines whether a data read (R/C=1)
or a convert (Ii/5:0) is in progress. Either chip enable
(CE) or chip select (5's) signal may be used to initiate
analog-to-digital conversion if R/C is low. In the IBM-
DACA, both CE and CS are already enabled in the original
design, which means only the R/C signal controls the analog-
to-digital conversion. To trigger the analog-to-digital
conversion, the start convert signal is ANDed with the ADC
CE signal from the external distribution panel board, and
its inverted signal is fed to the R/C pin of AD574K. The
ADC CE signal from the distribution panel can only delay the
analog-to-digital start conversion when it is low, but it
can not guarantee a synchronized hardware trigger of the
analog-to-digital conversion. Thus, a true hardware
triggered analog-to-digital conversion is not possible in
the original design.

In this modification, we have enabled a true hardware
trigger of the analog-to-digital conversion by disconnecting
the line between the R/C (pin 5 of AD574K) and the start
conversion signal line (pin 3 of 028) on the printed circuit
board. Lines were extended from both sides of the

disconnected point to a jumper daughter board which was

 

 

5V

GATE 0 (11)

IL___

 

GATB l (14)

,L_

 

 

I cm (15)

/ :Disconneq
\ :Dlsconne
X 3 Discoona

“Slim 5 . 1

the IBM‘DA:

lOl

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

5V 1.023 111120151 pin 8)
\
/<
Clock 0(9)
GATE 0 (11)
/ COUNTER 0
OUT OM) \ 027 in 5
‘— P
\
/<
CLOCK 1(15)
GATE 1 (14) COUNTER 1
( our 1(13) V 027 pin 1
/\
CLOCK 2(18) V 051 pin 9
cm 2 (m) A
OUT 2 17) .
/ COUNTER 2 ( X 027 pun 3

 

 

 

 

/ : Disconnect line and extend a line from right side to the jumper board
\ : Disconnect line and extend a line from hell side to the jumper board
I : Disconnect line and extend lines from both ends to the jumper board

Figure 5.11. Modification of the timer/counter circuit of

the IBM-DACA.

installed or
options (a 1
jumper is p]
work with a
unmodified I
another 'tr
conversion

requires 2
conversion.
exceed a pu
than 25 us
single trig
multivibrat
Pulse with
Source, reg
0f the mu

Conversion

(b) Modifi

The Int.
ls‘bit cox
caSledded 1
design of
the Clock
SYstem cl

Olltput of

102

installed on the IBM-DACA to implement the switching
options (a hardware or software trigger of the ADC). If a
jumper is placed between these two pins, the IBM-DACA will
work with a software trigger as it does normally with an
unmodified IBM-DACA board. If the R/C pin is connected to
another triggering pulse source, then the ADC start
conversion will be controlled by that pulse. The AD574K
requires 25 .us to complete a lZ-bit analog-to-digital
conversion. Therefore, the external trigger source must not
exceed a pulse width of 25 as. If a low-going pulse longer
than 25.ns is used, multiple conversions will be made on a
single trigger pulse. To prevent this, we used a monostable
multivibrator (74L8121 or 74L8123) which generates a single
pulse with (a 5 ,us pulse width on. every low-going trigger
source, regardless of its pulse width. This low-going pulse
of the multivibrator is used. to trigger' the ,ADC start

conversion.

(b) Modification of 8253-5 Timer/counter Circuit.

The Intel 8253-5 timer/counter on the IBM-DACA has three
16-bit counters (16). However, counter 0 and counter 1 are
cascaded to provide a single 32-bit counter in the original
design of the board. All three gate inputs are disabled and
the clock input of the counter O is connected to a 1.023 MHz
system clock. The second clock input is connected to the

output of counter 0 for cascaded operation of the two

 

1;_

 

19

 

38

Pin cc
1- Bl
4- 74
7- 74
11-01
1H)
17-C.
zo-d
2:1-Q
28-7
29-1
112-1
35.4

38-

Fizure 5.

103

1913171315141312111393 73543 21

 

'1
1

Hill
1111f

 

 

IIHIIIIIJIHIIIIH

 

 

 

 

IHIIHHIIHFHTI

38373635343332313029282726-252423222120

P1n connections

1- 81(7413123 pin 1)
4- 741.314 pin 3(optionai)
7- 741504 pin 3(opuonal)

u-cowzsa-o pin 11)
14-Delay(U27 pin 1)
17-Count 011111127 pin 3)
211-111(7415123 pin 4)
23-oz(7415123 pin 3)
28-741314 pin 6(opt10na1)
294741.304 pin 6(optional)
32-0ut 2(8253-5 pin 17)
35-C1k 1(8253-5 pin 15)

138-Ground

2- 82(7413123 pin 9)
5- 741.314 pin 5(opuona1)
8- 741804 pin 5(optional)

12-01(8253-5 pin 14)
15-Rate(1127 pin 5)
18-Sofl. trigger(023 pin 3)

zr-qumsm pin 13)
24-741514 pin 2(optional)
27-741304 pin 2(op1ional)

so-om 0(3233-5 pin 13)
33-1.oz3 mwn pin 9)
33-cn 2(3253-3 pin 13)

Figure 5.12. Jumper board pin layout.

 

3- 741514 pin 1(op1iona1)
6- 741.504 pin 1(optiona1)
9.10- +5V

13-02(8253-5 pin 16)
16-Count in(1151 pin 9)
19-Ground

22-§2(741.8123 pin 12)
25-741814 pin 4(optional)
28-741304 pin 4(0ptional
31-0ut 1(8253-5 pin 13)
34- C11: 0(8253-5 pin 9)
awn/Emma pin 3)

counters. T
users for th
In our modi
inputs, and
counter wori
from these
board for u:
By enabli
the three <
channel ca:
Also, the I
from the 1
board for
outside of
hex invert
conditioni

board.

2° Softwa:

T0 mani
DACA boa]
main Pro
BOPland
MicroSOf
Written

varlOUS

 

104

counters. Thus, only one clock input is available to the
users for their own configurations as shown in Figure 5.11.
In our modification, all three clock inputs, three gate
inputs, and three counter outputs were enabled to make each
counter work independently. Also, the lines were extended
from these timer input/output ports to the daughter jumper
board for user configuration (Figure 5.12).

By enabling all three clock inputs, three gate inputs, and
the three outputs of the timer/counter, each timer/counter
channel can be configured according to the user’s needs.
Also, the Delay-out, Rate-out, Count-in, and Count-out lines
from the distribution panel were extended to the jumper
board for easy access to timer/counter control lines from
outside of the IBM-DACA board. Optional installation of a
hex inverter and a Schmitt trigger for control and signal
conditioning gives even more flexibility to the IBM-DACA

board.

2. Software Development.

To manipulate the extended functions of the modified IBM-
DACA board, menu-driven software has been developed. The
main program was written in TURBO PASCAL (Version 4.0,
Borland International) and MACRO ASSEMBLER (Version 4.0,
Microsoft) to optimize the ADC throughput. This program was
written for general purpose use so as to be utilized in

various scientific applications. It is a complete package

 

 

 

to acquire
compatible pl
time, either
the screen.
user for sp
used with 31
will allow
data acquis
Set-up i
the lower h
mode of the
data-read 1
channel, t
the rate a
trigger no
the unmod
modified ]
the user '
be select.
With a he
an assem
fastest C
Source.
bethen .
(mode 2-
RIG line

and the

 

105

to acquire data and manipulate data on an IBM-PC or
compatible personal computer. Data can be displayed in real
time, either in numerical format or in a graphics plot, on
the screen. If desired, source code can be modified by the
user for specific applications. This program can also be
used with an unmodified IBM-DACA. However, the modifications
will allow extended modes of operation of the IBM-DACA for
data acquisition.

Set-up information for data acquisition is disPlayed in
the lower half of the main menu screen to show the operation
mode of the IBM-DACA. In set up, the user can implement the
data-read mode, the number of data points to read, the input
channel, the input signal voltage range, the polarity, and
the rate of data read. Mode 0 is reserved for the software
trigger mode, which is the default data acquisition mode of
the unmodified IBM-DACA. All other modes are for the
modified IBM-DACA and require a hardware trigger source. If
the user wants to use the unmodified IBM-DACA, mode 0 must
be selected. Mode 9 is reserved for fast data acquisition
with a hardware trigger of the ADC start conversion and uses
an assembly language subroutine. This mode gives the
fastest data read speed, which is dependent on the trigger
source. If mode 0 is selected, a jumper must be placed
between R/C and the software enable pin. If another mode
(mode 2-9) is selected, a jumper must be placed between the
R/C line and the Q output of the monostable multivibrator,

and the trigger pulse source must be fed to the B input of

 

 

 

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6420.

the nultivib:
The use
channels. A
other than u
each timer)
operation.
individual 1'
operation a
operation
counters a1
modes can
independen‘
graphics m

with Epson

3. Results

To test
sine ane
mode 0 (
meXimum 5
line). I!
use high
SamPles/
board ti
in Count
the ADc

 

 

the multivibrator.

The user can also program the three timer/counter
channels. A timer/counter set up window will pop up if modes
other than mode 0 are selected so that the users can program
each timer/counter channel for their desired mode of
operation. Each counter/timer of the Intel 8253-5 is
individually programmed by writing a desired mode of
operation and a count number. There are six modes of counter
operation for each. counter/timer. But if more than two
counters are cascaded, then very complex and sophisticated
modes can be obtained. The plotting routine employs device
independent graphics to plot data on various types of
graphics monitor. The hard copy of the plot can be obtained

with Epson printer.

3. Results and Discussion.

To test the performance of the modified IBM-DACA we used a
sine wave of 160 Hz (6250.»s/cycle) as the input source. In
mode 0 (software trigger of the ADC start conversion) a
maximum speed of 2130 samples/s was obtained (Figure 5.13.A
line). In the hardware trigger modes (modes 2-8) which still
use high level language for data read, a throughput of 2500
samples/s was obtained (Figure 5.13.A dotted line). The on-

board timer/counter output, which was programmed to operate

in counter mode 2, was used as a hardware trigger source for
the ADC start conversion. A 400.ps pulse interval gave the

fastest and most satisfactory performance.

 

 

 

In mode 9
read data
conversion,
maximum spet
output with
source (Fig‘
accurate t
because if
samples/s i
with true

obtained .

throughput

C. Conclus

In cone
for lumin.
0f the pu
disadvant
mechanics
i“Strumer
)1) Flue]

the sac

m°difiCa
(2) Unli

there is

 

 

 

108

In mode 9, which uses an assembly language subroutine to
read data and a hardware trigger source for the ADC
conversion, the throughput was increased dramatically. A
maximum speed of 18520 samples/s was obtained when a timer
output with a 27 ..us pulse interval was used as the trigger
source (Figure 5.13.8 dotted line). But this data has no
accurate timing relationship with the trigger source,
because if each pulse is used for ADC trigger then 37037
samples/s throughput must be obtained. An optimum speed,
with true timing relationship with trigger source, was
obtained with a 62 ,ys pulse interval, which gave a

throughput of 16130 samples/sec (Figure 5.13.3 line).
C. Conclusions.

In conclusion, the instrument designed in our laboratory
for luminescence measurements shares most of the advantages
of the pulsed source phosphorimeter, but does not share the
disadvantages of the conventional rotating-can type
mechanical phosphoriscope. The major advantages of this
instrument over the conventional phOSphorimeters are:

(1) Fluorescence, RTP, and RTP lifetimes can be measured on
the same instrument without additional instrumental
modification.

(2) Unlike the rotating-can type mechanical phosphoriscope,

there is no limitation on the time discrimination procedure.

 

 

 

 

Delay time 1
without adjl
(3) It is 1
frequency ,
detector de
accurately.
(4) It gi
lifetime m
lived phos
rotation 5
window can
(5) It giv
than a pul
(6) Final
IBM-KT co:

aCquired :

The 111,
digital .
30ftWare.
this mod
three Ch

utiliZed

109

Delay time and gate time can be easily changed by software
without adjustment of the rotation speed of the chopper.

(3) It is less dependent on the precision of the modulation
frequency of_ the excitation source. The chopper phase
detector detects every closing edge of the exciting source
accurately.

(4) It gives equally good. performance in RTP and RTP
lifetime measurements for short-lived phosphors and long-
lived phosphors. By changing the delay time, gate time or
rotation speed of the chopper, an optimum detection time
window can be obtained.

(5) It gives higher reproducibility and less RF interference
than a pulsed source, due to a chopped continuous source.
(6) Finally, this instrument is controlled by a low cost
IBM-XT compatible personal computer. Luminescence data are

acquired and manipulated easily on site.

The modified IBM-DACA allows initiation of analog-to-
digital conversion by a hardware trigger as well as by
software. A dramatic increase in throughput was observed by
this modification. using the hardware trigger. .Also, all
three channels of the on-board timer/counter can be fully

utilized by the user for complex modern data acquisition.

1. Vo-Dinh
Chem., 1971
2. Cline 1
Chem., 198
3. Nithipa
39, 109.
4. Lab Me
Ohio, 1984
5. Wiegan
Instrumen
6- Aubane
7. Hieftj
3- Savit:
1627.
9~ Brifl
Prentice.
10» Malm
f°r Scie
CA. 1981
11. Ingl
Prehtice
12~ Win1
13. Fig

44. 948

110

CHAPTER V REFERENCES
1. Vo-Dinh, T.; Walden, G. L.; Winefordner, J. D., Anal.
Chem., 1977, 49, 1126.
2. Cline Love, L. J.; Skrilec, M.; Habarta, J. G., Anal.
Chem., 1980, 52,754.
3. Nithipatikom, K.; Pollard, B. D., Appl. Spectrosc., 1985,
39, 109.
4. Lab Master User’s Guide, Scientific Solutions, Solon,
Ohio, 1984.
5. Wiegand, P. M.; Trischan, K. K.; Crouch, S. R., Anal.
Instrumentation, 1985, 14(2), 127.
6. Aubanel, E. E.; Oldham, K. 8., Byte, 1985, Ebb., 207.
7. Hieftje, C. M., Am. Lab., 1988, Oct., 110.
8. Savitzky, A.; Golay, M. J. E., Anal. Chem., 1964, 36,

1627.

9. Brigham, E. 0., The Fast Fourier Transformation,
Prentice-Hall, Englewood Cliff, N.J., 1975.
10. Malmstadt, Enke, Crouch, Electronics and Instrumentation

for Scientist, The Benjamin/Cummings Publishing, Menlo Park,

CA, 1981.
11. Ingle, .1. D.; Crouch, S.R., Spectrochemical Analysis,

Prentice-Hall, Englewood Cliff, N.J., 1988.
12. Winefordner, J. D., Acct. Chem. Res., 1969, 2, 361.

13. Fisher, R. P.; Winefordner, J. D., Anal. Chem., 1972,

44, 948.

14. IBM Pe

Adaptor TecI
15. Data Ac
1984.

16. Micros
Peripherals

1985.

111

14. IBM' Personal Computer Data .Acquisition and Control

Adaptor Technical Manual, IBM Corp, New York, 1984.
15. Data Acquisition Databook, Analog Device, Norwood, MA,
1984.

16. Microsystem component Handbook Microprocessors and
Peripherals, Intel Corp, Santa Clara, CA,

1985.

The phos
state is
associated
chemical s
is a chara
environmer
diagnostic
character:

Since t]
known to

f0110wing

Where I :

Where It

The Pho

require,

 

 

 

112

CHAPTER VI

ROOM-TEMPERATURE PHOSPHORESCENCE LIFETIMES

The phosphorescence decay lifetime of the excited triplet
state is important in studies of kinetic processes
associated with the triplet state of compounds in a given
chemical system. The lifetime of the excited triplet state
is a characteristic property of a molecule and its molecular
environment. Phosphorescence lifetimes frequently provide a
diagnostic tool since compounds having very similar spectral
characteristics can have greatly different lifetimes.

Since the phosphorescence decay of any single component is
known to be first order, it can be represented by the

following equation:

dI/dt = -kI (6.1)

where I is the phosphorescence intensity and k is the decay

rate constant. Integration of equation 6.1 yields
I = Io exp(-kt) (6.2)
where Io represents the phosphorescence intensity at t=0.

The phosphorescence lifetime, 1 , is defined as the time

required for the intensity of phosphorescence emission to

 

 

 

 

 

decrease t
excitation

given by

and the phc

of its ini'
However,

contains

equation 6

The backg:
current,
signal, e
818118.13.
The

temperat,
1ifetime:
temperat'
State by
minimize
TheSe
phospho:

descrip1

113

 

decrease to l/e of its initial intensity following

excitation (1). As a result, the phosphorescence lifetime is

given by

1’ = l/k (6.3)

and the phosphorescence intensity at this time equals to 1/e

of its initial intensity.

However, experimentally observed phosphorescence intensity

contains a finite background or blank intensity as in

 

equation 6.4.

I(t) = Io exp(-kt) + B (6.4)

The background signal is a convolution of the detector dark
current, instrumental offset, ground loops, light scatter
signal, etc., with solvent or substrate and sample impurity
signals.

The phosphorescence lifetimes of compounds at low
temperature (77 K) have been well studied compared to RTP
lifetimes. In the frozen state at liquid nitrogen
temperature, collisional quenching processes of the triplet
state by molecular oxygen and other impurities is greatly
minimized, and the temperature of the sample is constant.

These conditions give reliable and consistant

 

Phosphorescence lifetimes which can be used as a qualitative

descriptor of the luminescent species. However, RTP

 

 

 

 

lifetimes
environment
Because th
dependent a
type, dies
the are 1
system.
This ch
determinat
finally, a

RTP) spect
A. Method:

The a<
constant
lifetime
common me
monitorir
exciting

After
starts t
USUally,
instanta
entails
requirec'

SOUTCQ 1

 

 

114

 

lifetimes of compounds are extremely sensitive to
environmental conditions, especially for solution RTP.
Because the phosphorescence decay lifetime is strongly
dependent on the molecular environment (temperature, solvent
type, dissolved oxygen, other concomitant species, -etc.),
the RTP lifetimes of compounds can vary from system to
system.

This chapter describes general methods of RTP lifetime
determination, a new proposed decay kinetics model, and
finally, experimental data of nficellar-stabilized RTP (MS-

RTP) spectra and the decay kinetics experiment of PAHs.
A. Methods for Determining Decay Lifetimes.

The accurate measurement of the phosphorescence decay
constant and the subsequent calculation of the corresponding
lifetime of the triplet state is very important. The most
common methods for measuring excited state lifetimes involve
monitoring the time dependence of the sample emission while
exciting it with a pulsed or modulated source.

After source excitation, the phosphorescence intensity
starts to decay exponentially according to equation 6.2.
Usually, the source excitation function is not an
instantaneous impulse as shown in Figure 6.1. Instead, it
entails a rather smooth lagged tail. A delay time is

required to discriminate phosphorescence signal against this

source tail, stray light, and fluorescence. Actual sampling

 

 

 

Figure (

 

 

 

115

 

 

i u
I ' ’ 1t ,
10 l ,
1(t) = 10 End-kt)
1:»

 

 

 

 

 

 

t. t+ ‘6 Time

Figure 6.1. Schematic representation of typical RTP decay

curve and data acquisition process.

 

 

 

 

of the phos
time, ta, 3
There
luminescenc
detection 1
(PMTs) are
photodiode:
For
oscillosco
was the st
photon cor
with micrc
The de
luminescer
detector,
affect. o'
luminesce
caPRCitor
carefully
fast enou
Amrlifie,
reSPOnSe
Phosp]
data re<
deCay l
imPTOper

t° error

 

 

116

of the phosphorescence decay signal is done during the gate
time, tg, after delay time ta.

There are many different methods for measuring
luminescence decay with variations in excitation source,
detection device, and display. Generally, photomultipliers
(PMTs) are used to measure the luminescence signal, but
photodiodes may be usedif an intense laser source is used.

For recording the luminescence decay signal, an
oscilloscope coupled with a camera or photosensitive paper,
was the standard choice until recently. But nowadays, single
photon counting instruments and transient digitizers along
with microcomputers are more common.

The detection-display system may distort the observed
luminescence decay (2). Distortions are introduced by the
detector, cabling, amplifiers, etc. These distortions may
affect' observed lifetimes seriously when a short-lived
luminescence signal is to be measured. Resistors and
capacitors used in PMTs and amplifiers must be chosen
carefully. The RC time constants of these systems must be
fast enough to trace the fast decaying luminescence signal.
Amplifiers must have a high slew rate and wide bandwidth
response characteristics.

Phosphorescence lifetimes also depend on the method of
data reduction and deconvolution for the calculation of
decay lifetimes. Many' of the simple techniques such as
improper background correction and signal sampling can lead

to erroneous interpretation of the data. Popular methods for

 

 

 

the determ
exponential
the Gugger
recently,
The line
popular or

following

The decay
In I(t) v
from the
Ideally
lime f0;
indicatix
Criteriol
linear 1.
infinity
experime
function
function
imPUTitj
deCay s
Therefm

SUbtrac.

 

117

the determination of phosphorescence lifetimes from single
exponential decays involve the linear least-squares method,
the Guggenheim method, the phase-plane method, and more
recently, the rapid lifetime determination (RLD) method.

The linear least-squares method (3) is probably the most
popular one. By taking the logarithm of equation 6.2, the

following equation is obtained.

In I(t) = -kt + 1n Io (6.5)

 

The decay constant, k, can be obtained from the slope of the
ln I(t) vs time plot, and the decay lifetime is calculated
from the negative reciprocal of the slope.

Ideally, a plot of In I(t) vs time should yield a straight
line for single component decay. The straight line
indicating a single exponential decay can be used as a
criterion to judge the validity of the obtained data. The
linear least-squares method assumes that as time approaches
infinity, the observed signal level decreases to zero. But
experimentally, the observed signal level is not only a
function of phosphorescence signal intensity, but also a
function of dark current, amplifier offsets, emission from
impurities, etc. As a result, the experimentally observed
decay signal always contain a finite value of background.
Therefore, low noise, a stable base line, and background

subtraction are required for good results.

 

¥

 

 

 

For simp]
background:
method, met
such that t

dt.

If backgr<
taking the

backgrounc

A Plot 0
the slope
initial j
The Gu1
need £01
-However’
The fit1
aCCUrate
Should I
accurac3

acquisi1

 

118

For simple exponential decays with a constant but unknown
background, the Guggenheim method (4) is popular. By this
method, measurements of decay signal are performed in pairs

such that each pair is separated by a constant time interval

dt.

It = Io exp(-kt) + B (6.6)

It+dt = Io exp{-k(t+dt)} + B (6.7)

If background is constant in both intensity values then by

taking the difference in intensity values one can remove the

background term.

It - Itedt = Io exp(-kt) {1-exp(-kdt)} (6.8)

ln(It - Itedt) = -kt + ln{Io(l-e'kdt)} (6.9)

A plot of ln(It-It.at) versus time should be. linear. From
the slope and intercept values one can obtain lifetime and
initial intensity values.

The Guggenheim method is popular because it eliminates the
need for acquiring base line, and is simple and fast.
However, the Guggenheim method has several disadvantages.
The fitting region and dt must be properly selected for
accurate results. Margerison (5) suggested that the decay

should be followed'over 3-4 lifetimes, depending on the

 

accuracy of the individual measurements. If the data

acquisition period is shorter than 3-4 lifetimes, smaller dt

 

 

 

 

values mus‘
reconstruC'
equation 6
available.
Bacon a
the treatn
plane me
intensity.
phase-pla1
and the
Additiona
selection
guess of
The ma]
(8) is a
an31YSis.
time int
Start-to.
into 12hr
obtained

from the

Where N

deCay d

 

 

119

values must be used (6). Also the linear fit I(t) and the
reconstruction of I(t) using the best fit parameters and
equation 6.4 is complicated since the base line B is not
available.

Bacon and Demas (7) modified the phase plane method for
the treatment of luminescence lifetimes. The modified phase-
plane method permits direct calculation of initial
intensity, base line, and decay lifetimes. Furthermore, the
phase-plane method is more forgiving in the fitting ranges
and the noise levels than the Guggenheim method.
Additionally, the phase plane method requires no operator
selection of optimum fitting parameters, and no initial
guess of the parameters is required.

The more recent rapid lifetime determination (RLD) method
(8) is a significantly faster method allowing for real time
analysis. In the RLD method the data are collected at equal
time intervals, and all data points from the- experimental
start-to-stop time must be used. The decay data are divided
into three equal parts, and the sums; SO, S1, and 82 are
obtained from each portion. Decay lifetime can be calculated

from the following equations:

Y = ($1 - $2) / (SO - SI) (6.10)

Lifetime = -N dt / ln(Y) (6.11)

whene N is the number of data in each divided part of the

decay data, and dt is the fixed time interval between data

 

 

 

points. Ba:

from the ft

Ballow
RLD methoc
The RLD m
squares m
found to
calculate
reconstru
errors. 1
“0 warni
Parameter

In thh
method p
method

Spectrom

the less

 

 

120

points. Base line and initial intensity can be calculated

from the following equations.

A = ($0 - Sl)2 / (SO - 281 + 82) (6.12)
Io = A (1 - Y1/3) / (1 - Y) (6.13)
B = (SO - A) / N (6.14)

Ballow and Demas (9) performed an error analysis of the
RLD method for the evaluation of single exponential decays.
The RLD method was comparable to the weighed linear least-
squares method in terms of precision and accuracy, and was
found. to be 10-800 times faster. Also, the .RLD method
calculates the base line and initial intensity for ready
reconstruction of a decay curve, and calculation of fit
errors. The disadvantage of the RLD method is that it gives
no warning of more complex decays or no statistical
parameters for the evaluation of the goodness of the fit.

In this research, both linear least-squares method and RLD
method were used for RTP lifetime determination. The RLD
method was primarily used with home-built luminescence
spectrometer, and linear least-squares method was used with

the less noisy and stable Perkin-Elmer LS-5B spectrometer.

 

 

 

 

B. RTP De«

Solutions.

1. Decay K

Studies
the quencl
solution.
RTP is pr

several a

(i) The 1
located i
RTP cannc
(ii) Que]
mainly a
This ass
Which a
Observed
If QUen<
the tri
quenche,
c“Cent:
limitin
assumpt
(iii) 1

miCella

121

B. RTP Decay Lifetimes of Organic Compounds in Micellar

Solutions.
1. Decay Kinetics Model for Micellar Stabilized RTP.

Studies of micelle dynamics were performed to understand
the quenching and decay kinetics of luminescence in micellar
solution. In this section, a new decay kinetic model of MS-
RTP is proposed. To successfully apply this proposed model,

several assumptions are made as follows.

(i) The detectable RTP comes mainly from triplet molecules
located inside the micelle. This assumption is valid because
RTP cannot be observed without the micelle.

(ii) Quenching of the triplet state of the molecule occurs
mainly at the micellar surface and in the aqueous phase.
This assumption is based on our experimental results in
which as the concentration of quencher increases the
observed triplet lifetime approaches a steady-state value.
If quenching occurs mainly inside or outside the micelle,
the triplet lifetime must decrease continuously as the
quencher concentration, increases. But with a fixed micelle
concentration the micellar surface area is also fixed to a
limiting value for triplet quenching. This supports the
assumption of micellar surface quenching.

(iii) The probe molecules must be far more soluble in the

micellar phase than in the aqueous phase. This condition is

 

necessary
(iv) The c

that self-

The p
partitioni

phase and

where P‘
outside
mainly c:

the micel

Triplet

Surface

A3 a re

‘dIMP'1

 

 

122

necessary for application of the steady-state approximation.
(iv) The concentration of the phosphorescent probe is so low

that self-quenching of the excited state is unimportant.

The proposed decay model is shown as follows. The
partitioning of a phosphorescent probe P‘ between an aqueous
phase and micelle (M) is given by

MP‘ ;::::2 M + P‘ (6.15)
where P‘ and MP‘ denote the triplet probe located inside and
outside the micelle, respectively. The triplet emission
mainly comes from the phosphorescent probe located inside
the micelle.

MP‘ —————+ MP + hv (6.16)

Triplet quenching by' quencher Q' occurs at the micellar

surface and in the aqueous phase.

As a result, the rate of disappearance of MP'I is given by

-d[MP"]/dt = k-[MP'1-k+[MllP’]+k-p[MP‘]+k-q[MP‘][Q] (6.19)

 

 

 

and the ra‘

-d[P‘]/dt

If the phi

micellar s

[P'], the

From equa

[P

Substitu1

'dIMP‘]/.

Thus. th

Process,

of m»,

1...,

This e«
micelle

COncent

 

 

 

123

and the rate of disappearance of P‘ is given by

-d[P‘]/dt = k.[M][P‘] + kqlP'JIQ] - k-[MP‘] (6.20)

If the phosphorescent probe P" is far more soluble in the

micellar solution than in aqueous phase, such that [MP‘] >>

[P’], the steady-state approximation can be applied.

-d[P‘]/dt = 0 (6.21)

From equation 6.20 and 6.21 we obtain

[P*] = k-[MP'] / (k+[M] + kq[Ql) (6.22)

Substitution of equation 6.22 into 6.19 yields

*dEMP‘l/dt=[MP‘][k-+k-p+k-q[Q]'{k’k-[Ml/(k*[M]+kq[Q] (6.23)

Thus, the disappearance of MP' follows a first-order kinetic
process. Therefore, the observed disappearance rate constant

of MP‘, ko, which is reciprocal of lifetime 1 is given by

k0 = 1/1 = k_+k.p+k.q[Q]-{k+k-[M]/(k+[M]+kq[Ql)} (6.24)

This equation shows that at a fixed concentration of
micelle, the observed lifetime decreases as.the quencher

concentration increases until [Q] reaches the saturated

 

 

 

 

value [Q]:
concentrat
to the de

inside the

2. Experi:

Reagents:
Sodium
water (0
(99.99%,
sodium d.
grade, E]
in this
Baker),
(J.T. B:
ethanol.

sample I

Apparfitl
RTP a1
inStrum.
°Perati
detail
aequisi
Spectrc

IBM-pg

 

 

 

124

value [Q13 at the micellar surface. At a very low quencher
concentration, the observed rate Constant ko will be equal
to the decay rate constant of the triplet probe located

inside the micelle, k-p.

2. Experimental Section.

Reagents:

Sodium sulfite (J.T. Baker) was recrystallized from warm
water (0.5 ml/g) by cooling to 0 C. Thallium(I) nitrate
(99.99%, Aldrich), sodium bromide (99.9%, J.T. Baker),
sodium dodecyl sulfate, SDS (99%, Sigma), and methanol (GR
grade, EM Science) were used as received. The analytes used
in this research, naphthalene (Eastman), pyrene (J.T.
Baker), 2—bromonaphthalene (97%, Aldrich), and biphenyl
(J.T. Baker), were all recrystallized once from absolute
ethanol. Distilled and deionized water was used for RTP

sample preparation throughout the experiment.

Apparatus:

RTP and RTP lifetimes were measured by both the home-built
instrument and. the iPerkin Elmer' LS-SB spectrometer. The
operation of the home-built instrument is described in
detail in chapter V. For the Perkin Elmer LS-SB data
acquisition“ the chart recorder output (1V max) of the
spectrometer was amplified 10 times and digitized by the

IBM-PC data acquisition board (see chapter V) which was

 

 

 

 

 

installed
for this P
and analys

measuremen

Sample Pre
Stock 3
bromonaph‘
of analyt.
volume wi
thallium
dodecyl s
micellar
was used
An. a1
into a 11
a minimu:
made by ,
thallium
ml V01um
final V0
After
Cuvette
°xY8en
intensi1

aPPI‘Opr;

 

 

installed on an IBM-XT compatible computer. Software written
for this purpose was used for luminescence data acquisition
and analysis. The range of delay time and gate time for RTP

measurements were 0.03 - 0.05 ms and 1-3 ms, respectively.

Sample Preparation and General Procedure:

Stock solutions of naphthalene, pyrene, biphenyl, and 2-
bromonaphthalene were made by dissolving appropriate amounts
of analytes into 10 ml methanol and diluted to 100 ml final
volume with water» For the external heavy’ atom source,
thallium nitrate stock solution (0.25 M) was used. A sodium
dodecyl sulfate stock solution (0.5 M) was prepared for the
micellar solution, and sodium sulfite stock solution (0.4 M)
was used for deoxygenation of the sample solution.

An aliquot of methanolic analyte solution was pipetted
into a 10 ml volumetric flask and solvent was evaporated to
a minimum volume using nitrogen gas. Sample solutions were
made by adding the appropriate amounts of stock solutions of
thallium nitrate, SDS, and sodium sulfite into previous 10
ml volumetric flask. These solutions were diluted to 10 nfl.
final volume with the distilled and deionized water.

After thorough maxing, the solution was transferred to a
cuvette with a Teflon stopper. The progress of chemical
oxygen scavenging was monitored by measuring the RTP
intensity development at the emission maximum with the

appropriate excitation wavelength on the spectrometer.

 

 

 

 

 

Spectra we:

state and

3. Results

Micell
method f0
solution
because a
process t

deoxygena

Air Tighi
A tigh'
0f air w;
shows RT
0f a. ch
sample s
heavy at
After t
solutio,
the emi:
3 minut
The
into th

AS fre

 

126

 

Spectra were obtained after the RTP intensity reached steady

state and remained the same for at least 5 minutes.

3. Results and Discussion.

Micellar stabilized RTP (MS-RTP) is the most popular
method for solution RTP. Deoxygenation of the surfactant
solution was the most troublesome aspect of the MS-RTP,
because of a lot of foam formed during the deoxygenation
process by inert gas. This can be eliminated by chemical

deoxygenation using sodium sulfite.

Air Tight Capping of the Cuvette.

A tight seal of the sample cuvette to prevent any leakage
of air was necessary throughout RTP measurements. Figure 6.2
shows RTP development of 2-bromonaphthalene after addition
of a chemical scavenger, sodium sulfite, into a micellar
sample solution. The concentrations of surfactant (SDS) and
heavy atom (TlNOa) were 0.05 M and 1.5x10'2 M, respectively.
After the addition of 0.01 M sodium sulfite into the
solution, the development of RTP intensity was monitored at
the emission maximum for 20 min. RTP began to show up after
8 minutes and reached a steady-state maximum after 15 min.

The dotted line of Figure 6.2 shows the leakage of air
into the sample solution with no capping the sample cuvette.

As fresh oxygen enters into the solution, RTP fluctuates

 

 

 

 

 

127

cm

mu

sud: osoacnasmoGOEOAQIN «0

on

25.: mth
v"

1.1

 

Nu

 

On

 

acoEQoA0>op mam

.osau

0“ 9” “hamflh

 

I

can

can

OQM

06V

095

com

. com

 

 

 

coo“

AlflafinNIENUITEM

 

 

sinusoidall
oxygen and
sample cuv

very stabl

Optimum C:
Variou
solution
atom (Tl‘
sulfite
SDS and
RTP inter
The opt:
0.8x1o-2
603. Be]
decrease
reaction
intensit
which a'

micella:

The Eff
The ;
(CMC)
screenf
expect,

SOluti.

128

sinusoidally, indicating a new chemical equilibrium by
oxygen and sodium, sulfite. With an air tight seal of the

sample cuvette with a Teflon‘tOp, the RTP intensity remained

very stable with time.

Optimum Concentration of Sodium sulfite.

Various amounts of sodium sulfite were added into a
solution of a fixed amount of naphthalene, SDS, and heavy
atom (Tl‘), to find an optimum concentration of sodium
sulfite for chemical deoxygenation. The concentrations of
SDS and Tl’ were 3.5x10‘2 M and 1.5x10‘2 M, respectively.
RTP intensities were measured 15 min after solution mixing.
The optimum concentration range of sodium sulfite was
0.8x10"2 - 1.2x10“2 M, as shown in the upper graph of Figure
6.3. Below this optimum concentration, the RTP intensity
decreased due to the incomplete and slow deoxygenation
reaction. Above this optimum concentration range, the RTP
intensity decreased due to high sodium ion concentrations,
which at such levels could displace thallium ions from the

micellar surface.

The Effect of SDS Concentration on RTP Intensity.

The reported value of the critical micelle concentration
(CMC) of SDS is 8.2x10'3 M (10). Thus, the effective
screening of the triplet sample molecules by the micelle is
expected at surfactant concentrations above the CMC. Into a

solution of a fixed amount of Tl‘, sodium sulfite,

 

800'

'1.

l 4

( 6
e s
xtmcmufi 0>320m m. m
8 t
i n
F 01

0 <
0 0 0
6 4 2

xfimcuacm o>52om

 

129

800-

600-

400‘

Relative intensity

200m

 

0 w’ l *1 ‘T
OJ) (L4 (18

 

r’ r* ' l ' ‘T
1.2 1.6 2.0
Sodium sulfite conc.(x100,M)

800-

600-

400-

Relative intensity

2004

 

 

O I I T r r F

0.0 1.0 2.0 3fo ' do ' 5:0 ' {a r 7fo t are 3.0
[305] x100,M

Figure 6.3. The effect of sodium sulfite and SDS on the RTP

intensity of naphthalene.

 

 

 

naphthalen
optimum cc
in the 1
intensity
4.5x10‘2 1
The red
be explai
of heav:
concentra
concentr:
will re31
singlet :
dynamic
affect t
that ‘th.
more dii
less co
more po

higher

MS-RTP

Exci
biphen.
6.5. T-
avoid
cOncen

9“<1 0.

130

naphthalene, various amounts of SDS were added to find the
optimum concentration of SDS. The maximum RTP was observed
in the 1.5x10'2 - 4.5x10"2 M concentration range. RTP
intensity began to decrease at SDS concentrations above
4.5x10"2 M, as shown in the lower graph of Figure 6.3.

The reduced RTP intensity at a high SDS concentration can
be explained in several ways. It is expected that the number
of heavy atoms per micelle» ‘will decrease as the
concentration of SDS increases. As a result, the effective
concentration of the heavy atom will be decreased, which
will result in reduced spin-orbital interaction between the
singlet molecules and heavy atoms. Also the size, shape, and
dynamic motion of the micelle ‘will change, which could
affect the stability of triplet molecules. It was reported
that the removal of dissolved oxygen by sodium sulfite is
more difficult at a higher surfactant concentration than at
less concentrated SDS concentration (11). Also, there are
more possibilities of RTP quenching by impurities of SDS at

higher SDS concentration.

MS—RTP Spectra of Polyaromatic Hydrocarbon Molecules.
Excitation and emission spectra of naphthalene, pyrene,
biphenyl, and 2-bromonaphthalene are shown in Figure 6.4 and
6.5. The concentrations of lumiphors are kept at 5x10'5 M to
avoid any undesired quenching processes due to high sample
concentrations. MS-RTP spectra were obtained in 0.05 M SDS

and 0.01 M sodium sulfite with 15 mM TlNOa.

 

 

 

430
Figure
bromont

excita‘

   

mmama
a

190
O

 

230 242 254

mmm

>5 ufllflhln U>nb<J

 

II

0 on
0 II
2

m m m m m

Fr natives: N>u r4...

 

RELAY IVE INTENSI I"

RELAY IVE INTENSITY

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Figure 6 . 4 . Exc itat ion and emission spectra of 2-
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excitation .spectra and C and D are emission spectra.

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Excitation and emission spectra of naphthalene

(A,C) and biphenyl (B,D). A and B are excitation spectra and

C and D are

emission spectra.

 

 

Z-Bromo
527, and 5
has two d
showed
bromonaph
and blue
showed f
Excitatic
polyaromz

6.1.

Table 6.

Com]

Nap]

“Suns
Alt]

Soluti

133

2-Bromonaphthalene showed three broad RTP peaks at 497,

527, and 562 nm. Pyrene strongly absorbs light at 337 nm and

has two distinct RTP peaks at 595 and 653 nm. Naphthalene

showed a very similar spectral response as 2-

bromonaphthalene. RTP peaks of naphthalene are more sharp

and blue shifted than that of 2-bromonaphthalene. Biphenyl

showed featureless broad peaks at 450, 474, and 504 nm.

Excitation and emission spectral characteristics of

polyaromatic hydrocarbon compounds are summarized in Table

6.1.

 

Table 6.1. MS-RTP Spectral Characteristics of Selected PAHs.

 

 

 

Compound Name Excitation (nm) Emission peaks (nm)
Naphthalene 293 481, 513, 552
Pyrene 337 595, 655
2-Bromonaphthalene 298 497, 527, 567
Biphenyl 281 450, 474

 

MS~RTP Lifetimes of Polyaromatic Hydrocarbon Molecules.
Although triplet lifetimes of organic compounds at low
temperature are well established, RTP lifetimes in micellar

solution may vary from system to system. This is attributed

 

 

 

to the fac
in dynami
quenchers
be used f
better, i
decay kin
Almgrer
micellar
It was
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assumed
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ihospho:
length
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cases.
in the

DOne

134

 

to the fact that triplet molecules in micellar solution are
in dynamic equilibrium with various sources of triplet
quenchers. This is a serious problem if RTP lifetimes are to
be used for analytical purposes. To understand this problem
better, it was necessary to study the microenvironment and
decay kinetics associated with the triplet state.

Almgren et al (12) proposed a decay kinetic model in the
micellar system using SDS and nitrate as triplet quencher.
It was found that the phosphorescence lifetime depends
strongly on the exit rate of the probe molecule from the
core of the micelle. The exit rate of 1-bromonaphthalene
from SDS micelle was determined as 2.5x10‘4 3‘1. They
assumed that the main quenching of the excited state occurs
in the bulk aqueous phase.

Bolt and Turro (13) studied the exit rates of
phosphorescent detergent probes with varying hydrocarbon
length from host micelles using Co(III) hexamine as an
aqueous soluble triplet quencher. The log of the exit rate
was a linear function of the number of methylene in the
probe alkyl chain.

Cline Love et a1 (14) extended a kinetic model of micelle-
analyte interactions to several limiting cases. But they
failed to present experimental data to support their model
due to experimental difficulties involved in the limiting
cases. The limiting cases of their model involved quenching
in the bulk aqueous phase as well as inside the micelle. But

none of the previous models accounts for the quenching

 

 

 

 

 

 

 

 

 

process a
external 1

MS-RTP
sensitive
PAHs are
concentre
concentr:

increase

Table 6.

 

Compor

Napht
Z-Bro
Biphe

PYrel

* All
[Nazso
calcul

Coeffi

 

135

process at the micellar surface and the effect of the
external heavy atom source.

MS-RTP lifetimes of PAHs in micellar solution are very
sensitive in: environmental conditions. MS-RTP lifetimes of
PAHs are found to increase as increasing surfactant
concentration up to 0.05 M. At higher surfactant
concentrations, they decreased indicating the possible

increase in impurities in surfactant solution.

Table 6.2. MS-RTP Lifetimes of Selected Arenes.

 

 

Compound Excitation(nm) Emission(nm) Lifetime(ms)‘
Naphthalene 290 511 1.25:0.07
2-Bromonaphthalene 293 527 0.82:0.08
Biphenyl 280 475 1.12:0.07
Pyrene 337 595 8.31:0.05

 

* All sample concentrations were 5x10'5 M, [SDS] = 0.05 M
[NazSOa] = 0.02 M, [TlNOa] = 0.025 M. Above lifetimes were
calculated with linear least-squares method and correlation

coefficients were above 0.998 in all cases.

 

 

 

 

MS-RTP lii
MS-RTP li:
are shown
These M
reported
RTP 1ife1
several <
Kalyanasr
lifetime:
Their re
ms range
values.
The
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to envi1
oxygen,
sample
Figur
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line,
used t:
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over t
naphth
With (

RTP lj

 

 

 

136

MS-RTP lifetimes of PAHs were measured at room temperature.
MS-RTP lifetimes of selected PAHs under optimum conditions
are shown in Table 6.2. -

These MS—RTP lifetimes are considerably longer than other
reported lifetime values. Cline Love et al (15) reported MS-
RTP lifetimes of selected PAT-Is. Their reported values for
several compounds rarely exceed 1.2 ms. On the other hand,
Kalyanasundaram et al (16) observed much longer MS-RTP
lifetimes of several PAHs in various surfactant solutions.
Their reported MS-RTP lifetime values lie between 1.2 - 20
ms range, which are, in some cases, much longer than our
values.

The differences in MS-RTP lifetimes among different
systems indicates that RTP lifetimes are extremely sensitive
to environmental conditions. The concentrations of dissolved
oxygen, heavy atom, temperature, and other impurities in the
sample solution directly affect the observed RTP lifetime.

Figure 6.6 shows triplet decay data of naphthalene (upper)
and pyrene (lower). Dotted lines are raw data and solid
lines are simulated data. The RLD method generates base
line, decay lifetime, and initial intensity which can be
used to simulate the decay data. The RLD method works well,
as can be judged visually in the overlapped data plotted
over the noisy raw data. The calculated RTP lifetime of
naphthalene in 0.02 M SDS and 0.04 M TlNOs was 0.486 ms. But
with decreased concentrations of heavy atoms, much longer

RTP lifetimes were observed (Table 6.2). The RTP lifetime of

 

 

 

 

 

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Pyren,

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napht

 

 

 

137

 

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Figure 6.6. MS-RTP decay of 5x10’5 M naphthalene (upper) and
pyrene (lower). Solutions were deoxygenated with 0.01 M
sodium sulfite, and the concentrations of SDS were 0.05 M
for both, the concentrations of T1’ were 0.04 M for

naphthalene and 7.5x10‘3 M for pyrene.

 

 

 

 

 

 

pyrene in
ms, which
five time
The e'
investiga
lifetime
The RTP
the RTP .
as tempe‘

6.3.

Table 6.

Temp

15
20
25
30

35

* Sam]
sulfit

0.035

 

138

pyrene in 0.03 M SDS and 7.5x10'3 M Tl‘ solution was 11.2
ms, which is 10 times (longer than Cline Love’s value, but
five times shorter than Kalyanasundaram’s value.

The effect of temperature on the MS-RTP lifetime was
investigated. The changes in RTP intensity and triplet
lifetime were monitored at different solution temperatures.
The RTP intensity of pyrene decreased from 656 to 468, and
the RTP lifetime also decreased by 19% from 6.02 to 4.89 ms

as temperature increased from 15.0 to 35 h as shown in Table

6.3.

Table 6.3. The effect of temperature on RTP of pyrene.

 

 

Temp. (C) RTP intensity RTP lifetime (ms)‘
15 656 6.02:0.03

20 590 5.3630.04

25 532 5.02t0.04

30 502 4.9310.06

35 468 4.8920.06

 

* Sample solutions were deoxygenated with 0.02 M sodium
sulfite and concentrations of SDS and Tl‘ were 0.05 M and

0.035 M, respectively. Excitation=337 nm, emission=595 nm.

 

 

 

 

 

 

 

 

 

These .
intensity
consistan
control c

The eff
was also
source. '
micellar
sensitiv
lifetime

measured

Table 6

‘

[Tl*]x

139

These data shows that temperature affects both the RTP
intensity and the lifetime significantly. For accurate and
consistant RTP lifetime data, it seems that temperature
control of the sample solution is a necessary condition.

The effect of the external heavy atom on the RTP lifetime
was also investigated using thallium ions as the heavy atom
source. The use of an external heavy atom is very common in
micellar stabilized RTP. Usually, heavy atoms increase the
sensitivity of RTP but decrease the observed triplet
lifetimes. The RTP intensity and lifetime of pyrene were

measured as increasing the thallium ion concentration.

Table 6.4. The Effect of Heavy Atom on RTP Lifetime.

 

[Tl*]x102(M) RTP Intensity Lifetime(ms) ko(=1/lifetime,s‘1)

f

0.75 345 11.7030.04 86
1.50 488‘ 9.35:0.05 107
2.25 635 8.30:0.05 121
3.00 717 7.0110.07 143
3.75 - 763 5.8530.07 171
4.50 783 5.65:0.08 177

 

* [Pyrene] = 5x10'5 M, [SDS] = 0.05 M, [NaZSOB] = 0.025 M.

 

 

 

 

 

The RTP
concentra‘
rapidly (
thallium,
other ha
continuoc
trend we.
lifetime:
concentr‘
did not
was for:
sulfite
It w
functior
lifetimt
(Figure
atom is
interce
2560 a1
Other 1
It h
lifetil
and or
even i
m°tion
intere

inter;

   

 

 

140

The RTP intensity increased up’ to 0.06 M thallium ion
concentration, while the fluorescence intensity decreased
rapidly (Table 6.4). At higher concentrations above 0.06 M
thallium, precipitation took place in the solution. On the
other hand, the RTP lifetimes of pyrene decreased
continuously up to 0.05 M thallium concentration. The same
trend was observed with naphthalene even though the RTP
lifetimes were rather unstable at lower heavy atom
concentrations. The use of silver ion instead of thallium
did not work due to a very low solubility of AngOa, which
was formed by the reaction of dissolved silver ions and
sulfite ions.

It was found that the decrease in RTP lifetime is a
function of heavy atom concentration. The plot of reciprocal
lifetime, ko, versus thallium ion concentration, is linear
(Figure 6.7) according to the equation 6.24, where the heavy
atom is treated as a triplet quencher. From the slope and
intercept of the plot, k-q and k-p were calculated to be
2560 and 65, respectively. This result is very similar to
other triplet quenchers, such as nitrite (12).

It has been known that heavy atoms reduce phosphorescence
lifetimes through spin-orbital interactions (17). The spin
and orbital motions of the electrons are not independent,
even in atoms with very small nuclear charge. The orbital
motion of. the electron induces a magnetic field which
interacts with its spin magnetic momentum. This spin-orbital

interaction leads to a change in the direction of the spin-'

 

 

 

 

 

141

 

 

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142

angular momentum of one electron, thereby changing a singlet
state into triplet state, and vice versa.

The external heavy atom effect may take place in two ways:
one, by a complex formation with heavy atom, and another, by
a long range interaction through a statistical distribution
of the latter molecules around the phosphorescing molecule
(18). It has been shown that the rate constants of both 81 +
T1 and T1 9 So processes are increased (19). This is evident
from the reduced RTP lifetimes of the probe molecules at
high heavy atom concentrations. Furthermore, it has also
been demonstrated that T1 9 So radiationless transition is
not enhanced much and the observed reduction in the triplet
lifetimes is mainly due to an increase in the radiative
transition probability (20).

While various mechanisms to explain the enhanced spin-
orbital interaction and the reduced triplet lifetime have
been proposed, they are still not well understood (21). The
various schemes which have been previously proposed share a
common factor in that the singlet state is ultimately
responsible for introducing reduced phosphorescence
lifetimes. That is, the triplet state of the emitting
molecule is mixed with the singlet state of the other. The
source of the singlet state can be the same molecule (22),

the perturber (23), or* a. charge transfer state of the

molecule-perturber complex (24).

 

 

 

 

4. Conclu

RTP 111
of vario1
magnitud
heavy at
temperat
results
experime
analytiq

The
quenche
treated
atoms
surface
reactic

Place

decay

and ot

very d

condi1

It
lifet
diffe
drast
emplc

in u

 

143

4. Conclusions.

RTP lifetimes of PAHs in micellar solution were a function
of various environmental factors. A major contributor to the
magnitude of the observed RTP lifetimes was the external
heavy atom. Also, the method of deoxygenation and solution
temperature affect observed RTP lifetime significantly. This
results in. different RTP lifetimes under' different
experimental conditions, which is undesirable from an
analytical point of view.

The similar behavior of heavy atoms to other triplet
quenchers was striking since heavy atoms are not usually
treated as triplet quenchers. It is not clear whether heavy
atoms actually’ quench 'triplet molecules at the micellar
surface, but it seems quite probable that a main contact
reaction between the probe molecule and the heavy atom takes
place at the micellar surface. We tried to test the proposed
decay kinetics model using other heavy atoms such as silver
and other triplet quenchers without heavy atoms. But it was
very difficult to obtain consistant RTP data under the above
conditions.

It is recommended that the use of hp instead of RTP
lifetime will give a more valid criterion in identifying
different compounds in MS-RTP. The MS-RTP lifetimes may vary
drastically" depending on that experimental conditions
employed, but knp, which is the triplet decay rate constant

in the micelle will give rather consistent values.

 

 

 

 

 

 

 

1. Winefc
2. Dema:
Academic
3. Bouti
51, 1384
4. Gugge
5. Marge
C. H., T
6. Laid
New Yor
7. Baco
8. Woo.
Chem.,
9- Bal.
10. 01
Chem.,
11. Di
58, 14
12 Al
Soc.,
13. 1
4029.
14. ¢

Chem

 

144

CHAPTER VI REFERENCES

1. Winefordner, J. D., Acct. Chem. Res., 1969, 2, 361.

2. Demas, J. N., Ekcited State Lifetime Measurements,
Academic Press, New York, 1983.

3. Boutilier, G. D.; Winefordner, J. D., Anal. Chem., 1979,
51, 1384.

4. Guggenheim, E. A., Philos. Mag., 1926, 2, 538.

5. Margerison, D., Comprehensive Chemical Kinetics, Banford,

 

C. H., Tipper, C. F. H., Eds.; Elsevier, New York, 1969.

6. Laidler, K. J., Chemical Kinetics, 2nd Ed.; McGraw-Hill,
New York, 1965.

7. Bacon, J. B.; Demas, J. N., Anal. Chem., 1983, 55, 653.
8. Woods, R. J.; Scypinski, S.; Cline Love, L. J., Anal.
Chem., 1984, 56, 1395.

9. Ballew, R. M.; Demas, J. N., Anal. Chem., 1989, 61, 30.
10. Cline Love, L. J.; Habarta, J. G.; Dorsey, J. G., Anal.
Chem., 1984, 56, 1132A.

11. Diaz Garcia, M. E.; Sanz-Medel, A., Anal. Chem., 1986,
58, 1436.

12 Almgren, M.; Grieser, F.; Thomas, J. K., J. Am. Chem.
Soc., 1979, 101, 279.

13. Bolt, J. D.; Turro, N. J., J. Phys. Chem., 1981, 85,

 

4029.

14. Cline Love, L. J); Habarta, J. G.; Skrilec, M., Anal.

Chem., 1981, 53, 437.

 

 

 

 

 

 

15. Skril
1559.
16. Kalya
Phys. Lei
17. McGy
Chem. Ph.
18. Ebar
Japan, 1
19. Hum
Lett., 1
20. E181
42, 794
21. Cha
42, 194
22. let
32, 596
23. Ho:

245 M“

 

 

 

145

15. Skrilec, M.; Cline Love, L. J., Anal. Chem. 1980, 52,
1559.

16. Kalyanasundaram, K.; Grieser, F.; Thomas, J. K., Chem.
Phys. Lett., 1977, 51, 501.

17. Mcnynn, S. P.; Sunseri, H.; Christodouleas, N., J.
Chem. Phys., 1962, 37, 1818.

18. Ebara, N.; Yajima, Y.; Watanabe, H., Bull. Chem. Soc.
Japan, 1979, 52, 2866.

19. Humphry-Baker, K. R.; El-Sayed, M. A., Chem. Phys.
Lett., 1978, 58, 207.

20. Eisenthal, K. B.; El-Sayed, M. A., J. Chem. Phys., 1965,
42, 794.

21. Chaudhurl, N. K.; El-Sayed, M. A., J. Chem. Phys., 1965,
42, 1947.

22. Isubomura, H.; Mulliken, R. S., J. Am. Chem. Soc., 1966,
82, 5966.

23. Hoijtink, G. J., Mol. Phys., 1960, 3, 67.

24. Murrel, J. N., Mel. Phys., 1960, 3, 319.

 

 

 

 

 

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from th:
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146

CHAPTER VII

ROOM-TEMPERATURE PHOSPHORESCENCE 0F COMPOUNDS IN MIXED

ORGANIZED MEDIA: SYNTHETIC ENZYME MODEL-SURFACTANT SYSTEM

Room-temperature phosphorimetry (RTP) is quite different
from the classic low temperature phosphorescence technique
'which is typically performed in glass matrices at liquid
nitrogen temperature (1). Since the discovery of RTP,
numerous techniques have been developed to induce RTP from
various molecules. These include solid-state RTP (2),
micellar-stabilized RTP (3), sensitized RTP (4),
cyclodextrin-enhanced RTP ( 5 ) , and colloidal or
microcrystalline RTP (6). The probability of observing RTP
(especially in solutions) is enhanced in a rigid molecular
environment due to a reduced quenching effect.by oxygen or
other impurities, and in the presence of a heavy atom, due
to an increase in the rate of intersystem crossing.

‘Photophysical and photochemical properties of organic
molecules included in the cavity of cyclodextrins (CDs) have
been of considerable interest in the past decade (7).
Cyclodextrins form complexes with hydrophobic organic and
organometallic molecules in aqueous solution. Although there
is no direct proof for a fixation of the guest molecules
within the void space of the cyclodextrin, the complexes are

usually regarded as inclusion compounds, host-guest

 

 

 

 

 

 

compounds
and hydrt
(8). Whe1
molecule:
lifetime
from que
The
azaparac
capable
a vari:
azapara'
2,11,20
tetrafl
host tc
Specifi
functiq
the we
quarte
In t
naPhtt
bromo:
methyj
bindi
forma
molec

surf:

147

compounds, in which hydrogen bonding, van der Waals forces,
and hydrophobic interactions are the main binding forces
(8). When lumiphores are included inside the cyclodextrin
molecules, the resulting RTP shows enhanced intensity and
lifetime because the cyclodextrin protects the lumiphores
from quenchers (9).

The synthetic macrocyclic enzyme model compounds,
azaparacyclophanes (APCs), can also act as inclusion hosts

capable of molecular organization by forming complexes with

 

a variety of hydroPhobic molecules (10). Water-soluble
azaparacyclophane (APC), N,N,N’,N’,N",N",N’",N’"-octamethy1-
2,11,20,29-tetraaza[3.3.3.3]paracyclophanetetraammonium
tetrafluoroborate (methyl-APO), is an excellent inclusion
host toward certain organic substrates. A unique substrate
specificity was observed due to its cavity size and
functionality (11). The macrocyclic cavity is surrounded by
the wall, which is formed by four benzene rings and four
quarternary ammonium residues around the macrocyclic ring.
In this chapter, RTP results of anionic, 6-(p-toluidiny1)-
naphthalene-Z-sulfonate (TNS) and the neutral compound, 2-
bromonaphthalene in premicellar surfactant solutions using
methyl-APO and cyclodextrin as host, _ are described. The
binding ability and selectivity of methyl-APO in complex
formation with anionic and neutral polyaromatic hydrocarbon

molecules in a mixed organized media, methyl-AFC -

surfactant, are also discussed.

 

 

 

 

 

 

 

 

 

 

A. EXPERI

1. Materi

Reagents
Sodiun
water.
bromide
(99%, S:
l-napht]
6-tolui'
(Aldric
Methano
Purific
sample
Th
tereph
tetraf
methyl
used
Distij

SYnth.

APpar
A
Spec1

Spec‘

 

 

148

A. EXPERIMENTAL SECTION

1. Materials and Methods.

Reagents:

Sodium sulfite (J.T. Baker) was recrystallized from warm
water. Thallium(I) nitrate (99.999%, Aldrich), sodium
bromide (99.9%, J.T. Baker), sodium dodecyl sulfate, SDS
(99%, Sigma), 2-bromonaphtha1ene (97%, Aldrich), 8-anilino-
l-naphthalene sulfonic acid, ANS, ammonium salt (Aldrich),
6-toluidino—2-naphtha1ene sulfonic acid, TNS, potassium salt
(Aldrich), and B-cyclodextrin(Sigma) were used as received.
Methanol (GR grade, EM Science) was used without further
purification. Distilled and deionized water was used for RTP
sample preparation.

The compounds, d ,a‘-dibromo—p-xylene(98%, Aldrich),
terephthaloyl chloride (97%, Aldrich), trimethyloxonium
tetrafluoroborate (Aldrich), LiAlH4 (Aldrich), and
methylamine (40% in water, Matheson Coleman 8:. Bell) were
used to synthesize the methyl-APO as described below.
Distilled. benzene was ‘used as a solvent throughout the

synthesis experiment.

Apparatus:
All spectra were obtained with a.IPerkin-Elmer LS-SB
spectrometer. The chart recorder output (1 V maximum) of the

spectrometer was amplified 10 times and digitized by an IBM-

 

 

 

 

 

PC data a
computer.
luminesce
The rang

0003-000

Sample I
The m
highly <
,by Tabu
deoxyge
M) to t
To a
lumiphc
solutic
distil
soluti
and th
With
Spectl
RTP 11

for a

2. s!

In

°Cta1

 

149

PC data acquisition board installed on the IBM-KT compatible
computer. Laboratory' written software was used for the
luminescence data acquisition and data manipulation (12).
The range of delay and gate times for RTP measurements was

0.03-0.05 ms and 1-3 ms, respectively.

Sample Preparation and General Procedure.

The macrocyclic compound, methyl-APO, was synthesized in
highly dilute conditions according to the reported procedure
,by Tabushi et a1 (11). The sample solutions for RTP were
deoxygenated by adding sodium sulfite solutions (0.01 - 0.04
M) to the sample solution as reported by Garcia et a1 (13).
To a 10 mL volumetric flask, appropriate aliquots (of
lumiphor, sodium sulfite, heavy atom, and surfactant
solutions were added and diluted to 10 mL final volume with
distilled and deionized water. After thorough mixing, the
solution was transferred to a cuvette with a Teflon stopper,
and the RTP intensity was monitored at the emission maximum
with the appropriate excitation wavelength on the
spectrometer. Uncorrected spectra were obtained after the
RTP intensity reached the steady state and remained the same

for at least 5 minutes.
2. Synthesis of Azaparacyclophane.

In this research a simple APC , N,N,N’,N’,N",N",N’",N’"-

octamethyl-Z,11,20,29-tetraaza[3,3,3,3]paracyclophanetetra-

 

 

 

 

 

 

 

 

ammonium
model has
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150

ammonium tetrafluoroborate (methyl-APO), was chosen as a
model host compound. For the synthesis of methyl-APO, N,N’-
dimethyl-p-xylenediamine was synthesized first (Figure 7.1).
Macrocyclic ring was obtained by the condensation of N,N’-
dimethyl-p-xylene with terephthaloyl chloride in a highly
dilute condition. For the analysis of the compounds, Varian
EM360 nmr spectrometer and Nermag RIO-10 mass spectrometer

were used.

N,N’-Dimethy1-p-xy1enediamine (1).

Gaseous methylamine generated by dropping aqueous
methylamine solution (40 %) into the solid KOH, was passed
through a drying tube (KOH) and trapped as a liquid in the 2
L three-neck flask which was cooled by dry ice in acetone.
Thus trapped methylamine solution was collected up to 200 mL
(150 g). To this methylamine solution, 66 g (0.25 mole) of
p-xylene bromide in 600 mL of THF was slowly added under
gentle stirring for 6 h at -15 - -20'C. After an additional
2 h stirring, THF and excess amounts of methylamine were
evaporated, and the residue was made strongly alkaline with
30% aqueous NaOH solution. The product was extracted three
times with 200 mL ether. The HCl gas, which was generated by
dropping conc. H2804 into conc. HCl, was introduced into the
combined ether solution to obtain the precipitate (2HCl
salt) of N,N’-p-xy1enediamine. The final product was
obtained by recrystalization from the 3:1 (v/v) ethanol-

water solution (yield 62 X). The N,N’-p-xylenediamine was

 

 

 

 

 

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Figure 7.1. Synthesis of N,N,N’,N’,N".N".N.’".N’"-octamethy1
-2,11,20,29-tetraaza[3,3,3,3lparacyclophanetetraammonium

tetrafluoroborate.

 

 

recovered
strong a
ether. 1

product

N,N’ ,N".
azal3,31
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152

recovered when needed by dissolving it into water under
strong alkaline (pH 12) and by extraction with anhydrous
ether. Evaporation of the solvent gives a yellow oily

product 1.

N,N’,N",N’"-Tetramethy1-1,12,19,30,tetraoxo-2,11,20,29-tetra

aza[3,3,3,3]paracyclophane (2).

 

Ring closing was done under highly dilute conditions to
prevent unwanted polymer byproducts. Distilled benzene (2 L)
was placed in a three-neck flask and refluxed under
stirring. Reactant solutions of terephthaloyl chloride (1.5
g, 0.007 mole) and N,N’-p-xylenediamine (2.5 g, 0.015 mole)
in each 250 mL dropping funnel were added dropwise to the

hot benzene solution for 8 h, whose dropping rates were

 

maintained as equally as possible. After this addition was
complete, the mixture was refluxed for an additional 2 h.
The resulting hot solution was filtered to remove the salt
of diamine and evaporated under vacuum. Residues were
collected using chloroform. The final product 2(white solid)

was obtained by recrystalization in dioxane (yield 11 %).

N,N’,N",N’"-Tetramethyl-Z,11,20,29-tetraazal3,3,3,3]para
cyclophane (3).

A mixture of 200 mL dioxane and 2.2 g LiA1H4 was placed in
a 250 mL flask and refluxed under stirring. To this mixture
0.7821 g of 2 was added in small portions for 30 min and

refluxed for 2 days. To the reaction mixture, 3 mL NaOH

 

153

(15%) and 6 mL water were added slowly to convert the excess
LiAlHe into Li(OH)2, which is a white solid in dioxane.
After an additional 1 h refluxing, solution was filtered to
remove the white solid. The final product 3 (white crystals)
was obtained by recrystalization in methylene chloride
(yield 20 %). The nmr and mass spectrum of 3 are shown in

Figure 7.2 and Figure 7.3, respectively.

N,N,N’,N’,N",N",N’",N’"-Octamethyl-Z,11,20,29-tetraaza
[3,3,3,3]paracyclophanetetraammonium tetrafluoroborate (4).
0.32 g of 3 and 0.4 g of trimethyloxonium tetrafluoborate
were dissolved in a 200 mL dioxane and stirred for 6 h at
room temperature. The product precipitated from the solution
as a white powder and was filtered and combined with
additional crop, which was obtained by the concentration of
the filtrate solution one fifth by volume. Recrystalization

from 1:1 (v/v) acetonitrile-water gave a colorless crystal

of 4 (yield 55%).

 

 

 

 

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156

B. RESULTS AND DISCUSSION

The interesting characteristics of enzymes have stimulated
chemists to design and‘ synthesize various kinds of enzyme
models which can show enzyme-like behavior (14). Substances
capable of functioning as enzyme models or receptors should
have binding sites or molecular cavities and functionalities
so as to be able to form inclusion complexes. The substrate
specificity of enzymes is controlled largely by the specific
reaction pathways, the geometry of the substrate, and the
shape and size of the substrate.

The methyl-APO molecules used in this study have a cavity
surrounded. by four jpositively charged. active sites. The
unique molecular shape of methyl-APO resembles a "square

box"

(Figure 7.4), and the cavity size is 5.5-7 A wide and 6
A deep (15). The box-like opening of the methyl-APO can vary
in size, due to the single-bond character .of’ the four
quarternary nitrogen atoms and adjacent hydrocarbon chains,
to include guest molecules of various size by "induced—fit
binding” (16).

Association constant or complex formation constant between
host and guest molecules can be measured from steady-state
fluorescence intensities. The underlying theory for
employing steady-state fluorescence intensities to calculate
equilibrium constants have been reported by Benesi (17) and

Bright (18). For a simple 1:1 host-guest complex, an

equilibrium will exist between the fluorescent substrate, S,

 

 

 

 

157

 

 

 

 

-035;er co

2.6-TNS 1.8-ANS

Figure 7.4. Simplified structural representation of host

molecule, methyl-AFC (upper), and anionic guest molecules.

 

 

 

 

158

and the host macromolecule M.

M + S :::::: MS (7.1)
The equilibrium constant, K, is expressed as

K = [MS] / [M][S] (7.2)

Substituting mass balance expressions for S and M, one

obtains
K = [M8] / {([Mli-[MS])([S]1-[MS])} (7.3)

where [M11 and [$11 are the initial concentrations of the
host and substrate, respectively. If the condition is made

so that [M]1 >> [[MS] then equation 7.3 can be written as
K = [MS] / [M]1([S]1-[MSl) (7.4)

We assume that the fluorescence enhancement of the probe in
the observed spectrum, after addition of host, is due only
to the presence of the MS complex.

The quantum yield expression for the complex is given by

Q = F / k[MS] (7.5)

 

 

 

 

 

159

where Q is the quantum yield for complex MS, F is the
fluorescence intensity of MS, and k is an instrumental
constant. By substitution of equation 7.5 into equation 7.4

and rearrangement, one obtains following final relation.
[S]: / F = (kQ)‘1 + (KkQ)'1([M]1)"1 (7.6)

This equation is now in the form, y=ax+b. Thus, a reasonable
estimation of K can be obtained from a plot of [S]1/F versus

l/[M]1, by simply dividing the intercept by slope.

Fluorescence Enhancement of Lumiphors by APC.

The fluorescence probe molecules, ANS and TNS, are
substituted naphthalene family that have found extensive use
as fluorescence probes due to their highly sensitive nature
towards hydrophobic binding (Figure 7.4). They normally show
very weak fluorescence in aqueous solutions, but are highly
fluorescent when bound to bovine serum albumin or to several
proteins (19). The binding ability of the methyl-APO
molecule was tested with ANS and TNS in aqueous solutions.

Figure 7.5 shows the change in fluorescence spectrum of
guest molecules on addition of the host molecule, methyl-
APC. The fluorescence intensity of TNS (2.5X10‘5 M) in water
was negligible (bottom), but it increased by a factor of
twenty upon the addition of B-cyclodextrin (1X10" M) with
an emission maximum at 470 nm. Upon addition of methyl-AFC

(1X10‘4 M), the fluorescence intensity of TNS was enhanced

 

 

 

 

 

 

 

160

 

1000

900 -

700 -

500 -

RELATIVE INTENSIIV

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RELATIVE INTENSITY

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o ‘ ’ ...l. 17 l ._J_.._____L______..L ....
330 377 40‘ ‘31 43. ‘35 512 339 588 393

MANELENEHHOOQ

Figure 7 . 5 . Fluorescence enhancement of ANS (2 . 5X1 0‘ 5 M ,
excitation=375nm, upper spectrum) and TNS (2.5X10‘5 M,
excitation=337nm, lower spectrum) on addition of 1X10" M of

b-cyclodextrin (dotted line) and methyl-AFC (upper solid

line).

 

 

 

 

161

more than twice that with R-cyclodextrin (B-CD), and the
peak maximum blue shifted to 460 nm. Also with the methyl-
APC, the fluorescence peak became narrower than that with
B-CD. The same trend was observed when ANS was used as a
guest molecule instead of TNS. Upon addition of B‘CD (1X10"
M) to the ANS (2.5){10‘5 M) solution the fluorescence
intensity of ANS increased by a factor of two, but when the
same concentration of methyl-APC was added, the fluorescence
intensity increased by a factor of five. The fluorescence
peak blue shifted 8 nm with K-CD, and 25 nm with methyl-APC
when compared to the emission maximum of ANS alone.

Plotting the fluorescence intensity of guest molecules
against the reciprocal of the concentration of APC according
to the equation 7.6, produces a straight line as shown in
Figure 7.6. The association constant of ANS was calculated
from the slope and intercept value to give 1.6X104/mole.
This value was much greater than the association constant of
ANS with B-CD. The association constant of ANS with B’CD was
reported recently by Catena et a1 (20) to be 110/mole at 25
‘c.

The spectral changes are due to the changes in the
microenvironment of the ANS or TNS molecules. In both cases,
methyl-APO showed a larger blue shift and a greater
fluorescence enhancement than B-CD. These data suggest that
methyl-AFC molecules provide stronger and more hydrophobic

binding sites for TNS and ANS molecules than B—CD.

 

 

 

 

 

162

 

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163

RTP Enhancement of Lumiphors by Host.

If the host. molecules. can. provide an. environment to
protect excited triplet state molecules from quenching, it
is expected that the resulting phosphorescence will
increase.

Sample solutions were made for the observation of RTP with
various amounts of methyl-APO or B-CD. The TNS solutions for
RTP were deoxygenated with sodium sulfite (0.04 M) and,
thallium nitrate (0.02 M) was used as an external heavy atom

source. With the above conditions we could not observe any

 

RTP of TNS. However, RTP of TNS began to show up when
premicellar concentrations of SDS were added to the solution
mixture. RTP enhancement of TNS (2.5X10'5 M) are shown in
the upper spectrum of Figure 7.7. The RTP intensity of TNS
increased by a factor of two when methyl-APO (0.1 mM) was
added to the sample solution. containing the premicellar
concentration of SDS (5 mM), but it decreased slightly when
the same concentration of B-CD was added to the solution.
There were no noticeable spectral changes of the resulting
phosphorescence when either host was added.

In the case of .2-bromonaphthalene under the similar
experimental conditions, except for the heavy atom source
(NaBr was used instead of TlNOa), the RTP intensity of 2-
bromonaphthalene also increased on addition of the host
molecules. But again, the methyl-APO showed better RTP

enhancement as shown in the lower spectrum of Figure 7.7.

_‘

 

 

 

164

1000

 

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no r

700 '-

RELAIIVE INIENSIIY

 

 

 

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450 400 510 540 570 600 630 660 880 720 750
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Figure 7.7. RTP spectrum of TNS (2.5x10'5 M, upper spectrum)

and 2-bromonaphthalene (2.5x10'5 M, lower spectrum) ‘upon

addition of 0.1 mM solutions of fl-CD (dotted line) and APC
(upper solid line). The bottom solid lines in both spectra

were obtained with SDS only. Excitation: TNS=337; ANS=293nm.

 

 

165

The RTP of 2-bromonaphthalene could be observed without an
external heavy atom ‘by careful sample purification and
solution deoxygenation, but the RTP intensity in_that case
was much weaker than that with additional external heavy
atom. Although the fluorescence of ANS was enhanced by the
addition of nmmhyl-APC, we could not observe any RTP from
ANS under similar experimental conditions.’

The critical micelle concentration (CMC) for’ SDS is
reported to be 8X10"3 M (21). Also, the CMC of SDS was found
to increase on addition of B-CD in aqueous solution due to
the association of the SDS molecules with 8~CD. This results
in a decrease in the amount of available surfactant monomers
for micelle formation (20). The concentration of SDS
employed was 5 mM, so we eXpect some premicellar aggregates
of SDS in a solution without any host. However, we should
not expect any micelles in the above solutions containing
host molecules. Although methyl-APC was not the only
substance responsible for the observation of RTP, it appears
that methyl-APC enhances RTP by efficient organization of
lumiphors and SDS. The RTP of TNS at SDS concentrations
above the CMC still showed enhanced intensity with methyl-
APC.

The enhancement of RTP intensity on the addition of
methyl-APC, strongly indicates that SDS molecules aggregate
around the TNS-APC complex through electrostatic and
hydrophobic interaction. This provides a favorable

microenvironment that stabilizes the triplet state of TNS

 

 

 

166

molecules. It is quite surprising that the RTP of TNS
decreased even though fluorescence of TNS increased in the
aqueous solution on addition of B-CD. It is believed that
the decrease in phosphorescence intensity of TNS on addition
of B-CD is due to a preference for the aqueous bulk phase
over the neutral cyclodextrin by TNS molecules. Also, the
number of available surfactant monomers for TNS in aqueous
bulk phase is expected to decrease due to the aggregation of

surfactant molecules around the B-CD.

The Effect of Host, Heavy Atom, and SDS on RTP.

The effect of host concentration on RTP was studied at
fixed concentrations of SDS, lumiphor, heavy atom, and
sodium sulfite. The concentration of host was varied from 0
to 0.6 mM and the concentration of SDS was maintained at 5
mM. As increasing the concentration of methyl-APC, the RTP
intensity of TNS steadily increased up to. 0.3 mM and
decreased thereafter. The same trend was observed with 2-

bromonaphthalene. On the other hand, the RTP intensity of

TNS continuously decreased when increasing the B-CD

concentration as shown in the upper graph of Figure 7.8. The
decrease in RTP intensity of TNS above 0.3 mM may be due to
an impurity of methyl-APC and decrease in the number of SDS
molecules available for methyl-APC molecules.

The increase in the SDS concentration with the fixed
amount of heavy atom and sodium sulfite enhanced the RTP

intensity of lumiphors up to an SDS concentration of 0.1 M.

 

 

 

 

167
700?
...:
5004
.00:
soo~

200d

Relative RTP intensity

100‘

x—x CO
H APC
l ' T ' r ' r r
0 10 20 30 40 50 60
[HOST] (x1 05.14)

 

400-
350-

J
300-
250d
200-

150-

Relative RTP intensity

100-

50-

 

 

0 '7 f T I 1' r g g T T I' r I T Y F t T rfi

o , so . 100 150 200
[303] (x103.M)

Figure 7.8. The effect of host (upper graph) and SDS (lower

graph) on the RTP intensity of TNS (2.5x10f5 M).

 

 

 

 

168

Beyond this the RTP intensity began to decrease. This type
of change in RTP intensity was also observed in micellar
stabilized RTP of many PAHs. But, the inflection point of
SDS concentration at which RTP intensity starts to decrease
normally lies in the 0.04-0.05 M range, which are much lower
than that of methyl-APC - SDS mixed organized system.

The exact mechanism of these RTP changes is not clear at
this time, but it seems that the aggregation of SDS around
the methyl-APC plays a major role. Adsorption of surfactant
monomers on a hydrophilic solid surface such as silica has
been observed by studying the fluorescence decay of pyrene
dissolved in surfactant-silica media (23). The preposed
models on the basis of the adsorption data admit the
existence of condensed (or organized) molecular assemblies
on the surface, either in the form of micellar-like
aggregates (hemimicelles) or in the form of a more extended
lamellar phase (24).

The effects of heavy atoms on the RTP were quite
interesting. No RTP of TNS was observed from anionic TNS
with bromide ions as an external heavy atom source. In the
case of nonionic 2-bromonaphthalene, both Br' and Tl‘
enhanced RTP, but the bromide ions ‘enhanced the RTP
intensity more than twice that of thallium ions as shown in
Figure 7.9. These data indicate that the effective
enhancement of RTP by heavy atoms through intersystem
crossing requires a closer approach of heavy atoms to the

lumiphors. It is expected that thallium ions cannot approach

 

 

 

 

169

 

 

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170

close to the methyl-APC due to the electrostatic repulsions
between positively charged sites of the methyl-APC and
thallium ions. However, anionic bromide ions can approach
closely to the methyl-APC due to attractive forces between
them. It is worth noting in Figure 7.9 that the RTP
development of 2-bromonaphthalene by bromide ions took more
time than with the thallium ions due to the electrostatic
interactions of bromide ions with. anionic SDS molecules
around the methyl-APC.

Sodium sulfite has proven to be an excellent oxygen
scavenger in solution (13) even. though the reaction of
sulfite ions with oxygen is a slow process in this system.
The RTP development with time directly indicates the oxygen
consumption process in the sample solution. It appears to be
a two-step process in both the bromide and the thallium
system. These can be also observed in a highly concentrated
micellar solution. The oxygen consumption by‘sulfite ions
should be a diffusion controlled process. Also the diffusion
process of oxygen in the bulk phase and inside the SDS-APC-
lumiphor complex will be different. These data suggest that
the solution is composed of two different microenvironments

for lumiphors.

RTP Decay of Lumiphors.
Since the RTP lifetime is extremely sensitive to a

molecules’s microenvironment, information about the

 

171

molecular interaction of the lumiphors in the APC-surfactant
mixed media can be obtained from the RTP decay lifetimes.

RTP lifetimes of TNS and 2-bromonaphthalene were measured
in aqueous solutions containing a heavy atom and 5 mM SDS.
RTP lifetimes were calculated with the linear least-squares
method. Figure 7.10 shows the change in RTP decay of TNS on
addition. of the host. The, RTP lifetime of TNS in SDS
solution without any host was 124122.us with a correlation
coefficient of 0.999 (dotted curve). In the 0.2 mM B'CD
solution, the RTP lifetime of TNS was decreased slightly
(lower curve) to 117122 us, as expected from the decreased
RTP intensity upon addition of B-CD. But when 0.2 mM methyl-
APC was added to the TNS solution, the RTP lifetime of TNS
increased (upper curve) to 406120 .113 with a correlation
coefficient of 0.999. The RTP decay of 2-bromonaphthalene
under similar experimental conditions except for the heavy
atom (Br' instead of T1‘), showed increased lifetime on
addition of host. The RTP lifetime of 2-bromonaphtha1ene was
increased from 412:11,us to 455312.us upon addition of 0.1
mM B'CD and increased further to 5293.36 us with 0.1 mM
methyl-APC.

Differences in decay lifetimes can generally be attributed
to changes in the deactivation pathways of the excited state
or changes in the interaction of the excited state with the
surroundings (25). The increase in RTP lifetime of lumiphors
on the addition of methyl-APC indicates that the

microenvironment of lumiphors in the methyl-APC - surfactant

 

 

 

172

 

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173

mixed media stabilizes the excited triplet state of

molecules.

D. CONCLUSIONS

The comparative RTP study of anionic and neutral lumiphors
in a mixed organized media, surfactant-host, has
demonstrated that synthetic enzyme model molecules can
provide a favorable microenvironment for lumiphors through
electrostatic and hydrophobic interactions. Since the host
enzyme model molecules are synthetic in nature, a highly
selective microenvironment can be obtained by designing host
molecules for specific purposes. Consequently, the synthetic
enzyme model molecules could open a new avenue for chemical
analysis due to their inherent potential for controlled
specific host-guest interaction.

In the future, such synthetic enzyme model molecules may
find appl ications in luminescence , chromatography ,
biological immunoassay, flow injection analysis, and many

other research areas.

 

 

174

CHAPTER VII REFERENCES

(l) Hurtubise, R. J. Anal. Chem. 1983, 55, 669A.

(2) Ward, J. L.; Walden, G. L.; Winefordner, J. D. Thlanta
1981, 28, 201.

(3) Cline Love, L. J.; Skrilec, M.; Habarta, J. G. Anal.
Chem. 1980, 52, 754.

(4) Donkerbroek, J. J.; Elzas, J. J.; Goojier, C.; Frei, R.
W.; Velthorst, N. Talanta 1981, 28, 717.

(5) Scypinski, S.; Cline Love, L. J. Anal. Chem. 1984, 56,

322.

(6) Weinberger, R.; Cline Love, L. J. Appl. Spectrosc. 1985,

39, 516.
(7) Ramamurthy, V.; Eaton, D. F. Acct. Chem. Res. 1988, 21,

300.

(8) Cramer, F.; Mackensen, G. Angew. Chem. Intern. Ed. Engl.

1966, 5, 601.
(9) Turro, N. J.; Bolt, J. D.; Kuroda, Y.; Tabushi, I.

Photochem. Photobiol. 1982, 35, 69.

(10) Tabushi, I.; Kuroda, Y.; Kimura, Y. Tetrahedron Lett.

1976, 37, 3327.

(11) Tabushi, I.; Kimura, Y.; Yamamura, K. J. Am. Chem. Soc.

1981, 103, 6486.
(12) Kim, Haidong.; Zabik, M. J.; Crouch, S. R., Appl.

Spectrosc. 1989, 43, 608.

 

 

175

(13) Diaz Garcia, M. E.; Sanz-Medel, A. Anal. Chem. 1986,
58, 1436.

(14) Tabushi, Iwao Tetrahedron 1984, 40(2), 269.

(15) Tabushi, I.; Yamamura, K.; Nonoguchi, H.; Hirotsu, K.;

Higuchi, T. J. Am. Chem. Soc. 1984, 106, 2621.

(16) Murakami, Y.; Nakano, A.; Akiyoshi, K.; Fukuya, K., J.

 

Chem. Soc. Perkin Trans I, 1981, 2800.

(17) Benesi, H. A.; Hildebrand, J. H., J. Am. Chem. Soc.,
1949, 71, 2703.

(18) Bright, F. V.; Keimig, T. L.; McGown, L. 8., Anal.
Chim. Acta, 1985, 175, 189.

(19) Brand, L.; Gohlke, J. R. Ann. Rev. Biochem. 1972, 41,
843.

(20) Catena, G. C.; Bright, F. V., Anal. Chem, 1989, 61,
905.

(21) Cline Love, L. J.; Habarta, J. G.; Dorsey, J. G. Anal.
Chem. 1984, 56, 1132A.

(22) Georges, J.; Desmettre, S. J. Colloid. Interface Sci.
1987, 118, 192.

(23) Levitz, P.; Van Damme, H. J. Phys. Chem. 1986, 90,
1302.

(24) Cases, J. M. Bull. Mineral 1979, 102, 684.

(25) Nelson, C.; Patonay, 6.; Warner, I. M. Appl. Spectrosc.

 

1987, 41, 1235.

 

 

 

 

176

CHAPTER VIII

CONCLUSIONS AND FUTURE PROSPECTS

The ultimate goal of this research was to develop a new
RTP methodology using the synthetic enzyme model compounds,
the azaparacyclophanes. To carry out this goal two
additional areas had to be studied. These were luminescence
instrumentation for RTP and RTP lifetime measurements, and
the decay kinetics study of triplet state molecules.

In this final chapter, the three major research projects

are reviewed to draw conclusions. Luminescence

instrumentation is discussed first, then the RTP lifetime
study of PAHs in micellar solution follows. Finally, the RTP
study of PAHs in mixed organized media, APCtSDS system, is

described. Also in each section, future prospects for that

particular research area is discussed.

A. Luminescence Instrumentation.

The intended goal of this instrumentation, was to modify a
commercial spectrofluorometer, and to develop a computer-
controlled, versatile, and flexible luminescence data
acquisition and analysis system. This would allow
semiautomatic luminescence data (fluorescence, RTP, RTP

lifetime) acquisition and analysis with minimal cost.

 

 

 

 

 

 

 

177

These goals were carried out successfully (1,2), and the
details are documented in chapter V. This luminescence
instrument shares most of the advantages of the pulsed
source phosphorimeter, but does not share the disadvantages
of the conventional rotating-can type mechanical
phosphoriscope. The major advantages of this instrument over
the conventional phosphorimeters are:

(1) Fluorescence, RTP, and RTP lifetimes can be measured on
the same instrument without additional instrumental
modifications.

(ii) Unlike the rotating-can type mechanical phosphoriscope,
there is no limitation on the time discrimination procedure.
Delay time and gate time can be easily changed by software,
without adjustment of the rotating speed of the chopper.
(iii) This instrument gives equally good performance in RTP
land RTP lifetime measurements for short-lived and long-lived
phosphors. By changing the delay 'time, gate time, or
rotational speed of the chopper, one can obtain an optimum
detection time window.

(iv) This instrument gives a more reliable reproducibility
and less RF (radio-frequency) interference than a pulsed
source, due to a chopped continuous source.

(V) Finally, this instrument is controlled by a low-cost
IBM-XT compatible personal computer. Luminescence data are
acquired and manipulated easily on site.

The biggest and most persistent problem of this instrument

was an unstable Xenon-arc lamp. In the worst case, the

—;

 

 

 

 

 

178

source intensity fluctuated up to 20% of its maximum
intensity. We could not replace the source because that
spectrofluorometer model (SPF-500) had been discontinued by
the manufacturer. Also, the use of the slow ADC board and a
computer did not allow the measurement of fluorescence
lifetimes.

The instrument designed for this research can be improved
in several ways with extra investment. Replacement of the
unstable high pressure lamp with a high power pulsed laser
will give much more power. Also, a fast ADC board and
computer will enable the measurement of fluorescence
lifetimes. The PMT can be replaced by photodiode array for a
high speed multidimensional luminescence data acquisition.

In the software part, a spectral library routine could be
added for spectral data base applications, such as, compound
identification from the obtained spectrum by spectral peak
matching. An ideal intelligent instrument could be build by

the incorporation of artificial intelligence (3).
B. RTP Lifetimes of PAHs in Organized Media.

MS-RTP lifetimes of PAHs measured in this system were
found to be significantly longer than those of other
literature values. Similar results were obtained by other
researchers in this field (4). The reason for these longer
RTP lifetimes may be explained in several ways. It was found

that the RTP lifetimes of PAHs change from system to system

 

 

 

 

 

 

 

179

depending on the experimental conditions employed. Any
comparisons based on the numerical values of the RTP
lifetimes could lead to a serious misinterpretation of the
obtained lifetime data.

Heavy atoms were found to affect observed RTP lifetimes
significantly. Today, the use of heavy atoms in RTP is very
common. Heavy atoms usually increase the RTP sensitivity
while reducing triplet lifetimes. Since oxygen is such an
efficient triplet quencher, the deoxygenation method also
affects the observed. RTP lifetimes significantly. Sodium
sulfite has proven to be a very efficient oxygen scavenger
for RTP solution.

Unlike conventional frozen phosphorescence samples, every
component in the RTP solution is in dynamic equilibrium. As
a result, temperatures showed profound effects on the
observed RTP. For accurate and reproducible results, precise
temperature control of the sample was necessary.

The rapid lifetime determination (RLD) method for the
calculation of lifetimes gave good results with an unknown
background and noisy' data. Even. though. statistical
parameters which can judge goodness of the data fit are not
available, regeneration of decay data by simulation with the
obtained background, initial intensity, and lifetime value,
enables direct comparison of the regenerated data with raw

data. This RLD method is very fast, and may be adapted for

real-time lifetime analysis.

 

 

 

 

 

 

180

Since RTP lifetimes are highly dependent on the
experimental conditions, there should be some kind of
normalized conditions for the RTP lifetime experiment.
Unless some normalization of the RTP lifetime data which
were obtained in different systems is made, the analytical

usefulness of RTP lifetimes is questionable.

C. Application of Synthetic Enzyme Model Compounds in

Chemical Analysis.

The importance of organized media has been well understood
during the last two decades. The applications of macrocyclic
compounds and various ‘types of organized media. are now
emerging rapidly in chemical analysis (5,6).

Initial attempts were made to synthesize octopus-like
azaparacyclophanes which carry negative or positive charges
at their long tails. But their synthesis was not easy to
accomplish. As a result, basic azaparacyclophane without
long tails (methyl—APC) was chosen for testing and
evaluating for RTP applications.

The basic model compound was found to be an excellent host
for anionic as well as neutral compounds. The binding
constants of several anionic compounds are several orders
higher than that of well known cyclophanes. These were
attributed to the strong electrostatic and hydrophobic
interactions between APC and PAH. Although methyl-APC

strongly binds many PAHs, the open ends at the top and

 

 

 

181

bottom. of the methyl-APC made included lumiphors to be
exposed to outside quenchers. This prevented any observation
of RTP in the solution. containing only' methyl-APC. The
addition of premicellar concentrations of surfactant seemed
to cover up both open ends of the APC-PAH complexes, thus
inducing RTP. This result suggests that octopus-like
compounds might be better for RTP in this sense.

It was found that close approach or complex formation of
heavy atoms to lumiphors is necessary to induce an
analytically useful RTP signal. Also, electrostatic
interaction between the heavy atoms and the organized
assembly plays a very important role to induce a so-called
external heavy atom effect (7).

Immediate use of APCs in chemical analysis is prohibited
by tedious synthetic process required, because they are not
available commercially at this time. The necessity of highly
dilute conditions and low yield of the product in the
synthesis of azaparacyclophanes required labor intensive
devotion. But as more scientists recognize their importance
in chemical analysis they will be available commercially
sometime in near future.

The potential application of water-soluble APCs in
analytical chemistry may be wide ranging, including
applications in luminescence and. [chromatography. One
particularly interesting application of charged APCs would

be as a separation medium in capillary electrokinetic

chromatography.

 

 

 

 

 

182

CHAPTER VIII REFERENCES

1. Kim, Haidong; Zabik, M. J.; Crouch, S. R., Appl.
Spectrosc., 1989, 43, 608.

2. Kim, Haidong; Zabik, M. J.; Crouch, S. R., Appl.
Spectrosc., 1989, 43, 611.

3. Wade, A. P.; Crouch, S. R., Spectroscopy, 1989, 3, 24.

4. Kraus, P. R., Ph.D. Dissertation Thesis, Dept. of
Chemistry, Michigan State University, 1988.

5. Werner, T. C.; Cummings, J. G.; Seitz, W. R., Anal.
Chem., 1989, 61, 211.

6. Shaksher, Z. M.; Seitz, W. R., Anal. Chem., 1989, 61,

590.

7. Kim, Haidong, Crouch, S. R.; Zabik, M. J., Anal. Chem.,

1989, 61, Nov. 1 issue.

 

 

 

 

APPENDIX A

SOURCE CODE OF

TPLIB

(Library routines for Turbo Pascal)

 

 

 

 

 

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213

0
’

ADCR.ASM

.0

’

;This is a assembly subroutine for EMISPEC.PAS.

;This assembly routine collects spectrum data and stores
;data into Buffer data array. Data type is specified by

;DataType(1=Fluorescence,2-Phosphorescence,3-Lifetime).

’

 

Code Segment ‘
Assume CS:Code
Public ReadData
;Procedure ReadData(Var Buffer:IntBuff; Var Count,DataType:Integer);

ReadData Proc Near

Push 8? ;Save BP on stack
Mov BP,SP ;Now 8? has stack pointer
Cli ;Disable interrupt.
Les DI,[BP+8] ;Get Count
Mov CX,ES:[DI] ;Store it into CX
Les DI,[BP+4] ;Get data type
'Mov AL,ES:[DI] ;Move it into AL
Les DI,[BP+12] ;Get Buffer address
Cmp AL,2 ;If DataType-Z then
Je Phs ;Jump to phosphorescence
Cmp AL,3 ;If DataType-3 then
Fl Je Ltm ;Jump to lifetime routine
5:
Nov DX,1814 ;Start conversion by'
Nov AL,0 ;writting any byte to
Out DX,AL ;A/D start port(1814)
FDone:
Mov DX,1812 ;Check if data ready
In AL,DX ;by checking ADC control
Cmp AL,128 ;port.
Jb FDone ;Hait until done.
Inc DL ;Get lower byte first
In AL,DX
Mov ES:[DI],AL ;Store it into Buffer
Inc DI ;Next location
Inc DL ;Get higher byte
In AL,DX .
Mov ES:[DI],AL ;Store it into Buffer
Inc DI ;Next location
Loop Fls ;Continue Count times
Jmp Exit
Phs:
Mov DX,1817 ;Start conversion by sending
Hov AL,48 ;arm counters command to
Out DX,AL ;timer control port
PDone:
Mov DX,1812 ;Check if data ready
In AL,DX ;by checking ADC control
Cmp AL,128 ;port.
Jb PDone ;Hait until done.
Inc DL ;Get lower byte first
In AL,DX

Mov ES:[DI],AL ;Store it into Buffer

 

 

 

214

 

 

Inc DI ;Next location
Inc DL ;Get higher byte
In AL,DX
Mov ES:[DI],AL ;Store it into Buffer
Inc DI ;Next location
Loop Phs
Jmp Exit
Ltm:
Hov DX,1817 ;Initialize timer OUT4
Hov AL,228 ;to low
Out DX,AL
Hov DX,1814 ;Send A/D start conversion
Mov AL,0 , ;command for reset
Out DX,AL
Reset:
Mov DX,1812 ;Check data ready
In AL,DX
Cmp AL.128
Jb Reset ;Hait until done
Mov DX,1814 ;Reset by inputting
In AL,DX ;higher data byte
,Mov DX,1812 ;Now enable A/D external
Mov AL,132 ;start conversion.
Out DX,AL
Hov DX,1817 ;Load and arm
Mov AL,108 ;counter 3 and 4.
Out DX,AL
LDone:
Hov DX,1812 ;Check if data ready
In AL,DX ;by checking ADC control
Cmp AL,128 ;port.
Jb LDone ;Hait until done.
Inc DL ;Get lower byte first
In AL,DX
Mov ES:[DI],AL ;Store it into Buffer
Inc DI ;Next location
Inc DL ;Get higher byte
In AL,DX
Hov ES:[DI],AL ;Store it into Buffer
Inc DI ;Next location
Loop LDone ;Repeat DF.NumData times
Mov DX,1817 ;Disarm counter 3
Nov AL,196
Out DX,AL
Exit:
Sti ;Enable interrupts.
Pop 8P ;Restore BP
Ret 12 ;Three variables(3x4-12).
ReadData Endp
Code Ends

End

 

APPENDIX C

SOURCE CODE OF

IBM-DACA

 

 

 

 

 

 

 

 

 

      

                   

 

 

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223

° Procedure FastRead(Var Data: DataArray; Var NumData,
Channel: Integer);

This is a assembly subroutine for IBMDAC.PAS and

9
i
; Reads NumData with hardware A/D conversion trigger.
9

LSDATA.PAS.

code segment
assume cs: code
public getfast

getfast proc
push
mov
les
mov
les
mov
les
. cli
conv:
mov
mov
out
inc
mov
out
dec
wait:
in
and
cmp
jne
inc
mov
out
mov
in
mov
inc
inc
in
mov
inc
loop
sti
mov
POP
ret
getfast endp
code ends
end

near

bp

bp. 59
di,[bp+4]
bx,es:[di]
di,[bp+8]
cx,es:[di]
di,[bp+12]

dx,02E2H
al,1
dx,al

dx

al,bl
dx,al

dx

al,dx

al,l

al,0

wait

dx

al,0

dx,al
dx,22E2H
al,dx
es:[di],al
di

dx

al,dx
es:[di],al
di

conv

sp.bp
bp
12

;near call start at 4th byte
;get channel in bx

;variable pointer is 4.bytes
;get numdata in cx

;get data array address in eszdi
;clear interrupt

;AI-ctrl register

;set new data flag to prevent overrun
;by software enabling A/D

;now higher byte

;get channel

;set channel

;set dx to AI-ctrl register

;read A/D status

;mask out all except A/D done
;check A/D conversion done
;wait until done

;point dx to AI-ctrl higher byte
;to skip channel set

;enable data read

;AI-read register

;read lower byte datum

;store into data array

;next data location

;next higher byte

;read higher byte datum
;store into data array

;set next data array location
;repeat NumData times
;restore interrupt

 

 

 

 

APPENDIX D

SOURCE CODE OF

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