• TABLE OF CONTENTS
HIDE
 Title Page
 Dedication
 Acknowledgement
 Table of Contents
 Abstract
 Introduction
 Overview of phosphorescence
 An evaluation of cellulose as a...
 Biochemical and drug analysis by...
 Summary and future considerati...
 Appendices
 References
 Biographical sketch














Title: Room temperature phosphorescence
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Permanent Link: http://ufdc.ufl.edu/UF00099225/00001
 Material Information
Title: Room temperature phosphorescence some diagnostic studies in its application to biochemical and drug analysis
Physical Description: viii, 135 leaves : ill. ; 28 cm.
Language: English
Creator: Bateh, Ricky P., 1956-
Publication Date: 1982
Copyright Date: 1982
 Subjects
Subject: Phosphorescence   ( lcsh )
Drugs -- Spectra   ( lcsh )
Chemistry thesis Ph. D
Dissertations, Academic -- Chemistry -- UF
Genre: bibliography   ( marcgt )
non-fiction   ( marcgt )
 Notes
Thesis: Thesis (Ph. D.)--University of Florida, 1982.
Bibliography: Bibliography: leaves 131-134.
General Note: Typescript.
General Note: Vita.
Statement of Responsibility: by Ricky P. Bateh.
 Record Information
Bibliographic ID: UF00099225
Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: alephbibnum - 000334790
oclc - 09526455
notis - ABW4433

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Table of Contents
    Title Page
        Page i
        Page ii
    Dedication
        Page iii
    Acknowledgement
        Page iv
    Table of Contents
        Page v
        Page vi
    Abstract
        Page vii
        Page viii
    Introduction
        Page 1
        Page 2
    Overview of phosphorescence
        Page 3
        Page 4
        Page 5
        Page 6
        Page 7
        Page 8
        Page 9
        Page 10
        Page 11
        Page 12
        Page 13
        Page 14
        Page 15
        Page 16
        Page 17
        Page 18
        Page 19
        Page 20
        Page 21
        Page 22
    An evaluation of cellulose as a substrate material for room temperature phosphorescence
        Page 23
        Page 24
        Page 25
        Page 26
        Page 27
        Page 28
        Page 29
        Page 30
        Page 31
        Page 32
        Page 33
        Page 34
        Page 35
        Page 36
        Page 37
        Page 38
    Biochemical and drug analysis by room temperature phosphorescence
        Page 39
        Page 40
        Page 41
        Page 42
        Page 43
        Page 44
        Page 45
        Page 46
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        Page 112
        Page 113
        Page 114
        Page 115
        Page 116
        Page 117
        Page 118
    Summary and future considerations
        Page 119
        Page 120
    Appendices
        Page 121
        Page 122
        Page 123
        Page 124
        Page 125
        Page 126
        Page 127
        Page 128
        Page 129
        Page 130
    References
        Page 131
        Page 132
        Page 133
        Page 134
    Biographical sketch
        Page 135
        Page 136
        Page 137
        Page 138
Full Text











ROOM TEMPERATURE PHOSPHORESCENCE: SOME DIAGNOSTIC
STUDIES IN ITS APPLICATION TO BIOCHEMICAL AND
DRUG ANALYSIS











BY

RICKY P. BATEH


A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY


UNIVERSITY OF FLORIDA
1982















Scientific research is a social activity. . To
understand the nature of science, we must look at the
way scientists behave towards one another, how they are
organized, and how information passes between them. The
young scientist does not study formal logic, but learns
by imitation and experience a number of conventions that
embody strong social relationships--learns to play a role
in a system by which knowledge is acquired,- sifted, and
eventually made public property.

John Ziman



When we abandon the pursuit of truth for any reason,
whether it is because it is dangerous or because we are
lazy or for any other reason, then we become parasites,
robbing society and giving nothing in return. This is
why academic people have always to be so hard on each other.
You will find out that we are ruthless in exposing each
others' errors and eliminating those of our number who
let the standard fall. We have to because the scholar
who abandons the truth is a menace to our whole community.

Arthur Lewis















This dissertation is dedicated to my wonderful family.
My father, forever inquisitive, has relayed to me the impor-
tance of communication between professional personnel and
the layperson. My mother, always supportive of my career
decisions, has bestowed upon me the ultimate power of con-
templation. My brother, a friend indeed, has shown contin-
uous dedication to my professional achievements. Last but
not least, my sister, brother-in-law and niece have provided
me with the happiness necessary to attain the highest spiri-
tual level.














ACKNOWLEDGEMENTS

The author would like to thank Dr. J. D. Winefordner

for his support and encouragement throughout these research

endeavors. His liberal attitude towards graduate research

has been favorably applied towards my development as a

practical scientist. The JDW group has been, and always

will be, an interesting group with which to work. The

author would also like to acknowledge the assistance of

the three postdoctoral factoti, Jimmie Ward, Edward Voigt-

man and Ben Smith. He is most grateful to Jeanne Karably

for preparation of this work in its present form and to

Jeff Pate for preparation of the artwork.














TABLE OF CONTENTS

PAGE

ACKNOWLEDGEMENTS .............................. ..... iv

ABSTRACT ......................................... vii

CHAPTER

1 INTRODUCTION .............................. 1

2 OVERVIEW OF PHOSPHORESCENCE ............... 3

Introduction ............................ 3
Studies in Low Temperature
Phosphorescence ..................... 5
Studies in Room Temperature
Phosphorescence ................ ...... 7
Diagnostics ............................. 10
Instrumentation ....................... 10
Procedural Considerations ............. 10
Summary ............................... 22

3 AN EVALUATION OF CELLULOSE AS A SUBSTRATE
MATERIAL FOR ROOM TEMPERATURE
PHOSPHORESCENCE ......................... 23

Introduction ............................ 23
Experimental ............................ 24
Reagents and Materials ................. 24
Procedure ............................. 24
Results and Discussion .................. 26
Background on Papermaking ............. 26
Diagnostical Studies on Cellulose
Products ............................ 28
Phosphorescence background .......... 28
Fibrillation study .................. 33
Filter paper comparison ............. .33
Filter-paper lots comparison ........ 36
Conclusion .............................. 36
Note .................................... 36

4 BIOCHEMICAL AND DRUG ANALYSIS BY ROOM
TEMPERATURE PHOSPHORESCENCE ............. 39

Introduction ............................ 39








Biochemical/Drugs .................... 40
Pharmaceutical Formulations ........... 59
Experimental ........................... 61
Reagents and Materials ................. 61
Sample Preparation .................... 61
Biochemicals/drugs .................. 61
Pharmaceutical formulations ......... 66
Procedure ................ ...... ...... 66
Results and Discussion .................. 72
Biochemical/Drug Survey ............... 72
Xanthines .......................... 72
Indoles ............................ 75
Benzoic acid derivatives ............ 78
Others .............................. 84
Pharmaceutical Analysis .............. 110
Xanthines ........................... 110
Analgesics .......................... 110
Others .............................. 114
Summary ................................. 114

5 SUMMARY AND FUTURE CONSIDERATIONS ......... 119

APPENDICES

1 GLOSSARY FOR CHAPTER 3 .................... 121

2 ANCILLARY STUDIES ....................... ... 123

Herbicide Survey .......................... 123
Introduction .......................... 123
Procedure ............................. 123
Results and Discussion ................ 123
Therapeutic Drug Monitoring ............. 125
Introduction .......................... 125
Reagents and Materials ................ 125
Procedure .................. ........... 126
Results and Discussion ................ 126

3 COMPANY LIST .............................. 128

4 DATA FOR LINEAR REGRESSION ANALYSIS ....... 130

REFERENCES ....................................... 131

BIOGRAPHICAL SKETCH ................... .. .......... 135














Abstract of Dissertation Presented to the Graduate
Council of the University of Florida in Partial
Fulfillment of the Requirements for the Degree
of Doctor of Philosophy

ROOM TEMPERATURE PHOSPHORESCENCE: SOME DIAGNOSTIC
STUDIES IN ITS APPLICATION TO BIOCHEMICAL AND
DRUG ANALYSIS

By

Ricky P. Bateh

August, 1982

Chairman: J.D. Winefordner
Major Department: Chemistry

Room temperature phosphorescence (RTP) involves the

measurement of phosphorescence irradiated by molecules

illuminated with a source of ultraviolet light. In RTP,

analyte molecules are adsorbed onto solid substrate mate-

rials, such as filter paper and silica gel, and analyzed

for their characteristic phosphorescence emission.

In these studies, RTP test methodologies and appli-

cations of RTP are evaluated for their effectiveness in

demonstrating the analytical utility of the technique.

Improvements are made in sample-compartment design and in

sampling procedures currently used in RTP. A list of

compounds exhibiting RTP is made to add to the present

compilation of molecules known to phosphoresce at room








temperature. Trends in phosphorescence intensities are

indicated for groups of "homologous" compounds; generaliza-

tions are made for such trends, but no mechanisms are

described. Finally, RTP is used for the determination of

a variety of drugs in pharmaceutical formulations, over-

the-counter preparations and blood serum. While RTP has

been shown to be a viable method of chemical analysis,

real-sample applications have been limited. Thus, these

latter studies represent significant accomplishments in

RTP test methodologies. The procedures reported here are

quite simple and specific for the determination of a variety

of compounds in real samples.


viii













CHAPTER 1
INTRODUCTION

In the past decade, room temperature phosphorescence

(RTP) has received considerable attention as a method of

analysis for biochemicals, drugs and polynuclear aromatic

hydrocarbons. With the introduction of RTP as a new analy-

tical technique, it is necessary to evaluate the procedure

for appropriate test methodologies and applications. Sev-

eral workers (1-3) have reviewed the fundamental inter-

actions responsible for room temperature phosphorescence,

some analytical methodologies and a variety of applications.

In a continuation of the recent work on RTP, this

research was designed to optimize existing RTP methodolo-

gies, to evaluate the selection of an appropriate solid

substrate for use in RTP, to compile a list of molecular

species that exhibit room temperature phosphorescence under

specified conditions and to evaluate the analytical utility

of RTP in real-sample analysis. The first project involved

a design-modification of the multiple-sampling bar device

(4) and a rigorous evaluation of the sampling procedure

used in RTP. The latter study resulted in a major change

in procedure which allowed for a '3-fold reduction in anal-

ysis time (from u30 min to "10 min for 4 samples). Follow-

ing the procedure-optimization study, an evaluation of





2


cellulose as a substrate material for RTP was made to

facilitate selection of cellulose-based substrate materials

which would enhance phosphorescence of analyte molecules.

Once the RTP system was optimized, an in-depth study was

initiated to establish a data base of molecules exhibiting

RTP. In addition, several groups of "homologous" com-

pounds were studied to determine the effects of a variety

of substituents on phosphorescence intensity. In applying

RTP to real-sample analysis, it was shown that RTP could

be used quite-successfully in analyzing for a variety of

active ingredients (drugs) in pharmaceutical formulations,

over-the-counter preparations and in blood serum.













CHAPTER 2
OVERVIEW OF PHOSPHORESCENCE

Introduction

Luminescence is characterized by the emission of light

following its selective absorption. Fluorescence and

phosphorescence are two types of photoluminescence which

can occur following excitation by any form of electromag-

netic radiation.

In order to differentiate between absorption, fluores-

cence and phosphorescence processes, one should refer to

the modern theory of molecular structure. Consider a mole-

cule possessing a series of closely spaced energy levels

that can be excited from a lower to a higher energy level

by absorption of a discrete quantum of energy. A simpli-

fied Jablonski diagram (Figure 1) shows the processes which

a molecule can undergo. Absorption can take place and

results in a transition from the ground electronic state to

a higher electronic state. These ground to excited singlet

transitions are responsible for the ultraviolet-visible

absorption spectrum of a molecule. During excitation,

excess energy can be rapidly dissipated via radiationless

decay (internal conversion) and the lowest vibrational

level of the first excited state (Sl) is attained. The

transition from the first excited singlet (SI) to the









Singlet System


Triplet System

Internal
Conversion




Intersystem
Crossing -


Figure 1. Simplified energy level diagram of a
polyatomic molecule.







ground state (SO) gives fluorescence. If a molecule in

its excited state undergoes instead of internal conversion,

intersystem crossing to the triplet state (T), then relax-

ation from the first excited triplet state (TI) to the

ground state (SO) gives phosphorescence. In general,

fluorescence takes place in a shorter time frame and at

shorter wavelengths than phosphorescence. Molecules in the

metastable triplet state are more susceptible to radiation-

less decay (collisional deactivation), and as a result,

only molecules held in rigid media phosphoresce. For

phosphorescence, two general categories of rigid media

involve either dissolution of the analyte molecule in an

appropriate solvent and freezing in liquid nitrogen (77 K)

or adsorbing onto solid substrate materials such as silica

gel and filter paper at room temperature (298 K).

Studies in Low Temperature Phosphorescence

The conventional approach to phosphorimetry involved

the study of low temperature phosphorescence (LTP). In

1888, Wiedemann (5) first reported the phenomenon of phos-

phorescence by observing dyestuffs adsorbed in gelatin at

193 K. Some 55 years later, Lewis and Kasha (6) suggested

that phosphorescence could be used as a means of chemical

analysis. Kiers, Britt and Wentworth in 1957 (7) demon-

strated the first use of phosphorescence as a method of

analysis of organic molecules by showing that mixtures of

phosphorescent molecules could be spectrally-resolved via

selectivity brought about by differences in excitation and







emission wavelengths. By taking advantage of differences

in luminescence lifetimes of the molecules, they were able

to use temporal resolution as a further means of charac-

terizing the molecules.

In the subsequent 10 years, several laboratories

evaluated the use of LTP as an analytical technique by

using a variety of Dewar flask systems (77 K) in immersion-

cooling and conduction-cooling modes (8-14). Immersion

cooling was the first choice because liquid nitrogen was

transparent and non-luminescent in the wavelength range of

200-800 nm. However, there were numerous disadvantages to

this configuration, the two most important of which being

poor precision and accuracy of the measurement due to

irreproducible positioning of the sample cell and the trans

lucent nature of the frozen solution in the sample cell.

Other disadvantages included light loss due to the multi-

layered quartz system, flicker of excitation/emission radi-

ation due to refractive index changes caused by convection

of the coolant as it warmed, and additional flicker noise

due to bubbling of the coolant at nucleation sites caused

by dust or scratches inside the Dewar flask. Overall, the

immersion cooling technique was a time-consuming, laborious

method of analysis and improvements were sought in the

conduction cooling method.

Early designs of conduction cooling systems performed

poorly due to poor thermal contact between the sample cell

and the copper rod and due to fogging of the viewing area.








In addition, a vacuum, necessary to attain the required

low temperature, had to be broken to introduce a new

sample. To compensate for these limitations, two new

designs were recently evaluated (15,16). Although these

designs facilitated sample introduction and decreased sam-

pling time by -4-fold, the lowest temperature that could

be attained was b85 K for the immersed copper mass and

1100 K for the flow-through conduction cooling system. On

the whole, low temperature measurements were difficult to

make and the possibility of routine applications or. auto-

mation of phosphorimetry using LTP was remote.

Despite the limited success of LTP, it could be to

one's advantage to consider the more simple RTP system.

The immediate advantage of a RTP system is the absence of

cryogenic equipment necessary in LTP. While the simplicity

of the RTP system could lend itself to automation, it is

less sensitive but more selective than LTP.

Studies in Room Temperature Phosphorescence

The first observation of RTP of organic molecules

adsorbed onto solid substrate materials occurred in 1941

where samples were placed in solid boric acid (17). The

same phenomenon was also observed when samples were prepared

in a rigid polymer matrix such as poly(methyl methacrylate)

(18). In 1957, Szent-Gyorgyi (19) proposed spectrophos-

phorimetric analysis on paper and thin layer chromatograms

by dipping the sheets in liquid nitrogen and then irradi-

ating them with an ultraviolet light source. Roth, in







1967 (20), using a mercury lamp (254 nm) to irradiate

chromatograms, observed luminescence lasting 3-10 sec after

termination of the exciting light. While these observations

suggested phosphorescence rather than long-lived fluores-

cence, no quantitative work was done. Schulman and Walling,

in 1972 (21), rediscovered RTP when they observed phosphores-

cence radiating from a variety of ionic organic molecules

adsorbed on paper, silica, alumina and other supports.

This phenomenon was most pronounced when filter paper was

the substrate material and the solvent was strongly acidic

or basic. The heavy atom effect (addition of a "heavy

atom," e.g., iodide or silver, to enhance phosphorescence

intensities) was first studied by Kasha in 1952 (22) and

applied to analytical phosphorimetry in 1963 by McGlynn and

coworkers (23).

Early work in RTP involved the use of circular filter

paper discs placed in sample holder tips (Figure 2) (24-26).

The utility of RTP as an analytical tool had in some ways

been hindered by the continued use of this method. The

preparation of single samples on the tips was relatively

time consuming in that each sample or blank had to be dried

individually. Batch preparation of the samples was not

always convenient since for reasons of precision, the timing

of the drying and measurement of these samples was critical.

In the past, additional lack of precision had been attri-

buted to possible problems of positioning the filter paper

discs and/or tips in the sample compartment of the instrument.












Front View








Cover Plate







Side View


I I
I I





0`


Figure 2. Sampling tip for room temperature
phosphorescence studies.







In order to overcome the drying time and positioning prob-

lems and improve the uniformity of the treatment of multi-

ple samples, a multiple-sampling bar (Figure 3) was evalu-

ated (27). While this new device complements the sampling

procedure in RTP quite well, there are additional methods

to consider in evaluating its overall effectiveness in RTP.

The present work has continued the evaluation of the multi-

ple-sampling bar for a variety of applications in RTP.

Diagnostics

Instrumentation

All RTP measurements were made with a spectrofluoro-

meter fitted with a xenon arc lamp, a laboratory-constructed

phosphoroscope (Figure 4) (4) for bar-RTP (27) and a potted

photomultiplier tube. A ratio photometer supplied high

voltage to the photomultiplier tube in addition to serving

as a dc amplifier. All line voltages were regulated with

an ac regulator. A block diagram of the system configura-

tion is shown in Figure 5. The components of the RTP sys-

tem are listed in Table 2-1. All operating conditions were

the same throughout all of the RTP studies.

Procedural Considerations

In order to compensate for some of the inefficiencies

encountered with the original bar-RTP work, the sampling

procedure and the position of the flush port were changed.

The original sampling procedure consisted of placing filter

paper discs (%0.25 in dia.), obtained by punching filter

paper with a standard office paper punch, under the cover





























-Cover Plate
Paper Discs


Figure 3. Multiple-sampling bar for room temperature
phosphorescence studies.
























Figure 4. Laboratory-constructed phosphoroscope
assembly:

A. Base plate
B. Synchronous motor
C. Aligning block
D. Drive pulley
E. 0-ring
F. Shutter can
G. Pulley
H. Ball bearing
I. Sample compartment housing
J. Slit holder
K. Slit




13

II


0


























0 O

o 0

.0 0


C 0 +
0 E


j 0
I-
C 0
4. ) .VE

O C

o o. 0




0o m 0 0 ,

o 0- 0- o
0.. -4 U 4-l H



z0 0 xl r 0 -3 I
CS 0 WO 0 CH
S4-)U 0 05
-1 O 0 0-.

+ )C 00n 00 -o

-0 00 4 ) 0 . O 0U
o O s FH e *He






0 C C C cr I



*-H Lf / o ,



Cn



0-
3rkF Ff~~
b d a o o
*^i~kr r Uda
O d C l



























aJ a
0









Table 2-1.



Item


Lamp power
supply


Xenon arc lamp


Spectrofluoro-
meter



Sample
compartment



Phosphoroscope




Sampling bar



Photomultiplier
tube


Ratio photometer




X-Y recorder


16



Instrumentation for RTP Studies


Model
(Operating conditions) Source

LPS 251 HR (7.5A, Schoeffel
20 V)


901C-0011 (150 W) Canrad-Hanovia


Aminco-Bowman SPF American
4-8202 Slits- Instrument
4,3,2,2,3,2, mm Co.


American
Instrument
Co.


Laboratory- Reference 4


constructed
(200 Hz)


Laboratory-
constructed


1P21 (750 V)



4-8912 (R2, SV 100)




1620-827


ac regulator ACR 3000


Reference 27



Hamamatsu



American
Instrument
Co.


American
Instrument
Co.


Sorenson


ac regulator


ACR 3000





17

plate (with 4 holes each %0.25 in dia.) of the bar and then

the cover plate was tightened into place on the bar with 4

screws. Samples were spotted onto the paper discs in 5 pL

volumes with a Micro/pettor (Scientific Manufacturing

Industries). After spotting, the bar was placed under an

infrared lamp (12 min at '600C with the lamp at a height of

%8 in) so that the samples could dry. After drying, the

bar was transferred to the sample compartment (equipped

with a phosphoroscope can with a chopping rate of 200 Hz)

and was allowed to equilibrate under a flow of dry argon

gas (15 min at u20 L/min via a back-side flush). The dry-

ing and flushing steps were necessary to eliminate moisture

and oxygen, both of which quench phosphorescence. Sample

phosphorescence signal levels increased over a period of

a14 min at which time a plateau was reached for -2 min.

Measurements were made on this plateau.

In evaluating the previous procedure, it was thought

that the use of the infrared lamp to dry the samples would

alter the paper characteristics (see Chapter 3) and/or

would affect the chemical stability of the analyte molecules.

These effects have in fact been confirmed in one laboratory

(28). It was found that in the presence of acid, base or

heavy atom perturbers (Pb 2, T1 Ag+ and I ), heating

could cause oxidation of the hydroxyl groups on the surface

of the cellulose substrate material (resulting in a greatly

diminished RTP signal) or could cause oxidative breakdown

of certain analyte molecules. Thus, in order to eliminate








such undesirable effects during lamp-drying, an alternative

approach to dry the samples was investigated. First, the

flush-gas inlet port was changed from the back side of the

sample compartment to a front-surface flush position (Fig-

ure 6). Next, this modified configuration was compared to

the lamp-dry/back-side flush configuration. It was found

that the lamp-drying step could be eliminated without any

large effects on the sample signal levels (Table 2-2).

Both drying and equilibration could now take place in the

sample compartment without the use of an infrared lamp to

dry the samples. Nitrogen was then compared to argon as

the flush gas. The decision to try nitrogen over argon

was a financial one. Argon was purchased in tank-form

while nitrogen was available in-house as boil-off from a

liquid nitrogen reservoir. From the data in Table 2-3,

there is no large difference in signal levels if one sub-

stitutes nitrogen gas as the flush gas.

Thus, in the modified procedure, after spotting the

samples, the bar is placed into the sample compartment

where the samples are allowed to dry for 7 min under a flow

of dehumidified nitrogen gas (%20 L/min). During the dry-

ing process, sample phosphorescence signal levels increase

over a period of %7 min at which time a plateau is reached

for '2 min (Figure 7). Measurements are made on this

plateau.











































Figure 6. Modified sample compartment lid for use
with the RTP-sampling bar in a front-
surface flush configuration.



















o
*H 0


0





4 --



00
mi










| ,- 0 :






OI- oO
H u










N-






0 03
00 00




















o Lo <

,!IlSN31NI 3AII 93
\ ^ i'^
\ ~ ^ r- W Bl
\ :- rl
\~ C a- ^
\ 'r ^
\ *r-i i
\ 4-1 3
I O r mc
\ ~ ki-
l I 0; >

\ ^o
-- 1 --- \ --- l -- U o
0~ 0 0 M
m Q m F^

A11SN3NI 3AlV~13








Table 2-2.


Comparison of the Lamp-dry/Back-side Flush
Procedure to the Front-surface Dry/Flush
Procedure in RTP


Procedure RPI(A) Total Time (min)(B)

144 138
Lamp-dry/ 144 141
Back-side 144 13530
Flush 141 138

x= 141

111 114
Front-surface 114 114
Dry/Flush 108 114 10
108 114
111 117
x= 113

(A) RPI (relative phosphorescence intensity) using a PABA
test solution (50 pg/mL). Argon used as flush gas.
(B) Total time to process 4 samples.



Table 2-3. Comparison of Argon and Nitrogen as a Flush
Gas in RTP



Gas RPI(A)


Argon





Nitrogen


108 111
111 114
105 114
108 114
x= 111


114
129
111
117
= 121


(A) RPI (relative phosphorescence intensity) using a PABA
test solution (50 pg/mL). Both gases used in front-
surface flush configuration with a 7 min flush time.





22


Summary

For all practical purposes, the instrumentation,

operating conditions and sampling procedure have, up to

this point, been optimized. However, the selection of a

suitable support material requires further consideration.

While filter paper appears to work well, there are literally

hundreds of such products on the market. An evaluation of

cellulose (used in making filter paper) as a substrate

material is discussed in the next chapter.














CHAPTER 3
AN EVALUATION OF CELLULOSE AS A SUBSTRATE MATERIAL
FOR ROOM TEMPERATURE PHOSPHORESCENCE

Introduction

The ultimate success of RTP as a method of chemical

analysis depends on the selection of a suitable support

material. In the past, support materials such as cellulose,

silica gel and sodium acetate have been used in RTP measure-

ments of a variety of organic compounds (1-3). -Of the

three major support materials, cellulose appears to offer a

considerable advantage in that there exists a wide-range of

specialty papers with varying characteristics. The major

disadvantage of cellulose is the presence of a broadband

phosphorescence background (at 400-600 nm) (29-31). At

this laboratory, several filter papers have been evaluated

and physical and chemical treatments to minimize the phos-

phorescence background of the papers have been unsuccessful

(32,33). In the present study, several cellulose pulps are

evaluated as support materials to determine if there exists

a successful combination of physical/chemical characteris-

tics of cellulose pulp for RTP; treated filter paper is

also evaluated to find a possible source of the background

phosphorescence. For the reader's convenience, a glossary








(34) (Appendix 1) is included to explain terms (used in

this chapter) common to the pulp and paper industry but

otherwise unfamiliar to the layperson.

Experimental

Reagents and Materials

The following companies kindly provided the respective

materials: Buckeye Cellulose (grade 503 cotton linters

pulp); ITT Rayonier (Cellunier-P wood pulp); Southern

Cellulose (grades 270, 277 and 282-R cotton linters pulps);

Eaton-Dikeman (613 and 631 filter papers); Schleicher &

Schuell (S & S 903 filter paper-lots W01, W02, W12, W92,

W93, W94). Diethylenetriaminepentaacetic acid (DTPA) and

p-aminobenzoic acid (PABA) were purchased from Sigma Chemi-

cal Co. and were used without further purification. Sodium

hydroxide, periodic acid, ether, dioxane and potassium

iodide were used as received from Mallinckrodt, Inc. Abso-

lute ethanol, from U.S. Industrial Chemicals Co., was used

to prepare solutions with purified water obtained from a

Barnsted NANOpure system.

Procedure

Following the selected treatments (Table 3-1), the

sheets of S & S 903 filter paper were allowed to air-dry

in a photographic darkroom for 12 hr. Next, 0.25-in dia-

meter filter paper discs obtained with a standard office

paper punch were placed under the cover plate of the bar,

and the plate was screwed down onto the discs. Using a

"Micro/pettor" (Scientific Manufacturing Industries), 5 pL








of blank (1 M KI, 1 M NaOH in 50/50 v/v ethanol/water) or

5 wL of analyte (50 ug/mL PABA in 50/50 v/v ethanol/water)

were spotted onto the paper discs. The bar was then placed

in the sample compartment where the discs were allowed to

dry for 7 min under a flow of dehumidified nitrogen gas.

For each evaluation, 8 or 16 independent measurements were

made for both blank (Xex/Xem, 320/475 nm) and PABA (Xex/Aem,

296/432 nm). The blank excitation and emission wavelengths

were chosen to give the largest signals which are similar

to the substrate background phosphorescence (31). Blank

signals were u40% lower at the wavelengths set for PABA.

For the lot-analysis (Table 3-4) of S & S 903 filter

paper, all lots were treated with a DTPA soak for 24 hr,

rinsed for 3 min in water, and allowed to air-dry for 12 hr

in a photographic darkroom.

For the handsheet evaluation (Table 3-2), handsheets

(basis weight 1903% g/m thickness 0.55% mm) were made

from each source of cellulose pulp by two different methods.

The first handsheet was made from a sample of each pulp

as received from the processing plant. The second hand-

sheet was made from a sample of each pulp that was mechan-

ically beaten for 1 hr.

The sampling procedure for the lot-analysis and hand-

sheet evaluation followed the same sequence as previously

described.








Results and Discussion

Background on Papermaking

In order for one to understand the results obtained

in this study, one should have a fundamental knowledge of

the papermaking process and more specifically, a knowledge

of the types of cellulose used in making filter paper pro-

ducts. This fundamental knowledge will allow a more criti-

cal evaluation of the substrates used in RTP. However, a

detailed description of the papermaking process is beyond

the scope of this chapter, and so interested readers are

urged to consider reading some of the classical books on

pulp and paper technology (35-38). In addition, more

recent texts give updated information on testing procedures

used in paper analysis (39-41).

Generally speaking, purified cellulose can be obtained

from two major sources -- cotton and wood. Wood is com-

prised mainly of cellulose (t55%) and to lesser extents

of hemicelluloses and lignin (the fractions of each depen-

dent upon the type of wood). Cellulose is essential for

papermaking while the hemicelluloses can be beneficial in

making different types of paper; lignin, on the other hand,

is undesirable due to its effects on sheet formation and is

removed during chemical pulping and bleaching. The final

percentage of cellulose found in wood pulps can reach up

to 90% for specific pulping methods. Cotton fibers are

approximately 95% cellulose with minor amounts of waxes








and pectins and very little lignin. Chemical processing

of cotton fibers can give yields of 99+% cellulose.

Two broad classes of woods of commercial value to the

pulp and paper industry are softwoods (pines, spruces, firs

and cedars) and hardwoods (oaks, gums, beeches, birches

and eucalyptuses). The major types of fibers found in

softwood trees are the springwood fibers and the summer-

wood fibers. Paper sheets made with a high percentage of

springwood fibers (flexible fibers with flat surfaces that

pack more closely together) are relatively stronger, more

dense and less porous. The best source of springwood fiber

is central Canadian softwood pulp (%75% springwood). Hard-

woods have much shorter fibers than softwoods and as a

result do not bond well (sheet has low tensile strength),

but they do promote good sheet formation (fewer gaps in

sheet).

The two types of fibers found on most varieties of

cottonseeds are the lint (staple) fibers and the linters.

Lint fibers are used mostly in the textile industry, while

cotton linters are processed into pulp for papermaking or

for chemical derivatization products. Comparisons of the

two cotton fibers show distinct differences. The lint

fibers can grow up to 30 mm in length with cell wall thick-

nesses of up to 3 pm; cotton linters average 4 mm in length

with cell wall thicknesses of up to 10 pm. Lint fibers

with thin cell walls and wide lumens collapse when dried

and thus add strength and density to a sheet of paper.








Cotton linters, on the other hand, with thick cell walls

remain round on drying and impart bulk and porosity to

paper. Because of these characteristics, cotton linters

pulp is used extensively in filter paper applications (42).

Diagnostical Studies on Cellulose Products

This study began with a selection of cellulose pro-

ducts with properties complementing the theories (2) of

hydrogen bonding and/or electrostatic interactions giving

rise to rigid adsorption of organic molecules onto surfaces

of solid supports for which RTP of the compounds could then

be observed. The selection included an extremely "pure"

wood pulp (Cellunier-P), an equally "pure" cotton linters

pulp (Buckeye Cellulose 503), additional cotton linters

pulps (Southern Cellulose 270, 277 and 282-R) with tailored

properties and several "good" commercially available filter

papers (Eaton-Dikeman 613 and 631 and S & S 903).

Phosphorescence background. The first set of experiments

(Table 3-1) was set up to evaluate the phosphorescence

background of cellulose. Contrary to popular belief, the

vast majority of filter paper companies marketing products

in the clinical fields do not add special (luminescent)

chemicals (optical whiteners, sizing agents, etc.) to their

pulps. The desired characteristics of the papers are met

through chemically treating (caustic cooking/extracting and

inorganic bleaching followed by extensive washings with

purified water) and physical manipulation (refining, beat-

ing, etc.) of the pulps (43). Thus, filter papers are








relatively pure with the content of cellulose approaching

100%. However, a reasonable question concerns the magni-

tude of the phosphorescence of cellulose when "bone dry"

(31). Lloyd and Miller (44) have observed that highly puri-

fied cotton does not phosphoresce and that the phosphores-

cence may be attributed to trace contaminants absorbed on

the cotton. Similarly, Atalla and Nagel (45) have observed

that trace amounts of transition metals incorporated into

the crystalline domains of cellulose fibers may be respon-

sible for laser-induced fluorescence in cellulose. More

recently, Timell (46), Huwyler and coworkers (47,48),

Delmer and coworkers (49), Waterkeyn (50) and others (51-

55) have pointed out that filter paper cannot be "pure"

cellulose because cotton fibers contain trace amounts of

hemicelluloses and lignin. These hemicellulosic fractions

(mainly B-1,3-glucans) increase at the onset of secondary

wall formation and contain species that luminesce.

The various soaking treatments (results in Table 3-1)

were structured to test the presence of trace metals and

hemicelluloses and/or lignin in cellulose pulp. The

chelating agent DTPA was used in an attempt to remove trace

amounts of transition metals that were possibly involved

in contributing to the phosphorescence background in the

filter paper. While the background phosphorescence of the

blank was reduced slightly, the analyte signal improved

significantly. This treatment gave no improvement for

blank levels over past procedures (33). However, owing


























- "4 CD CD ( l -4 r 0
+ +








1 ,- ,. C Cf (X Cl
S+I























oo oo t O N :t (q
't -I t -1 C c4 i-4


M
O


CY)

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C7





4J



0
4J



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c+




'-4


ot
ct




















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00

4

0





























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i-H

















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*H










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:F C ) C) + +
0 C) 0)













)







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d)






4-1
i-
a,

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to possible interactions of DTPA's multi-charged structure

with PABA and to the gaps in the paper structure being

filled (see Figure 8), this treatment showed a greater

effect on the analyte phosphorescence signal. Unfortu-

nately, the soaking with DTPA gave no clues as to whether

trace metals were involved in the phosphorescence back-

ground of cellulose.

After Millson's work (29) an Eimac lamp (250 W-xenon

arc) and sunlight were used to try to bleach out "impuri-

ties" in the filter paper. This prolonged illumination

was actually detrimental to the paper as analyte signals

decreased due to possible disruption of the surface

structure.

The remaining treatments were used to evaluate the

presence of extractable hemicelluloses (and/or lignin) in

cellulose pulp. While none of the treatments unequivocally

confirmed the presence of such materials, the dioxane soak-

ing indicated (by a large decrease in background phosphores-

cence) that an extractable hemicellulosic (and/or lignin)

fraction may have been present. Periodic acid oxidation

(53) almost destroyed surface hydroxyl groups (to form

aldehyde groups), and no net analyte signal or background

was observed. Thus, while hemicelluloses (and/or lignin)

appear to be responsible for the background phosphorescence,

complete removal of these groups apparently disrupts the

surface structure, and analyte phosphorescence does not

occur (52).















S-..Ho H H U "o "0 0" (a)

H-0-H H.,H H'P H0 O
SH H. H H


H ,0. H H H H
H 0



O. ,O. HO..T H: HO
." 0. "oH, ".H (b)

H H. H N H. ,H H. H H.
o o o o 0o

0, o0.. 0. 0 0
.H H .H .H 0H .H .H H .H (C)
0' 0 0 '






HOOC-CH2 CH2-COO-
\+ /C COO (d)
NIN-CH2-CH2-NH-CH2-CH2-NH\ (
-OOC-CH2 CHz-COO- CH2-COOH


Figure 8. Simulation of hydrogen bonding between
two cellulose molecules:

a. Loosely through water molecules
b. Tightly through a monolayer of
water molecules
c. Directly
d. Loosely through the transposition
of DTPA into (a).








Fibrillation study. In the fibrillation study (Table 3-2),

two handsheets (one more highly fibrillated than the other)

were made from unbeaten and beaten pulps. Beating cellulose

pulps over a period of time decreases the average fiber

length and increases the average exposed surface area.

Therefore, with more hydroxyl groups exposed on the sur-

face, more complete adsorption of organic molecules on the

support material takes place and an enhancement in phos-

phorescence should be seen. But, at the same time, fibril-

lation increases the exposure of the hemicellulosic material

located within the inner matrix of the cellulose fibers.

As a result, a larger phosphorescence background should be

seen. In fact, the results (Table 3-2) obtained for

Cellunier-P, Buckeye Cellulose 503, Southern Cellulose 270,

277 and 282-R appear to reflect on these generalizations.

While the degrees of fibrillation have not been confirmed

for Cellunier-P and Buckeye Cellulose 503, one would expect

Buckeye Cellulose 503 cellulose pulp to be more fibrillated

than the Cellunier-P pulp. The trend for the three Southern

Cellulose pulps appear to follow in order with grade 270

being less fibrillated than grade 277 which in turn is less

fibrillated than grade 282-R.

Filter paper comparison. Eaton-Dikeman filter papers-613

and 631 were compared to S & S 903 filter paper (see Table

3-3) to determine whether paper porosity affects the inter-

action of the cellulose surface groups with analyte mole-

cules. An optimal filter paper should allow the bulk of
























0 0 0 z- '0
tq t *t ui rn

















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f -o in o







CD LO t) L) cD

-1i1




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rn M
























o LOn Ln C
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C,,









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or










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rt
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to ) *)


U 4 ) CCt


C H C- C3





C i Z l
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41o o >
t a +j > u





* to j *
. -H CC *,S 0





P3 C ccM
Ci 0 m C O

o _l r C o v







w L am z,
CC CCV


to C '0
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the analyte to remain upon the surface to accommodate

effective adsorption and/or interaction. Thus, "slow"

filter papers are the best choices. While the Eaton-Dike-

man 613 and 631 papers gave larger analyte signals than

the untreated S & S 903 paper, the DTPA-treated S & S 903

paper gave substantially larger signals. The discussion of

DTPA filling in the gaps of S & S 903 now becomes apparent

(by the larger signals) as the analyte molecules are

"trapped" in the DTPA-cellulose matrix (56).

Filter-paper lots comparison. The various lots (see Table

3-4) of S & S 903 filter paper are consistent in quality

for use in RTP applications. On a statistical basis (57)

(Duncan's multiple range procedure with a=0.01, using a

completely randomized design with 105 degrees of freedom

and mean square values of 0.058 and 130 for blank and ana-

lyte signals, respectively), the W94 (1980) and W93 lots

are significantly different (for analyte signals) from the

other lots of paper. For blank signals, the W94 (1980) and

W12 lots are significantly different from the other lots

of paper.

Conclusion

In view of the results obtained in this study, it is

clear that cellulose-based support materials cover a wide

range of products; nevertheless, for RTP applications, it

is practical to assume that the difference in performance

between the poorest paper and the best paper is consider-

ably less than an order of magnitude. Thus, researchers


























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o 0 c0 C) 0m 0D








t^ roMb b


cc,





co C) m C1 CD
to N N


ca




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' --








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u a
S-4
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oa
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a- C)>





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evaluating RTP for analytical studies should select support

materials giving the highest signal-to-noise ratio. If one

chooses to use filter papers (out of convenience of avail-

ability), then a pre-treatment is recommended to enhance

its adsorption characteristics. If indeed the background

phosphorescence can not be reduced in cellulose-based

products (and it seems unlikely it can be significantly

reduced), time-resolved phosphorimetry may effectively

correct for such interference. As a matter of choice, all

subsequent studies in RTP were made using DTPA-treated

S & S 903 filter paper as the substrate material.

Note

The author is indebted to Mr. Whitten Bell (Buckeye
Cellulose), to Mr. Fred Mathis (Southern Cellulose) and
to Mr. Harvey Wilson (ITT Rayonier) for samples of cotton
linters and wood pulps and for technical information and
advice. Gratitude is extended to Mr. Steve Ritchie and
to Mr. Bob Edwards (ITT Rayonier, Fernandina Beach, FL)
for samples of wood pulps and for the informative tour of
the pulp mill. Special thanks is given to Mr. Bradd Levine
(Eaton-Dikeman) and to Mr. Lothar Jeschke (Schleicher &
Schuell) for samples of filter paper. The author is also
indebted to Mr. John Baumgardner (Eaton-Dikeman) for making
the handsheets of paper, to Dr. Rajai Atalla (Institute
of Paper Chemistry, Appleton, WI), and especially to Dr.
Ralph Berni (USDA-Southern Regional Research Center, New
Orleans, LA) for paper analysis and for helpful suggestions.
An additional note of appreciation is extended to all
others in the pulp and paper industry for their cooperation
on this project.













CHAPTER 4
BIOCHEMICAL AND DRUG ANALYSIS BY ROOM
TEMPERATURE PHOSPHORESCENCE

Introduction

Recent studies in RTP have shown that a wide range of

organic molecules exhibit phosphorescence at room temper-

ature when adsorbed on a variety of suitable support mater-

ials (2). Several studies have led to developments whereby

RTP can now be applied to solving chemical problems and can

be used to provide additional information on the phenomenon

of phosphorescence of organic molecules. Compounds exhib-

iting RTP are usually grouped in classes of ionic or polar

organic molecules; under special circumstances, certain

nonpolar organic molecules (polynuclear aromatic hydro-

carbons) exhibit RTP. The largest group of compounds

exhibiting RTP is the azines, which includes quinolines,

isoquinolines, quinoxalines, quinazolines, indoles, inda-

zoles, benztriazoles, benzimidazoles and related benzyl-

derivatives of these classes. Other groups of compounds

exhibiting RTP include purines, the related xanthines,

benzoic acid derivatives and related species.

While documentation is available for those compounds

exhibiting RTP in the aforementioned groups, there is much

overlap in the area and very little mention is given to








those compounds which do not phosphoresce or which phos-

phoresce weakly and therefore can not be used in analytical

studies. Furthermore, most documentation describing RTP

as a useful analytical technique goes unsupported in the

literature. Only in remote cases does one find the appli-

cation of RTP to real-sample analysis (58-60).

In this project, groups of molecules were studied to

find trends in phosphorescence among related compounds.

When a group of compounds was found to exhibit RTP, then

appropriate real samples were selected and analyzed by RTP.

It was obvious that not all groups of compounds could be

studied, so selections were made from the azine group, the

purine group and the benzoic acid derivative group.

Although care was taken to select only those compounds not

previously studied, the selection process was a difficult

task; however, overlapping studies were kept to a minimum.

Biochemicals/Drugs

The first group of compounds selected to be studied

was the purine/xanthine group. This group was selected

for interests in future studies in evaluating the use of

RTP in therapeutic drug monitoring (Appendix 2). The

major compounds in this group include caffeine, theobromine

and theophylline. Caffeine (1,3,7-trimethylxanthine) is a

central stimulant and is found in large quantities in

coffee, tea and colas. Theobromine (3,7-dimethylxanthine)

is used therapeutically as a vasodilator and is principally

found in hot chocolate. Theophylline (1,3-dimethylxanthine)








is found in tea and is used therapeutically to treat

patients suffering from apnea, asthma and various stages

of chronic obstructive pulmonary disease. A summary of

these and related compounds and metabolites can be found in

Figure 9. The rest of the group will be covered in another

section.

The indoles were the second group of compounds studied.

Along with several biologically-important compounds, this

group includes 5-hydroxyindoleacetic acid, 5- hydroxytryp-

tophan, indole, indoleacetic acid, indole carboxylic acid,

indomethacin, melatonin, serotonin and tryptophan. Sero-

tonin serves as a chemical messenger; the presence of high

levels of this compound in serum or urine indicates active

stages of a malignant carcinoid tumor. Compounds related

to serotonin are 5-hydroxytryptophan, a precursor, and

5-hydroxyindoleacetic acid, a degradation product. Indole

is used in the perfume industry. Indoleacetic acid is a

plant growth regulator. Indomethacin, because of its

anti-inflammatory and analgesic effects, is useful for

treating certain types of arthritis. Melatonin is a skin

pigment factor. Tryptophan is an essential amino acid

because it is not synthesized by the human body. Figures

10 and 11 illustrate the structural similarity between

these compounds.

The third group studied contains a benzene ring as

the central structural element. Included in this group are

a variety of compounds shown in Figures 12-16. Benzoic










o CH3
H 3C N


CH,
Caffeine
1,3,7-trimethylxanthine


3-methylxanthine

0
II H
HO C .. --N

CH,
Theophylline
I,3-dimethylxanthine


0
I H


HCO N

1-methyluric acid


Theobromine
3,7-dimethylxanthine
0
II H 0


O H

Uric acid


0 N



H
Xanthine


Figure 9. Chemical structures of several xanthines and
uric acids.




43





Z
z
I Z' Z

a, 5 o |
O I :

0 I0



o o
I r




I
0 o
I a
2:
0 O


\/ 0

o 7=\ o -


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-O -
I-o o

1 0
-r- c













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-J
_J
x
X
O 0
o m
.0 0

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0
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0

z
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I



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FI -
o


I w
-r


L-

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\/ 0








acid, methylparaben, propylparaben and vanillin function

as pharmaceutic aids. Two compounds, p-aminobenzoic acid

and p-aminohippuric acid, serve as diagnostic aids in

studying pancreatic and renal function, respectively. p-

Aminobenzoic acid is also commonly used as a sunscreen

agent. p-Acetamidobenzoic acid is sometimes used as an

antidepressant drug. p-Hydroxybenzoic acid functions as a

reactive intermediate for organic dye synthesis. Sulfo-

salicylic acid is used as an analytical reagent for the

determination of protein (albumin) in urine. Acetylsali-

cylic acid and salicylamide, often used in combination, are

common analgesics. Sulfacetamide functions as an anti-

microbial agent while p-aminosalicylic acid is an anti-

tubercular agent. Salicylic acid, a keratolytic, is an

active ingredient in medicated cleansers used for the

treatment of acne. Probenecid functions as an uricosuric.

Lidocaine and procaine are common local anesthetics.

A group of miscellaneous compounds was also studied.

Chloroquine and primaquine, two antimalarial drugs, and

naphazoline, a vasoconstrictor, are shown in Figure 17.

Propranolol and dibucaine, a heart drug and a local anes-

thetic, respectively, are shown in Figure 18. A group of

antihypertensive agents (chlorthiazide, diazoxide, hydro-

chlorthiazide, reserpine and trichlormethiazide) and an

antibacterial agent (tetracycline) are shown in Figures

19 and 20.

































cC
V)




cn





Cd

*H










u
0
N





O l
C)



U >



0H
4-i

iN



( 0
au













0M

-I,











r
0

















I
0
I 0
0 o
0


0


0

N
Z
LJ
0



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LU

1
a-


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0
0
U







2:
z





I
0D



"T


O
0
N
z



OL
x
0


I
I







0

I

O
0
z


Q
0
S









COOCH3


OH


METHYLPARABEN

COOCH2CH2CH3


OH


PROPYLPARABEN


COOH


NH2


p-AMINOBENZOIC


Figure 13. Chemical structures of several benzoic
acid-related compounds.


ACID





































0







m

u
U)
q-







U)
Sui















tl





uC
c-)











r-( *,

U)
C) <






u)
4-1








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CL,


















0o_
80


I
0 Nc
0 -
C-)


rO
7-)
I
0

I
o7-


I
0
0
0
0
IO

8>=
"<


8-T
0-


0
0o



I









(CH3CH2CH2)2NSO.- COOH


PROBENECID


CHO

-I
N OCH3
OH


VANILLIN


Chemical structures of a uricosuric
(top) and a pharmaceutic aid(bottom).


Figure 15.








=H3
-NHCOCH2 N(C2H5 2
:H3



LIDOCAINE


H2N- COOCH2 CH2(CH )2



PROCAINE


Chemical structures of two common
local anesthetics.


Figure 16.










HNCH(CH2)3N(C2H)2
CH3

CHLOROQUINE


INH



NAPHAZOLINE

CH3
HNCH(CH2)3NH2

CH30j 0

PRIMAQUINE

Figure 17. Chemical structures of two anti-
malarial drugs(top and bottom) and
a vasoconstrictor(middle).







OH
OCH2CHCH2NHCH(C H)2





PROPRANOLOL


0 (CH,)3 CH,


CONHCHC H2 N(C2H 52


DIBUCAINE


Chemical structures of a heart drug
(top) and a local anesthetic(bottom).


Figure 18.









LJ
0

N

I n


IA 0
rN 0 0




o o


H




--W-r- I-r *
I I d>







z Ao
I "T
C) < -


oJ
g0 ^

C)T
I "










CH H ~
H H OCH3
CH COO1 OOC / OCH3
OCH OCH


RESERPINE








HC/CH N(CH
HO ,CHi N(CH3)2
OH

1 n VC coNH2
OH o OHOHO

TETRACYCLINE

Figure 20. Chemical structures of an antihyper-
tensive drug(top) and an antibacterial
agent (bottom).












HO0


3,4-Dihydroxymandelic acid
Dopa
Dopamine
Epinephrine
Homogentisic acid
Homovanillic acid
Isoproterenol
Metonephrine
3-Methoxytyramine
Norepinephrine
Tyrosine
Vanillylmandelic acid
Normetonephrine


X, X2
CHOHCOOH OH
CH2CH(N)COOH OH
CHH2NH2 OH
CHOHCH2NHCH, OH
CH2COOH OH
CH2COOH OCH3
CHOHCH2NHCH(CH)2 OH
CHOHCH2NHCH3 OCH3
CH2CH2NH2 OCH3-
CHOHCH2NH2 OH
CH2CHNH-,COOH
CHOHCOOH OCH3
CHOHCH2NH2 OCH3


Figure 21. Chemical structures of a series of catechol-
related compounds.















Acetophenazine

Chlorpromazine

Fluphenazine

Perphenazine

Prochlorperazine

Promazine

Promethazine

Thiopropazate

Thioridazine

Trifluoperazine

Triflupromazine

Trimeprazine


XI
(CH,)2CH2-N N-CH2CH2OH

(CH2)2CH2N(CH,)z

(CH,)2CHz,-rN -CHCH2OH

(CH) 2CH2-N -CHCH2OH

(CH2),CH-N_-N -CH3

(CH) C H2 N (C H,),
CH3
CHzCHN (CH3)2

(CH,)2 CH2-N rN-CH2CH 2OCCH,
CH,
CH,CHTO

(CH,),CH,-rNN-CH,

(CH2)2CHN (CH,),
CH,
CHC HCH CHN (CH,),


X2
COACH3

CI

CF,

CI

CI


CI

SCH3

CF,

CF,


Figure 22. Chemical structures of a series of
phenothiazines.








Two other groups of compounds (Figures 21 and 22),

studied quite-extensively by LTP (61,62), were studied

here at room temperature. However, with just a few excep-

tions, these compounds did not exhibit RTP under the

specific test conditions used in these studies. These

results will be discussed in a later section.

Pharmaceutical Formulations

Upon completion of the RTP studies with the diverse

selection of compounds, optimum conditions were established

for which each compound or groups of compounds could be

analyzed by RTP (see Table 4-3). Next, samples of compounds

were considered for real-sample applications. For practical

considerations, several drugs and their respective pharma-

ceutical formulations or over-the-counter preparations were

selected as working systems.

Oral administration of drug substances, via solid

dosage forms (e.g., tablets and capsules), is the most

frequent route used in distributing drugs in biological

systems. Large-scale production of such substances requires

the presence of a variety of other materials to complement

the active ingredient. Other additives may also be used

in formulations to enhance physical appearance, improve

stability or aid in drug distribution. These supposedly

inert additives must be considered when assay procedures

are established for quality control protocol of manufac-

tured formulations.








In recent years, generic brands of drugs have attained

widespread use in response to consumer groups' participation

in regulatory agencies (63). Generic brands of drugs offer

practitioners a wide selection of products for which thera-

peutic dosage regimens can be determined for individual

patients. Specific formulations are used to achieve the

desired pharmacodynamic/pharmacokinetic responses in

patients. Because generic manufacturers often use a

diverse group of diluents, binders, lubricants, coloring

and flavoring agents, preservatives, etc., in their formu-

lations, a specific procedure is needed for the quantitation

of active ingredients in the formulations.

Current U.S.P. procedures (64) for analyzing a variety

of drugs substances in pharmaceutical formulations are

rather cumbersome and as a result, many pharmaceutical

manufacturers are evaluating the use of high performance

liquid chromatography (HPLC) and luminescence methods of

analysis for quality control procedures.

In this section, RTP is used for the analysis of a

variety of drugs in pharmaceutical formulations and over-

the-counter preparations (see Table 4-2). The selection of

formulations and preparations to be analyzed was made by

consulting the American Drug Index (65). Under the various

generic names of drugs, a wide range of preparations was

selected to represent different matrices within which the

drugs were contained. The selections were made to illu-

strate the selectivity advantage of RTP as a method of

analysis of complex mixtures.








Experimental

Reagents and Materials

The analytical reagents used in these studies were

previously described in Chapter 3. All other chemicals

are listed in Table 4-1. Commercial preparations used in

these studies were purchased at a local hospital pharmacy

(Shands Teaching Hospital and Clinics, Gainesville, FL)

and are listed in Table 4-2.

Sample Preparation

Biochemicals/drugs. For the RTP studies of the compounds

listed in Table 4-1, standard stock solutions (200-400

pg/mL) were prepared by dissolving accurately weighed por-

tions of the compounds in an ethanolic solution (50/50 v/v

ethanol/water). Next, the samples were measured in four

steps to determine the optimum solvent system to use for

RTP studies. The four basic solvent systems were (1)

ethanolic (2) ethanolic + 1 M KI (3) ethanolic + 1 M NaOH

(4) ethanolic + both 1 M KI and 1 M NaOH. The addition of

1 M KI served as a heavy atom perturber. The 1 M NaOH

addition served to "ionize" the molecules to allow for a

more effective interaction (electrostatic or hydrogen bond-

ing) of the molecules with the substrate material. An

optimal solvent system was selected by comparing phosphores-

cence intensities (using a 100 pg/mL test solution) obtained

using the various solvent systems. Where applicable, by

using solubility data (66), the amount of ethanol in each

optimum solvent system was reduced to minimize the effects








Table 4-1. Sources of Chemicals Used in RTP Studies


Compound Source

p-Acetamidobenzoic acid (A)
Acetaminophen (A)
Acetophenazine (B)
Acetylsalicylic acid (A)
Allopurinol (A)
Alloxan (A)
p-Aminobenzoic acid (A)
p-Aminohippuric acid (A)
Aminophylline (A)
Aminosalicylic acid (A)
Amitriptyline (B)
Anisindione (B)
Atropine (A)
Azathioprine (A)
Benzoic acid (A)
Caffeine (A)
Calmagite (A)
Chloramphenicol (C)
6-Chloropurine (A)
Chloroquine (A)
8-Chlorotheophylline (A)
Chlorpheniramine (B)
Chlorphentermine (C)
Chlorpromazine (D)
Danthron (E)
Dexbrompheniramine (B)
Dexchlorpheniramine (B)
2,6-Diaminopurine (A)
Diazoxide (B)
Dibucaine (F)
Dihydroxymandelic acid (A)
1,3-Dimethyluric acid (A)








Table 4-1-continued.


Compound Source

1,7-Dimethylxanthine (A)
Diphenhydramine (A)
Dopa (A)
Dopamine (A)
Dyphylline (A)
Ephedrine (A)
Epinephrine (E)
Ethosuximide (C)
Fluphenazine (B)
Folic acid (A)
Folinic acid (A)
Furosemide (G)
Gitalin (B)
Gossypol (A)
Griseofulvin (B)
Guaiacol glyceryl ether (A)
Homogentisic acid (A)
Homovanillic acid (A)
Hydrochlorthiazide (F)
p-Hydroxybenzoic acid (A)
5-Hydroxyindoleacetic acid (A)
5-Hydroxytryptophan (A)
B-Hydroxyethyltheophylline (A)
Indole (A)
Indole carboxylic acid (A)
Indoleacetic acid (A)
Indomethacin (A)
Inosine (A)
Inosinic acid (A)
Isoproterenol (A)
Lidocaine (A)
Mefenamic acid (C)








Table 4-1-continued.


Compound Source

Melatonin (A)
6-Mercaptopurine (A)
Metanephrine (A)
Methotrexate (A)
3-Methoxytyramine (A)
Methsuximide (C)
Methylparaben (A)
1-Methyluric acid (A)
3-Methyluric acid CA)
1-Methylxanthine (A)
3-Methylxanthine (A)
7-Methylxanthine (A)
Naphazoline (F)
Nicotinic acid (A)
Normetanephrine (A)
Orphenadrine (E)
Oxtriphylline (C)
Oxymetazoline (B)
Perphenazine (B)
Phenacetin (B)
Phenazophridine (C)
Phenylalanine (A)
Phenylephrine (A)
Phenylpropanolamine (A)
Phenytoin (C)
Phytic acid (A)
Prazosin (H)
Primaquine (A)
Primidone (I)
Probenecid (A)
Procaine (A)
Prochlorperazine (D)








Table 4-1-continued.


Compound Source

Promazine (J)
Promethazine (J)
Propranolol (I)
Propylparaben (A)
Purine (A)
Pyrilamine (A)
Quinidine (A)
Quinine (A)
Reserpine (F)
Riboflavin (A)
Saccharin (A)
Salicylamide (A)
Salicylic acid (A)
Scopolamine (A)
Serotonin (A)
Sulfacetamide (B)
Sulfosalicylic acid (A)
Tetracycline (A)
Theobromine (A)
Theophylline (A)
Thiopropazate (K)
Thioridazine (L)
Thyroxine (A)
Tolnaftate (B)
Trichloromethiazide (B)
Trifluoperazine (D)
Triflupromazine (M)
Trihexyphenidyl (B)
Trimeprazine (D)
1,3,7-Trimethyluric acid (A)
Tripelennamine (A)
Tryptophan (A)








Table 4-1-continued.


Compound

Tryosine
Uracil
Uric acid
Vanillylmandelic acid
Vanillin
Xanthine


Source

(A)
(A)
(A)
(A)
(A)
(A)


(A) Sigma
(B) Schering
(C) Warner-Lambert
(D) Smith Kline French
(E) Riker
(F) Ciba-Geigy
(G) United States Pharmacopeia
(H) Pfizer
(I) Ayerst
(J) Wyeth
(K) Searle
(L) Sandoz
(M) Squibb





of ethanol on the cellulose substrate material (53). Table

4-3 lists those compounds (used in this study) that phos-

phoresce and the solvent systems used to observe the maxi-

mum phosphorescence intensities.

Pharmaceutical formulations. In the analysis of the

commercial preparations listed in Table 4-2, samples were

prepared for assay by dissolution/dilution in/with an







appropriate solvent system. Standard stock solutions were

prepared by dissolving accurately weighed portions of stan-

dards in an appropriate solvent system. Standard solutions

were prepared daily by mixing appropriate volumes of the

stock solution with the solvent system. All solutions,

when not being analyzed, were stored under suggested condi-

tions to insure stability of the reagents. For analysis of

representative samples, 16 or 20 tablets (contents of

capsules) were weighed and powdered with a mortar and

pestle, and 4 portions (equivalent to 100 pg/mL of active

ingredient in a total volume of 5 or 10 mL) were dissolved

in the appropriate solvent system. This procedure was

repeated for each solid sample. For the analysis of liquid

preparations, appropriate volumes (to give final concentra-

tions of 100 pg/mL of active ingredient) of the samples

were diluted with the appropriate solvent system. Four

different test solutions were prepared for each pharmaceu-

tical formulation or commercial preparation; each test

solution contained 100 vg/mL of active ingredient.

Procedure

All bar-RTP measurements were performed as previously

described in Chapter 2. Quantitation was achieved by

comparing relative phosphorescence intensities of samples

to those of standards. For the analyses, linear ranges

were established and linear regression analysis was performed

on each set of data.
























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Table 4-3. Optimum Solvent Systems for
Compounds Exhibiting RTP


Compound Solvent System

p-Acetamidobenzoic acid (1)
Acetylsalicylic acid (5)
p-Aminobenzoic acid (1)
p-Aminohippuric acid (1)
Aminophylline (2)
Aminosalicylic acid (5)
Azathioprine (4)
Benzoic acid (4)*
Caffeine (2)
6-Chloropurine (4)*
Chloroquine (1)
2,6-Diaminopurine (4)
Diazoxide (4)*
Dibucaine (1)
1,7-Dimethylxanthine (2)
Dyphylline (2)
Folinic acid (3)
Homogentisic acid (4)*
Hydrochlorthiazide (4)*
p-Hydroxybenzoic acid (1)
B-Hydroxyethyltheophylline (2)
5-Hydroxyindoleacetic acid (3)
5-Hydroxytryptophan (2)
Indole (3)
Indole carboxylic acid (3)
Indoleacetic acid (3)
Indomethacin (3)*
Melatonin (3)
6-Mercaptopurine (4)*
Methylparaben (3)
1-Methylxanthine (2)








Table 4-3-continued.


Compound

3-Methylxanthine
7-Methylxanthine
Naphazoline
Oxtriphylline
Prazosin
Probenecid
Procaine
Propranolol
Propylparaben
Reserpine
Salicylamide
Salicylic acid
Serotonin
Sulfacetamide
Sulfosalicylic acid
Tetracycline
Theobromine
Theophylline
Trichloromethiazide
Tryptophan
Tyrosine
Vanillin
Xanthine


Solvent System

(2)
(2)
(2)
(2)
(3)*
(4)
(3)
(3)@
(3)
(4)
(5)
(5)
(3)


(5)
(4)
(2)
(2)
(4)*
(2)
(2)
(4)
(2)


(1) water
(2) water + 1 M KI
(3) ethanolic + 1 M KI
(4) ethanolic + both 1 M KI and 1 M NaOH
(5) 2/98 v/v ethanol/water + both 1 M KI and 1 M NaOH
* Phosphorescence observed but not analytically useful;
i.e., signal levels <10 x blank level.
@ 2 M KI







Results and Discussion

Biochemical/Drug Survey

Xanthines. For the biochemical/drug survey, the two major

groups studied were the xanthine group and the benzene-

based group. Selected compounds in the xanthine group are

summarized in Figure 23. For the series of compounds, the

phosphorescence intensities follow the trend: dyphylline >

caffeine > theobromine > 1,7-dimethylxanthine > theophyl-

line 7-methylxanthine > 3-methylxanthine > l-methylxan-

thine s xanthine. In general, when a substituent other

than hydrogen occupies position X4, no phosphorescence is

observed. For the uric acid series, where there is a

double-bonded oxygen atom at position X4, no double bond

(dashed line in Figure 23) exists and a hydrogen atom

(dashed 'H') is present. B-Hydroxyethyltheophylline, a

theophylline derivative with a hydroxyethyl group at posi-

tion X3, exhibits the most intense RTP of the xanthine

series. Aminophylline and oxtriphylline, ethylene-diamine

and choline salts of theophylline, respectively, exhibit

RTP characteristic of the theophylline base.

For the xanthine structure, group substitution affects

phosphorescence intensity. Group substitution appears to

be most effective at position X3; substitution at position

X2 appears to be more effective than at position X1. Thus,

a methyl group at positions X1, X2 or X3 (X1 < X2 < X3 in

terms of effectiveness) gives an enhancement in phosphores-

cence when compared to the phosphorescence obtained when









0



x2

Caffeine
8-Chlorotheophylline
1,3-Dimethyluric acid
1,7- Dimethylxanthine

Dyphylline
1-Methyluric acid
3-Methyluric acid
I-Methylxanthine
3-Methylxanthine
7-Methylxanthine
Theobromine
Theophylline
1,3,7-Trimethyluric acid
Uric acid
Xanthine


Xi X2 X3
CH, CH, CH,


CH,
CH,
CH,
CH,
CH,
H
CH,
H
H
H
CH,
CH,
H
H


CH,
CH,
H
CH,
H
CH3
H
CH,
H
CH,
CH,
CH,
H
H


X4


H Cl
H =0
CH, -
CHUHHPH -
H =0
H =0
H -
H

CH -


Figure 23. Chemical structures of a series of
compounds in the xanthine group. A
hydrogen atom occupies X4 unless other-
wise noted.








a hydrogen atom occupies either of the positions. At this

time, there are no plausible explanations for the varied

effects of methyl group substitution at X1, X2 or X3 on

the observed RTP intensities. In theory, the presence of

methyl groups (electron-donating groups) in lieu of hydro-

gen atoms at these positions (X1, X2' X3) may contribute to

enhanced resonance stabilization of the molecules. This

resonance stabilization may ultimately affect the stability

of the triplet states of the molecules which in turn could

explain the observed trends in phosphorescence intensity.

Additional molecular stabilization appears to occur

when hydroxyl groups are present on the xanthine structure.

This added stabilization may be attributed to the inter-

actions (electrostatic and hydrogen bonding) of the hydroxyl

groups on the molecules with the hydroxyl groups on the

surface of the cellulose substrate material. Then, as would

be expected, B-hydroxyethyltheophylline and dyphylline

exhibit more intense RTP than caffeine. However, one would

expect dyphylline (with 2 hydroxyl groups) to exhibit more

intense RTP than 3-hydroxyethylthcophylline. In this case,

the reverse is true. Thus, in addition to the presence of

methyl and hydroxyl groups on the xanthine structure, there

are other unknown factors affecting the observed trends in

phosphorescence intensity.








With 8-chlorotheophylline and the uric acid series,

no RTP is observed. The presence of electron-withdrawing

species (Cl and 0) at position X4 may contribute to

resonance destabilization of the molecules which in turn

could destabilize the triplet states of the molecules so

that no RTP is observed. No other xanthine derivatives

with electron-withdrawing groups (at positions other than

X4) were available to further test the destabilization

theory.

While the observed trends of phosphorescence intensity

within the xanthine groups are definitive, there is only a

20-fold difference in phosphorescence intensity in going

from the weakest phosphor (xanthine) to the strongest

phosphor (B-hydroxyethyltheophylline). Overall, this group

of compounds represents an interesting series for persons

wishing to study phosphorescence mechanisms. In contrast

to observed differences in phosphorescence intensity, the

xanthines give essentially the same RTP spectrum. A

generalized RTP spectrum of the xanthine group (giving

nominal wavelengths for the series) is represented in

Figure 24.

Indoles. The differences in phosphorescence intensity

within the indole group are more pronounced than within the

xanthine group. The phosphorescence characteristics of the

indole group are apparently determined by the relative

solubility of the compounds in the solvent system. The

first group (5-hydroxytryptophan, serotonin, and tryptophan)
























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is more soluble in water than the second group (5-hydroxy-

indoleacetic acid, indole, indoleacetic acid, indole

carboxylic acid, indomethacin and melatonin). The enhanced

solvation (by water) of the molecules in the first group

could allow the molecules to interact (via electrostatic

interactions or hydrogen bonding) more effectively with the

hydroxyl groups on the surface of the cellulose substrate

material. These solvation effects could enhance the

stability of the molecules adsorbed on the solid substrate

whereby the triplet states become stabilized and large

phosphoresence intensities are observed. On the other hand,

the compounds in the second group are poorly solvated by

water and the molecules can not participate in strong inter-

actions with the cellulose substrate materials. The mole-

cules of the second group exhibit RTP but do so at much

lower levels (%25 times lower) than the first group.

While the two groups drastically vary in exhibiting

RTP, there is little variation in phosphorescence intensity

within each group. Despite the structural similarity

between the two groups, there are differences in the RTP

spectra. Figures 25 and 26 are RTP spectra obtained from

compounds in the first and second groups, respectively.

Nominal wavelengths are shown on each spectrum because

there is a lack of spectral resolution within each group.

Benzoic acid derivatives. For this diverse group of

compounds, several generalizations can be made. Using

benzoic acid as the starting material, the addition of























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certain groups to the benzene ring ortho and/or para to

the carboxyl group greatly enhances the phosphorescence

of the resultant compound. Group substitution meta to the

carboxyl group on the benzene ring yields a species that

exhibits RTP but the phosphorescence intensity is much

lower than that of a compound with ortho/para substitution.

Groups that are effective in providing resonance stabili-

zation of the benzene ring can accommodate favorable inter-

actions (electrostatic or hydrogen bonding) of the mole-

cule with the cellulose substrate material. Appropriate

groups include acetate, aldehyde, amide, hydroxyl and

methoxy groups and amino and carboxy functions. The groups

provide resonance stabilization of the benzene ring as well

as contain atoms which can participate in hydrogen bonding

or electrostatic interactions.

The most intense RTP is observed when an amino group

is para to carboxy functions or when a hydroxyl group is

ortho to an amino function or to a carboxy function. For

the compounds studied in the group, trends in phosphores-

cence intensities can be expressed by the following

relationships: p-aminobenzoic acid salicylamide >

salicylic acid = sulfosalicylic acid > acetylsalicylic

acid = procaine > p-aminosalicylic acid > sulfacetamide -

vanillin > p-hydroxybenzoic acid > methylparaben p-amino-

hippuric acid > p-acetamidobenzoic acid s propylparaben >

probenecid. Lidocaine and benzoic acid do not exhibit

RTP under the conditions used in these studies.








The difference in phosphorescence intensity between

probenecid (weakest phosphor) and p-aminobenzoic acid

(strongest phosphor) is a factor of 1v00. The phosphores-

cence intensity of p-aminobenzoic acid is %4 times that of

sulfacetamide. RTP spectra obtained for some of the

compounds in the benzoic acid derivative group are illus-

trated in Figures 27-34. In a mixture of strong phosphors,

e.g., p-aminobenzoic acid and salicylamide, it is not

possible to spectrally resolve the two components even

though the excitation and emission maxima of the compounds

differed. In general, it is difficult to resolve any one

component in a multicomponent mixture of compounds in the

benzoic acid group.

Others. The RTP studies of the remaining compounds yielded

variable results. For compounds of similar chemical struc-

ture, it was found that compounds forming colored solutions

upon dissolution in an appropriate solvent do not exhibit

RTP while their respective chemical analogs, forming color-

less solutions upon dissolution, do exhibit RTP. Two

specific sets of compounds exemplify this point. In the

first set, chloroquine exhibits intense RTP while prima-

quine, forming an orange-yellow-colored solution upon

dissolution, does not phosphoresce. Folinic acid exhibits

RTP; folic acid and methotrexate form yellow-colored solu-

tions upon dissolution and do not phosphoresce. Folic acid,

however, has been found to exhibit RTP when adsorbed on

sodium acetate (67). The fact that folic acid does not








exhibit RTP when adsorbed on a cellulose-based support

material further complicates the understanding of the

phenomenon of RTP. Does the presence of color affect the

observation of RTP? This is a complex question that can

not be answered by the studies reported here. However,

colored materials generally absorb light in the visible

region of the light spectrum and this could be a reason for

the lack of RTP. One other example of the presence of color

and a lack of RTP involved the study of herbicides in

Appendix 2. Most of the herbicides yielded yellow-colored

solutions upon dissolution and did not exhibit RTP.

Several miscellaneous compounds were studied by RTP

but no generalizations can be made regarding chemical

structure and resulting phosphorescence intensities. As a

matter of documentation, selected RTP spectra of various

compounds are shown in Figures 35-42.

Two groups of compounds studied here gave results

unlike other compounds with similar chemical structures.

The catechol group (Figure 21) and the phenothiazine group

(Figure 22) did not exhibit RTP. A few compounds in the

catechol group exhibited RTP, but the intensity levels

obtained from those compounds were not analytically useful.

At this time, there is no available documentation to

explain the results obtained for the catechol and pheno-

thiazine groups. However, the compounds in question

have been found to phosphoresce at low temperatures.
































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