Group Title: new method of analysis based on room temperature phosphorescence
Title: A New method of analysis based on room temperature phosphorescence
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Title: A New method of analysis based on room temperature phosphorescence
Physical Description: x, 92 leaves. : illus. ; 28 cm.
Language: English
Creator: Wellons, Stephen Lawrence, 1948-
Publication Date: 1974
Copyright Date: 1974
 Subjects
Subject: Phosphorimetry   ( lcsh )
Chemistry, Analytic   ( lcsh )
Chemistry thesis Ph. D
Dissertations, Academic -- Chemistry -- UF
Genre: bibliography   ( marcgt )
non-fiction   ( marcgt )
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Thesis: Thesis -- University of Florida.
Bibliography: Bibliography: leaves 87-90.
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General Note: Vita.
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Volume ID: VID00001
Source Institution: University of Florida
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Resource Identifier: alephbibnum - 000582513
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A NEW METHOD OF ANALYSIS BASED ON
ROOM TEMPERATURE PHOSPHORESCENCE















By

Stephen Lawrence Wellons


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
1974

















DEDICATION


Without the love, devotion, and confidence of my

wife, Susie; my mother, Lucy; my father, LaVerne; and

my mother and father-in-law, Helen and William Howard,

the work presented in this dissertation would not have

been possible. To them goes my eternal gratitude and

to them I dedicate this manuscript.

















ACKNOWLEDGEMENTS


The listing of people to whom I owe thanks for

their support is far too long to include them all here.

First, I want to thank especially Ronald Anthony Paynter

for his enthusiastic assistance in helping in this pro-

ject. Secondly, I wish to thank Dr. James Dudley

Winefordner for his support and counsel. I also want

to thank my other counselors, Dr. Willis Bagley Person,

Dr. Martin Thorvald Vala, Dr. Gerhard Martin Schmid, and

Sherlie Hill West for their patient and sincere assis-

tance.

Finally, I want to thank the entire faculty of the

Chemistry Department of the University of Florida for

giving me this opportunity and their support.













TABLE OF CONTENTS


ACKNOWLEDGEMENTS...................................

LIST OF TABLES .....................................

LIST OF FIGURES ....................................

Abstract ...........................................

Chapter

I INTRODUCTION................................

II THEORETICAL CONSIDERATIONS OF PHOSPHORES-
CENCE........................................

III EXPERIMENTAL CONSIDERATIONS.................

Instrumentation.............................

Chemicals and Reagents.......................

Procedure...................................

Choice of Substrate.......................
Choice of Solvent.........................
Sample Preparation.......................
Methods of Drying ........................
New Design of Sample Cell Assembly........

IV RESULTS AND DISCUSSION .......................

General Comments.............................

Weakly Phosphorescent Compounds .............

Intensely Phosphorescent Compounds...........

Lifetime Effects ............................

iv


Page

iii

vi

vii

ix



1


7

17

17

18

19

20
20
21
21
28

34

34

35

35

63












Chapter Page

Spectral Effects.......................... 64

Comparison with Older Methods ............ 69

Rigidity Studies........................... 72

V FUTURE STUDY .............................. 75

General................................... 75

Substrate Modifications ................... 75

Clinical Applications...................... 77

Air Pollution Applications................ 77

Other Luminescence Studies ................ 80

VI SUMMARY.................................. 82

APPENDIX......................................... 84

LITERATURE CITED.................................. 87

BIOGRAPHICAL SKETCH .............................. 91













LIST OF TABLES


Table Page

1. Phosphorescence Characteristics of Filter
Paper..................... ................... .. 21

2. List of Compounds That Did Not Emit Phos-
phorescence at Room Temperature in lM
NaOH on Filter Paper .......................... 36

3. Spectra Characteristics of Compounds That
Were Weakly Phosphorescent at Room Temper-
ature......................................... 37

4. Room Temperature Phosphorescence Charac-
teristics of Several Ionic Organic Molec-
ules Adsorbed on Filter Paper Using In-
frared Drying...................... ........... 38

5. Room Temperature Phosphorescence Charac-
teristics of Several Ionic Organic Molec-
ules Adsorbed on Filter Paper Using Des-
iccator Drying................................ 41

6. Comparison of Phosphorescence Intensities
at Room Temperature and at 77 K................ 73















LIST OF FIGURES


Figure Page

1. Electronic transitions resulting from the
absorption of a photon........................ 9

2. Drying apparatus used in room temperature
phosphorescence studies ...................... 25

3. Effect of drying time on intensity of room
temperature phosphorescence signal (5 mM
6-methylmercapto-purine in 1M NaOH).......... 27

4. Schematic diagram of sample holder for
filter paper circles used in room temper-
ature phosphorescence studies ............... 30

5. Detailed schematic diagram of boxed area
in Figure 4 of room temperature phosphor-
escence sample holder, designed specifically
to handle in filter paper circles.......... 32

6. Room temperature phosphorescence analytical
curve for 4-amino-benzoic acid in 1M NaOl
absorbed on filter paper at 600C............. 44

7. Room temperature phosphorescence analytical
curve for 4-amino-2,6-dihydroxyl pyrimidige
in IM NaOH absorbed on filter paper at 60 C.. 46

8. Room temperature phosphorescence analytical
curve for 2,6-diamino-purine in IM NaOH ab-
sorbed on filter paper at 60 C............... 48

9. Room temperature phosphorescence analytical
curve for 6-methylmercapto-purine in lM NaOH
absorbed on filter paper at 60 C............. 50













10. Room temperature phosphorescence analytical
curve for sulfanilamide in IM NaOH absorbed
on filter paper at 60 C........................ 52

11. Room temperature phosphorescence analytical
curve for sulfaguanidine in lM NaOH absorbed
on filter paper at 600C......................... 54

12. Room temperature phosphorescence analytical
curve for tryptophan in IM NaOH absorbed on
filter paper at 60 C............................ 56

13. Room temperature phosphorescence analytical
curve for 5-acetyl ur8cil in lM NaOH absorbed
on filter paper at 60 C........................ 58

14. Room temperature phosphorescence analytical
curve foi 2-thio-6-amino-uracil in 1M NaOH
absorbed on filter paper at 600C............... 60

15. Room temperature phosphorescence analytical
curve for 4-hydroxyl-3-methoxy-benzaldehyde
(vanillin) in 1M NaOH absorbed on filter
paper at 60 C.................................. 62

16. Temperature effect on phosphorescence spectra
of tryptophan (1 mg/ml in 1M NaOH)............. 66

17. Temperature effect on phosphorescence spectra
of 6-methylmercapto purine (1 mg/ml in IM
NaOH)........................................ . 68

18. Room temperature phosphorescence spectra of
Hyland Normal Control Serum in lM NaOH and
filter paper background.......................... 79


viii


Figure


Page











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



A NEW METHOD OF ANALYSIS
BASED ON ROOM TEMPERATURE PHOSPHORESCENCE



By

Stephen Lawrence Wellons

March, 1974



Chairman: James Dudley Winefordner
Major Department: Chemistry


The present study indicates that room temperature

phosphorescence has tremendous potential for analysis of

a wide variety of ionic organic molecules and that it can

be used for quantitative and qualitative identification

purposes. The substances tested are representative of a

wide variety of aromatic carboxylic acids, amines, thiols,

or phenols. Many of the compounds are of biological im-

portance or pharmaceutical interest.

The findings indicate that this method provides a

sensitive, selective, and accurate means of identifying

certain aromatic molecular species which are ionic. Al-

though there is no strong theoretical explanation for











this phenomena, it is believed that the ionic state of

the molecules results in great molecular rigidity via

adsorption to the substrate, which reduces radiationless

decay due to collisional deactivation.

Room temperature phosphorimetry offers many advantages

as an analytical method. It has good sensitivity with

the potential to be much more sensitive. It is inexpen-

sive and safe in the sense that liquid nitrogen or cry-

ogenic equipment is not required. It is convenient and

does not require expensive chemicals or high purity sol-

vents. This technique is a rapid method of analysis

which could be automated. The procedure established is

fairly simple and would be easy for a technician to

learn.

Room temperature phosphorescence definitely merits

future study. The technique developed should find many

applications to real systems, particularly for biological

and pollution samples. Studies that are of importance

for future research include the investigation of better

substrates, automatic sampling, the influence of pH and

solvent, and the analysis of species in blood serum, urine,

and air pollution particles. Finally, it would seem that

time-resolved phosphorimetry should provide additional

selectivity in the measurement of real samples.













CHAPTER I


INTRODUCTION


Molecular phosphorescence spectroscopy or phosphor-

imetry is a relatively new luminescence method for molec-

ular analysis. It is usually applied to unsaturated or-

ganic molecules with their n-bonding arranged in a con-

jugated manner. Previously, intense phosphorescence has

been almost exclusively observed at low temperature (less

than 1500K). As a result, earlier research in phosphor-

imetry as a method of analysis has been performed at low

temperatures, principally in liquid nitrogen because this

liquid is the most convenient to use.

The existence of phosphorescence has been known for

a number of years since it was first reported in 1888 by

Wiedemann (1). Schmidt (2) in 1896 observed the increase

of phosphorescence intensity when measured at low temper-

atures. It was first suggested as a method of analysis by

Lewis and Kasha in 1944 (3). Its utility as a method was

not realized until publication of a study by Keirs, Britt,

and Wentworth in 1957 (4). In the last ten years, its use

as an analytical technique has undergone phenomenal growth.

Applications of phosphorescence to routine analytical

problems may be found in a variety of areas: coal tar












fractions, antimetabolites, and carcinogenic hydrocarbons,

to name a few (5, 6, 7).

As a method of analysis, phosphorescence compares

favorably with competing electronic spectroscopic techniques,

mainly molecular fluorescence and ultraviolet absorption.

The standard criteria used for comparison of analytical spec-

troscopic methods are i) breadth of application, ii) selec-

tivity, iii) sensitivity, and iv) accuracy and precision.

(i) Most unsaturated organic compounds show measurable

ultraviolet absorption, but not all of them re-

emit the absorbed radiation as measurable fluor-

escence or phosphorescence. Therefore, absorp-

tion spectroscopy has a greater breadth of ap-

plication than either fluorimetry or phosphor-

imetry. However, all three methods are suitable for

many compounds for qualitative and quantitative deter-

mination. Thus, the selection of the method which

should be preferred can be decided on the spec-

troscipic characteristics of the system of in-

terest.

(ii) In regard to selectivity, phosphorimetry is the

superior method of analysis. Just as in fluor-

imetry, the two spectra obtained, excitation and

emission, can be utilized for identification. The











phosphorescence spectra are usually more charac-

teristic than the fluorescence spectra and are

therefore frequently better suited for identifying

compounds. Because of the wide range of measur-

able lifetimes of phosphors, time and phase reso-

lution can also be used for identification and

separation of complex mixtures methods which are

not possible in absorption spectrometry and which

are instrumentally difficult in fluorimetry (8).

(iii) Luminescence methods, in general, result in ten

to a thousand times lower detection limits and

greater sensitivity than absorption techniques.

Because the scatter of source radiation in phos-

phorescence is much lower than in fluorimetry,

wider monochromator slits and apertures can be

used. Relative limits of detection determined in

fluorimetry and phosphorimetry vary widely from

compound to compound depending on whether noise

levels, absorption coefficients, and quantum

yields are favorable. McGlynn in 1963 (9) on

the basis of three compounds, and in 1966,

Sauerland and Zander (10) on a wider basis, con-

cluded that spectrophosphorimetry and spectro-

fluorimetry had comparable sensitivity. Since










then, Winefordner and co-authors have greatly im-

proved the sensitivity of compounds measured by

phosphorimetry. The use of a rotating sample cell,

more stable source, open ended capillary, and lower

solvent background has increased sensitivity by

several orders of magnitude (8), making phosphor-

imetry the most sensitive technique.

(iv) All three spectrometric techniques have comparable

accuracy and precision. For example, the relative

standard deviation of phosphorescent measurements is

less than 1% using the rotating sample cell with
-5
10- molar solutions (8).

Thus, phosphorimetry with samples maintained at low

temperatures is now an important and established technique

for trace analysis. The major disadvantage of phosphorimetry

is the rather inconvenient need of low sample temperatures.

The need for such low temperature requirements results in

greater expense and complexity of analyses due primarily to

the added cost and use of liquid nitrogen or other coolants.

Also, such low temperatures make the automation of phos-

phorescent analysis virtually impossible. Therefore, if

phosphorimetry could be carried out at higher temperatures

with the same sensitivity and selectivity advantages, then

it might find greater use in real applications.












Phosphorescence has been observed at room temperature,

but at that temperature it is usually very weak and of

little analytical interest. For example, phosphorescence

at room temperatures has been reported in gaseous biacetyl

vapor (11), in liquid biacetyl solutions (12), in solid

boric acid or sugar solutions (13), or in very rigid plas-

tics such as polymethyl methacrylate (14). None of these

matrices are suitable for quantitative analyses. They are

either too specialized as in the case of biacetyl or too

difficult and time consuming as in the other cases. Polymer

solutions also are subject to interference from their own

ultraviolet absorption and phosphorescence emission.

In 1972, Schulman and Walling observed intense phos-

phorescence of several molecules at room temperatures

(15, 16). They reported that when certain organic substances

were ionically adsorbed on silica gel, alumina, paper, or

asbestos and thoroughly dried, efficient triplet state emis-

sion occurred. These studies dealt only with some of the

qualitative aspects and no attempt was made to develop

analytical applications of this phenomenon.

Thus, this dissertation will deal principally with

the development and evaluation of a method an analysis using

room temperature phosphorescence of ionic organic molecules

adsorbed on a substrate, primarily filter paper. This dis-

sertation will demonstrate that room temperature phosphorescence





6





offers a fast, economical, and convenient method of analy-

zing a variety of organic molecules, many of pharmaceutical

or biological importance.













CHAPTER II


THEORETICAL CONSIDERATIONS OF PHOSPHORESCENCE


In 1935, Jablonski (17) was the first to discuss

the theoretical basis of phosphorescence. Later, Lewis

and Kasha (3, 18) firmly established the theory explain-

ing this phenomenon. Since that time, experimental

measurement and calculation have thoroughly verified

their work. Their discussion of the theory pertinent

to this manuscript will be restricted to the luminescence

phenomena of an organic species in solution. Other

sources of phosphorescence, such as from physical defects

in crystals, will not be discussed here.

Consider a liquid solution of an organic compound

that exhibits strong ultraviolet or visible absorption.

If that solution is irradiated with electromagnetic

radiation of the wavelength of the absorption, then it

will emit isotropic radiation of a longer wavelength at

room temperature. This is commonly known as fluorescence.

Next, suppose that the solution is cooled to low temper-

atures or somehow becomes a rigid matrix. If this solid

solution is again subjected to ultraviolet or visible

radiation of the species' absorbing wavelength, it will

7












again fluoresce isotropically as well as emit radiation

of a wavelength longer than the fluorescence; this

latter emission will have a lifetime that is several

orders of magnitude longer than the lifetime of fluor-

escence. The long-lived phenomenon is called phosphorescence;

this radiation will generally be in the near ultraviolet

or visible region of the electromagnetic spectrum (between

350 nm to 600 nm) and the lifetime of phosphorescence will

be of the order of milliseconds to seconds. Phosphor-

escence decay follows first order kinetics.

Fluorescence and phosphorescence processes will

be considered for a single unsaturated organic molecule.

Figure 1 is a modified Jablonski diagram showing some

of the energy levels of a typical unsaturated organic

molecule. For the moment, consider only the electronic

energy levels of a molecule with a pair of electrons in

the ground state, denoted as S Each electron is given

a spin angular momentum quantum number of either +' or

-. In Figure 1, electrons having a +' value are shown

as arrows pointed upwards, and those of -' are pointed

downward. The Pauli exclusion principle requires two

electrons in the same orbital to have opposite spins,

as shown in the box next to So in Figure 1.










LTi S2


L -=f- L
2iZIITF _ F TY rs



;Ly







---._^^::^^EJ^'___


Symbol Process


T [EE
ZL


0

Rate Range of Rate1
Symbol Constants, sec (9)


I Absorption
II Internal Conversion
III Vibrational Relaxation
IV Intersystem Crossing
F Fluorescence
P Phosphorescence


1015 1016


kll >1012
kI 104 1012
kF 106 10
k- 10-2 104


Fig.l.--Electronic transitions resulting from the absorption
of a photon. Absorption and emission of photons is
indicated by straight lines. Nonradiative transitions
are indicated by broken lines.


=1












If a photon of the appropriate energy is absorbed

by the molecule, then one of these electrons may be

promoted to a higher energy level. The promotion process

may occur in one of two manners. In the first case,

the electron's spin angular momentum quantum number is

not changed. Thus, the spin multiplicity, defined as

one plus twice the absolute value of the sum of the

spin angular momentum quantum numbers of the electrons,

will be unity. States having a spin multiplicity of one

are called singlet states (S). Note that the multiplicity

of the ground and excited states in this case will be

the same. In the other case, the spin of the promoted

electron changes. Because an electron's spin quantum

number may have only two values, the promoted electron

now has the spin quantum number the same as the one

remaining in the ground state. The multiplicity of this

condition is three and called a triplet state (T). It

follows from Hund's Rule that the first triplet state

(T1) is of lower energy than the first excited singlet

state (SI).

However, usually only singlet-singlet (So+S1)

promotions are observed. This can be explained from

the first order quantum mechanical treatment of electron











states; transitions are allowed only between states of

the same multiplicity. This is only an approximation

and singlet-to-triplet absorption is observed, although

very weakly, i.e. as Sklar reported in the absorption

spectrum of benzene in 1937 (19). In this case, the

So-S1 absorption is about 105 times more intense than

the So-T1 absorption. Because the latter absorption

will not be pertinent to the work described in the man-

uscript, it will not be considered further.

Fortunately, there are other more efficient ways of

populating the triplet state than direct radiational

excitation. Those molecules excited to one of their

excited singlet states can sometimes be partially de-

excited to the triplet state via several different

processes. Consider a molecule that is in one of its

higher excited singlet states (S2,S3,...). It may reach

a lower state of the same multiplicity through a crossing

point of their potential energy curves. This process

is called internal conversion and is indicated as process

II in Figure 1. In the absence of intermolecular

collisions, internal conversion is restricted to excited

electronic states because the energy differences between

individual excited electronic states are much smaller

than between ground and excited states, and so vibronic











coupling between the first excited electronic state and

the ground state is much less likely to occur, preventing

complete radiationless deactivation.

The excess energy resulting from the vibrational re-

laxation and internal conversion is dissipated through

vibrational motions of less rigid parts of the molecules.

Internal conversion and vibrational relaxation are ex-

tremely rapid processes (rate constants of ca. 101 sec );

as a result, most molecules that are photon excited to

high electronic states, return nonradiationally to the

lowest vibrational levels in the lowest excited electronic

state.

Once in the lowest excited singlet level, the molecule

can deactivate itself completely by emitting a photon.

This singlet-singlet (S oS1) emission process is known

as fluorescence (process F in Figure 1). The molecule

may also be nonradiationally deactivated through inter-

molecular collisions, or quenching. A third alternative

is that the electron in the excited singlet state may

"cross over" to the next lowest triplet state. This

process is called intersystem crossing (process IV in

Figure 1). The rate constant for this process is about

10 sec-1 but varies over a wide range depending on (i)












the energy difference between the singlet and triplet

systems and (ii) on the degree of mixing of singlet

character in the triplet state. Again, first order

quantum mechanics forbids transitions between states

of different multiplicities. But this is not true

if there is significant spin-orbit coupling or mixing

of the singlet and triplet levels. Kasha in 1950

measured the rates for intersystem crossing in (I *)

systems and found them to be about 10 sec- (20)

which is about 105 times slower then vibrational or

internal conversion; thus, intersystem crossing will

occur almost exclusively at the lowest excited singlet

and triplet states.

The triplet state may be deactivated in several

ways. First, the electron may undergo reverse inter-

system "cross-over" to the singlet system. If the

molecule is then radiationally deactivated, then the

emission is referred to as delayed fluorescence. The

process occurs in the dye eosin and is also known as

E-type fluorescence. Also, the triplet state of the

molecule may be nonradiationally deactivated or quenched.

Typical quenchers are solvent molecules or impurities,

e.g., dissolved oxygen. A third and desired possibility














is that the triplet state will deactivate emitting a

photon. This process is called phosphorescence and is

indicated in Figure 1 as process P.

Several important conclusions can be drawn from the

varying orders of the rate constants. Phosphorescence

and fluorescence emission spectra will usually be single-

banded because kf and k are so much smaller than ki

or k II. Kasha's rule specifies that only the prevailing

lowest state of a given multiplicity of any molecule is

capable of emission.

The structure of luminescent-type compounds may

have a large effect on the kinetic processes involved

in the deactivation of the excited species. Straight

chains or non-rigid aromatic molecules generally show

poor luminescence properties. These types of compounds

are particularly susceptible to vibrational relaxation

processes, and easily dissipate their electronic energy

externally without emission. An example of the importance

of rigidity is givjn in the comparison of fluorescein

and phenolphthalein.











0 0





c coo- coo-
COO COO









fluorescein phenolphthalein

These compounds are identical in structural geometry

except for the central ring. Because phenolphthalein has

two hydrogen atoms instead of the rigidly held oxygen

atom, molecular twisting is much easier than in fluorescein.

Fluorescein emits a very intense luminescence radiation

in liquid solution, whereas phenolphthalein emits only very

weakly (21).

Temperature and viscosity effects on luminescence

process are also very important. With few exceptions,

strongly absorbing substances in low viscosity solutions

have not been observed to phosphoresce at room temperature

because the quenching of the excited molecules by inter-

molecular collisions occurs within the lifetime of the











excited state (about 10-7 sec).

In 1966, McCarthy thoroughly examined the effect on

phosphorescence in solution as a function of temperature

(22). He concluded that values near unity for the phos-

phorescence quantum yield would never be obtained at

even moderate solution temperatures unless solvents

of very high viscosity near 2730K were readily available.

However, McCarthy noted that such a development would be

of considerable analytical importance. A very important

conclusion that one must draw is that phosphorimetry re-

quires a rigid matrix. While Schulman and Walling did

not discover a high viscosity solvent, they did observe

a very practical method of achieving rigidity at room

temperatures.














CHAPTER III


EXPERIMENTAL CONSIDERATIONS


Instrumentation


An Aminco-Bowman spectrophotofluorimeter (Silver

Spring, Maryland 20910) with a Aminco-Keirs rotating can

phosphoroscope attachment was used for all measurements.

The excitation and emission motor-driven scanning mono-

chromators each contained a grating ruled 600 grooves per

mm and blazed at 300 nm and 500 nm respectively. Their

spectral band passes were approximately 6 nm per mm slit

width. A Hanovia 901-C-ll 50 W xenon lamp with the Aminco

ellipsoidal source condensing system was used as a continuous

source. A relatively steady lamp intensity was provided by an

Aminco 422-818 D.C. power supply delivering about 7.5 A to

the xenon arc lamp. An encapsulated RCA 1P21 photomul-

tiplier tube of spectral response type S-4 (300 nm to 600

nm) served as the detector (23). The photomultiplier tube

was continuously maintained at 700 V, 10 mA maximum, by a

Keithley 244 high voltage supply (Cleveland, Ohio 44139).

Phototube signals were amplified with a low noise nanoam-

meter designed by O'Haver and Winefordner (24). Spectra

17












were recorded on an Aminco 1620-827 X-Y recorder with

wavelength scan speeds of about 2 nm/sec with a nanoam-

meter time constant of 0.5 sec. The lamp intensity was

monitored with a J.E.M. Powermaster CdS-902 photo cell

(Pioneer Electronics and Research Corporation, Forest

Park, Illinois 60130) at 500 nm and displayed on a

Keithley 220 d.c. vacuum tube voltmeter. The monochroma-

tor wavelengths were calibrated with a mercury pen light

using the technique described by Udenfriend (25).

Temperature and relative humidity (R.H.) measure-

ments were made with a Taylor, Mason's Form, hygrometer

(Scientific Products, Evanston, Illinois 60204) using

wet- and dry-bulb thermometers, 2 F per division. Also,

a Serdex recording hygrothermometer (Bacharach Industrial

Instrument Co., Pittaburgh, Pennsylvania 15230) was used;

it had a bimetal strip, 20F per division, and a tissue

membrane 2% R.H. per division. Both instruments had an

accuracy of + 3% R.H.


Chemicals and Reagents


The analyte samples were obtained from the sources

listed in the Appendix and were used without further

purification.

Solvent solutions of acid and base were prepared












from distilled, filtered, and deionized water. No

detectable phosphorescence background was found from

these solvents whether at room temperatures or at liquid

nitrogen temperatures.

All glassware was prepared by soaking for more than

24 hours and then by washing in a solution of Lakeseal

laboratory glass cleaner (Peck's Products Co., Ithaca,

New York 14850). After rinsing, the glassware was sub-

merged overnight in 5-6 N nitric acid. This was followed

by rinsing thoroughly more than six times with distilled

water and by drying in an oven at 1100-1200C. Until

use, the clean glassware was stored in dust-proof con-

tainers.


Procedure


Because no other reports of this phenomenon as an

analytical tool have been made, great care was taken in

developing a reproducible method of preparing and test-

ing the samples. Although Schulman and Walling observed

room temperature phosphorescence on a variety of sub-

strates, silica, alumina, paper, asbestos, and (more

weakly) glass fibers, they reported that filter paper

seemed to give the best results (16).












Choice of Substrate. As a result, the selection

of a rigid support for analytical determinations was

restricted mainly to commercially available filter papers.

Fifteen different kinds were tested for their phosphor-

escence background and the results are given in Table I.

Eaton Dikeman 613 yielded the lowest phosphorescence

signal and was used in all further studies.

A high grade chromatography paper and Metrical

millipore filters (Gelman Instrument Company, Ann Arbor,

Michigan 48106) were also tested. The Metrical filters

lacked structural stability when treated with 1M NaOH

or 1M HC1 and were not considered further. The phosphor-

escence characteristics of the chromatography paper are

also given in Table I.

Choice of Solvent. The selection of IM NaOH as the

principal solvent was made for several reasons. Earlier

work on polynuclear acids by Schulman and Walling (15, 16)

showed that excess aqueous NaOH resulted in the strongest

phosphorescence signals. Because most of the compounds

selected for study included a variety of acids and bases,

many with amphoteric characteristics, other solvents were

considered. However, the study of several compounds in

different solvents indicates no preference of acid or base.









TABLE 1. Phosphorescence Characteristics of Filter Paper


Companya-Type


Excitationb
(nm)


Emissionb
(nm)


Relative Phosphor-
escence Signalsc


Curtin 7822-Ad

Curtin 7760

E-D 613

E-D 615

Reeve Angel-201

Reeve-Angel-202

S+S 604

S-P F-2402

S-P F-2406

Whatman 1

Whatman 30

Whatman 40

Whatman 41

Whatman 42

Will 13021

Will 13061


305

300(316,460)

294(320)

36

317

310

292

295

290

290

294(316)

294(320)

294(316)

300(322)

295(320,390)

316


aKey: Curtin = Curtin Scientific
E-D = Eaton Dikeman
S+S = Schleicher and Schuell
S-P = Scientific Products
Will = Will Scientific

bExcitation and emission peak wavelengths. Excitation wave-
lengths in ( ) are shoulders.

CAll signals taken with respect to E-D 613 (set equal to
unity), the filter paper used in the present studies.
dChromatography paper, grade #1.


1.8

3.0

1.0

4.8

5.0

1.5

3.0

1.8

3.0

2.6

5.0

2.0

3.5

1.6

10.

11.












For example, a 5 mM solution of 2-amino-6-methyl-

mercapto-purine dissolved in 1M NaOH, HC1, and deionized

water gave equivalent phosphorescence signals. Moreover,

solutions of 1M HC1 weakened the filter paper and the filter

paper circles were difficult to handle without disintegra-

tion. Also, almost all of the compounds to be studied

were readily soluble in 1M NaOH.

Sample Preparation. Circles (7 cm diameter) of

Eaton-Dikeman 613 filter paper were cut into in circles

using a conventional paper hole puncher. The paper

circles were suspended by their edges vertically with

small Micro-gator clips (Mueller Electric Co., Cleveland,

Ohio 44114) in a clothesline fashion. The sample solution

was allowed to run off the tip of the needle of a 50 pl

Hamilton syringe (Whittier, California 90607) until it

came onto contact with the filter paper circle; 5 pl

of sample was placed on each filter paper circle. Five

was chosen because it was a convenient quantity that gave

even and reproducible distribution of the solution.

Methods of Drying. Drying of the samples was es-

sential; previously reported by both Schulman and Walling

(15, 16) and further shown in this laboratory that

moisture on the paper sharply decreased the phosphorescence











signal. Drying initially was accomplished by 1) air

drying for at least 1 hr and 2) then placing the circles

in a desiccator for at least 80 min for final drying.

However, this procedure took more time (total time -

at least 140 min) and involved more sample handling than

was felt desirable. Hot air blowers and other methods

of drying, including ovens, were tested but most of

these proved to be too destructive to the paper circle

or the sample. Infrared lamps were gentle and very

effective. The temperature of the drying area was con-

trolled by varying the distance between the lamps and

the samples. The final arrangement is shown in Figure

2. In order to reduce fluctuations of the temperature

from air currents, the drying apparatus was set up in

an isolated corner of the room. Two large sheets of

fiber board were placed on the third and fourth sides

of the drying area. This simple method worked surpris-

ingly well. The phosphorescence of a sample could be

measured in as little as 15 min after spotting. The

sample needed to be handled only once, and its intensity

would be equivalent to samples dried by the first method.

Figure 3 is a typical graph showing the effect of drying

time on phosphorescence intensity.























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The relative humidity and temperature in the

region occupied by the drying rack was measured at

numerous points. Temperature, measured with two ther-

mometers and the Serdex hygrothermograph, was found to

be 600 + 30C. The relative humidity, measured with a

Taylor hygrometer and the Serdex hygrothermograph, was

determined to be less than 5%.

New Design of Sample Cell Assembly. In order to

measure the phosphorescence of the filter paper circles

in the Aminco-Bowman spectrophotofluorimeter, a number

of changes in the sample cell were necessary. The rod

designed to hold the samples is shown is Figure 4. In-

itially a strip of Scotch tape with a h in hole was

used to hold the paper circles. A more rigid brass

plate had a hole drilled 1/64 in smaller than a paper

circle. This held the sample in place and gave maximal

illumination. The rod, B, and cylinder C, were adjusted

to a depth and angle that gave maximum phosphorescence,

then tightened and sealed.

So that the sample would not absorb moisture during

measurement, the sample cell was flushed with warm dry
air at a rate of 15 Imin-1 The volume surrounding the
air at a rate of 15 1mm The volume surrounding the


























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33







sample cell with the sample holder in place was estim-

ated to be about 0.5 1. The air was filtered and dried

with silica gel in a drying tube and was heated to

650C in a 18 in length of copper pipe ( in outside

diameter) wrapped with a Briskeat heating tape prior to

entering the sample cell assembly.
















CHAPTER IV


RESULTS AND DISCUSSION


General Comments


A wide variety of vitamins, purines, pyrimidines,

catecholamines, and sulfa drugs were selected for study

in I11 NaOH on filter paper. The purpose of this particular

selection of compounds was to determine if an improved

method of analysis could be developed for biological or

pharmaceutical applications. Also selected were several

compounds for which low temperature phosphorescence had

not been observed. These would indicate whether the

breadth of application of room temperature phosphorimetry

could be extended beyond that of conventional low tem-

perature phosphorimetry. Included in this selection

were a number of compounds which had been studied pre-

viously in this laboratory, enabling comparison with

low temperature studies under similar experimental con-

ditions.

Those compounds that did not emit measurable phos-

phorescence when dissolved in 1M NaOH and dried on filter

34











paper are listed in Table 2.


Weakly Phosphorescent Compounds


Several of the compounds, certain purines or

pyrimidines, were weakly phosphorescent. These are

listed in Table 3 with their spectral characteristics and

signal-to-noise ratios. Further study on these compounds

was not considered analytically useful at the present

time.


Intensely Phosphorescent Compounds


About one-quarter of the molecules studied emitted

intense phosphorescence at room temperature using the

procedure previously described. A summary of their

analytical figures of merit is given in Table 4. For

comparison and completeness, Table 5 shows the earlier

work carried out in this laboratory by room temperature

phosphorimetry using desiccator drying techniques (26).

Figures 6 through Figure 15 show the quality of analy-

tical measurements using this technique. In all cases,

phosphorescence measurements were taken within at least

two orders of concentration above the calculated limit

of detection. The slope of the linear portion of each











TABLE 2



List of Compounds That Did Not Emit Phosphorescence
at Room Temperature in IM NaOH on Filter Paper.


adenosine

cytidine

guanosine

thymine

thymidine

uracil

uridine

folic acid

riboflavin

niacinamide

inosital

epinephrine

norepinephrine

tyrosine

6-phenyl-amino-purine

6-bromo-purine

5-amino-uracil

6-amino-uracil


2-amino-pyrimidine

2,4-dichloro-pyrimidine

2-amino-4-methyl-pyrimidine

2-amino-4,6-dihydroxyl-pyrimidine

2-amino-4-carboxylic-5-chloropyrimidir

4,6-dihydroxyl-pyrimidine

vitamin B1 (thiamine hydrochloride)

vitamin C (ascorbic acid)

vitamin D calciferoll)

vitamin K1

vitamin K3

vitamin KS

vitamin H (Biotin)

pyridoxime hydrochloride

sulfathiazole

sulfamerazine

sulfamethozine

sulfadiazine

















TABLE 3. Spectra Characteristics of Compounds
That Were Weakly Phosphorescent at
Room Temperaturea


Excitation
Wavelength


Emission
Wavelength


adenine 300 469 40

cytosine 301 405 30

guanine 282 427 40

4,5-diamino- 357 524 20
uracil

2-thio-4,6-dioxy- 342(300)d 467 70
pyrimidine


aRoom temperature phosphorescence of these compounds is
not considered at present time to be analytically useful.

bExcitation and Emission wavelengths are accurate to 5 nm.


cMeasurements made on a 5mM solution in 1 NaOH. Signal-
to-noise calculated on basis of analyte intensity less
blank divided by 5% of blank intensity.


shoulder


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analytical curve was computed using a linear regression

analysis program on the logarithms of the intensity and

concentration values. The linear correlation coeffecients

for most of these plots were equal to or higher than 0.999.

The limit of detection was determined to be the theoretical

concentration for a signal-to-noise value of two. The

percent relative standard deviation of the blank was

calculated to be + 5% using a least squares error analysis.


Lifetime Effects


Lifetime measurements were attempted on a number

of the room temperature phosphors. However, the nanoam-

meter had a limiting time constant of 0.05 sec. Also,

using the Aminco-Bowman spectrophotofluorimeter, it is

physically very difficult to measure lifetimes shorter

than 0.1 sec accurately. Most of the compounds studied

had phosphorescence lifetimes at room temperatures that

appeared to be 0.1 sec or shorter and as a result, it

was not possible to accurately measure them with this

instrument. Schulman and Walling also examined the

lifetimes of several molecules by flash photolysis and

found them all to be in the msec range (16). Tryptophan,











which at 770K had a decay lifetime of 6.3 sec was

measured at room temperature to have a lifetime of

0.6 + 0.1 sec. Most of these compounds listed in

Tables 4 and 5 have lifetimes longer than 0.5 sec

at low temperatures. Obviously, and as expected from

theoretical considerations, higher temperatures greatly

shorten phosphorescence lifetimes.


Spectral Effects


Another important result observed was a red shift

in the phosphorescence emission spectra at room temper-

ature as compared with 770C. This effect was noted

earlier (26). A shift of about 12 nm for tryptophan,

and 20 nm for 6-methylmercapto-purine can be seen in

Figures 16 and 17. Each set of spectra was produced

from the same solution under identical experimental

conditions. These shifts are not isolated cases as can be

seen from a comparison of the values given in the

Appendix and Table 4. For the family of purines studied,

there was a red shift ranging from 20 to 40 nm between

770C and room temperature phosphorescence emission max-

ima. Because the literature values may have been derived

for different environmental situations, they should be
























U
) C





c) 0

a) 0 t-
-' cz r-

0z ?
o~ C




0Z v






0 -H
0
0
0 0,
0: 0 eC
C)-0 0 -
o c
00


0f k~


~a0




bib


Q) -4 0
0 41
00. Z3-
00 C, 4-*
cC) c7











































AIISN31NI


30NIOS3J OHdSOHd























0


4 -
uZ





-H 0
UC
-4 0



00



,0 -



0U 0



0 .- ;
mo
0 Q) 44

u & E
a1) CO a)



0 0 4.
S00 -


S1v



CO)


BO





*-


X
,E5
C.


C cc oo


CO d CT
-H



U
x


0
M


o
7-






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OZH* -.4





68







71
0

z -
0


























C\(
-.














x
IIl




S-O








AliSN31Nil 3 N3 S 3atOH d 90Hd










used with some caution. When more than one value was

available, the one selected was nearest to the present

conditions.


Comparison with Older Methods


The four analytical criteria mentioned earlier, i)

breadth of application, ii) selectivity, iii) sensitivity,

and iv) precision and accuracy, will now be considered

in order to evaluate this method of analysis. Because

room temperature phosphorimetry is in a very early stage

of development, consideration should be given to its

potential in addition to its present usefulness.

(i) This study has shown that room temperature phos-

phorimetry, using aqueous IM NaOH as the solvent

and filter paper as the substrate, is applicable

to a wide variety of compounds: acids, bases,

phenols, etc. Also, those compounds that show

E-type delayed fluorescence, e.g., eosin Y, can

be measured with this technique. At present,

the area of application is limited to those com-

pounds that form stable ions under these exper-

imental conditions. However, with further study,

the potential exists that room temperature phos-

phorimetry could be extended to all those compounds











known to show strong phosphorescence at low

temperatures or delayed fluorescence at room

temperatures.

(ii) Room temperature phosphorimetry offers good

selectivity. As is the case in low temper-

ature phosphorimetry or fluorimetry, sub-

stances can be differentiated using both their

emission and excitation spectra. Further sep-

aration of phosphors is possible using time or

frequency resolution techniques. Parameters,

such as substrate, solvent, and pH can be more

conveniently varied at room temperatures than

at 770K.

(iii) Tables 4 and 5 show that this technique offers

good sensitivity. The limit of detections

extends from 20 ppb for eosin Y upwards to the

Ug/ml concentration region. Also, only a very

small sample size of Si is needed. Conven-

tional phosphorimetry requires a minumum of

20 ul and generally about 1 ml. Fluorimetry

usually requires about 1 ml of solution. As

a result, the absolute sensitivity of room

temperature phosphorimetry is enhanced by a











factor of 200 over that of these conventional

methods. An absolute limit of detection of

0.1 ng for 4-amino-benzoic acid was achieved

using this technique. This compares favorably

with the earlier results of Aaron and Winefordner.

A fluorimetric detection limit of 10 ng and

phosphorimetric detection limit of 1 ng for

this compound was reported (27).

(iv) The percent relative standard deviation of 5 %,

two orders of magnitude above the detection

limit, is quite good. It indicates that the

use of filter paper and the present drying

techniques are reproducible. It is also pos-

sible that the percent relative standard devi-

ation can be reduced greatly with more sophis-

ticated drying and handling techniques. Room

temperature phosphorimetry, however, does not

have some of the problems encountered at low

temperatures. These include the variability

in the cracking or snowing of the solid matrix,

the noise from the wobbling capillary tube, or

ice formation in the liquid nitrogen dewar. Thus,

it is possible that greater precision could be











achieved than is possible at low temperatures.

There is another important advantage that room

temperature phosphorescence has as an analy-

tical technique. It is a very fast technique;

with the present drying procedure, samples

can be handled at the rate of one sample per

minute from the start of drying to the com-

pletion of measurement.


Rigidity Studies


The main purpose of this dissertation was to develop

a new analytical technique using room temperature phos-

phorescence and to evaluate its usefulness. Another re-

lated and very interesting aspect would be the establish-

ment of a strong theoretical basis for this phenomenon.

Although no major attempt was made to solve this problem,

several observations were made that are relevant to the

discussion of the mechanism of room temperature phosphor-

escence.

A comparison of several of the analytes was made

between their phosphorescence intensity when adsorbed on

filter paper at room temperature and when frozen in a

rigid matrix at 770K. This comparison is given in Table 6.











TABLE 6. Comparison of Phosphorescence Intensities
at Room Temperature and at 77K


Compound Max. Intensity Max. Intensity
at 770K / at 3300K

4-amino-benzoic acid 6

6-methylmercapto-purine 8

2-amino-6-methyl-mercapto-purine 19

4-amino-2,6-dihydroxyl pyrimidine 50

2,4-dithio-pyrimidine 1.8

sulfaquanidine 110

tryptophane 10

5-acetyl-uracil 220

2-thio-6-amino-uracil 26

vanillin 1.6



aAll compounds prepared in IM NaOH. Concentrations are
approximately 5 mM.











Both intensities were measured under the same experimental

conditions, with exception of the sample holder. The room

temperature phosphorescence was measured using the appara-

tus described in Chapter III. The low temperature phos-

phorescence was measured using a 6 mm O.D. 1 mm I.D.

rotating quartz capillary suspended in a quartz dewar

filled with liquid nitrogen described by Winefordner (8).

Therefore, the relative intensity ratios in Table 6 are

indicative of the degree of rigidity with which the mo-

lecule is being held on the filter paper.

A trend that is apparent in this comparison is that

the molecules that have the most ionic sites show the

greatest rigidity. Vanillin and 2,4-dithio-pyrimidine

are doubly charged in strong alkaline solution. On the

other hand, compounds like sulfaquanidine and 5-acetyl-

uracil which are expected to be the least ionized in strong

base, show the greatest decrease in their relative phos-

phorescence intensities at room temperature. It should

be noted that because both of these compounds exhibit

strong phosphorescence (S/N n 100 at 10 pg/ml) in alka-

line solution, binding forces other than ionic (perhaps

hydrogen bonding), may be involved in maintaining the

required rigidness.












CHAPTER V


FUTURE STUDY


General


As is the experience with the development of any new

technique of new device, there have been, in this study,

both successes and some disappointments.

The areas of success include the development of a

versatile technique that is rapid and reproducible. An-

other success is the demonstration that room temperature

phosphorimetry can reach low detection limits.

Primary among the disappointments was the failure

to reduce the phosphorescence background of the blank.


Substrate Modifications


Future study in room temperature phosphorimetry will

make significant advances if substrate background can be

reduced. A reduction of background noise of two orders

of magnitude could easily be made before scattered light

or instrumental noise would become the limiting factor.

Such a reduction would result in the achievement of un-

usually low detection limits for many of the compounds stud-

led.










Filter paper is made up of purified a-cellulose

fibers, a-cellulose is a high molecular weight (50,000

to 500,000) polysaccharide composed of long straight

chains of B-D-glucose arranged in S(1-4) linkages. There

are three free alcoholic hydroxyl groups per sugar unit

in a single chain of cellulose. Cellulose fibers are

organized in bundles of parallel chains, cross-linked

by hydrogen bonding (28,29). Neither D-glucose nor pure

cellulose have an ultraviolet absorption spectra or give

fluorescence or phosphorescence (30). Thus, the phos-

phorescence background must be coming from some by-

product or residue left from the processing of the wood

chips. The problem of reducing blank background can be

approached from several different angles. One approach

is further chemical treatment of the filter paper to re-

move, destroy, or mask the impurity. Another is to find

some other cellulose product with no or little background.

A third is to use something else besides paper for a sub-

strate. A major advantage of filter paper is its superior

handling qualities, inertness to strong alkali, and con-

venience. Other substrates might include silica gel,

alumina, cotton fibers, and glass fibers.











Clinical Applications


New methods of analysis should be attempted on urine

or blood serum using the drugs and metabolites already

shown to give room temperature phosphorescence and others,

in particular, amphetamines, barbituates, etc. Please

note that in serum analysis, room temperature phosphorimetry

offers a technique with almost no interference from normal

blood. The comparison of serum phosphorescence and back-

ground is given in Figure 18. Thus, this method offers

the potential of an easy, rapid, sensitive analysis with

few or no pre-treatment steps.


Air Pollution Applications


Room temperature phosphorimetry appears to have real

use in airborne organic particulate studies. Many organic

compounds could be separated from the airborne particulates

by thin-layer or paper chromatagraphy and then determined

by direct room temperature phosphorimetry. Thus, small

volumes of polluted air could be analyzed quickly. Be-

cause of the carcinogenic potential of many of the air

pollutants, e.g., aromatic amines, ring-carbonyl compounds,

and aldehyde precursors, this application of room temper-

ature phosphorimetry would be important.




















,--,
Z



vi
4-I


OH
414

L0 0







VI a
CCCC








u 4o
S441

n C )
0 Q)U












0 0 0 L)
uo r










o .) -
0o c C
42 C
in 0



















co
00
cC C3

















-6
3 3
Ml'i<^ -




79












0
0


0



0
O


,iISNDINI 13ON-20S&l !JOHd OHd










Other Luminescence Studies


Because these are so many parameters involved in a

study of this nature, it was impossible to study all of

them or even to investigate thoroughly any given one.

One parameter that has not been investigated thus

far is the use of non-aqueous solvents. Theoretically,

if one changes the solvent, then the pH and ionization

can be varied to a different range. In this way, one

could conceiveably change the strength of ionic bonding

to the substrate and control the phosphorescence of the

analyte and its interference.

One of the principal reasons for pursuing this in-

vestigation was the belief that if phosphorimetry could

be inexpensively automated, it would be more readily

accepted by industry and clinical laboratories as a

useful analytical technique. While an automated pro-

cedure was not developed, it should be obvious that it

is a realistic possibility. Further development of an

automatic sampling system could be adapted to the Aminco

spectrophotofluorimeter. The use of an enclosed drying

chamber with controlled humidity should improve the

sample precision.

A better understanding of the theoretical basis for









room temperature phosphorescence is another important

area of future study. A theoretical understanding would

allow systematic optimization of conditions to get the

strongest bonding, greatest rigidity, and highest phos-

phorescence quantum yields.

As reported by Walling and Schulman (15, 16) and

verified in this laboratory, phosphors which are thoroughly

dried exhibit room temperature phosphorescence that is

independent of oxygen quenching. At the present time,

there is no reasonable mechanism explaining how ionic

bonding or any other kind of bonding prevents oxygen mol-

ecules from deactivating the triplet state. Rigidity

alone can not explain this anomaly satisfactorily. For

example, phosphors embedded in solid polymer glasses

gradually lose their luminescence properties at room

temperatures because molecular oxygen permeates the

polymer to quench the phosphorescence of the additive

(31). Therefore, this particular aspect of room temperature

phosphorescence should be considered a very important

project for future study. The understanding of how

oxygen quenching can be prevented could have application

in the other luminescence techniques as well.












CHAPTER VI


SUMMARY


The present study indicates that room temperature

phosphorescence has tremendous potential for analysis of

a wide variety of ionic organic molecules and that it

can be used for quantitative and qualitative identi-

fication purposes. The substances tested are represen-

tative of a wide variety or aromatic carboxylic acids,

amines, thiols, or phenols. Many of the compounds are

of biological importance or pharmaceutical interest.

The findings indicate that this method provides a

sensitive, selective, and accurate means of identifying

certain aromatic molecular species which are ionic. Al-

though there is no strong theoretical explanation for

this phenomenon,it is believed that the ionic state of

the molecules results in great molecular rigidity via

adsorption to the substrate, which reduces radiationless

decay due to collisional deactivation.

Room temperature phosphorimetry offers many advan-

tages as an analytical method. It has good sensitivity

with the potential to be much more sensitive. It is in-

expensive and safe in the sense that liquid nitrogen or

cryogenic equipment is not required. It is convenient

82










and does not require exotic chemicals or high purity

solvents. This technique is a rapid method of analysis

which could be automated. The procedure established

is fairly simple and would be easy for a technician

to learn.

Room temperature phosphorescence definitely merits

future study. The technique developed should find many

applications to real systems, particularly for biological

and pollution samples. Studies that are of importance

for future research include the investigation of better

substrates, automatic sampling, the influence of pH and

solvent, and the analysis of species in blood serum,

urine, and air pollution particles. Finally, it would

seem that time-resolved phosphorimetry should provide

additional selectivity in the measurement of real samples.












APPENDIX

SOURCES AND LOW TEMPERATURE PHOSPHORESCENCE
CHARACTERISTICS OF ANALYTE MOLECULES USED IN
ROOM TEMPERATURE PHOSPHORESCENCE STUDIES.


Excitation Emission References
Compounds Maximum, Maximum,
nm nm

6-benzylamino-purinea 286 413 (32)

6-methyl-purinea 272 405 (32)

6-chloro-purinea 273 419 (32)

6-bromo-purinea 273 420 (32)

6-methylmercapto-purinea 298 445

2,6-diamino-purinea 288 410 (32)

6-amino-purinea (adenine) 278 406 (32)

2-amino-6-methylmercapto 321 456 (32)
purinea

2-amino-6-oxypurinea 285 410 (33, 34)
(guanine)

uracila 270 410 (35)

thyminea 270 430 (35)

cytosinea 270 400 (35)

2-amino-purimidinea 310 399 (36)

2,4-dithio-pyrimidinea 276,(322) 420 (36)

2-thio-6-amino-uracila 285 412 (36)











Excitation
Maximum,
nm


Emission References
Maximum.
nm


4-amino-2,6-dihydroxyl-
pyrimidine

2-amino-4-methyl-
pyrimidinea

2-amino-5-chloro-4-
carboxylic acid-
pyrimidinea

2-amino-4,6-dihydroxyl-
pyrimidinea

4,5-diamino-uracila

5-amino-uracila

6-amino-uracila

2-thio-4,6-djoxy-
pyrimidine

4,6-dihydroxyl-pyrimidinea

sulfanilamidea

sulfathiazolea

sulfamerazinea

sulfamethazinea

sulfaguanidinea

sulfadiazinea

pteroylglu amij acid
(folic acid)

riboflavin (vitamin B2)a

thiamine (vitamin Bl)a


(255),303 408


Compounds


(36)


(36)

(36)


(36)


(36)

(36)

(36)

(36)


(36)

(37,38,39)

(39)

(38)

(38,39)

(38)

(38,39)

(40)


(40)

(40)










Compound


Excitation
Maximum,
nm


calciferol (vitamin D2)a

vitamin Kla

vitamin K3a

vitamin K5a

pyridoxine hydrochloridea
(vitamin B6 HC1)

niacinamidea

4-amino-benzoic acida

vanillinb

epinephrinea

tyrosinea

tryptophan

uridinea

thymidinea
(2-deoxyribose)

cytidinea

adenosinea
a a
guanosinea
norepinephrine
5-acetyl-uracil


Emission
Maximum,
nm


References


(40)

(41)

(41)

(41)

(40)


(40)

(40,42,43)



(44)



(45,46)

(35)

(35)


(35)


aSource: Nutritional Biochemicals Corp., Cleveland, Ohio 44101

Source: Eastman Chemicals, Kingsport, Tennessee 37662

principal maximum, shoulder in brackets.

w = weak
















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17. Jablonski, A., Z. Physzik, 94, 38 (1935).

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19. Sklar, A. C., J. Chem. Phys., 5., 669 (1937).

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23. American Instrument Co., Silver Spring, Maryland,
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29. Hamilton, J. K., and Mitchell, R. L., "Kirk-Othmer
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30. Sober, H. A., ed., "Handbook of Biochemistry,
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33. Udenfriend, S., and Zaltzman, P., Anal. Biochem., 3,
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