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I'

  
   
   

 
 

MEI-2&3}! ‘
Michigan Smw
University

 

ABSTRACT
Molecular Fluorescence Quantum Efficiency as a Function

of Excitation Wavelength, Solute Concentration,
Temperature and Acidity

By

Thomas Francis Majchrowski

With the development of a versatile, computerized spectrofluorimeter,
the effects of various factors on the fluorescence process can be
evaluated in a relatively short time. This study was undertaken to determine
the effects of excitation wavelength, solute concentration, temperature
and acidity on the quantum efficiency of quinine sulfate and anthracene
in various solvents.
The excitation wavelength affected the quantum efficiencies of the
quinine sulfate in acid solutions and the anthracene solutions in ethanol
and in benzene. The quantum efficiencies of the quinine sulfate solutions
in acidic media increased from 20-lOO% with increasing excitation wavelengths
of 3l3-365 nm. The quantum efficiencies of the anthracene solutions increased
from l0-36% with increasing excitation wavelengths from 331-342 nm. The
quinine sulfate solutions in water show no significant dependence of
quantum efficiency with increasing excitation wavelengths from 300-320 nm.
The quinine sulfate system shows no dependence of quantum efficiency
on solute concentration, whereas a dependence seems to exist for the
anthracene solutions. A 20-30% increase in quantum efficiency was observed
with 331 nm exciting light between anthracene solutions of the order of

4

l0- M and lo.5 M. However, problems due to inner-filter effects and

.i

isstru"£nt'
:er'inite CI

Tefpi
a::arent e‘.

.. .4: j
Lil-18C I

:':e :um ne
seen bet-wee.

A“ +'
:CluthflS.

Thomas Francis Majchrowski
instrumental sensitivity and reproducibility must be overcome before
definite conclusions can be made.

Temperature over a relatively narrow range of l3° (23°-l0°) has no
apparent effect upon the quantum efficiencies of any of the solutions
studied.

The acidity of the solvent did affect the quantum efficiencies of
the quinine sulfate solutions. A 20% decrease in quantum efficiency is
seen between the solutions of quinine sulfate in water and the acid
solutions. Comparison between the acid solutions of l.0 N and 0.l N H2504

shows no significant change.

11

MOLECULAR FLUORESCENCE QUANTUM EFFICIENCY AS A FUNCTION
OF EXCITATION WAVELENGTH, CONCENTRATION, ACIDITY AND TEMPERATURE

By

Thomas Francis Majchrowski

A THESIS

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

MASTER OF SCIENCE

Department of Chemistry

1971

The a
Dr. Andrew
for constr
assistance

t: my wife,

ACKNOWLEDGMENT

The author would like to express his appreciation and thanks to
Dr. Andrew Timnick for his assistance in this study, to J. F. Holland
for construction of the spectrofluorimeter, and to D. C. Jones for his
assistance in gathering data. Special appreciation is also expressed

to my wife, Pat, for bearing with me through these times.

TABLE OF CONTENTS

INTRODUCTION

THEORY

FLUORESCENCE
The Process of Fluorescence
The Intensity of Fluorescence
The Kinetic Aspects of Fluorescence
Fluorescence in Relation to Molecular Structure
The Effect of Molecular Structure on Fluorescence Quantum
Efficiency
Other Processes Competing with Fluorescence
The Measurement of Fluorescence
The Excitation Spectrum
The Fluorescence Emission Spectrum
Units for Fluorescence Spectra

FLUORESCENCE QUANTUM EFFICIENCY
Absolute Techniques for Measuring Quantum Efficiency
The Relative Technique for Measuring Quantum Efficiency
Results of Past Work

EXPERIMENTAL
Purification Procedures
Preparation of Solutions
Instrumentation
Experimental Procedure
RESULTS AND DISCUSSION
GENERAL
Dependence of Quantum Efficiency on Excitation Wavelength
Dependence of Quantum Efficiency on Solute Concentration
Dependence of Quantum Efficiency on Temperature
Dependence of Quantum Efficiency on Acidity of Solvent
CONCLUSION
LITERATURE CITED
APPENDICES
APPENDIX I
APPENDIX II

APPENDIX III

86
88
91

l-\

Table I.

Table 11. l
Taale 111. l
C
Table IV. I
a
Table V. T
R
Table VI. 7.
R<

Table VII. Tl

Table VIII.“

0.
Tab16111. Th
Tablex. The
Table“. Th

Table “I, The
Sul
a k

1.0

Table
“V- Typl

LIST OF TABLES

Table I. Comparison of Quantum Efficiencies

Table II. The Quantum Efficiency of Quinine Sulfate in
1.0 N H2504 at Room Temperature

Table III. The Quantum Efficiency of Quinine Sulfate in
0.1 N H2504 at Room Temperature

Table IV. The Quantum Efficiency of Quinine Sulfate in Water
at Room Temperature

Table V. The Quantum Efficiency of Anthracene in Ethanol at
Room Temperature

Table VI. The Quantum Efficiency of Anthracene in Benzene at
Room Temperature

Table VII. The Quantum Efficiency of Quinine Sulfate in

Table VIII.

Table IX.

Table X. The Quantum Efficiency of Anthracene in Ethanol at 10°C

1.0 N H2504 at 10 C

The Quantum Efficiency of Quinine Sulfate in

0.1 N H2304 at 10°C

The Quantum Efficiency of Quinine Sulfate in Water at 10°C

32

58

59

60

64

65

73

74

76
77

Table XI. The Quantum Efficiency of Anthracene in Benzene at lO°C

Table XII. The Comparison of the Quantum Efficiencies of Quinine
Sulfate in 1.0 N H2504 Between Integration Performed over
a Wavelength Interval and a Frequency Interval

Table XIII.Typical R-S Absorption Values for Quinine Sulfate in

1.0 N H SO

2 4
Table XIV. Typical R-S Absorption Values for Anthracene in Ethanol

LIST OF FIGURES

Figure Page
1. The Process of Absorption and Luminescence 7
2. Reciprocal Rates of Transition lO
3. A Typical Spectrofluorimeter 20
4. The Basic Components of the Spectrofluorimeter 46
5. The On Line Fluorescene System 47

6. Typical Excitation and Absorbance Spectra of Quinine Sulfate

Solutions 55
7. Typical Excitation and Absorbance Spectra of Anthracene

Solutions 56
8. Dependence of the Quantum Efficiency of Quinine Sulfate in

1.0 N H2504 on Excitation Wavelength and Solute Concentration 61
9. Dependence of the Quantum Efficiency of Quinine Sulfate in

0.1 N H2304 on Excitation Wavelength and Solute Concentration 62
10. Dependence of the Quantum Efficiency of Anthracene in Ethanol

on Excitation Wavelength and Solute Concentration 66
ll. Dependence of the Quantum Efficiency of Anthracene in Benzene on

Excitation Wavelength and Solute Concentration 67
12. Dependence of the Quantum Efficiency of Quinine Sulfate in

1.0 N H2804 on Temperature 78
13. Dependence of the Quantum Efficiency of Quinine Sulfate on

Acidity of the Solution 79

14. Typical Relative Emission Spectra of Quinine Sulfate Solutions 89

15. Typical Relative Emission Spectra of Anthracene Solutions 90

II S

nor any

Man has
existence on
tne aurorae.
daring the P
Caoes). ln '
mentioned. I
of light fro:
It wasn't uni
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Studies of th
Robert Boyle i
filth white lig
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incident light
the solution 1!

10”99" wavelem

INTRODUCTION

. some things though they are not in their nature fire
nor any species of fire, seem to produce light.” (1)

Aristotle

Man has observed luminescent processes since the beginning of his
existence on earth. Early man was undoubtedly awed by lightning and
the aurorae. The first written reference to luminescence was published

during the period 1500-1000 B.C. in the Chinese Shih Ching(Book of

 

Oades). In it the mysteries of the firefly and the glowworm are
mentioned. Around 300 B.C. Aristotle wondered about the strange emission
of light from fish as is shown by the above excerpt from his writings.

It wasn't until after the Dark Ages, however, that man began to study

the phenomenon to gain a better understanding of it.

About 1600, Nicolas Monardes wrote of the unusually intense light
blue color of an aqueous extract of wood called "lignum nephriticum“.
Studies of this solution by Athanasius Kircher, Francesco Grimaldi,
Robert Boyle and Isaac Newton led to the conclusion that upon illumination
with white light the solution was blue by reflection and yellow by
transmission. Not until 1852 did George Stokes identify the light as
luminescence. Stokes used optical filters and prisms to show that
incident light of one spectral region was absorbed and transformed by
the solution into an emitted light of a different spectral region of

longer wavelength. He observed that this luminescence emission terminated

abruptly w‘r
e’ission fr
phenomenon

The lun
Cascariolo
and discove
that the st
the“ glowec
after Casca
nave Cert-e t
glowed 10ng
‘0 be Calle

In 1888
between flu.
to 1Indude |
Phenomena 0‘
l2).

Untj] Qt
DropErtieS n
the fie]
fl

d We
domphotom
exact Stu die
fluorimetry .
moresCent s
to fluor‘i’Scer

could be Stuc

2
abruptly when the incident light was removed. This was similar to the
emission from the mineral fluorspar. Stokes therefore called the
phenomenon fluorescence.

The luminescence of solids was first reported in 1603 by Vincenzo
Cascariolo of Bologna. He heated a powdered natural barite with coal
and discovered that the cooled porous cake glowed at night. He found
that the stone apparently absorbed light from the sun during the day and
then glowed for hours in the dark. Many men studied similar solids
after Cascariolo. Since then, microcrystalline solid luminescent materials
have come to be called phosphors. Because Cascariolo's original phosphor
glowed long after excitation, long-lived luminescence emission has come
to be called phosphorescence.

In 1888 a German physicist, Eilhard Wiedemann, noticed similarities
between fluorescence and phosphorescence and introduced the term luminescence
to include both phenomena. He defined luminescence as "all those
phenomena of light which are not caused solely by a rise in temperature"
(2).

Until quite recent times, the instrumentation for studying luminescent
properties was not fully developed and hence most of the knowledge in
the field was gained by non-exact observations. With the development of
fluorophotometers and spectrofluorimeters, scientists could perform more
exact studies concerning the phenomenon. The first applications of
fluorimetry to analysis dealt with the determination of naturally
fluorescent substances. Later, with the discovery of reactions leading
to fluorescent products, many substances which normally did not fluoresce
could be studied. As the instrumentation and technique improved, more

was learned. The effects of such factors as solute concentration,

3
solvent, temperature, acidity, excitation wavelength, impurities and
photodecomposition led to a better understanding of the process.
With the advent of quantum theory, the energy exchange processes
involved in fluorescence could be explained. To account for the
efficiency of the emission-absorption process, Einstein considered all

the individual factors which contribute to the efficiency. Simply

stated, he defined the quantum efficiency as the ratio of the energy
emitted to the energy absorbed. Scientists, thus by measuring the
number of quanta of energy emitted and absorbed in fluorescence under
varying conditions, could study the quantitative effects of these variables.
Initially it was believed that the quantum efficiency for a particular
transition was constant when concentration or excitation wavelength was
varied. The early works of Melhuish (3) and Weber and Teale (4,5)
seemed to verify this. Those few results which introduced doubt about
the accepted theory were usually discredited because of proposed experimental
errors. Lately, the accepted conclusions of these early workers have
again been questioned. Chen (6,7), Borresen (8), Moss (9), and
Eisenbrand (10), along with others, have detected possible errors in
the earlier findings and propose that variables such as concentration,
solvent polarity, temperature and excitation wavelength affect the quantum
efficiency. Presently, those such as Fletcher (11,12,13), and Dawson
and Windsor (14) believe that the earlier results are still valid within
experimental accuracy, and that those disagreeing are overlooking certain
factors, including chemical impurities, the various mechanisms for
deactivation of the excited state and instrumental parameters.
Since a versatile, computerized spectrofluorimeter has recently been
developed, the effects of various factors on the fluorescence process can

be evaluated in a relatively short time. This study was undertaken to

4
determine the effects of solute concentration, acidity, temperature,
and excitation wavelength on the quantum efficiency of quinine sulfate

and anthracene in various solvents.

FLJORESCENL

The Process
Lumine
bv a noiecu

excited sin
excited tri
decay of th

When a
WHY 01‘ fl
light that '
S‘Jbstance w‘

however, doe

Whereh ls P
light. FOr
Sufficient t
this State,

kinetic, 0r '

At room

ground Sta te

Singlet State

000nm] abSOl

THEORY

FLUORESCENCE

The Process of Fluorescence

 

Luminescence is most conveniently defined as the radiation emitted
by a molecule, or an atom, after it has absorbed energy to rise to an
excited singlet or triplet state. Luminescence due to decay of an
excited triplet state is called phosphorescence, while the more rapid
decay of the excited singlet state is called fluorescence.

When a beam of light passes through a substance, it may either be
partly or fully absorbed, reflected, transmitted, or scattered. The
light that is reflected, transmitted, or scattered goes through the
substance with a minimum of interaction. The light that is absorbed,

however, does so in discrete units of energy or quanta
E = hv (I)

where h is Planck's constant and v is the frequency of the absorbed
light. For visible or ultraviolet light, the energy of these quanta is
sufficient to raise an electron in a molecule to an excited state. From
this state, the energy may be converted into rotational, vibrational,
kinetic, or chemical energy, or re-emitted as quanta of lower energy.

At room temperature, the most highly populated state of a molecule
is the lowest vibrational level of the ground electronic state. In the
ground state of most molecules each electron is paired with another in a
singlet state, So. When light, or radiant energy, of a frequency within
a normal absorption band of a molecule is absorbed, one of the paired

5

6

electrons is raised to the first excited singlet state, S], or possibly
to a second excited state, 82, with or without a change in the vibrational
energy level. The excess vibrational energy of the excited molecule is
usually dissipated through a radiationless process so that the molecule
drops back to the lowest vibrational level of the first excited singlet
state. From this state, the electrons may return to the various vibrational
levels of the ground state by emitting light of less energy than was
absorbed. This process is normal fluorescence. If after excitation no
energy is lost before emission, the light emitted is of the same frequency
as that of the light absorbed. This process is known as resonant
fluorescence.

If all the electrons originally excited by absorption of radiant
energy return to the ground state by emission of fluorescence, then one
quantum of fluorescent radiation is emitted for every quantum of radiation

absorbed. The quantum efficiency of this process

quanta emitted (2)
quanta absorbed

 

¢f

is unity. A portion of the excited electrons may return to the ground
state by other processes such as conversion to the triplet state, by
collision with solvent molecules or with molecules of another solute,
or possibly by photochemical change. The quantum efficiency of this
process is then less than unity. Figure l is a schematic energy level
diagram showing the possible processes which may occur on absorption of
radiation.

The intensities of various fluorescence bands vary considerably due
to the position of the nuclei of the atoms in the molecule at the moment
0f absorption or emission. The actual time required for photon

~15

absorjation is 10 seconds. This is relatively short compared to the

 

 

Triplet
State T1

Figure 1.

A = absorption of light

 

 

"in

 

 

 

 

 

 

 

 

 

 

 

 

 

Vv

VI.

V»

(t.
<‘v

B = radiationless deactivation, i.e., quenching

Second excited state

52

First excited state

51

Ground State

So

The Process of Absorption and Luminescence (15)

C = rapid radiationless transitions, i.e., internal conversion

0 = fluorescence

E = singlet-triplet crossover

= phosphorescence

= triplet quenching processes

tine require
occur. T'ner
geometry and
n fins cond
fitrational

vibrational

Horational l
sdution, the
rafid througf
be solvent n
etess vibrat

mxurs in 10

The inter
by definition,

Qaanta, mUItTp

Where
F = the tc
Io the in
= the co
= the ce'
c = the mo]

P: the flu

8
time required for vibrational levels to change, and for nuclear motion to
occur. Therefore, immediately after excitation, a molecule has the same
geometry and is in the same environment as it was in the ground state.
In this condition, the molecule can either emit a photon from the same
vibrational level to which it was excited or it can undergo changes in
vibrational levels before emission. Usually the molecule undergoes
vibrational deactivation before emission. This occurs because, in
solution, thermal relaxation of a vibrationally excited molecule is quite
rapid through transfer of excess vibrational energy from the solute to
the solvent molecule. This process is so efficient that all of the
excess vibrational energy of the excited state is lost. The process

-13 -11

occurs in 10 to 10 seconds.

The Intensity of Fluorescence

 

The intensity of fluorescence emission, for the ideal case, is,
by definition, equal to the intensity of the light absorbed, measured in

quanta, multiplied by the fluorescence quantum efficiency
F = [Ion-io'ebcna (3)

where
F = the total fluorescence intensity in quanta per second
I = the intensity of the exciting light in quanta per second
c = the concentration of the solute in the solution
b = the cell path length in a linear unit
8 = the molar absorptivity

o = the fluorescence quantum efficiency,

conside‘

’te Klm

Tne
lifetimé
Figure 2
kinds oi
for the
with ful

lni
ground 5
are con:
STStems
CONVErSj
DIESEnt)

The
10‘5
which oc.

which 0c<

9
In dilute solutions, where only a small fraction of the exciting
light is absorbed, and self-absorption of the fluorescence radiation is

considered to be negligible, Equation 3 is simplified to
F = [10(2.3 ch)¢]. (4)

For any given solute, when instrumental geometry, exciting frequency,
intensity, temperature, and solution composition are kept constant, the
detector response is directly proportional to the concentration of the

solute since
F = KC . (5)

The Kinetic Aspects of Fluorescence

 

The process of fluorescence is intricately dependent upon the
lifetimes, transition rates and rate constants of many other processes.
Figure 2 is a simplified scheme showing the various excited states, the
kinds of transitions possible, and the appropriate mean time, in seconds,
for the occurrence of these processes. Radiative transitions are shown
with full lines and non-radiative processes are shown with dotted lines.

Internal conversion from the lowest excited singlet state to the
ground state and intersystem crossing from upper excited singlet states
are considered negligible competition with fluorescence emission. Some
systems do not attain a triplet state in solution. The extent of internal
conversion from the lowest excited singlet state to the ground state is
presently not known.

The range in the lifetimes of the fluorescence transition 10- to
10.6 sec., is due to competition between the allowed n*+n transitions,

9

which occur in about 10' sec., and the less probable n*+n transitions,

which occur in about lO- sec. The size of the singlet—triplet split,

n
"(io‘

a =1ic
n' = ini
n = int
the

9' = int
“PP

9: int
the
kom = QUEI
f: fluc

p = Dhos

10

 

 

 

 

 

 

, i T 52
I 0
n. I
42) l
l
'7 -9 l I ‘“ S1
9 (10 ) ' '
P (10'2 to : kQ[Q] l n (e) f (10'9 a (10“5)
10+2) l l to 10-6
.L l. s:
SO

Figure 2. Reciprocal Rates of Transition (17)

light absorption

internal conversion from upper excited state

internal conversion from the lowest excited singlet state to
the ground state

intersystem crossing from upper excited singlet states to the
upper triplet state

intersystem crossing from the lowest excited singlet state to
the lower triplet state

quenching processes

fluorescence

phosphorescence

a phenomeno
5-, transi
have small
the greater
not result
Except
first order
reciprocals
has a suffi
ouencning b
processes.
often much
be consider
wiH be kQ[
the concent
In the
Will'ch the r

51'”9191‘. Sta

In this equ.

FEdSUred in
quanta_

If [S]:
Steady state

There the k'

11
a phenomenon to be discussed later, controls this competition. The
n*+n transitions have large singlet—triplet splits. The n*+n transitions
have small singlet-triplet splits. The smaller the singlet-triplet split,
the greater is the probability of intersystem crossing which may or may
not result in phosphorescence emission.

Except for light absorption, most of the processes considered are
first order reactions. These processes have rate constants equal to the
reciprocals of the mean times quoted. The lowest excited singlet state
has a sufficiently long lifetime to permit chemical reaction or luminescence
quenching by encounter with other molecules. These are second order
processes. However, since the concentration of quenching molecules is
often much greater than that of the excited molecules, the process can
be considered pseudo-first order. For this process, the rate constant
will be kQEQ], in which kQ is the bimolecular rate constant and [Q] is
the concentration of quenching molecules.

In the process of fluorescence emission a steady state develops in
which the rate of production of excited molecules in the first excited

singlet state is equal to the rate of light absorption, Ia

Ia = const = 2300 ecIO. (6)

In this equation, Ia is measured in einstein liter-1 sec"1 and I0 is

measured in einstein cm'2 sec—2. One einstein is equal to 6.023 X 1023
quanta.
If [5]] is the concentration of excited singlet molecules when the

steady state has been achieved
Ia = (kf + kn + k9 + 2kQ[Q])[S]] (7)

where the k's are the first order rate constants for processes already

described a
by various 1
The in
Mstributio
rxlecules i
investigate
sdute conc
except the

first order

The to

F*='
where ; '
'f 15

Rearrai

NhEn El

Mthljfetir
Since 1
then

THIS 15 anot

12
described and £kQ[Q][s]] is the sum of the rate of disappearance of S]
by various quenching processes.

The internal conversion rate represented by kn accounts for energy
distribution throughout the solute molecule as well as the solvent
molecules in close contact with the solute. If it is desired to
investigate quenching by only one particular solute, the remaining
solute concentrations must be kept constant. Then, all the kQ[Q] terms,
except the one for the quencher being studied,are included in a composite

first order rate constant kn'. In this case
q=<c+kn+kg+qmnmpo (m
The total rate of emission, F*, is
* = = : '
F kdhl q” (n+kn+kg+qmnmp% o)
where of is the fluorescence quantum efficiency.

Rearranging Equation 9, the fluorescence quantum efficiency is
af=chf+g +g+k&M) om

When excitation is terminated, fluorescence decays exponentially

with lifetime T, and

r =1/(kf+kn' +kg+kQIQl) (H)

Since the mean radiative lifetime for fluorescence is

T' 1/k

f on

then

This is another form of expressing fluorescence quantum efficiency (18).

luoreSCence
/_____

SeveFBI
fluorescence
First: a
of resonant S
iluoresces.
Second]!
absorfltlon be
as hydrogen b
spectrum 0CCU
spectrum. Th‘
shows this er”
As the number
to nine (naph‘
ultraviolet tc
Thirdly,
solution is us
giving fluores
Compounds havi
show a very lo
transitions ar
the first exci
l

ifetime than

tends to enhan.

...t -
n tranSitior

Splits. This ;

triplet splits

Unsubsti tuted a

13

Fluorescence in Relation to Molecular Structure

 

Several general rules which interrelate molecular structure with
fluorescence can be stated (19).

First, a multiple-conjugated double bond system with a high degree
of resonant stability is an absorbing structure which frequently
fluoresces.

Secondly, increased conjugation shifts the longest wavelength
absorption band to longer wavelengths. In the absence of effects such
as hydrogen bonding or ionic dissociation, the fluorescence emission
spectrum occurs immediately to the long wavelength side of the absorption
spectrum. The series benzene, naphthalene, anthracene and naphthacene
shows this emission shift to longer wavelengths with increasing conjugation.
As the number of conjugated double bonds increase from three (benzene)
to nine (naphthacene), the fluorescence emission curve shifts from the
ultraviolet to the infrared.

Thirdly, the high efficiency of prompt fluorescence in liquid
solution is usually associated with compounds having the n*-n transition
giving fluorescence emission from the first excited singlet state.
Compounds having the n*-n transition from the first excited singlet state
show a very low efficiency of prompt fluorescence. Because the n-n*
transitions are less intense than the n-n* transitions for excitation,
the first excited state resulting from the n-w* transition has a longer
lifetime than the state resulting from the n-n* transition, a factor which
tends to enhance intersystem crossing from the state resulting from the
“-n* transition. The n*-n transitions also have small singlet-triplet
splits. This phenomenon will be discussed later. However, small singlet-
triplet splits also increase the probability of intersystem crossing.

Unsubstituted aromatic hydrocarbons all undergo the n-n*, n*-n transitions.

They almos
mentiOHEd
When
an n-r" tr
depends, i
which cont
non-polar
In this ex
states of
transition
Subst
electrons,
Electron w
reduce or 1
'i-.-* trans'
ketones, ca
“95le flu:
Elect!
ellhance TM
fluorescem;
h"llll'd $010
Finall
increases f
and HUOI‘es.
60d SimIIar
joined by M

FluoreSCei n

14
They almost all show some degree of fluorescence. The benzene series,
mentioned previously, is an example of such a system.

When a hetero atom is introduced into a ring, the excitation may be
an n-n* transition. Whether the n-n* or n-n* transition predominates
depends, in many cases, on the polarity of the solvent. For quinoline,
which contains a ring nitrogen atom, the n-n* transition predominates in
non-polar solvents whereas in polar solvents the n-n* transition predominates.
In this example, the emitted fluorescence originates from the excited
states of two different electrons, with the electron involved in the
transition dependent on the polarity of the solvent.

Substituent groups, such as carbonyl with its non-bonded pair of
electrons, may introduce the n-n* exciting transition in a molecule.
Electron withdrawing groups, such as the carboxylor nitrate group, may
reduce or completely eliminate fluorescence emission due to reduced
n-n* transitions in the aromatic ring. Thus, simple aromatic aldehydes,
ketones, carboxylic acids, and amides are either non-fluorescent or
weakly fluorescent.

Electron-donating groups, such as phenolic hydroxyl or amine,
enhance n-n* transitions in aromatic rings and therefore increase
fluorescence emission. Most phenols and aromatic amines fluoresce in
liquid solution.

Finally, it is often stated that an increase in molecular rigidity
increases fluorescence efficiency in liquid solution. Phenolphthalein
and fluorescein display this effect. Both contain three benzene rings
and similar structural units, but two of the rings in fluorescein are
Joined by an oxygen bridge which introduces more rigidity in the molecule.

Fluorescein fluoresces while phenolphthalein, without the oxygen bridge,

doeS "0"

redUCes 10‘
.- e ct
fiEL£4£E—-'

The Q

:r kf is m
efficienC)’
greater th

The r
transition
Any factor
band will .
excited Sll
maximum mo'
intensity.
in the life
quantum eff
'n'hen symmet
long wavele

decrease in
The ra‘
the extent c

the lowest e

jt' Intersy

f0”16W forl

n" u

the triplet s

15
does not. The increased rigidity minimizes internal vibration and thus

reduces loss of energy from the excited state by internal conversion.

The Effect of Molecular Structure on Fluorescence Quantum Efficiency

 

The quantum efficiency of fluorescence is given by Equation 10

¢f = kf/Q‘f + kn. + k9 + kQEQD' (10)

If kf is much greater than the other rate constants, the quantum
efficiency will approach unity. If the other rate constants are much
greater than kf, the quantum efficiency will approach zero (20).

The rate constant, kf, is a measure of the probability of an electronic
transition between the lowest excited singlet state and the ground state.
Any factor affecting the intensity of the longest wavelength absorption
band will also affect the rate of spontaneous emission from the lowest
excited singlet state. Since the intensity is dependent upon the

maximum molar absorptivity, c , as c

max max changes, so will the emitted

intensity. As Emax decreases, there will be a corresponding increase

in the lifetime of the excited state and a decrease in kf. Since the
quantum efficiency is directly proportional to kf, it would also decrease.
When symmetry and/or orbital overlap restrictions are introduced into the
long wavelength transition, the value of Emax should decrease. A

decrease in quantum efficiency would likewise be expected.

The rate constant kg, the rate of intersystem crossing, depends upon
the extent of singlet-triplet mixing and the energy separation between
the lowest excited singlet state and the triplet state immediately below
it. Intersystem crossing in which there is a change of multiplicity is
formally forbidden by spectroscopic selection rules. Considerable state

mixing does occur, however, in organic molecules. For these molecules,

the triplet states are not pure, but have some degree of singlet

character. The
state, the more
Besides state r
split. The 5mg
greater will be
arises because
coupling betwel
vibrational Te.
SPlit is small
for a transiti.
interSystem Cr.
is large, a 13
case, the Prob.

The Exten
dependent uDOn
Spin'Orbltal c
SjngIEt‘trlple
with the “61d
along With the
so-called "hea
conditiOnS, tn

Another j
rate of intErg‘
spins win Enh.

reductj0n in f

TemPEratUl

16
character. The greater the amount of singlet character in the triplet
state, the more probable the process of intersystem crossing becomes.

Besides state mixing, another factor affecting k is the singlet-triplet

9
split. The smaller the energy difference between the two states, the
greater will be the probability of intersystem crossing. This situation
arises because intersystem crossing is brought about by vibrational
coupling between a vibrational level of the triplet state and the lowest
vibrational level of the excited singlet state. If the singlet-triplet
split is small, only a modest change in nuclear configuration is required
for a transition to occur between the two states. The probability of
intersystem crossing is large in this case. If the singlet-triplet split
is large, a large nuclear configurational change is required. In this
case, the probability of intersystem crossing is small.

The extent to which singlet-triplet mixing occurs in a molecule is
dependent upon the electromagnetic field surrounding an electron.
Spin-orbital coupling becomes larger with an increasing potential field.
Singlet-triplet mixing should also increase. If a n electron interacts
with the field near the nucleus of a heavy atom, singlet-triplet mixing,
along with the rate of intersystem crossing, will increase. This is the
so-called "heavy atom effect” in molecular spectroscopy. Under these
conditions, the fluorescence quantum efficiency decreases.

Another interesting effect is that of a paramagnetic species on the
rate of intersystem crossing. In general, species with unpaired electron
spins will enhance intersystem crossing. This results in a corresponding
reduction in fluorescence intensity and fluorescence quantum efficiency.

Temperature is an important factor affecting the rate of radiationless

deactivation of an excited state. During the lifetime of an excited

state, a molecc
solution. In 5
is the temperai
is increased.

of excitation 4

decrease.
Ether Processes

An excitec
other than that
include interns
discussed. 39“
Quenching, and

Collisiona
is a bimolecu]a
holecrjle and ti
rate of dlffusi
The dIIIUSiOn (
Sph“Tl-C511 molec

SOlVent and IS

VhEre R is the
in ”1595 (is).
the species 10v

There are
energy may OCCU

electron transf.

17
state, a molecule will undergo many collisions with other molecules in
solution. In some of these collisions there is an exchange of energy.
As the temperature of the solution is raised, the number of collisions
is increased. This may increase the probability of radiationless loss
of excitation energy. The fluorescence quantum efficiency then may

decrease.

Other Processes Competing with Fluorescence

 

An excited state may lose acquired energy by several processes
other than that leading directly to fluorescence emission. These processes
include internal conversion and intersystem crossing, processes already
discussed. Several other processes include collisional and noncollisional
quenching, and photochemical reactions.

Collisional quenching, sometimes referred to as external conversion,
is a bimolecular process which depends on the contact between the excited
molecule and the quencher. It is a diffusion controlled process. The
rate of diffusion of any species depends on the viscosity of the medium.
The diffusion controlled collision rate constant for the case of large
spherical molecules depends on the temperature and viscosity of the

solvent and is given by
kc = 8 RT/3000 n (14)

where R is the gas constant, T is the temperature andri is the viscosity
in poises (16). If a process is diffusion controlled, the lifetime of
the species involved must be greater than 10'9 seconds.

There are two mechanisms by which collisional loss of excitation
energy may occur: enhancement of intersystem crossing by the quencher or

electron transfer (21).

Oxygen and
of intersystem CI
to occur by fonne
transfer charactl

The overall reaC‘

in which 1A" TS ‘
paramagnetic quer

The concept
The excited moler
The ion pair can
in dissociation,
may be formed.

In a polar s
undergo character

my be Sumarlled

F*

mm” H is th.

Noncomsim
coupling in'CEraCt-
If the d0n0r and a
energy transfer he

one emits. If the

equihIbii'um state

energy has been tr

18
Oxygen and other paramagnetic species quench fluorescence by enhancement
of intersystem crossing. The actual intersystem crossing process appears
to occur by formation of a transitory complex of pronounced charge
transfer character between the excited solute and the quencher (22).
The overall reaction is believed to be as follows

1A* + 302 + (n+0 ‘

2 )* 3 3

_, *-
A + 02

in which 1A* is the excited solute and oxygen is a representative
paramagnetic quencher.

The concept of quenching by electron transfer is relatively new (23).
The excited molecule can react with the quencher to form an ion pair.
The ion pair can then dissociate and dissipate the excess energy thermally.
0n dissociation, either singlet or triplet ground states of the molecule
may be formed.

In a polar solvent the radical ions F' and 0+ can be solvated and
undergo characteristic radical ion reactions in solution. This mechanism

may be summarized by the following sequence of reactions

- + solvent - +
F* + Q T F '0 F solv + Q solv (16)
g/ \.
3
F + o F + Q

in which F* is the excited solute and Q is the quencher.

Noncollisional energy transfer most likely arises from a vibrational
coupling interaction between the excited states of the donor and acceptor.
If the donor and acceptor have a common vibrational frequency, resonant
energy transfer may occur. This transfer may occur back and forth until
one emits. If the acceptor has a lower vibrational level than the
equilibrium state of the donor, there will be no resonance. Once the

energy has been transferred to the acceptor it can undergo rapid internal

conversi 0‘

its excitr

deactivat
The

efficienc,

In this e

Stern-Vol
Cher
either re
which is
in their
deoendenc
DTEdomina
is usual]
Cherhical
Enough En
bOnds. T
Interacti
entities.

W

The

ShOwn in

PUrpOSe Si

19
conversion to the lowest vibrational level. The acceptor will remain in
its excited state until it either emits fluorescence or undergoes another
deactivation process.
The Stern-Volmer quenching equation gives the ratio of quantum
efficiency without quencher to that with quencher. It is
¢f° kQIQl

___.= 1 + = l + k[Q] (17)
f kf + kn + k9

 

In this equation ¢f° refers to the quantum efficiency determined without
quenching and of is the quantum efficiency with quenching. k is the
Stern-Volmer quenching constant (16).

Chemical reactions also compete with fluorescence. These may be
either reversible or irreversible reactions. An excited state reaction
which is reversible is the ionization and dissociation of molecules
in their excited singlet states. These molecules may show a strong pH
dependence. As the pH is varied one ionic form or the other will
predominate. The fluorescence emission from each of these ionic forms
is usually quite different. One of the most important irreversible
chemical reactions is photodecomposition. This process occurs when
enough energy is absorbed by the molecule to break internal molecular
bonds. The process is enhanced if the solvent molecules are capable of
interacting with the photodecomposition products to form other stable

entities.

The Measurement of Fluorescence

 

The measurement of fluorescence requires a system similar to that
shown in Figure 3. This figure shows a schematic diagram of a general

purpose spectrofluorimeter.

20

® detector

“Ti——

a,

4L ‘

4—... | sample with fluorescence
emission F7 i

monochromator

¢
“10 (— O "9 source
l

excitation monochromator

 

 

 

 

 

 

Figure 3. A Typical Spectrofluorimeter

This SYS'
visible and u
to select the
emission mono
analyze the e
to the excita
exdting ligh

Using th
enission spec
to the wavele
be exciting
the excitatio
enission mono

When a s
slit width, t
not only of t
being measure
Such apparent
Spectra. The

made With res

1h - .
‘E-EEELEQEIE

frequencsr of

ire
QUEIlcy' Tl

.nonochmma tor

21

This system consists of a source of polychromatic light covering the
visible and ultraviolet regions of the spectrum, an excitation monochromator
to select the required frequency for excitation, a sample container, an
emission monochromator and a photomultiplier-detector combination to
analyze the emitted light. The fluorescence is analyzed at right angles
to the excitation beam in order to overcome interferences from the
exciting light.

Using the system described in Figure 3, either an excitation or
emission spectrum can be obtained. By setting the fluorescence monochromator
to the wavelength of a fluorescence band and varying the wavelength of
the exciting light, the excitation spectrum is obtained. By setting
the excitation monochromator at an absorbing wavelength and varying the
emission monochromator, the emission spectrum is obtained.

When a simple, single-beam spectrofluorimeter is used at constant
slit width, the excitation and emission spectra obtained are a function
not only of the absolute fluorescence characteristics of the substance
being measured, but also of the characteristics of the particular instrument.
Such apparent Spectra are frequently gross distortions of the true
spectra. These spectra have to be corrected before comparisons can be

made with results from other instruments.

The Excitation Spectrum

 

The apparent excitation spectrum is a plot of Ioeof against the
frequency of the exciting light. To obtain the true excitation spectrum,
the intensity of the exciting light must be determined as a function of
frequency. This may be accomplished by shining a source of known intensity
from a standard lamp through the entrance slit of the excitation

monochromator and monitoring the intensity of the selected and known

frequency
device SUC
actinonete
The outpul
the number

screen an
The FTUOH

The a
photomult‘
through tl
because 01
tube with
within the
The actua‘,
to Obtain

The n
by the fol
directed i
Proceeds t
ilght is t
counter.
for the Se'
the deSirec
by a Ser‘ies
resphnSe Cu

OTTCe U

22
frequency transmitted through the monochromator with some calibrating
device such as a sensitive thermopile, a calibrated phototube, an
actinometer or a fluorescent screen of constant quantum efficiency.
The outputs from the thermopile or the phototube must be converted to
the number of quanta whereas outputs from the actinometer and the fluorescent

screen are directly related to the number of quanta incident on them.

The Fluorescence Emission Spectrum

 

The actual emission spectrum obtained from the output of the
photomultiplier tube which monitors the intensity of the light coming
through the exit slits of the emission monochromator is usually distorted
because of the variation of the quantum sensitivity of the photomultiplier
tube with frequency or wavelength, the wavelength dependent light losses
within the monochromator, and the varying band width of the spectrometer.
The actual emission spectrum must be corrected for these three factors
to obtain the true emission spectrum.

The monochromator-photomultiplier assembly response can be calibrated
by the following procedure. Radiation of the desired frequency is
directed into the monochromator through a slit of known width and
proceeds to the photomultiplier tube where the output is recorded. The
light is then directed into an actinometer or some other form of quantum
counter. Thus the number of quanta emitted from the lamp is obtained
for the selected frequency. This procedure is repeated stepwise through
the desired frequency range. Then the output of the phototube is corrected
by a series of multipliers derived from the manufacturer's phototube
response curve to yield a constant reading at all frequencies.

Once the assembly is calibrated in this manner, the true emission

spectrum can be obtained.

“MB for F1007“

Parker (15
interval rather
spectrum. Firs
spectrum obtain
relationship to
vavelength rath
emission bands .
region. Finall;
area under the <
efficiency. Th1
less energy.

Although tr.
frequency give n

unit may not be
Energy is direct

where n is the nc

not directly PFOF
htpl

“V9 ‘v‘ by thl

in Equation 20 g”

23

Units for Fluorescence Spectra

 

Parker (15) stated three reasons for using quanta per unit frequency
interval rather than energy per unit wavelength interval for the emission
spectrum. First, in using energy per unit wavelength, the fluorescence
spectrum obtained is a distorted version. It does not bear the correct
relationship to the corresponding absorption band. Secondly, when
wavelength rather than frequency is used for the horizontal scale, the
emission bands are unnaturally bunched together in the short wavelength
region. Finally, when energy units rather than quanta are used, the
area under the curve is not proportional to the fluorescence quantum
efficiency. This occurs since quanta of lower frequency carry proportionately
less energy.

Although the emission curves plotted using the unit quanta per unit
frequency give nice symmetrical curves in many cases, the use of this

unit may not be totally correct. According to Equation 1
E = hv. (1)
Energy is directly proportional to frequency. Quantizing Equation 1 gives
E = nhu (18)

where n is the number of quanta. In this case the number of quanta is

not directly pr0portional to the frequency, v. Rearranging Equation 18,

-L
n-hv- (19)
Replacing v by the relationship
v =.§ (20)
in Equation 20 gives
_ EX

 

24

The number of quanta is directly proportional to the wavelength. Therefore,
it seems that if the fluorescence is measured in terms of quanta, then
the horizontal scale should be in units of wavelength for the emission
spectrum.

However, within the experimental accuracy thus far attained in
quantum efficiency studies, there appears to be no difference in quantum
efficiency values when the emission is plotted either in terms of frequency

or wavelength.

FLUORESCENCE QUANTUM EFFICIENCY
Absolute Techniques for Measuring Quantum Efficiency

As stated previously in Equation 2, fluorescence quantum efficiency
is defined as the ratio of quanta of light emitted to the quanta of
light absorbed. Though this relationship is quite simple, it is difficult
experimentally to determine the quantum efficiency precisely of many
fluorescing substances.

Through the years, there have been many methods developed for
measuring fluorescence quantum efficiency. The most direct method, and
the first to be applied, was that of Vavilov (24). This method involves
the comparison of the intensity of the fluorescence emitted from the
front surface of a sample with the intensity of the exciting light
scattered from a completely reflecting matt surface.

Forster and Livingston in 1952 enclosed the sample in an integrating
sphere (25). The sphere was adapted to the measurement of quantum
efficiency. The intensities of the fluoresced and absorbed light were
determined separately with a thermopile. The quantum efficiency was

determined from the relationship

 

 

where

F:
IF =
GE and
GF =
e
35 and
ant
A = t
s:
2E and
}‘F=ti
Gilmor

standard sca

compare fluo

quanta reach'
surface, The
over the samp
“”hga monoc
The emission 1
Used as the N
fbrthe transn
iihh and the

Quantum Efficie

 

 

 

 

where
F = the change in thermopile response corresponding to the
substitution of the solvent cell for the sample cell

IF = the intensity of fluorescence

(D
II

F the transmission coefficients of the thermopile filter for the

exciting and fluoresced light

aF = the molar absorptivities of the exciting and fluoresced light
A = the difference in thermopile output corresponding to the

substitution of the solvent cell for the sample cell

the wavelengths of the exciting and fluoresced light.

Gilmore, Gibson and McClure used magnesium oxide, M90, as the
standard scattering substance of quantum efficiency unity with which to
compare fluorescent light (26). Their method assumes that the number of
quanta reaching the sample is the same as the number falling on the_MgO
surface. There is a slight correction for transmission loss in the window
over the sample, however. Relative spectral emission.curves were determined
using a monochromator with a photomultiplier mounted at the exit slit.

The emission spectra were plotted point by point. A galvanometer was

used as the readout meter. In this method, corrections had to be made

for the transmission window, index of refraction of the solvent, scattered
light and the galvanometer response. The final equation used to calculate

quantum efficiency was

 

vhere

Jeand 35

Se and S‘ =

1n =
hnz-l)‘]/2=

The a
nwhoyt
indeX of re
Therefore,
graphicall y

In 19!
quantum eff-
COhQElred the

26

 

o Safarfat o”2(1 / )d
¢f : 25 0 _§. . \) —-——-——c . J"? C 1 2V vijz (23)
03 a fa vlfff(2n-l) ISd
\) V \J \) \)
where
0e and DS = the galvanometer readings for the emitted and scattered

radiation respectively

v = the frequency of the exciting light

far = the reflectivity factor for the M90 surface at frequency va
fa = the transmission factor for the window between the emitter

and the phototube for the M90 readings

faC = the window transmission over the MgO

f C = the window transmission over the sample

f t = the transmission factor for the window between the emitter

and phototube for sample readings

Sa and Sv = the relative spectral sensitivity of the phototube for the
M90 and sample readings
I = the actual fluorescence intensity

\)

(2n2-l)-1/2= the index of refraction correction.

The authors found that only about a 2% error was introduced into the
result by taking the average of the window transmissions and of the
index of refraction corrections over the frequency range of the emitter.
Therefore, only u{v2(IV/v)dv and dqusvlvdv need to be evaluated
graphically every time.

In 1957, Weber and Teale developed yet another method for calculating
quantum efficiencies (4). Their technique, as with the previous techniques,

compared the fluorescence with the scattering of a standard material of

quantum efficiency unity. These techniques are all known as absolute

27
methods for evaluating quantum efficiency. Weber and Teale used a
dilute solution of glycogen as the scatterer. Large uncertainties in
results were found using MgO and other standard scatterers. This was
due to the difficulty in calculating changes in the spacial distribution
of the radiation taking place when the diffusion screen was substituted
for the fluorescent solution. By using purely dipolar scattering from
a solution of macromolecules, the geometry of the diffusion standard
could be made equivalent to that of the fluorescing solution.

The final equation obtained by Weber and Teale for quantum efficiency was

 

(dF/dA) (3 + P )f(xo)
Axo+0 f
of = (24)
(dS/dA)A (3 + Ps)f(Ax)
Xo+0
where
PS and Pf = the linear polarizations of the radiation in any direction
normal to the exciting light of the standard and fluorescing
solutions
Axo = the natural absorbance per unit path length for the exciting
light of wavelength X0
f(Xo) = a response factor characterizing the response of the detector
to light of wavelength A0
f(AA) = a response factor characterizing the response of the detector
to a wide band
(dF/dA)A
Ao+0
and
(dS/dA)AAO+O = the change in fluorescence and scattering intensity

respectively as the absorbance goes to zero.

The values of (dF/dA)A and (dS/dA)A can be obtained by
AO+O x040

28
plotting F and 5 against A. The slope of the linear part of the plot
can give the value of the derivative.

This technique developed by Weber and Teale is one of the most
widely used methods for determining absolute quantum efficiencies today.
Hercules and Frankel (27) have suggested using "Ludox”, a suspension of
colloidal silica in H20, as the standard scatterer. They claim it is
better than glycogen because it is more easily available in pure form
and is much more stable in solution. They also claim to obtain better
precision in their quantum efficiency calculations with "Ludox" (7%) than
with glycogen (10%) and M90 (20%). Dawson and Windsor (14) used
l'Hospitals rule to replace the differentials in Equation 26 and added

a correction for the indices of refraction to obtain

2
0'76(F/AF)AF+O f(Xo)nF

= 2 . (25)
(S/AS)AS+O f(AX)nS

The number 0.76 is the correction factor for the difference in spacial

 

¢f

distribution of light emitted from the fluorescent and light scattering

solutions. They claim that (F/AF)A and (S/AS)A can be much more

F+0 5+0
accurately extrapolated to zero concentrations than the zero concentration

limits of dF/dA and dS/dAA can be estimated.

AX0+0 Ao+0

Fdrster (28) derived an expression giving the intensity of fluorescence,
measured from the front surface of a cuvette, in terms of the molar
absorptivity of the solution and the geometry of the cuvette. However,
F6rster neglected the possibility of reabsorption of fluorescence. If
there is an overlap in the absorption and emission spectra, fluorescence
emission may be reabsorbed and then emitted again. The re-emitted radiation

may add to the primary fluorescence escaping from the front face of the

cuvette. Budo and Ketskeméty (29) made corrections to F6rster's equation

29
for the reabsorption of fluorescence. However, they did not consider
corrections for shifts in the region of absorption away from the front
face of the cuvette when the solution is diluted. Melhuish (3) made the
equation more exact by considering these shifts.

Melhuish rearranged the modified equation for fluorescence intensity
to obtain an expression for the absolute fluorescence quantum efficiency.
The equation is so complex and unwieldy that very few people have used it.

Melhuish did, nevertheless, provide an immeasureable aid to scientists
who later used the relative method for determining quantum efficiency.

He obtained the value for the absolute quantum efficiency of 5 X 10'3 M
quinine bisulfate in 1.0 N H2504 as 0.546. This value is used as the

standard in most relative techniques.

The Relative Technique for Measuring Quantum Efficiengy

 

With the development of more reliable instruments for measuring
fluorescence, and because of the laborious techniques necessary in
determining absolute quantum efficiencies, a new method of obtaining
fluorescence quantum efficiency has become popular. This method is a
relative technique.

The integrated area under the fluorescence emission spectrum is
proportional to the total intensity of the fluorescent light emitted by
the solution. The intensity is proportional to the product Ioecbof.
Therefore, if the fluorescence emission spectra of two solutions are
measured with the same instrument, and at the same exciting intensity,

10, the ratio of the two fluorescence intensities is given by

——=—————————-——( ). (26)

 

30

If the absorbances of the two solutions are measured, the ratio of the
fluorescence quantum efficiencies can be derived. If one of the substances
has a known quantum efficiency, such as quinine bisulfate, the value of
the other quantum efficiency can be obtained by comparison (15).

With the relative method, the many correction factors necessary in
the methods for determining absolute quantum efficiencies are theoretically
not necessary. The only correction factor that has been added to Equation

26 is that for refractive index. The equation then becomes

t2 F2

F1

1

DI _‘>

n
($2

- .(27)
2

In measuring relative fluorescence quantum efficiencies precautions
must be taken to avoid errors due to inner filter effects, oxygen
quenching, non-monochromatic exciting light and the other processes
already mentioned which compete with fluorescence.

Inner filter effects have no influence on the primary process of
emission, but simply reduce the intensity of the observed fluorescence
by absorption of the exciting light or of the fluorescent light within the
substance being observed. Inner filter effects are quite concentration
dependent. As the concentration of an absorbing solution is increased,
more of the exciting light is absorbed nearer the front of the cell.

Thus, less light reaches that part of the cell where the fluorescence

is measured by the usual right-angle arrangement. Most right-angle
measurements are made with light originating from the middle of the cell.
Distortion of the emission spectrum may also be produced by the presence
of a second solute which absorbs the emitted radiation.

The presence of oxygen dissolved in a solution causes the quenching
Process which have been previously discussed. A pathway for non-radiative

deactivation of the excited state is provided by the oxygen.

31
If the exciting light is not monochromatic, large errors may be
introduced into quantum efficiency measurements. For example, the mercury
line at 313 nm is weakly absorbed by anthracene, with the absorbance of
the mercury line at 253.7 nm being over 100 times as great. If the
313 nm line, isolated by filters, is used for excitation, the presence
of only one percent of the 253.7 nm line will be more than sufficient to

double the fluorescence intensity observed.

Results of Past Work

 

The quantum efficiencies of a great number of fluorescent compounds
have been investigated by many people using many different techniques.
Table I gives a summary of the quantum efficiencies obtained for quinine
and some of its derivatives, anthracene, fluorescein, chlorophyl a and
chlor0phyl b by several authors. This is a representative sampling of
reported quantum efficiencies.

An interesting item to note from Table I is the variation in quantum
efficiency for the same compound. The varience is not limited to one of
the compounds. Differences between 10—50% can be seen for each
compound listed. There seem to be factors affecting the comparability
of data. The problem may be that various factors such as concentration,
fluorescence quenching, temperature and the precision of the technique
used to obtain quantum efficiency are not controlled properly. There
may also be other problems inherent with the techniques used which haven't
been discovered. This is a relatively new field of study. It may be
quite possible that there are unknown factors affecting the results.

There is a controversy today between those who believe quantum
efficiency is constant with varying wavelengths of excitation and those
who believe quantum efficiency is dependent upon the wavelength of

excitation. It is interesting to look at Table I in this regard.

32

Table I. Comparison of Quantum Efficiencies

 

 

 

Compound Conc. Solvent Exc. Wave. of Ref.
Quinine bisulfate 5 x 10'3 M 1.0 N H2504 366 nm 0.546 3
Quinine - 1.0 N H2504 366 nm 0.54 14
Quinine - 1.0-N H2504 254 nm 0.55 14
Quinine - 1.0 N H2304 313 nm 0.48 14
Quinine - 1.0 N H2304 334 nm 0.56 14
Quinine bisulfate 5 x 10'3 M 0 1 N H2504 366 nm 0.50 3
Quinine sulfate - 0.1 N H2804 250 nm 0.582 32
Quinine sulfate - 0 l N H2504 350 nm 0.577 32
Quinine 7.42 x 10'5 M 0.1 N H2504 250 nm 1.02 7
Quinine 7.42 x 10'5 M 0.1 N H2804 313 nm 1.00 7
Quinine 7.42 x 10‘5 M 0.1 N H2504 345 nm 0.98 7
Quinine 7.42 x 10‘5 M 0.1 N H2804 348 nm 0.99 7
Quinine 7.42 x 10'5 M 0.1 N H2504 366 nm 1.09 7
Quinine 7.42 x 10'5 M 0.1 N H2804 380 nm 1.20 7
Quinine 7.42 x 10'5 M 0.1 N H2504 390 nm 1.23 7
Quinine sulfate 1.7 x 10'6 M 0.1 N H2504 318 nm 0.55 11
Quinine sulfate 1.7 x 10‘6 M 0.1 N H2504 365 nm 0.545 11
Anthracene - Ethanol 366 nm 0.27 14
Anthracene 3 x 10‘3 M Ethanol 366 nm 0.27 3
Anthracene - Ethanol - 0.30 4
Anthracene 4.4 x 10'5 M Ethanol - 0.30 30
Anthracene 1.5 x 10'6 M Ethanol 311 nm 0.295 11

Anthracene
Anthracene
Anthracene
Anthracene
Anthracene
Anthracene
Anthracene
Anthracene
Anthracene
Anthracene

Anthracene

Fluorescein
Fluorescein
Fluorescein
Fluorescein
Fluorescein
Fluorescein
Fluorescein
Fluorescein

Fluorescein

Chlorophyl
Chlorophyl
Chlorophyl
Chlorophyl

9)

O)

Q)

33

Table I (Continued)

.5 x 10'6 M

x 10'3 M

.5 x 10'6 M

.5 X 10- M

6
.5 x 10‘6 M

.78 ug/ml

x 10'5 gms/cc

X 10-

20 x 10’6 M
14 x 10'6 M
10 x 10'6 M
6.2 x 10‘6 M

Ethanol

Ethanol

Ethanol

Ethanol

Ethanol

Ethanol

Ethanol

Benzene

Benzene

Benzene

Benzene

0.1 N

o
L.

N

gms/cc 0.1 N

O O O C)
O OH O 0
Z

NaOH
NaOH
NaOH
NaOH
NaOH
NaOH
NaOH
NaOH
NaOH

Methanol

Methanol

Methanol

Methanol

366
366
366
340
376

436
436

313
365
435

T1111

nm

nm

nm

nm

nm

nm

[1111

nm

nm

nm

000000 00000 00000

GOO

GOO

.306
.27
.31
.31
.31
.31
.22
.27
.256
.29
.31

.93
.84
.85
.77
.79
.93
.86
.84
.92

.15
.14
.18
.18

11

31
11
31
27
33
14

27

24
15
25
25
27
14
l4
14

25
25
25
25

Chlorophyl
Chlorophyl
Chlorophyl
Chlor0phyl
Chlorophyl
Chlor0phy1
Chlorophyl
Chlorophyl
Chlorophyl
Chlorophyl
Chlorophyl
Chlorophyl
Chlor0phyl
Chlorophyl
Chlorophyl
Chlorophyl
Chlorophyl
Chlorophyl
Chlorophyl
Chlorophyl
Chlorophyl
Chlorophyl
Chlorophyl

34

Table I (Continued)

5.5 x10‘6 M
2.7 x10”6 M
21.0 x10‘6 M
7.2 x10'6 M
2.3 x10"6 M

6

9.7 X 10- M

15.0 x 10'6 M

6.5 x10'6 M
12.0 x10‘6 M
9.0 x10'6 M

Methanol
Methanol
Ethyl ether
Ethyl ether
Ethyl ether
Acetone
Methanol
Methanol
Methanol
Methanol
Ethyl ether
Ethyl ether
Methanol
Methanol
Methanol
Methanol
Methanol
Ethanol
Ethanol
Ethanol
Ethanol
Ethanol
Ethanol

436
628
644
662
698
436
628
644
662
436
644

nm

nm

nm

nm

nm

nm

nm

nm

nm

nm

nm

OOOOOOOOOOOOOOOOO

000000

.21
.21
.15
.20
.22
.21
.29
.11
.043
.048
.090
.074
.24
.24
.25
.26
.11
.24
.21
.24
.24
.11
.11

25
25
25
25
25
25

25
25
25
25
25
25
25
25
25
25
25
25
25
25
25

35

Weber and Teale (5 ) in 1958 studied the absolute quantum efficiencies
of a wide variety of organic compounds with different exciting wavelengths.
They found no variation of quantum efficiency with wavelength for any of
the compounds. The substances they studied included anthracene in
ethanol, fluorescein in 0.1 N NaOH and chlorophyl a and chlorophyl b in
hexane. They studied anthracene for the range of exciting light between
210-310 nm, fluorescein between 210-530 nm, and the chlorophyls between
220-690 nm. However, no data were given to verify their results. There
is no way to check their work.

In 1967, Chen (7), using a relative method for determining quantum
efficiencies, studied quinine in 0.1 N H2504. He found that there was a
change of quantum efficiency with variations in the wavelength of
excitation. His values vary between 1.02 and 1.23 over a range of 140 nm.
Chen pointed out that the excitation spectrum of acidic quinine doesn't
match the absorption spectrum. This would imply that quantum efficiency
is dependent upon the excitation wavelength. This is contrary to the
vast majority of fluorescent organic compounds. The deviations were
noted in the long wavelength region. The quantum efficiencies Chen
reported are clearly higher with excitation at the low energy end of the
long wavelength excitation band than at shorter wavelengths. Below
340 nm there is no difference in the excitation and absorption spectra.
There is also no difference in the quantum efficiencies obtained with
wavelengths of excitation in this region.

This deviation in the spectrum was also noticed earlier by Bdrreson
(8) and Moss (9). Bdrreson obtained a ratio of quantum efficiencies for
quinine in 0.5 N H2304 of 1.47 for wavelengths of excitation of 345 nm

and 250 nm. Moss reported a ratio of 1.6 to 1.9 for the same conditions.

36

Both men used the technique of Weber and Teale. They concluded that the
quantum efficiency of quinine was dependent upon the wavelength of
excitation.

Chen also observed that the emission spectrum was dependent upon
the wavelength of the exciting light. Changing the excitation wavelength
for quinine sulfate from 340-390 nm resulted in a shift of the emission
peak from 458 to 466 nm, an 8 nm shift. This shift couldn't be explained
by non linearity of the phototube response. The same results were
obtained when the emission intensities were kept constant and the input
energy varied. This same effect was observed for 6-methoxyquinoline, the
parent compound of quinine. Thus, Chen concluded that the changes were
due to properties inherent with the electronic structure of the parent
nucleus.

Chen suggested that the reason the deviations were found in the
excitation spectrum, that the emission peak shifted, and that the
quantum efficiencies were dependent upon the wavelength of excitation
was that there was emission arising from two different excited states
simultaneously. He believed that the absorption spectrum of quinine in
acid contained a weak hidden transition at long wavelengths. This could
be caused by emission from two corresponding states. These two states
would have to be the lowest lying singlet states S1 and 32. His main
support for this conclusion comes from polarization studies. The
polarization of the emission spectrum for quinine is not constant. This
indicates that the emission arises from two oscillators, and that the
oscillators are not mutually parallel. Also, the polarization observed
in the bulk of the absorption band of quinine in glycerol is low (0.34)
compared with the theoretical maximum (0.50) for the case in which a

single anisotropic absorption oscillator is parallel to a single linear

37
emission oscillator. This behavior is explained if the emission arises
from the two singlet states, S], and $2, simultaneously.

In 1969, Fletcher (13) examined several samples of quinine sulfate
and quinine bisulfate from various sources to see if the deviations in
the reported quantum efficiencies depended upon the source. The
absorbance and fluorescence excitation spectra of all the samples were in
satisfactory agreement with one another.v All the samples, except one,
showed good agreement in quantum efficiency. The reproducibility was
to 2%. One commercial solution did give a value that was 20% low. It
was believed that there were possible impurities in this sample. Fletcher
concluded that the deviations between reports for quantum efficiency
couldn't be due to the sources of quinine.

Fletcher also evaluated the quantum efficiency relationship with
wavelength of excitation. Two values for the quantum efficiency of
quinine sulfate that he reported were 0.55 for a 313 nm exciting light
and 0.545 for a 365 nm exciting light. He found no unexpected deviation
from a constant value. He suggested that some of the conflicting results
in the literature could be due to the common practice of using different
spectral band widths for absorbance measurements than those used for
emission measurements. This is possible because of the usual procedure
of measuring sample absorption with one instrument and sample emission
with another. The measurement of accurate quantum efficiencies requires
that the sample absorption be known for the same spectral band width,
wavelength and type of light source as used for the excitation of
fluorescence.

The shift of fluorescence emission with changing wavelength of
excitation reported by Chen was also observed by Fletcher (12). Once

the emission peak begins to shift, Fletcher states that excitation

38
spectra have no real significance unless one is looking at the whole
emission band with an instrument that has a linear phototube response.
If a linear response instrument examines only a portion of the emission
band, a false change in the recorded light emission can occur. This
happens when the emission peak shifts out of the range of the emission
monochromator setting.

Fletcher doesn't believe Chen's explanation for the shift in quinine.
He believes fluorescence can only come from the 51 state. Transitions
between excited states of the same multiplicity are so rapid that all
the excited electrons should be in the S1 state for emission.

Fletcher postulates that the shift is due to emission from a single
energy level of the molecule in either of two or more different average
geometrical arrangements. This is suggested in that all molecules which
exhibit the shift, such as quinine, 6-methoxyquinoline, 2-aminopurine and
luminol have rotatable auxochromes or chromophores. Similar or parent
compounds without rotatable auxochromes do not show this shift. The
conformation of the rotatable auxochrome will affect the intensity and
wavelength location of the absorption bands. The extent of electron
cloud overlap, and the consequent degree of n-resonance depends upon
the relative geometrical position of the auxochrome with respect to the
chromophore. Electronic energy levels are very dependent upon the
conformation of molecules having rotatable auxochromes.

Dawson and Windsor (14) have also performed quantum efficiency studies.
Their values for quinine appear to show a slight variation with the
wavelength of the exciting light. The value at 313 nm (0.48) is definitely
lower than the other value obtained at the other wavelengths (0.55).
However, they disregarded this value, and claimed that the quantum
efficiency is constant because of the results from the other exciting

wavelengths.

39

From their study of fluorescein in 0.1 N NaOH, Dawson and Windsor
also claimed that the quantum efficiency was independent of wavelength
of excitation. The average values they reported at each wavelength do
appear to vary, however. The variation is only slightly greater than
their experimental precision of :_0.04. There is definitely a difference
in the values, though.

An interesting item that Dawson and Windsor noticed was that the
quantum efficiency of quinine increases with sulfuric acid concentration.
Their data show an increase from 0.50 in 0.1 N H2804 to 0.54 in 1.0 N H280

4
to 0.60 in 3.0 N H2304. It is believed that this increase is due to
different ionic forms of the quinine existing under the different acid
concentration conditions.

Eisenbrand (10) also noticed this variation in quantum efficiency
for quinine between 1.0 N H2804 and 3.0 N H2504. He found no variation
of quantum efficiency between 0.01 and 0.2 N H2504. The values were

6-13% higher in 3.0 N H SO than in 1.0 N H SO however. Again, the

2 4 2 4’
existence of different ionic forms was postulated to cause this difference
in quantum efficiency.

It is worth noting here that Dawson and Windsor used a modified
Weber and Teale technique. Their values agree to within 13% with values
published by Melhuish (3). However, there is a large discrepancy between
their values and those published by Weber and Teale (4). The values
Dawson and Windsor obtained for anthracene and fluorescein are 6-l8% lower
than the values reported by Weber and Teale. In both cases, a variety of
wavelengths of excitation were used. It is difficult to explain this.
Possibly Weber and Teale didn't correct for the problems of non-uniform
light scattering by the glycogen solution. The problems with this have

been mentioned earlier. Dawson and Windsor used "Ludox", which is

claimed to be a much more reliable and stable scattering material.

‘11:; ‘4 ,

40

There has been very little research into the effect of concentration
on quantum efficiency. This is unfortunate considering the wide range
of concentrations used in the reported studies. It is highly possible
that a part of the variation in quantum efficiencies can be attributed
to a concentration dependence.

Forster and Livingstone (25) have studied chlorophyl a and chlorophyl b
at different concentrations. They noticed a concentration dependence.
From their data, it can be seen that as the concentration decreases,
there is a considerable increase in quantum efficiency. This same
phenomenon occurred in several solvents.

Though there are no published reports for the concentration dependence
of quinine and its derivatives, for anthracene nor for fluorescein,
the data from Table I should give some information in this regard.

The wide variation in reported quantum efficiencies and the lack of
stating the concentration in many cases makes it difficult to study the
concentration effect upon quinine and its derivatives.

The same problem exists with fluorescein. The concentration is
not stated in most cases. However, Forster and Livingston (25) did
study fluorescein and state the concentrations of the solutions used. The
data show an increase in quantum efficiency of from 0.77 to 0.79 with
concentrations varying from 4 X 10‘5 gms/cc to l X 10'5 gms/cc. This is
a small difference. It does exist, however.

Anthracene is much more amenable to study. Melhuish (3) and Chen (7)

3 M anthracene in ethanol.

5

report a quantum efficiency of 0.27 for 3 X 10'

Medinger and Wilkinson (30) report a value of 0.30 for 4.4 X 10' M

anthracene in ethanol. Fletcher (11) and Himel and Mayer (31) report a

value of 0.31 for 1.5 X 10'6

general trend toward larger quantum efficiencies with more dilute solutions.

M anthracene in ethanol. Again, there is a

-2.-m1

' ' ‘..r as
11,—...- _

41

These data seem to point to a concentration dependence of fluorescence
quantum efficiencies. With a decrease in solute concentration, there is
less probability of radiationless deactivation by collision with other
solute molecules resulting in an increased probability of fluorescence.
With this increase of fluorescence comes a corresponding increase in
quantum efficiency. For this case, it is assumed that the solvent
molecules do not offer a path for radiationless deactivation of the
excited state.

There seem to be many problems which are inherent in determining
quantum efficiencies. The data in Table I seem to show a concentration
dependence of quantum efficiency. If this is true, the extrapolation
to infinite dilution techniques, such as that of Weber and Teale, should
give different values than the relative techniques which are performed
on more concentrated solutions. The relative methods themselves should
give different reported results depending upon the concentration of the
solutions studied.

The evidence is not complete yet, but if the wavelength of excitation
affects fluorescence and quantum efficiency, then the wavelength of
excitation needs to be specified for a comparison of results between
laboratories.

Himel and Mayer (31) have tabulated several other factors which may
affect the determination of quantum efficiency. Inner-filter effects may
cause a decrease in light reaching that portion of the cell from which
fluorescence is measured. There would be a corresponding decrease in
quantum efficiency. Concentration quenching or oxygen quenching is
quite possible. Again, the quantum efficiency would decrease. Temperature
has a strong effect on quantum efficiency. The lower the temperature, the

larger the quantum efficiency for most compounds. The behavior of solvent

42
may affect the quantum efficiency. The solvent may absorb or fluoresce.
In either case, the quantum efficiency would be affected. The difference
in refractive index between the standard and fluorescing solutions
affects the values of quantum efficiency. Since it is possible to have
different ionic species present for a substance under different conditions
of acidity, there are possibilities of different species emitting at
each different pH. The quantum efficiencies should differ in this case
also. There are also instrumental problems. Fluctuation in light
intensity may result in unequal illumination of the excitation monochromator
slits. If the slits are illuminated totally at one time and only partially
at another time, the fluorescence and quantum efficiency will vary. If
the sample cells are not properly aligned each time, variations in the
measured fluorescence would again result in different quantum efficiencies.
Finally, if an instrument isn't free of scattered light, there may be
intensification of emission readings at the wavelength of the scattered
light. There should be a corresponding increase in quantum efficiency
also.

From the previous discussion, it appears evident that many factors
affect the determination of quantum efficiency. The deviation in reported
values to date probably is caused by lack of correction for these factors.
In order to obtain reproducible quantum efficiencies between laboratories,
it is important that all the factors are controlled or corrected. When
this is accomplished, a better understanding of quantum efficiency

measurements should result.

EXPERIMENTAL

PURIFICATION PROCEDURE

Anthracene

 

The anthracene (Matheson, Coleman and Bell) used in this study was
recrystallized three times from benzene and vacuum dried; m.p. 216°C,
literature value 216°C (34). A carbon and hydrogen analysis was
performed in the Michigan State University School of Natural Resources.

Anal. Calc for C H C, 94.34; H, 5.66. Found for C H C, 94.24;

1410‘ 1410‘

H, 5.68.

Quinine Sulfate

 

The quinine sulfate (Mallinkrodt, N.F. powder) used in this study
was recrystallized three times from distilled water and vacuum dried;
m.p. 234°C, literature value 235°C (35). A carbon, hydrogen and nitrogen
analysis was performed in the Michigan State University School of Natural
Resources. Anal. Calc for anhydrous (C20H24N202)2-H2304: C, 64.32;

H, 6.75; N, 7.50. Found: C, 62.71; H, 6.81; N, 7.48. Consideration
of the carbon analytical result indicates that the purified quinine

sulfate was 98% in the dihydrate form (for the dihydrate C, 61.3).
Solvents

The absolute ethanol (Rossville Gold Shield Alcohol, 200 Proof) and
the benzene (Matheson, Coleman and Bell, Chromatography Reagent, 99+
mol %) were used as solvents without any purification. The stock solution

of concentrated sulfuric acid (Allied Chemical, Baker and Adamson Quality,

43

44
95.5-96.5%) was boiled and 1.0 N and 0.1 N sulfuric acid solutions were
prepared from it with triply distilled water.
The solvents were analyzed for absorbance on a Cary 14 spectr0photometer
and for fluorescence with the spectrofluorimeter. No detectable absorbance

or fluorescence was observed.
PREPARATION OF SOLUTIONS

All solutions were prepared from stock solutions by successive
dilutions of 10 ml of the previously prepared solution to 100 ml with

the appropriate solvent in a volumetric flask.

Anthracene Solutions

 

Stock solutions of 0.0183 g of anthracene diluted to 100 ml with
absolute alcohol and 0.0218 g of anthracene diluted to 100 ml with

benzene were prepared. The series of solutions prepared from the ethanol

3 6

stock solution ranged from 1.03 X 10' M to 1.03 X 10' M. The series

of solutions prepared from the benzene stock solution ranged from

3

1.22 x 10' M to 1.22 x 10'6 M.

Quinine Sulfate Solutions

 

Stock solutions of 0.0385 g of quinine sulfate diluted to 100 ml

with 1.0 N H2S04, 0.3924 g of quinine sulfate diluted to 100 ml with

0.1 N H2504, and 0.0390 g of quinine sulfate diluted to 100 ml with triple

distilled water were prepared. The series of solutions prepared from

4 6

the 1.0 N H SO stock solution ranged from 4.91 X 10' M to 4.91 X 10' M.

2 4
The series of solutions prepared from the 0.1 N H2504 ranged from

3 6

5.01 X 10' M to 5.01 X 10' M. The series of solutions prepared from

4

the water stock solution ranged from 4.98 X 10' M to 4.98 X 10'6 M.

45
INSTRUMENTATION

A computerized spectrofluorimeter designed and constructed by
J. F. Holland (36) of Michigan State University was used in this research.
Figure 4 illustrates the basic components of the spectrofluorimeter.

Light from a source, such as an Osram xenon arc lamp, is focused
by lenses through the entrance slit of a Bausch and Lomb grating monochromator.
Light of a selected wavelength is then directed through focusing lenses
to a vibrating mirror assembly. The vibrations of the mirror reflect .
the light alternately through the reference and sample cells. The cells
used for measurement are standard clear-window 10 mm quartz cells made
especially for fluorimetry. The amount of light absorbed by the sample
is measured by a Rhodamine B quantum counter and photomultiplier tube as
a difference in the quanta transmitted through the reference and sample
cells in line with the incident exciting light. The amount of fluorescence
emission is monitored at right angles to the incident exciting light
through anouther Bausch and Lomb grating monochromator by another
photomultiplier tube.

The outputs of the spectrofluorimeter are monitored by a PDP 8/1
computer. Figure 5 is a block diagram describing the computer oriented
part of the system. Five signals are fed through a multiplexer-analog
to digital converter combination into the computer from the spectrofluorimeter.
Two signals, Eex and Eem’ are voltages which are related to the wavelengths
of the excitation and emission monochromators. Two current signals,
i1 and i2, are proportional to the output of the quantum counter when
light passes through the sample and reference cells respectively. The
last signal, i3, is a current which is proportional to the emission

photomultiplier output.

xenon
arc
lamp

46

 

 

 

 

 

excitation monochromator

 

N513

L——“: ——:a
y \
oY \
.I‘t 1.
F \
I \\\
r1 ‘1
ll \h
I
I i b
1 I '
O-+D-<I~*Oa,/ 1
focusing —!"'
lenses 3?
' cell
to? compartment
vibrating mirror k_-.
assembly \,

 

emission monochromator

Figure 4.

L4

G; <9

photomultiplier tube

filter

quantum counter

 

 

 

focusing lenses
r00

photomultiplier tube

 

 

Basic Components of the Spectrofluorimeter

47

 

 

 

 

 

 

 

 

 

 

 

Eex Eex’ voltage proportional to hex
O L__.
i1 and i2 i1 and 12, currents proportional to
I 0i quantum counter outputs for reference
and sample alternately
Eg_ia__, i3, current proportional to emission
E photomultiplier output
1 - .
F41 Eem’ voltage proportional to Aem
l multiplexer

 

 

analog-digital converter

 

 

D tel etype

 

PDP-8/I

 

 

plotter

 

 

Figure 5. On Line Fluorescence System

48

The computer serves both as a data acquisition terminal and also
performs the necessary manipulations of the data for the pr0per output.
Output from the computer may be either punched on tape, printed or
plotted.

A computer program was developed to treat or use the acquired data.
From i1 the R value, which was related to the intensity of light which
had passed through the reference solution, was obtained. From i2 the
S value, which was related to the intensity of light which had passed
through the sample solution was obtained. A value for R-S, which was
related to the number of quanta of light absorbed by the sample, was
computed.

From i3, F, which is related to the number of quanta emitted by the
sample, was derived.

The F values were integrated through the emission wavelength range
of the substance. This integration gave a number proportional to the
total number of quanta emitted by the sample. This value was divided by
the R-S value, the number which was proportional to the number of quanta
of light absorbed by the sample, to obtain the quantum efficiency.

Several other instruments were used in this study. Some of the
absorbance measurements were made on the Cary Model 14 recording
spectrophotometer. A Beckman Model 76 pH meter, equipped with a
combination electrode, was used for all pH measurements. Refractive
index measurements, at room temperature, were made with an Abbey type

Bausch and Lomb refractometer.

49
EXPERIMENTAL PROCEDURE

Cleaning of Cells

 

Before use the cells were cleaned with detergent and rinsed with
water. This cleaning procedure was adequate since no residual fluorescence
could be detected after cleaning. After cleaning, the sample cell was
rinsed by portions of the sample solution and then filled with another
portion of that solution. Similarly, the reference cell was rinsed with

the appropriate solvent and then filled before measurements were made.
Special Treatment of Samples Before the Scan

Since quinine sulfate is not affected by oxygen quenching, only the
anthracene solutions were purged with nitrogen gas, presaturated with
the proper solvent, for ten minutes to remove the oxygen. For low
temperature studies the solutions, in flasks, were cooled to 10 :_1°C

in an ice bath.

Procedure for Obtaining the Emission Scan

 

Both monochromators were synchronized at 300 nm and at 650 nm
through an initial calibration procedure. Any wavelength range could
then be selected by typing the initial and final wavelengths of this
range on the teletype. The computer only registered the desired data
through this range.

With the 4.91 X 10-5 M quinine sulfate in 1.0 N H2504 comparison
standard in the sample cell, the emission spectrum was scanned through
380-610 nm using an excitation wavelength of 365 nm. The computer
stored the quantum counter output which corresponded to the currents
i1 and i2 and the emission photomultiplier output at small selected

wavelength intervals which corresponded to the current i3. The quantum

50
efficiency, 0.550, for the comparison solution was then typed into the
computer.

Once the reference quantum efficiency was stored in the computer,
each of the solutions to be studied was scanned over the desired emission
wavelength range at selected excitation wavelengths. The excitation
wavelengths chosen either corresponded to an absorption peak for the
solute or to wavelengths used for excitation by previous investigators.

The quinine sulfate solutions in sulfuric acid were scanned from
380-610 nm at excitation wavelengths of 365, 334 and 313 nm.

The quinine sulfate solutions in water were scanned from 340-560 nm
with excitation wavelengths of 320 and 300 nm.

The anthracene solutions in both benzene and ethanol were scanned
from 360-500 nm using excitation wavelengths of 342 and 331 nm.

The comparison standard was rerun between every two or three scans
to check the reproducibility of the instrument.

For the low temperature study, refrigerated ethylene glycol was
circulated continuously through the cooling channels of the cell
compartment. The cell compartment could be maintained at 10 : 1°C.

A quantum efficiency for the comparison standard had to be evaluated
at the low temperature. This value was used as the reference quantum

efficiency for all low temperature studies.
Procedure for Obtaining the Excitation Scan

As with the emission scan, both monochromators were synchronized
at 300 nm and 650 nm through an initial calibration procedure. Then a
command was typed into the computer which set it in an excitation mode
of operation. Any excitation wavelength range could then be selected,
Just as with the emission scan, by typing the initial and final wavelengths

of this range on the teletype. An emission wavelength was selected.

51

Each of the solutions was scanned through an excitation range of
250-400 nm. The anthracene solutions were scanned with the emission
monochromator set at 400 nm. The quinine sulfate solutions were scanned
with the emission monochromator set at 450 nm.

The computer once again acquired data corresponding to the quantum
output, i1 and i2, and the emission photomultiplier output i3. From
these stored data the excitation spectrum, the absorption spectrum, and

an absorption corrected excitation spectrum could be obtained.

RESULTS AND DISCUSSION

GENERAL

The quantum efficiencies, corrected for refractive index effects, of
quinine sulfate in 1.0 N H2504, 0.1 N H2304 and water and of anthracene
in ethanol and in benzene, are listed for both room temperature (23°C)
and low temperature (10°C) studies in Tables II-XI. The solutions were
corrected for refractive index against the standard solution as shown
in Equation 27. The correction factors are 0.993 for the 0.1 N H2S04,
0.991 for the water, 1.02 for the ethanol, and 1.25 for the benzene
solutions. The results listed in these tables are average values obtained
over a period of three months by a relative technique for obtaining quantum
efficiencies. During this time several different sets of sample solutions
were prepared. The average deviation of the quantum efficiencies is 10%.
The same general trends were noticed for each set of solutions.

3M

Melhuish (3) obtained a value for the quantum efficiency of 5.0 X 10'
quinine bisulfate in 1.0 N H2504 of 0.546. This value was obtained by an
absolute method for evaluation of quantum efficiency described previously.
The fluorescence emission was measured using a front surface technique.

Other investigators (31,33) have used quinine bisulfate in 1.0 N H2504
as their reference standard and have used 0.546 as their reference quantum
efficiency. However, the modern relative techniques for obtaining quantum
efficiency use the right-angle technique of fluorescence measurement. A

3

solution of quinine bisulfate as concentrated as 5.0 X 10' M doesn't

allow the light to penetrate deeply enough into the sample for measurement

52

53
of the resulting emission by the right-angle technique. This is an
example of the inner-filter effects described earlier. Thus, less
dilute solutions of quinine bisulfate in sulfuric acid have to be used
as the comparison reference solution.

Quinine sulfate instead of quinine bisulfate in sulfuric acid can
be used as the reference solution (7,14). In acid solution, quinine
sulfate and quinine bisulfate give identical absorbance curves, and
excitation and emission spectra. It is believed that both substances
exist in similar forms in acid solutions and have identical quantum
efficiencies.

In this study, 4.91 x 10'5

M quinine sulfate in 1.0 N H2504 was
used as the reference standard solution. This solution concentration is
in the optimum range of concentration to obtain the best instrumental
sensitivity. The fluorescence emission of this solution, also, does not
appear to be hindered by inner-filter effects. A reference quantum
efficiency of 0.550 was chosen because the value was reasonably close to
that obtained by Melhuish (3) for the quinine sulfate system.

Figure 6 shows absorbance and excitation spectra of 4.91 X 10'4 M
quinine sulfate in 1.0 N H2504. There appears to be two "humps" in the
excitation spectrum between 325 and 400 nm. It is believed that the
inner-filter effect, which is a prominent occurrence in concentrated
solutions, causes this phenomenon. A large fraction of the exciting light
may be absorbed before it penetrates the sample to the region from which
emission is monitored. The amount of emission detected is thus less than
that actually caused by the exciting light. The absorbance spectrum
suggests the above conclusion. In the region where the "hump" occurs in

the excitation spectrum, the maximum absorbance is also found. The

quinine sulfate solutions of the order of 10'5 M or less do not exhibit

54
this"hump"in the excitation spectrum because the light travels a path of
more uniform intensity through more dilute solutions.
Figure 7 shows a similar effect for 1.03 X 10"4 M anthracene in
ethanol. In this case, one of the excitation peaks, the 359 nm peak, is
considerably smaller in relation to the other peaks than it is in the

1.03 x 10'5

M solution. Once again the lowering of the fluorescence
emission occurs in a region of maximum absorbance.

Because of the inner-filter effect, care must be taken in analyzing
any results from fluorescence emission studies for more concentrated
solutions.

Figures 6 and 7 also show the initial regions of the fluorescence
emission spectra of quinine sulfate and anthracene in relation to the
respective excitation and absorbance spectra. For quinine sulfate in acid
solution, there is very little overlap of emission and absorbance spectra.
There is a large excitation region through which emission effects may be
studied. The overlap of the excitation, absorbance, and emission spectra
is much more pronounced for anthracene. Exciting light of wavelengths less
than 350 nm must be used for emission studies of anthracene solutions to
prevent emission and reabsorption interference. For this reason, exciting
light of only 342 and 331 nm was used to study the quantum efficiencies of
the anthracene solutions.

The same problem exists for the solutions of quinine sulfate in
water. There is a shift in the fluorescence emission peak in water for
quinine sulfate to shorter wavelengths. The excitation, absorbance and
emission spectra overlap from 340 nm through the longer wavelengths.
Therefore, only excitation wavelengths of 320 and 300 nm were used in the

quantum efficiency studies of quinine sulfate in water.

55

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57

The Dependence of Quantum Efficiency on Excitation Wavelength

 

Tables II-IV list the quantum efficiencies of quinine sulfate in
1.0 N H2804, 0.1 N H2304 and water at room temperature at various excitation
wavelengths. Figures 8 and 9 graphically illustrate the effect of excitation
wavelength on the quantum efficiency of quinine sulfate in acid solutions.
Both figures show the same trend. As the wavelength of excitation
increases from 313-365 nm, the quantum efficiency of each solution increases
also. These results agree with the results of Chen (7), Borresen (8)
and Moss (9), and disagree with the results of Fletcher (11), Dawson and
Windsor (l4) and Eastman (32).

The quantum efficiency of the 4.91 X 10'4 M quinine sulfate solution
in 1.0 N H2804 changes from 0.374 with a 313 nm exciting light to 0.522
with a 365 nm exciting light. This is an increase of 39% over the initial
quantum efficiency. In 0.1 N H2S04, the quantum efficiency of the
5.01 X 10'4 M quinine sulfate solution increases from 0.389 to 0.559, or
44%. The same effect is observed at the other concentrations. In the
1.0 N H2S04, the 4.91 X 10.5 M quinine sulfate solution shows an increase

6

from 0.375 to 0.550, or 47%, and the 4.91 X 10' M solution shows an

increase of 29% from 0.280 to 0.361. In the 0.1 N H SO the 5.01 X 10’5 M

2 4’
quinine sulfate solution shows an increase of 40% from 0.382 to 0.536 and
the quantum efficiency increases from 0.107 to 0.219, or 100%, for the
5.01 X 10'6 M quinine sulfate solution.

From Table IV, a much smaller, almost negligible effect is seen
for the dependence of the quantum efficiencies of the solutions of quinine
sulfate in water on excitation wavelength. This may be due to the narrow
range of exciting wavelengths which could be used in this study. A
wider range of excitation wavelengths can not be used because of the

strong overlap of the excitation, absorbance and emission spectra above

340 nm.

Table II.

58

The Quantum Efficiency of Quinine Sulfate in 1.0

at Room Temperature (23°C)

Concentration (

4.91
4.91
.91
.91
.91
.91
.91
.91

h-h-b-b-b-b-b

.91

X
X
X

10
10
10
10
10
10
10

-4
-4
-4
-5
-5
-5
-6

Fluorescence emission scan:

moles
liter

 

)

380-610 nm

Excitation Wavelength (nm) Quantum

365
334
313
365
334
313
365
334
313

OOOOOOOOO

N H SO

Efficiency

.522
.474
.374
.550
.507
.375
.361
.348
.280

59

Table III. The Quantum Efficiency of Quinine Sulfate in 0.1 N H2804 at

Room Temperature (23°C)

Fluorescence emission scan: 380-610 nm

 

Concentration (Tglgi) Excitation Wavelength (nm) Quantum Efficiency
5.01 x 10‘4 365 0.559
5.01 x 10'4 334 0.494
5.01 x 10'4 313 0.389
5.01 x 10'5 365 0.536
5.01 x 10‘5 334 0.506
5.01 x 10‘5 313 0.382
5.01 x 10'6 365 0.219
5.01 x 10‘6 334 0.199
5.01 x 10‘6 313 0 107

60

Table IV. The Quantum Efficiency of Quinine Sulfate in Water at Room
Temperature (23°C)

Fluorescence emission scan: 340-560 nm

 

Concentration (T?l::) Excitation Wavelength (nm) Quantum Efficiency
4.98 x 10'4 320 0.337
4.98 x 10'4 300 0.334
4.98 x 10‘5 320 0.434
4.98 x 10‘5 300 0.424
4.98 x 10"6 320 -
4.98 x 10'6 300 -

61

 

 

 

 

 

 

 

~ 8
0.50 - A
0.40 ~

I “c
0.30 —
if
Concentrations
0.20 - _4
A = 4.91 x 10 M
B = 4.91 x10"5 M
0'10 T c = 4.91x10'6 M
0.00 a 1 1 1 1
310 320 330 340 350 360 370

Excitation Wavelength (nm)

Figure 8. Dependence of the Quantum Efficiency of Quinine Sulfate in
1.0 N H2S04 on Excitation Wavelength and Solute Concentration.

Fluorescence emission scan: 380-610 nm.

0.500

0.400

0.300

0.200

0.00

Figure 9.

 

 

 

 

 

 

._
x 0
Concentrations
A = 5.01 x10'4 M
7 B = 5.01x10‘5M
c = 5.01x10’6 M
1 1 1 1 .1
310 320 330 340 350 360 370

Excitation Wavelength (nm)

Dependence of the Quantum Efficiency of Quinine Sulfate in
0.1 N H2504 on Excitation Wavelength and Solute Concentration.

Fluorescence emission scan: 380-610 nm.

63

There is a possible correlation which may give a reason for this
behavior of the quantum efficiency of quinine sulfate with changing
excitation wavelength. From Figure 6, the excitation spectrum.within the
range 313 to 365 nm, shows a general increase in the intensity of the
fluorescence emission. The absorbance curve also increases through this
range. It is difficult to notice this since the intensities are not on the
same scale, but the intensity of the excitation spectrum may increase
more rapidly through this range than the extent of absorbance. If this is
the case, with quantum efficiency defined as the number of quanta emitted
divided by the number of quanta absorbed, and since the number of quanta
emitted appear to be increasing more rapidly than the number of quanta
absorbed, then an increase in quantum efficiency may be expected. For
real meaning to this conclusion, the relationship between emission intensity
and the number of quanta emitted and between the absorbance and the
number of quanta absorbed must be known.

Between 300 to 320 nm, the range within which the quantum efficiencies
of the solutions of quinine sulfate in water were studied, the excitation
spectrum shows a very small increase as does the absorbance curve. 0n
the basis of the previous discussion, not as large a change, if any, would
be expected in the quantum efficiencies obtained between these two
wavelengths.

Tables V and VI list the quantum efficiencies of solutions of anthracene
in ethanol and in benzene as the excitation wavelength is varied..

Figures 10 and 11 graphically illustrate the effect excitation wavelength
has on the quantum efficiencies of these solutions.

5 6

The 1.03 X 10- M and 1.03 X 10- M solutions of anthracene in ethanol

5 6

and the 1.22 X 10' M and 1.22 X 10' M solutions of anthracene in benzene

display the same effect as the quinine sulfate solutions in acid. There

64

Table V. The Quantum Efficiency of Anthracene in Ethanol at Room
Temperature

Fluorescence emission scan: 360-500 nm

 

Concentration (T?1::) Excitation Wavelength (nm) Quantum Efficiency
1.03 x 10'4 342 0.241
1.03 x 10'4 331 0.268
1.03 x 10‘5 342 0.225
1.03 x 10‘5 331 0.196
1.03 x 10"6 342 0.119
1.03 x 10'6 331 0.088

65

Table VI. The Quantum Efficiency of Anthracene in Benzene at Room
Temperature

Fluorescence emission scan: 360-500 nm

 

Concentration (Tglgi) Excitation Wavelength (nm) Quantum Efficiency
1.22 x 10'4 342 0.197
1.22 x 10‘4 331 0.268
1.22 x 10’5 342 0.233
1.22 x 10’5 331 0.213
1.22 x 10"6 342 0.170
1.22 x 10‘6 331 0.165

66

 

 

 

 

0.30
\A
B
0.20 — /
d”r
0.10 .. ”//C
0.00 ’4 1 #-
320 330 340 350
Excitation Wavelength (nm)
Figure 10. Dependence of the Quantum Efficiency of Anthracene in

Ethanol on Excitation Wavelength and Solute Concentration.

Fluorescence emission scan: 360-500 nm. A = 1.03 X 10‘4 M;

5 6

B = 1.03 X 10- M; C = 1.03 X 10‘ M.

 

 

 

 

 

 

 

0.30
::::::::::=><:::::::B
0.20 - A
¢ 0
0.10 —
0.00 ' ' <-
320 330 340 350

Excitation Wavelength (nm)

Figure 11. Dependence of the Quantum Efficiency of Anthracene in
Benzene on Excitation Wavelength and Solute Concentration.

Fluorescence emission scan: 360-500 nm. A = 1.22 X 10'4 M;

5

B = 1.22 x 10' M; c = 1.22 x 10'6 M.

68
is an increase in quantum efficiency with use of exciting wavelengths of

331 and 342 nm. As was the case with the solutions of quinine sulfate in
water, a wider range of exciting wavelengths could not be used in this
study because of the strong overlap of the excitation, absorbance and
emission spectra above 350 nm. For the anthracene in ethanol solutions,

5

the 1.03 X 10' M solution shows an increase in quantum efficiency of

6

from 0.196 to 0.225, or 15%. The 1.03 X 10' M solution displays an

5

increase of 36%, from 0.088 to 0.119. The 1.22 X 10' M solution of

anthracene in benzene shows an increase in quantum efficiency of

6 M solution shows no

0.213 to 0.233, or 10%, while the 1.22 X 10'
significant change.

Figure 7 shows between 331 and 342 nm, a slight increase in the intensity
of the excitation spectrum for anthracene in ethanol as well as a corresponding
increase in the absorbance curve. Therefore, the same effect of excitation
wavelength on quantum efficiency as was seen with the quinine sulfate in
acid solutions may be expected with anthracene also, but to a smaller
extent.

Figures 10 and 11 seem to show an anomaly, however. The effect of
excitation wavelength on quantum efficiency is reversed for the 1.03 X 10.4 M

4 M anthracene in benzene solutions.

anthracene in ethanol and 1.22 X 10—
The quantum efficiency of the first solution seems to decrease from

0.268 to 0.241 between 331 and 342 nm. The second solution also shows a
decrease of 0.268 to 0.197. The inner-filter effect explains this

anomaly. Figure 7 shows a strong absorbance by anthracene in ethanol

at 342 nm. Therefore, in more concentrated solutions there is an increased
probability of the inner-filter effect causing less fluorescence and

also a lower quantum efficiency than is expected at this wavelength.

This same explanation is true for the concentrated solutions of anthracene

in benzene.

69
The examples mentioned here show the typical effect of excitation
wavelength on the quantum efficiencies of the solutions studied.
For the quinine sulfate system in acidic media, at concentrations in

the order of 10-5

M or less, where the inner-filter effect is negligible,
the quantum efficiencies vary considerably (20-100%) with increasing
wavelengths of excitation from 313 to 365 nm.

Water solutions of quinine sulfate exhibit no effect on quantum
efficiency with increasing excitation wavelengths from 300 to 320 nm.

The anthracene system in ethanol and benzene shows a variation in
quantum efficiency with increasing excitation wavelengths from 331 to

5 M or less. The magnitude

342 nm for concentrations in the order of 10'
of this effect is smaller for this system (a 10—40% increase) than it is

in the quinine sulfate system.

The Dependence of Quantum Efficiency on Solute Concentrations

 

Figures 8, 9, 10 and 11 seem to show very little dependence of the
quantum efficiencies of quinine sulfate solutions on concentration, but
a dependence of the quantum efficiencies of anthracene solutions on
concentration does seem to exist.

Figure 8 shows three distinct quantum efficiency curves for the three

different concentrations of quinine sulfate in 1.0 N H2304. However, the

4

difference in values is less than 5% between the 4.91 X 10' M and

5

4.91 X 10' M solutions. This is within the 10% range of experimental

precision and thus this quantum efficiency change is not significant.

The inner-filter effect may be causing the lower quantum efficiency

readings of the 4.91 x 10‘4

5

M quinine sulfate solutions in relation to

values of 4.91 X 10' M solution.

70

The quantum efficiency values of the 4.91 X 10'6 M quinine sulfate
solution seem to vary considerably from the more concentrated solutions.
This may not be totally true, however. This concentration is within the
limit of sensitivity of the spectrofluorimeter-computer combination. The
problem stems from the inability to measure accurately and reproducibly
values of R and S which are close in value to get the small R-S differences
proportional to the quanta of exciting light absorbed by the sample and
the inability to measure accurately low F values which correspond to the
quanta of light emitted by the sample. Therefore, until instrument
performance is perfected, the quantum efficiencies obtained for concentrations
of solutions in the range of 10'6 M must be questioned.

Figures 10 and 11 suggest a concentration dependence of the quantum
efficiencies of solutions of anthracene in ethanol and in benzene. Using
a 331 nm exciting light, the anthracene in ethanol solutions show a change

in quantum efficiency of 0.196 to 0.241, or 22%, between the 1.03 x 10'4 M

and 1.03 X 10'5 M solutions. In benzene, there is a difference in quantum
efficiency of 0.213 to 0.268 or 26% between the 1.22 x 10'4 M and
1.22 x 10'5 M solutions. whiie this is a significant change, and it

indicates a concentration effect, part of this change is undoubtedly due
to the inner-filter effect. Once again, the decrease in quantum efficiency

6 M is questionable because of

in going to solutions of the order of 10'
the uncertainties in measuring low values of R-S and F.

At 342 nm there is a greater probability of inner-filter effects in
the more concentrated solutions. As stated previously in discussing
excitation wavelength effects on quantum efficiency, this is the reason
for the strange behavior in the quantum efficiency relationship between

4

the solutions of anthracene on the order of 10' M and 10"5 M in both

ethanol and benzene shown in Figures 10 and 11. Therefore, no comparison

71
can be made with use of the 342 nm exciting light for the effect of
concentration on quantum efficiency.

It seems that the quinine sulfate solutions show very little
dependence of quantum efficiency on solute concentration, while the
anthracene solutions show a dependence. Before more definite conclusions
can be claimed, instrumental sensitivity, and reproducibility, must be
improved and inner-filter effects must be minimized.

One method of lessening inner—filter effects for concentrated solutions
is to use 1 mm fluorescence cells instead of 10 mm cells. With the smaller
cells, the exciting light travels through a much shorter distance to
excite that part of the solution from which emission is monitored.
Therefore, the probability of uniform excitation of the solution in the
cell is increased.

The problem of instrumental sensitivity and reproducibility can be
partially overcome by using a more stable light source than is now being
used. Xenon arc lamps give out a continuium of radiation and thus are
useful sources. However, the arcs tend to wander. If the arc wanders too
much, there is non-uniform illumination of the entrance slit of the
excitation monochromator and hence non-uniform illumination of the
sample. Either a more stable lamp is necessary or very small entrance
slits must be used to decrease the effect of the wandering light.

The problem of obtaining values of quantum efficiencies of dilute
solutions may be partially alleviated through the use of some form of

quantum counter to monitor the low intensity fluorescence emission.

The Dependence of Quantum Efficiency on Temperature

 

Tabulated values for the quantum efficiencies of quinine sulfate and

anthracene in various solvents at room temperature (23°C) can be found

72
in Tables II-VI. Values for the quantum efficiencies of these materials
at 10°C can be obtained from Tables VII-XI.
A comparison of quantum efficiency values at 23°C and at 10°C seems
to show very little difference for both the quinine sulfate and the
anthracene solutions. Figure 12 shows the typical relationship of quantum
efficiency of a series of quinine sulfate solutions of concentration

4 M to 4.91 x 10'6

4.91 X 10' M in 1.0 N H2504. The maximum deviation
between quantum efficiencies obtained at 23°C and at 10°C is less than 5%.
Since the reproducibility of the instrument is only 10%, the small change
which is noted is insignificant.

To study any temperature effect in solution, temperature ranges
between the freezing point of the solution and higher temperatures should
be used. _

Since benzene freezes around 5°C, a lower limit of 10°C was set for
this temperature study. Room temperature was the upper limit since lack
of time prevented the extension of the temperature range for these studies.

Since the temperature range studied was only l3°, it is reasonable

that no great effect should be observed.

The Dependence of Quantum Efficiency on Acidity of the Solvent

 

The pH of various concentrations of quinine sulfate in 1.0 N H2504
was 0.6, in 0.1 N H2504 was 1.5, and in water was 5.8.

Figure 13 illustrates the typical behavior of the quantum efficiency
of the quinine sulfate solutions with changing acidity of the solvent.
This figure shows the variation of quantum efficiency of quinine sulfate
solutions of approximately the same concentration in 1.0 N H2504,

0.1 N H2504 and water.

Table VII. The Quantum Efficiency of Quinine Sulfate in 1.0 N H280

at 10°C

Concentration (

4.9l
4.91
4.91
4.91
.91
.91
.91
.91

h-D-b-b-b

.91

X
X
X

10
10
10
10
10
10

-4
-4
-4
-5
-5
-5

Fluorescence emission scan:

moles
liter

 

)

73

Excitation Wavelength (nm)
365
334
313
365
334
313
365
334
313

380-610 nm

Quantum

000000000

4

Efficiency

.511
.464
.368
.540
.490
.366
.352
.344
.278

Table VIII. The Quantum Efficiency of Quinine Sulfate in 0.1 N H2S0

at 10°C

Concentration (

5.
5.

5

01 x 10'

01 X 10-

.01
.01
.01
.01
.01
.01
.01

X
X

10-
10-
10-
10-
10-
10-
10-

Fluorescence emission scan:

moles

liter
4

 

4
4
5
5
5
6
6
6

)

74

Excitation Wavelength (nm)
365
334
313
365
334
313
365
334
313

380-610 nm

Quantum

000000000

4

Efficiency

.551
.490
.386
.530
.500
.378
.216
.196
.104

75

Table IX. The Quantum Efficiency of Quinine Sulfate in Water at 10°C

Fluorescence emission scan: 340-560 nm

 

Concentration (T?l::) Excitation Wavelength (nm) Quantum Efficiency
4.98 x 10'4 320 0.330
4.98 x 10'4 300 0.328
4.98 x 10‘5 320 0.430
4.98 x 10'5 300 0.420
4.98 x 10'6 320 -
4.98 x 10’6 300 -

76

Table X. The Quantum Efficiency of Anthracene in Ethanol at 10°C

Fluorescence emission scan: 360-500 nm

 

Concentration (T112?) Excitation Wavelength (nm) Quantum Efficiency
1.03 x 10’4 342 0.222
1.02 x 10‘4 331 0.266
1.03 x 10‘5 342 0.271
1.03 x 10‘5 331 0.241
1.03 x 10‘6 342 0.088
1.03 x 10'6 331 0.054

77

Table XI. The Quantum Efficiency of Anthracene in Benzene at 10°C

Fluorescence emission scan: 360-500 nm

 

Concentration (T1126) Excitation Wavelength (nm) Quantum Efficiency
1.22 x 10'4 342 0.181
1.22 x 10‘4 331 0.229
1.22 x 10'5 342 0.206
1.22 x 10'5 331 0.192
1.22 x 10'6 342 0.164
1.22 x 10"6 331 0.160

Figure 12.

78

 

 

 

 

 

 

 

1 1 1 1 1
300 310 320 330 340 350 360
Excitation Wavelength (nm)
Dependence of the Quantum Efficiency of Quinine Sulfate on

Temperature. Fluorescence emission scan: 380-610 nm.
A = 4.91 x 10'5 M quinine su1fate in 1.0 N H2504; B = 4.91 x
10‘4 M quinine sulfate in 1.0 N H2S04; c = 4.91 x 10'6 M

quinine sulfate in 1.0 N H2504. ————— 23°C. —--——--10°C

79

 

 

 

 

0.0 1 1 l 1 1 1
300 310 320 330 340 350 360 370

 

Excitation Wavelength (nm)

Figure 13. Dependence of the Quantum Efficiency of Quinine Sulfate on

Acidity of the Solution. Fluorescence emission scan: 3804610 nm.

-—— 4.91 x 10'4 M quinine sulfate in 1.0 N H2504. pH 0.6

- -5.01 X 10'4 M quinine sulfate in 0.1 N H2504. pH 1.5

mm4.98 x 10‘4 M quinine sulfate in water. pH = 5.8

80

Only a slight difference, less than 5%, is shown between the quantum
efficiencies of the quinine sulfate solutions in 1.0 N H2304 and 0.1 N H2504
so thus they are considered to be the same. There appears to be a significant
difference, approximately 20%, between the quinine sulfate solutions in
water and the acid solutions.

Chen (7) and Eisenbrand (10), along with others, have seen a dependence
of the quantum efficiency of quinine sulfate on solvent acidity. Chen
observed a dependence between 0.1 N H2504 and 3.0 N H2504. Eisenbrand
did not observe a change in quantum efficiency until he varied the

acidity from 1.0-3.0 N H 504. In the present study, there was no variation

2
between the 1.0 N and 0.1 N H2504 solutions, but there was a variation
between the acid solutions and the water solutions.

It has been postulated by many investigators (7, 10, 14) that the
change in the intensity of the fluorescence emission, and the corresponding
change in quantum efficiency, for the quinine sulfate is due to different
ionic forms existing in solutions at the different pH's. There are two
possible positions on the quinine sulfate molecule for protonation. One
possible position is the nitrogen atom located in the quinoline ring. The
other position is a nitrogen atom located in a side-chain ring.

In a highly acidic solvent, the quinine sulfate molecule exists in
the protonated form. Protons are attached to both nitrogen atoms. Thus,
in 1.0 N and 0.1 N H2504, the substance should exist in its protonated
form. That the structural form is the same for the molecule in both acid
concentrations can be deduced from the similarity of the excitation,
absorbance, and emission spectra of these solutions. The quantum
efficiencies do not even differ between these solutions.

For the quinine sulfate in water the pH is 5.8. This is different, by

a factor of 4 pH units, than the acid solutions. Since the water

solutions are not very acidic, the two nitrogen atoms are only partially

81
protonated. The excitation, absorbance and emission spectra of quinine
sulfate in water solutions differ from those of the acid solutions.
This difference may be attributed to ionic species which are different

from those existing in acidic solutions.

CONCLUSION

A study of the effects of excitation wavelength, solute concentration,
temperature, and acidity on the quantum efficiency of quinine sulfate in
1.0 N H2504, 0.1 N H2504 and water and anthracene in ethanol and in benzene
was undertaken.

Excitation wavelength seems to affect quantum efficiency considerably.
As the wavelength of excitation increases from 313-365 nm, the quantum
efficiency of the quinine sulfate solutions in acid also increases
Increases in quantum efficiency range from 20-100%. Quinine sulfate
solutions in water do not show a significant change in quantum efficiency
over the narrow range of excitation wavelengths studied. However; as
excitation wavelength increases from 331-345 nm, the quantum efficiencies
of the anthracene solutions in ethanol and benzene increase also. The
increases in quantum efficiency were considerably lower for anthracene
than for the quinine sulfate solutions. The increases range from 0-15%.

The study of the effect of solute concentration on quantum efficiency
seems to point to an independence of quantum efficiency for quinine sulfate,
but a dependence for anthracene. However, problems of inner-filter effects,
instrumental sensitivity and reproducibility must be overcome before
definite conclusions can be made.

Varying the temperature of the quinine sulfate and anthracene solutions
from 23°C to 10°C seem to have no effect on the quantum efficiencies of
the solutions. This may be expected over the narrow temperature range

studied. For future work, a larger temperature range is suggested.

82

83

The acidity of the solvent did affect the quantum efficiencies of the
quinine sulfate solutions. For the acid solutions, where the pH varied less
than one unit, there was no change seen in the quantum efficiencies.
However, there is a 20% change between the water solutions of quinine
sulfate and the acid solutions. The pH of the water solutions varied by
four units from the acid solutions. It is believed that different ionic
species, exist at the different acidities and control the amount of
fluorescence emission and the quantum efficiency of the solutions.

Unfortunately, due to instrumental problems, not as much was
accomplished in this study as desired. Once the reproducibility and
sensitivity of the instrument is improved, more exact answers may be
obtained concerning the effect of the many factors mentioned in this thesis

on quantum efficiency.

LITERATURE CITED

10.
11.
12.
13.
14.
15.
16.
17.

18.
19.
20.

LITERATURE CITED

Leverenz, H. W., "An Introduction to the Luminescence of Solids",
Dover Publications, Inc., New York, 1968, p xi.

5515,, p. xii.

Melhuish, W. H., J. Phys. Chem., 55, 229 (1961).

Weber, G. and Teale, F. W. J., Trans. Faraday Soc., 55, 646 (1957).
Weber, G. and Teale, F. W. J., Trans. Faraday Soc., 55, 640 (1958).
Chen, R. F., Nature, 555, 69 (1966).

Chen, R. F., Anal. Biochem., 15, 374 (1967).

Borresen, H. C., Acta. Chem. Scand., 15, 2089 (1965).

Moss, 0. W., Clin. Chim. Acta., 5, 283 (1960).

Eisenbrand, J., Z. Anal. Chem., 115, 170 (1961).

Fletcher, A. N., J. Mole. Spectros., 55, 221 (1967).

Fletcher, A. N., J. Phys. Chem., 25, 2742 (1968).

Fletcher, A. N., Photochem. Photobiol., 5, 439 (1969).

Dawson, W. R. and Windsor, M. W., J. Phys. Chem., 25, 3251 (1968).
Parker, C. A. and Rees, W. T., Analyst., 55, 588 (1960).

Parker, C. A. and Rees, W. T., Analyst., 51, 85 (1962).

Parker, C. A., "Photoluminescence of Solutions”, Elsevier Pub. 00.,
Amsterdam, New York, 1968, p. 69.

5515,, p. 72.

1515,, p. 428.

Hercules, D. M., ”Fluorescence and Phosphorescence Analysis",
Interscience Publishers, John Wiley and Sons, New York, 1966, p. 24.

84

21.
22.
23.
24.
25.
26.

27.
28.

29.
30.
31.
32.
33.
34.

35.
36.

85

 

 

Ibid., p. 33.

 

Vavilov, S. J., Z. Physik, 55, 266 (1924).

Forster, L. S. and Livingstone, R., J. Chem. Phys., 55, 1315 (1955).
Gilmore, E. H., Gibson, G. E., and McClure, D. 5., J. Chem. Phys.,
15, 829 (1952).

Hercules, D. M. and Frankel, H., Science, 151, 1611 (1960).

F6rster, T., "Fluoresezenz Organischer Verbindungen", Vanderbeck and
Ruprecht, Gottingen, 1951, p. 143.

Budo, A. and Ketskeméty, J., J. Chem. Phys., 55, 595 (1956).
Medinger, T. and Wilkinson, F., Trans. Faraday Soc., 51, 620 (1965).
Himel, C. M. and Mayer, R. T., Anal. Chem., 55, 130 (1970).

Eastman, J. W., Photochem. Photobiol., 5, 55 (1967).

Drushel, H. V., Sommers, A. T., Cox, R. C., Anal. Chem., 55, 2166 (1963).
Weast, W. C., ed., "Handbook of Chemistry and Physics", The Chemical
Rubber Co., Cleveland, 1966, p. c 121.

1515., p. c 529.

Holland, J. F. and Timnick, A., Paper 115, The Pittsburg Conference on

Analytical Chemistry and Applied Spectroscopy, Cleveland, 1971.

APPENDICES

APPENDIX I

THE COMPARISON OF QUANTUM EFFICIENCIES OF QUININE SULFATE IN 1.0 N H2504
BETWEEN INTEGRATION PERFORMED OVER A WAVELENGTH INTERVAL AND A FREQUENCY

INTERVAL

As stated previously it appears from Equation 22 that if fluorescence
is measured in terms of quanta, then the horizontal scale for the emission
spectrum should be in units of wavelength. Most work today is performed
with a horizontal scale in units of frequency. Parker (15) states reasons
for using the frequency units.

Quantum efficiencies were determined in this study for quinine sulfate
in 1.0 N H2504 by using both a wavelength interval and a frequency
interval for integration of the emission curve. Table XII shows the results.

There is no significant difference in the quantum efficiencies
obtained by using either units for the interval of measurement of the
emission curve with the precision now obtainable with existing spectro-
fluorimeters. Once the standard is recorded over the desired emission
range, in whatever units desired, then all other recordings are made in

terms of the same units.

86

87

Table XII. The Comparison of the Quantum Efficiencies of Quinine Sulfate
in 1.0 N H2504 between Integration Performed over a Wavelength
Interval and a Frequency Interval

Fluorescence emission scan: 380-610 nm

Concentration ($%%§%- Excitation Wavelength (nm) Quantum Efficiency

Values for a wavelength interval
4

4.91 x 10' 365 0.520
4.91 x 10'4 334 0.470
4.91 x 10’4 313 0.370
4.91 x 10'5 365 0.550
4.91 x 10‘5 334 0.504
4.91 x 10‘5 313 0.374
4.91 x 10'6 365 0.345
4.91 x 10'6 334 0.360
4.91 x 10‘6 313 0.272
Values for a frequency interval
4.91 x 10'4 365 0 522
4.91 x 10'4 334 0.474
4.91 x 10‘4 313 0.372
4.91 x 10'5 365 0.550
4.91 x 10'5 334 0.509
4.91 x 10'5 313 0.375
4.91 x 10'6 365 0.361
4.91 x 10'6 334 0.352
4.91 x 10'6 313 0.208

APPENDIX II

Since no reference to the complete emission spectra for the various

solutions was made in the text of this thesis, typical complete emission

spectra are shown in the following figures.

88

89

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iofi x —m.¢ .E: opmiomm

 

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90

 

 

//\V° \u'N
./ \

 

 

ov—‘—\'
1 \m 1
360 430 500
Excitation Wavelength (nm)
Figure 15.

Typical Emission Spectra of Anthracene Solutions. Fluorescence

emission scan: 360-500 nm. 1.03 X 10"4 M anthracene in ethanol.

1 - excitation wavelength = 342 nm; 2 = excitation wavelength =

331 nm.

APPENDIX III

The R-S value, which is proportional to the amount of the exciting light
absorbed by the sample solution has been discussed previously. Typical
values of R-S for quinine sulfate and anthracene are given in the following _

tables.

91

92

Table XIII. Typical R-S Absorption Values for Quinine Sulfate Solutions

 

in 1.0 N H2504
Concentration (Tglgi) Excitation Wavelength (nm) R-S
4.91 x 10'4 365 650
4.91 x 10"4 334 520
4.91 x 10'4 313 420
4.91 x 10'5 365 100
4.91 x 10'5 334 90
4.91 x 10‘5 313 60
4.91 x 10"6 365 20
4.91 x 10'6 334 10
4.91 x 10‘6 313 12

93

Table XIV. Typical R-S Absorption Values for Anthracene in Ethanol

 

Concentration (Tglgi) Excitation Wavelength R-S
1.03 x 10'4 342 470
1.03 x 10'4 331 280
1.03 x 10'5 342 80
1.03 x 10’5 331 50
1.03 x 10'6 342 10
1.03 x 10'6 331 10

 

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