Group Title: new phosphorimetric sampling system and subsequent temperature studies /
Title: A new phosphorimetric sampling system and subsequent temperature studies /
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Permanent Link: http://ufdc.ufl.edu/UF00099381/00001
 Material Information
Title: A new phosphorimetric sampling system and subsequent temperature studies /
Physical Description: x, 169 leaves : ill. ; 28 cm.
Language: English
Creator: Ward, Jimmie Lee, 1954-
Publication Date: 1980
Copyright Date: 1980
 Subjects
Subject: Phosphorimetry   ( lcsh )
Chemistry thesis Ph. D
Dissertations, Academic -- Chemistry -- UF
Genre: bibliography   ( marcgt )
non-fiction   ( marcgt )
 Notes
Thesis: Thesis (Ph. D.)--University of Florida, 1980.
Bibliography: Bibliography: leaves 165-168.
Statement of Responsibility: by Jimmie Lee Ward.
General Note: Typescript.
General Note: Vita.
 Record Information
Bibliographic ID: UF00099381
Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: alephbibnum - 000100398
oclc - 07412728
notis - AAL5859

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A NEW PHOSPHORIMETRIC SAMPLING SYSTEM AND
SUBSEQUENT TEMPERATURE STUDIES





BY


JIMMIE LEE WARD


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


UNIVERSITY OF FLORIDA


1980















ACKNOWLEDGEMENTS


I would like to express feelings toward all of those

with whom I have worked over the past four years. My

association with these people has been an education in

itself. I would like to express my special appreciation

to Dr. Esther LueYen-Bower for her time, patience and en-

couragement directed at helping me along the way, to

Dr. Gary L. Walden for his countless hours of friendship

and exchanging ideas, to Mr. David L. Bolton for his as-

sistance with the PDP 11/34, and most of all to Dr. James D.

Winefordner, without whom this could never have been

possible.

Mere thanks and appreciation are not enough for my

wife and parents. Their sacrifices for me can never be

repaid.
















TABLE OF CONTENTS


CHAPTER PAGE

ACKNOWLEDGEMENTS . . . . . . . ii

LIST OF TABLES . . . . . . . v

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

ABSTRACT . . . . . . . . . ix

I INTRODUCTION . . . . . . . . 1

II BACKGROUND INFORMATION . . . . .. 6

Historical Background . . . . 6
Theoretical Background . . . . 9

III STUDIES IN ROOM TEMPERATURE PHOSPHORIMETRY 28

Filter Paper Background. . . . .. 32
Nonreproducible Sample Positioning . 47

IV TWO NEW LTP DEVICES BASED ON CONDUCTION
COOLING. . . . . . . . .. 59

Experimental . . . . . ... .62
Results and Discussion . . . ... 72

V STUDIES IN LUMINESCENCE WITH VARYING
TEMPERATURE. . . . . . . .. 77

Preliminary Experiments with
Phosphorescence. . . . . ... 80
Effects on Fluorescence. . . . . 98

VI CONCLUSIONS AND FUTURE WORK. . . . .. 131

Room Temperature Phosphorimetry. ... . 131
Conduction Cooled Phosphorimetry . .. 132
Temperature Studies. . . . . .. 133









CHAPTER

APPENDICES

A MECHANICAL DRAWINGS OF PARTS MADE BY THE
MACHINE SHOP FOR THIS PROJECT . . .

B FORTRAN PROGRAM USED IN COLLECTING
FLUORESCENCE LIFETIME AND INTENSITY
DATA . . . . . . . . .

REFERENCES . . . . . . . .

BIOGRAPHICAL SKETCH . . . . . .


PAGE
















LIST OF TABLES


TABLE PAGE

1 FRACTION OF MOLECULES IN STATE Tj HAVING
SUFFICIENT THERMAL ENERGY TO ALLOW
REVERSE INTERSYSTEM CROSSING. . . .. 17

2 SOME OF THE SUBSTRATES TESTED FOR RTP. ... . 29

3 RESULTS OF SOAKING PAPER SUBSTRATES IN
SOLVENTS. . . . . . . . .. 40

4 RESULTS OF FLUTING SOLVENTS THROUGH PAPER
SUBSTRATES. . . . . . . . ... 41

5 RESULTS OF HEATING S&S 604 AT 120 C
FRACTION OF SIGNAL LEFT AFTER HEATING 43

6 RESULTS OF HEATING S&S 903 AT 120 C
FRACTION OF SIGNAL LEFT AFTER HEATING . 44

7 RESULTS OF HEATING PAPER AT 250 C
FRACTION OF SIGNAL LEFT AFTER HEATING . 46

8 RESULTS OF LOD MEASUREMENTS AS A MEANS OF
COMPARISON FOR THE TWO RTP SYSTEMS. ... 57

9 COMPARISON OF ANALYTICAL FIGURES OF MERIT FOR
PHOSPHORIMETRY BY CONVENTIONAL IMMERSION
COOLING (IC-77K), BY CONDUCTION COOLING
(CC-100K) AND BY THE NEW FLOW COOLED
DEVICE (FC-85K) . . . . . ... 73

















v
















LIST OF FIGURES


FIGURE PAGE

1 Schematic representation of the progress of
this research. . . . . . . . 3

2 Diagrammatic representation of SO, Sl, and
T1 . . . . . . . . 12

3 Analytical chemists representation of the
energy diagram for processes involved in
fluorescence and phosphorescence ... 14

4 Old Type RTP Attachment . . . . ... 35

5 RTP sample holding fingers. . . . .. 37

6 The new RTP sample holding bar. . . .. 49

7 Modified sample compartment bottom,
phosphoroscope and slit holder necessary
to receive the conduction cooling device 52

8 Modified sample compartment lid for use with
the new RTP sampling bar . . . . 55

9 The old immersion type sampling system. . 61

10 The first conduction cooling device ..... 65

11 The second flowthrough type conduction
cooling device . . . . . . .. 67

12 Diagram showing the dewar flask, conduction
cooling arrangement. . . . . .. 70

13 Sample temperature vs time as the level of
LN2 in the dewar flask drops ...... 79

14 Block diagram of the apparatus used in
measuring phosphorescence vs temperature 82

15 Photoanodic current vs time as temperature
varies . . . . . . . ... 85










16 Temperature curves for Lepidine in:
(a) 50:50 ethanol:water and
(b) 15:85 ethanol:water. . . . . . 88

17 Temperature curves for Lepidine in:
(a) 50:50 ethanol:water, (b) 70:30
ethanol:water, and (c) 100:0 ethanol:
water. . . . . . . . . . 91

18 Photoanodic current at the relative maximum
vs ethanol:water ratio . . . . .. 93

19 Cooling curves for (a) Lepidine and
(b) 4-chloroquinoline in 50:50 ethanol:
water. . . . . . . . . ... 96

20 Experimental apparatus for determination of
fluorescence lifetimes and intensities . 101

21 A plot of fluorescence lifetime vs temperature
for Nupercaine hydrochloride with no heavy
atom . . . . . . . . . 103

22 A plot of fluorescence lifetime vs temperature
for Nupercaine hydrochloride in 0.1 M I . 105

23 A plot of fluorescence lifetime vs temperature
for Nupercaine hydrochloride in 0.1 M Ag+. 107

24 A plot of fluorescence lifetime vs temperature
for Riboflavin with no heavy atom. ... .109

25 A plot of fluorescence lifetime vs temperature
for Riboflavin in 0.1 M I .. . . .. 111

26 A plot of fluorescence lifetime vs temperature
for Riboflavin in 0.1 M Ag . . . ... 113

27 A plot of fluorescence lifetime vs temperature
for Riboflavin in 0.1 M T1 . . . ... 115

28 A plot of fluorescence signal vs temperature
for Nupercaine hydrochloride with no heavy
atom . . . . . . . . . 117

29 A plot of fluorescence signal vs temperature
for Nupercaine hydrochloride in 0.1 M I . 119

30 A plot of fluorescence signal vs temperature
for Nupercaine hydrochloride in 0.1 M Ag+. 121


FIGURE


PAGE








FIGURE PAGE

31 A plot of fluorescence signal vs temperature
for Riboflavin with no heavy atom ... 123

32 A plot of fluorescence signal vs temperature
for Riboflavin in 0.1 M I . . . ... .125

33 A plot of fluorescence signal vs temperature
for Riboflavin in 0.1 M Ag+ . . . .. .127

34 A plot of fluorescence signal vs temperature
for Riboflavin in 0.1 M T1+ . . . .. .129

35 Mechanical drawing of the RTP sampling bar 140

36 Mechanical drawings of the raised block and
top plate used in adapting the RTP bar to
the Aminco. . . . . . . . ... 142

37 Mechanical drawing of the side extension/
bottom cover for the Aminco . . . .. .144

38 The new phosphoroscope . . . . ... .146

39 Drawings of the bottom extension and bottom
plate used with the Aminco. . . . .. .148

40 Modified slit assembly . . . . ... .150

41 The first conduction cooling device. ... .152

42 The second conduction cooling device ... 154

43 Diagram showing the dewar flask, conduction
cooling arrangement . . . . ... .156

44 Sample compartment lid used with conduction
cooling devices . . . . . ... 158


viii















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

A NEW PHOSPHORIMETRIC SAMPLING SYSTEM AND
SUBSEQUENT TEMPERATURE STUDIES

By

Jimmie Lee Ward

June 1980

Chairman: James D. Winefordner
Major Department: Chemistry

Three new sample holding devices are presented for use

with the Aminco-Bowman Spectrophotofluorometer. One of

these is a device designed to improve both rotational and

translational nonreproducibility in the positioning of

filter paper discs used in room temperature phosphorimetry

(RTP). The purpose of this is to decrease limits of de-

tection (LOD) by increasing the signal to noise ratio

(S/N). Although the precision is improved in the position-

ing of the filter paper, there is no effect on S/N or LOD

since the limiting source of noise is the varying background

of the filter paper itself. The washing and baking of the

filter paper as a means of decreasing this background are

discussed; however, both models are unsuccessful.

The other two sample holding devices are intended to

increase precision in sample tube positioning and thus








decrease LOD by low temperature phosphorimetry (LTP). Little

improvement is found over the LOD by more conventional

sampling systems, but the time required to run each sample

is reduced by up to a factor of six by the newer systems.

This, along with their greater convenience, makes the new

devices attractive for routine applications of LTP.

One of the new LTP devices allows temperature to be

varied over the range 100 K to 298 K. This is used in an

attempt to verify theoretical predictions on the behavior

of phosphorescing molecules with changing temperature. This

attempt is unsuccessful possibly due to failure of the

instrument to achieve sufficiently low temperatures.















CHAPTER I
INTRODUCTION


During the winter quarter of 1978, a short term

project in room temperature phosphorimetry (RTP) was begun,

which eventually led to several larger projects. The

overall scheme is represented in Figure 1. The original

project was to see what, if anything, could be done to

reduce the problems of low signal to noise and high

phosphorescence background in RTP on paper substrates. The

approach taken was to attempt the lowering of background

and related noises of the paper substrate itself. (The

approach of finding a background free substrate had al-

ready been tried and had failed.)4

The first step taken in attempting to remove the

paper's background was to try to wash away the source of

the background. Strong washes of acids and bases, alcohols,

hexane, benzene, chloroform, methylene chloride and water

were used to rinse and/or soak filter papers. The only

cases that showed any substantial decrease in background

were the soaking of the paper in strong base and O.1MH2SO4;

however, after soaking for sufficient time to reduce the

background, it was discovered that the maximum phospho-

resence of an analyte molecule adsorbed on the treated

paper was also reduced by a proportional amount.

1



























C
U)

(1)

--4


4-1

44



0






0
CD
41



0










41
4-4
0-
'-I






r4
Cr
Cn

ct
.r-
ar

(U










)i)


-1l
HO









The next step was to try to reduce the paper's back-

ground by heating the paper. Several filter paper samples

were heated in a 120 C oven, and samples were removed and

tested at intervals from 30 minutes to 3 hours. After

about 1 hour, the background began to decrease, but the

paper became brittle and analyte signals also began to de-

crease. The experiment was repeated with an oven tempera-

ture of 250 C. After 45 minutes in the oven, the paper

background and analyte signals observed were both reduced

to 50% of their original levels. After 2 hours, the filter

paper samples began to char.

After combining the washing and heating techniques, it

was decided that whatever was the cause of the phospho-

resence background in the paper was also responsible for

the matrix allowing RTP of molecules adsorbed on the paper.

In the light of this, it was decided that it should be

left for the paper companies to develop an improved low

background substrate for RTP. A publication appealing to

those who might develop such a substrate is presented as

a short communication in Talanta.5

Since the background and related noises could not be

reduced, the next approach was to try to improve precision

related to the positioning and optical alignment of the

sample. In the past, samples were suspended on one end of

a long rod.2 Slack in and bending of this rod were major

factors in the poor precision of RTP (often as high as

30% relative standard deviation). A new sampling system









was devised which held the sample rod rigidly at points

above and below the sample. This sampling system also

lended itself well to low temperature phosphorimetry (LTP),

whose long quartz tubes also suffered from positioning

problems. Two conduction cooling devices were developed to

allow the use of the new system for LTP.6'7

During the evaluation experiments, it was discovered

that the temperature of the first LTP device could be varied

continuously from 100 K to ambient. The phosphorescence

signals and lifetimes of several samples in solution have

been measured as a function of temperature. The result of

changing temperature on the heavy atom effect has also been

studied. It is hoped that the results of these experiments

will eventually show that phosphorimetry may be used as an

analytical tool at temperatures substantially higher than

were previously considered useful. This would then allow

the development of a convenient phosphorimeter for routine

analysis, not dependent on liquid nitrogen.















CHAPTER II
BACKGROUND INFORMATION


Historical Background


The first reported observation of phosphorescence from

organic molecules was made by Wiedemann in 1888. Weidemann

observed a long lived photoluminescence from several or-

ganic dyes adsorbed on gelatin. During the next 50 years,

numerous other researchers worked with phosphorescence,

leading to work done by G. N. Lewis et al. during the
10-13
1940's. In 1944, Lewis and Kasha proposed the triplet

theory for the phosphorescence of organic molecules,11

based on work by Lewis et al.,10 and Terenin.14 Gouy

balance type measurements made independently by Lewis et al12,13

and Evans 15 found that paramagnetism and phosphorescence

from illuminated samples simultaneously ". fade away

according to the same rate law."9 With the development of

electron spin resonance (ESR), paramagnetism was verified

in phosphorescing samples by Hutchison and Mangum.16'17

McClure offered indirect proof of the triplet theory and

restated the theory as it is accepted today.18

The first report of the use of phosphorescence as a

means of analysis for organic molecules was made by Kiers,

Britt and Wentworth in 1957.19 They found that spectral








resolution of components in mixtures was possible as a

result of selectivity by the molecules for different excita-

tion wavelengths and resulting selectivity from different

emission wavelengths. In addition, the lifetime of the

decay of phosphorescence varies from molecule to molecule

adding a third form of selectivity.

The method of phosphorescence as used by Kiers, Britt

and Wentworth, and others never caught on as a widely-used

analytical tool as a result of several problems. Phospho-

resence had only been observed from solid samples resulting

from either the adsorption of the molecule on some polymer

matrix or the freezing of the sample in some suitable

solvent to form a rigid matrix. In either case, the

formation of a rigid matrix is imperative to the observa-

tion of molecular phosphorescence, in that the probability

for collisional (radiationless) deactivation of the triplet

state must be minimized.

As a matter of convenience, the method of freezing a

solution of the molecule of interest became the method of

choice; however, in order to minimize collisions in the

sample, it was felt necessary to cool the sample to liquid

nitrogen temperature (77 K). As a result, the typical

matrix was a snowy, or frosted,glass, since few solvents

form transparent matrices at 77 K. Use of the snow matrix

was fine for qualitative studies, but inhomogenieties

arising from scattering of radiation by interfaces in the

sample resulted in gross imprecision in quantitative studies.









Hollifield and Winefordner20 and Zweidinger and Winefordner21

found that spinning the snowy sample in an NMR spinner type

device could compensate for inhomogenieties in the snow.

Spinning the sample improved precision somewhat, but align-

ment of the spinning device and precession of the spinning

sample proved to create new precision problems.

Aside from the expense, hazards and inconvenience in-

volved in handling liquid nitrogen, several instrumental

problems have arisen with the use of liquid nitrogen as a

coolant for phosphorimetry. The conventional means of

cooling a sample is the immersion of a quartz capillary

containing the sample solution into liquid nitrogen.

Phosphorescence measurements are made while the sample is

immersed. In order to prevent absorption of excitation or

phosphorescence radiation as well as interference arising

from luminescence of the dewar flask containing the liquid

nitrogen, it is necessary that the dewar be constructed of

high quality optical grade quartz. The resulting expense

can be considerable. In addition, any imperfections or

scratches in the dewar flask can give rise to boiling

surfaces for the liquid nitrogen. Bubbles passing through

optical paths are a great source of noise and error. Other

problems involved in the immersion cooling of sample solu-

tions are discussed elsewhere.22

The alternative to frozen samples is adsorption on a

solid polymer matrix, originally gelatin. In their work,

Lewis etal. used samples prepared in solid solution with








boric acid. These matrices were, however, impractical for

routine work since such preparations are quite tedious.

In 1957, Szent-Gy6rgyi found that samples adsorbed on

filter paper and TLC plates were observed to phosphoresce
23
when immersed in liquid nitrogen.23 Thus, a new support

matrix came into use for proteins and polyaromatic

hydrocarbons.

In 1967, Ross observed long-lived phosphorescence from

chromatograms irradiated by ultraviolet at room temperature.24

In 1972, Schulman and Walling rediscovered the phenomenon

and began exploring the analytical possibilities of room

temperature phosphorescence of samples adsorbed on filter

paper and many other supports.25,26 Since that time, room

temperature phosphorimetry has been studied by a number of
3, 25-44
research groups25-44 and has been shown to be a highly

selective, quite sensitive method for the analysis of

several organic compounds. The history of room temperature

phosphorimetry has been more completely covered in other

places.22


Theoretical Background


In their lowest energy, or ground state, organic

molecules contain an even number of paired electrons which

are distributed among discrete electronic energy levels.

The two electrons of each pair have opposite spin, so the

net electronic spin (S) for the molecule is zero. The

multiplicity (2S +1) is one; hence, the molecule is said to








be in a singlet state, SO. The subscript zero indicates

that this is the lowest singlet, or ground state. However,

if the molecule is in an excited state, the excited elec-

tron can now remain paired (of opposite spin), or become

unpaired (of like spin) with respect to the remaining

ground state electron. In the paired case, the total spin,

S, remains zero and the multiplicity is one. This singlet

state is denoted Sl, the subscript indicating that this is

the first excited singlet. In the unpaired case, the total

spin is one (S = 1/2 +1/2), and the multiplicity becomes

three, a triple (T1). When a molecule is excited from

SO to SI, the excited electron may change spin, allowing the

molecule to pass from S1 to T1 (intersystem crossing). This

process is illustrated in Figure 2.

If a triplet state is produced, the molecule can de-

excite to SO by emitting a photon. The resulting radiation

is phosphorescence. If the molecule is excited to S1, but

T1 is not produced, the molecule can still deexcite to SO

by photon emission. This radiation is called fluorescence.

Generally, excitation of the molecule to singlet states

above S,l i.e., S2, S3, etc., has little effect on the

fluorescence or phosphorescence, since states higher than

S1 readily deexcite to S1 through nonradiational means.

For each transition which results in radiation

(fluorescence or phosphorescence), there is a corresponding

radiationless transition. Each of these radiationless

transitions is illustrated by a dashed arrow on Figure 3.

















M


43
CI











r, 4
Ca












(t
C-4r
C.










o N


r-I
4-4HQ
U)
-cl

4J





UC)
a)

4-4
C
C
Cld
C

CIC







me
-Jo
cd









Z7)2
4d0









.ri -H
C) 4







Ill
-4 C4



4 0
C'
CC



*7 4-4



.1i u
244
H C)
C'0
C)

C
Co





12



































<----































Figure 3. Analytical chemists representation of the
energy diagram for processes involved in fluorescence and
phosphorescence.


A = absorption
VR = vibrational relaxation
F = fluorescence
IC = internal conversion


ISC = intersystem crossing
P = phosphorescence
CD = collisional deactiva-
tion of the triplet
E = energy




















sl









E













so


VR


VR
ISC
1 I I I
I i II
I III
IIII I
11111


IA F I I C P I CD
ItI I

I lil \
1111 I I

III III

ill ill* I
I I I I I I
I11I l;Ili
III IIII
IIII


@ YJ Y


T1








Each electronic energy level (So, SI, TI, etc.) has

several vibrational energy levels (see Figure 3); there-

fore, a variety of photon energies can be used to excite

the ground state molecule to various vibrational levels of

S It is this variety of excitation energies that is
responsible for the observation of broad band excitation

spectra. Once in SI, the excited electron tends to drop to

the lowest vibrational level of S1 through a process known

as vibrational relaxation, or it may undergo intersystem

crossing and then vibrational relaxation to the lowest

vibrational level of T1. Similarly, upon fluorescence or

phosphorescence emission, the excited electron may de-

excite to any of the vibrational levels of SO, resulting in

broad band emission spectra. Since it is usually considered

that electrons remain in the lowest vibrational level of SO

at room temperature, it is also assumed that vibrational

relaxation occurs in S. after deexcitation. Notice (in

Figure 3) that T1 is of lower energy than SI. This is

physically observed since the radiative transition T1 S.

(phosphorescence) is of longer wavelength than S1 SO

(fluorescence) or S. S1 (excitation).45

Although absorption from SO directly to T1 has been
46,47
observed,4647 the process is spin forbidden, and as a

result the transition probability is very small.9 For

this reason, SO 0 S1 absorption followed by intersystem

crossing is considered the major means of populating T1,








and SO -T1 has been neglected on Figure 3 and from further

discussion.

In order for the reverse process of intersystem cross-

ing (reverse intersystem crossing) to occur, the electron

must have sufficient thermal energy to be excited to a vi-

brational level of T1 of equivalent total energy to S1. If

some molecule has a SO S1 absorption wavelength of 300 nm

and a T1 S emission wavelength of 450 nm, assuming only

0-0 vibrational level transitions, the energy difference

between S1 and T1 (ES IT) is given by:




AE 1.99 x 10-25Jm 1.99 x 0-25Jm
S-T 7 -7
1 1 3.00 x10 m 4.50 x10 m


= 2.21 x 1019J [eq 1].



From this, using Boltzmann statistics, the fraction of the

population of T1 having enough thermal energy to allow

reverse intersystem crossing (FRIC) is given by



gu -(2.21x0-19 J/1.38 x 10-23JK T)
FRIC g e [eq 2],



where gu and g. are the statistical weights of the upper

and lower states, respectively. Table 1 shows the FRIC

calculated at several temperatures. Based on this informa-

tion, we see that even at very high temperatures for con-

densed phase phosphorescence measurements, reverse









intersystem crossing is negligible compared to other

processes. For this reason, reverse intersystem crossing

is not shown on Figure 3, nor shall it be considered in

further discussion.



TABLE 1

FRACTION OF MOLECULES IN STATE T1 HAVING
SUFFICIENT THERMAL ENERGY TO ALLOW
REVERSE INTERSYSTEM CROSSING


(NEAREST ORDER
T (K) RIC OF MAGNITUDE)


77 10-91

100 10-70

273 10-26

298 10-24

398 10-18


Kinetic Approach


Assuming the model given in Figure 3, and using the

approach of Forster,48 it is possible to express the quanti-

ties of fluorescence and phosphorescence lifetimes and

quantum efficiencies in terms of the kinetic expressions

arising from the rate constants for each process shown in

Figure 3.

When a molecule is excited from SO to SI, the rate of

excitation (kA) is usually given by:








k BS SI E [eq 3],
A S0 --S 1 [eq 3],


where BS S is the Einstein induced absorption coefficient
-1
-1 -2 -1 -1
in (Js m Hz ) S and E is the irradiance of the
-1 -2 -1
source in (Js m 2Hzl ). The change in the population of
d[S1]
SI( dt ) can then be expressed:


d[S1
dt kA[SO] kIC[S] -AS1 Sg [S



kSl TI [S11 [eq 4],


where kic is the rate constant for internal conversion,

AS1 $SO is the Einstein coefficient for the spontaneous

emission of fluorescence and kS T1 is the rate constant

for intersystem crossing. Actually, attributing only one

rate constant to the process of internal conversion is

somewhat misleading since kic is actually a collection of

several rate constants. For the sake of simplification,

we shall assume that processes such as dimerization and

photochemical reactions are negligible. This assumption

made, internal conversion becomes collisional in nature.

The term collision is used loosely in this work to describe

any diffusion controlled second order process. The decrease

in population of S1 resulting from internal conversion is

given:








-k's ,1 [S] [C1] k'S ,C2 -k'S1C3 [S] [C3]


. -k',Cn [S ] [Cn] [eq 5].



If k' n is the second order rate constant for deactiva-

tion of S1 via collisions with particle Cn, then the number

of terms in [eq 5] is determined by the number of possible

collisional partners, e.g., solvent molecules, etc. Based

on this, kic from [eq 4] actually becomes:


kic = k'SIC1 [C ] +k'S [C2] +k'1C3 [C3


+ . k' [Cn] [eq 6].


All other rate constants from [eq 4] are first order.

If we define the fluorescence quantum efficiency (YF)

as the fraction of absorbed radiation reemitted as

fluorescence, we see:

As, s, Sl AS 1 SO [Sl]
AS1 S0 A1

B = [eq 7].
F BSO S 1 E SO kA [So [eV 7].

d[S1]
Using a steady state approximation, where = 0, and

solving [eq 4] for AS S0 [Sl], we find



As S0 [SI] = kA[SO] kic [S] -k T [S1 ] [eq 8].








Substituting into [eq 7]:


kA[SO]- kI[S1] -kS- T[SI
YF= k [S 1 [eq 9a]
A 0

or as it is more conventionally expressed:

AS1 SO[S
Y A +k [eq 9b].
SS k S1+TI +kIc


If the radiative fluorescence lifetime (rF) is defined as

the time required for the fluorescence intensity to fall to
48-50
l/e, it can be expressed:


1
TF = +As +k [eq 10].
kIC S1 0 S SI S T1

Using the same approach for phosphorescence, the
d[T1]
change in population of T( dt ) can be expressed:


d[T1]
dt = kS TI[S1 -AT1 [T] -kcD [T [eq 11],



where AT +SO is the Einstein coefficient for the spontane-

ous emission of phosphorescence and kCD is the rate constant

for collisional deactivation of T1. By analogy to kic and

[eq 6]:



k = k' TC1[C1 +k' T [C2 +k'T C[C3
CD T2 T1 ,C3


+ . + k' T Cn [Cn]
1' n


[eq 12].








If the phosphorescence quantum efficiency (Yp) is

defined as the fraction of absorbed radiation reemitted as

phosphorescence:


AT 1 S [Ti] AT -S [TI
Y BS S1 E S = kA [e 13]
0 1

d[S]
Again assuming steady state conditions, where 0
d[Tl] dt
and dt = 0, we may solve [eq 11] for AT1+ SO[TI] and

[eq 4] for kS Tl[S1 to obtain:



AT1 ST 1 = k TS1 kcD[T1] eq 14];


and


kSITISl11 = kA[SO] kICSl] -AS +3sS i [eq 15].


Substituting [eq 15] into [eq 14], and the result of that

into [eq 13], we find:


kA[S0] kIC[S 1 -AS1 s0[S] kCD [T
Y kA[SO] [eq 16a]


or as it is more conventionally expressed:


AT SO ks1 T1
Y= I 0 [eq 16b].


r


T1 S0 CD S1 T1 S1 )S0 NIC








If, as in the case of fluorescence, we define the

observed phosphorescence lifetime (T ) as the time re-

quired for the phosphorescence to decay to l/e, then



T AT 1 +k [eq 17].
AT1S0 +CD


In the above discussion, no consideration has been

given to induced emission from S1 or T1. Since by

definition:

3
Bu = Auz [--7] [eq 18],
8Thv


where Bu is the Einstein coefficient for induced emission,

Bu E << Auz for low irradiances and is therefore negligible.

Also by definition, the Einstein coefficients A S

AT S0 and B S1 are temperature independent. Consider-

ing that, all radiative constants are temperature indepen-

dent. Looking at [eq 9a] and [eq 10], we see that tempera-

ture dependence can only enter YF and TF through kic and

kSl T Similarly, any temperature dependence in Y and

must come from kic, kl T 1 and kCD (see [eq 15] through

[eq 17]). Since temperature dependent intersystem crossing

typically occurs from an excited singlet to a higher

excited triplet, e.g., S1 -T2, and since this process is

rarely observed, we shall consider that intersystem cross-

ing, particularly k T 1, is temperature independent. If
b1 1
this assumption is valid, kI and kCD must both be tempera-

ture dependent.








Until now, we have considered kiC and kCD to be en-

tirely collisional in nature. Strictly speaking, this is

not true. Quenching processes have been shown to have some

temperature independent nature; however, the temperature

independent processes have also been shown to contribute
49
only on the order of 0.1% of the total quenching process.4

For this reason, we shall continue to consider quenching as

a diffusion controlled second order process. In order to

further describe the processes involved, it is necessary

to expand the individual collisional components of kic and

kCD. To do this, we will now consider the sample case of

quenching of Sl by the colliding species Cn. In [eq 5],

this was described by k'S,C n[S [Cn] ; k's cn must now

be expanded to:

-(Ws /CKT)
k' = k" e n [eq 19],
SI',n S1,Cn


where W ,n is the work function for effective collisional
SIVCn
quenching, and K is Boltzmann's constant.

If we assume that colliding particles in the quenching

process act as hard spheres and that the energy of activa-

tion for the quenching process is much less than KT (where
50-52
T is temperature, in k), k" C can be expressed:

(rS + r )2
k" = p r _Tn [eq 20],
SVC n 3 rS rC








where P is the fraction

ing, q is the viscosity

radii of the respective

to vary between 0.6 and

we shall approximate P=

that r
r1 rn


of collisions effective in quench-

in poises and rS and r are the
1 n
particles. The value of P has been shown
49
1;4 so for the sake of simplicity,

1. Furthermore, if we consider


4KT
k" n 3
S1,Cn 3n


[eq 21].


Viscosity, n, also varies greatly with temperature;

however, since this problem has been dealt with in great
52,53
detail elsewhere, 3 and since at this point n can only

be approximated at very low temperatures, no further con-

sideration will be given to this matter.

If we now combine [eq 19] and [eq 21], we find:


-T (W s /KT)
SJ, 4- T e 'C
k n 3 e
SI',C 3ri


[eq 22].


Assuming the dominating collisional species is the solvent,

kiC becomes:


-(WS /KT)
S4T S1'Cn
kIC 3n e


and YF becomes:

-(W /AT)
(4KT e 'C
kA[SO]- 3 e n


Y
F


[S 1 [Cn]) -kS 1TI [S


[eq 23],


kA[Se]
[ec 24a],












AS1 SO
-(W s,/KT)
SC


A 4KT
AS1+SO + ( e


[C ]) +kS T


[eq 24b];


and TF becomes:


[eq 25] .


(W C /KT)
Sr',C


4KT
(- e
3n


[C ]) +ASl S+kST


Using the same approach for collisional deactivation from

the triplet, Yp can be expressed:


1C/KT)
r i


[S1] [Cn )


4 -(W
kA [S] e
p = A
P kA [SOi


-(W
4KT
-AS1 0 s [S] 3t e
kA [SO]


TC n/KT)
T1,Cn


[T1] [Cn])


[eq 26a],


or


Y
F


T =
F










Y =
P


[AT1 + (4~O
I S 0 (3n


AT+ SO k S 1 T1


- (WT n /KT)

[Cn]) -k 1
ksI T1


4cT
[As +SO + 3 e


-(W /CT)
S1'Cn


and Tp becomes:


Tp =
P


[eq 27].


4T (W /KT)
A T4T [C
A1 SO+ 3 e [C0]


Thus, it is possible, if values for all of the rate constants

are known, to predict fluorescence and phosphorescence

quantum efficiencies and lifetimes as a function of

temperature.


Heavy Atom Effect


The intensities and lifetimes of phosphorescence and

fluorescence radiation have been observed to change when

influenced by the presence of some atom or group of atoms.

In general, the presence of the additional species results

in one (ormore) of three effects. First, the S1 T1 transi-

tion might be enhanced, resulting in an increase in Y


[eq 26b];


[C ] )]











and a decrease in YF. Second, the radiative T1 -S0

transition might be enhanced. This would result in an

increase in the peak intensity of phosphorescence with a

decrease in Tp but no change in Yp, i.e., there is no change

in the integral of phosphorescence intensity as a func-

tion of time. Third, internal conversion and/or colli-

sional deactivation of the triplet might be enhanced,

resulting in decreases of YF' Tp, Yp and Tp.

These effects have been observed under two specific

sets of circumstances and, as a result, are called the in-

ternal heavy atom effect and the external heavy atom effect.

The internal heavy atom effect is observed when some species,

usually a halogen, is substituted onto the molecule of

interest. The external heavy atom effect is observed when

a perturbing species, usually ethyliodide, I or some

paramagnetic metal ion, is introduced into a solution

containing the molecule of interest.54

The exact mechanism responsible for these effects is

not known; however, in the case of the internal heavy atom

effect, it is believed that charge transfer processes re-

sulting from the inductive effect of the substituent are

responsible. Similarly, in the external heavy atom effect,

close proximity of species such as ethyliodide and I are

thought to induce charge transfer.55 In the case of para-

magnetic ion induced heavy atom effects, it is believed that

the close proximity of the paramagnetic atom alters the

singlet-triplet mixing characteristics of the molecule

through some spin-orbit coupling mechanism.'55















CHAPTER III
STUDIES IN ROOM TEMPERATURE PHOSPHORIMETRY


The attractive features of room temperature phospho-

rimetry (RTP) over low temperature phosphorimetry (LTP) are:

(i) the analytical procedure, involving solid substrates at

ambient temperature rather than glasses in small cells at

liquid nitrogen temperature, is simpler; (ii) degassing of

solvents is not necessary; and (iii) the use of cryogenic

equipment is avoided.

There is, however, one major limitation to RTP, i.e.,

virtually all substrates that produce significant analyte

phosphorescence signal levels also exhibit a broad band

phosphorescence background (~400-600 nm). This background

is often of the same order of magnitude as analyte signals

from relatively high concentrations. In the past, workers

at the University of Florida, as well as others, have evalu-

ated substrates for RTP on a rather random, haphazard basis

(see Table 2) with relatively little success. For a wide

variety of substrates, analyte signals may vary over a wide

range for a given amount of analyte, but the ratios of

analyte to signal to blank signal and of signal to noise

rarely vary by more than a factor of 2 or 3. The only

exceptions to this are those cases where the substrate is

















r-U C1 r 0 0
-I I N 'r (N

vN VO tf :
m~2 (( d~ H~ (


N CN


-H u

.1 0
.4J 4
Hl 1)
a) Hr u
U H



> 4.J (i

9 r m


(N
0
(N


(N

m H(N i) CN
U LSUUU



00 0000 W $-
I .E.0.0.0 2) e
cc m En In CI C (a ( m
,a aa a a 1 a a14 1 a4 4 C:4 :


w cj
C4 04
(d to
P4 04


1.4 (4 .4 (.4

Q4 04 04 04
(a 0 0 fa
a4 4 04 a,


0

0





Ec


< E






E t

44


0


0


couccoccuu uo uuuuoo 0 0


















m

H f


N


Ln
N


H ^ H H ~


~. -a. ~


a4-a

10 0
pmc
m (d

(L1 0 10

HW HE

mQ fa o *H
04 04E-4 CO 4


>1


o ,

& a4


O O
0 0 0
-H


cU


; 0 0

S 4-U )

W V cn


Z Z 0 0 ti Z P Z









ul

ci
0
a)


o
4-1

nl
-Q
0



10
rox


c,
-H



>-p
N


N H


in

o <
0 -10

(N 0 1
1n


po -
(0 L -


-H

0)
p

a) C >,
14 -1
fd s
P & fi




















CN







Z Z Z Z Z


-1



114
0
Q)

C u


Cd O
4C in
-1-1 cl
iti l i


eg
'0
Cd




O

cr
Cd
0
U

(N









completely ineffective and analyte signals are not

detectable.

One minor problem in RTP has been the poor precision

resulting from nonreproducible sample positioning. This

was a result of the manner in which the sample was sus-

pended on one end of a rather long aluminum rod.2 This

chapter addresses itself to both of the above problems.


Filter Paper Background


In order to systematize studies to find improved RTP

substrates, two approaches could have been taken. Firstly,

an ordered progression of many possible substrates could be

evaluated for effectiveness, but this approach is not much

of an improvement over the previous, random approach since

the number of possible substrates approaches infinity.

Secondly, a single, effective substrate could be chosen,

and studies could be conducted, directed at improving this

substrate. The latter of the two approaches seemed more

attractive, and filter paper was chosen as the single sup-

port easiest to work with. Even among filter papers, the

variety is almost endless (see Table 2), so the field was

further limited to 2 particular filter papers. (Schleicher &

Schuell papers 604 and 903 were chosen since they exhibited

the largest analyte signals and highest signal to noise of

all papers previously studied, by a narrow margin.)








Experimental


All RTP measurements were made on an Aminco-Bowman

Spectrophotofluorometer (American Instrument Company,

Silver Springs, MD) fitted with a 150 W xenon arc lamp

(Schoeffel, Westwood, NJ), and Aminco-Keirs rotating can

phosphoroscope and a potted 1P21 photomultiplier tube

(Hamamatsu, Middlesex, NJ). An Aminco Ratio Photometer

supplied high voltage to the photomultiplier tube in addi-

tion to serving as a D.C. amplifier. All line voltages

were regulated with a Sorenson 1001 A.C. Regulator

(Sorensen, So. Norwalk, CT).

The standard Aminco phosphorescence sample compartment

was used with a laboratory constructed RTP attachment

(see Figure 4) as described by Paynter et al.2 Six aluminum

sample holders, or "fingers," were used to hold 1/4 in.

diameter filter paper discs (see Figure 5). The filter

paper discs were prepared by punching filter paper with a

standard office paper punch. These discs were placed under

the coverplates of the fingers and were tightened into

place by 2 screws.

Once the filter paper was in the finger, samples and/

or heavy atoms were spotted onto the paper in 3 or 5 pL

volumes with a SMI micropettor (Scientific Manufacturing

Industries, Emeryville, CA). After spotting, all samples

were dried 10 minutes under an infrared heat lamp. While

still under the heat lamp, the fingers were slipped onto
































Figure 4. Old Type RTP Attachment. The 6-3/4" long
1/4" diameter aluminum shaft was tapped to 1/4-20 and held
in the center of the brass cylinder by means of 2 nuts.
A 1/8" diameter hole at the bottom of the shaft kept the
set screw for the RTP finger from slipping.

















s"


35





3h











4r
III








I I-- I









HI .I 1/".i















I I2-5/16.
I I I~d
I I














-2-1/2"
o.d.


-i


































Figure 5. RTP sample holding fingers. 1/4" diameter
filter paper discs were clamped under the cover plate and
exposed through a circular window. (Shown twice actual
size.)











-- 5/8"-K


Front View


1-1/4"



4,


15/64"


Cover Plate


1/4"


Side View


--H 5/128"


J5/8"



5/8"

I








the rod of the RTP attachment and were tightened into

place by means of a set screw. The assembly was then im-

mediately transferred to the sample compartment, where it

was allowed to cool for 10 minutes under a flow of dry

nitrogen gas. (During the cooling process, signals in-

creased over a period of ~9 minutes at which time a plateau

was reached for about 2 minutes. Measurements were made on

this plateau. After the plateau, signals began to fall off

as moisture was collected by the sample support.)


Washing


The simplest cause of the phosphorescence background

could have been nothing more than some adsorbed impurity

common to each manufacturer's process. In an attempt to

eliminate that possibility, LueYen-Bower rinsed 5-1/2 in.

diameter filter paperdiscs (Eaton-Dikeman 613) with 10 mL

each of 1M NaOH, lM HNO3, ethanol methanol, deionized water

and acetone. She found essentially no change except in the

cases of acetone and distilled water which gave on the order

of 30-50% background reduction. In the hope that a 10 mL

rinse on 5-1/2 discs simply was not enough to completely

rinse away an impurity, several additional experiments were

performed.

All rinses were done in 2 ways. First, 1/4 in. discs

of filterpaper were prepunched to fit the sampling fingers

of the instrumentation. These discs were soaked for ap-

proximately 18 hours in beakers containing 20mL of the








solvent being used. After soaking, the discs were air

dried and used as normal filter paper.

The second rinse method closely resembled paper

chromatography. Strips of filter paper, 1-1/2 in. x 3 in.,

were hung in 250 mL beakers containing =1/2 in. of solvent.

Solvents were allowed to elute up the paper for approxi-

mately 18 hours. The paper strips were allowed to air dry,

then 1/4 in. discs were punched from just above the solvent

line.

Several discs were produced for each of the solvents

used by both rinsing methods. With these discs, all

phosphorescence measurements were made in triplicate ac-

cording to the following scheme. Two discs of each type of

paper (S&S 604 and S&S 903) were placed in the sample

fingers. One of these was spotted with 5 iL of 1M NaOH/NaI,

and background was measured from both discs by normal RTP

procedures. After background measurements, each of the

discs was spotted with 5 pL of 100 ppm Privine HC1. The

paper was again I.R.-dried and phosphorescence of the sample

was measured. The results of these experiments are listed

in Table 3 and Table 4.

From the results in the two tables, we find that there

is no advantage of one rinse method over another. In addi-

tion, there is no improvement of the rigorous rinse methods

over the quick rinses made by LueYen-Bower.

Since there was an improvement over samples with no

rinsing by those rinsed with water and acetone, several


























>u


























>0
H




































z
z
rf
C-1


























>


CN H H- r- N H- C N 0


C

Z
H













0



0









rn
U)











F41


000 0 (A
z 0 Cd

U r-, c rH X H OH 4
S 4 Q) (N ()
a 0u 00000o o


H 0o


l 0








r-i 0


c H H N i H-i r








SH --I H H H H 0 -q


N H- Hrl (N -r CN 0








S-l l N rl 0 l







N H-l il lr N


Hl 0








r- O







- 0








c,


S0 Z
4-1 r-1

U H

















-i r-l r-1 lN r- -A -I CD







(N -4 r-i -A N -4 -A (N 0 -4


-1 0



- 0



HO


Z
F-. r--
>0








HP




F
a



























H4
c-i
























Z
HtO
0Z
:a:






























m


(N -4 r-


- r-4 i- ( N 0


-4 '-i -4 (N r -4 -4 0O


0








Hi 0


-4 -1 N -4 0 C -







N~~~Ncq 0


O r


0 0r
u C c


UM Kio


0

(NO



( -,


-l4 r-l r-4 ( r 0








discs were rinsed first with acetone and then water, using

the soaking method. The results showed no improvement over

each of the 2 solvents individually.

It should be noted that the appearance of the paper

support did not change by washing, with 3 exceptions.

Washing with NaOH and H2SO4 caused the filter paper to turn

a brownish color and become very brittle. Washing with

HC1 caused some yellowing and brittleness in the paper.


Heating


When washing the filter paper failed to reduce the

background sufficiently, it was thought that the background

might be due to some impurity more inherent than simply an

adsorbed impurity. As a result, heating the paper was

considered as a possible means of background reduction.

Filter paper discs were prepunched for use with RTP

sample fingers. Half of the discs were treated with 5 _L

of a solution IM in NaOH and 1M in NaI. All discs were

placed in a preheated oven and sample discs were removed

at 1/2 hour intervals. Upon removal from the oven, discs

were put into the RTP fingers and allowed to cool in the

instrument under a nitrogen flush for 10 minutes, after

which background measurements were made. Once the back-

ground was measured, 5 pL of 100 ppm Privine HC1 was added.

After 10 minutes of I.R. drying and 10 minutes of cooling

under dry nitrogen flush gas, phosphorescence signals were

measured for Privine HC1. The results of these experiments

are shown in Tables 5 and 6.

















TABLE 5

RESULTS OF HEATING S&S 604 AT 120 C
FRACTION OF SIGNAL LEFT AFTER HEATING


0 1

1/2 1

1 1/2

1-1/2 1/3

2 1/3

2-1/2 1/3


1 1

1 1/2

1/2 1/10

1/3 0

1/3 0

1/3 0


1

1/2

1/10

0

0

0
















TABLE 6

RESULTS OF HEATING S&S 903 AT 120 C
FRACTION OF SIGNAL LEFT AFTER HEATING


0 1 1 1 1

1/2 1 1 1/2 1/2

1 1/2 1/2 1/10 1/10

1-1/2 1/3 1/3 0 0

2 1/3 1/3 0 0

2-1/2 1/3 1/3 0 0








No improvement was found in the Privine HC1 phospho-

rescence over the background in either case (treated paper

or untreated paper); however, after 1/2 hour, the treated

paper had turned visibly brown, and by 1-1/2 hours, it had

turned black and had a pasty texture. After 1-1/2 hours,

the untreated paper had yellowed and was quite brittle.

This experiment was repeated at an oven temperature of

250 C using only untreated paper. At the higher tempera-

ture, 15 minute time intervals rather than 30 minute in-

tervals were used for sampling (see Table 7). The results

very closely followed the results at 120 C. After

45 minutes, phosphorescence intensities had dropped to

50% of their original values. After 2 hours heating time,

the paper discs had begun to char and turn black around

the edges. After an additional 15 minutes of heating,

signal levels fell to zero.

As one final check, several filter paper disks were

soaked in each of the solvents shown in Table 3 except

H2SO4 and NaOH. These discs were then heated at 120 C for

1/2 hour. Blank signals and Privine HC1 signals were meas-

ured for these discs as well as for a set of unprepared

discs. As a result, an improvement in signal to noise of

a factor of two was found after all of the paper pretreat-

ment. This showed no advantage over simply rinsing 3 in.

filter paper circles with 10 mL of acetone or water.

Furthermore, this did not approach the 1-2 order of

















TABLE 7

RESULTS OF HEATING PAPER AT 250 C
FRACTION OF SIGNAL LEFT AFTER HEATING



S&S 604 S&S 903

PRIVINE PRIVII
[ME (hr) BLANK HC1 BLANK HC1

0 1 1 1 1

1/4 1 1 1 1

1/2 3/4 3/4 3/4 3/4

3/4 1/2 1/2 1/2 1/2

1 1/3 1/3 1/3 1/3

1-1/4 1/3 1/3 1/3 1/3

1-1/2 1/4 1/4 1/4 1/4

1-3/4 1/4 1/4 1/4 1/4

2 1/4 1/4 1/4 1/4








magnitude of improvement needed to really make RTP more

viable as an analytical method.

As one final step in trying to improve the filter paper

background, a handsheet of the raw cellulose material used

in making S&S 903 was obtained from Schleicher & Schnell.

This handsheet exhibited the same background as the S&S 903

filter paper. In addition, washing and heating of the hand-

sheet gave the same results as treatment of the finished

paper. Purified a-cellulose did not give background sig-

nals of the level observed in the raw material handsheet,

nor did it induce RTP from Privine HC1. What is done to

the a-cellulose in making the handsheet is not known. As

a result, it was decided that improvement of the filter

paper background could not practically be done in our

laboratory but could better be done by the paper manu-

facturers or someone with facilities for making their own
5
paper.


Nonreproducible Sample Positioning


Since the RTP sample holding systems used in the past

(see Figures 4 and 5) were susceptible to rotational posi-

tioning imprecision as well as bending of the sample holding

rod, it was decided that the construction of a new sample

holding device should be undertaken in hopes of improving

sample to sample precision. The new device (illustrated in

Figure 6) is based on a flat aluminum bar with an elongated

cover plate suitable for simultaneously holding 4 samples.





































Figure 6. The new RTP sample holding bar. Four
samples are held under the cover plate for simultaneous
treatment.








Since timing for I.R. drying and cooling is critical in

RTP precision, multiple samples on the same device should

allow very consistent treatment at least for those samples

on the bar at the same time.


Experimental


All instrumentation and sample treatment was the same

as for previous RTP measurements; however, the Aminco-Kiers

phosphoroscope attachment would not allow the extension of

the new device into the sample compartment. As a result the

Aminco SPF sample compartment was modified to receive the

RTP bar as shown in Figure 7. Dimensions and construction

drawings of all new parts (including the RTP bar and sample

compartment modifications) are given in Appendix A. One

side of the sample compartment bottom plate (A) was extended

2 in. to serve as a mount for the synchronous drive motor

(B). The phosphoroscope can was driven by a rubber belt (C)

about pulleys (D) and (E); the belt passes through a slot

in the sample compartment side. The phosphoroscope pulley

(E) and can (F) were machined from one piece of aluminum

stock and were dimensioned to press fit the roller bearing

(G) (New Departure number Z993LDF). A 2 in. by 2 in. by

1-3/4 in. bottom extension (H) with a 1-1/2 in. diameter

hollow center was needed as a means of holding the bar and

allowing vertical motion for adjustment for multiple samples.

The bottom of extension (H) was covered by a small plate (I).

This plate had a rectangular slot whose length lay at 450






























Figure 7. Modified sample compartment bottom,
phosphoroscope and slit holder necessary to receive the
conduction cooling device. (A) sample compartment bottom
plate, (B) synchronous drive motor, (C) pulley belt,
(D) drive pulley, (E) phosphoroscope pulley, (F) phos-
phoroscope can, (G) bearing, (H) bottom extension/rod
holder, (I) slotted bottom, (J) slit holder, (K) cell
slit, (L) sample compartment wall.

















Li


I H

B I








from the axis of both excitation and emission monochromators.

The slot prevented rotational motion of the RTP bar. Since

the Aminco cell slit holder would not fit within the inner

diameter of the phosphoroscope can, an aluminum slit holder

(J) was constructed to hold the Aminco SPF cell slits (K)

just on the outside of the can. This allowed the slits to

be at the same distance from the monochromator mirror as

with the Aminco system. All parts except the phosphoro-

scope and slit holder were machined from phenolic resin.

A new sample compartment lid (Figure 8) was constructed to

hold the new RTP bar. This lid also had a slot at 450 from

the excitation and emission optical axis for prevention of

rotational motion. In addition, a raised block was located

next to the slot. A spring-loaded ball and plunger type set

screw in the raised block snapped into depressions on the

back of the RTP bar, allowing vertical repositioning for

multiple sample use.

In order to locate positions on the cover plate where

holes were to be drilled for samples, a strip of blueprint

paper was taped over the RTP bar and cover plate. The bar

was then positioned in the sample compartment and exposed

to 425 nm excitation from the Aminco in each of the vertical

sample positions. The blueprint paper (still on the bar)

was then developed by exposure to ammonia vapor. Small

white spots appeared where the 425 nm light exposed the

paper for 10 minutes. These spots were drilled as sample

holes in the cover plate.































Figure 8. Modified sample compartment lid for use
with the new RTP sampling bar. A ball and plunger set
screw in the small raised top portion was used in adjust-
ing the vertical position.









Reagents


Aminophylline was used as supplied by Ely Lilly and

Company (Indianapolis, IN). The diphosphate salt of

Chloroquine was used as received from Sigma Chemical Com-

pany (St. Louis, MO). Analytical grade paraaminobenzoic

acid (PABA) was purchased from Fisher Scientific Company

(Fairlawn, NJ). Solutions were made using deionized water

and absolute ethanol as solvents. During all measurements

sample compartments were flushed with dry nitrogen gas.


Results and Discussion


In order to check whether in fact precision of

positioning was improved, a small disc of uranyl glass was

prepared. This disc was then mounted on first the old RTP

device and then the new RTP bar. Each of the devices was

positioned in the Aminco sample compartment, uranyl glass

phosphorescence was measured and the device was removed

successively 16 times. The per cent relative standard

deviation (% RSD) for the uranyl glass on the old RTP

device was 5%, and 3% for the new RTP bar, so some improve-

ment was achieved by the new device.

In order to check for improvement in real sample type

situations, limits of detection (LOD) were measured for

4 samples. The results of these experiments are given in

Table 8.

A lack of real improvement in the LOD and % RSD

values indicates that the real precision problem was not

















TABLE 8

RESULTS OF LOD MEASUREMENTS AS A MEANS OF
COMPARISON FOR THE TWO RTP SYSTEMS


SAMPLE LOD % RSD BLANK % RSD SAMPLE

O N O N O N

Aminophylline 5.5 1.5 8.1 2.4 12 8.7

Chloroquine 3 0.2 6 6.9 8.3 3.9
2 PO4

paraamino- 0.5 0.1 9.7 5.3 7.2 8.3
benzoic acid


O = old system
N = new system





58


really as much in the positioning of the sample as in im-

precision of the filter paper itself from one sample disc

to another. Again this raises the point that for RTP to

become a really viable, widely used technique, improvement

in the precision and magnitude of the background of the

substrate is imperative.















CHAPTER IV
TWO NEW LTP DEVICES BASED ON CONDUCTION COOLING


From its outset, low temperature phosphorimetry (LTP)

of organic molecules in aqueous solvents has been plagued

with problems of inhomogeneously frozen samples and non-

reproducible sample cell positioning.56,57 Like the past

RTP systems, LTP samples have been suspended into the

sample compartment from the top (see Figure 9). The usual

approach to minimizing sample inhomogeneity is to cool the

sample very slowly by immersing over a long period of time.

As an additional means of increasing precision, nuclear

magnetic resonance sample spinners have been used to spin

phosphorimetric samples.2 Precession of the quartz sample

cell tube during spinning, however, adds to the positioning

precision problem.

In order to solve these problems, it is necessary to

devise some means of cooling the sample cell at a uniform

and reproducible rate, without the need for manually lower-

ing the tube over a long period of time. In addition, the

sample should be firmly held in a given position with

respect to optical components of the instrumentation. The

new RTP system, discussed in Chapter III, was ideally suited

to this last point, so two new LTP devices based on conduc-

tion coolingwere devised, compatible with the sample











compartment modifications for the RTP device. It should be

pointed out that the concept of conduction cooling is not

new (refer to Winefordner, Schulman, and O'Haver58 for a

discussion of several previous conduction cooling approaches);

however, conduction cooling has not been used for analytical

phosphorimetric measurements, possibly because of the lack

of a commercial conduction cooling device and/or the lack

of a suitable design in the published literature.


Experimental


Apparatus


Conventional, immersion cooling, LTP measurements were

made on an Aminco-Bowman Spectrophotofluorimeter (American

Instrument Co., Silver Springs, MD) fitted with an Aminco

150 W xenon arc lamp, an Aminco-Keirs rotating can phos-

phoroscope and a potted Hamamatsu (Middlesex, NJ) 1P21

photomultiplier tube. An Aminco Ratio Photometer supplied

high voltage to the photomultiplier tube in addition to

serving as a D.C. amplifier. The standard Aminco phos-

phorescence sample compartment was used with an optical grade

quartz dewar flask suspended within the phosphoroscope. The

actual sample cell was a quartz tube approximately 10.5 in.

long with 3 mm o.d. 1 mm i.d.

Conduction cooled LTP measurements were made on an

Aminco-Bowman Spectrophotofluorimeter fitted with a Schoeffel

(Westwood, NJ) 150 W xenon arc lamp and a potted Hamamatsu

1P21 photomultiplier tube. A Princeton Applied Research








(Princeton, NJ) model 280 high voltage power supply was used

with the photomultiplier tube. Signals were processed with

a D.C. amplifier such as that described by O'Haver and
59
Winefordner.

The first conduction rod (see Figure 10) was machined

from copper and was 3/4 in. in diameter. A 1 in. diameter

phenolic resin sleeve (A) covered the portion of the rod

resting within the bottom extension, and as further insula-

tion a cavity of dead air was trapped between the sleeve and

the bottom extension. A bar (B) extended 3 in. below the

sample compartment into a dewar flask of liquid nitrogen

(LN2). A slot (C) near the top of the bar served as an

optical window through which excitation and emission radia-

tion passed. The sample cell slip fitted into the conduction

rod from the top. An Aminco fluorescence sample compartment

cover, with the handle removed, served as the sample com-

partment cover. Sample tubes were inserted into the con-

duction rod through the screw hole left by the removed

handle.

The second conduction cooling device (see Figure 11)

was machined from copper stock 1 in. in diameter and

3-3/4 in. long. This rod was hollowed, leaving a cavity

3/4 in. in diameter, extending 2-13/16 in. from the open

end. The center of the other end was drilled 13/16 in.

deep to clear a 3 mm o.d. sample tube. At 3/8 in. down the

side from the closed end, a 5/16 in. slot was milled 1/2 in.

deep. This slot served as the optical window. Two 3/16 in.

































Figure 10. The first conduction cooling device.

A. phenolic resin insulation sleeve
B. copper conduction bar
C. optical window
D. quartz sample tube
































Figure 11. The second or flowthrough type conduction
cooling device.








holes were drilled into the closed end, extending into the

cavity, after which two 3/4 in. long pieces of 1/8 in. i.d.

copper tubing were silver soldered into the holes. The

open end was finally closed by a 3/4 in. diameter, 1/4 in.

thick plug, having a 1/4 in. by 1/4 in. by 7/8 in. tab

placed parallel to the back of the optical window. Tubing

placed over the two copper tubes served as the LN2 inlet

and outlet.

The LN2 inlet was gravity fed from the bottom of a

specially built dewar flask (see Figure 12). The outer por-

tion of the flask had a series of raised ridges, serving as

bellows to prevent the breaking of the feed through tube on

contraction of the inner surface. The LN2 outlet tube was

led back into the top of the dewar flask allowing the LN2

to recirculate. The top of the flask was covered by a cap

with a pressure relief valve to minimize frost formation

in the dewar flask.

The sample cells were of the same quartz tubing as used

in conventional LTP but pieces were cut to 3 in. in length

and sealed at one end.

Temperature measurements were made using an Omega

Engineering (Stamford, CT) type WTJ-8 thermocouple with an

Omega Engineering model MCJ Electronic Ice Point. Thermo-

couple potentials were measured on a Keithley (Cleveland,

OH) model 610 BR electrometer.






























Figure 12. Diagram showing the dewar flask, conduc-
tion cooling arrangement.

A. dewar flask
B. drain to conduction cooling device
C. liquid nitrogen
D. cotton padding
E. frost cap
F. return tube from conduction cooling device
G. funnel for liquid nitrogen addition





F


/E


/ B








Reagents


Lepidine (99%) and 4-chloroquinoline (98%) were from

Aldrich Chemical Company, Inc. (Milwaukee, WI). Amino-

phylline was used as supplied by Eli Lilly Company

(Indianapolis, IN). The diphosphate salt of Chloroquine

was used as received from Sigma Chemical Company (St. Louis,

MO). Solutions were made using deionized water and redis-

tilled reagent grade ethanol as solvents. During all

measurements sample compartments were flushed with dry

nitrogen gas collected from LN2. Very dry nitrogen is

important in preventing frost from forming on optical

surfaces and moving parts in the conduction cooled system.


Procedure


In the case of conventional "immersion cooling" LTP,

the quartz dewar flask was completely filled before each

sample. Approximately 50 pL of sample was drawn into the

sample cell tube. The tube was then lowered into the sample

compartment until the lower end of the tube was about 1 mm

above the surface of the liquid nitrogen. This position was

held for 30 s, at which time the tube was lowered by in-

crements of =5 mm every 15 s. When the tube had been

lowered "50 mm into the LN2, it was then slowly lowered the

remaining distance to the measurement position. The timing

on this final lowering was not critical to precision. Care

was taken to insure that the same optical surface of the








sample cell faced the excitation radiation each time. Since

the sample was actually immersed in LN2, the sample

temperature was 77 K.

In the case of conduction cooled LTP, 25 vL of sample

were introduced into the sample cell tube by a syringe fitted

with a 3 in long, blunt-end, 17 gauge, stainless steel needle.

The sample cell was then inserted into the conduction cool-

ing rod and rotated until a given surface of the cell was

exposed to excitation radiation. The speed of the insertion

was not critical to precision. The sample temperature after

insertion into the first conduction cooling device was

100 K. The second device gave a sample temperature of 85 K.

For all measurements, the rotating can phosphoroscope

chopping rate was 200 Hz. All glassware was cleaned with

No-Chromix(R) (Godax Laboratories, New York, NY).

Limits of detection (LOD) were taken to be that con-

centration giving a signal three times the standard devia-

tions of sixteen blanks.


Results and Discussion


In order to evaluate the new systems, analytical fig-

ures of merit were determined for 4 compounds mentioned

above. The results for this study are given in Table 9.

Wavelength data for each compound show that the wavelength

maxima shift with temperature. The direction of this shift

varies from compound to compound.






73








S4-

O C)


S, m o .- .
V U U










!-4 o
0 4 "4 .
4 I
HO









E" d d o d

>. q -
u D
HH H 42








00 C l
O U C )

0 0 CD





u 0 0 0 H




0 0 0 0


0r0 H
Ln co a V)
0 4 0 0 0 0 0 2


So m m
HE -Uan in C 0
E ( 0 c L 4 o
U )







H 0-1 0 0. 0 4
0 0 o a c




C40r14 - (0 CC
0 C0
OH C- 40 0 4 C


HJZ 0 W H 0 CC 4J C) H
E-' CN I' H IC 0 C) M C)


S 0 ID 42 0 C) CC



I S 0)




0 42I C) 42 C) 0i








For three of the compounds, lower LOD's were obtained

by the second conduction cooled system than by either of the

other low temperature systems. For each of these compounds,

the precision of the second conduction cooling system was

better, or at least comparable, to the precision of the

other two systems. This, considered with the fact that the

signals remain high at the reduced temperature, explains

the improved results. However, in the case of Lepidine,

the precision was good but the actual signal level of the

sample was low compared to the blank. This led to similar

LOD's for the three methods.

Ten consecutive samples measured by the immersion cooled

system required approximately 200 mL of LN2. The initial

step of simply cooling the first conduction rod down to

100 K required about 400 mL of LN2. After the initial cool

down, the first conduction cooled system required about

200 mL for ten consecutive samples. In addition, it was

only necessary to add LN2 after every third sample for con-

duction cooling, while good precision required that the

quartz dewar flask in immersion cooling be refilled for

each successive sample. Prior to running samples, the bottom

fed dewar flask was filled with LN2 (=1.5 L). When the

second conduction device reached the operating temperature

(85 K 2 K), the dewar flask was refilled and covered.

This filling lasted for approximately 40 min. of operation.

During this time, temperature variations were less than

1/2 degree.








Considering the time required to add LN2 and prepare

samples, the maximum sample turnover rate by immersion

cooling was one sample each 6 min, limited by the 3 min

immersion time. Allowing the same sample preparation time

and one-third the LN2 addition time, the maximum sample

turnover rate for conduction cooling was only slightly over

2 min, limited by 45 s to 1 min required for the sample

to cool and reach maximum phosphorescence intensity after

insertion.

As in the first device, sample turnover was about one

sample per minute, plus sample preparation time, but re-

duced LN2 handling makes the second device more attractive.

A large LN2 reservoir designed for use with this system

would make phosphorimetry quite practical for routine use

in the lab.

Considering these facts, the conduction cooling systems

would seem to have more promise for use in routine types of

analysis. Furthermore, there was no special technique or

practice required in order to maintain reasonable precision

by conduction cooled LTP, while precision in immersion cooled

LTP was largely operator dependent.

Immersion cooled LTP systems might be improved by the

development of mechanical or automated devices for increased

ease of sample introduction. While such a device might not

improve precision, it would certainly remove much of the

operator dependence. In addition, the development of care-

ful sample insertion techniques should improve the precision








in conduction cooled LTP. The most obvious way to improve

the conduction cooled system, at this stage, seems to lie

in design improvement for the conduction cooling rod, in

order to achieve lower temperatures and possibly higher

signal-to-noise ratios. Joule Thomson refrigerators are a

possible approach, but cost and cooling rate obviate their

use in routine analytical LTP.

The present conduction cooling systems offer some

temperature variation versatility since change in the LN2

level in the dewar flask allows the temperature to be

changed. This temperature control is now being used as a

means of studying such properties as quantum efficiencies

and lifetimes as a function of temperature.

The flow-through cooling approach maintained the advan-

tages of simplicity and uniform freezing rate exhibited by

the previously described conduction cooling device, while

maintaining a lower temperature for better signal levels.

The new device added another advantage over the first con-

duction device in that the temperature was more stable.

This stability was a major factor in the improvement of

precision seen in the newer device.















CHAPTER V
STUDIES IN LUMINESCENCE WITH VARYING TEMPERATURE


As mentioned in the previous chapter, the first con-

duction cooling device was found useful in performing

measurements with varying temperature. Actually this is a

benefit that arose from a disadvantage of using the device

for measurements at a fixed temperature. Early in the use

of the conduction cooling device, it was found that the

temperature of the sample was critically dependent on the

level of LN2 in the dewar flask into which the device was

immersed. Temperature variations of up to 100 were found

to occur in the span of only a few minutes. With the

temperature dependence of luminescence in mind, the adverse

effects of such temperature fluctuations on precision could

be significant.

To make measurements with changing temperature, how-

ever, the picture is much brighter. When the dewar flask

is kept full for 15 to 20 min. the conduction cooling bar,

and thus the sample, reaches its minimum temperature of

100 K. If, at this point, no further LN2 is added, the

temperature of the sample begins to increase as the level

of LN2 in the dewar falls with evaporation. If time is

measured from the last addition of LN2, a plot of sample

temperature vs time can be made as in Figure 13. The



























Figure 13. Sample temperature vs time as the level of
LN2 in the dewar flask drops. Zero (0) minutes corresponds
to the time at which the last LN2 was added.








reproducibility of this temperature curve is reasonably

good (within 5%). As the level of LN2 falls on the bar of

the conduction cooler, the rate of temperature change de-

creases gradually over the first 20 min. At about 20 min.

the level of LN2 falls below the bottom of the bar, so

there is no contact between copper and LN2. After this

time, the temperature of the sample increases linearly

until the sample begins to melt.


Preliminary Experiments with Phosphorescence


The possibility that the gradual increase in sample

temperature could be used to study the effects of temperature

on luminescence led to phosphorescence measurements as the

sample warmed.


Experimental Procedure and Results


The compound Lepidine, 4-methylquinoline, was selected

as a first sample since its phosphorescence at 77 K is

quite intense and long lived. The apparatus used in meas-

uring phosphorescence intensity vs temperature was essen-

tially the same as for evaluation of the conduction cooling

device as described in Chapter IV, with the addition of a

Texas Instrument strip chart recorder. (See Figure 14 for

a block diagram of the apparatus.)

A closed-end quartz tube was filled with -50 iL of

100 ppm Lepidine. The tube was then inserted into the

sample compartment before the addition of LN2. The dewar














'a


0
u


U)



0
4
0
a














.0

04




0










-4
oC
c



























0
a


















r-H
'0















0
Ca























cQ
Ca
r0

c)








CJ
a













-Hl
a


a









4-C


cD
EO





82









I /














0--.7




C r






0i





5
r'C

o zI
CU C

~L 0
o 4a) 0L


CI 0 Cr
rl L ~UCa c








flask beneath the sample compartment was next filled by

means of a nylon funnel and a short length of plastic tub-

ing. Thermocouple potentials were manually recorded at

short intervals (approximately every 30 s) directly onto

the phosphorescence plot being made by the strip chart

recorder. These potentials were later converted to

temperatures.

When the conduction cooler and sample reached their

minimum temperatures, LN2 was slowly poured into the funnel

until stable phosphorescence signal and temperature were

observed for several minutes. When this stability was

achieved, no further LN2 was added, and again thermocouple

potentials were recorded as the sample slowly warmed. The

resulting plot of photoanodic current from the photo-

multiplier vs time is shown in Figure 15. Since initially

both the photoanodic current (phosphorescence) and tempera-

ture changed rapidly, the response times of the instruments

made correlation of signal vs temperature impossible on

cool-down; therefore, all plots of current vs temperature

were made as the sample warmed.

In the region around 175 K, it was noticed that the

phosphorescence signal increased with time even though

temperature also increased with time. In order to deter-

mine whether this increased signal was actually phospho-

rescence from the sample or background emission arising from

the solvent (possibly triboluminescence as the structure of

the frozen solvent changed), a 10:90 ethanol:water blank was













0n






Q4
00
) *








40 r
1--









4
a ..
So
C










*0
02
0202

-4 H 'I


S0-


0) 4


02C
a, 0
a1) C





> .
41 -C








c4

02 -H 0
a















.H
4.1 C00













ro 0



*0
4.)


S-r4







*H > H
02








40 '
rO 11
0m2






s.c

2rl-*
C; a-
(U *r

1- i O
3 C (
ar ) -








treated in a manner identical to the Lepidine sample. No

signal above dark current was observed in the region around

175 K (see Figure 15). In order to observe the effect of

solvent composition on the increased signal at 175 K,

measurements of signal vs temperature were made for Lepidine

in solvent compositions of 10:90 through 100:0 ethanol:water

at increments of 5% ethanol

As the solvent changed, the shapes of the temperature

curves were also observed to change. Changing the solvent

composition from 10% ethanol to 50% ethanol caused the

position of the increase in signal to shift toward higher

temperatures. The magnitude of the local maximum in signal

relative to the smallest signal observed at a temperature

lower than that of the local maximum was also found to

increase with increasing ethanol fraction. In Figure 16,

temperature curves for Lepidine in 50:50 ethanol:water

and 15:85 ethanol:water are shown. The lowest signal on

the 15:85 curve prior to 175 was at 160 K and was 1.1 nA.

The signal increased by 0.50 nA as the temperature in-

creased to 175. Dark current was 0.37 nA. The lowest

signal on the 50:50 curve was 3.6 nA at 173 K and the peak

occurred at 188 K with a signal of 6.0 nA (an increase of

2.4 nA). The intensity of light observed at the local

maximum was found to be larger at higher ethanol fractions

(up to 35%). For solvents with more than 35% ethanol the

magnitude of the signal increase was found to be smaller.





























Figure 16. Temperature curves for Lepidine in:
(a) 50:50 ethanol:water and (b) 15:85 ethanol:water.


































































140 160 130


T (K)


1000 -


100 -


10 -I


0.1
100


2 60 2O


I








For ethanol concentrations greater than 50%, no further

signal increases were observed with increasing temperature.

In Figure 17, representative curves are given for Lepidine

in ethanol:water mixtures above 50% in ethanol. Figure 17

also serves to illustrate that, although the maximum in-

tensity at 100 K decreases as the ethanol fraction increases

above 50%, the temperature at which the signal makes its

first major drop increases significantly. A plot of the

peak intensity for the local maximum vs ethanol:water ratio

is given in Figure 18.

Questions arose as to whether the signal increase was

peculiar to Lepidine, and,if not, were the shapes of the

curves sample dependent. In order to answer these questions,

treatment identical to that of Lepidine was given to the

compounds Chloroquine diphosphate and 4-chloroquinoline.

These compounds were chosen because they have emission wave-

lengths similar to that of Lepidine. If the shapes of

temperature curves of compounds with similar phosphorescence

characteristics at 77 K are significantly different, it is

possible that they could be resolved by changing the

analysis temperature.

The effect of changing the fraction of ethanol in the

solvent was quite similar for all three compounds. Relative

maxima occurred at the same temperature in each case for

both Lepidine and 4-chloroquinoline. The effect was not

well pronounced in the case of Chloroquine. Although peaks

occurred at the same temperatures for Lepidine and































Figure 17. Temperature curves for Lepidine in:
(a) 50:50 ethanol:water, (b) 70:30 ethanol:water, and
(c) 100:0 ethanol:water.




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