• TABLE OF CONTENTS
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 Title Page
 Acknowledgement
 Table of Contents
 List of Tables
 List of Figures
 Abstract
 Introduction
 Phosphorescence theory and...
 Experimental system and proced...
 Results and discussion
 Summary and future work
 Appendices
 References
 Biographical sketch














Title: Pulsed source time resolved phosphorimetry for quantitative and/or qualitative analysis of drugs
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Title: Pulsed source time resolved phosphorimetry for quantitative and/or qualitative analysis of drugs
Physical Description: ix, 74 leaves. : illus. ; 28 cm.
Language: English
Creator: Harbaugh, Karen Frances, 1945-
Publication Date: 1973
Copyright Date: 1973
 Subjects
Subject: Drugs -- Analysis   ( lcsh )
Phosphorimetry   ( lcsh )
Drug adulteration   ( lcsh )
Chemistry thesis Ph. D
Dissertations, Academic -- Chemistry -- UF
Genre: bibliography   ( marcgt )
non-fiction   ( marcgt )
 Notes
Thesis: Thesis--University of Florida,1973.
Bibliography: Bibliography: leaves 71-73.
Additional Physical Form: Also available on World Wide Web
General Note: Typescript.
General Note: Vita.
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Bibliographic ID: UF00097577
Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
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Resource Identifier: alephbibnum - 000577616
oclc - 13996451
notis - ADA5314

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Table of Contents
    Title Page
        Page i
    Acknowledgement
        Page ii
    Table of Contents
        Page iii
        Page iv
    List of Tables
        Page v
    List of Figures
        Page vi
        Page vii
    Abstract
        Page viii
        Page ix
    Introduction
        Page 1
        Page 2
        Page 3
        Page 4
        Page 5
    Phosphorescence theory and measurement
        Page 6
        Page 7
        Page 8
        Page 9
        Page 10
        Page 11
        Page 12
        Page 13
        Page 14
        Page 15
        Page 16
        Page 17
    Experimental system and procedure
        Page 18
        Page 19
        Page 20
        Page 21
        Page 22
        Page 23
        Page 24
        Page 25
        Page 26
        Page 27
        Page 28
        Page 29
        Page 30
        Page 31
        Page 32
        Page 33
        Page 34
        Page 35
        Page 36
        Page 37
        Page 38
        Page 39
        Page 40
        Page 41
    Results and discussion
        Page 42
        Page 43
        Page 44
        Page 45
        Page 46
        Page 47
        Page 48
        Page 49
        Page 50
        Page 51
        Page 52
        Page 53
        Page 54
        Page 55
        Page 56
        Page 57
        Page 58
        Page 59
        Page 60
        Page 61
        Page 62
        Page 63
        Page 64
    Summary and future work
        Page 65
        Page 66
    Appendices
        Page 67
        Page 68
        Page 69
        Page 70
    References
        Page 71
        Page 72
        Page 73
    Biographical sketch
        Page 74
        Page 75
        Page 76
        Page 77
Full Text








PULSED SOURCE TIE RESOLVED PHOSPHORIHETRY FOR QUANTITATIVE
ATD/OR QUALITATIVE ANALYSIS OF DRUGS








By
KAREN FRANCES HAR3AUGH


A DISSERTATION PRESENTTED 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


1973












A MOiM bEiD Ur"HENT S


For the good things I have accomplished and learned

so far in my life, I owe a great debt of gratitude to a

number of people. The gifts of joy and wonder they have

given me are absolutely priceless. Though they are not

listed by name, they are remembered and loved.

Others have shown me that our world is less than

ideal, and these worthwhile lessons .I also acknowledge.

It is largely because of them that I sincerely hope I can

add to the world's balance on the side of goodness.











TABLE OF CONTENTS

Page

ACKNOWLEDG ETTS . . . . . . . . . ii

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

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

ABSTRACT. . . . . . . . . . . .. viii

Chapter

I. INTRODUCTION. . . . . . . . . 1

II. PHOSPHORESCENCE THEORY AND 'EASUREMENT. . 6

Phobphorescence Theory . . . . . 6

Phosphorescence Lifetimes. . . . . 9

Pulsed Source Phosphorescence Measurement. 12

III. EXPERIMENTAL SYSTEM AND PROCEDURE . . . 18

Instrumentation . . . . . ... 18

Instrumental Procedure . . . . .. 28

Data Reduction . . .... . . . 32

Drugs and Reagents . . ... . . 39

IV. RESULTS AND DISCUSSION. . .. . . . 42

Noise and Precision. . . . . .. 42

Quantitative Analysis of Mixtures via Time
Resolved Phosphorimetry . . . . 51

Qualitative Analysis via Time Resolved
Phosphorimetry. . . . . . 62


iii












Chapter

V. SUDI~ILABY AMID FUTUYZE WORK . . ..

APPENDICES


Appendix I. . . . . . . . .... ..

Appendix II . . . . . . . . . ..

LIST OF REFERE:NCES. . ..............

BIOGRAPHICAL SKETCH ................ .


Page


. . 65












LIST OF TABLES

Table Page

1. Specifications and Operating Conditions for
the Flashtube Excitation Source . . . 22

2. Comparison of Curve Fit and Graphical Methods
for Data Reduction. . . . . . . 38

3. Illustration of the Precision Obtainable with
the Pulsed Source Phosphorimeter. . . 50

4. Phosphorescence Lifetimes (s) of Drugs in
Various Solvents. . . . . . . 54

5. A Comparison of the Relative Initial
Phospnorescence Intensities of Selected
Drugs in Various Solvents . . . . 56

6. Results of Quantitative Analysis of Drugs by
Pulsed Source, Time Resolved Phosphorimetry 59

7. Result of Quantitative Analysis of Drugs by
Pulsed Source, Time Resolved Phosphorimetry,
with Spectral Isolation . . . . . 61











LIST OF FIGURES

Figure Page

1. Modified Jablonski diagram showing the possible
transitions in the excitation and relaxation of
an organic molecule . . . . . . . 7

2. Plot of relative response (P /I ) versus
phosphorescence lifetime,r,p 0
according to Equation 13. . . . . . 17

3. Block diagram of the instrumental components of
the pulsed source phosphorimeter. . . . 21

4. Diagram of the Xenon flashlamp plexiglass
housing .. . . . . . . . . 23

5. Spectral irradiance of Xenon Novatron 599
Flashtube as a function of wavelength . . 25

6. Circuit diagram of the impedance matching
device between the signal average and the
flashlamp power supply. . . . . . . 29

7. Diagram of the events that occur during one
cycle of excitation and observation for pulsed
source time resolved phosphorimetry . . . 30

8. Experimental decay curve for a single
component sample (codeine, 0.4 mg/ml in EPA). 35

9. Semi-logarithmic plot of phosphorescence
intensity versus time for a single component
sample (codeine, 0.4 mg/ml in EPA). . . . 34

10. Experimental decay curve for a binary mixture
(morphine and codeine in ethanol) . . . 35

11. Semi-logarithmic plot of phosphorescence
intensity versus time for a binary mixture
(morphine and codeine in ethanol) . . . 37

12. Chemical structures of selected
phosphorescent drugs. . . . . . . 40








Figure Page

13. Variation in peal: to peak noise levels with
increasing amplification and load resistance 453

14. Comparison of (A) sourceless noise to (B)
phosphorescence decay of ethyl morphine
(0.0032 mg/nl in ethanol). . . . . . 44

15. Plot of the signal to noise ratio, S/N, versus
increasing sweep counts, N . . . . . 46

16. Plot of the signal to noise ratio versus
concentration of Amobarbital in chloroform . 48

17. Plot of the signal to noise ratio versus
concentration of Phenobarbital in chloroform 49

18. Phosphorescence emission spectra of drugs in
chloroform showing peak positions. . . . 53

19. Analytical curves for (A) Amobarbital, (B)
Quinine, (C) Cocaine, (D) Morphine, in
chloroform . . . . . . . . . 57

20. Analytical curves for (A) Codeine, (B) Ethyl
Morphine, and (C) Morphine, in ethanol . . 58


vii










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



PULSED SOURCE TIME RESOLVED PHCSPHORIMETRY FOR QUA2NTITATIVE
AND/OR QUALITATIVE ANALYSIS OF DRUGS

By

Karen Frances Harbaugh

December, 1973


Chairman: James D. Winefordner
Major Department: Chemistry

A pulsed source time resolved phosphorimeter has been

described and its use for qualitative and quantitative

phosphorescence analysis of drugs in single and multicomponent

mixtures has been demonstrated. This system offers the

advantages that both qualitative identification and

quantitative determinations may be performed on certain

compounds without prior physical separation by use of

spectral and temporal resolution under various environmental

conditions.

A Xenon flashlamp, which provides high spectral

irradiance in the region most phosphors absorb, was

utilized as the excitation source in the experimental

system. A monochromator was used to spectrally isolate


viii









the components of interest, and a signal average was used

to monitor the decay of phosphorescence intensity as a

function of time. Based on differences in phosphorescence

lifetimes, the graphical separation of components was

accomplished by a semilogarithmic plot of phosphorescence

intensity versus time. Quantitation was achieved via

the proportional relationship between the initial phosphores-

cence intensity and the concentration of the species in

solution. Qualitative identification was accomplished by

the changes in the characteristic phosphorescence lifetimes

with changes in environmental conditions.











CHAPTER I


INTRODUCTION

Phosphorescence is a luminescence process whereby

light is emitted by a molecule following the absorption of

radiation of higher energy. It was first discussed by

Jablonski in 1935 [1]. The qualitative possibilities of

phosphorescence were suggested by Lewis and Kasha in 1944,

with considerable theoretical discussion [2], and Keics,

Britt and Wentworth evaluated phosphorescence as a quanti-

tative tool for chemical analysis in 1957 [3]. They showed

that a binary mixture of structurally similar compounds

could be resolved by judicious selection of excitation and

emission wavelengths and by choice of the delay before the

observation time after termination of the exciting radiation.

This was the first use of temporal resolution for chemical

analysis by phosphorimetry.

Time resolution in phosphorimetry utilizes the dif-

ference in molecular lifetimes just as spectral resolution

utilizes the differences in excitation and emission wave-

lengths. Phosphorescence lifetimes, for a given set of

environmental conditions, area unique characteristic of a

molecule, along with the excitation and emission spectra.









Should the spectral information of two (or more) molecules

be so similar that delineation of components is impossible

by spectral means, the molecules may be phosphorimetrically

resolved temporally, if their phosphorescence lifetimes

differ sufficiently.

Because most molecules generally absorb between 250

and 400 nm and emit between 350 and 500 nm, conventional

phosphorimetry, in which only excitation and emission

spectral resolution is utilized, is at best a rather

non-selective method of analysis. However, the phosphores-

cence lifetime- of molecules can differ greatly and the

influence of various solvents, particularly those containing

heavy atoms, such as iodide, upon the measured phosphores-

cence lifetimes can be even more striking. Therefore

time resolved phosphorimetry should be a powerful tool

possessing all of the sensitivity advantages of conventional

phosphorimetry as well as the selectivity advantages due to

time resolved. Fitzgerald and Winefordner [4] have shown by

information theory how time resolution plus spectral reso-

lution can provide up to 450 times the analytical information

provided by conventional phosphorimetry alone.

The initial time resolution studies made use of a

spectral continuum source with a mechanical shutter for

termination of exciting radiation. Keirs et al. [3]1 used a

mechanical phosphorescope which included a continuous light






3

source and a modified Becquerel type rotating disk.

St. John and Winefordner [5] later used an Aminco-Bowmnan

spectrophotofluorometer fitted with a manual guillotine

shutter and a dc readout coupled with a logarithmic con-

verter to time resolve mixtures of slow decaying phosphors

(of the order of seconds). Hollifield and Winefordner [6]

described a single disk mechanical phosphoroscope which

measured phosphorescence intensity at several intervals

along the emission decay curve.

The continuum source, mechanical shutter phosphoro-

scopic instruments, however, have certain limitations that

can be overcome by means of a pulsed source gated detector

system. The shutter transit time, and excitation and emission

viewing times are inflexibly linked in the former, and

limited by mechanical considerations (see [7]). Winefordner

[8] was first to suggest the advantages of pulsed source

gated detector instrumentation for time resolved phos-

phorimetry of fast phosphors (of the order of milliseconds)

in 1969, and O'Haver and Winefordner [7,9], had already put

continuous (CW) and pulsed source phosphorimetry on a sound

theoretical basis. Fisher and Winefordner [10] recently

compared conventional phosphorimeters to a unique pulsed

source system and demonstrated the advantages of the

instrument for the time resolution of selected phosphors.

Flash lamps have a greater spectral radiance in the region










where most phosphors absorb, which could lead to greater

sensitivities for certain phosphors, especially the fast

decaying species. Also, with portions of the excitation

and observation cycle independently variable, selectivity

is greatly increased.

O'Donnell, Harbaugh, Fisher and Winefordner [11]

used a modified version of this instrument to demonstrate

the utility of time resolved phosphorimetry for the analysis

of synthetic mixtures of halogenated biphenyls, Since this

time, however, no other applications of pulsed source time

resolved phospiorimetry have been shown.

The use of drugs, both legitimate and illicit, in

our time is extremely widespread and analysis of pure drugs

and of drug levels in human fluids is an important part of

the effective use and/or control of pharmaceuticals. Be-

cause conventional phosphorimetry has already proven to be

a sensitive analytical method for drugs which phosphoresce

[12-16], the application of time resolved phosphorimetry to

drug analysis should greatly extend the usefulness of

phosphorimetric drug assay.

The purpose of this research is to show the

versatility of a pulsed source, gated detector time resolved

phosphorimeter as applied to the analysis of selected drugs

in synthetic mixtures. The advantages and limitations are






5

illustrated as well as the quantitative and qualitative

possibilities. The feasibility of drug analysis in real

samples is demonstrated.











CHAPTER II


PHOSPHORESCES:CE THEORY AND IEASURI-ENT


Phosphorescence Theory


Absorption of radiation by an unsaturated organic

molecule promotes an outer electron from the lowest vi-

brational level of the ground state, where it normally

exists at room temperatures or below, to one of a number

of higher energy levels, depending on the energy of the

absorbed light. The molecule will eventually regain its

ground .state configuration by giving up this energy to

the environment via a combination of various deactivation

steps, which are illustrated by a modified Jablonski

diagram in Figure 1. The excited electron will first re-

turn to the lowest excited singlet level via vibrational

relaxation, internal conversion, and tunneling (for a de-

tailed discussion of these processes, see [17]). From the

excited singlet state, the molecule may return to the ground

state by internal conversion, whereby the molecule loses

its energy to the environment by nonradiative means. In

molecules with a large number of vibrational modes in the

ground state, as with aliphatic compounds, this process is

favored. Aromatic molecules, having rigid ring structures,





V
S2








S- S
V

T1




P /C
A / 01











Fig. 1.-=odified Jablonski diagram showing the possible


Transtio Tie of tr siio, seconds

A = Absorption. 1015

V = Vibrational relaxation1of

C = Internal conversion 10-13

F = Fluorescence 10-8

S = Intersystem crossing 10- 10-

P = Phosphorescence 10 10








generally have an energy gap that precludes internal con-

version and tunneling, and so luminescence becomes possible.

The molecule will fluoresce if a quantum of light is emitted

as the molecule drops to the ground state energy level.

This process is easily observed at room temperature.

Alternatively, the electron in the excited singlet

state may undergo a change in spin and occupy an excited

triplet state. This transition, called intersystem crossing,

is forbidden classically but allowable quantum mechanically

via spin-orbit coupling, which results in mixing of the

spin and orbital moments, so that the process has a finite,

though small probability. Spin orbit coupling is enhanced

by the presence of heavy atoms and paramagnetic ions, al-

though these are not a prerequisite for the phenomenon.

Once in the triplet state, the molecule may again

relax to the ground state configuration by internal con-

version, intersystem crossing back to the excited singlet

state with subsequent fluorescence (delayed fluorescence),

or it may phosphoresce by emitting a quantum of light of

lower energy than would be emitted via fluorescence.

The absorption of light energy and the return of the

electron to the first excited singlet state via vibrational

relaxation and internal conversion is much faster than the

subsequent transitions. Intersystem crossing and fluores-

cence have comparable rate constants and their transition

times are therefore about the same. Once the triplet state








is populated, though, reverse intersystem crossing is

favored over phosphorescence at room temperatures in low

viscosity liquids, and so luminescence from the triplet

state is rarely observed under these conditions. At low

temperatures, however, and in rigid media, the molecule can

maintain the triplet state configuration long enough for

phosphorescence to occur because collisional and vibrational

processes, which contribute heavily to the conversion back

to either the excited or ground singlet states, are at a

minimum.


Phosphorescence Lifetimes

As noted above, phosphorescence is in competition

with internal conversion and reverse intersystem crossing

for depopulation of the triplet state. Letting T represent

the number of molecules in the triplet state configuration,

then the rate of loss of this species with time is

dT= T+ k + ktT = k T + kT (1)
dt p ic ts p

where t is time and k k ic, and kts are the rate constants

for phosphorescence, internal conversion between the triplet

and ground states, and intersystem crossing from the triplet

state back to the first excited state, respectively. The

rate constants for all Dossible deactivation steps determine

the mean observable phosphorescence lifetime in the following








way,

i = -1 (2)
kp + k (2)

In the absence of competing nonradiative processes for de-

activation of the triplet state, Equation 2 reduces to


0 1 (3)
p

which is the inherent (natural) mean radiative lifetime, a

theoretical quantity independent of environmental para-

meters. Unlike the inherent mean lifetime, the mean

observable lifecime.is dependent upon temperature, matrix,

and impurities that may be present in the sample or solu-

tion. These effects can be at least partially controlled

to optimize analytical conditions.

The mean observable phosphorescence lifetime (here-

after referred to as the lifetime or phosphorescence life-

time) may vary with environmental temperatures above 2000 K,

but will do so negligibly at lower temperatures (for example,

see [18]). A change in lifetime with temperature is not

easily utilized analytically because it is difficult to

regulate temperatures in the low range, and the amount of

change may not be significant. The liquid nitrogen used to

maintain a temperature of 770 K for phosphorimetric analysis

eliminates the variation in lifetime with temperature as

well as maintaining a rigid matrix to reduce collisional de-

activation of the triplet state.







11
Phosphorescence spectra tend to exhibit much less depen-

dence on the dipole moment and hydrogen bonding properties of

the solvent than do fluorescence spectra [19] The observable

lifetimes, however, can and do change considerably with a

change in solvent characteristics [11, 20-2] due to the

effects which the solvent may have on the nonradiative rate

constants.

Heavy atoms will increase the spin-orbit coupling

coefficient of the wave function, thereby increasing the

probability of singlet-triplet intersystem crossing. Thus,

solvents containing a heavy atom will cause an increase in

. ki, and, accordingly, a decrease in qp. (These pertur-
i p
bations often remove the exponentiality of the decay, however,

so care must be taken that the nonexponential character

will not interfere [25,26]).

An increase in the polarity of the solvent can

decrease the energy separation between the excited triplet

or singlet state and the ground state when the molecule is

more polar in the excited state than in the ground state.

If this occurs, internal conversion becomes more probable,

I k. increases, and p again decreases. In general, then,
i 1 p
longer phosphorescence lifetimes are observed in nonpolar

solvents. Changes in solvent polarity will have a larger

effect on n->T1* transitions carbonyll type) than on->TTf*

transitions (aromatic ring type), because the carbonyl group










is more polar itself, and thus more susceptible to polar

interactions.

The ring structure and molecular substituents will

also affect the lifetime observed, but the degree and di-

rection of change depends to a great extent on the primary

molecular system itself. Generally, an increase in the size

of the fT system will result in a red shift in the spectrum

and a decrease in 1 Electron donating substituents also
p
tend to cause a red shift, but the direction of the change

in phosphorescence lifetime depends on the position of the

substituent [23,27].

The above statements apply generally to the factors

influencing lifetimes. In addition to the above general

considerations, other environmental conditions may also

affect the phosphorescence lifetimes which are observed.


Pulsed Source Phosohorescence 1,'easurement


The observed phosphorescence intensity for a phosphor,

as a function of time after termination of the exciting

radiation, is an exponential decay of the form


It = exp (- ) (4)

where It is the phosphorescence intensity at any time t,

I is the initial phosphorescence intensity (at t = 0),

.and T is the phosphorescence lifetime. In a mixture of









phosphors, the total phosphorescence intensity, I tT, is

given by

tT = ItA + ItB (5)

where the subscripts A and 3 refer to the different

components. For a multicomponent mixture of independently

absorbing and emitting phosphorescent species,


ItT = Iti exp (- t-) (6)

Time resolved phosphorimetry is based on Equation 6, and

is a means of separating the total intensity into its

individual components.

In order to accomplish quantitative analysis by time

resolution (measurement of phosphorescence signal as a

function of time), a strict relationship must exist between

the radiant flux observed, P and the concentration of a

given phosphor in a single or multicomponent sample. The

radiant phosphorescence flux emitted, P is related to the

radiant flux absorbed, Pabs, by


P = P 0abs (7)
p abs p

where 0 is the ratio of radiant flux emitted to radiant

flux absorbed. It is assumed in Equation 7 that there is

no pre-filter or post-filter effects and that only the

analyte absorbs and emits.









The radiant flux absorbed is given by


Pabs = o Pt = P (1 exp (-2.3 abc)) (8)

where P is the radiant flux (either CV or pulsed source;

however, for the latter case, P0 is the average radiant

flux) incident upon the absorbing sample, Pt is the radiant

flux transmitted, a is the molar absorption (extinction)

coefficient of the absorber, b is the absorption path length

(assuming a rectangular cell), and c is the concentration

of the analyte, in moles per liter. If(2.3 abc) is less

than 0.01, as is the case for dilute solutions of the

analyte, then

Pabs = P (2.3 abc) (9)

and,

P = P (2.5 abc)0 (10)
p o p

Thus a linear relationship does exist between the concentra-

tion of the phosphorescent species and the radiant flux

emitted as phosphorescence as long as the above assumptions

are true and as long as the analyte is dilute. Also, it is

necessary to assume a steady state condition of excitation

and emission.

In time resolved phosphorimetry, phosphorescence

intensity is measured at various delay times. Because P0

decreases with time after termination of the exciting pulse,

P will also decrease with time, and so the slope of an
P








analytical curve will decrease with increasing delay times.

This fact is used to advantage in the multiple analytical

curve method of time resolved phosphorimetry [35].

O'Haver and Winefordner [7] derived equations which

relate the operating parameters for a rotating can

phosphoroscope to the observed signal; these authors later

extended their considerations to the pulsed source gated

detector system [9]. They reported that the integrated

luminescence intensity, Pp, measured with a gated detector

for a single cycle is given by

t d t
[exp(- )][1-exp(- -)][1-exp(- -)]
P = I[ p (11)

[l-exp(- -)]

where I is the initial phosphorescence intensity, td is

the delay time between excitation and observation, t is

the observation (measurement) time, and q is the phosphores-

cence lifetime. Because the duration of the flash is small

compared to the lifetime of the phosphor (tf is microseconds

compared to in milliseconds or seconds), Equation 11

reduces to
t t
[exp(- -)][l-exp(- --)]
Pp = Io tf (12)
[1l-exp(- c)]
-I-r









This equation can be simplified further if the observation

time, t and therefore the total cycle time (tf + td +

t + t ) is much larger than '. Therefore,
p a
td
exp(- --)
P = Io t tt- -, (13)

which describes the phosphorescent species measured by the

instrumental system described in this work.

Some of the advantages of the pulsed source system

are apparent from an examination of these equations. It is

possible to achieve the enhancement of the signal of one

phosphor over that of another with a different lifetime by

the adjustment of the parameters td, t., and tc [17].

This exemplifies a major advantage of a pulsed source

system over a mechanical rotating disk or can arrangement,

because these parameters are not individually adjustable in

the latter. Figure 2 is a plot of the relative response

(P /1 ) versus lifetimes calculated with Equation 13 and

with typical parameter values, as stated in the legend.

Curve A illustrates the increased sensitivity pulsed source

phosphorimetry offers for fast phosphors in the presence of

long-lived species. On the other hand, curve B of Figure 2

shows how the influence of a short-lived species can be

eliminated by proper choice of a delay time. This is one

of the techniques that adds to the versatility of time re-

solved phosphorimetry.






17











io-2 (A)

10-3_


10 -

l-5
S10 (B'
10-6




10-4 10- 10-2 10-1 1 10
Phosphorescence lifetime (seconds)

Fig. 2.-Plot of relative response (P /1 ) versus
phosphorescence lifetime,, o
according to Equation 15.

The parameters used to calculate the curves are:

Curve A: tf = 1.2x10-6 s, t = 10 s, td = 5.2xl0-3 s.

Curve B: t = 1.2x10-6 s, t = 10 s, td = l.OxlO-2 s.











CHAPTER III


EXPERIMENTAL SYSTEM AITD PROCEDURE


Instrumentation


Pulsed source spectroscopy is not a recent develop-

ment. Physical chemists have been using this technique to

study fluorescence and phosphorescence decay characteristics

since Backstrom and Sandros first used such a system in

1958 [28]. Many workers since that time have improved upon

the technique (for example [29-31]); most workers have

utilized gated photomultipliers. Winefordner [8] suggested

the advantages of using this technique for phosphorimetric

analysis, and Fisher and Winefordner [10] built a time re-

solved pulsed source gated detector phosphorimeter and

compared it to conventional phosphorimetry. A time resolved

phosphorimeter utilizing a pulsed source has also been

described by Hamilton and Nagvi [532]. Hattori and Kato [335]

have reported time resolution studies using a continuum

source with a rotating disk to effect time variation of

pulsed irradiation of the sample. Mathiasch [34] also used

a continuum source but with a time averaging computer to

measure lifetimes of weak phosphors.









The system developed by Fisher and Winefordner [10]

has been modified for this work. For completeness, a full

description of the experimental set-up will be given. A

block diagram of the entire system is shown in Figure 3.

The components represented by dashed lines are options

available but were not used for this study. These compo-

nents could be used for a routine analysis to reduce costs,

for example, the use of emission filters in place of a
monochromator, or to facilitate data handling, as with the

use of a log converter or computer.

A Xenon Novatron 599 flashtube (Xenon Corporation) is

used as the source, and is more intense than the flashtube

previously used [535]. The specifications and recommended

operating conditions are listed in Table 1. (Flashtube

theory is briefly outlined in Appendix I and discussed more

fully in [36].) The voltage necessary for the triggering

pulse, as well as for the discharge in the lamp, is supplied

by a Model 457 Hicropulser power supply (Xenon Corporation).

For this work, 8 kV was applied across the lamp. This is

enough above the lamp threshold voltage to provide repro-

ducibility and good spectral radiance, but is below the

maximum value allowable in order to prolong the lamp life.

The flashtube is mounted in a plexiglass housing,

which is illustrated in Figure 4. The housing in turn

rests on a steel mount designed so that its distance from









Component
Designation

A

B
C


D


KEY TO FIGURE 3


Component

Xenon Nodel 457 Nicropulser (Xenon Corp.,
Medford, Mass.)
Xenon Novatron 599 Flashtube (Xenon Corp.)
Modified Aminco SPF sample compartment
(American Instrument Co., Silver Spring,
Md.)
Heath Company EU-700 scanning monochromator
(Heath Co., Benton Harbor, Michigan)
RCA IP28A photomultiplier tube in a Heath
EU-701-30 Photomultiplier Module
Keithley 244 High Voltage Po-.:er Supply
(Keithley Instruments, Cleveland, Ohio)
Load box (see text)
Tektronix 26A2 Differential Amplifier with
2601 Mainframe Power Supply (Tektronix,
Inc., Beaverton, Oregon)
Biomac 1000 Signal Averager (Data Labora-
tories, Ltd., Mitcham, Surrey, England)
Sargent Model SR strip chart recorder
(E.H. Sargent and Co., Birmingham, Alabama)
Impedance matching device (see text)
Log converter
Paper tape punch
Computer
Filter
Multiplier phototube with power supply







21





F










D J








FL T---

0
I
I --



P' .I

I
L-------
A



S!

i









Fig. 5.-Block diagram of the instrumental components of the
pulsed source phosphorimete'r.












TABLE 1

SPECIFICATIONS A2TD OPERATING CONDITIONS FOR THE FLASHTUBE
EXCITATION SOURCE


Xenon Novatron 599 Flashlamp
(Xenon Corp., Medford, Mass.)

Arc length

Maximum energy per pulse

Repetition rate

Rise time

Pulse width

Average maximum watts

Peak power

Life


3.8 cm

5 J
0-60 Hz

500 ns

1.2 us

300 W (at 60 Hz)

5 x 106 W

5 x 104 pulses














9 i


A= side view
B= back view
C= top view
D= flashlamp
E= " plexiglass
F= stainless steel base
G= banana lead socket
H= clip


B 2

G@

















--u---j

1 1- 3%"----


C -T



3-3/4"


Fig. 4. Diagram of the Xenon flashlamp plexiglass
housing.









the sample compartment can be adjusted. A quartz lens 2 in

in diameter and with a 4 cm focal length is placed between

the lamp and sample cell to focus the lamp discharge image

on the sample to obtain maximum irradiance.

The flash repetition rate is controlled by a signal

from the signal average (discussed later) and was in no

case greater than 0.3 Hz. This allowed sufficient recovery

time for the lamp between flashes to insure greater repro-

ducibility, avoid spurious discharges, and prolong lamp life.

The output of a used (but not exhausted) flashtube

is shown in Figure 5. To obtain the spectrum, the lamp was

fired repeatedly while the monochromator was scanned across

the wavelength range. The source spectrum is not corrected

for photomultiplier response, which falls off below 300 nm.

Also, a Corning 754 UV filter is in place between the flash-

tube and the sample compartment to reduce stray light above

400 nm.

The quartz sample tube (25 cm x 5 mm i.d.) is placed

in a modified Aminco SPF sample compartment (American

Instrument Co.) which is fitted to a Heath Company EU-700

scanning monochromator by means of an adapter plate. The

compartment holds a quartz dewar flask (American Instrument

Co.), and the sample tube is held in position by the modi-

fied Varian spinner assembly described by Lukasiewicz,

Rozynes, Sanders and Winefordner [37]. The sample, how-

ever, was not rotated.
















S0 00
4)


4-








-r4










400 350 300
Wavelength

Fig. 5.-Spectral irradiance of Xenon Novatron 599 Flashtube as a function of
wavelength.









The scanning monochromator is a single-pass f/6.8

Czerny-Turner unit, with reciprocal linear dispersion of
O
about 20 A/mn which will cover the region from 190-700 nm

with an IP28A multiplier phototube. Resolving power is

about 0.1 nm. Light through the monochromator is detected

by an RCA IP28A multiplier phototube housed in a suitable

light-tight compartment (EU-701-30 Photomultiplier Module,

Heath Co.). A high voltage power supply (Hodel 244,

Keithley Instruments) provides 800 volts to the phototube.

Because the differential amplifier (Hodel 26A2,

Tektronix, Inc.) required a voltage input, a load box with

variable load resistors was used to develop a voltage from

the anode current of the multiplier phototube. This voltage

was then amplified by the differential amplifier (which is

powered by the Tektronix 2601 mainframe power supply); the

resulting voltage is detected by a signal average. Care

must be taken that the load resistor used is not so large

that it will increase the instrumental time constant enough

to distort the phosphorescence decay signal. Because the

measuring system contains stray capacitance, C, to ground

as well as the load resistance, R, the measured voltage

will decay exponentially with a time constant of

t = R C (14)

where t is in s, R is inn_, and C in F. This time constant

should be an order of magnitude smaller than the shortest








phosphorescence lifetime measured in order to minimize dis-

tortion of the measured decaying signal. (The measured

lifetime would be longer than the phosphorescence lifetime

if the effect of the time constant of the measurement device

is significant.)

The signal average (Biomac 1000, Data Laboratories,

Ltd.) has many modes of operation [38]; however, only

signal averaging was necessary in this work. The delay

time between termination of the excitation flash and obser-

vation of the phosphorescence decay, the time of observation

(the sweep time, and the delay time between observation

of the phosphorescence and the initiation of the next flash

are variable: 0.32 ms to 5.12 s for the delay before; 5 ms

to 81.92 s for the sweep time, and 40 ms to 655.4 s for the

delay after, all in increments of two. (The delay after

observation may also be zero.) In addition, the number of

sweeps to be averaged is variable from 1 to 16,384, in

increments of two; a "counts per address" control allowed

scale adjustment for the oscilloscope display (and thus

for the output).

Output from the signal average is recorded by a

potentiometric recorder (Model SR, 3. H. Sargent and Co.);

the observed phosphorescence signal is plotted versus time.

The recorder is calibrated with a time mark generator for

an accurate time base. A log converter could have been









used between the signal average and the recorder to produce

a straight line, with the slope inversely proportional to

the lifetime, rather than an exponential decay; however,

this refinement was not deemed necessary for the present

studies.

The flashlamp power supply is triggered by a signal

from the signal average at the beginning of each observa-

tion cycle, and the two are interfaced with a small

impedance matching device. The electronic diagram of this

system is given in Figure 6.


Instrumental Procedure

The events occurring during one excitation and

observation cycle are illustrated in Figure 7. The signal

average initiates the cycle with a trigger pulse to the

flashlamp power supply. During the time width of the flash,

tf,phosphors in the sample are elevated to the triplet

state, as evidenced by their phosphorescence and represented

by the dashed line. When the flash terminates, the

phosphorescence intensity decays exponentially. After a

preset delay time, t (initiated at the beginning of the

flash, unlike td in Equations 11 through 15) the signal is

monitored for some chosen time, t and then a delay after,

ta, is used to lengthen the cycle if needed. This cycle is

repeated for the necessary number of sweeps, and the






























Fig. 6.-Circuit diagram of the impedance matching device
between the signal average and the flashlamp
power supply.




T =D40D3 transistor

R =10 K

C = .001 F


V =+ 9V (dry cell)


















I \ ___-- t --*
I




<-------------^ -------------- 4




Fig. 7.-Diagram of the events that occur during one cycle
of excitation and observation for pulsed source
time resolved phosphorimetry.


tf = Flash duration

t = Delay between excitation flash and observation
td = Delay between initiation of flashlamnp dis-
charge and observation time

t = Observation time (sweep time)

ta = Delay after observation

t = Total cycle time
c









resulting "averaged" decay curve is plotted by the recorder

for subsequent data reduction.

The delay before the observation time is necessary

to eliminate initial scatter associated with the flash or

to minimize luminescence signals due to fluorescence or

fast phosphorescence when measuring a phosphor with a long

lifetime; this delay is at least 0.32 ms. The sweep time

used for proper lifetime determination should be at least

5 xT'. This allows the phosphorescence intensity to decay

to within 2 per cent of zero luminescence, which is neces-

sary to establish an accurate baseline for the lifetime

calculations. It is possible to adjust the sweep time to

measure fast phosphors in the presence of longer decaying

species. The observation time in this case should be at

least five times the lifetime of the short phosphor, but

need only be long enough to establish a straight line portion

for the long-lived phosphor in order to allow subtraction

of the phosphorescence of long-lived species from the total

phosphorescence signal at the various delay times.

The delay after is used to maintain the flash rate

below 0.3 Hz and to allow the phosphorescence signal to

decay to near zero so that the initial intensity does not

steadily increase with successive excitation flashes. For

accurate quantitative work, the total cycle time should

always be at least five times the lifetime of the longest

lived phosphor.










Data Reduction

Various methods of data reduction for time resolution

of single and multicomponent mixtures have been described by

Fisher [35]. Only the methods employed in this work will be

described here.

A typical recorder tracing of the phosphorescence

signal as a function of time is illustrated in Figure 8.

By drawing a smooth curve through the tracing, average values

at various delay times can be plotted on semi-logarithmic

graph paper, as shown in Figure 9. The initial intensity,

I is measured by extrapolating the straight line of the

semi-logarithmic curve back to t = 0. The lifetime, 'T0,

is equal to the time it takes for the phosphorescence signal

to change from I to 36.8 per cent of I The points of

decay of a single component solution may also be fitted to

an exponential curve of the form y = y exb (compare to

Equation 4) by a calculator (Wang 600 Series, Wang Labora-

tories, Inc., Tewksbury, Mass.) to determine Io (yo) and

Tp(- -); this method was used to determine the phosphores-

cence lifetimes of the compounds of interest, and to

calculate the initial phosphorescence intensities of stan-

dard solutions for use in analytical curves.

Multicomponent curves were analyzed graphically. A

decay curve of a binary mixture is shown in Figure 10; the



















4-


4,
H



O



CO














I I I I
26 58

tie Ins

ig 8.1orina dea urefra igecopnn
sapl (cden,0. g/ni.EA)




















T = 49
i40- o
: = 44 ms
0





0 20 0

0


0

I I I
20 40 60 80
time, ms


Fig. 9.-Semi-logarithmic plot of phosphorescence intensity
versus time for a single component sample
(codeine, 0.4 mg/ml in EPA).





















4



















21 37 53
time, ms



Fig. 10.-Kx-erimental decay curve for a binary mixture
(morphine and codeine in ethanol).










corresponding semi-logarithmic plot is shown in Figure 11.

The decay curve at large delay times results in a straight

line which can be extrapolated back to t = 0 to find the

initial phosphorescence signal for the long-lived species.

When the contribution from this component is subtracted

from the original curve, the result is a straight line

representation of the fast phosphor. Again, extrapola-

tion back to t = 0 provides the initial phosphorescence

signal for the short-lived component. The respective life-

times are determined from the proper straight line sections.

When the lifetimes of the two components are quite similar,

as with morphine and codeine in this example, then the

measured lifetimes may be in error. The effect of this

error on the calculated initial phosphorescence intensity

and therefore on the quantitative results is best determined

by calculation of relative percent error of a determination;

this is discussed in a later section. For a ternary

mixture, this deletion procedure is repeated.

Because the curve fit method is used for lifetime

calculations as well as for the determination of initial

intensity of standard solutions, while the graphical method

is used for the calculation of the initial intensity of

components in mixtures for comparison with these standards,

it is important to know how the two methods correlate. In

Table 2 the initial intensities and lifetimes obtained by

















I = 88
OOB
r-i 0
STB = 135 ms
-P \

o 0
S\ (B)
SO0
g 100-
0
0 0

S 50- O IoA 58
A O
O TA = 43 ms
50- 0
I IO

(A) O



20 40 60
time, ms


Fig. ll.-Semi-logarithmic plot of phosphorescence intensity
versus time for a binary mixture (morphine and
codeine in ethanol).











TABLE 2

COMPARISON OF CURVE FIT ANTD GRAPHICAL METHODS FOR DATA
REDUCTION


Percent
Error


Percent
CV Error


Curve fit 55.4 6.6 11.0 .81 .03 3.5

Graphical 53.9 6.7 12.4 .83 .054 4.1


The number of sweeps was 8 for each case. The component
measured was quinine (0.092 mg/ml in chloroform).


I = -nitial phosphorescence intensity

C6 = Relative standard deviation


T = Phosphorescence lifetime










the curve fit and graphical methods for quinine (.092 mg/ml

in chloroform) are comparable by both methods. The

graphical method offers the advantage of visibly indicating

any nonexponential character of a decay.

Quantitative analysis is accomplished by comparing

the initial intensity of the unknown to a standard plot of

initial intensities versus concentration (measured in the

same manner as the unknown), as determined for single

component standards. In this procedure, it is assumed that

the compounds in a mixture act independently.


Drugs and Reacents


The drugs used in this study were reagent grade

(Applied Science Labs, Inc., State College, Pennsylvania);

all drugs were ordered in accordance with BITDD regulations

and were used as received. Of the drugs ordered, those

found to be phosphorescent, and thus suitable for this

work, are illustrated in Figure 12.

Phosphorimetric quality anhydrous ethanol was pre-

pared in the laboratory according to the procedure outlined

in Appendix II. The chloroform is Certified A.C.S. Spectra-

analyzed (Fisher Scientific Company, Fair Lawn, New Jersey),

and was distilled once on a glass bead packed 6 ft column

to eliminate some of the phosphorescence background. The

ethyl ether, anhydrous (Nallinckrodt Chemical Works,

















OH

Morphine


Codeine


W O 'OC2 H5


OH

Ethyl Morphine


C2H5 N
CH2 0

HC
"(C H3)2

Amobarbital


(C2H5)2


NH2


Procaine


Phenobarbital


CH3
H| I
H C-C-N H2



0H


Amphetamine


CH3
N



C6 H5 C-O-C2H5
0


Meperidine


CH3
H I
H -C-N-C H3



e th he t aine




Methamphetamine


OH 0
I H H H H I1
H CC --N--C H2 HC-C C-C-O-C H3

CH30C (CH2.2 N C3 C-o-c-C6H5

H H H -C CH
CH H H .H
CH2
Quinine Cocaine


Fig. 12.-Chemical structures of selected phosphores-
cent drugs.










St. Louis, Missouri), and cyclohexane (Hatheson Coleman and

Bell, Los Angeles, Calif.) were used as received. The EPA

used was a commercially prepared mixture (American Instru-

ment Co.).

Stock solutions were prepared by weighing a 1 mg

portion of the appropriate druS, and filling with solvent

to the mark in a 5 or 10 ml volumetric flask. Though these

small flasks could contribute to the error of analysis, they

were used because a very limited amount of each drug was

available. Serial dilutions for standard determinations

were made for each drug.













CI~t=IER IV


RESULTS AND DISCUSSION


Noise and Precision


The detection limit and accuracy of an analytical

method depends largely on the noise encountered in the

measurement, and the precision with which measurements can

be accomplished. Noise levels of this experimental system

were measured in the absence of sample or source irradiation,

and were found to be appreciable. In Figure 15 the

measured peak-to-peak noise level is shown at various

values of the differential amplifier and load resistor;

512 sweeps were performed by the signal average. As ex-

pected, the noise level increases with increasing amplifi-

cation, but it is not linear, as the increase in signal is

assumed to be. Noise is the main limitation in achieving

low detection limits, but Figure 13 shows that the relative

signal to noise ratio is slightly better at higher amplifi-

cation.

The major portion of the noise is due to dark current

shot noise from the photo detector and amplifier drift

and/or pickup noise. In Figure 14, the significance of the

instrumental noise level to an analysis is shown. An







(B) Load Resistance, 43
10 53 100



40-






SO-0


0O'
0 B



I 20-
0


0




10-












(A) Amplification, V


Fig. 13.-Variation in peaI: to peak noise levels with in-
creasing amplification and load resistance.

Curve A: 53 load resistance, varying amplification.
Curve B: 500V amplification, varying load resistance.























4Z,










(B)
-p





0















time


Fig. 14.-Comparison of (A) sourceless noise to (B)
phosphorescence decay of ethyl morphine (0.0052
mg/ml in ethanol).










analytical decay curve of ethyl morphine (0.0032 mg/ml in

ethanol) is compared to the sourceless noise level. There

is no significant contribution to the noise by the source

or sample variations.

In determining the lowest concentration level

detectable by an instrumental system, the important con-

sideration is the signal to noise ratio, S/N. The limit

of detection is commonly defined as the concentration of

species which provides a signal equal to twice the

peak-to-peak noise level observed. For a given solution,

the theoretical improvement in S/N with increasing sweep

counts on the signal averaSer (N) is shown in Figure 15,

curve A. Also shown in Figure 15, curve B, is the S/N

improvement for ethyl morphine (0.056 mg/ml in ethanol) with

increasing sweep counts. The improvement for the sample is

thus not ideal.

An increase in the number of sweeps does reduce the

noise level but by less than 47, where N is the number of

sweeps. There are disadvantages to extending the analysis

time by increasing sweep counts. The time of analysis could

become unreasonably long, especially in the case of slow

phosphors. The flash lamp has a limited lifetime, and is

rather expensive. Also, over a large number of sweeps, the

lamp intensity would change (as the lamp ages) making suc-

cessive analyses imprecise,














15-



-sH
CO



0 (A)
0-
o












40 80 120

Number of sweeps, N


Fig. 15.-Plot of the signal to noise ratio, S/N, versus


Curve A: Theoretical ("N) improvement in S/N.

Curve B: S/N improvement for ethyl morphine
(0.056 mg/ml in ethanol).
(0.055 mg/mi in ethanol).









In Figures 16 and 17 the increase in S/N with in-

creasing concentration is illustrated for amobarbital in

chloroform, and for phenobarbital in chloroform. If the

number of sweeps were the same for all concentration levels,

the curves would be linear, and a limit of detection for

each species could be calculated by an extrapolation of the

line to S/IN = 2. However, in this study, N was increased

for the lower concentrations in order to improve the pre-

cision and so a linear extrapolation is not valid because

of the uncertain improvement in S/N. For meaningful de-

tection limit v .lues a more detailed evaluation of the

signal to noise ratio at given sweep counts is necessary.

This was not ascertained and so detection levels for the

drugs used in this study are not given in this work.

Precision depends on the same noise sources as are

present in determining detection limits. Confidence in the

results of any analytical technique requires that the pre-

cision be good, although good precision does not necessarily

insure good accuracy. The reproducibility of measurement

by the experimental system used in this work is demonstrated

in Table 3. In part A, high and low concentrations of

quinine in chloroform (a snowed matrix) and in EPA (a 5:5:2

volume ratio of ether, isopentane and ethanol which forms a

clear glass) were measured several times, with the results







48







15-



OO
0

10-


o
0
+I I















10-3 0-2 10-1

Concentration of Amobarbital, mg/ml


Fig. 16.-Plot of the signal to noise ratio versus
concentration of Amobarbital in chloroform.



































.6 .12
Concentration of Phenobarbital, mg/ml


Fig. 17.-Plot of the signal to noise ratio versus
concentration of Phenobarbital in chloroform.







TABLE 3


ILLUSTRATION OF THE PRECISION OBTAINABLE WITH THE PULSED SOURCE PHOSPHORIMETER
Concentration T Percent Percent
mg/ml N Solvent ~ o Error I c- Error SC
0.28 10 C 52.4 2.8 5.4 0.85 0.02 2.1 32
A 0.003 10 C 63.7 6.5 10.1 0.94 0.06 6.4 52
0.25 8 EPA 66.5 6.3 9.4 0.98 0.04 4.1 32
0.02 10 EPA 48.9 6.5 12.9 1.11 0.05 4.6 32
0.092 8 C 55.4 6.6 11.0 0.81 0.03 3.8 32
0.092 8 C 57.7 8.8 15.2 0.82 0.04 4.9 16
B 0.092 8 C 57.9 8.9 15.4 0.80 0.05 6.2 8
0.28 10 C 45.0 1.1 2.4 0.88 0.02 2.3 32
0.51 11 EA 58.8 4.2 7.1 1.11 0.05 3.0 32
C 0.003 10 C 66.3 2.7 4.1 1.94 0.06 6.0 52
Sections A and C: the sample is renewed between determinations.
Section B: the sample is not renewed between determinations.
Quinine is the phosphor used in all cases.
N = number of determinations.
I = mean initial phosphorescence intensity.
G= relative standard deviation.
7= mean phosphorescence lifetime.
SC= sweep counts.
Solvents used are:
C = chloroform.
EA = ethanol.
EPA = 5:5:2 volume ratio of ether, isopentane and ethanol.









as shown. As expected, the lower concentrations show

poorer precision, because the signal to noise ratios are

lower.

Because a signal average is used, it is expected

that an increase in the number of sweeps would improve

precision. This is indicated in part B by the improvement

of precision with an increase from 16 sweeps to 52, re-

sulting in about the same relative standard deviation as

in part A. Although it is reasonable to expect further

increase in precision with an increase in the number of

sweeps, this is not always desirable because time of

analysis becomes significant at long observation times when

a large number of sweeps are made, and, as already indicated,

noise levels in measurement may still be significant at high

sweep counts.


Quantitative Analysis of Hixtures via Time
Resolved Pho:.-horimetry

The purpose of this study is to show how time re-

solved phosphorimetry can be applied to the measurement of

drug mixtures. The drugs chosen to demonstrate quantitative

analysis of binary mixtures are not necessarily the best

possible ones to demonstrate time resolution nor are the

mixtures chosen comprehensive in terms of type of specific

mixtures which can be measured. These drugs do, however,

illustrate that time resolution is applicable to drug










analysis and can overcome difficulties of temporally

separating spectrally similar molecules. With pulsed

source, time resolved phosphorimetry, a mixture of drugs,

whose phosphorescence spectra overlap, can be quantitatively

determined without a prior physical separation step neces-

sary for conventional phosphorimetry, which would be time

consuming if not difficult or impossible to accomplish in

many cases. The fact that a physical separation step is

not needed prior to analysis is the major advantage of this

technique.

In time resolution phosphorimetry it is first neces-

sary to measure the spectra and lifetime of the phosphores-

cence of each species present in the sample. From this

data, it is apparent which drugs can be spectrally resolved,

which can be temporally resolved, and which can be resolved

by a combination of these techniques. The phosphorescence

emission spectra are illustrated in Figure 18. Because

phosphorescence spectra do not change appreciably with

changes in solvent, the phosphorescence peaks obtained with

chloroform are representative of the peaks obtained in the

other solvents. The lifetimes, however, vary significantly

with a change in the matrix, as shown in Table 4.

A third important consideration in time resolution

studies is the relative phosphorescence signals of the

components being analyzed. For the best results, the










Pheno-
barbital
(385)


300


Cocaine (415)

Procaine (445)



S\C


400


500


Wavelength,


Fig. 18.-Phosphorescence emission spectra of drugs in
chloroform showing peas: positions.


600










TABLE 4

PHOSPHORESCE3TCE LIFETILMES (s) OF DRUGS


IN VARIOUS SOLVENTS


Drug

Meperidine

Amphetamine

He thamohe t amine

Amobarbital

Cocaine

Phenobarbital

Procaine

Quinine

Ethyl morphine

Morphine

Codeine


Emission
peak, nm

590

390

395
400

415

385

445

515

510

510

515


EPA.


5.9

ins

.0015

2.2

1.8

ins

1.0

.045

.028

.040


DEE


7.5

9.6

.0016

2. 4

1.65

2.9

1.1

.040

.020

.059


.55

.52

.55
.0015

.82

.77

1.45

.85

.033

.014

.028


i2

i.

i:


1:




i.


ns ins

ns ins

ns .0016

- 2.2

ns 1.7

ns ins

ns ins

ns ins

ns ins

.041 .041


= 5:5:2 volume ratio of ether, isopentane, ethanol

= anhydrous ethanol

= chloroform

= cyclohexane

= diethyl ether

= Drug was at least partially insoluble in the
solvent, and so was not run.


EPA

EA

C

CH

DEE

ins










phosphorescence signal of the components being measured

should be about the same, because the species with small

phosphorescence signals compared to the other will be lost

in the noise of the observed phosphorescence signal. The

relative initial phosphorescence intensities of the drugs

in the various solvents are sho.rwn in Table 5.

Quinine, morphine, ethyl morphine, and codeine

exhibit severe overlap of emission spectra, and all

combinations of these four drugs are temporally resolvable

except codeine and ethyl morphine. The best results would

probably be atained in ethanol solvent, because the life-

time differences are comparable to those in chloroform and

greater than in EPA, but the relative intensity for

morphine is higher in ethanol. Analytical curves for

several drugs in chloroform and ethanol are shown in Figures

19 and 20. The lower sensitivity of morphine in chloroform

results in a lower slope of the analytical curve; this could

be due to some quenching effect. Time resolution of

morphine from a mixture of codeine, morphine and ethyl

morphine is also possible, as shown in Table 6.

Time resolution of morphine in a mixture of morphine

and ethyl morphine, and morphine in a mixture of morphine

and codeine illustrates the usefulness of the technique

with phosphors having similar lifetimes. These results

also illustrate the usefulness of time resolution











TABLE 5

A COMPARISONT OF THE RELATIVE INITIAL PHOSPHORESCENCE
INTENSITIES OF SELECTED DRUGS IN VARIOUS SOLVEN'-TS

Log I arbitrary units: EA C DEE EPA

Amphetamine 3 4 3.5

Methamphetamine 2.8 4 -

Amobarbital 6 6 5.6 6

Cocaine 4.6 5.3 4 4

Phenobarbital 5 4.6 5 4.6

Procaine 4 4 --

Quinine 3.95 4.5 4

Ethyl morphine 3.7 3.5 3.3

Morphine 3.7 2 4

Codeine 3.8 3.7 3.5

EA = Ethanol

C = Chloroform

DEE= Diethyl ether

EPA= 5:5:2 volume ratio of ether, isopentane and
ethanol

















10_

0


) C




S102-
0 D









1-1 10






10-9 10-1


Concentration, mg/mi

Fig. 19.-Analytical curves for (A) Amobarbital, (B)
Quinine, (C) Cocaine, (D) Morphine, in chloroform.












1O


A


/ 0O


10-2 i0-1

Concentration, mg/ml




Fig. 20.-Analytical curves for (A) Codeine, (3) Ethyl
Horphine, and (C) Horphine, in ethanol.


P-)
-P


H
10-'



0
O
02
P)
0




C 0)-
0
0



P-I












TABLE 6


RESULTS OF QUANTITATIVE ANALYSIS CF DRUGS BY PULSED
TIHE RESOLVED PHOSPHORIHrTRY


Mixture


Solvent


Amount
Present
(mg/ml)


Amount
Found
(mg/ml)


Relative
Error


SOURCE,


Percent


Morphine EA 0.067 0.068 1.6

Codeine EA 0.023 0.022 4.3

Morphine EA 0.11 0.12 9.0

Ethyl
morphine EA 0.052 0.042 20.0

Morphine EA 0.11 0.16 45

Codeine +
Ethyl
morDhine EA -

Morphine EA 0.16 0.16 0

Cuisine EA 0.062 0.058 7.0










phosphorimetry for the analysis of drugs and drug metabo-

lites in the presence of each other (codeine and ethyl

morphine are both metabolites of morphine).

Time resolution of a short and long-lived species

is illustrated by the analysis of morphine and quinine.

This particular combination also has a potential practical

application because quinine is used as a diluent for

heroin; morphine,being the immediate metabolic product of

that drug, in vivo, if adequately separated from urine,

could be determined in the presence of the quinine which

is most likely extracted along with the morphine.

Amobarbital and phenobarbital overlap enough to

prevent spectral isolation; however, time resolution can be

used to determine both barbituates. Conversely, it would

be desirable to time resolve amphetamine and methamphetamine,

but their lifetimes do not differ enough in any of the

solvents used to allow it. Cocaine and phenobarbital

similarly do not show a great enough difference in lifetimes

to allow a temporal resolution. If they did, it could be

done at the point of maximum overlap between the two peak

emission wavelengths, or at each emission peak to maximize

the signal of interest while at the same time eliminating

a portion of the "interferent."

A combination of spectral and temporal resolution

analysis is shown in Table 7. Although it is possible to











TABLE 7

RESULT OF UAtCTTITATIVE ANALYSIS OF DRUGS BY PULSED SOURCE,
TIHE RESOLVED PHOSPHO2I-.ETRY, WITH SPECTRAL ISOLATION


Solvent


Amount
Present
( g/ml)


Amount
Found.
(ms/ml)


Relative Percent
Error


Mixture


Morphine C 0.038 0.042 10

Cocaine C 0.021 0.018 14










perform a quantitative analysis by spectral isolation alone,

the measurement of the phosphorescence lifetime provides

additional qualitative information if the nature of the

drug is unknown. Measurement of a nonexponential decay at

a given wavelength could also show that more than one drug

species is present in the measured sample.


Qualitative Analysis via Time Resolved Phosphorimetry


Because excitation and emission spectra and phosphores-

cence lifetimes of a molecule in a given environment are

characteristic of that molecule, and because these parameters

may change to a different degree for different molecules

with a change in environmental conditions, time resolved

phosphorimetry could be a useful technique for molecular

identification. From an examination of Table 4, it is

obvious that, of these drugs, any single component sample

could be identified qualitatively with spectra and lifetime

data except for meperidine and the amphetamines, and ethyl

morphine and codeine. (It could well be, however, that

with an extension of solvent data, these combinations would

be resolvable and therefore identifiable under some other

environmental conditions.) Furthermore, qualitative

analysis could be accomplished on multicomponent samples

as long as the species are spectrally and/or temporally

resolvable.









As an example of the feasibility of drug identifica-

tion by time resolved phosphorimetry, a qualitative unknown

(prepared by a colleague) was analyzed and found to contain

procaine and quinine. The emission spectrum shows peaks at

445 nm and 510 nm. The lifetime of the species at 510 nm

was found to be 0.85 s and was 1.45 s at 445 nm. In addi-

tion to procaine and quinine, the sample also contained

morphine, but at a lower concentration than the quinine.

Because the relative intensity of morphine is much lower

than quinine in chloroform, the morphine signal is not

visible and its presence went undetected. This points out

a limitation of time resolved instrumentation for qualita-

tive and quantitative work. Hinor components will not be

easily seen unless they happen to be well isolated spectrally.

A second unkno:.n mixture was found to contain

quinine, procaine, and a component that did not fit any of

the data in Table 4 (e = 430, 1V= 1.6 s). This third

component turned out to be caffeine, and would have been

identifiable had the data for it been catalogued. It is

likely that for qualitative work (such as on drug tablets

in a state laboratory), the information on all (or most) of

the possible components will be at hand, so that this would

not be a major problem. This sample also contained

morphine, which was not detected for the same reasons given

above.







64

An important consideration for both qualitative and

quantitative analysis is that, if a component does not

phosphoresce, or does so weakly, it will not interfere,

unless its concentration is large enough to cause pre-

filter effects in absorption.












CHAPTER V


SUMMARY A:D FUTURE WORK


A pulsed source phosphorimeter has been described and

its use for qualitative and quantitative analysis of drugs

in single and multicomponent mixtures has been demonstrated.

This system offers the advantages that both qualitative

identification and quantitative determinations may be per-

formed on certain compounds without prior physical separation

by use of spectral and temporal resolution under various

environmental conditions.

A flashlamp was utilized as the excitation source in

the experimental system. Spectral isolation of the

components of interest was accomplished by a monochromator.

A signal average was employed to monitor the phosphores-

cence intensity as a function of time. The initial

phosphorescence intensity and the phosphorescence lifetime

were determined from a plot of the log of the measured

intensity versus time. Temporal resolution was performed

by a graphical delineation of components and subsequent

determination of individual intensities and lifetimes.

Quantitation is achieved via the proportional relationship

between the initial phosphorescence intensity and the concen-

tration of the species in solution.










There is much yet to be done with this system. The

noise level of the time resolved phosphorimeter is unusually

high. An optimization of this system with respect to noise

levels would result in much improved limits of detection.

An investigation into the application of this technique to

real samples, whether they be the qualitative identification

or pure or mixed drug forms (as tablets and powders) or the

quantitative analysis of drug levels in human fluids, is a

logical extension of this study. Also a study on variations

of the pulsed source technique with instrumentation improve-

ments (greater irradiance of the sample by means of mirrors

behind the source, or specific cycle programming to opti-

mize the procedure for a particular component) would improve

sensitivity and selectivity of the method. The use of a

computer for data reduction would facilitate numerical

computations.

Dedication of this technique to a specific analytical

problem could allow the simplification of instrumentation,

with an appropriate reduction in cost. This method would

then be more commercially feasible, although it is a

powerful research tool as it is.


























APPENTDT CES













APPE11DIX I


General Theory of Flashlamp Operation

A flashlamp converts electrical energy into radiant

energy by discharging a high voltage capacitor through a

tube of inert gas. Xenon is used most often as the fill gas

because it has a high cross-sectional area for efficient

collisional excitation by high speed electrons, and low

excitation and ionization potentials.

Flashlamps emit high spectral irradiance in the

visible and ultraviolet regions. Thus they have considerable

analytical utility in luminescence spectrometry because most

luminescent species absorb in the ultraviolet region.

The electrical discharge is initiated by a high

voltage trigger pulse. The energy dissipated is given by

J = 1 C V2 (A-l)

where J is in joules, C is the capacitance in pF, and V is

the voltage in kV. The duration of the flash is given by

t = RC (A-2)
7 R C1

where tf is the duration time, C is again capacitance, and

Rf is the effective arc resistance, which depends on arc

length, tube diameter, type and pressure of the fill gas,

and the voltage.








69

Xenon flash tubes with a quartz envelope have an

effective color temperature of about 7,0000 K, as do DC

xenon arcs, but the emissivity is much greater, especially

at the peak of the flash.













APPENDIX II


Preparation of Phosphorimetrically Pure Ethanol


Phosphorimetrically pure anhydrous ethanol is necessary

in order to obtain good analytical results, free from an

interfering background. Commercially prepared absolute

ethanol is unsuitable because it contains interferents

either as a result of the preparation method (as with a

benzene azeotrope) or due to phosphorescing denaturants.

A modification of the Lund and Bjerrum method [39]

for the preparation of anhydrous alcohols was employed to

prepare phosphorimetrially clean anhydrous ethanol. Into

a 2 1 round bottom flask is placed 1.5 1 of 95 per cent

ethanol, 10 g or more of magnesium turnings, and several

g of iodine. The mixture is refluxed for at least 6 h

during which time the water is converted to Mg(OH)2, with

the evolution of hydrogen and the formation of the

hydroxide quite visible. The reaction is as follows,

Mg + 2 EtOH = H2 + Mg(OEt)2 ,

Mg(OEt)2 + 2H20 = Mg(OH)2 + 2EtOH .

The magnesium must be kept in excess. The iodine catalyzes

the reaction and is reduced during the refluxing.

After refluxing, the alcohol is distilled. The middle

cuts show an appreciable decrease in phosphorescence back-

ground.













LIST OF REFERE::CES


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

2. G. N. Lewis and M. Kasha, J. Am. Chem. Soc., 66, 2100
(1944).
3. R. J. Keirs, R. D. Britt, Jr., and V. E. Ventworth,
Anal. Chem., 29, 202 (1957).

4. J. J. Fitzgerald and J. D. Winefordner, Rev. Anal.
Chem., in press.

5. P. A. St. John and J. D. Winefordner, Anal. Chem., 39,
500 (1967).
6. H. C. Hollifield and J. D. Winefordner, Chem. Instr.,
1, 31 (1969).
7. T. C. O'Haver and J. D. Winefordner, Anal. Chem., 538,
602 (1966).

8. J. D. Winefordner, Accts. of Chem. Res., 2, 361 (1969).

9. T. C. O'Haver and J. D. Winefordner, Anal. Chem., 38,
1258 (1966).

10. R. P. Fisher and J. D. Winefordner, Anal. Chem., 44,
948 (1972).

11. C. M. O'Donnell, K. F. Harbaugh, R. P. Fisher and
J. D. Winefordner, Anal. Chem., 45, 609 (1975).

12. J. D. Winefordner and H. W. Latz, Anal. Chem., 35,
1517 (1965).
13. J. D. Winefordner and H. Tin, Anal. Chem. Acta., 32,
64 (1965).

14. Ibid, 31, 259 (1964).

15. W. J. McCarthy, P. A. St. John and J. D. Winefordner,
"Phosphorimetry as a Heans of Chemical Analysis," in
S. Udenfriend, ed., Fluorescence Assay in Biology and
Medicine. Academic Press, NT.Y., 1970.










16. H. C, Hollifield and J. D. Winefordner, Talanta, 12,
860 (1965).
17. J. D. Winefordner, S. G. Schulman and T. C. O'Haver,
Luminescence Spectronetry in Analytical Chemistry.
Wiley-Interscience, New York, 1972.

18. D. M. Grebenshchikov, N. A. Kovrizhnykh and S. A.
Kozlov, Opt. Spektrosk., 31, 755 (1971).

19. S. G. Schulman, Fluorescence News, Vol. 6, Number 4,
Hay, 1972.

20. K. F. Harbaugh, C. H. O'Donnell and J. D. Winefordner,
Anal. Chem., 45, 381 (1973).
21. J. J. Aaron, J. J. Mousa and J. D. Winefordner, Talanta,
20, 279 (1973).

22. R. J. Lukasiewicz, J. J. Mousa and J. D. Winefordner,
Anal. Chem., 44, 963 (1972).

23. J. E. Kent, H. F. O'Dwyer and R. J. Stephens, Chem.
Phys. Let., 10, 201 (1971).

24. D. H. Haaland and G. C. Nieman, J. Chem. Phys., 59,
1013 (1973).

25. G. C. Giachino and D. R. Kearns, J. Chem. Phys., 52,
2964 (1970).

26. S. P. NcGlynn, H. J. Reynolds, G. W. Daigre and N. D.
Christodoyleas, J. Phys. Chem., 66, 2499 (1962).

27. N. C. Yang, D. S. McClure, S. L. Murov, J. J. Houser,
R. Dusenbery, J. Am. Chem. Soc., 89, 5466 (1967).

28. H. J. T. Backstrom and K. Sandros, Acta. Chem. Scand.,
12, 823 (1958).

29. R. G. Bennett, Rev. of Sci. Instr., 31, 1275 (1960).

30. H. L. Bhaumik, G. J. Clark, J. Snell and L. Ferder,
Rev. of Sci. Instr., 36, 37 (1965).
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Rev. of Sci. Instr., 41, 107 (1975).

32. T. D. S. Hamilton and K. Naqvi, Anal. Chem., 45, 1581
(1973).









33. S. Hattori and Y. Kato, J. Molec. Spec., 59, 432
(1971).
34. B. Mathiasch, Anal. Let., 4, 519 (1971).

35. R. P. Fisher, Ph.D. Dissertation, University of
Florida, 1971.
36. Amglo Corporation, Flashtube Tngineering Manual,
Chicago, Ill.

37. R. J. Lukasiewicz, P. A. Rozynes, L. B. Sanders and
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38. Biomac 1000 Operating Manual, Data Laboratories, Ltd.,
Surrey, England.

39. H. Lund and J. Bjerrum, Ber., 64B, 210 (1931).













BIOGRAPHICAL SKETCH


Karen F. Harbaugh was born in Andalusia, Alabama,

on January 8, 1945. After attending public schools in

Washington state, California, Alabama, and Florida, she

graduated from Renton High School in Renton, Washington.

She obtained a Bachelor of Science degree from the

University of Florida in December, 1966. Graduate studies

were begun immediately thereafter, but were temporarily

set aside when she went to work for Shell Chemical Company

in Deer Park, Texas. She returned to graduate school at

the University of Florida in January, 1970, and has worked

toward a doctorate in chemistry since that time.










I certify that I have read this study and that in
my opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.




,Gerhard H. Schmid
Associate Professor of Chemistry



I certify that I have read this study and that in
my opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.




Willis 3. Person
Professor of Chemistry



This dissertation was submitted to the Graduate Faculty of
the Department of Chemistry in the College of Arts and
Sciences and to the Graduate Council, and was accepted as
partial fulfillment of the requirements for the degree of
Doctor of Philosophy.


December, 1973


Dean, Graduate School










I certify that I have read this study and that in
my opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.




G'erhard M. Schmid
Associate Professor of Chemistry



I certify that I have read this study and that in
my opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.


Willis B. Person
Professor of Chemistry


This dissertation was submitted to the Graduate Faculty of
the Department of Chemistry in the College of Arts and
Sciences and to the Graduate Council, and was accepted as
partial fulfillment of the requirements for the degree of
Doctor of Philosophy.


December, 1973


Dean, Graduate School










































M 7 2 2 5 8 1 4




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