Flash Photolysis and Pulse Radiolysis. Contributions to the Chemistry of Biology and Medicine

240 Pages·1983·18.047 MB·English

by  R. V. Bensasson, E. J. Land and T. G. Truscott (Auth.)

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Book
FOSTER
Nuclear Magnetic Resonance in Biological
Research Journal
Photochemistry and Photobiology
Flash Photolysis
and Pulse Radiolysis
Contributions to the Chemistry
of
Biology and Medicine
R. V. BENSASSON
Laboratoire de Biophysique
Inserm U. 201, CNRSERA 951
Museum National d'Histoire Naturelle
61 rue Buffon, 75005 Paris, France
E. J. LAND T. G. TRUSCOTT
Paterson Laboratories Department of Chemistry
Christie Hospital and Holt Radium Institute Paisley College
Manchester M20 9BX Renfrewshire PA1 2BE
England Scotland
PERGAMON PRESS
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Copyright © 1983 R. V. Bensasson, E.J. Land,
T. G. Truscott
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First edition 1983
Library of Congress Cataloging in Publication Data
Bensasson, R. V.
Flash photolysis and pulse radiolysis
Bibliography: p.
Includes index.
I. Flash photolysis. 2. Pulse radiolysis.
3. Biological chemistry —Technique. I. Land, E.J.
II. Truscott, T. G. III. Title. [DNLM: 1. Bio-
chemistry—Methods. 2. Photolysis. 3. Spectrum
analysis. QU 4 T873f]
QP519.9.F52B46 1983 574.19'2'028 82-19052
British Library Cataloguing in Publication Data
Bensasson, R. V.
Flash photolysis and pulse radiolysis
I. Photobiology I. Title
II. Land, E.J. III. Truscott, T. G.
574.19'153 QH515
ISBN 0-08-024949-3
Printed in Great Britain by A. Wheaton 9 Co. Ltd., Exeter
Preface
Life was initiated and is maintained through This text is not a comprehensive review of all
radiation absorption. Some thousands of millions of contributions of flash photolysis and pulse radiolysis
years ago, solar ultra-violet radiation, unfiltered by to all biological molecules of importance; our aim is
the reducing primitive atmosphere of our planet, rather to choose some examples. In particular we
ionizing radiations emitted by radioactive elements consider porphyrins, among which are chlorophylls,
and heat of volcanoes were available to synthesize essential pigments of photosynthesis, and also com-
complex organic molecules and to trigger the pre- pounds present in the blood; polyenes, involved in
biotic chemical evolution leading to anaerobic life. photosynthesis and vision; quinones, cytochromes,
Photosynthesis appeared later when the earth's nicotinamides and flavins involved in electron trans-
atmosphere became oxidizing and developed, shielded port in chloroplasts and/or mitochondria; DNA and
from the ultra-violet radiation by an ozone layer. proteins, the main cellular targets damaged by radi-
At that time "heterotrophs", organisms living on ation, and a number of drugs used in phototherapy,
materials prefabricated by the ultra-violet radiation, radiotherapy and general medicine. This text is
disappeared and were replaced by "autotrophs", oriented towards the younger research worker in
organisms able to incorporate porphyrins and to these fields and the senior scientist in related fields.
photosynthesize their own food by using visible Although the techniques discussed in this text
light. Photosynthesis provided then, and still pro- were initiated more than 25 years ago they are not
vides, the energy needed for all kinds of living organ- yet used as much in biology as other spectroscopic
isms. The sun also allows many living organisms to methods, namely spectrofluorimetry, electron spin
recognize their environments. However, ionizing, resonance and nuclear magnetic resonance. Until
ultra-violet or visible radiation has deleterious effects about 1968 these techniques were restricted to a
on life. It can damage the main biological molecules time resolution in the region of 10 microseconds
and lead to an alteration of their activity or to (1 jxs is 10~6 s). However, they have now been ex-
cellular death. This interaction of radiation with tended to the nanosecond (1 ns is 10~~9 s) and pico-
biomolecules, either beneficial or deleterious to second range (1 ps is 10~12 s), the time during which
life, is the focal point of many scientific disciplines light travels 0.3 mm.
using different techniques. Among those techniques, We wish to express our sincere thanks to col-
flash photolysis and pulse radiolysis are very power- leagues with whom we have discussed various sections
ful. They allow the study of transient species such of the manuscript. These include B. Alpert, R.R.
as excited states, radicals and solvated electrons Birge, J. Butler, J. Cadet, R.J. Cogdell, H. Frauen-
produced by the interaction of radiation with bio- felder, P.F. Heelis, M.F. Hipkins, B. Honig, L. Lind-
molecules. These techniques have not only led to qvist, P. Mathis, M. Ottolenghi, M. Rougee, R. Santus
the clarification of a number of problems related and A J. Swallow.
to photo biology and radiobiology, but have also
We also thank members of the Medical Illustra-
helped to elucidate biochemical reactions which
tions Department at the Christie Hospital and Holt
are not radiation-induced by selectively generating
Radium Institute for the figures and Miss M.G. Camp-
certain types of free radicals present in normal
bell of Paisley College for the typing. We thank the
metabolic processes.
Centre National de l'lnformation Chimique, Paris,
which provided bibliographic information.
V
Chapter 1
Introduction
Page No. Page No.
1.1 Flash Photolysis and Pulse Radiolysis 1 1.5 Properties of Excited States 11
1.5.1 Extinction Coefficients of Triplet-Triplet
Transitions 11
1.2 Transient Species 2 (a) Singlet Depletion Method 11
(b) Energy Transfer Method 12
1.5.2 Extinction Coefficients of Singlet-Singlet
1.3 Comparison of Effects of Light and Ionizing Transitions 13
Radiation 4 1.5.3 Singlet-Triplet Intersystem Crossing Quantum
1.3.1 Generation of Solute Excited States . . .. 5 Yields 13
1.3.2 Generation of Solute Radicals 6 (a) Heavy Atom Fluorescence Quenching
Method 13
(b) Comparative Method Using Laser Flash
1.4 Assignment of Transient Species 8 Photolysis 14
1.4.1 Kinetics 8 1.5.4 Triplet Reactions 14
1.4.2 Solvent Polarity 9
1.4.3 Energy Transfer 9 1.6 Properties of Radicals 16
1.4.4 Light Emission 10 1.6.1 Extinction Coefficients of Radicals . . .. 16
1.4.5 Effects of Oxygen or Nitrous Oxide . . .. 11 1.6.2 Radical Reactions 16
1.4.6 Complementary Pulse Radiolysis and
Flash Photolysis Data 11 1.7 Properties of Carbocations and Carbanions ... 18
1.1. FLASH PHOTOLYSIS AND PULSE electrically as a function of time at a single wave-
RADIOLYSIS length, using a continuous source of light to monitor
the sample. About a decade later (Matheson and
The technique of flash photolysis*, introduced in Dorfman, I960; McCarthy and MacLachlen, I960;
1949 (Norrish and Porter, 1949; Porter, 1950) is a Keene, I960; Boag and Steel, 1960) the same prin-
potent means for studying the initial physicochemical ciple was applied to the study of the initial effects of
effects of light upon matter. The technique consists ionizing radiation, the exciting light flash being re-
of the irradiation of a sample with a very short high placed by a source of high energy radiation (pulse
intensity flash of light, the resultant changes in light radiolysis).
absorption being followed either photographically The principle of both flash photolysis and pulse
over a wide wavelength range by means of a second radiolysis with photoelectric detection is illustrated
weaker flash triggered a given time after the high in Fig. 1.1. The analysing lamp provides a continuous
intensity photolysing flash, or, more often, photo- beam of light which passes through the sample,
* The word photolysis implies the breaking of chemical bonds. However, in flash photolysis often no bonds are actually
broken. Nevertheless, the term 'flash photolysis' is in common usage even when bonds are not broken and is used throughout
this text.
1
2 FLASH PHOTOLYSIS AND PULSE RADIOLYSIS
usually at right angles to the path of the exciting microseconds (1 JUS = 10~"6 s) or so. More recently,
pulse of radiation. The short pulse of radiation results pulsed lasers have become available, allowing first
in the formation of transient species. A monochroma- nanosecond (1 ns = 10~9 s) and subsequently, when
tor is used to select wavelengths at which the transi- mode-locked, picosecond (1 ps = 10~12 s) time
ent intermediates absorb. The transmission of the resolutions. Linear accelerators or Van de Graaf
generators delivering short pulses of fast electrons
pulse of light or
ionising radiation lasting several microseconds down to a few nano-
seconds, are by far the most common sources of
ionizing radiation used in pulse radiolysis (with
energies normally between 1 and 15 MeV). Linear
accelerator pulses actually consist of series of fine
structure pulses lasting several tens of picoseconds.
This property of accelerator pulses can be used to
attain picosecond time resolutions. The detection
wavelength range currently attainable using either
oscilloscope technique is 0.2 — 3 jum. The maximum time resolu-
Fig. 1.1. Principle of flash photolysis and pulse radiolysis. tion attainable depends on the type of detector used
Reprinted with permission from Keene in 'Pulse Radiolysis' which in turn is dependent upon the particular wave-
Ed. Ebert et al Copyright 1965 by Academic Press Inc. length being studied. Developments in the instru-
(London). mentation used have been reviewed by Porter and
West (1974) and West (1977) for flash photolysis and
sample before, during and after the pulse of radiation
Dorfman (1974) for pulse radiolysis.
is thus scanned by the photodetector which converts
changes in light intensity to electrical signals which
are displayed on the oscilloscope. The transmission
changes are then normally converted to optical 1.2. TRANSIENT SPECIES
densities. By carrying out such measurements at a
variety of wavelengths a transient absorption By far the most common radiation-induced un-
spectrum may be mapped out. In spectral regions stable intermediates detectable by flash photolysis or
where the transient and original material overlap a pulse radiolysis are electronically excited states or
difference spectrum is obtained, the change in optical- free radicals. Carbocations, carbanions, unstable
density observed being: metal ions, isomers or conformers are also formed in
certain cases.
AOD = (e-e)cl, The excited states normally encountered are either
t G
singlet states, which contain no unpaired electrons, or
where e is the extinction of the ground state, e is triplets, which contain two unpaired electrons (Fig.
Q %
the extinction of the transient, c is the concentration 1.2). Triplet states are generally longer-lived than
of ground state converted to transient and 1 is the
optical path length. Three other detection methods
have been used less widely in flash photolysis and
pulse radiolysis, namely the observation of electrical
conduction, electron spin resonance and polaro-
graphy of the radiation-induced transients.
Since the introduction of flash photolysis and
pulse radiolysis both the pulsed sources of radiation
and the detection systems employed have continued
to be improved as regards the time resolution attain-
able and the wavelength range over which measure-
ments can be carried out. The first types of sources
used in flash photolysis were gas-filled discharge
lamps having durations of a minimum of a few Fig. 1.2. Excited state energy levels and transitions.
INTRODUCTION 3
singlet excited states. Singlet and triplet excited states Although many triplet excited states have intrinsic
sometimes decay by giving out light, this radiation lifetimes of seconds as measured trapped in rigid
being referred to as fluorescence and phosphores- glasses at liquid nitrogen temperatures, in fluid solu-
cence respectively. Transitions between electronic tion at room temperature, due to quenching reactions
states of like multiplicity (internal conversion, IC) are they tend to have experimental lifetimes somewhere
spin-allowed. Transitions between states of different in the microsecond—nanosecond range. Excited
multiplicity (intersystem crossing, ISC) are spin-for- singlet states, on the other hand, always have life-
bidden, although usually some crossover does occur. times shorter than one microsecond. We are normally
The Jablonski diagram (Fig. 1.2) illustrates these and concerned with n to n* and n to 7r* transitions and
other processes. The approximate durations of the the usual terminology for the corresponding excited
processes shown in Fig. 1.2 are as follows: states, 17T7T* and 1n7r* for singlets and 37T7r* and 3n7r*
for triplets will be used (see, for example, Turro,
1978).
-> Sn absorption 10"15s Free radicals, either charged or neutral, contain
s -> Sl internal conversion 10"11 - 10" -14 single unpaired electrons and are in general highly
n reactive in fluid solution. They can be stabilized by
-> internal conversion 10~7 - 10" "9s
sl S0 trapping in an inert medium at liquid nitrogen
-11
sl -* S0 + hVt fluorescence io-7 - 10" temperature. As far as organic free radicals are con-
intersystem crossing lO"8 - 10" -14 cerned, aromatic free radicals tend to be more stable
sl Tn
T -> T, internal conversion 10"11 - 10" -14 than aliphatic radicals due to increased resonance
n 1 stabilization. The relationship between some of the
Ti -> S0 intersystem crossing 10+1 - 10" "3s different types of aromatic radicals that can be form-
-> phosphorescence 10+1 - 10" -3 ed from simple aromatics is illustrated below:
Ti so+hPv s
Benzyl
C^Hs CH
2
(X = CH, Ar = CH)
3 6 5
Phenoxyl
CHO*
6 s
(X = OH, Ar = CH)
6 5
Anilino
CH NH*
6 5
(X = NH, Ar = CH)
2 6 5
4 FLASH PHOTOLYSIS AND PULSE RADIOLYSIS
One of the major contributions of pulse radiolysis molecules, since only a few electron volts are required
and flash photolysis has been the discovery of the to produce chemical change in a single molecule. A
hydrated electron, e^, in the early 1960s. Pulse wide spectrum of MeV energies of such radiations is
radiolysis of water and flash photolysis of anions in possible and these have different penetrations
water led to the observation of a broad structureless through matter.
transient peak around 720 nm which, on the basis of The effects produced by light (ultra-violet and
earlier theoretical predictions and experimental visible) and ionizing (high energy) radiation are
deductions, was assigned to e"~. Solvated electrons initially quite different. With light irradiation the
aq effects are produced by the specific absorption of
have since been observed by pulse radiolysis and
photons by molecules having an absorption band in
flash photolysis in a wide variety of solvents in-
the appropriate region. In most cases one is dealing
cluding alcohols (A^ *OH 630 nm), ammonia (X^H^
with a solution, and in such cases it is usually the
1500 nm) and ethers (A^o 2 3 00 nm) (Dorfman, solute which absorbs UV or visible radiation and is
1973). The absorption maxima of the electron in chemically changed. With high energy irradiation,
hydrocarbons is also in the infra-red region. The however, the absorption of photons is independent
solvated electron is the smallest anionic free radical of chemical nature and is, as described above, a means
and is a strong reducing agent. Hydrated electron re- of generating fast electrons within the medium as a
activities have been determined for hundreds of whole. In any dilute solution, therefore, it is always
compounds, many of biological interest. the solvent that absorbs most of the high energy
In a few cases it has been possible to observe by radiation and is consequently chemically changed
pulse radiolysis and flash photolysis a dissociative initially. Nevertheless, both light and ionizing radi-
ionization of the solute giving a carbocation or a dis- ation ultimately give rise to similar, and in some cases
sociative electron attachment by the solute giving a the same, short-lived chemical intermediates, i.e.
carb anion. excited states and/or free radicals. The mechanism
through which these chemical intermediates are form-
ed is different, however.
1.3. COMPARISON OF EFFECTS OF LIGHT
AND IONIZING RADIATIONS* The quantitative effects of light are measured by
the quantum yield, 4>, which may be defined as the
The effects of ionizing radiation and light are number of molecules undergoing a particular process
commonly termed "radiation chemical,, and "photo- (for example: fluorescence, phosphorescence or
chemical" effects, respectively. Although light is, of singlet-Hriplet intersystem crossing) per number of
course, a form of radiation, the term "radiation photons absorbed by the system. If, for every photon
chemistry" is normally reserved for chemical re- absorbed, a molecule undergoes a particular photo-
actions initiated by high energy ionizing radiation — chemical process, the quantum yield for the process is
that is, radiation with a wavelength shorter than one. If other processes compete, the quantum yield
about 100 nm. Such radiations include X-rays, y rays will be less than one.
and corpuscular radiations such as fast electrons. The chemical effects of ionizing radiation are
Since X-rays and y rays, by one mechanism or an- measured in G values. G is defined as the number of
other, give rise to fast electrons within the medium radicals, excited states or molecular products pro-
being irradiated, the end product of X, y or fast- duced or transformed in an irradiated system absorb-
electron irradiation is often the same. These fast ing 100 eV of energy.* The G-values for the species
electrons initially have appreciable energy and in produced in the primary interaction with ionizing
slowing down form many more secondary electrons radiation are usually up to ~ 3; much higher G values
which in turn are capable of ionizing several thousand indicate the presence of chain reactions.
* The distinction between light and ionizing radiation is arbitrary because ultra-violet and, in some cases, even visible, light can
lead to ionization. Nevertheless, the terms 'light* and 'ionizing radiation' are commonly used in photochemical and radiolytic
terminology, respectively.
t The absorbed dose is often expressed in rads (1 rad = 100 ergs per gram = 6.242 X 1013 eV per gram), or, more recently,
Grays (1 Gy = 100 rads).
INTRODUCTION 5
1.3.1. Generation of Solute Excited States (i) Geminate ions, accounting for ~ 90% of the
total, which recombine within a few nano-
The formation of solute excited states by ionizing seconds at room temperature. Here the
radiation may occur by four different mechanisms: positive and corresponding negative ions
direct excitation, ion recombination, energy transfer formed together never escape each other's
from excited solvent and subexcitation electrons influence.
(Singh, 1972). As examples, we now discuss some
aspects of the mechanism of formation of solute (ii) Non-geminate (free) ions, accounting for
excited states in the common solvents hexane and ~ 10% of the total, which recombine homo-
benzene, in particular via the predominant mecha- geneously over times greater than a few nano-
nism of ion recombination. Light-induced solute seconds. Here the positive and negative ions
excitation will be compared. We shall consider first formed together escape each other's influence
of all anthracene (A) in the non-polar aliphatic and recombine over microsecond time scales.
hydrocarbon solvent hexane, comparing ionizing
irradiation with light excitation. With a dilute solu- It has been suggested by some, for example Beck
tion in hexane, the high energy radiation is absorbed and Thomas (1972), that singlet excited hexane (life-
practically exclusively by the solvent: time < 1 ns) is also involved to some extent in the
formation of excited anthracene in such solutions.
CH -AAAA— CH;+ + e~ Perhaps the important point to be emphasized here,
6 14 6 4
over which there is no debate, is that ionizing radi-
Hexane, unlike benzene which will be discussed later, ation produces both singlet and triplet excited states
does not have any relatively long-lived excited states, in hexane, many of the triplet solutes being formed
so the main process is solvent ionization. Since independently of the singlet excited state of the
electrons are not readily solvated in non-polar solute. This fact enables one to use high energy irradi-
solvents, the electrons rather either recombine with ation to form and study the triplet excited states of
the parent ion CHi 4 or add on to A: molecules which have zero or very low intersystem
6
crossing efficiencies without the use of a triplet
e~ + A-> A" sensitizer. A large number of biological molecules
(e.g. many polyenes) fall into this category, as will
Some of the hexane positive ions likewise react with become apparent in subsequent chapters in this book.
anthracene: In the case of near—UV excitation of anthracene
in hexane, light absorbed by anthracene leads to
CHiV A->CH + A*+ various singlet excited states of anthracene alone. The
6 6 14
solvent does not absorb in the near—UV and so is not
Lowest^ excited anthracene triplet (T) and singlet (S) excited. All singlet excited states of anthracene
states are then formed by fast recombination of except the lowest are extremely short—lived and
A + A and CH + A . Some further anthra- rapidly relax to the first excited singlet state. Trip-
6 14
cene triplets are ^lso formed via intersystem crossing. let—excited anthracene is only then formed via inter-
At room temperature all the above processes occur system crossing.
mainly within a few nanoseconds of energy deposi- Secondly, we consider the high energy irradiation
tion within the medium, although a small proportion of anthracene in the non-polar aromatic hydrocarbon
of the recombinations take place over rather longer solvent benzene. Here the picture is slightly different.
times. Basically, two types of ions are involved: This is because benzene^, unlike hexane, has its own
t Throughout the text we will refer to the lowest excited singlet and triplet states as simply the excited singlet and triplet
states unless otherwise stated.
X The purity of the benzene is critical to the lifetime of solute excited states (Cundall et aL, 1968; Land, 1969).
6 FLASH PHOTOLYSIS AND PULSE RADIOLYSIS
easily accessible and reasonably long-lived singlet and excited states and high yields of free radical ions.
triplet excited states. The mechanism here contains The basic reason for this is that in polar solvents, e.g.
some of the following reactions: water or alcohols, the initially formed ions, in
particular the electrons, are solvated and stabilized by
CH->\/\/U CHe+ + e~ the solvent and tend not to recombine to form
6 6 6
excited states. In non-polar solvents most of the
e + CgHg • CfcHs initial positive ions never escape the influence of the
corresponding negative ions (electrons) formed at the
CH + CH6+ • 2CH (singlet and/or triplet same time, whereas in polar solvents nearly all
6 6 6 6 6
positive and negative ions initially formed escape
excited states) each other's influence. In general, therefore, solute
excited states are best studied by pulse radiolysis in
lCH* +A ^CeHe + 'A* non-polar solvents and solute radicals, or radical ions,
6 6
are best studied in polar solvents. Unfortunately, due
3CH* +A CH + 3A* to solubility factors, it is not always possible to study
6 6 6 6
1 y^* » 3 A* solute excited states in non-polar solvents (for ex-
ample, biliverdin, see 2.3.5). In such cases other
Although the singlet excited lifetime of benzene is somewhat more polar solvents such as acetone can be
~ 20 ns, the triplet excited lifetime is only ~ 3 ns in used. In this solvent approximately equal amounts of
pure benzene (Bensasson et aL, 1971). Thus it is solute excited states and radicals are produced (Arai
necessary to have a relatively high concentration of and Dorfman, 1965), which can lead to somewhat
anthracene present to scavenge all the triplet- ambiguous data.
excited benzene. In fact, the lifetime of triplet-
excited benzene becomes longer when the benzene is 1.3.2. Generation of Solute Radicals
diluted with hexane or cyclohexane. Thus, if one is
studying a solute at low concentration, dilution of Since water is such an important biological sol-
the benzene with hexane or cyclohexane results in vent, it is not surprising that a great deal of work has
higher yields of excited solute. been devoted to the study of its chemistry induced
Ultra—violet light irradiation of anthracene in by ionizing radiation. This radiation produces, in
benzene, on the other hand, leads to basically the < 10—15 s, excited molecules, H0*, cations,
2
same series of reactions as found for anthracene in H0 , and electrons, e~~, ejected as quasi-free
2
hexane. particles able to ionize other molecules. The ejected
Since high energy-induced solute excited states are electrons, after losing their energy by ionizing and
formed via excitation or charge transfer from the exciting molecules along their path, are finally
solvent, the rate of formation of excited solute is in thermalized and solvated in a time of the order of
general faster for higher solute concentrations. Con- 10-12s:
sequently, the shorter-lived the solute excited state
being studied, the higher the solute concentration e fc- p ^ p
necessary in order to form sufficient concentrations therm aq'
of it for observation. This limitation as regards the The H0 + ions deprotonate by a very fast re-
2
study of very short-lived solute states by pulse radio- action:
lysis does not apply to flash photolysis.
Another proviso in the study of solute excited H0 + + H0 —• OH* + H0+
2 2 3
states by pulse radiolysis is that the solute must be
soluble in a solvent of low polarity. It is a fairly in 1.6 X 10~14 s, a reaction even faster than the re-
general rule in high-energy radiation chemistry that combination of H0 and e~~. The very fast dis-
2
non-polar solvents tend to support high yields of sociation of excited water molecules gives H and
solute excited states and low yields of free radical OH*, but the yield of primary free radicals by this
ions (which escape geminate ion recombination), dissociation is very minor in comparison with the
whereas polar solvents tend to support low yields of yield of the ionization process. Within 10—9 s of high