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LOW TEMPERATURE LUMINESCENCE SPECTROSCOPY OF SOME
AROMATIC HYDROCARBONS AND RELATED COMPOUNDS
by
Brian Sinclair Causey
A thesis submitted
for the degree of
Doctor of Philosophy
in the
University of London
Department of Chemistry Imperial College of Science and Technology London SW7 2AY February 1979
LOW TEMPERATURE LUMINESCENCE SPECTROSCOPY OF SOME
AROMATIC HYDROCARBONS AND RELATED COMPOUNDS
by
Brian Sinclair Causey
A thesis submitted
for the degree of
Doctor of Philosophy
in the
University of London
Department of Chemistry Imperial College of Science and Technology London SW7 2AY February 1979
ABSTRACT
The good selectivity due to the high resolution spectra obtained
in the Shpol'skii effect has been utilised for qualitative fingerprinting
with some quantitative measurement of various carcinogenic aromatic
hydrocarbons plus some related nitrogen heterocyclics in coal tars,
pitches, used oils and polluted waters. These applications have been
compared with more standard, familiar, analytical procedures such as
gas chromatography, thin layer chromatography and high pressure liquid
chromatography with electrochemical detection. The necessity for only
minimal simple column chromatography, solvent extraction or micro-
sublimation sample clean up is shown.
Truly precise, quantitative, analysis with only partially selective
excitation sources has been found to be only possible for a limited
number of polynuclear aromatic hydrocarbons (P.A.H.). With others,
and especially more polar species, problems were encountered with inner
filter effects, matrix effects and intermolecular energy transfer.
A fundamental study of the mode of appearance of non-phonon luminescence
lines has been undertaken to illucidate the nature of these intermolecular
forces. In conjunction with a concentration study a unique variable
temperature conduction cell, designed to help obtain data to develop
quasi-linear spectroscopic theory and also to monitor crystallisation
plus phase change temperatures, has been linked with a time averaging
facility with an oscilloscope read-out to give a more precise data
acquisition and storage system.
Phototautomerism, dimerisation and conformation changes have been
put forward as reasons for the broadening of quasi-lines among simple
nitrogen heterocyclics.
On studying other potentially useful matrices for analysis such
as silicates, polymers and gels, hydrogen bonding has been found to
play a significant role and to limit spectroscopic information.
Finally some electrophoretically separated proteins have been
monitored for natural fluorescence In polyacrylamide gels.
2
3
Contents
CHAPTER I MOLECULAR LUMINESCENCE SPECTROSCOPY Page
1.1. Introduction 10
1.2. Historical Survey 10
1.3. Quasi-linear Spectroscopy - The Shpol'skii Effect 11 1.4. Radiative Emission 12
1.5. Optical Density 18
1.6. Matrix Isolation 20
CHAPTER II INSTRUMENTATION
2.1. Corrected Spectra 22
2.2. Phosphorescence 22
2.3. Commercial Instruments 23
2.4. Shpol)skii Spectrofluorimeters 28
2.5. High Resolution Spectrofluorimeters j1
2.6. Detection 32
CHAPTER III CARCINOGENS and the Aromatic Hydrocarbons in Perspective
3.1. Cancer
3.2. Hydrocarbon - D.N.A. Interaction
3.3. Metabolism
3.4. Environmental Pollution
46
50
54 55
CHAPTER IV P.A.H. ANALYSIS in Coal Tars and Pitches
4.1. World Energy Problem in Relation to Coal 60 4.2. Energy and associated P.A.H. Production 61 4.3.1. Coal Tar Analysis 62
4.3.2. Analytical Techniques 63
4.3.3. Chromatography with Fluorescence Detection Methods 64 4.4. Direct Spectrofluorimetric Analysis 66 4.5. Experimental 67 4.6. Results and Discussion 69
4.7.1. Conclusions 75 4.7.2. Multiple-site Structure 75 4.8. High Pressure Liquid Chromatography 77 4.9.1. High Molecular Weight Species 8o
4.9.2. Summary 86
4
CHAPTER V DETERMINATION OF P.A.H. COMPOUNDS in Oil Samples utilising
the Shpol'skii Effect
5.1. Introduction 87
5.2. Chemical Constituents of Petroleum 87
5.3. General Methods 88 5.4. Applications of Low Temperature Luminescence 89
Spectroscopy utilising the Shpol'skii Effect
5.5. Experimental 89
5.5.1. Instrumentation 89
5.5.2. Solvents 90
5.5.3. Oil Samples 90
5.5.4. Column Chromatography 90
5.5.5. Thin Layer Chromatography of Oil Samples 91
5.6.1. Quantitative Analysis 97 5.6.2. Quantitative Results 97 5.7. Results Summary 97 5.8. Discussion 98
CHAPTER VI A RAPID ROUTINE METHOD FOR QUANTITATIVE DETERMINATION
of Benzo(a)pyrene in Water by Low-Temperature Spectro-
fluorimetry
6. Introduction 100 6.1. Previous Applications of Low-temperature Spectrofluori-
metric Methods for the Determination of P.A.H. in
Water. 101 6.2. Experimental 102
6.2.1. Apparatus 102
6.2.2. Materials and Reagents 103
6.3. Procedure 103
6.3.1. Recovery of B(a)P from Distilled Water by Extraction
Procedure. 103
6.3.2. Determination of B(a)P by Low-temperature Spectrofluori-
metry 104
6.3.3. Limit of Detection of B(a)P by Procedures employed. 107
6.3.4. Total Analysis Time. 107
6.3'.5. Results 107 6.3.6. Experiments on Quenching Effects 107
5
Chapter VI
6.3.7. Analysis of Some Water Samples 108 6.4. Conclusions 113
CHAPTER VII HALF-BAND WIDTH STUDY OF NON-PHONON LUMINESCENCE
(NPL) LINES
7.1. Exciton States in Crystals 116
7.2. Mixed Substitutional - Solid Crystals 119
7.3. Shpol'skii Matrices 120
7.4. Alkane Matrices 121
7.5. Some Observations of the Effect of Concentration and
Temperature on the Width of Non Phonon Luminescence
(NPL) Lines of Several P.A.H.'s 122
7.5.1. Introduction 122
7.5.2. Experimental 123
7.5.3. Reagents 124
7.5.4. Study of the effect of Variation in Temperature on
Q.L.S. at 77 K 124
7.5.5. Temperature Results 125
7.5.6. Concentration Effects 130
7.5.7. Discussion 133
7.5.8. Resume 147
CHAPTER VIII INSTRUMENTATION FOR TIME AVERAGING and TEMPERATURE
STUDIES
8.1.1. Correlation 148
8.1.2. D.C. Amplification 149
8.1.3. Integration 149
8.1.4. Signal-to-Noise Enhancement 150
8.2.1. Photon Counting 151
802.2. Experimental 151
8.3. Lock in Amplifiers 154
8.4.1. Signal Averaging 154
8.4.2. Method 156
8.4.3. Gate and Sweep 156
Chapter VIII
8.4.4. An Evaluation of Detection Systems
8.5. Alternative Refractor Plate Systems
8.6. Conclusions
8.7. Variable Temperature Cell
8.8. Temperature Studies
CHAPTER IX NITROGEN HETEROCYCLICS
9.1. Spectroscopy Effects Associated with Nitrogen Hetero 181 -cyclics
9.2. Experimental 182
9.3. Instrumentation 184
9.4. Summary of Spectral Shift Effects 185
9.5. Carbazole Emission Characteristics 185 9.6.1. Energy-Transfer Effects for Benzo(f)quinoline 191
9.6.2. Photochanges for Benzo(f)quinoline 194
CHAPTER X OBSERVATIONS USING OTHER MATRICES
10.1.1. Specifically-adsorbing Silica Gels 199 10.1.2. Preparation of the Gels 200
10.2. Room Temperature Phosphorescence 205
10.3. Proposed Mechanism for Benzo(f)quinoline Phosphorescence 208
at Room Temperature
10.4. Polyacrylamide Gels 209 10.5. Experimental 210
10.6. Summary 214
6
160
171
172
173
175
REFERENCES 215
7
ACKNOWLEDGEMENTS
My sincere thanks must go to my supervisor, Dr Gordon F. Kirkbright
and to Dr Clausius de Lima for the introduction to this area of research.
Much appreciated assistance in other specific areas has been given by
Dr Jones, Reader in Structural Chemistry, Bradford University along with
Dr Drake with the coal work; Dr Silvano Monarca from Perugia University
in Italy (on a World Health Organisation Fellowship) and Dr Rob Young
of the Public Health Laboratories, Imperial College, with the water work;
to Adrian Shaw for chromatographic separations on oil samples and to all
the staff in the Chemistry Stores plus Workshops for their invaluable
aid.
Special thanks are due to all members of the Analytical group both
past and present along with my other College friends. I acknowledge
the considerable effort of Mrs U.O. Fowler for typing, correcting and
organising the thesis manuscript.
Finally my deepest gratitude goes to my fiancee, Jenny, and her
multitude of friends.
The Science Research Council has financed the whole of this work
which was carried out from September 1975 to July 1978 and is entirely
original except where due reference is made.
DEDICATION
This thesis is dedicated to
my parents,
William Leslie Causey and
Mary Ann Innes Sutherland
8
'The grand aim of all science is to
cover the greatest number of empirical
facts by logical deduction from the
smallest number of hypotheses or
axioms.'
Einstein
9
1.2. Historical Survey
Knowledge of the luminescent properties particularly of proteins
predates modern science but the earliest report was of an extract of
the wood'lignum nephriticum' in 1565 by Monardes(1). In 1746 Berconi2)
observed phosphorescence of his hands after vigorous washing and exposure
to strong sunlight. Indeed this term for long lasting luminescence
was coined in the early 1500's after the Greek word meaning 'light
bearing'; the same root being later used in 1611 for the element
phosphorous. It was after Brewster in 1833 had detected red emission
from chlorophyll,however,that . Stokes(3) used the term fluorescence
to describe the blue white light from 'fluorspar' in 1852 and went on to
show that proteinaceous materials were both fluorescent and phosphorescent.(4)
These early works before 1900 have been well documented by Harvey(5).
10
CHAPTER I
MOLECULAR LUMINESCENCE SPECTROSCOPY
1.1. INTRODUCTION
The term 'luminescence' encompasses an entire range of emission
phenomena where the frequency of the emitted radiation is independent
of the exciting energy. This energy can take many forms including electro-
chemi-, thermo-,tribo-,sono-, bio- and Xray initiated luminescence.
• luminescence is produced in many different ways, fluorescence
and phosphorescence have become particularly important as physical,
organic and analytical tools, especially in the ultra violet and visible
spectral regions. Not only do they serve to identify a specific
substance in a complex chemical mixture but they can also be used in
conformational and configurational analysis. In problems of dimerisa-
tion, adsorption, in many photoprocesses such as isomerisation and
phototautomerism and kinetically by virtue of their known lifetimes
they help in the mechanistic study of complex reactions and enzyme
metabolic processes. Indeed, with flow cytometry fluorescence has
helped delve more deeply into the processes of photobiology and cell
function itself.
'Modern' scientific studies of low temperature luminescence
started with the monitoring of the phosphorescence of dyes and alkaloids o (6)
in gelatin matrices at -80 C .
11
Early studies, especially by DewarC7) are well reviewed by
Nicholls(8) in 1911 and other literature is adequately referenced
in a thesis by _ . De Lima(9), London University. Also reviewed
therein is the application of the Shpol'skii effect in low temperature
luminescence spectroscopy of polycyclic aromatics.(10,11)
1.3. Quasi-linear Spectroscopy - The Shpol'skii Effect
A molecule is a quantum system with a discrete set of characteristic
energy states, but the electronic spectra of molecules usually have the
form of broad bands. In practice one studies the spectra of a collection
of a large number of molecules which are undergoing various perturbations.
Thus for structural spectroscopic studies of aromatic polyatomic molecules
one uses the condensed phase in the form of liquid and solid solutions
of pure and mixed crystals. The individual molecules in crystals are
held together by van der Waals forces which only slightly affect the
free molecule electronic spectra. In the limit of vanishing inter-
molecular forces, obviously, the molecular crystal will become an oriented
gas. This was the situation in a solid solution of coronene at low
concentration in n-hexane, which allowed Shpol'skii and co-workers(12)
in 1952 to observe sharp quasi-linear spectra.
These lines have been shown to correspond to purely electronic
transitions, i.e. to transitions in which the vibrational state of the
lattice does not change. In cases of -weak coupling of the electronic
and lattice vibrational transitions, these quasi-lines are accompanied par-
ticularly on the long wavelength side of the fluorescence lines and on the
short wavelength side of the absorption lines by a diffuse band the
'phonon-wing' (see Chapter VII).
The phonon wing arises from electronic transitions in the P.A.H.
accompanied by concomitant changes in the vibrational state of the
lattice. In the case of strong coupling between the electronic
transitions of the P.A.H. and the vibrational transitions of the lattice,
only the phonon wing is observed. The existence of several slightly
different, but well defined types of guest (analyte)-host in the alkane
matrix corresponding to different - al isomers of the alkane
molecule is thought to be responsible for the 'multiplet' structures
of the quasi-lines in many Shpol'skii spectra.
12
From an analytical aspect molecular rotation is restricted at
low temperature and thus the P.A.H.'s which have a rigid planar
framework will give rise to exceptionally sharp lined spectra.
Moreover, non-radiative processes are suppressed leading to an
intensification of fluorescence and the appearance of the intercom-
binationally 'forbidden' T -+ So emission.
1.4. Molecular Transitions
The spectra of polyatomic molecules are very much more complex
than those of atoms and diatomic species. There are 3n-6 vibrational
modes, where n is the number of atoms in the molecule, moments of inertia
become large and rotational fine structure is usually no longer resolvable
in the gas phase or extremely complex. As can be seen simply from a
Jablonski term diagram the proliferation and extensive overlapping of
states in polyatomic molecules profoundly influences their photochemical
behaviour. Radiationless transitions are often very rapid and the
identification of excited states and the assignment of transitions is
difficult. Recently deeper inroads into molecular orbital and group
theory and development of experimental techniques such as laser photolysis
are beginning at last to bring some understanding to the spectroscopic
theory of complex molecules.
Electronic states of polyatomics are partially classified by their
multiplicities and if linear these can be further characterised by the
quantized component of the orbital angular momentum. It is most
convenient, although less precise than group theory symmetry methods,
to discuss electronic transitions in terms of the initial and final
orbitals of the electron involved in a transition. The probability of
an optical transition between 2 states is given by the transition moment
integral m = j yliµ dT. If the value of this integral is not zero the
transition is allowed. The selection rules, which state the conditions
when this probability is reduced, are only approximate because of the
difficulties in evaluation of this integral. There are two factors
which effect this probability, namely (1) spin, and (2) symmetry.
1.41. Spin The usual perturbing factor is the spin orbit coupling
between the electron spin and orbital angular momentum. This phenomenon
leads to the possibility of observation of singlet-triplet transitions
because
13
0 0 *T =
a.T + Xs CX is a mixing coefficient)
H '~ X _ s so T dT ~. ET - Es J
where ET and Es are the triplet and singlet state energies. Hso is the
Hamiltonian operator for the spin orbit perturbation.
Hso = kX(LS) where L and S are the orbital and spin angular momentum
operators respectively while X represents the spin orbit coupling factor
which depends on the field developed by the nucleus.
If the potential energy of the system is time independent the sum of
the potential V and kinetic energy T is identical with the Hamiltonian
in classical mechanics. Thus H = E the conservation of energy where
H _ 2m (pa+ pb+ pc) +V(abc) and p is the momentum. The quantum mechanical
expression from substitution of 2,i./ N wherever pN appears which results is
thus 2 2 2 2
H=- h r a 2 +-2 +d2 +V(abc) 87m da db dc
2 However d2 etc are just operators with instructions to act on the wave
da t
function o4 the molecule concerned. The basic expression HV = Et
of quantum mechanics results, 2
where H = PE Hi+ E r and is the Hamiltonian operator. i mn mN
With rigorous mathematics(13) one can then extend this to give a linear
combination of atomic orbitals or the free electron molecular orbital
theory(14) of molecular transition states.
1.42 Symmetry Forbidden The transition moment integral depends also
on the symmetry of the orbitals t
and 'b as well as the spin factor, µ
and, unlike spin forbidden transitions in molecules containing light
atoms, symmetry forbidden transitions are usually observable but are
of low intensity and the symmetry changes are affected by vibrational
motions.
A change may be momentum forbidden if there is a large change in
the linear or angular momentum. Also the transition moment is reduced
for g-g, u-u, parity rules which applies fairly strongly to aromatic
hydrocarbons in particular.
14
Some of the more frequently encountered ways of designation'
photochemical transitions are shown in Table 1
Absorbance Max. 165nm 256nm 304 nm
Extinction Coef. L :15000 160 18 Ethylene Benzene Formaldehyde
Group Theory1Blū1A,9 1g2u 15 1
Enumerative o-PS1 Sō51 ō S1
Mullikon V(---N V4--N (1t-N
Kasha 11-1T 1T-~T1 n->TT+
Platt 1 B-1A 1Lb 1A 1u -1A
M.O. Theory c l o Ī ITt compIGA oT pi aT Ī I PTT'"
TABLE I PHOTOC HEMICAL TRANSITIONS
The perimeter free electron orbital model introduced by Platt(16)
is useful for the classification of the it electronic states of the
cata condensed hydrocarbons, (C4N+2H2N+4
where N is the number of
benzenoid rings) in which no C atom belongs to more than two rings
and also the peri-condensed hydrocarbons in which certain carbon atoms
are at the junction of three rings. For this model if a perfectly
allowed i7-'7* transition has an oscillator strength FA then any other will have its oscillator strength given by a series of probability
factors F = fsfofmfpFA fs for a spin forbidden transition —10-5
seconds (s) for 2nd row elements. The momentum forbidden factor is
10-1 -' 10-3 s for condensed ring systems. Parity forbiddeness fp
is about 10-1 s for most relevant transitions.
Different types of transition and — molar extinctions, f numbers
and intrinsic lifetimes are given (Figs. 1.1, 1.2 and 1.3).
Tr• IB 'L̀
1 } allowed (' IV)
.lj 'L► } forbidden ('U)
Singlet-triple-
15
Fig. 1.1 Orbital functions derived from tho six 2p, orbitals of six carbon atoms • in the benzene molecule. Tho number of nodal planes through the z axis increase
with energy. Keo corresponds with the 'A15, ip, with the 1132., ' pa with ' Bl „ and tpa with 1E,„. Tho shaded areas represent negative parts of the ;gave function.
. Singlet-aingtel --.-
1
log (max
5.
logf • log T sr 0
4 1
—8
3 2 — 7
2 3 0
2 4 b
• 4 0 5
-1 G
3
— 2 —2 7 -1
0 I-3 —4
--8
9 1
Se -. 81
lB
'L►
allowed ('1V) S. » T, (ketones, nitroso)
}forbidden ('U) (pyrazino, phenazine; nitro)
Fig. 1.2 Different types of transition (Kasha and Platt classifications) and approximate molar extinctions, f numbers and intrinsic emission lifetimes.
'EI.,, 'B, Sa
181., IL., SI
'B1 , 'L„ Sa
180 nm 200 nm
2G0 nm
~410 24 ,, S► •
Fig. 1.3 Energy level diagram for benzene using group theory and Platt symbols.
16
It is not possible to generate the B1µ and B2µ forms of excited
state from the totally symmetric hexagonal ground state designated
as 1 A1g by the change in electric dipole moment vector which represents
the effect of electromagnetic radiation. The restriction of the
symmetry selection rule can be broken down by molecular vibrations
which change the hexagonal symmetry and obviously therefore effects
the resolution and diffuseness of the spectral bands.
Fig. (1a) shows the position of the nodal planes and the sign of
the wave function in different regions for the states Ba, Bb, La, Lb
in anthracene.
Each nodal plane cuts the molecule twice, so that for- the B states
(Q = 1) there are two cuts, and for the L states (Q = 2n+1) there are
2(2n+1) cuts which is equal to the number of C atoms and C-C bonds.
The suffixes a and b refer to the 2 alternative positions for the nodes
relative to the molecule, a where the nodal planes bisect the C-C bonds
and b where otherwise the nodal planes pass through the C atoms.
The Ba state corresponds to a strong dipole oscillation in the
molecular plane, polarized I to the long molecular axis while the Bb
state corresponds to a strong oscillation polarized parallel to this axis.
The La .and Lb states correspond to weaker dipole oscillations with
polarizations I and parallel to the long molecular axis respectively.
The lowest excited state in the polyacenes is the triplet state 3 La.
The lowest excited singlet state may be either 1Lb (benzene, naphthalene,
phenanthrene) or 1 L (anthracene, and higher linear polyacenes).
The Group theory necessary for matrix isolated molecules in static
crystal fields is solved for molecules of any symmetry isolated at a
site in a crystal field of any symmetry As the potential barrier
to rotation of the molecule relative to the host crystal is raised,
certain symmetry operations become decreasingly 'feasible' in the sense
of Longuet-Higgins. Using correlation methods the symmetry species
of the rotational states are determined from the free rotation limit
to any of librational limits. Examples of linear symmetric and spherical
rotors are given. Becker(i9) in fact uses such a standpoint to describe
molecular fluorescence theory in detail.
In Chapter Vila deeper consideration of solid state theory, excitons
and the homogeneous and heterogeneous broadening effects of Shpol'skii
non phonon lines will be considered.
9 WV\IN.AAP..1
Dissoc.orion 73 lsOmerzoton
DiSSOC.0110r1
Isom
D.ssoc ,aon
isc,merizo!on , s CrerriC‘J.
rCla5n
S
1,vvvvvvvv„
Dissoc■cmon
J 8 < 2
isorner.zohon =
7 1; - 11 " 13 Crelrn7:0Cani 1
O 1 11 :, 0
li
0 2 4 5 7
I I I
ro4:01, Ye 10 12 14 15
processes rcd,c1.ocoess)
Figure lb Jablonski diagram showing principal decay processes in polyatomic molecules (specific unimolecular rate, sec'
111 :0%wpm-in (161 '1 S . S,. (2) AI,,r la', S. (3, Vabra;:ana: r!,I.n .10 'IS,.
t4) F:aore,..rice 10' (('>5, •-• S. II Ilaia%ale..ut„r apaensliang so, Oa hair mail con% cr,,a n and ,bratcnit: relaxation 116") S S,. 17) In:ctn.:I comer:a:an and irational relaxation 1')S4 IN ITI>Cr> t,r, crosaaag S, T2, T,.
Pti 1ntar*aacm erosaang (2, S.--• T. 'probably too slaw to compete agaght O)). 110a ,6 'I So
COr.so-oor. and brational relaxation 110' 2) T„—• Tt . ((2) l'haaaphorasccni.c113'-10 - ') T, (I 31 Ab‘orption 1111”) 7, 1141 Išrirlolceu;,r qucncl ng14„; (SAT, S,.
5) Interspatem crossinl; and brational relaxation (I0'-10-') 71-0 S..
18
1.5. Radiative Emission
Although there are many intriguing photochemical and photophysical
happenings after a molecule is excited, five main processes which an
excited state may undergo are radiative transitions including lasing,
radiationless energy loss, electronic energy transfer, chemical reactions
and finally more subtle isomerisations and tautomerisms.
The photochemically interesting spectral region for us is between
2000 and 70001, 5 -, 1.43 pm-1, 5 x 10-4 - 1.43 x 104cm 1 corresponding to an energy range from
E2000- 143 kcal to E14000- 20 kcal
Solar ultraviolet below 270nm. however is absorbed by the ozone and air.
Because energy necessary for breaking chemical bonds between C and C or
C and 0 amounts to 80 kcal/mole and 88 kcal/mole respectively only
radiation with an energy higher than these values (X s -325nm) can disrupt
these bonds in a photolytic reaction. These important subtle photochemical
changes will be expanded in the more relevant chapter ( 1X Excitation
to a number of singlet levels according to the Einstein relationships
occurs thus Btu = Bud = c2Aue/8rr byd
.
If the refractive index of the surrounding medium is n for light of
frequency v then A = 8n h 3n3 /c2 But. Aut, being the probability of
emission from excited molecules during one second (s), so Auf= 1/Tut.
T is the mean radiative lifetime
Tut = 1/kF namely IF1 1/e of its value.
kf is 1st order rate constant for fluorescence.
This assumes that the probability of emission and return to the
lower state is independent of the presence of other excited molecules.
At any time t, the number (no.) of molecules emitting per second is
proportional to the no. of excited molecules, N, which are present
dN (3-7 = - kF N on integration N = No exp - kFt
or the rate of emission Q is given by
Q = - dt = QQ exp - kFt
19
Several formulae have been derived to relate the radiative lifetime
Tut
to To to the absorption which occurs over a band
gt is the degeneracy of the lower state
gu is the degeneracy of the upper state
ō is the maximum of the absorption band
and e is the molecular extinction coefficient
one due to Forster states
)3 7 = 2.88 (= 87 2303c) x 10-9n2 r (2v - v ) edv O V
Rough values of lifetime can be estimated from To z 10-4/ems (secs).
The observed lifetime of an excited state T is less than the
radiative lifetime To and is determined by all the c'..aactivation processes
T = 1/Ek1 wherē Etc. is the sum of all the rate constants for unimolecular
deactivation of the excited state.
The fluorescent quantum yield OF
is the
no. of quanta emitted, si ~ 'so -+ hv} kF, Hence T = T
o OF
Si + Q -' so +Q
0F kQ CQ 7 kQ CQ ] = 1 + = 1 +
16F Ek. E k.
00
f = 1 + Ksv [Q]
Pf Ksv is the important Stern-Volmer constant equivalent to kQT and some
comparative plots between photoacoustic radiationless energy and solution
luminescence emission have been made (Chapter X) for quenching.
J,.B. Birks in his three volumes of 'Organic Molecular Photophysics'
gives an absolutely rigorous approach to the above processes and selection
rules governing molecular transitions of P.A.H. together with delving deeply (20-22) into dimer, excimer and •exciton state theory.
-2 2 1 8rr 2303 c o
n ~. g
J E dv
TO N gu
no. of quanta absorbed in exciting so~ si k. 1
20
1.51. Optical Density
For low optical densities in the conventional right angle
illumination and viewing of the sample most frequently employed in
luminescence studies, the radiant power of luminescence can be
obtained from the expression P = light absorbed Ia x OF
P = Io fl - exp (-2.3 ect)10F = Io(1 - 10-gic')OF originally developed by Lothian.
A A _ Now PA =
0 A EAcA + e c 1 (Io It ) B B
When there are other absorbing components B, C, D etc in the solution
whether or not they phosphoresce, the exciting light reaching (A) the
analyte is less thus
2 f eAcA + eBcB + ... PA 0PAIo.(2.3 eAcAd - (2.3) eAcA 21
but in a very dilute sample solution the second and further terms will
become minimal.
In a front surface case this effect is also less but the non-linearity
of luminescence growth intensity at high concentration and re-absorption,
can also be called inner filter effects. Remedies are basically,
(i) high intensity selective excitation
(ii) dilution and careful sample preparation. However there
obviously is a signal sample decrease here:so more amplification
and consequently noise must be taken care of by more sophisticated
detection electronics like time averaging and photon counting,
Chapter VIII;
(iii) a standard additions analytical procedure to compromise for
matrix effects, (Chapter VI).
1.6. Matrix Isolation
As can be seen from the Jablonski diagram (p. 17) the rates of the
intermolecular processes which compete with fluorescence decay depend
upon the nature of the molecule, the first excited state and its
surroundings. The P.A.H. are a group with high fluorescence efficiencies
21
from a (Tr 71-*) excited singlet state. From an analytical viewpoint
the low temperature will enhance signals and thus sensitivity by
reducing diffusional quenching though in glasses at 77K this and
electron tunneling(~3) are still fairly prominent. Oxygen quenchin 24'~5)
will also be reduced and minimisation of line broadening(26) will aid
selectivity. These advantageous effects of low temperature luminescence
have led to the paralleled development of matrix isolation photo-
chemistry initially originated in the 1940's by Lewis•~P, extended
to rare gas matrices by Pimantel(28) and subsequently utilised by Jacox
and Milligan(29). Fairly sophisticated continuous flow liquid helium
cryostat vacuum assemblies are currently being used by two active
groups in this country. Turner in Newcastle is working in the infra
red region on inorganic species while Rest and Salisbury(30 X31āt
Southampton University are working on characteristic ultra violet
emission spectra of organics particularly styrenes and organometallics
plus the detailed effects of oxygen leading to new charge transfer
absorption bands for benzene.
Wehry, Mamatov et al.(31) in the United States have now taken the
whole field one step further by producing last year for the first time
Shpol'skii spectra for 1,2 benzanthracene, matrix isolated by vapour
deposition in n paraffins followed by an annealing process.
In our studies two main types of cell are used; namely a high
volume adaptation of a commercially available cold finger dewar cell
and a 'home built' copper conduction system both of which operate at
liquid nitrogen temperatures although later a unique variable temperature
cell was designed and constructed (Chapter VIII) which operates at any
temperature from + 160°C to - 196°C and therefore allows for annealing
of samples.
22
CHAPTER II
INSTRUMENTATION
2.1. CORRECTED SPECTRA
A true fluorescence spectrum is plotted as the relative quantum
intensity F(v) (in relative number of quanta per unit wavenumber
interval) against wavenumber v (in cm-1). Spectrometers are presently
being continually developed to record accurately and swiftly the true
corrected fluorescence spectrumC33'34)
A fully compensated spectrofluorimeter (Figures 2.1.1) U) requires corrections for temporal and spectral variations in output of the Hg
or xenon arc source, dispersion differences and light losses in the
monochromators and wavelength response of the photomultiplier; thus
necessitating the use of reference signals, 2 photomultipliers and a
rhodamine B quantum counter. Alternatively it entails constant
instrumental calibration curves obtained by
(i) measurements with a calibrated tungsten filament lamp for the
visible, with thermopiles and/or with
(ii) standard fluorescent solutions.
2.2. PHOSPHORESCENCE
The luminescence spectra of organic molecules at low temperature
are usually a superposition of various emissions. An appropriate
delay time following excitation shut off will thus allow the separation
of both fluorescence and phosphorescence. This may be done as in the
original phosphoroscope of Becquere135)using 2 circular discs, by the
use of two chopperV6)or one and an electronic gate as shown (Fig.2,21 537)
In this system the mechanical light chopper has a diameter of 25 cms
and rotates at 25 Hz. Its forty holes produce a series of square light
pulses of 500 p-sec. duration, which are used to excite the luminescent
sample. A trigger unit mounted on the chopper produces the reference
trigger pulses (Fig.2Z.D) which have a fixed relationship with respect
to the excitation pulses. The trigger pulses activate a linear gate
Brookdeal 415 and after a delay time T a gate signal of duration T.
200 µ-secs. is chosen so that the gate signal will occur during the time
when the excitation is off. The gate signal opens during its '0N' time
23
channel 2 of a dual scaler and all pulses solely due to delayed
emission or phosphorescence are recorded whereas total luminescence is
monitored by photon counting in channel 1.
2.3. COMMERCIAL INSTRUMENTS
Various commercial luminescence spectrometers are available,
notably from Baird Atomic, Shimadzue, Perkin Elmer including one new
microprocessor controlled instrument with a vidicon detector and the
American Instrument Company (S. Iver Springs, Maryland, U.S.A.). The
optical layout of our Aminco Bowman instrument from the above company
is shown (Fig. 2.3 ) This system was continually used especially
for initial screening and phosphorescence studies but did not have the
required resolution or sensitivity for most of the low temperature
Shpol'skii work recorded. The latter's most recent top model, the ani.a.o
S.P.F. 1,000 CS has in fact been shown to give a corrected emission
Shpol'skii spectrum of coronene inf-heptane at 77 K concentration
1 µg ml-1 in a one mm internal diameter cell and uses a 500 watt xenon
arc source backed by an ellipsoidal mirror plus an extra photomultiplier
for the correction channel«°) The lay-out of another particularly(FT1.4-2•i) impressive and very flexible unit from Glen Creston instruments is also
shown. In particular, it has a superb modular cell assembly system
that scads to great flexibility which is often lacking in commercially
orientated production models. Light from a xenon arc source usually
(500 or 1,000 watt) is dispersed by the excitation monochromator and
may be polarised before it reaches the beam splitter - a coarse (8 grooves/mm
reflection. grating with opposing facets ruled at equal angles to the
incident light. This and subsequent optics assure identical treatment
of the two light paths, a pre-requisite for accurate optical comparison.
In the simplest mode, a reference spectrum of the source is recorded.
The light path is represented in Figure2;% by the solid line and the
dashed line from the beam splitter to the reference detector. In the
transmission mode the intensity of light transmitted by the sample is
recorded, uncorrected for incident light. This is the path shown as
the solid line, the dashed line to the sample, and the dash-dot line to
the transmission. Unfortunately such sophisticated instrumentation was
not available in our laboratory and we found anyway that for our high
resolution purposes of monitoring quasi-lines a laboratory modular
Referenco signals 31--- - --- Electrical signals Main light path
Velodyno system I Excitation
monochromator M1
Xenon are
REF 2
Wave no. signal to recorder
Cam correction
Emission monochromator M2
Atten. 3
Y—• •
Velodyne system 2
\t REF 2 .,,,wAI,.M .A , rVN,.,,.«......(w.,,.W,.,M..I , •. 6,.4Vi.
Chopper
Atten. 1
REF
lotnr PM I
Phasing signals for ampiificrs
PM2 Rhodamine screen
D3
Mechanical linkage
Fig. a.l.t A fully compensated spectrot uoriin ter. Corrections of fluctuations and variation in apectralout.put of the xenon are, light losses in the monochromators and wavelength re;ponao of the photomultiplier, are made by use of reference signals, two photumultipliers, and a rhoQnminu quantum- -
24
L = Light source. M3, M3 = Kizer D247 quartz-prism
monoch rom :tors. Di, Dr = Chopper-discs driven by syn-
chronous motors. B — Silica-plate beam splittcr.
F = Silica cell (0.5 mm) containing fluorescent-screen solution.
P, = Monitoring photomultiplier. P3 = Fluorescence - phosphoresc-
ence photomultiplier. • Q = Fused-quartz Dewar flask
containing sample cell.
FIG,2,1,2 Sp,ectrophosphorimetcr
PHOTO -MULTIPLIER
i I—
XENON LAMP 1.
EMISSION 3 MONOCHROMATDn WATER FILTER
DRIVE 4 DRIVE
RECORDING SYSTEM
`3
5 EXCITATION
rAONOCHROM.
14 1 LIGHT CHOPPER
15
i
REFERENCE TRIGGER
--J L. —
DETECTION SYSTEM
12
11
I I I I I I
I I , 'delay t►me I
1 ,I •1 I 1 I I ,
I , 1--I I I •gate I
•time I
i
!r fi •
r--i 4C I I
t f~
4D
'GATE SIGNAL
TOTAL LUMINESCENCE IN CHANNEL 1
EXCITATION
REFERENCE TRIGGER PULSE
44
- 4B
PHOSPHORESCENC 11 IN CHANNEL 2 4E
Figure 1.1J Schematic diagram of the luminescence spectrometer
25
Figure Separation of total luminescence and phosphore_ceact•.
SLIT I
XENON ARC LAMP ORATING G-I
PHOTOHULTIPLIER TURRET (SLIT 7)
PHOTOMULTIPLIER SHUTTER AND FILTER HOUSING
GRATING G-2 EMISSION MONOCHROMATOR EXCITATION
MONOCHROMATOR
MR-I MR-4
MIRRORS MIRRORS MR-2 MR-3
Gi
SLIT 4 SLIT 5
CELL SLIT G
SLIT 3 LI, HT TRAP Et DESICCANT CHAMBER SLIT 2 FHOTOMULTIPLIER TUBE
Figure 23 Optical Unit Showing Position of Slits (Am.++c f3.u, ^ms's
SIZE, ARRANGEMENT AND PLACEMENT OF SLITS
(Width of Slits in Millimeters)
See Figure 10
Mono- chromator
Slit 1 Cell Slit 2 Cell Slit 3'X Cell Slit 4* Cell Slit 5
Mono- chromator Cell Slit 6
Photo- multiplier Shutter Slit 7
Arrangement No. 1 + • 1 0.5. a 3 - 3 0. 5 1 0.5
Arrangement No. 2 2 1 3 3 1 2 1
Arrangement No. 3 3' 2' 3- 3' 2 3` 2
Arrangement No. 4 4 3 4 4 . 3 4 3
Arrangement No. 5"* 5 4 5 • 5 4 5 5
*These slits serve as baffles to reduce instrumental scatter. +Highest resolution, recommended for identification.
**Highest sensitivity, recommended for trace analysis.
Ts.cerwrioN MONOC.0,0,AA1001
IMAM
REFERENCEE ocrecten %.„:,• I, • 4.
•
• 014 :'-----"--"-14.uTTEI •Ovra31 ..... %...z
c.......... 's - .. imual .4. -.
SITT - ........b, LC/ gamot-/t
r I. ,/,' ..,=..t......__...4-1-,9-.",...., 1 . .., 1 cif.i...i..1.;,-;,' t, - i ' \ ! . . i . : ......0, . I A i i \ I
Crhq..00,11,E a TRANrowSS,ON DETECTOR
• . • :
CMJSSOO MONOC..101.”..1.0.1
2.MLPANA.ToN
1.0,71
• VOt DETECT:Xi
no En. era lurid
EPEE AMP
LEI, Ili TO
01.1,.CE
SMOOT ?ilk; ALOOF
I EMSI
ISON 1 i,Ar:11IS PM cure I LIM 101.0
i ' I
relOTOU tur,,ttil
-COWIT I IC)
PlIF AMP FOLIAGE
y.F
COUNTER CCL.ITER 4/,Ne CLCCK
[Oct
( cr., ..11,
SCALING AtiO
LATCHES
1 WiSSION I I TRANSMIS
PM ILT.1 1 PM UOE
- - ' 'PHOTON' r,:...,1 • • •co,.„,T
- PREAMP iv 'LACE
T■2■rsrnill.nee Ab-sor3r.ce l'-:des. the dale lines represent tte eztra to record Abso•baote; lines denote logic Dow coo-.. noon to both er odes.
s..TY3T riNs7.1
PHOTON -COUNT PRE AMP
Ft Of ER. CII TvcF.
IN uPPENIt
TO
DISPLAY
-A .-1sLIODTHar.,i
Ezrorlo
OVER PLACE
SCALtHC AND
LATCHES
27
fiat FLUDROLOG Light paths, tight traverses all paths shown whene.er the source is lit and shutters open, but choice of mode determines operational detectors. Fig an 25 Reference d Trans- . - - •• mission Modes. Dashed line
represents logic for Reference mode, dotted Use that of Transmission rnsde, and send line that common to both.
Fig1.7 Emission and Ernittance Modes. The solid lines denote the se-quences in both modes. The dashed lines repre-sent the reference input for Emittance, and the dotted line the conflp• ration for uncorrected Emission.
FiglS Pelative Eluor,s-cence Efficiency Mode: This diagrams the elec-tronics which assemble data according to the formula EAft-1).
28
assembled spectrofluorimeter was indeed a more flexible system
particularly for the continual optimisation and cell changes required
while researching into the Shpol'skii effect.
2.4. SHPOL'SKII SPECTROFLUORIMETERS
Most Russian workers(41,42) use a spectrograph I.S.P.-51 which has
three glass prisms and produces a dispersion of 0.65 nm mm-1 at 400 nm
although a high transmission type with reciprocal linear dispersion
of 2.0 nm/mm at 410 nm and a relative aperture of 1:4 has often been
used.(43'44) It is also apparent that most of their systems utilise
mercury vapour discharge lamps for excitation.(45) The PRK 2-4 types
are at high pressure (1 atmosphere) contained in a quartz surround
envelope and rated at 200 or 275 watts respectively.
Personov's initial 1,000 watt DRSh Hg lamps and xenon arcs via
an excitation monochromator have been superseded in his most recent
studies by laser excitation.(46) Ting and Kung(47) also in a laser
study have used a Spectra-Physics 125A lie-Ne model whereas Wild et al.
utilised an argon ion laser (coherent radiation type 52) having u.v.
mirrors for coronene. The 351.1 line was separated from the 3638
line by a small quartz prism and had an intensity of about 10 mW.
Some initial pilot studies in our laboratory with a 3 mW helium-
cadmium laser (Electro-Photonics/Liconox, Model 401/301) in which its
principal emission can be selected at 325 nm or 441 nm by changing the
appropriate mirrors, have been conducted. Using this laser in the
former 325 nm mode with power output of 3 mW some gain in detection
limit for coronene, benz(a)anthracene and dibena,h)anthracene was
obtained but not until using the 7 mW output at the 441.6 nm laser line
with perylene was a significant enhancement in both signal intensit;,~~a1L1
signal to noise and signal to background observable. The latter point
is particularly important regarding the high scattering background usually
produced when using Hg or xenon excitation for front surface spectroscopy.
It is also relevant that the latter laser line does in fact exactly
overlap the longest wavelength (0,0) band absorption of perylene thus
confirming the necessity really for a tunable dye laser. Farooq's(48)
studies have recently confirmed that the P.A.H. have very narrow (Fig.2.9)
quasi-linear absorption throughout the u.v. and blue spectral regions
Compound
Coronene *
3,4-Benzopyrene $
1,2-Benzanthracene *
1,2,5,6-Dibenzanthracene *
Perylene *
3,4,8,9-Dibenzopyrene
IUPAC
(a)
(a)
(ai)
(ah)
Luminescence emission wavelength/nm
445.o5 403.00
383.75 394.25 443.95
449.15
150-W Mercury- xenon arc vapour
discharge . lam •
10-3 10-4t
lo-3t
10-3t
l0-3§
10-4H
Excitation source
He - Cd laser
325 rim
5x10-
1 x 10-4 5 x 10-4
3 x 10-3
441 nm
2 x 10-3 7 x 10-4
2 x 10-3 2 x 10-4t
Table III (RQC 1.3 )
Detection limits, p.p.m., obtained at 77 K with different excitation sources for P.A.H. compounds
* In hexane
t 300 nm interference filter
$ In octane
§ 250 nm interference filter
II 300 or 325 nm interference filter
d
Wavelength /11m
Fig. 2.9 Luminc::ccncc Excitation Spectra at 77Z &c-c.lLf-
A, cr roncne (1r r1 in Hcpt?ne)
B, -ben opyrene (10- ~M in octane)
C, pyren(.! (10-511 in octane)
30
31
and thus shown why we had only limited success utilising aluminium
electrodeless discharge lamps due to poor overlap of the fine atomic
lines with the necessary absorption molecular quasi-lines and lack of
general power output. Although limited success in exciting a 10-7 molar
benzo(a)pyrene solid solution was achieved by using molecular CN emission.
Unfortunately ultra violet lasers are very low on power output at
the present state in their technology apart from various fixed wavelength
long life Liconix He-Cd Blue Lasers which can give 15 mw, 4 nW or
2.5 mW in the a.v. at 325 nm with lifetimes greater than 6,000 hours
now typical. Presently the only feasible analytical continuous wave -
dye laser system for our analytical studies would be the Molectron
Spectroscan 10 with varying power output between 360 nm 740 nm and
the company offers a frequency doubling accessory to allow operation
360 nm -y down to 258nm. However this leads to huge decreases in intensity.
The shortest wavelength dye system recorded to date is when using
p-terphenyl in cyclohexane or ethanol pumped by a nitrogen laser which
thus allows the dye system to give reasonable power output from 360 nm
down to 336 nm. For spectral widths of a few angstrom a prism may be
inserted into the laser cavity whereas a grating or Fabry Perot etalon
will give exceptionally fine line widths. Ferguson and Maue(49) have
useu a tunable dye laser system, dipheny2oxazole in toluene, tunable
between 355 nm 385 rim with a line width of 0.2 nm, for site dependent
Shpol'skii studies on anthracene in an n-heptane matrix. Line narrowing
with stimulated emission of perylene in n-octane at liquid helium
temperatures using a N2 pumped dye laser(50) similar to the one described
by HUnach(51) has peen reported. Moreover, the general utility of
lasers in the analytical molecular spectroscopy of P.A.H. has been
assessed recently by Winefordner et al.(52) and Richardson et al.,
the latter quoting picomolar detection limits.C53)
2.5. HIGII RESOLUTION SPECTROFLUORIMETER
The use of different types of monochromators with various optical
assemblies, apertures and resolution lead us to the conclusion that for
vibrational analysis or for fundamental studies, it is necessary to
have high resolution s 1.0 nm/mm and this will usually have to be a •
laboratory made modular assembly for the Shpol'skii effect.
.32
However, considering the limited applicability of the Shpol'skii
effect a compromise may often have to be established between the
purchase of such a high resolution instrument and its utility.
A reciprocal dispersion of 1.0 - 2.0 nm/mm and a low f number,
say f6, will be satisfactory for routine purchase. Our modular system
(Fig.2.10 illustrated diagrammatically) however utilised a Rank-Hilger
Monospek 1000, with an aperture of f8. This is a plane grating
symmetrical Czerny-Turner assembly in which the radiation transmitted
through the entrance slit is collimated and directed towards the
diffraction grating by a mirror. A Jobin-Yvon grating 1200 lines mm-1
blazed at 300 nm disperses the incident radiation which is then focused
by another mirror (12" x 6") on to the exit slit. The diffraction
grating motor drive velocity could be selected at any of 10 velocities
from 0.5 nm min 1 to 50 nm min 1 and the reciprocal linear dispersion
with this grating was 0.82 nm min -1 at the exit slit. Both slit widths
were adjustable by a micrometer vernier wheel calibrated in 0.005 nm/
division, slit heights being adjusted via diaphragms whose Hartmann
number could be recorded. The low dark current photomultiplier, 50 nm
EMI 6256S had 10 mm of effective cathode area an S type response, a
spectro•sil window thus extending the useful range down to about 165 nm,
and a very high gain, ca. 108. (See Plate I, p.34)
2.6. DETECTION
(i) Analog
The signal was primarily amplified using a microammeter RCA,
Model WV-84c) before passing through a back off unit constructed and
incorporated because of the all too familiar presence of a large back-
ground. A Servoscribe model RE 511.20 recorder was used for spectral
read out.
Most Shpol'skii spectra were monitored with a medium pressure Hg
vapour discharge lamp of 125 watt output; initially a Phillips MBW/u
from which the Woods glass envelope was always removed, but later, due
to discontinuation of this line, a similar GEC model was used for the
water samples (Chapter VI). In this work an ellipsoidal mirror was
also used to focus as much light as possible on to the preliminary
r—j EHT
P
ci PC •
F TL
S
UD SA j
1 I
0
r
T C R^
PS Power supply
EEIT . Photomultiplier power supply
P Photomultiplier
M Mirrors
G Grating
C Cell
L - Lenses
F Filter
CR Recorder for thermocouple
CRZ Spectrum Recorder
SA Signal Averager
O Oscilloscope
PA Pre-Amplifier
PC Phosphoroscope Cam
B Refractor Plate
T Trigger
r1+.r2 Rheostates for cell and window heaters.
Fig. 2.10
35
interference filter system whereby various areas of spectral radiation
could be chosen for excitation. (Chapter V)
Some comparative work with a 150 W xenon arc lamp (Osram XBO W/1)
powered by a Perkin-Elmer model 150 unit operating at 20 V d.c. was
carried out, this source being particularly useful when various Hg lines
overlaid or interfered with compound luminescent lines.
(ii) Digital
Photon counting for measuring luminescence intensities use electronic
detection in which high speed circuitry counts individual photon pulses.(54,55)
It has proved to have several advantages over conventional analog detection
being especially suitable for weak signals though its full potential can
only be utilised in a background compensated situation, especially for
front surface scattering matrices. The method has an excellent long
time stability and effectively eliminates drifts usually encountered in
analog systems. We utilised a photon counter Model 300, EDT Research,
London, with a PMT cooling chamber to test the elimination of dark current
and the efficiency of photon counting on discriminating various noise
types.
Finally,.a time averaging facility Unimax 4000, Data Laboratories
Limited, Mitcham, Surrey, utilised with various rapid scan devices,
modulation trigger systems and in conjunction with a phosphorescence
arrangement (Chapter VIII) was investigated.
Sample-handling Systems
(i) Cold finger cell
The commercially available Dewar flask sampling system (American
Instrument Co. Maryland) used for low temperature studies on our Aminco
Bowman Spectrophotometer was incorporated into our high resolution system
together with a high volume adaptation of this (Quartz Fused Products Ltd,
Weybridge, Surrey) like that used in excitation studies (Fig. 2.13).
Sample volumes'of 0.3 mis — 0.5 mls were then pipetted into silica
cells of length 200 mm, internal diameter (i.d.) 3 mm and wall thickness from 0.6 1 mm, depending on the freezing rate required (Table II ii)
_3b
—v
9
2,12 Schctr.atic diagram of instrument assembly employed: 1. lamp power supply; .i. xenon arc latnn; :t, rotating sector; 4, (;rating monochromator: 5, samle cell and housing; li, photo-multinlier detector; 7, photor,:itipler po..er supply; S, a.c. amplifier; 9, porcntio:netric chart recorder, 13.13 and 13, biconvex lenses.
2,13. 1.w..--tcruperatute cell as,^inbl;: (see text for hey to co nvoncnts). •
37
(ii) Copper conduction cell FIG.2.14
The construction of this new sample cell was based on systems proposed
by ParkerC331 and Svishchy ev,(56) without the inconvenience of the latter
with which it is necessary to warm the whole system in order to change
the sample. Also for biologically active samples as often used in our
environmental studies of P.A.H., aflatoxins, pesticides etc this system
must be especially useful practically with less possibility of contamination.
The copper reservoir A, surrounded with polystyrene H as insulator acts
as the coolant sink which can be utilised in conjunction with a liquid
nitrogen siphoning system to give continuous flow operation with no
scan interruptions. A vacuum space B, at the head of the reservoir
decreases heat transmission while the sample cell (D,E,F and G when
assembled) is attached by means of four copper or brass screws, g. The
sample compartment capacity of 0.5 mis was later reduced in the variable
temperature adaptation (Chapter VIII). Two soldered syringe tips (i.d.
2 mm) provided the ideal connection for filling and flushing the cell
and allowed for contraction of the sample volume to prevent bubble
formation on the illuminated surface. This sample compartment was
isolated from the ambient temperature by a narrow quartz microcell E,
which had been flushed and filled with dry argon at low pressure
(6 ~ 10 torr) to prevent fogging. This chamber was used as a window
and a thin cork or car gasket was found preferable to the rubber initially
used, to seal it against the copper jacket.
Positioning of the chamber using an aluminium front plate F,
painted matt black to minimise troublesome surface scatter effects, by
nylon screws, gave good alignment. These screws are very important
because, being slightly flexible yet poorly conducting, they allow freezing
of gels or snow matrices without cracking the vacuum chamber. Finally,
incorporation of a sample defroster consisting of a single turn of nichrome
wire (2.18 n m-1), G, heated at approximately 4 volts a.c. via a rheostat,
completed the cell arrangement.
All room temperature work apart from later comparative spectra on
gels and silicates was conducted on the Aminco Bowman double monochromator
instrument some results of which were correlated with opto acoustic
spectroscopy run on a single channel instrument constructed by Dr Adams'
group in this department.(57)
Table Ilii
Comparison of mean cooling rates for the two sample
cells employed
Sample cell Mean cooling Ratio of intensities of rate/K min 1 components of coronene
doublet
(I445.15nm(I443.44nm)
Silica tube :
0.6 mm. wall thickness 720 3.3 `!' 0-3
1.0 mm wall thickness 540 3.7 ± 0.3
Copper cryostat cell 75 4.05 ± 0.1
r 214 Cp; lier crvo't::t samp::: c'1l assembly Ver key. sec tcx j.
38
Table II. iii
Reproducibility of results obtained with silica-tube and copper cryostat cells
Compound IConcentration/M ,Solvent Luminescence emission wavelength,/nm
i Relative standard deviation,
Silica-tube cell
Copper cryostat p_ v cell
Coronene 2.5 x 10-5 Hexane 445.05 9.7 1.5 Coronene 2.5 x 10-7 Hexane 445.05 12 2.5
3,4-Benzopyrene 1 x 10-7 Octane 403.00 4 1.3 3,4-Benzopyrene 1 x 10
-9 Octane 403.00 18 11
4o
High resolution characteristic quasi-line spectra (Fig.2.15 ) and
detection limits (Table .iv) for twenty compounds which are of
particular interest in our later environmental samples were primarily
determined. Also illustrated is the first recorded quasi-line spectra
of indene in n-octane which is favourably compared with indene,
matrix isolated in argon at liquid helium temperaltures (Fig.216)
The detection limits quoted are impressive and an improvement on
traditional chromatographic methods. The absolutely characteristic
fingerprints of isomers is also important considering the different
mutagenic properties for different geometric arrangements of the
aromatic rings. The completely different spectra for benzo(a)pyrene
and benzo(e)pyrene illustrated is an apt example.(Figs.2.17and2i )
Hg 437.75
449.75 453.0
454.5 455.25
1149 367.0
369.75 462.25) -371
471.5
477.75- 492.5 495.0
486-0
439.0
444.25
454.75 458.25
462.5
468.0 ~.471 25 --472.25
477.5 478-75
480.5
485.5
v m
352.25
Luminēscence tnten5tty
42
Table II (iv)
Compound
I'u P AC
Hexane Octane Ng ml 1
Det. limit
., braalcefs
Pyrene 371.75 5 x 10-3 Coronene 445 1 x 10-3 Perylene 451 1x10-2 _ 4x10-8M 1,12-Benz(ghi)perylene 419
3,4-Benzo(k)fluoranthene 403.25 1.5 x 10-3 1,2-Benzo(e)pyrene 388.25 1 x 10-3
3,4-Benzo(a)pyrene 402.4 403 1.25 x 10-4
1,2-Benzanthracene 383.75 2 x 10-3
3,4,9,10-Dibenz(ah)pyrene 431.5 3 x 10-4 Ovalene 480.6
Methylcholanthrene 392.55 5 x 10-3 Chrysene 365 Indenopyrene 465 3 x 10-2 1,2,5,6-Dibenzanthracenes 394.25 5 x 10-3 1,2,3,4-) ( 395.25 3 x l0
-4
1,2,4,5-)Dibenzopyrenes ( 395.5 1 x 1O_4 3,4,8,9-) ( 449.25 1 x 10
Nitrogen Heterocyclics Phos.
Quinolines 458.92 Molar -~ 10-7
Isoquinoline 487.7
5,6-Benzo(f)quinolines 456.56 10-6
(Carbazole 343 Fluor) 10-7
INDENE £CONC 10-4 M)
OCTANE (77°K P8LYCRYSTALLINE
SNOW
300
METHYLCYCLOHEXANE GLASS
43
MATRLX EFFECT ON THE RESOLUīION OF THE
ULTRA VIOLET LUMINESCENCE EMISSION OF INDENE
ARGON aE 10K
4 0 i A H.
Fig. 2.11 Benzo(a)pyrene. Flulrescence 10-5 M solution in n-octane.
T3 et.wz6 tie)
45
c.
46
CHAPTER III
CARCINOGENESIS AND THE AROMATIC HYDROCARBONS IN PERSPECTIVE
Section (a)
3.1. CANCER
It is not really surprising that the virologist sees cancer
as a response to a tumour virus, the developmental biologist points
to an abnormal cell differentiation, the geneticist discerns an
effect caused by a genetic mutation, the chemist talks of stereo-
chemistry, the physicist of bioelectronic energy transfer and the
mathematician of the quantum mechanics of life. They are all
relevant to a small degree but nobody can yet see the whole framework
because the cancer puzzle is a whole series of puzzles . interlaced.
Two characteristics o' cancer cells, lack of control over growth and
tendency to invade, imply a third, namely the ability of cancer cells
to pass the malignant properties on to their progen.:y, cell division
after cell division.
In some animals viruses can be a cancer causq as demonstrated for
chicken sarcomas by Peyton Rous of the Rockefeller Institūte in 1911.
The normal manner in which tumour viruses act is not totally clear
but may be much more insidious than simple 'contagion', possibly latent
in our cells there are genes for an 'RNA tumour virus'; this dormant
coding being activated by perturbing chemicals or radiation. Undoubtedly
cancer is caused by alterations in a cell's DNA, so that a key group
of genes no longer functions correctly. Our own immune self-defence
system however is not so effective as we age, or against these cancer
cells which can camouflage themselves by hiding their surface antigens.(58)
Weaver(59) states that a key piece in the cancer picture is the
cause for cell differentiation and hence has been researching into gene
'turn-on' by demonstrating that light triggers sporulation in moulds
by pigment activation.
An analogous factor in cancer causes is simply our bodies' inevitable
imperfection. Nature has endowed us with this degree of incompleteness
to allow for improvement of our genes and rapid evolution. However the
K )enipyrene
O©O
O •0ethyltholanthrene
Figure31. Carcinogenic polycyclic hydrocarbons.
CH3
1.2,3,4-Tetramethylphenanthrene . Dinethyianthracene
Deniphenanthrene /aI
'Denzanthracene
47 .
CARCINOGENS
.t-Dibendanthraccne
NH2
o-Toluidine 2,4-Diaeinotolucua
CH3
2.4,6-Trinethylaniline
112N CH —CH
o-Tolidine 4-Amirostllben 4,4•-Hcthyienedizniline
NH2 71H3
0211 I:H2
2-Haphthylaiino R-Diphcnylanine
1702
Scnzidine
2-Amlxnminrt 2-Aminofluorene 2-Phenanthrjlaalne
Figure,. Carcinogenic aromatic amines.
48
corollary to that must be a small but significant tendency for a
series of detrimental rather than constructive changes which could
and now all too commonly does, free the cell from its normal restraints
with dire results. What triggers a normal cell to lose control
(break all its normal bonds) and become malignant may be a somewhat
selective process but is almost certainly a continuously varying
function of our environment.
The WHO organisation estimated that 60 -, 90%(60) of all human
cancer is due to trace chemicals in air, water and food. Even skin
cancers, once again a pigment activation problem due to the sun's
radiation, is a continual reminder of the environment's potential
hostility. The latest Surgeon General of the United States data
unequivocally support the link between cigarette smoking and lung
cancer.(61) Cigarette tars(62,63) not only contain P.A.H.(64)
but also many less well characterised but potentially toxic nitrogen,
sulphur and oxygen heterocyclics as well as co-carcinogen (promoters)
of which very little is known. These chemical constituents are not
far removed from the very first chemical causations noted by Sir Percival
Potts 200 years ago of soot, leading to scrotal cancer of poorly washed
chimney sweeps. In 1918 confirmation was provided that repeated
application of coal tar(65) to the ears of rabbits produced skin cancer.
Then (66) characterised some specific carcinogens
namely benzo(a)pyrene and dibenz(a,h)anthracene. Unfortunately other
industries also subjected their workers to health hazards. Thus the
identification of carcinogenic P.A.H. in mineral oils was reported by
the Medical Research Council's Carcinogenic Action of Mineral Oils
Committee in 1968(67) but these developments did not prevent cancer
occurrence continuing to arise in workers using oils such as tool setters(68)
(69) (70) and jute workers. The coal gas industry has an even worse record.
Many of these causations are not solely P.A.H. but concomitant
enhancement referred to as 'co-carcinogenesis', a most striking example
being the thousandfold increase of benzo(a)pyrene activity due to the
simple aliphatic dodecane reported by Bingham and Falk.(71)
Our own studies on hydrocarbon solutions in tetrahydrofuran show
peroxide formation leading to enhanced fluorescence and therefore suggests
Aza-pyrene
;I-AzsfI rrand'cnn
Benz (a)acridine
$urz[c)ccridnre
Dibenz(a,j]acridine
'pitapzf,a,h]acridinc
1a6)lil1:'o1yryefle hylroca ben: (+++vinyl four t inr!s or Ipnre identifies+ in tars e•btainad II ton cioareues nodlorun.( in urban ntmospher as_
Cigarettes ('Yo tar) Urban ot 'osp!lergs o . (no t%') C ō n iū Ō . n o n b ri a
L~ o ō 0 y o '
o to W ū D'ū
0.96
0.05
0.52
0.47
6.0
0.03
13.2
4.1
(Leeds) 14 (Rome) 5.8
(Rome) 4.2
(Rome.) 10.2
(Leeds) 21 (Rome) 5.8
(London) 20-39 0.24 0.28 (Leeds) 47.
(Hambtug) 134
(London) 12-26 0.12 0.59 (Leeds) 26
(Rome) 2.3 (Hamburg) 115
0.0 0.04
0.02 0.09
0.05 0.03 (London)12-46 (Leeds) 40
0.03 0.09 (London) 2-6
(Leeds) 9
(London) 4-20 (Leeds) 9
0.03 0.07 (Rome) 9.4
0.08 0.11
0.07 0.13
017 0.40
ilczirzo(a)pyrene
e,tzo(e)pyrcnc
.b?nz[a.h]anthracene
0mz(ajnnthracene
Ciirysene
F)ueranthene
Cerytcne
iicnzo[3, h, i] peryl ene
/t,nilranthrene
CCcConena
Oento[%Jfluorar..thene
Oa.szo[k] fluorantheno
Qsnzo[b]!luoranthene
rndeno[1,2,3c.d]pyrene Imm•
E
rn▪ ▪ • •
•
(Rome)13
• (Rome) 3.0 1
(7-me
-
thyl de.rs4:lrIVC) 0.2
++ -I-
(7-methyl derivative) 0.6
0.27riq + per 100 0.04
cigarettes
0 01)•7 per 100 ....0.03 cigarettes
49
trace -35
4 0.4- 21.6
1.3-- 11.G
4 0.9- 15
0.2- 17 5.7
1-25 5.0
0.7 trace -5
6.0 2-35
trace O.2.n -3
2.0 2.3- i 2.2
0.8- 4.4
0.5- 20
2.3- 7.4
1.9- 8.2
+ + +
50
that many of the environment's photochemical P.A.H. products could be
of more danger than their precursors particularly remembering the now,
well documented peroxidation step in the metabolism of these compounds
(p. 53) .Fig. 3.6)
3.2. HYDROCARBON - DNA INTERACTION
Data generated on structure activity studies led to the Pullmans(72,73)
in France from molecular orbital calculations on thirty seven unsubstituted
P.A.H. concluding that for carcinogens the energy of activation at the
K regions must not exceed a certain value while that at the L regions
must exceed another specific value.
Arco and Argus(74) also showed that molecular
geometry was another of many important factors. In fact, polycyclic
carcinogens can initiate neoplastic changes without the Pullman
limitation. Bui-Hoi(75) already envisaged the importance of van der
Waals forces in key cellular interactions and it is obvious that covalent
bond formation, hydrogen and charge transfer bonding, dipole interactions,
resonance, dispersion and exchange resonance forces along with triplet
state interactions must all play a part. Ionisation of uanine due
to alkylation of the base possibly by amines may also lead to anomalous
pairing in DNA but intercalative binding of P.A.H. with DNA which has
been well described and documented (74'76) is of prime importance here.
Thus Boyland and Green(77) have shown on a molecular model of DNA that
planar P.A.H.'s such as benzo(a)pyrene and dibenzo(ah)anthracene can
be accommodated between the base pairs by slight untwisting of the sugar
phosphate backbone. This steric accommodation would then be stabilised
by polarisation bonding between the hydrocarbons and purines and by a
hydrophobic effect involving the entire double helix strand. Definitive
confirmation of Boyland's interpretation was made by elegant flow
dichroism experiments in Japan(78) illustrated overlea40 Moreover
the polarisation bonding between the alternately stacked hydrocarbon
molecules and bases promotes plane parallel molecular adlineation and,
hence, keto-enol type lactam -i lactim tautomerism in the latter.(79)
The complexing ability of P.A.H. and indeed N heterocyclics is dependent
on pH and is sensitive to the presence of inorganic ions and small polar
molecules(8o) which is consistent with the polyelectrolyte nature of DNA
Pholomulliplier E1
51
A. Hydrocorbon intercalated parallel to the bases. E, > E ii, therefore Ac is negative
cleoronce 05 mm.
flow line
flow line
polarizer,
1
plane of polarization
polarizer,
plane of polorizotion
oscillatory plane porollel to flow line
Monochromator
Monochromator
oscillations in plane ea*
perpendicular to flow line I non polorized ~C1Tj r,1 ( p7 \ light beam ~
___.. .____ - .__.. - l -.O •
planes of electric vectors cross section of
DNA double he/ix-
Pholomulliplier
B. Hydrocarbon bound externally and oriented perpendiculor to the bases. El < E it therefore Ac is positive
,..<1 i....„,•_,,,...„ !—!.- '
`
I 'mai iQl 1̀ Pholom.ritialier
!~I;l~ti!~ , I ll: _
~~~~ lLali~ll; i,i'
--- o/G",A &Evote
Menochramolcr
oscillations in plane perpen- d~culor to floo line _
._
cross section of hydrecCrAsn bound /tea tosur /ace clGNd flow line plane of
polar ization
Monochrerrator
PhatomuItipl:er
it
oscillatory plane porollel to flow line
plan cf polanzohon
Frc.3.3'rinriple of the d.:zertnination of differential dichroi=nr of polyey'clk hydrocarbons. complexed Avid' DNA. In (A) is shovrn why the nr EAT ivy vi n of _lc indieatr::, interenl-•rtion parallel to the bas v; in (B) the bai of a 1.05iti" ..Ne value, indicative of external coinploxing pacalk-i to the 1,-ngthu-ie axis of DNA, is depicted.
Cytosine Guanine
:._,c) I
:. 1 ro'• *e's ''rc;
C;
';;ICtit{~
52
A 3
htc3.4 SrZrntalir. rc Pwsmtotion aef po;,:E1e, d+ofia1 orient: Iions of bulyCuirhsr aromalie molecules in voWCblesiog uith I)\.1. In (.1) is sr(.t ;light uWtuithng of Iho !wheal faaticf)unr and intercalation of the aromatic compound into flat space arisen, parallel to the base-pairs; in (B) is shown external binding to I)\TA without. disturbance of the double helix, the planes of the aromatic molecules lying more or less parallel to the helix axis.
Cal (I Adenine Thyninc ZZ!
. '- J .Iryr:l~~t i
3,4,8,9-Dibenzopyrene Tricycloquinozoline
A g 1a1o3.5 Geometric similarity of polyrutcicar aromatic carcinogens with purine-
1)yriuddhne base-pairs in ])NA. (A) 3..1,S,9-Dibenzopyrene and adenine'-thyminr. . (B) Tricyclo,tuinazoline and
cytosine-guanints.
_.
1-lydroclIruon
Diol cnoxidcs'
Tctrahydrotctrol
Fi:;. 3. 6TJ,c mel al:()1i"m of Ll'Il7.r.J(a)pyrcnc by JnjCro50111~1 cllzyml';; to dihYllrotliols i\nd to d ivlr:I'llx i(les.
53
,_,_,J-Dibendanthracene
Ieiter - ittf
PJ~
H
I Microsomes + NADPH - 02
COCH3
„.COCH3
OH
t.- •Benzanthracene
Rat liver soluble fraction + PAPS + Mg?'
N..,,COCI13
0.S03 H
-Fio.3 ) The metabolic activation of _'-acctviaralnofluo eue in rat liver. PAPS== 3'-pho dsoadenosyl- 5•-phn:phoalpha tc.
54
plus the intercalative model for the complexing. A final noteworthy
observation is the potent quenching of the phosphorescence emission
of frozen aqueous solutions of DNA by traces of benzo(a)pyrene.C81)
The stereochemical and tautomeric effects aesignated above
will be further expanded in Chapter IX since analogous molecular properties
give rise to Shpolskii structural effects.
3-3. METABOLISM
Metalbolism as is shown for benzpyrene (Fig. ) via the
aihydrodiols anti diolepoxides is necessary fpr inducement of cancer.
This metabolic activation is usually due to hydroxylase enzyme
systems localised in the microsomal fractions of liver and other
tissues. (69'82) Although mutagenic and carcinogenic processes
obviously both originat in the cell as genetic coding systems other
properties of a specific 'at risk' organic chemical may overlap somewhat
and can be either or both.
CH3 2-Methyl... -bentphenanthrene 9,10-Dimethyl- , henAthrccene
a 3cnzt~yrene
Fig.3. LL) Struclures of some carcinogenic pelvcyclic aromatic itvc!ro':whons (earlier nomenclature) shoving Ii and L regions.
20-Methylcholanthrene
55
Fairly recently Ames(eX3~Z.84) has developed a sensitive system for
measuring the mutagenic activity of microsome-activated carcinogens
using specially developed strains of Salmonella typhinurium some of
which detect DNA base pair substitution and therefore react selectively
to alkylating agents while others detect frameshift mutations. Mutant
colonies are detected by their growth on a nutrient medium that lacks
histidine.
3.4. ENVIRONMENTAL POLLUTION
Estimates indicate that in a highly industrialized country, e.g.
U.S.A., as much as 75 - 80% of the cancer incidence is of environmental origin. Large quantities of synthetic organic compounds from their
very nature are present in our everyday life such as azo dyes, hair
dyes, solvents in inks and paints, pesticides, automobile emissions
and others are disseminated into the environment as wastes from
industrial plant; coal, oil, heavy industry etc. In fact any charring
or combustion process involving organic matter is liable to produce
carcinogens; indeed, recently, a multitude of publications on the
dissemination of P.A.H. in sediments have occurred including a historical
record and a global distribution survey both by Nase ,03 Hitas.(85,86)
There have been suggestions that at least some P.A.H. can be synthesized
by algae (Borneff et a1.C87 )), by plants (Graef and Diehl(88)), or by
various bacteria (BrisotS89), Knorr and Schenk(90), De Lima-Zanghi(91),
Zobell(92)). Then it was shown by Hase and Hitss(93) that bacteria
accumulate P.A.H. but do not synthesize them. They may also originate
from petroleum(94'95), but this would have to be indirectly since the
P.A.H. homologedistribution in sediments has been shown by Youngblood
and Blumer(96) to be monotonically decreasing with increasing number
of alkyl-carbons whereas Spears and Whitehead(97) showed P.A.H. mixtures
from petroleum to be deficient in the unsubstituted species. In situ
chemical aromatisation such as geological diagenesis of specific
terpenoids and pigments may produce one or two P.A.H.'s (Blumer(98))
but this is obviously not a major source.
Combustion is probably the most common source particularly of the
carcinogenic P.A.H. in nature (National Academy of Sciences 1972(99)).
56
Although prairie and -forest fires contribute, anthropogenic sources
particularly due to the burning of fossil fuels must be the main cause _
of the high fall-out near urban areas. Many workers have reported
the presence of P.A.H. in polluted air sorbed on to particulate matter100-103)
and usually characterised as primary or secondary. The former is found
in sizes between 1 pm and 20 pm being produced directly by physical or
chemical processes characteristic of a specific emitter whereas the
latter particular matter type ranges from molecular clusters of the
order of 5 nm to particles with diameters of several micrometers.(104)
Obviously careful sampling and statistical analysis of such environmental
types is of prime importance(105) due to widespread differences. A
common distribution pattern as shown in the bar graph(105) usually
results however.
13001
1500
Fip~.'3.7Bar graph of total conte- trdlions of PAH in urban (column 7.. small town (column 2), and rura (column 3) areas.
30 -
L PNENANIwn[3.[ .[ 0204.}PTRC CC l2.3 0• ENE SM[M110[ HI
Thus it is not surprising that the National Cancer Institute studies
show cancer mortality to be most pronounced in urban and industrialized
areas particularly where the regional density of the petrochemical
industry is high. Badger et al. studied the pyrosynthesis of P.A.H.
and proposed(107,108) mechanistic rodtes from 400 - 750°C dependent
upon an initial pyrolysis which produces smaller cracked unstable
molecules or radicals which react further to produce more complex
structures. Other workers(109) tried to show that P.A.H. synthesised
330
0 0 0
z [30 .
ac
0
I-a
z U 300 - z O O
l . GO \.;
rq comp.) ``:f \> ::
!q tuts( amorti\ ~`;: ` j_-- A OCIDEF
METHart '
A 8 CDEF
iSoi JTYLEI:E
400
300
200
tCO
COMBUSTION
CO DUSTION )ao °RELATIVELY COMPLETE" 300 °RELATIVELY R COMPLETE"
300
200E-
lob
•
ROPA`;E
500—
400
300i
!Go ■, fin;'---~
A b C O E F o~
---
: A O CDEF
800—
-' 000r
1 400
20C. I'
A S CLEE
A tli flt.CE►:E 0 C P:ZO io) FYREr:t'
0 PYR? t: Z E GEr:Z0 )PY4=_hc
C īLUOR.'.!ITALN.. r PF.PrLcr:r.
r l = , 3.8 1̀1•: Al of ti:_ I,.-:;o urb ,rr n: int- rc!.I r+ci n
pur c:rni:, of f;. _1
I
58
in a flame during incomplete combustion is independent of the fuel used
to a large extent (Table in Fig. 3.8i With the lag time in carcinogenesis, usually 5 - 20 years, the
recent mushrooming in the numbers of new synthetic organic chemicals
and in particular their more common occurrence in ecosystems, air,
food, and surface waters, the acute problem of establishing maximum
permissible doses and concentrations of individual carcinogens taking
into account the nature and duration of contact with them, becomes
clear and of hasty necessity.
The detecting, identifying, tracing and correlating of the sample
types and origins responsible for such environmental pollution is also
of utmost urgency.
Thus it appears that in the near future a strategy for controlling
the ever growing problem of 'environmental cancer' must in fact be
formulated together with associated legislation. Detecting presumptive
carcinogens identifying them and tracing their movements in the environ-
ment by means of screening based on bacterial mutagenesis is necessary.
Then by determining associated metabolites etc in human urine samples
it may be possible to determine which of the presumptive carcinogens
represent specific risks to.selected people. The final but intimately
important step of regulating environmental emissions will no doubt be
the most difficultjF~5 .3.),4.t)
60
4
20.-
V.' 08-W
0 6—
01
Economic gizW►7iz, employment and energy
100
80
10 ū • 8
Oil end gas
1 Tool en:.gy input to 69 // etectricty genrYotIon qtr //
l / / Cool// // 1
/ / /
1 / p /, r Hydroelectric
c 1
f Y Y 1; `T 2
X'r 1 • / 1 u /. I u ti _ Ge tie:met/ I
.s I-0—
02
/ 1 / 1
/ 1
i 1
/ 1 / 1
/ 1
/ 1 • / 'Soto? / 1 /
Figure. 3,9_ Energy input to electricity 0II- generation by utilitin,- in the USA 1910
Sc I 1
U 1 rr,0 r3ot) ryfu 19E:0 2000
in
20i0
. 6o
CHAPTER IV
'P.A.H.' ANALYSIS IN COAL TARS AND PITCHES
4.1. WORLD ENERGY PROBLEM IN RELATION TO COAL
The present world energy situation is characterized by total
consumption at an average rate of about 7.5 terawatts of thermal energy which is roughly equivalent to 8 billion tons of coal per annum. Presently 5.5 of this 7.5 terawatts is supplied by oil and gas and this has increased steadily over the last 30 years.(110,111)
Oil systems are most accessible and easy to handle, can easily adjust
to market and end-user requirements have a low capital cost investment
and are more socially acceptable especially in an environmental sense
in comparison with coal or nuclear sources.
For Western Europe the projected unconstrained energy usage is
3.5 terawatts for the year 2000; this would decrease to 3 terawatts with efficient conservation measures. The partitioning of this energy
supply would be 21% from coal, 17% from nuclear power, 50% from oil
and 12% from renewable resources including about 5% from solar energy.
However, these overall needs are rather optimistic as domestic coal
production cannot be developed rapidly enough in Western Europe particu-
larly with increasing environmental legislation on sulphur and nitrogen
oxides, other specific pollutants including aromatic hydrocarbons and
with the additional problems of scrubbing processes and surface land
reclamation. About 85% of all coal reserves and resources exist in
the U.S.A., the Soviet Union and China, indeed 1.2 billion tons of coal
production for 1985 has been advocated in the latest statements of United
States energy policy. In Western Europe, Britain and West Germany have
70 billion tons of coal-equivalent or roughly 65 terawatt years; thus,
even at the present rate of energy consumption of 2 terawatts per year
this appears to be an uncomfortably low stockpile.
Nuclear power is really no more promising because energy available
from light reactors using all the available '5 million tons of uranium' is roughly 35 terawatt years. The demand for this premium uranium is becoming increasingly more competitive with an inevitable parallel
61
cost escalation though fast breeder reactors and fuel recycling plants
give some promise. West Germany has already heavily committed itself
to a nuclear power programme and by the year 2000 A.D. will obtain one-
third of its energy needs from this source together with one-third from
hard coal and one-third from oil plus gas supplies. Longer term energy
forecasting is speculative but there is the real potential of two virtually
inexhaustible 'clean' energy supplies; namely, nuclear fusion and solar
power with its many lesser known though 'dispersive' alternatives such
as energy farming to produce energy from biomass fermentation or enzyme
biocatalytic hydrogen production.(112)
But, for the moment, with the rapid utilisation of crude oil as an
energy source and a petrochemical feedstock, there is an obvious necessity
for the most efficient use of this natural resource together with an
associated development of other fossil fuels. Two main problems arise
here, namely converting any alternative into a form usable by current
refineries but also dealing with the problems of environmental impact.
4.2. 'ENERGY AND ASSOCIATED P.A.H. PRODUCTION
The dangers to public health of fossil fuel processing and indeed
any widespread combustion methods especially the internal combustion
engine have already been widely demonstrated in the previous chapter.
Correlation by Hites, Laflamme, and Farrington, (85)
of the combustion of fossil fuels and related P.A.H. production with
the sedimentary record shows a levelling off but at a high level since
the peak coal production period at the beginning of this century.
The high incidence of cancer mortalities and nasal tract carcinomas
in those states of the U.S.A. with petroleum industries is yet another
indication of the environmental problems facing power policy planning.(113)
PAH
abundance
_L 1 1850 1810 1890 1910 1930 1950 1970
Fig.~rl . Total relative un-substituted PAH abun-dance observed in three dated sections of a sedi-ment core from Buzzards Bay. Massachusetts (open circles), and calculated PAH production (closed circles) as a function of time.
62
Year
Table 2t(Energy produced from various of year.
fuels (
10'5 Btu)
Hydro- electric
and associated PAH production as a function
Year Coal Wood
Energy (in
Oil Gas
No- clear
PAH
Arbi- - Normal- trary izcd
1850 0.22 2.1 .2.3 9
1860 0.52 2.7 3.2 12 1870 1.0 2.9 3.9 15 1880 2.0 2.8 . 4.8 18 1890 4.0 2.5 0.15 0.25 6.6 25 1900 7.0 2.0 0.25 0.25 0.25 9.1 34
1910 13 1.8 1.0 0.58 0.58 15.2 57
1920 1 5 1.6 2.7 0.78 0.78 17.7 67
1931) 14 1.4 5.8 0.76 1.9 17.7 67
1940 13 1.3 7.8 0.90 2.7 17.4 66
1950 13 1.2 13 1.6 6.0 19.4 73
1960 Il) 1.0 21) . 1.7 13 19.0 72
1971) 14 0.84 29 2.6 23 0.21 26.4 10O
4.3.1. Coal Tar Analysis
All crude fossil fuels are a complex wide boiling range group of
hydrocarbons. Depending on their source these can contain significant
levels of nitrogen, sulphur and oxygen containing components. Shale-
oil contains much higher levels of nitrogen compounds than typical crude
oil (rock-oil), thus this material may require different processing
and/or pre-treatment. With coal, it requires that the ratio of carbon
to hydrogen be changed so that the processing can be undertaken with
current facilities. The carbon to hydrogen ratio for various fuels
is ca. 6.6:1 for a crude oil, 17:1 in lignite, and 15:1 in bituminous
coal.(114)
63
Thus the analytical requirements for fossil fuels will depend
on their utilisation. If combustion is the end energy source require-
ment then separation and individual identification of useful components olra;jvP.t.11 ,
may well be necessary. j. Further, knowledge of the composition of tar
is also necessary before the processes which occur during the preparation
of pitch-based electrode binders(115,116)
and carbon fibres the exposure
of tar-based protective coatings and timber preservatives and the utility
of road tars, can be understood. Analytical methods developed for coal
tar should also be applicable in assessing the potential of new methods
of obtaining chemicals from coal(117-119), either by novel carbonization(120-122
methods or by solvent extraction(123-125).
Ideally they should also help
in investigating the structure of the coal from which the tar is produced,
and continually monitoring carcinogenic materials.
Crude coal tar is formed as a by-product of high temperature
carbonization of coal at coke oven plants and gas works giving approxi-
mately 7.5 gallons per ton of coal.
Tars with a specific gravity (s.g.) of between 1.14 and 1.22 consist
principally of P.A.H. of 2 to 6 rings. Coke oven tars however contain only small amounts, less than 5% of paraffinic hydrocarbons and phenols
and are relatively rich in unsubstituted P.A.H.s.
The tar produced in continuous vertical retorts at gas works is in
contrast of relatively low s.g. i.e. between 1.06 and 1.12 and it may
contain up to 20% of phenolic compounds and up to 20% of paraffinic
material the remainder being composed of mono- and polynuclear aromatics.
4.3.2. Analytical Techniques
Coal tar is a highly aromatic multi-component system of around
104 acidic, basic and neutral compounds, of which about 103 have been
positively identified(126,127) by repeated separation and purification.
Art. alternative analytical procedure is to fractionate by solvent (128,129) cimowla p
extraction or gel permeationt then analyse the more volatile
fractions by gas chromatography and/or mass spectrometry individually
or in combination.
High resolution glass capillary or pyrolysis gas chromatography
with a mass spectrometric finish and computer graphics read-out are
the most sophisticated techniques used.
64
The high molecular weight fractions may then be studied by liquid
chromatography, spectroscopy or by a statistical structural
analysis of some physical property such as molar volume.
Edstrom and Petro fractionated P.A.H. by gel permeation chromatography
and from elution curves of thermal residues of coal tar pitch concluded
that separations occur as a complex function of molecular size, shape and
polarity.(i30) High molecular weight portions of low temperature coal
tars in tetrahydrofuran passed rapidly through the Sephadex type gel(131)
while low molecular weight materials pass more slowly because of the
diffusion into the pores in the beads of the support material. A
similar phenomenon has been observed with sephacryl in the work reported
in this thesis.
Proton magnetic resonance has also been well used ever since
Richards et al.(132).demonstrated that the shape of the broad-line
proton resonance spectrum of coal at low temperature varied with the
carbon content. Studies have also been conducted with N.M.R.,(133)
which is much less sensitive than P.M.R., and this, together with high
pressure liquid chromatography, electron spin resonance and mass
spectrometry are reviewed in detail in relation to coal tar analysis
by Bartle.(134)
4.3.3. Chromatography with Fluorescence Detection Methods
Time consuming early methods for trace aromatic hydrocarbon analysis
in natural products involving chromatography and solvent partitioning
have been reviewed.(135) Sawicki, Stanley and Johnson,(136) however,
eliminated the tedious extraction procedure by monitoring direct light
emission from plates of cellulose or plastic sheets. The separated
spots are treated with between 0.25 to 1 ;it of various solvents in small
increments and both excitation and emission spectra were recorded. The
detection limit for pyrene, benzo(a)pyrene, anthracene and benz:anthracene
varied between 0.5 and 50 ng. Interesting nitrogen heterocyclics such
as the benzoquinolines, methyl dimethyl and ethyl plus some benz:(c)acridines
were also identifiable in a coal tar pitch basic fraction.
A similar methodology, but utilising Whatman No.4 paper impregnated
with paraffin oil as the chromatographic medium, was suggested by Maly(137)
65
semiquantification being achieved by a spot diameter measurement.
After a complicated extraction of coal tar Matsushita and co-workers(138)
finally obtained 93 separate fluorescent spots. Liquid-liquid partition
with cyclohexane, dimethylsulphoxide,20 vol. % HCl, 5% NaOH water and
benzene was followed by a two-dimensional dual-band thin layer chromato-
graphic separation with spectrofluorometric determination of the scraped
spots at 365 nun and 253 nm. Benzo(e)pyrene, chrysene,benzo(a)anthracene,
benzo(a)pyrene, benzofluoranthenes(b)(k)(f)(j), indeno(1,2,3-cd)-pyrene,
benzo(g,h,i)perylene, dibenzo(a,h)pyrene dibenzo(a,i)pyrene, anthracene,
fluoranthene, pyrene, perylene, coronene,anthanthrene, naphthacene,
peropyrene, benzo(b)chrysenegn1tribenzo(a,e,i)pyrene were all identified
with benzo(a)pyrene(B(a)P) being determined at 7400 p.p.m. in the coal
tar.
Ra en(i39) has used an interesting combination of low temperature
luminescence in conjunction with TLC on layer materials of polytetra-
fluoroethylene fluoroglide 200 and a new microcrystalline nylon Aviamide-6,
for the separation of P.A.H. and related heterocyclics. Ten aromatic
hydrocarbons and heterocyclics together with a mixture that contained
the ten were chromatographed on thin layers of Aviamide-6-fluoroglide
20 (4:1) and developed with n-propanol. The resulting chromatograme were
observed with radiation at 254 or 366 nm under liquid nitrogen; eight
of the compounds were resolved with the separation presumably dependent
on the differing solubilities in N propanol. Oxidation of the tar with
air until its softening point reaches 85oC has been claimed to reduce
the B(a)P content to 78 p.p.m. supposedly with the formation of a B(a)P
quinone which is much less carcinogenic.(140) Other methods of reducing
the Ei:a) P content(141) involve a higher rate of pyrolysis or microbiological action. In conjunction with this are B(a)P concentrations of 0.021 to
1.18 pg per cubic metre(142) monitored at a coke oven battery, this
being 3 orders of magnitude higher than for normal ambient air; thus
prompting Malk(143) to suggest the establishment of an upper limit as a
hygiene standard.
~44)
Many gas chromatographic methods nave e been reviewed including a gas solid method especially suggested by Frycka(145) to
66
resolve the problem of separating phenanthrene, anthracene and carbazole
in tar products. This particular problem can also be solved by using
the specificity of quasi-linear spectroscopy (Chapter IX).
4.4. DIRECT SPECTROFLUORIMNTRIC ANALYSIS
Progress towards elucidation of the effective structures present
in coals is likely to be aided by techniques which enable individual
polycyclic hydrocarbon molecules to be identified in extracts derived
with the minimum of degradation. While fluorescence spectrometry has
been widely applied(146447) to the study of polycyclic aromatic
hydrocarbons (P.A.H.)_ solution electronic excitation gives rise to
broad-band absorption and emission spectra in many solvents.(4$)
Thus, in mixtures of P.A.H., interference caused by overlapped excitation
or emission spectra and by intermolecular quenching reactions (which
influence the quantitative dependence of fluorescence intensity on the
concentrations of the species present) generally necessitates preliminary
separation of compounds by chromatography or solvent extraction for
identification. Nevertheless Kershaw(149) very recently used luminescence
at room temperature as a very sensitive qualitative indicator of twelve
different aromatic ring systems present in coal liquids. It is surprising
more applications of luminescence spectroscopy have not been published
as a decade ago Zander(~50) suggested the potential of low temperature
phosphorescence in the characterisation of coal tars but some of this
work lacked sufficient resolution and thus selectivity. As we have
noted however when some P.A.H. are frozen in the crystalline Shpol'skii
matrix of n-alkane solvents (formed at 77 K) well resolved line spectra result.
For complex mixtures of P.A.H. and other materials such as coal
extracts quantitative measurements by Shpol'skii spectroscopy present
problems.(148,151,152)
Thus, unless the freezing process is very
rapid (and even then unless concentrations are low) microcrystalline
aggregates of solute may occur in the solvent matrix. Further,
fluorescence quantum efficiencies of solute molecules in crystallites
usually differ from those isolated in the solvent matrix. Moreover,
intermolecular energy transfer proceeds with very high efficiency in
the solid state; this magnifies the effect of quenching and sensitization
67
phenomena.(i53) In coals, extracts and tars, inhomogeneous micellar
aggregates of polar aromatic compounds may form in very dilute alkane
(i.e. non-polar) solvents. Under these conditions, only species
resistant to deactivation and quenching (of which benda)pyrene is a
good example) will be seen clearly in Shpol'skii spectra. Concentrations,
therefore, must be adequate for detection, yet low enough to minimise
solute aggregation.(154,155)
Although quasi-linear spectroscopy has been applied in the U.S.S.R.
to many real sample situations including oils, ointments, car exhausts,
soots, dusts, plant emissions and cigarette combustion, as reviewed
by Farooq,(48) only occasional reference has been made to the complexities
of coal tar analysis, probably for the reasons stated earlier. Utkina(135)
in the Proceedings of a conference on spectroscopy in the U.S.S.R.
mentioned coal tars while other reports have been vague and/or contrasting.
Thus Khesina et al.(157) when analysing combustion tars and soots by
standard additions stated the need for prior chromatography was eliminated
whereas Jager(158) reckoned that very poor precision was achieved without
this pre-treatment.
4.5. EXPERIMENTAL
The coal samples (obtained through Mr E. Bradburn, N.C.B. Yorkshire
Regional Geological Service, Doncaster) originated in seams from three
English coalfields, and formed part of the well-established Hirsch
series, stored under nitrogen. For coals from each seam, samples of
85-98% purity were available by manual sorting into three groups of
macerals: vitrinite(V), exinite(E) and inertinite(I). Approximately
1 g of each of the nine finely ground maceral samples was refluxed for
6 hours with 20 ml carbon disulphide (B.D.H. AnalaR grade). After the
hot solution had been filtered and bulked with several washes of the
filter-cake, the solvent was carefully distilled off and the extract
air-dried for 15 minutes at 333 K. In order to enhance the solvent power of n-hexane for the larger P.A.H.'s, the extracts were first
dissolved in the minimum of AnalaR-grade cyclohexane (previously eluted
through a silica-gel column to remove any P.A.H. impurities). These
solutions were then diluted with n-hexane, so that the concentrations
of the original extracts in the final solutions were all below 0.1 g dm-3.
430 440 453
Cannock Wood exinite
E
370 403 410 tro Wavelength / n:n
Chistet vitrinite Chistet inertinite
411 4L0 4E0 440 420 130runWavelength / n
I 453 470
Wovelength/nm
0 C
A
B
A
0 Markham Main
Cxinite
A
400 £13 420 433 440 4S3
Wovetength / nm
ANALYSIS OF COAL TAR EXTRACTS F:1
68
69
A 20 g sample of an electrode-binder coal-tar pitch (obtained from
Mr C.R. Mason, B.S.C. (Chemicals) Ltd) was similarly stirred and heated
to boiling with 50 ml 3yclohexane (treated as before); after the solution
had cooled, filtration gave a 0.2% pitch solution. Separate 0.2 ml
portions were then examined in 10 ml n-hexane and 20 ml n-octane.
Shpol'skii luminescence spectra in the 370-480 nm region were recorded
at 77 K on a luminescence spectrometer described previously.(152)
Light from the excitation source (a 150 W xenon-arc continuum lamp or
125 W mercury-vapour discharge lamp) was focused via a condenser lens
system on to the frozen sample matrix. The sample cell was the copper
cryostat described elsewhere (p.3q) The appropriate excitation
band was selected by means of interference filters. Luminescent
radiation was detected by an EMI 6252S photomultiplier tube mounted
at the exit slit of a high-resolution monochromator (Rank Hilger Ltd,
Monospec 1000; aperture f8; linear reciprocal dispersion of 0.8 nm mm-1).
4.6. RESULTS AND DISCUSSION
Table 4.2 lists eleven P.A.H. positively identified in the spectra
of the coal macerals, examples of which are shown in Figs. 4.1 and 4.2.
Letters above the peaks (and above the peaks for the vNNire extract in
Fig. 4.3) correspond to those of the P.A.H. in Table 4.2.
Uncertainties in peak assignments can arise when emission lines
from different compounds overlap. This is true, for example, around
445 nm with perylene and coronene in an n-hexane matrix, but in n-heptane
coronene can be distinguished either by an intense luminescence peak at
426 nm or by its intense phosphorescence at 563 nm. For the pitch samples
(Fig. 4.9), the use o;f an alternative excitation wavelength (xenon
arc instead of mercury-vapour discharge lamp) suppressed or increased
emission from different P.A.H.'s (e.g. to facilitate distinction between
benz(a)pyrene and benzofluoranthenes); it also enabled peaks from the
light source to be distinguished from those of the sample. The presence
of certain P.A.H. (chrysene, benz(e)pyrene and phenanthrene) may be
confirmed by their characteristic phosphorescence, which is also quasi-
linear. Spectra at higher wavelengths, especially those in n-h xane,
displayed lines at 453-454 nm, attributed to green phosphorescence of
simple heterocyclics such as quinoline and isoquinoline, or a combination
of these.
CANNOCK WOOD EXINITE F. 4+.a
isita
370 400 410 4-20 430 ...,,wavelength rim
440 450
3,
c A
. 380 390 400 410
MARKHAM MAIN VI Ī RINI i E
44000
1:100 '1:9
04.
game
•
OMB _
•
•
/
010
0.111
1:9 ••1:9 1:9 1: 9 •
ANALYSIS OF COAL TAR AND PITCH EXTRACTS (r-A'L -~ l
KENT ;YO RKSHJRE STAFFO ;DSHIRE PITCH ; CH ISLET :MARKHAM MAIN CANNOCK WOOD
NO 5 SEAM BARNSLEY SEAM SHALLOW SEAM
8-5% • 96% 00% $9 % 98 90% EXT. VIT. • , . IN. EXINITE ,VITRINITE EXT.
• A / / / / # /
98% VIT.
CORONENE OVALENE B PERYLENE C •BENZ(ghi)PERYLENE • D PYRENE E QENZ(a)PYRENE F DIBENZ(u i )PYREN E G CHRYSENE H BENZ(a )ANTHRACENE _ I. 910 DIMETHYLAN ī HRACENE J BENZ(k)FLUORANTHENE K DIBENZ(ah)PYRENE L
SOLVENT -MATRIX RATIO 1 CYCLOHEXANE: HEXANE
w /w EXTRACTED
N CB COAL RANK CODE
6.3 0.5 0-3 ' 1.4 0.3 1 1.1 0•4 501 601 ; 702 70 2 ' 90 2 .
73
Requirements for effective application of the Shpol'skii technique
in this field include availability of specific reference compounds (so
that extrapolation of our data to indicate possible average structures
is not feasible) and absence of excess concentration of any component.
Had sufficient sample been available, removal by chromatography of some
of the P.A.H.'s present in higher concentrations and fractionation of
the extracts might well have enabled more compounds to be identified.
In particular, the background continuum and slight broadening of peaks
seen in the Chislet vitrinite spectrum, probably caused by micro-
crystallite quenching reactions, might be reduced in simpler chromato-
graphic fractions of the extract. Further identifications would be
aided by greater variation in solvent matrix and by application of
selective and/or more intense excitation.
It is also relevant that P.A.H. of surprisingly high molecular
weight (up to 400) are present in field-desorption mass spectra(159)04)
of the same maceral samples. Such highly condensed aromatic systems
are not so readily detected in coal extracts with low-ionization-
voltage electron-impact mass spectroscopy.(160,161) Rings as large
as ovalane(162) and coronene(163) have figured in attempts to illustrate
coal and asphaltene structures(164) but it is interesting to have new
direct evidence for their presence in'at least small quantities.
Quasi-linear luminescence spectra for organic material from lower
Cretacean agillite deposits have been reported(165) to contain peaks
characteristic of perylene, benzo(g,h,i)perylene and benzo(a)pyrene.
In the pitch spectra in n-hexane with (a) zenon source and
(b) mercury source, benzc(a)pyrene and benzo(g,h,i)perylene were
readily identified, but also indicated the presence of pyrene;
the partially broadened emission at 445.1 nm could be caused by quenching
or a re-absorption which is prevalent at concentrations around 10-6
M.
The presence of benzc(a)pyrene is confirmed in the spectrum measured in
n-octane with a mercury lamp, while bena<j)fluoranthene and dibenz(a,i)-
pyrene can also be detected in the mercury-lamp spectra from both
matrices. The estimated concentration of benzo a)pyrene was ^-4 x 10-7M,
i.e. -,4 x 10-5 M in the original cyclohexane extract. Of the many
fluorescent compounds known to be present in the cyclohexane pitch
extract, the number of polycyclics identified from Shpol'skii spectra
(seven) is admittedly small. Again, subdivision of the extract by
FIG.4r4 FIELD • DESORPTION MASS 'SPECTRA RA
VITRINITE
rt-r.-'- ,._.,....4 1~ ,i~~~~ 1~ i1~ f~i~ ~; i•'~ •'tiA~ ,' ! .•,I I ,~ Iry~ ? ' 1' 1
ITf '.~ -.l,~.r Jr r•,-.,~~•r+';-~t'h~=n-~-r-•~~,-'•~—',. ALL., _rt'i -.. 1 . W • 1•"f-- •-r1';t• .' .. ':-mai #r a 1$1 d1 H, 111 $• 111 ~ .TI 10. N, 401 NI 799 700 !e11 eL 9,1
1K. 600•0. 61011 • f$
M.M. VITRINITE •..
41.
11•
4,.
11
1.
li
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► Jill 'li,lil,ie
e .0
ISO 1104
I1 ,i
~:.~~~ I ; li!i ~ l°! ~ 1~7 ,~ i ~ ►
I~~;~I': !r; .. '! • "':.J, '1r~.~ i !II„IN I ;:i.., ! I ►.,~ ~ ~► LIIIr..li~..l~ Irr
.n a51 $1111 115. 11U ate 711 9 rc, 1104. .11 160
O. 1-. . 00
GO lR
RU Mr $11
11f1. a.111.1. • IR
1 I •
.1•
111
.I• I,•
INERTINITE Chislet
00
41.
10
l0
. • • _ . ...,,rn., i•.•. ..., ,.-;. vt r•1 -. r00 4-r 07,7•• r-rd~(•••r+r1—r
10 U/ 100 H1 110 00$ 550 .I. •T, 501 e$$ 11
6$ l0 eff0 0060.0, 1,1,11 r 60
400
r
75
chromatography would favour identification of further species.
Further it would be advantageous to use a more selective narrow-line
excitation source such as a tunable dye laser or light from a 1000 W
xenon arc which has been primarily selected by a monochromator.
4.7:1. Conclusions
Luminescence spectrometry utilising the Shpol'skii effect provides
an effective means of identifying small quantities of P.A.H. present
in complex coal and pitch extracts and this has potential as a finger-
print. It provides evidence for the presence of a range of aromatic
systems with molecular weights up to 400 and (taken with gas-liquid-
chromatographic, mass-spectrometric and N.M.R. spectroscopic evidence)
is suggestive that these well-defined individual compounds are present
in the original coal. Since the Shpol'skii technique is especially
sensitive to the highly carcinogenic benz(a)pyrene (at the 0.1 p.p.m.
level), and is not unruly expensive, it could provide a means for
monitoring this compound in the environment e.g. exhaust fumes and
smokes without recourse to very complicated analytical separation
methods.(166)
4.7.2. Multiple-site Structure
Multiplet quasi-line structure is aptly illustrated by the splitting
of the benzo(a)pyrene (B(a)P), 0-0 peak even in a real sample situation
when using different paraffin matrices (Fig. 4,5) Several crystal
modifications of the lattice including possible rotational isomers(9)
causing crystal field perturbations or more simply, different emitting
centres like micellar microcrystalline aggregates leading to varying
solute (analyte) dipole alignment(167,168) are just two propce_ck
theories. However all peculiarities of the multiplets cannot be
explained solely by the presence of different centres while (169) P. Y Y P polymorphism
has been discounted(170) altogether. A detailed analysis of the
structure of some complex multiplets reveals that peak frequency intervals
coincide with the frequencies of certain optical crystal modes(171) and
the reasons are still a matter for conjecture. Certainly at temperatures
less than 100 K a and 0 modifications of hexane have been shown to
'co-exist',(172) this explaining the increase in the multiplicity of the
V
SOLVENT=DECANE • HEXANE HEXANE FILTER: .300 300 3 0 0
Xenon.
FIG"4,5 EFFECT OF SOLVENT ON THE SPLITTING OF . THE B(a)P 403 nm Peak
403 XO2-3 PITCH
HEPTANE OCTANE OCTANE 275
or 250 250 300 300
B(k
Pu re B(a) P
77
phosphorescence spectrum of quinoline and the prominent reason for our
splitting of the B(a)P in n-heptane is probably a similar phenomenon
to the pseudo liquid sites postulated by Pfister(170) to explain the
sharpest of all quasi-line spectra namely for coronene. in n-heptane.
Some strange modification in the lattice of the latter results in
greater local symmetry and less intermolecular interaction in certain
sites.
The fourth peak at 403.45 nm in the real pitch spectrum is due
to benzo(k)fluoranthene (B(k)F) which occurred at 403.4 nm in n-octane.
The analysis of both these hydrocarbons is quantified in Chapter VI.
Therein the polluted water analyses often give this similar B(k)F to
B(a)P distribution whereas no B(k)F was detected in the used oil samples
analysed in the next chapter. This suggests that the B(k)F/B(a)P ratio
may be indicative of pollution sources but obviously further distribution
surveys particularly on the automobile production of P.A.H.'s and from
carbon black in tyre composites needs to be undertaken.
Crystal field theory and the idea of Shpol'skii inclusion sites
will be exemplified in Chapter VII suffice to conclude here that certain
P.A.H. preferentially give spectra in different solvents and even within
that framework different sites lead to various fluorescent, phosphorescent
yields and other photophysical energy dissipative pathways. Herein
lies the feasibility for an exceptional degree of selectivity, alongside
fine tuning of sensitivity whereby by varying solvent ratios and with
the use of selective., polarised and/or laser excitation one may manipulate
the analytical system at will.
4.8. HIGH PRESSURE LIQUID CHROMATOGRAPHY
Unfortunately this promising analytical technique cannot, unlike
its widely used counterpart gas chromatography, be coupled to a mass
spectrometer nor has it sufficient resolution (or sensitivity when
using conventional ultra violet detectors) in complex samples as commonly
tackled in environmental P.A.H. chemistry. In an investigatory com-
parative study reasonable separation was achieved with a 5 component P.A.H. mixture using Gunasingham'.s(173) high pressure liquid chromatograhic
arrangement with special electrochemical wall jet cell detection at a
glassy carbon electrode. The resulting fairly sensitive detection of
Fig.4.6(i High Resolution Gas Chromatography Using a Glass Capillary (60 metres x•0.25 mm) aia.
N) S a m plc GOC,- 2.40°C.... ' 2°G
• •
b
A
•
e - o-xylene f - phenol g - o-cresol h - biphenyl
a - benzene b - toluene c - ethyl benzene d - m +p xylene
79
t) J
Fig. 4.6(i) High Resolution Gas Chromatograph using a Glass Capillary (60 metres x 0.25 cros in dia.) coated with a phenyl methyl silicone stationary phase (O.V. 17). Pf.N.
8o
P.A.H. electrochemically is of interest in itself and undoubtedly
results as suggested by Tobias(174) via free radical formation. When
a real pitch sample was analysed however the resulting lack of selectivity
was aptly demonstrated (Pe:6..11-3 _).With the success of a micro capillary
cell fluorescence detector from Perkin Elmer an interesting combination
might result by coupling the high resolution Shpol'skii spectral technique
shown throughout this thesis, with an efficient clean up plus rapid
separation technique such as high pressure liquid chromatography. A
stop flow system with a low volume cell would however be required.
A conceivable design of very low volume yet large surface area has
already been designed and constructed by Faroog(48) for another purpose,
namely. the monitoring of quasi-linear absorption. F,)S. k.~~~ll~l S~O.0 Fte ~oork
re5o1,..t", N C- CYN,G+.v..Ule ,..~~~ c̀ ci5 arc) M“\--0 cyrk )h 4.9.
4.9.1. High Molecular Weight Species
Very little work has been undertaken on the high molecular weight
species shown to be present by mass spectrometry in many pitches which .
are exceptionally difficult to charaterise by traditional chromatographic
or the more sophisticated high resolution or high pressure liquid techniques.
It was thus attempted to use molecular fluorescence spectrometry in an
attempt to characterise these.
(175) _ Yamado ,I,E haw shown how various structural analyses
provide not only useful information on chemical structure but also
on the solubility of pitch.
Indeed the pitch fractions supplied in solid form were only soluble
in tetrahydrofuran although sample (2) also had a limited solubility
in chloroform. The average molecular weight structures were between
400 and 800 since alkane soluble components had been removed.
It was attempted to obtain their room temperature and possible
quasi-linear spectra as these extracts were soluble in tetrahydrofuran
which has earlier been shown to produce sharp line spectra.(9) Only
limited information was obtained. Nevertheless this is still extremely
useful because these higher molecular weight species are thought to
control agglomeration and binding properties of the coal so they are
vital new technology for the future quest of improved environmentally
acceptable yet still economically viable gasification processing of coals.
2.,•47 Gan2m
IOW
/
J 1 1 1
a Vci wyn C7
•
82
Fluidised bed techniques are just onA example of improved technology
from intensive efforts conducted especially by the National Coal Board.
The longer wavelength luminescence of sample (2) compared with
sample (1) (Fig./hi-4 suggests that it contains a higher proportion of
higher molecular weight species with larger more condensed aromatic
ring systems. It was initially supposed that species of molecular weight
500, 750 etc might be dimers and trimers etc of B(a)P but this is very
difficult to confirm as not even the B(a)P monomer molecules give a
distinct spectra in tetrahydrofuran probably due to some lattice
distortion via peroxy radical attack. Attempts to employ cyclopentane
with larger clathrate like 'hole sizes' ( see p.0.1) at 77 K were not
Successful; this results from the limited solubility of these heavy
end fractions and their great tendency to adhere in micellar clusters,
in fact the ultra violet spectra obtained are very reminiscent of
polymeric emission. This problem will be magnified by the probable
presence of 'polar' interlocking groups thus forming ethers, furans,
phenols, thiols, carbazoles, chlorinated P.A.H.'s etc. The effect
of polar and oxygen linkages has already been shown to lead to loss
of Shpol'skii structure in the vitrinite sample4140-lowever, some
structure around 470 to 500 nm region and an intense phosphorescent
band from 450 nm to 520 nm (Fig. 4.9) confirm the presence of many
stable aromatic Ti ring systems of high molecular weight but even more
efficient extraction and separation is needed before accurate character-
isation can be made. In the solvent extraction systems used the resulting
percentage solubility yields are tabulated (4.3). For vitrinites
the highest pyridine extraction efficiency was associated with the
lowest rank grade. The aromaticity and size of coal molecules, the
openness of general structure and the ability of a solvent to break
down van der Waals' forces or to swell the coal 'molecular-sieve'
analogous to polyacrylamide or sephacryl (see Chapter X), so that
solvent has access to smaller interstitial molecules obviously controls
the solvent extraction dissolution properties.
Most difficulty of Shpol'skii structural resolution in the 400 -
500 nm range was found for vitrinites because they are acidic, polar
extracts with higher oxygen content fractions. These latter factors
are particularly prominent in enhancing hydrogen bonding plus electronic
FIG 4.8' Pi fch in cyclohexane (1m 2 5 ml THE )
478
489
Table 4.3
Solvent analysis of the coal series; percentages extracted by
pyridine and carbon disulphide.
Colliery Coal type % pyridine soluble
% CS2 soluble
I Py W, CS2
Chislet No. 5 seam exinite 18.8 6.3 3.0
vitrinite 34.6 0.5 69.2
inertinite 1.2 0.3 4.0
Markham Main exinite 4.3 1.4 3.1 Barnsley seam
vitrinite 16.4 0.3 54.7
inertinite 8.8 2.6 3.4
Cannock Wood exinite 4.3 1.1 3.9 Shallow seam
vitrinite 12.8 0.4 32.0
inertinite 1.2 0.3 4.0
84
FLG. 4.9 LUMINESENCE EMISSION (at 77*K) of a HIGH MOLECULAR WEIGHT PITCH 1 inT.IHF
460
86
energy transfer processes which interfere with the normal production
of Shpol'skii spectra (Chapter VIII). The final noteworthy observation
was the exceptionally high perylēne content obtained for vitrinites.
4.9.2. summary,
With the extra correlation studies done by Drake and Jones(176) on
these coal tar extracts one can confirm that the P.A.H. composition of
the tars appears to be similar to their distribution in petroleum. Other
workers(177) using low molecular weight data have already noted some
similarities. As for the heavy end pitches, these may well be analogous
to petroleum asphaltenes existing as a combination of a cross-linked
polymeric suprastructure with a complex mass of heterocyclic clusters
alongside alkyl substituted aromatics making up the complex infra-
structure whose strongest electrostatic type intermolecular interactions
can only be broken down by prolonged solvent action other than more
drastic processing methods.
87
CHAPTER V
DETERMINATION OF P.A.H. COMPOUNDS IN OIL SAMPLES UTILISING
THE SHPOL'SKII EFFECT
5.1. INTRODUCTION
Incomplete combustion of coals, oils, shale tars and other fuels
is responsible for a great proportion of the P.A.H. compounds found
in the environment. Such sample types together with tobacco smoke
condensates, sewage liquor extracts, pitches plus raw coal extracts
have formed the preliminary basis for an assessment of the potential
utility of quasi-linear low temperature luminescence spectroscopy as
an analytical technique.
Although it has proved possible to effect general characterisation
of fairly complex pitches and coal extracts by this method even to the
point of identificatic.i of some individual compounds, only approximate
quantification of up to ca. ten of the lower molecular weight P.A.H.'s
has been possible without extensive prior separation. The emphasis
of this chapter lies on the examination of oil samples with the need
for preliminary column chromatography prior to the determination of
polynuclear aromatic hydrocarbons (P.A.H.).
5.2. CHEMICAL CONSTITUENTS OF PETROLEUM
The ultimate goal of petroleum chemistry in its analytical aspects
is to be able to resolve the individual hydrocarbons and other related
constituents in oil samples. This diverse mixture of hydrocarbons
includes paraffins, naphthenes, unsaturates and the aromatics which
are of particular importance in the context of this thesis.
The ring analysis of kerosenes and gas oils gives values which
differ widely for paraffinic and naphthenic or asphaltic products.
The percentage of paraffinic side chains is as high as 70 to 80 in
kerosenes and gas oils from paraffin and mixed-base crude oils. The
percentage of rings is small in such distillates with the average number
of rings/molecule being less than 2. Waxes containing 1, 2, 3-naphtha
rings plus isoparaffins and finally asphaltenes which are made up of
highly condensed aromatics together with the bulk of non-hydrocarbon
material of the original fraction, make up the heavier molecular weight
constituents.(178)
88
5.3. GENERAL METHODS OF ANALYSIS .
Various methods are presently used for the determination of
P.A.H. compounds in petroleum products on a routine basis. High
pressure liquid chromatography on microparticular silica gel is now
widely employed for screening purposes usually with ultra violet
absorption for detection, although fluorimetric detection permits
higher sensitivity using a low volume, flow through, capillary cell.(179)
However, the main analytical schemes employed by commercial oil
companies is based on the procedure described by Grimmer.(180)
A sample (ca. 5 g) is spiked with an internal standard (perylene
or pyrene) before being extracted into dimethyl sulphoxide, with re-
extraction into cyclohexane followed by solvent removal by evaporation;
the sample is then eluted through a silica column and then subjected
to a prolonged (more than 20 hours) gel permeation separation on
Sephadex prior to a gas liquid chromatographic finish using a 10 mm
by 2 metre packed column at 270°C. The total analysis time is
ca. thirty hours.
i►u14,l the British Petroleum Company(181) have reduced this analysis
time to seven hours by using high pressure liquid chromatography
rather than gel chromatography; this is not always as efficient
however for separating lubricating oils. The extraction efficiencies
and percentage recoveries are estimated using radioactively labelled
C1'' B(a)P.
Shell Company(181) resort to preliminary solvent extraction
with dimethylsulphoxide and cyclohexane for preliminary sample clean
up before refractive index screening followed by a GLC/mass spectrometry
detection procedure. (181)
A typical application of Grimmer's procedure has been shown to
allow the quantification of fourteen P.A.H.'s in high proteins, foods
and fats as well as oils.(182)
More relevant to the work in our laboratories is the determination
of compounds in used engine oils by high pressure liquid chromatography(16`)
and the use of the fluorescence spectra to characterize high boiling
petroleum distillates.(184) Many other P.A.H. determinations in
natural products are referenced in the review of Simpouli.(144)
89
5.4. APPLICATIONS OF LOW TEMPERATURE LUMINESCENCE SPECTROMETRY
UTILISING THE SHPOL'SKII EFFECT
Numerous applications of P.A.H. detection have been reported by
uti ising the Shpol'skii method and these include the quantification
of B(a)P in not only industrial and natural products,(i85) paraffin
types(186-188) and oils(189) but also in domestic octane(190) at
concentrations in the 0.4 x 10-11 g/ml ± 30% range.
In a detailed study Serkovskaya(i91) has described the column
chromatography of various oils in medicinal ointments using both silica
gel ASK (0.2 - 0.3 mm) or alumina with elution by petroleum ether
(40/70) and chloroform (2:1) necessary before using a Shpol'skii
luminescence determination. Previous attempts have also been made
to tabulate both thin layer separation results with ordinary low
temperature luminescence methods on plates notably by Hood and WinefordnerC192)
In this chapter an analytical procedure has been derived to allow
for the quasi-linear spectral analysis of messy oil samples for P.A.H.'s
by utilising simple preliminary column chromatography; some correlation
with thin layer chromatographic data on the fractions has also been
attempted.
5.5. EXPERIMENTAL
5.51. Instrumentation
All luminescence measurements were made with the spectrofluorimeter
arrangement described earlier employing a frontal surface illumination
technique (angle of incidence 40°) via a focussed 125 watt Hg vapour
discharge lamp excitation source through an interference filter.
Detection was accomplished with a high resolution monochromator of
reciprocal linear dispersion 0.8 nm mm-1 and an EMI 6256S photomultiplier
whose output was led to a potentiometric chart recorder (Servoscribe
Model RE 511.20 after D.C. amplification).
The sample was injected into a copper cell in the form of an
n-paraffin solution which after cooling (540°K min-1
) resulted in a
frozen film matrix. Luminescence of the thin layer plates and eluted
fractions was examined using a 125 watt Phillips Hg vapour lamp mounted
in a viewing box. The thin layer plates used were of the small 'test'
variety (2.5 cms x 7.0 cms) and were prepared from a mixture of Merck
90
Kieselguhr (60) HF 254 and alumina HF 254 (60E) 1:2 w/w.
An adapted 10 ml burette (1.0 cms i.dia.) packed with a solid
phase slurry of alumina activity 1, neutral, medium grade with mobile
phase elution of cyclohexane initially followed by benzene sufficed for
the column chromatography.
5.52. Solvents
Solvent reagents were of spectrograde purity but n-octane was
additionally purified by eluting down an activated alumina column to
remove unsaturated phosphorescent impurities and the cyclohexane was
bidistilled before use.
5.53. Oil Samples
Three oil samples were investigated in detail. These were:
(i) Super Visco-static 20-50 motor oil as an unpolluted background
oil monitor (pale ochre in colour with a 'clean' appearance). 1 ml of
this was diluted to 10 ml with n-hexane.
(ii) A six month old used car oil tapped off the sump of a three-
year old 1000 cc car engine (BLMC Mini S4-~(') 1-t:FDP) This was 'black' in colour and distinctly 'unclean' in appearance. 1 ml of this was once
more diluted to 10 ml with n-hexane.
(iii) A motorway sampled oil originally found as an 'environmental
hazard sample' on some sheep's wool at the side of the M6 motorway
junction 7. This was extracted into cyclohexane and 1 ml of this 'brown'
sample diluted to 10 ml with n-hexane. A 3 ml sample of this solution was then chromatographed alongside 3 ml portions of samples (i) and (ii) on separate columns.
5.54. Column Chromatography
The three oil samples (3 mis) were placed on individual chromatographic
columns and eluted initially with cyclohexane; six fractions each of
15 ml were collected. The mobile phase was then changed to benzene and
the elution continued until a further seven fractions of ca. 10/20 ml
each, had been collected from all three columns.
The gradual changes in the luminescence exhibited by the sample
fractions were observed by viewing these in the ultra violet light monitoring
91
box. Fluorescence and the yellowish colour of the eluted fractions
in the visible region gradually diminished until a colourless weakly
fluorescent sample was obtained by the sixth extract.
The eluant was then changed to the more polar benzene and the
fraction monitored as above.
5.55. Thin Layer Chromatography of Oil Sample No. (iii)
Thin layer plates spotted with one to five µL of 10% n-hexane
solutions of selected fractions (1, 2, 3, 7, 8, 11) showing luminescence
under the ultra violet viewing box were then developed in an n-octane:
benzene solvent mixture (ratio 1:1 v/v) as the mobile phase and observed
under ultra violet light.
Two main groups of component spots were seen to be present, one
group with refractive index, Rf factor of ca. 0.6 to 0.7 (e.g. fractions
one and two) with a:second major group with lower refractive index
factors of ca. 0.2 to 0.3 (e.g. fractions seven and eight). As a
result of monitoring the luminescence on thin layer (TLC) plates
fractions two and seven were chosen for further detailed spectrofluori-
metric investigation.
Fraction two was made up into two different Shpol'skii solvents,
namely a tenfold dilution with n-hexane (2a) and a tenfold dilution
with n-octane (2b).
Fraction seven was diluted tenfold with n-hexane only (7a).
All these sample fractions were then monitored for characteristic
P.A.H. luminescent spectral peaks at 77 K to enable qualitative identification ('Fig. 5.1). Thus Figures 5.2 to 5.5 show quasi-line spectra of all the oil samples and background blanks. The used car
oil shows high proportions of benzo(a)pyrene, benzo(ghi)perylene and
benzo(a)anthracene, while the motorway kerosene oil type shows even
more P.A.H.'s including pyrene, benzo(a)pyrene, benzo(e)pyrene,
dibenzo(a,i)pyrene, methylcholanthrene, benz (a)anthracene, benzo(ghi)-
perylene. A surprisingly close qualitative comparison and even semi-
quantitative estimation can be made of the polluted oil scan with that 155
of the synthetic mixture of eight P.A.H.'s (Figures 5.4 and 5.5).
92
FIG 5,1 ' PA,I-1` LUMINESCENCE OF USED CAR SUMP OIL (sample ii)
at 77°K (sensitivity all 200 my )
B(ulP
403nm
FILTER
375
300
431.5 n m
431.55n m Q I B ENZ ANTHRAC ENE .75
V
419nm
403nm
BENZO Ighi iPERYLE NE 406nm
415.2nm
DI BEÑZ.OPY REN ES
CO R ONIENE PERY LEN E.
BuP Mutti 'Diet in
431.5 nm n hexane
FIG. 5.2 Benzene Etuate (1 mt -10 mt ri-hexane) of (ii )
USED CAR OIL [ E H T 2000 vottsl (375 Fitter)
H Ln
I Hg
I
Hci
G
FIG. 5,3
I MO-IOW/AY OIL SAMPLE:-CYCLOHEXANE ELUATE FRACTION 2 1rril-110mt HEXANb. cp I
•.•., • H=CORONENE .I=1,12BENZPERYLENE J =34S9DIBENZPYRENE
CO 0
E tn co)
Fraction 2 OIL SAMPLE D .!FIG 5,4 r,
0 Hg C
D Zit i D al
E=DIjENZ (ai)PYRENE F =BEN Z (a)A N T H RAC E N E (3= BENZO (e)PY RENE
Octune:Cyclohexane 9:1 325 Exctn. 500 rnV
1'rorn ref. (51 t'or cv̂r ,•,vr.~ .a~~• rec.~ 9«M~12 r6',u- ,̀ . NNW
LOO
13 h
Wavelength/nm Fig. 5.5 1vmissior spectra of a synthetic mixture itt n-nclanr, - tyc1uhexaoe at 77 K: (a) pyretic;
(b) 1,2h'r5mthraeene; (r) 3-tnrthrlcholanthrenc; (d) l , _' ,S ,G-rlihrnr,+nthrtrcne; (c) 1,2,1,5-dihcnzopy-rcnc; (f) 3,4-ben7opyrene; (g) 3,1,9,10-dibcnzopyrene; and (h) 3,4.H,9 -dibarzopyrcrc
C
96
tn O 07 0 °a O
A Cl O Ō
to 0.4 N A .1.i; o c
C 1459 11 f iii d e
cH
C c,d,e
rn
O fM
N N tn
U)
9 O n h ID v
O 0 C,
97
5.6.
5.61. Quantitative Analysis
Apart from the above suggested relative ratioed comparison
a better estimate of the benzo(a)pyrene content of the oil was achieved
by determination against a standard calibration curve. This was
done as follows: although the majority of the B(a)P was found in the
second and third cyclohexane extract traces were also found from
screening in the others. 1 ml of each of the six extracts was there-
fore combined. 1 ml of this mixed solution was then diluted to 10 ml
with n-octane and the B(a)P concentration found by a straightforward
standard additions procedure. Thus a fairly precise quantification
of the B(a)P could be made. Trials on recoveries of the B(a)P from
spiked blank oils indicated ca. 95/ recovery in the first six cyclohexane
extracts. A better method for extraction efficiencies would be to use
radioactively labeller' B(a)P as suggested in British Petroleum's modifi-
cation of the Grimmer procedure.
5.62. Quantitative Results
Oil sample (300 mg) eluted down column initially
(a) Estimation
From ratioed comparison with synthetic sample of P.A.H.
B(a)P concentration is ca. 5 µg/m1 of oil. (b) Accurate Quantification
From standard additions B(a)P 9 ± 2 pg/ml. (c) Without Pre-clean up
A direct method was then tried for the old car oil sample
(ii) JKB 435 P without preliminary column clean up. 10 mg of the crude
darkened oil was made up to 5 ml with n-octane, this master solution was spiked with various concentrations of standard B(a)P plus coronene
for a combined standard additions internal standard method using the
300 filter.
Results direct combined
B(a)P 2.5 pg/ml 6.5 ± 1 µg/ml
5.7. RESULTS SUMMARY
Cyclohexane and benzene represent non-polar and slightly polar
solvents, respectively, in the common elutropic series of increasing
polarity solvents :- petroleum ether, cyclohexane, carbon tetrachloride,
98
trichtoromethylene, toluene, benzene, methylene chloride, chloroform,
ether to the very polar methanol and water used in ;2, dimensional TLC
separation of P.A.H.'s in water Samples. Apart from this useful balance
of eluting power we elected to utilise cyclohexane and benzene because
it was found that they have the most minimal effect (at 10% conc.)
on the quality of the quasi-linear spectra obtained on later low tempera-
ture matrix formation with n-paraffins. Although we have still obtained
good Shpol'skii spectra of coal sample types after carbon disulphide and
chloroform extraction; the final concentration of these solvents was < 2%.
From an examination of the luminescent spectra obtained for the
solutions of fraction 2(10% in n-hexane and n-octane) and fraction 7
(10% in n-hexane) from oil sample (iiX and their TLC results it appears
that predominantly, the smaller ring size, low molecular weight P.A.H.
compounds travel faster through both column and thin-layer chromatographic
systems. This simple semi-separation of a complex oil sample on an
ordinary column allows maximum utility; of the Shpol'skii technique.
5.8. DISCUSSION
The specific spectral identification properties possible by
monitoring quasi-linear luminescence spectra
A_ good qualitative, and semi-quantitative, monitor of many
of the most environmentally important P.A.H.'s can be made by the above
procedure, without resorting to HPLC which still in many instances has
difficulty in resolving isomers of benzopyrene as well as the dibenzopyrenes.
The fact that eleven P.A.H.'s were analysed :by GLC using an FID
detector on this same sample by Public Health Laboratories in Civil
Engineering Department of Imperial College and that the same hydrocarbons
would be separated and detected by three dimensional TLC work seems
initially to discount the credibility of our equivalent Shpol'skii
procedure. However, when one considers 'the state of the art' as
regards the fairly crude excitation employed here, i.e. not nearly
enough excitation intensity is obtained unless one utilises fairly
broad half band pass spectral filters (like 375 filter, Fig.5.1 ) for
these multicomponent samples the potential with the added selectivity
available by changing solvent matrices for environmental screening work
is good. Even though some difficulties in quantification are encountered
99
necessitating the use of a standard additions procedure for acceptable
reproducibility and precision, similar difficulties exist in most
techniques for P.A.H. analysis. Thus in GLC non-specificity resulting
from the use of a flame ionisation detector and large backgrounds
necessitate extra time consuming pre-separation clean up with better
detection using integrated area techniques. The intense difficulty
of separating some vitally important isomers except perhaps for the
newest liquid crystal phases,(193) the virtual incapability to characterise
high molecular weight species and the virtual neglect of many related
heterocyclics in these sample types must be areas where the selectivity
of the spectroscopic Shpol'skii technique may well make significant
gain in the next decade of development particularly with the expected
paralleled development of laser and electronic detection technology.
100
CHAPTER VI
A RAPID ROUTINE METHOD FOR QUANTITATIVE DETERMINATION OF
BENZO(a)PYRENE IN WATER BY LOW-TEMPERATURE SPECTROFLUORIMETRY
6. INTRODUCTION
Prior to the seventies only about one hundred different organic
compounds had been identified in water whereas the number has now
considerably escalated to fifteen hundred of which more than one third
of these have been detected in drinking waters throughout the world.
Indeed today over fifteen thousand chemicals are currently in production
with more than five hundred additional ones introduced each year thus
creating a mammoth task for the analytical chemist and the environmental
scientist in particular as many of these traces are biologically active.
In 1964 the potential carcinogenic hazard of polycyclic aromatic
hydrocarbons (P.A.H.) in water supplies was noted by the W.H.O. Expert
Committee on the Prevention of Cancer(194) (World Health Organisation,
1964) and later, in 1970 and 1971, this organisation recommended that,
for the safety of consumers, the concentration in treated surface water
of six P.A.H.** chosen as pollution indicators should not collectively
exceed 200 ng/1.(195496)
The recommended analytical method for the determination of these
P.A.H. was originally developed by Borneff and Kunte(197) and involves
the use of two-dimensional thin-layer chromatography. Some limitations
of this method(198) have recently led to research into the use of
alternative chromatographic techniques, e.g. gas chromatography(199)
and high-performance liquid chromatography;200,201) in order to
develop a more suitable method for routine application.
The use for this purpose of low-temperature spectrofluorimetry
utilizing the Shpol'skii effect(202), previously studied in this department
is proposed here. This technique is capable of high selectivity and
sensitivity in the determination of P.A.H. compounds at 77 K and can
permit their determination in mixtures without lengthy initial chromatographic
** These are: benzo(a)pyrene; fluoranthene; benzo(ghi)perylene; benzo(k)fluoranthene; benzo(k)fluoranthene and indeno(1,2,3-cd)pyrene.
101
separation procedures.(204,205)
The method has already become widely
used in many laboratories in the USSR where recently it has been
recommended for the analysis of carcinogenic aromatic hydrocarbons,
especially benzo(a)pyrene, (B(a)P). The widespread distribution of
B(a)P throughout the biosphere has been amply demonstrated during the
past twenty years by many workers who have been concerned about its
carcinogenic activity; it is one of the most carcinogenic P.A.H.
compounds (International (International Agency for Research on Cancer,
1973; National Academy of Sciences, 1977; Hoffmann and Wynder, 1977)
and for these reasons the determination of B(a)P in the environment
has often been used to provide an index of P.A.H. pollution of the (209-213) (214-216)
water and air environments. In 1973, B(a)P with benzo(k)fluor-
anthene (B(k)F), was chosen as an indicator of the carcinogenic hazard
presented by airborne particulates and its determination was undertaken
by the International Union of Pure and Applied Chemistry(217) (IUPAC, 1974).
Recently, on the basis of the data of animal experiments to establish
doses of B(a)P which did not produce cancer, Shabad(2i8) calculated a
maximum permissible concentration (MPC) of B(a)P in water of 0.3 ng/l.
6.1. PREVIOUS APPLICATIONS OF LOW-TEMPERATURE SPECTROFLUORIMETRIC
METHODS FOR THE DETERMINATION OF P.A.H. IN WATER Muel and Lacroix(219) were the first to utilize low-temperature
spectrofluorimetry to determine the B(a)P content of drinking water
samples by using the standard addition method, but this compound was
undetectable in the small volume water samples examined. Jger and
Kassowitzova(220) determined the concentration of B(a)P in drinking
water down to concentration of 3 ng/ml with a relative error of 40% and
found that the accuracy was greatly influenced by the presence of other
organic compounds. Snow samples and soils were analysed for P.A.H. by Gurov and Novikov, (221) and Stepanova et al. C222) p developed a procedure
for the quantitative analysis of a mixture of B(a)P and other P.A.H., in sewage and other industrial exhausts, based on preliminary TLC
separation and low-temperature spec trofluorimetric quantification.
In a more recent and detailed investigation, Khesina and Petrova(223)
employed this low-temperature technique in the determination of B(a)P
and seven other P.A.H. in extracted waste water, after a preseparation
102
by column chromatography. Other work concerned with the determination
of B(a)P in environmental waters by this method for public health
purposes has been reviewed by Andelman and Suess(211) and recently by
Andelman and Snodgrass.(210)
In this thesis is described the quantitative application of low-
temperature spectrofluorimetry using the Shpol'skii effect as a rapid
routine method for the screening of B(a)P in the aquatic environment,
without prior separation or after a rapid pretreatment procedure.
6.2. EXPERIMENTAL
6.21. Apparatus
The basic assembly of the apparatus employed was similar to that
previously described (Chapter II).'
A 1 metre grating monochromator (Rank HAger Ltd, Monospek 1000)
with an aperture of f8 and a reciprocal linear dispersion of 0.8 nm mm-1
at the exit slit was employed in conjunction with a 50 mm EMI 6256S. P.M.T.
The signals were amplified using a microammeter (RAC, Model WV-84C)
before being recorded directly on a potentiometric chart recorder
(Servoscribe, Model RE 511.20). A 125 watt mercury lamp (G.E.C., MBW/U)
was used as excitation source. The 375 and 325 nm wavelengths of
excitation were isolated by means of interference filters. An adapted
Aminco cold finger dewar-flask sample cell was used. A coil of nichrome
wire was positioned round the transparent quartz base of the dewar.
This wire was heated by passing a low a.c. current through it in order
to minimize frosting and thus avoiding light scattering. Alternatively
dry nitrogen can be circulated or a vacuum may be maintained in the
sample compartment to achieve the same result. A further source of
light scattering, caused by bubbling of liquid nitrogen, may be
eliminated by the addition of a small amount of liquid helium to the
liquid nitrogen.(224)
Silica tubes (3 mm i.d., 5 mm o.d.) were used as sample cells.
A study of the relative standard deviation of two different kinds of
cells has already been conducted in this laboratory (Chapter II). The
copper cell discussed therein gave significantly better precision at the
ng level. The dewar cell system was chosen for these screening trials,
however, because of its rapidity and commercial availability. However
a multisample copper cell could provide a useful apparatus for routine
analysis.
103
6.22. Materials and reasents
For the extraction procedure 5 1 separating funnels were used in
conjunction with a stirrer; the cyclohexane (AnalaR, BDH) was distilled
twice at 30°C under vacuum in a rotating evaporator (BUchi). The
recovered cyclohexane was dried with anhydrous Na2 SO4 previously washed
with cyclohexane. Acetone (AnalaR, BDH) was used to clean all glass
apparatus.
fl-octane (AnalaR, BDH) purified * by percolating through activated
silica gel (60-120 mesh) and cyclohexane (spectrosil for U.V. spectroscopy,
Hopkin and Williams) provided, in a 9:1 solution, a suitable solvent-
matrix for studying the quasi-linear luminescence spectra of the pure
P.A.H. solutions and of the water sample extracts. Samples of pure
polycyclic aromatic hydrocarbons were available from the sources acknowledged
(Chapter II).
6.3. PROCEDURE
6.31. Recovery of B(a)P from distilled water by extraction procedure
Six extraction trials using a known quantity of B(a)P and distilled
water samples were carried out in order to study the reproducibility and
the percentage recovery of the entire procedure. The efficiency of a
similar technique has been already studied by Monarca and has been
shown to give for B(a)P and other,P.A.H. higher and more reproducible
recoveries, than an alternative adsorption technique based on the use
of a microreticular resin.(225)
A 15 1 sample of distilled water was divided into six 2.5.1 aliquots.
250 ng of B(a)P, as a 500 ng/ml standard solution in acetone, was added
to each and mixed in order to obtain a final concentration of B(a)P in
water of 100 ng/l. A stirrer was used for 10 min. to extract each
sample in a 5 1 separating funnel covered with an aluminium foil with
* n-octane for analytical use has been analysed by Fedonin et al.(226)
by using quasi-linear luminescence spectra and has been shown to
contain B(a)P as an impurity.
1o4
125 ml of redisilled cyclohexane. The emulsion was allowed to stand
for 1 hour and the cyclohexane layer was separated and the separating
funnel was washed with three 5 ml portions of cyclohexane. The
combined cyclohexane solutions were dried through a prewashed anhydrous
Na2804 layer and evaporated to small volume by distillation under
vacuum at 3000 in a rotating evaporator. The concentrated solution
was transferred to a 10 ml volumetric flask, evaporated to dryness
with a purified N2 stream and made up to volume with a solution of
purified n-octane-cyclohexane mixture (9:1 v/v).
0.5 ml of the 500 ng/ml B(a)P standard solution in acetone was
transferred to two 10 ml flasks, dried using N2 stream and diluted with
n-octane-cyclohexane mixture (9:1 v/v) to provide the standards for
the recovery experiment.
The quasi-linear luminescence spectrum at 77 K of each solution was recorded using a V.ivelength of 375 nm for excitation and the
quantitative determination was carried out at the characteristic
403.0 nm peak of the B(a)P.
The widespread occurrence of the P.A.H. in the air of the
laboratory and from other sources and the possible contamination of
the samples from other interfering substances requires that all
operations are undertaken with extremely clean glassware. Therefore
all glassware was cleaned with acetone and detergents and then carefully
rinsed with distilled water prior to the extraction procedure.
This cleaned apparatus was allowed to remain in contact with a
solution of potassium permanganate for 12 hours and prior to use was
rinsed with distilled water(197). The graduated flasks were cleaned
with detergents and then with acetone and finally with the octane-
cyclohexane solution solvent.
6.32. Determination of B(a)P by low-temperature spectrofluorimetry
An octane-cyclohexane solvent (9:1 v/v) was employed, as previously
recommended, for the quantitative determination of B(a)P using its
quasi-linear luminescence emission spectrum at 77 K. This was recorded using an excitation wavelength of 375 nm at a scanning speed of
2.5 nm mm-1 with a 0.1 mm spectrometer slit (0.08 nm spectral half band
pass). The intensity of the luminescence was measured at the 403.0 nm
105 •
Lun-itlescence Intonsity (a. u.) for HoP FD c) _.L
c) I.) . i--- ---i
•
0 0
- 0
0 -
0)
Figure 6.1
/ /
//
/ /
/
106
(3 if:: r.r,•.)
600
•
450
CV3
(1)
0 300 (i)
• 0 CI) Cl)
•J
r) L..3c..0. - (325 ii:n) /
/ /
/ /
(32n :u) /
/ /
/
/ /
/
150 /
/
/ /
(37; v)
0•• 01 2-5 7.5 15
25
Concentration, ng m1 1 Figure 6.2
107
maximum. Experiments were undertaken to determine the range of
concentration over which a linear relationship was obtained between
the signal intensity of 403.0 nm. and the concentration of B(a)P, in
the octane-cyclohexane solvent. As shown in Figure 6.1 good linearity
was obtained in the range of concentration 10-8 - 10-6 and furthermore
good precision can be obtained for measurements in the 1 ng/ml - 25 ng/ml
range which is required for analysis of real water samples (Figure 6.2).
6.33. Limit of detection of B(a)P by procedures employed
The limit of detection of B(a)P in octane-cyclohexane solution
for the proposed procedure was found to be 0,5 ng/ml. The limit of
detection of B(a)P in water samples was 0.1 - 0,2 ng/1 (for 5 - 5.5 1 water samples) by direct comparison with a calibration curve prepared from B(a)P in octane-cyclohexane and 0,8 - 1.5 ng/1 (for 5 - 2.5 water samples) by the standard addition method described below.
6.34. Total analysis time
The total analysis time, from the extraction to the determination
was less than 2 hours per sample; the sample analysis rate may be
improved by processing several samples simultaneously.
6.35. Results
The recovery experiments for the six samples taken through the
extraction and measurement procedure indicated a mean recovery of
B(a)P of 92.3% with a relative standard deviation of 0.21. A second
extraction of the 2.5 1 water samples resulted in recovery of an
additional 4% of B(a)P; thus for routine analysis a single extraction was considered satisfactory.
6.36. Experiments on quenching effects
Experiments concerned with evaluation of the importance of any
quenching effects by other P.A.H. on the low temperature luminescence
emission of B(a)P were carried out with synthetic mixtures at concen-
tration ratios near to those usually encountered in real water samples.
A solution of B(a)P, indeno(1,2,3-cd)pyrene (IP) and fluoranthene
(FL) prepared in the concentration ratio 1:1:20, according to Borneff
(227) • , has shown that there is no evident quenching effect
over the concentration range for B(a)P from 10-8M to 10-7M, whereas
at 10-6M the average depression of the B(a)P luminescence response
at 403.0 nm in this mixture was ca. 20% (Figure 6.] ).
The interference from benzo(k)fluoranthene (B(k)F), observed
in room temperature spectrophotofluorimei'ry (IUPAC, 1973), was not
observed in this work at low temperature. B(k)F shows maximum
luminescence emission at 403.3 nm when an excitation wavelength of
375 nm is employed; although this is close to that of B(a)P at equal
concentrations B(k)F shows only ca. 10% of the B(a)P response at
403.0 (Figure 6.2).
108
A synthetic mixture
ratio of 1:1:20 gave approximately
containing only B(a)P, Ip and FL.
between B(a)P and B(k)F appears to
in water samples according to some
IP, B(k)F and FL at a concentration
the same response as a mixture
The concentration ratio of 1:1
be about the maximum encountered
authors.(219,220,227) It is
of B(a)P,
therefore recommended that a preliminary rapid screening procedure
of the samples be undertaken using excitation at 325 nm, as this
wavelength results in the appearance of two different peaks at 403.3
(B(k)F) and 403.0 (B(a)P) and thus. permits prior knowledge of the
approximate concentration ratio of the two compounds. The spectra
obtained are illustrated in Figures 6.3, 6.4.
6.37. Analysis of Some Water Samples
Standard Additiōn Method
The extracts of the samples examined were diluted with 1 ml of
octane-cyclohexane (9:1) solvent solution, excited at 375 (and 325) nm
and the luminescence emission intensities obtained at 403 nm were
compared with those obtained from a calibration curve prepared for B(a)P
in octane-cyclohexane in order to determine the approximate B(a)P
content. The sample solutions were then diluted appropriately to
a suitable concentration range and the B(a)P concentration in each
was determined by the standard additions method. For this purpose
the sample was diluted to 2 ml with the octane-cyclohexane solution
and divided into four aliquots of 0.5 ml which were transferred to
100—
90
109
80- 0
th 0
• 70 :D •
?' 60 co CD 4-,
▪ 50 -
0 (I) 40- O.)
I 30
20-
'10
0 402.0 403.0 404-0 405-0
V■favu!ength, rim
Figure 6.3.
• 2(dP 4O3 nn
ak)F 403.3nm
403 403-3
4PNEL SYNTHETIC MIXTURE
4033 REAL SAMPLE
2
•
300
111
four 2 ml volumetric flasks. A known amount of a standard solution
of B(a)P (10-7M) was added to three of these aliquots. After the
addition of the same volume of cyclohexane (0.2 ml) each of the four
solutions was diluted to 2 ml with the octane-cyclohexane solution
(Table61).
Table61.
Standard Addition Method for B(a)P
Solution Sample, ml
B(a)P solution
(10-7M) ml Cyclohexane
ml Octane- cyclo- hexane ml
Total volume
ml
X 0.5 0.0 0.2 1.3 2.0
A 0.5 0.3 0.2 1.0 2.0
B 0.5 o.6 0.2 0.7 2.0
C 0.5 0.9 0.2 0.4 2.0
River and Rain Water
In order to compare the Borneff and Shpol'skii methods, four
extracts of water samples obtained from an independent source were
examined. These had been subjected to the following extraction and
purification procedure.
Two 2.5 1 samples of the same rain water and two 2.5 1 samples
of the same river water (Rhine river) were extracted with 125 ml of
cyclohexane; one of each type of sample was purified by the micro-
sublimation technique previously used by other authors for the rapid
measurement of B(a)P and B(k)F in air.(215)
Drinking Water
4 1 of drinking water taken in our laboratory were extracted by the procedure described above. No pre-cleanup procedure was
carried out.
Results
Having constructed the calibration curve for the samples (Figure 6.5)
it was found that in comparison with a calibration curve prepared from
pure B(a)P in octane-cyclohexane the background in these samples did
u) •
160--
140 -Jr
120
c~3
> :G, 100-
C) 80 -a) C) Co
•~) 60-
40
20T-
r
0 2 4 I I
10 12 14 16 18 20 0
6 8 I 1 I
Concentration, ng m1-1
113
not affect the quantitative determination of B(a)P and that the same
water samples, with and without pre-purification by microsublimation
gave approximately the same results, the amount of B(a)P found in
uncleaned samples being slightly higher than in the others, perhaps
because of probable losses in the pretreatment step.
In the drinking water sample the concentration of B(a)P was below
the limit of detection by the standard additions method, therefore
the determination was carried out by direct comparison with the B(a)P
calibration curve prepared from pure B(a)P in octane-cyclohexane.
However, in cases in which it is known beforehand that the occurrence
of fluorescence quenching is unlikely because of the low organic
material content in the solution, as in drinking water, direct comparison
with this calibration curve is satisfactory for screening purposes.
6.4. CONCLUSIONS
The examination of the results achieved shows the promise of
this method for screening analysis of environmental waters. The
reproducibility of the method is adequate and may probably be increased
by improved sample cell design.
The required time of total analysis is very short especially for
drinking water samples which usually do not need precleaning procedures
and may be reduced further by using a simultaneous multiple extraction
system and a multiple sample cell.
In the preliminary scanning of the concentrated sample (2) extract
traces of various other hydrocarbons were detected, namely indenopyrene
(typical Shpol'skii spectra, Figure 6.6) benzo(ghi)perylene, pyrene
and . _ -benzanthracene.
Fluoranthene is best detected by its quasi-linear phosphorescence
spectrum in n-heptane solvent at its maximum 541 nm peak wavelength however.
Herein lies the flexibility of the Shpol'skii technique whereby
by changing solvent, excitation conditions or monitoring phosphorescence,
selective tracing of different hydrocarbons can be made.
Preliminary trials with sewage liquor extracts have suggested some
interferences and thus the microsublimation purification or TLC may then
be most useful. Fig_6,7
F cc I N DENO PY RENE
4631 471.1
< 10 After Filtration
total PAH (ng/1)
50 100
1 H 111111
River I 95
Active carbon and prechlorination
Alum and Polyelectrolyte
After Clarification I < 10
Finished Water I < 10
60
50
Figure (.' Removal of PAHs during treatment
115
116
CHAPTER VII
HALF-BANDWIDTUDY OF NON-PHONON LUMINESCENCE LINES (NPL)
7.1. EXCITON STATES IN CRYSTALS
Aromatic molecules form molecular crystals in which the molecules
are held together by weak van der Waals forces. A first approximation
to a molecular crystal is a system of N oriented but non-interacting
molecules, described as an 'oriented gas'. If one molecule of this
system is excited, the energy of the system would be unchanged by
moving the excitation to any other molecule of the system. There is
thus an N-fold degeneracy associated with the excitation of one molecule
in the 'oriented gas'. In a real crystal, the excited molecule interacts
with the unexcited molecules by coulombic and electron-exchange inter-
actions. Due to this finite intermolecular interaction it is not
possible to construct a true stationary state of the undeformed crystal
with the excitation energy localised at only one molecule. If 6,:E is
the energy of interaction between the molecules, then due to the uncertainty
principle the lifetime AT of a molecular excitation against migration to
a new site in the crystal is of the order of h/ LE.
Any intermolecular interaction obviously removes the N-fold
degeneracy of the system. Waves of excitation having different
crystal momenta and spreading over the entire crystal are formed;
these correspond to the exciton states of the crystal. Expressed
alternatively, the electronic excitation energy or exciton may migrate
through the crystal until it is emitted transferred to an impurity
molecule or degraded radiationlessly. The theory of the exciton
states of aromatic crystals was initiated by Davydov(228) though
modern expansion has been instigated by Frenkel and Wagner in particular,(~~9)
and others including Craig,(230) Knox,(231) Rice(232)
and Robinsor_.(233)
Thus the exciton may also be viewed in terms of a bound electron-
hole pair travelling through a crystalline media in a state of total
wave vector k. There exist two extremes, the Frenkel (tight-binding)
used for molecular crystals with the Wannier weak binding option being
more precise for weak binding in insulating crystals. These two
117
extreme types differ in the degree of separation of the electron in
the conduction band from the hole in the valance band. A simplified
model based on Birks' observations on P.A.H.'s will suffice here
however. Many aromatic crystals, such as naphthalene and anthracene
comprise two groups of translationally inequivalent molecules, i.e.
there are two molecules per unit cell. If *a and Vb are the wave-
functions of these molecules, the factor group wavefunctions of the
unit cell are
a = f2 ( ra + 4/b ) •
*0 =-2; (Va - *b)
These two exciton states arise from one molecular state tIr
because there are two inequivalent sites a and b in the unit cell.
If an electronic transition occurs in a free molecule, its transition
moment M is represented by a vector in a definite direction with
respect to the molecular axes. The polarization of the corresponding
transitions to the exciton states a and p is found by taking the sum,
or difference, of vectors at sites a and b in the unit cell. If an
electronic transition occurs in a free molecule, its transition moment
M is represented by a vector in a definite direction with respect to
the molecular axes. The polarization of the corresponding transitions
to the exciton states a and S is found by taking the sum, or difference,
of vectors at sites a and b pointing in the direction of the transition
within the molecule. The difference in energy of the transitions to
the exciton states a and S corresponds to the interaction energy between
the molecules at the two sites. If this is due to dipole-dipole
interaction, the energy difference is proportional to M2/r3 multiplied
by a dipole-dipole orientation factor where r is the intermolecular
separation. Thus, in a molecular crystal in which there are two
molecules per unit cell, each excited electronic state in the molecule
produces two exciton states in the crystal. The energy separation,
or Davydov splitting factor between these states corresponds to the
interaction energy of the molecule with translationally inequivalent
molecules. Transitions to the two exciton states are polarized parallel
to the symmetry axes of the crystal, and the ratio of their intensities
118
is described as the polarization ratio. The energy transitions to
the exciton states are of the form
Ec -Eo +A ±B C3)
Eo is the corresponding energy transition in an isolated molecule,
A, which may be either negative or positive, in the spectral shift
parameter, due to the solvent shift and to interactions with trans-
lationally equivalent molecules and 2B is the Davydov splitting factor.
In a molecular crystal in which there are more than two molecules per
unit cell e.g. benzene with four molecules per unit cell, equation (3)
becomes
Ec = Eo + A + B . (4 )
where B. corresponds to the jth factor group of the unit cell.
The magnitude of this splitting factor depends on the transition
moment M. For allowed electric dipole S('A-'Bab) transitions (see
Chapter I) 2B --20,000 cm-1 for the anthracene p('A-IL~) transitions
and the naphthalene a('A-'Lb) transitions 2B ^'200 cm , and for the
singlet triplet ('A-3 La) transitions 2B x-10 cm-1. Thus the Davydov
splitting factor for each of the vibronic (0-0, 0-1, 0-2 etc) bands of
an electronic absorption band system depends on the vibronic transition
moment.
The average time during which one molecule in a crystal is in
an excited state is of the order T = We where e is the intermolecular
interaction energy. At the end of this time there is a high probability
of finding the excitation on a neighbouring molecule. Typically this
interval may lie in the range 10-10 to 10-i3 second. These times are
in the range corresponding to the periods of intramolecular vibrations.
If the intermolecular interaction is sufficiently large, such that the
excitation transfer time is considerably less than that of a vibrational
period, the molecules will remain fixed in their ground state equilibrium
configurations throughout, and the excitation transferred will be
electronic in nature. At the other extreme occurs the situation of
a small interaction energy and a correspondingly long transfer time
relative to the vibrational motion. The vibrations couple strongly
with the free molecule electronic levels. Here the nuclear framework
adopts a new equilibrium configuration before the excitation is transferred
so that that which is transferred is vibronic in nature. It includes
119
both the movement of electronic and vibrational energy. The rate
of transfer in this case is less than that corresponding to the purely
electronic intermolecular interaction energy. If the transfer times
are even longer corresponding to the periods of lattice vibrations with
frequencies up to 150 cm-1 then deformation of the lattice must occur
in the process of excitation transfer. The transfer time is then
further increased by the lattice distortion and the excitation acts
as if localised because its displacement through the crystal now takes
place very slowly. This is similar to the processed excitation of
impurity molecules in low concentration mixed crystals as these are
localised because of the absence of resonance interactions with the
host. The various properties of the systems however depend on the
degree of coupling, discussed in detail by Simpson and Peterson.(234)
7.2. MIXED SUBSTITUTIONAL-SOLID CRYSTALS
The emission spe:'.tra of dilute mixed crystals in which the
absorption is by the host and the emission is by the guest have provided
both evidence to support the existence of energy transfer processes
and details of the nature of energy transfer. The occurrence of such
phenomena is aptly demonstrated through the appearance of delayed
fluorescence attributed to the formation of singlet excitons from
the interaction of long lived triplet excitons.(235) Host crystals
are chosen on the basis of their transparency in the wavelength region
corresponding to the absorption spectrum of the guest and on their
forming satisfactory substitutional solid solutions. Electron spin
resonance studies have confirmed for instance that naphthalene at
least when in its triplet state forms perfectly aligned solid solutions
with durene(236,237) thus giving information on symmetries and frequencies
of spectrally active vibrations in excited electronic states. At 4.2 K
values < 1 cm-1 are commonplace. These narrow bandwidths have made
possible studies on the intramolecular mechanisms of electronic relaxation
in large molecules such as naphthalene and anthracene. The near-
resonance intramolecular coupling between a quasi-continuum of vibrational
levels of one electronic state and the discrete well separated levels
of an energetically higher-lying electronic state leads to a broadening
of the absorption linewidths of the discrete levels and the possibility
of additional structure. The broadening of the absorption linewidths
120
of the higher excited states relative, to the lowest excited state of
the one multiplicity appears to be a general feature of the aromatic
compounds. From studies on many P.A.H. and aza aromatics only azulene
is a notable exception. Frequently low temperature mixed crystal
studies provide the only means of obtaining the luminescence spectrum.
An exceptional example of this is the phosphorescence spectrum of
1,5-naphthyridine in durene which illustrates multiple sites, activity
of phonon modes and Herzberg-Teller vibronic origins. The emitting
state is a 3 Bu(7rr*) which is very intense because of spin-orbit coupling
with the I Au(nrr*) state and by spin-orbit vibronic coupling with the
higher l Bu(rrrr*) states. The lack of an inplane two-fold axis of
symmetry leads to two orientations and hence at least two sites for
the 1,5-naphthyridine in durene. The site splitting is 56 cm-1.
7.3. SHPOL'SKII MATRICES
Unfortunately, However, in mixed-crystal systems the upper energy
levels of the aromatic guest molecules frequently overlap the manifold
of energy levels of the _ _ host molecule, so that it is not often
possible to study transitions to the upper states of a guest molecule
in mixed crystals of the type described. Moreover, it is often not
possible to find suitable host crystals especially if the guest molecule
is a large and/or complicated aromatic species but because the conditions
of isomorphism do not apply in the exceptional Shpol'skii matrices which
are weakly interacting host crystals for aromatic solutes it is possible
to study the upper excited states of these solutes as if they were a
dilute cold gas with minimal interferences. They also have the additional
advantage that host absorptions lie above 65,000 cm-1 so that most
excited states can be studied. Shpol'skii matrices are usually formed
by n-paraffins although quasi-linear structure has been reported in
tetrahydrofuran,(238) carbon tetrachloride,(9) benzene, (239) cyclohexane;239)
iso-octane,(24o) methylcyclohexane,(241) methylcyclohexanol(241) plus
various polymers such as polyethylene.(242) How such a wide variety
of differing chemical and molecular structure arrangements can give
rise to sharp spectra is indeed an unusual phenomenon but the fact that
quasi-lines have now been obtained for more than 500 compounds suggests
that it is .actually of fairly common occurrence. Synmorphism i.e. analogy
of shape has been put forward as a major hypothesis combined with
121
'clathrate' like channel complexes. The canal complexes of urea are
a well known example of inclusion compounds. While urea ordinarily
crystallizes in the tetragonal system, when crystallized together with
a normal paraffin the urea is re-arranged into a hexagonal structure
and this allows a separation of normal from branched paraffins as only
the former with their straight zigzag chains turn out to fit without
hindrance in a channel of 51 diameter. Similar 'lock and key' effects
have resulted in the "sharpening" of quasi-line structure in P.A.H.
in equivalently dimensional paraffins. Many exceptions exist however
and recent additional Russian studies have shown that for benzene
homologues and other heterocyclics, the synmorphism of the matrix
molecules with the luminescent 'impurity' analyte is NOT always the
determining factor, whereas the essential role of energy transfer from
the matrix to the radiating analyte is confirmed (Chapter IX). Physical
properties are particularly important in these systems so that spatial
correspondences can play the role of chemical reactivity with the
cavities required to accommodate the 'analyte' often being imprinted
during crystallization (analogous to memory effects of specifically
adsorbing silica gels imprinted during gelation and subsequent drying,
Chapter X). Different prints can be re-established after careful
annealing also.
7.4. ALKANE CRYSTALLIZATION
Hexane and octane crystals are triclinic with space group (Pi)
with one molecule per unit cell. The alkanes are packed in parallel
layers especially well defined in both cases for the [101] planes
with an interplanar distance of about 3.5 A very similar to the 'spiral coil' separation in DNA.
The structure of heptane is not accurately known however it is triclinic
with 2 molecules in the unit cell having non parallel axes.(243)
Substitution of two host molecules is necessary in the case of peri-
condensed planar aromatics like coronene where the solute thickness
of 3.4 Ā fits in all alkane solvent matrices.(244)
122
7.5. SOME OBSERVATIONS OF THE EFFECT OF CONCENTRATION AND TEMPERATURE ON THE WIDTH OF NON-PHONON LUMINESCENCE LINES OF SEVERAL POLYNUCLEAR
AROMATIC HYDROCARBONS
7.51. INTRODUCTION
The Shpol'skii effect, in which well resolved fine structure is
observed in the luminescence emission spectra of polynuclear aromatic
hydrocarbons (P.A.H.) in selected n-alkane solvents at 77 K, may be
employed for the identification of these compounds in multicomponent
samples such as coal extracts, pitches, oils and waters with only minimal
pre-separation (Chapters IV, V, VI).
Certain difficulties however are encountered in the routine
application of this technique to quantitative analysis owing to the
occurrence of quenching interactions which restrict the linear dynamic
range of the technique and lead to interference effects in the deter-
mination of particular P.A.H. compounds in the presence of others.
We have therefore undertaken a study of the mode of appearance of
quasi-linear, non-phonon luminescence lines (N.P.L.) of P.A.H. compounds
at 77 K in n-paraffin solvents in order to investigate some of the factors
which contribute to their line-width and intensity.
The natural line width of an optical transition from a ground state
So to an excited state Si of lifetime TH is measured at 2-maximum
intensity and is given by the relationships vH = 1/ TH 27c.
Neglecting obvious instrumental parameters, such as the optical
resolution of the spectrometer employed, and apart from the Heisenberg
uncertainty principle, the other principal factors affecting the spectral-
width and shape of N.P.L. lines of P.A.H. compounds are:
(a) Temperature dependent effects such as electron-phonon(245,246)
interactions. The net result of the interaction of electronic motion
in a molecular impurity centre with vibrations of the crystal lattice
is, however, not just the broadening of the N.P.L. line but the appearance
of an adjacent broad phonon background emission.
This can be shown theoretically(247) by making use of standard
non-stationary perturbation theory and the Born-Oppenheimer adiabatic
approximation, as employed to interpret the Mossbauer effect and the
luminescence spectra of Inorganic Phosphors.
123
When the coupling becomes strong the phonon band becomes more
intense; this also occurs when the temperature is increased. In
the latter case, however, we also observe a decrease in intensity and
broadening and shift of the N.P.L. line. Such temperature studies are
being conducted in -h laboratory (with a variable temperature Cu cell),
and should aid in refinement of the interpretation of the nature and
origin of spectra obtained by the Shpol'skii effect.
(b) Discrete physical effects in the crystalline solvent, such as the
occurrence of interlaced spirals, folding and polytypism, which have
all been shown to be strikingly prevalent in the lower members of the
n-paraffins,(248,249) or to a lesser degree viscosity, polarity and
dielectric constant effects which may lead to dipole interactions
especially for excited state molecules.
(c) Interaction of the differently oriented molecules with their
immediate environment; these interactions may occur between the P.A.H.
molecules themselves, especially at high concentrations.
Richards and Rice,(250) in a study of absorption transitions of
coronene (in n-heptane), and anthracene and benzo(ghi)perylene in
n-hexane have shown that even in these favourable conditions their N.P.L.
linewidths were at least 3 to 4 orders of magnitude greater than the
natural linewidth due to the non-ideality of the crystal lattice and
its perturbing contribution to A V and k.
This chapter presents the results of an investigation of the
occurrence of variation in half-width of non-phonon luminescence lines
of several P.A.H. compounds in n-alkane solvents at 77 K with variation
in solute concentration; a possible interpretation of these effects is
also presented which draws on earlier work in this and related fields.
Some temperature studies are also reported.
7.52. Experimental
Apparatus The basic assembly of the spectrometer employed to
study the low temperature luminescence emission spectra of the P.A.H.
compounds examined was similar to that described previously.
An interference filter (peak 300 nm with a spectral 2-band width
of 30 nm) was used to select exciting light radiation from a 125 watt
124
medium pressure Hg vapour lamp (Philips type MBW/21). This radiation
was focussed into a light-tight sample cell compartment using two
silica lens (45 mm diameter and.75 and 50 mm focal length).
Luminescence emission spectra were recorded photoelectrically
using a 1 metre Czerny-Tuer mounted grating monochromater (Rank-Hilger
Monospek 1000, f8, reciprocal linear dispersion at exit slit 0.8 nm mm-1)
operated at 2000 V and an EMI 6256S photomultiplier by a Brandenburg EHT
supply. Slow scan speeds of 2.5 A/min. and entrance and exit slit-widths
of 0.1 mm, corresponding to a spectral half-band width of 0.08 nm, were
used for linewidth measurements throughout.
A commercially available dewar flask low temperature sampling
system,(American Instrument Co., Maryland) similar to that described
previously was employed. Sample cells of transparent fused silica
tubing (length 200 mm, i.d. 3 mm, wall thickness 0.5 - 1 mm) were used with this system. -Liquid samples in these cells were plunged into
liquid nitrogen contained in the dewar flask so as to achieve rapid
freezing, and the flask was then placed in the sample cell compartment
so that the incident radiation was slightly defocused at the surface
of the frozen sample.
For the temperature studies a unique variable temperature copper
conduction cell (described in detail in Chapter VIII) was utilised.
7.53. Reagents
The solvents employed were n-heptane and n-octane. The unsaturated
impurities in the n-octane solvent were reduced to a minimum by passing
over an activated purified alumina column.
Samples of pure polynuclear aromatic hydrocarbons were available
from the sources acknowledged in an earlier publication. In this
study the following compounds were employed: coronene, benzo(a)pyrene,
perylene, benzo(e)pyrene, benz(a)anthracene and dibenzo(ah)pyrene.
7.54. Study of the Effect of Variation in Temperature on Quasi-linear
Spectra at 77 K
For the temperature studies on benzo(a)pyrene, perylene and coronene
a unique, variable temperature copper cryostat cell was constructed.
125
By varying the applied voltage to the 'thermocoax cable' heater silver
soldered on to the back of the Cu cell assembly described earlier, any
temperature between 50°C and - 196°C could be selected. A thin wire
iron-constanto;n thermocouple gave a continuous monitor of the sample
surface temperature and equilibration for 3 to 5 minutes was made
before recording the_spectra either by slow wavelength scanning or via
the repetitive optical scanning and time averaging device. The latter
arrangement is described in detail in the next chapter and utilises a
rotating perspex block driven by a synchronous motor mounted in front
of the exit slit of the spectrometer.
7.55. Temperature Results
plotted graphical results show that in Shpol'skii systems the
effect of temperature increase on the more strongly coupled perylene
molecules leads to a large line shift and increase in phonon background
alongside the non-phc.lon line broadening for both the 0-0 purely
electronic and 0-1 electronic vibronic transitions, whereas the shift
and increase in the phonon band are much less for benzo(a)pyrene, a
weakly coupled system. A tenfold increase in background intensity
for perylene was also noticed on increase in concentration from 10-7M to 10-4M and at 10-4M concentration the background is roughly twice that
observed for benzo(e)pyrene. (Fig.7.1-7.4).
It has been reported that the phonon background follows a Gaussian
profile with broadening at a rate by -/T (255,256) but truly non-phonon
lines broaden at a rate sv >k2 (though for inorganic crystal phosphors
by is much less. Particularly near liquid helium temperatures the
intensity should decrease exponentially as exp - (T/9D)2 where the
important factor is the Debye temperature 9D, and the shape of the lines
can be envisaged as a superposition of a Gaussian and Lorentzian line
profile with an increasing proportion of the latter as the temperature
steadily increases above 77 K.
The noted convergence between the coronene doublet together with a
progressive reduction in the 443.36 nm / 444.76 nm line ratio with
increasing temperature observed here has led us to attribute this to
a redistribution of energy between crystal sites.
-1956 C FIG. (7.1 ) TEMPERA-CURE BROADENING PROFILES for 3,4 BENZPYRENE 4.03,(0-0) LINE
403 z ' 4.02 404 107 M in OCTANE OSCILLOSCOPE TRACES After T IME AVERAGING
Wuvetength i n A 404 • 403 492
o 0-
4 8 12 1 G 2C1 24 30 VOLTS
I 8
7
6
!c,
2
1
F1 6UFE 7.2
5
4
127
A TEMPERATURE STUDY OF THE 403 n m (0-01 '\ BENZ(a)FYRENE ;LINE
= HALF BAND WIDTH x = SAMPLE TEMPERATURE
~o =PEAK SHIFT I= INTENSITY
_O 44
-156 \ , 10
-160
-164
-168
-172
-176
-180
-184
- 16
i✓2
-195
-196 Ō ~4 a 1'2 16 20 ? 28 3?_ 36 40 44 48 50 VOLTS
128
-FIG 7.3)TEMPERATURE CHARACTERISTIC S of the 451.8nm (0-1) PERYLENE in n-OCTANE LINE
°C TEMP o =1/2 BAN D WIDTH r, =SAMPLE TEMP RATURE / 4
-108 PEAK SHIFT x 44 / / = INTENSITY I p x
I
I,
■
I
40
36
32
28
24
20
16
12
8
4
116
-124
-132
-140
• -14
156
-164
-17
-180
-188
1a9
FIG. 7 4 LORENTZIAN GAUSSIAN I Io I.Io exp[--b2 (you l2]
1ta2 ( - v0) 2
PERYLENE IN OCTANE
0-1
•
130
7.56. Concentration Effects
The presence of a broad diffuse phonon background often overlying
some mercury or xenon continuous scattered background (particularly for
weak concentrations less than 10-6 M whereupon the background also
becomes increasingly sloping) can lead to fairly large errors in the
measured half band width L. Therefore careful judicious choice of
a baseline method must be made so for B(a)P, for instance, after many
studies including digital counting at various wavelengths a diagonal
baseline between 402.2w and 404.2 was found to be the best although the
method used in the water work gave fairly similar precision. A particu-
lar advantage in these studies was the fact that we are monitoring the
luminescent emission as the phonon background can become fairly substantial
and asymmetrical in absorption studies. Another difficulty is encountered
when multiplets occur; unless fully resolved these can result in variations
of Av for specific lines. For example in the case of dibenza,`lpyrene
Cv = 4.5 ± 0.5 A at 10-6
Molar for the 431.5 nm peak but energy transfer(Fig.7.̀
and some degree of merging can occur from the 431.25 nm and 431.75 nm
side peaks. In the case of benzo(a)anthracene some more drastic
phenomena possibly microcrystallite formation occurs at high concentration
as the line distributions completely alter.
Perylene (Figure 7.6 )
Before studying the effect of the presence of perylene on the line
width observed for B(a)P in n-octane the former hydrocarbon was itself
monitored in the same solvent with excitation at 250 nm. No change in
w was observed although the rather high & value obtained of 9.5 ± 0.5 4
indicated that n-octane was not the most ideal matrix for this hydrocarbon;
hexane in fact is the best Shpol'skii host although A; values are still 0
relatively large (> 5 A) due to strong coupling with the lattice. This
coupling together with the small band pass of the 250 nm filter of 20 nm
leads to a loss of sensitivity for the perylene calibration curve which
also shows a small degree of concavity between 10-6M and 10-5M probably
due to pre absorption which shows itself again to produce a slight
positive deviation at even higher concentrations (> 10-4M).
431,5 nm
'II
43125 n ; fi
431.75
Fig (7,5) Slow scan 2.5$ /min specfrum across the finely re soived triplet of 34910 Di benzopyrene
Cone,
10~6 1a-5 ~o -G 10 10-7
132
Fig (7,6) Luminescence i ntensity vs.concentrat ,e
for Pe rylene 45 1Am peak
133
Benzo(a)pyrene, Benzo(e)pyrene and Coronene (Figures 7.6 - 7.13)
Loss of linearity in the calibration curve at higher solute concen-
trations becomes steadily more marked for benzo(e)pyrene, coronene and
benzo(a)pyrene in that order. Combined with this these P.A.H. compounds
also show an almost exponential increase in line half width with
increasing concentration. This effect is also particularly pronounced
for benzo(a)pyrene; the half-width in this case increases to greater
than 8 A at 10-4M concentration. The onset of line broadening in these cases occurs at concentrations between 10
-6M and 10-5M. The coronene half-
0 width values < 2.5 A are in fact the narrowest Shpol'skii lines we have
monitored; special pseudo liquid sites have been postulated for these
exceptionally sharp lines (p.r3G) 1.9-)
Benzo(a)pyrene and perylene mixtures in n-octane (Figure 7.14)
Because of the pronounced effects monitored with benzo(a)pyrene
it was decided to investigate the effect of perylen on .the Shpol'skii
luminescence of this hydrocarbon. An equimolar (10-7) mixture was examined; this showed a ca. 50% decrease of the B(a)P 403 nm peak
fluorescence intensity signal and yet no measurable change in the
half-width value for this line was detected.
These results suggest a simple inner filter effect due to the
absorption of incident radiation by the 402.6 nm perylene absorption
band.
7.57. Discussion
The deviation towards the concentration axis of the luminescence
growth curves observed at high concentration for these front surface
illuminated samples may be due to pre-absorption effects( 2 However,
the onset of broadening of the N.P. lines corresponds to the loss of
linearity of the calibration curves. This suggests the operation of
intermolecular interactions of the van der Weals type between analyte
species.
According to classical intermolecular theory(257) the forces between
molecules having permanent electric moments are made up of
(a) purely electrostatic interactions between the permanent charge
distributions of the two molecules which vary as 1/r2(where r is the
intermolecular separation distance) and are.therefore distinctly long
range forces;
41~'
Or' Fig (7.7) St owiy scanned spectrum of in n-octcine 13e nzopyrene
-o o
t>
4,4
4.3
4.1 2 10
4.0 10
135
4 10
4.2
10 3
U,-, .14-6 10 10 -4
M o lc r i ty Fig (7,FJ) \'cri Lion of lumin.:sc ncc intensity x und
. 1-1a1 f- band width I of 12 Denzct2yreno Garot,) pv-c.'.
Wi th conc.entfction,
10 -3
444.76nm
4
Fig (7.9) Slowty scanned spectrum 2-5 A /min of Cōronene /heptane
: 42•? nm
10
2.6
137
2.5
2.4
2.3
2.2
24
2.0
1-0
10 8 ~ō' 166 105 . 10
Fiq.(7io) Vctriution of luminescence intensity x and halt -- . - hwnd vtid~h I of .Coronene peak with conc2ntr-
at i o n,
1J
1.9.
29 .ted C
12.8
•1
10-1
,02
~0
105 1,r n3
2.6
2.5
2.4
23
22
2•}
2.0
138
F i c1.i~i~i ,'c. -iat ion of! UIT:in? =ccnce i n ten i ty x und hci i t -- b~~i ~! ',;k uh I of Corone 2 444.3 r wi th conccnir. at ion,
E29 3
3 -OR 3 n 19 E29
9
3E + 29
3 32v
3a « lu
50,000
40,000
30,000 —
20,CC
SINGLETS TRIPLETS
CO:.ONENE
GROJNZI STATE Al9
10,000
4
•
u
1E
139
v } 0
z
Figure-?:11 Singlet and Triplet Energy Levels of Coronene.
c.
c
72
70
6.8
6.6
6.4
6.2
60
58
56
5.4
5.2
5.0
c r
v C a
-r
x104
3 10
0
411.
76
74
140
10-8 10-7
iol(tr :tV
x —~
4.8
4-6
4.4
42
4.0
3.8
3.6
3.4
32_
3.0
Fig (7,1?) Vctriation of Lumir: SCCrice intensity (x.) and width , ne 403 ci k with lP•.'1 ntrat i C i
141
FIG. 7.13 BROADENING OF RiP 0-0 LINE
3
• (4) 4.1 It4)
CZ, 4, Half-band %,v1dth in A • 4".• J. 4.1 441 tZ.) 4:-.
tt) a:,
CD CD CD CD, Lurninesc.enc.c Intensity
143
(b) the Debye energy of induction representing the interactions between
the permanent charge distribution of 1 molecule and the moments induced
in the other molecule. These inductive forces vary as 1/r4, 1/r5, 1/r6
depending on the type of species involved.
(c) The London dispersive energy between 2 induced charge distributions
giving rise to short range forces which vary as 1/r7 and a potential
energy between the molecules which are attracted that decreases with
the sixth power of their separation. The quantum mechanical treatments(258)
of I.M.F. differs in 2 main respects:
(1) the orientations of the molecules are only partially specified by
the quantum numbers;
(2) there is often the possibility of resonance forces; which can play
an important role between two similar non-polar molecules and for which
there is no classical analogy.
Previous studies(259,260)
have shown that inductive resonance
interactions (o 1/R6) between the excited and unexcited molecules are
responsible for the radiationless energy transfer occurring efficiently
among biological compounds such as tryptophan, chlorophyll etc and
manifested in phenomena such as concentration depolarization, concentration
quenching of fluorescence and sensitized fluorescence. However, Ermo]cev(261)
deduced that observed sensitized phosphorescence(262) in crystalline
media at low temperature could only be interpreted in terms of exchange-
resonance interactions between a triplet donor molecule and an unexcited
acceptor molecule theoretically studied by Dexter(263)
(k ,A e-2R/L).
Exchange-resonance interactions must also take place from a singlet
excited molecule surrounded by unexcited singlet molecules of an acceptor
having a lower fluorescent level, or between like molecules if it has
a 3 level fluorescent system. These interactions are also most likely
at high acceptor concentrations such as could occur in liquid scintil-
lators or Shpol'skii systems(264) with molecular aggregation or in
crystals. Crystal and free molecule effects interfere with the
rates of electronic relaxation and thus the linewidths and crystal site
symmetry effects modify rules governing electronic transitions. This
leads to modern crystal theories, e.g. the Frenkel-Exciton Theory which
evaluates the excitation exchange integrals by expanding the intermolecular
potential energy function V in a series of inverse powers of R. But if
144
the molecules are neutral and possess transition dipole moments as in
the case with P.A.H. compounds then only the first non-vanishing
dipole-dipole interaction term is retained and this varies as 1/R3.
11,- 4-1Simpson(234) also found a similar relationship and the dependence
of coupling strength for resonance force transfer of electronic energy
in van der Waals solids. In fact in the lowest excited singlet state
of crystalline perylene the intermolecular interactions are comparable
to the electronic band width and thus, according to the Simpson and
Peterson criterion, the electronic-vibrational coupling falls in the
strong to intermediate category compared with a weak coupling for
benzo(a)pyrene.
Our results indicate some rather special properties of the B(a)P
n-octane Shpol'skii system. B(a)P has a three level system involved
in electronic excitation processes with energy values ideal for inter-
system crossing and at low temperature vibrational relaxation of S1* is
severely limited. In fact lifetime studies of microcrystalline B(a)P
are not inherent to the free molecule.
These facts lead us to postulate the possibility, of some almost
'sandwich-type' complex excimer being forced between an excited B(a)P
molecule in a good Shpol'skii site and an unexcited B(a)P molecule
which may have been unable to find a potential 'good' site due to the
lower solvent to solute ratio at high concentrations
Molecules of n-octane per / ml 3.7 x 1020
Molecules of B(a)P per ml at 10-7M = 6 x 101 10-4M = 6 x 1016
The nearest approach of molecules even in a unit cell for single
crystal P.A.H.'s is around 3.5 1 from x-ray Crystallographic Data(265)
and from various quenching studies in solid glasses and crystalline
media at 77 K;12 Ā to 15 A was the maximal interaction distance. This
suggests a likely range to base our semi empirical correlation curve
attempts of I.M.F. versus the N.P.L. broadening curve, which does in
fact best fit a 1/R3 rather than 1/R6 or exponential relationship.
This is an interesting correlation especially considering the utility
of a similar type of equation for explaining enhancement of phosphorescence
by aggregation of dye molecules.(266)
In the splitting of the
excited singlet state in general for parallel oblique and in line dipoles
145
O
10 9 8 7 6 5 4 3
Fig .7.15 Attempted cor retution using
Interaction c--c 1 1 R 3 x
11 ft b a • ei
146
pE = 2IMI2/R3 (cos a+ 3 cos29) where pE = E(S"-E(S'))
is the transition moment of 1 molecule, R is the distance between centres
of 2 molecules and a is the angle between the polarisation axes of the
molecule.
(a) A/ l
1 ~ A I \ I
I/
7----///% a 1 \ 1
/ \ /
I \ I
Case (b) can then lead to fluorescence quenching and there is
enhancement of phosphorescence for the aggregate. This may be
analogous to our high concentration effects which we believe result
from the occupation of not so well defined lattice sites by 'excessive
analyte molecules' or a 'sudden crush' in the layered paraffin matrix
which must allow more scope for dipole disalignments especially if
this effect is so severe as to form aggregates,267'268) microcrystals(153)
or sandwich dimers(269) (site effect dependency of intersystem crossing
for an anthracene-n-heptane Shpol'skii matrix was postulated by Ferguson
and Mau(49) who ruled out intermolecular energy transfer as improbable
for their dilute concentrations < 10-5M).
Further studies including phosphorescence are being undertaken on
dibenzo(ai)pyrene which has shown a more severe loss of linearity in
its calibration curve than benzo(a)pyrene. Both these molecules are
in fact potent carcinogens which operate by intercalation between the
base pairs of nucleic acids with which they seem to have a particular
analogous overall geometric area.
147
Their reactivity might be similar to the renowned reactivity of
the lower triplet state of dye molecules which as with chlorophyll can
also exchange singlet to triplet energy via molecular aggregation.
7.58. RESUMĒ
The implications of this work go further than trying to quantify
and extend the Shpol'skii effect as an exceedingly sensitive and
selective trace analytical technique for monitoring many of the main
environmental carcinogenic hydrocarbons in real samples with minimal
prior separation. More fundamentally this spectroscopic technique
allows a detailed study of the transfer and storage of energy in organic
molecular crystal systems which may in the future be extended to biopolymers
as the transfer and trapping of excitation in mixed crystals has important
parallels in the field of photobiology. Franck and Teller(~70) showed
the correlation with photosynthesis whereby light is absorbed by chemically
inactive molecules that transfer the excitation to a 'chemical reaction
centre' where the energy is stored and utilised at will. A few hundred
of the molecules serve a 'reaction centre' which thus plays a role
equivalent to that of the impurity molecule trap in a dilute mixed
crystal. The energy transfer is believed to occur through an exciton
mechanism(271) and thus here there must also be a futuristic but feasible
method for harnessing solar power.
148
CHAPTER VIII
INSTRUMENTATION FOR TIME AVERAGING AND TEMPERATURE STUDIES
8.U. Correlation
As limits of detection are lowered and weaker physical effects
are utilised to provide information the problem of discriminating
an analytically useful signal from extraneous unwanted signals becomes
increasingly difficult. Therefore we must make maximum use of their
two main distinguishing features, namely,
(i) the unique yet reproducible spectra of 'true signal' data,
(ii) the time occurrence or phase coherence of the signal frequency
components which can be controlled in a predictable manner.
Typically
Signal
0
Amplification
with
band width
control
Multiplication
by a
reference
signal
± Integrator
Reference
signal O
Reference
channel
control
Most modern S/N enhancement techniques involve a multiplication-integration
operation which is really signal correlation, i.e. multiplying one signal
by a delayed version of a second signal and integrating or time averaging
the product. When this time-averaged product is evaluated over a range
of relative displacements a correlation pattern is generated. If
correlation is carried out with continuous functions, it can be mathe-
matically described by the following integral
1 r +x CCab (± T) = lim 2x ; a(x)b (x ± T) dx
X -+ m -x
where CCab is the correlation pattern of the two signals a(x) and b(x)
and T is their relative displacement.
149
Practically correlation is done,on sampled waveforms and is then
best described by the following summation
CCab (± nAx) _ Exa (x)b (x Ax), n = 0,1,2,...
where Ax is the sampling interval. The relative displacement is n Ax
and is identical to T. Correlation of two signals is equivalent to
multiplying their Fourier transform
CCAB (f) = A(f). B(f).
This equation, however, may also be used to describe electronic
filtering, where the effect of a particular filter on a waveform can
also be described as a cross-correlation between the waveform and the
Fourier transform of the transfer function. The mathematics is quite
complex involving both real and imaginary Fourier transformations.
8.12. D.C. Amplification
• In the simplest d.c. amplifiers used to amplify our photomultiplier
output the time constant T is determined by the resistor capacitor
product (RC) which determines the noise band width, Af. Hence
Af (4T)-1 = 4 RC-1
Various difficulties accrue with this detection system because of the
many extra d.c. signals such as the dark current, the background current
and the noise, etc which can only be partially removed by the use of
a backing-off device.
8.13. Integration -Table 8.1..
Integration improves S/N ratios because the frequency components
that make up a signal add in-phase and the noise frequencies add
randomly as they are not, in general, phase related.
Integration of d.c. signals is accomplished in two basic ways.
Active and passive low pass filters can be used where the RC time
constant is typically much larger than the integration time. The
other main approach is to use an integrating digital voltmeter such
as those employing voltage-to-frequency converters. Two modes of
integration, namely constant time and constant charge, were investigated
in detail with de Lima, both being shown to lead to improved precision
of analytical procedure.
Table 8.1
Relative standard deviations obtained in measurement of luminescence
of benzo(a)pyrene in octane at 77 K with instantaneous and d.c. integration techniques of signal registration.
Concentration/M
1 x 10--4
1x10-5
Relative standard deviation, %
Direct read-out Integration Integration for fixed to constant time period charge
2.5 3.0 - 3.o 3.o
5 x l0-6 9.o 4.6 0.9 5x10-7 25.o 3.o 1.4 1 x 10-7 9.4 0.7 5 x l0-8 5.5 1.6
2.5 x 10-8 6.o 4.4 2.5 x l0-9 4.4 6.o
8.1,4. Signal-to-Noise Enhancement
One can enhance the S/N ratio observed in a measurement by
(1) Low Pass Filtering, or
(2) d.c. integration.
Difficulties still arise due to the measurement bandwidth being
centered at d.c. (OHr), which is the region of maximal 1/f noise and
thus the range of signals to which integration may be applied is
essentially limited to d.c. or relatively slowly changing signals
because otherwise the long term drifts introduced would interfere.
Severe distortions can also occur when applying these techniques to
continuous signals where the parameter of interest is being scanned
as a function of time. Thus the development of lock-in amplifiers
and boxcar integrators which can overcome these limitations to a large
extent has considerably intensified during the last decade.
150
151
8.21. Photon Counting
The current pulse output of our photomultiplier tube was directly
integrated by counting.
The output of the PMT is amplified with a pulse amplifier and
then all pulses greater in amplitude than a pre-set discrimination
level are counted for an accurate preset. time interval. This thus
becomes the integration time. For a random phenomenon such as
photoelectron emission, the standard deviation of the total integrated
count is the square root (IC) of the total count, thus the precision
should improve as the /Time as with other integration methods.
Photon counting generally allows long integration times with
little or no interference from 1/f noise an additional advantage from
a clear signal point of view but perhaps time inefficiency will limit
this applicability in analysis. Background effects must also be taken
I -
into account and so a more realistic equation due to F~Nkl;N~Hor1;cka,..,~j~„m, r
was found to agree more closely with experiment,
(R T)2 R T2 namely S/N =
s s
(2RB/R6)2 2RB2
where Rs is the signal count rate, RB is the background count and T
is the total counting time.
8.??. Experimental
A simple single-channel system (Model 300, EDT Research, London)
having a capacity of 107 counts was used for the photon counting
experiments and the output from the photomultiplier was developed
across a 50 ohm load resistor. A pulse-pair resolution of 10 NS
was obtained with this system. The EMI 6256S photomultiplier tube
used for the photon counting experiments had low dark current (at
ambient temperature ca 40 counts s-1 and at dry ice temperature ca
4 counts s-1). A specially constructed cooling chamber based on the
design of Sharp(273) was used for cooling the PMT during experiments.
A constant flow of dry nitrogen was circulated around the PMT to avoid
condensation of atmospheric moisture during the cooling or the warming
cycle.
152
Figure 8.1. shows the luminescence growth curves obtained for B(a)P, utilising both photon counting with five second counting times alongside analogue signal registration. Both readouts correspond to the net difference between the signals obtained at the luminescence maximum (403.0 nm) and for the background at 400.0 nm. A similar range of linearity is observed with each system (i.e. in the concentration* range 10-7 - 10-9M). Although the growth curves might suggest that an improvement in detection limit is obtainable by photon counting, the determination of the relative standard deviation for replicate measure-ments of low concentrations indicates that, in these high background situations, the limit of detection for the compounds investigated is similar for photon counting and analogue techniques but the overall precision obtainable with photon counting is superior. Experiments with various counting times indicated that that necessary for significant improvement in detectability was unacceptable for routine practical application.(Tzbles ō.2,8.3)
Table 8.2
Precision of photon counting and analogue signal registration for luminescence of P.A.H. compounds at ?7 K
Relative standard deviation, % Compound Concentration Photon counting Analogue
Benzo(a)pyrene 5 x 10 - 46 32
1 x 10-9 2.0 25
1 x 10-8 1.3 5.3
1 x 10-7 0.5 3.9
1 x 10-6 0.2 1.6
1 x 10-5 0.7 1.4
Dibenzo(ai)pyrene 1 x 10-9 44 44
1 x 10-8 5.0 6.2
1 x 10-7 o.6 4.4
1 x 10-6
0.3 4.0
1 x 10_5
o.6 2.2 1 x 10-4 0.9 1.1
153 •
Molarity
Fig. Comparison of analytical growth curves for .benzopyrene obtained with photon-counting and analogue signal registration: A. proton count-ing (cou::t. obtained for 5 s count ti=e); and B, analogue read-out (my).
10-4
100 10-10 10-9 10-a 70-7 10-5 10-5
`e 104 V
ō 103
J i02
101
Table 8.3
Variation in signal to noise ratio with counting time
for benzo(a )pyrene
Counting time/s Signal to noise ratio
5 10
10 15
15 19
30 26
60 36
8.3. LOCK IN AYPLIFIERS
The lock in amplifier is an exceptional example of the advances
in electronic processing of small signals.(274) it is an instrument
using some time averaging for the extraction of the amplitude of a.c.
signals which are present with large noise levels. In operation the
a.c. signal is amplified and then rectified (or demodulated) by a
switching signal of the same frequency as the information signal so
that a d.c. output is obtained. The d.c. output is proportional to
the a.c. amplitude of the information signal which is at a single
frequency and receives essentially no contribution from all other
"noise" frequencies. Some signal averaging takes place in the process
as the d.c. output signal is low-pass filtered. In fact a simple
RC filter is the simplest kind of signal averager.
8.41. Signal Averaging (A, Plate 2) (6155)
The main purpose of signal averaging is to improve the reproducibility
and precision of the measurement of a waveform. This is done by
accumulating and superimposing a large number of repetitive measurements
so that the signal increases with slope 2 with the number of repetitions
whereas random fluctuations will algebraically add to zero. Therefore
it is imperative to ensure that (a) the signal is as pure an additive
component as possible. Obviously the averaged signal can be no better
than the best error free signal; hence, one must remove inherent
additive systematic error contributions as much as possible by good
154
156
design of the apparatus anS. then, if still required, e.g. a single
beam instrument, by a separate background or blank measurement.
Unwanted signals include drift, d.c. offset, dark current and background
intensity which can be removed only by signal averaging if they can be
separately measured or isolated from the measurement. (b) The
averaging must be carried out over a sufficiently large number of
repetitions to ensure a random distribution of fluctuations.
8.42. Method
The method of signal averaging is to sample and digitize a signal
at frequent intervals and store the array of measurements from a single
sweep in a multichannel memory. In this way after a large number of
sweeps the signal or regularly occurring information will build up to
a large value whereas random fluctuations will accumulate less rapidly
and add to a small value (theoretically negligible) in comparison.
Thus if enough measurements are taken to obtain a r-presentative
average signal then the S/N will be improved in proportion to /N.
If however there is a large coherent noise component present it will
add along with the signal and there will be no net gain by signal
averaging.
8.43. Gate and Sweep
A given signal may be averaged by overlaying successive sweeps
N times so that each point on the waveform is measured N times. Each
point, however, has some finite time period which is called the gate,
and a single sampling of one point is really an average over the gating
time or dwell time.
One can also decrease the noise by integrating during the dwell
time which will decrease the high frequency noise in proportion to /r
where T is the dwell time. Selection of the optimum sampling time
for a given application is determined by the trade-off between the S/N
ratio improvement required and the time inefficiency that can be tolerated.
The narrower the gatewidth the greater the resolution, but also the
greater the number of repetitions required for a given output S/N ratio.
Many salient features are illustrated in the graphical results illustrated.
..'
157
388.1
388.1
Readout after 4 K averaging
384.6
Fig. 8.2. Benzo(e)pyrene Fluorescence. Analog spectrum with
refractor plate in place but at right angles.
158
Fig. 8.3. Dibenzo(ah)pyrene (10-7M) Time Averaged (1 K)
readout (a)
(b) 8 K readout
40 0 nm 85
Transmission
370nm
Fig. 8.4 Transmission Characteristics of a Perspex
(14 mm) Refractor Block
- 5% T
340 nm
160
8.41i. An Evaluation of Detection Systems
Experiments on the use of signal averaging were made with a
1,000-channel signal-processing system (Unimax 4000, Data Laboratories
Ltd, Mitcham, Surrey). This apparatus has an on-line digital processing
system for the storage and analysis of waveforms in both the time and
frequency domains. The instrument is modular and has four basic units.
The first unit is a memory with 1024 words and the second is a sweep
timer, which generates timing pulses so as to control the rate at which
the data is sampled during analysis. Delay times after each sweep
(from 1 µs to 999 ms) and before each sweep (from 1 ms to 999 µs) can
be selected. A programme unit is provided in which not only single
channel averaging is possible but also multi-channel averaging, as four
single-channel inputs, which can be averaged simultaneously, are avail-
able. A display control module provides for the selection of the
number of sweeps in intervals of 2x from 1(2°) to 16 384(214).
In order to produce rapid repetitive scanning of small wavelength
ranges in the luminescence emission spectra various assemblies were
utilised. Primarily, an oscillating transparent refractor-plate
mechanism; the operation of which is similar, in principle, to that
of those proposed by McWilli m,(275) Roldan(276) and Snelleman et al.(277)
was constructed. This initial assembly was located immediately behind
the exit slit within the monochromator.
A frequency of oscillation of about 5 Hz giving a maximum angle
of incidence of about 12° was used in the experiments. As the refractor
plate, a polished, polymethylmethacrylate (perspex) block of path length
25 mm sufficed being transparent from 345 nm throughout the visible
region (Figure 8.4). This plate was driven by an oscillator circuit
constructed in our workshops but similar to that described by Snelleman
et al.(277) by which the amplitude of oscillation could be controlled.
A simple pre-amplifier (of gain 50) was constructed and used between
the photomultiplier and the signal averager. The signal being averaged
was continuously observed at a display oscilloscope (Hewlett-Packard,
Model 175A) and after averaging was either photographed or point plotted
by using a potentiometric chart recorder. Although these studies gave
a more precise detection system some practical difficulties arose;
161
namely, the phosphor bronze strips on the oscillator had only a
limited lifetime (usually between 4 and 5 hour and often became
'wallowy' which led to imprecise overlay of repetitive scans leading
to further loss of resolution and inefficient averaging, thus it was
often noticed that after a few scans the resolution was better than a
prolonged average. Another deficiency was the limited wavelength
scan of 2 - 3 nms; this was not really sufficient to make the system
analytically useful from a time point of view, especially considering
the long times involved in the averaging process.
The evaluation of the lateral displacement d is not necessarily
. as straightforward as many publications state. This is so because
when a parallel plate is placed in a diverging beam of a point source,
a point image will result only if two conditions are satisfied.
(1) The plate must be perpendicular to the axis of the beam.
(2) The beam angle must be such that all the rays can be considered
to be paraxial. When the plate is tilted with respect to the beam axis
there is no single image that one can speak of. In fact, for every
point source two virtual astigmatic line images are produced. These
images are parallel and perpendicular to the plane of incidence and are
known as the 'tangential' and saggital images. Besides, for angular
positions of the plate for which the paraxial approximation is not
satisfied the presence of severe spherical aberration will cause further
degradation of the image. The positions of these virtual images can
all be calculated from applied optics. (278 )
Our main interest is simply the lateral displacement, d, at an
angle of incidence, 9, for a material of refractive index, N, and
thickness, t, at the angle of incidence, 9, (Fig. 8.5)
(a) therefore d = t sin 9 ' cos 9
\ 1 " N 'cos 9 which reduces to
l (b) d = to
N N - 1 "\; for small incident angles (less than 10o
),
when the scan will be virtually linear. Thus our perspex refractor
plate ND = 1.49 of 25 mm thickness at maximum oscillation of ± 13°
gave a limit of 3 nm on the wavelength range scanned.
A very similar system was obtained by changing the demountable
perspex block and replacing it with two fluorescence cuvettes which 143
were then filled with glycerol (N = 1.43). This leads to a very flexible
162
system whereby by changing the liquid various scan amplitudes can be
achieved and also automatic light filtering may be achieved by the use
of coloured liquids; some defocusing was noticed thus the Hg triplet
was not completely resolvable but by grinding some curvature on the
perspex and slight defocusing of the incident light complete resolution
of these triplets was achieved (Figure 8.6).
With the initial oscillator scan system 16 x 1024 sweep counts
using a sweep interpoint time of 120 µsecs would take 55 minutes.
The great advantage of using refractor plates to create a rapid
scan, however, is their convenience; unlike vibrating slits or mirrors
little adaptation to the usual monochromator arrangement is needed as
the refractor block is just an inexpensive addition. Therefore we
decided to persevere with a perspex refractor plate but to use a syn-
chronous motor to rotate the block completely, thus enabling a fast
but large (more than 18 nm) scan width. A 3000 r.p.m. motor (Evershed-
Vignoles Limited, London W4) allowed a fivefold time efficiency improve-
ment over the oscillating plate system. Thus 16 K sweeps required
ca 10 minutes with this system and 1 K just 39 seconds. This rotating
block therefore performs 10 resolutions in 55.55 psecs so using a 7 sec
interpoint time this leads to a total sweep time equal to 1024 x AT,
namely 7.168 milliseconds. (B in Plate 2).
This total gate width represents a total angle of 129.6° used to
derive information; really only 74° of that, corresponding to approxi-
mately 12.8 nm, is of practical use, partial and then total cut-ofi' occurring over the other portions (Figure 8.7).
To achieve this a very last interpoint time of 4 µsecs was required
but even 6 µsecs proved to be too rapid for the averager which then produced an extremely noisy spectrum (Figure 8.8).
Obviously the frequency components of the signals were changed (Fig.8.9)
using this faster scanning system and so the pre-amplifier had to be
re-optimises, between 5.5 and 8.2 kHz being found to be the best for
the rapid risetimes involved other difficulties of transmission cut ofi
at angles greater Lhan 50° and the variations of refractive index with
wavelength also became apparent during trials with this system. Also
owing to the large depth of the block a substantial increase in the
d in mm
d.t sin81-~cos0 ncos9Ō
r
d teCn n1
10° 20° 30° 40° 50° 60° 70° 80° 90° ® in °
24 22 20 18 1G 14 12 10 8 6 4 2
0
Fig. 8.5 Comparison of lateral displacement equations for a refractor plate incident / 0 , thickness t
and R.I. = n.
Ca) Cb)
Fluorescence cells filled with glycerol as refracting medium
ordinary oscillating perspex plate
4
Fig. 8.6
(c)
Improved resolution of oscillating curved thin refractor plate.
Notice that wobble of phosphor bronze strip leads to some noise and non reproducible forward and backward scans.
rim
change in separation of Al doublet
(c,x
)X x i
X' X
(d)/
x
(a)
x
(b)
X
X
11
9
8
7
6
5
4
2
1
380 384
388 392 396 400 404 408 412
,changing incident L on diffraction grating
Fig. 8.7. The effect of changing central wavelength of monochromator on the separation of an Al Hollow Cathode Lamp doublet. This shows clearly the regions of (a) linearity, partial (c) and total (d) cut off as well as the variations of transmission at various incident Ls (b).
r
10 µ,sec
1
Fig. 8.8. Scan of an Aluminium Doublet (396.2 nm and 394.6 nm)
using various
slits 0.0125 nm, IIIT 1400 volts,
Pre amplifier 5.5 kHz !1
Double range of
2 rotations of
perspex block
166
N
15 µsec
J
0.8 K Hz 3.1 K Hz
I
167
5.5 8.2 10.8
Fig. 8.9. Effect of Pre-amplifier on Mercury Triplet calibration
of rotating Perspex refractor plate.
168
Figure 8.10. Variation of Scope Readout of Time Averaged Benzo(e)pyrene) Spectrum with different grating /s
Central X on
monochromator
while averaging
(lx) * 388.1 nm (0-0) line
169
Figure 8.11. Time Averaged B(a)P signals showing the inversions in
magnitude the Hg 404.7 ratio with change of concentration
B(a)P 403 • 10-7 10-7
10-6 B(a)P
Hgt
Figure 8.12. Coronene Phosphorescence using Time Averager and
Rotating cam simultaneously
170
171
optical path length of the monochromator was introduced, 8.5 mms; this
led to loss of resolution of the multiplet. A 2 mm plate introduced
0.66 mm displacement in a 0.75 metre grating, i.e. 0.09% of f the focal
length but in our case in obtaining a large scan this was increased to
0.85% of f. This was corrected in the stationary mode so the mono-
chromator could give normal spectra by grinding a curved surface on the
perspex block using a 150 mm diameter shallow iron tool (Carrington
Optical Engineering Co., London). A slurry of silicon carbide on a
rotating chuck sufficed for the grinding, polishing being done with
cerium oxide powder.
However, beyond a certain angle of incidence severe aberrations
took place and because the speed of the motor could not be varied,
the system was thus limited. A large refractor plate also cuts down
on signal transmission (ca. 4% is lost at each surface) especially at
high angles of incidence. According to Fresnel's formulae this can
be > 25% loss for 0 - 45°. No improvement in detection limits was
achievable either; thus refractor plates, unless one could use thin
slabs of high refractive index but of good optical clarity and trans-
mission (e.g. diamond or some silicones), appear to be rather limited
here in single beam front surface spectroscopy.
8.5. ALTERNATIVE REFRACTOR PLATE SYSTEMS
Shaklee and Rowe(279) have used a spectrometer for studying the
optical properties of solids in which wavelength modulation was achieved
by an oscillating refractor plate based on the technique suggested by
Dreors.(280) Perregaux and Ascarelli(281) meanwhile observed that with
a single beam system in wavelength modulated reflectivity a large
contribution to the signal was due to the wavelength derivative of the
'Io' incident intensity. Therefore, in order to realise the full
potential of the wavelength modulation technique care must be taken to
eliminate the contribution of the incident light intensity to the
modulated signal. The latest utility of such a plate was as a method
of automatic wavelength calibration in a computer controlled multielement
atomic fluorescence/emission spectrometer by Malmstadt and Spillman.(282)
This was achieved via direct control over the quartz plate's rotation
by monitoring the output of the P.M.T. tube as a function of plate
position, whereby the computer can use feedback.
172
Most uses though entail derivative spectroscopy(283) which, by
sacrificing some sensitivity allows extra structural information to
be achieved.(284) Snelle,mānhas reported that with a thin quartz
plate (12 mm thick) by taking the second derivative high background
emissions may in some cases be eliminated although the actual signal
intensity is reduced by ca one third with that attainable utilizing
a disc chopper. O'Haver with Green(285) recently reviewed nil methods
including electronic, mechanical and numerical for obtaining differentiation
of signals.
8.6. CONCLUSIONS
The major inhibition to improved detection limits throughout
these various digital detection readout systems, however, has been the
continual presence of phonon band background which is complicated and
overlaid at low analyte concentrations by scattered radiation. This
is always a difficulty, especially in front surface emission spectroscopy
but particularly in this technique where polycrystalline samples are
examined. This is magnified in our experimental system by the use
of rather crude, fairly wide band pass excitation via interference
filters.(F.'s "3.11 0.x•1 53.00
Various alternatives for improvement would be
(a) by the use of polarisation techniques. Even with polaroid film
on the perspex scanner, a 33% reduction in Hg scatter was achieved at
445 nm but signal intensity is also greatly reduced.
(b) A second monochromator. This severely limits the light intensity
reaching the sample and thus more powerful 500 watt
excitation lamps are also required.
(c) The better, though expensive, alternative, is the use of a tunable
dye laser. Pilot studies have already been recorded (Chapter II).
(d) A possible method of reducing background effects is to use a rotating
interference filter as shown by Hieftje.(286)
(e) A background correcting integrating circuit interfaced with the
readout. A feasible system constructed by Anino and Jordan(287) for
use in chromatographic baseline problems could well be adapted to tackle
some of the background problems encountered here.
173
(f) Undoubtedly a corrected automated spectrofluorimeter is required
in the complex ultra violet region.
8.7. VARIABLE TEMPERATURE CFT.T,
It was decided that a variable temperature cell used in conjunction
with time averaging would be very useful for a wide variety of practical
and theoretical reasons, namely
(a) Accurate temperature control thus allowing
(b) variable sample preparation including 'optimisation of' freezing
rate and annealing to which Shpol'skii systems are particularly sensitive.
(c) To study the fundamental nature of non-phonon luminescence lines
and their interdependence upon temperature.
(d) To determine line shapes and broadening processes which via
utilising an accurately temperature controlled cell would allow more
meaningful results in conjunction with our precise time averaging readout
detection system.
(e) To monitor and accumulate data on crystallisation, phase and glass
transition temperatures.
(f) To study dimerisation, excimer and microcrystallite formation, all
of which are very susceptible to variations in temperature.
(g) Future uses on various different matrices including polymeric
emission and particularly with the idea of following protein conformation
changes and enzyme reactions.
(h) To observe combined photochemical changes by the effect of both heat
and ultra violet light.
Most of these studies are physical in nature but many may have
analytical potential and they are all extremely relevant to improving
our knowledge of molecular luminescent properties.
Various practical considerations in the design of such a multi-
purpose cell had obviously to be considered. Thus it had to be robust
yet optically good, easy to dismantle and handle. Above all,
flexibility particularly as regards handling of different sample types,
silicates, gels, films, liquids, powders etc was of utmost priority.
Figure 8.15 shows the cell which incorporated many of the improved
adaptations of the copper conduction cell described in detail in Chapter II.
FIG 8,.13 VA RI ABLE TEMPERATURE - COPPER CONDUCTION CELL
174
r
0.75 I
0 11 45
I T
., O
ii •
.---1.25`' 3
QUARTZ ,MICROCELL
THERMOCOAX CABLE
1I
. f1
. .
1
17 5' I I I I I I
I it, 0 .
■
SYRINGE FILLING TUBES
Y
175
Namely, syringe type connectors allowing easy filling and flushing
without contamination yet flexible dismantling to accommodate silicates,
powders, strips of polyacrylamide gels or indeed with the removal of
the microcell altogether, pieces of doped perspex. Heating was achieved
by silver soldering a multiply 'concertinered' strip of thermocoax
wire (Phillips type SI.x ) Thermocoax is a special mineral
insulated co-axial heating cable consisting of a centre core along which
the current is passed, a concentric, layer of magnesium oxide and an
overall sheath of stainless steel. By soldering into the copper with
silver a very efficient heat transfer was achieved. Neat clips attached
to carefully stripped portions of the central cores allowed contact
with a rheostat via attached wires.
An applied voltage of forty volts allowed an equilibration
temperature of ca. -140°C to be attained with the cell body filled with
liquid nitrogen. When used in its Shpol'skii mode, i.e. with the quartz
microcell attached, very good insulation was needed between the aluminium
mask on one side and the copper frame on the other. A thin loop of
asbestos wool was used for the primary purpose with car gasket material
being found most suitable for the latter although cork or rubber could
also be used.
8.8. TEMPERATURE STUDIES
Glasses have an arbitrarily 'frozen in' configurational disorder.
This disorder is established when the liquid is cooled quickly past
its glass transition temperature, Tg (the temperature where its viscosity
passes 1013 poise). However, glass formation occurs over a range.
Large molecules at such viscosities neither flow nor rotate, and so
observed changes in viscosity are thought to be due to a reorientation
of parts of the molecules to more stable positions, i.e. configurational
relaxation.
Some glassy media even at 77°K where molecular rotational relaxation
and diffusion rates are very slow, still exhibit small structural
changes.
For any glass the Tg value is a temperature at which a sudden
change in viscosity occurs. At temperatures 30°C or more below Tg,
176
glass relaxation effects are of negligible importance. Thus, if the
'coolant is liquid nitrogen' one would like tō choose for analysis
purposes solvent glasses having Tg > 110°K. Unfortunately, Tg data
for organic glasses are rare. Ethanol has Tg 90 - 95°K, methylpentane
Tg = 88°K but propanol is good, 120°K, and dimethylhexane is 103°K.
Free radicals, cations, anions, electrons and complexes produced photo-
chemically or by ionizing radiation in organic glasses decay at rates
which depend on the nature and temperature of the trapping matrix as
well as the guest species, but there is as yet very little definitive
knowledge as to what property or properties of the matrix control the
decay rates. For instance the initial decay rate of ethyl radicals
in 3 methyl pentane glass at 77°K (25% in ca. 3 hr) is much longer
than that in methyltetrahydrofuran (25% in ca. 20 minutes) and the
half-life of the trapped electron varies similarly.
It is known that small changes in the local environment effect
the lowest triplet state of benzene. It is probable that the radiation-
less rate constant is being changed. This is largely dependent upon
the Franck-Condon overlap factor, which itself may vary with the
vibrational frequencies or with a distortion of the molecules. This
latter point is emphasised by the distortion of benzene when placed
in a site of symmetry lower than D6h. Thus certain freezing methods
for glasses can trap molecules in strained sites which may then be
altered by annealing. An interesting structure is thus observed for
carbazole in the next chapter by annealing procedures.
Studies on the viscosity of methyltetrahydrofuran at 77°K have
demonstrated that the macroscopic properties responsible for such
viscosity are not closely related to the molecular processes necessary
to allow radical decay(289). Other workers have also recently shown
the consequences Of electron tunneling and diffusion in the temperature
dependent recombination fluorescence of photoionized indole and
NNN.'N'-tetramethyl-p-phenylenediamine in organic glasses ranging from
ethanol, propanol to methylcyclohexane and 2-methyltetrahydrofuran.(290)
The initial intensity and decay rate of the recombination fluorescence
decreases as the u.v. irradiation temperature is increased from temperatures
below the glass transition temperature Tg of the matrix. This is
177
interpreted in terms of electron tunneling to the cation in which the
tunneling barrier height or electron trap depth increases slightly
(0.05 - 0.2 eV) with increasing irradiation temperature. By considering
how the matrix polarity affects the degree of electron trap depth
deepening as well as the depth relative to the excited singlet level
of the solute, it is possible to understand the difference in magnitudes
and their changes for the initial decay rate and the recombination
fluorescence. At temperatures 10 - 30 K above Tg,depending on the
matrix polarity diffusive recombination dominates that occurring by
tunneling and produces a peak in the fluorescence,unless the electron
trap depth has dropped below the excited singlet of the solute.
These studies are of particular importance as regards our work on
tryptophan and indoles etc in polyacrylamide matrices in the final
chapter of this thesis. Thus photoionization thresholds for tryptophan
and indole are comparrble by this method and may help to explain to
some degree our difficulties in achieving luminescent structure with
these polar amphoteric molecules in comparison with indene for instance
for which a quasi-linear spectrum was obtained.
Environmental non-uniformity has especially striking effects upon
the phosphorescence of a solute if the compound possesses two low lying
triplet states separated by a small energy difference. This situation
is widespread in heterocyclics and carbonyl compounds, in which low
lying (nut) and (Tut) triplets are often separated by very small energies.
This is encountered when xanthene is dissolved in EPA or mixed hydro-
carbon-alcohol solvents whereupon two distinct phosphorescences with
different decay times are observed because some xanthene molecules are
entrapped in a hydrocarbon region while others are ensconced in an
'alcohol' domain. Effects of this type have been reported
for some very common phosphorescent molecules including indole, tryptophan,
2-naphthol and pro flavin.
Problems of microenvironmental heterogeneity are most serious
when both solute and solvent are polar. This is made worse when a
complex mixed solvent such as EPA ethanol, is used as the solvent.
Even in relatively simple solvent mixtures such as 80% decanol 20%
cyclohexane we have monitored various inflexion points in the slow
178
cooling curves indicative of slight structural phase changes. Thus
it is extremely useful analytically to be able to anneal the samples
so sometimes enabling the analyte molecules to take up less strained
positions within the 'matrix' host resulting in a cleaner vibrational
spectrum. Crystallinity is another important aspect of this work as
many polymers and glasses have varying often small degrees of short
range order. Birefringence studies using a polarising microscope
indicates this degree quite well while more detailed studies on polymers
may be undertaken by X-ray diffraction.
We have also found that even our polycrystalline matrices typical
of Shpol'skii systems undergo various subtle phase changes. For
example cyclohexane and tetrahydrofuran both show inflexion points
before and after their crystallisation temperatures. Iso-octane also
reorientates itself at 150°C to give a polycrystalline snow which then
shows quasi-line spectra whereas above 150°C the matrix is more glassy
in appearance. (This more open structure also allows, incidentally,
much more efficient oxygen diffusive quenching). A similar effect
was recently reported for fast and slow freezing of iso-octane leading
to slightly adjusted structure of quinoxaline quasi-linear spectra,
because of low and high temperature (respectively) modifications.
In methylcyclohexane slow freezing also gives a crystalline matrix
but here rapid freezing leads to the production of a glass matrix
similarly with decanol where inflexion-points are shown monitored
with the thermocouple.F;95 g.u, $•«).
Differential thermal analysis would allow a more detailed analysis
of the latent heats of fusion of paraffinic matrices, as they solidify
and undergo phase changes.
final T
-191° C
cell warms on transfer
slow freeze
-196°C
FIG. 8.14.
TEMPERATURE PROFILES FOR
TE T RAHYDROFURAN IN THE
COOLED COPPER CELL .
fast) freeze
179
•-446 •(b) " B ir
Coronene ottP i) ne in Decano[ •
_102.8°-
181
CHAPTER IX
NITROGEN HETEROCYCLICS
9.1. SPECTROSCOPIC EFFECTS ASSOCIATED WITH NITROGEN HETEROCYCLICS
In earlier chapters some specific applications of low temperature
luminescence spectroscopy utilising the Shpol'skii effect have been
demonstrated; it appears to be not only useful as a selective analytical
method but in the monitoring of intermolecular forces as well, thus
allowing further understanding of energy dissipative processes.
Obviously, one may also investigate intramolecular vibrational effects
and matrix isolated free radicals. In this chapter, however, particular
unique advantages of Shpol'skii quasi-linear spectroscopy are shown.
Namely, this method has enabled the author to trace the fine spectral
shifts associated with introduction of nitrogen atoms into an aromatic
polynuclear system, thus allowing corresponding energy correlations of
particular reactive sites.
A nitrogen atom introduced into an aromatic ring does not disturb
the 77 conjugation significantly and has negligible effect on the shape
of the molecule and the dimensions. In larger polynuclear ring systems,
however, the nitrogen atom leads to 'flipping', leading to non-
planar conformers particularly of larger heterocyclics, whose three
dimensional stereochemistry is presently being well documented, plus
some degree of resonance exchange. In fact, on studying simple
nitrogen heterocyclic molecules many points of controversy which are
pertinent to the fundamental origin of quasi-linear spectra arise.
Thus, many heterocyclics give a complicated multiplet so for measurement
of spectral line shifts to derive electronic data it is necessary to
utilise the most intense absorption and emission resonance line;
this often may require selective excitation optics. Secondly,
whereas the ordinary aromatic analogues are mostly planar structures,
heterocyclic stereochemistry shows the multitude of possible conformer
permutations possible so that molecules such asacridan in fact undergo
constant 'flipping' about the nitrogen atom, between various spatial
arrangements. This may result in some blurring of energy levels and
associated broadening of quasi-lines. Also, the nn lone pair orbital
present on the nitrogen atom will not be quite in true resonance with
182
the rest of the IT conjugation. From the viewpoint of chemical
analysis, apart from the need for very selective excitation to obtain
the best results, as different sites may preferentially undergo phos-
phorescence decay, heterocyclics usually have a lower fluorescence
quantum yield because of singlet triplet deactivation often due to the
participation of an excited n-n* state. Indeed when entrapped in a
hydrophilic domain, inversion can take place with rr-n* becoming the
lowest excited state. Traces of polar molecules may also lead to
H-bonded complexes with the scavenging lone pair on the nitrogen atoms.
This then can once more lead to broadening effects in quasi-lines as
can microcrystal or dimer formation.
Many of these effects have led us to a greater understanding of the
molecular behaviour involved and thus it is possible to manipulate the
system to obtain maximal structural spectroscopic information. The
corollary of this is that with studying the shifts and/or deformation
of quasi lines information regarding the electronic and structural
properties of the molecule can be derived. It was therefore decided
to investigate the fine shifts associated with the electronic effects
of nitrogen introduction into aromatic ring systems.
9.2. EXPERIMENTAL
A wide range of nitrogen heterocyclics was chosen for this study
to compare two ring systems, naphthalene with quinoline, isoquinoline
and the double nitrogen containing quinoxalines. Three-ring staggered
systems, the parent analogue of which is phenanthrene, with the hetero-
cyclic analogues being the benzoquinolines of which only Benzo(f)-
quinoline, Benzo(h)quinoline were readily available to us in pure
zone refine form.
The linear three-ringed system comparison of anthracene with
heterocyclics including phenazine and carbazole gave rather different
results which will be mentioned later.
Finally four-ringed systems, in particular the benzoacridines, were
compared with their 'P.A.H.' analogue, benz(a)anthracene. Hexane was
the most universally acceptable solvent especially for the simple
alkaloids and their analogous P.A.H.'s which was the main theme of this
work. Certainly pentane for anthracene could be used and tetrahydrofuran
becomes particularly effective when the more polar heterocyclics lead to
Quinoline
7AZAINDOLE
Tryptophun
ji,--,c1-k,p14N `OH
Purine
~N
Quinōxiline
Phonanfhridine
PHOīOīAUTOMER l' a •• - 'e ; c
l
FIG 9,1
.9 E.
163 Isoquinoline.
„1.6.
•9~
Carbuzole
/Benzoquinolino 7,a tes)
.77
Acridine Q.9 y. .7 14-
1 1.7 1 Phenazine
183
184
dissolution solubilisation difficulties. The optimum concentrations
of 10-5 Molar was used in recording fluorescence spectra without inter-
ference from formation of microcrystals although an initial recording
at 10-4 Molar is often necessary for clear identification of the main
luminescent regions on initial scanning.
It was then attempted to obtain a cross survey of the general
luminescent properties of more biochemically interesting heterocyclics
such as the indene, indole, tryptophan combination. The latter
amphoteric amino acid is only soluble in acid salts or gels whereas
carbazole had to be dissolved in octane by the addition of toluene
ca. 100 µl per 10 mls of octane). Similar but more pronounced diffi-
culties with drugs like benzotriazole and lophine led to the need for
the use of 50% octanol as solvent. Hydroxypenicillin was monitored in
decanol both at room temperature with the Aminco-Bowman spectrofluorimeter,
monitoring the broad band emission at 380 nm and in a low temperature
glass on the high resolution instrument. No fine structure was observed.
Knowledge of the molecules lipophilicity as expressed by the log of its
partition coefficient between octanol and water proved to be very useful
in deciding potential solvent combinations for some of the rarer hetero-
cyclic compounds.
Reagents such as the paraffin solvents were carefully dried over
sodium wire and all made up samples were stored over calcium hydride
particularly effective in scavenging for water traces especially in
n-alkane solvents.
9.3. INSTRUMENTATION
Although the basic instrument utilised was the same in these
heterocyclic studies as earlier (p 32
some modifications were
found to be necessary. Thus, although it was possible to record
phosphorescence of quinoline and isoquinoline by measuring total
luminescence it was found necessary to use a phosphoroscope cam rotating
slowly to clean up the benzoquinoline phosphorescence which was overlaid
and obscured to some degree by Hg scatter lines. The alternative use
of a xenon arc source led to considerably broader less intense emission
with larger background emission.
185
An arrangement as described for. monitoring the phosphorescence
of phenanthrene by time averaging using an Aminco rotating cam and the
cold finger cell was utilised. This cell, but with five fine capillary
tubes holding aqueous phase tryptophan samples, had to be utilised
because otherwise freezing leads to shattering of the normal cell types
due to the expansion of aqueous phase glasses.
9.4. SUMMARY OF SPECTRAL SHIFT EFFECTS (Table 9.1).
The larger shifts associated with quinoline and benzo(f)quinoline
with respect to naphthalene and phenanthrene compared with those due to
isoquinoline and benzo(h)quinoline shows that in the former pair the
nitrogen atom is located at the most reactive sites with the acceptor
properties of the nitrogen atom being most pronounced in those positions.
One factor from the energy viewpoint which is immediately apparent
from the AS shifts of the singlet levels is the uniformity and close
similarity of these shifts for quinoline, benzo(f)quinoline, and benzo-
(e)acridene of 168 cm-1, 176 cm-1, and 190 cm-1 relative to their
respective P.A.H. analogues namely naphthalene, phenanthrene, and benz (a)-
anthracene (Table 9.1). These studies in fact can thus lead to an
interesting electronic energy analysis particularly of those molecules
containing various substituents and heteroatoms but which are still
based on the four ringed benzo(a)anthracene moiety. This as illustrated
in Chapter III is the optimum framework size for interference with
pyrimidines. But unlike the P.A.H.'s which act via epoxy radical
activation, nitrogen heterocyclics have an inbuilt polar lone pair which
can attach itself directly to proteinaceous material, allowing energy transfer
and thence intercalation, dimerisation and proton transfer. Important
future correlation studies on these and other heterocyclics such as the
sulphur analogues will soon no doubt be made.
9.5. CARBAZOLE EMISSION CHARACTERISTICS (Fig. 9.2 ab)
As clearly illustrated in the low temperature luminescence spectra
of carbazole various phenomena are occurring. Namely at 10-4 molar
concentration in the non polar n-octane paraffin in which carbazole
was only just soluble the chances of microcrystallite formation on
freezing to 77 K are enhanced.
Indeed the fairly broad band 41 A (A, half band width) was monitored
in.the carbazole emission spectrum. This seems undoubtedly due to
168 522
a►
0 495
245
Table 9.1. Effect of Nitrogen atom on Electronic Levels
of Aromatic Analogues
2 Rings
Isoquinoline
31,887
315.26
21,782
459.67
31,719 21,755
Quinoxaline 465
21,505
3 Rings
Phenanthrene 345.5 462 Ā
-Benzo (f) quinoline
-B.(f)Q
-B.(h)Q
28,843
344.6 456.56
29,019
176 181.8
6o 6o
4 Rings 0
-Benz(a)anthracene 384 595 A
26,020 16,800 cm-1
-Benz(e)acridine 190
-Benz(a)acridine 30
186
Naphthalene
Quinoline AS 4313.6 459.1
-1
S intr
Tet Aem AT
315.26 470.36
r 31,719 21,260~
Table 9.2 Results Tabulated
ROOM 77 K 77 K FLUORESCE, PHOSPH.
Napthalene 315.26 470.36 Quinoli ne 313.6 458 92 isoquinoline 487-7 Quinozaline 344 342, 6 Benzotriazole 332 330 Benzo(f)quinoline 340 345 45656 Tryptophan 340 346 458 Carbazole 34 6, 343' 404
Hydroxy pen icitlin 375 380
Phenanthrene 346.26 359.35
Lophino 390
I n dole 332
187
188
excimer possibly dimer emission, because on tenfold dilution plus the
addition of 100 µ1. of toluene, which highly solubilizes carbazole a
multiplet quasi-linear structured emission appeared at 348.3 nm with
just a small band at 342.8 nm where the former excimer emission had
been recorded, consistent with the idea that good Shpol'skii emission
characteristics are only produced when monitoring monomer species (Fig.9.2b).
The effect of heat via our special copper cell assembly''. on. the -
proposed carbazole dimer band at ca. 343 nm for the concentrated solution was a small blue shift, considerable decrease in intensity and an interesting
multiple splitting effect.
Indeed the thermodynamic energy savings to be taken into account
show the importance of the degree of dimerisation as a function of
temperature. Thus for polynaphthalene and polyvinylcarbazoles the
broader emission consistent with the maximum dimer/monomer ratio is at
approximately - 60°C. For the smaller heterocyclic diazophenanthrene
molecules it is around - 110°C (296). The relative degree of isomerisation,
tautomerisms, radical formations and proton transfers could be also due
to the relative energies of the solute :imorse, curves and the electron
potential well depth in various matrices. This has already been shown
to influence recombination luminescence of indole and can tell us
whether a diffusive mechanism is operating or whether electron tunneling
is prominent (p.11 G)
For nitrogen heterocyclics such as carbazole and diazaphenanthrene
z
L aa X
nil
the interaction energy between dimers ICH consists of
(1) the interaction energy of the permanent dipoles of the two molecules
in the dimer,
(2) the difference of the solvation energy of a dimer and two monomers.
pH can in fact be calculated from point dipole interactions thus
'°Z'6 0.1112T3 qÆ aouaosoaonTg DUT2400-1.1 UT OTOZI2(a.MD
C.947E --(-1 4 N OP(S
More structure as temp.f to 1501)K
rtizr 4 volts f l
339nm
CARBAZOLE (10-4 NO in n-octane
191
AH = 4/R3 where p is the dipole moment which for diazaphenanthrene
in the excited state is 1.15 Debye units and in the ground state is
3.93 D.(297)
In fact further information on the enthalpy and entropy changes
on dimerisation could be obtained by a plot of log K versus 1/T where
K = ~D]Z and is most easily obtained from an isobestic point at
various temperatures of the excitation spectra.
In the present luminescent emission studies on carbazole the
broader bands were always made up of several narrower quasi like lines,
this structure only becoming experimentally apparent on better solvation
and/or dilution or by 'breaking up' the aggregates temporarily by supplying
thermal energy. Apart from microcrystallite formation the general lack
of resolution with more complicated heterocyclics is probably also due
to interaction of the nitrogen lone pair with the matrix often with
proton participation and the complex stereochemistry leading to constant
molecular flipping between energetically closely equivalent conformations
leading to a large number of slightly different exciton energies. This
extra structure is also more easily identified in ethyl carbazole.(298)
9.61. Energy-Transfer Effects for Benzo(f)Uuinoline (Fig. 9.3, 9.4)
In the quasi-linear spectra of benzo(f)quinoline there are different
multiplicities for the fluorescence (triplet) and phosphorescence (singlet)
spectra in the same solvent and at the -same temperature. Spin orbital
interactions will be the main reason for this. Namely in the paraffin
crystalline lattice the analyte benzo(f)quinoline molecules are probably
located in three different site types. So due to the different disposition
of paraffin molecules around the luminescent centres in the crystalline
matrix (equivalent to different solvent environments) spin-orbitl
interaction in the centres is different which changes the possibility of
intersystem crossing to the phosphorescent level.299'300) It was further
observed that the phosphorescence was intensified in the branched iso-
octane solvent and a shoulder suggested the possibility of an extra
phosphorescent site. In contrast in methylcyclohexanol benzo(f)quinoline
shows mostly fluorescence.
QUASI-LINEAR FLUORESCENCE 5,6 BENZOQUINOLINE / HEXANE
351 345.8
' Figure 9.3
FIG,9.4 QUASI-LINEAR PHOSPHORESCENCE
5,6 BENZOQUINOLINE in nHEXANE . 1+56.56
194
9.62. Photochanges for Benzo(f)quinoline
After approximately thirty minutes irradiation the structured
fluorescence of the benzo(f)quinoline turned into a broader merged
band somewhat decreased in intensity and shifted fairly considerably
to the red. This is highly analogous to shift effects noted for
azaindole and described by Kasha as due to phototautomerism via a
biprotonic transfer giving rise to a form of hydrogen bonded dimer.(3°1)
It was further noticed that the height of the triplet level for
benz6(f)quinoline is higher for a solvent containing an odd number
Of carbon atoms whereas the singlet levels show no such trend.
An obvious extension of the tautomeric and hydrogen transfer effects
just discussed can obviously be made in various areas of important
interest. Namely, the quinoxalines and indoles particularly tryptophan
which were both.found to give blue fluorescence and green phosphorescence
at similar wavelengths to benzo(f)quinoline. Purine emission is moved
to the blue however around 320 nm but is still interestingly structured
and dependenton the resonance of the planar lactim tautomer.(302)
It was also attempted to study the chlorophylls as an example of the porphyrinic macrocycle vibrations which would lead to mote insight on
the energy transfers of such moities as pyrrole rings. However, although
it was possible to induce temporarily some fine structure on the room
temperature emission of hexane solutions of mixed chlorophylls (p.19?)
by controlled µ litre addition of water it was not possible to observe
quasi-line emission at 77 K. In fact liquid helium temperatures have
195
Table 9.3
Compound Hexane Octane µg ml-1
Det. limit
Pyrene 371.75 5 x 10-3
Coronene 445 1 x 10-3 Perylene 451 1x10-2 - 4x10-8M 1,12-Benz(ghi)perylene 419 3,4-Benzo(k)fluoranthene 403.25 1.5 x l0-3
1,2-Benzo(e)pyrene 388.25 1 x 10-3
3,4-Benzo(a)pyrene 402.4 403 1.25 x 10-4 1,2-Benzanthracene 383.75 2 x 10-3
3,4,9,10-Dibenz(ah)pyrene 431.5 3 x 10-4
Ovalene 480.6 Methylcholanthrene 392.55 5 x 10
-3
Chrysene 365 Indenopyrene 465 3 x 10-2 l,2,5,6-Dibenzanthracenes 394.25 5 x 10-3 1,2,3,4-) ( 395.25 3 x 10-4 1,2,4,5-)Dibenzopyrenes 3,4,8,9-)
( 395.5 ( 449.25
1 x l0-4 1 x 10
Nitrogen Heterocyclics Phos.
Quinolines 458.92 Molar 10-7 Isoquinoline 487.7 5,6-Benzo(f)quinolines 456.56 ti 10 6
(Carbazole 343 Fluor) 10-7
ci 0 0
ISOQU%NOLI NE
196
Figure 9,5
Fig.9.6 Chlorophyll in n-Hexane plus additions of H2 O
FLUORESCENCE ON AMINCO
198
been shown to be required probably once more because of micellar
aggregation. A photomultiplier particularly sensitive in the red is re-
required to detect porphyrinic emission extending from 630 - 750 nm.
Liquid nitrogen temperatures are sufficient in the quasi-line
study of some zinc,(3o3) magnesium,(303) copper,(304) palladium,(305)
platinum(305) and vanadyl porphyrins(3o6) although often minute trace
solutions of concentrations (less than 10-9 molar) only are required.
The latter vanadyl complexes are of particular importance in tracing the
origin of petroleum.
In conclusion it can be seen that hydrogen bonding can play an
exceedingly important role in electron transfer and hence light emission
properties. Fundamental changes in the latter can occur as if H bonding
and radical formation leads to a colloidal aggregate system leading to
polymer like emission characteristics.
cAS
199
CHAPTER X
OBSERVATIONS USING OTHER MATRICES
INTRODUCTION
This study was initiated in order to discover the feasibility
of using other matrices to attain useful analytical luminescence data
and if possible very selective information as from the Shpol'skii
clathrate-like systems. Although the latter effect is observed at
low temperature, much of the work reported in this chapter was
conducted at room temperature; the connection between the two systems,
however, is real and interesting.
10.1.1. Specifically-Adsorbing Silica Gels
Silica gels are used universally in analytical and organic chemistry
due to their selective adsorptive power, inertness, ready availability,
low cost and simplicity of use. (Many supplementary separation media
are continually being developed including polymers with polyamide and
glycosilic linkages such as cellulose acetate, sephadex, sepharose,
dextran and sephacryl. The latter gel media are particularly effective
for high molecular weight material and biopolymers because their mode
of operation relies on selectivity via molecular size distributions so
very large molecules are excluded from the 'beads' of gel whereas smaller
molecules diffuse into the various pores, thus being relatively retarded
by the percolation effect).
Silica attains its optimal properties for the separation of
substances often by the manner of drying: air dried, stored, activated
gels or those treated chemically all show different sorption activities.
In this study the coagulation of the silica gels is brought about
in the presence of certain quantities of a 'printing' organic molecule
as initially suggested by Dickey,307 although here the effect has been
monitored for the first time very sensitively by fluorescence.
For the activation it is necessary that the substance chosen can
interact with the oligosilicic acids of the sol. The sensitization
seems to be only possible with substances of a minimum molecular size,
aromatic and basic in nature which must be soluble and sufficiently
stable in an acid medium at pH 3.
200
In fact if we could attain specific adsorption of proteins on
silicates or polyacrylamide but also could induce footprints using
.P.A.H.'s then one could test these energy-site-interactions and possibly
effect their removal in an industrially feasible process. For the
printing of the information contained in an organic molecule into the
silica gel it obviously is not necessary to take over all the 'letter'
i.e. elements capable of interaction with the silanol groups. Furthermore,
and of more concern regarding relations between structure and physiological
effect of compounds, it is not even necessary to take over certain elements
but just a certain number in the correct distances to give a stable
multicentered bond. Two patented applications of this effect are
adsorbents initially printed with nicotine to act as tobacco filters
and enzyme reactor beds of polyacrylamide used to immobilise biocatalysts.
This improved the half-life of the entrapped cells but moreover by
reinduction of cells with a sixteen hour cycle under growth conditions
allows the biocatalyst to be reactivated to a level greater than that
originally present.
10.1.2. Preparation of the Gels
The preparation of the gels with specific adsorption properties
toward a certain substance is accomplished by acidifying sodium silicate
solutions and then dosing these with a specific compound. Various acids
were utilised and in all cases a sodium silicate solution density of
1.57 g/ml was utilised.
Acetic Acid Method
6 mis of sodium silicate were diluted with 30 mis of water and then stirred very quickly and thoroughly on addition of 6 mis of glacial acetic acid and finally 25 mis of a water solution of the printing substance
usually at a concentration of 5 x 10-6 molar.
Oxalic acid is also useful in that it guarantees a constant pH -,5 throughout the gelation. Rapid mixing is necessary to prevent premature
precipitation of the silica. The mixture usually becomes translucent
after 6 - 8 hours, coagulates after about ten hours and finally solidifies after one week. However, noticeable blue fluorescence in our case with
benzo(f)quinoline (which also required ethanol instead of water for
dissolution) was not observed until the gel had dried to some extent
(usually requiring more than two weeks), whereupon the gel shrinks
201
appreciably. This is also a necessary condition for the specific
adsorption effect although the drying process may be accelerated by
•grinding followed by oven-drying. Silicates dosed with the dyes fluores-
cein and rhodamine showed similar behaviour with more intense luminescence
after drying. When hydrochloric acid is used in the process, needles of
sodium chloride require to be removed by sieving. The gel is extracted
with methanol in a Soxhlet apparatus until no more activating substance
can be found in the extract (20 - 30 litres of methano1/10 g of gel).
Even after extraction a certain amount of activator is retained; this
can be detected spectrophotometrically by dissolving the gel in concentrated
sodium hydroxide. It is this chemically inextractable material which has
puzzled many workers308. Initial attempts to obtain a luminescent finger-
print from this retained material showing vibrational fine structure
similar perhaps to the clathrate held molecules in Shpol'skii matrices
were unsuccessful as the silica surface scattered much of the incident
radiation.
The specific adsorption is a measure of the increase in adsorption
capacity of activated gels with respect to analogously prepared blank gels
which can be ascertained by thin layer or column chromatography or by the
'batch' method. In the latter method a precisely weighed quantity of
activated or 'control' gel is equilibrated with a known volume of a
solution containing the substance to be evaluated at a certain concentration.
The decrease in the fluorescence signal and hence the concentration can. be
related to the adsorption capacity. An adsorption isotherm may thus be
obtained by equilibrating several gel portions with separate solutions
of higher concentration (see diagrams 10.1, 10.2). Saturation was
found to occur at 1.7 x 10-4M in the adsorption isotherm at approximately
the some concentration that the fluorescence calibration curve flattens
out due to dimerisation, although this is always complicated with right-
angle illumination by pre-absorption. effects. This is a problem which
opto-acoustic spectroscopy might overcome and could be a factor why
initial pilot studies on the effect of quenching fluorescein dye
emission with KI gave a 20-fold decrease in fluorescence signal but
only a corresponding 2-fold increase in the opto-acoustic signal obtained.
Comparative Stern-Volmer plots are given (Fig.l0.3). Unfortunately it is
stank get extracts B(f) Q
at pH 7
a
10'3 1P 5 10-4
t0
X 10-0 • ,.
specifically adsorbecl
• gel • isotherm x 100•
10
• EM] 10
7.
t~ t
Figure. 10,1. Calibration and Adsorption Curves for • Benzo(t)quinoline
10'
Figure 10.2 S pecific adsorption Monitored fizorimetrically
a-5x104M W.4 - pH 2 EtOH
d 5x1011eM pH3 after 5mins
b FL• - emission 520 (ex 48 6) b' B( f) 0: emissi bn 430 (excl.\ 390 )
Relative S ignai Intensity Fluorescein Dye
M
o=OPTOACOUSTIC SPECTROSCOPY
= FLUORESCENCE Sr'ECTROSCOPY
FI C URE 10.3 204
STERN --VOLNiER PLOTS
8
6
2 K .Quenc hQr
C7 Conceli'rqt-I
10 1 [M] Ō To--2
10-2
205
not possible to make,a direct comparison of the photophysical'pathways
in monomer species due to the present lack of sensitivity in the latter
technique. Indeed opto-acoustic spectroscopy may well become an ideal
complementary technique allowing a detailed study of some particularly
important amino-acids and carotenoids which have high radiationless
deactivation and may also permit a more detailed study of phonon, exciton
mechanisms. Unlike some previous studies the technique of molecular
emission spectroscopy used in our work, here, gave a very sensitive
monitor of the adsorption effects. Cross-correlation was also achieved
by monitoring the actual emission spectra from the front surface of the
silica particles. Although these exhibited no fine structure, the
emission was very intense.
Lloyd3 has recently shown the enhanced emission of benzoquinolines
and acridine on a microparticulate silica substrate which was used for
detection in high pressure liquid chromatography. This he attributed
to proton donation by the silica to the first and highly basic excited
states of these nitrogen heterocyclic molecules.
In our study we not only monitored intense blue fluorescence but
also intense and long-lived green phosphorescence emission of the benzo-
(f)quinoline dosed silica after evaporation of the solvent (Fig. 10.4).
The use of low temperature conditions gave only a small increase in the
intensity of this emission and a slight narrowing of the wide phosphorescence
band.
10.2. ROOM TEMPERATURE PHOSPHORESCENCE
The room temperature phosphorescence of adsorbed ionic organic
molecules was first reported a decade ago by Roth310 and later by Schulman
and Welling;311 the effect was typically observed only after solvent
evaporation from samples. There has recently been an upsurge of interest
in the analytical potential of the phenomenon which has also this year
been observed for compounds adsorbed on sodium acetate matrices.j12
Of particular interest are compounds possessing an indole nucleus which
have been shown to exhibit room temperature phosphorescence after
evaporation from alkaline-ethanolic solutions. Only the strongly
adsorbed molecules phosphoresce; those weakly physically adsorbed do not.
The most common explanation is that the matrix holds the adsorbed compound
rigidly and thereby restricts vibrational motions necessary for non radiative
Hy 435.8 n FIG.10.4 ROOM TEMPERATURE
FRONT SURFACE LUMINESCENCE
OF BENZO(f)QUINOLINE DOSED
SILICA GELS.
i
390nr nm
207
decay from the triplet state. Because salts and sugars can increase
the lifetime of this phosphorescence it has been suggested313 that an
alternative mechanism may beentrapment within channels or interstices
of the matrix thus decreasing permeability to the quenching effects of
oxygen; the overall triplet decay constant kT for an organic molecule
can generally be expressed as
kT = kr +kln, +kq [0,2]
where kr, ku, and kq are the radiative, non radiative and oxygen-quenching
rate constants, respectively. Evidence from our studies seems to
favour the latter mode of action. Certainly some workers have elaborated
in speculation regarding the role of hydrogen bonding, which helps to
align molecules to allow maximum molecular overlap but is not a primary
cause of the room temperature phosphorescence. This phenomenon has
also been observed in our laboratory for pesticides on paper substrates
and will be reported elsewhere.
Before outlining a possible mechanism for the preponderance of
intense green room temperature phosphorescence of benzo(f)quinoline
[B(f)Q] after heating B(f)Q-dosed silica gel some rapid tests were
carried out on quinolines.
A solution of benzoquinoline (l0-3 molar) prepared in r -hexane
fluoresced pale blue on excitation with ultra-violet light at
room temperature (using 250 nm int. filter).
At 77 K some green phosphorescence was also noticed but on warming
the bulk solution this once more reverted to pale blue fluorescence.
However, a few microcrystals had been deposited towards the top of the
silica tube and these showed intense green phosphorescence even at
room temperature: On addition of droplets of ethanol, from a dropping
pipette this emission changed to an intense blue. This was also the
case with the bulk solution on addition of a few drops of ethanol to
the hexane. Conclusions which may be drawn from these observations
are:
(i) A hydrogen bonded complex may remove the perturbation effect
of the nitrogen lone-pair and lead to blue fluorescence from the
lowest u-n' excited singlet state.
(ii) With no hydrogen bonding, isolated molecules will show
green phosphorescence due to intramolecular intersystem crossing.
208
(iii) Similarly, microcrystalline aggregate formation leads to
even more lone-pair perturbations, intermolecular intersystem
crossing and enhanced phosphorescence (for exciton mechanisms,
see Chapter VII).
(iv) This increase in population of the triplet state can lead
to increased chemical reactivity chemisorption, photoreduction
in the case of adsorbed molecules, possibly because of the ease
of formation and stabilisation of free radicals.
10.3. PROPOSED MECHANISM FOR BENZO(F)QUINOLINE PHOSPHORESCENCE AT
ROOM TEMPERATURE
Amorphous silica which constitutes the gel existing possibly in a
linear polymer array
[Si(OH)6] +H2O H2O [SiCOH)5 ] +OH
[(2x2 0)si(OH)5 ]- [(Ho)4 si,,, ,.=si (OH)4 ]2- +2H20
and varies between polysilicic acid units with little silicon and colloidal
Si.02 with high silicon content. On gelation and subsequent drying, I
however, the — Si — OH groups can lose some water which is physically I 1
adsorbed. Persistent silanol groups attached as — Si — OH. 0..._H can I
be cone^ted by stronger heating to give a siloxane lattice moiety,
— Si —0•- i — , whose pores can be widened by this activation. The
active silica gel sites are thus acidic and attract the highly basic
first excited states of the benzo(f)quinoline possibly initially via
ethanolic solvent groupings.
H . o n o o ...
— OH.O ,H NH
5 2
After solvent evaporation the weak hydrogen bonding may be broken
and the silica gel pores widen. The benzoquinoline molecules thus
reorganise and take up the energetically most favoured (if available)
sites. Some may become strongly chemisorbed directly to the silica
surface while others may group in microcrystals excluded from the pores
or in the form of dimers or aggregates trapped inside the lattice inter-
stices, this constituting the 'specifically adsorbed' quantity of material.
ti 209
These will, via intersystem crossing, give highly intense green
phosphorescence emission, the intensity depending on the actual con-
formational arrangements within the different sitese This is analogous
to the site dependency of phosphorescence observed for benzo(f)quinoline
within crystalline n-paraffin matrices.
1 I I — Si ---*— 0 --
\\J■4eN 1 L.)
0 ---
One of various
Isolated B(f)Q
2 B(f)Q molecules energetically favourable molecule may overlayed leads to staggered conformations undergo intra- greater N perturbation
molecular I.S.C. and intermolecular I~SoC.
Obviously whether the B(f)Q molecules are trapped within clathrate-like
'holes' and channels or held chemically and/or physically on the silica
surface will lead to differing ageing phenomena. The importance of this
'adsorbed' state chemistry is emphasised by the asbestos-mediated membrane
uptake of benzo(a)pyren2 as monomer reported recently as a mechanism of
cocarcinogenicity.
10.4. POLYACRYLAMIDE GELS
Polyacrylamide gels are now widely used in a number of techniques
in analytical biochemistry. They are easily formed and yield stable
and hydrophilic gels with pore sizes dependent on the mode of polymerisation.
High resolution electrophoresis for proteins and other macromolecular
separations may be conducted using short columns or thin layers of such
gels. Detection of these materials is then achieved by dye-staining
and direct transmission densitometry. We have found, however, that
polyacrylamide, specifically prepared, is largely transparent to ultra--
210
violet light and can provide an appropriate medium for separation of
real protein samples while still allowing in situ study of tryptophan
moieties utilising their characteristic fluorescence. This can then
allow a sensitive, yet practically simple, method of studying conformational
changes and/or photophysical energy pathways.
10.5. EXPERIMENTAL
Initial experiments on trapping fluorescein dye into a polyacrylamide
matrix were reasonably successful. The initial polymerisations took
place without bisacrylamide and this led to a brittle polymer which gave
a higher degree of scattered light. The fluorescein fluorescence was
fairly broad but not completely uniform and was shifted to long wavelength
compared with fluorescein adsorbed on silica gel.
Polyacrylamide gels used for electrophoresis are generally formed by
addition (free radical) polymerisation of acrylamide (crosslinking co-
monomer) by one of two methods:
(a) Photochemically using riboflavin and ultra-violet light as initiator.
Obviously though this would introduce fluorescence at ca. 520 nm and
this procedure was therefore avoided.
(b) Chemically using ammonium persulphate as initiator and sulphites as
catalysts.
We have found large differences in background light absorption and
emission characteristics of the gels depending on their mode of preparation.
It was eventually found that polyacrylamide containing 8% by weight of
total monomer and 2% by weight of cross linking bisacrylamide, together
with a minimum of sulphite (< 1%), produced a virtually scatter-free
gel, i.e. 30 times less scattering at 250 nm than one-containing 5%
cross linking agent. A great reduction in the 410 nm emission usually
associated with gels prepared for electrophoresis was also observed
(Fig. 10.5).
Practically, polymer samples would be directly moulded within exist-
ing cuvettes (for the Aminco) or cut into discs for the high resolution
instrument. in fact 6 inch long, 2 cm diameter 'gel snake' polyacrylamide
columns containing denatured electrophoretically separated rabbit muscle
were found particularly easy to handle. 6 one inch sections could be
211
Figure 10.5 Tryptophan emission from elect rophoreses, of rabbit muscle in potyacrytamide
4.10 n m
reduced polyucr y tumide
emission
FIG, 10.6 ROOM TEMPERATURE LUMINESCENCE OF BE NZ(A)ANTHRACENE ,DOSED POLYME T HACRYLATE _
213
monitored in turn by placing them in the normal 4-sided cuvettes.
The protein samples exhibited blue-pale fluorescence and green phos-
phorescence which intensified with time. Unfortunately,surface.
scatter from the frozen polymer surface also became larger. Using
250 nm excitation, fluorescence was detected at ca. 335 nm and
phosphorescence between 450 to 460 at 77 KJ3enz (a)anthracene was also
monitored in perspex. (Fig. 10.6).
That future work should be concentrated in this area of possible
hydrophilic matrices for the observation of structured luminescence
is further confirmed by the most recent studies both from Russica314
where weak but structured phosphorescence of some amino acids and
other polar organics have been observed in inorganic crystalline
hydrates, particularly of aluminium and beryllium. But also from
the Ames Laboratory, Iowa, U.S.A., where workers have used an adaptation
of Yersonov's laser l method of selective excitation to attain quasi
linear spectra of P.A.H.'s in glycerol water matrices at liquid helium
temperature.315
)p
500 450 400 050 .t, am
Fig .ttii) Phosphorescence spectra of acetophenone in methylcyclohexane (1), methylcyclohexanol (2) (reduced by a factor of 5), and methylcyclohexanone (3).
500 950 400 350 - Jt,am
Fig.014Traces of phosphor-escence spectra of benzoic acid in cyclohexane (1) and
methylcyclohexane (2).
•
Jo .
•
500 450 900 050 Aoun
Fig. Li) Phosphorescence spectra of benzaldehyde in cyclohexane (1) and methylcyclohexanol (2) (reduced by a factor of 4) .
500 450 400 030 l nm •
Fig- iii) Phosphorescence spectra of benzaldehyde in cyciohexanone (1) and methylcyclohexanone (2).
Phenol, benzylamine, and N-methylbenzylamine lumi-nesce only in methylcyclohexanol.
Comparing the phosphorescence spectra obtained for benzene derivatives in different organic solvents,
215
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