low temperature luminescence spectroscopy of some … · 2016. 7. 15. · 2.5. high resolution...

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


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


9.3. 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

j I !

► 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


Cannock Wood exinite 4.3 1.1 3.9 Shallow seam

vitrinite 12.8 0.4 32.0


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- cyclohexane 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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