Table Of ContentOther Titles of Interest
BAKER and BETTERIDGE
Photoelectron Spectroscopy: Chemical and Analytical Aspects
BECKEY
Field Ionization Mass Spectrometry
DAMANY et al.
Some Aspects of Vacuum Ultraviolet Radiation Physics
ELWELL and GIDLEY
Atomic Absorption Spectroscopy
EMSLEY and LINDON
NMR Spectroscopy using Liquid Crystal Solvents
JACKMAN and STERNHELL
Applications of Nuclear Magnetic Resonance Spectroscopy
in Organic Chemistry 2nd edition
SCHEINMANN
An Introduction to Spectroscopic Methods for the Identification
of Organic Compounds (2 vols.)
SOBELMAN
An Introduction to the Theory of Atomic Spectra
FLUORESCENCE AND PHOSPHORESCENCE
SPECTROSCOPY : PHYSICOCHEMICAL
PRINCIPLES AND PRACTICE
by
STEPHEN G. SCHULMAN, Ph.D.
Department of Pharmaceutical Chemistry
College of Pharmacy, University of Florida
PERGAMON PRESS
OXFORD · NEW YORK · TORONTO · SYDNEY · PARIS · FRANKFURT
U.K. Pergamon Press Ltd., Headington Hill Hall, Oxford
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U.S.A. Pergamon Press Inc., Maxwell House, Fairview Park,
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CANADA Pergamon of Canada Ltd., 75 The East Mall,
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AUSTRALIA Pergamon Press (Aust.) Pty. Ltd., P.O. Box 544,
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75240 Paris, Cedex 05, France
FEDERAL REPUBLIC Pergamon Press GmbH, 6242 Kronberg-Taunus,
OF GERMANY Pferdstrasse 1, Federal Republic of Germany
Copyright © 1977 S. G. Schulman
All Rights Reserved. No part of this publication may be
reproduced, stored in a retrieval system or transmitted in any
form or by any means: electronic, electrostatic, magnetic
tape, mechanical, photocopying, recording or otherwise,
without permission in writing from the publishers
First edition 1977
Reprinted 1979
Library of Congress Cataloging in Publication Data
Schulman, Stephen Gregory.
Fluorescence and phosphorescence spectroscopy.
(International series in analytical chemistry; v. 59)
1. Fluorescence spectroscopy. 2. Phosphorescence
spectroscopy. 1. Title.
QD96.F56S38 1976 543'.085 75-46591
ISBN 008-020499-6
In order to make this volume available as economically ana
rapidly as possible the author's typescript has been reproducea
in its original form. This method unfortunately has its typo-
graphical limitations but it is hoped that they in no way
distract the reader.
Printed in Great Britain by A. Wheaton & Co. Ltd, Exeter
To Joke and Christina
for their love, encouragement,
tolerance and infinite patience.
PREFACE
The past two decades have witnessed the emergence of
fluorescence and phosphorescence spectroscopy to become among
the most useful of tools in experimental biology and chemistry.
The availability of low to moderate cost commercial instrumen-
tation, in recent years, has made these analytical methods
accessible to virtually all laboratories. No other instrumental
methods available at comparable cost, can equal or surpass
fluorimetry and phosphorimetry in analytical sensitivity. Con-
-9
centrations of luminescing materials as low as 10 molar are
routinely determined. This aspect is particularly desirable
in the biomédical sciences where low concentrations of drugs,
metabolites and toxins in blood serum and urine must routinely
be monitored.
Although there are many excellent reference works currently
available on luminescence spectroscopy, many of these tend to
be either theoretically oriented, with heavy emphasis on quantum
mechanical interpretation of spectra of simple molecules as
they relate to molecular electronic energy states, or instru-
mentally oriented, with emphasis on electronic and optical
aspects of instrument design. Most practitioners of fluores-
cence and phosphorescence spectroscopy, currently, are either
analytical chemists or biologically oriented scientists with
limited background in quantum mechanics, electronics, optics,
and higher mathematics in general. This group of experimenters
is usually constrained to the use of commercially available
instrumentation in which there is no great deal of variability
in fundamental design from one manufacturer to another. More-
over, the luminescing molecules of interest to this group are
often extremely complicated drugs and metabolites which do not
lend themselves to detailed understanding by rigorous quantum
mechanical treatments.
vii
viii Preface
Yet it is important that the limits of instrumental capa-
bility and at least a qualitative picture of the relationships
between molecular electronic structure, environmental inter-
actions and luminescence spectra can be understood by all
practitioners in order to maximize sensitivity, selectivity,
interpretation and overall reliability of data taken in fluores-
cence and phosphorescence spectral measurements. This book
then, is written with the analytical chemist and biological
scientist in mind and represents an attempt to make the in-
strumental, and especially the structural and environmental
aspects of luminescence spectra intelligible to the reader with
a general college background in chemistry and physics.
The author expresses his gratitude to Ms. Carolyn B. Gran-
tham, Dr. A.C. Capomacchia, Dr. D.V. Naik, Mr. R.J. Sturgeon,
and Mr. Peter F. Eisenhardt for their invaluable comments and
assistance with the preparation of this manuscript.
Gainesville, Florida Stephen G. Schulman
July, 1976
CHAPTER 1
PHOTOPHYSICAL PROCESSES
IN ISOLATED MOLECULES
Fluorescence and phosphorescence occur in molecules as a
result of and subsequent to a series of physical phenomena,
normally beginning with the absorption of light. These phe-
nomena as well as fluorescence and phosphorescence are derived
from the electromagnetic nature of light, the details of molec-
ular structure (especially molecular electronic structure) and
the nature of the environment of the luminescent molecule. It
should be fairly obvious that an appreciation of these phenom-
ena is necessary for the understanding of the relationships
between molecular structure and luminescence spectroscopy to
chemical and biological problems. This section then, will deal
with molecular electronic structure, the interactions of light
with charged particles in molecules and the events that follow
the absorption of light in molecules. For the sake of simplic-
ity, these arguments will deal only with isolated molecules.
The effects of environmental interactions of molecules on
electronic spectra will be considered in a later chapter.
Molecular Electronic Structure
The interactions between atomic electrons to form chemical
bonds are due to the valence electrons and orbitals comprising
the partially filled outer shells of atoms. The electrons
occupying the filled inner shells of atoms which belong to
molecules are localized upon the atoms from which they origi-
nated and contribute only very weakly, through their repulsive
properties, to molecular electronic structure. Because it is
the valence electrons which are responsible for the electronic
spectra of molecules in the visible and ultraviolet regions of
the spectrum as well as for chemical reactivity, our considera-
tion of molecular electronic structure will be confined to
1
2 S. G. Schulman
those features which arise from the valence electrons and Or-
bitals of molecules.
A chemical bond or occupied molecular orbital may be
thought to originate from the overlap of occupied atomic Or-
bitals. The geometry of the overlap is used to classify the
type of chemical bonding, and the filling of the molecular
orbital is governed by the Pauli exclusion principle (i.e.
a maximum of two electrons can occupy one orbital). Some of
the various types of molecular orbitals are described below.
g-3onds
The overlap of two atomic orbitals along the line joining
the nuclei of the bonded atoms results in a σ-bond. A σ-bond
can accommodate two electrons, in accordance with the Pauli
exclusion principle. The distribution of charge in a σ-bond
is strongly localized between the two bonded atoms. Although
each atom participating in the σ-bond contributes one atomic
orbital to the formation of the σ-orbital, the two electrons
occupying the σ-orbital may originate, one from each atom or
both from the same atom. In the former case, the σ-bond is
called a covalent bond, and in the latter case, it is called
a coordinate covalent bond.
If the two atoms joined by the covalent bond exert unequal
attractions upon the electron pair comprising the σ-bond, the
electron pair will spend more time near one atom than the other.
In this case, the more strongly attracting atom is said to be
more electronegative than the other, and the bond is said to be
a polar covalent bond. Because the electronic charge in a σ-
bond is localized along the line between two atoms, electronic
repulsion and the exclusion principle prevent the formation of
more than one σ-bond between any two atoms in a molecule.
Electrons engaged in σ-bonding are usually bound very tightly
by the molecule. Consequently, a great deal of energy is re-
quired to promote these electrons to vacant molecular orbitals.
Photophysical Processes in Isolated Molecules 3
This means that molecular electronic spectra involving σ-
electron transitions occur well into the vacuum ultraviolet and
are not of interest in conventional luminescence spectroscopy
which is concerned with the general region between the near
ultraviolet and the near infrared (i.e. 200-1000 nm).
π-Bonds
The overlap of two atomic orbitals at right angles to the
line joining the nuclei of the bonded atoms is said to result
in a π-bond. π-Bonding is a weaker interaction than σ-bonding
and consequently, usually occurs secondarily to σ-bonding. The
formation of π-bonds always involves atomic p or d orbitals,
but never s orbitals. In a π-bond, the distribution of elec-
tronic charge is concentrated above and below the plane con-
taining the σ-bond axis. While σ-electrons are strongly
localized between the atoms they bind, π-electrons, not being
concentrated immediately between the parent atoms, are freer to
move within the molecule and are frequently distributed over
several atoms. If several atoms are σ-bonded in series, and
each has a p or d orbital with the proper spatial orientation
to form a π-bond with the others, rather than an alternating
series of localized two atom π-bonds being formed, a set of π-
orbitals are formed which are spread over the entire series of
atoms. These π-orbitals are said to be delocalized. In some
cyclic organic molecules, π-delocalization extends over the
entire molecule. These compounds are said to be aromatic and
are the molecules of primary interest in fluorescence and
phosphorescence spectroscopy.
Because π-electrons are not concentrated between the bonded
atoms, they are not as tightly bound as σ-electrons. Hence,
their electronic spectra occur at lower frequencies than do σ-
electron spectra. For molecules containing isolated π-bonds,
the transitions involving π-electrons are still in the vacuum
ultraviolet or at the limit of the near ultraviolet. Molecules
containing delocalized π-electrons usually have π-electron
4 S. G. Schulman
spectra in the near ultraviolet while the superdelocalized π-
systems, the aromatic molecules, have π-electron spectra which
range from the near ultraviolet for small molecules, to the
near infrared for large ones·
Nonbonded Electrons
With the exceptions of most transition metal complexes and
a very few stable free radicals, enough σ- and π-bonds will
encompass any atom in a molecule, to engage in bonding any
electrons which were unpaired in the valence shell of the iso-
lated atom. In all atoms of the periodic table which have more
than four electrons in the valence shell (e.g. nitrogen), how-
ever, there are electrons in the valence shell which are already
paired. The orbitals occupied by these electrons are already
filled, although the valence shell, in the isolated atom, is not
complete. These electrons are unavailable for conventional
covalent bonding and yet have energies comparable to other
electrons in the same shell. Consequently, they are called
nonbonding or n-electrons. When the atom is engaged in bonding,
the valence shell electrons involved in σ-bonding will drop in
energy, well below the energy of the n-electrons. The energy
of the electrons involved in π-bonding (if any) will usually
drop below the energy of the n-electrons but not quite as much
as in the case of the σ-electrons. Because the n-electrons are
higher in energy than either the σ- or π-electrons, they must
be considered as potential contributors to the spectral fea-
tures of molecules possessing them. While the n-electrons
originate from pure atomic orbitals, the difference in repul-
sion experienced by these electrons as a result of the differ-
ence in electronic environment of a molecular atom compared
with that of an isolated atom infer that the n-orbitals will
have more of the properties of localized molecular orbitals
than of atomic orbitals. This is indeed found to be the case.
The bonding geometries of atoms in molecules are influenced at
least as strongly by the number of nonbonded electron pairs as
by the number of bonded electron pairs around them.
