Table Of ContentLuminescence Applications
in Biological, Chemical, Environmental,
and Hydrological Sciences
In Luminescence Applications; Goldberg, M.;
ACS Symposium Series; American Chemical Society: Washington, DC, 1989.
In Luminescence Applications; Goldberg, M.;
ACS Symposium Series; American Chemical Society: Washington, DC, 1989.
383
A C S SYMPOSIUM S E R I E S
Luminescence Applications
in Biological, Chemical, Environmental,
and Hydrological Sciences
Marvin C. Goldberg, EDITOR
U.S. Geological Survey
Developed from a symposium sponsored
by the Division of Environmental Chemistry
at the 193rd Meeting
of the American Chemical Society,
Denver, Colorado
April 5-10, 1987
American Chemical Society, Washington, DC 1989
In Luminescence Applications; Goldberg, M.;
ACS Symposium Series; American Chemical Society: Washington, DC, 1989.
Library of Congress Cataloging-in-Publication Data
Luminescence applications in biological, chemical, environmental, and
hydrological sciences.
Marvin C. Goldberg, editor
"Developed from a symposium sponsored by the
Division of Environmental Chemistry at the 193rd Meeting
of the American Chemical Society, Denver, Colorado,
April 5-10, 1987."
p. cm.—(ACS Symposium Series, 0097-6156; 383).
Includes index.
ISBN 0-8412-1560-X
1. Luminescence spectroscopy—Congresses
2. Biochemistry-Technique—Congresses
3. Environmental chemistry-Technique—Congresses
I. Goldberg, Marvin C., 1933- . II. American Chemical
Society. Division of Environmental Chemistry. III. Series
QP519.9.L84L86 1989
543'.0858—dc19 88-39131
CIP
Copyright © 1989
American Chemical Society
All Rights Reserved. The appearance of the code at the bottom of the first page of each
chapter in this volume indicates the copyright owner's consent that reprographic copies
of the chapter may be made for personal or internal use or for the personal or internal
use of specific clients. This consent is given on the condition, however, that the copier
pay the stated per-copy fee through the Copyright Clearance Center, Inc., 27 Congress
Street, Salem, MA 01970, for copying beyond that permitted by Sections 107 or 108 of the
U.S. Copyright Law. This consent does not extend to copying or transmission by any
means—graphic or electronic—for any other purpose, such as for general distribution, for
advertising or promotional purposes, for creating a new collective work, for resale, or for
information storage and retrieval systems. The copying fee for each chapter is indicated in
the code at the bottom of the first page of the chapter.
The citation of trade names and/or names of manufacturers in this publication is not to
be construed as an endorsement or as approval by ACS of the commercial products or
services referenced herein; nor should the mere reference herein to any drawing,
specification, chemical process, or other data be regarded as a license or as a conveyance
of any right or permission to the holder, reader, or any other person or corporation, to
manufacture, reproduce, use, or sell any patented invention or copyrighted work that may
in any way be related thereto. Registered names, trademarks, etc., used in this publication,
even without specific indication thereof, are not to be considered unprotected by law.
PRINTED IN THE UNITED STATES OF AMERICA
In Luminescence Applications; Goldberg, M.;
ACS Symposium Series; American Chemical Society: Washington, DC, 1989.
ACS Symposium Series
M. Joan Comstock, Series Editor
1988 ACS Books Advisory Board
Paul S. Anderson Vincent D. McGinniss
Merck Sharp & Dohme Research Battelle Columbus Laboratories
Laboratories
Daniel M. Quinn
Harvey W. Blanch
Universit f Iow
University of California—Berkele
Malcolm H. Chisholm James C. Randall
Indiana University Exxon Chemical Company
Alan Elzerman E. Reichmanis
Clemson University AT&T Bell Laboratories
John W. Finley
C. M. Roland
Nabisco Brands, Inc.
U.S. Naval Research Laboratory
Natalie Foster
Lehigh University W. D. Shults
Oak Ridge National Laboratory
Marye Anne Fox
The University of Texas—Austin Geoffrey K. Smith
Rohm & Haas Co.
Roland F. Hirsch
U.S. Department of Energy
Douglas B. Walters
National Institute of
G. Wayne Ivie
Environmental Health
USDA, Agricultural Research Service
Michael R. Ladisch Wendy A. Warr
Purdue University Imperial Chemical Industries
In Luminescence Applications; Goldberg, M.;
ACS Symposium Series; American Chemical Society: Washington, DC, 1989.
Foreword
The ACS SYMPOSIUM SERIES was founded in 1974 to provide a
medium for publishing symposia quickly in book form. The
format of the Series parallels that of the continuing ADVANCES
IN CHEMISTRY SERIES except that, in order to save time, the
papers are not typeset but are reproduced as they are submitted
by the authors in camera-ready form. Papers are reviewed under
the supervision of the Editors with the assistance of the Series
Advisory Board and are selected to maintain the integrity of the
symposia; however, verbatim reproductions of previously pub
lished papers are no
research are acceptable
types of presentation.
In Luminescence Applications; Goldberg, M.;
ACS Symposium Series; American Chemical Society: Washington, DC, 1989.
Preface
LUMINESCENCE YIELDS DATA
that often cannot be provided by any
other methodology. This book is a compilation of a wide variety of
original research contributions. Substantial information is given on the
use of luminescence techniques to understand specific cell responses and
the chemical mechanisms of cell action. An examination of natural
environments is presented in the form of specific studies that
characterize materials in both solid and liquid form and give information
on the respective reaction
Advanced research on
luminescence studies, as well as both active and passive use of
luminescence, is included.
Developments in laser design, microelectronics, and computers over
the past 20 years have resulted in a renaissance for fundamental and
analytical applications of molecular luminescence. Twenty years ago the
conventional wisdom was that, although luminescence is inherently very
sensitive for detection of emitters, the generally broad, featureless
emission band and the frequent interferences from trace contaminants
made interpretation of luminescence spectra difficult. It became
necessary to isolate samples of unknown materials to such a high degree
that luminescence measurements did not contribute much additional
material. Today, new techniques have made luminescence spectroscopy
indispensable as a sensor of chemical species in environments as diverse
and complex as biochemical fluids, chromatographic columns, the
Earth's atmosphere and waters. Furthermore, luminescence allows the
determination of internal energy states and follows how they change with
time on a scale that can be as short as one picosecond. Such
measurements are crucial for fundamental investigations of the dynamics
of chemical processes.
For analytical applications, many techniques have greatly improved
our ability to analyze multicomponent systems. These include the
coupling of luminescent detectors with chromatographic columns and
other separation devices; combining lifetime and polarization data with
spectral measurement; three-dimensional plotting of the total
excitation-emission intensity matrix; synchronous scanning of the
excitation and emission wavelengths, tagging specific molecules in a
mixture with unique fluorescent labels; and using tunable lasers to
induce emission from electronic states that are characteristic of
ix
In Luminescence Applications; Goldberg, M.;
ACS Symposium Series; American Chemical Society: Washington, DC, 1989.
particular molecules. Chemi-excitation of luminescent molecules is
another versatile way to impart additional sensitivity and specificity to
the analysis of multicomponent systems. Compounds such as amines
and amino acids have been derivatized and made to react with oxalates
and hydrogen peroxide after high-performance liquid chromatography
separations. The chemiluminescent reactions permit detection at
femtomole levels.
Fluorescence analysis has been extended to many nonfluorescent
species by the development of a wide range of derivatizing agents that
form a fluorescent product. This approach has been especially useful
with biochemical molecules, many of which are not natural fluorophores.
Luminescence lifetimes are measured by analyzing the rate of
emission decay after pulsed excitation or by analyzing the phase shift and
demodulation of emission from chromophores excited by an amplitude-
modulated light source.
now allow luminescence lifetimes to be routinely measured accurately to
nanosecond resolution, and there are increasing reports of picosecond
resolution. In addition, several individual lifetimes can be resolved from
a mixture of chromophores, allowing identification of different
components that might have almost identical absorption and emission
features.
In studies of molecular dynamics, lasers of very short pulse lengths
allow investigation by laser-induced fluorescence of chemical processes
that occur in a picosecond time frame. This time period is much less
than the lifetimes of any transient species that could last long enough to
yield a measurable vibrational spectrum. Such measurements go beyond
simple detection and characterization of transient species. They yield
details never before available of the time behavior of species in fast
reactions, such as temporal and spatial redistribution of initially
localized energy in excited molecules. Laser-induced fluorescence
characterizes the molecular species that have formed, their internal
energy distributions, and their lifetimes.
Biological applications of luminescence make use of resonance
energy transfer as a microscopic ruler to measure distances between
chemical groups in complicated biological structures. Electronic
excitation of a fluorophore can be dissipated either by fluorescent
emission or by nonradiative resonant energy transfer to an acceptor
molecule. The transfer rate depends on the distance between donor and
acceptor groups. Fluorescence lifetimes are shortened as the fraction of
energy dissipated nonradiatively increases. Resonant energy transfer is
particularly significant over the distance range of 2 to 5 nm, which is also
the range of typical protein diameters and membrane thicknesses. As a
result, fluorescent lifetime measurements of natural and derivative
fluorophores are used to determine distances between binding sites on
χ
In Luminescence Applications; Goldberg, M.;
ACS Symposium Series; American Chemical Society: Washington, DC, 1989.
proteins and chromophore distances in biological aggregates. Such
measurements have produced important results regarding the
conformation and structures of several proteins and geometric details of
protein-membrane interactions. Particularly valuable is the fact that
luminescence measurements of molecular conformation can often be
made in situ, yielding unique information not possible from X-ray
diffraction about molecules that change shape when crystallizing or do
not form crystals at all.
Many biologically important processes are related to changes in
activity caused by environmental changes at metal binding sites on
proteins. The normally bound ions Ca2+ and Mg2+ can be replaced by
the transition metal ion fluorescent probes Tb3+ and Eu3+, whose
luminescent lifetimes are very sensitive to their chemical environment,
especially the degree of hydration Thei emissio lifetime b d
to track environmenta
reacting or responding to external influences. There are many other
instances where the sensitivity of chromophore lifetimes to their
chemical surroundings has been used to probe the details of
environmental conditions around a chromophore, such as solvation,
micelle structures, and the structures of dissolved complexes.
The reactivity of large and complex molecules is often closely related
to their size and shape. Luminescence polarization sometimes offers a
way to determine these properties. Electronic excitation of a
chromophore with polarized light will produce polarized emission.
However, any rotation of the molecule after excitation but before
emission will cause some depolarization of the emission. Depolarization
and lifetime measurements can be used together to determine rates of
rotation of chromophoric molecules that can be related to a molecule's
rotational diameter. Studying the temperature behavior of depolarization
can yield additional information about a molecule's shape. Observing
how rotational rates change under different conditions can indicate
corresponding changes in the shape of a molecule, such as denaturation
of proteins at high temperatures or complex formation at high
concentrations.
Luminescence science has developed into a powerful tool for studying
nature in macro- and microenvironments. At present, there are many
scientific advances being reported that incorporate fluorescence
technology into the research regimen. I expect this trend to continue as
the scientific community becomes more cognizant of the knowledge to
be gained by use of luminescence techniques.
The purpose of the symposium on which this book is based was to
report original research advances that use luminescence as a basic
investigative tool. Because these applications transcend the field of
chemistry, a multidisciplinary group of prominent scientists contributed
xi
In Luminescence Applications; Goldberg, M.;
ACS Symposium Series; American Chemical Society: Washington, DC, 1989.
to the symposium to enrich it with a variety of applications. Researchers
in the biological, chemical, and physical sciences have been in the
forefront of those employing luminescence methodology and have
brought some innovative uses of luminescence to bear on their research.
This book presents excellent research results in the biological, chemical,
environmental, and hydrological sciences. Each chapter addresses an
important application of luminescence and advances its particular
subject discipline.
Acknowledgments
I wish to acknowledge the assistance of the many people who
contributed to this publication. Patricia M. Negomir's help in
organizing this book was greatly appreciated John B Weeks gave
material assistance to th
an excellent job of editing the chapters. Patricia A. Griffiths, Linda S.
Britton, and their staff supplied typing assistance that was invaluable.
Ranee Velopoldi contributed to the design of the dust jacket. Finally, I
wish to thank all of the contributing authors for being patient while
awaiting publication of their material and for doing such an outstanding
job in their respective fields.
MARVIN C. GOLDBERG
U.S. Geological Survey
Box 25046, MS 424
Lakewood, CO 80225
September 26, 1988
xii
In Luminescence Applications; Goldberg, M.;
ACS Symposium Series; American Chemical Society: Washington, DC, 1989.
