EPA/600/4-91/011
April 1991
MOLECULAR OPTICAL SPECTROSCOPIC TECHNIQUES
FOR HAZARDOUS WASTE SITE SCREENING
by
DeLyle Eastwood
Lockheed Engineering and Sciences Company
Las Vegas, NV 89119
Contract No. 68-03-3249
Tuan Vo-Dinh
Oak Ridge National Laboratory
Oak Ridge, TN 37831
IAG No. DW-89933900-0
Project Officer
William H. Engelmann
Advanced Monitoring Systems Division
Environmental Monitoring Systems Laboratory
Las Vegas, NV 89193-3478
ENVIRONMENTAL MONITORING SYSTEMS LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U. S. ENVIRONMENTAL PROTECTION AGENCY
LAS VEGAS, NEVADA 89193-3478
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NOTICE
The information in this document has been funded wholly or in part by the
U. s. Environmental Protection Agency under contract No. 68-CO-0049 to
Lockheed Engineering and Sciences Company and under interagency Agreement No.
DW 8893 3900-01 to the U. S. Department of Energy (Oak Ridge National
Laboratory). It has been subjected to the Agency's peer and administrative
review, and it has been approved for publication as an EPA document. Mention
of trade names or commercial products does not constitute endorsement or
recommendation for use.
ii
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ABSTRACT
The U.S. Environmental Protection Agency is interested in field screening
hazardous waste sites for pollutants in surface water, ground water and soil.
This report is an initial technical overview of the principal molecular
spectroscopic techniques and instrumentation and their possible field-
screening applications at hazardous waste sites. The goal of this overview is
to describe the power and utility of molecular spectroscopic techniques for
hazardous waste site screening and to define the main strengths, weaknesses
and applications of each major spectroscopic technique. These spectroscopic
methods include electronic (ultraviolet-visible absorption and luminescence)
and vibrational (infrared absorption and Raman scattering) techniques. A
brief discussion is also given for some other techniques that rely on
spectroscopic detection (colorimetry and fluorometry as well as immunoassay
and fiber-optic chemical sensors). Hyphenated techniques such as high-
performance liquid chromatography and gas chromatography - Fourier transform
infrared spectroscopy are discussed for applications where the simultaneous
detection of the whole spectrum, rather than single wavelength detection, is
involved.
The report is organized as follows: The Introduction (Section 1) is
followed by a general Conclusions section (Section 2) that surveys in tabular
form the applicability of each spectroscopic technique for field and
laboratory use, together with classes of pollutants measured, advantages,
limitations, sensitivity, and current field applicability. The cost of
instrumentation and analysis and the time needed for analysis are briefly
addressed, and broad guidelines are given for three categories of
iii
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instrumentation, portable, field deployable and semi-field deployable.
sections 3 through 8 discuss the specific spectroscopic areas in more detail.
Each section presents a brief outline of the spectroscopic principles and
instrumentation for the particular spectroscopic technique and describes the
state-of-the-art approach. Advantages, limitations, sensitivities and
examples of specific techniques and their applications to environmental
pollutants are also discussed. Conclusions are given for each spectroscopic
technique at the end of each section. The reference section (Section 9),
contains all references cited, as well as a cross section of the definitive
literature. This bibliography is intended to give the reader an introductory
background for general principles and field applications of molecular
spectroscopic techniques. The Appendix consists of a set of figures that
address some of the major spectroscopic methods, including luminescence
techniques such as fluorescence emission, synchronous fluorescence, room
temperature phosphorescence, infrared methods, and surface-enhanced Raman
spectroscopy.
iv
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TABLES
Number Pacte
1 Characteristics of spectroscopic 8
Techniques for Field Analysis
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LIST OF ABBREVIATIONS AND SYMBOLS
ABBREVIATIONS
ASTM
CCD
CARS
EPA
FIA
FOCS
FTIR
GC
GC-MS
HPLC
IR
K
LC
LC-SLM
LESC-LV
LT
NRS
ppb/ppm
PAH
PBB
PCB
RIA
RR
SERS
SFC
SNR
SL
SPR
TIR
TLC
UV-VIS
American Society for Testing and
Materials
Charge-Coupled Device
Coherent Anti-stokes Raman spectroscopy
(U.S.) Environmental Protection Agency
Fluoroimmunoas s ay
Fiber-optic Chemical Sensors
Fourier Transform-Infrared spectroscopy
Gas Chromatography
Gas Chromatography-Mass Spectrometry
High Performance Liquid Chromatography
Infrared Spectroscopy
Degrees Kelvin
Liquid Chromatography
Liquid Crystal Spatial Light Modulator
Lockheed Engineering & Sciences Company
- Las Vegas
Low Temperature
Normal Raman Spectroscopy
part per billion/part per million
Polyaromatic Hydrocarbons
Polybrominated Biphenyls
Polychlorinated Biphenyls
Radioimmunoassay
Resonance Raman
surface-Enhanced Raman spectroscopy
Supercritical Fluid Chromatography
Signal to Noise Ratio
Synchronous Luminescence
Surface Plasmon Resonance
Total Internal Reflection
Thin Layer Chromatography
Ultraviolet-Visible Spectroscopy
Excitation Wavelength
Emission Wavelength
vi
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ACKNOWLEDGEMENTS
One of the authors (DeLyle Eastwood) wishes to acknowledge Russell L.
Lidberg and Gail Gibson for helping with the luminescence figures and with
completion of the final version of the text and Table 1. She also wishes to
thank Clare L. Gerlach for organization of the technical references and for
assistance in the preparation of the final manuscript. Marianne L. Faber was
responsible for technical editing of this manuscript. Edward J. Poziomek was
the official reviewer of the document and is thanked for his thoughtful
comments on the scope, organization and technical content of the document.
Donald F. Gurka provided FTZR figures for this report and reviewed the FTZR
and Raman sections as well.
One of the authors (Tuan Vo-Dinh) wishes to thank the U.S. Department of
Energy for continuing support (EPA Contract No. DW89933900-0 and DOE Contract
No. 1824-B124-A1).
Both of the authors wish to thank the many spectroscopists who provided
references or comments for this report and, in particular, Linda J. Cline-Love
for her informal review comments and the reviewers for the external EPA
Project Report, Earl L. Henry and Linda B. McGown.
vii
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CONTENTS
ABSTRACT ...............................
TABLES ................................. V
LIST OF ABBREVIATIONS AND SYMBOLS .................... vi
ACKNOWLEDGEMENTS ...........................
INTRODUCTION ............................ 1
CONCLUSIONS ............................ 4
ULTRAVIOLET-VISIBLE ABSORPTION SPECTROSCOPY ............ 16
INTRODUCTION ......................... 16
THEORY ............................ 16
INSTRUMENTATION ........................ 17
PRACTICAL APPLICATIONS .................... 18
CONCLUSIONS .......................... 20
REFERENCES .......................... 22
ULTRAVIOLET-VISIBLE LUMINESCENCE SPECTROSCOPY ........... 24
INTRODUCTION ......................... 24
THEORY ............................ 25
INSTRUMENTATION ........................ 27
TOTAL LUMINESCENCE SPECTROSCOPY ................ 28
SYNCHRONOUS LUMINESCENCE SPECTROSCOPY ............. 29
ROOM -TEMPERATURE PHOSPHORESCENCE ............... 33
CHEMILUMINESCENCE ....................... 36
CONCLUSIONS .......................... 37
REFERENCES .......................... 41
INFRARED ABSORPTION SPECTROSCOPY .................. 48
INTRODUCTION ......................... 48
THEORY ............................ 48
INSTRUMENTATION ........................ 49
FOURIER TRANSFORM INFRARED SPECTROSCOPY ............ 51
CONCLUSIONS .......................... 55
REFERENCES .......................... 57
RAMAN SPECTROSCOPY ......................... 61
INTRODUCTION ......................... 61
THEORY ............................ 61
RESONANCE RAMAN SPECTROSCOPY ................. 65
SURFACE-ENHANCED RAMAN SPECTROSCOPY .............. 66
CONCLUSIONS .......................... 70
REFERENCES .......................... 71
SPECTROSCOPIC IMMUNOASSAY TECHNIQUES ................ 76
INTRODUCTION ......................... 76
DISCUSSION .......................... 76
CONCLUSIONS .......................... 79
REFERENCES .......................... 80
FIBER OPTIC CHEMICAL SENSORS .................... 81
INTRODUCTION ......................... 81
DISCUSSIpN .......................... 82
CONCLUSIONS .......................... 85
REFERENCES .......................... 87
APPENDIX A ............................... 1
TYPICAL ENVIRONMENTAL POLLUTANT SPECTRA FOR SOME MAJOR
SPECTROSCOPIC TECHNIQUES ...................... 1
viii
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SECTION 1
INTRODUCTION
The U.S. Environmental Protection Agency (EPA) is interested in field
screening of hazardous waste sites for pollutants in surface and ground water
as well as soil. Major reasons for this interest are to achieve improved cost
effectiveness and to expedite remedial investigations at Superfund sites and
thus reduce the time lag between sampling and the receipt of analytical data,
which can often amount to 30 days or more. Field analytical screening can
also help to confine a detailed field investigation to those areas of a site
which are truly contaminated and thus reduce the number of samples sent to the
analytical laboratory, thereby providing more comprehensive environmental
studies as well as more relevant data with reduced cost and time.
Detailed characterization of many chemical pollutants in environmental
samples from waste sites can be performed using analytical techniques such as
liquid or gas chromatography and mass spectrometry. For many applications,
these procedures are needlessly time consuming and expensive, often, optical
spectroscopic methods and experiments that are field deployable or portable
provide attractive alternatives that permit large number of samples to be
screened, characterized, and prioritized in the field with little or no sample
preparation. These screening techniques permit rapid response and consider-
able cost savings because detailed analyses are required only for a selected
subset of samples. Spectroscopic techniques may sometimes provide information
on unusual sample types, or on nonvolatile compounds that are of high-
molecular weight or thermally labile. For functional groups or geometrical
isomers, these techniques may also provide specific information that could not
be obtained by more common EPA-approved methods such as gas chromatography.
spectroscopic techniques may also offer advantages for in situ measurements
-------
(with fiber optics), remote measurements, flow-through analyses, and
nondestructive testing.
Each spectroscopic technique has certain advantages and disadvantages.
Some may be more widely applicable, may be more feasible for field deployment
using current technology, or may be more specific or sensitive for trace
identification or classification. All of the techniques discussed in this
report have the potential for field application either by themselves or in
conjunction with appropriate separation or chromatographic steps. Recent
rapid advances in computer hardware and software, chemometrics, and pattern
recognition algorithms, although beyond the scope of this report, have also
been combined with advances in spectroscopic instrumentation to improve the
analysis of complex environmental pollutant mixtures and extract maximum
information from data sets.
The main objective of this report is to provide a technical overview and
assessment of the principal molecular spectroscopic techniques and
instrumentation with applications for field screening at hazardous waste
sites. These methods currently include UV-visible absorption and luminescence
(electronic) spectroscopy as well as infrared absorption and Raman
(vibrational) spectroscopy. For each method, a brief outline of the
spectroscopic principles and instrumentation considerations is given to
familiarize the reader with the present state-of-the-art approach.
Advantages, limitations, sensitivities, and examples of specific techniques
and their applications to environmental analyses are also discussed.
This report is intended to cover the most important spectroscopic
techniques that haye potential for field applications. Specific highlights
are also given for adjunct techniques such as colorimetric and fluorometric
analysis with chemical derivatization, spectroscopic immunoassay techniques,
and fiber optic chemical sensors. The range of possible applications of
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spectroscopic methods for field analysis is very broad and might include uses
for identification, classification, semiguantitation, and quantitation. A
direct comparison with other types of field analysis is beyond the scope of
this report.
This report is meant as a technical assessment and source document. This
document can provide a basis for early decision making on potential
spectroscopic techniques for field surveying. It is not the intention of this
document to compare specific instruments or procedures, although some
references are included for information. It gives only a broad overview of
time and cost considerations for various instrumental analytical procedures.
The authors consider that this report will serve as a useful source of
technical information and will contribute to the appreciation of the
usefulness of molecular spectroscopic techniques for hazardous waste site
screening. The extensive reference sections, although not comprehensive,
gives the reader access to background material related to field applications
of molecular spectroscopic techniques. This document can serve as a bridge
leading to more detailed reviews for specific decision making.
Conclusions are given for each spectroscopic technique at the end of its
section (Section 3 through 8). In the general conclusions (Section 2) a table
summarizes the applicability of each spectroscopic technique for field and
laboratory use, together with advantages, limitations, sensitivity, current
field availability and estimated cost and time. The figures in Appendix A
illustrate some of the main spectroscopic approaches as applied to specific
classes of pollutants. It is hoped that this overview will allow an
appreciation of the power and utility of molecular spectroscopic techniques
for hazardous waste site screening.
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SECTION 2
CONCLUSIONS
Field screening at hazardous waste sites for pollutants in surface water,
ground water, and soil is of importance because it can expedite remedial
investigations. Molecular spectroscopic analytical methods applied to field
screening provide an attractive alternative to standard EPA techniques such as
chromatographic and mass spectrometric procedures. Spectroscopic approaches
can provide valuable qualitative and quantitative information with substantial
savings of time and money. Instruments and methods are developing rapidly in
this growing area, which can greatly improve environmental analytical
technology.
Spectroscopic methods that are portable or field deployable permit
samples to be screened and prioritized in the field with little or no sample
preparation. Spectroscopic methods can sometimes provide information on
unusual sample types or on non-volatile compounds that are of high molecular
weight or that are thermally labile. These techniques also are advantageous
for in situ or remote measurements, real-time flow-through analysis, and
nondestructive testing. All of the spectroscopic methods have specific
advantages and shortcomings and have potential applicability for particular
environmental problems. Table 1 summarizes the advantages, limitations, and
sensitivities with examples of specific techniques and their application to
environmental pollutants. This table also includes definitions of portable,
field-deployable and semi-field-deployable instruments and includes relative
estimates of cost and time factors.
-------
Ultraviolet-visible absorption spectroscopy is a mature technique that
has good quantitative accuracy for single compounds after separation, or for
simple mixtures. If it is used in conjunction with high-performance liquid
chromatography using an optical multichannel analyzer as a detector, the
entire spectrum for each chromatographic peak can be recorded. Its
sensitivity is moderate and its specificity is low. Colorimetric reagents can
greatly increase the specificity of the method and improve sensitivity by
moving the spectrum of the reaction product into the visible region with high
absorption coefficients, ultraviolet-visible absorption spectroscopy is most
useful for unsaturated compounds (aromatic or heterocyclic).
Ultraviolet-visible luminescence (fluorescence and phosphorescence), when
applicable, can be the most sensitive spectroscopic technique for trace and
ultratrace analysis, especially with laser excitation. It is useful in
aqueous solutions to the part per billion to part per trillion level.
Specific techniques most useful in the field include synchronous luminescence
and room temperature phosphorescence. Luminescence is applicable to most
polyaromatic compounds and their derivatives and can be made applied to many
other compounds by using fluorometric reagents for chemical derivatization
reactions. It can also be used with high performance liquid chromatography as
a multichannel detector. Luminescence is much more selective for
identification or classification purposes than ultraviolet-visible absorption
but less selective than infrared or Raman spectroscopy. Its selectivity can
be enhanced using various excitation and emission wavelengths and by time or
phase resolution methods, and indirect detection methods such as fluorescence
quenching or energy transfer.
infrared absorption spectroscopy (dispersive and Fourier transform) has
been used in field applications, especially for monitoring air pollutants
using a gas cell, for characterizing oil or hazardous chemicals where
structural information from group frequencies is useful, and where sensitivity
-------
is not the critical factor. Infrared devices are also useful as real-time
detectors with GC-FTIR and for specific quantitation applications such as oil
and grease. Disadvantages include the need for sample preparation in order to
eliminate water which is the major interferent, some difficulties related to
quantitation and the mediocre sensitivity of the technique. Lately, more
compact, rugged instruments along with better sample preparation and signal
processing techniques that are designed to increase the sensitivity of this
method have made it more attractive for field use.
Raman spectroscopy complements infrared spectroscopy because it also
provides structural information but with different selection rules. Raman
spectroscopy is not sensitive to water and can use visible or near-infrared
optical techniques. Until recently, Raman was considered to have several
disadvantages for field use including complex instrumentation, need for laser
excitation and relatively low sensitivity. These disadvantages have been
«
reduced by the advent of more compact Raman spectrometers, smaller and more
rugged lasers, and special, more sensitive Raman techniques. The most
promising Raman technique for field use is surface-enhanced Raman spectroscopy
in which Raman scattering efficiency can be enhanced by factors of as much as
10* for some compounds when a chemical is adsorbed on a special roughened
metal (Cu, Ag, Au) surface. Although this technique may be promising for
future field applications, it is not yet fully understood or developed and may
not apply to all chemicals. The advantage of the technique is that it has the
potential to combine the sensitivity of luminescence with structural
information similar to that provided by infrared spectroscopy.
A comparison of the main spectroscopic techniques is given in Table 1 at
the end of this section.
Ancillary techniques that rely on spectroscopic detection and that
greatly enhance the utility of spectroscopic methods include colorimetry,
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fluorometry, immunoassay, and some fiber optic chemical sensors. Fiber optic
sensors may also use some change in the optical properties of the fiber or
cladding or may be used as probes for most of the spectroscopic techniques
discussed.
Spectroscopic techniques are being used with increasing frequency for
field screening, allowing rapid response and reduced costs for environmental
monitoring programs. Such techniques also help to optimize sampling efforts
and help to prioritize samples for more detailed analysis. Some spectroscopic
methods can be used in place without sampling, e.g. fiber optic chemical
sensors, whereas others can be used with portable instrumentation or field
deployable instruments set up in a mobile laboratory. Recent instrumentation
developments, such as more compact lasers, miniaturized optical hardware, new
types of detectors, increased use of fiber optics, and better computer
software for spectral data processing and pattern recognition have increased
the utility of these • spectroscopic methods.
Further research and development efforts are needed to improve the field
applicability of current and new spectroscopic analytical techniques, to make
instruments more portable and compact. Also, new techniques that employ
field-ready instruments need to be accompanied by detailed analytical
protocols, appropriate standards, calibration criteria and appropriate quality
assurance for specific pollutant classes. Field spectroscopic instruments and
methods are a rapidly improving and growing analytical area which can greatly
improve environmental analytical technology.
A better appreciation of the conclusions, relative to the applicability
of these spectroscopic techniques, can be obtained by reviewing Table 1.
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I.
CBARACTBHIETICB OF SPBCTROSCOPIC TECHNIQUES POR FIELD ANALYSIS
OV-V1S ABSORPTION
00
CURRENT RELATED LAB
FIELD TECHNIQUES
APPLICABILITY ADVANTAGES LIMITATIONS SENSITIVITY APPLICABILITY 4 SENSORS
VOLYAKOHATIC COHPOUHDS
(PAC«>
DYES
COLORIMETRIC REACTION
PRODUCTS
MATURE TECHNIQUE
I NSXIIU HENTATI OH
READILY AVAILABLE
GOOD QUANTITATIVE
ACCURACY fOR SINGLE
COMPOUNDS AND SINPLE
MIXTURES
FEN INTERFERENCES
BY NOMAROMATICS
SPECTRAL DATA
AVAILABLE
UMSPEClriC
(COMPARED lt> IR AMD
LUMINESCENCE)
EXTENSIVE SAMPLE
PREPARATION
QUANTITATION MAY BE
AFFECTED BY SOLVENT.
POLARITY OR MEDIUM,
CHEMICAL COHPLBXATION
MODERATE SENSITIVITY
ppa - ppb IH FAVORABLE
CASES
PORTABLE
-HAND-HELD COLORIMETER
-COLORXHETRIC KITS
FIELD DEPLOVABLB
INSTRUMENTATION WITH
MULTICHANNEL DETECTORS
HPLC DETECTORS
UV-VIS TECHNIQUES
- PT
- DERIVATIVE
LT MATRIX ISOLATION
RZFIECTAHCE
PH01OACOUSTIC
6PECTROSCOPY
PIBER OPTIC
COLCRIMETRIC SENSORS
MULTICHANNEL DETECTORS
- DIODE ARRAYS
- CCO*
CONTINUED
-------
I. CONTINUED
VO
CURRENT RELATED LAB
riELO TECHNIQUES
APPLICABILITY ADVANTAGES LIMITATIONS SENSITIVITY APPLICABILITY t SENSORS
POLYARONATIC COHPOUHDS
FLUORESCENT DYES
FLUOROHSTRIC REACTION
PRODUCTS
ream
PHENOLS
PESTICIDES
SENIVOLATtLES
NONVOLATILES
PETROLEUM OILS
HOST SENSITIVE HETHOD
FOR TRACE AND
ULTRATRACE ANALYSIS
NHEN AVPLICKBLE
INSTRUMENTATION
REAO1LT AVAILABLE
NO INTERFERENCE BY
HATER
FEN INTERFERENCES BY
NONAAOHATICS
SOHB STRUCTURAL
SPECirlCITT
- ENHANCED BY SPECIAL
TECHNIQUES
VERY SELECTIVE
- ENHANCED BY TIKE AMD
WAVELENGTH
VARIABILITY
CAN DISTINGUISH
GEOMETRICAL I6OHERS
L1HITED TO COHPOUNDS
HITH FAIRLY MICH
LUMINESCENCE YIELDS
(USUALLY FACl. UNLESS
DERIVATIIED)
RELATIVELr UNSPECIFIC
FOR STRUCTURAL
INFORMATION
(COMPAAEO TO IR)
QUAMTITATION
COMPLICATED BY
DIFFERENCES IN QUANTUM
YIELDS, QUENCHING,
HI CSOEHVI RONHENTS
LIMITED REFERENCE
SPECTRA AVAILABLE
EXCELLENT SENSITIVITY
ppt> (pptrtlllon OR
LESS HITH LASER
EXCITATION)
DEPENDENT ON QUANTUM
YIELDS
PORTABLE INSTRUMENTS
AVAILABLE
FIELD DEPLOYABLB
INSTRUMENTS AVAILABLE
FLOH-THRODCH OIL-HATER
MONITORS AMD HPLC
HITH MULTICHANNEL
DETECTORS
FRONT SURFACE - RTF
LUMINESCENCE
TECHNIQUES
- FLUORESCEHCC
- PHOSPHORESCENCE
- SYNCHRONOUS
- TIME AND PHASE
RESOLUTION
- POLARIZATION
- RT ANO LX
- ID
- MICROSCOPY
FIBER OPTIC
rUIOROHETRIC SENSORS
MULTICHANNEL DETECTORS
- DIODE ARRAYS
- CCDl
FLUORESCENCE QUENCHING
OR ENERGY TRANSFER
- INDIRECT HAYS
TO MEASURE
NONLUHIHESCEHT
HOLEULES
CONTINUED
-------
1 CONTINUED
CURRENT RELATED LAB
FIELD TECHNIQUES
APPLICABILITY ADVANTAGES LIMITATIONS SENSITIVITY APPLICABILITY t SENSORS
INCREASED SPECIFICITY
FOR INDIVIDUAL PRO
OH PAC CLASSES IN
COMPLEX MIXTURE
PETROLEUM OILS
CREOSOTES
INCREASED SPECIFICITY
LESS SPECTRAL
OVERLAP
CLASSIFICATION OP PAH.
BY NUMBER OP R1HCS
USEFUL FOR SCREENING
COHDINB WITH OTHER
LUMINESCENCE
TECHNIQUES
DECREASE IH
SENSITIVITY WITH
NARROWER BANDPASS ES
AND WAVELENGTH OFFSET
LOSS OP VIBRATIONAL
STRUCTURE IH SPECTRUM
NEED DUAL SCANNING
MONOCKROMATORfl
NEED POLYCHROMATIC
SOURCE
COOD SENSITIVITY
SLIGHTLY LOVER THAN
FLUORESCENCE EMISSION
DEPENDENT ON
INSTRUMENTAL
CONDITIONS
DEPENDENT ON STOXES
SHIFT OP COMPOUND
PORTABLE INSTRUMENTS
UNDER DEVELOPMENT
FIELD DEPLOYABLE
INSTRUMENTS AVAILABLE
LT MEASUREMENTS
TIME AMD PHASE
RESOLUTION
DERIVATIVE
REMOTE MONITOR UNDER
DEVELOPMENT
SYNCHRONOUS
PHOSPHORESCENCE
— ——— ^ ^— —— — ^— — — ^—^— — ROOM TBNTEIUTVIIE rKOBrnUKEaiEHCE |HTr| — — — — ^— ^— — — — ^ — ^—
CURRENT RELATED LAB
FIELD TECHNIQUES
APPLICABILITY ADVANTAGES LIMITATIONS SENSITIVITY APPLICABILITY * SENSORS
MOST LUMINESCENT PAC*.
PCB., PAH>
DIRECTLY OR HITH HEAVY
ATOM PERTURBED
EASY SAMPLE PHEP
ELIMINATES SCATTER
MID FLUORESCENCE
BACKGROUND
LONGER LIFETIMES THAN
FLUORESCENCE
NO NEED FOR CRYOGENIC
INSTRUMENTATION
USEFUL FOR SCREENING
ADDITIONAL SELECTIVITY
DUE TO PERTURBER
OXYCEH NAY QUENCH
IN SOLUTION
LESS STRUCTURE THAN
LTP
SUBSTRATE/TECHNIQUE
DEPENDENT
QUANTITATION MAY BE
COMPLICATED
LIMITED CORRECTED
SPECTRA AVAILABLE
COOD SENSITIVITY
ppb IH FAVORABLE CASES
DEPENDENT ON QUANTUM
YIELD OP COMPOUND
DEPENDENT OH
EFFICIENCY OF
PCRTURBBR
PORTABLE INSTRUMENTS
UNDER DEVELOPMENT
FIELD DEPLOYABLE
INSTRUMENTS AVAILABLE
PRONT SURFACE
RIGID MEDIUM
- FILTER PAPER
- TLC PLATE
DdSIHETRY
EASY SAMPLE PREP
CAN COMPARE HITH LT
TECHNIQUES FOR
OPTIHI1ATION
TIME RESOLUTION
TLC
ORGAN I I ED MEDIUM
- MICELLE SOLUTION
- CYCLODEKTHIN
CONTINUED
-------
1 CONTINUED
CURRENT RELATED LAB
FIELD TECHNIQUES
APPLICABILITY
LUMINESCENT PAC«
PCB>
ADVANTAGES LIMITATIONS SENSITIVITY APPLICABILITY t SENSORS
HIGHER SENSItlVITY,
SPECIFICITY THAN RT
VIBRATIONM. STRUCTURE
SIMILAR TO RAMAN
O.UUITITATIOH OVER
« ORDERS Or HACHITUDE
DISTINGUISH ISOHERfi
VERT SELECTIVE
- BRKAMCED BY TIME AMD
WAVELENGTH
VARIABILITY
CRYOGENIC APPARATUS
HORE COMPLICATED
NEED SKILLED OPERATOR
LESS REfERENCE
SPECTRAL DATA THAN RT
SOME AHALYTES MATRIX
DEPENDENT
EXCELLENT SENSITIVITY
pptrillion IN OPTIMAL
CASES
IMPROVED WITH LASER
LIMITED SEMI-FIELD
DEPLOY ABILITY
LT TECHNIQUES
- SHPOLSKII SPECTRA
- LASER-LINE HARROWING
- SITE SELECTION
- MATRIX ISOLATION
LOU TEMPERATURES
77 K TO 4 K
CONTINUED
-------
TABU 1 CONTINUED
APPLICABILITY
ORGANIC MID INORGANIC
DETERMINATION OP
SPECIFIC FUNCTIONAL
GROUPS
APPLICABILITY
ORGANIC AND INORGANIC
DETERKIHATIOK OP
SPECIFIC PUHCTIOHAL
CROUPE
ROUTINELY USED FOR
REAL-TIME CC AND
VAPOR ANALYSIS
ADVANTAGES
HIGHLY SPECIFIC
STRUCTURAL DATA OH
GROUP FREQUENCIES
NATURE TECHNIQUE
INSTRUMENTATION MIDELY
AVAILABLE
SPECTRAL LIBRARIES
AVAILABLE
AOVAHTAGES
HIGHLY SPECIFIC
STRUCTURAL DATA ON
CROUP FREQUENCIES
INSTRUMENTATION WIDELY
AVAILABLE
REAL-TINE FLOM THROUGH
VAPOR APPLICATIONS
- GC-FTIR
SPECTRAL LIBRARIES
AVAILABLE
LIMITATIONS
MID/LOU SENSITIVITY
MATER IS INTERFERENT
REQUIRES SPECIAL
OPTICS/SOLVENTS
QUAMTITATIOH
DIFFICULTIES
WEAK OPTICAL SOURCES
AMD DETECTORS
LIMITATIONS
LESS SENSITIVE THAN
LUMINESCENCE
REQUIRES SPECIAL
OPTICS/SOLVENTS
CAM TOLERATE SOME
MATER (BACKGROUND
SUBTRACTION)
ORGAHICS DETECTION
1-10 ppthoutind IN
HATER
SENSITIVITY
LESS SENSITIVE THAN
UV-VIS ABSORBANCE
MUCH LESS SENSITIVE
THAN FLUORESCENCE
ppthouiand TO ppn
IN FAVORABLE CASES
SENSITIVITY
MORE SENSITIVE THAN
DISPERSIVE III
- SIGNAL AVERAGING
ppm TO •ubppa IN
FAVORABLE CASES
CURRENT
FIELD
APPLICABILITY
PORTABLE AND FIELD
INSTRUMENTS AVAILABLE
PORTABLE UNIT WITH
CAS CELL
OUAHTITATION OF GREASE
AND OIL
ATM ATTACHMENTS FOR
SOLIDS, OILS
CURRENT
FIELD
APPLICABILITY
FIELD AND SEMI-FIELD
OEPLOYABLE
- WITH OR WITHOUT CC
- VOLATILES /SEMI VOL
ADAPTABLE TO USE
WITH SFC
RELATED LAB
TECHNIQUES
t SENSORS
FTIR
GC/LC-FT1R
RELATED LAB
TECHMZQUES
4 SENSORS
CC/LC-FTIR
MATRIX ISOLATION
- LT FOR SENSITIVITY
HICROSCOPX
CONTINUED
-------
TABLS 1 COMTIHUED
U)
APPLICABILITY
SINGLE COMPOUNDS
SIMPLE MATRICES
ORGANICS OVERTONES
CURRENT RELATED LAB
FIELD TECHNIQUES
ADVANTAGES LIMITATIONS SENSITIVITY APPLICABILITY 4 SENSORS
SOURCES AND OPTICAL
MATERIALS BETTER THAN
HID-IR
OPTICALLY GOOD SENSOR
HATER I ALE-
CM DISTINGUISH MAJOR
COMPONENTS OP SIMPLE
MATRIX
FEWER INTERFERENCES
THAU NID-IR
LESS SPCCTRAL
STRUCTURE THAN MID-IK
- OVERTONE OVERLAP
- LESS sptcxpiein
> INTERPRETATION
COMPLICATED
HOT USEFUL FOR COMPLEX
MATRICES
SIGNAL PROCESSING AMD
PATTERN RECOGNITION
REQUIRED
LOW SENSITIVITY
10 - 1 ppthoutand
PORTABLE NEAR- III
INSTRUMENT WITH FIBER
OPTIC PROBE
CHARACTERISATION OP
OIL
BULK CHEMICAL
ANALYSIS
SURFACE/POLLUTANT
INTERACTION STUDIES
NEAR IR SENSORS
PROCESS CONTROL
CONTINUED
-------
TABU 1 CONTINUED
CURRENT RELATED LAB
APPLICABILITY
ORGANIC AMD INORGANIC
AQUEOUS SOLUTIONS
BIOLOGICAL MATRICES
POLYMERS
APPLICABILITY
HANV POLLUTANTS
DEMONSTRATED FOR I
- PVRIDIHE
- HYDRAIINB
- PAH»
- PESTICIDES
FIELD TECHNIQUES
ADVANTAGES LIMITATIONS SENSITIVITY APPLICABILITY t SENSORS
SPECIFIC AS IB FOR
STRUCTURAL INFORMATION
DIFFERENT SELECTION
MULES - COMPLEMENTS III
FEWER INTERFERENCES
THAN IR IN VIS OR
HEAR-IR REGIONS
HATER AND GLASS NOT
INTERFERENCES
GOOD OPTICS AND
SOLVENTS AVAILABLE
CAN HANDLE UNUSUAL
SAMPLE SHAPES/SUES
ADVANTAGES
SPECIFIC IN STRUCTURAL
INFORMATION
MORE SENSITIVE THAN
NORMAL RAMAN
AS SENSITIVE AS
LUMINESCENCE IN
FAVORABLE CASES
NO INTERFERENCE BY
HATER
|EEE ALSO IIRS)
FLUORESCENCE INTERFER-
ENCE IN UV-VIS
REQUIRES LASER SOURCE
RELATIVELY COMPLEX
INSTRUMENTATION
REQUIRES SKILLED
OPERATOR
NOT AS NATURE AS IR
RELATIVELY POOR LIMITS
OF DETECTION
MODERATE SENSITIVITY
1000 - 20 ppa
SENI-FIELD DEPLOYABLB
INSTRUMENTS UNDER
DEVELOPMENT
RESEARCH IN I
- AQUEOUS SOLUTIONS
- BIOLOGICAL MATRICES
- POLYMERS
SPECIAL RAMAN
TECHNIQUES
- SERB
- RESONANCE
- CARS
- MICROPROBES
- MICROSCOPY
LT APPLICATIONS
CURRENT RELATED LAB
FIELD TECHNIQUES
LIMITATIONS SENSITIVITY APPLICABILITY I SENSORS
RELATIVELY NEW TECH.
SURFACE/SUBSTRATE
MATERIAL DEPENDENT
REPRODUCIBILITY
REQUIRES LASER AND
SPECIAL SUBSTRATE
NOT ALL ANALYTES
ENHANCED EQUALLY
FEW SPECTRAL LIBRARIES
(SEE ALSO NRS)
GOOD SENSITIVITY FOR
SELECTED ANALYTES
ppn. - ppb IN
FAVORABLE CASES
FIELD DEPLOYABLE
INSTRUMENTATION UNDER
DEVELOPMENT
RESEARCH TO OPTIMIXB
TECHNIQUES
MICROSCOPY
HICROPROBES
SURFACE STUDIES
FIBER OPTIC SENSORS
HPLC
(UKDER DEVELOPMENT)
MULTICHANNEL DETECTORS
CONTINUED
-------
TABLE 1 CONTINUED
Ul
CURRENT RELATED LAB
FIELD TECHNIQUES
APPLICABILITY ADVANTAGES LIMITATIONS SENSITIVITY APPLICABILITY 1 SENSORS
PACe ABSORBING IN UV
PHENOLS
SPECIFIC IN STRUCTURE
HAY ELIMINATE
FLUORESCENCE
BACKGROUND
(SEE ALSO NRS)
ONLY CHROHOPHORE
VIBRATIONS ENHANCED
LI HI TED TO UV
ABSORBING COMPOUNDS
- HMNLT PkCll
OUANTITATIOH DIFFICULT
HOI COMPARABLE TO
OTHER RAMAN
TECHNIQUES
UV LASER SOURCE
COMPLEX
INSTRUMENTATION
(SEE ALSO NRS|
FAIR SENSITIVITY IN
FAVORABLE CASES WITH
CHROHOPHORE VIBRATIONS
HAMY PRACTICAL
DIFFICULTIES
CHROMOPHORS
CHARACTERIIATIOH
BIOLOGICAL APPLICATION
DEFINITIONS OF PORTABLE. FIELD DEPLOY ABLE. AMD SEMI-FIELD DEPLOXABLE AS USED IN THIS TABLE AREI
PORTABLE I
BATTERY POWERED
ONE PERSON CAR CARRY
LITTLE SAMPLE PREP. ( <10 HIM.)
< $10,000.
INSTRUMENT COST
ANALYSIS COST < (10.
FIELD OEPLOYABLEl
GENERATOR POWERED
COMPACT, TNO PEOPLE CAN LIFT ISEVERAL INSTRUMENTS IN MOBILE LAB)
RELATIVELY SIMPLE SAMPLE PREP. | <1 HR.)
INSTRUMENT COST ilo,OOO. TO SIOO.QDD.
ANALYSIS COST tit. - 1100.
DEFINITIONS OF ABBREVIATIONS AS USED IN THIS TABLE AREi
ATR
CARS
CCD
rriR
cc
HPLC
IR
LC
LT
N«S
Attenuated Totil Reflectance
Coherent Anti-Stake* Raaan Spectroicopy
Charae-Coupled Device
Fancier Trenafora-Infrated Spec trot copy
Gae Chraaatography
High Performance Liquid Chronatography
Infrared Epactroacopy
Liquid Chroaatoqrephy
Low Tenperetate
Normal Haaan Spectroacopy
SEHI-FIELD DEFLOTABLEl
CAN FIT IK MOBILE LAB
COMPLEX OR FRAGILE INSTRUMENT
OFTEN CONSIDERABLE SAMPLE PREP. ( >t HB.)
INSTRUMENT COST > SIOO.OOO.
ANALYSIS COST > flOO.
PAC Polyarooatic Conpounde
PAH Polyerooatic Hydrocarbane
PCS Polychlorlnated Slphenyla
ppb/ppn pact par billion/part par Billion (n
RTP Rood Temperature Phoepnoreecanca
SERS Surface-Enhanced Kaaan Spacecoacopy
SFC Supercritical Fluid Chroaato?raphy
TLC Thin-Layer Chroaetography
UV-VIS Ultraviotet-Viaibla Spectroecopy
-------
SECTION 3
ULTRAVIOLET-VISIBLE ABSORPTION SPECTROSCOPY
INTRODUCTION
Ultraviolet-visible (UV-vis) absorption spectroscopy has long been
accepted as a mature -workhorse- technique especially suited for quantitative
analysis in samples of limited complexity (Wehry, quoted in Bjorseth, 1983).
THEORY
The theory involved in absorption spectroscopy has been discussed in many
standard texts' such as Murrell (1963), Burgess and Knowles (1981) and will be
mentioned only briefly here. Most of the organic molecules that absorb
strongly in the UV-vis are unsaturated, aromatic, or heterocyclic aromatic
molecules. The electronic transitions involve the excitation of one electron
from a bonding n to an antibonding n molecular orbital. Most polycyclic
aromatic hydrocarbons (PAHs) have spectra of this type containing considerable
vibrational fine structure, but the spectra of their polar derivatives have
much less well-resolved structure in liquid solution. For some heterocycles
and carbonyls, an unshared electron pair occupies a nonbonding orbital on the
heteroatom or carbonyl oxygen. One of the nonbonding electrons can sometimes
be promoted to an antibonding n orbital (n to *•). This transition, although
formally forbidden by quantum mechanical selection rules, can occur, and this
n to rr* transition is then often at longer wavelengths and much less intense
than IT to n* transitions.
16
-------
As described above, the light absorbed corresponds to electronic
transitions from the ground state to different excited states of the molecule,
usually in the 10'" second tine span. Usually, a double beam instrument is
used and light is simultaneously transmitted through the sample and through a
reference cell containing solvent. The basic equation for light transmission
(sometimes called the Beer-Lambert Lav) can be expressed as:
where
I = intensity of the transmitted radiation
I, = intensity of incident radiation
£ = molar absorptivity (LlT'cm'1)
C = molar concentration (ML'*)
d - thickness of sample (cm)
Absorbance, transmittance and intensity can then be related as:
absorbance = A = Iog1( -1 = GCd
transmittance * T • —
INSTRUMENTAT ION
(TV- vis double beam absorption spectrophotometers , usually using deuterium
sources for the UV spectral region and tungsten sources for the visible region
and photomultiplier or photodiode detection, have been discussed in many
standard texts (Burgess and Knowles, 1981) and will not be treated in detail
here. The increased availability of multichannel detectors such as photodiode
array detectors (PDAs) with millisecond responses has led to increased use of
these devices with high performance liquid chromatography (SPLC) or
supercritical fluid chromatography (SFC) on a real-time basis. These rapidly
developing techniques have potential for increased use in field laboratories.
17
-------
PRACTICAL APPLICATIONS
Polynuclear aromatic hydrocarbons (PAHs), in particular, have well-
structured UV-vis absorption spectra which are useful for identification
purposes. UV-vis absorption has the disadvantages of relatively low
sensitivity and selectivity, as compared with luminescence techniques. UV
absorption spectroscopy can be useful for nonfluorescent compounds or for dyes
absorbing in the near UV or visible region of the spectrum. Recent uses have
generally included low-temperature matrix isolation or Shpol'akii applications
(Meyer, 1971; nakhimovsky, 1989), (much more commonly used with UV-vis
luminescence for analytical applications) which are not practical for field
use, or computer derivatization, which enhances the observed spectral
structure for the analyst.
Second derivative spectrometers have been available for almost two
decades (see Eager, 1973; Hawthorne, 1980). Haas et al. (1988) have developed
and field-tested a portable spectrometer for second derivative absorption
spectrometry for the screening of aromatic contaminants in ground water.
Benzene and other aromatics such as phenols have been analyzed at the ppm
(pg/mL) level in a shallow ground-water well by using a prototype instrument
with a 5-m fiber optic probe, in comparison, a more sensitive fluorescence
method, also in the second derivative mode, can monitor benzene and phenols to
the ppb (ng/mL) level (Vo-Oinh, in Eastwood, 1981). The second derivative
absorption technique has limited applicability for complex mixtures because so
many compounds absorb in the same region, but may be satisfactory for
relatively simple mixtures at the ppm level.
Colorimetric techniques using chemical derivatization to move the absorp-
tion spectrum of the complex into the visible region have long been used for
quantitation. For screening purposes, a field kit has recently proven useful
for PAHs, and a separate kit is available for polychlorinated biphenyls
16
-------
(PCBs). The field method for analysis of aromatics at the sub-ppm level
developed by Hanby (1988) uses the Friedel-Crafts reaction. An alkyl halide
extractant and the polyaromatic pollutant form electrophilic aromatic
substitution products with the Lewis acid catalyst, which also serves as the
dehydrant necessary for this Friedel-Crafts reaction. These products are very
large molecules that have a high degree of electron delocalization. Hence,
they are intensely colored and can be compared with appropriate color charts.
A variation of this method can be used for analysis of trichloroethylene.
Hanby (1988) states that this method is more accurate than direct-injection
gas chromatography, but that it has the disadvantage of being relatively
cumbersome in the chemicals and equipment required.
Several kits have been developed that rely on colorimetric spot tests or
sensitive papers for selected environmental analyses (Leichnitz, 1979; Hach,
1988). These established techniques have limited applicability to complex
environmental samples but can be used for field-monitoring specific major
pollutants, especially when they have been verified by other techniques.
Hand-held colorimeters have proven useful, but their applicability does not
extend into the UV region. Colorimetric techniques are also being applied to
fiber-optic chemical sensors, but the required chemistry is more complex
because the reagent should be stable for long periods of time, and must be
immobilized on the fiber. In addition, the chemical reactions involved should
be easily reversible for some sensors, yet other sensors, designed to be
integrating probes, would require nonreversible reactions.
woolerton et al. (1988) discussed a qualitative KWIK-SKRENE colorimetric
method for PCBs in dielectric oils or soil. This method can detect
approximately 10 ppra of Aroclor 1260, but the chemical equipment may be cum-
bersome and some interferences may occur.
19
-------
Colorimetric detection is also used with bioassay and immunoassay
techniques. Sellers (1979) reported the detection of numerous pesticides
including Baygon*, carbaryl, diazinon, Dursban', and malathion in water to 10
ppm or less using enzyme test tickets and pretreatment with bromine. The
method works by measuring the inhibition of the enzyme cholinesterase in
solution by organophosphate and carbamate pesticides with colorimetric
detection (absence of color indicating a positive test). The bromine serves
to improve the sensitivity of diazinon, Dursban*, and malathion by converting
them from phosphorothionates to phosphates by oxidative desulfurization. The
tickets are stable under field conditions between 4«C and 40»C for as long as
8 months.
This discussion does not attempt to give a comprehensive survey of the
colorimetric literature; it presents highlights and references for
colorimetric reactions and biomarker reactions that use colorimetric
detection.
CONCLUSIONS
UV-vis absorption is a mature technique for quantitating and semi-
quantitating pollutants after separation or in relatively simple mixtures.
Because it is relatively insensitive (as compared to UV-vis luminescence),
relatively unstructured (as compared to infrared), and subject to interfer-
ences, at least in the UV region, it is most useful for pollutants where
alternative luminescence techniques do not exist or for compounds that absorb
strongly in the visible region, such as non-fluorescent dyes or colorimetric
reaction products.
Hand-held colorimeters are potentially useful for screening, but
currently do not extend to the UV region. The use of UV-intensified diode
20
-------
optical multichannel devices for absorption detection is increasing for EPLCs
and for real-tine, flow-through applications.
For field use, colorimetric applications, either as sensitive papers,
tubes, or spot-tests, or as colorimetric fiber-optic sensors deserve more
attention. However, these methods are usually designed to apply to specific
pollutants and therefore will lack general applicability. Although
colorimetric reagents were extensively discussed in the older literature, many
had unstable chemistries, numerous interferences, or were not sufficiently
specific. For example, metal chelate reagents often produce products that
have similar spectra for many different metals in addition to the target
metal. Also, some potentially useful colorimetric reagents which are not
commercially available or have undesirable characteristics (instability,
toxicity, mutagenicity, etc.) rendering their use by unskilled personnel
difficult. Seawater may also be an interferent for some colorimetric methods
(Eastwood, private communication). Colorimetric reagents were formerly not
required to be stable for long periods of time under field conditions. A new
look at colorimetric reagents to supplement fluorometric reagents is needed,
if better colorimetric sensors are to be developed.
21
-------
REFERENCES
This reference section contains all references cited and an additional
selection of definitive references that should provide the reader with a basic
understanding of ultraviolet-visible absorption spectroscopy.
Burgess, C. and Knowles, A., Eds., Techniques in Visible and Ultraviolet
spectrometry. Vol. II. chapman and Hall, London, 1981.
Chemetrics Catalog, 1988/1989.
Duquette, P. H., Guire, p. E., and Swanson, H. J., "Fieldable Enzyme
immunoassay Kits for Pesticides,* Proceedings, First international symposium
on Field Screening Methods for Hazardous Waste Site Investigations, Las Vegas,
NV, 1988, pp 239-242.
Eastwood, o., "Use of Luminescence spectroscopy in oil Identification," in
Modern Fluorescence Spectrogcopy. E. L. Wehry, Ed., Vol. 4. 1981, pp 251-275.
Haas III, J. W., Lee, E. Y., Thomas, C. L., Gammage, R. B., "Second -
Derivative ultraviolet Absorption Monitoring of Aromatic Contaminants in
croundwaters," Proceedings, First International symposium on Field Screening
Methods for Hazardous Waste Site Investigations, Las Vegas, NV, 1988, pp 105-
106.
Hach Company, PR/2000 soectrophotometer Handbook. Bach, 1988.
Hager, R. N., Anal. Chem., Vol. 45, 1973, pp 1131-1132.
Hanby, J. D., "A New Method for the Detection and Measurement of Aromatic
Compounds in Water," Proceedings, First International Symposium on Field
Screening Methods for Hazardous Waste Site Investigations, Las Vegas, NV,
1988, pp 389-394.
Hawthorne, A. R., Amer. Ind. Hyg. Assoc. J., Vol. 41, 1980,
p 915.
Herzberg, G., Molecular spectra and Molecular Structure; Vol. 3s Electronic
Spectra and Electronic Structure of Polyatomic Molecules; Van Nostrand
Reinhold, NY, 1966.
Jungreis, E., spot Test Analysis. John Wiley & Sons, NY, 1985.
Leichnitz, K., Ed., Detector Tube Handbook; Air Investigations and Technical
Gas Analysis with Drager Tubes. 4th Edition, 1979.
Meyer, B., Low Temperature Spectroscopy. Elsevier, NY, 1971.
Miller, J. N., Edf.; standards in Fluorescence Spectrometrvi chapman and Hall,
London, 1931.
Murrell, J. N., The Theory of the Electronic Spectra of Organic Molecules;
John Wiley & Sons, NY, 1963.
22
-------
Nakhiroovsky, L. A., Lamotte, M., and Jouaset-Dubien, J.f Handbook of Low
Temperature Electronic Spectra of Polvcyelic Aromatic Hydrocarbons. Elsevier,
NY, 1989.
Orodpour, M., Anderson, K. w., and Anderson, J. C., "Analysis of Two Component
Systems Utilizing Second Derivative UV-Visible Spectroscopy," presented at the
Pittsburgh Conference, Atlanta, GA, March 1989, poster.
Ruch, W. E., Ed., Chemical Detection of Gaseous Pollutants; &n Annotated
Bibliography. Ann Arbor Science, Ann Arbor, HI, 1966.
Sellers, D. R., "Feasibility of Monitoring Pesticide Breakthrough from
Charcoal Columns," U.S. Army Medical Research and Development Command Final
Report, 1979.
Henry, E. L., "Optical Spectrometric Techniques for Determination of
Polycyclic Aromatic Hydrocarbons," in Bjorseth, A., Handbook of Polvcyelic
Aromatic Hydrocarbons. Marcel Dekker, NY, 1983.
Woollerton, G. R., Valin, s., and Gibeault, T.f "The Kwik-skrene Analytical
Testing system Description of a Tool for Remediation of PCS spills,"
Proceedings First international Symposium on Field screening Methods for
Hazardous Waste Srce Investigations, Las Vegas, NV, 1988, pp 387-388.
Vanderlaan, M., Watkins, B. and stanker, L., "Immunochenical Quantification of
Dioxins in industrial chemicals and soils," Proceedings, First international
symposium on Field Screening Methods for Hazardous Haste Site Investigations,
Las Vegas, NV, 1988, pp 249-250.
zander, M., "Ultraviolet Absorption and Luminescence spectrometry: an Overview
of Recent Developments," chapter 6 in chemical Analysis of Polvcvclie Aromatic
Compounds. T. Vo-Dinh, Ed., John Wiley & Sons, NY, 1989, pp 171-200.
23
-------
SECTION 4
ULTRAVIOLET-VISIBLE LUMINESCENCE SPECTROSCOPY
INTRODUCTION
Ultraviolet-visible (UV-vis) photoluminescence (fluorescence or phosphor-
escence) has become a well-established technique for field and laboratory
screening and for specific guantitation applications using both scanning and
filter spectrofluorometers. Luminescence has the advantages of very high sen-
sitivity for some classes of pollutants, good selectivity, relative freedom
from interferences by water and non-fluorescing chemicals, ease of sample
preparation, and availability of field-deployable instrumentation.
*
Disadvantages include: wide variability in fluorescence yields, matrix and
microenvironment effects, and quenching effects. Since the mid-1970s, the
United states Coast Guard has been using standard fluorescence techniques in
mobile laboratories for oil spill identification (U.S. coast Guard, 1977).
These procedures also served as the basis for American Society for Testing and
Materials (ASTM) methods such as ASTM D3650-78. The EPA in Ediaon, NJ has
developed similar methods for monitoring oils and hazardous chemicals at
spills and at Super fund sites (Remeta and Gruenfeld, 1987). In addition to
aroma tic 3, PAHs, and oils, luminescence has been shown to be useful for
pollutants such as phenols, PCBs, and some pesticides, heterocycles, and metal
complexes.
These methods are still being used, for the most part with standard flu-
orescence emission techniques, significant developments have taken place in
sources, such as lasers and miniaturized lamps, and in detectors, with near-
infrared detectors becoming more common. Of special importance is the
24
-------
increased use of photodiode arrays, and more recently, CCDa, which have
allowed real-time spectra to be generated for use with hyphenated techniques
such as BPLC, SFC, and time-resolved measurements. Increased use of computer
automation, signal processing, and computerized library search routines,
together with commercially available spectrofluorometers that yield corrected
fluorescence spectra, has allowed for intercomparison of fluorescence spectral
data. Libraries of corrected fluorescence spectra have, until recently, been
unavailable, with exceptions such as Berlman (1971) and Brownrigg et al.
(1979).
THEORY
Conventional luminescence spectroscopy uses either a fixed wavelength
excitation (!„) to produce an emission spectrum or, less commonly, a fixed
emission wavelength (X..) to record an excitation spectrum. Excitation
«,
spectra are occasionally used for comparison with UV-vis absorption spectra
for the identification of unknown compounds.
In the last section, the Beer-Lambert Law was stated to be I = I.10"68*
where I was the transmitted light. The following discussion is based on
Killer (1981):
J, a1.*, = I.*i
-------
I = 2.303 If €Cd
r
Assuming the assumptions above are valid, this equation ia frequently
used to show that:
(1) the intensity of the fluorescence is directly proportional to the
intensity of the exciting light, so that the more intense the
light source, the more intense the fluorescence (hence the
advantage of lasers assuming that photodecomposition does not take
place);
(2) fluorescence intensity for a compound depends on € at the exciting
wavelength as well as on the quantum yield;
(3) the intensity of fluorescence is directly proportional to
concentration of the analyte.
The assumptions and approximations involved in deriving this equation are
often overlooked.
For organic molecules in solution or solid state, the following general
theory applies: the essence of photoluminescence spectroscopy is that a
molecular sample, excited by light from an external source, emits light at
different wavelengths (usually longer than the excitation wavelength) as
fluorescence or phosphorescence. In luminescence spectroscopy, the observed
transitions are electronic transitions with vibrational structure; the
selection rules for the vibrational structure are the same as those in Raman
spectroscopy, that is, allowed vibrational transitions are those which
correspond to changes in polar izability of the molecule.
Usually, on excitation with ultraviolet or visible light, the decay of
the molecule to the lowest excited singlet is radiationless . Radiative
/
transitions from the lowest excited singlet to different vibrational levels of
the ground state give rise to fluorescence emission. The lifetimes for these
fluorescence decays are usually from 10*" to 10"' seconds. Mixing of the
26
-------
singlet states with the lowest multiplet (usually triplet) states permits
radiative transitions (phosphorescence) from the lowest triplet state to the
ground state to occur. This transition is usually forbidden to occur due to
spin-selection rules. The lifetime of the phosphorescence decay can vary from
10** seconds to several seconds. Phosphorescence, as will be discussed later,
is easily quenched because of its longer lifetime and therefore is usually
observed only at low temperatures, in rigid matrices or in organized media, or
in the absence of oxygen. Because light must be absorbed and then re-emitted
radiatively, in competition with radiationless deactivation processes,
molecules with high luminescence quantum yields on excitation in the near-
ultraviolet or visible regions are normally those with aromatic or
heterocyclic structures (with extended IT or conjugated-bonding). In-depth
discussion of the theory of photoluminescence is available in standard texts
such as Parker (1968), Becker (1969), Miller (1981), and Vo-Dinh (1984).
INSTRUMENTATION
For most commercial spectrofluorometers, xenon arc lamps are used as
excitation sources, although mercury or other line sources or lasers with
fixed or tunable wavelengths may be used. Although some types of lasers, such
as diode lasers, nitrogen-dye lasers and even excinter lasers, are being made
more compact and rugged to be more suitable for field deployment, there are
also disadvantages for field use such as relatively high coat, relative
fragility and frequent lack of tunability. Probably lasers should be used
only when their special features are required or when factors such as limited
sample size or restricted detection volume are involved. For scanning
spectrofluorometers, the continuous spectrum of the light source is dispersed
by using an excitation monochromator, which can be scanned mechanically to
choose the excitation wavelength region with a selected bandpass. The emitted
light originating from the sample is usually detected at right angles to the
exciting light by an emission monochromator coupled to a detector.
27
-------
Photomultiplier tubes are the most commonly used detectors, but multichannel
detectors are gaining popularity. Further discussions of instrumental
considerations for photoluminescence are available in standard texts (Miller,
1981; schulman, 1985). A portable luminescence device with a fiber optic
probe has recently become commercially available for field measurements using
a fixed excitation wavelength (Environmental systems Corporation, 1988) and is
being modified to allow synchronous luminescence measurements. This
instrument was used for sensitized fluorescence spot tests of PAHs (Vo-Dinh
and White, 1986).
TOTAL LUMINESCENCE SPECTROSCOPY
Total luminescence (also called contour luminescence or excitation-
emission arrays) contains all of the information in the excitation and
emission spectra of the mixtures. A total luminescence spectrum can be
computer-derived by using consecutive emission spectra generated at different
excitation wavelengths. The complexity of the observed arrays, which are
generally instrument dependent and often contain more information than is
needed, has led to relatively little use of this technique. Christian et al.
in Wehry (1981) showed that such arrays could be generated in seconds using a
videofluorometer. Warner et al. (1979) discussed the design of such a system
and multicomponent data reduction schemes. Although the feasibility of
analyzing environmental samples by BPLC with this technique has been demon-
strated, it has not been used in the field. Denton et al. (1987, 1988, 1989)
has demonstrated CCDs in spectroscopic detection. This technology promises to
greatly improve spectroscopic instrumentation. When used for complex
mixtures, it should be combined with appropriate feature extraction techniques
to eliminate redundant information and to select the most important features.
It is most useful for feasibility studies on new analytes or sample types.
28
-------
SYNCHRONOUS LUMINESCENCE SPECTROSCOPE
Synchronous luminescence (SL) spectroscopy, introduced by Lloyd (1971),
generates spectra which are more simplified cross-sections of excitation-
emission arrays. These spectra can be rapidly and easily produced by
commercial spectrofluorometers. SL procedures have advantages over conven-
tional luminescence for environmental screening procedures because they reduce
or eliminate the frequent overlap of various emissions from the numerous
compounds in complex mixtures. Another unique feature of the SL technique is
the capability to provide spectral information in one measurement scan, for
PAH compounds with different numbers of fused rings. This technique has been
discussed in detail by Vo-Dinh, in Wehry (1981) and elsewhere.
With synchronous spectroscopy, the luminescence signal is recorded while
both X.. and XH are simultaneously scanned (Lloyd, 1978; Vo-Dinh, 1978). A
s
constant wavelength interval is maintained between the excitation and the
emission monochromators throughout the spectrum. As a result, the intensity
of the synchronous signal Zu, can be written as a product of two functions as
follows:
where:
k = a constant
c = concentration of the analyte
EE • excitation function
Ea = emission function
For a single molecular species, the observed intensity Xa is simplified,
often to a single peak, and the bandwidth of the peak is narrower than for the
conventional emission spectrum. Even for broad and featureless excitation and
emission spectra of molecules such as phenol, the synchronous signal will be
narrow. This feature can significantly reduce spectral overlap in multi-
29
-------
component mixtures, correlation of the signal wavelength position with the
structure of the compounds becomes easier. For example, the spectrum of an
aromatic compound with a larger number of rings occurs generally at a longer
wavelength than the spectrum of a compound with a smaller number of rings.
With conventional spectroscopy, this basic rule cannot often be utilized
advantageously due to severe spectral overlap. By confining each individual
spectrum to a narrow and definite spectral band, the synchronous method offers
the possibility of identifying specific compounds or a class of compounds in a
mixture.
A synchronous spectrum can be visualized on an excitation-emission matrix
(EEM) as a 45° line parallel to, and to the red of, the scattered excitation
light (an emission spectrum would be represented as a horizontal line on the
same matrix). For a small wavelength offset, similar to the stokes shift, the
resulting spectrum contains only a few peaks corresponding to zero-zero
transitions of fluorescent compounds in the mixture. For pattern recognition
in complex mixtures, larger wavelength offsets may also be used on an
empirical basis, variable SL, scanning the excitation and emission
monochromators at different speeds, was used by Kubic et al. (1980) to get
other cross-sections of the contour array. Another version of SL uses a
constant AE rather than AX, which is more theoretically significant.
Synchronous techniques work especially well to produce simple spectra for
PAH mixtures whose components have spectra with well-defined vibronic bands.
The technique may be less specific for spectra with asymmetric or unstructured
peaks or where the zero-zero transition is not strong.
SL offers instrumental simplicity. Devices intended for conventional
fixed-excitation spectra can often be employed for synchronous measurements
with little or no modification. Several spectrometers are available with pro-
vision for interlocking the excitation and emission monochromators. A variety
30
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of environmental samples has been analyzed to illustrate the applicability of
the SL techniques for screening PAH compounds in waste waters. (Abbott et
al., 1986).
The simple method of SL analysis opens up many possibilities for
monitoring organic pollutants by luminescence spectroscopy. The SL technique
can be applied to obtain not only spectroscopic fingerprints from complex
samples, such as oil spills as discussed by Eastwood in Wehry (1981), but also
specific information of analytical interest regarding individual pollutants or
pollutant classes. The synchronous fluorescence technique has already been
used for some field applications and, because of its simplicity, has immediate
applicability for use in mobile field laboratories and potential applicability
for use with portable instruments that have fiber optic probes. Vo-Dinh and
Abbott (1984) have successfully used this technique to rank relative amounts
of different PAH classes as compared to other more standard methods of
analysis. A protocol for total PAH characterization and quantitation based on
fluorescence emission and synchronous spectra is now under development at EPA-
Las Vegas (Eastwood et al., 1989).
The derivative technique, usually but not always the second derivative,
can be used to enhance the selectivity of photoluminescence techniques
including synchronous fluorescence. In this mode of data representation, the
signal produced is proportional to the second-derivative (d1) of the spectrum
with respect to wavelength. Second-derivative signals can be obtained by
numerical differentiation, modulation techniques, or direct electronic
differentiation. (Green and O'Haver, 1974). In the d1 mode the measurement
is of the rate of change of curvature of a peak. Broad peaks are eliminated
in the recording, but sharp spectral features are intensified; hence, this
technique provides improved compound selectivity and quantitation.
31
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The synchronous and derivative techniques can be combined for many of
environmental sample analysis applications. Purcell et alr in Cline-Love and
Eastwood (1985), successfully separated the SI, peaks of phenol, o-cresol, m-
cresol, and p-cresol by taking the fourth derivative (applying the second
derivative twice) of the SL system.
Luminescence spectroscopy is especially well-suited for determination of
chemicals that fluoresce in complex mixtures, due to the multidimensional
nature of the information available in the luminescence spectrum. For
analytical purposes, luminescence excitation and emission spectra, lifetimes,
polarization, and quenching or enhancement by perturbers are some of the
independent dimensions of analytical information.
AS an example, fluorescent compounds with highly overlapping emission and
excitation spectra may have different fluorescence lifetimes so that their
relative intensity contributions can be resolved in the lifetime domain. Even
with HPLC analysis, separation of PACa in complex real-world samples can be
difficult. McGown (1989) has used the fluorescence lifetime information
provided by phase-modulation fluorescence to strengthen the quantitative
analysis of fluorescence detection for HPLC. For on-line detection,
fluorescence lifetime heterogeneity of chromatographic peaks are used as an
indicator of the purity of the peaks.
Phase-resolved fluorescence spectroscopy (POTS) has not yet been used in
the field, but is under consideration for use with HPLC and with fiber-optic
remote sensors. Commercial instrumentation for PRFS is compact enough to fit
in a mobile laboratory.
These techniques are most valuable in situations where the variations in
the composition of complex samples (containing a large number of organic com-
pounds, e.g., oil spills [Eastwood in Wehry, 1981], exhaust soot, by-product
32
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water [Vo-Dinh in Wehry, 1981]) fail to provide significant changes in the
conventional fluorescence spectrum.
ROOM TEMPERATURE PHOSPHORESCENCE
Phosphorescence has an advantage in that many interferences, such as
fluorescence emission as well as Rayleigh and Raman scattering, can be
eliminated either by wavelength or lifetime selectivity. Although low-
temperature luminescence techniques such as laser-excited Shpol'skii, line-
narrowing fluorescence, or matrix isolation spectroscopy appear to be more
suitable for laboratory applications, room temperature phosphorescence (RTF)
enhancement is gaining increased interest for field applications (Vo-Dinh,
1984). RTP techniques usually require adsorption on solid substrates or
stabilization in organized media such as micelles (Cline-Love and Skrilec,
1981) or cyclodextrins (Warner in Eastwood and Cline-Love, 1988) to avoid
quenching by intermolecular collisions or by oxygen processes that might lead
to the deactivation of the phosphorescent triplet state.
The following is a brief discussion of the theory of phosphorescence (for
further discussion see Vo-Dinh [1984]). From the first excited singlet Slf
the molecule may undergo transition to some vibrational level of the triplet
manifold via a mechanism known as intersystem crossing (ISC), intersystem
crossing is possible because of the coupling of the electron spin with the
orbital angular momentum which produces a quantum mechanical mixing of states
of different multiplicities. The molecule then relaxes to the lowest vibra-
tional level of Tt by radiationless vibrational relaxation processes. From Tt
the molecule may return to the different vibrational levels of the ground
state so either by a radiationless deactivation process or by the emission of
a photon (phosphorescence). A less common emission process, delayed
fluorescence, can also occur by repopulation of the st state by thermal
activation or by triplet-triplet interaction.
33
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Because of its 3pin-forbidden nature, phosphorescence emission exhibits
typically longer decay tines than the spin-allowed fluorescence process, in
liquid solutions at room temperature, bi- and mono-molecular quenching proc-
esses usually cause nonradiative deactivation of the triplet state. The pre-
sence of oxygen, an efficient triplet quencher, is also a major contributor to
the radiationless deactivation of the triplet level. The radiationless deac-
tivation process for most molecules in the triplet state is so efficient that
phosphorescence can normally be observed only when the solution is frozen into
rigid matrices (Birks, 1970). Conventional methods in phosphorimetry,
therefore, involve: (1) careful preparation of oxygen-free solutions, <2)
insertion of analyte compounds into polymer samples, or (3) use of rigid
matrices of frozen organic solvent. The first two techniques involve tedious
and time-consuming preparation, the third requires experiments at low
temperatures, usually 7*7 to 4.2 K.
Intense phosphorescence at room temperature has been observed from
various salts of polyaromatic compounds (FACs) adsorbed on solid supports,
such as silica, alumina, paper, and asbestos (Shulman and Hailing, 1972; Vo-
Dinh, 1984). This type of phosphorescence is assumed to originate from
surface-adsorbed molecules, because none could be observed from finely ground
samples of free crystalline compounds. Numerous ionic compounds were found to
show strong phosphorescence, especially when they were spotted onto substrates
following dissolution in strongly acidic or basic solvents. As a consequence,
the ionic state of the molecules may have resulted in an increased molecular
rigidity via adsorption to the substrate, thus reducing the effect of
collisional deactivation. Hydrogen bonding was also found to be responsible
for phosphorescence of adsorbed compounds at room temperature.
Unlike conventional phosphorimetry, RTP does not require cryogenic equip-
ment and involves simple experimental steps such as: (1) substrate
34
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preparation (optional pretreatment). (2) sample delivery, (3) drying process,
and (4) spectroscopic measurement.
The versatility of sampling procedures is one of the main advantages of
the method. Various sample collection methods are possible, including
spotting, leaching, swipe techniques, and liquid filtration. The use of
filter paper or filter membranes as direct sample support broadens the type of
samples that can be monitored and is advantageous in performance,
reproducibility, convenience and low cost.
Although some luminescent chemicals such as PCBs have higher phosphor-
escence than fluorescence quantum yields, others such as PAHs require the
enhancement of the triplet emission by the external heavy atom effect, which
perturbs the electronic levels of the analyte and hence increases ISC between
the singlet and triplet states, leading to enhanced phosphorescence. Numerous
heavy atom salts such as thallium and lead acetate, silver perchlorate, cesium
or methyl iodide and sodium bromide have been found to be efficient in
enhancing the phosphorescence quantum yields of PAHs selectively (Vo-Dinh and
Hooyman, 1978). RTP signals can be selectively enhanced by several orders of
magnitude by pretreating the matrix or premixing the sample with the heavy
atom perturber.
Two types of external heavy-atom perturbers are commonly used for RTP.
Type A perturbers such as thallium acetate form phosphorescent ground state
charge-transfer complexes with the aromatic compound. Type B perturbers such
as methyl iodide do not associate with the compound. Type B perturbers
normally give multi-exponential decay curves, whereas Type A perturbers,
depending on the concentration of the perturber, will give mono- or bi-
exponential decay curves. Because the perturbers not only increase the
phosphorescence quantum yields but do so with different enhancement factors,
depending on perturbers used and their concentrations, the selectivity of the
35
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technique can be increased. (Vo-Dinh and Hooyman, 1978; Jakovljevic, 1978;
White and Seybold, 1977).
synchronous phosphorescence may also be used with the optimum AX
determined by the singlet-triplet energy difference, which might range from
100 to 300 ran.
These RTP techniques have been successfully used to analyze for indi-
vidual chemicals in complex environmental mixtures containing polyaromatic
hydrocarbons such as benzo(a)pyrene, benzo(e)pyrene, chrysene, fluoranthene,
fluorene, pyrene and phenanthrene. (Vo-Dinh, 1984; cline-Love and Skrilec,
1982; Bower and Winefordner, 1978; Ford and Hurtubise, 1979).
CHEMILUMINESCENCE
Chemiluminescence occurs when a chemical reaction produces an electron-
ically excited state to emit light on returning to the ground state. As
recently shown by Fernandez-Gutierrez and Kunoz de la Pena in Schulman (1985),
the Chemiluminescence intensity can be expressed as:
where:
Z = rate of the chemical reaction
4>a = Chemiluminescence quantum yield of the excited product
number of emitted photons
number of reacting molecules
Chemiluminescence can be measured as a function of time with the maximum
intensity usually proportional to the concentration of analyte, or by mixing
the reactants in a flowing system.
-------
Usually, for inorganic analyses, concentrations between UP* and 10'*
can be measured, requiring relatively simple equipment, chemilurain-
escence has been most often used for biochemical analyses, such as adenosine
triphosphate (ATP) using the oxidation of luciferin catalyzed by the
luciferase enzyme, or for air pollutant analysis. The development of ehemi-
luminescence for analytical purposes has been limited because few molecules
demonstrate this phenomenon in solution and also chemiluminescence methods
tend not to be very selective. Chemiluminescence reactions may be catalyzed
by low levels of ions. Chemiluminescence assays have therefore been developed
for ions such as Cu1* and Co1* based on their catalytic effect on the oxidation
of luminol. several other inorganic ions or gases have also been determined
by chemiluminescence methods involving luminol or luciferin.
CONCLUSIONS
uv-vis luminescence (fluorescence and phosphorescence), when applicable,
is potentially the most sensitive spectroscopic analytical method, especially
when laser excitation is available. Therefore, luminescence is the method of
choice for field use for trace or ultratrace analysis for classes of
pollutants with appreciable luminescence yields, such as most polyaromatic
compounds (PACs), both aromatic and heterocyclic. At higher levels, or where
extensive sample preparation is needed, other methods become competitive with
luminescence. Luminescence is especially applicable for water samples, where
little or no sample preparation may be involved, and for real-time flow-
through applications either direct or with BPLC multichannel detection.
As compared to uv-vis absorption, luminescence is potentially more
sensitive and subject to fewer interferences. As compared to infrared and
Raman, this technique is more sensitive, but spectra are less specific and
structured, vibrational structure in luminescence spectra ia found, however,
for certain classes of compounds such as PAHs. Additional spectral structure
37
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can be introduced by use of rigid media, low temperature, site-selective laser
excitation, or derivative techniques. Specificity in complex mixtures without
separatory or chromatographic procedures can be improved by techniques such as
synchronous luminescence, luminescence lifetime techniques or special low
temperature methods.
Fluorometric reagents can be extremely sensitive and specific, but many
have the same problems as colorimetric reagents, namely interferences, lack of
specificity, and chemical instability, especially for use with fluorometric
fiber-optic sensors. Other possible disadvantages include fluorescence
background such as that from fulvic or humic acids, scatter from excitation
light, quenching, or self-absorption. Quenching and self-absorption can
usually be avoided by dilution and scatter can be minimized by use of RTP,
life-time discrimination, or appropriate filters. Fluorescence from fulvic or
humic acids is usually a problem only at the low ppb level and then could be
avoided by separatory methods, time-resolved techniques, or background
subtraction.
Field-deployable scanning and filter spectrofluorometers have been
available and in use for some time, primarily for petroleum oil and PAH
analyses, and by hydrologists who use fluorescent dyes to trace ground water
movement. At least one portable fluorescence instrument has been developed,
and another portable spectrofluorometer with a fiber optic probe and
synchronous capability is under development. The main obstacle has been the
apparent lack of a market, because, until recently, attention was not focused
on thia promising technique for environmental applications. Now ASTM and EPA
analytical methods are being developed for PAHs, PCBs, and phenols, which
should stimulate the use of luminescence in environmental analysis.
Classes of compounds for which fluorescence is especially applicable
include petroleum oils and PAHs. Fluorescence has also been found useful for
38
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other polyaromatic compounds including phenols, some pesticides and
heterocycles. RTF is potentially applicable to important pollutants such as
PCBs and PBBs, and chlorinated dibenzodioxins and dibenzofurans.
For field use, fluorescence can be used to characterize oils and
creosotes, quantitate total PAHs and rank relative amounts of different PAH
classes using synchronous techniques. PCBS and some pesticides can be
detected and quantitated by RTF, possibly in conjunction with thin layer
chromatography
-------
procedures. Research efforts are also desirable to advance several of these
promising low-temperature approaches.
Chemiluminescence is a sensitive but specialized technique that should be
utilized along with other luminescence procedures, when applicable.
Analytical protocols need to be developed for more naturally fluorescing
species and better fluorometric reagents for nonfluorescing species,
especially for use with spot tests for field instruments and for fiber optic
chemical sensors (FOCS). Better portable spectrofluorometers need to be
developed with capability for synchronous measurements. Also needed are
smaller lasers (the development of which is proceeding rapidly), especially
for the UV range, better UV optical fibers having higher transmittance in the
UV, miniaturized sources, and other optical components. More research is
needed in solid surface spectroscopy to better understand the theory in order
to optimize experimental conditions and improve phosphorescence yields for
RTF.
-------
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Shimadzu, shimadzu Application Data (brochure), RF Spectrophotometry 3,
"Simultaneous Determination of Polycyclic Aromatic Hydrocarbons by Synchronous
Excitation Spectroscopy; Determination of Perylene, Anthracene, Fluorene, and
Carbazole," shimadzu corporation, no date.
Shulman, E. M. and Walling, C., Science, Vol. 178, 1972, p 53.
Siegel, N. and Haug, A., "Aluminum Interaction with Calmodulin - Evidence for
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Sogliero, G., Eastwood, D., and Ehmer, R., "Some Pattern Recognition
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pp 5-16.
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6 Sons, NY, 1984.
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46
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47
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SECTION 5
INFRARED ABSORPTION SPECTROSCOPE
INTRODUCTION
Vibrational apectroscopy has traditionally been of importance for
qualitative identification because of its specificity. Many sharp peaks may
be present in vibrational spectra, which can be related to molecular
structure. The frequencies of most molecular vibrations occur generally in
the infrared region of the electromagnetic spectrum. These vibrations may be
detected and measured either directly in an infrared (IR) spectrum or indi-
rectly in a Raman inelastic scattering spectrum.
THEORY
Quantum mechanical selection rules permit only discrete vibrational energy
levels. The selection rule for a vibration to be infrared active is that the
vibration must result in a change in the electric dipole moment. The number
of modes of vibration is (3N - 6) for a molecule consisting of N atoms (3N - 5
if the molecule is linear). For large molecules there are thus many
vibrational transitions. Many vibrations can, however, be localized to
particular bonds or groupings, such as the -C=O (carbonyl) group, forming the
basis of characteristic group frequencies. Also, vibrational modes are
observed relating to the skeletal vibrations of the molecule.
A simplified example can be given for a diatomic molecule (with 2 atoms
of mass nij and m,) connected by an elastic spring with force constant k. This
system can be considered to represent a harmonic oscillator in the classical
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physics approach. According to this model, the vibrational frequency v of the
bond connecting the two atoms can be approximately described by the formula:
"" 2/r
where:
k = force constant of the spring
u = reduced mass of the molecule
A quantum mechanical treatment of the same system will show that a
harmonic oscillator will have the energy levels E, given by E, - (v+l/2)hv,
where v = 0,1,2,3... is the vibrational quantum number, h is Planck's constant
and v is the classical vibrational frequency of the harmonic oscillator. The
vibrational selection rules are v = 0 or ± 1. In the real world, oscillators
k
will be found to have anharmonic components, which can be thought of, in the
classical physics approach, as due to inelasticity or friction in the spring.
See treatment in Griffiths and de Haseth (1986).
INSTRUMENTATION
A common dispersive grating instrument for measuring IR spectra is the
double-beam spectrophotometer. The light source is usually a heated filament
(or metal carbide rod) that emits a continuous spectrum of a "black body"
exhibiting considerable intensity in the IR region. The excitation beam is
split in two with one beam passing through the sample and the other through a
reference cell. Both beams then pass through a chopper, which allows altern-
ate sampling of the two beams. These beams then pass through a monochromator
to the detector, which measures the difference in intensities between the two
beams. Two types of infrared detectors have been commonly used: quantum
detectors and thermal detectors such as the Golay detector and the
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pyroelectric bolometer. Because thermal detectors respond relatively slowly,
solid-state semiconductor quantum detectors such as mercury cadmium telluride
(MCT) or PbS and PbSe are increasingly used. For good sensitivity, lead
sulfide must be chilled with a thermoelectric cooler below ambient
temperature, and NCT must be maintained at 77 K.
An ZR spectrum usually consists of a plot of the absorbance as a function
of wavenumber (cm*1 ) and is characterized by the positions of the maxima of
each of the absorption bands VM expressed in cm"1 . Within the ZR region of
the spectrum, the range from 1400 to 4000 cm*1 is especially useful for
identifying frequencies for characteristic groups, e.g., -c=o. The region
from 600 to 1400 cm*1 contains many bands, including those from skeletal
vibrations, so that it is especially useful for "fingerprinting" of aliphatic
and aromatic hydrocarbons and petroleum oils. Dispersive mid-ZR spectroscopy
has long been used by organic chemists for structural determinations. Zts use
as an analytical procedure has never been fully developed due mainly to its
/
lack of sensitivity. Qualitative (e.g., for oil identification, Grant and
Eastwood, 1983) and a few specific quantitative (e.g., oil and grease) methods
have already been employed for field use.
The near ZR region from roughly 780 nm to 1600 nm (12,800 to 6250 cm"1)
contains many broad, overlapping harmonic and overtone peaks. Computer
pattern recognition and signal processing techniques are used to deconvolute
the broad spectral peaks. This spectral region has recently proven useful for
process control and for monitoring selected environmental pollutants
(including the use of near ZR sensors), but would prove less useful for trace
analysis, because it is sensitive only to major components, down to 1 to 0.1%.
The specificity of ZR is an advantage in comparison with UV-vis absorp-
tion and luminescence spectroscopies. Disadvantages include relatively low
sensitivity due to relatively weak bands, the fact that ZR band strengths may
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not appear to be directly proportional to concentration if measurements are
made at low spectral resolution, and special optical and solvent requirements.
In particular, absorption bands of water in IR spectra are a serious
limitation of this technique for trace analysis for hazardous waste
applications. FTIR spectral subtraction techniques using a circle cell can be
used down to the part per thousand level in water, but often ppm or lower is
desired.
FOURIER TRANSFORM INFRARED SPECTROSCOPY
Fourier transform IR (FTIR) spectroscopy uses an interferometer in
conjunction with Fourier transform mathematical techniques and a dedicated
computer. This technique is especially useful for real-time or flow-through
gas phase measurements although it is also applicable to solids, films and
liquids.
The theory behind FTIR was discussed by Griffiths and de Baseth (1986).
Most interferometers are based on the same principle as the Hichelson
interferometer, which divides a beam of light into two parts and recombines
them after a path difference has introduced interference between the beams.
Intensity variations can be measured by the detector as a function of path
difference. The interferometer consists of two plane mirrors, one of which is
moved with a drive mechanism in a direction perpendicular to the plane of the
other fixed mirror, with a beam splitter between them.
For a monochromatic light source, the theory can be described briefly as
follows: The optical path difference between the beams introduced by the
fixed and movable mirrors is called the retardation 6. The amplitude of the
interferogram after detection and amplification is proportional to the
intensity of the source, the beam splitter efficiency, the detector response,
and the amplifier characteristics.
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1(5) is the modulated ac component of the intensity, usually called the
interferogram.
For a monochromatic source and an ideal interferometer, the equation for
the interferogram ia:
1(6) = 0.5l(7)Cos2/rv6
For a polychromatic source and a nonideal interferometer, where the
beamsplitter efficiency, detector efficiency, etc. are given as a function of
wavelength, 1(6) can be expressed as:
1(6} = 0.5H(v> I(v)Cos2;iv6
vhere: H(7) - wavelength-dependent correction factor
B(v") is the parameter describing the intensity of the source as a
function of wavenumber v as modified by the instrumental characteristics.
B(v") = 0.58(7)1(7) = single beam spectral intensity
so that:
1(5) =» B(v*)Cos2TnT6
where: 1(6) = cosine Fourier transform of B(v")
Usually, the moving mirror ia scanned at a constant velocity, v (cm/sec)
so that the retardation at t seconds can be thought of as:
6 = 2vt
then:
For a continuous polychromatic source:
d6
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Other factors as discussed by Griffiths and de Haseth (1986) include
resolution, phase errors, and beam divergence.
The simultaneous measurement of all spectral elements by an
interferometer is called the mutiplex or Fellgett's advantage, allowing for
rapid spectral acquisition. The mutiplex advantage can be expressed as a
sensitivity advantage, comparing the signal-to-noise ratio (SNR) of a spectrum
measured on a Fourier transform instrument to the SNR of the same spectra on a
dispersive spectrometer. If the resolution, acquisition time, and all other
instrumental conditions are equivalent, the SNR for the FT instrument will be
increased by a factor equal to tf, where M is the number of resolution
elements assuming detector dark noise is dominant. The time advantage of an
FT spectrometer, allowing complete spectra to be recorded in milliseconds, is
even greater, being directly proportional to M. A smaller advantage is
Jaquinot'3 advantage, which stems from increased throughput of Fourier
spectrometers as compared to IR grating spectrometers. The increased
throughput is dependent on the optics and resolution used, so that the total
advantage of the FTIR over dispersive instruments is small in this regard.
Another, the Connes advantage, is that the IR spectral frequency in the FTIR
is precisely referenced by laser lines. This feature enables the spectra to
be independent of the instrument and useful for interlaboratory comparison and
spectral library collection.
Griffiths and coworkers have compared FT and grating IR spectrophoto-
meters from a theoretical viewpoint in terms of the multiplex advantage of FT
spectrometers, relative optical throughput, and the comparative performance of
the detectors used with each type of system (Griffiths et al., 1977).
Recently, FTIR, especially used as GC-FTIR for complex environmental
samples, has become accepted as a laboratory technique. Gurka (1988) has
developed standard protocols for EPA analytical applications.
S3
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For satisfactory GC-FTIR analysis, the interface is most important. Most
commonly, heated light pipes have been used. Basically this involves a long
narrow flow cell coated with gold, with IR-transparent windows and with
transfer lines for the GC effluent. Matrix-isolation capillary GC-FTIR has
been commercially developed, and a real-time cold finger cryotrapping approach
has been reported by Griffiths. In their present form, these systems would be
complex instrumentally for field use, but offer advantages for higher
sensitivities and lower detection limits, as compared to the more common GC-
FTIR systems that use light pipes.
Although GC-FTIR offers advantages for field analysis of volatile pol-
lutants, analytical techniques for nonvolatiles by hyphenated-FTIR techniques
are in a less advanced stage of development. HPLC-FTIR techniques are
currently being studied by the EPA. Supercritical fluid chromatography (SFC)
has also been combined with FTIR, and Liebman et al.(1989) have reported the
possibility of combining such analytical techniques with fiber optics and
expert systems for field use. Although some of these methods may remain pri-
marily laboratory methods, FTIR and hyphenated FTIR methods are increasingly
being considered for field conditions. Fateley et al. (1989) has recently
explored the use of long path length FTIR in a mobile field laboratory to
measure air emissions of volatile organic compounds from soil.
Computers are integral parts of FTIR instruments, although they are being
increasingly used with dispersive IR instruments also. Although extensive IR
library data bases exist, many of the spectra for dispersive IR are old and
are not equivalent to spectra analyzed under modern spectroscopic and sampling
conditions. Yet, for many field applications, dispersive IR spectrometers
with a minicomputer-based data system may also be used at lower cost. The
choice of the type of instrument is best decided by the users to satisfy their
particular needs.
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in general, the use of an FTIR spectrometer is strongly favored for most
measurements in which the time to acquire data is limited by the type of
experiment (e.g., on-line gas chromatography-infrared (GC-IR) or for
techniques in which the time needed for absorption spectroscopy is very long,
as with absorption spectroscopy of samples with very high absorbance).
FTIR has several other advantages over conventional methods. It is much
less susceptible to stray radiation. In addition, because a computer is
already used to obtain the Fourier transform, it is easy to add multiple scans
to improve the signal-to-noise ratio (for Poisson's distribution-type noise,
noise adds up as the square root of the number of scans, whereas signal adds
linearly). Digital subtraction (useful for subtracting water background to
obtain infrared spectra in aqueous solutions) can produce difference spectra.
This method has advantages in obtaining infrared spectra in aqueous solutions.
Although IR is a. very specific technique, like other absorption methods,
it lacks the potential real-time sensitivity for ultratrace measurements as
compared to UV-vis luminescence. Special techniques have been used to improve
detection limits of IR including tunable laser diode infrared absorption
spectroscopy. FTIR, in particular, is now sensitive enough for many
environmental applications.
CONCLUSIONS
Dispersive and FTIR instruments have already been used in the field,
especially for total hydrocarbon and oil quantitation and characterization.
The advantages of IR are largely that it is a mature technique with
commercially available spectral libraries and that it is specific for
characterization, because functional groups can be identified even for unknown
compounds. Quantitation is also performed, although some difficulties have
been reported. Disadvantages have included relative lack of sensitivity,
55
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solvent interferences, and need for special solvents and optics. One of the
greatest limitations of IR for in situ field analysis of liquids and solid
wastes is interference from water, which necessitates more complicated sample
preparation. For this reason it has been used in the field mainly for vapor
phase applications (either remotely or with long-path length gas cells).
EPA has used infrared primarily as GC-FTIR, with vapor phase detection in
a light pipe, or for front surface measurements on a cryostat cold finger.
Fate ley et al. (1989) are also studying FTZR for soil gas emissions with vapor
phase detection. Liebman (1988) has reported FTZR detection of pollutants
combined with supercritical fluid extraction (SFE) and fiber-optic light-pipe
interfaces. ZR used with HPLC and flow-through applications is less
developed. For EPA, the future seems to hold increased use of GC-FTIR and
vapor phase long-path length ZR, including field deployment. FTZR, with
attenuated total reflectance (ATR) attachments, is also applicable for
characterization of bulk pollutants and front surface measurements on complex
matrices such as oil-soaked soils. Portable ZR instruments exist and are used
in the field especially for gas analysis, but they should be miniaturized to a
greater extent. Near-IR portable instruments and sensors are under
development, but are mainly useful for relatively simple, highly concentrated
mixtures, for process control, and for oil characterization. Mid-IR sensors
are limited by the lack of inexpensive fiber optic materials for the mid-lR
region and by interference from water.
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This reference section contains all references cited and an additional
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Spartz, H.L., Witkowski, M. R., Fateley, J. B.f Jarvis, j. M., White, J. S.,
Paukstelis, J. V., Hammaker, R. M., Fateley, W. G., Carter, R. E., Thomas, M.,
Lane, D. D., Harotz, G. A., Fairlesa, B. J., Holloway, T., Hudson, J. L., and
Gurka, D. F., "Evaluation of a Mobile FT-IR System for Rapid VOC
Determination; Fart 1: Preliminary Qualitative and Quantitative calibration
Results,* American Environmental Laboratory, November 1989, pp 15-30.
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SECTION 6
RAMAN SPECTROSCOPE
INTRODUCTION
Raman spectroscopy has been used much less than IR spectroscopy due to
its relatively complex instrumentation, relatively low sensitivity and
interferences in the visible from fluorescence. Recent improvements in near-
infrared lasers sources to reduce interferences from fluorescence and in
signal processing have made Raman techniques potentially more promising for
field applications. Raman spectra are due to inelastic scattering processes
and the Raman-active vibrations are those which cause changes in the
polarizability of the, molecule. Therefore, Raman spectroscopy is
complementary to IR because Raman-active vibrations are often IR-inactive and
vice versa.
THEORY
In general, scattering is produced when electrons in a molecule oscillate
under the influence of an applied electromagnetic wave. The extent of the
scattering depends on the polarizability of the electrons in the molecule
(i.e., the dipole moment induced by the electric field).
When a molecule with a spherical, symmetric electron cloud is placed
between the plates of a charged condenser, the electrons are pulled toward the
positive plate and the protons toward the negative plate. The molecule is
said to be polarized, and has an induced dipole moment. Representing the vec-
tor of the electric force of the external field as E, and the induced dipole
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moment oriented parallel to the direction of E as H, \L can be defined as /J
oE, where a is the polarizability of the atom. Using Cartesian coordinates
to resolve the electric field, this equation can be rewritten as:
For an asymmetric molecule, a may be different for the x, y, and z directions;
for the anisotropic case, the polarizability is described by a tensor
P. • «„ E. + a., ET + a_ B.
V, ' «„ E. * °w ET * an E.
P. • a» E. * a., ET * a« E.
where a.m, a^, . . . a,,, are proportionality constants between p. and E,, ^, and
E,, . . .p. and E,, etc.
when light of frequency v0 impinges on a molecule, the electronic cloud
of the molecule develops an induced frequency. An induced dipole moment
vibrates at frequency v., and its amplitude is proportional to the polariz-
ability of the molecule. As a result, the molecule emits Rayleigh radiation,
the frequency of which is v0.
The polarizability of the molecule depends on its size, shape, and orien-
tation; and it can be viewed as a polarizability ellipsoid. The polariz-
ability ellipsoid may be modified as a result of change in the shape of the
molecule due to vibrations of the atomic nuclei. Therefore, radiation from
the molecule contains not only v.. the exciting frequency, but also the sum
and difference of the exciting and vibrational frequency v. i.e., v.. = v. + v.
and va = v. - v..
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The frequency shifts v.. and v. are called anti-Stokes and Stokes Raman
lines (or bands), respectively. The stokes Raman radiation is of lower
frequency (energy) than the anti-Stokes and is usually the only spectrum
recorded. Anti-Stokes bands are due to thermally excited ("hot") states and
will decrease in intensity at lower temperatures.
In Raman spectroscopy a powerful laser source scatters light
inelastically from the sample. The scattered light, usually collected at
right angles, reaches a spectrometer: preferably one with high stray light
rejection, high resolution, and high throughput.
Raman instruments require a powerful source, preferably monochromatic.
Most Raman spectrometers currently use argon or krypton ion lasers. Light
scattering increases as v4, leading to a gain in Raman intensity at higher
frequencies (shorter wavelengths). For resonance Raman analysis of
pollutants, usually UV sources are required. For this application excimer
lasers such as KrF or frequency doubling or tripling a visible light source is
required.
Raman spectrometers require high resolution, low stray light
spectrometers; usually double or triple monochromators are used. Concave
holographic gratings also are more nearly perfect and improve throughput and
stray light rejection. Other optics and detectors are similar to those needed
for luminescence measurements.
A near infrared solid-state laser source is now used on one commercial FT
Raman system. This avoids fluorescence background, an important consideration
for most Raman measurements, at the cost of some sensitivity.
Raman suffers from relative insensitivity (20 - 1000 ppm even with argon
ion laser excitation) as compared to OV-vis fluorescence and absorption,
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although the optical and solvent problems are reduced if excitation in the UV
or visible is involved. Water, for example, is not an interferent. If lasers
with excitation wavelengths in the visible or UV region are used, background
fluorescence may be a serious problem, although there are ways to avoid this
(such as use of a large quantity of a fluorescence quencher, or time
resolution).
For environmental use the relative insensitivity of the normal Raman
technique coupled with the greater instrumentation complexity required for its
performance has so far limited this technique to a few feasibility studies:
its use in the field still appears to be premature.
Tilotta et al. (1987) demonstrated a new signal processing approach which
involved optical multiplexing with Hadamard transform Raman spectroscopy in
the visible and near-IR spectral region. This spectrometer utilizes a liquid
crystal spatial light, modulator (LC-SLM) in its exit focal plane, in this
instrument, the dispersed radiation is focussed onto an encoding mask where
resolution elements are transmitted or rejected by absorption, depending on
electronically controlled encodement which makes individual elements of the
mask opaque or transmitting. This instrument allows the rejection of Rayleigh
scattering and allows spectral and background subtraction with simultaneous
observation of multiple Raman levels for pollutants such as 2-nitropropane.
Although this relatively new technique has not yet been field tested, it
offers obvious advantages of increased experimental simplicity and time
savings for on-the-fly or time-resolved studies.
As stated by Gerrard and Bowley (1988), for most common analytical appli-
cations involving routine qualitative and quantitative analysis of unknowns,
better established methods are available that are more rapid, are more
sensitive, or have larger data bases. In general, other techniques are
preferred to normal Raman spectroscopy, when available, for environmental
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analytical applications unless special, more sensitive Raman techniques can be
employed such as resonance Raman (using a tunable UV laser) or surface-
enhanced Raman, or unless the special sampling advantages of Raman are needed.
RESONANCE RAMAN SPECTROSCOPY
Normal Raman spectroscopy (MRS) is induced by excitation far removed from
any electronic transitions of the molecule, i.e., a virtual transition. In
the Resonance Raman (RR) effect, the frequency v. is allowed to be near an
electronic absorption v^.. In this preresonance region, a marked enhancement
of intensity can result due to a mechanism involving a single excited
electronic state, in this case the intensity I of the resonance Raman signal
can be given with several simplifying approximations (see discussion in Carey,
1982) as:
f v' * v1 T
c~(v. ± vTit >• r ;.
IK*-O 1
A smaller term arising from the vibronic mixing of two excited states is not
shown. This is the resonance Raman effect, and the advantage is in its great
sensitivity and selectivity as a tool for investigating chromophore structure,
because only vibrational modes directly associated with the chromophore have
their intensities enhanced. Normal modes, which have a large shift in
equilibrium geometry upon electronic excitation, produce intense resonance
Raman features.
The enhancement in the RR spectrum is typically between 10' and 104. The
RR effect can be obtained at concentrations of 10*4 M'L*1 or less. At these
concentrations normal Raman spectra are usually undetectable. Thus, RR
provides a means of selectively probing vibrational frequencies of a
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chromophore with a sensitivity approaching that of ultraviolet absorption
spectroscopy.
Raman, like ZR, has great specificity, but differs from IR in two
important aspects. Raman is well suited to condensed phase samples and UV-RR
has the sensitivity for analyses at the ppb level for some strong Raman
scatterers (chromophores) without sample concentration. The vibrational
information obtained is complementary to ZR because the selection rules are
different and may be especially useful for skeletal vibrations and some
specific vibrations such as the -C=N stretch. The technique is still immature
and requires relatively cumbersome equipment such as an excimer laser and
Raman shifter or a crystal to frequency-double a tunable visible laser source.
The Raman technique offers a large linear dynamic range, and water can be used
as a suitable solvent.
Mann and Vickers (1988) applied ultraviolet resonance-enhanced Raman (RR)
spectroscopy to hazardous waste analysis both directly and in conjunction with
HPLC. some hazardous pollutants that have been analyzed by Raman include
phenols, dimethylsulfone, sulfate and bisulfate.
Asher et al. (1983) found that PAHs, for example, could be studied with
excitation far enough in the UV region that fluorescence background was not
obtained. The disadvantage from the environmentalist's point of view is that
only the chromophore is excited. Most pollutants do not have a suitable
chromophore in the visible region, and UV lasers are still expensive and
complex to operate under field conditions.
SURFACE-ENHANCED RAMAN SPECTROSCOPY
Surface-enhanced Raman spectroscopy (SERS) appears to be the most promis-
ing Raman technique for ultratrace environmental analysis, and consequently it
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deserves more detailed discussion. SERS was first observed by Fleischnann et
al. (1974) from pyridine molecules adsorbed on silver electrode surfaces that
had been roughened electrolytically by oxidation/reduction cycles.
Enhancements in the Raman scattering efficiency have been observed by
factors of as much as 10* in favorable cases when a compound is adsorbed on or
near special metal surfaces (Jeanmaire and Van Duyne, 1977). This enormous
enhancement of the normally weak Raman scattering process helps to overcome
the relatively low sensitivity of Raman spectroscopy. Its disadvantages are
that it is still not well understood; electromagnetic and chemical sorption
effects both appear to be involved, and the effect does not occur to the same
extent for all analytes.
The electromagnetic effect is associated with large local fields caused
by electromagnetic resonances occurring near metal surface structures. A
major contributor is surface plasmons associated with collective excitations
of surface conduction electrons in metal particles (Chang and Furtak, 1982).
These plasmons are excited by the incident radiation (Ritchie, 1957). At the
plasmon frequency the metal becomes highly polarizable, resulting in large
local surface fields that increase the Raman emission intensity, which is
proportional to the square of the applied field at the surface. Other types
of electromagnetic enhancement effects include the lightning-rod effect, where
electromagnetic field lines near high curvature points on the surface become
concentrated, and the image effect, where the surface is polarized by dipole-
induced fields in adsorbed molecules.
Electromagnetic enhancement mechanisms are: (1) long range in nature,
because the dipole, fields in polarizable metal particles vary as the inverse
cube of the distance to the center of the particle; (2) generally independent
of the adsorbed molecule and dependent on the electronic structure of the
substrate and the roughness of the surface.
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The chemical sorption effect is associated with the overlap of metal and
adsorbate electronic wave functions, which can lead to ground-state and light-
induced charge transfer processes. The chemical effect relating to SERS is
short range (0.1 to 0.5 nm), depending on the adsorption site, the geometry of
bonding, and the energy levels of the adsorbate molecule. Although the
contribution of charge-transfer processes to SERS may be large in some cases
(xlO-10'j, the chemical enhancement mechanism is restricted by its
specificity.
The SERS effect is similar to normal Raman scattering in that the inten-
sity of the scattered light is linear with that of the incident light and is
depolarized. This effect seems to occur only under specific experimental
conditions relating to the dielectric constant and morphology of the surfaces.
Silver surfaces give the strongest enhancement effects, followed by Cu, Au,
Pt, and Ni. The roughening of the surfaces is also critical and depends on
the type of surface preparation. Microspheres, posts, metal islands,
colloids, and metal-coated cellulose have been used. For spheroidal silver
particles, diameters ranging from 10 to 100 nm are optimal. For roughened
silver electrodes the surface protrusions are generally between 25-500 nm.
The SERS effect for silver island substrates was found to occur with the
first monolayer of adsorbate molecules. SERS spectra of pyrene adsorbed on
silver-coated quartz posts were found to show some peak shifts (e.g., 1582
cm'1 vs 1597 cm'1 for NRS of pyrene in solution). This indicates that these
vibrations were affected by adsorption to the metal surface. For large
molecules, only chemical groups close to the SERS-active surface may be
enhanced.
Vo-Dinh and coworkers (1984) evaluated the SERS technique for
environmental applications using practical SERS-active substrate materials
based on silver-coated Teflon microspheres deposited on glass and filter
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paper. A wide variety of organophosphorus chemicals including methyl
parathion, fonofoxon, cyanox, diazinon, formothion, and dimethoate were
investigated (Alak and Vo-Oinh, 1987). SERS analyses were reported of several
chlorinated pesticides including carbophenothion, bromophos, dichloran,
linuron, chlordan and 1-hydroxychlordene (Alak and Vo-Dinh, 1988). The
detection limits for these pesticides were measured at nanograo and
subnanogran levels. The results achieved with these chemicals are of great
analytical interest because these chemicals are difficult to detect by other
techniques, such as luminescence spectroscopy, due to the low luminescence
quantum yields of these compounds. A mixture of structurally related
compounds and a soil sample contaminated with pesticides were analyzed by SERS
to illustrate the selectivity of this new technique as a screening tool for
environmental applications (Alak and Vo-Dinh, 1987). Vo-Dinh (1989) has
demonstrated the SERS spectrum of complex environmental samples (diesel
particulate samples from the National Institute for Standards and Technology)
containing six PAR compounds as spotted on various SERS-active substrates.
Carrabba et al. (1987) have reported using SERS to analyze for compounds
such as hydrazines used for rocket fuels. They currently are developing a
prototype field-deployable SERS using utilizing electrochemical roughening of
silver electrodes. A fiber-optic system was also recently developed for SERS
in situ analysis using a silver-coated, microparticle-based sensing probe
(Bello and Vo-Dinh, 1990).
The use of silver colloids for SERS measurements in solutions has been
investigated widely (Tran, 1984; Sheng et al., 1986; Laseraa et al., 1987;
Ahern and Garrell, 1987). Recently, Fateley and coworkers have developed the
SERS colloid technique for HPLC detection (Freeman et al., 1988).
Measurements of SERS in the near IR region have also recently been
investigated (Chase and Parkinson, 1988).
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CONCLUSIONS
Raman spectroscopy, because it is a relatively under-utilized technique
involving complex instrumentation and laser excitation, is probably the
farthest from field deployment. Normal Raman spectroscopy has structural
information overlapping with, and complementary to, IR. It has the advantage
that water and glass are not interferents and that solid samples and unusual
sample shapes and sizes can be accommodated. NRS is also relatively
insensitive with detection limits between 20 and 1000 ppm. Techniques with
greater sensitivity, rivaling luminescence, are resonance Ramam (RR), which
requires even more complex instrumentation (excimer lasers or frequency-
doubling crystals and Raman shifters) and surface-enhanced Raman.
SERS is a promising technique in terms of its potential sensitivity and
specificity. It could also be used with sensor technology. One of its
disadvantages is that the intensity of the Raman signal is dependent on the
substrate surface and material and also on the specific analytes being
studied. Also, SERS is a new technology, not yet fully understood and
implemented. Although there is interest in developing a field-deployable
instrument, only a few research groups can so far obtain reliable,
reproducible, and sensitive SERS results even in the laboratory. Pollutants
which have been successfully studied by this technique include pyridine,
hydrazine, and other rocket fuels, PAHs, and organochlorine and
organophosphate pesticides. Extensive research efforts are currently being
undertaken to develop the potential of this relatively new analytical
technique.
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This reference section contains all references cited and an additional
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Williamson, J. H., Bowling, R. J., McCreery, R. L., "Near-Infrared Raman
spectroscopy with a 783-nm Diode Laser and CCD Array Detector," Appl.
Spectroac., Vol. 43, No. 3, 1989, pp 372-375.
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SECTION 7
SPECTROSCOPIC IMMUNOASSAY TECHNIQUES
INTRODUCTION
Inununoassay-iiranunological methods, offering the capability of excellent
selectivity and specificity through the process of antibody-antigen recogni-
tion, have revolutionized many aspects of chemical and biological sensor
technologies down to ultratrace levels. Although radioimmunoassay (RIA),
utilizing radioactive labels, has been most widely used, it has disadvantages
for field use, such as cost of instrumentation, short shelf life of some
radioisotopes and hazards of handling radioactive materials under field
conditions. Fluoroimmunoassay (FIA) using fluorescent labels and related
assay methods has great potential advantages for field use with a spot test
approach and has been discussed elsewhere in great detail (Smith et al., in
Wehry Vol. 3, 1981; Karnes et al. in Schulman, 1985).
DISCUSSION
The theory of immunoassay is beyond the scope of this report, but
considerable chemistry and biochemistry is involved for the development of
each test. Suitable derivatives of the pollutant must be developed that can
be complexed to proteins so that specific antibodies can bind to them with
high affinities. Conjugates of these derivatives must be prepared with
properties to allow rapid bonding to immobilized antibodies and good recovery
and stability of enzyme activity.
76
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A survey of all aspects of luminescence immunoassay is given by Karnes et
al. (1985). This rapidly moving field was earlier surveyed by Smith et al.,
in Wehry (1981). Karnes et al. (1985) stated that FIA sensitivities were
typically in the 10'" M range. FIA techniques are not yet as sensitive as RIA
procedures, although they can be made more sensitive using laser excitation.
Currently, commercial instrumentation for FIA is limited to simple analyses
not requiring extremely high sensitivities. One approach to improve FIA
sensitivity would be to use time-resolved fluorescence or phosphorescence
detection. Use of image detectors for spectral or spatial resolution would
also reduce sample analysis time.
FIA can also be used with fiber optic chemical sensors. Vc-Dinh et al.,
(1987) have developed immunochemical fiber optic sensors for specific
environmental pollutants such as benzo(a)pyrene.
Colorimetric immunoassay tests are also specific and sensitive although
not quite as sensitive as FIA and RIA. Colorimetric portable immunoassay kits
for pesticides such as paraoxon were recently discussed by Duquette et al.
(1988). In this case the analyte competes with an enzyme-analyte conjugate
for a limited number of immobililized antibody sites. This test can detect
paraoxon at one microliter in water with positive results indicated by color
development in ten minutes. This test is operable in salt water and fresh
water and is stable under field conditions for as long as one year. This
assay format could be modified to measure other environmental pollutants such
as PCBs.
Three other examples of field-portable Colorimetric immunoassay
(biomarker) procedures were recently reported by White and Van Emon (1989) and
are discussed below.
77
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Westinghouae Bioanalytic Systems (Rockville, MD.) has developed a rapid
test for the wood preservative pentachlorophenol which is sensitive to 3 ppb
and takes only 30 minutes to perform. A pentachlorophenol-enzyme conjugate
competes with free pentachlorophenol for binding to the immobilized antibody.
The enzyme binds on a colorimetric substrate with absorbances being measured
on a battery-operated field portable colorimeter. This method was
successfully demonstrated at a Superfund site in Hew Brighton, MN.
Antox corporation developed an enzyme-linked immunosorbent assay (ELISA)
for toxic light aromatics including benzene, toluene, and xylene (BTX), which
is claimed to be sensitive to the ppm level. The BTX screening assay uses a
polyclonal antibody-coated tube as the solid phase. This test is still under
evaluation; phenols do not appear to be an interferent but the antibody may
cross-react with alkylated aromatics.
A third assay described by Brady et al. (1989) for aldicarb, requires
minimum sample preparation and analysis time of 2.5 hours. The assay response
is linear over the range of 16 to 2000 ng.
As summarized by White and Van Emon (1989), immunoassay techniques have
important advantages and disadvantages. The advantages include relative
sensitivity, specificity, cost-effectiveness, and speed in comparison with
more common 6C-HS analysis. They can be used as rapid field-portable
semiquantitative or quantitative methods, to process large number of samples,
to reduce time for sample preparation, and as screening methods.
Disadvantages include the fact that these methods usually can not be used
for unknown chemicals or chemical classes or on complex mixtures of unknown
compounds. Also, they are subject to interferences and cross-reactivities
with compounds other than the target analyte. They are not real time methods;
sample preparation and time for reaction is needed. They may have a limited
78
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dynamic range. Also, considerable lead tine can be involved in developing
immunoassay procedures.
CONCLUSIONS
Fluoroimmunoassay (FIA) and colorimetric immunoassay techniques can be
extremely sensitive (especially FIA) and specific (perhaps too specific, they
usually cannot be used for class detection). Because of their specificity
individual reagents are usually required for each pollutant, when, in many
situations, broad classes of pollutants are of interest. Nevertheless,
immunoassay can be made field-portable and applicable for pollutant
monitoring. These techniques will be most needed where simpler fluorescence,
colorimetric, or fluorometric procedures do not apply and when extreme
specificity is needed. They also can be used with FOCS, in some cases.
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REFERENCES
This reference section contains all references cited and an additional
selection of definitive references that should provide the reader with a basic
understanding of spectroscopic inununoassay techniques.
Brady, J. F., Fleeker, J. R., Wilson, R. A., and Mumma, R. O., "Enzyme
Immune-assay for Aldicarb," in Biological Monitoring for Pesticide Exposure. R.
G. H. Wang, c. A. Franklin, R. C. Honey cutt, and J. C. Re inert, Eds., ACS
Symposium Series 382, ACS, Washington, DC, 1989.
Collins, W. P., Ed., Alternative Immunoassavs. John Wiley & Sons, NY, 1985.
Duquette, P. H., Guire, p. E., and Swanson, H. J., "Fieldable Enzyme
inununoassay Kits for Pesticides," Proceedings First International Symposium on
Field screening Methods for Hazardous Waste Site Investigations, 1988, pp 239-
242.
Hammock, B. D. and Mumma, R. O., and Recent Advances in Pesticide Analytical
Methodology. J. Harvey, Jr. and G. Zweig, Eds., Am. Chem. Soc., Washington,
DC, 1980.
Karnes, H. T.,-O'Neal, J. s., and Schulman, S. G., "Luminescence Immunoassay,"
in Molecular Luminescence Spectroscoov; Methods and Applications. Part 1. S.
G. Schulman, Ed., John Wiley & Sons, NY, 1985, pp 717-780.
Lin, J. N., Kopeckova, P., Ives, J., Chuang, H., Kopecek, J., Herron, J., Yen,
H. R., Christensen, D., Andrade, J. D., "Remote, Continuous, Multichannel
Biochemical sensors Based on Fluoroimmunoassay Technologies," Proceedings
First International Symposium on Field Screening Methods for Hazardous Waste
Site investigations, 1988, pp 251-252.
smith, D. S., Hassan, M., and Nargessi, R. D., "Principles and Practice of
Fluoroimmunoassay Procedures," in Modern Fluorescence Spectroscopv 3. E. L.
Wehry, Ed., Plenum, NY, 1981, pp 143-192.
van Emon, J. M., Seiber, J. N., and Hammock, B. D., in Analytical Methods for
Pesticides and Plant Growth Regulators. Vol. XVII. Academic Press, 1989, pp
217-263.
van Emon, J. M., Seiber, J. N., and Hammock, B. D., in ACS Symp. Ser., Vol.
276, 1985, pp 307-316.
Vo-Dinh, T., Tromberg, B. J., Griffin, G. D., Ambrose, K. R., Sepaniak, M. J.,
and Gardenhire, E. M., Appl. Spectrosc., Vol. 41, 1987, p 735.
white, R. J. and van Emon, J. M., "Report on Immunochemical Techniques for
Identifying and Quantifying organic compounds in Biological and Environmental
Samples," U.S.E.pA. Report EPA/600/X89/288, 1989.
80
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SECTION 8
FIBER OPTIC CHEMICAL SENSORS
INTRODUCTION
Fiber-optic chemical sensors (FOCS) offer a means of expanding the role
of spectroscopy in environmental monitoring. The need for in situ, low-cost,
real-time monitoring of organic and inorganic pollutants in ground water and
surface waters has been one of the main forces driving the development of
fiber optic sensors, particularly FOCS. The toxic pollutants of interest
cover a wide range of contaminants from organic chlorides, phenols, and heavy
metals from industrial and hazardous waste sites to aromatic hydrocarbons from
leaking underground storage tanks to organophosphates from Department of
Defense installations.
FOCS can be made very sensitive and specific to satisfy regulatory
requirements. Other advantages of FOCS include their small physical size,
geometric flexibility, and the possibility of real-time, multiple analyses in
situ. Disadvantages include short operational and storage life (due to
complex chemistries) and lack of ruggedness. Another disadvantage is the
relatively narrow spectral range of inexpensive, commercially available
fibers, although better UV and IR fibers are being developed. The present
lack of performance and calibration standards for interfiber reproduciblity
represents another problem to be overcome by developing peer-approved ASTM
standards. Recent advances in spectrochemical instrumentation, laser
miniaturization, biotechnology, and fiber optics research have provided
Bl
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opportunities for novel approaches to the development of sensors for the
detection of environmental pollution by toxic chemicals.
DISCUSSION
Fiber optic sensors may be divided into two general classes. The first
class of sensors uses the waveguide, usually an optical fiber, as a simple
lightpipe taking light to and from a sensing device, such as a microcell
containing a dye that exhibits changes in absorbance or luminescence with
varying concentrations of a substance or a physico-chemical parameter (e.g.,
CO,, o,, or pH). For this class of sensors, one important factor involves the
light transmission efficiency through the fiber. The second category of
sensors involves an intrinsic change in the properties of the optical fiber
itself, which serves as the sensing element. This category may be further
subdivided into two subcategories: evanescent field sensors and surface
plasmon resonance sensors.
Fiber optics sensors can use either bifurcated or single-strand fibers.
in a device based on bifurcated design, separated fibers carry the excitation
and emission radiation. In the single-fiber device, a dichroic filter or
mirror-pinhole assembly is generally used to separate the excitation and
emission radiation. When compared to multiple (bifurcated) fiber designs,
sensors that utilize a single optical fiber to transmit excitation radiation
to the sample, and the emission radiation from the sample to the detector,
have the advantages of good signal collection efficiency and small size. The
small-diameter attribute is important for environmental applications, since it
may be important to fit a number of different sensors down a one or two inch
monitoring well. Single-fiber sensors, however, exhibit high optical
background levels because a significant amount of excitation radiation, which
is reflected at the fiber tip, is not efficiently rejected by spatial
filtering. Attempts to minimize this back-scatter radiation problem include
82
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the use of high rejection ratio double monochromators, and grinding fiber
faces at angles that minimize the reflected optical background. Disadvantages
of these correction techniques include the relatively low throughput of double
monochromators and the difficulty of producing small quartz surfaces with
precise angles.
Optical fibers utilize total internal reflection (TIR) to achieve propa-
gation length with very low loss, but another complementary feature of TIR can
be used in optical sensing for surface sensitivity. Evanescent-field spectro-
scopy is an extension of the well-known internal reflection method. The
evanescent-field technique uses an optical waveguide as the transmitting
medium in place of the crystal medium often used in internal reflection
spectroscopy. When light is reflected at a dielectric interface, i.e., at the
interface between two materials of different refractive indices, the energy
associated with the light is not totally confined to the material in which the
incident and reflected waves are propagated. There is a drastic decrease of
energy away from the reflected point into the second material. This field is
known as the evanescent field, because energy cannot be propagated in this
direction. It decays within a distance comparable with the wavelength of the
light.
Sensors can also be based on the application of the surface plasmon
resonance (SPR) principle. It is possible to arrange a dielectric/metal/
dielectric sandwich such that, when light impinges on a metal surface, a wave
is excited within the plasma formed by the conduction electrons of the metal.
A surface plasmon is a surface-charge-density wave at a metal surface. A
plasmon resonance is induced in the surface of a metal conductor by the impact
of light reflected/off the metal surface. The critical angle is naturally
very sensitive to the dielectric constant of the medium immediately adjacent
to the metal, and this characteristic therefore lends itself to exploitation
for analytical sensors. For example, the metal can be deposited as, or on, a
83
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grating; upon illumination with a wide band of frequencies, the absence of
reflected light can be observed at the frequencies at which the resonance
matching conditions are net.
The most common FOGS reagents are organic chemicals whose reaction
products are suitable for colorimetric or fluorometric measurements. A number
of such sensors have been developed (Wolfbeis in Schulman, 1988). Angel
(1989) recently field-tested a colorimetric sensor for trichloroethylene.
Much of the required chemistry for such sensors is discussed in the older
chemical literature. Many of the chemical reagents are not reliable under
field conditions or are not designed for stability for the time required for
an in situ sensor (3 months to 1 year). -Recently fluoroimmunoassay FOCS have
been tested by Vo-Dinh et al. (1989) using enzyme coatings. Klainer et al.
(1988) recently developed a gasoline sensor based on changes in the refractive
index of a proprietary cladding material, due to absorption of the light
aromatics in the gasoline by the cladding.
The current status of FOGS have been discussed in a number of recent
references by Wolfbeis in Schulman (1988), Eccles and Eastwood (1988), Klainer
et al. in Wolfbeis (1990), Vo-Dinh et al. in Wolfbeis (1990) and others.
Feasibility studies have been performed on many pollutant sensors including
chloroform, gasoline, pH, ammonia, benzo(a)pyrene, aluminum, cyanide,
actinides, and sulfur dioxide, only a few of these sensors have proven rugged
enough for long-term field testing and commercialization. Progress is needed
in the area of remote sensors because of the tremendous need for continuous
environmental monitoring and the large potential cost savings provided by
remote sensors in contrast to more conventional analytical techniques.
84
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CONCLUSIONS
Although there is no reason FOCS cannot eventually prove satisfactory for
environmental monitoring and screening applications, there may be delays
because of the many practical and engineering problems that must be solved
before FOCS can be rugged, reliable, and inexpensive enough for general field
deployment and commercialization. If chemical sensors rather than remote
spectroscopy approaches are to be used, a wide variety of reliable chemical
sensors must be developed for the major pollutants. The technology requires
better UV fibers and sources, or else chemical reactions to move spectral
responses into the visible region. Other important areas for research and
development include: better instrumentation with miniaturization of all
components of the FOCS system; peer-accepted calibration, characterization and
performance standards through organizations such as the American Society of
Testing and Materials (ASTM); improved reagent immobilization and polymer
membrane technology: and improved methods and systems of optical coupling such
as improved molded optics or nonimaging optics. Development of satisfactory
FOCS requires interdisciplinary research teams, which might include analytical
chemists, polymer and dye chemists, molecular spectroscopists, optical
physicists, optical and mechanical engineers, material scientists, and
hydrologists. Appropriate instrumentation and laboratory support facilities
are also required. Currently, few of the research groups developing FOCS have
such complete teams.
FOCS can be thought of as light pipes, combined with detection by
colorimetrie or fluorometric reagents or by measuring changes in some optical
property of the fiber or the protective cladding. Although of great potential
long-term applicability to ground-water monitoring, some sensors are closer to
field testing and commercialization than others, some FOCS are currently
being field tested and evaluated (pH, temperature, conductivity, alkali
metals, and gasoline). Other FOCS are still under development and may require
85
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several years of research and development as well as engineering improvements
before they can be applied in the field. For some sensors, immobilization of
the coatings and reversibility, stability, and specificity of the reagents are
still problems to be addressed in the research laboratory. At present, vapor-
phase sensors are more developed than true liquid-phase sensors.
The small sizes of fiber optic sensors could allow smaller diameter
(1/2") monitoring wells to be drilled at substantial cost savings. Despite
the problems which remain to be overcome, remote fiber optic sensors have an
important future in continuous environmental monitoring of ground water.
In addition to FOCS, in situ spectroscopy, using fiber optic probes, is a
possibility for most of the spectroscopic techniques discussed in this report.
86
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REFERENCES
This reference section contains all references cited and an additional
selection of definitive references that should provide the reader with a basic
understanding of fiber optic chemical sensors.
Bright, F. V., Litwiler, K. S., "Multicomponent Fluorometric Analysis Using a
Fiber-optic Probe," Anal. Chen., Vol. 61, 1989, pp 1510-1513.
Burgess, L. w., Fuh, M. S., and Christian, G., "Use of Analytical Fluorescence
with Fiber optics," Progress in Analytical Luminescence, ASTM STP 1009, D.
Eastwood and L. J. cline-Love, Eds., American Society for Testing and
Materials, Philadelphia, 1988, pp 100-110.
Carroll, M. K., Bright, F. V., Hieftje, G. M., "Luminescence originating in an
Optical Fiber," Appl. Spectrosc., Vol. 43, No. 1, 1989, pp 176-178.
Dessy, R. E., "Waveguides as Chemical Sensors," Anal. Chem., Vol. 61, No. 19,
1989, pp 1079-1094.
Eastwood, D., Lidberg, R., Simon, S., and Vo-Dinh, T., "An Overview of
Advanced Spectroscopic Field Screening and In-Situ Monitoring Instrumentation
and Methods" proceedings of the chemistry for the protection of the
Environment Conference, Lublin, Poland, 1989.
Eccles, L. A.f and Eastwood, D., "Rationale for In Situ Environmental
Monitoring with Fiber Optics," SPIE Meeting, FACSS, Boston, 1988.
Ferrell, T. L., Arakawa, E. T., Gammage, R. B., James D. R., Goudonnet, J. P.,
Reddick, R. c., Haas, J. w., and Wachter, E. A., "Fiber-Optic Surface-Enhanced
Raman System for Field Screening of Hazardous Compounds," Proceedings First
International symposium on Field Screening Methods for Hazardous Waste site
investigations, 1988, pp 41-42.
Guided wave, "Laboratory Determination of octane Number in Gasoline by NIR
Analysis," Application Brief IB2-289, Guided Wave, El Dorado Hills, CA, 1989.
Herron, N., Cardenas, D., Hankins, W., Curtis, J., Simon, S., and Eccles, L.,
"Modification, calibration, and Field Test of a Chloroform Specific Fiber
optic Chemical Sensor," Chemical Research Service, 1990.
Klainer, S. M., Dandge, D. K., Goswami, K., Eccles, L. A., and Simon, S. J.,
"A Fiber Optic Chemical Sensor (FOCS) for Monitoring Gasoline," In Situ
Monitoring with Fiber Optics, Part 3, a U.S. Environmental Protection Agency
pub., EPA/600/X-88/259, 1988, pp 1-39.
Klainer, S. M., Goswami, K., Dandge, D. K., Simon, S. J., Herron, N. R.,
Eastwood, D., and Eccles, L. A. "Environmental Monitoring Applications of
Fiber Optic chemical Sensors (FOCS)," chemical Sensors, o. s. Wolfbeis, Ed.,
CRC, Boca Raton, in Handbook of Fiber-Optic. FL, 1990.
Milanovitch, F. P., Garvis, D. G., Angel, s. M., Klainer, s. M., and Eccles,
L. A., "Remote Detection of organochloridea with a Fiber Optic Based sensor,"
Analytical instrumentation, 15(2), 1986, pp 137-147.
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Murphy, E. M., and Hostetler, D. D., -Evaluation of Chemical Sensors for In
Situ Ground-Water Monitoring at the Hanford Site, "Pacific Northwest
Laboratory Report PNL-6854 (UC-403), Prepared for the U.S. Department of
Energy by Battelle Memorial Institute, March 1989.
Olsen, K. B., Griffin, J. w., Nelson, D. A., Matson, B. S., and Eschbach, P.
A., "Prototype Design and Testing of Two Fiber-optic Spectrochemical Emission
Sensors," Proceedings Field Screening Methods for Hazardous Haste Site
Investigations, 1988,
pp 117-125.
Seitz, W. R., "Chemical sensors Based on Fiber Optics," Anal. Chem., Vol. 56,
No. 1, 1984, pp 16-34.
seitz, w. R., "Chemical Sensors Based on Fiber Optics," Sensors, August 1985,
pp 6-9.
Seitz, W. R., "In Situ Detection of contaminant Plumes in Groundwater,"
submitted for publication in USA CRREL.
smith, D. S., Hassan, M., and Nargessi, R. D., "Principles and Practice of
Fluoroiromunoassay Procedures," in Modern Fluorescence Spectroscopy 3. E. L.
Wehry, Ed., Plenum, NY, 1981, pp 143-192.
Vo-Dinh, T., Tromberg, B. J., Griffin, G. D., Ambrose, K. R., Sepaniak, M. J.,
and Gardenhire, E. M., Appl. Spectrosc., Vol. 41, 1987, p 735.
wolfbeis, o.s., "Fiber Optical Fluorosensors in Analytical and clinical
Chemistry," Molecular Luminescence Spectroscopv. Methods and Applications;
Part 2. S. G. Schulman, Ed., John Wiley & Sons, NY, 1988, p 129.
Wolfbeis, O.S., "Fluorescence Optical Sensors in Analytical Chemistry," Trends
in Analytical Chemistry, Vol. 4, No. 7, 1985, pp 184-188.
Wolfbeis, O.S., "The Development of Fiber Optic Chemical Sensors by
Immobilization of Fluorescent Probes," Applied Fluorescence Technology, Vol.
1, No. 1, 1989, pp 1-6.
Zhang, Y., seitz, w. R., Grant, C. L., and Sundberg, D., "A clean Amine-
containing Poly (vinyl chloride) Membrane for In Situ Optical Detection of
2,4,6-Trinitrotoluene," Anal. Chim. Acta, Vol. 217, 1989, pp 217-227.
Zhang, Y. and Seitz, w. R., "Single Fiber Absorption Measurements for Remote
Detection of 2,4,6-Trinitrotoluene," Anal. Chim. Acta, Vol. 221, 1989, pp 1-9.
88
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APPENDIX A
TYPICAL ENVIRONMENTAL POLLUTANT SPECTRA FOR SOME MAJOR
SPECTROSCOPIC TECHNIQUES
Number PAGE
A-l Excitation and emission fluorescence spectra of benzo(a)pyrene. 2
A-2 Room-temperature fluorescence of pyrene, benzo(e)pyrene and 3
benzo(a)pyrene.
A-3 Room-temperature synchronous fluorescence of No. 6 and 4
No. 2 fuel oils.
A-4 Fluorescence spectra of phenol 5
(a) Excitation and emission fluorescence spectra
(b) Synchronous fluorescence spectrum
(c) Second-derivative synchronous fluorescence spectrum
A-5 Second-derivative synchronous fluorescence spectrum of phenol and 6
p-cresol (Avl=3 nm)
A-6 Room-temperature phosphorescence spectra of acridine with and without 7
silver nitrate as a heavy-atom perturber.
A-7 Room-temperature phosphorescence spectra of fluoranthene using several 8
heavy-atom agents.
A-8 Room-temperature phosphorescence spectra of a multicomponent mixture 9
of fluorene (FLu), phenanthrene (Phe), chrysene (chy), Benzo(e)pyrene
(BeP), Dibenzanthracene (DBA) and pyrene (Py), using excitation at
295 nm and 300 nm.
A-9 Room-temperature phosphorescence of uranyl ion complexed with a 10
proprietary complex (Uraplex J.
A-10 Low-temperature (77 K) phosphorescence of 2-chlorobiphenyl, 4-chloro- 11
biphenyl and biphenyl (note 2nd order LT-fluorescence of biphenyl).
A-ll Low-temperature (77 K) phosphorescence of 4-chlorobiphenyl and 12
4-bromobiphenyl.
A-12 Low-temperature (77 K} phosphorescence of Aroclor 1221 and 13
Aroclor 1248.
A-13 Schematic of GC-FTIR instrumentation. 14
A-14 FTIR vapor phase absorbance spectra of DDT 15
(dichlorodiphenyltrichloroethane). (D. Gurka, EPA-EMSL-LV)
A-15 GC-FTZR vapor spectra for 2,3,6-trichlorotoluene. 16
(D. Gurka, EPA-EMSL-LV)
A-16 GC-FTIR spectra search comparison {D. Gurka, EPA-EMSL-LV) 17
(a) Soil extract identified as p-chlorotoluene
(b) Authentic vapor phase IR spectrum of p-chlorotoluene.
A-17 Schematic of laser-Raman spectrophotometer. 18
A-18 Surface-enhanced Raman scattering (SERS) spectrum of perylene. 19
A-19 surface-enhanced Raman scattering (SERS) spectrum of methyl 20
parathion. The €47.1 nm line of a krypton laser was used for
excitation. A silver-coated microsphere substrate was used.
-------
LESC-LV
BENZO[a]PYRENE
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Ul
EXCITATION
EM - 42B nm
.9,4.3 nm
300
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400
WAVELENGTH (nm)
Figure A-l. Excitation and emission fluorescence spectra of benzo(a)pyrene.
-------
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BENZO[a]PYRENE
350 375 400
425 450 475 500
WAVELENGTH (nm)
525 550
Figure A-2. Room-temperature fluorescence of pyrene, benzo(e)pyrene and
benzo(a)pyrene.
-------
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111
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350 400 450 500
WAVELENGTH (nm)
550
600
Figure A-3. Room-temperature synchronous fluorescence of No. 6 and Ho. 2
fuel oils.
-------
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250 300 350
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Figure A-4. Fluorescence spectra of phenol
(a) Excitation and emission fluorescence spectra
(b) synchronous fluorescence spectrum
(c) second-derivative synchronous fluorescence spectrum
-------
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Figure A-5. Second-derivative synchronous fluorescence spectrum of phenol and
p-cresol (AX=3 run)
-------
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B = WITHOUT Ag*
I I I I I I I I I I I I I
tl 1
500
600
700
800
WAVELENGTH (nm)
Figure A-6. Room-temperature phosphorescence spectra of acridine with and
without silver nitrate as a heavy-atom perturber.
-------
ORNL-OWG 78-16839
in
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I I I I I I I I I I I I I
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Figure A-7. Room-temperature phosphorescence spectra of fluoranthene using
several heavy-atom agents.
-------
ORNL-DWG 78-16834
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BeP
DBA
X = 330 um
295 um
chy
400
500 600
WAVELENGTH (nm)
700
Figure A-8. Room-temperature phosphorescence spectra of a multicomponent
mixture of fluorene (FLu), phenanthrene (Phe), chrysene (chy),
Benzo(e)pyrene (BeP), Dibenzanthracene (DBA) and pyrene (Py),
using excitation at 295 nm and 300 nm.
-------
LESC-LV
z
Ul
H
Z
URANYL
EX = 420 nm
BP = 4.3,0.9 nm
210 ng/mL in URAPLEX
RT - PHOSPHOR.
450 475 500 525 550 575
WAVELENGTH (nm)
600
625
650
Figure A-9. Room-temperature^phosphorescence of uranyl ion complexed with a proprietary
complex (Uraplex ).
-------
Z
UJ
UJ
U
Z
UJ
o
CO
UJ
DC
O
I
o.
CO
o
2-CI BIPHENYL
LESC-LV
EX = 270 nm
8P a 4.5,0.9 nm
15 ug/mL in HEPTANE
LT • EMISSION
77 K
4-CI BIPHENYL
BIPHENYL
380 414 448 481 515 549 583 616 650
WAVELENGTH (nm)
Figure A-10. Low-temperature (77 K) phosphorescence of 2-chlorobiphenyl,
4-chloro-biphenyl and biphenyl (note 2nd order LT-
fluorescence of biphenyl).
11
-------
CO
Z
111
111
o
Z
111
o
CO
III
cc
o
Z
o.
CO
o
Z
a.
4-CI BIPHENYL
LESC-LV
EX = 270 nm
BP = 4.5,0.9 nm
15 ug/mL in HEPTANE
LT • EMISSION
77 K
4-Br BIPHENYL
380 414 448 481 515 549 583 616 650
WAVELENGTH (nm)
Figure A-ll. Low-temperature (77 K) phosphorescence of 4-chlorobiphenyl and
4-bromobiphenyl.
12
-------
LESC-LV
CO
z
01
IU
o
z
UJ
o
IU
cc
o
X
Q.
CO
O
X
a.
AROCLOR 1221
EX = 270 nm
BP = 4.5,0.9 nm
15ug/mL in MCH
LT - EMISSION
77 K
AROCLOR 1248
380 414 448 481 515 549 583 616 650
WAVELENGTH (nm)
Figure A-12. Low-temperature (77 K) phosphorescence of Aroclor 1221 and
Aroclor 1248.
13
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IR SOURCE
INTERFEROMETER
MCT DETECTOR
MIRROR
LIGHT PIPE
MIRROR
GC
DETECTOR
MIRROR
INLET
GAS CHROMATOGRAPH
Figure A-13. schematic of GC-FTIR instrumentation.
-------
.52 -i
.41 -
ui
o
Z .30
<
CD
K
O
CO
CD .19
.08 -
.03
4000
CCI3
-1- ~T~
3600 3200
2800 2400 2000 1600
WAVENUMBERS
-T- T~
1200 800 400
Figure A-14. FTIR vapor phase abaorbance spectra of DDT (dichlorodiphenyltrichloroethane)
(D. GUrka, EPA-EMSL-LV)
-------
3.0 -i
g 2-°
OQ
DC
O
m
S 1.0 -
0.0 -
4000
3000 2000
WAVENUMBERS
1000 750
Figure A-15. GC-FTIR vapor spectra for 2,3,6-trichlorotoluene. (D. Gurka, EPA-EMSL-LV)
-------
8-OOOOn
Z
o
I
Z
Ul
\u
O
Z
<
03
CC
O
CO
CD
1.2999
(a)
V/vJ
3200
1.4000-1
Z
o
I
•Z
UJ
IIJ
O
Z
<
o
E
O
Ifl
GO
-0.1399
2400 1600
WAVENUMBERS
800
3200
2400 1600
WAVENUMBERS
800
Figure A-16.
GC-FTIR spectra search comparison (D. Gurka, EPA-EMSL-LV)
(a) soil extract identified as p-chlorotoluene
(b) Authentic vapor phase IR spectrum of p-chlorotoluene.
17
-------
MONOCHROMETER
SAMPLE
COMPARTMENT
DETECTOR
Figure A-17. Schematic of laser-Raman opectrophotometer.
-------
8
ORNL-DWG 86-10164
±! 6
"E
3
£T 5
a
4
t 3
(/)
z
UJ
SERS SPECTRUM
PERYLENE
0
200
I I
I I I
I
I
I I
I
400 600 800 1000 1200
RAMAN SHIFT (cm'1)
1400
1600
1800
Figure A-18. surface-enhanced Raman scattering (SERS) spectrum of perylene.
-------
K>
(A
I
V)
z
Ul
oc
ui
IA
PARATHION, METHYL
S
(CH3O)2*P-O
SILVER-MICROSPHERE SUBSTRATE
EXC. 647.1 nm (Kr LASER)
200 400 600 800 1000 1200
RAMAN SHIFT (cm-1)
1400
1600
1800
Figure A-19. surface-enhanced Raman scattering (SERS) spectrum of methyl parathion.
The 647.1 nm line of a krypton laser was used for excitation.
A silver-coated microsphere substrate was used.
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