Rapid screening of polycyclic aromatic compounds and fingerprinting of complex samples by laser-excited fluorescence spe...

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Title:
Rapid screening of polycyclic aromatic compounds and fingerprinting of complex samples by laser-excited fluorescence spectrometry
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v, 98 leaves : ill. ; 28 cm.
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English
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Mellone, Anthony, 1957-
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Subjects / Keywords:
Polycyclic aromatic compounds   ( lcsh )
Fluorescence spectroscopy   ( lcsh )
Laser spectroscopy   ( lcsh )
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bibliography   ( marcgt )
theses   ( marcgt )
non-fiction   ( marcgt )

Notes

Thesis:
Thesis (Ph. D.)--University of Florida, 1989.
Bibliography:
Includes bibliographical references (leaves 94-97).
Statement of Responsibility:
by Anthony Mellone.
General Note:
Vita.

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University of Florida
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RAPID SCREENING OF POLYCYCLIC AROMATIC COMPOUNDS
AND FINGERPRINTING OF COMPLEX SAMPLES BY
LASER-EXCITED FLUORESCENCE SPECTROMETRY

By

ANTHONY MELLONE


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF
THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


1989















ACKNOWLEDGMENTS


I gratefully acknowledge the sage advice and saintly patience

shown me over the last five years by Jim Winefordner, without whom this

work could not have been accomplished. I cannot begin to express my

appreciation of all the members of Jim's group, who have not only helped

to make my time in graduate school more productive and enjoyable, but

have, each in his or her own way and without exception, enriched my

life. I would, however, like to thank those who helped to make my

endeavors more productive. Benny Smith and Ed Voigtman were always

willing to help in any way, both materially and spiritually. Brad Jones

and Jorge Vera were also helpful in experimental designs. Mike Mignardi

and Mark Click helped often in taming the computers. I will always have

fond memories of the time shared with Ben Womack, Paul Johnson and Tom

Manning.

I owe much to those who have inspired me over the years to seek

graduate education. My parents have always stressed the value of

education. My mother never lost faith in me. My father never tired of

encouraging me forward. Dr. Popham, an instructor at SEMO, and Dr.

Matthews, my boss at Washington University, were both inspirations to

me. I am deeply indebted to Ingrid, my wife. She has shown me

patience, kindness and understanding, not to mention 70 words per

minute.













TABLE OF CONTENTS

Page

ACKNOWLEDGMENTS......................................................ii

ABSTRACT .............................................................iv

CHAPTERS

1 INTRODUCTION ..............................................1

Polycyclic Aromatic Compounds .......................1
Analysis of Polycyclic Aromatic Compounds............3
Screening Techniques...............................10

2 LASER-EXCITED FLUORESCENCE OF AROMATIC MOLECULES
IN A GRAPHITE FURNACE .................................... 14

Introduction....................................... 14
Experimental ........................... ...........15
Results and Discussion .................. ............18

3 SURVEY OF COMPLEX SAMPLES BY GRAPHITE FURNACE,
LASER-EXCITED FLUORESCENCE ................................36
Introduction........................................36
Experimental. ......................................37
Results and Discussion..............................40

4 LASER-EXCITED FLUORESCENCE FINGERPRINTS OF
ENVIRONMENTAL MATERIALS WITH A PULSED LASER,
GATED PHOTODIODE ARRAY SYSTEM ............. ..............77

Introduction...................... ................. 77
Experimental....................................... 79
Results and Discussion..............................81

5 SUMMARY AND FUTURE WORK.................................. 89

REFERENCES............................................................94

BIOGRAPHICAL SKETCH ................................................. 97















Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy


RAPID SCREENING OF POLYCYCLIC AROMATIC COMPOUNDS
AND FINGERPRINTING OF COMPLEX SAMPLES BY
LASER-EXCITED FLUORESCENCE SPECTROMETRY

By

Anthony Mellone

December, 1989

Chairman: James D. Winefordner
Major Department: Chemistry

Polycyclic aromatic compounds (PACs) are widespread contaminants

in our environment. They are encountered as complex mixtures in real

samples due to the nonspecific processes by which they form. Routine

methods for PAC analysis are costly and time-consuming. The aim of this

work was to develop and evaluate laser-excited fluorescence of aromatic

compound vapors produced in a graphite furnace (GFLEF) by direct

atomization of untreated complex samples as a screening method for PACs

and as a fingerprinting technique for environmental and other complex

samples.

Untreated samples were placed into the graphite furnace. The

furnace temperature was slowly raised. As components vaporize according

to volatility, sequential fluorescence spectra were collected by a

photodiode array. These spectra were plotted as fluorescence intensity

versus wavelength versus scan number, producing a three-dimensional plot

representative of the sample. Scan number was correlated to furnace










temperature. A continuous wave (cw) laser was used to excite

fluorescence. The system was used for PAC screening and fingerprinting

of crude oils, particulate matter and petroleum products. Limited

thermal resolution and broad fluorescence features precluded the

identification of individual components in complex mixtures.

A second experimental system with improved imaging of the graphite

furnace enabled use of the system over a wider temperature range.

Fingerprinting and PAC screening capabilities of GFLEF were evaluated by

analysis of a wide array of samples types including used and unused

cooking and motor oils, dairy products, fruits, meat, paper, tobacco,

vitamin formulations and over-the-counter medications.

An experimental system employing a pulsed dye laser and gated

photodiode array enabled even greater extension of the useable

temperature range by discrimination against furnace wall emission. By

tuning the laser to an atomic absorption line, atomic fluorescence

information could be included into the fingerprints for complex samples.

The various GFLEF systems provided rapid and simple screening for

PACs and fingerprints for complex samples. The high temperatures

attainable with the graphite furnace enabled the direct analysis of

solids and resinous materials often too difficult to analyze by

alternate methods.


















CHAPTER 1
INTRODUCTION


Polycyclic Aromatic Compounds

Polycyclic aromatic compounds (PACs) are widespread contaminants

in the environment that are produced mainly by the burning of fossil

fuels and refuse. The industrialization of modern society resulted in a

greater dependence on fossil fuels. Concurrently, refuse production

increased from both commercial and domestic sources. PACs are,

therefore, closely linked to our industrialized society and desire for

consumer products and a high standard of living. Table 1-1 is an

estimation of worldwide PAC production and sources (1).

Polycyclic aromatic compounds consist of two or more fused

aromatic rings, and may contain substituted alkyl groups. Heteroatoms

may be incorporated into the rings or substituted groups. Most PACs are

produced by the incomplete burning of organic material. At temperatures

of 500-800 OC, organic compounds are cracked to smaller free-radical

fragments. This process is pyrolysis. These fragments then recombine

to form larger, more stable aromatic compounds in a process known as

pyrosynthesis. The amount and types of PACs produced vary greatly.

Under ideal conditions of temperature and oxygen content, organic

material is combusted to CO2 and water to a greater degree (2).



















Table 1-1. Worldwide PAC Productiona


Source

Heating and power generation

Industrial Processes

Incineration and open burning

Vehicular transportation



Total PAC emissions

a. From reference (1).


Emission (tons/year)

260,000

105,000

135,000

4,500


504,500












Generally, lower temperatures yield higher concentrations of

substituted PACs. Higher temperatures yield higher concentrations of

unsubstituted PACs. The organic starting material and other combustion

conditions will effect the pyrolysis products. Nitrogen, sulphur and

oxygen, when present, can be incorporated into PACs (2,3). Materials

containing PACs usually have extremely complex compositions because

pyrolysis is an unspecific process.

Polycyclic aromatic compounds are formed to a much lesser degree

by natural processes. They have been identified in coal, crude oil,

sediments and airborne particulates. Sources of these naturally

occurring PACs include volcanic activity, forest fires and the

degradation of organic material in the processes forming fossil fuels.

Many members of the PAC class of compounds are known or suspected

carcinogens. The Environmental Protection Agency (EPA) has recognized

many PACs as carcinogens (4). The carcinogenic effect of these PACs in

microgram quantities and their prevalence in the environment dictate

close monitoring of their presence and investigations of their sources

and health effects in order to regulate their detrimental effects.



Polycyclic Aromatic Compound Analysis

Because of the carcinogenic nature of many PACs, there is intense

analytical interest in the development of methods to analyze specific

components of the complex samples in which PACs normally occur.

Interest in PAC analysis has focused on the analysis of polycyclic

aromatic hydrocarbons (PAHs), a subset of the PAC family of compounds.












PAHs contain only carbon and hydrogen, and are usually present in much

higher concentrations than PACs that may contain heteroatoms.

By far, the most popular method of analyzing PACs is by gas

chromatography (GC). Advantages of GC include high resolution,

availability of universal and element-specific detectors, availability

of cross-linked polymeric and chemically bonded stationary phases that

are stable at high temperatures and compatibility with mass

spectrometers.

Gas chromatographic analysis requires that the analyte be volatile

and thermally stable at the temperature used. These conditions are

generally met for PACs of seven or fewer rings (5). High resolution GC

methods make them the techniques of choice for separation and

quantitation of PACs in complex samples (6).

High-performance liquid chromatography (HPLC) is also routinely

used for PAC analysis and is suitable for analysis of PACs soluble in

the mobile phase. This includes most PACs of less than 13 rings (5).

Advantages of HPLC include the separation of thermally labile compounds,

compatibility with absorbance, fluorescence and electrochemical

detectors and the availability of many suitable solvents and mixed

phases (7). These mobile and stationary phases make possible many

different separation mechanisms that can be used to separate analytes

based on physical and chemical differences. The spectroscopic and

electrochemical detectors used with HPLC are selective and sensitive.

The limited resolution of HPLC usually makes more than one separation

necessary. The initial separations can be extractions, other forms of

chromatography or preliminary HPLC fractionation (8).










5

The areas of GC and HPLC applicability overlap, but generally the

methods are complementary. These two methods, with mass spectrometric

confirmation of peak identifications, are often used for the most

complete analysis of complex samples such as air and diesel particulate

matter, petroleum products and coal tar (8-10).

Supercritical fluid chromatography (SFC) has experienced

tremendous growth as a method for PAC analysis. This growth in the last

decade is due to the introduction of capillary SFC columns and the

availability of commercial SFC instrumentation (11). A 1981 reference

(3) treats SFC in about two pages while a 1989 reference (11) devotes an

entire chapter to PAC analysis by SFC.

Supercritical fluid chromatography offers easier detector coupling

and better separations than HPLC plus a wider mass range than GC and has

been used to identify components in automobile exhaust (12), petroleum

products (13) and coal tar (14). Presently, most applications of SFC to

PAC analysis have been aimed at method and instrument development.

Thus, the full potential of SFC is yet to be realized.

Paper and thin layer chromatography have also been used for PAC

analysis, but suffer from poor reproducibility and sensitivity (15).

Absorption and luminescence spectra of PACs, although specific,

are broad and featureless. If a sample contains more than two or three

individual PACs, identification may be difficult. To detect PACs in

complex samples, a separation is required prior to spectroscopic

detection. Spectroscopic techniques, thus, are widely used as

chromatographic, particularly HPLC, detectors (3). Fluorescence has at

least two advantages over absorption for PAC analysis. First, lower












detection limits are achieved because of a lower background. Second,

selectivity is higher because the excitation wavelength can be selected

based on the requirements of a particular analysis. Lasers are used

extensively because they are a well-collimated, intense excitation

sources.

At low temperatures, absorption and fluorescence peaks of PACs are

narrowed, which greatly increases the usefulness of spectroscopic

techniques for PAC identification. UV/visible absorption in solid

matrices has been used to identify PACs (16). Matrix isolation Fourier

transform infrared spectroscopy (17) and low temperature fluorescence

(18) also have been used to selectively detect PACs in simple mixtures.

Allowing molecular vapors to expand through a small orifice into a

vacuum is another approach to low temperature spectroscopy. In a free

jet expansion, molecules are quickly cooled. As a result, spectra

exhibit the characteristically narrow absorption and fluorescence peaks,

allowing selective detection of PACs in mixtures (19). If a dye laser

is used as an excitation source, analytes may be selectively ionized by

the absorption of two or more photons. The ions may be detected as a

current across electrodes. Alternatively, the ions can be detected by a

mass spectrometer, greatly increasing the amount of analytical

information available (20). This form of low temperature spectroscopy

has the advantages of fast cooling, high resolution and compatibility

with gas chromatography and mass spectrometers.

Improved selectivity is accomplished by synchronous luminescence

spectroscopy by taking advantage of absorption and luminescence

information simultaneously. This is done by scanning the excitation












source and emission detector together with a constant energy or

wavelength difference (21). Signals are obtained only for compounds

that have the selected energy or wavelength difference between

excitation and emissions transitions. Lifetime-selective and phase-

resolved fluorescence detectors have been used in HPLC determination of

PACs (22).

Other specialized luminescence techniques for PAC analysis take

advantage of selective phosphorescence and fluorescence enhancement or

quenching (23). While fluorescence detection is the most widely used

spectroscopic technique for PAC analysis, other techniques used in

special cases are gaining in popularity.

Raman spectroscopy detects inelastically scattered photons. Like

infrared spectroscopy, Raman spectroscopy has excellent selectivity for

chemical groups. Surface-enhanced Raman was first observed in 1974 for

molecules adsorbed on metal surfaces, particularly silver (24). The

advantages of surface-enhanced Raman spectroscopy are ease and

versatility of sample handling, applicability to water systems and the

ability to investigate adsorption dynamics. Raman spectroscopy has been

used to selectively detect components in environmental samples (25).

Photothermal spectroscopy is an absorption technique which is applicable

to large, irregular solid samples (26) as well as to HPLC detection

(27). The numerous variations of this up-and-coming technique as well

as theoretical aspects have been described by T. Vo-Dinh (28). Nuclear

magnetic resonance (NMR) is most helpful in the elucidation of unknown

structures and has also been used to selectively detect PACs in mixtures

extracted from environmental samples and tobacco smoke condensates (29).












Mass spectrometry (MS) has been an indispensable tool for PAC

identification and quantitation since 1951 (30). High resolution

chromatographic retention data combined with mass spectral information

can often provide an unambiguous identification of components of complex

mixtures. Structure elucidation is often possible based on the

fragments present in the spectrum. "Soft" ionization techniques such as

chemical ionization produce mainly molecular ions, yielding molecular

weight information. Selective ionization of isomers can be achieved in

a free jet expansion by the use of lasers (20). Mass Spectrometry is

compatible with GC, capillary supercritical fluid chromatography and

capillary electrophoresis. Secondary ion, fast atom bombardment, laser

desorption and field ionization desorption are vaporization methods for

thermally labile compounds. The most used method for unambiguous PAC

analysis in complex mixtures is GC separation followed by electron

impact ionization MS. Although MS is usually less sensitive than

fluorescence for PAC analysis, its high specificity and compatibility

with GC make it one of the most useful methods for PAC analysis.

The complex samples in which PACs are present often contain

thousands of different PACs in widely varying concentrations. For a

given sample type and target PAC, appropriate methods exist for

quantitative and qualitative analysis. Common to all techniques are

time-consuming sample pretreatment steps. These steps may, beyond the

initial extraction, involve further extractions, adsorption

chromatography, sample preconcentrations, derivatization and HPLC

fractionation. These procedures aim to remove interference and reduce

the number and increase the concentrations of components to manageable












and detectable levels. For every pretreatment step, confidence in

conclusions about the original sample is reduced. Precise results are

not necessarily accurate. Problems include evaporative loss of volatile

components, reactions with oxidants and surface-catalyzed photochemical

reactions that sometimes result in products and mixtures exhibiting a

greater carcinogenic nature. Extraction efficiencies have been shown to

vary widely for different PACs and for certain compounds in different

sample types (31). These degradation and loss complications are

affected by the particle size and the microscopic nature of the surface

to which PACs are adsorbed (32).

Since biological tests are performed using the same extracted,

fractionated samples, it follows that misconceptions may exist about the

composition and biological effects of PAC-containing samples.

Unacceptable variability in PAC quantitation, in some cases order of

magnitude differences, have been reported for identical aliquots

analyzed by different methods (33). In the same study, compounds

detected by HPLC were not confirmed by GC-MS. The problem of

identifying particular compounds is due, at least in part, to the above

complications as well as variability among different lots of the same

stationary phases which can, on occasion, change the elution order of

PACs, differences in mass spectra dependent upon instrumental parameters

and a lack of standards available for confirmation of peak assignments.

In general, PAC analysis is tedious, time consuming, uses very

expensive, state-of-the-art equipment and requires much technical

expertise. The most selective and sensitive techniques are also the

most sophisticated.















Screening Methods

Due to the complex nature of PAC-containing samples, the

variability of results and expense in both time and equipment required

for analysis of individual components of these samples, some

investigators have questioned the necessity and wisdom of current

methods for routine analysis (1,34). They suggest that such

sophisticated methods are not cost- or information-effective.

Two solutions to this problem are possible. Samples can be

characterized by analysis of one PAC and inferences made concerning the

concentrations of other PACs. Alternatively, a method measuring total

PAC content irrespective of isomers could be used. Both of these

solutions are screening methods.

Screening methods are techniques that provide incomplete

information about a sample in a rapid and simple manner. Screening

methods indicate the absence or presence of a few compounds or class of

compounds. They are helpful in characterizing samples and for

prioritizing samples for further analysis by alternate techniques if

more specific information is desired.

If the characterization provided by the screening method is unique

to a particular sample, it can be used as fingerprint for that sample.

Fingerprints can be used to differentiate samples and can be helpful,

for example, in tracing the origin of pollutants such as an oil spill.

Numerous problems are associated with screening methods. Methods

that are sensitive to PACs irrespective of isomers yield nonspecific

information from which no inference concerning health effects can be










11

made. Methods that rely on measurement of target compounds that are in

some manner representative of the very large and complex group of PACs

that comprise nearly all real samples suffer from lack of reliability in

the choice and analysis of the target compound and in the inferences

made concerning the concentrations of other components. Target

compounds are referred to as proxy, indicator, surrogate or signature

compounds.

The most commonly used target compound for proxy PAC analysis is

benzo(a)pyrene (BaP). This, apparently, is due to the carcinogenic

nature of BaP, its presence in most PAC-containing samples and the

wealth of information available in the literature concerning the health

effects and analytical considerations of BaP.

Mixing of PACs from various local sources takes place to the

extent that there is a high degree of correlation among PAC species in

the atmosphere for a given locale (35). Any one compound is therefore a

good index of the concentrations of the others. In another study, PAC

concentrations relative to BaP were found to vary depending on the

location of the sampling station, the time of day and the weather, with

location showing the most significant variations (36). In addition, the

concentrations of PACs adsorbed to particles vary with particle size and

emission source (37). Since size is a determining factor in retention

of particles in the respiratory tract, this consideration cannot be

overlooked in determining PAC impact on health.

Another problem with proxy methods is that any inaccuracy in

determining the proxy compound would result in inaccurate inferences

about the concentrations of other components. For example, the C20H12












isomers BaP, benzo(k)fluoranthene (BkF), benzo(b)fluoranthene,

benzo(e)pyrene and perylene are among the most commonly encountered

PACs. They are also difficult to separate chromatographically. It is

likely that BkF, a noncarcinogenic PAC that exhibits a nearly identical

fluorescence spectrum as BaP, has in some cases been erroneously

measured as the carcinogen BaP (38).

As stated earlier, extraction efficiencies and degradation losses

of PACs vary greatly among the various PACs and for PACs in different

sample matrices. Field tests have shown that tracers introduced at the

time of sampling could not be used to account for these losses due to

the variability of recovery of the tracers, ranging from near 0 to 500%

(39). P.W. Jones estimated that 80% of all PAC studies have failed to

take these variances into account (1). This indicates that preliminary

studies that would be required to choose a proxy PAC and to ascertain

the relative concentrations of other PACs in order to infer their

concentrations from subsequent proxy studies may not yield reliable

information.

After careful characterization of a particular emission source,

reliable proxy PAC analysis should be possible; however, a universal

proxy PAC would not be possible. Since considerable synergism for

biological activity exists among PACs, mixtures often exhibit greater

carcinogenic activity than would be expected from their individual

carcinogens (40). It is, therefore, important to correlate PAC

concentrations as well as health effects with proxy PACs, data which are

totally lacking for most PACs.










13

The aim of this work was to develop laser-excited fluorescence of

aromatic compound vapors produced by direct electrothermal atomization

of untreated complex samples in a graphite furnace (GFLEF) as a

nonspecific screening method for PACs and as a fingerprinting technique

for environmental and other complex samples.

















CHAPTER 2
LASER-EXCITED FLUORESCENCE OF AROMATIC MOLECULES
IN A GRAPHITE FURNACE


Introduction

Many PACs are classified as EPA priority pollutants because they

are known or suspected carcinogens. The two main sources of PACs in the

environment arise from incomplete combustion of fossil fuels for energy

production and from incomplete combustion of refuse. Interest in

carcinogenic compounds led to the development of many gas and liquid

chromatographic techniques for analysis of specific compounds in such

samples as crude oils, petroleum products, diesel particulates and urban

dust (3). These methods generally involve elaborate extraction and

fractionation schemes prior to analysis.

Recently, several groups have reported the use of electrothermal

atomizers for the study of the ultraviolet absorption of molecular

vapors. Tittarelli et al. found that by monitoring ultraviolet

absorption at 190 nm as a crude oil sample was vaporized from 140 to 900

OC, spectral fingerprints characteristic of the samples could be rapidly

obtained (41). In later work (42), a photodiode array was used to

monitor absorption in the wavelength range of 190 to 355 nm. Plotting

the sequential spectra produced a three-dimensional plot characteristic

of the sample vaporized. These representations were also used as

fingerprints for the samples, and had more distinguishing












characteristics than earlier two-dimensional plots. Absorption of

ultraviolet light by vapors produced in a graphite furnace has also been

used to characterize pollutants in water and sediments and as a

fingerprinting technique for various oils, petroleum products and soaps

(43). The high temperatures obtained with a graphite furnace

facilitated the direct analysis of heavy oils, resinous materials and

other complex samples often too difficult for analysis by gas

chromatographic techniques.

In this work, a novel system involving laser-induced fluorescence

of PACs in a graphite furnace was investigated as a screening method for

PACs and as a fingerprinting technique for environmental samples and

other complex mixtures. Direct analysis of crude oil, petroleum

products and solid materials was accomplished without sample

pretreatment.



Experimental

A block diagram of the experimental system is shown in Figure 2-1.

The laser was a He-Cd continuous wave (cw) laser (Model 4120N, Liconix,

1390 Borregas Ave., Sunnyvale, CA, 94089) with a maximum output of six

mW at 325 nm. The laser beam passed through a 1.5 mm hole drilled

through a polished aluminum off-axis parabolic mirror before passing

lengthwise through the inside of the graphite tube.

The graphite furnace was an atomic absorption spectrometric system

(Model CTF555, Instrumentation Laboratories, Thermo-Jarrell Ash, 590

Lincoln St., Waltham, MA, 02254). The controller had adjustments for

six temperature settings and allowed selection of the ramp rate between















PLOTTER


COMPUTER


PRRRBOLIC
MIRRORS


PHOTODIODE


ARRRY


SPECTROGRAPH


BERM STOP


GRAPHITE
FURNACE


Block diagram of the experimental system.


Figure 2-1.












the six stages. The furnace was set to slowly ramp from room

temperature to 50 C over one minute to dry the sample, then ramped

linearly from 50 to 500 OC over approximately two minutes as

fluorescence spectra were collected. The furnace was contained in a

small enclosed housing, which allowed volatization of the sample to

occur in an inert environment of argon. To monitor the temperature, the

housing also contained a thermocouple in contact with the graphite tube.

The fluorescence was collected at 1800 relative to the laser

excitation. The cone of fluorescent light exiting the graphite tube was

collimated by an off-axis parabolic mirror. Loss of signal by light

escaping back through the 1.5 mm hole was approximately 0.3%. The

collimated light was focused on the entrance slit of the spectrograph by

a second off-axis parabolic mirror.

The flat field spectrograph (Instruments SA, Inc., 173 Essex Ave.,

Metuchen, NJ, 08840) has a dispersion of 24 nm/mm, a focal length of 0.2

m and a spectral range of 200 to 800 nm. A 1024 pixel intensified diode

array (Model TN 6100, Tracor Northern, 2551 West Beltline Highway,

Middleton, WI., 53562) was mounted to the spectrograph with a

laboratory-constructed flange, which held the array in the focal plane.

The diode array head contained a Peltier effect thermoelectric cooler

capable of cooling the head to -20 C, which reduced the dark current.

A dedicated computer controlled data acquisition, storage, processing

and output. Generally, the array was exposed for 2 s before being read

and stored. Spectra were displayed on the screen and stored after

automatic subtraction of a restored background spectrum. Additional

software, Quadra (Tracor Northern), allowed for the generation, scaling












and plotting of three-dimensional representations of fluorescence

intensity versus wavelength versus scan number of sequentially

accumulated spectra. Scan number was correlated to time and, therefore,

furnace temperature.



Results and Discussion

To determine the sensitivity of the system, analytical calibration

curves were prepared by placing 100 p1 of standard concentrations

ranging from 100 to 0.1 pg/ml in methanol into the graphite furnace. A

drying cycle was employed to evaporate the solvent. The appearance time

of the sample has been found to depend linearly on the boiling point of

the compound (44). Figures 2-2 through 2-5 show typical vaporization

cycles for several standards. The temperature ramp rates were chosen as

a compromise between the increased sensitivity of fast ramp rates and

the increased thermal resolution of slower ramp rates. Small variations

in ramp rates have little effect on the appearance of the three-

dimensional plot. Since all plots have the same temperature scale, ramp

rate affects the number of spectra taken during the vaporization cycle,

assuming the scan rate remains unchanged. For instance, the plot of a

standard vaporized at a 30% greater ramp rate would appear the same if

the number of scans displayed was reduced from 55 to 39.

For preparation of calibration curves, spectral information was

collected and stored in a slightly different form. The software allowed

the user to define a spectral region of interest. This region was

chosen to correspond to the fluorescence peak of a particular compound.

As the sample vaporized, only the area above baseline of the region of




































400



300o
300


200 -



100


650 600 550 500 450 400 350

WAVELENGTH (NM)


Figure 2-2. Vaporization plot for anthracene.


>-
I-

Un
z
LLU
z
F-


LL 20000


I-

-r












20




















-z 500
-. .400


10000



- 200
Scr- ___ _______

100
0
650 600 550 500 450 400 350

WAVELENGTH (NM)










Figure 2-3. Vaporization plot for benzo(b)fluoranthene.












21


















> 10000 -- ------------ 500


-- ------ -___ 00
-0 0 -0-_


2- 400


--3000
50000 50 40 40





WAVELENGTH (-M)






Figure 2-4. Vaporization plot for pyrene.200
100

0--
650 600 550 500 450 400 350

WAVELENGTH (NM)












Figure 2-4. Vaporization plot for pyrene.











22

















500
I-
20000--


I 400



S- --- -2
F-


30000

a- ma--""" -"" I-----"'""""""" '""" "

650 600 550 500 450 400 350

WAVELENGTH (NM)








Figure 2-5. Vaporization plot for coronene.
Figure 2-5. Vaporization plot for coronene.












interest was stored. A representation of the area of the fluorescence

peak versus time is called a histogram. The area under the histogram

peak was proportional to the volume under the peak in the three-

dimensional representation but required much less memory. For the six

compounds for which standard curves were prepared, the log-log plots

were linear over three or more orders of magnitude. The limits of

detection (LODs) were calculated as three times the standard deviation

of the blank measurement divided by the slope of the analytical

calibration curve (counts/ng). The slopes of the analytical calibration

curves, the linear dynamic ranges and the LODs for six PACs are shown in

Table 2-1. The major limitation to lower LODs is the high standard

deviation of the blank measurement. Uncertainty in the background

subtraction causes the low concentration points to deviate from the

linear calibration curve. At higher concentrations, the curves bend

over, especially for strongly absorbing compounds due to prefilter,

postfilter and self-absorption effects. Also, furnace emission

interfered with compounds fluorescing at longer wavelengths and/or

vaporizing at higher temperatures. These detection limits were about 10

times higher than those reported in previous work in this laboratory by

Kirsch (45). However, Kirsch (45) used more powerful lasers and the

fluorescence collection angle was not limited by baffles in the graphite

tube housing. In addition, averaging pixels produced a high signal-to-

noise ratio at the expense of spectral resolution. Detection limits

obtained in this work were comparable to the approximate determination

limits reported by Shekiro and Skogerboe (44) for absorbance

measurements of PAC vapors. If detection limits were calculated using



















Table 2-1 Limits of Detection (LOD) Obtained by Laser Fluorescence
Spectrometry of Vaporized Polycyclic Aromatic Hydrocarbons (PAH)


PAH

Pyrene

Benzo(b)fluoranthene

Benz(a)pyrene

Coronene

Anthracene

Fluoranthene


LOD (ng)

34

47

73

79

103

138


Linear Dynamic Ranpea


Orders of magnitude

Slope of log-log calibration curve.


Slope

0.99

1.04

1.04

0.97

1.03

1.01












the same criteria as Shekiro and Skogerboe (44), the detection limits

were three to five times lower than those shown in Table 2-1.

To demonstrate the thermal resolution of the system, standard

mixtures were vaporized. Figure 2-6 shows the thermal resolution of two

PACs. Standard compounds evolved over about a 25 s, or 145 oC interval.

In the thermal dimension, the FWHM for most of the compounds tested is

about 9 s, or about 50 C. Since the PACs fail to undergo sharp

vaporization transitions into the vapor phase, the thermal resolution

capabilities are limited. The highest number of compounds thermally

resolved was three. Vapor phase PAC spectra exhibit peak widths on the

average of 60 to 90 nm FWHM. This lack of sharp features and limited

thermal resolution precluded the identification of individual components

of a complex mixture. It also seems likely that components in a sample

matrix vaporize at slightly higher temperature than the pure materials,

further complicating the extraction of qualitative information.

Finally, to demonstrate the fingerprinting capability of the system,

three samples of crude oil, three samples of particulate matter and

several types of petroleum products were analyzed. The resulting three-

dimensional maps all contained distinguishing features. Samples

producing similar plots could be differentiated by the relative

intensities or peak widths of the various features. Figure 2-7 through

2-14 show plots obtained from the vaporization of some of these samples.

The simplicity and speed of laser-excited fluorescence of aromatic

molecular vapors produced in a graphite furnace, the characteristic

fingerprints obtained and the ability to analyze solids and resinous










26

materials indicate that it will be a useful method for PAC screening and

fingerprinting.



































I-

z
Ld
20000
z

LU
>


I-
LJ
ar


500


400


o0c7


600 500 400


WAVELENGTH (NM)















Figure 2-6. Vaporization plot for a standard mixture of anthracene and
perylene, showing the thermal resolution of two PACs.



































500

50000
-n 400


-Z \300
Uo
> K
2> 200

-j"


0
600 500 400

WAVELENGTH (NM)


Figure 2-7. Vaporization plot for Kirkuk crude oil.









































0500
I-






Iii

200 S,-,
<
-j




600 500 400

WAVELENGTH (NM)









Figure 2-8. Vaporization plot for SRM 1582 crude oil.



































500


400


300
o7.
SK.


600 500 400

WAVELENGTH (NM)


Figure 2-9. Vaporization plot for E.S. Sider crude oil. Note the
broader peaks in the 200-300 C range compared to the previous figure.


- 50000

z
w
LU
z

LUJ
>

I-
-I
LUJ
IC




































>-
i0000
in
z
Lu
z


W 500(

I-


-J
Q'2


500


o c
K.


, ,0 ..-,-,.I ,,, ....i ,,,i .
0A 111.l ii ii ii r --- I
^'-^^^^^^^'^


600 500 400

WAVELENGTH (NM)











Figure 2-10. Vaporization plot for SRM 1580 shale oil.


0


0







































4000




2000 20
,.,_ 200


-~~~ ~~ ,.^^ l^ J- .-?-- -

600 500 400

WAVELENGTH (NM)





Figure 2-11. Vaporization plot for regular gasoline.


500


400





































500



u-i
400


L-
z 300
- 5000



- --
200



-0


600 500 400

WAVELENGTH (NM)






Figure 2-12. Vaporization plot for SRM 1649 urban dust.






























500


400


300
"3 O


600 500 400
WAVELENGTH (NM)


Figure 2-13. Vaporization plot for SRM 1648 urban dust.
intense fluorescence in the 200-300 C range compared to
figure.


Note the more
the previous


5000


































500


400


L


600 500 400

WAVELENGTH (NM)






Figure 2-14. Vaporization plot for SRM 1650 diesel particulate matter.
Note the narrower peaks in the 200-300 OC range compared to the previous
two figures.


I-
- 4000
z







-J
2000


>
Ix
wJ


.


j

















CHAPTER 3
SURVEY OF COMPLEX SAMPLES BY GRAPHITE FURNACE,
LASER-EXCITED FLUORESCENCE


Introduction

Preliminary investigations into the use of laser-excited

fluorescence of aromatic molecules in a graphite furnace indicated that

it was a sensitive method for screening of PACs in complex samples such

as crude oils, particulate matter and petroleum products. The three-

dimensional plots obtained by direct vaporization of untreated samples

were useful as distinguishing fingerprints for the samples analyzed.

Most of the PACs in our environment result from the use of fossil

fuels for energy production and from the burning of refuse. These PACs,

therefore, are associated with particulate matter in the atmosphere,

water and in sediments.

Despite the fact that most PACs are produced by combustion of

fuels and refuse, significant human exposure to PACs occurs as a result

of the use of consumer products such as tobacco and the ingestion of

PAC-containing foods as well as from exposure from air and water.

For many people, the major risk from PAC exposure may be from PACs

in food. Of particular concern are smoked products such as smoked meats

and cheeses. Regardless of PAC exposure from other sources, people

ingest with their food amounts of PACs exceeding an acceptable, safe

level (46).













Because of the variability in diets and food preparation,

benzo(a)pyrene (BaP) exposure from food may be the most significant

exposure, even for tobacco users. In the same study, average daily

intake of BaP in water and air was found to be within the allowable

daily intake (46). For example, one charcoal-broiled steak may contain

an amount of BaP equivalent to that from the smoke of 22.5 packs of

cigarettes (47). Evidence concerning PAC health effects comes largely

from occupational and community air pollution studies, while the effects

of ingested PACs have been largely ignored (46).

Another PAC contamination that is often overlooked is used oil,

often discarded haphazardly by the general public. Of the PACs produced

by an engine, about 85 percent is retained in the crankcase oil and

about 15 percent is emitted into the atmosphere as part of the exhaust

(48). Total PAC concentration of used motor oils has been found to be

as high as 15 g/l (49).

To a certain extent, lack of information concerning the PACs in

foods and used oils is due to the tedious and time-consuming procedures

involved with PAC analysis. In this work, the applicability of GFLEF

for PAC screening in a wide array of consumer products and in used oil

was demonstrated. Distinguishing fingerprints for foodstuff, oils, used

oil and over-the-counter medications were also obtained by GFLEF.



Experimental

The experimental system used for the survey of consumer products

was the same as that described in Chapter 2, with two modifications

(Figure 3-1). First, a graphite furnace with more accurate and flexible










38

temperature control was employed (Shimadzu, model GFA-4A, Kyoto, Japan).

Temperature was controlled by a microprocessor. Temperatures and ramp

rates for up to six stages could be chosen. Temperature control was

achieved by a current sensor for temperatures below 1100 OC. A silicon

photocell monitored furnace wall emission for temperature control

feedback for temperatures exceeding 1100 OC. This allowed temperature

cycles to be tailored to different sample types with greater ease than

with the previous furnace. In addition, the three-dimensional plots

obtained were more reproducible due to better temperature control.

Second, the off-axis parabolic mirrors were abandoned in favor of

a flat mirror with a four mm hole drilled at a 450 angle and a lens.

The excitation laser passed through the hole in the mirror and into the

graphite furnace. The cone of fluorescent light exiting the graphite

tube housing at 1800 relative to the laser excitation was folded by the

mirror onto a lens that focused the light onto the entrance slit of the

spectrograph. The advantages of this arrangement are lower cost, easier

alignment of optical components and better imaging of the graphite

furnace. Since a better image of the furnace emission is obtained, it

can more effectively be masked, reducing the background emission from

the furnace wall collected by the photodiode array. This was important

because of the higher temperatures used and lower PAC concentrations

encountered in some of the samples in this work.

For solid samples, approximately two to three mg were placed

inside the furnace. The tobacco samples were blended by the retailer

and were composed of a mixture of tobacco from various sources. These

















PHOTODI ODE
ARRAY


SPECTROGRAP


COMPUTER


PLOTTER


LENS


BEAM

STOP


MIRROR


GRAPHITE TUBE


Block diagram of the experimental system.


LASER


Figure 3-1.










40

samples were powdered in a mortar and pestle in order to obtain a small,

representative sample for analysis. Liquid samples were pipetted

directly into the furnace. For oils, milk and juices, the volume

vaporized was 5.0 pi. No sample pretreatment was involved in the

analysis with the exception of grinding the tobacco, which is not a

pretreatment in the usual sense of extraction or fractionation. After

the vaporization cycle, the furnace temperature was raised to an

empirically determined temperature high enough to prevent memory

effects, usually 1000 OC.



Results and Discussion

Fourteen tobacco samples were analyzed by GFLEF. The samples were

numbered one through fourteen. Sample numbers one through ten were

blended pipe tobacco samples. These samples were blended by the

retailer with tobacco from various sources. Many of the mixtures have

components in common. Sample numbers eleven through fourteen were four

different brands of cigarette tobacco. All four cigarette tobacco

samples and one of the pipe tobacco samples produced unique fingerprints

that could be used to identify the samples. The remaining nine pipe

tobacco samples were divided into three groups of two samples and one

group of three samples. The plots obtained from the vaporization of the

nine samples for which unique plots were not obtained could be used to

differentiate the samples from other samples outside the group. This

was the basis of the grouping. Examples of tobacco fingerprints are

shown in Figures 3-2 and 3-3.












Various investigators have reported the presence of PACs in food

products such as vegetable oils (50), dairy products (51), fruit and

meat (52). Figure 3-4 shows the plot obtained from the vaporization of

beef fat. Figure 3-5 shows the vaporization cycle of a sample of beef

hot dog, showing fluorescence from components vaporizing at higher

temperatures than the beef fat. If, however, this fingerprint was

plotted with a different intensity scale, fluorescence similar to that

seen in the beef fat vaporization was visible. Figure 3-6 is the

vaporization cycle for a beef hot dog plotted on a different intensity

scale to show the low intensity contributions from the beef fat.

Vaporization plots were also obtained for four fruit samples.

GFLEF screening revealed PAC content in all four samples. In addition,

plots obtained from various forms of the same fruit exhibited similar

spectral characteristics. Figure 3-7 is the vaporization plot of canned

orange juice concentrate. This plot is very similar to the plot for

fresh orange juice (Figure 3-8) except that the fluorescence intensity

is greater. Figure 3-9 shows the vaporization cycle for an exterior

orange peel sample. Although components of this sample vaporize at a

higher temperature, the spectral characteristics are similar to the

spectral features of the other two orange samples. For comparison, a

sample of canned grape juice concentrate was analyzed (Figure 3-10). A

comparison of Figures 3-7 and 3-10 reveals that the fluorescence

intensity and the intensity versus furnace temperature profiles of the

two samples are virtually identical. However, spectral features differ.
































400

2000
Lf
z








100w 0
w









0
30C 700

WAVELENGTH (NM)


Figure 3-2. Vaporization plot for tobacco #1.


































2000




1000
-4
i000 -1


300


300 700


WAVELENGTH (NM)









Figure 3-3. Vaporization plot for tobacco #8. Note the difference in
the intensity vs temperature profiles between this plot and the previous
plot.
































400


500


00 0c

,K


300 700


WAVELENGTH (NM)


Figure 3-4. Vaporization plot for beef fat.
































00400
1 0000

z 0
z









300 700

WAVELENGTH (NM)


Figure 3-5. Vaporization plot for beef hot dog.































400


200


1)


300 700


WAVELENGTH (NM)




Figure 3-6. Vaporization plot for beef hot dog plotted on a different
intensity scale to show the lower intensity contributions from the beef
fat visible at lower temperatures.











47
























500
S1000-
S400
- ,1 0 000


500
200
4: -

100
rrO

300 700

WAVELENGTH (NM)


Figure 3-7. Vaporization plot for orange juice concentrate.












48























600


400








Iit
0 j200 -

o lrr loo
300 700

WAVELENGTH (NM)





Figure 3-8. Vaporization plot for fresh orange juice. Note the
spectral and intensity vs temperature similarities with the previous
figure.


I



























600


2000 -


yo.,


300


WAVELENGTH (NM)


Figure 3-9. Vaporization plot for orange peel. Note that the spectral
characteristics are similar to the previous two figures, however, these
features appear at higher temperatures.

































-600


1000
-1
I



500




00 700
0I

300 700


-4 00
L ChN


WAVELENGTH (NM)





Figure 3-10. Vaporization plot for grape juice concentrate. Note the
different spectral characteristics compared to the three previous
figures.










51

This demonstrates the advantage of the three-dimensional technique GFLEF

with photodiode array detection in providing distinguishing

fingerprints. One might wonder how GFLEF would be helpful in

distinguishing grape juice from orange juice. However, the technique is

sensitive enough to provide fingerprints for dilute solutions or small

amounts of residue.

Marked differences in the fingerprints for whole milk, American

cheese, butter and margarine are apparent (Figures 3-11 through 3-14).

The characteristic features of these plots indicate that fingerprinting

of different brands would be possible, particularly for cheeses.

Five different refined vegetable oils were obtained from a local

retail grocer. Corn, safflower, soybean, olive and canola oils all

exhibited PAC fluorescence. The low intensity of the fluorescence

indicated low PAC concentration. Because the fluorescence intensity was

low, photodiode array background noise contributed significantly to the

plots. The noisy, low-intensity plots could not be used as fingerprints

to differentiate the five vegetable oils. Figure 3-15 is a vaporization

plot for a corn oil sample.

While GFLEF could not be used to fingerprint refined vegetable

oils, the method was sensitive to changes that occur in the composition

of oils as the oils are used in food preparation. Two samples of

vegetable oil were obtained from a local restaurant. One sample was

unused fry oil (Figure 3-16). The other sample was taken from a deep

fryer (Figure 3-17). The used oil exhibits additional PAC fluorescence

not present in the unused oil. The origin of the additional PACs











52

















600



Z 1000
400
o-
z


300 L
S500 r

0200

0 illilll-100
300 700

WAVELENGTH (NM)


Vaporization plot for whole milk.


Figure 3-11.

































700



W E60r
-O

z


5000 400
z



300
< -



0 IIIII-r I 100
300 700

WAVELENGTH (NM)


Figure 3-12. Vaporization plot for American cheese.


































300 oC
~".


300 700


WAVELENGTH (NM)


Figure 3-13. Vaporization plot for butter.




































600




400




200


..-. -'-J

0
300 700

WAVELENGTH (NM)


Figure 3-14. Vaporization plot for margarine.
peaks at 250 OC compared to the previous plot.


Note the lower intensity




























S-4
- 200
z
- -4
Lj j 10 0
I- ?

rr
0
300 700

WAVELENGTH (NM)


f- i400
oO& c'

,


Figure 3-15. Vaporization plot for corn oil.



































700


K


200


300 400 500 600
WAVELENGTH (NM)





Figure 3-16. Vaporization plot for unused fry oil.



































200 -

I


J
-1
H
A
i
100 -J
i
4
-i
i


700


600


0
oK


200


300 400 500 600
WAVELENGTH (NM)





Figure 3-17. Vaporization plot for used fry oil. Note the additional
features near 300 C compared to the previous figure.










59

present in the used oil may come from the pyrolysis of components of the

oil or food, or from incomplete removal of heavily used oil when the oil

was changed.

In order to simulate the changes that occur in heavily used oil,

corn oil was heated in a cast iron frying pan on high heat for one hour.

Figures 3-18 and 3-19 are plots obtained from the vaporization of the

unused and used corn oil, respectively, plotted with the same intensity

scale. Again, no conclusions can be drawn about the origin of PACs in

the used oil. They may occur as a result of pyrolysis of components of

the oil or residue from the considerable previous use of the pan in food

preparation. However, the significantly higher PAC content of the

synthetically aged corn oil indicates that the PACs are pyrolysis

products of corn oil components.

Crude oils contain PACs, as do the many of the petroleum products

refined from crude oil. One of the potentially important uses for GFLEF

is as a fingerprinting technique for petroleum products. GFLEF has

provided a distinguishing fingerprint for every crude oil and petroleum

product analyzed to date. In order to further investigate the

fingerprinting capabilities of GFLEF, several brands of motor oil were

obtained from a local retail merchandiser. Plots obtained by GFLEF

could be used as fingerprints to selectively identify different brands

of motor oil. Fingerprints of Castrol 30W, Quaker State 30W and Penzoil

10W-40 motor oils are shown in Figures 3-20 through 3-22, respectively.

As noted previously, used motor oils contain additional PACs as a

result of use. Figure 3-22 is a fingerprint of unused Penzoil 10-40

motor oil. Figure 3-23 is a fingerprint of the crankcase oil of an





































350


1000
-I





500 j
-4
3i


0
300


yoC)


WAVELENGTH (NM)





Figure 3-18. Vaporization plot for corn oil, plotted with the same
intensity scale as the next figure.












61























-4 600



I---
00








300 700
WAVELENGTH (N)
-12 0
-j



300 700

WAVELENGTH (NM)


Figure 3-19. Vaporization plot for used corn oil.












62



















S- --350

- 2000
z 4
u -
o 250


I-



-J

00


300 400 500 600

WAVELENGTH (NM)


Figure 3-20. Vaporization plot for Castrol 30W motor oil.





























1


1
5000






0
300
-1
I


0,
300


350


/250

o c
K


400 500 600


WAVELENGTH (NM)







Figure 3-21. Vaporization plot for Quaker State 30W motor oil. Note
the difference in spectral characteristics between this plot and the
other motor oil fingerprints.





























350


5000 !
-4


-I
j
i


1


o0


300 400 500 600

WAVELENGTH (NM)


Figure 3-22. Vaporization plot for Penzoil 10W-40 motor oil. The
spectral characteristics and intensity vs temperature profile of this
plot differs from the other motor oil samples.




































350


-i
1
-i


4
I
4

2000 ~
-t
4
-I


o0


300 400 500 600

WAVELENGTH (NM)












Figure 3-23. Vaporization plot for crankcase oil immediately after a
fresh change of Penzoil 10W-40 motor oil.



































350


4000




12000


2000 -
-i
-4
1


K


300 400 500 600

WAVELENGTH (NM)












Figure 3-24. Vaporization plot of Penzoil 10W-40 after 1000 miles of
driving.


L










67

automobile immediately after a fresh change of Penzoil 10W-40 motor oil.

The differences between Figures 3-22 and 3-23 are a result of incomplete

removal of the old oil. Figure 3-24 is a fingerprinting of the same

crankcase oil after 1000 miles of driving. The compounds responsible

for the additional features of Figure 3-23 have increased in

concentration during the 1000 miles of use. While PAC appearance in

used oil may not be a direct result or cause of oil wear, PAC content

detected by GFLEF should be a useful method to monitor oil wear. The

changes that occur in PAC content in oil during use are cumulative,

detectable by GFLEF and could be correlated to the useful life of motor

oil.

Several paper samples were analyzed by GFLEF. All samples

exhibited PAC fluorescence. The vaporization plots were not useful as

fingerprints. Based on the plots, paper samples could be grouped into

only two categories. White paper such as typing, computer and notebook

paper all exhibited low intensity fluorescence. As with the refined

vegetable oil vaporization plots, photodiode array noise contributed

significantly to the plots and masked any spectral characteristics that

might help distinguish samples. Figure 3-25 is a plot obtained from the

vaporization of a sample of computer printer paper. The second group of

paper products included unbleached paper such as brown paper used in

paper bags, institutional brown paper towel and cardboard. The plots

obtained from the vaporization of this group of paper products failed to

exhibit differences needed to use the plots as fingerprints. Figure 3-

26 is a vaporization plot for a sample of brown paper towel.











68























800
>- 100

Ln
z 600
w
z 500

b--
L 50 4 O 00



200

100
300 700


WAVELENGTH (NM)


Figure 3-25. Vaporization plot for computer paper.





































4000 6
500
z
'400
z

,> 2000 3 0 0 o C



100-


0-
A0 VE IIE H 1 |
300 700

WAVELENGTH (NM)


Figure 3-26. Vaporization plot for brown paper towel.










70

Reproducibility problems were also encountered in the analysis of

paper samples. In order to obtain reproducible plots, the sample had to

be finely divided, care had to be taken to assure that the entire sample

was in contact with the furnace wall and the furnace temperature had to

be raised slowly. If the entire sample was not in contact with the

furnace wall, components that should have vaporized at lower

temperatures were vaporized instead at higher temperatures, causing

irreproducible fluorescence signals. This problem was more severe with

samples that were not finely divided. During an investigation of

appropriate temperature ranges and ramp rates, an interesting

irreproducibility was observed with paper samples. Figure 3-27 is the

vaporization plot obtained using a fast ramp rate from 50 to 800 C.

Under these conditions, plots sometimes contained features in the 500 to

600 OC temperature range. Although the intensity of these additional

features varied greatly, when present, the temperature of the appearance

of the features and their spectral characteristics were reproducible.

In addition, the fluorescence spectral characteristics were different

from the fluorescence characteristics of components detected at lower

temperatures or with slower ramp rates. Using faster ramp rates, the

composition of the vapor at furnace temperatures of 400 to 500 C was

different from that observed at lower temperatures and different from

that observed in the same temperature range when slower ramp rates were

used. This indicated a more complicated process than components

vaporizing at higher temperatures than they would have under conditions

normally used. Possibly, components of the paper sample that were not

vaporized at lower temperatures due to the fast temperature ramp rate


































800


600


500
1000 400


300


100
0 lI l I l l I
300 700

WAVELENGTH (NM)





Figure 3-27. Vaporization plot for brown paper towel using a faster
ramp rate. Note the additional features in the 500-600 OC range.










72

were pyrolyzed at higher furnace temperatures instead. Since components

of complex samples analyzed generally vaporize at temperatures below 500

C, pyrolysis should not complicate the screening of those samples for

PAC content. In addition, if pyrolysis does occur but can be made to be

reproducible, the fingerprinting capabilities of GFLEF will not be

affected and may even extend the applicability of GFLEF fingerprinting

to materials that contain organic material but no PACs.

The PAC class of compounds includes many compounds with beneficial

uses. Many hormones, amino acids, pesticides and drugs are PACs.

Vaporization plots were obtained from two vitamin formulations and two

over-the-counter pain medications. Figure 3-28 through 3-31 are plots

obtained from the vaporization of a multivitamin, vitamin C, aspirin and

ibuprofen, respectively. This method is potentially useful for

fingerprinting and monitoring the uniformity of such products.

The versatility of GFLEF with photodiode array detection in PAC

screening and fingerprinting of a wide array of samples types has been

demonstrated. Liquid and solid samples were analyzed with no sample

pretreatment in less than five minutes per sample. Appropriate

temperature ranges and ramp rates are easily ascertained and ensure

reproducible plots and credible PAC screening.





























5000 1
1

-i


300 700


WAVELENGTH (NM)







Figure 3-28. Vaporization plot for a multivitamin.


600


































S600


ooo
S4000



-LJ
- 1000

w iVL 300 C

020000 P

-100
0 Il l ] 1 1 1
300 700

WAVELENGTH (NM)


Figure 3-29. Vaporization plot for a vitamin C formulation.




































2000


i



0 T
300 700


00 C


WAVELENGTH (NM)









Figure 3-30. Vaporization plot for aspirin.


-600


500
































1 600

S 400
- 500
z
S20400


S2002 30 o c




S-1 1 100
0-
300 700

WAVELENGTH (NM)


Figure 3-31. Vaporization plot for ibuprofen.

















CHAPTER 4
LASER-EXCITED FLUORESCENCE FINGERPRINTS OF ENVIRONMENTAL
MATERIALS WITH A PULSED LASER,
GATED PHOTODIODE ARRAY SYSTEM



Introduction

Analysis of crude oil, petroleum products, particulate matter,

cooking oil, food and tobacco by laser-excited fluorescence of molecular

vapors in a graphite furnace with photodiode array detection was shown

to be a simple and reliable method for PAC screening in these complex

samples. In addition, GFLEF provides reproducible, characteristic plots

useful as fingerprints for individual samples.

Using the previously described continuous wave (cw) experimental

systems, compounds that vaporized at higher temperatures or fluoresced

at longer wavelengths gave higher limits of detection due to

interference from furnace wall emission. This problem is most severe at

higher temperatures and at the higher wavelength, or red, end of the

detected wavelength range. Furnace wall emission also made interfering

contributions to fingerprints obtained from the vaporization of complex

samples. This interference was greater with samples exhibiting low

intensity fluorescence and with resinous samples such as crude oils,

which scattered furnace wall emission toward the detector to a greater

extent than samples with less complex matrices. As furnace temperature

rose, furnace wall emission increased, eventually saturating the












photodiode array. The temperature at which furnace wall emission

saturated the photodiode array varied depending on the alignment of the

optics, how well the emission was masked and how much scattering the

sample caused. Generally, photodiode array saturation occurred at

approximately 700 to 1000 C, thus limiting the usable temperature range

of the method.

Another limitation of the cw laser system was that the lasing

wavelength was not tuneable. Although 325 nm is an effective wavelength

to excite PACs with three or more rings, this lack of tunability

inherently limits the flexibility of the method.

A GFLEF experimental system utilizing a tuneable, pulsed laser and

gated photodiode array would have several advantages over the cw system.

A pulsed excitation source and gated photodiode array would allow

discrimination against background emission. This would provide

fingerprints with less contribution from background emission such as

furnace wall emission and stray light. The useable temperature range

would also be extended significantly. Tunability would enable the

choice of an appropriate excitation wavelength based on the analytes of

interest. By tuning the laser to an atomic absorption line, atomic

fluorescence information could also be used in fingerprinting complex

samples. For a given set of samples, a wavelength that provides the

most distinguishing fingerprints could be employed.

The focus of this work was to investigate these and other

prospective advantages of a pulsed laser, gated photodiode array GFLEF

experimental system. The system was used to obtain improved

fingerprints and to detect molecular as well as atomic fluorescence.















Experimental

A block diagram of the experimental system is shown in Figure 4-1.

The laser was a flashlamp dye laser (model CMX-4, Chromatix, 560 Oak

Mead Parkway, Sunnyvale, CA, 94086) with about one mW average power (one

kW peak power) in the wavelength range used. The dye used was rhodamine

6G (Eastman Kodak Co., Rochester, NY, 14650). The laser output passed

through a four mm hole drilled in a flat mirror at a 450 angle before

passing lengthwise through the inside of the graphite tube.

The graphite furnace assembly was manufactured by Instrumentation

Laboratories (Model CTF555, Thermo-Jarrell Ash, 590 Lincoln St.,

Waltham, MA, 02254). The operational characteristics of this furnace

were described in Chapter 2.

The fluorescence was collected at 1800 relative to the laser

excitation. The cone of fluorescent light exiting the graphite tube

housing was folded by the mirror onto a lens that focused a 1:1 image of

the graphite furnace onto the entrance slit of the spectrograph. The

spectrograph was described in Chapter 2.

An intensified photodiode array manufactured by Princeton

Instruments, Inc., (model IR4-1024, P.O. Box 2318, Princeton, NJ,

06580) was mounted to the spectrograph with a laboratory-constructed

flange. Data collected by the photodiode array was stored and processed

by a PC's Limited 286 computer supplied by Princeton Instruments.

An air-acetylene burner and an additional lens aided in tuning the

laser to atomic absorption lines. A standard solution was aspirated

into the flame as the laser was tuned, while fluorescence was monitored















































Beam
stop


Trigger photodiode


Figure 4-1. Block diagram of the experimental system. PDA, photodiode
array.












using the same detection system employed with subsequent graphite

furnace atomization. This configuration simplified the change from

laser tuning to sample analysis.



Results and Discussion

The most obvious advantage of the pulsed laser, gated photodiode

array system was a reduction of furnace wall emission compared to the cw

system. This background emission occasionally interfered with

fingerprinting of oil samples, where furnace wall emission was scattered

to a greater extent than with standards. With the pulsed laser, gated

photodiode array system, furnace wall emission was not apparent. Figure

4-2 shows the three-dimensional plot obtained from the vaporization of a

crude oil sample using the pulsed laser system. The photodiode array

controller was triggered by a photodiode positioned to detect a small

fraction of the laser pulse scattered from the back side of the mirror.

The gate for data collection was set to correspond temporally with the

excitation pulse. Thus, fluorescence was collected only during the one

us excitation pulse. The furnace wall emission and stray light

contributions to the plot were reduced by the same factor as the duty

factor of the laser. Using a laser pulse repetition rate of 15 Hz,

these background contributions should be reduced by a factor of about

66,000.

One possible advantage of the pulsed system was selectivity based

on the fluorescence lifetime of PACs. However, the laser pulse width,

the narrowest useable photodiode array gate and the smallest gate delay

all were too large to allow selective PAC detection based on



































611E4.





30500








170E3.y '
0
80 240 400 550 710 870 t

Wavelength (nm)







Figure 4-2. Vaporization plot for crude oil. Note the absence of
furnace wall emission.










83

fluorescence lifetime. In addition, the photodiode array, when

operating in the delayed gate mode, could collect spectra at a maximum

rate of only three Hz. These properties precluded taking advantage of

this potential capability of a pulsed, gated system. The laser and

photodiode array would require precision on the order of, at most,

several nanoseconds and a high pulse rate to achieve sensitive

fluorescence lifetime selectivity.

Advantages of the wavelength tunability of the dye laser were also

realized. The flashlamp dye laser could be tuned to an excitation

wavelength that produced the most distinguishing fluorescence

fingerprints for a given group of samples. A novel additional use for

the pulsed laser, gated photodiode array system was as a diagnostic tool

for laser-excited fluorescence of atoms produced in a graphite furnace.

Photomultiplier tubes generally used for atomic fluorescence have

greater sensitivity and linear response than photodiode arrays, so

sensitivity degraded compared to previous laser-excited atomic

fluorescence studies (53). However, the multi-wavelength nature of the

photodiode array provided a unique look at some of the processes

occurring in the graphite furnace.

As previously noted, oil samples caused increased furnace emission

to be scattered to the detector, a phenomenon which could not accurately

be accounted for by standards. The photodiode array allowed assessment

of the extent of this problem and whether it significantly interfered

with detection and quantitation of atomic species. In order to simulate

a situation in which an atomic fluorescence determination was hindered

by furnace wall emission, an oil sample containing iron was vaporized












with the laser tuned to the fringe of an iron absorption line. Figure

4-3 is the photodiode array scan exhibiting maximum fluorescence

intensity of the iron 375.5 nm line with the laser tuned to the

absorption maximum at 296.7 nm. Figure 4-4 is the photodiode array scan

exhibiting maximum iron fluorescence from a similar plot of the same

sample for which the laser was detuned to 296.5 nm. Severe interference

from the furnace wall emission is apparent. The photodiode array

allowed the assessment of the nature and extent of any spectral

interference that might be encountered. This information would be

useful in obtaining a reliable background for baseline subtraction. In

spectra and plots obtained with the Princeton Instruments photodiode

array, furnace wall emission apparently falls off at about 710 nm.

This, however, is an artifact of the software, which was written to

control a 1024 diode array instead of the 700 diode array used.

The thermal evolution, laser-excited atomic fluorescence system

was also useful as a fingerprinting technique for complex samples such

as oil. Figure 4-5 shows a three-dimensional plot obtained from the

vaporization of a sample of used jet engine oil. Fluorescence of

molecular and iron vapors contributed to the fingerprint for this

sample. In addition, this plot could be used to confirm that the

organic matrix had been vaporized prior to atomization of the iron.

The pulsed laser, gated photodiode array system provides

fingerprints with greatly reduced background contributions. Furnace

wall emission did not saturate the photodiode array, even at furnace

temperatures of approximately 2600 C required to vaporize iron.










































240 400 550 710
Wavelength (nm)


870


Figure 4-3. Photodiode array scan exhibiting maximum iron fluorescence
from the vaporization plot of jet engine oil. Excitation was at 296.7
nm.


506E4.


133E3.
80

























850E3.







>>
I1
a
a


-1

0


V










4BOE3.


Ou e4u 4UU 550 710 870

Wavelength (nm)







Figure 4-4. Photodiode array scan exhibiting maximum iron fluorescence
from the vaporization plot of jet engine oil with off-line (296.5 nm)
excitation.


,,,











87





















869E4.





A
e


:




127E.
80 240 400 550 710 870

Wavelength (nm)


Figure 4-5. Vaporization plot of jet engine oil.










88

Wavelength tunability imparts flexibility to the technique not present

with the previous cw system. This flexibility includes the ability to

add atomic fluorescence information to the fingerprinting of complex

samples based on PAC content, enhancing the capability to differentiate

samples.















CHAPTER 5
SUMMARY AND FUTURE WORK



Polycyclic aromatic compounds are widespread contaminants in the

environment. They are produced mainly by the burning of fossil fuels

for energy production. Many PACs are known or suspected carcinogens and

mutagens. They are usually found as components of complex samples and,

therefore, present a difficult challenge to the important role of the

analytical chemist. Due to interest in PACs and the presence of these

compounds in complex samples, many chromatographic and spectroscopic

techniques have been and continue to be developed that provide compound-

specific information on PAC content. Unfortunately, these methods often

involve elaborate, time-consuming sample pretreatment, sequential

determination of target compounds and the use of standards for

confirmation of identification. With each step in the analysis,

confidence in the analytical result decreases. In general, PAC analysis

is tedious, time-consuming, uses very expensive state-of-the-art

equipment and requires highly trained technical experts. The most

selective techniques are also the most sophisticated.

Recently, the efficiency of current methods of analysis has been

questioned. Methods that can provide information on the presence of

PACs in a rapid and simple manner would be useful for routine PAC

screening. In addition, if the characterization of the sample is unique

to a particular sample, it can be used as a fingerprint for the sample.












Fingerprints can be used to differentiate samples and, thus, would be

helpful in tracing the origin of pollutants such as an oil spill.

The aim of this work was to show the usefulness of laser-excited

fluorescence of aromatic compound vapors produced by direct atomization

of untreated complex samples in a graphite furnace as a screening method

for PACs and as a fingerprinting technique for environmental and other

complex samples. The untreated samples were placed in a graphite

furnace and were vaporized by a gradual temperature gradient. As the

components were vaporized according to volatility, sequential

fluorescence spectra were collected by an intensified photodiode array.

These sequential spectra were plotted in the three-dimensional format of

wavelength versus fluorescence intensity versus scan number. Scan

number could be correlated to furnace temperature.

The GFLEF system was used to provide fingerprints and screen for

PACs in complex samples. Crude oil, petroleum products, urban dust

particulates, diesel particulates, standards and standard mixtures were

analyzed by GFLEF. Polycyclic aromatic compounds exhibit broad,

featureless fluorescence spectra. They also have a significant vapor

pressure over a broad temperature range and fail to undergo a sharp

transition into the vapor phase. Thus, GFLEF was able to provide

specific identification of components for only simple standard mixtures

of two or three components. However, the speed, simplicity and

sensitivity of the method to PACs allowed rapid screening for PACs in

complex samples and fingerprinting of the samples based on PAC content.

Analysis time was less than five minutes per sample.












A diverse array of sample types were analyzed to evaluate the

performance of the technique as a routine screening and fingerprinting

method. Petroleum products, tobaccos and many types of foodstuffs and

over-the-counter medications could be selectively identified based on

the fingerprints for the samples. The useful lifetime of cooking and

engine oils could be monitored due to the buildup of PACs during use.

Samples that were difficult to fingerprint generally had low PAC levels.

With these samples, such as unused refined vegetable oils, photodiode

array noise contributed significantly to the vaporization plots.

Vaporization of paper indicated that at higher temperatures, pyrolysis

may contribute to the vaporization plots.

A similar experimental system utilizing a flashlamp-pumped dye

laser and a gated intensified photodiode array was developed to

investigate the advantages of such a system. Fingerprints devoid of

background emission were obtained due to the discrimination by gateable

detection against continuous background emission. Reduction of furnace

wall emission, the most intense background contribution, greatly

extended the useable temperature range. This, in conjunction with

wavelength tunability, enabled the inclusion of atomic fluorescence

information into the fingerprints of complex samples.

Another use for the pulsed, gated system was as a diagnostic tool

for methods normally using discrete detectors. The system was used to

evaluate spectral interference encountered in atomic fluorescence

normally detected using a spectrometer and photomultiplier tube.

Laser-excited fluorescence of molecular vapors produced in a

graphite furnace was a useful PAC screening technique and fingerprinting












method for environmental and other complex samples. Optimum

experimental parameters such as sample size, temperature ranges and ramp

rates were easily and quickly ascertained without prior knowledge of

sample composition. Analysis time for samples was less than five

minutes and required no sample extractions, fractionation or cleanup of

any kind. In order to obtain reliable, reproducible fingerprints, care

was taken to obtain homogeneous, representative samples for analysis.

The complexity of samples in which PACs are found and the variability of

component concentrations facilitated fingerprinting, in contrast to the

hindrance that sample complexity usually imparts to PAC analysis. The

high temperatures attainable in a graphite furnace enabled direct

analysis of solids and resinous materials often too difficult to analyze

by alternate methods.

The informing power of this method would be increased greatly by

coupling it with a method that increases selectivity for specific

compounds. Low temperature and lifetime-selective techniques hold the

most promise in this respect. If the vapors produced by the furnace

underwent a supersonic expansion, narrowed absorption and fluorescence

peaks would allow identification of specific components. Alternatively,

the vapors from the furnace could be condensed onto the cold finger of a

helium refrigerator for subsequent low temperature fluorescence

spectroscopic analysis. Both of these methods are capable of cooling

analytes to a temperature facilitating selective identification of

components in mixtures by low temperature fluorescence.

Selective detection of components with long fluorescence lifetimes

is also possible. Requirements for lifetime selectivity include narrow










93

laser pulses, detector gate widths and gate delays in the nanosecond or

sub-nanosecond range. Increased spectral selectivity would enable the

investigation of the conditions and products of pyrolysis by which PACs

are produced. Studies of the matrix, adsorption and pyrolysis effects

that contribute to the appearance time of the components responsible for

the various features of the vaporization plots would also be possible.

Increased selectivity would require additional experimental system

complexity and detract from the usefulness of GFLEF as a non-selective

PAC screening technique, but would also engender other avenues of

application.














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