EPA-600/7-78-182
September 1978
Sensitized Fluorescence for the
Detection of Polycyclic
Aromatic Hydrocarbons
by
E.M. Smith and P.L Levins
Arthur D. Little, Inc.
Acorn Park
Cambridge, Massachusetts 02140
Contract No. 68-02-2150
Task No. 10302
Program Element No. EHB537
EPA Project Officer: Larry D. Johnson
Industrial Environmental Research Laboratory
Office of Energy, Minerals, and Industry
Research Triangle Park, NC 27711
Prepared for
U.S. ENVIRONMENTAL PROTECTION AGENCY
Office of Research and Development
Washington, DC 20460
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TABLE OF CONTENTS
Page
I. SUMMARY 1
II. INTRODUCTION 2
III. DISCUSSION 5
1. Model Analytes 5
2. Fluorescence Sensitizers 7
3. Instrumentation and Substrates 11
4. Environmental Samples 12
5. Attempted Purification of Sensitizers 19
IV. SUGGESTED APPLICATIONS 23
V. SPOT TEST ANALYSIS PROCEDURE 24
1. Principle 24
2. Range and Sensitivity 24
3. Interference 24
4. Precision and Accuracy 24
5. Apparatus 24
6. Reagents 24
7. Procedure 24
8. Calculations 25
9. Stability 26
VI. REFERENCES 27
iii
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LIST OF FIGURES
No. Page
1 Singlet - Singlet Energy Transfer 3
2 Equipment and Procedure for Spot Test 9
3 Fluorescent Emission of Naphthalene - PAH 10
4 Phenanthrene Fluorescence Spectra 20
5 Phenanthrene Emission Spectra 22
LIST OF TABLES
No. Page
1 Model Analytes for Sensitized Fluorescence of PAH 6
2 Fluorescence of Sensitizers 7
3 Comparison of LRMS Data and Sensitized Fluorescence
Results on Chemical Waste Incineration Samples 14
4 Level 1 LC Evaluation Mixtures 18
iv
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I. SUMMARY
A fluorescent spot test has been devised for polycyclic aromatic
hydrocarbons (PAH) based on the sensitization of the inherent fluore-
scence of such compounds. The basic procedure involves spotting a filter
paper with a small amount of the sample solution, adding naphthalene, in
solution, to the spot and visually observing the fluorescence under
illumination with a simple ultraviolet light source. On filter paper
10 pg of PAH in a spot of 0.25 cm diameter can generally be visualized
when treated with naphthalene. In the case of benzo[a]pyrene, 1 pg has
been detected.
This method has been shown to be specific for PAH with minimum
interference from other compounds. The method may be used to estimate
the general level (factors of 10) of PAH in samples to aid in decisions
for further more specific analyses.
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II. INTRODUCTION
This project was initiated to determine whether the phenomenon of
sensitized fluorescence could be utilized in the analysis of polynuclear
aromatic hydrocarbons (PAH) as a class. A major objective was to develop
a simple procedure for detection of PAH at much lower levels than current
methods based on fluorescence analysis. This procedure, requiring only
instrumentation readily available to most laboratories, would provide a
low cost screening technique to determine whether environmental assessment
samples contained levels of PAH such that more detailed analyses should be
undertaken.
The PAH are inherently fluorescent materials and are known to exhibit
sensitized fluorescence. The two processes of directly excited fluore-
scence and sensitized fluorescence are presented in a simplified energy
level diagram in Figure 1.
Compound A, as depicted, absorbs energy (i) and is raised to various
excited singlet energy levels. An energy release is made vibrationally
until the lowest excited singlet state is achieved (t). The energy
is released from this state to the various vibrational levels of the
ground state in the form of fluorescent emissions (i) . When compound B
is present, a vibrational coupling interaction can occur between the
excited states of Compound A and Compound B , resulting in resonant
energy transfer and fluorescent emissions of Compound B will be observed.
As can be seen in the diagram, Compound B must have a common vibrational
frequency with Compound A and its lowest vibrational level of the excited
singlet state must be at lower energy than the corresponding level for
Compound A. The transfer of energy is most efficient when the acceptor
(B) is present in an extremely low molar ratio to the donor (A) .
Notable examples of sensitized fluorescence are naphthacene in benz[a]-
f *y \
anthracene
respectively.
f *y \ /*3\ / —f\
anthracene and in anthracene at molar ratios of 10 and 10 ,
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Excited Singlet
States
Vibrational Coupling
I
Ground Electronic
State
Absorption
f
/
0
V
l
*
8
c
CD
O
CD
O
LL
1
Fluorescence
Compound A
Compound B
FIGURE 1 SINGLET - SINGLET ENERGY TRANSFER
-3-
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In most studies involving directly excited fluorescence, the limit
of detection of PAH has been on the order of 10 ng/mL in solution or,
in the case of thin layer chromatography, 10 ng/spot. With sensitized
fluorescence it was considered likely that by using the analyte as the
"minor" constituent of an appropriate mixture, the limit of detection
4 6
could be reduced on the order of 10 to 10 -fold as noted in the above-
mentioned sensitized fluorescence systems.
Among aromatic hydrocarbons, both the absorption and fluorescence
shift to longer wavelengths (lower energies) with increasing conjuga-
tion; both are also at longer wavelengths for linearly conjugated com-
pounds than for corresponding non-linear isomers. From the energy con-
siderations, then, lower molecular weight aromatic compounds should be
sensitizers for PAH of higher molecular weight (anthracene-naphthacene
system) and in the case of isomers, a non-linearly conjugated aromatic
compound should sensitize a linearly conjugated compound (benzfa]anthra-
cene-naphthacene system).
Some lower molecular weight aromatic compunds might sensitize the
fluorescence of many PAH and thus be of general use for PAH detection in
a screening type of test.
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III. DISCUSSION
1. Model Analytes
The PAH of interest in environmental assessment are those
containing three or more fused rings. The known carcinogenic PAH
contain at least four fused rings. In the benzenoid series, the low
molecular weight analytes would be the isomers phenanthrene and anthra-
cene of which only anthracene fluoresces in the visible. Higher molecular
weight PAH, e.g., pyrene and chrysene, show fluorescent peaks in the
ultraviolet but still have bands in the visible. PAH containing even
more condensed rings all exhibit fluorescence in the visible region of
the spectrum.
Similar spectral emission qualities are found in the methylene
bridged PAH, e.g., fluorene derivatives. Fluorene and the benzofluorenes
fluoresce in the ultraviolet and compounds with more fused rings emit
in the visible region.
In order to meet the objective of keeping the method simple,
only materials known to fluoresce in the visible spectral region were
studied as analytes. In Table 1 are shown the compounds selected as
representative analytes, their molecular structure, corrected fluorescent
emission peaks and carcinogenicity rating . These compounds were
chosen because of their range of molecular weights and their availability.
(One factor that complicated this study is that many PAH recognized as
carcinogens are no longer readily available.)
Although only non-heterocyclic compounds were tested as analytes
in this study, the heterocyclic compounds bridged to aromatic ring
structures are considered as analytes that can also be detected by
sensitized fluorescence. The lower molecular weight members that
fluoresce only in the ultraviolet region should be efficient sensitizers
for the higher-molecular weight compounds, e.g., carbazole for the
benzocarbazoles.
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TABLE 1
MODEL ANALYTES FOR SENSITIZED FLUORESCENCE OF PAH
Compound
Anthracene
Structure
M.W. Emiition Peaki(4) Carcinogenicity'5'
nm
178 378,400,422
Pyrene
202 370, 382, 392
Fluoranthene
202 465
Perylene
Benz[a]pyrene
Coronene
Dibenzo[a,i] pyrene
252
252 392,416
300 425, 442, 450
302 420,450
-H-+
-, Not Carcinogenic +++, Highly Carcinogenic
-6-
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2. Fluorescence Sensitlzers
In accord with the energy considerations mentioned earlier,
lower molecular weight aromatic hydrocarbons were selected as potential
fluorescence sensitizers. These included benzene, naphthalene, fluorene
and phenanthrene, all of which absorb and fluoresce in the ultraviolet
region of the spectrum. Their fluorescence emission bands are shown in
Table 2. It should be noted that phenanthrene is indicated to have some
fluorescence in the visible portion of the spectrum. As will be discussed
later, a reasonable assumption is that a small amount of impurity present
is responsible for the visible emission attributed to phenanthrene.
TABLE 2
Fluorescence of Sensitizers
Emission
Compound Wavelength (nm)
Benzene 255 - 300
Naphthalene 300 - 365
Fluorene 302-370
Phenanthrene 348 - 407
In keeping with the objective for simplicity, observations of
sensitized fluorescence were made with the unaided eye. Some compari-
sons of detection were made with a fluorescence spectrophotometer
although the major use of such instrumentation was in checking the
purity of analytes and sensitizers. Furthermore, since the sensiti-
(1 2 3)
zation is reported to be much more efficient in the solid state ' '
than in solution, the study was directed toward simple procedures for
producing small amounts of solid sensitizer/analyte mixtures.
Benzene, being a liquid at room temperature, was tested once in
the solid state to confirm the presumption of its sensitizing effect.
A solution of 10 pg perylene in lyL benzene was frozen on a glass micro-
scope slide. Excitation (254 cm) of the mixture resulted in the
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fluorescence of the perylene. Neither solid benzene (lyL = 900yg) nor
pyrene (100 pg) exhibited fluorescence alone.
Naphthalene was found to sensitize the fluorescence of all the
model analytes, both in the form of crystalline mixtures on microscope
slides and as mixtures in small spots on filter paper. The sensitiza-
tion occurred with 10 pg amounts of the PAH and even 1 pg in the case
of benz[a]pyrene (BaP). At that level, however, the fluorescence dis-
appeared in about one minute, presumably due to loss of the BaP, either
due to photoxidation or vaporization. At the 10 pg level the fluorescence
of dibenzofa,i]pyrene was very strong. The other analytes, however, were
considered to be at about the minimum level of detectability. The
naphthalene itself gave a weakly visible blue-white background that might
be due to impurity or to scatter of the blue wavelengths that passed
the filter of the 254 nm source. Because of the low intensity level
of this background light, the exact nature of the background was not
determined.
The naphthalene - PAH sensitized fluorescence detection was
found to be readily accomplished in the form of a spot test on filter
paper. A IpL solution of the sample was applied to a small area (0.25 cm
diameter) in the center of the paper and the same volume of sensitizer
solution (60yg/yL) then added. Sensitizer and analyte background spots
were applied (lyL each solution) to areas on either side of the mixture.
As mentioned before, the naphthalene does have a visible background; at
10 pg and even 100 pg the fluorescence of model analytes alone was not
evident.
The simplicity of the system is illustrated in Figure 2 showing
the equipment and application of the test spots. Disposable capillary
micropipets rated at lyL are used for sample application. Pencil marks
are drawn on the filter paper around the spot application areas. To
minimize spot size, the sample spots are allowed to dry before application
of the sensitizer spots. (The reverse order of application works just
as well.) Figure 3 schematically indicates the appearance of the filter
paper when illuminated at 254 nm.
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Filter paper
pipets
pipettor
Pipetting
solution
by capillary
action
Application
of solution
to test area
FIGURE 2 EQUIPMENT AND PROCEDURE
y
-------
Sensitizer
Mixture
Sample
FIGURE 3 FLUORESCENT EMISSION OF NAPHTHALENE - PAH
10
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The sensitivity of the system is even greater than originally
expected in that the mole ratio of sensitizer to analyte is on the order
of 10 in the case of 10 pg of four-ring PAH. At increased loadings of
analyte, e.g., 100 pg, the intensity of fluorescence is indeed greater.
The same effect is noted with lower amounts of sensitizer, e.g., 6pg
naphthalene with 10 pg analyte. However, the volatility of naphthalene
is such that with 6pg the fluorescing mixture disappeared in less than
two minutes at room temperature. With 60yg of naphthalene, the fluore-
scence persisted on the order of 10-15 minutes.
Although naphthalene worked well as a sensitizer for all the
model analytes, higher molecular weight aromatic compounds were con-
sidered as potentially capable of greater sensitivity and selectivity
because of their closer approach to the PAH structures. Fluorene and
phenanthrene appeared especially suitable because their inherent fluore-
scence was only in the ultraviolet spectral region and they did not sub-
lime so readily as naphthalene at room temperature. However, studies
in an attempt to use these sensitizers showed that all available samples
of both fluorene and phenanthrene exhibited intense visible fluorescence
in the solid state. The fluorescence, ascribed to PAH impurities in the
materials, interfered in the detection of 10 pg amounts of the model
PAH analytes.
Microscale purification of fluorene by paper chromatography
indicated that it would sensitize the fluorescence of fluoranthene very
well. Some efforts were undertaken to obtain pure fluorene and phenanthrene
for use as sensitizers and the measures are described in a following
section (111,5). The purification never reached the desired level of
complete removal of fluorescent contaminants.
3. Instrumentation and Substrates
When it became evident that the sensitized fluorescence would
allow visual observation of picogram quantities of PAH, emphasis was
placed on developing a spot test technique that would require only
simple instrumentation. For ultraviolet exposure of the samples, a
11
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Chromatovue Cabinet, Model C-5, (Ultraviolet Products, Inc., San Gabriel,
California) was found to be generally effective. This unit contains both
254 and 365 nm lamps of which the 254 nm source was used for the naphthalene
PAH fluorescent detections. The filter in this unit does, however, pass
some wavelengths in the blue that are scattered by the sensitizer spot
and substrate. Hand-held units such as the same firm's Model SL 2357
Mineralight do not have the necessary intensity and in addition transmit
portions of the wavelengths in the red as well as the blue. These
hand-held units can, however, be used with high (100 pg) amounts of PAH.
From private communication with other users of 254 nm lamps it was
determined that the 254 nm filters deteriorate with usage - indeed
measurements of the output of the Chromatovue unit indicated only
2 2
200 yW/cm three inches from the filter compared to claimed 760 yW/cm
eight inches away with a new unit.
Whatman #42 filter paper was selected as the most generally
useful substrate after experimentation with quartz plates, glass slides,
Eastman Type 301R2 Silica Gel chromatogram sheets, both Whatman #42
and Whatman #41 papers, Millipore Type GS filter and a transparent
Teflon film. Some Whatman #42 lots have been noted to take on a green
fluorescence on exposure to the 254 nm light, but that has not been
found to interfere with the test for PAH.
4. Environmental Samples
Samples representative of those collected in environmental
assessment studies were tested with the naphthalene reagent in order
to explore the specificity and sensitivity of the method. Most of the
samples had been previously analyzed for PAH by mass spectrometry or
were so analyzed during the course of this study. The samples consisted
of:
A. Effluents from thermal destruction of chemical wastes.
B. Particulates collected on filters from exhaust stacks.
C. Medicated shampoo (sold over-the-counter) labeled as
containing 1% coal tar extract.
12
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D. Fly-ash from power plant burning low-sulfur coal.
E. Mixtures prepared to test EPA Level 1 liquid chromatographic
(LC) procedure.
F. 1,3,5-Trinitrotoluene (representing potential interferences).
A. Thermal Destruction Samples
A chemical waste thermal destruction study had been per-
formed under a separate EPA contract and the following samples available
from that study were examined.
a. Pentane extract of sorbent trap sample from API
separator bottoms .
b. Combined knock-out trap, probe wash and filter sample
from pyrolysis zone sample of rubber manufacturing
waste .
c. Methylene chloride extract of ash from styrene tars .
d. Pentane extract of sorbent trap sample from fuel oil
(8)
background run .
e. Methanol extract of sorbent trap sample from hot zone
(8)
incineration of PVC waste .
f. Methylene chloride soluble portion from the dry
(8)
impinger of the fuel oil background run .
g. Methylene chloride soluble fraction from probe wash
(8)
and filter, incineration of PVC waste.
Solutions of each of the above were prepared at 10 ng/yL and 100 pg/pL
for testing of lyL spots on Whatman #42 paper. At the higher level, all
samples exhibited sensitized fluorescence. At 100 pg/spot the API oil
waste sorbent trap sample did not show the sensitization; the other
effluents did give the fluorescence but at lower intensities than did the
10 ng spots. The correlation with the low resolution mass spectral data
i
is shown in Table 3.
13
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TABLE 3
Comparison of LRMS Data and Sensitized
Fluorescence Results on Chemical
MW > 178
'. PAH by LRMS
low
moderate-high
moderate
high
(no data)
none
(no data)
Waste Incineration
Samples
Sensitized Fluorescence
10 ng spot
moderate
high
high
high
weak
0
0
100 pg spot
0
moderate
weak
weak
—
—
—
Sample
a
b
c
d
e
f
g
Another indication of the level of PAH present was the
direct fluorescence of the solutions. Samples a through e fluoresced
at 10 ng/uL and samples b and d still fluoresced at 100 pg/yL. All
sensitized fluorescence was observed as blue-white although the non-
sensitized spot of 10 ng of b gave a weak yellow fluorescence of itself.
B. Filter Samples
The particulates on filters (glass fiber) were from
three processes:
a. Exhaust stack filter from EPA Method 5 sample train
fO\
used in incineration of PVC waste
b. Similar filter used in incineration of nitrochloro-
(Q)
benzene with #2 fuel oilv .
c. Filters taken from the stack of a home-type oil
burner using #2 fuel.
Filters a and b were very lightly colored (tan) and represent stack
samples taken after water scrubbers.
14
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Since only small portions of the filters from a and b
above were available, extracts were made by wicking benzene or methylene
chloride along a wedge-shaped piece of each lying on a microscope slide.
In the case of sample a, no solid was found at the tip nor was there
any evidence of color migration along the wedge. Naphthalene applied
to the tip did not show any sensitized fluorescence. In the case of
sample b, birefringent crystals formed on the slide just beyond the tip
of the wedge as the solvent evaporated. These crystals were not
fluorescent of themselves nor did they show any fluorescence when treated
with naphthalene.
The filters of sample c were sufficiently large to process
by Soxhlet extraction with methylene chloride and an ensuing level 1 LC
separation. A blank filter was carried through the same procedure to
allow an estimate of the amount of organic matter extracted from the
particulates on the filter. The weights were obtained by difference
between the combined weights of weighing cups, thimbles, and filters
before and after extraction and were as follows:
Filter 1 Filter 2 Blank Filter
Gross 5.7 mg. 4.1 mg. 3.8 mg.
Net 1.9 mg. 0.3 mg.
The three extracts were initially reduced in volume to 25 mL of which
lyL was tested with naphthalene. The filter samples did show positive
sensitization and the blank showed none.
The extracts were next processed through Level 1 LC and
fractions 2, 3, and 4 were brought to 10 mL volumes. Naphthalene pro-
duced strong blue-white fluorescence with lyL spots each of LC fractions
3 and 4. GC/MS was run only on the number 3 fractions and confirmed
i
the presence of PAH such as anthracene, chrysene and higher-molecular
weight species.
15
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C. Coal Tar Extract Shampoo
A medicated shampoo, labeled as containing 1% coal-tar,
fluoresced intensely (yellow) under 254 or 365 nm radiation as did a
hexane extract of 600 mg of the emulsion. The extract, reduced to 1 mL,
was subjected to the Level 1 LC separation with fractions 2, 3, and 4
being collected and brought to 10 mL final volume each. Fraction 2 was
clear and a lyL portion showed no enhanced fluorescence with the
naphthalene reagent. Fractions 3 and 4 were yellow colored and highly
fluorescent. Fraction 3 was diluted 10 -fold before a lyL spot showed
no visible fluorescence of itself. This spot did show sensitized
fluorescence when treated with naphthalene although the result was
estimated to be at the lower limit of detectability. LRMS of fractions
3 and 4 showed the presence of PAH ranging from fluorene (m/e 166)
through the dibenzoperylenes (m/e 352).
This sample was used to evaluate the potential use of the
fluorescent spot test in estimating concentration of PAH which might be
detected in environmental samples. Certain assumptions were made, e.g.,
that the amount of PAH in fraction 3 was two times that in fraction 4
(an estimate based on the visible fluorescent intensity of the fractions)
That estimate plus the assumption that the lyL spot from the final
dilution represented 1 to 10 pg of PAH led to the following calculation:
Fraction 3: 1 to j-° PS x 10 mL x 1 x 105 = 1 to 10 mg
Fraction 4: 0.5 to 5 mg
Combined 1.5 to 15 mg
The original mass extracted was 600 mg and the estimated
PAH content of the shampoo, then, would be in the range of 0.25 to 2.5%
by weight.
16
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D. Fly Ash Sample
A fly ash sample from a midwestern power plant was
available for testing. This fly ash represented the residue from a low-
sulfur coal. A small amount of the solid on a microscope slide was
treated directly with the naphthalene sensitizer in benzene; no fluore-
scence was observed when the naphthalene crystallized.
E. Organic Level 1 LC Test Mixtures
Mixtures of organic chemicals were available which had
been prepared for evaluating Level 1 LC procedures. The solutions were
made up in pentane to contain 500 ppm of the compounds listed in Table 4.
TABLE 4
Level 1 LC Evaluation Mixtures
n-pentane
n-octane
n-nonane
n-decane
n-undecane
n-tridecane
n-pentadecane
n-heptadecane
cumene
phenol
n-methylaniline
acenaphthene
hexadecane
4,4'-dichloro-
biphenyl
tetrachloroethane
cumene
benzaldehyde
2-ethylhexanol
o-nitrotoluene
quinoline
dihexylether
The initial reason for considering these mixtures was to examine them
for possible interferences in the sensitized fluorescence procedure.
However, when it became evident that mixture b contained a fluorescent
species, the evaluation changed to center on it. Mixtures a and c
(UiL spots of them) did not show any fluorescence alone or in combination
with naphthalene. A lyL spot from mixture b did fluoresce and the
fluorescence was enhanced with naphthalene. Portions of the mixture
were spiked with anthracene, fluoranthene, perylene, pyrene, and coronene
so that 10 pg/yL of the analytes would be present. Treatment with
naphthalene of these PAH-spiked solutions of sample b gave rise to fluore-
scence of the same intensity as seen with the unspiked mixture.
17
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Although the fluorescence could not be directly attributed
to any of the components of the mixture because of the molecular
structure, the possiblility of contamination was considered. Since
acenaphthene and 4,4-dichlorobiphenyl are relatively low-molecular weight
aromatic compounds, the original materials used to prepare the mixture
were checked in solid form for fluorescence and noted to emit with yellow
and blue-white colors respectively. Solutions of each were then prepared
in methylene chloride approximating the concentration of each in the
original mix, 300 ng/yL. At this concentration, a lyL spot from the
acenaphthene solution was non-fluorescent of itself but strongly fluore-
scent (yellow) where sensitized with naphthalene. The equivalent
dichlorobiphenyl spot fluoresced alone and was strongly sensitized
by naphthalene.
Each of the compounds (100-400 ng) was analyzed by GC/MS.
Using capillary columns, both acenaphthene and 4,4-dichlorobiphenyl did
not reveal any species that would account for the observed fluorescence.
The acenaphthene was seen to contain a small amount of material of
m/e 178 (anthracene and/or phenanthrene). No higher-molecular weight
aromatic compounds were found in the dichlorobiphenyl. It was concluded
that both components must have contained PAH as contaminants and in the
solid state each acted itself as sensitizer for the fluorescence of the
impurities. It was also concluded that the acenaphthene and dichloro-
biphenyl did not interfere with the detection of the impurity by naphthalene.
F. Nitroaromatic Compounds
Nitroaromatic compounds are known to interfere in fluore-
scence analysis by a quenching mechanism . Trinitrotoluene (TNT) was
used as a model of such compounds to determine its effect on the naphtha-
lene-sensitized fluorescence of anthracene. At high concentrations,
2yg TNT to 10 pg anthracene in 60yg naphthalene, the fluorescence of
2
anthracene was indeed quenched. But at 1 ng TNT (still 10 times the
anthracene concentration) , the sensitized fluorescence of the anthracene
was definitely observed. These compounds are therefore viewed as minor
interferences.
18
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5. Attempted Purification of Sensitizers
As indicated in Section 111,2, low molecular weight PAH were
also considered potential sensitizers for fluorescence, particularly
phenanthrene and fluorene. GC/MS and fluorescence analysis of solid
phenanthrene and fluorene showed that both contained anthracene as the
principal visible fluorescent contaminant. Figure 4 demonstrates the
effect of the emission spectra of the anthracene in phenanthrene. In
solution (0.01 mg/mL benzene) the emission is that of phenanthrene
with a shoulder at 400-405 nm attributed to the presence of a trace amount
of anthracene. The emission curve obtained on the solid phenanthrene
is really that of anthracene, a case of sensitized fluorescence.
High performance liquid chromatography (HPLC) was investigated
as a method to purify fluorene and phenanthrene. Initially, size
exclusion chromatography (SEC) on a y-Styragel column was attempted,
using tetrahydrofuran as solvent. Detection of components was
with a differential refractometer. Solids obtained from different
fractions collected during SEC were observed for fluorescence, or lack of
it, under 254 and 365 nm radiation. The solids that were recovered
always exhibited fluorescence, indicating a lack of adequate separation
of undesired impurities from the bulk samples. A reverse phase column
system, using a Partisil 10-ODS column, appeared to give good separation
using small volume injections (lOyL). However, attempts to use such a
system for preparative work failed due to the small capacity of the column,
the amount of sample involved, the loss of solids on evaporation, and
eventual loss of the separating capability of the column due to over-
loading.
Partial success was achieved with a liquid-liquid partitioning
process reported for removing anthracene from phenanthrene. The
phenanthrene was dissolved in cyclohexane and the solution then treated
with concentrated sulfuric acid. Six sulfuric acid extractions reduced
the ratio of anthracene to phenanthrene drastically but never sufficiently
to render the mixture nonfluorescent. Continued extractions appeared to
19
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— Solution
Solid
t
330
350
370 390 410 430 450 470 490
510
FIGURE 4 PHENANTHRENE FLUORESCENCE SPECTRA
20
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result in chemical changes in the mixture. These results are presented
in Figure 5 where the fluorescence emission spectrum for the starting
material, the phenanthrene after six treatments, and the phenanthrene after
17 treatments are shown.
Fluorene was similarly partially purified by the partioning
between sulfuric acid and cyclohexane. Results similar to those found
with anthracene were observed.
The process with both compounds, however, was not adequately
reproducible. Therefore, the sensitization of PAH fluorescence by
naphthalene was made the method of choice.
21
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— • — Prior to Extraction
Instrument Sensitivity 1x
After Extraction (6 Times)
"~""~~""" Instrument Sensitivity 33x
_ __. After Extraction (17 Times)
Instrument Sensitivity 10x
350
400
450
500
Wavelength, nm
FIGURE 5 PHENANTHRENE EMISSION SPECTRA
22
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IV. SUGGESTED APPLICATIONS
In its present form the sensitized fluorescence spot test is use-
ful for screening environmental assessment samples for the presence of
PAH at least as low as 10yg/L (pg/yL) in solution. The absence of
sensitized fluorescence might well indicate that additional analyses
for PAH are not necessary; on the other hand, a positive fluorescence
test might indicate that GC/MS analyses should be performed to determine
the exact nature of the PAH detected.
The positive identification of an individual PAH could be made
based on the emission spectrum of the analyte-sensitizer mixture. The
identification could be done with lower quantities of PAH than have been
detected, e.g., with TLC procedures. The instrumentation would consist
of a fixed wavelength excitation source and a monochromator for the
emission spectra.
With the use of fluorescence spectrophotometers, the purity of the
analyte might not be so critical since the fluorescence due to the trace
contaminant would be known. The increase in intensity due to addition
of PAH in an analyte could be measured readily.
23
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V. SPOT TEST ANALYSIS PROCEDURE
1. Principle; The fluorescence of a polycyclic aromatic hydro-
carbon is greatly enhanced when it is present in trace quantities
(10 to 10 mole ratio) in a solid aromatic hydrocarbon of lower
molecular weight, e.g., anthracene in naphthalene. In the case of isomers,
the less linearly conjugated one is a sensitizer for the more linearly
conjugated one(s), e.g., phenanthrene for anthracene.
2. Range and Sensitivity; Many PAH can be detected at 10 pg in
the presence of 6 to 60 yg of naphthalene. Benz[a]pyrene has been
detected at 1 pg.
3. Interferences; highly-nitrated aromatic compounds are known
to quench fluorescence of PAH. At trace levels, however, that effect
is probably less likely than the transfer of energy to PAH.
4. Precision and Accuracy; Concentrations of PAH can be estimated
within a factor of 10 in the sensitized fluorescent spot test by 1:10
serial dilutions of the sample.
5. Apparatus; (Sources are those used during study and equivalent
sources are acceptable)
5.1 Ultraviolet source, 254 nm (Chromatovue Model C5)
5.2 Filter paper (Whatman #42)
5.3 Pipets (Drummond Microcaps, lyL)
6. Reagents;
6.1 Naphthalene (Fisher Scientific Catalog //N-134, "Certified"
60yg/yL.
6.2 Benzene or methylene chloride (Fisher Scientific,
Spectroanalyzed Grade).
7. Procedure; This sensitized fluorescence spot test presupposes
that the sample has been obtained in an organic solvent either by direct
solution, extraction, or a separation procedure such as liquid
chromatography.
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7.1 With pencil, mark three circles on filter paper each
approximately 0.25 cm in diameter.
7.2 With the paper supported so that marked spots are not in
contact with any other surface, apply lyL of sample
solution to central portion of each of two marked spots.
Allow to air dry, keeping spots from contacting other
surfaces.
7.3 Similarly apply lyL of naphthalene reagent solution to
remaining blank circle and to spot containing sample.
7.4 Observe spots under 254 nm, viewing either side of
substrate. Note whether differences in intensity or
color exist between sample-reagent spot and either
spot alone. Any difference indicates sensitized fluore-
scence. (At 1 ng PAH the fluorescence of the sample
spot itself should not be evident.)
Since the limits of detection are 1 to 10 pg PAH/spot for sensitized
fluorescence and approximately 10 ng/spot for non-sensitized fluorescence,
the results of the spot test procedure can be used to make the following
estimates of PAH contents in the yL of sample.
a) non-fluorescent when treated with sensitizer. : £ 1 pg
b) weakly fluorescent when treated with sensitizer : 1-10 pg
c) strongly fluorescent when treated with sensitizer
but not fluorescent alone : ^ 100 pg
d) fluorescent without sensitizer : ^ 10 ng
From such estimates, the decision to proceed with further analyses can
be made. In the case of strong sensitized fluorescence, or fluorescence
without sensitization, a better estimate of concentration may be made
by directly testing dilutions of the sample solution.
i
8. Calculations; In order to determine the PAH level in the
sample solution, the observation should be made on successive 1:10
dilutions until the sensitized fluorescence is no longer observed. Under
the conditions for the test—a lyL sample volume and 10 pg as the lowest
detectable amount of PAH—the concentration of PAH in the solution can be
25
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calculated (within a factor of 10) as follows:
C = 1 x 10(n'6) g/L
where n = number of 1:10 dilutions.
(The above formula is derived from the more explicit one:
C = 10 * 10:12S xlO-1).
1 x 10 L
For example, a solution sample that was diluted eight (8) times to reach
the point of no recognizable sensitized fluorescence would contain
1 x 10(8~6) = 100 g/L.
Since the sample solution used in this test may be an extract, LC
fraction, aliquot of another solution, or derived in some other way
from an original environmental assessment sample, the appropriate factors
must then be applied to compute the PAH content of that original sample.
9. Stability; The naphthalene sensitizer solution, kept in a
tightly-stoppered dark brown bottle, has been found to be stable over a
one-year period.
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VI. REFERENCES
1. Hercules, D., Ed., Fluorescence and Phosphorescence Analysis,
Principles and Applications, Interscience, N.Y., 1966.
2. Sawlckl, E., Talanta, 16.1231 (1969).
3. Lipsett, F.R., and Dekker, A., Can. J. Phys. 30_ 165 (1951).
A. Porro, T.J., Anacreon, R.E., Flandreau, P.S., and Fagerson, I.S.,
J. Assoc. Off. Agr. Chem. 56_ 607 (1973).
5. Particulate Polycyclic Organic Matter, National Academy of Sciences,
1972.
6. Practical Fluorescence, Theory, Methods and Techniques, G.C. Guilbault,
Marcel Dekker, Inc., New York, 1973.
7. Facility Report No. 2, EPA Contract No. 68-01-2966, Subcontract No.
A82870DNB-L, November 1976.
8. Facility Report No. 5, EPA Contract No. 68-01-2966, Subcontract No.
A82870DNB-L, July 1977.
9. Facility Report No. 6, EPA Contract No. 68-01-2966, Subcontract No.
A82870DNB-L, June 1977.
10. Lamey, S.C. and Maloy, J.T., Separation Science J3, 391 (1974).
27
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TECHNICAL REPORT DATA
(Please read Instructions on i/ie reverse before completing)
1. REPORT NO.
EPA-500/7-78-182
3. RECIPIENT'S ACCESSION NO.
4. TITLE AND SUBTLE Sensitized Fluorescence for the
Detection of Polycyclic Aromatic Hydrocarbons
5. REPORT DATE
September 1978
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
8. PERFORMING ORGANIZATION REPORT NO.
E.M. Smith and P.L. Levins
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Arthur D. Little, Inc.
Acorn Park
Cambridge, Massachusetts 02140
1O. PROGRAM ELEMENT NO.
EHB537
11. CONTRACT/GRANT NO.
68-02-2150, Task 10302
12. SPONSORING AGENCY NAME AND ADDRESS
EPA, Office of Research and Development
Industrial Environmental Research Laboratory
Research Triangle Park, NC 27711
13. TYPE OF REPORT AND PERIOD COVERED
Task Final; 7/76-8/78
14. SPONSORING AGENCY CODE
EPA/600/13
is.SUPPLEMENTARY NOTES J.ERL-RTP project officer is Larry D. Johnson, Mail Drop 62, 919/
541-2557.
16. ABSTRACT
The report describes a fluorescent spot test, devised for polycyclic aroma-
tic hydrocarbons (PAH), based on the sensitization of the inherent fluorescence of
such compounds. On filter paper, 10 pg of PAH in a spot of 0.25 cm diameter can
generally be visualized when treated with naphthalene. In the case of benzo(a)pyrene,
1 pg has been detected. This method has been shown to be specific for PAH with mini-
mum interference from other compounds. The method may be used to estimate the
general level (factors of 10) of PAH in samples to aid in decisions for further more
specific analyses.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS
c. COSATI Field/Group
Pollution
Aromatic Polycyclic Hydrocarbons
Fluorescence
Naphthalene
Analyzing
Pollution Control
Stationary Sources
13B
07C
20F
14B
18. DISTRIBUTION STATEMENT
Unlimited
19. SECURITY CLASS (This Report)
Unclassified
21. NO. OF PAGES
31
20. SECURITY CLASS (This pagt)
Unclassified
22. PRICE
EPA Form 2220-1 (9-73)
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