United States
Environmental Protection
Agency
Industrial Environmental Research
Laboratory
Research Triangle Park NC 2771 1
EPA-600/7-79-207
August 1979
Evaluation of Sensitized
Fluorescence for
Polynuclear Aromatic
Hydrocarbon Detection
Interagency
Energy/Environment
R&D Program Report
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into nine series These nine broad cate-
gories were established to facilitate further development and application of en-
vironmental technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in related fields.
The nine series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
6. Scientific and Technical Assessment Reports (STAR)
7. Interagency Energy-Environment Research and Development
8. "Special" Reports
9. Miscellaneous Reports
This report has been assigned to the INTERAGENCY ENERGY-ENVIRONMENT
RESEARCH AND DEVELOPMENT series. Reports m this series result from the
effort funded under the 17-agency Federal Energy/Environment Research and
Development Program. These studies relate to EPA's mission to protect the public
health and welfare from adverse effects of pollutants associated with energy sys-
tems. The goal of the Program is to assure the rapid development of domestic
energy supplies in an environmentally-compatible manner by providing the nec-
essary environmental data and control technology. Investigations include analy-
ses of the transport of energy-related pollutants and their health and ecological
effects; assessments of, and development of, control technologies for energy
systems; and integrated assessments of a wide-range of energy-related environ-
mental issues.
EPA REVIEW NOTICE
This report has been reviewed by the participating Federal Agencies, and approved
for publication. Approval does not signify that the contents necessarily reflect
the views and policies of the Government, nor does mention of trade names or
commercial products constitute endorsement or recommendation for use.
This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.
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EPA-600/7-79-207
August 1979
Evaluation of Sensitized Fluorescence for
Polynuclear Aromatic Hydrocarbon
Detection
by
T. R. Smith
TRW Defence and Space Systems Group
One Space Park
Redondo Beach, California 90278
Contract No. 68-02-2689
T. D. 104
Program Element No. INE624
EPA Project Officer: Larry 0. 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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CONTENTS
Abstract 1v
Tables v
Figures v1
1.0 INTRODUCTION 1
2.0 EXPERIMENTAL CONDITIONS 4
3.0 SPOT TEST PROCEDURES 6
3.1 Results 6
3.2 Procedures 6
4.0 DETECTION LIMIT AND SAMPLE ANALYSIS 8
4.1 Results 9
4.2 Interferences 9
4.3 Methods of Identification by GC/MS 12
4.4 Combustion Effluents 13
4.5 Coke Oven Effluent Samples 13
4.6 Spot Test Versus GC/MS: Detection Limit 14
5.0 SUGGESTIONS FOR FUTURE STUDY 32
6.0 CONCLUSION 37
7.0 REFERENCE 38
111
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Abstract
A flourescent spot test used for detecting the presence of polynuclear
aromatic hydrocarbons (PAHs) has been evaluated as a screening technique
for samples to be analyzed by gas chromatography/mass spectrometry (GC/MS).
The spot test is based on the phenomenon of sensitized fluorescence and
is capable of easily detecting 100 picograms of PAH in a 1 microliter
sample, a level of sensitivity adequate for the screening of combustion
effluent samples.
Two interferences were observed: 1) Samples which are highly colored
required dilution to allow viewing of the fluorescence level and 2) samples
containing substantial amounts of phthalate esters produced false positive
results. No false negative results were observed in this study.
It is our conclusion that the spot test procedure is adequate for the
screening of combustion effluent samples prior to GC/MS analysis.
1v
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Tables
1. Results of spot test analysis of standards 10
2. Results of spot test analysis of samples from
industrial sites 11
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Figures
1. Total Ion chromatogram of a combustion effluent sample .... 15
2. Mass chromatogram of m/e 252 16
3. Mass chromatogram of m/e 202 17
4. Total 1on chromatogram of a typical coke oven sample 18
5. Mass chromatogram of m/e 202 19
6. Mass spectrum - scan number 454 20
7. Mass spectrum - scan number 476 21
8. Mass spectrum - scan number 738 22
9. Mass chromatogram of m/e 252 23
10. Mass spectrum - scan number 710 24
11. Total ion chromatogram of detection limit test sample .... 25
12. Mass chromatogram of m/e 202 26
13. Mass chromatogram of m/e 252 27
14. Mass chromatogram of m/e 300 28
15. Mass chromatogram of m/e 302 29
16. Mass chromatogram of m/e 202 30
17. Mass chromatogram of m/e 252 31
18. HPLC of benzene, chloronaphthalene and benzo(a)pyrene .... 34
19. HPLC of naphthalene, fluorene, phenanthrene
and benzo(a)pyrene 35
20. HPLC of coke oven sample 36
v1
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1.0 INTRODUCTION
This document represents the final report on the evaluation of the
Polynuclear Aromatic Hydrocarbon Spot Test. This report is submitted in
partial fulfillment of the requirements for EPA contract No. 68-02-2689,
Technical Directive 104. The work was conducted by TRW under the direction
of EPA Task and Project Officer, Dr. L. D. Johnson.
The original fluorescence spot test discussed in this report was
developed by Arthur D. Little, Inc. under contract to the EPA and is based
on the phenomenon of sensitized fluorescence. The objective of this effort
was to determine if the test could be used as a screening aid for samples
submitted for PAH analysis by gas chromatography/mass spectrometry (GC/MS).
The procedure can potentially provide a low cost screening technique to
predetermine the presence of PAHs.
This test was evaluated specifically for its detection of PAHs. The
importance of PAHs as a class originate from the serious health hazard
posed by certain components of this class of compounds. Specifically,
benzo(a)pyrene and dibenzo(a,i)pyrene are compounds within this class
which have been shown to be potent cancer causing agents. The potential
danger of contracting cancer is incurred when these compounds are inhaled
into the lungs or come into contact with the skin. Therefore, it is
important that these compounds be detected and identified in effluents
which could come into contact with the population.
The analysis schemes thus far implemented for the determination of
PAHs encompass several analytical approaches including nonspecific
analysis, detailed quantitative and qualitative analysis, and specific
analysis for a single PAH. The limits of detection have been reported to
be at the 10 picogram level in some instances. The analysis times range
from 15 minutes to several hours with the evaluation if the test
results occupying an equal amount of time. The PAH spot test is a survey
technique which provides information as to the presence or absence of PAH
as a class and no specificity is provided. The level of detection is in
the picogram range and the analysis time is on the order of 15
minutes. Gas chromatography/mass spectrometry can provide qualitative
and quantitative information on the PAHs present in an effluent. Sample
1
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analysis time is typically between 1 to 2 hours with the data
evaluation consuming at least an equal amount of time. The limits of
detection for the GC/MS analysis normally approaches 5 nanograms per
PAH.
For general purpose determination of PAH in complex mixtures the use
of GC/rlS is unexcelled. The gas chromatograph is used to separate the
complex mixture into individual component peaks and the mass spectrometer
is used as a detector for both qualitative identification of the PAH and
for quantitative determination. For simple cases where only a few PAH
are present, the use of packed gas chromatographic columns 1s recommended.
Packed columns are generally easier to use, require a shorter time for
analysis and are cheaper than capillary columns. When highly complex mixtures
of PAH are present or significant interferences occur, the use of capillary
columns becomes necessary. Chromatographic column selection is usually
dependent on the volatility and polarity of the PAH to be determined.
The major limitation of the gas chromatograph is the volatility of
compounds to be analyzed. In order to be determined by GC/MS, PAHs must
produce good chromatographic peaks at temperatures below 300°C. At or
above 300°C even the most stable GC column liquid phases produce high
background in the mass spectrometer. This GC temperature limitation means
that high molecular weight PAHs (above molecular weight 300) cannot be
reliably determined by this technique. For this reason some PAHs which
produce fluorescence in the spot test may not be analyzable by GC/MS.
As discussed above, the,analysis of samples by GC/MS for the routine
detection of PAHs expends costly GC/MS instrument time as well as the time
needed to interpret the data. The PAH spot test is proposed to serve
as a screening tool for the GC/MS analyst. This pretest will serve as a
go/no go analysis. That is, if the test indicates the presence of PAH,
the sample will be run via GC/MS. If no fluorescence is observed it will
not require analysis. Since the PAH spot test requires only about 15
minutes of effort and no instrument time, implementation of this test
should result in a significant decrease in PAH analysis time and cost.
Clearly, this is contingent upon several factors. First, for maximum
benefit to the combustion environmental assessment program sensitivity
should equal or exceed that of GC/MS for other projects the sensitivity
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requirement varies with the job. Second, interferences which would
result in false negative findings must be either identified and taken
into consideration or they must be nonexistent. The experiments reported
here were designed to evaluate these possibilities.
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2.0 EXPERIMENTAL CONDITIONS
Phenanthrene and benzo(a)pyrene were chosen as model analytes to test
the spot test procedure. This choice was based on the facts that:
t Phenanthrene and benzo(a)pyrene are representative of PAHs
which are likely to occur in combustion samples.
• The range of aromatic fused ring compounds detectable by
GC/MS is covered by these two compounds.
• Any difference in sensitivity of the spot test to PAHs should
be discernable by the comparison of the response of these
two components,
» and lastly, because of benzo(a)pyrene's carcinogenicity, it
is critical that the presence of this compound be detected
by the spot test at low concentrations.
An ultraviolet Chromatovue Cabinet, Model C-70, was used for ultraviolet
exposure of the samples. This unit contains both 254 and 365 nm lamps of
which the 254 nm source is used for the naphthalene-PAH fluorescent
detections. Whatman 142 ashless filter paper was used as a substrate for
the sample analysis.
The phenanthrene and benzo(a)pyrene used for the prelimenary study
were obtained from Analabs, Inc., North Haven, Conn. The solvent, methylene
chloride, v/as obtained from Burdick and Jackson, Inc., MusKegon, MI.
The sensitizer, solution, naphthalene was prepared at a concentration of
60 yg/yfc 1n methylene chloride.
The gas chromatographic/mass spectrometric analyses were done on a
duPont model 321 GC/MS. The key operating parameters of the GC/P1S were
as follows:
Column - 2 m. X 3mm ID glass packed with 3% OV-101 on 100-120
Chromosorb WHP
Carrier Gas - Zero grade helium at 30 ml/min
Oven Temperature - 100°C to 295°C programmed at 8°C/min
Injector Temperature - 300°C
Transfer lines and separator Temperature - 270°C
Mass Range - 40-450 AMU
Scan Function up - 1.9 seconds
down - 0.0 seconds
4
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hold at top - 0.0 seconds
hold at bottom - 0.1 seconds
The high performance liquid chromatograph (HPLC) used for this report
was a duPont model 850 equipped with a variable wavelength ultraviolet
spectrophotometer. The wavelength monitored on all chromatograms was
254 nm. For all separations a Zorbax-ODS, 4.6 mm ID X 25 cm, liquid
chromatographic corumn was used. All solvents were obtained from Burdick
and Jackson, Inc.
The samples used to determine applicability of the spot test procedure
to combustion effluent samples were provided by ongoing EPA sponsored
projects. Specific sample type identifications are presented in the results
section of this report.
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3.0 SPOT TEST PROCEDURE
3.1 PREPARATION OF SAMPLES
No preparation of the sample is required. The sample should be in a
solution to allow easy application to the test substrate. Solids and gases
can be analyzed with some modification of the application technique
3.2 PROCEDURE
A Chromatovue ultraviolet cabinet Model C-70 was used to expose the
samples. The 254 nm lamp source was used. A 7.0 centimeter circular
ashless Whatman #42 filter was used as the support for the samples. The
sensitizer used was naphthalene at a .concentration of 60 yg/pJi in methylene
chloride.
The spot test is carried out as follows:
1. Draw three circles in pencil on a piece of Whatman #42
filter paper as close to one another as feasible.
2. Using a 10 y£ syringe, spot the filter paper with the
sensitizer (naphthalene). 1 mlcrollter of sensitizer
should be spotted on the leftmost circle and 1 microliter
of sensitizer should be spotted on the center circle.
The spots should not be allowed to overlap. The
.substrate should be supported so as not to touch any
surface.
3. Allow the solvent to evaporate from the filter paper.
4. 1 microliter of sample is then applied to the rightmost
and center circles. Again allow the solvent to evaporate
and do not allow the substrate to touch any surface
until the solvent has evaporated.
5. The filter paper is placed in the Chromatovue cabinet.
6. Compare the spots visually with the unaided eye.
7. If the center spot (sample plus sensitizer) fluoresces
brighter than the sensitizer spot (leftmost), the presence
of PAHs is suspected. Also, any significant difference in
color between the sensitizer only spot versus sensitizer/
sample spot is also considered a positive indicator for
PAHs.
8. Samples which give positive results by the spot test should
be submitted for GC/MS analysis to identify the source of
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the fluorescence.
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4.0 DETECTION LIMIT AND SAMPLE ANALYSIS
In order to determine the level of detection attainable with the
equipment available, a set of standards were prepared and tested.
Phenanthrene and benzo(a)pyrene were chosen as the test standards. As
previously discussed, phenanthrene and benzo(a)pyrene were chosen because
both are considered representative of the PAHs likely tc occur in
combustion effluent samples. Their differences in structure may also cause
differences in their detectability by the PAH spot test. Additionally,
the range of aromatic fused ring compounds detectable by GC/MS is covered
by these two compounds and the presence of benzo(a)pyrene, because of its
carcinogenicity, must be detectable by the spot test at low concentrations.
The levels of standards prepared and the results of the analyses are
presented in Table 1. The limit of detection of the spot test is
extropolated to be approximately 100 plcograms for these two materials.
That is, a 1 micro!Her sample containing 100 picograms of PAH is
easily detected. This observation differs by an order of magnitude
from the detection limit of 10 picograms previously reported . The
difference in the reported detection limit is probably due to the point
at which the investigator decides to declare a significantly distinguishable
difference in the fluorescent intensity of the sensitizer alone versus the
sensitizer/sample spot. Since this decision is subjective it was decided
to choose a level that was easily discernable by personnel not familiar
with the test. A lower detection limit is achievable but it would require
the operator to make judgements based on subtleties of intensity and hue
and would require extensive operator training and experience.
Another factor which must be taken into consideration when detection
limits are compared is the ultraviolet lamp intensity. Any difference in
lamp intensity will result in a difference of fluorescence intensity and
consequently, the detectable difference between the sensitizer only and
the sensitizer/sample spots would be concommitantly effected. It is an
accepted fact that lamp intensity varies with age and it is not unusual
to encounter significant differences in lamp intensities in an apparatus
such as the Chromatovue C-70. Differences in lamp intensity as a source of
variation of the detection limit was not investigated as a part of this
task.
8
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4.1 RESULTS
A series of industrial samples which gave negative results for both
the PAH spot test and GC/MS analysis were spiked with a known amount of
benzo(a)pyrene (100 pg/v«.) and retested by the spot test. As expected, the
samples after spiking yielded a positive indication by the PAH spot test.
Over 20 samples from industrial sites have been tested by both
GC/MS and the fluorescence spot test. A partial listing of these samples
and their results are given in Table 2. Approximately 10% of the samples
which gave a positive result of the spot test showed no PAHs by GC/MS. As
will be illustrated below, the spot test has a level of detection which is
lower than that of GC/MS, when the GC/MS is used in the traditional scanning
mode. Taking this fact into consideration, 1t is not unreasonable to
expect a small percentage of the samples to prove positive by the spot test
and negative by GC/MS. But what is more important is the fact that in all
cases where the spot test gave negative results the GC/MS also found no
PAHs. That is, no false negatives were encountered.
4.2 INTERFERENCES
The effectiveness of the PAH spot test is lessened when a highly
colored sample is encountered. If the sample is opaque or highly colored,
the sample must be diluted. This dilution is to allow the operator to
view the fluorescence of the sample without interference from the sample's
color. The detection limit of the spot test is increased proportionatly
as the sample is diluted.
An example of this interference is demonstrated by the combustion
effluent samples studied. It is not unusual for combustion effluent
samples to posses a deep yellow color. When tested, the yellow color of
the sample combined with the blue color of the naphthalene sensitizer
forms a purple-blue sensitizer/sample spot. This purple-blue color of the
sensitizer/sample spot is different in color from t'ne blue sensitizer only
spot. Since it has been noted that in some cases a difference in hue is
an indication of PAH presence, this interference may be the source of
some confusion and result in unconfirmed positives.
When the sample is highly colored, it must be diluted so that the color
does not interfere with the fluorescence. For samples where color was a
9
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Table 1
Results of spot test analysis of standards
Phenanthrene
Benzo(a)pyrene
Amount
80 ng
8 ng
160 pg
80 pg
Test Results
Positive
Positive
Positive
Negati ve
Amount
490 ng
49 ng
147 pg
98 pg
49 pg
Test Results
Positive
Positive
Positive
Negative
Negative
10
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Table 2
Results of spot test analysis of samples from Industrial sites
Coke Oven Extracts
Spot Test GC/MS
Sample
A
B
Positive
Positive
Positive
Positive
A
B
C
Combustion Effluent Samples
(Commercial Oil Burner)
Negative Negative
Positive Positive
Positive Positive
A
B
C
D
E
F
G
H
Combustion Effluent Samples
(Coal Fired Power Plant)
Negative Negative
Negative Negative
Positive Negative
Negative Negative
Negative Negative
Negative Negative
Negative Negative
Positive Negative
11
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source of interference, the dilution was achieved by adding more sensitizer
to both the sensitizer/sample and sensitizer only spots. The sensitizer was
added equally to both spots until the sample color no longer interfered.
This was done to maintain the total amount of sample constant. As of this
writting, no more than 3 m1crol1ters of sensitizer has been required.
For samples that are highly colored, it is also suggested that the samples
not only be examined immediately after application to the filter paper,
but also after the sample has been exposed to the UV source for a total of
5 minutes. This delay allows for a clearer distinction between the
sensitizer and sensitlzer/sample spot. During this 5 minute period the
fluorescence of the naphthalene diminishes and if there are PAHs present
in the sample, the rate of fluorescence decay will be more rapid in the
sensitizer only spot. Another suggested technique is to also view the
sample under the 365 nm source. In some cases this has been found to
enhance the differences in the spots.
4.3 METHODS OF IDENTIFICATION BY GC/MS
When using GC/f1S polynuclear aromatic hydrocarbons are best identified
as isomer classes based on molecular weight. Once a chromatographic
separation has been achieved and the large quantity of mass spectral data
is stored in a computer based system, individual mass chromatograms for
specific molecular weights can be displayed. After it is established
which classes of PAH compounds are present in a given sample, standards
of the individual components or relative retention time data can be used to
identify specific isomers.
PAHs typically show very strong molecular ions (the mass representative
of molecular weight) in their mass spectra because of the stability of their
aromatic ring system. This strong molecular ion allows for a very simple
identification of molecular weight even in complex mixtures. The
disadvantage of this strong molecular ion production is that isomeric
PAHs produce spectra which are virtually identical. The mass spectrometer
is a rather poor device for distinguishing isomers since once ionized a
given compound will seek its most thermodynamically stable form. Since
isomeric PAHs produce similar or identical mass spectra, the determination
of structure is best accomplished by comparision of their GC retention
times to standards. That is, for qualitative identification of specific
12
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isomers, standard addition of known PAH compounds is made to each sample.
An increase in GC peak intensity without broadening is used as confirmation
of identification. Once a single isomer of a given molecular weight is
identified, relative retention time data can be accurately applied for
identification of isomers where standards are not available.
4.4 COMBUSTION EFFLUENTS (COAL BURNING PO'-JER PLANT)
Figure 1 is an example of the total ion chromatogram of a combustion
effluent sample which was analyzed by both GC/MS and the spot test. Both
the GC/MS and PAH spot test yielded negative results. Examination of the
mass chromatogram for m/e 252 (Figure 2) indicates that benzo(a)pyrene,
perylene, etc. were not detected by GC/MS. Also, the mass chromatogram
for m/e 202 (Figure 3) shows no indication of fluoranthene, pyrene, etc.
This technique of using selected mass values to search GC/MS data for PAH
is used routinely in our laboratory. By using mass chromatograms, GC/MS
data can be rapidly searched for the most common PAHs. This technique is
less time consuming than displaying the mass spectra for all of the peaks
observed in a GC/MS run.
The data displayed in Figures 1-3 are typical of samples run from this
combustion source. Some samples of the series gave positive results on the
spot test but all proved to be negative by GC/MS.
4.5 COKE OVEN EFFLUENT SAMPLES
When coal is pyrolyzed by a burning or coking process, significant
amounts of PAHs are produced. In the burning process most of the organic
material is destroyed through oxidation, producing water and carbon
dioxide. In the coking process, however, significant amounts of organics
are evolved and emitted to the atmosphere. For these reasons the coke oven
samples were an essential inclusion into the experimental scheme.
Figure 4 is a GC/MS total ion chromatogram of a typical coke oven
sample. This sample was shown to contain PAHs by the spot test. Presented
in Figure 5 is the mass chromatogram for the m/e 202 of this sample. In
this figure, peaks containing m/e of 202 are evident. The possible
identities of these peaks include the PAHs, fluoranthene and pyrene. An
examination of the mass spectra of these peaks which are shown in Figure 6,
13
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7, and 8 will more clearly aid in defining their identity. These mass
spectra confirm the presence of pyrene and fluoranthene. The mass
chromatogram of the 252 ion is illustrated in Figure 9. Two peaks are
clearly shown which have significant abundance at m/e 252. Since the
benzopyrene isomers have a molecular weight of 252 and elute in this
region of the chromatogram, they serve as likely candidates. Examination,
of the mass spectrum of the peak eluting at scan 710 (Figure 10) confirms
the presence of at least one benzopyrene isomer.
4.6 SPOT TEST VERSUS GC/MS: DETECTION LIMIT
Figure 11 is the GC/MS total ion chromatogram of a sample which gave
a strongly positive PAH spot test result. The amount of sample injected
onto the GC column was 3 m1crol1ters. This is 3 times the amount
of sample used for the spot test. Figures 12 through 15 are the mass
chromatograms for the m/e's 202, 252, 300, and 302, respectively. These
represent the ions which are indicative of the PAHs typically present in
these samples. In Figure 16, the area of the m/e 202 peak maximizing at
scan 447 (15:00 minutes) is 53K counts. Extrapolation of this number to
an area still distinguisable in the mass chromatograms suggests that the
sample size could be reduced from 3.0 fefc to 0.3 y£, an effective dilution
of 10, and still be detectable by GC/MS. Figure 17 is the mass chromatogram
of the 252 ion including the areas calculated for the components containing
this ion. The previously noted extrapolation is also applicable in this
case. For the purposes of comparison, this will be considered the limit of
detection for the GC/MS analysis. That is, the sample, if diluted by more
than a factor of 10, would not contain an amount of PAH that could clearly
be detected by GC/MS.
In order to compare the aforementioned results to the detection limits
of the PAH spot test, the sample was diluted and the resultant dilutions
subjected to the spot test. The sample when diluted by a factor of 100
still gave a positive result by the spot test. The subsequent level of
PAHs in the sample after a one hundredfold dilution was previously
extrapolated to be below the detection limit of the GC/MS. Consequently,
using this sample as an example, the limit of detection of the PAH spot
test has been demonstrated to be more than adequate for the purpose of
determining which samples are to be further analyzed by GC/MS for PAHs.
14
-------
tea. a
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Figure 1. Total ion chromatogram of a combustion effluent sample.
-------
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Figure 2. Mass chromatogram of m/e 252.
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-------
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-------
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N •
It*
QUAH: A •. l.» BASE: 0 M. 3
252
1136*.
252.W5
* «.5M
6:4«
13:2*
26:40
33:28
SCAN
TII1E
Figure 13. Mass chromatogram of m/e 252.
-------
r\>
<»
100.
300
HAS5 CHBOWTOGIAM DATA: BS07CIB II
04/10/79 I5:0I:0« CAL1: DGMte II
SAIffLE: 3UL BS07CIB
RANGE: G 1.1122 LABEL: N 9. 4.0 QUAN: A «. !.• BASE: U 20. 3
SCANS 1 TO 1122
208
131
y
320
3
1
ill
t
728
627
1"
IT in
tj.
1
2M
6:4*
13:20
20:00
8W
26:48
340.
300.090
* 0.500
1096
SCAN
TItlE
Figure 14. Mass chromatogram of m/e 300.
-------
HASS CHNHATOGRAn
M/10/79 15:«1:M
SAHHE: 3UL BS07CIB
BANGE: G 1.1122 LABEL: N
». 4.« (MAN
DATA: BS07C1B II
CALI: DC0410 II
• BASE: U 28. 3
SCANS 1 TO 1122
ro
351.
SCAN
TINE
Figure 15. Mass chromatogram of m/e 302.
-------
MASS GHROttTOGBAH DATA: BS07CIB II
M/I9/79 I5:«1:M CALI: BCMie II
SAHTLE: 3UL BS07CIB
RANGE. G 1.1122 LABEL: N 1,,4.9 QUAH: A 1. 1.9 BASE. 0 ». 3
7584.
~ 912.
467
SCANS 4M TO 5»
7584.
2t2.«6«
* «.5M
13:2*
16:49
52» SCAH
17:20 TINE
Figure 16. Mass chromatogram of m/e 202.
-------
HASS CHBOMATOGRAh
04/10/79 15:01:00
SAMPLE: 3UL BS07CIB
BANCE: G 68*. 740 LABEL:
DATA:
CALI:
BS07CIB 1467
BC04I0 II
SCANS 650 TO 800
N 1. 4.8 QUAN:
1.0 BASE: U 20. 3
100.
252
6784.
252.075
0.500
SCAN
26:40 Tlffi
Figure 17. Mass chromatogram of m/e 252.
-------
5.0 SUGGESTIONS FOR FUTURE STUDY
Other alternatives exist to the screening technique proposed in the
Arthur D. Little, Inc. report. High performance liquid chromatography
(HPLC) is a technique that can provide a semi-qualitative and quantitative
analysis for PAH in a time span as short as 15 minutes. As with the
gas chromatograph, the identity of the eluting components are assigned by
retention time and spiking. Specific detection techniques such as
ultraviolet absorption and fluorescence can be employed to screen for PAHs.
The selectivity of these detectors are extremely useful when analyzing
complex samples. Setting the UV or fluorescence detector at wavelengths
specific for the PAH(s) provides for an enhanced response of the components
of interest over other components in the sample matrix.
Figures 18 through 20 are high performance liquid chromatograms of
PAH standards and a coke oven sample. Figure 18 is a standard mixture
of benzene, chloronaphthalene and benzo(a)pyrene. Under these conditions,
20% water 1n methanol at a flow of 1 milliter per minute on a 25 cm
ODS column, benzo(a)pyrene elutes 1n approximately 35 minutes while
benzene elutes in 7 minutes. Figure 19 1s the chromatogram of naphthalene,
fluorene, phenanthrene and benzo(a)pyrene. In this case 1t takes 70
minutes for benzo(a)pyrene to elute from the column. This longer analysis
time allows for better Identification of components in complex mixtures.
Figure 20 1s a coke oven sample run under the same conditions as the
standard 1n Figure 19. As can be seen, this provides an acceptable
separation of a very complex sample. The presence of fluorene and
phenanthrene can be easily distinguished.
These examples are typical of the results that can be obtained using
HPLC and a UV spectophotometer detector.
Unlike nonaqueous reverse phase chromatography (NARP), conventional
reverse phase high performance liquid chromatography involves the use of
nonpolar chromatographic stationary phase (e.g. c-18 bonded to an inert
support) and a polar solvent system (e.g. methanol/water). In the case of
PAH compounds, the use of this type of system for separation of PAHs above
300 molecular weight can result in excessive analysis time. This is due
in part to the limited solubility of PAHs in the solvent systems used.
Substituting a less polar solvent system for the polar solvent systems
32
-------
5.0 SUGGESTIONS FOR FUTURE STUDY
Other alternatives exist to the screening technique proposed in the
Arthur D. Little, Inc. report. High performance liquid chromatography
(HPLC) is a technique that can provide a semi-qualitative and quantitative
analysis for PAH in a time span as short as 15 minutes. As with the
gas chromatograph, the identity of the eluting components are assigned by
retention time and spiking. Specific detection techniques such as
ultraviolet absorption and fluorescence can be employed to screen for PAHs.
The selectivity of these detectors are extremely useful when analyzing
complex samples. Setting the UV or fluorescence detector at wavelengths
specific for the PAH(s) provides for an enhanced response of the components
of interest over other components in the sample matrix.
Figures 18 through 20 are high performance liquid chromatograms of
PAH standards and a coke oven sample. Figure 18 1s a standard mixture
of benzene, chloronaphthalene and benzo(a)pyrene. Under these conditions,
20% water in methanol at a flow of 1 milliter per minute on a 25 cm
CDS column, benzo(a)pyrene elutes 1n approximately 35 minutes while
benzene elutes in 7 minutes. Figure 19 1s the chromatogram of naphthalene,
fluorene, phenanthrene and benzo(a)pyrene. In this case 1t takes 70
minutes for benzo(a)pyrene to elute from the column. This longer analysis
time allows for better identification of components in complex mixtures.
Figure 20 1s a coke oven sample run under the same conditions as the
standard 1n Figure 19. As can be seen, this provides an acceptable
separation of a very complex sample. The presence of fluorene and
phenanthrene can be easily distinguished.
These examples are typical of the results that can be obtained using
HPLC and a UV spectophotometer detector.
Unlike nonaqueous reverse phase chromatography (NARP), conventional
reverse phase high performance liquid chromatography involves the use of
nonpolar chromatographic stationary phase (e.g. c-18 bonded to an inert
support) and a polar solvent system (e.g. methanol/water). In the case of
PAH compounds, the use of this type of system for separation of PAHs above
300 molecular weight can result in excessive analysis time. This is due
in part to the limited solubility of PAHs in the solvent systems used.
Substituting a less polar solvent system for the polar solvent systems
32
-------
routinely used in reverse phase chromatography Increases the solubility of
the PAHs. Consequently, with the use of methylene chloride and acetonitrile
as a solvent system, coronene (m/w 300) and decacyclene (m/w 454) can be
eluted in approximately 8 and 17 minutes, respectively. Thus with NARP,
components which cannot be analyzed by GC/MS can be handled easily and
in a relatively short analysis time. It is recommended that HPLC be
investigated as an additional method for the analysis of PAHs.
33
-------
START
CO
figure 18. High performance liquid chromatogram
of A. benzene, B. chlorobenzene and C. benzo(a)pyrene.
-------
Ut
cn
. I
4
___ |__
] __3..
-O-
-5O-
Figure 19. HPLC of A. naphthalene, B. fluorene,
C. phenanthrene and D. benzo(a)pyrene.
-------
CO
CT>
Figure 20. HPLC of coke oven sample
A. fluorene; B. phenanthrene.
-------
6.0 CONCLUSION
It is clear from the data presented 1n this report that the spot test
has proven acceptable as a screening tool for samples submitted for GC/MS
analysis of PAHs. Two requirements were to be met by this screening
procedure. One, the detection limit of the PAH spot test must be below
that of the GC/MS and two, the interferences that prevent accurate results
from the spot test must be defined. These issues have been addressed.
That is, the detection limit of the screening technique (spot test) is
below that of the accepted analysis technique (GC/MS). Also, interferences
occuring from the inherent color of the sample were discussed.
It was shown that unconfirmed positives can be obtained from several
sources. A unconfirmed positive result will occur when a sample contains
a level of PAHs which is above the detection limit of the spot test and
below the detection limit of the GC/MS. Another source of unconfirmed
positives can be caused by the interference of the sample color with the
fluorescence of the sample.
Another source of false positives which must not be overlooked are
cases in which the fluorescence is caused by PAHs which have a molecular
weight above 300. GC/MS will not prove adequate as a method of analysis.
Clearly, high performance liquid chromatography must be used in cases where
high molecular weight PAHs are suspected. Indeed, it may prove beneficial
to analyze all samples which prove positive by the spot test by HPLC for
high molecular weight PAHs and not only those which prove positive by the
spot test and negative by GC/MS.
37
-------
7.0 REFERENCE
1. Sensitized Fluorescence for the Detection of Polycyclic
Aromatic Hydrocarbons, Report No. EPA-600/7-78-182, NTIS
No. PB 287-181 IAS, September 1978.
38
-------
TECHNICAL REPORT DATA
(Please read Inunctions on the reverse before completing)
REPORT NO.
EPA-600/7-79-207
2.
3. RECIPIENT'S ACCESSION NO.
TITLE AND SUBTITLE
Evaluation of Sensitized Fluorescence for Polynuclear
Aromatic Hydrocarbon Detection
REPORT DATE
August 1979
6. PERFORMING ORGANIZATION CODE
. AUTHOR(S)
T.R. Smith
8. PERFORMING ORGANIZATION REPORT NO.
.. PERFORMING ORGANIZATION NAME AND ADDRESS
TRW Defence and Space Systems Group
One Space Park
Redondo Beach, California 90278
10. PROGRAM ELEMENT NO.
INE624
11. CONTRACT/GRANT NO.
68-02-2689, T.D. 104
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 PE
Task Final; 1-6/79
PERIOD COVERED
14. SPONSORING AGENCY CODE
EPA/600/13
5. SUPPLEMENTARY NOTES
919/541-2557.
project officer is Larry D. Johnson, Mail Drop 62,
16. ABSTRACT rphe repOrt gives results of an evaluation of a fluorescent spot test for
detecting the presence of polynuclear aromatic hydrocarbons (PAHs) as a screening
technique for samples to be analyzed by Gas Chromatography/Mass Spectrometry
(GC/MS). The test is based on the phenomenon of sensitized fluorescence and is
capable of easily detecting 100 picograms of PAH in a 1 microliter sample, a level
of sensitivity adequate for screening combustion effluent samples. Two interferences
were observed: highly colored samples require dilution to allow viewing of the fluor-
escence level, and samples containing substantial amounts of phthalate esters pro-
duce false positive results. No false negative results were observed in the study.
The test is adequate for screening combustion effluent samples prior to GC/MS
analysis.
7.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b. IDENTIFIERS/OPEN ENDED TERMS
COS AT I Field/Group
Pollution
Combustion Products
Screening
Aromatic Poly eye lie Hydrocarbons
Fluorescence
Sensitizing
Pollution Control
Stationary Sources
Sensitized Fluorescence
13B
21B
14B
07C
20F
13H
"S. DISTRIBUTION STATEMEN1
Release to Public
19. SECURITY CLASS (ThisReport)
Unclassified
21. NO. OF PAGES
43
20. SECURITY CLASS (This page)
Unclassified
22. PRICE
EPA Form 2220-1 (9-73)
39
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