EPA-600/4-82-025
DETERMINATION OF POLYNUCLEAR AROMATIC HYDROCARBONS IN
INDUSTRIAL AND MUNICIPAL WASTEWATERS
by
Paul E. Strup
Battelle Columbus Laboratories
Columbus, Ohio, 43201
Contract No. 68-03-2624
Project Officer
James E. Longbottom
Physical and Chemical Methods Branch
U.S. Environmental Protection Agency
Cincinnati, Ohio, 45268
RECEIVED
FEB 29 1980
iurt AGENCY
LIBRARY, REGION V
ENVIRONMENTAL MONITORING AND SUPPORT LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
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DISCLAIMER
This report has been reviewed by the Environmental Monitoring and
Support Laboratory, U.S. Environmental Protection Agency, and.approved
for publication. Approval does not signify that the contents necessarily
reflect the views and policies of the U.S. Environmental Protection
Agency, nor does mention of trade names or commercial products constitute
endorsement or recommendation for use.
ii
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FOREWARD
Environmental measurements are required to determine the quality
of ambient waters and the character of waste effluents. The Environmental
Monitoring and Support Laboratory-Cincinnati conducts research to:
Develop and evaluate techniques to measure the presence "and
concentration of physical, chemical, and radiological pollutants
in water, wastewater, bottom sediments, and solid waste.
Investigate methods for the concentration, recovery, and identi-
fication of viruses, bacteria, and other microbiological organisms
in water. Conduct studies to determine the responses of aquatic
organisms to water quality.
Conduct an Agency-wide quality assurance program to assure
standardization and quality control of systems for monitoring
water and wastewater.
Under provisions of the Clean Water Act, the Environmental
Protection Agency is required to promulgate guidelines establishing
test procedures for the analysis of pollutants. The Clean Water Act
Amendments of 1977 emphasize the control of toxic pollutants and
declare the 65 "priority" pollutants and classes of pollutants to be
toxic under Section 307(a) of the Act. This report is one of a series
that investigate the analytical behavior of selected priority pollutants
and suggests a suitable test procedure for their measurement.
Dwight G. Ballinger
Director
Environmental Monitoring and Support Laboratory
iii
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ABSTRACT
A method for the determination of 16 polynuclear aromatic hydrocarbons
(PAH) in wastewater was developed. This method, based on the use of high
performance liquid chromatography with ultraviolet and fluorescence de-
tection, is readily suited for detection of condensed ring aromatics in
many types of aqueous samples. Aqueous sources included in this study
are flyash wash water, municipal sewage, and industrial effluent samples.
Precision and accuracy of the method were estimated from the results
of five wastewater samples spiked at levels between 0.1 and 250 ppb for
the various PAH compounds. Recoveries were generally 85% or better
from these wastewater sources.
Storage of several spiked wastewater samples for 0 and 7 days at
various temperatures, pH and chlorine levels resulted in a matrix of
recovery data for the various PAH species. This data indicated that
the highest recovery for PAH in wastewater are obtained at a pH level
near 7, in the absence of any chlorine with the analysis being completed
as soon after collection as possible.
This report was submitted in fulfillment of Contract No. 68-03-2624
by Battelle Columbus Laboratories under the sponsorship of the U.S.
Environmental Protection Agency. This report covers a period from
November 1, 1977, to March 1, 1979, and work was completed as of
March 1, 1979.
iv
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CONTENTS
Foreward ill
Abstract iv
Figures vi
Tables '. . vii
1. Introduction 1
2. Objective 2
3. Technical Approach 3
4. Literature Review 4
5. Experimental Procedures 6
Analytical methods evaluation 6
Gas chromatography (GC) 6
High performance liquid chromatography (HPLC) 6
Solvent stability studies 7
Extraction studies 8
Preservation studies 8
LC clean-up studies 9
Wastewater studies . H
6. Results and Discussion 14
Analytical methods evaluation 14
Gas chromatography 14
High performance liquid chromatography ... 14
Sensitivity 19
Variance of detector response 23
Variance in retention times 23
Solvent stability studies 23
Extraction studies 22
Preservation studies 27
LC clean-up studies 31
Wastewater analysis 31
7. Summary and Recommendations 53
References 54
Appendix A 55
Appendix B 65
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FIGURES
Number Page
1 Alumina Lc clean-up scheme used for PAH 10
2 Silica Gel LC clean-up scheme used for PAH 12
3 GC capillary column separation of Group A PAH 16
4 GC capillary column separation of Group B PAH 17
5 Chromatogram of HPLC separation of 16 PNA standards . . 18
6 HPLC separation of 16 PAH standards on HC-ODS 21
7 HPLC separation of water effluent extract from plastic
industry (1st liter) 33
8 HPLC separation of water effluent extract from plastic
industry (2nd liter) 34
9 HPLC Chromatogram of spiked water effluent extract from
plastic industry 35
10 HPLC Chromatogram of spiked raw sewage 38
11 HPLC Chromatogram of unspiked raw sewage ....... 39
12 HPLC Chromatogram of spiked treated sewage 40
13 HPLC Chromatogram of unspiked treated sewage 41
14 HPLC Chromatogram of spiked flyash wash 44
15 HPLC Chromatogram of unspiked flyash wash 45
16 HPLC Chromatogram of spiked flyash wash settling pond . 47
17 HPLC Chromatogram of unspiked flyash wash settling pond 48
vi
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TABLES
Number Page
1 GC capillary column retention times for PAH on
30 meter SE-30 ". . 15
2 PAH detection limits 20
3 HPLC retention times for PAH on reverse phase columns. . 22
4 Variation in fluorescence detector response 24
5 Variance of retention time 25
6 Solvent stability studies ... 26
7 Extraction studies 28
8 Preservation studies 30
9 Preservation studies Data and ANOVA analysis 32
10 PNA recovery 31
11 Plastics industry wastewater 37
12 Raw sewage wastewater 42
13 Treated sewage wastewater 43
14 Flyash wash wastewater 46
15 Flyash settling pond wastewater 50
16 Average recoveries for PAHs from wastewater samples. . . 51
A-l High performance Liquid Chromatography of PAHs 63
vii
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SECTION 1
INTRODUCTION
The objective of this program was to establish and verify pro-
cedures for the analysis of 16 priority pollutant PAHs in complex
aqueous media. Data obtained during this study will be used to aid in
the selection of appropriate test procedures which will ultimately be
used by the EPA in monitoring water and wastewater pollutants. These
procedures should employ simple sample treatment as well as common lab-
oratory instrumental and analytical approaches.
Sixteen polynuclear aromatics were studied in this program with
regard to solvent stability, extraction efficiency, and preservation.
These 16 PAHs were: acenaphthene; benzo(ghi)perylene; fluorene; phen-
anthrene; dibenzo(ah)anthracene; indeno(1,2,3-cd)pyrene. These com-
pounds were combined into two groups for simultaneous study, in order
to simplify initial analyses.
Further studies were then conducted to evaluate the extraction and
analytical procedures that had been developed during this initial phase.
These studies were performed using wastewaters obtained from various
sources.
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SECTION 2
OBJECTIVE
The objective of this study was to develop an optimized analytical
method for the determination of 16 priority pollutant PAH in aqueous
media and to validate the method on a variety of aqueous effluents.
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SECTION 3
TECHNICAL APPROACH
The successful completion of this program involved the fulfillment
of certain directives set forth in the contract by EPA. An extensive
literature review was first conducted to evaluate the previous work in
the area. Subsequent work was directed toward determination and then
full evaluation of an appropriate measurement technique, which best
satisfied the requirements for sensitivity and selectivity, as well as
the considerations of sample cost, i.e. equipment, time, and training,
which would be needed for the method. The stability of the PAH compounds
in water miscible solvents and their instability in chlorinated and
unchlorinated buffered water at different pHs and storage temperatures
were studied over the prescribed time periods. Extraction efficiency of
two organic solvents was also studied for the standard compounds. The
remainder of the program involved the study of the sample preparation
and clean-up steps which would be necessary to eliminate sample inter-
ferences. The complete method was then applied to several representative
wastewater samples and an assessment was made of the precision and
accuracy of the complete procedure.
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SECTION 4
LITERATURE REVIEW
A literature review was conducted to establish the most attractive
approach to the analysis of PAH in water. Although there is a large
volume of literature on the subject, for the purposes of this, report
we will present only a few of the most important references. Our dis-
cussions will focus on the methodologies used for preservation, extrac-
tion, cleanup, and determination of the PAH's.
Preservation of PAH in water has not apparently been studied in
detail in any single investigation. In one report the fate of PAH in
water was studied, with special emphasis on the effect of UV light (1).
In this study benz(a)anthracene and benzo(a)pyrene were used as model
compounds since they are among the most reactive PAH's. The conclusions
reached by these authors include:
The PAH's are primarily adsorbed on particulate in natural
water samples.
The PAH's are degraded to quinones when exposed to UV light
above a certain threshold intensity .
The degradation of PAH by UV light is not significantly
affected by pH or ionic strength.
A second study (2) has evaluated the effect of chlorination on
PAH stability. This study concluded that PAH's can be degraded by
added chlorine, especially at low pH and at elevated temperature.
At low temperature and neutral or high pH the effect of chlorination
is greatly reduced.
There are several methods reported for extraction of PAH from water
including solvent extraction (1-7), polyurethane foams (8), and XAD-2
resin (9). In general good recoveries have been obtained using all of
these techniques, primarily because of the extremely hydrophobic nature
of the PAH's. Solvents used for extraction include benzene (1),
isooctane (3), and methylene chloride (2,4,6,7). At least one report
(7) concluded methylene chloride was the solvent of choice because
of its good extraction properties and ease of work up.
There have been two methods reported for cleanup of PAH extracts:
a) solvent-solvent partition (4,7) and b) adsorption (column or thin
layer) chromatography (3,4,8). One study (4) compared the recoveries
using these two techniques and found them to be equivalent. The solvent
partitioning method involves 1) extraction of the PAH's into isooctane,
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partitioning of PAH's into DMSO or nitromethane, and 3) dilution of
the extract with water and back extraction into isoctane. Absorption
chromatography has been performed using alumina (3), acetylated cellulose
(8), and silica gel (4). Silica gel and alumina appear to be the best
for this application since they are readily available in pure form and
can be produced with uniform activity.
For the determination of PAH's GC/FID (1,2,8,9), GCMS (3,4,6),
TLC (7,10), and HPLC (11,12) have been employed. TLC does not appear
to have sufficient resolution to separate all sixteen if the priority
pollutant PAH's and GCMS is considered to expensive for the purposes
of this study. Therefore the candidate methods appear to be GC/FID
and HPLC with a fluorescence detector. Although no direct comparisions
of these two techniques for PAH analysis were found it appears that HPLC
with fluorescence detection is more sensitive than GC/FID whereas
GC/FID may be somewhat more readily available. The largest unknown
about both techniques is the degree of resolution which can be achieved
and whether or not all sixteen PAH's can be resolved. Separation of
all sixteen PAH's in a single chromatographic run has not been previously
reported. A comparison of some of the characteriztics of these two
techniques is given below.
GC
Use of capillary column 20-30 meters
will give 50-60K plates
Commercial coatings limited
FID-universal detector
May require extensive sample clean-up
* Sensitivity advantage for lower
MW compounds
FID-linear response over wide range
of concentrations
* Possible one analysis for all PAHs
PAH identification by retention time
only (with ready facility for GC-MS)
* Automation easy
Investment low ($10K)
Sample destroyed
HPLC
Packed columns (reverse or
normal phase)
Commercial coating limited
UV or Fluorescence detector
selective
May require less extensive
clean-up
Greater sensitivity for selected
compounds UV and/or fluorescence
UV-linear response over wide
range of concentrations
* May require several fraction-
ations for all PAHs
PAH identification by retention
time plus UV absorbance or
fluorescence
Automation more difficult
Investment medium ($30K)
Sample not destroyed
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SECTION 5
EXPERIMENTAL PROCEDURES
ANALYTICAL METHODS EVALUATION
The objective of this phase of the study was to ascertain which of
the two chromatographic techniques (GC or HPLC) is most appropriate for
the determination of the sixteen priority pollutant PAH's.
Gas Chromatography (GC)
Glass capillary gas chromatography was investigated as an analytical
technique for the 16 PAH's. Various glass capillary columns were in-
vestigated using the following GC conditions:
Instrument - Hewlett-Packard 5730A
Detector - FID
Detection temperature - 300°C
Injector temperature - 300°C
Injection mode - 2 pi split 10:1
Column temperature - Group A - 2 minutes isothermal at
40°C and then 40° - 260°C at 8°/min.
Group B - 2 minutes isothermal at
80°C and then 80° - 260°C at 8°/min.
A spectra-Physics 4000 chromatographic data system was used to determine
peak areas.
Detection limits for each compound were determined, based on a
signal to noise ratio of 10:1.
High Performance Liquid Chromatography (HPLC)
Both normal and reversed phase forms of HPLC were investigated for
the separation of PAH's. For detection of the PAH's both fluorescence
(at various excitation and emission wavelength settings) and UV (fixed
at 254 nm) were evaluated.
Stationary phases were packed in 4.6 m.m. I.D. x 25 cm stainless
steel columns and were either purchased commerically or slurry packed
usitag conventional techniques.
Solvents were filtered through .22 viM Millipore filters and degassed by
boiling for a few minutes prior to use on the HPLC. The chromatographic
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apparatus employed was as follows:
Solvent delivery system - Altex 420 microprocessor
controlling dual Altex 100A
pumps.
Injector - Rheodyne 7120 with a 20 vil loop.
Detectors - Fluorescence - Schoeffel FS970
UV-LDC UV-3 at 254 nm.
Data system - Hewlett Packard Model 3385A.
SOLVENT STABILITY STUDIES
The purpose of this phase of the study was to determine which water
miscible organic solvents are Best for the preparation of standard
solutions of the PAH's for QA/QC purposes.
Solvent stability studies were conducted using DMSO and acetone as
the solvents of choice. These solvents were selected because they are
both water miscible and readily dissolve PAH.
Four ml of acetone and DMSO solutions of PAHs at a concentration of
20 ppm were sealed in glass ampules. The PAHs were separated into 2
groups to facilitate the separation and quantitative analysis. These
groups were: (A) naphthalene, anthracene, pyrene, chrysene, acenaphthene
and dibenzo(ah)anthracene; and (B) phenanthrene, fluorene, benz(a) an-
thracene, fluoranthene, benz(a)pyrene, and acenaphthylene.
Benzo (k+b)fluoranthenes were not included in these studies due to
an insufficient quantity of these standards. Because of supplier back-
log, indeno(l,2,3-cd)pyrene and benzo(ghi)perylene were not incorporated
into these initial groups and were analyzed independently.
The sealed ampules were kept in the dark, at room temperature for
30, 60, and 90 day time periods. Following chromatographic calibration,
3 ampules from each group were opened at the appropriate time interval.
Analysis of PAHs in acetone was conducted using capillary GC using the ..
conditions described previously.
Because of solvent tailing, analysis of PAHs in DMSO was performed by
HPLC using the following conditions.
Column - 250 x 4.6 mm Spherisorb ODS 5y particle diameter
Gradient - 50% acetonitrile in water to 100% in Acetonitrile in .
50 minutes.
Flow - 1.0 ml/min
Solvent delivery system - Altex 420 microprocessor controlling
dual Altex 1QOA pumps.
Injector - Rheodyne 7120 with a 20 yl loop
Detector - UV-LDC UV-3 at 254 nm
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Quantisation for both GC and HPLC was conducted by peak area
integration using either a Spectra Physics 4000 or Hewlett-Packard
8085A integrator. Calibration curves were prepared by appropriate di-
lution of a standard stock solution which were run at suitable sen-
sitivity levels. Peak areas of interest were compared to the calibration
curves and reported as percent recovery.
EXTRACTION STUDIES
The purpose of this phase of the study was to determine the extrac-
tion efficiencies of the various PAH's under a variety of conditions,
in order to select an optimized extraction protocol.
Extraction studies were conducted to evaluate the extraction ef-
ficiencies of methylene chloride and 15 percent methylene chloride in
hexane for PAH's in water at pHs 2,7, and 10.
One ml of standard solution in acetone was added to 500 ml of ap-
propriately buffered water to yield an approximate concentration of
40 ppb for each of the PAH in a 1000 ml separatory funnel. Certified
pH 7 and pH 10 buffers were purchased from VWR. The buffers used are
as follows:
pH 2 - NaH2P04 in phosphoric acid, 0.05 M Ionic Strength
pH 7 - sodium and potassium phosphate, 0.05 M Ionic Strength
pH 10 - sodium borate and sodium carbonate, 0.05J1 Ionic Strength
PAH's were extracted three times using 30 ml portions of the ap-
propriate solvent. The extract was then dried with MgS04, filtered, and
the MgS04 washed with an additional 30 ml of solvent. The resultant
extract was reduced in volume to one ml using micro Kuderna-Danish
evaporation. Extractions were done in triplicate for pH 2 and 10 and
quadruplicity for pH 7.
Triplicate analysis on each of the above extracts was conducted
using a capillary column GC equipped with a Hewlett-Packard 7671A auto-
matic sampler.
Benzo(b)fluoranthene was not included in these studies due to an
insufficient quantity of this standard.
PRESERVATION STUDIES
The purpose of this phase of the study was to examine the effects
of sample storage under a variety of pH, temperature, and oxidant con-
ditions in order to develop protocols for sampling and sample storage.
Preservation studies were conducted to evaluate the stability of
PAH's in water for selected conditions. These conditions were: pH 2,
7, and 10; chlorine 0 and 2 ppm; temperature, 'ambient and 4°C. The
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three pH levels were achieved using the buffers described in the £x-
traction Section. The two ppm chlorine level was achieved by spiking
the sample with 2 ml. of a solution containing "1500 ppm of calcium
hypochlorite.
- - »
One ml of standard PAH solution in acetone was added to 500 ml dis-
tilled water, (to achieve a concentration of 40 ppb for each PAH) which
had previously been buffered and/or chlorinated in accordance with the
above matrix. Each matrix was performed in two groups as in the previous
studies with the addition of benzo(k)fluoranthene and indeno(l,2,3-cd)-
pyrene to Group A and benzo(ghi)perylene to Group B. All matrix weries
were stored in the dark for a period of 7 days; one series at-ambient
temperature, the other at 4°C. Each condition was run in duplicate.
Following the 7 day storage period, each solution was extracted
with methylene chloride as described in the Extraction Studies section.
The storage bottle was also rinsed with each 30 ml portion of methylene
chloride prior to being used for extraction in the separatory funnel.
Each extract was then dried, and reduced in volume as described above.
Duplicate injections were made onto a capillary column GC equipped with
a Hewlett-Packard 7671A automatic sampler. Quantitation was performed
by peak area integration using a Spectra Physics 4000 integrator. Cal-
ibrations were performed every 24 hours with periodic calibration checks
at four run intervals.
LC CLEAN-UP STUDIES
The purpose of this phase of the study was to evaluate several
clean-up techniques in order to determine which technique gives best
recoveries for the various PAH's.
Recoveries of PAH's from two LC clean-up procedures were investigated.
Each LC separation scheme was performed by applying 0.5 ml of a 200 ppm
cyclohexane solution of representative PAH's. The PAH's in solution
were chosen on the basis of representative ring number and relative
elution volumes. These PAH's were: naphthalene, anthracene, fluoran-
thene, chrysene, and dibenz(a,h)anthracene.
The alumina LC column was prepared by placing 10 g of alumina
(3% H20 w/w) in a 25 x 1 cm column, and wetting with 15 ml pentane. As
the pentane reached the top of the alumina, 3/4" of Na^SO, was added.
The column was washed with 20 ml of methylene chloride ana then recon-
ditioned with 20 ml of pentane. The PAH extract is then placed on the
head of the column and eluted with 25 ml of pentane and 25 ml of methylene
chloride. The latter methylene chloride fraction contained the PAH's.
The LC scheme is presented in Figure 1.
The silica gel LC column was prepared by slurry packing 10 g of
silica gel (Davidson Grade 923, 100-200 mesh) with methylene chloride
into a 1 x 25 cm LC column. The column is then washed with 40 ml of
pentane. The sample extract is then placed on the head of the silica
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10 g ALUMINA-DRY PACKED 25 cm COLUMN
15 ml PENTANE 3/4"
20 ml
CH2CL2
20 ml PENTANE
?LE
25 ml PENTANE
25 ml CH2CL2 (PAH FRACTION)
Figure 1. Alumina LC clean-up scheme used for PAH.
10
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gel and eluted with 25 ml of pentane and 25 ml of 40% methylene chloride
in pentane. This latter fraction contained the PAH's of interest. This
LC scheme is presented in Figure 2. After allowing the first 25 ml of
pentane to pass through the LC column, successive 10 ml fractions were
collected and analyzed by HPLC using UV detection at 254 nm. No volume
reduction was performed since the initial concentration of PNAs was
chosen to eliminate this step.
WASTEWATER STUDIES
The purpose of this phase of the study was to evaluate the analyti-
cal protocols, developed on the basis of information collected in
earlier phases of this study, on actual wastewater samples, and to make
modifications as required.
Based on our extraction efficiency studies the following procedure
was initially employed for the analysis of actual wastewater samples
as supplied by EPA as well as samples collected by our laboratories.
Extract immediately three 1 liter aliquots of each waste-
water using 3 x 60 ml methylene chloride.
Spike six 1 liter aliquots of each wastewater with PAH com-
pounds prior to extraction, with 3 aliquots being stored
for 7 days at 4°C.
Pour extract through drying column containing sodium sulfate.
Reduce volume of extract to 1.0 ml using Kuderna - Danish
concentrator apparatus.
Solvent exchange with cyclohexane and perform silica gel
clean-up technique (Figure 2).
Concentrate the PAH fraction to 2-3 ml using Kuderna- -Danish
concentrator apparatus.
Solvent exchange with acetonitrile and adjust the extract
volume to 1.0 ml.
Analyze by injecting 5 yl into the HPLC equipped with an
HC-ODS column and fluorescence detector at Xex 280 and
Xem >389.
Quantitation was performed by comparing the peak areas for the
individual PAH species to those of a standard made up to the concentra-
tion representing 100% recovery.
Following discussions with EPA personnel the extraction procedure
was modified so that all concentration steps were performed using a
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10 g SILICA GEL-SLURRY PACKED IN 1x25 cm COLUMN
40 ml, PENTANE
SAMPLE ADDED
25 ml PENTANE
25 ml 40% CH2C12 IN PENTANE
PAH FRACTION
Figure 2. Silica Gel LC clean-up scheme used for PAH.
12
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hot water bath rather than a tube heater. Also a 254 nm UV detector
was coupled to the fluorescence detector to achieve lower detection
limits for naphthalene and acenaphthylene. The revised procedures are
described in detail in Appendix A.
The following water samples were selected for analysis:
A plastics manufacturing industry (Samples supplied by
EPA-Source Unknown).
Raw municipal sewage water (Columbus, Ohio).
Treated municipal sewage water prior to chlorination
(Columbus, Ohio).
Flyash Wash from coal fired power plant (Columbus, Ohio).
Flyash settling pond prior to runoff (Columbus, Ohio).
All wastewater samples were collected by our laboratories except
the plastics manufacturing industry wastewater which was supplied by
EPA.
Extractions were conducted immediately on three 1 liter aliquots
of each wastewater as received. In addition, six 1 liter aliquots
were spiked with PAH at a level at least 5 times background prior to
extraction, 3 aliquots being stored for 7 days at 4°C.
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SECTION 6
RESULTS AND DISCUSSION
ANALYTICAL METHODS EVALUATION
Gas Chromatography
Experimental GC conditions were based on our considerable ex-
perience with analysis of PAHs utilizing capillary columns. Although
PAH separation in this study was achieved using an SE-30 capillary
column, equal resolution has been achieved using capillary columns
coated with SP-2100, OV-101, and SE-54. Examples of these separations
are shown in Figures 3 and 4, with retention times given in Table 1.
The conditions used for GC analysis are as follows:
30 meter SE-30 glass capillary
FID detector - 300°C
Injector - 200°C
2 yl split injection 10:1 at 40°C Group A: 80°C Group B
Isothermal for 2 min then programmed 8°C/min to 260°C
Instrument detection limits for the various PAHs, using capillary
GC, are listed in Table 3. Due to the rather poor peak shape, and re-
sulting poor detection limits and resolution, for the high molecular
weight PAHs (e.g. indeno-pyrene) capillary column GC was not pursued
further as an analytical approach. However, use of specially coated
capillary columns could reduce these problems and for the lower
molecular PNAs (e.g. napthalene, anthracene, etc.) GC is probably a
very good approach.
High Performance Liquid Chromatography
Normal and reverse phase HPLC were investigated for the separation
of the PAHs of interest. There is much in the literature describing the
separation of a limited number of PAH using reverse phase HPLC (11,12);
however, no references could be located on the separation of all 16 PAH
of interest using a single stationary phase.
Initial HPLC reverse phase separations were conducted using a
250 x 4.6 mm Spherisorb ODS 5 y column using a 75-100% MeOH in water
gradient elution in 30 min. This gradient gave acceptable resolution
for PAHs in Group A but failed to separate fluorene and phenanthrene
in Group B. Although using a longer gradient elution time (i.e. 40 min)
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TABLE 1. GC CAPILLARY COLUMN RETENTION TIMES FOR
PAH ON 30 METER SE-30
Group A Group B
Compound RT (min) * Compound RT (min) *
Napthalene 9.53 Acenaphthylene 9.65
Acenaphthene 14.30 Fluorene 11.51
Anthracene 20.70 Phenanthrene 13.95
Pyrene 21.98 Fluoranthene 17.21
Cyrysene 25.81 Benz(a)anthracene 21.40
Benz(a)fluoranthene 30.93 Benz(a)pyrene 28.37
Indeno(1,2,3-cd)pyrene 43.49 Benz(ghi)perylene 42.10
Dibenz(a,h)anthracene 43.84
* Elutlon conditions for Group A and B were different so that
retention times cannot be directly compared.
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Anthracene
NJ
o
Pyrene
Chrysene
Benz(k)fluoranthene
Indeno(l,2,3-cd)pyrene
Dlbenz(a,h)anthracene
Figure 3.
GC capillary column separation of Group A PAH.
16
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10..
Acenaphthylene
Fluorene
Phenanthrene
Fluoranthene
20
Benz(a)anthracene
I
30
Benz(a)pyrene
Benz(ghi)perylene
MIN
Figure 4. GC capillary column separation of Group B PAH.
17
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B(a)P
IP
DiB(ah)A/B(ghi)perylene
Figure 5. Chromatogram of HPLC separation of 16 PNA standards.
18
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achieved this separation, acetonitrile in water proved to be more selec-
tive and gave a flatter base line.
Moderate success on separation of all 16 PAHs was achieved using a
gradient elution of 50% acetonitrile to 100% acetonitrile in 50 minutes.
An example of this separation is shown in Figure 5. However, the impor-
tant isomer pair, benz(a)anthracene/chrysene as well as dibenz(a,h)-
anthracene/benz(ghi)perylene, are not completely resolved. This latter
pair could be resolved using an isocratic step program, but these con-
ditions still left benz(a)anthracene and chrysene unresolved.
Several normal phases for the separation of PAH were investigated.
These phases included Lichrosorb Si60, alumina, and Lichrosorb NH2-
Hexane was the mobile phase in all cases. The results of these studies
indicated that there were not enough efficiency or selectivity to separate
all 16 PAH.
Further investigation of various reverse phase columns indicated
that the separation of all 16 PAHs could be achieved using a Perkin-
Elmer 10 v, 0.26 x 25 cm HC-ODS column. Figure 6 shows the separation
of all 16 consent decree PAHs using a single stationary p"b:as. The con-
ditions used for this separation were 40% acetonitrile in water isocratic
for 5 minutes followed by a gradient elution from 40-100% acetonitrile
in 25 minutes at a flow rate of 0.5 ml/min. Fluorescence detection was
used with excitation at Xex 280 nm and a cut off filter of Xem >389 nm.
PAH retention times using the Spherisorb and HC-ODS columns are given in
Table 3.
Sensitivity
Since all 16 PAH compounds can be resolved using the Perkin-Elmer
HC-ODS reverse phase column, it is possible to optimize a fluorescence
or UV detector for the best signal to noise ratio for each compound.
Fluorescence at Xex 280 nm, Xem >389 nm gives good sensitivity for most
of the larger ring PAH compounds but does not have good sensitivity for
the smaller ring compounds like naphthalene and acenaphthylene.
Although these studies were carried out using fluorescence at Xex
280 nm, Xem >389 nm, samples were spiked at levels approximately 25 times
the minimum detection limit (i.e. 25 x the detection limit in ng on
column assuming a 1000:1 concentration factor and 5 microliter injection
volume), making the choice of wavelength setting of little consequence.
However, for increased sensitivity for the smaller ring PAH compounds a
UV detector at 254 nm can be placed in series with the fluorescence de-
tector. Sensitivities obtained using these wavelengths are listed in
Table 2. Detector response was found to be linear to 25 x these sensi-
tivities in terms of nanograms on column which covered the range in which
subsequent samples were spiked.
Details of the recommended analytical procedure for the analysis of
PAH in industrial wastewater, based on the results of these studies is
19
-------
TABLE 2. PAH DETECTION LIMITS
Capillary GC
FID
Compound 10:1 split
(ng)
Naphthalene
Acenaphthylene
Acenaphthene
Fluor ene
Phenanthrene
Anthracene
Fluoranthene
Pyrene
Benz (a) anthracene
Chrysene
Benz (b) f luoranthene
Benz (k) f luoranthene
Benz (a) pyrene
Dibenz (a, h) anthracene
Benz(ghi)perylene
Indeno (1 , 2 , 3-cd) pyrene
1.0
1.5
.5
2.0
2.0
2.0
1.0
2.0
2.0
4.0
6.0
3.0
10.0
10.0
uv
254
(ng)
2.5
5.0
3.0
0.5
0.25
0.10
0.50
0.10
0.20
0.20
1.0
0.30
0.25
1.0
0.75
0.30
Fluorescence
Xex 280, Xem >389
" (ng)
20.0
100.0
4.0
2.0
1.2
1.5
0.05
0.05
0.04
0.5
0.04
0.04
0.04
0.08
0.2
0.1
Minimum detectable quantity in nanograms injected at a signal to noise
ratio of 10:1
20
-------
S9
i
£
e ' _.
5
|
m
Napthalene
Acenaphthylene
Acenaphthene
3
F
r; = i Phenanthrene
Anthracene
Fluorene
Ftuoranthene
Pyrene
B(a)A
B(b)F
Chrysene
B(k)F
B(a)P
DiB(ah)A
IP
B(ghi)perylene
c
Figure 6. HPLC separation of 16 PAH standards on HC-ODS.
21
-------
TABLE 3. HPLC RETENTION TIMES FOR PAH ON
REVERSE PHASE COLUMNS
Column: Spherisorb ODS
4.6 x 250 nm
Gradient: Acetonitrile/H20
50%-lOOZ 50 min
Flow: 1 ml/min
Column: Perkin-Elmer HC-ODS
2.6 x 250 mm
Gradient: Acentonitrile/H20 in
402-5 min
402-100Z-25 min
Flow: 0.5 ml/min
Retention time
Retention time
Compound
Naphthalene
Acenaphthylene
Acenaphthene
Fluorene
Phenanthrene
Anthracene
Fluoranthene
Pyrene
Benz (a) anthracene
Chrysene
Benz (b) f luoranthene
Benz (k) f luoranthene
Benz (a) pyrene
Dibenz ( a , h) anthracene
Benz (ghi) pery lene
Indeno(l, 2, 3-cd) pyrene
(Minutes)
11.45
13.00
14.91
15.21
16.66
17.46
19.58
20.41
23.63
23.63
27.56
28.01
28.85
33.06
33.06
31.30
(Minutes)
16.17
18.10
20.14
20.89
22.32
23.78
25.00
25.94
29.26
30.14
32.44
33.91
34.95
37.06
37.82
39.21
22
-------
given in Appendix A. Excitation, emission, and adsorption spectra for
the 16 PAH compounds of interest are shown in Appendix B for use as
reference spectra as well as use in evaluation for determining optimum
detection wavelengths.
Variance of Detector Response
The variance of the fluorescence detector response was checked to
determine if there was any affect on detector response (e.g. dissolved
oxygen in the HPLC mobile phases) over a period of time.
Spectral grade acetonitrile and high purity water were passed through
a .22 yM Millipore filter prior to boiling each mobile phase for a period
of 5 minutes. After allowing them to cool, each mobile phase was poured
into its respective 'container and the HC-ODS column equilibrated by going
through one gradient elution with no injection. Following equilibration,
a normal separation was performed by injecting a PAH solution. A further
PAH standard separation was performed at the end of the day. These
standard analyses continued for a period of 3 days which is approximately
the duration before the mobile phases need to be replenished. The peak
areas for each PAH component were averaged for the six runs and the stan-
dard deviation from the average determined. The results which are listed
in Table 4 indicate that over a period of 3 days, there is only a slight
variance in the detector response ; and since recoveries are calcu-
lated using daily calibrations, these variances would not substantially
affect analytical results.
Variance in Retention Times
Absolute retention times for individual PAH compounds was found to
vary somewhat day to day. Although a microprocessor was used to control
the entire gradient and reconditioning of the column prior to injection,
these variances nevertheless occurred.
The initial gradient concentration of 40% acetonitrile in water was
held for a period of 10 minutes to obtain column equilibrium prior to in-
jection. Holding this concentration :'for 30 minutes before injection
yielded no decrease in the variance. Thus, for shorter total analysis
time a 10 minute reconditioning time was used prior to sample injection.
prior to sample injection.
The variances which were obtained on the six PAH standard solutions
over a period of 3 days are listed in Table 5.
SOLVENT STABILITY STUDIES
Results of the solvent stability studies are shown in Table 6.
An ANOVA analysis of this data indicates that acetone and DMSO are
virtually equally suitable solvents for the PAHs studied for a time
period of 90 days. ANOVA analysis of the data indicated a significant
difference favoring acetone over DMSO for fluoranthene however examination
23
-------
TABLE 4. VARIATION IN FLUORESCENCE DETECTOR RESPONSE
*
Relative percent standard deviation
Compound Xex 280/Xem >389
% (n = 6)*
Naphthalene ' ±3
Acenaphthylene ±3
Acenaphthene ±3
Fluorene ±3
Phenanthrene ±2
Anthracene ±1
Fluoranthene ±2
Pyrene ±2
Benz(a)anthracene ±1
Chrysene ±1
Benz(b)fluoranthene ±1
Benz(k)fluoranthene ±3
Benz(a)pyrene ±7
Dibenz(a,h)anthracene ±2
Benz(ghi)perylene ±5
Indeno(l,2,3-cd)pyrene ±2
* Over a 3 day period.
24
-------
TABLES. VARIANCE OF RETENTION TIME
Compound Average retention time Standard deviation
Time (min) (n « 6)
Time (min)
Naphthalene
Acenaphthylene
Acenaphthene
Fluorene
Phenanthrene
Anthracene
Fluoranthene
Pyrene
Benz (a) anthracene
Chrysene
Benz (b) f luoranthene
Benz (k) f luoranthene
Benz(a)pyrene
Dibenz (a , h) anthracene
Benz (ghi) perylene
Indeno (1,2, 3-cd) pyrene
16.17
18.10
20.14
20.89
22.32
23.78
25.00
25.94
29.26
30.14
32.44
33.91
34.95
37.06
37.82
39.21
±0.31
±0.27
±0.50
±0.24
±0.24
±0.23
±0.24
±0.24
±0.23
±0.22
±0.20
±0.19
±0.20
±0.18
±0.19
±0.18
25
-------
TABLE 6. SOLVENT STABILITY STUDIES
ON
Compound
Naphthalene
Acenaphthene
Anthracene
Pyrene
Chrysene
Indeno (1,2, 3-cd) pyrene
Dibenz(a,h)anthracene
Acenaphthylene
Fluorene
Phenanthrene
Fluor an thene
Benz( a) anthracene
Benz (a) pyrene
Benz(ghi)perylene
0 day
100+5*
98+6
100i0.6
100+0 . 6
100+0
101+3
101+2
99+2
100+1
100+1
100+1
100 ±0
10011
100+4
30 day
99±5
97+7
97±0.6
99+2
97±0.6
99+2
99 ±4
101+3
101+2
100±3
98+3
100+3
103+5
101±3
DMSO
60 day
101±2
104+5
99+1
99±0.6
99±0
97±0.6
101±3
98+2
101 ±2
98+2
95 ±2
96±2
95+2
95±2
90 day
95+2
100+3
97±0.6
100±2
99±2
99+3
100±3
102±2
102+1
102+1
100+1
102±0.6
103+3
101+3
Average
99+4
.100+5
98±2
100+1
99±1
99+2
100+2
100+3
101±2
100+2
98+3
100±3
100+4
99+3
0 day
100+1
10010.6
100+2
99i2
100+4
9912
98+3
10014
10017
100+3
100+3
10014
100+5
10013
30 day
96+9
95 16
9815
9915
99+6
99 ±2
9516
10014
10212
103 ±4
101+2
10313
89110
99±2
Acetone
60 day
99+2
98 ±2
96i2
100+2
10012
97+6
9519
103+2
10411
10211
100+0.6
98+5
102±2
10013
90 day
10411
10213
100+5
9813
10410.6
9815
9817
10412
10513
10113
10114
95±2
97 ±8
9912
Average
100l4
9914
9814
9913
101 14
9814
9716
10213
10314
10213
10112
99 ±5
97 ±8
10012
* Average percent recovery ± standard deviation of triplicate analyses.
-------
of the data shows this to be an artifact due to the high precision of
the assay.
EXTRACTION STUDIES
Extraction studies were conducted to evaluate the extraction ef-
ficiencies of methylene chloride and 15% methylene chloride in hexane
for PAH in water at pH 2, 7, and 10. Results of these analyses are shown
in Table 7. ANOVA analysis of the extraction data indicates, as shown
in Table 7, that methylene chloride is a significantly better extraction
solvent for virtually all of the PAHs. For several of the PAHs, anthra-
cene, pyrene, benz(b)fluoranthene, indeno(l,2,3-cd) pyrene, and dibenz-
(a,h) anthracene, pH also had a significant effect, according to the
ANOVA analysis. Inspection of the data indicates that in these cases
pH 10 gave much lower recoveries than pH 7 or pH 2.
On the basis of these data it is apparent that methylene chloride is
the extraction solvent of choice, and that while pH 10 can lead to lower
recoveries, pH 7 and pH 2 generally give good recoveries.
PRESERVATION STUDIES
Preservation studies were conducted to evaluate the stability of PAH
in water under selected conditions. These conditions include pH 2, 7,
and 10; 0 and 2 ppm chloride; and room temperature (RT) and 4°C.
Distilled water which had previously been buffered and/or chlorinated
in accordance with the analytical matrix was spiked with PAH standard
solution and stored for 7 days. Each storage condition was run in dup-
licate.
Following the 7 day storage each solution was extracted, concentrated
to 1 ml using a Kuderna-Danish evaporator, and analyzed.
The results of these analyses which are corrected for extraction
efficiencies are given in Table 8. These data have been presented in
alternate format, along with the ANOVA results in Table 9.
The most curious results of these analysis is the zero percent re-
covery of naphthalene at RT, pH 7, 0 ppm Cl, and acenaphthylene at RT,
pH 2, and 2 ppm Cl respectively. These analysis were repeated with fresh
stock solution and stored for 7 days under identical conditions. Results
of these analysis were identical, i.e., 0 percent recovery for these two
compounds. It is possible that chlorine is degrading the acenaphthylene
in some way but there seems to be no plausible explanation for the naph-
thylene results.
27
-------
TABLE 7. EXTRACTION STUDIES
K>
oo
15% MeCl2/Hexane
Naphthalene
Acenaphthene
Anthracene
Pyrene
Chrysene
Benz (k) f luoranthene
Indeno (1 , 2 , 3-cd) pyrene
Dibenz (a , h) anthracene
Acenaphyhylene
Fluorene
Phenanthrene
Benz (a) anthracene
Benz (a) pyrene
Benz (ghi) perylene
Fluoranthene
pH 2
66+8*
8119
87±10
95116
72+31
63125
96132
64120
9119
9718
106+15
9717
72+6
57113
102+11 ,
pH 7
66111
76113
77+12
88H4
79110
64+18
88+17
60110
99114
107122
103+25
83+14
74116
61122
97117
pH 10
5719
70+9
66+9
75111
6718
59+7
56+19
41111
93+14
11018
119112
104112
91+24
6712
129129
MeCl2
pH 2
102±5
100±5
100±7
100+3
103+10
104H4
106+13
97+11
81+2
9110.5
9012
9011
8612
8912
89+1
pH 7
100+7
99+9
100±7
10014
100+10
10115
100+4
99+2
7112
8012
8813
92+6
101+7
9116
8914
ANOVA Analysi;
pH 10 Solvent pH
89l8 +
101l8 +
91+4 +
84i2 0
78+1 +
63+4 +
64+11 0
59+6 +
69+1 +
79+3 +
88+3 +
9013 +
8917 +
88+4 +
9111 0
0
0
+
+
0
+
+
+
0
0
0
0
0
0
0
* Average percent recovery + standard deviation for triplicate or quadruplicate analyses.
+ Statistically significant differences at the 95% confidence level.
0 No significant difference at the 95% confidence level.
-------
From the data presented in Table 9 it is obvious that added chlorine
reduces stability to some extent, although this is highly dependant on
the particular compound and the pH level, as will be discussed later.
In general stability was lower at RT than at 4°C although for benzo(k)-
fluoranthene, indeno(1,2,3-cd)pyrene, and dibenz(a,h)anthracene room
temperature gave slightly better stability at RT. The effect of pH on
stability varied from compound to compound, although normally stability
increased in the order pH2
-------
TABLE 8. PRESERVATION STUDIES
o
Naphthalene
Acenaphthene
Anthracene
Pyrene
Chrysene
Benz (k) f luoran thene
Indeno (1,2, 3-cd ) py r ene
Dibenz (a , h) anthracene
Acenaphthylene
Fluorene
Phenanthrene
Fluoranthene
Benz (a) anthracene
Benz(a)pyrene
Benz(ghi)perylene
' Average
RT
0 Cl
77±5*
86±5
8317
85±12
63±12
80±14
77±13
73111
74±2
73±2
7012
61±5
61±1
44110
94±6
73
RT
2 Cl
89±9
79±7
75±13
86±6
88±10
91114
88113
88111
0
6411
5912
6011
911
2613
6312
64
pH
4°C
0 Cl
8119
9019
8518
9115
79110
80114
75113
75111
7512
8010.5
7315
6711
7411
11215
10619
78
2
4°C
2 Cl
6115
5415
3917
5813
59110
54114
46113
61111
513
6912
6017
6412
1011
2614
94113
50
RT
0 Cl
0
6919
2317
8414
84111
6917
75116
7419
7312
7612
7213
6114
73112
8417
8616
67
RT
2 Cl
9317
6619
2217
7917
53110
8215
8514
78116
1712
5012
6914
4514
5816
3517
8416
61
PH
4°C
0 Cl
8917
9719
8817
8616
83110
7315
7814
7012
7312
6812
6913
6114
6516
8617
8819
78
7
4°C
2 Cl
9718
10019
4517
9714
77110
6516
7314
6018
6812
7313
7213
6514
6416
4617
8816
73
RT
0 Cl
10418
10218
10014
11114
10913
12514
142111
124118
7811
7013
6613
6412
7413
11117
11015
95
RT
2 Cl
8318
7718
2514
8613
9111
10514
117111
8816
5211
4813
4513
4412
5015
7217
5715
69
PH
4°C
0 Cl
11118
10218
10014
11112
10511
12116
133111
10516
7214
5716
5713
5512
6113
8919
88110
91
10
4'C
2 Cl
10118
98111
3814
93110
95113
10314
102112
9018
8414
71110
6113
5912
6313
8917
8418
82
* Average recovery 1 standard deviation for duplicate samples.
-------
LC CLEAN-UP STUDIES
The percent recoveries of the representative PAHs from each LC
separation scheme are listed in Table 10 below.
TABLE 10. PNA RECOVERY
Silica Alumina
% Recovery Total % Recovery Total
Elution Volume*
Naphthalene
Anthracene
Fluoranthene
Chrysene
Dibenz(a.h)
anthracene
10
<0.5
0.5
0.5
1.0
<0.5
20
93.0
10A.5
96.5
97.0
92.0
30
<0.5
<0.5
<0.5
<0.5
<0.5
93
105
97
98
92
10
100
86
78
38
0.5
20
<0.5
13.0
18.0
53.0
83.0
30
<0.5
<0.5
<0.5
<0.5
<0.5
100
99
96
91
83
*. 40 percent methylene chloride in hexane.i
From the above data, it is apparent that either LC separation
scheme would be suitable as a clean-up procedure. However, silica gel
gave somewhat higher recoveries for the larger molecular weight com-
ponents as well as exhibiting a considerable faster flow rate during
the separation procedure. Thus, the use of silica gel was favored over
alumina as the stationary phase for these clean-up procedures. A 25 ml
elution volume was considered to be optimum, based on this data.
WASTEWATER ANALYSIS
As stated in the Experimental Section, five water samples were
selected for analysis using the method outlined. Analysis of the plastic
industry wastewater and raw sewage were conducted using the Spherisorb
ODS column while subsequent wastewaters were analyzed using the Perkin-
Elmer HC-ODS column.
Initial wastewater analysis were conducted by HPLC using a Spher-
isorb ODS reverse phase column and selective fluorescent wavelength de-
tection based on the work of Das and Thomas (12). Quantitation of benz-
(a)anthracene/chrysene using this method involves the use of the ratio
of the response at Xex 250 and Xem >370 to the response at Xex 280 and
>389.
Analysis of the plastic industry wastewater indicated this source to
have a large number of components as well as having a rather high
fluorescent background level. A chromatogram illustrating this high
background is shown in Figure 7. Triplicate injection of a single
sample extract gave good repeatability; however, comparison of different
aliquots from the same sample gave erratic peaks which made quantitation
31
-------
TABLE 9. PRESERVATION STUDIES DATA AND ANOVA ANALYSIS
K)
Compound
Naphthalene
Acenaphthene
Anthracene
Pyrene
Chrysene
Benzo (k) f luor anthene
Indeno (1,2, 3-cd) pyrene
Dibenz (ah) anthracene
Acenaphthylene
Fluorene
Phenanthrene
Fluoranthene
Benz (a) anthracene
Benz (a) pyrene
Benz (ghi) pery lene
Average Stability
pH 2 pH 7 pH 10
77
77
71
80
72
76
72
74
39
72
66
63
39
52
89
70
83
45
87
54
' 72
78
71
' 58
i 6?
; 71
58
65
63
86
100
95
57
100
100
108
118
96
72
50
57
55
62
90
85
ANOVA Average ANOVA Average
Analysis Stability Analysis Stability
pH 4°C RT TCMP 0 ppm 2 ppm
f 90
+ 90
+ 66
0 89
0 83
+ 83
0 85
0 77
+ 64
-1- 70
0 65
+ 62
+ 56
+ 75
0 91
6r +
67 +
55 +
89 0
81 0
92 +
97 +
87 +
49 +
63 0
63 0
55 +
54 0
62 +
68 +
77
91
79
94
87
91
97
87
74
70
68
62
68
88
95
87
79
41
83
77
83
85
78
38
55
61
56
34
49
78
ANOVA
Analysis
Chlorine
f .
+
+
+
+
+
+
0
f
0
+
+
+
+
f
+ Significantly differenct at 95Z confidence level.
0 No significant difference at 95Z confidence level.
-------
iTrtPT.
'£. ifl
-rtfr- ZEftO \
o. 2
Figure 7. HPLC separation of water effluent extract from plastic industry
(1st liter).
33
-------
Figure 8. HPLC separation of water effluent extract from plastic Industry
(2nd liter).
34
-------
11 . -2
r
1 3 , .3
Figure 9. HPLC chromatogram of spiked water
effluent extract from plastic industry.
j f.. 47
35
-------
difficult. Retention times between spiked and unspiked samples agreed
for some PAH species but the wavelength ratio Xex 250 - Xem 370/ Xex 280 -
Xem 389 varied widely from the experimentally determined values. Ex-
amples of these variations are illustrated by comparison of Figure 7
with that of Figure 8. This variation occurred even among the spiked
samples and the high recoveries observed are probably due to this wide
variation in background PNA levels.
A possible explanation for these differences is that one aliquot may
have contained more particulate material than another. Although the
bulk sample bottle was shaken before removing the aliquot for extraction,
there may have been differences in particulate content between aliquots.
Since PAH is known to be distributed primarily onto the particulate,
this is an important aspect in interpretation of the results.
Results of the plastic industry wastewater analysis are listed in
Table II.
Subsequent wastewaters were homogenized prior to subdivision into
1 liter aliquots to eliminate these variations in background levels.
However, the initial supply of plastic industry wastewater was exhausted
and homogenization was not performed on this sample.
Raw municipal sewage from Columbus, Ohio, was obtained as the sec-
ond wastewater to be studied. Initial extraction studies indicated a
fluctuating background similar to that observed with the plastic industry
effluent. However, homogenization of this sample produced a more con-
sistent background on subsequent extractions. For real samples an al-
ternate solution would be to collect a 1 liter sample, representative of
a particular source, and extract the entire sample.
Analysis indicated the presence of chrysene at 2.4 ppb to be the PAH
present in greatest concentration; BaP was present at 0.07 ppb.
Results of these analysis are listed in Table 12. Typical chromatograms
of spiked and unspiked sewage extract are shown in Figures 10 and 11.
The detection limits (DL) for each sample were calculated based on the
concentration required for a given compound to yield a peak two times
greater than the baseline fluctuation (due to compounds eluting in the
vicinicity of the compound of interest) for that sample.
Treated municipal sewage water prior to chlorination was obtained as
the third wastewafter sample. Again homogenization was performed before
aliquots were taken. Sample extracts indicated a lower and more uniform
background than had been obtained with the raw sewage. Analysis indi-
cated the presence of fewer PAH compounds than the untreated sewage as
well as a lower concentration for chrysene at 0.3 ppb. However, an
increase in the concentration of fluoranthene was detected from 0.5 ppb
for the untreated sewage to 0.7 ppb for the treated.
Good PAH recoveries from spiked treated sewage were obtained except
for naphthalene. These low results are due to the fact that the
36
-------
TABLE 11. PLASTICS INDUSTRY WASTEWATER
Compound
Naphthalene
Acenaphthylene
Acenaphthene
Fluorene
Phenanthrene
Anthracene
Fluoranthene
Pyrene
Benz (a) anthracene
Chrysene
Benz (b) f luoranthene
Benz (k) f luoranthene
Benz (a) pyrene
Dibenz (a , h) anthracene
Benz(ghi)perylene
Indeno (1 , 2 , 3-cd) pyrene
Spiked
level
(ppb)
70
450
25
23
3.8
7.9
2.2
6.9
0.66
6.8
0.24
0.62
0.30
1.7
3.4
1.4
JJpiked
Unspiked
%
recovery
0 days 7 days
76 ± 14
134 ± 15
192 ± 28
152 ± 27
183 ± 16
181 ± 35
123 ± 23
223 ± 72
208 ± 45
155 ± 26
105 ± 13
221 ± 24
108 ± 12
107 ± 13
127 ± 9
133 ± 18
68 ± 19
117 ± 13
181 ± 37
156 ± 27
179 ± 33
213 ± 48
182 ± 21
168 ± 25
223 ± 31
169 ± 25
135 ± 7
156 ± 38
122 ± 15
115 ± 12
126 ± 18
119 ± 13
Background
ppb
<4.0
<20
15.9 ± 5.3
0.7 ± 0.4
4.2 ± 2.3
7.8 ± 3.4
1.0 ± 0.06
4.8 ± 2.2
0.45 ±0.21
1.05± 0.30
<0.03
0.88±0.30
<0.03
<0.06
<0.15
<0.08
-DL
ppb
4.0
20.0
0.8
0.4
2.1
3.0
0.23
0.93
0.07
0.1
0.03
0.03
0.03
0.06
0.15
0.08
37
-------
ca
t
< it irr -
-j i j-j
f|
60
s
a
a
a.
a
-------
3.3-5,
19. 13
Figure 11. HPLC chromatogram of unspiked raw sewage.
39
-------
Figure 12. HPLC chromatogram of spiked treated sewage.
40
-------
J.
i .35
3.>i3
4.22
-- 1:8$
-- 12.31
14.96
-- 13.75
i *f «si
21: os
21.73
22.44
'-,43 r+3
«i -W » *5 I
S4:E>
* 25.86
STOP
Figure 13. HFLC chronatogram of unspiked treated sewage.
41
-------
TABLE 12 .
RAW
SEWAGE WASTEWATER
Spiked
Compound
Naphthalene
Acenaphthylene
Acenaphthene
Fluorene
Phenanthrene
Anthracene
Fluoranthene
Pyrene
Benz (a) anthracene
Chrysene
Benz(b) f luoranthene
Benz (k) f luoranthene
Benz (a) pyrene
Dibenz (a , h) anthracene
Benz (ghi) pery lene
Indeno (1,2, 3-cd) pyrene
Spiked
level
(ppb)
70
450
25
23
3.
7.
2.
6.
0.
6.
0.
0.
0.
1.
3.
1.
8
9
2
9
66
8
24
62
30
7
4
4
0 days
108 ±
119 ±
93 *
101 ±
115 ±
114 ±
133 ±
127 ±
102 ±
81 ±
132 ±
112 ±
114 ±
80 ±
81 ±
98 ±
Z
recovery
7 days
9
5
4
7
8
7
12
16
7
3
24
15
9
6
8
2
113
103
85
115
129
108
134
113
109
93
133
116
" 90
89
81
93
±
±
±
±
±
±
+
+
+
+
±
+
+
+
±
+
7
5
5
13
12
7
10
12
9
9
19
14
10
6
12
7
Unspiked
Background
PPb
<4
<20
0
0
0
1
0
2
0
0
0
<0
<0
<0
<0.8
<0.4
.74 ±
.3 ±
.47 ±
.0 ±
.19 ±
.4 ±
.10 ±
.10 ±
.07 ±
.06
.15
.08
0.15
0.15
0.14
0.4
0.08
1.05
0.04
0.04
0.01
MDL
PPb
4.0
20
0.8
0.4
0.3
0.3
0.02
0.2
0.02
0.2
0.02
0.02
0.03
0.06
0.15
0.08
42
-------
TABLE 13 . TREATED SEWAGE WASTEWATER
Spiked
Compound
Naphthalene
Acenaphthylene
Acenaphthene
Fluorene
Phenanthrene
Anthracene
Fluoranthene
Pyrene
Benz (a) anthracene
Chrysene
Benz (b) f luoranthene
Benz (k) f luoranthene
Benz(a)pyrene
Dibenz (a, h) anthracene
Benz (ghi) perylene
Indeno (1,2, 3-cd)pyrene
Spiked
level
(ppb)
20
250
11
6
5
11
0
2
0
2
0
0
0
0
0
0
.6
.1
.0
.3
.3
.3
.64
.0
.3
.14
.21
.4
.42
.96
recovery
0 days 7 days
38 ±
79 ±
79 ±
77 ±
83 ±
82 ±
127 ±
88 ±
74 ±
89 ±
88 ±
86 ±
101 ±
86 ±
86 ±
106 ±
3
8
3
5
6
6
6
4
6
16
12
4
7
3
6
6
24 ±
88 ±
86 ±
75 ±
89 ±
88 ±
124 ±
89 ±
71 ±
92 ±
86 ±
89 ±
98 ±
89 ±
90 ±
97 ±
5
9
7
8
9
7
7
5
7
9
9
7
8
5
5
3
Unspiked
Background
ppb
<4
<20
1.8
<0
<0
<0
0.7
<0
<0
0.3
<0
<0
<0
<0
<0
<0
± 1.0
.4
.3
.3
± 0.05
.2
.02
± 0.1
.02
.02
.03
.06
.15
.08
MDL
ppb
4.
20
1.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0
0
4
3
3
05
2
02
1
02
02
0.03
0.
0.
06
15
0.08
43
-------
2
g
O
I
P-
I
G,
OC<
-------
= =. y
=1=- 13,33
Figure 15. HPLC chromatogram of unspiked flyash wash.
45
-------
TABLE 14. FLYASH WASH WASTEWATER
Spiked
Compound
Naphthalene
Acenaphthylene
Acenaphthene
Fluorene
Phenanthrene
Anthracene
Fluoranthene
Pyrene
Benz (a) anthracene
Chrysene
Benz (b) f luoranthene
Benz (k) f luoranthene
Benz (a) pyrene
Dibenz (a, h) anthracene
Benz (ghi)perylene
Indeno (1 , 2 , 3-cd) pyrene
Spiked
level
(ppb)
40
250
11.6
6.1
5.0
11.3
0.3
2.3
0.64
2.0
0.3
0.14
0.21
0.40
0.42
0.96
0 days
87 +
88 ±
91 ±
86 ±
92 ±
92 ±
90 ±
94 ±
91 ±
92 ±
87 ±
86 ±
85 ±
89 ±
89 ±
85 ±
recovery
7 days
6
5
4
5
8
6
6
5
3
7
7
6
5
7
5
9
85 ±
85 ±
87 ±
76 ±
94 ±
87 ±
89 ±
88 ±
86 ±
86 ±
80 ±
86 ±
87 ±
85 ±
82 ±
83 ±
6
7
5
7
9
6
7
6
10
9
8
9
4
6
8
7
Unspiked
Background
ppb
<4
<20
<0.8
<0.4
<0.24
<0.3
0.02 ± 0.01
<0.1
<0.01
<0.1
<0.01
<0.01
<0.01
<0.02
<0.05
<0.02
MDL
ppb
4
20
0.8
0.4 .
0.24-
0.3
0.01
0.1
0.01
0.1
0.01
0.01
0.01
0.02
0.05
r
0.02
46
-------
~p> 15.7
5
17.77
29. tJS
Figure 16, HPLC chromatogram of spiked flyash wash
settling pond.
47
-------
- - 1 i,
Figure 17.
HPLC chromatogram of unspiked
flyash wash settling pond.
48
-------
concentration of naphthalene was close to the detection limit and the
integrator did not integrate the sloping shoulders on this small peak.
Since recoveries were calculated using peak area, a low result was ob-
tained. Probably peak height would be a more accurate method for
quantitation whenever the detection limit of the PAH compound is ap-
proached. Since our data was collected using a chromatographic data
system it is not possible to calculate recoveries on the basis of peak
heights since many of the peaks were offscale on the attenuation set-
ting used. Results of these analysis are listed in Table 13. Typical
chromatograms of spiked and unspiked treated sewage are shown in
Figures 12 and 13.
Flyash wash water from a coal fired power plant was collected as
the fourth wastewater. The flyash which is collected by the electro-
static precipitators is received into large hoppers. These hoppers are
emptied every few hours by slurrying the flyash with water which drains
into a primary settling pond. The flyash wash samples were collected
just at the outfall of the primary settling pond.
No difficulties resulted during the homogenization and subsequent
analysis of this wastewater. Fluoranthene was the only PAH apparently
present in this sample. As the water contact time with the flyash is
relatively short, this is not surprising.
Results of these analyses are given in Table 14. Examples of
spiked and unspiked chromatograms are shown in Figures 14 and 15.
An overflow.secondary settling pond adjacent to the primary settling
pond above was the source of the fifth wastewater. As the sample was
collected in late summer, the water in both ponds was relatively low.
Thus the primary settling pond water would not have overflowed into the
secondary for quite some time. A surface grab sample of this water
was obtained at the edge of the pond.
No difficulties resulted during the homogenization and subsequent
analysis of this wastewater. Again, fluoranthene was the only PAH
apparently present in this sample although at a slightly higher
concentration than that found in the primary wash.
Results of these analysis are given in Table 15. Examples of spiked
and unspiked chromatograms are shown in Figures 16 and 17.
The recovery data after seven'days storage for each of the last
four wastewater samples has been combined in Table 16 to give an average
recovery for each of the PAHs. The plastics industry sample data has
not been included because of the problems encountered with sample
homogeneity.
Based on the data obtained for the several wastewater samples it is
obvious that the PAHs were stable for seven days using the recommended
preservation techniques and that the sample matrix did not seriously
49
-------
TABLE 15. FLYASH SETTLING POND WASTEWATER
Spiked
Compound
Naphthalene
Acenaphthylene
Acenaphthene
Fluor ene
Phenanthrene
Anthracene
Fluoranthene
Pyrene
Benz (a) anthracene
Chrysene
Benz (b) f luoranthene
Benz (k) f luorantehen
Benz (a) pyrene
Dib enz ( a , h) anthracene
Benz (ghi)perylene
Indeno ( 1 , 2 , 3- cd) pyrene
Spiked
level
(ppb)
40
250
11.6
6.1
5.0
11.3
0.3
2.3
0.64
2.0
0.3
0.14
0.21
0.40
0.42
0.96
recovery
0 days 7 days
87 ±
90 ±
96 ±
97 ±
93 ±
85 ±
115 ±
87 ±
93 ±
86 ±
.88 ±
90 ±
92 ±
90 ±
94 ±
97 ±
17
5
6
8
5
5
12
6
4
8
6 .
9
9
8
6
8
83 ±
89 ±
90 ±
93 ±
88 ±
86 ±
113 ±
86 ±
87 ±
87 ±
. 83 ±
88 ±
87 ±
86 ±
85 ±
89 ±
5
6
5
7
8
6
14
6
6
5
5
6
5
4
6
6
Unspiked
Background
ppb
<4.0
<20
<0.8
<0.4
<0.24
<0.03
0.03 ± 6.01
<0.1
<0.01
<0.1
<0.01
<0.01
<0.01
<0.02
<0.05
<0.02
MDL
PPb
4
20
0.8
0.4
0.24
0.03
0.01
0.1
0.01
0.1
0.01
0.01
0.01
0.02
0.05
0.02
50
-------
TABLE 16. AVERAGE RECOVERIES FOR
PAHs FROM WASTEWATER SAMPLES
Compound Recovery
Napthalene 76+37*
Acenapthalene 91+8
Acenapthene 87+2
Fluorene 90+18
Phenanthrene 100+19
Anthracene 92+10
Fluoranthene 115+19
Pyrene 94+12
Benz (a) anthracene 88+_15
Chrysene 90+4
Benz(b)fluoranthene 95+25
Benz(k)fluoranthene 94+14
Benz(a)pyrene 91+4
Dibenz(a,h)anthracene 87+2
Benz(ghi)perylene 85+4
Indeno(l,2,3-cd)pyrene 91+8
* Average recoveries for four wastewater samples after seven days
storagei the standard deviation of the four measurements.
51
-------
affect PAH recovery, since the recoveries for distilled water (e.g. pre-
servation studies) and actual wastewater samples are virtually identical.
The only serious problem encountered during the wastewater analysis was
the problem of sample homogeneity, as discussed previously for the plas-
tics industry sample. It appears that there are two alternative solutions
to this problem: 1) thoroughly homogenize the sample before subsampling
it or 2) obtain a representative sample from a particular source and
analyze the entire sample.
52
-------
SECTION 7
SUMMARY AND RECOMMENDATIONS
Several important points can be drawn from this analytical methods
study for PAH in wastewater. These are:
* Sample homogenization is required in order to obtain
a sample of uniform PAH content. Alternatively the
entire sample can be extracted.
* For best preservation of PAHs in water, the pH sould be
near 7, stored at near freezing temperatures and free
chlorine should be removed by addition of a reducing agent.
Methylene chloride is a suitable extraction solvent
for PAHs in water at pH 7 or pH 10.
All 16 consent decree PAHs can be separated by HPLC on a
single stationary phase using a .Perkin-Elmer HC-ODS
column (2.6 x 250 mm I.D.).
* With proper wavelength selection all consent decree
PAHs can be quantitated at sub-ppb levels.
Although the silica LC clean-up procedure probably
removes the majority of the organics in the wastewater
extract, a large number of components can still remain
in the collected PAH fraction. This was observed in
some of the wastewaters. Thus there is a. need for
unambiguous peak identification. This could be achieved
through the use of a stop-flow scanning spectrofluorimeter
or by obtaining a UV or fluorescent spectra on collected
fractions.
PAH solutions are stable in DMSO and acetone for at least
90 days.
53
-------
REFERENCES
1. McGinnes, P.R. and V.L. Snoeyink. Determination of the Fate of
Polynuclear Aromatic Hydrocarbons in Natural Water Systems.
OWRR A-045-I11, Illinois Water Resources Center, Urbana, 111.
55pp., 1974
2. Harrison, R.M., R.A. Wellings. Effect of Water Chlqrinations upon
Levels of Some Polynuclear Aromatics Hydrocarbons in Water.
Env. Sci. Tech., 10(12), 1151-1156, 1976.
3. Pancirov, R.J. and R.A. Brown. Polynuclear Aromatic Hydrocarbons
in Marine Tissues. Env. Sci. Tech. 11(10), 989-992, 1977.
4. Bjorseth, Alf. Analysis of PNA's in Air and Water. In Control of
Specific Toxic Pollutants, Proceedings, Air Pollution Control
Association, Feb. 13 -16, 1979.
5. Haenni, E.G. and J.W. Howard. DMSO A Superior Analytical Extraction
Solvent for PNA's and for Some Highly Chlorinated Hydrocarbons.
Journal of the AOAC 45(1), 67-72, 1962.
6. Hites, R.A. and K. Biemann. Water Pollution: Organic Compounds in
the Charles River, Boston. Science 178, 158-160, 1972.
7. Achieson, M.A.,R.M. Harrison, R. Perry, and R.A. Wellings. Factors
Affecting the Extraction and Analysis of PNA's in Water. Water
Research 10, 207-212, 1976.
8. Baser, D.K. and J. Saxena. Monitoring of PNA's in Water II.
Extraction and Recovery of Six Representative Compounds with
Polyurethane Foams. Env. Sci. Tech. 12(7), 791-794, 1978.
9. Junk, G.A., C.D. Chriswell, R.C. Chang, L.D. Kissinger, J.J. Richard,
J.S. Fritz, and H.J. Svec. Applications of Resins for Extracting
Organic Compounds from Water. Anal. Chem. 282, 331-337, 1976.
10. Castex, H., Identification of PNA's by Paper Chromatography and
Spectrofluorimetry. Revue De L' Institut Francais Du Petrole
29(2), 155-172, 1974.
11. Soedigo, S., W.W. Angus, and J.W. Flesher. HPLC of PNA's and Some
of Their Derivatives. Anal. Biochem. 67, 664-668, 1975.
12. Das, B.S. and G.H. Thomas. Fluorescence Detection in HPLC of
PNA's. Anal. Chem. 50(7), 967-973, 1978.
54
-------
APPENDIX A
POLYNUCLEAR AROMATIC HYDROCARBONS
METHOD 610
1. Scope and Application
1.1 This method covers the determination of certain polynuclear aromatic
hydrocarbons (PAH). The following parameters may be determined by
this method:
Parameter
Acenaphthene
Acenaphthylene
Anthracene
Benzo(a)anthracene
Benzo(a)pyrene
Benzo(b)fluoranthene
Benzo(ghi)perylene
Benzo(k)fluoranthene
STORET No. Parameter
STORET No.
34205 Chrysene 34320
34200 Dibenzo(ah)anthracene 34556
34220 Fluoranthene 34376
34526 Fluorene 34381
34247 Indeno(l,2,3-cd)pyrene 34403
34230 Naphthalene 34696
34521 Phenanthrene 34461
34242 Pyrene 34469
1.2 This method is applicable to the determination of these compounds
in municipal and industrial discharges. It is designed to be
used to meet the monitoring requirements of the National Pollutant
Discharge Elimination System (NPDES). As such, it presupposes a
high expectation of finding the specific compounds of interest.
If the user is attempting to screen samples for any or all of the
compounds above, he must develop independent protocols for the
verification of identity.
1.3 The sensitivity of this method is usually dependent upon the level
of interferences rather than instrumental limitations. The limits
of detection listed in Table I represent sensitivities that can
be achieved in wastewaters.
1.4 This method is recommended for use only by experienced residue
analysts familiar with High Performance Liquid Chromatography
(HPLC) or under the close supervision of such qualified persons.
2. Summary of Method
2.1 A 1-liter sample of wastewater is extracted with methylene chloride
using separatory funnel techniques. The extract is dried and
concentrated to a volume of 10 ml or less. HPLC conditions are
described which allow for the accurate measurement of the compounds
in the extract.
55
-------
2.2 If interferences are encountered, the method provides a selected
general purpose clean-up procedure to aid the analyst in their
elimination.
3. Interferences
3.1 Solvents, reagents, glassware, and other sample processing hardware
may yield discrete artifacts and/or elevated baselines causing
misinterpretation of gas chromatograms. All of these materials
must be demonstrated to be free from interferences under the
conditions of the analysis by running method blanks. Specific
selection of reagents and purification of solvents by distillation
in all-glass systems may be required.
3.2 Interferences coextracted from the samples will vary considerably
from source to source, depending upon the diversity of the industrial
complex or municipality being sampled. While general clean-up
techniques are provided as part of this method, unique samples may
require additional, clean-up approaches to achieve the sensitivities
stated in Table 1.
3.3 The extent of interferences that may be encountered using liquid
chromatographic techniques has not been fully assessed. Although
the chromatographic conditions described allow for a unique resolution
of the specific PAH compounds covered by this method, other PAH
compounds may interfere.
4. Apparatus and Materials
4.1 Sampling equipment, for discrete or composite sampling.
4.1.1 Grab sample bottle - amber glass, liter or quart volume.
french or Boston Round design is recommended. The container
must be washed and solvent rinsed before use to minimize
interferences.
4.1.2 Bottle caps - Threaded to screw on sample bottles. Caps
must be lined with Teflon. Foil may be substituted if
sample is not corrosive.
4.1.3 Compositing equipment - Automatic or manual compositing
system. Must incorporate glass sample containers for the
collection of a minimum of 250 ml. Sample containers must
be kept refrigerated during sampling. No tygon or rubber
tubing or fittings may be used in the system.
4.2 Separatory funnel - 2000 ml, with Teflon stopcock.
4.3 Drying column - A 20 mm ID pyrex chromatographic column with coarse
frit.
4.4 Kuderna-Danish (K-D) Apparatus
56
-------
4.4.1 Concentrator tube - 10 ml, graduated (Kontes K-570050-1025
or equivalent). Calibration must be checked. Ground glass
stopper (size 19/22 joint) is used to prevent evaporation of
extracts.
4.4.2 Evaporative flask - 500 ml (Kontes K-57001-0500 or equivalent).
Attach to concentrator tube with springs. (Kontes K-662750-
0012).
4.4.3 Snyder column - three-ball macro (Kontex K503000-0121 or
equivalent).
4.4.4 Snyder column - two-ball micro (Kontes K-569001-0219 or
equivalent).
4.4.5 Boiling chips - extracted, approximately 10/40 mesh.
4.5 Water bath - Heated, with concentric ring cover, capable of
temperature control (±2°C). The bath should be used in a hood.
4.6 HPLC Apparatus:
4.6.1 Gradient pumping system, constant flow.
4.6.2 Reverse phase column, HC-ODS Sil-X, 250 mm x 2.6 mm ID
(Perkin Elmer No. 809-0716 or equivalent).
4.6.3 Fluorescence detector, Aex 280 nm and Xem >389 nm.
4.6.4 UV detector, 254 nm, coupled to fluorescence detector.
4.6.5 Strip chart recorder capatible with detectors, 250 mm (A
data system for measuring peak areas is recommended).
4.7 Chromatographic column - 250 mm long x 10 mm ID with coarse fritted
disc at bottom and Teflon stopcock.
5. Reagents
5.1 Preservatives:
5.1.1 Sodium hydroxide - (ACS) 10 N in distilled water.
5.1.2 Sulfuric acid - (ACS) Mix equal volumes of cone. H2SC>4
with distilled water.
5.1.3 Sodium thiosulfate - (ACS) Granular.
5.2 Methylene chloride, Pentane, Cyclohexane, High Purity Water-HPLC
quality, distilled in glass.
5.3 Sodium Sulfate - (ACS) Granular, anhydrous (purified by heating at
400°C for 4 hrs.).
57
-------
5.4 Stock standards - Prepare stock standard solutions at a concen-
tration of 1.00 Vg/yl by dissolving 0.100 grams of assayed
reference material in high quality acetone or other
appropriate solvent and diluting to volume in a 100 ml ground glass
stoppered volumetric flask. The stock solution is transferred to
ground glass stoppered reagent bottles, stored in a refrigerator,
and checked frequently for signs of degradation or evaporation,
especially just prior to preparing working standards from them.
5.5 Acetonitrile - Spectral quality.
5.6 Silica gel - 100/120 mesh desiccant (Davison Chemical grade 923^or
equivalent). Before use, activate for at least 16 hours at 130°C
in a foil covered glass container.
6. Calibration
6.1 Prepare calibration standards that contain the compounds of interest,
either singly or mixed together. The standards should be prepared
at concentrations covering two or more orders of magnitude that will
completely bracket the working range of the chromatographic system.
If the sensitivity of the detection system can be calculated from
Table I as 100 Pg/1 in the final extract, for example, prepare
standards at 10 ug/1, 50 yg/1, 100 ug/1, 500 ug/1, etc. so that
injections of 1-5 yl of each calibration standard will define the
linearity of the detector in the working range.
6.2 Assemble the necessary high pressure liquid chromatographic apparatus
and establish operating parameters equivalent to those indicated in
Table I. By injecting calibration standards, establish the sensitivity
limit of the detectors and the linear range of the analytical systems
for each compound.
6.3 Before using any clean-up procedure, the analyst must process a
series of calibration standards through the system to validate
elution patterns and the absence of interferences from the reagents. -
7. Quality Control
7.1 Before processing any samples, the analyst should demonstrate through
the analysis of a distilled water method blank, that all glassware
and reagents are interference-free. Each time a set of samples is
extracted or there is a change in reagents, a method blank should
be processed as a safeguard against chronic laboratory contamination.
7.2 Standard quality assurance practices should be used with this method.
Field replicates should be collected to validate the precision of
the sampling technique. Laboratory replicates should be analyzed to
validate the precision of the analysis. Fortified samples should be
analyzed to validate the accuracy of the analysis. Where doubt
exists over the identification of a peak on the chromatogram, con-
firmatory techniques such as fraction collection and GC-mass
spectroscopy should be used.
58
-------
8. Sample Collection, Preservation, and Handling
' 8.1 Grab samples must be collected in glass containers. Conventional
sampling practices should be followed, except that the bottle must
not be prewashed with sample before collection. Composite samples
should be collected in refrigerated glass containers in accordance
with the requirements of the program. Automatic sampling equipment
must be free of tygon and other potential sources of contamination.
8.2 The samples must be iced or refrigerated from the time of collection
until extraction. Chemical preservatives should not be used in the
field unless more than 24 hours will elapse before delivery to the
laboratory. If the samples will not be extracted within 48 hours of
collection, adjust the sample to a pH range of 6.0-8.0 with sodium
hydroxide or sulfuric acid and add 35 mg sodium thiosulfate per part
per million of free chlorine per liter.
8.3 All samples must be extracted within 7 days and completely analyzed
within 30 days of collection.
9. Sample Extraction
9.1 Mark the water meniscus on the side of the sample bottle for later
determination of sample volume. Pour the entire sample into a
two-liter separatory funnel. Check the pH with wide-range paper
and adjust to within the range of 5-9 with sodium hydroxide or
sulfuric acid.
9.2 Add 60 ml methylene chloride to the sample bottle and shake 30
seconds to rinse the walls. Transfer the solvent into the separatory
funnel, and extract the sample by shaking the funnel for two minutes
with periodic venting to release vapor pressure. Allow the organic
layer to separate from the water phase for a minimum of ten minutes.
If the emulsion interface between layers is more than one-third the
size of the solvent layer, the analyst must employ mechanical
techniques to complete the phase separation. The optimum technique
depends upon the sample, but may include stirring, filtration of
the emulsion through glass wool, or centrifugation. Collect the
methylene chloride extract in a 250-ml Ehrlenmeyer flask.
9.3 Add a second 60-ml volume of methylene chloride to the sample bottle
and complete the extraction procedure a second time, combining the
extracts in the Erlenmeyer flask.
9.4 Perform a third extraction in the same manner. Pour the combined
extract through a drying column containing 3-4 inches of anhydrous
sodium sulfate, and collect it in a 500-ml Kuderna-Danish (K-D)
flask equipped with a 10 ml concentrator tube. Rinse the
Ehrlenmeyer flask and column with 20-30 ml methylene chloride to
complete the quantitative transfer.
9.5 Add 1-2 clean boiling chips to the flask and attach a three-ball
Synder column. Prewet the Snyder column by adding about 1 ml
59
-------
methylene chloride to the top. Place the K-D apparatus on a
steaming hot (60-65°C) water bath so that the concentrator tube is
partially immersed in the hot water, and the entire lower rounded
surface of the flask is bathed in steam. Adjust the vertical
position of the apparatus and the water temperature as required to
complete the concentration in 15-20 minutes. At the proper rate
of distillation the balls of the column will actively chatter but
the chambers will not flood. When the apparent volume of liquid
reaches 1 ml, remove the K-D apparatus and allow it to drain for
at least 10 minutes while cooling. Remove the Snyder column and
rinse the flask and its lower joint into the concentrator tube
with 1-2 ml of methylene chloride. A 5-ml syringe is recommended
for this operation. Stopper the concentration tube and store
refrigerated if further processing will not be performed immediately.
9.6 Determine the original sample volume by refilling the sample bottle
to the mark and transferring the liquid to a 1000 ml graduated
cylinder. Record the sample volume to the nearest 5 ml.
9.7 If the sample requires clean-up before chromatographic analysis,
proceed to Section 10. If the sample does not require clean-up,
or if the need for clean-up is unknown, analyze an aliquot of the
extract according to Section 11.
10. Clean-up and Separation
10.1 Before the silica gel clean-up technique can be utilized, the
extract solvent must be exchanged to cyclohexane. Add a 1-10 ml
aliquot of sample extract (in methylene chloride) and a boiling
chip to a clean K-D concentrator tube. Add 4 ml cyclohexane and
attach a micro-Snyder column. Prewet the micro-Snyder column by
adding 0.5 ml methylene chloride to the top. Place the micro-K-D
apparatus on a boiling (100°C) water bath so that the concentrator
tube is partially immersed in the hot water. Adjust the vertical
position of the apparatus and the water temperature as required to
complete concentration in 5-10 minutes. At the proper rate of
distillation the balls of the column will actively chatter but the
chambers will not flood. When the apparent volume of the liquid
reaches 0.5 ml, remove the K-D apparatus and allow it to drain for
at least 10 minutes while cooling. Remove the micro-Snyder column
and rinse its lower joint into the concentrator tube with a min-
imum of cyclohexane. Adjust the extract volume to about 2 ml.
10.2.1 Prepare a slurry of lOg activated silica gel in methylene chloride
and place this in a 10 mm ID chrmatography column. Gently tap the
column to settle the silica gel and elute the methylene chloride.
10.2.2 Preelute the column with 40 ml pentane. Discard the eluate and
just prior to exposure of the sodium sulfate layer to the air,
transfer the 2 ml cyclohexane sample extract onto the column,
using an additional 2 ml of cyclohexane to complete the transfer.
60
-------
10.2.3 Just prior to exposure of the sodium sulfate layer to the
air, add 25 ml pentane and continue elution of the column.
Discard the pentane eluate.
10.2.4 Elute the column with 25 ml of 40% methylene chloride/60%
pentane and collect the eluate in a 500 ml K-D flask
equipped with a 10 ml concentrator tube. Elution of the
column should be at a rate of about 2 ml/min.
10.2.5 Concentrate the collected fraction to less than 10 ml by K-D
techniques as in 9.5, using pentane to rinse the walls of
the glassware. Proceed with HPLC analysis.
11. High Performance Liquid Chromatography HPLC
11.1 Table I summarizes the recommended HPLC column materials and
operating conditions for the instrument. Included in this table
are estimated retention times and sensitivities that should be
achieved by this method. An example of the separation achieved by
this column is shown in Figure 1. Calibrate the system daily with
a minimum of three injections of calibration standards.
11.2 To the extract, add 4 ml acetonitrile and a new boiling chip, then
attach a micro-Snyder column. Increase the temperature of the hot
water bath to 95-100pC. Concentrate the solvent as above. After
cooling, remove the micro-Snyder column and rinse its lower joint
into the concentrator tube with about 0.2 ml acetonitrile. Adjust
the extract volume to 1.0 ml.
11.3 Inject 2-5 yl of the sample extract with a high pressure syringe.
Record the volume injected to the nearest 0.05 yl, and the re-
sulting peak size, in area units.
11.4 If the peak area exceeds the linear range of the system, dilute the
extract and reanalyze.
11.5 If the peak area measurement is prevented by the presence of
interferences, further clean-up is required.
11.6 The UV detector is recommended for the determination of naphthalene
and acenaphthylene and the fluorescence detector is recommended for
the remaining PAHs.
12. Calculations
12.1 Determine the concentration of individual compounds according to
the formula:
Concentration, yg/1 = t'
(v.) (vs)
61
-------
where A * Calibration factor for chromatographic system in
nanograms material per area unit,
B = Peak size in injection of sample extract, in area units
V. = volume of extract injected (yl)
V = Volume of total extract (pi)
V = Volume of water extracted (ml)
s
12.2 Report results in micrograms per liter without correction for
recovery data. Whenduplicate and spiked samples are analyzed, all
data obtained should be reported.
13. Accuracy and Precision
Data is not available at the present time.
BIBLIOGRAPHY
"Determination of Polynuclear Aromatic Hydrocarbons in Industrial and
' Municipal Wastewaters". Report for EPA Contract 68-03-2624 (In prepar-
ation) .
62
-------
TABLE A-l HIGH PERFORMANCE LIQUID CHROMATOGRAPHY OF PAH's
Detection
Compound Retention time (min) UV
Naphthalene
Acenaphthylene
Acenaphthene
Fluorene
Phenanthrene
Anthracene
Fluoranthene
Pyrene
Benzo (a) anthracene
Chrysene
Benzo (b) f luoranthene
Benzo (k) f luoranthene
Benzo (a) pyrene
Dibenzo (a ,h) anthracene
Benzo (ghi) perylene
Indeno (1,2, 3-cd) pyrene
16.17
18.10
20.14
20.89
22.32
23.78
25.00
25.94
29.26
30.14
32.44
33.91
34.95
37.06
37.82
39.21
2.5
5.0
3.0
0.5
0.25
0.10
0.50
0.10
0.20
0.20
1.0
0.30
0.25
1.0
0.75
0.30
Limit (ug/l)f
Fluorescence
20.0
100.0
4.0
2.0
1.2
1.5
0.05
0.05
0.04
0.5
0.04
0.04
0.04
0.08
0.2
0.1
HPLC conditions: Reverse phase HC-ODS Sil-X 2.6 x 250 mm Perkin-Elmer
column; isocratic elution for 5 min. using 40% acetonitrile/60% water,
then linear gradient elution to 100% acetonitrile over 25 minutes;
flow rate is 0.5 ml/min.
j- Detection limit is calculated from the minimum detectable HPLC response .
being equal to five times the bacground noise, assuming an equivalent
of a 2 ml final volume of the 1 liter sample extract, and assuming an
HPLC injection of 2 microliters.
63
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For Conditions, See Table I.
64
-------
APPENDIX B
UV and Fluorescence Spectra of Sixteen Selected PNA's
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72
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