United States
Environmental Protection
Agency
Municipal Environmental Research
Laboratory
Cincinnati OH 45268
EPA-600/2-80-030
March 1980
Research and Development
&EPA
Analytical
Procedures for
Determining Organic
Priority Pollutants in
Municipal Sludges
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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 ENVIRONMENTAL PROTECTION TECH-
NOLOGY series. This series describes research performed to develop and dem-
onstrate instrumentation, equipment, and methodology to repair or prevent en-
vironmental degradation from point and non-point sources of pollution. This work
provides the new or improved technology required for the control and treatment
of pollution sources to meet environmental quality standards.
This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.
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EPA-600/2-80-030
March 1980
ANALYTICAL PROCEDURES FOR DETERMINING ORGANIC
PRIORITY POLLUTANTS IN MUNICIPAL SLUDGES
by
J. S. Warner, G. A. Jungclaus, T. M. Engel,
R. M. Riggin, and C. C. Chuang
Battelle Columbus Laboratories
Columbus, Ohio 43201
Contract No. 68-03-2624
Project Officer
Richard A. Dobbs
Wastewater Research Division
Municipal Environmental Research Laboratory
Cincinnati, Ohio 45268
MUNICIPAL ENVIRONMENTAL RESEARCH 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 Municipal Environmental Research
Laboratory, U. S. Environmental Protection Agency, and approved for publica-
tion. 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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FOREWORD
The Environmental Protection Agency was created because of increasing
public and government concern about the dangers of pollution to the health
and welfare of the American people. Noxious air, foul water, and spoiled
land are tragic testimony to the deterioration of our natural environment.
The complexity of that environment and the interplay between its components
require a concentrated and integrated attack on the problem.
Research and development is that necessary first step in problem
solution and it involves defining the problem, measuring its impact, and
searching for solutions. The Municipal Environmental Research Laboratory
develops new and improved technology and systems for the prevention, treat-
ment, and management of wastewater and solid and hazardous waste pollutant
discharges from municipal and community sources, for the preservation and
treatment of public drinking water supplies, and to minimize the adverse
economic, social, health, and aesthetic effects of pollution. This
publication is one of the products of that research; a most vital
communications link between the researcher and the user community.
This report describes analytical procedures developed for the deter-
mination of semivolatile organic priority pollutants in municipal sludge
at levels down to 0.01 yg/g.
111
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ABSTRACT
An analytical procedure was developed for the determination of 54
semivolatile organic priority pollutants in sludge at levels down to 0.01
yg/g wet weight. The procedure involved extraction with methylene chloride
or chloroform, cleanup of groups of compounds having common properties,
and in most cases analysis of the fractions by GC-MS using high-resolution
glass capillary columns and selected ion searches. The final analyses
involved the analysis of three separate fractions, namely benzidines, phenols,
and neutrals. The benzidines were determined by HPLC analysis using an
electrochemical detector instead of by GC-MS because GC-MS sensitivity for
these compounds was too low. Quantitation in the GC-MS analyses involved
the internal standard method applied to selected ion responses. Relative
response factors obtained from the analysis of standard solutions were
used as correction factors.
The cleanup steps were considered the most critical parts of the
program. Benzidines were cleaned up from neutral and acidic components by
a simple acid-base extraction procedure. Phenols were cleaned up from
neutral components by acid-base extraction. Fatty acids, a major inter-
ference, were separated from phenols by gel permeation chromatography using
Bio-Beads S-X8. Neutrals were cleaned up from acidic components by acid-base
extraction. Triglycerides, a major interference in the neutrals, were
removed by gel permeation chromatography using Bio-Beads S-X8. Saturated
hydrocarbons, another major interference in the neutrals, were removed by
adsorption chromatography using activated silica gel. This step also re-
moved highly polar components.
The procedure was applied to the analysis of sludge spiked with the
priority pollutants of concern at a level of 0.05 yg/g, wet weight basis
(1 yg/g dry weight basis for 5% solids sludge). Recoveries were generally
good and in many cases were greater than 50 percent. For many of the
phthalates and aromatic hydrocarbons, recoveries of several hundred percent
were obtained indicative of their presence in the starting sludge. Some of
the chloroethers, nitro compounds, and phenols were not recovered possibly
because of retention by particulate material in the sludge , degradation
during the 24-hour equilibration period, or loss in the relatively large
amount of caustic wash required to remove the fatty acids. Good recoveries
were obtained for benzidines even at 0.25 yg/g dry weight.
IV
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CONTENTS
Foreword ill
Abstract iv
Figures vi
Tables vi
1. Introduction 1
2. Conclusions 3
3. Recommendations 4
4. Statement of the Problem 5
5. Overall Approach 6
6. Experimental Studies 9
Extraction 9
Removal of Acids from Neutrals 11
GPC Studies 12
Sephadex LH-20 Chromatography 12
BioBeads Chromatography 14
Silica Gel Cleanup 18
Packed Column GC-MS Analysis 18
Analysis of Phenols 18
Determination of Pesticides 19
Analysis of Spiked and Nonspiked Samples 19
Appendix S-100 Method for Semivolatile Organic Components 33
v
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FIGURES
Number page
1 Scheme for determination of semivolatile priority pollutants
in sludge 8
2 Elution profiles of whole sludge extract on (a) Bio-Beads
S-X12, (b) Bio-Beads S-X8, and (c) Bio-Beads S-X4 15
3 Elution profiles of (a) whole sludge extract, (b) fatty acids,
and (c) triglycerides on Bio-Beads S-X8 16
TABLES
Number Page
1 EPA Semivolatile Organic Priority Pollutants 2
2 Yields From Various Extractions Methods 10
3 Repetitive Homogenization-Extraction of Sludge With DCM. ... 11
4 Test Solvent Systems For Sephadex LH-20 Fractionations .... 13
5 Capacity Factors of Reference Compounds Eluted with Methylene
Chloride from Bio-Beads S-X8 17
6 Comparison of Packed Column and Capillary Column GC Analysis
for Determining the Recovery of Priority Pollutants from
Digested Municipal Sludge 20
7 Recovery of Priority Pollutants from Water 23
8 Recovery of Priority Pollutants from Digested Municipal Sludge 26
9 Recovery of Priority Pollutants from Raw Sludge 29
VI
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SECTION 1
INTRODUCTION
The U.S. Environmental Protection Agency has designated 114 organic
chemicals as priority pollutants and is setting limits on the discharge
levels permitted for these pollutants in wastewater, sludge, landfill
leachate, etc. In order to establish programs for compliance with the
discharge limits satisfactory analytical methodology is needed for use by
the dischargers and by EPA. The objective of the study described in this
report was the development of efficient methodology for the determination
of 54 of the semivolatile organic priority pollutants in municipal sewage
sludge.
Those 54 chemicals were comprised of 11 acids (phenols), 2 bases
(benzidines), 16 polycyclic aromatic hydrocarbons, 6 phthalates, 2 nitros-
amines, 3 chloroalkyl ethers, 8 chlorinated hydrocarbons, and 6 miscellane-
ous neutral compounds. They are listed in Table 1. The detection limit
desired was 0.3 yg/g of sludge on a dry weight basis. The sludge of
interest included both primary and activated sludge prior to treatment by
an anaerobic digester and the digested sludge.
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TABLE 1. EPA SEMIVOLATILE ORGANIC PRIORITY POLLUTANTS
1.
2.
3.
4.
5.
6.
7.
1.
2.
3.
1.
2.
3.
4.
Polycyclic Aromatic Hydrocarbon
Acenaphthene
Acenaphthylene
Anthracene
Benzo(a)anthracene
Benzo(b)fluoranthene
Benzo(k)fluoranthene
Benzo(g,h,i)perylene
Benzo(a)pyrene
9. Chrysene
10. Dibenzo(a,g)anthracene
11. Fluoranthene
12. Fluorene
13. Indeno(l,2,3-cd)pyrene
14. Naphthalene
15. Phenanthrene
16. Pyrene
Phthalates
Bis(2-ethylhexyl) phthalate
Butylbenzyl phthalate
Diethyl phthalate
4. Dimethyl phthalate
5. Di-n-butyl phthalate
6. Di-n-octyl phthalate
Chlorinated Hydrocarbons
2-Chloronaphthalene
1,2-Dichlorobenzene
1,3-Dichlorobenzene
1,4-Dichlorobenzene
5. Hexachlorobenzene
6. 1,2,4-Trichlorobenzene
7. Hexachlorobutadiene
8. Hexachlorocyclopentadiene
Chloroalkyl Ethers
1. Bis-(2-chloroethyl) ether 3. Bis-(2-chloroisopropyl) ether
2. Bis-(2-chloroethoxy)methane
Nitrosamines
1. N-Nitrosodiethylamine
2. N-Nitrosodiphenylamine
Miscellaneous Neutrals
1. 4-Bromophenyl phenyl ether
2. 4-Chlorophenyl phenyl ether
3. 2,4-Dinitrotoluene
1. 4-Chloro-3-methylphenol
2. 2-Chlorophenol
3. 2,4-Dichlorophenol
4. 2,4-Dimethylphenol
5. 4,6-Dinitro-2-methylphenol
6. 2,4-Dinitrophenol
4. 2,6-Dinitrotoluene
5. Isophorone
6. Nitrobenzene
Acids
7. 2-Nitrophenol
8., 4-Nitrophenol
9. Pentachlorophenol
10.. Phenol
11. 2,4,6-Trichlorophenol
Bases
Benzidine
2. 3,3'-Dichlorobenzidine
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SECTION 2
CONCLUSIONS
The analysis scheme developed works very well in most cases for the
analysis of neutrals, phenols, benzidines, pesticides, and PCBs in raw and
digested sludge. The combination of extractions, gel permeation chromato-
graphy, and adsorption chromatography used for cleanup is an effective
approach. The use of HPLC with an electrochemical detector is highly
sensitive and selective for the determination of benzidines. The computer-
ized GC-MS system used with glass capillary columns is highly effective
as a sensitive and selective method for detecting and quantitating nearly
all of the other semivolatile organic priority pollutants.
The primary remaining problems are (1) poor recoveries of nitrophenols,
(2) poor recoveries of some of the more polar neutrals, namely, the nitro
compounds, chloroethers, and isophorone, and (3) poor quantitative re-
producibility. These problems could be caused by degradation during the
24-hour equilibration step, retention by the particulate material of the
sludge, loss to the aqueous phase facilitated by soaps during caustic
extraction, or alteration of separation patterns caused by large amounts
of fatty acids. A systematic study should be made of each of these possible
causes. Such a study could be carried out very efficiently without its
being a major effort by using fatty acid and priority pollutant standards
and determining recoveries by GC or HPLC.
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SECTION 3
RECOMMENDATIONS
Determine the recoveries and reproducibilities of recovery of chloro-
ether and nitrobenzenes, with and without the addition of sludge extract-
ables, in each of the following cleanup steps: (a) caustic extraction,
(b) Bio-Beads S-X8 chromatography, and (c) silica gel chromatography.
Determine the recoveries and reproducibilities of recovery of
nitrophenols with and without the addition of sludge extractables, in each
of the following cleanup steps: (a) Bio-Beads S-X8 chromatography, (b)
caustic extraction, and (c) methylene chloride extraction of the acidified
caustic extract.
Determine the effect of equilibration time on the recoveries and
reproducibilities of recovery of nitrophenols and polar neutrals from
spiked sludge.
Determine the effect of adding salt or a water-miscible organic
solvent on the recoveries and reproducibilities of recovery of nitrophenols
and polar, neutrals from spiked sludge.
Use glass capillary column gas chromatography for the analyses of
neutrals required in the above studies using a Hall detector for chloro-
ethers and a thermionic detector in the nitrogen mode for the nitrobenzenes,
Use HPLC with a variable wavelength UV detector for the analyses of
nitrophenols required in the above studies.
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SECTION 4
STATEMENT OF THE PROBLEM
Sludge generally contains 1 to 8 percent solids on a dry weight basis
and those solids in turn contain 10 to 15 percent solvent-extractable
organic material. The desired detection limit of 0.3 yg/g on a dry weight
basis thus corresponds to 2 to 3 yg/g of organic extractable material. The
bulk of the extractable material is expected to be components such as
petroleum fuels, lubricating oils, asphalt, fats, fatty acids, and deter-
gents. The analysis problem can be equated to that of looking for a few
parts per million of a lipophilic component in crankcase oil or cooking
oil. In this perspective the complexity of the problem is immediately
apparent. Extensive cleanup is desirable; however, the broad range of
physical properties of the various priority pollutants makes it impossible
to separate them as a single group. A combination of cleanup methods,
detector sensitivity, and detector selectivity is needed.
If the GC-MS detection limit is 10 ng and one percent of the sample
extract, e.g. 2 yl out of a total of 200 yl, is injected, at least 100 mg
of total extractable material must be dealt with in the cleanup process.
If the maximum concentration of total material that can be handled
satisfactorily by the GC-MS system is 10 mg/ml (2 mg/200 yl) then at least
a 98 percent cleanup is required. If the GC-MS detection limit is higher,
an even greater degree of cleanup is required.
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SECTION 5
OVERALL APPROACH
The basic approach used for determining semivolatile priority
pollutants in sludge involved extraction with methylene chloride or chloro-
form, cleanup of groups of compounds having common properties, and analysis
by GC-MS using high-resolution glass capillary columns and selected ion
searches. The benzidines were an exception in that they were determined by
HPLC using an electrochemical detector according to the method of Riggin and
Howard*.
Several approaches to extraction were studied including freezedrying
followed by Soxhlet extraction, methanol-drying followed by Soxhlet extract-
ion, azeotropic drying and the extraction associated with it, and repetitive
equilibration with solvent. The latter method, involving homogenizing to
promote equilibration, centrifuging to promote phase separation, and
withdrawal of the organic layer with a syringe, was chosen as an effective
and convenient method. Also less decomposition would be expected using this
method because of the absence of heat.
The 54 compounds of concern are comprised of neutrals, phenols and
benzidines. A separate aliquot of the sludge sample was used for the deter-
mination of each of the three groups of compounds. The benzidines were
cleaned up from the acidic and neutral components present in a chloroform
extract by extraction into strong aqueous acid followed by neutralization and
reextraction into chloroform. Because of the difficulty of protonating
dichlorobenzidine, a very strong acid, 1.0.NH2S04, is required for this
step. The use of 0.1N H2S04 is entirely inadequate. All hydrocarbons,
triglycerides and fatty acids are removed in this process. The use of HPLC
and the selectivity of the electrochemical detector provide additional
discrimination from interferences.
The phenols were cleaned up from neutral and basic components present
in a methylene chloride extract by a combination of gel permeation chromato-
graphy (GPC) and acid-base extraction. The extract was first passed through
a GPC column of Bio-Beads S-X8, a porous styrene-divinylbenzene copolymer,
with methylene chloride as the eluting solvent. Higher molecular weight
components, primarily the triglycerides, long-chain hydrocarbons, and long-
chain fatty acids, elute prior to the phenols and were thus removed by this
R. M. Riggin and C. C. Howard, Anal. Chem. , 51, 210 (1979)
-------
step. The phenols, which were contained in a lower molecular weight
fraction, were cleaned up from the remaining neutral and basic components
by extraction into aqueous base followed by acidification and reextraction
into methylene chloride.
The neutrals in a separate methylene chloride extract were separated
from phenols and the large amounts of fatty acids by caustic extraction
using rather large volumes of 0.1N NaOH in 10% NaCl. This left a solvent
extract containing the neutral priority pollutants mixed with major amounts
of triglycerides, hydrocarbons, and more polar interfering neutral components,
Gel permeation chromatography, identical to that described for cleanup of
phenols, was applied to the neutrals also. In this case the triglycerides
eluted prior to the priority pollutants but separation of many of the hydro-
carbons from the relatively large phthalate esters was not possible. There-
fore, after the removal of the triglycerides by GPC, two additional GPC
fractions were collected, the first of which was cleaned up further by
adsorption chromatography on activated silica gel. The hydrocarbons were
eluted first from the silica gel and discarded, followed by the more polar
neutral components, the phthalates, chloroethers, nitroaromatics and
isophorone. Any highly polar compounds were left on the silica gel column.
The aromatic hydrocarbons and halocarbons were obtained in a second GPC
fraction which was analyzed by GC-MS without further cleanup.
The overall analysis scheme described above, involving the use of
three separate sludge samples for phenols, neutrals, and benzidines,
respectively, is outlined in Figure 1. The final analyses entailed HPLC-
EC analysis of a benzidine fraction and GC-MS analysis of a methylated
phenol fraction and combined neutral fraction. The largest group of
frequently occurring interfering compounds were the alkylbenzenes and
alkylnaphthalenes present in the neutrals. Since these compounds have
physical properties similar to those of dichlorobenzenes and naphthalene
they cannot be Temoved—thus the selectivity of the GC-MS system must be
relied upon for the determination of dichlorobenzenes and naphthalene in the
presence of such interferences. It should also be recognized however, that
the alkylbenzenes and alkylnaphthalenes may represent nearly as great an
environmental hazard as some of the priority pollutants and thus their
presence should be of interest.
The GC-MS system used employed high-resolution glass capillary columns
and selected ion searching. Such a system provided high sensitivity, high
resolution of priority pollutants from any remaining interferences, and
high selectivity of detector responses. Quantitation was based upon the
total area of the selected ions of a priority pollutant peak relative to the
total area of the selected ions of an internal standard peak. Relative
response factors obtained from the analysis of standard solutions were used
as correction factors in the calculations of priority pollutant
concentrations.
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SLUDGE
00
JOO g Wet Weight
Acidify With KHS04
Extract With CU2C1
Cleanup Using Bio-Beads
S-X8
Extract With 0.2 N NaOH
I
Acidify Aqueous Phase
With 6 N HC1
|
Extract With CH2C12
|
Methylate .Using C112N2
Analyze by CC-MS
PHENOLS
100 g Wet Weight
Acidify With K1ISO,
Extract With CH C12
Wash With 0.1 N NaOH
Fractionate Using Bio-Beads S-X8
Collect Two Fractions, GPC-1
and GPC-2
Cleanup GPC-1 Using Activated
Silica Gel
Combine GPC-2 and Cleaned up
GPC-1
Analyze by GC-MS
NEUTRALS
10 g Wet Weight
Dilute with 0.1 M Phosphate
Buffer, pH7
Extract With C1IC1 Preserved
With Ethanol
Extract With 2 N lUSO,
T
Neutralize Aqueous Extract
to pll 6-7 Usine 1 M Na PO,
Extract With ClICK
Add Methanol to CHC1., Extract
and Concentrate
Dilute With 0.1 M Acetate
Buffer, pll 4.7
J
Analyze by HPLC Using
Electrochemical Detector
BENZIDINES
Figure 1. Scheme for determination of semi volatile priority pollutants in sludge.
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SECTION 6
EXPERIMENTAL STUDIES
The experimental details of the analytical procedure developed for
this program are described in Appendix A. The various studies involved
in developing and evaluating the procedure are discussed below. Although
there was not sufficient time for a systematic study of recoveries of all of
the compounds from each step during the development of the procedure various
modifications were studied briefly.
EXTRACTION
Several methods of extraction were studied including freeze drying
followed by Soxhlet extraction, methanol-drying followed by Soxhlet
extractionsazeotropic drying and the extraction associated with it, and
repetitive equilibration with solvent. Since the extraction step was
studied prior to the development of efficient cleanup methods, the effective-
ness of extraction was based on the yield of total extractable material
obtained.
Data comparing the effectiveness of various extraction techniques for
both raw and digested sludge are shown in Table 2. The values given are
in weight per cent of dry weight of sludge. The raw sludge was 4.0% solids
by weight and the digested sludge was 2.8% solids by weight.
The weight per cent of extractables recovered by each method was based
on the residue weight obtained by evaporating the solvent from a 100-yl
aliquot of the extract.
The data suggest that extraction method 6, 7, 8, or 9 would be the best
choice for the most efficient removal of the total organic material from
sludge. Homogenization of the sludge with dichloromethane (Method 8) was
selected for subsequent studies. The method is very rapid and simple and
is carried out at room temperature. A Tekmar Tissumiser was used for the
homogenization. Only Teflon and stainless steel, which are easily cleaned
prior to each extraction, contact the sample. Following homogenization
the sample is centirfuged and the organic layer withdrawn with a syringe.
The process is repeated four times to give a total of five extracts which
are combined and dried over magnesium sulfate.
It is desirable to avoid methanol in the extracting solvent because it
must be removed prior to subsequent cleanup on silica gel. This additional
step would require extra effort and presents a potential loss of priority
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TABLE 2. YIELDS FROM VARIOUS EXTRACTIONS METHODS
Yield, mg per 1QQ mg of dry sludge
Description of Extraction Method RawDigested
1. Soxhlet extract with MeOH then 1:1 benzene-
MeOH 18.4 7.6
2. Freeze dry; Soxhlet extract with DCM3 18.7 6.9
3. Freeze dry; Soxhlet extract with 1:1 benzene-
MeOH 20.2
4. MeOH wash; Soxhlet extract with 1:1 benzene-
MeOH 15.0 9.0
5. MeOH wash; Soxhlet extract with DCM 17.4 8.3
6. MeOH wash; Soxhlet extract with MeOH then
fresh 1:1 benzene-MeOH 25.6 11.3
7. Azeotropic drying with EDC ; centrifugation 25.6 11.6
8. Homogenization with DCM; centrifugation 22.4 12.1
9. Homogenization with 1:1 DCM-MeOH; centri-
fugation 26.3 ,12.7
a. Dichloromethane (methylene chloride)
b. Ethylene dichloride
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pollutants as great as the possible increase recovery during the initial
extraction.
The effectiveness of each of the five homogenization-extraction steps
using dichloromethane (DCM) as the solvent was determined. For this study
lOOg of a wet digested sludge was extracted five times with dichloromethane.
The results, shown in Table 3, indicate that the first three extractions
remove 95 per cent of the total organic material obtained from the five
extractions. Therefore only three extractions were used in subsequent
studies.
TABLE 3. REPETITIVE HOMOGENIZATION-EXTRACTION
OF SLUDGE WITH DCM
Extraction
Step
1
2
3
4
5
Weight of
Total Extract, mg
106
79
15
4
6
210
Weight Per cent
of Total
50
38
7
2
3
100
REMOVAL OF ACIDS FROM NEUTRALS
The approach initially tried for obtaining the neutrals involved start-
ing with a single methylene chloride extract to obtain both a neutral
fraction and an acid fraction. The acids were removed from the neutrals
by extraction of the extract from lOOg of wet sludge with four 50-ml portions
of 0.2 £J NaOH. The combined aqueous layers were acidified and back ex-
tracted with methylene chloride to give an acid fraction containing fatty
acids and phenols. However, the sludge used contained very high levels of
fatty acids, 1 to 2% on a wet weight basis, which formed so much soap that
the caustic extraction was not efficient. Some of the netural components
stayed in the soap solution and some of the soaps stayed in the methylene
chloride. Emulsions were also a problem.
Because of the above problems the extraction scheme was modified by
greatly increasing the amount of dilute caustic used for washing out fatty
acids and by adding salt to the water to avoid emulsion formation. However,
the large amount of water used made it difficult to back extract the
nitrophenols into methylene chloride after acidification. Consequently a
separate sludge extract was used for obtaining a cleaned up phenol fraction
in which gel permeation chromatography (GPC) was used as the first cleanup
step as will be discussed below.
11
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The neutral fraction was further fractionated by GPC to give one fract-
ion containing the phthalates and the more polar priority pollutants and a
second fraction containing the aromatic hydrocarbons, halocarbons, and ethers.
Only the first fraction was cleaned up further by silica gel chromatography.
The two fractions were then combined in most cases to give only one final
neutral extract for GC-MS analysis.
GPC STUDIES
Sephadex LH-20 Chromatography
Sephadex LH-20, a modified dextran gel was studied for use as a gel
permeation fractionation step to clean up groups of compounds on the basis of
molecular size. Three major conditions had to be considered. First, the
solvent system had to be compatible with that of the sludge extract. Second,
since Sephadex LH-20 is less dense than many common organic solvents, densi-
ties of different solvents had to be taken into consideration. Third, the
solvent system chosen had to optimize separations between different sizes of
molecules.
Since the concentrated sludge extract was in methylene chloride, the
first choice was to use methylene chloride as the eluting solvent in the
fractionation step. Since the Sephadex LH-20 was less dense than methylene
chloride, this necessitated the use of an upward-flow liquid chromatographic
apparatus. A 750 mm x 15 mm I.D. upward flow column was obtained for this
purpose and packed with approximately 50 grams of Sephadex LH-20 swelled in
methylene chloride. The apparatus was found to be an effective means of
fractionating the sludge extracts but it was determined after actual applica-
tion of sludge extracts that large amounts of material remained on the col-
umn after elution of the priority pollutants. This indicated the need for
washing the packing with a solvent which would remove the adsorbed material
or using fresh Sephadex LH-20 for each sample. Either alternative necessi-
tated repacking the upward flow column for each sample application. Since
it took considerable time to pack the upward flow apparatus, a compatible
solvent system for use in a downward flow system was considered to be a time-
saving alternative.
The choices of solvent systems in which the sludge was relatively solu-
ble were somewhat limited. The sludge extract was soluble in such solvents
as methylene chloride, ethylene dichloride, and benzene. The extract was
moderately soluble in acetone, slightly soluble in n-butyl chloride and
cyclopentane, and insoluble in methanol, ethanol, and isopropanol. Studies
were also done to determine which mixtures of these and similar solvents
would allow the use of a downward flow apparatus and also would be compatible
with the sludge extract. Nine solvent systems were decided upon for evalua-
tion, and are shown in Table 4. The solvent systems which provided the best
conditions were 80:20 ethylene dichloride:n-butyl chloride and 70:30 methylene
chloride:n-butyl chloride. The former, which contained less n-butyl chloride,
was chosen for further study because it was more compatible with the sludge
extract.
It was also necessary to determine the amount of material that the gel
could efficiently handle. Varying amounts of sludge extract was applied to
12
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TABLE 4. TEST SOLVENT SYSTEMS FOR SEPHADEX LH-20 FRACTIONATIONS
Solvent System
Comments
70:30 Methylene Chloride:Isopropanol
90:10 Ethylene Bichloride:Cyclopentane
80:20 Ethylene Bichloride:Acetone
80:20 Ethylene Bichloride:n-Butyl Chloride
80:20 Ethylene Dichloride:Benzene
70:30 Methylene Chloride:n-Butyl Chloride
70:30 Methylene Chloride:Cyclopentane
70:30 Methylene Chloride:Benzene
70:30 Methylene Chloride:Acetone
Isopropanol caused precipitation.
Gave poor separation.
Gave poor separation.
Designated for further studies.
Benzene is restricted solvent.
Designated for further study.
Gave poor separation.
Benzene is restricted solvent.
Gave poor separation.
-------
the Sephadex LH-20 columns, and it was found that approximately 5 mg of
extractable material could be applied for each gram of dry Sephadex LH-20
without significantly overloading the column.
After this process of optimizing the column run conditions, several
standards containing various classes of priority pollutants were run on the
column. It became evident that very polar compounds such as phenols did not
elute from the column.
BioBeads Chromatography
BioBeads are porous styrene-divinylbenzene copolymers which do not have
severe adsorption problems. The process of optimizing solvent systems was
repeated, mainly concentrating on those solvent systems which were success-
ful when used with Sephadex LH-20. Toluene was suggested by the manufacturer
as a possible solvent for this application and was studied along with
acetone, ethylene dichloride, methylene chloride, and 50:50 methylene
chloride:acetone. Also studied were the different types of BioBeads
differentiated by increasing pore sizes and hence increasing size exclusion
limits. The grades studied were BioBeads S-X12, S-X8, S-X4 and S-X3, in
order of increasing size exclusion limits.
Toluene was found to be unsatisfactory as a solvent since it did not
display good separating abilities. All of the other solvent systems dis-
played good separation abilities. Acetone was rejected because it did not
dissolve the sludge extractables as well as the other solvents.
As the size exclusion limit of the BioBeads gel was increased its bed
volume and separation ability increased. This indicated a tradeoff between
good separation ability and short fractionation times. BioBeads S-X8
provided good fractionation capabilities and an acceptable analysis time
and was chosen for further study- Elution profiles of whole sludge extracts
on BioBeads S-X12, S-X8, and S-X4 are shown in Figure 2.
The column flow parameter was found to be an important factor in
optimization of this technique. The flow rate was optimized at approximately
4 to 8 ml/hour/cm2. Therefore, a 15 mm I.D. column, which has a cross-
sectional area of 1.8 cm , would have an optimum flow rate of 7 to 14 ml/
hour.
It was anticipated that certain classes of compounds, specifically fatty
acids, triglycerides, and saturated hydrocarbons, would be present in large
amounts in the sludge extracts and would subsequently cause problems in
detecting low levels of the priority pollutants. It was therefore necessary
to ascertain the efficiency of the BioBeads S-X8 system in separating these
interfering compounds from the priority pollutants. Figure 3 represents
three samples run on a 40 mm x 9 mm I.D. column packed with 10 grams of Bio-
Beads S-X8 in 50:50 methylene chloride:acetone. The first sample (Figure
3(a)) was whole sludge extract, the second sample (Figure 3(b)) was com-
posed of fatty acids, and the third sample (Figure 3(c)) contained tri-
glycerides. It was ascertained by subsequent gas chromatographic analysis
that the later eluting material, as indicated in Figure 3(a), was composed
14
-------
Elution Volume, ml
Figure 2. Elution profiles of whole sludge extract on (a) BioBeads S-X12,
(b) BioBeads S-X8, and (c) BioBeads S-X4
-------
10
(a)
0
10
Triglycerldes
Fatty Acids
Saturated Hydrocarbons
(b)
Fatty Acids
10
bO-
B
(c)
Trlglycerides
10 15
Elution Volume, rnl
20
25
Figure 3. Elution profiles of (a) whole sludge extract, (b) fatty acids,
and (c) triglycerldes on BioBeads S-X8
-------
of saturated hydrocarbons.
Capacity tests were also performed using BioBeads S-X8, and it was
estimated that a maximum of 5 milligrams of sludge extractables per gram
of BioBeads S-X8 could be applied without significantly overloading the
column.
The use of BioBeads S-X8 in the place of Sephadex LH-20 eliminated the
adsorption problems encountered in the latter. When whole sludge was run
through a BioBeads S-X8 column, virtually all of the material was recovered
from the gel. This was also evident when phenol standards were run through
the BioBeads S-X8 and no adsorbance problems were observed. Since material
did not seem to adsorb to this column it was possible to reuse this column
for more than one sample, thus saving the time necessary to pack new BioBeads
S-X8 columns and also eliminating the necessity of purchasing large amounts
of the gel. This also made the use of BioBeads S-X8 upward flow column
packed in methylene chloride more desirable since the need for repeated re-
packing of the column was eliminated. The size of the column was increased
to increase its capacity to handle up to 1 gram of extractable material.
The performance of the column in terms of efficiency and retention times
of reference compounds was studied. Approximately 2500 theoretical plates
were achieved for the elution of di-n-octyl phthalate. The capacity factors,
K', for a number of reference compounds are given in Table 5.
TABLE 5. CAPACITY FACTORS OF REFERENCE
COMPOUNDS ELUTED WITH METHYLENE
CHLORIDE FROM BIO-BEADS S-X8
Compound Capacity Factor
r
Di-n-octyl phthalate 0.40
Dimethyl phthalate 0.67
Phenylacetic acid 0.70
2,4-Dinitrophenol 0.76
Hexaethylbenzene 0.83
2-Nitrophenol 0.92
4-Nitrophenol 0.99
2,4-Dimethylphenol 1.01
Phenol 1.08
2-Chlorophenol 1.09
2,4-Dichlorophenol 1.11
Benzene 1.19
Dibenz(a,h)anthracene 1.19
Pyrene 1.31
Sulfur 1.80
It is of particular interest to note that the K' for sulfur is consid-
erably higher than that of the other compounds. Because of this the GPC
17
-------
cleanup works very well for the removal of sulfur which would otherwise
interfere with the GC-MS analysis.
SILICA GEL CLEANUP
It was necessary to include relatively large molecules in the fraction
taken from the BioBeads cleanup of the neutral fraction in order to analyze
for the larger phthalates. This necessitated a cleanup step to remove the
relatively large amounts of long-chain hydrocarbons that were contained in
that fraction. Silica gel chromatography was evaluated as a method to
fractionate this extract on the basis of compound polarity. Standards con-
taining a wide variety of compounds were run on silica gel, and the type of
solvent system needed to elute each compound was noted. Nonpolar compound
such as saturated hydrocarbons eluted with nonpolar solvent such as
petroleum ether. Addition of increasing percentages of methylene chloride
in petroleum ether eluted more polar molecules such as subsituted benzenes,
naphthalenes and polynuclear aromatic hydrocarbons. It was necessary to go
to 5% acetone in methylene chloride to elute polar neutral compounds such
as isophorone, nitro compounds, and the phthalates. Very polar molecules
remained on the silica gel.
PACKED COLUMN GC-MS ANALYSIS
In an effort to simplify the GC-MS analysis somewhat and utilize pre-
viously developed EPA methods, the use of packed GC columns instead of
a capillary column was studied very briefly. It was found that the sensitiv-
ities achieved using packed columns, in terms of GC-MS peak areas, were
generally poorer, by a factor of 5 to 10, than those achieved using capillary
columns. Resolution, of course, was much lower with the packed columns.
The results of a single comparison of the two techniques applied to the
determination of components in spiked sludge are given in Table 6. The
amounts found using a capillary column were somewhat higher than those
found with packed columns. In some cases where components were found by the
capillary technique none were found using packed columns. Additional studies
are necessary for making a more valid comparison. However, since the
confirmation of the identity of each component detected by a selected ion
search is based upon its full mass range spectrum, the superior resolution
of capillary column systems should give significantly fewer false negatives
in a matrix as complex as sludge extracts.
ANALYSIS OF PHENOLS
Methylation of phenols by diazomethane is used to improve their gas
chromatographic properties. Although the more acidic phenols, e.g. nitro-
phenols and trichlorophenol, methylate rather completely, the less acidic
phenols, e.g. dimethylphenol, chlorophenol, or phenol itself, are only
partially derivatized if at all.
One approach that was studied in an effort to achieve an improvement
in the analysis of phenols, was the use of a packed GC column, 1% SP-1240DA,
for the analysis of free phenols. The column gave good separation and good
peak shape for all of the phenols however the sensitivity was lower than that
18
-------
obtained with a capillary column. The packed column data for phenols in
Table 6 was obtained using 1% SP-1240DA. Despite the disadvantage of poorer
sensitivity the packed column approach offers the advantage of eliminating
the methylation step and therefore solves the problem of incomplete
methylation of the less acidic phenols.
Another approach to the derivatization problem is the use of a
different derivatizing agent that might be suitable for all phenols. With
this in mind we investigated briefly the preparation of pentafluorobenzyl
derivatives by reaction with pentafluorobenzyl bromide (PFBB) in the
presence of potassium carbonate and a crown ether. The method worked very
well for the less acidic phenols but did not work for nitrophenols at low
levels. Since the less acidic phenols can be determined satisfactorily as
free phenols using a capillary column and the more acidic phenols are readily
methylated by diazomethane this method was retained as the method of choice.
DETERMINATION OF PESTICIDES
The pesticides and PCBs included in the list of priority pollutants
are similar to the semivolatile neutrals in terms of solubility, polarity,
and size. Accordingly they would be expected to be found in the same
fraction as the neutrals in the analysis scheme developed on this program.
This means that the pesticides and PCBs could be determined at the same time
that the neutrals are being determined with no additional effort other than
the additional selected ion searches and calibration studies to establish
retention times and response factors.
In an effort to validate this concept a sample of digested sludge was
spiked with aldrin, eudrin, heptachlor, p,p'-DDT, p,p'-DDE, p,p'-DDD, y-BHC,
and Arochlor 1254 at a level of 10 yg/100 g wet weight and analyzed using
the procedure in Appendix A. All of the spiked components were found in the
GPC-2 neutral fraction. The recoveries were greater than 50% for each
component. The results indicate that separate analysis schemes for pest-
icides and PCBs are not necessary.
ANALYSIS OF SPIKED AND NONSPIKED SAMPLES
Samples of distilled water, raw municipal sludge, and digested
municipal sludge were spiked with all 54 of the priority pollutants of
concern and analyzed in triplicate. Similar analyses were performed for
nonspiked samples in triplicate. The spiking level varied from 2.5 to 6
yg/lOOg depending upon the concentrations in standard solutions obtained
from Supelco, Inc. The analytical procedures used was that described in
detail in Appendix A. The recoveries obtained are given in Tables 7, 8,and 9.
The recoveries from the water samples were very good for nearly all of
the components except for dibenzo(a,h)anthracene and some of the phenols.
This indicates that with a few exceptions the components are not being lost
by the cleanup procedure in the absence of interferences. Most of the
components are also recovered from raw or digested sludge, however, in many
cases, a meaningful value for percent recovery could not be calculated be-
cause of the very significant but highly variable amounts found in the
19
-------
TABLE 6. COMPARISON OF PACKED COLUMN AND CAPILLARY COLUMN
GC ANALYSIS FOR DETERMINING THE RECOVERY OF
PRIORITY POLLUTANTS FROM DIGESTED MUNICIPAL SLUDGE£
Amount Amount Recovered, pg/100
Added,
Compound'3 yg/100 ml
Bis-(2-chloroethyl) ether
1, 3-Dichlorobenzene
1,4-Dichlorobenzene
1 , 2-Dichlo rob enzene
Bis-(2-chloroisopropyl) ether
N-Nitrosodipropylamine
Nitrobenzene
Bis- ( 2- chloroe thoxy ) me thane
1, 2,4-Trlchlorobenzene
Naphthalene
Hexachlorobutadiene
2-Chloronaphthalene
2, 6-Dlnltro toluene
Dimethyl phthalate
Acenaphthylene
Acenaphthene
2 , 4-Dini tro toluene
Diethyl phthalate
Fluorene
4-Chlorophenyl phenyl ether
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
Packed
Column0
NAe
4.6f
NA
NA
NA
NA
1.9
5.7
ND§
ND
NA
NA
ND
ND
NA
NA
ND
ND
ml, Using Given GC Method
Capillary
Columnd
NA
2.1
2.1
2.1
NA
NA
NA
NA
6.7
12
1.1
3.4
NA
NA
ND
1.8
NA
NA
7.7
ND
-------
-TABLE 6. (Continued)
Amount Amount Recovered, pg/100 ml, Using Given GC Method
Compound"
N-Nitrosodiphenylamine
4-Bromophenyl phenyl ether
Hexachlorobenzene
Phenanthrene
Anthracene
Di-n-butyl phthalate
Fluoranthene
Pyrene
Butylbenzyl phthalate
Chrysene
Benzo (a) anthracene
Bis (2-e thy Ihexyl) phthalate
Di-n-octyl phthalate
Benzo (b ) f luoranthene
Benzo (k) f luoranthene
Benzo (a) pyrene
Benzo ( g , h , i) perylene
Indeno(l, 2, 3-cd) pyrene
Dibenzo (a , h) anthracene
Acids
2-Chlprophenol
Added,
yg/100 ml
5.0
5.0
5.0
5.0
5.0
5.0
2.5
2.5
5.0
2.5
2.5
5.0
5.0
2.5
2.5
2.5
2.5
2.5
2.5
5.0
Packed
Column0
NA
0.4
0.5
9.0h
NA
1.1
1.5
NA
ND
ND
NA
NA
ND
ND
ND
ND
ND
ND
1.3
Capillary
Columnd
NA
1.9
2.2
15
15
NA
6.0
7.7
NA
6.1
6.1
NA
NA
3.7
3.7
4.0
2.1
2.3
ND
1.2
-------
TABLE 6. (Continued)
Amount Amount Recovered, ug/100 ml, Using Given GC Method
Compound^
Phenol
2 , 4-Dimethylphenol
2,4-Dlchlorophenol
2,4, 6-Trichlorophenol
2-Nitrophenol
4-Chloromethyphenol
4-Nitrophenol
4 , 6-Dinitro-o-cresol
Pentachlorophenol
2,4-Dinitrophenol
Added,
IJg/lOO ml
6.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
Packed
Column0
7.0
2.0
3.0
3.0
ND
3.0
ND
ND
3.0
ND
Capillary
Columnd
11.0
1.6
6.1
5.1
ND
10
ND
ND
3.3
ND
The digested sludge used had a dry solids content of 2.5 g/100 ml and a total
lipids content of 0.5 g/100 ml.
b. The priority pollutant standards used were purchased from Supelco, Inc.
c. A2mx2mmI.D. glass column packed with 3% SP-2250-DB on 100-120 mesh
Supelcoport was used for neutrals and a 2 m x 2 mm I.D. glass column packed
with 1% SP-1240-DA on 100-200 Supelcoport was used for free phenols.
d. A 30 m x 0.2 mm I.D. glass capillary column coated with SE-30 was used for
neutrals and methylated phenols.
e. Not appropriate; only fraction GPC-2 and the acid fraction were analyzed using
the packed column; this component does not appear in these fractions.
f. Total for all three isomers.
g. Not detected.
h. Total for phenanthrene and anthracene.
-------
TABLE 7. RECOVERY OF PRIORITY POLLUTANTS FROM WATER
Co
Compound
Neutrals
Bis-(2-chloroethyl) ether
1 , 3-Dichlorobenzene
1 ,4-Dichlorobenzene
1,2-Dichlorobenzene
Bis-(2-chloroisopropyl) ether
N-Nitrosodipropylamine
Nitrobenzene
Bis-(2-chloroethoxy) methane
1 , 2 ,4-Trichlorobenzene
Naphthalene
Hexachlorobutadiene
2-Chloronaphthalene
2,6-Dinitrotoluene
Dimethyl phthalate
Acenaphthylene
Acenaphthene
2 ,4-Dinitrotoluene
Diethyl phthalate
Amount
Added,
Ug/100 ml
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
.0
.0
.0
.0
.0
.0
.0
.0
.0
.0
.0
.0
.0
.0
.0
.0
.0
.0
Amount Recovered, yg/100 ml
, in Given Sample
Unspiked
1
NDe
ND
ND
ND
ND
ND
ND
ND
ND
0.4
ND
ND
ND
0.2
ND
ND
ND
0.8
2
ND
'ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
0.2
3 Avg.
ND
ND
ND
ND
ND
ND
ND
ND
ND
0.3 0.2
ND
ND
ND
ND 0.1
ND
ND
ND
0.6 0.5
1
3
3
3
6
5
6
4
3
5
6
2
6
5
9
7
7
5
7
.3
.9
.9
.0
.4
.8
.8
.9
.0
.7
.8
.6
.6
.0
.3
.4
.3
.3
Spiked
2
1.3
2.8
2.8
2.9
1.1
1.1
4.3
2.6
3.5
4.0
4.7
3.2
ND
5.6
5.5
4.7
ND
5.8
3
(f)
(f)
(f)
(f)
(f)
(f)
(f)
(f)
(f)
(f)
(f)
(f)
(f)
(f)
(f)
(f)
(f)
(f)
Avg.
2.3
3.4
3.4
4.5
3.3
4.0
4.6
3.3
4.3
5.4
3.8
4.9
2.8
7.3
6.4
6.1
2.7
6.6
Average ,
Recovery,
%
46
67
67
89
65
79
91
65
85
104
75
98
56
144
128
121
54
121
-------
TABLE 7. (Continued)
NJ
-O
2
Compound
Acids
c
2-Chlorophenol
c
Phenol
c
2 ,4-Dimethylphenol
c
2 , 4-Dichlorophenol
d
2 ,4 ,6-Trichlorophenol
d
2-Nitrophenol
4-Chloromethylphenol
4 -Nitrophenol
4 ,6-Dinitro-o-cresol
Pentachlorophenol
2,4-Dinitrophenol
Bases
Benzidine
3,3' -Dichlorobenzidine
a. The priority pollutant standards
Amount
Added,
Amount Recovered, pg/100
Unspiked
Ug/100 ml 1
5.0
6.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
0.6
0.6
used were
b. (Avg. Recovered from Spiked Sample) - (Avg.
c. Determined as the free phenol
d. Determined as the methyl ester
e. Not detected
f. Neutral fraction lost
ND
0.9
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
purchased
Recovered
2
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
from
from
3 Avg.
ND
ND 0.3
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
Supelco, Inc.
Unspiked Sample)
Amount Added
ml, in Given Sample
Spiked
1 2 3 Avg.
0.7 0.9 ND 0.5
2.7 ND ND 0.9
ND ND ND —
4.0 0.6 2.2 2.3
4.8 ND 4.5 3.1
ND ND ND
6.4 ND 8.6 5.0
ND ND ND —
5.5 ND ND 1.9
4.7 0.1 3.1 2.6
ND ND ND —
0.4 0.4 0.4 0.4
0.4 0.6 0.6 0.5
•v i nn
A -LUU
Average
Recovery ,
%
10
10
—
46
62
—
100
—
38
52
—
67
83
-------
TABLE 7. (Continued)
N3
Ol
Compound
Neutrals
Fluorene
4-Chlorophenyl phenyl ether
N-Nitrosodiphenylamine
4-Bromophenyl phenyl ether
Hexachlorobenzene
Phenanthrene
Anthracene
Dl-n-butyl phthalate
Fluoranthene
Pyrene
Butylbenzyl phthalate
Chrysene
Benzo (a) anthracene
Bis ( 2-ethylhexyl ) phthalate
Di-n-octyl phthalate
Benzo (b) f luoranthene
Benzo (k) f luoranthene
Benzo(a)pyrene
Benzo (g,h,i)perylene
Indeno(l,2,3-cd)pyrene
Dibenzo (a, h) anthracene
Amount
Added,
Amount Recovered, yg/100 ml, in Given Sample
Unspiked
yg/100 ml 1
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
2.5
2.5
5.0
2.5
2.5
5.0
5.0
2.5
2.5
2.5
2.5
2.5
2.5
ND
ND
ND
ND
ND
0.6
ND
3.7
0.3
0.4
27
ND
ND
0.9
0.4
ND
ND
ND
ND
ND
ND
2
ND
ND
ND
ND
ND
ND
ND
0.6
ND
ND
0.3
ND
ND
0.1
ND
ND
ND
ND
ND
ND
ND
3 Avg.
ND
ND
ND
ND
ND
ND 0.2
ND
0.9 1.7
ND 0.1
ND 0.1
1.7 0.7
ND
ND
0.9 0.6
0.3 0.2
ND
ND
ND
ND
ND
ND
1
7.4
4.5
2.0
5.1
5.7
5.6
7.6
10.9
2.1
2.5
7.4
2.5
2.5
11.2
8.3
2.5
2.5
2.2
0.6
2.0
ND
Spiked
2
5.1
4.1
5.7
6.2
6.2
6.3
7.8
6.6
3.4
4.6
2.6
3.2
3.2
0.3
0.4
2.2
2.2
1.6
0.9
0.3
ND
3
(f)
(f)
(f)
(f)
(f)
(f)
(f)
(f)
(f)
(f)
(f)
(f)
(f)
(f)
(f)
(f)
(f)
(f)
(f)
(f)
(f)
Avg.
6.3
4.3
3.9
5.7
6.0
6.0
7.7
8.8
2.8
3.6
5.0
2.9
2.9
5.8
4.4
2.4
2.4
1.9
0.8
1.2
—
Average
Recovery ,
°/
125
86
77
113
119
116
154
142
108
140
86
114
114
104
84
96
96
76
30
48
—
-------
TABLE 8. RECOVERY OF PRIORITY POLLUTANTS FROM DIGESTED MUNICIPAL SLUDGE
NJ
Compound
Neutrals
Bis-(2-chloroethyl) ether
1 , 3-Dichlorobenzene
1 , 4-Dichlorobenzene
1 , 2-Dichlorobenzene
Bis- (2-chloroisopropyl) ether
N-Nitrosodipropylamine
Nitrobenzene
Bis- (2-chloroethoxy)methane
1,2, 4-Tr ichlorobenzene
Naphthalene
Hexachlorobutadiene
2-Chloronaphthalene
2 , 6-Dinitrotoluene
Dimethyl phthalate
Acenaphthylene
Acenaphthene
2,4-Dinitrotoluene
Diethyl phthalate
Amount
Added,
yg/100 ml
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
.0
.0
.0
.0
.0
.0
.0
.0
.0
.0
.0
.0
.0
.0
.0
.0
.0
.0
Amount Recovered, ng/100 ml,
Unspiked
1
NDf
ND
ND
ND
ND
ND
ND
ND
0.3
5.1
ND
ND
ND
ND
ND
1.4
ND
37
2
ND
9.3
9.3
9.3
ND
ND
ND
ND
2.8
32
ND
ND
ND
ND
ND
6.2
ND
0.4
3
ND
1.8
1.8
1.8
ND
ND
ND
ND
0.4
5.8
ND
ND
ND
1.0
ND
1.7
ND
ND
Avg.
3.7
3.7
3.7
1.2
14
— -
0.3
2.4
12
1
ND
0.1
0.1
0.1
ND
ND
ND
ND
1.0
3.7
0.5
0.7
ND
0.7
ND
0.6
ND
1.5
in Gfven Sample
Spiked
2
ND
10
10
10
ND
1.5
ND
ND
13
38
4.9
10.1
ND
5.2
ND
6.4
ND
5.0
3
ND
2.1
2.1
2.1
ND
5.4
ND
ND
6.7
12
1.1
3.4
ND
14
ND
1.8
ND
16
Avg.
4.1
4.1
4.1
—
2.3
—
—
6.9
18
2.2
4.7
—
6.7
—
2.9
—
7.5
Average
Recovery,
%
NM8
NM
NM
—
46
—
—
NM
NM
44
94
—
128
—
NM
—
NM
-------
TABLE 8. (Continued)
Compound
Neutrals
Fluorene
4-Chlorophenyl phenyl ether
N-Nitrosodiphenylamine
4-Bromophenyl phenyl ether
Hexachlorobenzene
Phenanthrene
Anthracene
Di-n-butyl phthalate
Fluoranthene
Pyrene
Butylbenzyl phthalate
Chrysene
Benzo (a) anthracene
Bis(2-ethylhexyl)phthalate
Di-n-octyl phthalate
Benzo (b)f luoranthene
Benzo (k) f luoranthene
Benzo (a) pyrene
Benzo(g,h, i)perylene
Indeno(l, 2, 3-cd) pyrene
Dibenzo (a , h) anthracene
Amount
Added,
yg/100 ml
5
5
5
5
5
5
5
5
2
2
5
2
2
5
5
2
2
2
2
2
2
.0
.0
.0
.0
.0
.0
.0
.0
.5
.5
.0
.5
.5
.0
.0
.5
.5
.5
.5
.5
.5
Amount Recovered, pg/100 ml, in Given Sample
Unspiked
1
1.9
ND
ND
ND
ND
8.1
8.1
7.4
3.8
4.8
150
3.2
3.2
690
190
0.7
0.7
ND
ND
ND
ND
2
6.6,
ND
ND
ND
ND
35
35
4.0
16
24
33
15.0
15.0
13
9.7
4.9
4.9
6.4
3.2
3.9
ND
3
ND
ND
ND
ND
ND
6.3
6.3
2.5
3.9
5.1
60
4-2
4.2
12
8.1
1.9
1.9
2.4
0.8
1.1
ND
Avg.
2.8
—
—
—
—
16
16
27
7.9
12
81
7.5
7.5
240
93
2.5
2.5
2.9
1.3
1.7
—
1
1.0
4.0
ND
0.7
ND
7.7
7.7
13.1
3.4
4.1
47
3.6
3.6
150
64
1.0
1.0
1.0
1.7
2.2
ND
Spiked
2
16.0
ND
ND
4.3
3.8
39
39
9.7
18
24
64
14
14
21
9
4.3
4.3
7.2
0.8
1.6
ND
3
7.7
ND
ND
1.9
2.2
15
15
32
6.0
7.7
400
6.1
6.1
100
66
3.7
3.7
4.0
2.1
2.3
ND
Avg.
8.5
1.3
—
2.3
2.0
21
21
18
9.1
12
170
7.9
7.9
90
46
3.0
3.0
4.1
1.5
2.0
—
Average
Recovery,
NM
26
—
46
40
NM
NM
NM
NM
NM
NM
NM
NM
NM
NM
NM
NM
NM
NM
NM
—
-------
TABLE 8. (Continued)
Compound
Amount
Added,
pg/100 ml
Amount Recovered, pg/100 ml, in Given Sample
Unspiked Spiked
Avg.
Avg.
Average
Recovery,
CO
Acids
2-Chlorophenol
Phenold
2,4-Dimethylphenol
2,4-Dichlorophenol
2,4,6-Trichlorophenol
2-Nitrophenol
d
4-Chloromethyphenol
e
4-Nitrophenol
e
4,6-Dinitro-o-cresol
e
Pentachlorophenol
g
2,4-Dinitrophenol
5.0
6.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
ND
ND
ND
ND
ND
ND
NU
ND
ND
ND
HD
15
ND
ND
ND
ND
ND
ND
ND
ND
NTJ
ND
0.4
ND
ND
ND
SID
ND
ND
ND
ND
ND
5.1
1.2 4.5
11.0 14
1.6
6.1
5.1
ND
10
ND
ND
3.3
ND
ND
5.1
3.2
ND
ND
ND
ND
1.6
ND
3.5
0.8
1.5
5.6
4.9
ND
2.9
ND
ND
2.6
ND
3.1
8.6
1.0
5.6
4.4
4.3
2.5
6.2
MM
20
112
86
50
Bases
Benzidine
3,3'-Dichlorobenzidine
0.6
0.6
ND
ND
ND
ND
ND
ND
0.4
0.3
0.4
0.2
1.2
0.6
0.7
0.4
117
67
The priority pollutant standards used were purchased from Supelco, Inc.
The digested sludge used had a dry solids content of 2.5 g/100 ml and a total lipid content of
0.5 g/100 ml.
(Avg. Recovered from Spiked Sample) - (Avg. Recovered from Unspiked Sample)
Amount Added
x 100
Determined as the free phenol
Determined as the methyl ether
Not detected
Not meaningful because of large amounts and/or wide variations in amounts found in unspiked
samples as well as in spiked samples.
-------
TABLE 9. RECOVERY OF PRIORITY POLLUTANTS FROM RAW SLUDGE
Compound
Neutrals
Bis- (2-chloroethyl) ether
1, 3-Dichlorobenzene
1 , 4-Dichlorobenzene
1, 2-Dichlorobenzene
Bis-(2-chloroisopropyl) ether
N-Nitrosodipropylamine
Nitrobenzene
Bis-(2-chloroethoxy)methane
1,2, 4-Trichlorobenzene
Naphthalene
Hexachlorobutadiene
2-Chloronaphthalene
2 , 6-Dinitrotoluene
Dimethyl phthalate
Acenaphthylene
Acenaphthene
2,4-Dinitrotoluene
Diethyl phthalate
Fluorene
Amount
Added,
yg/100 ml
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
.0
.0
.0
.0
.0
.0
.0
.0
.0
.0
.0
.0
.0
.0
.0
.0
.0
.0
.0
Amount Recovered, pg/100 ml, in Given Sample
1
NDf
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
0.7
5.0
Unspiked
2
ND
55
55
55
ND
ND
ND
ND
ND
65
ND
ND
ND
0.6
ND
21
ND
1.1
30
3
ND
15
15
15
ND
ND
ND
ND
0.4
30
ND
ND
ND
0.2
ND
5.9
ND
4.5
22
Avg.
33
33
33
—
—
—
—
0.1
32
—
—
—
0.3
—
9.0
—
2.1
17
1
6.1
0.2
0.2
0.2
ND
2.3
ND
7.2
2.0
7.5
1.0
ND
ND
6.1
ND
4.0
ND
3.5
8.0
Spiked
2
6.1
25
25
25
ND
6.7
ND
7.3
7.5
35
1.8
ND
ND
5.9
OT
26
ND
6.9
19
3
0.2
20
20
20
ND
6.2
ND
6.3
8.6
46
2.6
1.0
ND
6.0
ND
9.1
ND
7.2
27
Avg.
4.1
15
15
15
—
5.1
—
6.9
6.0
29
1.8
0.3
—
6.0
—
13
—
5.9
18
Average c
Recovery,
%
82
NM8
NM
NM
NM
102
—
138
118
NM
36
6
—
114
—
NM
—
76
NM
-------
TABLE 9. (Continued)
UJ
O
Compound
Neutrals
4-Chlorophenyl phenyl ether
N-Nitrosodiphenylamine
4-Bromophenyl phenyl ether
Hexachlorobenzene
Phenanthrene
Anthracene
Di-n-butyl phthalate
Fluoranthene
Pyrene
Butylbenzyl phthalate
Chrysene
Benzo (a) anthracene
Bis(2-ethylhexyl)phthalate
Dl-n-octyl phthalate
Benzo (b) f luoranthene
Benzo (k) f luoranthene
Benzo (a) pyrene
Benzo (g,h,i)perylene
Ind eno(l, 2, 3-cd) pyrene
Dibenzo (a , h) anthracene
Amount
Added,
g/100 ml
5
5
5
5
5
5
5
2
2
5
2
2
5
5
2
2
2
2
2
2
.0
.0
.0
.0
.0
.0
.0
.5
.5
.0
.5
.5
.0
.0
.5
.5
.5
.5
.5
.5
Amount Recovered, g/100 ml, in Given Sample
Unspiked
1
ND
ND
ND
ND
39
39
13
25
19
280
7.6
7.6
140
78
3.3
3.3
4.3
1.3
2.7
ND
2
ND
ND
ND
ND
27
27
53
34
35
180
22
22
44
48
5.7
5.7
5.9
4.8
5.5
ND
3
ND
ND
ND
ND
50
50
22
8.9
8.9
40
7.5
7.5
29
26
2.3
2.3
2.4
ND
ND
ND
Avg.
—
—
39
39
29
23
21
170
12
12
71
51
3.8
3.8
4.2
2.0
2.7
—
1
1.2
2.4
3.1
2.5
34
34
64
11
12
450
8.6
8.6
100
19
3.5
3.5
5.2
ND
ND
ND
Spiked
2
3.3
5.0
2.4
1.4
38
38
55
26
26
180
25
25
45
34
12
12
7.6
2.2
3.3
ND
3
3.2
3.3
2.4
2.4
68
68
14
14
13
45
9.1
9.1
44
30
4.5
4.5
5.9
2.5
3.3
ND
Avg.
2.6
3.6
2.6
2.1
47
47
44
17
17
225
14
14
63
28
6.7
6.7
6.2
1.6
2.2
—
Average
Recovery,
%
54
72
52
42
NM
NM
NM
NM
NM
NM
NM
NM
NM
NM
116
116
80
NM
NM
—
-------
TABLE 9. (Continued)
Amount
Added
Compound pg/100 ml
Acids
2-Chlorophenol
Phenold
2 , 4-Dimethylphenol
2 , 4-Dichlorophenol
2,4, 6-Trichlorophenol
2-Nitrophenole
4-Chloromethy] Dhenol
4-Nitropnenole
4, 6-Dinitro-o-cresol
Pentachlorophenol
2, 4-Dinitrophenole
Bases
Benzidine
3, 3-Dichlorobenzidine
5.0
6.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
1.2
1.2
a. The priority pollutant standards used
b. The raw sludge used had a dry
1.4 Mg/100 ml.
solids
c. (Avg. Recovered from Spiked Sample) -
Amount Recovered, pg/100 ml, in
Unspiked
1
ND
14
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
2
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
were purchased
content of
4.4
3 Avg.
ND
ND 5
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
from Supelco,
yg/100 ml and a
(Avg. Recovered from Unspiked
Amount Added
d. Determined as the free phenol
e. Determined as the methyl ether
f . Not detected
1
ND
16
ND
1.4
0.2
ND
2.9
ND
ND
2.6
ND
0.8
1.2
Inc.
Given Sample
Spiked
2
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
0.8
1.2
total lipid
Sample)
v i r\r\
x 1UU
3 Avg.
ND
14 10
WD
3.9 1.8
2.0 0.7
ND
ND 1.0
ND
ND
6.7 3.1
ND
0.8 0.8
1.6 1.3
content of
Average
Recovery,
%
—
NM
—
36
14
—
20
—
—
62
—
100
108
Not meaningful because of large amounts and/or wide variations in amounts found in unspiked
samples as well as in spiked samples.
-------
unspiked samples. There was a noticeable lack of success in recovering some
of the more polar neutrals and the nitrophenols. The poor recoveries could
have been caused by various factors including degradation during the 24-hour
period of equilibration with sludge, poor extraction from the sludge matrix,
loss to the aqueous phase during caustic extraction steps, and alteration
of separation patterns caused by large amounts of fatty acids. These are
discussed under Recommendations.
32
-------
APPENDIX
S100 METHOD FOR SEMIVOLATILE ORGANIC COMPONENTS
110 Scope and Application
111 This method covers the determination of 54 semi-volatile organic
priority pollutants. A complete list of these compounds is given
in Table 111.
112 The method is applicable to the measurement of these compounds
in raw and digested municipal sludge.
113 The method is capable in most cases of detecting 2 pg of a
priority pollutant per 100 ml of wet sludge (0.5 yg/g dry weight
basis for sludge containing 4% solids).
120 Summary
121 This method offers separate procedures for the analysis of the
acidic, neutral, and basic components. The procedure involves
the use of repetitive solvent extraction to efficiently recover
components from the sludge matrix. Gel permeation chromatography,
silica-gel chromatography, and acid-base extractions are used as
cleanup procedures to eliminate interferences. The acidic
(phenolic) and neutral components are determined by GC-MS analysis
using high-resolution glass capillary columns and selected ion
searches. The bases, benzidine and 3,3-dichlorobenzidine, are
determined by HPLC analysis using an electrochemical detector.
The overall analysis scheme is shown in Figure 121.
122 This method is recommended for use only by analysts experienced
in liquid chromatography, glass capillary column GC-MS analysis,
and trace organic analysis of environmental samples, or under the
close supervision of such qualified persons.
130 Apparatus and Reagents
131 For sample extraction, Section 150:
131.1 Tekmar tissuemizer
131.2 Solvents, distilled-in-glass grade:
a. Methylene chloride
b. Chloroform preserved with ethanol
33
-------
TABLE 111. EPA SEMIVOLATILE ORGANIC PRIORITY POLLUTANTS
2.
3.
4.
5.
6.
7.
8.
Polvcyclic Aromatic Hydrocarbon
1.
2.
3.
4.
1.
2.
Acenaphthene
Acenaphthylene
Anthracene
Benzo(a)anthracene
Benzo(b)fluoranthene
Benzo(k)fluoranthene
Benzo(g,h,i)perylene
3enzo(a)pyrene
9. Chrysene
10. Dibenzo(a,g)anthracene
11. Fluoranthene
12. Fluorene
13. Indeno(l,2,3-cd)pyrene
14. Naphthalene
15. Phenanthrene
16. Pyrene
Phthalates
1. Bis(2-ethylhexyl) phthalate
2. Butylbenzyl phthalate
3. Diethyl phthalate
4. Dimethyl phthalate
5. Di-n-butyl phthalate
6. Di-n-octyl phthalate
Chlorinated Hydrocarbons
2-Chloronaphthalene
1,2-Dichlorobenzene
1,3-Dichlorobenzene
1,4-Dichlorobenzene
5.
6.
7.
8.
Chloroalkvl Ethers
Hexachlorob enzene
1,2,4-Trichlorobenzene
Hexachlorobutadiene
Hexachlorocyclopentad iene
3is-(2-chloroethyl) ether
Bis-(2-chloroethoxy) methane
3. Bis-(2-chloroisopropyl) ether
Nitrosamines
1. N-Nitrosodiethylamine 2. N-Nitrosodiphenylamine
Miscellaneous Neutrals
1. 4-Bromophenyl phenyl ether
2. 4-Chlorophenyl phenyl ether
3, 2,4-Dinitrotoluene
1. 4-Chloro-3-methylphenol
2. 2-Chlorophenol
3. 2,4-Dichlorophenol
4. 2,4-Diaethylphenol
5. 4,6-Dinitro-2-methylphenol
6. 2,4-Dinitrophenol
Benzidine
Acids
4. 2,6-Dinitrotoluene
5. Isophorone
6. Nitrobenzene
7. 2-Nitrophenol
8. 4-Nitrophenol
9 - Pentachlorophenol
10. Phenol
11. 2,4,6-Trichlorophenol
Bases
3,3'-Dichlorobenzidine
34
-------
SLUDGE
OJ
100 g Wet Weight
Acidify With KHSO.
I *
Extract With CII2C12
Cleanup Using Bio-Beads
S-X8
Extract With 0.2 N NaOH
I
Acidify Aqueous Phase
With 6 _N HC1
Extract With CII2C12
Methylate Using CILN,,
Analyze by GC-MS
PHENOLS
100 g Wet Weight
Acidify With KHSO
Extract With CII Cl™
Wash With 0.1 N NaOH
r
Fractionate Using Bio-Beads S-X8
Collect Two Fractions, GPC-1
and CPC-2 i
Cleanup GPC-1 Using Activated
Silica Gel i
Combine GPC-2 and Cleaned up
GPC-1 i
Analyze by GC-MS
NEUTRALS
10 g Wet Weight
Dilute with 0.1 M Phosphate
Buffer, pll?
Extract With CIIC1 Preserved
With Ethanol
Extract With 2 N H2SO/(
Neutralize Aqueous Extract
to pll 6-7 Using 1 M Na P0/
Extract With C1IC1,
Add Methanol to CI1C1 Extract
and Concentrate
Dilute WJth 0.1 M Acetate
Buffer, pll 4.7
Analyze by 1IPLC Using
Electrochemical Detector
BENZTD1NES
Figure 121. Scheme for determination of semi volatile priority pollutants in sludge.
-------
c. Methanol
131.3 Glassware
a. Centrifuge tubes - 50 ml and 200 ml, with Teflon-
lined screw caps
b. Round-bottom flasks - 500 ml and 100 ml, with 24/40
joints
c. Vortex evaporator tubes - 15 ml
d. Separatory funnels - 125 ml with Teflon stopcocks
131.4 Rotating evaporator
131.5 Vortex evaporator
131.6 Reagents
a. Magnesium sulfate, anhydrous - conditioned at 450°C
b. Potassium bisulfate, anhydrous - conditioned at 450°C
c. Phosphate buffer - 0.1 M, pH 7
131.7 Syringe - 50 ml with 8-inch 15-gauge square-tipped needle
131.8 Microbalance
131.9 Aluminum foil pans - 25 mm
132 For removal of interferences, Section 160.
132.1 Reagents
a. Magnesium sulfate, anhydrous - conditioned at 450°C
b. Sodium hydroxide - 0.1 N in 10% NaCl
c. Sodium hydroxide - 20%
d. Hydrochloric acid - 6 IS
e. Trisodium phosphate - 0.4 M
f. Sulfuric acid - 2 Jfl
g. Diazald
h. Acetate buffer - 0.1 M, pH 4.7
132.2 Glassware
a. Separatory funnels - 60 ml, 125 ml, and 500 ml with
Teflon stopcocks
b. Micro diazomethane generating apparatus - obtained from
Paxton Woods Glass Shop, 7500 Brill Road, Cincinnati,
Ohio
c. Round-bottom flasks - 100 ml and 500 ml
d. Vortex evaporator tubes - 15 ml graduated, screw cap,
conical centrifuge tubes
e. Centrifuge tubes - 50 ml and 200 ml, screw cap
f. Chromatography column - 400 mm x 9 mm I.D. Lab-Crest
column with 100-ml reservoir, scintered glass frit,
Teflon stopcock, and Solv-Seal joints
132.3 Solvents, distilled-in-glass grade
a. Methylene chloride
b. Petroleum ether, b.p. 30-60°C
c. Acetone
d. Hexane
e. Ethylene dichloride
f. Chloroform preserved with ethanol
132.4 Silica gel - 100-200 mesh Davison grade 923, activated for
16 hours at 150°C
36
-------
132.5 Standard solutions
a. GPC calibration solution for neutrals - methylene
chloride containing 1 mg each of di-n-tridecyl
phthalate, di-n-octyl phthalate, 4-chlorophenyl phenyl
ether, dimethyl phthalate, pyrene, and sulfur per ml
b. GPC calibraton solution for acids - methylene chloride
containing 1 mg each of 4-phenylbutyric acid, 2,4-
dinitrophenol, 2,4-dichlorophenol, and sulfur per ml
c. DDA internal standard solution - 10 mg of deca-
deuteroanthracene in 100 ml of heptane
132.6 Rotating evaporator
132.7 Vortex evaporator
132.8 Gel-permeation chromatography system*
a. Chromatographic column - 1200 mm x 25 mm I.D. glass
b. Bio-Beads S-X8 - 200 g per column
c. Pump - capable of constant flow of 0.1 to 5 ml/min at up
to 100 psi
d. Injector - with 5 ml loop
e. Ultra-violet detector - 254 nm
f. Strip-chart recorder
132.9 Centrifuge capable of handling 50-ml tubes
133 For quantitation, Section 160:
133.1 GC-MS system
a. Capable of scanning from 50 to 450 a.m.u. every 2
seconds
b. Capable of producing a recognizable mass spectrum at
unit resolution from 10 ng of methyl stearate when the
sample is introduced through the GC inlet
c. Interfaced with a gas chromatograph equipped with an
injector system designed for splitless injection glass
capillary column work. All sections of the transfer
lines must be glass or glass-lined and deactivated
with Carbowax 20M
d. Interfaced with a computer data system having a
selected ion search program, i.e., a program capable
of searching a full mass range total ion chromatogram
for selected ions after the run is completed**
e. Glass capillary column - 30mx0.2mmI.D. coated
with SE-30
133.2 HPLC System
a. Pump - capable of constant flow of 0.1 to 5 ml/min at
up to 5000 psi
*For the processing of large numbers of samples a GPC Auto Prep 1001, avail-
able from Analytical Biochemistry Laboratories, Inc., or equivalent may be
used with an operations procedure that will give performance equal to that
described herein.
**The system should have capabilities which at least meet the requirements of
the EMSL guidelines contianed in "Sampling and Analysis Procedures for
Screening of Industrial Effluents for Priority Pollutants", April, 1977.
37
-------
b. High-pressure injector - with 50 yl loop
c. Chromatographic column - 4.6 mm I.D. x 25 cm stainless
steel, packed with Lichrosorb RP-2, 5 micron particle
diameter
d. Electrochemical detector - equipped with a thin layer
glassy carbon electrode
e. Strip-chart recorder - 1 to 10 volts full scale
f. Mobile phase - 50:50 acetonitrile: 0.1 M acetate buffer,
pH 4.7
133.3 Syringes - 10 yl and 100 yl
133.4 Standard solutions - 0.2 mg/ml of neutrals, 0.5 mg/ml of
phenols, and 0.1 mg/ml of benzidines in methanol.
140 Sampling and Preservation
141 Samples should be collected in 2000-ml wide-mouth glass containers
with clean Teflon-lined or foil-lined caps. The containers should
be heated in an oven at 450-500°C overnight to remove any traces'
or organic contamination before use. The containers should be
filled no more than two-thirds full with sample to minimize
breakage during freezing.
142 Samples should be refrigerated at 4°C immediately after collection
and extracted within 24 hours. If extraction within 24 hours is
not possible the samples should be frozen. Samples may be
stored for up to 30 days at -20°C or indefinitely at -75°C. In
order to prevent breakage during storage it is essential that the
container not be permitted to be slightly warmed and recooled.
150 Sample Extraction
151 Three separate samples are extracted, one each for neutral, acidic,
and basic fractions.
152 Neutral fraction
152.1 Place 100 g of homogeneous sludge into a 200-ml centrifuge
tube and acidify with 5 g of KHS04.
152.2 Add 100 ml methylene chloride to the centrifuge tube and
homogenize for one minute with a tissuemizer. As a safety
precaution in case of breakage place the glass tubes in the
metal holders of the centrifuge prior to homogenization.
152.3 Cap the centrifuge.-tubes tightly with Teflon-lined screw
caps and centrifuge to achieve good phase separation.
Remove the methylene chloride layer with a 50-ml syringe
and transfer to a 500-ml round-bottom flask.
152.4 Repeat extraction procedure two more times and combine the
methylene chloride layers.
152.5 Concentrate to 60-80 ml on a rotating evaporator at 35°C.
38
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153 Acid fraction
153.1 Extract 100 g of homogenized sludge following the procedure
of 152.1 to 152.4 and dry the combined extracts by shaking
with 2 g of MgS04.
153.2 Decant the extract into a 500-ml round-bottom flask and
concentrate to 50 ml on a rotating evaporator at 35°C.
Transfer concentrate to a 100-ml round-bottom flask and
concentrate to 8-10 ml. Transfer to a 15-ml vortex
evaporator tube and make up to 10 ml with methylene
chloride. Mix thoroughly. Obtain a residue weight of
100 yl of the 10-ml concentrate and calculate the total
amount of material in the sample. The residue weight is
determined by placing the 100 yl on a tared aluminum foil
pan, allowing the solvent to evaporate, and reweighing the
pan using a microbalance. Concentrate the extract further
as necessary on a vortex evaporator at 25°C to adjust the
final total volume to 1.0 ml for every 200 mg of material
in the sample. Centrifuge the concentrate to remove traces
of particulate material.
154 Basic fraction
154.1 Place 10 g of homogenized sludge in a 50-ml centrifuge tube
and dilute with 20 ml 0.1 M phosphate buffer (pH 7).
Add 10 ml of chloroform and homogenize the mixture for one
minute using a tissuemizer. Centrifuge to achieve a good
phase separation and remove the chloroform layer using a
50-ml syringe.
154.2 Repeat the above extraction two more times using 10 ml of
chloroform each time and combine the chloroform layers.
160 Removal of Interferences
161 The cleanup procedures described in this section are designed to
remove the major interfering classes of compounds found in sludge
extracts, namely triglycerides, fatty acids, and long-chain
hydrocarbons.
162 Neutral fraction - Base extraction is used to remove phenols and
fatty acids. Gel permeation chromatography is used to remove
triglycerides and to obtain an intermediate-sized molecule fraction
containing the higher alkyl phthalates and a small-molecule
fraction containing the remaining neutral compounds of interest.
Silica gel chromatography is used to remove saturated hydrocarbons
from the higher alkyl phthalates.
162.1 Transfer the extract from Section 152.5 to a 1000-ml
separatory funnel and add 200 ml of petroleum ether.
Extract three times with 400-ml portions of 0.1 N^ NaOH in
10% NaCl followed by two washes with 200-ml portions of 10%
NaCl and discard the aqueous layers. If an emulsion forms
39
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at the solvent interface, collect the emulsion in a
centrifuge tube and centrifuge to separate the layers.
162.2 Dry the final organic layer by shaking with 2 g of MgSCty.
162.3 Concentrate to 200 mg/ml as described in Section 153.2.
Centrifuge the concentrate to remove any traces of
particulate material.
162.4 Prepare a 1200 mm x 25 mm I.D. gel permeation chromatography
(GPC) column by slurry packing using 200 g of Bio Beads S-X8
that have been swelled in methylene chloride for at least
4 hours. Prior to initial use, rinse the column with
methylene chloride at 1 ml/min for 16 hours to remove any
traces of contaminants. Calibrate the system by injecting
5 ml of the GPC calibration solution for neutrals (132.5-a),
eluting with methylene chloride at 2 ml/min for at least
3 hours and observing the resultant UV detector trace. The
column may be used indefinitely as long as no darkening
or pressure increases occur and a column efficiency of at
least 1200 theoretical plates is acheived. The pressure
should not be permitted to exceed 50 psi. Recalibrate the
system daily.
162.5 Inject up to 5 ml of the neutral concentrate (from 162.3)
onto the GPC column and elute with methylene chloride at
2 ml/min for at least 3 hours. Discard the first fraction
that elutes up to a retention time represented by the
minimum between the di-n-tridecyl phthalate peak and the
di-n-octyl phthalate peak in the calibration run. Collect
as Fraction GPC-1 the next fraction eluting up to a re-
tention time represented by the minimum between the dimethyl
phthalate peak and the 4-chlorophenyl phenyl ether peak in
the calibration run. Collect as Fraction GPC-2 the
remaining eluate that elutes up to a retention time
represented by the minimum between the pyrene peak and the
sulfur peak in the calibration run.
162.6 Apply the above GPC separation to any remaining neutral
concentrate using only up to 5 ml at a time. Combine the
fractions to give one "Fraction GPC-1" and one "Fraction
GPC-2".
162.7 Prepare a 400 mm x 9 mm I.D. silica gel chromatography
column by slurry packing using 20 g of activated silica
gel suspended in 25% acetone in methylene chloride. Wash
the column, using gravity flow, with 50 ml of 25% acetone
in methylene chloride to remove any traces of impurities
and then wash it with 50 ml of petroleum ether to remove
the polar solvent. Care should be taken to avoid any
bubbles of air or solvent vapor in the column. Solvent
flow should be continued only until the solvent level is
within 1 mm of the top of the silica gel.
162.8 Concentrate Fraction GPC-1 to 2 ml using the procedures
described in Section 153.2 and add 2 ml of petroleum ether.
162.9 Apply the Fraction GPC-1 concentrate to the freshly
prepared silica gel column and open the stopcock to permit
flow until the liquid level is within 1 mm of the top of the
40
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silica gel. In a similar fashion rinse the sample onto the
column completely with two 1-ml portions of 50% methylene
chloride in petroleum ether.
162.10 Elute the column with 50 ml of 50% methylene chloride in
petroleum ether to remove interfering nonpolar and slightly
polar components. Discard this eluate.
162.11 Elute the column with 50 ml of 25% acetone in methylene
chloride and collect the eluate as the phthalate fraction.
The highly polar components remain on the silica gel which
is then discarded.
162.12 Combine the phthalate fraction with Fraction GPC-2 and
concentrate to 10 ml using the procedures described in
Section 153.2. Add 0.1 ml of the DDA internal standard
solution and 1.0 ml of ethylene chloride and concentrate
to 0.2 ml using a vortex evaporator. This is the final
neutral fraction used for GC-MS analysis.
163 Acid fraction - Gel permeation chromatography is used to remove
triglycerides and fatty acids. Acid-base extraction is used to
remove the remaining neutral and basic components.
163.1 Calibrate the GPC system described in Section 162.4 by
injecting 5 ml of the GPC calibration solution for acids
(132.5-b), eluting with methylene chloride at 2 ml/min
for at least 3 hours and observing the resultant UV detector
trace. Inject up to 5 ml of the acid concentrate (from
153.2) onto the GPC column. Elute with methylene chloride
at 2 ml/min for at least 3 hours. Discard the first
fraction that elutes up to a retention time represented by
the minimum between the 4-phenylbutyric acid peak and the
2,4-dinitrophenol peak in the calibration run. Collect as
the phenolic fraction the remaining eluate that elutes up
to a retention time represented by the minimum between the
2,4-dichlorophenol peak and the sulfur peak in the
calibration run.
163.2 Apply the above GPC separation to any remaining acid
concentrate using only up to 5 ml at a time. Combine the
phenolic fractions.
163.3 Concentrate the combined phenolic fractions to 2 ml using
the procedures described in Section 153.2. Transfer the
concentrate to a 50-ml centrifuge tube and add 20 ml of
hexane. Extract two times with 20 ml of 0.1 N_ NaOH in 10%
NaCl. Centrifuge if necessary to facilitate phase
separation. Combine the aqueous layers in a 200-ml
centrifuge tube and acidify with 1 ml of 6 _N HC1.
163.4 Extract the acidified aqueous phase two times with 20-ml
portions of methylene chloride. Centrifuge, if necessary,
to facilitate phase separation. Combine the organic layers
and dry over MgS04.
163.5 Add 0.1 ml of the DDA internal standard solution and 10 ml
of ethylene dichloride and concentrate to 3 ml using the
procedures described in Section 153.2.
41
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163.6 Methylate the phenols by bubbling diazomethane, generated
from Diazald, into the solution until it turns yellow.
Cap the sample and keep it at room temperature for 30
minutes. If the yellow color dissipates during this time
add more diazomethane.
163.7 Concentrate the sample to 0.2 ml in a vortex evaporator at
30°C. This is the acid fraction for GC-MS analysis.
164 Basic fraction
164.1 Transfer the chloroform extract from'154.2 to a 50-ml
centrifuge tube and extract twice with 10 ml 2 N^ H2SC-4
using a 50 ml syringe to withdraw the aqueous layer from
the bottom.
164.2 Combine aqueous layers in a 50-ml beaker containing a
magnetic stirring bar and neutralize by the dropwise
addition with stirring of 1 ml of 0.4 M Na3?04 followed
by the dropwise addition over at least a two-minute period
of 20% NaOH to pH 6-7 (approximately 7 ml will be required).
Do not allow the sample pH to ever exceed pH 8.
164.3 Transfer the neutralized aqueous extract to a 60-ml
separatory funnel and extract twice with 10 ml portions of
chloroform. Wash the combined chloroform extracts with 5
ml of distilled water. Add 5 ml of methanol to the
chloroform extract and concentrate to 0.2 ml using a vortex
evaporator at 25°C.
164.4 Dilute to 1 ml with 0.1 M acetate buffer (pH 4.7). This is
the basic fraction used for the analysis of benzidines by
HPLC.
170 Quantitation
171 The neutral and acidic (phenolic) components are quantitated by
GC-MS analysis using an SE-30 glass capillary column. The basic
components (benzidines) are quantitated by HPLC analysis using
an electrochemical detector.
172 Neutral and acidic fractions
172.1 Inject 2 yl of sample into the GC-MS using the splitless
mode with the injector at 270°C, column at 60°C, transfer
"line at 280°C, and helium carrier gas flow at approximately
2 ml/min. Hold the column temperature at 60°C for 5
minutes, then temperature program at 4 degrees per minute
to 270°C and hold at 270°C for 15 minutes. Scan m/e values
of 40-450 at approximately 30 scans per minute. Start data
acquisition four minutes after injection.
172.2 Locate the priority pollutant compounds in the GC-MS runs
by selected ion searches (see 133.1-d) (SIS). In this
method, the computer is instructed to search the full mass
range mass spectra for several specified m/e values which
are characteristic of the compound of interest. These
42
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TABLE 172.2.
GC-MS DATA USED FOR DETERMINING SEHIVOLATILE
PRIORITY POLLUTANTS
Compound
Approximate
Retention
Time, nin/^M.W.
Ions Used For Quantitation,
m/e (intensity)
Bis-(2-chloroethyl) ether
1 , 3-Dichlorobenzene
1 , 4-Dichlorobenzene
1,2-Dichlorobenzene
Bis-(2-chloroisopropyl) ether
N-Nitrosodipropylamine
Nitrobenzene
Isophorone
Bis- (2-chloroethoxy) me thane
1,2, 4-Trichlorobenzene
Naphthalene
Hexachlorobutadiene
Eexachlorocyclopentadiene
2-Chloronaphthalene
2 , 6-Dinitro toluene
Dimethyl phthalate
Ac enaphthal ene
Acenaphthene
2 , 4-Dinitro toluene
Diethyl phthalate
Fluor ene
4-Chlorophenyl phenyl ether
N-Nitrosodiphenylamine -^)
4-Bromophenyl phenyl ether
Hexachlorobenzene
Phenanthrene
Anthracene
Di-n-butyl phthalate
Fluoranthene
Pyrene
Butylbenzyl phthalate
Chrysene
Benzo (a) anthracene
3is(2-ethylhexyl) phthalate
Di-n-octyl phthalate
Benzo (b) fluoranthene
Benzo (k) fluoranthene
Eenzo (a) pyrene
Benzo (g ,h , i) perylene
Indeno(l ,2 ,3-cd) pyrene
Dibenzo(a ,h) anthracene
Neutrals
10.5
10.6
10.6
11.2
11.7
12.6
13.4
14.6
15.5
16.8
17.1
18.8
22.7
24.0
25.5
26.3
27.0
27.2
27.9
30.4
30.5
30.6
31.4
33.0
33.4
35.6
35.9
40.1
42.5
43.7
48.5
50.9
51.2
52.9
55.9
56.4
56.9
62.8
66. £
68.3
72.5
142
146
146
146
170
130
123
138
180
180
128
258
270
162
182
194
152
154
182
222
166
204
198
248
282
178
178
278
202
202
298
228
228
390
390
252
252
252
276
276
278
93(100), 63(99),
146(100) , 148(65) ,
146(100) , 148(65) ,
146(100). 148(65) ,
45(100), 77(19),
70(100), 130(30)
77(100), 123(50)
82(100), 138(15)
93(100), 95(32),
180(100), 182(97)
128(100) , 127(15)
225(100), 227(60)
237(100), 235(63),
162(100), 127(40)
165(100), 89(80)
163(100), 77(21)
152(100) , 151(23)
153(100) , 154(90)
165(100), 63(60)
149(100), 178(25)
166(100), 165(90)
204(100), 141(75)
169(100) , 168(72)
248(100), 250(95)
282(100) , 284(77)
178(100) , 176(17)
178(100) , 176(17)
149(100), 104(10)
202(100) , 101(25)
202(100) , 101(25)
149(100), 91(62)
228(100), 226(27)
228(100)
149(100), 167(38)
149(100) , 167(34)
252(100), 253(23)
252(100), 253(23)
252(100) , 253(23)
276(100)
276(100)
278(100)
95(31)
111(35)
111(35)
111(35)
79(12)
123(21)
272(12)
43
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TABLE 172.2. (Continued)
Compound
Approximate
Retention
Time, min£a) M.W.
Ions Used for Quantitation,
m/e (intensity)
(c)
2-Chlorophenol(-c)
Phenol
2,4-Dimethylphenol
2,4-Dichlorophenol
2,4,6-Trichlorophenol(Me)
ophenol(Me)( }
(d)
2-Nitrophenol(M<
4-Chloro-3-methylphenol
4-Nitrophenol(Me)(d)
4,6-Dinitro-o-cresol(Me)
Pentachlorophenol(Me)(d)
2,4-Dinitrophenol(Me)(d)
Decadeuteroanthracene
(c)
Phenols
11.0
11.7
16.9
18.1
22.6
22.9
23.3
25.8
33.8
34.9
35.5
Internal Standard
128
94
122
162
210
153
142
153
212
278
198
128(100),
94(100),
107(100),
162(100),
195(100),
77(100),
107(100),
107(100),
89(100),
237(100),
76(100),
130(33)
66(60),
121(97),
164(66)
197(95),
106(80),
142(69),
77(82),
165(61),
265(91),
151(69),
65(35)
122(85)
167(75)
92(65)
77(62)
153(12)
182(58)
280(70)
168(62)
35.9
188 188(100)
a.
b.
c.
d.
GC conditions: 30 m x 0.2 mm I.D. glass capillary column coated with SE-30;
hold the column temperature at 60°C for 5 minutes then program at 4 degree
per minute to 270°C and hold at 270°C for 15 minutes.
Decomposes upon injection in the GC to diphenylamine; therefore, it is
detected as diphenylamine.
Determined as the free phenol.
Determined as the methyl ether.
44
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selected ions are given in Table 172.2. Whenever a peak
is tentatively identified by SIS methods as a priority
pollutant, its full mass spectrum and retention time
should be studied manually for positive confirmation. The
area counts for the selected ions of each identified
priority pollutant and the DBA internal standard are then
obtained by the computer.
172.3 Quantitate the identified priority pollutants using the
following equation:
PP 10 I
X DDA X RF X W
where X = concentration of the priority pollutant in the
sludge in yg/g dry weight
PP = area counts obtained for the priority pollutant
DDA = area counts found for decadeuteroanthracene
RF = response factor of the priority pollutant
relative to that of DDA as determined by GC-MS
analysis of standard solutions
W = dry weight, g, of 100 g of wet sludge
Response factors for the particular GC-MS system used
should be determined at least weekly-
173 Basic fraction
173.1 Inject 50 yl of the final base extract into the sample loop
of the HPLC system described in Section 133.2. Use a flow
rate of 0.8 ml/min and operate the detector at 0.8 volts
versus a standard calomel electrode.
173.2 Quantitate the benzidine and 3,3'-dichlorobenzidine on the
basis of peak heights using the following equation:
H
_ sample 5.
A H , X W
std
where X = concentration of the benzidine in the sludge in
yg/g dry weight
TT
sample = peak height of the benzidine found in the sample
H = peak height of the benzidine obtained from 50 yl
of a standard solution containing 0.5 yg/ml of
each of the two benzidines
45
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W = dry weight, g, of 100 g of wet sludge
180 Quality Assurance
181 Sample processing
181.1 Process blanks - In order to assess any contamination
sources during sample extraction and cleanup, at least
one process blank (distilled water) should be run
concurrently with each set of 10 sludge samples.
The resulting neutral and acidic fractions may be
analyzed by GC alone instead of by GC-MS.
181.2 Spiked process blanks - In order to assess recovery
efficiencies of the extraction and cleanup procedures used,
in the absence of matrix effects, at least one spiked
blank should be run concurrently with each set of 10
sludge samples. The spike should contain 10 yg of each
of the priority pollutants of concern. The resulting
neutral and acidic fractions may be analyzed by GC
alone instead of by GC-MS. Recoveries of at least 25%
for each component should be achieved. Occasionally in
order to assess the lower sensitivity of the method a spike
containing only 2 yg per 100 ml should be used. At the 2 yg
level recoveries of at least 25% should not be expected for
all compounds.
181.3 Spiked sludge - In order to assess overall recovery
efficiencies, including matrix effects, and also assess
the reproducibility of the method, triplicate samples
of a representative sludge should be run with every 10
sludge samples. The sludge should be spiked with each
of the priority pollutants of concern at a level of 10 yg
per 100 ml. The spiked sludges should be equilbriated
at 4°C for 1 hour prior to extraction. Recoveries of
at least 10 percent for each component should be achieved.
Occasionally in order to assess the lower sensitivity of
the method a spike level of only 2 yg per 100 ml should be
used. At the 2 yg level recoveries of at least 10% should
not be expected for all compounds.
181.4 Replicate samples - In order to assess the reproducibility
of the method, one of every 10 sludge samples should be
run in triplicate.
182 GPC fractionation - The GPC system should be calibrated daily or
after every 10 runs to determine retention times and column
efficiency by injecting 5 ml of the GPC calibration solutions.
The column efficiency or number of theoretical plates (N) is
calculated from the retention time, t, and the peak width at half
height, W]_/2, obtained for the di-n-octyl phthalate peak on the UV
trace using the equation
46
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The column should not be used unless at least 1200 theoretical
plates can be achieved.
183 GC-MS analysis - Strict performance standards need to be main-
tained to ensure that the GC-MS system is providing adequate
sensitivity and high quality chromatograms and mass spectra.
183.1 Capillary column performance - Prior to installing the
glass capillary column in the GC-MS, evaluate the column
in a GC system designed for all-glass capillary column work.
With the helium carrier gas flow adjusted to 30-40 cm/sec,
the split ratio adjusted to 10:1 and, column oven
temperature set at 100°C, inject 2 yl of a column per-
formance test mixture containing 25 ng/yl each of
2,6-dimethylphenol, 2,4-dimethylaniline, n-decyl alcohol,
n-decyl aldehyde, n-tridecane, and n-tetradecane.
a. The acidity of the column is measured as the ratio
of the peak height of dimethylphenol to that of
dimethylaniline. A value of 0.5 to 2.0 is considered
acceptable.
b. The polarity of the column is determined from the
degree of tailing of the n-decyl alcohol peak. This
is evaluated by drawing a perpendicular from the apex
of the peak to the baseline and measuring, at one-
tenth peak height, the width from the front of the peak
to the perpendicular line and from the back of the peak
to the perpendicular line. If the width at the back of
the peak is greater than four times the width of the
front of the peak, the column is too polar for sample
analyses. Low polarity is particularly important for
achieving satisfactory sensitivity in the analyses of
polycyclic aromatic hydrocarbons and more polar
compounds.
c. The number of effective plates (Neff) is determined
from the n-tetradecane peak using the equation:
?
t
Neff = 5.5'
W
1/2
where tc = the corrected retention time of n-
tetradecane, and
^1/2 = tne Peak width at half height.
The number of effective plates of acceptable columns
must be at least 50,000. Only columns which meet all
of the above criteria are to be used for the GC-MS
analyses.
47
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183.2 Mass spectrometer tune up - Calibrate and tune the mass
spectrometer each day before samples are analyzed. After
tuning and calibration has been completed, admit
decafluorotriphenylphosphine (DFTPP) into the mass
spectrometer via the solids probe. Its spectrum should
be comparable to that described by Eichelberger, et al.
[Anal. Chem., ^16, 995 (1975)]. Retune and recalibrate
the instrument if necessary.
183.3 Capillary column GC-MS performance - Evaluate the
performance of the complete glass capillary column GC-MS
system each day prior to its use for the analysis of
neutral fractions. Using splitless injection techniques
and the standard sample analysis conditions inject 2.0 yl
of a heptane solution containing 20 ng each of
1-naphthylamine, 2-naphthol, 1-pentadecanol, DFTPP,
3-methylnonadecane, tridecylcyclohexane, n-eicosane, pyrene,
n-heneicosane, and methyl stearate. The total ion
chromatogram is used to assess the system performance.
a. The acidity of the system is measured as the ratio of
the peak areas of the naphthylamine and naphthol. It
must be between 0.5 and 2.0 to be acceptable.
b. The polarity of the system is measured by the peak
height of 1-pentadecanol, pyrene, or methyl stearate
relative to that of n-eicosane. In each case a value
of at least 0.2 must be obtained.
c. The sensitivity of the system is measured by the signal
to noise ratio obtained for pyrene. It must be at
least 20.
d. The resolution of the system is assessed by the
resolution achieved between tridecylcyclohexane and
3-methylnonadecane. The resolution should be
sufficient to give a valley between the two peaks which
is no more than 20% of the peak heights.
e* The high/low mass spectral balance of the system is
determined from the mass spectrum of DFTPP. The ratio
of the intensities of the 442 ion and the 198 ion should
be 0.4 to 0.7. This is a check on the earlier MS
tuneup.
183.4 Response factors and retention times - Once each week or
each time a substantal change in GC-MS performance is
observed, the response factors and retention times for the
. priority pollutants should be determined. This is
accomplished by analyzing 2 yl of standard solutions
containing 50 ng/yl of decadeuteroanthracene and each of
the neutral and acidic priority pollutants.
184 HPLC analysis - With each set of samples analyzed, analyze a
standard containing 0.5 yg/ml of each of the two benzidines.
The retention times and peak heights are used for the detection
and quantitation of the benzidines in the samples.
48
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
EPA-600/2-80-Q30
3. RECIPIENT'S ACCESSION-NO.
4. TITLE AND SUBTITLE
Analytical Procedures For Determining Organic
Priority Pollutants in Municipal Sludges
5. REPORT DATE
March 1980 (Issuing Date)
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
J. S. Warner, G. A. Jungclaus, T. M. Engel,
R. M. Riggin, and C. C. Chuang
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Battelle Columbus Laboratories
505 King Avenue
Columbus, Ohio 43201
10. PROGRAM ELEMENT NO.
1BC611
11. CONTRACT/GRANT NO.
68-03-2624
12. SPONSORING AGENCY NAME AND ADDRESS
Municipal Environmental Research Laboratory—Cin.,OH
Office of Research and Development
U. S. Environmental Protection Agency
Cincinnati, Ohio 45268
13. TYPE OF REPORT AND PERIOD COVERED
Final
14. SPONSORING AGENCY CODE
EPA/600/14
15. SUPPLEMENTARY NOTES
Project Officer: Richard A. Dobbs (513) 684-7649
16. ABSTRACT
An analytical procedure was developed for the determination of 54 semi-
volatile organic priority pollutants in sludge at levels down to 0.01 yg/g wet
weight. The procedure involved extraction with methylene chloride or chloroform,
cleanup of groups of compounds having common properties, and in most cases
analysis of the fractions by GC-MS using high-resolution glass capillary columns
and selected ion searches. The final analyses involved the analysis of three
separate fractions, namely benzidines, phenols, and neutrals. The benzidines
were determined by HPLC analysis using an electrochemical detector instead of
by GC-MS because GC-MS sensitivity for these compounds was too low. Quantitation
in the GC-MS analyses involved the internal standard method applied to selected
ion responses. Relative response factors obtained from the analysis of standard
solutions were used as correction factors.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.IDENTIFIERS/OPEN ENDED TERMS C. COSATI Field/Group
Organic Compounds
Activated Sludge
Primary Sludge
Chemical Analysis
Mass Spectroscopy
Gas Chromatography
Priority Pollutants
07C
18. DISTRIBUTION STATEMENT
Release to public
19. SECURITY CLASS (This Report)
UNCLASSIFIED
21. NO. OF PAGES
55
20. SECURITY CLASS (Thispage)
UNCLASSIFIED
22. PRICE
EPA Form 2220-1 (9-73)
49
U S GOVERNMENT PRINTING OFFICE 1980-657-146/5639
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