United States
Environmental Protection
Agency
Municipal Environmental Research
Laboratory
Cincinnati OH 45268
Research and Development
EPA-600/S2-80-196 Dec. 1980
Project Summary
GC/MS Methodology for
Measuring Priority Organics in
Municipal Wastewater
Treatment
Dolloff F. Bishop
An EPA-developed methodology for
measuring priority toxic organics in
municipal wastewaters and sludges is
based on GC/MS technology. Suc-
cinctly, the methodology separates the
purgeable priority organics from the
environmental sample by purging with
inert gas and trapping the organics on a
Tenax and silica gel trap. The organics
are then desorbed, identified, and
quantitated with packed column
GC/MS analysis. The extractable
organics are separated by extracting
with methylene chloride, first at pH 11
and then at pH2, and then the organics
in the base-neutral and acid extracts
are identified and quantitated by
packed column GC/MS analysis.
The basic methodology analyzes the
purgeable organics in municipal
wastewaters satisfactorily but
requires one modification in the equip-
ment. By substituting charcoal for the
silica gel in the trap used in the purge-
able procedure, all of the purgeable
priority organics are identified and
satisfactorily quantified. In the basic
methodology for extractable organics,
a few of the organics are not measured
well. Statistics on the analytical
recoveries are summarized for the
priority organics.
Municipal wastewaters and sludges
contain a wide variety of extractable
organics that can interfere in the
GC/MS analysis. Thus, the extracts
may require clean-up or organic separ-
ation before the GC/MS analysis.
Principal classes of organic interfer-
ences included lipids, fatty acids, and
saturated hydrocarbons. The
approaches to separate the desirable
priority organics from the interfer-
ences include acid-base separation,
molecular size separation, and polarity
separation. These approaches, applied
in various combinations, are described
as proposed methods for analysis of
priority organics in municipal sludges
and as additional procedures to lower
the detection limit for the organics in
municipal wastewaters.
This publication, which can be
purchased from the NationalTechnical
Information Service, presents a state-
of-the-art review on the current
GC/MS methodology for the analysis
of priority toxic organics in municipal
wastewater treatment. Both recently
published and unpublished literature
are summarized.
Basic Methodology
In GC/MS analysis of priority toxic
organics, the organics are divided into
purgeable and extractable classes. The
extractable class is further subdivided
into organic acids, base-neutral com-
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pounds, and a selected subclass of
pesticides and PCB's. In the EPA's basic
GC/MS methodology (1), the purgeable
organics are separated from the envi-
ronmental sample by classical purge
and trap procedures. The organics,
thermally desorbed from the trap* are
then analyzed by pack column GC/MS
techniques. The extractable organics
are extracted with methylene chloride,
first at pH 11 and then at pH 2. The acid
and base-neutral extracts are concen-
trated and then analyzed by packed
column GC/MS techniques.
Limitations
The basic methodology for purgeable
and extractable organics has been
extensively applied to raw municipal
wastewaters (2). The initial application
of the basic methodology on municipal
raw wastewater missed four of the
purgeable organics: chloromethane,
dichlorodifluoromethane, vinyl chlor-
ide, and bromomethane. With substi-
tution of charcoal for the silica gel in
the Tenax trap and purging at 49°C, the
modified approach identified all of the
purgeable priority organics and
exhibited the best overall recoveries
(~ 90%) for the analysis of priority
organics in municipal wastewaters.
The basic methodology does not
measure all of the extractable priority
pollutants well. N-nitrosodimethyl-
amme does not chromatograph effec-
tively under the conditions of the
method and is sufficiently volatile as an
extractable organic to result in poor
analyses. Hexachlorocyclopentadiene,
while successfully determined (3) in
some laboratories, has been missed by
others (2).
The neutral compound, 2-chloroethyl
vinyl ether, also was not detected (2) by
the basic methodology. Thermal decom-
position of 1,2-diphenylhydrazine to
azobenzene and N-nitrosodiphenyl-
amme to diphenylamine has also been
observed (4). Co-eluting pairs of
anthracene-phenanthrene, benzo(a)-
anthracenechrysene, and benzo(b)-
fluoranthene-benzo(k)fluoranthene on
the specified packed GC column are not
resolved by mass spectroscopy and,
therefore, are not distinguishable by the
methodology When desired, the use of
capillary GC columns (SP-2100 on 30-
m, wall-coated capillary) in place of the
packed column can eliminate the co-
elution problem for the three co-elution
pairs.
Finally, the bases (benzidines) have
been difficult to chromatograph at low
concentrations. An alternative high
performance liquid chromatography
(HPLC) method (5)(6) specifically for
benzidines has significantly lowered
detection limits. Verification of the
benzidines for legal purposes may,
however, require a GC/MS procedure.
The detection limits for the organics
depend on the sample matrix. Agency
estimates on detection limits in waste-
waters for the basic methodology are
typically 10 //g/L for most of the purge-
able and base-neutral priority organics.
Agency estimates for most of the acid
(phenols) organics in wastewaters are
typically 25 fjg/L.
Statistical Evaluation
Kleopfer, et al. (3) recently completed
a statistical evaluation of EPA's basic
methodology using data from seven
laboratories that analyzed both indus-
trial and municipal wastewaters. The
statistics (Table 1) indicate an overall
average recovery of about 90% of the
purgeables and about 80% of the acids
(phenols) from both distilled water and
wastewater analyses. Significant
wastewater matrix effects did not occur
for either the purgeables or the acids.
Indeed, when compared with distilled
water recoveries, the overall average
recoveries in the wastewater analyses
increased slightly for purgeables and
decreased slightly for acids. In the
purgeable and acid analyses, Kleopfer
found that the recoveries for specific
organics decreased significantly (purge-
ables, at the 99% confidence level;
acids, at the 95% level) as the volatility
of the organics increased.
For base-neutrals, pesticides, and
PCB's, the study revealed significantly
lower average recoveries in the waste-
water analyses (68% for base-neutrals,
59% for pesticides and PCB's) compared
with those in distilled water (84% for
base-neutrals, 78% for pesticides and
PCB's). The lower recovery in the waste-
water analysis was attributed to
increased reactivity of these classes of
priority organics. When Kleopfer
separated the base-neutral class into
more chemically reactive and less
chemically reactive groups, the statis-
tical analyses confirmed the greater
variability and poorer recoveries in the
more reactive grouping.
The quality control data (3) specified
in the methodology (1) revealed the
quality control limits (± 3a) for percent
recoveries on individual organics often
ranged from zero to several hundred
percent. This broad range for some of
the organics indicates that either the
basic methodology or the analytical per-
formance of the laboratories could be
improved. Nevertheless, as an
analytical tool for such a wide variety oi
organics, the methodology with proper
quality control is generally satisfactory
for the screening analysis of the
organics in wastewaters.
Table 1. Recoveries of Priority Pollutants
Priority pollutant fraction
Volatile (purgeables)
Acid (phenols)
Base-neutrals
Pesticides and PCB's
Recoveries
Method Standard*
P±Spc
90 ± 13
84 ± 13
84 ±25
78 ±11
(percent)
Sample Spike"
P±Spc
92 ± 15
76+ 19
68 ±21
59 ±11
"Method standard refers to recoveries by standard addition to distilled water.
"Sample spike refers to recoveries by standard addition to sample.
CP± Spare weightedaverages ofthe data points and are in units of percent recovery
± one standard deviation (Sp).
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Interferring Substances
Cryotrap Modification
Municipal sludges contain sufficient
interferences such that the Agency's
basic methodology is not successful.
The complex samples and those
samples where low detection limits (~ 1
/ug/L) are desired require alterna-
tive approaches or additional separation
and clean-up procedures. The Agency
has developed an interim procedure (7)
for purgeable analyses in sludges that
employs a modified purge and trap pro-
cedure with dilution of the sample to
5000 mg of solids in a modified purging
apparatus.
To improve analyses of purgeable
organics in sludge samples and to lower
their detection limits in all samples,
DeWalle and Chian (8) have modified
the purge and trap method (Figure 1) to
include an on-column cryotrap after the
Tenax column and ahead of a capillary
GC column. The cryotrap, cooled by
liquid nitrogen, captures the organics
during desorption from the Tenax
column. The cryotrap is then rapidly
warmed to focus and release the
organics into the capillary GC. The
improved resolution of the capillary
column along with the cryotrap is
claimed to reduce detection limits of the
purge and trap method. Possible future
improvements in the purge and trap
method to reduce the sludge matrix
effects include using salts (NazSCu
"salting out") or warming the sample
above ambient temperature to improve
the purgeability of the organics.
5-ml sample
I
Purging with N*
I
Adsorption on Tenax Trap
I
Desorption at 180°C with back flushing
I
On-column cryotrappmg of desorbed
organic with liquid nitrogen
I.
Release of organics and capillary
GC/MS Analysis
(30 m SE-54 WCOT column) External
standard method for
quantitation
Figure 1. Analysis of purgeable
organics by cryotrap
capillary GC/MS (2).
Conventional Reduction
Approaches
The large amount of extractable
organics in complex samples such as
sludges requires special extraction
techniques (9) and separation and
clean-up procedures before GC/MS
analysis. The principal classes or
organic interferences extracted from
raw municipal wastewater and sludge
samples are: lipids; fatty acids; and
saturated hydrocarbons. In the com-
plexsamples, the large amounts of
extractable interferences overwhelm
both the GC and the mass spectrometer.
To permit analysis, these interferences
must, therefore, be reduced in the exact
fractions before injection into the
GC/MS system. Three principal
conventional approaches are available
for this reduction: acid base separation;
molecular size separation by gel
permeation chromatography; and
polarity separation (silica gel chroma-
tography, etc.).
Acid-base separation is the
fundamental separation approach
behind the Agency's basic methodol-
ogy. In this methodology, base-neutral
extraction followed by acid extraction
divides the total amount of interfer-
ences between acid and base extracts,
separates the base-neutrals from the
acids, and thus, reduces the degree of
interference in each fraction injected in
the GC/MS detector. Acid-base
separation, however, may be applied at
many points in a separation scheme to
remove or separate acid compounds
from neutrals or bases in a complex
extract.
Molecular size separation is especial-
ly effective in removing the lipids, high
molecular weight fatty acids, and hydro-
carbons from the extract. These
materials apparently thermally decom-
pose in the GC system and create very
complex GC chromatograms. Large
amounts of these materials will also
reduce GC column life.
Separation due to polarity with silica
gel (9) or florisil (8) is used to separate
the saturated hydrocarbons from the
aromatic or polar priority organics. A
cesium silicate approach (8) has also
been used to separate the acids
(phenols) from the base-neutrals
priority organics and from neutral
interferences.
The separation or "clean-up"
approaches can be assembled in
various combinations to reduce the
interferences from extracted municipal
sludges. The high organic content of
sludges prevents efficient conventional
extraction for separation of the
organics. While work is ongoing to
evaluate continuous liquid-liquid
extraction, micro-extraction and
extractive steam distillation techniques
on sludges, homogenization-centrifuge
extraction, and modified soxhlet tech-
niques have demonstrated efficient
extraction capabilities. The homogen-
ization-centrifuge technique has been
adopted in the Agency's interim
procedures for the analysis of sludges
(7).
A method has been developed by
DeWaMe and Chian (8) for the analysis
of the extractable priority organics in
complex samples. The methodology
(Figure 2) uses an acid-neutral extrac-
tion followed by a base extraction; gel
permeation chromatograph (GPC)of the
acid-neutral extract into two fractions
and a discard that contains the large
interferences; florisil chromatograph of
one GPC fraction for separation of the
saturated hydrocarbons from those
priority neutrals in the fraction; and
cesium silicate for separation of the
acids (phenols) from the priority
neutrals in the second GPC fraction. The
phenol fraction from the silicate separa-
tion may be denvatized with diazo-
methane before GC/MS analysis or
analyzed by fused silica capillary
column GC without derivatization. The
method produces three neutral
fractions that may be combined into a
single extract before GC/MS analysisor
be analyzed separately. The method
uses capillary GC/MS techniques for
the final analysis. The pesticides and
PCB's are analyzed in the neutral
fraction. Alternative extraction
techniques under evaluation include
homogenization-centrifugation, liquid/
liquid extraction, and extractive steam
distillation.
Results
In their survey of 25 cities, the Muni-
cipal Environmental Research Labora-
tory uses the DeWalle and Chian proce-
dures for measuring the purgeable and
extractable organics in both wastewater
and sludge samples. The data indicate
detection limits for the priority organics
of about 1 /ug/L for the extractables and
< 1 /ug/L for the purgeables in waste-
water samples The data also suggest
detection limits of 5 to 10 /ug/L for the
-------
Sample
Extraction at pH 2 with
• Extraction at pH 12 with CH2CI2
Drying and concentration
Addition to pentane
GPC on Biobead S-X2
Drying and concentration
GC/MS analysis
(30-m capillary GS-SE54)
Internal standard quantification
1 1
Discard Concentre
(lipids) exchanf,
pentt
\
Flonsil se
\
I
Discard SO0/
(hydrocarbons) ett
Solvt
and i
tion and Cesium
je into separ
me
oaration
\ CH2CI2
'o pentane/ Ether
ler extract extract
?nt Exchange Concen- Concer
concentration tration
\
silicate
at ion
extract
itration
GC/MS analysis of neutrals
30-m capillary GC-SE54
Internal standard quantification
Methanol p
Partition
Concer
GC/MS
Internal standa
henol extract
to ChzClz
itration
analysis
rd quantifical
Figure 2. Analysis of extractable organics (2) with cleanup and capillary GC/MS.
organics in sludge samples. The method
is not fully satisfactory for all of the
priority organics in all the highly
variable sludge matrices. Losses of
individual organics occur either through
reaction with the matrix or losses in the
separation processes. At the present
time, insufficient data have been
assembled to provide statistical analysis
of the recoveries of the priority organics
by the method.
References
1. "Guidelines Establishing Test
Procedures for Analysis of
Pollutants, Proposed Regulation,"
Federal Register, 44(233):69526-
69558, December 3, 1979.
2. Levins, P. L, et al., "Source of Toxic
Pollutants in Influents to Sewage
Treatment Plants/'USEPA draft
report. Office of Water Planning
and Standards, Washington, D.C.,
November 1979.
Kleopfer, R. D., Dias, J. R., and Fair-
less, B. J., "Priority Pollutant
Methodology Quality Assurance
Review," USEPA, Region VII
Laboratory, Kansas City, KS 66115.
"Seminar on Analytical Methods
for Priority Pollutants," Denver, CO,
November 1977, Proceedings of
the Seminar, USEPA, Effluent
Guidelines Division, Washington,
D.C.
"Development and Application of
Test Procedures for Specific
Organic Toxic Substances in
Wastewater Category 7-Benzidine,"
EPA Contract 68-03-2624 (in prep-
aration).
"Guidelines Establishing Test Pro-
cedures for Analysis of Pollutants,
Proposed Regulations," Federal
Register. 44(233):6948-6949,
December 3, 1979.
"Interim Methods for the Measure-
ment of Organic Priority Pollutants
in Sludges," USEPA, Environmen-
tal Monitoring and Support Labora-
tory, Cincinnati, OH 45268,
September 1979.
DeWalle, F. and Chian, E.,
"Presence of Priority Organics in
Sewage and Their Removal in
Sewage Treatment Plants," Interim
Report, July 1,1978-May 31,1979,
Grant 806102, USEPA, Municipal
Environmental Research Labora-
tory, Cincinnati, OH 45268 (in
preparation).
Warner, J. S., et al., "Analytical
Procedures for Determining
Organic Priority Pollutants in Muni-
cipal Sludge,"EPA-600/2-80-030,
USEPA, Municipal Environmental
Research Laboratory, Cincinnati,
OH 45268, March 1980.
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The author Dolloff F. Bishop is also the EPA Project Officer (see below).
The complete report, entitled "GC/MS Methodology for Measuring Priority
Organics in Municipal Wastewater Treatment," (Order No. PB 81-127813;
Cost: $8.00 subject to change) will be available from:
National Technical Information Service
5285 Port Royal Road
Springfield. VA 22161
Telephone: 703-487-4650
The EPA Project Officer can be contacted at:
Municipal Environmental Research Laboratory
U.S. Environmental Protection Agency
Cincinnati, OH 45268
ft US GOVERNMENT PRINTING OFFICE. 1881 -757-064/0205
-------
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Environmental Protection
Agency
Center for Environmental Research
Information
Cincinnati OH 45268
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