united states
Environmental Protection
Agency
Environmental Monitoring and
Support Laboratory
Cincinnati OH 45268
Research and Development
EPA-600/S4-84-009 Feb. 1984
Project Summary
Analytical Procedures for Aniline
and Selected Derivatives in
Wastewater and Sludge
P.M. Riggin, S.V. Lucas, T.F. Cole, and M.A. Birts
A detailed evaluation of gas chroma-
tography (GC) and high performance
liquid chromatography (HPLC) for the
determination of anilines in aqueous
media has been conducted. The most
applicable procedure was found to be
GC with thermionic nitrogen-phospho-
rus selective detection (IMPD). This GC
method is capable of determining a
wide variety of anilines at low part per
billion (ppb) level in industrial aqueous
discharges, as well as effluents from
publicly owned treatment works (POTW's).
Method performance data for authentic
environmental samples are presented.
Analytical precision was generally 5-
15% Relative Standard Deviation (RSD)
and recoveries were generally 75% or
better.
This Project Summary was developed
by EPA's Environmental Monitoring and
Support Laboratory. Cincinnati, Ohio,
to announce findings of the research
project that is fully documented in a
separate report of the same title.
Introduction
Aniline and its substituted derivatives
(referred to as "anilines") are widely
produced for a variety of industrial and
commercial purposes, including dyestuff
and pesticide manufacturing. The United
States Environmental Protection Agency
(EPA) has as part of its mission the
responsibility for providing test procedures
for organic pollutants which are monitored
under such regulations as the Clean
Water Act (PL-92-500) or Toxic Substances
Control Act (TSCA). Since many of the
anilines have been placed on the priority
list for testing under TSCA, the need
arose for an analytical procedure for
determining a wide variety of anilines in
industrial wastewater effluents.
This report describes the development
and evaluation of GC and HPLC approaches
using selective detectors for analyzing
trace concentrations in selected industrial
wastewaters.
Results and Discussion
The method development effort was
conducted as a phased approach wherein
each aspect of the procedure was
evaluated. The critical elements evaluated
included HPLC determinative techniques,
GC determinative techniques, extraction
approaches, cleanup techniques, and
sample preservation. An optimized analyti-
cal method, using GC with NPD detection,
was devised and evaluated. Details of
each evaluation step are described in the
main report.
Extraction Methodology
Extraction efficiencies of the anilines
were determined in reagent water
buffered at pH 5 (0.01 M acetate buffer),
pH 7, (0.01 .M phosphate buffer), andpH
11 (0.01 M phosphate buffer). Methanol
solutions of the compounds were spiked
into one-liter volumes of the buffered
media in a separatory funnel at the
10 //g/L level. Three successive extractions
with 60-mL volumes of methylene chloride
were performed using two-minute extrac-
tion periods. The extracts were combined
and concentrated. Extraction recoveries
were determined by HPLC with 254 nm
ultraviolet (UV) detection. This extraction
approach was selected since existing
priority pollutant methods use similar
approaches.
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GC Column Evaluation
Retention time data for the various
anilines on various packed and glass
capillary columns were determined. The
10% Carbowax-2% KOH column gave
poor performance. Subsequently, a
chemically bonded Carbowax column
(Ultrabond R 20 M distributed by Supelco)
was evaluated. All of the anilines were
eluted from the Ultrabond" column.
However, very poor response was obtained
for aniline and4-bromoaniline(indicating
loss of these compounds on the column),
and a number of compounds coeluted.
The 3% SP-2250 column partially
resolved all of the anilines; however,
some peak tailing was evident, especially
for aniline. The 5% SP-2401 DB (contain-
ing KOH) and 1.5% SP-2250/1.95% SP-
2401 gave no improvement in peak
shape. SP-2250 was chosen as the best
available packed column fordetermination
of anilines.
However, each column failed to resolve
at least one compound pair. No column
completely resolved 3-chloro and 4-
chloroaniline although SE-54 appeared
to give the best separation for these two
compounds. SE-54 was selected for use
in the program, although other phases
(e.g., SE-52, SP-2100) were also considered
to be acceptable. These columns should
be considered for use when components
coeluting on SE-54 need to be quantified.
During the course of this program,
fused silica capillary columns (FSCC) of
suitable quality became available. Com-
pared to conventional glass columns,
FSCC are durable and easy to use.
Retention data for the anilines on two
FSCC's are given in Table 1.
Using both SE-54 and SE-30 FSCC, all
19 anilines can be resolved. SE-54 was
used as the primary column for the
method validation effort although SE-30
probably would have been just as
appropriate.
GC Detector Evaluation
The Hall electrolytic conductivity
detector (HECD), nitrogen selective mode
(NPD), and the photoionization detector
(PID) were evaluated in terms of sensitivity
using the SE-54 glass capillary column.
The HECD and NPD detectors gave
similar detection limits in several cases
although the NPD was more sensitive for
many of the compounds. In addition, the
NPD gave a more stable baseline and less
day-to-day variation in response. The PID
gave very low response for the nitro-and
dinitroanilines when the 9.5 eV source
was used. Although detection limits were
improved considerably when the 10.2 eV
Table 1. Retention Data for Anilines
Relative Retention Time
on Stated Column
Compound
An/line
2-Chloroaniline
3-Chloroaniline
4-Chloroaniline
4-Bromoaniline
2-Nitroaniline
2.4,6- Trichloroaniline
3, 4 -Dichloroaniline
3-Nitroaniline
2,4,5 -Trichloroaniline
4-Nitroaniline
4-Chloro-2-nitroaniline
2-Chloro-4-nitroaniline
2, 6-Dichloro-4-nitroaniline
2-Bromo-6-chloro-4-nitroaniline
2-Chloro-4.6-dinitroaniline
2, 6-Dibromo -4 -nitroaniline
2, 4 -Dinitroaniline
2-Bromo-4, 6-dinitroaniline
SE-54
1.0 (7.5)°
1.6(12.1)
1.9(14.6)"
2.0(14.7)"
2.4 (18.0)
2.9(21.9?
2.9(21.9?
3.0(22.7)
3.3 (24.5)
3.5 (26.3)
3.8 (28.3?
3.8(28.3?
4.2 (31.2)
4.3(31.9)
4.6 (34.8)
4.9 (37.1)
5.0 (37.6)
5.1 (38.4)
5.3 (39.8)
SE-30
1.0 (6.3)
1.1 (7.1)
1.4 (9.0l°
1.5 (9.1?
1.9(12.1)
2.1 (15.6)
2.5 (16.3)
2.6 (16.6)
2.9 (18.0)
3.2 (20.4)
3.4(21.7)
2.9 (22.0)
3.3 (24.8)
3.5 (26.0)
3.8 (28.8)
4.0 (30. 1)
4.2 (31.6?
4.2 (31.6?
4.5 (33.4)
* Actual retention time in minutes given in parenthesis.
b These compounds only partially resolved.
c These compounds essentially unresolved.
" These compounds essentially unresolved.
source was used, the higher energy
source would result in the detection of
virtually all aromatic compounds. There-
fore, the PID was not expected to be as
specific for anilines as the nitrogen
selective detection systems.
The selectiveness of the three detectors
were evaluated using an extract from a
POTW aqueous effluent. The PID detector
(10.2 eV) has a very complex chromato-
gram with a high baseline level. The
chromatographic response using NPD
and HECD were similar. NPD was
selected for use in the final method
because it was more selectivethan PID and
gave better baseline stability and day-to-
day repeatability than HECD.
Since the use of both packed and
capillary columns was to be considered in
the final method, the sensitivity of the
NPD for the anilines using 3% SP-2250
packed and SE-54 glass capillary columns
was determined.
HPLC Column Evaluation
Although several HPLC columns (e.g.,
Spherisorb ODS, Lichrosorb C18, Lichro-
sorb C8, Lichrosorb C2, and/j-Bondapak)
was evaluated, Dupont Zorbax ODS was
found to give the best resolution for the
anilines. Better separation was obtained
using 40% acetonitrile/60% acetate
buffer than using 50% methanol/40%
acetate buffer mobile phase. In addition,
the column back pressure was substan-
tially lower when acetonitrile was used.
Adjusting the pH of the acetate buffer from
4 to 5.5 in 0.5 pH unit increments
resulted in better separation between 2-
chloroaniline and 2-chloro-4-nitroaniline
(which coeluted at pH 4 and pH 4.5), but
resulted in poor separation between 2-
chloroaniline and 4-bromoaniline (which
coeluted at pH 5.0 and pH 5.5). Buffer
strength had little or no effect on the
separation. Based on these results, the
optimum HPLC separation system was
considered to be a Zorbax ODS column
with a 40/60 acetonitrile/0.2.M s°dium
acetate buffer, pH 4.
HPLC Detector Evaluation
Both UV and electrochemical (ampero-
metric) detectors were evaluated in terms
of sensitivity for the anilines. The UVdata
and electrochemical data for the anilines
are presented; all of the nitro-substituted
anilines absorb strongly in the 340-400
nm region, whereas the other compounds
show no appreciable absorbance above
300 nm. Consequently, the use of UV
detection at 350 nm to selectively detect
nitroanilines is feasible.
The electrochemical oxidation potentials
for the anilines ranged between 0.9 and
1.4 volts, with the dinitroanilines being
the most difficult to oxidize. Using HPLC
with electrochemical and UV detection,
only the nitro-substituted anilines were
detected at 340 nm, whereas all of the
anilines were detected at 254 and 280 nm.
In general, detection limits were 1-10
nanograms injected using UV detection,
which corresponded to a level of 0.1-1
jt/g/mL in the sample extract. |
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Extracts from POTW wastewater were
injected onto the HPLC-UV system with
the UV detector successively at 254, 280,
and 340 nm. In all cases, a very complex
chromatogram was obtained. Although
additional sample cleanup techniques
could have been used in an attempt to
reduce the background interference
level, the evaluation of HPLC analysis
was discontinued.
Evaluation of Cleanup
Techniques
Two extract cleanup approaches, acid
back extraction and adsorption chromatog-
raphy, were evaluated. Only the most
basic anilines (e.g., anilines and the
haloanilines) were extracted into 1N
HgSCU from methylene chloride. Therefore,
acid back extraction was not considered
further in this program, although this
procedure might be very effective when
only aniline or monohaloanilines are to
be determined.
Adsorption alumina, basic alumina,
and Florisil were evaluated using a
variety of elution schemes. Adsorption
alumina was found to give very poor
recoveries of the anilines even when 10%
methanol in ether was used as the
elution solvent. Addition of 1 % trimethyla-
mine to the 10% methanol in ether
solvent did not substantially improve
recovery of the anilines. Basic alumina
also gave poor recoveries for the anilines
when using either the activated or
methanol deactivated adsorbent. Aniline
recovery was less than 1% using this
elution scheme and was only slightly
higher (4%) using the methanol deactiva-
tion scheme.
The Florisil cleanup schemes gave
consistently better recoveries than did
the various alumina cleanup schemes.
However, the activated Florisil schemes
both gave poor recoveries for anilines and
the dinitroanilines.
On the basis of these results, the 5%
isopropanol deactivation procedure (elim-
inating the 10% CH2Cl2 in hexane
fraction) was selected for use in the final
method. The isopropanol deactivation
procedure was favored because it elimi-
nated the need to prepare adsorbent
beforehand and no fines were produced
in the deactivation procedure.
Sample Preservation
Evaluation
The water samples studied were as
follows:
O Reagent water
O POTW water
O Effluent from a coking operation
G Treated (biological treatment) waste-
water from a plant producing nitro-
anilines (wastewater No. 3)
O Treated (biological treatment) waste-
water from a plant producing aniline
(wastewater No. 4)
O Wastewater No. 4 to which 2 mg/L
of sodium hypochlorite was added.
Storage at pH 7 and 4°C were found to
give best stability of the anilines.
Method Validation
Based on the results of the experiments
described above, an analytical method
was selected for validation. This method
involved the following steps:
O Extraction of the wastewater at pH
11 with methylene chloride
O Concentration of the extract by
Kuderna-Danish concentration
O Cleanup of the extract using Florisil
deactivated with isopropanol
O Exchange of the final concentrate
into toluene
O Analysis by GC-NPD using an SE-54
FSCC as the primary column. SE-30
FSCC and 3% SP-2250 packed
columns are used as secondary
columns in the method.
All of the validation work described
below involved the use of a SE-54 FSCC.
Florisil fractions 1, 2, and 3 (50%
methylene chloride in hexane, 5% IPA in
hexane, and 5% methanol in hexane,
respectively) were combined prior to
concentration and analysis.
The analytical curves for the various
anilines were determined using reagent
water. All steps in the method, including
Florisil cleanup, were utilized in this
study. Recoveries for the majority of the
anilines were constant over the range
tested, approximately 3-300 x method
detection limit (MDL). However, poor
recoveries at less than 10 x MDL were
observed for aniline (29%), 4-chloroaniline
(7%), and 4-bromoaniline (23%). Never-
theless, all of the anilines were readily
detectable at the lowest spike level.
The performance of the method at
higher spike levels was evaluated for
reagent water, treated wastewater No. 3,
and treated wastewater No. 4. Spike
levels were approximately 100 /jg/L (20-
100 x MDL) in all cases. The results for
this evaluation are given. Recoveries
were generally 70-120% for all samples,
except for aniline (55%), and 4-chloroani-
line (49%) in distilled water. Percent
relative standard deviations were gener-
ally 5-15%.
Conclusions
The application of GC-NPD has been
found to be an effective method for the
determination of a wide variety of
anilines. This approach offers better
selectivity and sensitivity than HPLC and
other GC detectors. A conventional
solvent extraction scheme using methylene
chloride gave adequate recovery from
aqueous waste samples. A successful
adsorption column cleanup technique
was identified.
Capillary column chromatography
using an SE-54 FSCC offers better
sensitivity and selectivity than the
optimum packed column (3% SP-2250),
although the latter column is still useful
as an alternative approach. Theoptimized
analytical method developed gives good
precision and accuracy for a variety of
water types over a wide range of
concentrations. The method reported
herein is recommended as the method of
choice when a wide variety of anilines are
to be determined.
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R. M, Riggin. S. V. Lucas, T. F. Cole, and M. A. Bins are with Battelle Columbus
Laboratories. Columbus. OH 43201.
Stephen Billets was the EPA Project Officer (for present contact see below).
The complete report, entitled "Analytical Procedures for Aniline and Selected
Derivatives in Wastewater and Sludge," (Order No. PB 84-140 102; Cost:
$20.50, subject to change) will be available only from:
National Technical Information Service
5285 Port Royal Road
Springfield, VA 22161
Telephone: 703-487-4650
For information Fred K. Kawahara can be contacted at:
Environmental Monitoring and Support Laboratory
U.S. Environmental Protection Agency
Cincinnati. OH 45268
fttl.S. GOVERNMENT PRINTING OFFICE: 1984-759-015/7319
United States
Environmental Protection
Agency
Center for Environmental Research
Information
Cincinnati OH 45268
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