United States
Environmental Protection
Agency
Environmental Monitoring and
Laboratory
Cincinnati OH 45268
Research and Development
EPA-600/S4-82-022 May 1982
v
Project Summary
etermination of Benzidines in
Ylh(te^trial and Municipal
stewaters
x ,
Ralph M. Riggin and C. C. Howard
www.elsevier
A
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several
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decrease
to abou W-ppirroTBenzidine and DCB.
Apparent levels of 10-20 ppb of
benzidine were present in the dye
plant effluents. For one dye plant,
effluent benzidine was determined to
be 9 ppb and 12 ppb using two
different sets of chromatographic
conditions, thus supporting the belief
that benzidine is present at the level
stated.
Precision and accuracy of the
method were estimated from the
results for five wastewater samples
spiked at levels between 1 and 50 ppb.
For this group of samples the re-
coveries were 69 ± 15% for benzidine
and 76 ± 9% for DCB.
Storage of several wastewater
samples for (two or seven days) at 4° C
and pH 2 resulted in degradation of the
compounds in several cases, probably
due to oxidation or irreversible adsorp-
tion to paniculate matter. Therefore,
-.-Yl
COM I
to obtain accurate values for
» and DCB, it is believed to be
r to assay the sample as soon
llection as possible. For
Containing chlorine, a reduc-
such as sodium thiosulfate
added, since chlorine was
rapidly degrade benzidine
\iect Summary was devel-
Environmental Mon-
Support Laboratory,
OH, to announce key
'he research project that is
'uity documented in a separate report
of the same title (see Project Report
ordering information at back).
Introduction
Under provisions of the Clean Water
Act, the Environmental Protection
Agency is required to promulgate
guidelines establishing test procedures
for the analysis of pollutants. The Clean
Water Act Amendments of 1977
emphasize the control of toxic pollutants
and declare the 65 "priority" pollutants
and classes of pollutants to be toxic
under Section 307(a). This report is one
of a series that investigates the ana-
lytical behavior .of selected priority
pollutants and suggests a suitable test
procedure of their measurement.
It presents the results obtained under
Contract No. 68-03-2624, in which a
method was developed for the determi-
nation of benzidine, dichlorobenzidine,
and diphenylhydrazine in aqueous
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effluents. This work was conducted in
two phases, wherein Phase I consisted
of evaluation of various analytical
methodologies, and Phase II consisted
of validating the most promising method
using several aqueous effluent samples.
Data from the Phase I study, which were
presented in a report dated May 19,
1978, are also presented herein.
The objective of this study was to
develop an optimized analytical method
for the determination of benzidine,
dichlorobenzidine (DCB) and 1,2-di-
phenylhydrazine (DPH) and to validate
the method on a variety of aqueous
effluents.
The successful completion of this
program involved the fulfillment of
certain directives set forth in the
contract by EPA. An extensive literature
review was first conducted to evaluate
the previous work in the area. Subse-
quent work was directed toward deter-
mination and then full evaluation of an
appropriate measurement technique,
which best satisfied the requirements
for sensitivity and selectivity, as well as
the considerations of sample cost, that
is, equipment, time, and training, which
would be needed for the method. The
stability of the benzidine compounds in
water miscible solvents and their
instability in chlorinated and unchlorin-
ated buffered water at different pHs and
storage temperatures were studied over
the prescribed time periods. Extraction
efficiency of two organic solvents was
also studied for the standard compounds.
The remainder of the program involved
the study of the sample preparation and
clean up steps which would be neces-
sary to eliminate sample interferences.
The complete method was then applied
to several representative wastewater
samples and an assessment was made
of the precision and accuracy of the
complete procedure.
Analytical Methods
Development
Three analytical techniques were
evaluated for potential use in the
determination of benzidines: (1) direct
GC/AFD, (2) GC/ECD following derivati-
zation with a fluoroacy group! and (3)
HPLC/EC.
Direct GC/AFD
Gas chromatographic properties of
the three compounds were investigated
using a Hewlett Packard 5730 gas
chromatograph equipped with dual
FID/AFD. Columns were 6ft x 2 mm I.D.
glass, packed with a 3% loading of the
liquid stationary phase on Gas Chrom Q
100/120. The stationary phases eval-
uated were OV-1, OV-17, SP2250DB,
and OV-225, which represent a wide
range of polarities. GC/FID was used to
evaluate all four stationary phases and
GC/AFD was evaluated for the
SP2250DB stationary phase.
The four stationary phases investi-
gated represent a wide range of
polarities. Both OV-17 and SP2250DB
were found to give satisfactory results.
OV-1 was acceptable but some degree
of tailing was noted, especially at low
concentrations. OV-225 gave an un-
acceptable degree of column bleed.
The use of AFD f or the detection of the
benzidines was investigated using
SP2250DB. By operating isothermally
at 230°C, a sensitivity of about 1 ng on
column can be achieved for benzidine
and DCB; but under these conditions,
the DPH peak appears in the solvent
front.
It was noted for each column that two
peaks arose from the injection of DPH,
and furthermore, that the larger of the
peaks elutes much earlier than would
be expected for a reasonably polar
compound of this molecular weight. In
order to confirm the identity of the
eluted components, GC/MS was used,
employing a 30 meter SE-30 glass
capillary column.
A single sharp peak was observed
eluting at 210° from a temperature
programmed run. The mass spectrum
revealed it to be a component with a
molecular ion at 182 amu, whereas
DPH has a molecular ion at 184. The
component resulting from DPH injection
was suspected to be azobenzene (M.W.
182) which was confirmed by the
injection of pure azobenzene. It is
apparent, therefore, that DPH instan-
taneously decomposes to azobenzene in
the GC injection port.
GC/ECD
The use of various derivatization
reagents to form highly electron captur-
ing derivatives was investigated. The
following derivitization reagents were
evaluated: (1) trifluoroacetic anhydride
(TFA); (2) pentafluoropropionyl anhydride
(PFPA); (3) heptafluorobutyric anhydride
(HFBA); (4) triftuoroacetyl imidazole
(TFI); and (5) heptafluorobutryl imiazole
(HFBI).
Numerous problems were encountered
using this approach, the most serious of
which was the production of multiple
peaks and the incomplete reaction of
DPH with all of the reagents studied.
Benzidine reacted well with the HFBI
but not any of the anhydrides or the TFI.
DCB derivatized the best of the three
amines and failed to form a derivative
only with TFA. Similar results were
obtained using both the anhydrides and
imidazoles. The DCB-HFBA derivative
was formed with good yield, whereas
there was a relatively poor yield of about
10% of benzidine-HFBA derivative. No
underivatized amine was present in
either case, so some decomposition
must have occurred in the benzidine
derivatization reaction. Both benzidine
and DCB were successfully derivatized
using HFBI. As stated earlier, HFBI
derivatization of DPH was not suc-
cessful, since no derivative formation
was observed.
On the basis of these results, this
approach did not seem useful for the
analysis of benzidines and was not
further investigated, since an alternate
technique, HPLC/EC, was working
quite well. However, it appears that
HFBI would be the best derivatizing
reagent to use for benzidine and DCB, if
one wished to assay these compounds
by GC/ECD.
HPLC with Electrochemical
Detection
This approach proved useful almost
immediately, so following a short period
of column evaluation, a set of parameters
was selected which gave excellent
resolution, sensitivity, and reproduc-
ibility. Several reversed phase and ion
exchange columns were preliminarily
evaluated, including: Zorbax ODS and
CN, M Bondapak C-18, Lichrosorb RP-
18, RP-8, and RP-2, Spherisorb ODS,
and Whatman Partisil SCX. All the ODS,
CN, and RP-8 packings gave poor
efficiency for the amines—less than
2000 plates in all cases—although they
gave efficiencies of greater than 10,000
plates for anthracene, which is com-
monly used for determining column
efficiency. The Whatman SCX column
was very poor in that it gave badly tailing
peaks. The RP-2 column was found to
give excellent efficiency for the amines—
greater than 6000 plates. Better resolu-
tion of the DCB and DPH was achieved
using acetonitrile instead of methanol
under the conditions evaluated.
Based on the results mentioned
above, a set of HPLC conditions was
selected which gave optimum resolution
and sensitivity for the compounds of
-------
interest. These conditions are listed
below:
Column—RP-2 5 micron 25 cm x4.6
mm I.D.
Mobile phase—50% acetonitrile - 50%
sodium acetate buffer
0.1 M pH4.7
Detector—Electrochemical (glassy
carbon) 3 mm diameter,
Bioanalytical Systems
model LC2
Detector potential-—0.8V
Injection volume—25 microliters
Using the electrochemical detector,
the minimum detectable quantities
were 0.05 ng for benzidine, 0.1 ng for
DCB, and 0.3 ng for DPH at a signal to
noise ratio of five to one. Linear
response for the components is obtained
from 0.1 - 400 nanograms injected for
DCB and benzidine (a linear dynamic
range of 103). Above 400 nanograms, a
rapid fall from linearity is observed for
DCB and benzidine. Precision of peak
heights for eight replicate injections of
benzidine was ±2.8% at the 50 nano-
gram level.
The electrode potential (0.8V) was
chosen based on our study of response
versus electrode potential for each of
the compounds. This study shows that
full (diffusion controlled) response is
achieved for each compound at 0.8V
versus Ag/Ag Cl and above.
Solvent Stability Studies
The stability of the three amines in
water miscible solvents was studied
over a 90-day period. For DCB and
benzidine, acetonitrile and methanol
were used. Both components were
completely stable over the 90-day
period. DPH was found to decompose
completely in three days or less in all
solvents investigated, including benzene,
methylene chloride, methanol, tri-
ethylamine, acetonitrile, and acetic
acid. It is apparent that DPH standards
must be prepared fresh daily and thus
no further studies were conducted.
Extraction Studies
Extraction studies were conducted to
evaluate the extraction efficiencies of
methylene chloride and chloroform
preserved with 2% ethanol for the
benzidines in water at pHs 2,7, and 10.
Fifty microliters of standard solution
in acetone was added to 500 ml of
appropriately buffered water to yield a
concentration of 10 ppb for each of the
compounds in a 1000 ml separatory
funnel.
The benzidines were extracted with
50 and .then 30 mL of the appropriate
solvent. The extract was washed with
20 mL water, and the solvent was
exchanged to methanol by concentrating
to 5 mL on a rotating evaporator at 35° C.
Finally, the extract was concentrated to
2 mL on a vortex evaporator and
prepared for HPLC by dilution to 4 mL
with 0.1 M sodium acetate buffer.
The extraction methodology was
found to be a very delicate area since
numerous unanticipated problems
developed which led to low recoveries,
especially for benzidine and DPH. The
stability of DPH was found to be a major
problem which was not completely
solved.
The following are some of the
problems encountered:
1. Benzidine is substantially ad-
sorbed on Na2SC>4 if a normal
solvent drying step is employed.
The use of KaCOs corrected this
problem. Elimination of the drying
step was found to be useful when
using HPLC analysis.
2. Benzidine is heat labile so that
Kuderna-Danish concentration
techniques gave low recoveries.
The use of rotary evaporation
eliminated this problem.
3. Decomposition of benzidine oc-
curred when concentrating
methylene chloride or chloroform.
The addition of 15% MeOH prior to
concentration stabilizes the
benzidine.
Generally, chloroform at pH 7 was
found to give a more efficient extraction,
expecially for benzidine. As expected,
no benzidine was recovered at pH 2,
whereas the less basic DCB was
extracted at all pH values. DPH de-
graded readily in both aqueous and
organic media and although both
chloroform and methylene chloride
gave about 70% recoveries at pH 10, no
set of extraction parameters was found
which gave greater than about 70%
recovery. At pH 2, DPH was found to
degrade to benzidine to some extent.
Based on these data, it was concluded
that chloroform is a satisfactory solvent
for the extraction of benzidines.
Storage Stability Studies
The stability of the benzidines in
water under a variety of storage
conditions was investigated. Two
temperatures, 4°C and room tempera-
ture, three pH levels, 2, 7, and 10, and
two chlorine levels, 0 and 2 ppm, were
evaluated by preparing duplicate 500
mL samples spiked with 10 ppb of DCB
and benzidine or DPH. DPH was done
separately since it can degrade to
benzidine under certain storage con-
ditions. The solutions were stored in
amber glass bottles for 7 days prior to
extraction.
None of the amines were detectable
in the solutions to which chlorine was
added. A pH of 2 was found to give the
best results for benzidine and DCB.
However, at pH 2 DPH degrades to
benzidine, thus creating an undesired
artifact. This problem can be overcome
by employing pH 4.7 acetate buffer
where both benzidine and DCB are well
preserved, and DPH degrades to two
unidentified components, not to
benzidine. At pH 4.7, two peaks were
obtained which did not interfere with
DCB or benzidine, neither of which was
DPH (based on chromatographic reten-
tion times).
Based on these results, it was
concluded that a pH between 2 and 4 is
best for preservation of the benzidines.
Some artifacts due to DPH decomposi-
tion to benzidine are likely at pH 2 and
below if DPH is present in the sample.
Addition of a reducing agent (such as
NazSOa) to destroy chlorine is required.
DPH was found to be very unstable in
wastewater, specifically secondary
sewage. DPH, spiked at the 100 ppb
level, disappeared with a half life of
about 15 minutes in the presence of
oxygen and about 60 minutes with
oxygen removed. This result indicates
that DPH analysis in wastewater is
virtually meaningless, since the DPH
level determined cannot be directly
related to the DPH in the sample at the
time of collection.
Wastewater Studies
Based on the above studies, the
following procedure was applied to the
analysis of actual wastewater samples.
• Adjust pH of 500 mL to 1 Laliquotof
wastewater to pH 7 and serially
extract with 100, 50 and 50 mL
volumes of chloroform.
• Extract the benzidines from the
solvent with three 25 mLaliquotsof
1M H2S04.
• Neutralize the acid solution to pH 6-
7 and serially extract with 30, 20
and 20 mL volumes of chloroform.
• Wash the solvent with 20 mL
distilled water and exchange the
solvent to methanol while con-
centrating to 5 mL on a rotating
evaporator.
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• Concentrate the methanol extract
to 1.0 mL with a gentle stream of
nitrogen.
• Add 4.0 ml pH 4.7 acetate buffer
and analyze by HPLC with electro-
chemical detector.
The following water samples were
selected for analysis:
1. Surface water (Olentangy River)
2. Secondary sewage effluent (Col-
umbus, Ohio)
3. Primary sewage (Columbus, Ohio)
4. Final effluent from plant produc-
ing various organic chemicals
(such as nitrobenzene, nitrophenols,
o-dichlorobenzene, and chloro-
anilines).
5. Final effluent from plant produc-
ing benzidine based dyes.
6. Oxidation process stream from a
plant producing benzidine based
dyes.
Each wastewater was analyzed,
unspiked, in triplicate. Six 500 mL
aliquots of each sample were spiked
with benzidine and DCB at a level at
least five times background. Three
aliquots were analyzed immediately and
three were analyzed after storage, at pH
2 and 4°C, for a period of time—either
two or seven days. The pH 2 level was
attained by addition of 1M H2SC>4. One
gram per liter of sodium thiosulfate was
added to each sample prior to spiking.
The analytical results for all samples are
tabulated in Table 1.
Sample 2 was a secondary sewage
effluent spiked at the 4 ppb level.
Table 1. Data for Wastewater Analyses
Results obtained were comparable to
those for the surface water sample.
Storage of this effluent, spiked at the 4
ppb, for 48 hours at 4°C, and pH 2,
resulted in essentially no loss in
recovery. The shorter storage period
was used, since it was felt that the
stability of benzidine in dilute aqueous
solution was so unpredictable that
samples should be assayed as soon as
possible. Forty-eight hours appeared to
be the shortest time period which could
reasonably be expected for the comple-
tion of sample collection and transport
to the analytical laboratory.
Sample 3 was a primary sewage
sample and was spiked at the 1 ppb
level. This spike level was chosen in
order to validate the procedure at levels
approaching the detection limit, although
as a general practice higher spiking
levels were used so that accuracy could
be more easily determined and poor
recoveries would still result in a
measurable peak. As shown in Table 1,
recoveries were quite good and precision
was about ±10-20%. However, after
storage for seven days, low recovery
and poor reproducibility were observed
for DCB, but not for benzidine. This
result appears to indicate that DCB may
be irreversibly bound to particulate
matter, since this sample had a large
amount of suspended matter.
Sample 4 was a final effluent sample
from a plant producing various organic
chemicals such as nitrophenols, o-
dichlorobenzene, chloroanilines, and
nitrobenzenes. Samples 1-3 were
selected for this study because they
represent matrices which receive
inputs from a diversity of industrial
processes, and thus benzidine determi-
nation might be applied to such samples
in the future. Sample 4, on the other
hand, was selected because it represents
a matrix suspected of containing various
aromatic amines (anilines) and phenols
which would be likely interferences for
the HPLC/EC method (since they are
readily oxidized). Sample 4 was spiked
at the 1 ppb level. Recovery and
particularly precision for benzidine
were poor both before and after a
storage time of 48 hours. Recovery and
reproducibility for DCB were better than
for benzidine, both before and after
storage. The reason for this lack of
reproducibility for this sample is not
clear, although it may be due to
chemical oxidation, since benzidine is
much more easily oxidized than DCB.
Sample 5 was obtained from a plant
producing benzidine based dyes. This
sample was about one year old upon
receipt and had been stored at pH 2 and
4°C during that period of time. The
sample was diluted 10:1 with distilled
water prior to analysis. Recoveries were
very good before storage-but decreased
after storage. The drop in benzidine
level was particularly disturbing, since
there was a detectable level of benzidine
in the unspiked sample, which had been
stored for over a year. It seemed
possible therefore that the benzidine
Sample Sample
No. Description
Distilled water
1 Surface water
2 Secondary sewage**
3 Primary sewage
4 Final effluent**
for organic chemicals
Benzidine
DCB
Benzidine
DCB
Benzidine
DCB
Benzidine
DCB
Benzidine
DCB
Background Spike
(ppb) (ppb)
<.1 4
<1 4
<./ 4
<.1 4
<./ 4
<./ 4
.1 1.0
.1 1.0
<. 1 1.0
<.1 1.0
Recovery
0 days
70 ± 7*
50 ± 5
63 ± 6
48 ± 5
73 ± 8
63 ± 7
64 ± 5
55 ± 10
47 ±46
89 ± 8
7 days
—
—
—
60 ± 12
58 ± 6
71 ±21
19 ± 10
52 ± 17
60 ±21
plant
5 Final effluent** Benzidine
from plant producing DCB
benzidine based dyes
6 Process effluent** Benzidine
from oxidation process DCB
in plant producing
benzidine based dyes
*Mean ± standard deviation.
**Stored for 48 hours instead of seven days.
12 ±3
50
50
10
10
79 ±
78 ±
82 ±
98 ±
14
7
3
5
47 ±
23 ±
75 ±
95 ±
8
7
7
3
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background was due to matrix inter-
ferences, rather than benzidine. The
extract from Sample 5 was injected at
various electrode potentials to see if a
lower electrode potential would result
in a lower background for benzidine.
The apparent level of benzidine in the
unspiked sample decreased in going
from 0.8 to 0.6 volts. This indicates that
an interfering component, with an
oxidation potential greater than
benzidine, is present. However, it was
still not certain whether the benzidine
level at 0.6 volts was accurate. In order
to check this, a second set of HPLC
conditions was used. The value obtained
for benzidine using the original chroma-
tographic conditions (12 ppb) correlated
closely with the value obtained using
the alternate colum (9 ppb), thus
indicating that the peak probably is
benzidine.
The final sample, sample 6, run
through the analytical scheme, was an
oxidation process effluent from the
benzidine based dye plant from which
sample 5 was taken. This sample was
also diluted 10:1 with distilled water
before analysis. Due to a high back-
ground at 0.8 volts, benzidine was
determined at 0.6 volts. Recoveries for
benzidine and DCB spiked at the 10 ppb
level were excellent both before and
after storage for 48 hours at 4°C and pH
2.
An estimate of the precision and
accuracy of the method can be obtained
using the results for all five waste
samples. For these samples, the average
recovery was 69 ± 15% for benzidine
and 76 ± 9% for DCB.
and DCB are stable at pH 2 and pH
4.7. However, at elevated pH
benzidine is more readily degraded,
due to oxidative reactions.
Analysis of several wastewater
samples showed that the analytical
methods developed herein work
well for the analysis of benzidine
and DCB down to the 1 ppb level for
most samples. Dye plant samples
are a special case since those
utilized in this program all showed
apparently detectable 10-20 ppb
levels of benzidine. Confirmed
measurements on two HPLC
columns indicate that benzidine is
present at the levels stated, although
more definitive measurements
should be made.
Ralph M. Riggin and C. C. Howard are with Battelle Columbus Laboratories,
Columbus. OH 43201.
James E. Longbottom is the EPA Project Officer (see below).
The complete report, entitled "Determination of Benzidines in Industrial and
Municipal Waste waters," (Order No. PB82-196 32O; Cost: $10.50, subject to
change) will be available only from:
National Technical Information Service
5285 Port Royal Road
Springfield, VA 22161
Telephone: 703-487-4650
The EPA Project Officer can be contacted at:
Environmental Monitoring and Support Laboratory
U.S. Environmental Protection Agency
Cincinnati, OH 45268
Summary and
Recommendations
The conclusions drawn from this
study are as follows:
• DPH is too unstable in both organic
solvents and aqueous solutions to
permit meaningful analytical data
to be obtained.
• Benzidine and DCB are completely
stable for at least 90 days in both
methanol and acetonitrile when
stored in the dark in sealed ampules.
• Benzidine and DCB can be efficiently
extracted from water with chloro-
form at pH 7 and pH 10. At pH 2,
DCB is readily extracted but
benzidine is not.
• All three compounds are destroyed
when stored in aqueous solutions
containing 2 ppm of chlorine. In the
absence of chloroform, benzidine
*USGPO: 1982 — 559-092/3406
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Environmental Protection
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Center for Environmental Research
Information
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