EPA 600 K 93
METHOD 509. DETERMINATION OF ETHYLENE THIOUREA (ETU) IN
WATER USING GAS CHROMATOGRAPHY WITH A
NITROGEN-PHOSPHORUS DETECTOR
Revision 1.0
December 1992
D.J. Munch and R.L. Graves
T.M. Engel and S.T. Champagne
Battelle, Columbus Division
ENVIRONMENTAL MONITORING SYSTEMS LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
-------
-------
METHOD 509
DETERMINATION OF ETHYLENE THIOUREA (ETU) IN WATER
USING GAS CHROMATOGRAPHY WITH A
NITROGEN-PHOSPHORUS DETECTOR
1. SCOPE AND APPLICATION
1.1 This method utilizes gas chromatography (GC) to determine ethylene
thiourea (ETU, Chemical Abstracts Registry No. 96-45-7) in water.
1.2 This method has been validated in a single laboratory during
development. The method detection limit (MDL) has been determined
in reagent water (1) and is listed in Table 2. Method detection
limits may vary among laboratories, depending upon the analytical
instrumentation used and the experience of the analyst. In addition
to the work done during the development of this method and its use
in the National Pesticide Survey, an interlaboratory method
validation study of this method has been conducted.
1.3 This method is restricted to use by or under the supervision of
analysts experienced in the use of GC and in the interpretation of
gas chromatograms. Each analyst must demonstrate the ability to
generate acceptable results with this method using the procedure
described in Sect. 9.3.
1.4 When a tentative identification of ETU is made using the
recommended primary GC column (Sect. 6.7.1), it must be confirmed
by at least one additional qualitative technique. This technique
may be the use of the confirmation GC column (Sect,, 6.7.2) with the
nitrogen-phosphorus detector or analysis using a gas
chromatograph/mass spectrometer (GC/MS).
2. SUMMARY OF METHOD
2.1 The ionic strength and pH of a measured 50-mL aliquot of sample are
adjusted by addition of ammonium chloride and potassium fluoride.
The sample is poured onto an Extrelut column. ETU is eluted from
the column in 400 ml of methylene chloride. A free radical
scavenger is then added in excess to the eluate. The methylene
chloride eluant is concentrated to a volume of 5 ml after solvent
substitution with ethyl acetate. Gas chromatographic conditions
are described which permit the separation and measurement of ETU
with a nitrogen-phosphorus detector (NPD).
3. DEFINITIONS
3.1 ARTIFICIAL GROUND WATER -- An aqueous matrix designed to mimic a
real ground water sample. The artificial ground water should be
reproducible for use by others.
-------
3.2 CALIBRATION STANDARD (CAL) — A solution prepared from the primary
dilution standard solution or stock standard solutions and the
internal standards and surrogate analytes. The CAL solutions are
used to calibrate the instrument response with respect to analyte
concentration.
3.3 METHOD DETECTION LIMIT (MDL) — The minimum concentration of an
analyte that can be identified, measured, and reported with 99%
confidence that the analyte concentration is greater than zero.
3.4 -INTERNAL STANDARD (IS),-- A pure analyte(s) added to a sample,
extract, or standard solution in known amount(s) and used to
measure the relative responses of other method analytes and
surrogates that are components of the same sample or solution. The
internal standard must be an analyte that is not a sample
component.
3.5 FIELD DUPLICATES (FD1 and FD2) — Two separate samples collected at
the same time and place under identical circumstances and treated
exactly the same throughout field and laboratory procedures.
Analyses of FD1 and FD2 give a measure of the precision associated
with sample collection, preservation and storage, as well as with
laboratory procedures.
3.6 INSTRUMENT PERFORMANCE CHECK SOLUTION (IPC) -- A solution of one or
more method analytes, surrogates, internal standards, or other test
substances used to evaluate the performance of the instrument
system with respect to a defined set of criteria.
3.7 LABORATORY REAGENT BLANK (LRB) — An aliquot of reagent water or
other blank matrix that is treated exactly as a sample including
exposure to all glassware, equipment, solvents, reagents, internal
standards, and surrogates that are used with other samples. The
LRB is used to determine if method analytes or other interferences
are present in the laboratory environment, the reagents, or the
apparatus.
3.8 QUALITY CONTROL SAMPLE (QCS) — A solution.of method analytes of
known concentrations which is used to fortify an aliquot of LRB or
sample matrix. The QCS is obtained from a source external to the
laboratory and different from the source of calibration standards.
It is used to check laboratory performance with externally prepared
test materials.
3.9 STOCK STANDARD SOLUTION (SSS) — A concentrated solution-containing
one or more method analytes prepared in the laboratory using
assayed reference materials or purchased from a reputable
commercial source.
3.10 SURROGATE ANALYTE (SA) — A pure analyte(s), which is extremely
unlikely to be found in any sample, and which is added to a sample
aliquot in known amounts(s) before extraction or other processing
ant is measured with the same procedures used to measure other
sample components. The purpose of the SA is to monitor method
performance with each sample.
-------
4. INTERFERENCES
4.1 Method interferences from contaminants in solvents, reagents,
glassware and other sample processing apparatus may cause discrete
artifacts or elevated baselines in gas chromatograms. All reagents
and apparatus must be routinely demonstrated to be free from
interferences under the conditions of the analysis by running
laboratory reagent blanks as described in Sect. 9.2.
4.1.1 Glassware must be scrupulously cleaned (2). Clean all
glassware as soon as possible after use by thoroughly
rinsing with the last solvent used in it." Follow by
washing with hot water and detergent and thorough rinsing
with tap and reagent water. Drain dry, and heat in an
oven or muffle furnace at 400°C for 1 hr. Do not heat
volumetric ware. Thermally stable materials might not be
eliminated by this treatment. Thorough rinsing with
acetone and methylene chloride may be substituted for the
heating. After drying and cooling^ seal and store glass-
ware in a clean environment to prevent any accumulation of
dust or other contaminants. Store inverted or capped with
aluminum foil.
4.1.2 The use of high purity reagents and solvents helps to
minimize interference problems. Purification of solvents
by distillation in all-glass systems may be required.
4.2 Interfering contamination may occur when a sample containing a low
concentration of ETU is analyzed immediately following a sample
containing a relatively high concentration of ETU. Thorough
between-sample rinsing of the sample syringe and associated
equipment with ethyl acetate can minimize sample cross contamin-
ation. After analysis of a sample containing high concentrations
of ETU, one or more injections of ethyl acetate should be made to
ensure that accurate values are obtained for the next sample.
4.3 Matrix interferences may be caused by contaminants that are
coextracted from the sample. The extent of matrix interferences
may vary considerably from source to source, depending upon the
sample. Tentative identifications must be confirmed using the
confirmation column (Sect. 6.7,2) and the conditions in Table 1.
4.4 Studies have shown that persistent ETU decomposition is
circumstantially linked to free radical mechanism. Addition of a
free radical scavenger is necessary to prohibit any free radical
reactions.
5. SAFETY
5.1 ETU is a suspected carcinogen and teratogen. Primary standards of
ETU should be prepared in a hood. A NIOSH/MESA approved toxic gas
respirator should be worn when the analyst handles high concentra-
tions of ETU. Each laboratory is responsible for maintaining a
current awareness file of OSHA regulations regarding the safe
handling of the chemicals specified in this method. A reference
file of material data handling sheets should also be made available
-------
to all personnel involved in the chemical analysis. Additional
references to laboratory safety are available and have been
identified (3-5) for the information of the analyst.
6. EQUIPMENT AND SUPPLIES
6.1 SAMPLING CONTAINERS — 60-mL screw cap vials equipped with Teflon-
faced silicone septa. Prior to use, wash vials and septa with
detergent and rinse with tap and distilled water. Allow the septa
to air dry at room temperature, place in a 105°C oven for 1 hr,
then remove and allow to cool in an area known to be free of
organics. Heat vials at 400°C for 1 hr to remove organics.
6.2 GLASSWARE .
6.2.1 Concentrator tube, Kuderna-Danish (K-D) - 10-mL or 25-mL,
graduated. Calibration must be checked at the volumes
employed in the test. Ground glass stoppers are used to
prevent evaporation of extracts.
6.2.2 Evaporative flask, K-D - 500-mL Attach to concentrator
tube with springs.
6.2.3 Snyder column, K-D - three-ball macro to which a condenser
can be connected to collect solvent.
6.2.4 Vials - Glass, 5 to 10-mL capacity with Teflon lined screw
caps.
6.3 Boiling stones - carborundum, #12 granules, heat at 400°C for 30
min prior to use. Cool and store in a desiccator.
6.4 Water bath - Heated, capable of temperature control (±2°C). The
bath should be used in a hood.
6.5 Balance - Analytical, capable of accurately weighing to the nearest
0.0001 g.
6.6 Tube heater - Capable of holding 8 K-D concentrator tubes and
heating the mid-section of the tubes to 35-40°C while applying a
nitrogen stream.
6.7 GAS CHROMATOGRAPH - Analytical system complete with GC equipped
with a nitrogen-phosphorus detector, split/splitless injector for
capillary columns and all required accessories. A data system is
recommended for measuring peak areas. An autoinjector is
recommended to improve precision of analyses.
6.7.1 Primary column - DB-Wax or equivalent,10-m x 0.25 mm I.D.
bonded fused silica column, 0.25 urn film thickness.
Validation data presented in this method were obtained
using this column. Alternative columns may be used
provided equal or better peak separation and peak shape
are obtained.
-------
6.7.2 Confirmation column - DB-1701 or equivalent, 5 m x 0.25 mm
I.D. bonded fused silica column, 0.25 pm film thickness.
6.7.3 Detector - Nitrogen-phosphorus (NPD). This detector has
proven effective in the analysis of fortified reagent and
artificial ground waters. A NPD was used to generate the
validation data presented in this method. Alternative de-
tectors, including a mass spectrometer, may be used.
7. REAGENTS AND STANDARDS
7.1 REAGENT WATER — Reagent water is defined as water in which an
interference is not observed at the retention time for ETU at the
method detection limit. A Millipore Super-Q Water System or its
equivalent may be used to generate reagent water. Water that has
been charcoal filtered may also be suitable.
7.2 Methylene chloride, ethyl acetate — distilled-in-glass quality or
equivalent.
7.3 Nitrogen gas - high purity.
7.4 Extraction column, Extrelut QE - Obtained from EM Science (Catalog
No. 902050-1). Extrelut QE columns contain a specially modified
form of large pore Kieselguhr with a granular structure.
7.5 Ammonium chloride, granular, ACS grade — for pH and ionic strength
adjustment of samples.
7.6 Potassium fluoride, anhydrous, ACS grade — for ionic strength
adjustment of sample.
7.7 Dithiothreitol (DTT) (Cleland's reagent) - for use as a free-
radical scavenger (available from Aldrich Chemical Co.).
7.7.1 DTT in ethyl acetate, 1000 jug/mL - Prepare by adding i g
DTT to a 1-L volumetric flask and diluting to volume with
ethyl acetate. Store at room temperature'.
7.8 Propylene thiourea (PTU) - For use as a surrogate standard.
Prepared from carbon disulfide and 1,2-diaminopropane using the
procedure published by Hardtmann, et. al. (Journal of Medicinal
Chemistry, 18(5), 447-453, 1975).
7.9 3,4,5,6-Tetrahydro-2-pyrimidinethiol (THP) - >98% purity, for use
as an internal standard (available from Aldrich Chemical Co.).
7.10 ARTIFICIAL GROUND WATERS — Two artificial ground waters were used
to generate the validation data in this method. The first was used
to mimic a hard ground water, and the second used to mimic a ground
water with high organic content.
7.10.1 Hard artificial ground water — Absopure Natural Artesian
Spring Water obtained from the Absopure Water Company in
Plymouth, Michigan.
-------
7.10.2 Organic-contaminated artificial ground water — Reagent
water spiked with fulvic acid at the 1 mg/L concentration
level. A well -characterized fulvic acid, available from
the International Humic Substances Society (associated
with the United States Geological Survey in Denver,
Colorado), was used.
7.11 STOCK STANDARD SOLUTION (0.10 jug//iL) - The stock standard solution
may be purchased as a certified solution or prepared from pure
standard material using the following procedure:
7.11.1 Prepare stock standard solution by accurately weighing
0.0010 g of pure ETU. Dissolve the ETU in ethyl acetate
containing 1000 /jg/mL of DTT and dilute to volume in a
10-mL volumetric flask. Larger volumes may be used at the
convenience of the analyst. If ETU purity is certified at
96% or greater, the weight may be used without correction
to calculate the concentration of the stock standard.
Commercially prepared stock standards may be used at any
concentration if they are certified by the manufacturer or
by an independent source.
7.11.2 Transfer the stock standard solution into a Teflon sealed
screw cap vial. Store at room temperature and protect
from light.
7.11.3 The stock standard solution should be replaced after two
weeks or sooner if comparison with laboratory control
standards indicates a problem.
7.12 INTERNAL STANDARD FORTIFYING SOLUTION -- Prepare an internal
standard fortifying solution by accurately weighing 0.0010 g of
pure THP. Dissolve the THP in ethyl acetate containing 1000 /ig/mL
of DTT and dilute to volume in a 10-mL volumetric flask. Transfer
the solution to a Teflon sealed screw cap bottle and store at room
temperature. Addition of 50 ill of the internal standard fortifying
solution to 5 mL of sample extract results in a final internal
standard concentration of 1.0 /jg/mL.
7.13 SURROGATE STANDARD FORTIFYING SOLUTION - Prepare a surrogate
standard fortifying solution by accurately weighing 0.0010 g of
pure PTU. Dissolve the PTU in ethyl acetate containing 1000 /ig/mL
of DTT and dilute to volume in a 10-mL volumetric flask. Transfer
the solution to a Teflon sealed screw cap bottle and store at room
temperature. Addition of 5 M- of the surrogate standard fortifying
solution to a sample prior to extraction results in a surrogate
standard concentration in the sample of 10 fig/I and, assuming
quantitative recovery of PTU, a surrogate standard concentration in
the final extract of 0.10
7.14 INSTRUMENT PERFORMANCE CHECK SOLUTION - Prepare the instrument
performance check solution by adding 10 juL of the ETU stock
standard solution, 1.0 mL of the internal standard fortifying
solution, and 100 pi of the surrogate standard fortifying solution
to a 100-mL volumetric flask and diluting to volume with ethyl
-------
acetate containing 1000 /xg/mL of DTT. Transfer the solution to a
Teflon sealed screw cap bottle and store at room temperature.
8. SAMPLE COLLECTION. PRESERVATION. AND STORAGE
8.1 SAMPLE COLLECTION — Grab samples must be collected in 60-mL glass
containers fitted with Teflon-lined screw caps (Sect. 6.1).
Conventional sampling practices (6) should be followed; however,
the bottle must not be prerinsed with sample before collection.
After the sample is collected in the bottle, seal the bottle and
shake vigorously for 1 min.
8.2 SAMPLE PRESERVATION — ETU may degrade in some samples even when
the sample is refrigerated. No suitable preservation reagent has
been found other than mercuric chloride. However, the use of
mercuric chloride is not recommended due to its toxicity and
potential harm to the environment. Previously, mercuric chloride
- was used to prevent only biological degradation. Preservation tests
indicate that ETU is chemically stable in aqueous samples.
Biological degradation may occur only rarely in samples with
limited biological activity such as finished drinking waters.
8.3 SAMPLE STORAGE — The samples must be iced or refrigerated at 4°C
and protected from light from the time of collection until
extraction. Samples should be extracted as soon as possible after
collection to avoid possible degradation of ETU.
9. QUALITY CONTROL
9.1 Each laboratory using this method is required to operate a formal
quality control (QC) program. The minimum requirements of this
program consist of the following: an initial demonstration of
laboratory capability; measurement of the surrogate compound in
each sample; analysis of laboratory reagent blanks, laboratory
fortified blanks, laboratory fortified matrix samples, and QC check
standards.
9.2 LABORATORY REAGENT BLANKS — Before processing any samples, the
analyst must demonstrate that all glassware and reagent
interferences are under control. This is accomplished by analyzing
a laboratory reagent blank (LRB). A LRB is a 50-mL aliquot of
reagent water, fortified with the internal standard and the
surrogate compound, that is analyzed according to Sect. 11 exactly
as if it were a sample. Each time a set of samples is analyzed or
reagents are changed, it must be demonstrated that the laboratory
reagent blank is free of contamination that would prevent the
determination of ETU at the MDL. All interfering contaminants must
be eliminated before sample analyses are started.
-------
9.3 INITIAL DEMONSTRATION OF CAPABILITY
9.3.1 Select a representative ETU concentration about 10 to 20
times the MDL or at the regulatory MCL, whichever is
lower. Prepare a primary dilution standard in ethyl
acetate 1000 times more concentrated than the selected
concentration.
9.3.2 Using a syringe, add 50 ftL of the primary dilution
standard to each of a minimum of four 50-mL aliquots of
reagent water. Also add an appropriate amount of the
internal standard and surrogate to each sample. A
representative ground water may be used in place of the
reagent water, but one or more unfortified aliquots must
be analyzed to determine background levels, and the
fortified level must exceed twice the background level for
the test to be valid. Analyze the aliquots according to
the method beginning in Sect. 11.
9.3.3 Calculate the measured concentration of ETU1 in each
replicate, the average percent recovery (R), the -relative
standard deviation of the percent recovery (RSD), and the
MDL (1). Ground water background corrections must be made
before R and RSD calculations are performed.
9.3-.4 The mean recovery value of ETU, expressed as a percentage
of the true value, must fall within ± 30%, and the
relative standard deviation of the mean recovery should be
less than 30%. If these conditions do not exist, this
procedure must be repeated using four fresh samples until
satisfactory performance has been demonstrated.
9.4 The analyst is permitted to modify GC columns, GC conditions, or
detectors to improve the separations, identifications, or lower the
cost of measurement. Each time a modification is made, the analyst
is required to repeat the procedure in Sect. 9.3.
9.5 ASSESSING SURROGATE RECOVERY
9.5.1 All samples and blanks must be fortified with the
surrogate compound according to Sect. 11.1 before
extraction to monitor preparation and analysis of samples.
9.5.2 Surrogate recovery must be evaluated for acceptance by
determining whether the measured surrogate concentration
(expressed as percent recovery) falls within the required
recovery limits. Performance-based recovery criteria for
PTU has been generated from single-laboratory results.
Measured recovery of PTU must be between 70 and 130
percent.
9.5.3 If the surrogate recovery for a sample or blank is outside
of the required surrogate recovery limits specified in
Sect. 9.5.2, the laboratory must take the following
actions:
-------
(1) Check calculations to make sure there are no errors.
(2) Check internal standard and surrogate standard
solutions for degradation, contamination, or other
obvious abnormalities.
(3) Check instrument performance.
Reinject the extract if the above steps fail to reveal the
cause of the problem. The problem must be identified and
corrected before continuing. Reanalyzing the sample or
blank, if possible, may be the only way to solve the
problem.
9.6 ASSESSING THE INTERNAL STANDARD
9.6.1 The analyst is expected to monitor the internal standard
peak area in all samples and blanks during each analysis
day. The IS response for any sample chroiliatogram should
not deviate from the IS response of the most recent daily
calibration check standard by more than 30%.
9.6.2 If >30% deviation occurs with an individual extract,
optimize instrument performance and inject a second
aliquot of that extract. If the reinjected aliquot
produces an acceptable IS response, report results for
that injection. If a deviation >30% is obtained for the
reinjected extract, reanalyze the sample beginning with
Sect. 11, provided the sample is still available.
Otherwise, report results obtained from the reinjected
extract, but mark them as suspect.
9.6.3 If consecutive samples fail the IS response acceptance
criteria, immediately analyze a medium calibration check
standard. If the check standard provides a response
factor (RF) within 20% of the predicted value, then follow
procedures itemized in Sect. 9.6.2 for each sample failing
the IS response criteria. If the check standard provides
a response factor (RF) which deviates more than 20% from
the predicted value, then the analyst must recalibrate.
9.7 ASSESSING LABORATORY PERFORMANCE
9.7.1 The laboratory must analyze at least one laboratory
fortified blank (LFB) per sample set. The ETU fortifying
concentration in the LFB should be 10 to 20 times the MDL
or the regulated MCL. Calculate the percent recovery of
the ETU. If the recovery falls outside the control limits
(see Sect. 9.7.2), the system is judged out of control and
the source of the problem must be identified and resolved
before continuing analyses.
9.7.2. Until sufficient LFB data become available, usually a
minimum of 20 to 30 results, the laboratory should assess
its performance against the control limits described in
Sect. 9.3.4. When sufficient laboratory performance data
-------
become available, develop control limits from the mean
percent recovery (R) and standard deviation (S) of the
percent recovery. These data are used to establish upper
and lower control limits as follows:
Upper Control Limit = R + 3S
Lower Control Limit = R - 3S
After five to ten new recovery measurements are made,
control limits should be recalculated using only the most
recent 20 to 30 data points.
9.7.3 Each laboratory should periodically determine and document
its detection limit capabilities for ETU.
9.7.4 At least once each quarter, preferably more frequently,
each laboratory should analyze quality control samples.
If criteria provided with the QCS are not met, corrective
action should be taken and documented.
9.7.5 Each laboratory must analyze an unknown performance
evaluation (PE) sample at least once a year. ETU results
must be within acceptable limits established by the
Quality Assurance Research Division of the Environmental
Monitoring Systems Laboratory, U.S. Environmental
Protection Agency, Cincinnati, Ohio.
9.8 ASSESSING INSTRUMENT PERFORMANCE — Instrument performance should
be monitored on a daily basis by analyzing the instrument
performance check solution (IPC). The IPC contains compounds
indicates appropriate sensitivity and column performance. The IPC
components and performance criteria are listed in Table 4.
Inability to demonstrate acceptable instrument performance
indicates the need for remedial action on the GC-NPD system. A
chromatogram from the analysis of the IPC is shown in Figure 1.
The sensitivity requirements are set according the MDL. MDLs will
vary somewhat in different laboratories according to instrument
capabilities.
9.9 ANALYTE CONFIRMATION — When doubt exists over the identification
of a peak on the chromatogram, confirmatory techniques such as
chromatography with a dissimilar column, or an alternate technique
such as particle beam/HPLC/mass spectrometry (EPA Method 553) may
be used. A suggested confirmation column is described in Table 1.
9.10 ADDITIONAL QC — It is recommended that the laboratory adopt
additional quality assurance practices for use with this method.
The specific practices that are most productive depend upon the
needs of the laboratory and the nature of the samples.
10. CALIBRATION AND STANDARDIZATION
10.1 Establish GC operating parameters equivalent to those indicated in
Table 1. Ensure that the gas chromatographic system is working
properly by injecting the instrument performance check solution
(Sect. 7.14) and checking for proper peak shapes, reasonable
-------
retention times, and sufficient sensitivity. The GC system is
calibrated using the internal standard technique (Sect. 10.2).
10.2 INTERNAL STANDARD CALIBRATION PROCEDURE — This approach requires
the analyst to select at least one internal standard compatible in
.analytical behavior to the compound of interest. The analyst must
further demonstrate that the measurement of the internal standard
is not affected by method or matrix interferences. In developing
this method, THP (3,4,5,6-tetrahydro-2-pyrimidinethiol) was found
to be a suitable internal standard.
10.2.1 Prepare ETU calibration standards at five concentration
levels by adding volumes of the ETU stock standard
solution to five volumetric flasks. To each flask, add a
known constant amount of internal standard and dilute to
volume with ethyl acetate containing 1000 ng/ml of DTT.
One of the standards should be representative of an ETU
concentration near, but above, the MDL. The other concen-
trations should correspond to the range of concentrations
expected in the sample concentrates, or should define the
working range of the detector.
10.2.2 Inject each calibration standard and tabulate the relative
response for ETU to the internal standard (RR ) using the
equation:
where: Aa = the peak area of ETU, and
A}s = the peak area of the internal standard.
Generate a calibration curve of RR versus ETU
concentration in the sample in /ig/L.
10.2.3 The working calibration curve must be verified on each
working shift by the measurement of one or more
calibration standards. If the ETU response varies from
the predicted response by more than 20%, the test should
be repeated using a fresh calibration standard.
Alternatively, a new ETU calibration curve should be
prepared.
11. PROCEDURE
11.1 SAMPLE EXTRACTION
11.1.1 Pi pet a 50-mL aliquot of water sample into a sample bottle
(Sect. 6.1) containing 1.5 g of ammonium chloride and 25 g
of potassium fluoride. Seal bottle and shake vigorously
until salts are dissolved. Fortify the sample with 5 /iL
of the surrogate standard fortifying solution (Sect.
7.13).
11.1.2 Pour contents of bottle onto Extrelut column. Allow the
column to stand undisturbed for 15 min.
-------
11.1.3 Add 5 ml of 1000 ng/mL DTT in ethyl acetate to a K-D
concentrator tube equipped with a 500-mL flask.
11.1.4 Add 400 ml of methylene chloride in 50-75 ml portions to
the Extrelut column and collect the eluant in the K-D
apparatus (Sect. 11.1.3).
11.2 EXTRACT CONCENTRATION
11.2.1 Conduct the following work in a fume hood which is
properly vented. Add 1 or 2 boiling stones to the K-D
apparatus and attach a macro Snyder column. Prewet the
Snyder column by adding about 1 ml of methylene chloride
to the top. Attach a condenser to the Snyder column to
recover the methylene chloride as it escapes the column.
Place the K-D apparatus in a 65-70°C water bath so that
the K-D tube is partially immersed in the hot water, and
the entire lower rounded surface of the flask is bathed
with hot vapor. When the apparent volume of liquid
reaches 5 ml, remove the K-D apparatus and allow it to
drain and cool for at least 10 min.
11.2.2 Reduce the liquid volume in the K-D tube to approximately
1 ml by placing the sample in a tube heater at 35-40°C
under a stream of nitrogen. The tube heater heats the
solvent in the K-D tube at volume markings between 1 and
10 ml.
11.2.3 Dilute sample to 5 ml with ethyl acetate; rinse walls of
K-D tube while adding ethyl acetate. Immediately fortify
the sample with 50 nl of internal standard fortifying
solution (Sect. 7.12). Agitate sample to disperse
internal standard. Transfer sample to a GC vial and
determine ETU by GC-NPD as described in Sect. 11.3.
Samples should be protected from light and analyzed within
24 hours of extraction. Sample extracts can be stored for
up to 28 days, frozen at -10°C and protected from light.
11.3 GAS CHROMATOGRAPHY
11.3.1 Table 1 summarizes the recommended GC operating condi-
tions. Included in Table 1 are retention times observed
using this method. An example of the separations achieved
using these conditions are shown in Figure 1. Other GC
columns, chromatographic conditions, or detectors may be
used if the requirements of Sect. 9.3 are met.
11.3.2 Calibrate the system daily as described in Sect. 10. The
standards and extracts must be in ethyl acetate.
11.3.3 Inject 2 /iL of the sample extract. Record the resulting
peak size in area units.
11.3.4 The width of the retention time window used to make
identifications should be based upon measurements of
actual retention time variations of standards over the
-------
course of a day. Three times the standard deviation of a
retention time can be used to calculate a suggested window
size for a compound. However, the experience of the
analyst should weigh heavily in the interpretation of
chromatograms.
12. DATA ANALYSIS AND CALCULATIONS
12.1 Calculate the ETU concentration in the sample from the ETU relative
response (RRa) to the internal standard using the calibration curve
described in Sect. 10:2.2.
12.2 For samples processed as part of a set where the laboratory control
standard recovery falls outside of the control limits in Sect.
9.7.2, ETU data must be labeled as suspect.
13. METHOD PERFORMANCE
13.1 In a single laboratory, ETU recovery and precision data from
reagent water were determined at four concentration levels.
Results were used to determine the MDL and demonstrate method
range. These data are given in Table 2. Data from the
inter!aboratory method validation study of this method are also
available.
13.2 In a single laboratory, ETU recovery and precision data from two
artificial ground waters were determined at a single concentration
level of 10 pg/L. Results were used to demonstrate applicability
of the method to different ground water matrices. These data are
listed in Table 3.
14. POLLUTION PREVENTION
14.1 Although this method requires 400 ml methylene chloride extracting
solvent per sample, no pollution of the environment will occur due
to the recovery of the solvent during the extract concentration
procedure. Very little solvent will escape the fume hood. No
other solvents are utilized in this method except for the very
small amount of ethyl acetate needed to make up calibration and
fortification standards. These small amounts of solvent pose no
threat to the environment.
14.2 For information about pollution prevention that may be applicable
to laboratory operations, consult "Less is Better: Laboratory
Chemical Management for Waste Reduction" available from the
American Chemical Society's Department of Government Relations and
Science Policy, 1155 16th Street N.W., Washington, D.C. 20036.
15. WASTE MANAGEMENT
15.1 It is the laboratory's responsibility to comply with all federal,
state, and local regulations governing waste management,
particularly the hazardous waste identification rules, and land
disposal restrictions. The laboratory has the responsibility to
protect the air, water, and land by minimizing and controlling all
releases from fume hoods and bench operations. Compliance is also
-------
required with any sewage discharge permits and regulations. For
further information on waste management, consult "The Waste
Management Manual for Laboratory Personnel," also available from
the American Chemical Society at the address in Sect. 14.2.
16. REFERENCES
1. 40 CFR, Part 136, Appendix B
2. ASTM Annual Book of Standards, Part 31, D3694, "Standard Practice
for Preparation of Sample Containers and for Preservation,"
American Society for Testing and Materials, Philadelphia, PA, p.
679, 1980.
3. "Carcinogens - Working with Carcinogens," Department of Health,
Education, and Welfare, Public Health Service, Center for Disease
Control, National Institute for Occupational Safety and Health,
Publication No. 77-206, Aug. 1977.
4. "OSHA Safety and Health Standards, General Industry," (29 CFR
1910), Occupational Safety and Health Administration, OSHA 2206,
(Revised, January 1976).
5. "Safety in Academic Chemistry Laboratories," American Chemical
Society Publication, Committee on Chemical Safety, 3rd Edition,
1979.
6. ASTM Annual Book of Standards, Part 31, D3370, "Standard Practice
for Sampling Water," American Society for Testing and Materials,
Philadelphia, PA, p. 76, 1980.
-------
17. TABLES. DIAGRAMS. FLOWCHARTS. AND VALIDATION DATA
TABLE 1. PRIMARY AND CONFIRMATION CHROMATOGRAPHIC CONDITIONS
Analyte
Retention Time, min
Primary column
Confirmation column
ETU
THP (internal standard)
PTU (surrogate standard)
3.5
5.1
2.7
4.5
5.0
2.2
Primary conditions:
Column:
Carrier gas:
Makeup gas:
Detector gases:
Injector temperature:
Detector temperature:
Oven temperature:
Sample:
Detector:
Confirmation conditions:
Column:
Carrier gas:
Makeup gas:
Detector gases:
Injector temperature:
Detector temperature:
Oven temperature:
Sample:
Detector:
10 m long x 0.25 mm I.D. DB-Wax bonded fused
silica column (J&W), 0.25 m film thickness
He @ 30 cm/sec linear velocity
He @ 30 mL/min flow
Air @ 100 mL/min flow; H2 @ 3 mL/min flow
220°C
230°C
220°C isothermal
2 fil splitless; 9 sec split delay
Nitrogen-phosphorus
5 m long x 0.25 mm I.D. DB-1701 bonded fused
silica column (J&W), 0.25 m film thickness
He @ 30 cm/sec linear velocity
He @ 30 mL/min flow
Air @ 100 m:/min flow; H2 @ 3 mL/min flow
150°C
270°C
150°C isothermal
2 nl splitless; 9 sec split delay
Nitrogen-phosphorus
-------
TABLE 2. RESULTS FROM HDL AND METHOD RANGE STUDIES (a)
Fortified
Level ,
itg/i
5.0
10
25
100
Amt in
Blank,
W/L
0.492
ND (b)
ND
ND
n(d)
7
7
7
7
R(e)
97 (c)
102
94
97
S(f)
0.845
0.886
1.31
5.96
RSD(g)
17
9
6
6
-
MDL
2.7
- •
_
—
(a) Studies conducted in reagent water; average recovery of PTU surrogate
from seven fortified reagent water samples was 100% (RSD) was 8.5%).
(b) ND = not detected.
(c) Data corrected for amount detected in blank.
(d) n - number of recovery data points.
(e) R = average percent recovery.
(f) S = standard deviation.
(g) RSD - percent relative standard deviation.
-------
TABLE 3. RESULTS FROM MATRIX EVALUATION STUDIES (a)
Matrix
Hard (b)
Organic-contaminated (c)
(a) Samples were fortified
(b) Absopure Natural Artesi
Amt. in
Blank,
WI-
ND (d)
ND
with at the 10
an Spring water
n(e)
7
7
/ig/L level
obtained
R(f)
93
93
with
from
S(g)
0.372
0.253
ETU.
the Absopure
RSD(h)
4
3
Water
Company in Plymouth, Michigan.
(c) Reagent water fortified with fulvic acid at the 1 mg/L concentration
level. A well-characterized fulvic acid, available from the
International Humic Substances Society (associated with the United
States Geological Survey in Denver, Colorado), was used,,
(d) ND = not detected.
(e) n = number of recovery data points.
(f) R = average percent recovery.
(g) S = standard deviation.
(h) RSD = percent relative standard deviation.
-------
TABLE 4. QUALITY CONTROL STANDARD
Test
Sensitivity
Chromatographic
performance
Cone.
Analyte /*g/mL
Ethyl ene thiourea (ETU) 0.01
3,4,5,6-Tetrahydro- 1
2-pyrimidinethiol (THP)
Requirements (a)
Detection of analyte; S/N > 3
PSF between 0.95 and 1.05 (a)
PSF between 0.93 and 1.07 (b)
(a) PSF = peak symmetry factor. Calculated using the equation.
PSF
0.5 x W(%)
where w(^) is the width of the front of the peak at half height and W(^) is the peak
width at half height.
(b) PGF = peak Gaussian factor. Calculated using the equation.
1.83 x w(%)
. PGF -
where W(^) is the peak width at half height and W(l/10) is the peak width at tenth
height.
-------
*
•o
\ fl
V *
19
4)
(Q
cn
2
Li S
i V Uu
erf
F
r
fr
O
>•
o
i
o
o
g
o
•Q
o.
-------
------- |