600-4-85-018
DETERMINATION OF NEUTRAL NITROGEN-CONTAINING
PESTICIDES IN INDUSTRIAL AND MUNICIPAL
WASTEWATERS - FENARIMOL, MCK 264, MGK 326, AND PRONAMIDE
by
J.S. Warner, T.M. Engel and P.J. Mondron
Battelie Columbus Laboratories
Columbus, Ohio 43201
Contract No. 68-03-2956
Project Officer
Thomas Press ley
Physical and Chemical Methods Branch
Environmental Monitoring and Support Laboratory
Cincinnati, Ohio 45268
ENVIRONMENTAL MONITORING AND SUPPORT LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
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DISCLAIMER
The information in this document has been funded wholly or in part by the
United States Environmental Protection Agency under contract 68-03-2956
to Battelle Columbus Laboratories. It has been subject to the Agency's
peer and administrative review, and it has been approved for publication
as an EPA document.
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FOREWORD
Environmental measurements are required to determine the quality of
ambient waters and the character of waste effluents. The Environmental
Monitoring and Support Laboratory - Cincinnati, conducts research to:
o Develop and evaluate methods to measure the presence and
concentration of physical, chemical, and radiological pollutants in
water, wastewater, bottom sediments, and solid wastes.
o Investigate methods for the concentration, recovery, and
identification of viruses, bacteria and other microbiological
organisms in water; and, to determine the responses of aquatic
organisms to water quality.
o Develop and operate an Agency-wide quality assurance program to
assure standardization and quality control of systems for monitoring
water and wastewater.
o Develop and operate a computerized system for instrument automation
leading to improved data collection, analysis, and quality control.
This report is one of a series that investigates the analytical behavior
of selected pesticides and suggests a suitable test procedure for their
measurement in wastewater. The method was modeled after existing EPA
methods being specific yet as simplified as possible.
Robert L. Booth, Acting Director
Environmental Monitoring and Support
Laboratory - Cincinnati
111
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ABSTRACT
A method was developed for the determination of four neutral
nitrogen-containing compounds (fenaridfol, MGK 264, MGK 326, and pronamide)
in wastewaters. The method development program consisted of a literature
review; determination of extraction efficiency for each compound from water
into methylene chloride; development of a deactivated Florisil cleanup
procedure; and determination of suitable gas chromatographic (GO) analysis
conditions.
The final method was applied to wastewaters from manufacturers of
fenarimol and pronamide in order to determine precision and accuracy. The
wastewater from a manufacturer of fenarimol was spiked with the four
compounds at the 20 yg/L level. The wastewater from a manufacturer of
pronamide was spiked with the four compounds at the 500 yg/L level.
Recoveries for the four compounds were in the 74 to 110 percent range at
both concentration levels. Method detection limits (MDLs) for the four
compounds in distilled water were in the 2 to 6 yg/L range, but may be
higher in wastewaters due to interfering compounds.
This report was submitted in partial fulfillment of Contract No.
68-03-2956 by Battelle Columbus Laboratories under the sponsorship of the
U. S. Environmental Protection Agency. This report covers the period from
August 1, 1981 to September 30, 1983.
IV
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FIGURES
Number Page
1 GC-AFD Chromatogram of 100 ng Each of the Neutral
Nitrogen-Containing Compounds (Column 1) 9
2 GC-AFD Chromatogram of 200 ng Each of the Neutral
Nitrogen-Containing Compounds (Column 2) 10
3 Analytical Curve for Fenarimol 12
4 Analytical Curve for MGK 264 13
5 Analytical Curve for MGK 326 14
6 Analytical Curve for Pronamide 15
7 GC-AFD Chromatograms of Fenarimol Wastewater (a) Spiked
at the 20 yg/L Level with the Neutral Nitrogen-
Containing Compounds and (b) Unspiked 17
8 GC-AFD Chromatograms of Pronamide Wastewater (a) Spiked
at the 500 yg/L Level with the Neutral Nitrogen-
Containing Compounds and (b) Unspiked 18
9 El Mass Spectra of (a) Fenarimol Standard and (b)
Fenarimol Found in Fenarimol Wastewater 19
10 El Mass Spectra of (a) Pronamide Standard and (b)
Pronamide Identified in Pronamide Wastewater 20
vi
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TABLES
Number Page
1 Extraction of Neutral Nitrogen-Containing Compounds 7
2 Elution Orders and Recoveries of Neutral Nitrogen-
Containing Compounds 8
3 Data from MDL Determination for the Neutral Nitrogen-
Containing Compounds 11
4 Data from Analytical Curve Determination for the Neutral
Nitrogen-Containing Compounds 11
5 Recoveries from Validation of Neutral Nitrogen-Containing
Compounds Method 16
vii
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SECTION 1
INTRODUCTION
•
Fenarimol, MGK 264, MGK 326, and pronamide are neutral compounds
containing carbon, hydrogen, oxygen, nitrogen, and sometimes, chlorine.
They were combined into one group (neutral nitrogen containing compounds;
for method development purposes only.
Fenarimol (I) is a fungicide.
OH
Qfo
Its CAS registry number is 60168-88-9 and its IUPAC name is
2,4'-dichloro-(pyrimidin-5-yl)benzhydroyl alcohol. Other common synonyms
include "EL 222" and "Rubigam". The acute oral LD50 of fenarimol in rats
is 2500 ing/kg. It is unstable in sunlight. No additional information was
found during the literature search.
MGK 264 is used as an insecticidal synergist for pyrethroids and
is present in two isomeric forms (II) (III).
o
IE
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Its CAS registry number is 136-45-8 and its IUPAC name is
N-(2-«thylhexyl)-8,9,10-trinorborn-5-ene-2,3-dicarboxiaide. Common
synonyms include "Octacide 264", "Synergist 264", "Van Dyk 264", and
"Sinepyrin 222". Its LD50 in rats is 2800 mg/kg. The literature search
yielded descriptions of silica gel and solvent partition cleanup
procedures (1), and analyses by gas chromatography-electron capture
detector (GC-ECD) using various packed column types (1,2). Infra-red and
ultraviolet spectra were reported (3).
MGK 326 (IV) is a fly repellent.
1ST
Its CAS registry number is 113-48-4 and its IUPAC name is dipropyl
pyridine-2,5-dicarboxylate. Its other common synonyms include "R-326",
"MGK R-326", and "MGK Repellent 326". It is hydrolyzed by alkali and its
oral LD50 in rats is 6230 mg/kg. No information on analysis methods for
MGK 326 was found from the literature search.
Pronamide (V) is a herbicide used to control grasses in alfalfa,
lettuce, and other crops.
C!
<
Cl
Its CAS registry number is 23950-58-5 and its IUPAC name is N(l,l-
dimethylpropynyl)-3,5-dichlorobenzamide. Some of its synonyms include
"Kerb", "Kerb SOW", and "RH 315". The oral LD50 in rats for pronamide is
8350 mg/kg. The literature review revealed an analysis method by which
the pronamide is treated with acid to form methyl 3,5-dichlorobenzoate
which is subsequently analyzed by GC-ECD (4,5). No direct analysis method
for pronamide was found.
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The four compounds are neutral, relatively stable in water under
normal conditions, and contain nitrogen. For these reasons, the
selected approach to the determination of these compounds in water
included extraction from water with methylene chloride using separatory
funnel techniques, cleanup using Florisil adsorption chromatography and
analysis using packed column GC with an alkali flame detector (AFD).
The final method is included in Appendix A of this report.
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SECTION 2
CONCLUSIONS
EXTRACTION, CONCENTRATION, AND CLEANUP
The four neutral nitrogen-containing pesticides can be extracted
into methylene chloride from water and cleaned up on a two percent
deactivated Florisil column with greater than 85 percent recovery using
standard separatory funnel extraction techniques. Use of
Kuderna-Danish (K-D) equipment to perform extract concentrations did
not lead to significant recovery losses. The four neutral
nitrogen-containing pesticides elute from two percent deactivated
Florisil in acetone or less polar solvents. Recoveries greater than 85
percent are obtained. This cleanup procedure was used for wastewaters
from manufacturers of fenarimol and pronamide.
CHROMATOGRAPHY
Two packed GC columns, 3Z SP-2250 and 32 SP-2100, were found to be
acceptable for the GC-AFD analysis of these compounds. The 3Z SP-2250
column gave better peak shape for the four compounds and was designated
as the primary column. The 3% SP-2100 column was used as the secondary
column.
VALIDATION STUDIES
Recoveries of the four neutral nitrogen-containing compounds from
water at concentrations ranging from 10 to 1000 yg/L were generally
greater than 85 percent resulting in linear analytical curves. The
MDLs ranged from 2 to 6 Wg/L for all of the compounds. Recoveries of
the four compounds from wastewater from a manufacturer of fenarimol at
the 20 ug/L level and from wastewater from a manufacturer of pronamide
the 500 yg/L level were greater than 85 percent except for MGK 264 at
the 500 yg/L level (74 percent recovery).
GC-MS CONFIRMATION
Positive identifications of fenarimol and pronamide in wastewaters
were made by GC-MS.
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SECTION 3
EXPERIMENTAL
Studies were performed Co determine if techniques such as
separator? funnel extraction, Florisil adsorption chromatography
cleanup, K-D apparatus concentration, and packed column GC-AFD analysis
would be applicable to the determination of the four neutral
nitrogen-containing compounds in vastewaters. Since recovery data and
literature references indicated that these compounds are relatively
stable in water, stability studies were not performed.
EXTRACTION, CONCENTRATION, AND CLEANUP
One liter of spiked reagent water was placed in a two-liter
separatory funnel and the pH was adjusted to 7 by addition of 6 fll
sodium hydroxide or 6 If su If uric acid. The sample was spiked with
100 ug/L of each of the four compounds. The sample was extracted three
times with 60 ml each of methylene chloride. The combined extracts
were dried by passing them through 10 cm of anhydrous granular sodium
sulfate and then concentrated to one mL.
Florisil was activated by heating in a wide-mouth jar at 160-170°C
overnight. Florisil, 20 grams, was slurried in 100 mL of ethyl ether
containing 400 ul of reagent water. The slurry was transferred to a
chromatographic column. The solvent was allowed to elute from the
column. Petroleum ether, 25 mL, was added to the column and also
allowed to elute from the column. An additional 50 mL of petroleum
ether was added to the column. The above mentioned extract was added
to the petroleum ether. This solvent was eluted from the column and
collected (Fl). Nine additional 50 mL solvent elutions were collected:
six percent ethyl ether in petroleum ether (F2); 15 percent ethyl ether
in petroleum ether (F3); 50 percent ethyl ether in petroleum ether
(F4); ethyl ether (F5); six percent acetone in ethyl ether (F6); 15
percent acetone in ethyl ether (F7); 50 percent acetone in ethyl ether
(F8); acetone (F9); and six percent methanol in acetone (F10). Each
fraction was solvent exchanged to acetone and concentrated to 1 mL.
CHROMATOGRAPHY
Two columns, 32 SP-2250 on 100/120 mesh Supelcoport and 3Z SP-2100
on 100/120 mesh Supelcoport, were evaluated for the determination of
the four compounds by GC-AFD.
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VALIDATION STUDIES
The MDLs for the four compounds were determined by analyzing seven
replicate distilled water samples spiked at the 5 yg/L concentration
level.(6) The sample extracts were cleaned up using the Florisil cleanup
procedure prior to analysis. The amounts recovered were determined by
external standard calibration and the MDLs were calculated from these
data.
Distilled water was also spiked in duplicate at the 10, 50, 100,
500, and 1000 yg/L concentration levels and recoveries of the four
compounds were determined as described earlier. An analytical curve
was generated by plotting the amount spiked into the samples versus the
amount recovered from the samples.
A wastewater from a manufacturer of fenarimol and a wastewater
from a manufacturer of pronamide were used for method validation
studies. Seven aliquots of each wastewater were analyzed to determine
levels of interferences. The fenarimol wastewater was spiked with the
four compounds at the 20 yg/L level, processed and analyzed. The
pronamide wastewater was spiked at the 500 yg/L level and also
processed and analyzed. Seven replicate extractions were performed at
each concentration level.
GC-MS CONFIRMATION
Unspiked extracts from the fenarimol and pronamide wastewaters and
stock solutions of fenarimol and pronamide were analyzed by capillary
column GC-MS (electron impact (El) mode) to confirm tentative
identifications made of those two compounds.
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SECTION 4
RESULTS AND DISCUSSION
EXTRACTION, CONCENTRATION, AND CLEANUP
The four neutral nitrogen-containing pesticides were extracted
from distilled water into methylene chloride and cleaned up on two
percent deactivated Florisil with greater than 85 percent recovery.
Recovery data from duplicate extractions at the 100 yg/L concentration
level are given in Table 1.
TABLE 1. EXTRACTION OF NEUTRAL NITROGEN-CONTAINING COMPOUNDS
Compound
Fenarimol
MGK 264 (b)
MGK 326
Pronamide
Recovery* % (a)
93,
89,
90,
89,
102
103
107
105
(a) Results from duplicate analyses.
(b) Sum of two isomer peaks.
The four compounds eluted in Florisil fractions 5-9 (ethyl ether,
and six percent, 15 percent, and 50 percent acetone in ethyl ether, and
acetone). Elution order data are given in Table 2.
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TABLE 2. ELUTION ORDERS AND RECOVERIES OF NEUTRAL
NITROGEN-CONTAINING COMPOUNDS FROM FLORISIL
Recovery in Specified Fraction, %
Compound F5
Fenarimol
MGK 264
MGK 326
Pronamide 122
F6
80
110
F7
22
3
F8 F9 Total
102
113
30 70 100
122
(a) Elution solvents were 50 mL each of the following:
F5 - 100% ethyl ether
F6 - 6% acetone in ethyl ether
F7 - 15% acetone in ethyl ether
F8 » 50% acetone in ethyl ether
F9 - 100% acetone
CHROMATOGRAPHY
Both the 3% SP-2250 column and the 3% SP-2100 column were
satisfactory for the determination of the four neutral nitrogen-containing
compounds. The 3% SP-2250 column, however, gave better peak shape and was
chosen as the primary column. The following conditions were used for the
primary and alternate columns:
Column: 2m x 2.0mm I.D. 32 SP-2250 on 100/120
mesh Supelcoport or 2m x 2.0mm I.D. 3%
SP-2100 on 100/120 mesh Supelcoport
Detector: Alkali flame detector (AFD), 16 bead volts
Injector Temperature: 2508C
Detector Temperature: 300°C
Oven Temperature: 80°C isothermal for 4 min; programmed
from 80°C to 300*C at 8°C/min; 3008C
isothermal for 4 min.
Carrier Gas: Helium at 30 cc/min flow rate
Chromatograms obtained under these conditions are shown in Figures 1
and 2.
VALIDATION STUDIES
The MDLs for the neutral nitrogen-containing compounds ranged from 2
to 6 ug/L. Recoveries and MDL data are given in Table 3.
-------
Fenarimol
21.0
— r — | — t- •r~v— r""|-— r"i — r- i— 'j — ft— i — r—|- •?•••* — i — r--j- T -r**i"i — |- • r"« •••! •» " |
23.8 25.0 27.0 28.8 31.0 3310 33L0
Retention Time, Min.
Figure 1. GC-AFD Chromatogram of 100 ng Each of the Neutral Nitrogen-Containing Compounds
(Column 1)
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Fenarlmol
a 7
so
33
Retention Time, Min.
Figure 2. GC-AFD Chromatogram of 200 ng Each of the Neutral Nitrogen-Containing Compounds
(Column 2)
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TABLE 3. DATA FROM MDL DETERMINATION FOR THE NEUTRAL NITROGEN-
CONTAINING COMPOUNDS
Amount Spiked,
Compound yg/L
Fenarimol
MGK 264 (b)
MGK 326
Pronamide
5.0
5.0
5.0
5.0
Amount Recovered,
WK/L(a)
5.7 ±
4.8 ±
5.6 ±
5.1 ±
0.8
0.6
1.9
1.3
MDL, yg/L
2.4
1.8
6.0
4.1
(a) Average of seven replicate analyses.
(b) Sum of two isomer peaks.
Recoveries of the four compounds obtained during the analytical
curve studies were greater than 85 percent except at the 10 yg/ml
concentration level for fenarimol, MGK 264, and pronamide. The
resultant analytical curves were linear in the concentration range of
10 to 1000 yg/L. Recovery data are given in Table 4. The analytical
curves generated from the recovery data are shown in Figures 3-6.
TABLE 4. DATA FROM ANALYTICAL CURVE DETERMINATION FOR THE
NEUTRAL NITROGEN-CONTAINING COMPOUNDS
Compound
Fenarimol
MGK 264 (b)
MGK 326
Pronamide
Amount
10
6.6,9.3
6.6,8.3
8.6,9.8
5.6,9.2
Recovered at Given Spike Level.
50
41,49
49,43
48,43
42,60
100
92,100
89,100
90,110
89,110
500
480,420
470,400
500,420
470,410
, yg/L(a)
1000
990,920
1200,870
1200,890
1100,900
(a) Duplicate analyses.
(b) Sum of two isomer peaks.
11
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iooo r
900
• oo
i
700 J-
• 00 !-
Amount Recovered,
Ug/L
SOOf-
I
I
i.OOr
aoo
100
/:.
1 _L. 1.1,1
/
\
100 10O 3OO bOO 3OO «OO 7OO »OO 9OO 100O
Amount Spiked, ug/L
Figure 3. Analytical Curve for Fenarimol
12
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1OOO
• 00
• oo
700
• OO
Amount Recovered,
Ug/L 90°
4OO
300
200
10O •
10O 2OO 3OO 40O 300 «OO 7OO BOO »OO 1OOO
Amount Spiked, yg/L
Figure 4. Analytical Curve for MGK 264
13
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lOOOr
too •
• 00 •
70O •
• 00 *•
Amount Recovered,
Mg/L
4OO •
30O •
2OO •
100
2OO 3OO
90O «OO 7OO «OO »OO 10OO
Amount Spiked, ug/L
Figure 5. Analytical Curve for MGK 326
14
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10OO
• 00r
• 00
700
«oo
Amount Recovered,
Mg/L
300
JtOO
300 •
aoo •
100 >
^
tOO 200 3OO UOO 3OO «OO 7OO SOO 900 IOOO
Amount Spiked, ug/L
Figure 6. Analytical Curve for Pronamide
15
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Recoveries of the four compounds were greater then 85 percent
except for MGK 264 et the 500 yg/L level (74 percent recovery). Seven
one-liter aliquots of unspiked fenarimol vastevater were analyzed and
fenarimol was tentatively identified at the 1.8 ± 0.3 ug/L level.
Similarly, pronamide was tentatively identified in the pronamide
wastewater at the 210 ± 30 yg/L level. Recovery data were corrected
for the fenarimol and pronamide, levels in the wastewaters. Recovery
data from low and high level validations in wastewaters are given in
Table 5. Chromatograms of spiked and unspiked fenarimol wastewater
extracts and spiked and unspiked pronamide wastewaters are given in
Figures 7 and 8, respectively.
* TABLE 5.
»
Compound
Fenarimol
MGK 264 (b)
MGK 326
Pronamide
RECOVERIES FROM VALIDATION OF
COMPOUNDS METHOD
Wastewater(c)
1
2
1
2
1
2
1
2
Spike Level,
UR/L
20
500
20
500
20
500
20
500
NEUTRAL NITROGEN-<
Recovery, %(a)
98
96
96
74
110
95
100
86
:ONTAINING
Relative Standard
Deviation, 7.
4.4
4.4
22
3.5
6.6
3.7
4.9
3.3 .
(a) Average of seven replicate analyses.
(b) Sum of two isomer peaks
(c) 1 « Wastewater from a manufacturer of fenarimol.
2 * Wastewater from a manufacturer of pronamide.
GC-MS CONFIRMATION
The presence of fenarimol in the fenarimol wastewater and
pronamide in the pronamide wastewater were confirmed by GC-MS on the
basis of capillary column retention time and mass spectra. Mass
spectra of fenarimol and pronamide (in a standard mix and in unspiked
wastewater extracts) are given in Figures 9 and 10, respectively.
16
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MGK 326
PRONAMIDE
FENARIMOL
17 19 21
23 23 27
•inure*
2 9
3 1
I
33
I
33
(b)
FENARIMOL
i . , ...
23 23
»SBHTI*
—I—
29
17
I
2 1
27
31
33
33
Figure 7. GC-AFD Chromatograms of Fenarimol Wastewater (a) Spiked
at the 20 ug/L Level with the Neutral Nitrogen-Containing
Compounds and (b) Unsplked.
17
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FENARIMOL
21 33 23 27 29 31 33 33
• I•UTI•
(b)
17
21 23 23 27
29
31 33
Figure 8. GC-AFD Chromatograms of Pronamide Wastewater (a) Spiked
at the 500 ug/L Level with the Neutral Nitrogen-Containing
Compounds and (b) Unspiked.
18
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IM.I
90.1-
lis
ISO
2 9
291
243
I"1 f* I
230
r 22M2I.
tM.I
I 7
fo'
''
2«7
at
m/e, amu
2*1
(b)
9C1J8.
Figure 9. El Mass Spectra of (a) Fenarimol Standard and (b) Fenarimol
Found in Fenarimol Wastewater.
19
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MM-
•ill
2 Jii tfc-J!
JF
xr ai
75.1
tft.it
"H-« «fr« ,. U
T
1
1 ».
I7MM.
(b)
Figure 10.
m/e, amu
El Mass Spectra of (a) Pronamide Standard and (b)
Pronamide Identified in Pronamide Wastewater
20
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REFERENCES
1. Moore, J.B. Residue and Tolerence Considerations with Pyrethrum,
Piperonyl Butoxide, and MGK 264. In: The Natural Insecticide
Pyrethrum, Academic Press, Inc. New York and London, 1973.
pp 293-306.
2. Bruce, W.N. Detector Cell for Measuring Picogram Quantities
of Organophosphorus Insecticides, Pyrethrin Synergists, and
Other Compounds by Gas Chromatography. J. Agr. Food Cheat.,
15(1):178-181, 1967.
3. Gore, R.C., R.W. Hannah, S.C. Pattacini, and T.J. Porro.
Infrared and Ultraviolet Spectra of Seventy-six Pesticides
Journal of the AOAC. 54(5):1040-1082, 1971.
4. Adler, I.L, C.F. Gordon, L.D. Haines, and J.P. Wargo.
Determination of Residues of herbicide N-(l,1-Dimethylpropynyl)3-5-
Dichlorobenzamide by Electron Capture Gas-Liquid Chromatography.
Journal of the AOAC, 55(4):802-805, 1972.
5. Adler, I.L, L.D. Haines, and J.P. Wargo. Pronamide. In:
Analytical Methods for Pesticides and Plant Growth Regulators,
G. Zweig, ed. Academic Press, New York, London and San Francisco
1976. pp. 443-449.
6. Glaser, J. A., et al., "Trace Analysis for Wastewaters",
Environmental Science and Technology, 15, 1426 (1981).
21
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November 29, 1982
NEUTRAL NITROGEN-CONTAINING PESTICIDES
METHOD 633,1 __
•
1. Scope and Application
1.1 This method covers the determination of certain neutral nitrogen-
containing pesticides. The following parameters can be determined
by this method:
Parameter CAS No.
Fenarimol 60168-88-9
MGK 264-A 136-45-8
MGK 264-B 136-45-8
MGK 326 113-38-4
Pronamide 23950-58-5
1.2 This is a gas chromatographic (GC) method applicable to the deter-
mination of the compounds listed above in municipal and industrial
discharges as provided under 40 CFR 136.1. Any modification of
this method beyond those expressly permitted shall be considered a
major modification subject to application and approval of alternate
test procedures under 40 CFR 136.4 and 136.5.
1.3 The method detection limit (MDL, defined in Section 15)
for each compound is listed in Table 2. The MDL for a specific
wastewater may differ from those listed, depending upon the nature
of interferences in the sample matrix.
1.4 The sample extraction and concentration steps in this method are
similar to those in other 600 series methods. Thus, a single
sample may be extracted to measure the compounds included in the
22
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scope of the methods. When cleanup is required, the concentration
levels must be high enough to permit selecting aliquots, as
necessary, in order to apply appropriate cleanup procedures.
1.5 This method is restricted to use by or under the supervision of
analysts experienced in the use of gas chromatography 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 Section 8.2.
1.6 When this method is used to analyze unfamiliar samples for any or
all of the compounds above, compound identifications should be
supported by at least one additional qualitative technique. This
method describes analytical conditions for a second gas chroma-
tographic column that can be used to confirm measurements made with
the primary column. Section 14 provides gas chromatograph/mass
spectrometer (GC-MS) criteria appropriate for the qualitative
confirmation of compound identifications.
2. Summary of Method
2.1 A measured volume of sample, approximately 1 liter, is solvent
extracted with methylene chloride using a separatory funnel. The
methylene chloride extract is dried and concentrated to 1.0 ml.
Gas chromatographic conditions are described which permit the
separation and measurement of the compounds in the extract by
alkali flame detector gas chromatography (GC-AFD).l
2.2 This method provides an optional Florisil column cleanup procedure
to aid in the elimination of interferences which may be
encountered.
23
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3. Interferences
3.1 Method Interferences may be caused by contaminants in solvents,
reagents, glassware, and other sample processing apparatus that
lead to 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
Section 8.5.
3.1.1 Glassware must be scrupulously cleaned.2 Clean all
glassware as soon as possible after use by 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 15 to 30 min. Do not heat volumetric ware.
Some thermally stable materials such as PCBs may not be
eliminated by this treatment. Thorough rinsing with
acetone and pesticide quality hexane may be substituted for
the muffle furnace heating. After drying and cooling, seal
and store glassware in a clean environment to prevent any
accumulation of dust or other contaminants. Store inverted
or capped with aluminum foil.
3.1.2 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.
3.2 Matrix interferences may be caused by contaminants that are
coextracted from the sample. The extent of matrix interferences
24
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will vary considerably from source to source, depending upon the
nature and diversity of the industrial complex or municipality
sampled. The cleanup procedure in Section 11 can be used to
overcome many of these interferences, but unique samples may
require additional cleanup approaches to achieve the MDL listed in
Table 2.
4. Safety
4.1 The toxicity or carcinogenicity of each reagent used in this method
has not been precisely defined; however, each chemical compound
should be treated as a potential health hazard. From this
viewpoint, exposure to these chemicals must be reduced to the
lowest possible level by whatever means available. The 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.
5. Apparatus and Materials
5.1 Sampling equipment, for discrete or composite sampling.
5.1.1 Grab sample bottle - Amber borosilicate or flint glass, 1-
liter or 1-quart volume, fitted with screw caps lined with
Teflon. Foil may be substituted for Teflon if the sample
is not corrosive. If amber bottles are not available,
protect samples from light. The container and cap liner
25
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must be washed, rinsed with acetone or methylene chloride,
and dried before use to minimize contamination.
5.1.2 Automatic sampler (optional) - Must incorporate glass
sample containers for the collection of a minimum of
250 ml. Sample containers must be kept refrigerated at 4°C
and protected from light during compositing. If the
sampler uses a peristaltic pump, a minimum length of
compressible silicone rubber tubing may be used. Before
use, however, the compressible tubing should be thoroughly
rinsed with methanol, followed by repeated rinsings with
distilled water to minimize the potential for contamination
of the sample. An integrating flow meter is required to
collect flow proportional composites.
5.2 Glassware (All specifications are suggested. Catalog numbers are
included for illustration only.)
5.2.1 Separatory funnel - 2000-mL, with Teflon stopcock.
5.2.2 Drying column - Chromatographic column 400 mm long x 10 mm
ID with coarse frit.
5.2.3 Chromatographic column - 400 mm long x 19 mm ID with 250 mL
reservoir at the top and Teflon stopcock (Kontes K-420290
or equivalent).
5.2.4 Concentrator tube, Kuderna-Danish - 10-mL, graduated
(Kontes K-570050-1025 or equivalent). Calibration must be
checked at the volumes employed in the test. A ground
glass stopper is used to prevent evaporation of extracts.
26
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5.2.5 Evaporative flask, Kuderna-Danish - 500-mL (Kontes
K-570001-0500 or equivalent). Attach to concentrator tube
with springs.
5.2.6 Snyder column, Kuderna-Danish - three-ball macro (Kontes
K-503000-0121 or equivalent).
5.2.7 Snyder column, Kuderna-Danish - two-ball micro (Kontes
K-569001-0219 or equivalent).
5.2.8 Vials - Amber glass, 10 to 15 ml capacity with Teflon lined
screw-cap.
5.2.9 Erlenmeyer flask - 250-mL.
5.2.10 Graduated cylinder - 1000-mL.
5.2.11 Beaker - 250-mL.
5.3 Boiling chips - Approximately 10/40 mesh carborundum. Heat to
400°C for 4 hours or Soxhlet extract with methylene chloride.
5.4 Water bath - Heated, capable of temperature control (+20C). The
bath should be used in a hood.
5.5 Balance - Analytical, capable of accurately weighing to the nearest
0.0001 g.
5.6 Gas chromatograph - Analytical system complete with gas
chromatograph suitable for on-column injection and all required
accessories including syringes, analytical columns, gases,
detector, and strip-chart recorder. A data system is recommended
for measuring peak areas.
5.6.1 Column 1 - 180 cm long x 2 mm ID glass, packed with 3%
SP-2250 on Supelcoport (100/120 mesh) or equivalent. This
column was used to develop the method performance
27
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statements in Section 15. Alternate columns may be used in
accordance with the provisions described in Section 12.1.
5.6.2 Column 2 - 180 cm long x 2 mm 10 glass, packed with 3%
SP-2100 on Supelcoport (100/120 mesh) or equivalent.
5.6.3 Detector - Alkali-flame detector (AFD), sometimes referred
to as a nitrogen-phophorous detector (NPD) or a thermionic
specific detector (TSD). This detector has proven
effective in the analysis of wastewaters for the compounds
listed in the scope and was used to develop the method
performance statements in Section 15.
6. Reagents
6.1 Reagent water - Reagent water is defined as a water in which an
interferent is not observed at the method detection limit of each
parameter of interest,
6.2 Methylene chloride, methanol, petroleum ether, ethyl ether,
acetone, distilled-in-glass quality or equivalent. Ethyl ether
must be free of peroxides as indicated by EM Quant Test Strips
(available from Scientific Products Co., Catalog No. P1126-8 and
other suppliers). Procedures recommended for removal of peroxides
are provided with the test strips.
6.3 6N. Sodium hydroxide - Dissolve 24.0 grams NaOH in 100 mL of
reagent water.
6.4 6N.Sulfuric acid - Slowly add. 16.7 ml of cone. H2S04 (94%) to about
50 ml of reagent water. Dilute to 100 ml with reaqent water.
6.5 Sodium sulfate (ACS) Granular, anhydrous; heated in a muffle
furnace at 400°C overnight.
6.6 Florisil - PR grade (60/100 mesh). Purchase activated at 1250°F
and store in brown glass bottle. To prepare for use, place 150 g
28
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1n a wide-mouth jar and heat overnight at 160-17QOC. Seal tightly
with Teflon or aluminum foil-lined screw cap and cool to room
temperature.
6.7 Stock standard solutions (1.00 yg/uL) - Stock standard solutions
can be prepared from pure standard materials or purchased as
certified solutions.
6.7.1 Prepare stock standard solutions by accurately weighing
about 0.0100 grams of pure material. Dissolve the material
in distilled-in-glass quality methanol and dilute to volume
in a 10-mL volumetric flask. Larger volumes can be used at
the convenience of the analyst. If compound purity is
certified at 96% or greater, the weight can be used without
correction to calculate the concentration of the stock
standard. Commercially prepared stock standards can be
used at any concentration if they are certified by the
manufacturer or by an independent source.
6.7.2 Transfer the stock standard solutions into Teflon-sealed
screw-cap bottles. Store at 40C and protect from light.
Frequently check stock standard solutions for signs of
degradation or evaporation, especially just prior to
preparing calibration standards from them.
6.7.3 Stock standard solutions must be replaced after 6 months or
sooner if comparison with check standards indicates a
problem.
29
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7. Calibration
7.1 Establish gas chromatographic operating parameters equivalent to
those indicated in Table 2. The gas chromatographic system can be
calibrated using the external standard technique (Section 7.2) or
the internal standard technique (Section 7.3).
7.2 External standard calibration procedure:
7.2.1 For each compound of interest, prepare calibration stand-
ards at a minimum of three concentration levels by adding
volumes of one or more stock standards to a volumetric
flask and diluting to volume with acetone. One of the
external standards should be at a concentration near, but
above, the method detection limit. The other concentra-
tions should correspond to the range of concentrations
expected in the sample concentrates or should define the
working range of the detector.
7.2.2 Using injections of 1 to 5 yL of each calibration standard,
tabulate peak height or area responses against the mass
injected. The results can be used to prepare a calibration
curve for each compound. Alternatively, the ratio of the
response to the mass injected, defined as the calibration
factor (CF), can be calculated for each compound at each
standard concentration. If the relative standard deviation
of the calibration factor is less than 10% over the working
range, the average calibration factor can be used in place
of a calibration curve.
30
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7.2.3 The working calibration curve or calibration factor must be
verified on each working shift by the measurement of one or
more calibration standards. If the response for any com-
pound varies from the predicted response by more than ±10%,
the test must be repeated using a fresh calibration stand-
ard. Alternatively, a new calibration curve or calibration
factor must be prepared for that compound.
7.3 Internal standard calibration procedure. To use this approach, the
analyst must select one or more internal standards similar in
analytical behavior to the compounds of interest. The analyst must
further demonstrate that the measurement of the internal standard
is not affected by method or matrix interferences. Due to these
limitations, no internal standard applicable to all samples can be
suggested.
7.3.1 Prepare calibration standards at a minimum of three con-
centration levels for each compound of interest by adding
volumes of one or more stock standards to a volumetric
flask. To each calibration standard, add a known constant
amount of one or more internal standards, and dilute to
volume with acetone. One of the standards should be at
a concentration near, but above, the method detection
limit. The other concentrations should correspond to the
range of concentrations expected in the sample concen-
trates, or should define the working range of the detector.
7.3.2 Using injections of 1 to 5yL of each calibration standard,
tabulate the peak height or area responses against the
31
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concentration for each compound and internal standard.
Calculate response factors (RF) for each compound as
follows:
RF a (AsCis)/AisCs)
where:
As * Response for the compound to be measured.
Ais = Response for the internal standard.
Cis * Concentration of the internal standard inug/L.
Cs = Concentration of the compound to be measured in
yg/L.
If the RF value over the working range is constant, less
than 10% relative standard deviation, the RF can be assumed
to be invariant and the average RF can be used for calcu-
lations. Alternatively, the results can be used to plot a
calibration curve of response rations, As/Ais against RF.
7.3.3 The working calibration curve or RF must be verified on
each working shift by the measurement of one or more
calibration standards. If the response for any compound
varies from the predicted response by more than £10%, the
test must be repeated using a fresh calibration standard.
Alternatively, a new calibration curve must be prepared for
that compound.
7.4 Before using any cleanup procedure, the analyst must process a
series of calibration standards through the procedure to validate
elution patterns and the absence of interferences from the
reagents.
32
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8. Quality Control
8.1 Each laboratory using this method is required to operate a formal
quality control program. The minimum requirements of this program
consist of an initial demonstration of laboratory capability and
the analysis of spiked samples as a continuing check on
performance. The laboratory is required to maintain performance
records to define the quality of data that is generated.
8.1.1 Before performing any analyses, the analyst must
demonstrate the ability to generate acceptable accuracy and
precision with this method. This ability is established as
described in Section 8.2.
8.1.2 In recognition of the rapid advances occurring in
chromatography, the analyst is permitted certain options to
improve the separations or lower the cost of measurements.
Each time such modifications to the method are made, the
analyst is required to repeat the procedure in Section 8.2.
8.1.3 The laboratory must spike and analyze a minimum of 10% of
all samples to monitor continuing laboratory performance.
This procedure is described in Section 8.4.
8.2 To establish the ability to generate acceptable accuracy and
precision, the analyst must perform the following operations.
8.2.1 Select a representative spike concentration for each
compound to be measured. Using stock standards, prepare a
quality control check sample concentrate in methanol 1000
times more concentrated than the selected concentrations.
33
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8.2.2 Using a pipet, add 1.00 ml of the check sample concentrate
to each of a minimum of four 1000-mL aliquots of reagent
water. A representative wastewater may be used in place of
the reagent water, but one or more additional aliquots must
be analyzed to determine background levels, and the spike
level must exceed twice the backgorund level for the test
to be valid. Analyze the aliquots according to the method
beginning in Section 10.
8.2.3 Calculate the average percent recovery (R), and the
standard deviation of the percent recovery (s), for the
results. Wastewater background corrections must be made
before R and s calculations are performed.
8.2.4 Using the appropriate data from Table 3, determine the
recovery and single operator precision expected for the
method, and compare these results to the values measured in
Section 8.2.3. If the data are not comparable, the analyst
must review potential problem areas and repeat the test.
8.3 The analyst must calculate method performance criteria and define
the performance of the laboratory for each spike concentration and
parameter being measured.
8.3.1 Calculate upper and lower control limits for method
performance as follows:
Upper Control Limit (UCL) = R + 3 s
Lower Control Limit (LCL) = R - 3 s
where R and s are calculated as in Section 8.2.3. The UCL
and LCL can be used to construct control charts** that are
useful in observing trends in performance.
34
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8.3.2 The laboratory must develop and maintain separate accuracy
statements of laboratory performance for wastewater
samples. An accuracy statement for the method is defined
as R i s. The accuracy statement should be developed by
the analysis of four aliquots of wastewater as described in
Section 8.2.2, followed by the calculation of R and s.
Alternately, the analyst may use four wastewater data
points gathered through the requirement for continuing
quality control in Section 8.4. The accuracy statements
should be updated regularly.**
8.4 The laboratory is required to collect in duplicate a portion of
their samples to monitor spike recoveries. The frequency of spiked
sample analysis must be at least 10% of all samples or one sample
per month, whichever is greater. One aliquot of the sample must be
spiked and analyzed as described in Section 8.2. If the recovery
for a particular compound does not fall within the control limits
for method performance, the results reported for that compound in
all samples processed as part of the same set must be qualified as
described in Section 13.3. The laboratory should monitor the
frequency of data so qualified to ensure that it remains at or
below 5%.
8.5 Before processing any samples, the analyst should demonstrate
through the analysis of 1-liter aliquot of reagent water that all
glassware and reagents interferences are under control. Each time
a set of samples is extracted or there is a change in reagents, a
laboratory reagent blank should be processed as a safeguard against
laboratory contamination.
35
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8.6 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. Field duplicates may be
analyzed to monitor the precision of the sampling technique. When
doubt exists over the identification of a peak on the chromatogram,
confirmatory techniques such as gas chromatography with a
dissimilar column, specific element detector, or mass spectrometer
must be used. Whenever possible, the laboratory should perform
analysis of standard reference materials and participate in
relevant performance evaluation studies.
9. Samples Collection, Preservation, and Handling
9.1 Grab samples must be collected in glass containers. Conventional
sampling practices? should be followed; however, the bottle must
not be prerinsed with sample before collection. Composite samples
should be collected in refrigerated glass containers in accordance
with the requirements of the program. Automatic sampling equipment
must be as free as possible of plastic and other potential sources
of contamination.
9.2 The samples must be iced or refrigerated at 4°C from the time of
collection until extraction.
9.3 Adjust the pH of the sample to 6 to 8 with 6N^ sodium hydroxide or
6N^sulfuric acid immediately after sampling.
36
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10. Sample Extraction
10.1 Mark the water meniscus on the side of the sample bottle for later
determination of sample volume. Pour the entire sample into a 2-
liter separatory funnel. Check the pH of the sample with wide
range pH paper and adjust to 6 to 8 with 6 N sodium hydroxide or 6
N sulfurlc acid.
10.2 Add 60 ml of methylene chloride to the sample bottle, seal, and
shake 30 seconds to rinse the inner walls. Transfer the solvent to
the separatory funnel and extract the sample by shaking the funnel
for 2 minutes with periodic venting to release excess pressure.
Allow the organic layer to separate from the water phase for a
minimum of 10 minutes. If the emulsion interface between layers is
more than one third the volume of the solvent layer, the analyst
must employ mechanical techniques to complete the phase separation.
The optimum technique depends upon the sample, but may include
stirring, filtration of the emulsion through glass wool, centri-
fugation, or other physical methods. Collect the methylene
chloride extract in a 250-mL Erlenmeyer flask.
10.3 Add a second 60-mL volume of methylene chloride to the sample
bottle and repeat the extraction procedure a second time, combining
the extracts in the Erlenmeyer flask. Perform a third extraction
in the same manner.
10.4 Assemble a Kuderna-Danish (K-D) concentrator by attaching a 10-mL
concentrator tube to a 500-mL evaporative flask. Other
concentration devices or techniques may be used in place of the K-D
if the requirements of Section 8.2 are met.
37
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10.5 Pour the combined extract through a drying column containing about
10 cm of anhydrous sodium sulfate, and collect the extract in the
K-0 concentrator. Rinse the Erlenmeyer flask and column with 20 to
30 ml of methylene chloride to complete the quantitative transfer.
Once the flask rinse has passed through the drying column, rinse
the column with 30 to 40 ml of methylene chloride.
10.6 Add 1 or 2 clean boiling chips to the evaporative flask and attach
a three-ball Snyder column. Prewet the macro Snyder column by adding
about 1 ml methylene chloride to the top. Place the K-0 apparatus
on a hot water bath, 60 to 65°C, so that the concentrator tube is
partially immersed in the hot water, and the entire lower rounded
surface of the flask is bathed with hot vapor. Adjust the vertical
position of the apparatus and the water temperature as required to
complete the concentration in 15 to 20 minutes. At the proper rate
of distillation, the balls of the column will actively chatter but
the chambers will not flood with condensed solvent. When the
apparent volume of liquid reaches 1 ml, remove the K-0 apparatus
and allow it to drain and cool for at least 10 minutes.
10.7 Remove the macro-Snyder column and rinse the flask and its lower
joint into the concentrator tube with 1 to 2 mL of methylene
chloride. Add 1 or 2 clean boiling chips and attach a two-ball
micro-Snyder column to the concentrator tube. Prewet the micro-
Snyder column with methylene chloride and concentrate the solvent
extract as before. When an apparent volume of 0.5 ml is reached,
or the solution stops boiling, remove the K-D apparatus and allow
it to drain and cool for 10 minutes.
38
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10.8 Remove the micro-Snyder column and adjust the volume of the extract
to 1.0 ml with methylene chloride. Stopper the concentrator tube
and store refrigerated if further processing will not be performed
immediately. If the extract is to be stored longer than 2 days,
transfer the extract to a screw-capped vial with a Teflon-lined
cap. If the sample extract requires no further cleanup, proceed
with solvent exchange to acetone as described in 10.9. If the sample
requires cleanup, proceed to Section 11.
10.9 Add 1 or 2 clean boiling chips to the concentrator tube along with 10 ml
of acetone. Attach the two-ball macro Snyder column and prewet the
column with about 1 ml of acetone. Adjust the temperature of the water
bath to 85-95°C. Concentrate the solvent extract as before to an
apparent volume of 0.5 ml and allow it to drain and cool for 10 min.
Add a second 10 ml of acetone to the concentrator tube and repeat
the concentration procedure a second time. Adjust the final volume
of the extract to 1.0 ml with acetone.
10.10 Determine the original sample volume by refilling the sample bottle
to the mark and transferring the water to a 1000-mL graduated
cylinder. Record the sample volume to the nearest 5 ml.
11. Cleanup and Separation
11.1 Cleanup procedures may not be necessary for a relatively clean
sample matrix. The cleanup procedure recommended in this method
has been used for the analysis of various clean waters and
industrial effluents. If particular circumstances demand the use
of an alternative cleanup procedure, the analyst must determine the
elution profile and demonstrate that the recovery of each compound
of interest is no less than 85%.
39
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11.2 The following Florisil cleanup procedure has been demonstrated to
be applicable to the four neutral nitrogen pesticides listed in
Table 1.
11.2.1 Slurry 20 g of Florisil in 100 mL of ethyl ether and 400 uL
of reagent water. Transfer the slurry to a chromatographic
column (Florisil may be retained with a plug of glass wool).
Allow the solvent to elute from the column until the
Florisil is almost exposed to the air. Wash the column
with 25 ml of petroleum ether. Use a column flow rate of
2 to 2.5 mL/min throughout the wash and elution profiles.
Add an additional 50 ml of petroluem ether to the head of
the column.
11.2.2 Quantitatively transfer the sample from Section 10.8 to the
petroleum ether suspended over the column. Allow the
solvent to elute from the column until the Florisil is
almost exposed to the air. Elute the column with 50 ml of
50% ethyl ether in petroleum ether. Discard this fraction.
11.2.3 Elute the column with 50 mL of 100% ethyl ether
(Fraction 1) and collect in a K-D apparatus. Repeat
procedure with 50 ml 6% acetone in ethyl ether
(Fraction 2), 50 mL 15% acetone in ethyl ether (Fraction
3), 50 mL 50% acetone in ethyl ether (Fraction 4), and 100
mL 100% acetone (Fraction 5), collecting each in a separate
K-D apparatus. The elution patterns for the neutral
nitrogen compounds are shown in Table 1. Concentrate each
fraction to 1 mL as described in Section 10.6 and 10.7.
The fractions may be combined before concentration at
40
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the discretion of the analyst. Solvent exchange fraction
1 to acetone as described in Section 10.9 if the fractions
are not combined.
11.2.4 Proceed with gas chromatographic analysis.
12. 6as Chromatography
12.1 Table 2 summarizes the recommended operating conditions for the gas
chromatograph. Included in this table are estimated retention
times and method detection limits that can be achieved by this
method. Examples of the separations achieved by Column 1 and
Column 2 are shown in Figures 1 and 2. Other packed columns,
chromatographic conditions, or detectors may be used if the
requirements of Section 3.2 are met. Capillary (open-tubular)
columns may also be used if the relative standard deviations of
responses for replicate injections are demonstrated to be less than
6% and the requirements of Section 8.2 are met.
12.2 Calibrate the gas chromatographic system daily as described in
Section 7.
12.3 If the internal standard approach is being used, the analyst must
not add the internal standard to the sample extracts until
immediately before injection into the Instrument. Mix thoroughly.
12.4 Inject 1 to 5 uL of the sample extract using the solvent flush
technique.8 Record the volume injected to the nearest 0.05 uL, and
the resulting peak sizes in area or peak height units. An
automated system that consistently injects a constant volume of
extract may also be used.
12.5 The width of the retention time window used to make identifications
should be based upon measurements of actual retention time varia-
tions of standards over the course of a day. Three times the
41
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standard deviation of a retention time for a compound can be used
to calculate a suggested window size; however, the experience of
the analyst should weigh heavily in the Interpretation of
chromatograms.
12.6 If the response for the peak exceeds the working range of the
system, dilute the extract and reanalyze.
12.7 If the measurement of the peak response is prevented by the
presence of interferences, further cleanup is required.
13. Calculations
13.1 Determine the concentration of individual compounds in the sample.
13.1.1 If the external standard calibration procedure is used,
calculate the amount of material injected from the peak
response using the calibration curve or calibration factor
in Section 7.2.2. The concentration in the sample can be
calculated as follows:
(A)(Vt)
Concentration, yg/L = fv^TTv")
where:
A » Amount of material injected, in nanograms.
VT 3 Volume of extract injected in yL.
V-t * Volume of total extract in yL.
Vs a Volume of water extracted in ml.
13.1.2 If the internal standard calibration procedure was used,
calculate the concentration in the sample using the
response factor (RF) determined in Section 7.3.2 as
follows:
(As)(Is)
Concentration, yg/L = (A.S)(RF)(VO)
42
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where:
As « Response for the compound to be measured.
A-jS » Response for the internal standard.
Is » Amount of internal standard added to each extract
in ug.
V0 » Volume of water extracted, in liters.
13.2 Report results 'in micrograms per liter without correction for
recovery data. When duplicate and spiked samples are analyzed,
report all data obtained with the sample results.
13.3 For samples processed as part of a set where the laboratory spiked
sample recovery falls outside of the control limits in Section 8.3,
data for the affected compounds must be labeled as suspect.
14. GC-MS Confirmation
14.1 It is recommended that GC-MS techniques be judiciously employed to
support qualitative identifications made with this method. The
mass spectrometer should be capable of scanning the mass range from
35 amu to a mass 50 amu above the molecular weight of the compound.
The instrument must be capable of scanning the mass range at a rate
to produce at least 5 scans per peak but not to exceed 7 seconds
per scan utilizing a 70-V (nominal) electron energy in the electron
impact ionization mode. A GC to MS interface constructed of all-
glass or glass-lined materials is recommended. When using a fused
silica capillary column, the column outlet should be threaded
through the interface to within a few mm of the entrance to the
source ionization chamber. A computer system should be interfaced
to the mass spectrometer that allows the continuous acquisition and
storage on machine readable media of all mass spectra obtained
throughout the duration of the chromatographic program.
43
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14.2 Gas chromatographlc columns and conditions should be selected for
optimum separation and performance. The conditions selected must
be compatible with standard GC-MS operating practices.
Chromatographlc tailing factors of less than 5.0 must be
achieved.10
14.3 At the beginning of each day that confirmatory analyses are to be
performed, the GC-MS system must be checked to see that all DFTPP
performance criteria are achieved.9
14.4 To confirm an identification of a compound, the background
corrected mass spectrum of the compound must be obtained from the
sample extract and compared with a mass spectrum from a stock or
calibration standard analyzed under the same chromatographic
conditions. It is recommended that at least 50 nanograms of
material be injected into the GC-MS. The criteria below must be
met for qualitative confirmation.
14.4.1 The molecular ion and all other ions that are present above
IQ% relative abundance in the mass spectrum of the standard
must be present in the mass spectrum of the sample with
agreement to plus or minus 10%. For example, if the
relative abundance of an ion is 30% in the mass spectrum of
the standard, the allowable limits for the relative
abundance of that ion in the mass spectrum for the sample
would be 20-40%.
14.4.2 The retention time of the compound in the sample must be
within 6 seconds of the same compound in the standard
solution.
44
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14.4.3 Compounds that have very similar mass spectra can be
explicitly identified by GC-MS only on the basis of
retention time data.
14.5 Where available, chemical ionization mass spectra may be employed
to aid in the qualitative identification process.
14.6 Should these MS procedures fail to provide satisfactory results,
additional steps may be taken before reanalysis. These may Include
the use of alternate packed or capillary GC columns or additional
cleanup (Section 11).
15. Method Performance
15.1 The method detection limit (MDL) is defined as the minimum
concentration of a substance that can be measured and reported with
99% confidence that the value is above zero.H The MDL
concentrations listed in Table 2 were obtained using reagent
water.1 Similar results were achieved using representative
wastewaters.
15.2 This method has been tested for linearity of recovery from spiked
reagent water and has been demonstrated to be applicable over the
concentration range from 10 x MDL to 1000 x MDL.
15.3 In a single laboratory, Battelle's Columbus Laboratories, using
spiked wastewater samples, the average recoveries presented in
Table 3 were obtained after Florisil cleanup. Seven replicates of
each of two different wastewaters were spiked and analyzed. The
standard deviation of the percent recovery is also included in
Table 3.1
45
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TABLE 1. ELUTION CHARACTERISTICS OF THE NEUTRAL NITROGEN
COMPOUNDS ON 2% DEACTIVATED FLORISIL
Parameter
Fl
Elation In Specified Fraction(a)
F2
F3
F4
F5
Fenarimol
MGK 264
MGK 326
Pronamide
X
X
X
X
(a) Elution solvents are 50 mL each of the following:
Fl = 100% ethyl ether
F2 s 6% acetone in ethyl ether
F3 - 15% acetone in ethyl ether
F4 * 50% acetone in ethyl ether
F5 = 100% acetone (100 mL)
46
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REFERENCES
1. "Development of Methods for Pesticides in Wastewaters", Report for EPA
Contract 68-03-2956 (In preparation).
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, Pennsylvania, 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, August, 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. "Handbook for Analytical Quality Control in Water and Wastewater
Laboratories", EPA-600/4-79-019, IT. S. Environmental Protection Agency,
Environmental Monitoring and Support Laboratory - Cincinnati, Ohio 45268,
March,1979.
7. ASTM Annual Book of Standards, Part 31, D3370, "Standard Practice for
Sampling Water," American Society for Testing and Materials,
-Philadelphia, Pennsylvania, p. 76, 1980.
8. Burke, J. A., "Gas Chromatography for Pesticide Residue Analysis; Some
Practical Aspects," Journal of the Association of Official Analytical
Chemists, 48, 1037 (1965).
9. Eichelberger, J. W., Harris, L. E., and Budde, W. L., "Reference Compound
to Calibrate Ion Abundance Measurement in Gas Chromatography - Mass
Spectrometry", Analytical Chemistry. 47, 995 (1975).
10. McNair, H. M., and Bonelli, E. J., "Basic Chromatography", Consolidated
Printing, Berkeley, California, 52 (1969).
11. Glaser, J. A., et a!., "Trace Analysis for Wastewaters", Environmental
Science and Technology, ^5_, 1426 (1981).
47
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TABLE 2. CHROMATOGRAPHIC CONDITIONS AND METHOD
DETECTION LIMITS
Parameter
Pronamide
MGK 326
MGK 264
Retention
Column 1
19.9
21.9
23.0,and
23.5U)
Time (min)
Column 2
22.0
23.8
25. 5, and
27.5U)
MDL
(ug/L)
4
6
2
Fenarimol 30.6 32.2
Column 1 conditions: Supelcoport (100/120 mesh) coated with 3% SP-2250
packed in a 1.8 m long x 2 mm ID glass column with helium carrier gas at a
flow rate of 30 mL/min. Column temperature is programmed from 80°C to
300QC at 8°C/min with a 4 min hold at each extreme, injector temperature
is 250°C and detector is 300°C. Alkali flame detector at bead voltage of
16 V.
Column 2 conditions: Suplecoport (100/120 mesh) coated with 3X SP-2100
packed in a 1.8 m long x 2 mm ID glass column with helium carrier gas at a
flow rate of 30 mL/min. All other conditions as for Column 1.
(a) Two isomers of MGK 264 are resolved from each other.
48
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TABLE 3. SINGLE LABORATORY ACCURACY AND PRECISION^)
Parameter
Fenarimol
MGK 264
MGK 326
Pronamide
(a) Column
(b) 1 = Low
2 - Hig
Sample
TypeW
1
2
1
2
1
2
1
2
1 conditions
Background,
ug/L(c)
1.8
NO
NO
ND
ND
NO
ND
210
were used.
level relevant Industrial
h level relevant industrial
Spike
Level ,
vg/L
20
500
20
500
20
500
20
500
Mean
Recovery,
%
98
96
96
74
108
95
102
86
Standard
Deviation,
%
4
4
23
4
7
4
5
3
Number
of
Replicates
7
7
7
7
7
7
7
7
effluent.
effluent.
(c) ND » Not detected.
49
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MGK 264
Fenariaol
/
-i—i—•r--t—i—r- «—|—t-
29 27
-r-r- i
29
9 1
99 99
Retention Time, Min.
FIGURE 1. GC-AFD CHROMATOGRAM OF lOOng EACH OF THE NEUTRAL NITROGEN COMPOUNDS
(Column 1).
-------
Fenarlnol
ft 'V T-r- -r- t—r • •»—r
3 1
Retention Time, Mln.
FIGURE 2. GC-AFD CHROMATOGRAM OF 200ng EACH OF THE NEUTRAL NITROGEN COMPOUNDS
(COLUMN 2).
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