EPA/600/4-85/060
September 1985
SINGLE-LABORATORY VALIDATION OF EPA METHOD 8150 FOR THE ANALYSIS
OF CHLORINATED HERBICIDES IN HAZARDOUS WASTE
by
F. L. Shore, E. N. Amick, and S. T. Pan
Lockheed Engineering and Management Services Company, Incorporated
Las Vegas, Nevada 89114
Contract Number 68-03-3050
Task Monitor
D. F. Gurka
Quality Assurance Division
Environmental Monitoring Systems Laboratory
Las Vegas, Nevada 89114
ENVIRONMENTAL MONITORING SYSTEMS LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
LAS VEGAS, NEVADA 89114
-------
NOTICE
The information in this document has been funded wholly or in part by
the United States Environmental Protection Agency under Contract Number
68-03-3050 to the Lockheed Engineering and Management Services Company,
Incorporated, Las Vegas, Nevada. It has been subject to the Agency's peer
and administrative review, and it has been approved for publication as an
EPA document. Mention of trade names or commercial products does not
constitute endorsement or recommendation for use.
-------
FOREWORD
The Resource Conservation and Recovery Act of 1976 (RCRA) and its
amendments require the U.S. Environmental Protection Agency (EPA) to
regulate hazardous waste activities.1»2»3 Implementation and enforcement
of the RCRA requires analytical procedures that can provide data of known
precision and accuracy on analytes in hazardous waste samples. Reliable
data collected under prescribed quality assurance/quality control
procedures will allow the EPA to identify and delineate waste sites,
characterize waste composition, and detect environmental contamination
resulting from operations that generate hazardous wastes.
The document* Test Methods for Evaluating Solid Waste. Office of
Solid Waste Manual SW-846, was published to provide a compilation of
state-of-the-art methodology for evaluating RCRA solid wastes for
environmental and human health hazards. SW-846 Method 8150 for chlorinated
herbicides required validation as part of an ongoing program to evaluate
SW-846 methods. Detailed single-laboratory validation guidelines are
available as reported by the Association of Official Analytical Chemists
(AOAC) Committee on Interlaboratory Studies^ and the EPA report
"Validation of Testing/Measurement Methods"6 were followed, where
feasible, in this study.
-------
ABSTRACT
Method 8150, published in the second edition of Test Methods for
Evaluating Solid Waste. Manual SW-846, required optimization, ruggedness
testing, linearity determinations, precision tests, bias testing, gas
chr oma tography/mass spectrometric allowed the
determination of the herbicide analytes as the methyl derivatives in a
single, 20-minute GC run. The original Method 8150 procedure required
three packed columns for the ten target analytes.
Final ruggedness testing on the optimized procedure gave a mean
recovery of 89.3% with a standard deviation of 4.3%. The precision of the
method is excellent. Percent relative standard deviations (% BSD's) are
less than 10 (n * 20, each analyte) over a 10* linear range of concentra-
tion for MCPP and MCPA and over a 103 linear range of concentration for
the other target herbicide esters. Detection limits for electron capture
detection and mass spectrometric identity confirmation were determined and
found to be matrix dependent.
-------
CONTENTS
Page
Foreword iii
Abstract *v
Figures
Tables *
Abbreviations and Symbols xiii
Acknowledgment x*v
1. Introduction 1
2. Conclusions 3
3. Recommendations ^
4. Preliminary Studies 5
Introduction 5
Safety 5
Data and results 11
Conclusions 29
Recommendat ions 29
5. Ruggedness Tests 30
Introduction 30
Ruggedness testing of free acid herbicide extraction
and analysis 31
Ruggedness testing for herbicide ester hydrolysis
and analysis ^5
Ruggedness testing of optimized herbicide extraction
and analysis ^9
Conclusions 51
6. Optimization of Analytical Procedures 52
Introduction 52
Simplex optimization of chlorinated herbicides 52
Ester hydrolysis optimization 57
Conclusions 59
7. Linearity 60
Introduction 60
Experimental 60
Results and discussion 61
Conclusions 67
8. Precision 68
Introduction 68
Experimental 68
Results and discussion 68
Conclusions ^3
v
-------
Page
9. Bias Testing 74
Introduction 74
Experimental 74
Data and results 75
Conclusion 84
10. GC/MS Confirmation 85
Introduction 85
Experimental 85
Data and results 86
Conclusions 99
11. Quality Control 100
Introduction 100
Performance criteria 100
Conclusions 101
References 103
Append!x
A. Validated Method 8150, chlorinated herbicides by
methylation and GC/EC 106
-------
FIGURES
Number Page
1 The Mini Diazald® Apparatus and generation of diazomethane. ... 8
2 The diazomethane generating apparatus 10
3 Gas chroraatograra of methylated herbicides (I) 14
U Gas chromatogram of methylated herbicides (II) 16
5 Analysis report of methylated herbicide gas chromatography. ... 17
6 Gas chromatogram of methylated herbicides (III) 19
7 Gas chromatogram of pentafluorobenzylated herbicides 20
8 Block diagram of herbicide analysis 33
9 Gas chroraatograra of methylated herbicides, sample 4,
experiment 1 ^7
10 Three-factor sequential simplex worksheet for
herbicide method optimization 55
1 1 Hydrolysis of acid herbicide esters 58
12 MCPA GC/EC response 62
13 MCPP GC/EC response ^2
14 Dalapon GC/EC response 63
15 Dicamba GC/EC response 63
16 Dichlorprop GC/EC response 64
17 2,4-D GC/EC response 64
18 Sllvex GC/EC response 65
19 2,4,5-T GC/EC response 65
vii
-------
FIGURES (Continued)
Number Page
20 2,4-DB GC/EC response 66
21 Dinoseb GC/EC response 66
22 Solvent blank for precision measurements 70
23 Low concentrations: Dicamba, MCPP, MCPA, Dichlorprop, 2,4-D,
Silvex, 2,4,5-T, 2,4-DB, Dinoseb 70
24 Low concentration: Dalapon 71
25 High concentrations: Dicamba, MCPP, MCPA, Dichlorprop, 2,4-D,
Silvex, 2,4,5-T, 2,4-DB, Dinoseb 71
26 High concentration: Dalapon 72
27 Dicamba concentration bias 76
28 MCPP concentration bias 76
29 MCPA concentration bias 77
30 Dichlorprop concentration bias 77
31 2,4-D concentration bias 78
32 Silvex concentration bias 78
33 2,4,5-T concentration bias 79
34 2,4-DB concentration bias 79
35 Dinoseb concentration bias 80
36 Chromatogram, sample A (most concentrated spike) from kaolin
clay/still bottoms 82
37 Chromatogram, sample E (least concentrated spike) from kaolin
clay/still bottoms 82
38 Chromatogram, sample F, blank from kaolin clay/still bottoms. . . 83
39 Reconstructed ion chromatogram 87
viii
-------
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
ige
88
89
89
90
90
91
91
92
92
93
93
94
94
95
95
96
96
97
97
98
98
FIGURES (Continued)
GC/EC chromatogram of target herbicide esters
Dalapon electron impact mass spectrum
Dicamba electron impact mass spectrum
MCPP electron impact mass spectrum
MCPA electron impact mass spectrum
Dichlorprop electron impact mass spectrum
2,4-D electron impact mass spectrum
Silvex electron impact mass spectrum
2,4,5-T electron impact mass spectrum
2,4-DB electron impact mass spectrum
Dinoseb electron impact mass spectrum
Dalapon Finnigan INCOS FIT value vs. concentration
Dicamba Finnigan INCOS FIT value vs. concentration
MCPP Finnigan INCOS FIT value vs. concentration
MCPA Finnigan INCOS FIT value vs. concentration
Dichlorprop Finnigan INCOS FIT value vs. concentration
2,4-D Finnigan INCOS FIT value vs. concentration
Silvex Finnigan INCOS FIT value vs. concentration
2,4,5-T Finnigan INCOS FIT value vs. concentration
2,4-DB Finnigan INCOS FIT value vs. concentration
Dinoseb Finnigan INCOS FIT value vs. concentration
Quality control chart for GC/EC chlorinated herbicide analysis.
ix
-------
TABLES
Number Page
1 Free Acid Herbicides 13
2 Methyl Herbicide Standard Test Mixture Analysis 15
3 Recoveries (X) of Herbicides from Selected Matrices Using
Method 8150 and Capillary Gas Chromatography with a DB-5
Column (Single Determination, Methyl Derivatives) 21
U Analysis of Spiked Herbicides in Kaolin Clay (Type P) Using
Peak. Height Comparison (Methyl Derivatives) 22
5 Analysis of Spiked Herbicides in Kaolin Clay (Type P) Using
Peak Area Comparison (Methyl Derivatives) 23
6 Analysis of Freshly Spiked Herbicides in Kaolin Clay
(Type P) (Methyl Derivatives) 24
7 Analysis of Spiked Herbicides in Kaolin Clay (Type P)
Following 7 Days Storage at -38°C (Methyl Derivatives) .... 25
8 Recoveries (?) of Spiked Herbicides from Kaolin Clay (Type P)
Using Sonication with Methylene Chloride at Selected pH
Values (Single Determination, Methyl Derivatives) 26
9 Recoveries of Spiked Herbicides from Kaolin Clay (Type P)
Using Sonication at pH 7 with Selected Solvents (Single
Determination, Methyl Derivatives) 27
10 Recoveries of Spiked Herbicides from Kaolin Clay (Type P)
Using Selected Extraction Techniques and
Pentafluorobenzylation (Single Determination) 28
11 Design for Test of Experimental Conditions 30
12 Experimental Variables and Assigned Values for Herbicide
Method 31
13 Standard Spiking Solution (Acetone) 36
14 Percent Recovery of Herbicides for Experiment 1 Using
Conditions of Table 12 38
x
-------
TABLES (Continued)
Number Page
15 Differences for Herbicide Method, Experiment 1 39
16 Percent Recovery of Herbicides for Experiment 2 Using
Conditions of Table 12 40
17 Differences for Herbicide Method, Experiment 2 4 1
18 Percent Recovery of Herbicides for Experiment 3 Using
Conditions of Table 12 42
19 Differences for Herbicide Method, Experiment 3 43
20 Concentration of Standards Used in Ester Hydrolysis Experiment. . 46
21 Conditions Altered and Assigned Values for Herbicide
Hydrolysis Method 47
22 Percent Recovery of Herbicides from Herbicide Ester
Hydrolysis Method 47
23 Differences for Herbicide Ester Hydrolysis Method 48
24 Design for Test of Experimental Conditions, Experiment 4 49
25 Experimental Variables and Assigned Values for Herbicide
Method, Experiment 4 49
26 Percent Recovery of Herbicides for Experiment 4 Using
Conditions of Table 25 50
27 Differences for Herbicide Method, Experiment 4 51
28 Experimental Value Ranges for Simplex Optimization 53
29 Initial Vertices (Percent) for Simplex Optimization 53
30 Simplex Optimization Progress 54
31 Results for Initial Simplex Vertices 56
32 Conditions and Results for Each Vertex in Simplex Optimization. . 56
33 Concentration of Herbicide Ester Working Standard 57
34 Optimization of Herbicide Ester Hydrolysis by Methanol Addition
(Percent Recoveries) ^
xi
-------
TABLES (Continued)
Number Page
35 Linear Range and Detection Limits for Chlorinated Herbicides. . . 61
36 Results of Precision Determinations 69
37 Final Concentrations (ppb) of Analytes Added to Clay and
Clay/Still Bottom Samples (A-E) 75
38 Recoveries (%) for the Chlorinated Herbicides in Kaolin Clay
(Concentrations A-E) 81
39 Recoveries (%) for the Chlorinated Herbicides in Kaolin Clay/
Still Bottom Samples (Concentrations A-E) 81
40 Minimum Concentrations Required to Give Full Scan Mass
Spectra FIT Values of 800 (1-yL Injection) 99
xii
-------
ABBREVIATIONS AHD SYMBOLS
ABBREVIATIONS
RCHA - Resource Conservation and Recovery Act of 1976 and its
amendments.
% RSD - percent relative standard deviation.
2,4-D - 2,4-dichlorophenoxyacetic acid.
2,4-BB - 4-<2,4-dichlorophenoxy)butyric acid.
Dalapon - 2,2-dichloropropanoic acid.
Dicaraba - 3t6-dichloro-2-methoxybenzoic acid,
Dichlorprop - 2-(2,4-dichlorophenoxy)propionic acid.
Dinoseb - 2-Cl-methylpropyl>-4,6-dinitrophenol.
MCPA - 2-methyl-4-chlorophenoxyacetic acid,
MCPP - 2-(4-chloro-2-methylphenoxy>propionic acid.
Silvex - 2-(2t4,5-trichlorophenoxy)propionic acid.
2,4,5-T - (2,4,5-trichlorophenoxy)-acetic acid.
GC/MS - gas chromatoRraph-mass spectrometer system or analysis.
GC/EC - gas chromatography-electron capture detection,
ppb - parts per billion.
Diazald® - H-methyl-H-nitroso-p^toluenesulfonamide.
K-D - Kudema-Danish concentrator.
a - standard deviation
KHz - kilohertz
xiii
-------
ACKNOWLEDGMENT
The authors thank Professors M. E. Hunk and M. L. Parsons of Arizona
State University for helpful suggestions and evaluations of the project
work plan. T. L. Vonnahme, P. J. Marsden, and A. R. Bujold of Lockheed
Engineering and Management Services Company* Incorporated, provided helpful
suggestions and some preliminary work. We also thank Fred C. Hart
Associates for carrying out the diasomethane methylations.
xiv
-------
SECTION 1
INTRODUCTION
Four related procedures for the analysis of chlorinated herbicides
have been proposed by the FDA,7 ASTM,® and the BPA (Method 6159 and
Method 8150*). All of these procedures use diazomethane for methylation
of the free acid herbicides. This reagent is known to be toxic and
explosive10 and these procedures should only be attempted by an
experienced analyst. It is interesting to note that one publication
states that Dalapon is not methylated by diazomethane.11
Another methylating reagent, the boron trifluoride-methanol complex, is
known to give better results for chlorinated herbicides in at least some
cases.12**3 However, 2-methyl-4-chlorophenoxyacetic acid (MCPA), Dinoseb,
and Dicamba are not methylated by the boron trifluoride-methanol complex.®
Because analysis of these analytes is required by Method 8150, boron
trifluoride-methanol is not a suitable methylating procedure for this
study. As a possible alternate derivatization procedure, pentafluoro-
benzylation1* was also studied.
The extraction procedure of Method 8150 (and the ASTM Method) uses
large amounts of ether which creates a potential fire hazard. The FDA
procedure uses chloroform (a suspected carcinogen) for extraction. Use of
methyl t-butyl ether15 or methylene chloride following the Superfund
contract laboratory protocol1** seemed viable alternatives for extraction,
as did the mixed solvent acetone/hexane which would allow adjustment of
the solvent polarity to optimum.
Method 8150 uses three different packed column runs for GC of the
methyl esters; we hoped that a single capillary gas chromatography run
could be used. Generally, capillary columns give better chromatographic
resolution and less background detector noise than do packed columns.17
Thus, first priority was to test a capillary column for analysis of the
derivatives. Method 8150 states that microcoulombic detection is
preferred to electron capture detection for the methylated herbicides.
With the added resolution of capillary chromatography, the more sensitive
and widely available electron capture detection was deemed preferable.
1
-------
Method 615 is of more recent vintage than Method 8150 but much of the
same methodology is retained, and safety is discussed in more detail in
Method 615 (as a separate section). The methylation procedure has been
changed to more closely resemble the ASTM method with the addition of
methanol (said to give faster and more complete methylation18) and
distillation of diazomethane directly into the reaction mixture. Packed
column GC/EC is recommended. The validation of Method 8150 required the
steps summarized in this report and resulted in the optimized and
validated protocol given in Appendix A.
The project work plan consisted of:
1. Examination of Method 8150 and related literature.
2. Revision of the method using preliminary experiments.
3. Ruggedness testing to find important variables.
4. Simplex optimization to refine important variables.
5. Final ruggedness testing to test range of important variables.
6. Determination of linear dynamic range.
7. Precision and accuracy testing.
8. Preparation of optimized protocol.
9. Testing of protocol on real samples.
10. Revisions of the final protocol based on sample analysis.
2
-------
SECTION 2
CONCLUSIONS
• Method 8150 for analysis of chlorinated herbicides in hazardous waste
has been validated at the Environmental Monitoring Systems Laboratory, Las
Vegas, Nevada (EMSL-LV), as described in this report.
• The optimized protocol is valid for 9 of the 10 analytes. Dalapon is
not recovered using this procedure.
• Ruggedness testing of the optimized protocol gives a mean recovery of
89.3% with a 4.3% RSD for the range of experimental conditions tested.
• The validated protocol uses one capillary column GC run rather than
the three packed column runs proposed in the original method. This
results in considerable savings in analysis time.
• The validated protocol substitutes the less flammable methylene
chloride for ether in the extraction step.
• The estimated GC/EC detection limits are 0.1 to A ng/g for 8 of the
analytes. MCPP and MCPA have higher detection limits, 66 and A3 ng/g
respectively.
• The measured % RSD's of all analyte concentrations are below 10%.
Horwitz, et al^'^O suggests interlaboratory data should have variations
less than 16% if a single-laboratory variation of 8 to 11% has been
obtained.
• Quality control guidelines and the detailed protocol are presented to
support a future interlaboratory validation.
3
-------
SECTION 3
RECOMMENDATIONS
Method 8150 has been revised as shown in the Appendix A protocol. The
revised protocol has been single-laboratory validated and is now ready for
internal and external review. An interlaboratory comparison test using
the chlorinated herbicide analytes in selected matrices is the next
laboratory test of the procedure.
The pentafluorobenzylation procedure is quite promising as an
alternative method. The greater sensitivity of the analyte derivatives in
comparison to the methyl derivatives has promise in multiresidue screening
procedures. Single-laboratory validation of the pentafluorobenzylation
method for chlorinated herbicides is recommended. Although this extra
sensitivity may not be necessary for typically high level RCRA wastes, it
may prove valuable for lower level environmental samples (fish, soil, and
sediments).
4
-------
SECTION 4
PRELIMINARY STUDIES
INTRODUCTION
Experiments were designed to define the scope of the Method 8X50
modification. This section describes experiments on sonication extraction
with an organic solvent* capillary GC/EC optimization, pentafluorobenzyla-
tion, matrix selection, stability of herbicide spikes, and precision and
accuracy studies. The methylation and safety requirements are also
discussed.
SAFETY
General
Because diazomethane is a known carcinogen and a possible explosion
hazard, we decided to prepare and use diazomethane in a containment
facility using the following precautions:
1. Diazomethane is an explosion hazard; therefore, all generation
reactions were carried out in a hood behind an explosion shield.
2. All glassware used in the generation had Clear-Seal® joints or
their equivalent. GROUND-GLASS JOINTS SHOULD NOT BE USED. Glassware
used in the ether trap was fire-polished.
3. Wire brushes were not used in cleaning the Clear-Seal® apparatus
because they can scratch the inner surface of the glass and, thus,
increase the explosion hazard of this procedure.
4. Dioxane and other solvents that may freeze were not used because the
sharp edges of the crystals formed may cause an explosion.
5. Diazomethane is a volatile carcinogen. All contact with diazomethane
or Diazald® was avoided.
6. Ether is a flammable solvent. A funnel was used to pour the ether
carefully into the reaction vessel.
7. Compound data sheets were supplied to containment facility personnel
prior to work initiation on a given compound.
-------
8. To avoid contamination, separate glassware and supplies were used for
each sample preparation.
9. All sample preparations were localized in a hood demonstrated to be
free from volatile contaminants.
10. Contact of solutions with any surface besides clean glass or Teflon
was not permitted.
11. An analytical balance capable of accurately weighing to at least
+0.01 mg in the hood was required (Mettler AE 163 or equivalent).
12. All containers (inner and outer) removed from the containment
facility were wiped clean with ether before removal.
Generation of Diazomethane
Ethereal solutions of diazomethane were prepared using an Aldrich
"Mini Diazald® Apparatus" or equivalent and the following procedures.21
Most derivatization procedures required 2 mmole of diazomethane (0.5 g
Diazald® reagent) and the generation of large amounts of diazomethane
was avoided. This procedure is taken from Figure 1; the letters
identifying laboratory apparatus are shown in Figure 2.
1. The apparatus shown in Figure 2 was assembled except for the
separatory funnel (a). The ice water bath (b) that cools the
receiving flask (c) was supported by a lab jack so that the flask was
easily removed from the apparatus at the end of the distillation.
2. The warm water bath (d) for the reaction vessel (e) was heated with a
hot plate and allowed to come to temperature (65-70*C) before the
cold finger trap (f) was filled with dry ice/acetone.
3. The ether trap (g) for any excess diazomethane was made from a test
tube half filled with absolute ether, a cooled, fire-polished,
disposable pipet, and latex tubing.
4. After the apparatus was assembled, a solution of 2.5g of potassium
hydroxide in 4 niL water was added to the reaction vessel (e). Hext,
a mixture of 14 mL 2-(2-ethoxyethoxy)ethanol and 8 mL absolute ether
was added. The temperature of the warm water bath was constantly
monitored.
5. The separatory funnel (a) was charged with a solution of 0.5g Diazald®
in 45 mL absolute ether (unless specified otherwise).
6. The separatory funnel was placed on the reaction vessel (e) and the
stopcock was opened so that rate of addition of Diazald®/ether
solution approximated the rate of distillation. (This addition took
20 to 40 minutes.) More dry ice was added to the cold finger trap as
necessary.
6
-------
7. After all of the Diazald® solution was added, 10 mL of ether was
slowly added and the distillation continued until the distillate was
colorless.
8. The hot plate was turned off and the distillate was checked to verify
it as yellow. If there was no color, no diazomethane was collected.
Derivatization Reaction
1. The lab jack was lowered, the ether trap (g) was removed, and then
the ice bath (b) was removed.
2. The receiving flask was lowered and was supported on a cork ring.
3. Approximately equal amounts (usually 2 mL) of the ethereal
diazomethane were added to the standards or extracts using a cooled,
fire-polished, disposable pipet. The samples for derivatization were
provided in concentrator tubes. The standards were dissolved in
approximately 4mL 9:1 absolute ether/absolute methanol in volumetric
flasks unless otherwise specified.
4. The mixture was swirled to ensure complete mixing of the solutions.
The color was recorded. If the sample was not highly colored, the
derivatization mixture was yellow. THE SAMPLE WAS NOT STOPPERED
UNTIL AFTER STEP 6.
5. The color was checked and recorded fifteen minutes after the addition
of the ethereal diazomethane.
6. The derivatization mixtures were allowed to stand unstoppered
overnight in the hood unless otherwise directed.
7. The next day the vessels were stoppered with foil-wrapped corks of
the proper size and placed in a walking can for transport to the lab.
7
-------
»~
CO
•n
H*
OO
c
l-t
in
H
=r
n
X
H*
H.
H1-
R>
N
to
H*
Cl
>
¦o
-o
to
to
r*
P
w
w
3
rt
o
a
0
ft.
H-
to
1
rt
3*
0)
D
ro
o
tA
IO>
HI*
:£>» 9
» I 5 « I
j •«« 9 O
n5i?
"5
51 f!
2 " 5- * S
3 5 » S
1-5?
HiJ*
5'a a i
* _
i;E
* *' T»
5 2,s |1
1*
HBI*
>20
If
SeSj",
Ptiif
"fSsr
f'JOO
9 ? a sr m _
N H
M N M N N
I f S3
till;
*• TJr
silks'
MM
E 9, a
y ; 5
* 5 «•
£5 ?
?5t
9 5 7
??l£a
If.
re
J 5 a
i'K-3 x3
8 ®
?rl I
r» S
*3r *
iili
as*1*
s.si.8 a«
It*
3 vi tn
-------
Thu information it prwMmttd lo «MMt you in actuating our product it is «nt«nd«d • o* uk by prion*
We Op not guarantee l#*oret>** reayttt. an<]w«utLm( no liability in connection with its us* Thu jn»ormation is not int«o<3-
•d til lieenaa to operate under, or a recommendation to infringe. any patent covering any male**' o* w» Not (or drug,
houieftold or other uWi
AJdnch Chemical Company, loc
MO Watt Saint Paul Ave . MHwtuKM. Wl $3233 Telephone {414} 272-38S0 Cafile Aldrtcham TWX 910-26J 3052
Figure 1. (continued)
9
-------
A. Separatory Funnel
B. Ice Water Bath
C. Receiving Flask
D. Warm Water Bath
E. Reaction Vessel
F. Cold Finger Trap
G. Ether Trap
Figure 2. The diazomethane generating apparatus.
10
-------
DATA AND RESULTS
Physico-chemical and toxicity properties of the free acid herbicides
selected for Method 8150 are shown in Table 1. The LD50, water
solubility, and pKa values were collected from the literature to help in
understanding possible problems in the method development.
Because the packed column GC described in Method 8150 was judged to be
inadequate for chromatographically separating contaminants from analytes,
and because three different columns were required to analyze all analytes,
the first laboratory work was to find capillary GC conditions to separate
all methylated analytes. This was done on a DB-5 (J & W Scientific) GC
column as shown in Figure 3. Precision measurements (% RSD's) ranging
from 3.75 to 9.98 were obtained for analysis of a standard mixture as
shown in Table 2. Figures 4 and 5 show a sample chromatogran and analysis
report from the Tracor 540 GC/EC and the IBM CS9000 data system. An
alternative GC column, Supelcowax® (Supelco, Inc.) shows promise for
separating the methylated herbicides (see Figure 6).
The pentafluorobenzylated herbicides were prepared as a possible
alternative to the methyl derivatives of Method 8150. As shown in Figure
7, the gas chromatographic separation of the pentafluorobenzylated (PFB)
herbicides on DB-5 is superior to the methylated derivatives. Besides
improved separation, increased electron capture sensitivity is noted for
all PFB versus methyl derivatives of the analytes.
The matrix initially chosen for the optimization was "casting body"
clay. This matrix has been used for dioxin organic performance evaluation
samples. On analysis of unspiked casting-body clay using Method 8150 with
capillary GC/EC analysis, an impressive number of peaks was observed. The
manufacturer explained that lignite was a component of casting-body clay.
Lignite is decomposed organic material containing many carboxylic acids.
Three other matrices, desert soil (a sandy soil collected near Las Vegas,
Nevada), Ajax C (type C, kaolin calcined at 2000°C), and Ajax P (type P,
pure mineral kaolin), were evaluated as replacements as shown in Table 3.
Ajax P (Westwood Ceramic Supply, City of Industry, California) was
selected as most suitable for further study.
11
-------
Kaolin clay (type P) was spiked and analyzed in quadruplicate using
Method 8150 as shown in Tables A and 5 with peak height and peak area
quantitation giving about equal variation and recovery. Three of the
analytes were spiked at a higher concentration for a pesticide inter-
laboratory comparison study. The analysis of this sample was repeated to
verify the integrity of the sample after freezer storage for one week.
Because the stored samples gave only slightly higher recoveries, this
study serves to show the reproducibility of the method for these analytes
at this concentration.
An alternative to the Method 8150 ether extraction procedure is
sonication with a suitable organic solvent.^ The sonication extraction
procedure has been successfully tested by Battelle Memorial Institute in a
round-robin study^0 and verified by Research Triangle Institute for
sonication of solids.31 Sonications using methylene chloride were done
on spiked samples wet with three different buffers: pH 1 to closest
approximate Method 8150, pH 3 because the median pKa of the acids is
approximately 3 (Table 1) and pH 7 because Dinoseb is reported to be
extracted more efficiently at neutral pH.l*»32 The results are reported
in Table 8. The recoveries of the phenoxyacid herbicides were lower than
with Method 8150, but the best recovery of Dinoseb to that time was
obtained at pH 7. The procedure was repeated at pH 7 with several solvents
using a larger amount of anhydrous Na2S0^ prior to solvent addition;
those results are presented in Table 9. Recoveries are shown in Table 10
for the pentafluorobenzylation procedure tried on spiked clay samples
extracted by Method 8150 and by sonication with several solvents.
12
-------
Table ^. Free Acid Herbicides
Common Name(s) Systematic Name(s)
Rats Water Solubility
Structure LDgQfmg/kg) (mg/L. 25°C) pKa
37521 90020 26423
70021 4621 4.823
933020-21 502,00021 1 724
2900+80020 4.50021 1.9325
80020 71020 3.2826
5820 5220 4 6222
70022 82521 3.0723
93021 62021 3.2026
65021-22 14021 28423
50022 (30°C)23820 2.8424
2,4-D
2.4-DB
Dalapon
Dicamba
Dichlorprop
Dinoseb
MCPA
MCPP.
Mecoprop
Silvex,2,4,5-
TP. Fenoprop
2.4.5-T
2.4-dichlorophenoxy-
acetic acid
4-(2,3-dichloro-
phenoxy) butyric acid
2,2-dichloropropanoic
acid
3,6-dichloro-2-methoxy-
benzoic acid. 3.6-
dichloro-o-anisic acid
2 (2. 4-dichlorophenoxy)-
propionic acid
2-sec-butyl-4.6-dinitro-
phenol, 2-(1 methyl-
propyl)-4.6-dinitrophenol
2-methyl-4-chlorophenoxy-
acetic acid, (4-chloro-
o-toloxy)acetic acid
2-(4-chloro-2-methyl-
phenoxy) propionic acid
2-(2,4,5-trichlorophenoxy)-
propionic acid
(2.4,5-trichlorophenoxy)-
acetic acid
.CI
CI-^_^OCH,CO,H
CI och,ch,ch,co2h
CI
ch3cci2co,h
c'Oci
OCHj C0,H
CIOCH CO,H
CI CH,
°:N
f~\ CH CHjCHj
^ < CHj
0,N OH
c,"O'0CH'C0iH
CH,
OCH COjH
CH,
CnQ-OC
CL CH,t
CI-Q-OCH C02H
ci fcHl
CI^^-OCH,CO,H
C.^CI
-------
CO
R.
I
10
Minutes
-T-
15
20
instrumentation:
Column:
injection:
injection Temp:
Detection Temp:
Oven Temp:
Attenuator:
Perkin-Elmer. Sigma I E C Detector
DB-5 (J&W Scientific). 1 *jm. 0.25mm x 30 meter
1 uL splitless autosampler
220°C
350°C
110=C for 3 min., 40°/min. to 2003C, hold for 16 min.
6
Figure 3. Gas chromatogram of methylated herbicides (I).
U
-------
TABLE 2. METHYL HERBICIDE STANDARD TEST MIXTURE ANALYSIS
COMPOUND
CONCENTRATION
RETENTION
% RSD
ng/uL (as free Acid)
TIME (MIN)
n » 4
Dalapon
1.10
2.46
3.757.
Dicamba
0.83
8.42
5.93%
Dichlorprop
1.15
9.53
5.272
2,4-D
1.20
9.84
6.77%
MCPA
16.8
10.57
4.032
Silvex
0.19
12.19
7.32 %
2,4,5-T
0.18
12.84
9.98%
2,4-DB
1.66
14.62
4.61%
Dinoseb
0.44
14.88
7.65%
Instrumentation:
Perkin-Elmer, Sigma I E.C. Detector
Column:
DB-5 (J & W Scientific), llim,
0.25mm x 30 meter
Injection:
XViL splitless autosampler
Injection Temp:
220°C
Detection Temp:
350°C
Oven Temp:
110°C for 3 min., 40°/min. to
200°C, hold for 16
min.
Attenuator:
6
15
-------
35651-
c
3
o
o
c
&
(0
Q
5914
J
<
Q.
o
s
ML
* ^
> IT)
<75 *
-------
Channel RECALC Time: 16 :10:51 Date:MON 14 MAY 84
Sample name HERBICIDES
Data file DEMO:PANHB001
Method name PAN
Author ST PAD
Instrument TRACOR 540
Column 15M DB5
Notes
Run time 16.00 min. Delay time. . .0.00 min.
Acq. time 15:45:20 Acq. date . . .M0N 14 MAY 84
Start PW 10.00 sec.. End PW 10.00 sec.
Slope sens 1.00 uv/sec.
Area reject 500
# peaks found 32
AREA PERCENT REPORT
Peak
R.T. (min) R/S
Peak name
Area %
Area
Peak Ht.
BL
1
0.697
1.907
6972
884
BV
2
0.929
0.878
3210
303
VB
3
1.339
0.438
1601
387
BV
4
1.561
DALAPON
3.282
11999
2424
W
5
1.710
3.843
14051
808
VE
6
2.602
0.371
1358
299
BB
7
8.862
0.250
913
118
BV
8
9.388
DICAMBA
22.505
82279
25587
W
9
9.558
1.311
4792
1229
W
10
9.700
MCPP
1.867
6826
1715
VE
11
10.042
0.334
1221
133
EV
12
10.175
Dichlorprop
8.647
31614
8883
W
13
10.384
2,4-D
9.214
33686
9455
W
14
10.855
MCPA
6.312
23076
6523
W
15
11.247
0.293
1072
155
W
Figure 5. Analysis report of methylated herbicide gas
chromatography (see Figure 4 for conditions).
17
-------
AREA PERCENT REPORT
Peak
R.T. (rain) R/S
Peak name
Area %
Area
Peak Ht.
BL
16
11.545
0.248
906
147
W
17
11.732
0.409
1495
334
W
18
11.855
SILVEX
6.049
22115
5796
W
19
12.249
2,4,5-T
5.634
20598
5155
VB
20
13.281
2,4-DB
16.499
60321
13717
BV
21
13.452
DINOSEB
9.403
34377
7713
VB
22
14.534
0.150
547
87
BB
23
15.750
0.159
580
36
VB
TOTALS 100.000 365609
FIGURE 5. (continued)
18
-------
a.
o
CO
.o
JZ
U
= O
o
<
a.
a
2
S D
= 4
W3
H
10
*
-------
a
o
5 e-
E °
<0 £
u o
a 5
c
o
a
9
<7>
Q
CN
.O
©
M
o
c
1A
*
CN
Li)
CO
Q
4
CN
10
Instrumentation:
Column:
Injection:
Injection Temp:
Detection Temp:
Oven Temp:
Attenuator:
15
2<)"
r
25
30*
Minutes
Perkin-Elmer, Sigma I E.C. Detector
DB-5 (J&W Scientific), 1 pm 0.25mm. 30 meter
1 pL splitless autosampler
220°C
350°C
70°C for 1 min., 10°/min. to 240°C, hold for 17 min.
2
Figure 7. Gas chroma togram of pentafluorobenzylated herbicides.
20
-------
Compounds
TABLE 3. RECOVERIES (%) OF HERBICIDES FROM SELECTED MATRICES
USING METHOD 8150 AND CAPILLARY GAS CHROMATOGRAPHY WITH A DB-5
COLUMN* (SINGLE DETERMINATION. METHYL DERIVATIVES)
MATRIX
Kaolin Kaolin
Clay Clay
Solvent Desert (Type C) (Type P) Spike
Spike Soil (Calcined at 2000*C) (Pure Mineral) (ppb)
Dicamba
MCPP
Dichlorprop
2,4-D
MCPA
Silvex
2,4,5-T
2,4-DB
Dinoseb
103
114
109
94
122
105
98
95
0
80+
**
90
90
80
70
110
100
<5
100
144**
102
97
103
121
97
900
0
91
98
102
83
101
90
94
80
0
164
4099
248
252
3890
39
42
774
99
* Method 8150 with capillary GC/EC as shown in Figure 3.
Peak height comparisons.
** Coelution of contaminants.
21
-------
CLAY (TYPE P)
NATIVES)
ConcentratIon (ppb)
Medlan
Mean (ppb)
Standard
9*>X Confi-
Spike
Percent
Compounds
I
2
3
U
(ppb)
(Range)
Deviation
dence level
(ppb)
Recovery
Bicambn
15^.9
11*9.8
150.1*
156.9
15?.7
153.0+1.89*
3. ^
5-^
200.8
76.2
MCPP
3603.0
3603.0
30TT.1
3902.U
3603.0
35^9. ^6.57*
3^3.3
51*5.8
1*7^9.6
7l*.7
Dichlorprop
169.2
169.0
173.3
17S.3
171.3
172.Ul.9^
ii.ii
7.0
i?l«.6
60.u
2,U-D
111.6
106.3
118.8
118.1
11U.9
113.7±>.l8*
5.9
9.^
182.8
62.2
Silvex
26.06
2I4.I4T
26.66
27.06
26.36
26.06*3.07*
I.II4
1.8
38.62
67.5
1 f T>
OA O 1
1 C Or.
On AT
01 ¦) i,
OA
mo •} 14.^
O "37
•> A
1,1 l.A
hA . s
-------
TABLE 5. ANALYSIS OF SPIKBD HERBICIDES IN KAOLIN CLAY (TYPE P)
USING PEAK AREA COMPARISON* (METHYL DERIVATIVES)
Compounds
1
Concentration (ppb)
2 3 14
Median
(ppb)
Mean (ppb)
(Range)
Standard
Deviation
95* Confi-
dence Level
Spike
(ppb)
Percent
Recovery
Dicanba
153.0
150.9
153.1
155.6
153.1
153.2+0.02*
1.9
3.0
200.8
76.2
MCPP
3796.h
3010.6
1*123.0
14067.5
39^3.1
3996.1i+3.6l*
133.3
211.9
I47I49.6
814.1.
Dichlorprop
158.7
159.6
169.8
160.2
163.9
1614.1+3.00*
5-7
9.1
2114.6
76.14
2,l4-D
12?. 1
112.5
125.6
122.9
122.5
120.0+3.141*
5.7
9.1
102.0
66.1.
Silvex
2ii.66
23.20
25.70
25.77
25.18
214.03+3.63*
1.2
1.9
38.62
63.9
2,li,5-T
20.35
16.9^
20.81
20.03
20.58
19.714+7.05X
1.9
3.0
1.1.140
I4Y.6
2,l4-DB
700.1
655.1
765.6
685.li
725.5
721.5+7.11*
60.8
96. r
8?1.0
0Y.9
Dinoneb
20.2
13. '1
18.7
10.9
10.0
17.0+12.36*
3.0
14.0
103.3
IT. 3
•Analysis as in Table I4 using peak areas measured by the IBM C$9000.
-------
TABLE 6. ANALYSIS OF FRESHLY SPIKED HERBICIDES IN KAOLIN CLAY (TYPE P)*
(METHYL DERIVATIVES)
•Concentrate ior. (ppb)
Modi an
Moan (rpb)
Standard 955? Ccnfi-
Spike
Pc~overy
Compounds
1
2
3 >»
(pcb)
(HanRe)
'Jcviation denee Level
(ppb)
Percent
2yh-1)
U5I.0
397. U It31.9
UU1.5
lt33.6+1t.3fi*
26.1 1*1.5
C01.6
72.1
Lrilvcx
369.6
391.0
379.9 3B1..1
3^2.0
381.1+1.68*
9.0 U.3
It88.3
78.1
2,U,5-T
359.1
355.
337.6 3!t6.'t
351.1
3149.7+2.21J
9.7 15.'»
1*96.',
73.'t
•Lree Table U fcr conditions of Analysis
-------
TABLE 7. ANALYSIS OF SPIKED HERBICIDES IN KAOLIN CLAY (TYPE P)
FOLLOWING 7 DAYS STORAGE AT -38*C (METHYL DERIVATIVES)
Concent rat ion (ppb)
Median
Mean (ppb)
Standard
95% Confi-
Spike
Percent
Compounds
1
2
3
7,
(ppb)
(Range)
Deviat ion
dence Level
(ppb)
Recovery
1.86.6
l*3l*-5
1.66.5
1*53.8
160.2
1*60.3+3.52*
21.9
3l*.0
601.6
76.5
Gilvcx
398.0
l»l6.0
liOb.2
301*. 0
1*01.5
1*01.1*2.33%
13.3
21.1
1*00.3
02.2
2,1*,5-T
1*10.7
31*1.9
367.1
360.5
363.0
370.1+5.50*
29.1
1*6.3
1*96.5
71*.5
Table k for conditions of analysis.
-------
TABLE 8. RECOVERIES (%) OF SPIKED HERBICIDES FROM KAOLIN CLAY
(TYPE P) USING SONICATION WITH METHYLENE CHLORIDE AT SELECTED
pH VALUES* (SINGLE DETERMINATION. METHYL DERIVATIVES)
Herbicide
Compounds
7
PH
3
1
Spike
ppb
Dicamba
15
33
59
201
MCPP
36
60
**
4600
Dichlorprop
14
19
98
215
2,4-D
24
40
30
183
Silvex
35
42
61
39
2,4.5-T
8
15
31
41
2,4-DB
51
58
57
821
Dinoseb
62
46
45
103
* A 5.0g sample + 5.0 mL of phosphate buffer (or dil. HC1 for pH=l) plus
10-15g of anhydrous Na2S04, mixed by spatula. Sonicated 3 times
with 60 mL of methylene chloride for 3 minutes each. Combined extracts
concentrated and exchanged to hex&ne using a K.D. apparatus with a
final reduction to 2.0 mL by dry nitrogen stream. Derivatized by
diazomethane and GC/EC analysis using the DB-5 column (see Figure 3 for
conditions). Calculations based on area integration using the IBM
CS9000.
** Coelution of contaminant.
26
-------
TABLE 9. RECOVERIES OF SPIKED HERBICIDES FROM KAOLIN CLAY (TYPE P)
USING SONICATIOW AT pH 7 WITH SELECTED SOLVENTS*
(SINGLE DETERMINATION. METHYL DERIVATIVES)
Solvent
Herbicide
Compounds
Spike
ppb
CHoClo
1:1
CHoClo/hexane
1:1
acetone/hexane
Dicamba
201
76
80
11
MCPP
4750
<10
<10
<10
Dichlorprop
215
79
86
43
2,4-D
183
64
64
<10
MCPA
4758
80
73
46
Silvex
39
84
87
38
2,4,5-T
41
64
69
**
*-
1
o
00
821
93
103
86
Dinoseb
103
86
80
82
* See Table 8 footnote for description of the method. This study was
improved by using a larger amount of anhydrous Na2SO^ (25g).
** Coelution of contaminant.
-------
TABLE 10. RECOVERIES OF SPIKED HERBICIDES FROM KAOLIN CLAY (TYPE P)
USING SELECTED EXTRACTION TECHNIQUES* AND PENTAFLUOROBENZYLATION**
(SINGLE DETERMINATION)
Modified Modified Sonication Sonication Sonication
Method 8150 Method Sample Sonication 1:1 CH2CI2/ 1:1 hexane/
Herbicide
Compound
Spike
PPb
8150
Recoverv(%)
Spike
PPb
ch2ci2
PH7
acetone
PH7
acetone
PH7
Dalapon
170
97
170
92
80
100
Dicamba
161
57
201
86
98
85
MCPP
476
99
4750
91
101
98
Dichlorprop
258
—
215
98
96
99
2,4-D
219
75
183
30
18
10
MCPA
633
78
4758
95
10
103
Silvex
39
—
39
73
95
73
2,4.5-T
41
80
41
—
—
—
2,4-DB
821
—
821
42
91
104
Dinoseb
124
103
66
85
102
* Method 8150 was modified as described in Table 4. Sonication conditions
were the same as those in the Table 9 experiment.
** Following extraction, the solvent was exchanged to acetone (4mL) using a
stream of dry nitrogen. Aqueous 30% K2C03 (30 uL) and 20 uL of penta-
fluorobenzyl bromide were added. The tube was sealed with Teflon tape
and a screw cap and was heated in a water bath at 60*C for 3 hours.
The volume of the cooled tube was reduced to about 0.5 ml using a
stream of dry nitrogen and 2 iL of hexane was then added. The solution
was then reduced just to dryness under a stream of dry nitrogen and
redissolved in 2 mL of toluene/hexane (1:9). This solution was
chromatographed on 5% water-deactivated silica topped with anhydrous
Na2SO^. Elution of the analytes was done with 75:25
toluene/hexane. Analysis was done by GC/EC using the Tracor SAO gas
chromatograph and IBM CS9000 peak areas with a DB-5 (J & W Scientific)
1 iim, 0.25 mmx 15M column.
28
-------
CONCLUSIONS
1. Capillary GC/EC on DB-5 (J & W Scientific) provides superior
chromatographic resolution and requires less time than the packed
column GC/EC described in Hethod 8150.
2. Kaolin Clay (Type P) is a clean and generally nonretentive matrix
suitable for optimization of spiked sample analysis.
3. At the concentrations tested, Method 8150 gives very low (or 0)
recovery of Dinoseb. Even a spiked solvent blank gave 0 recovery.
This is probably a pH problem. (See Conclusion #6 for a solution to
this problem.)
4. Variable results were found for Method 8150 analysis for Dalapon.
This acid is highly water soluble, highly acidic, and the methyl ester
is quite volatile.
5. Better recoveries were obtained for analytes tested at higher
concentrations (2,4-D, 2,4,5-T and Silvex).
This suggests the recoveries of Method 8150 will be limited by the
sample preparation or analyte level and not the GC determination.
6. Extraction with methylene chloride using sonication is promising as a
replacement for the ether extraction of Method 8150. The procedure is
simpler and uses less solvent. The only acceptable recovery of
Dinoseb was obtained using this method.
7. Derivatization with pentafluorobenzyl bromide is a promising
alternative to methylation. All analytes can be derivatized. The
capillary GC on DB-5 (J & W Scientific) gives superior sensitivity and
resolution compared to that of the methyl derivatives. The enhanced
sensitivity is particularly important for MCPA and MCPP. The methyl
esters of these analytes give only about l/1000th the response of the
other methylated analytes in GC/EC. The pentafluorobenzyl derivatives
of all the analytes give similar responses in GC/EC. This method
requires more development to verify stability of the derivative and GC
separation from possible interferences. The derivatization time of
three hours does affect sample through-put.
RECOMMENDATIONS
Sonication extraction with an organic solvent and capillary GC/EC
promises to yield an improved method for free acid herbicide analysis.
This method would be much easier to optimize for all analytes and promises
to give better sensitivity than the involved extraction and packed column
GC of Method 8150. The pentafluorobenzylation procedure is an attractive
alternative to methylation with probably lower detection limits.
29
-------
SECTION 5
RUGGEDNESS TESTS
INTRODUCTION
A preliminary ruggedness test prior to simplex optimization is useful
to define the variables to be optimized by simplex. Ruggedness testing
does not result in optimization but can narrow the choices of conditions.
The ruggedness test design of W. J. Youden^* was used to test seven
variable conditions with only eight determinations by using two levels of
each variable (designated by a capital and lower case letter) which are
distributed as shown in Table 11. To determine the effect of changing
experimental condition 1 from A to a, the average analysis results of
samples 5 through 8 are subtracted from the average analysis results for
samples 1 through 4 yielding a ruggedness difference which indicates the
importance of varying condition 1. The importance of condition 2, changing
from B to b, is given by the 1, 2, 5, and 6 average results minus the 3,
4, 7, and 8 average results.
TABLE 11. DESIGN FOR TEST OF EXPERIMENTAL CONDITIONS
Experimental Values of Conditions in Determination Number
Condition
1
2
3
4
5
6
7
8
1
A
A
A
A
a
a
a
a
2
B
B
b
b
B
B
b
b
3
C
c
C
c
C
c
C
c
4
D
D
d
d
d
d
D
D
5
E
e
E
e
e
E
e
E
6
F
f
f
F
F
f
f
F
7
G
g
g
G
g
G
G
S
The standard deviation of each test analysis mean subtraction difference
per herbicide was calculated as 2/7 times the square root of the sum of the
squares of the differences as suggested by Youden.3*
30
-------
RUGCEDNESS TESTING OF FREE ACID HERBICIDE EXTRACTION AND ANALYSIS
We decided to perform three ruggedness tests, in each case testing
three sets of seven variable experimental conditions. In addition,
ruggedness differences were calculated for each analyte per sample and per
the mean of the samples. The experimental variables are shown in Table 12
for the general procedure diagrammed in Figure 8.
TABLE 12. EXPERIMENTAL VARIABLES AND ASSIGNED VALUES FOR HERBICIDE METHOD
Value for
Value for
Capital
Lower Case
Condition
No.
Letter
Letter
Letter
Experiment 1
pH of phosphate buffer
added to clay
1
A,a
1.0
3.0
Acetone:hexane ratio in
sonication
2
B,b
1:1
2:3
Analyte concentration
3
C.c
IX
3X
Beaker size used in
sonication
4
D,d
250 mL
400 mL
Base extraction or acid wash
of clay extract
Filter for clay extract
Solution for methylation
Volume of buffer or water
added to clay
pH of buffer or water
added to clay
Sonicator output setting
Sonication temperature
Solvent volume in sonication
Base extraction or acid
wash of clay extract
Amount of CH2N2
(molar excess)
E,e
F.f
G.g
Base Extraction
Celite Column
4 mL Hexane and
2 mL Isooctane
Acid Wash
Whatman No.
2 filter
paper
6 mL Hexane
Experiment 2
1 A,a 80 mL
2 B,b 5 (acetate buffer)
3 C.c 6
4 D,d 25*C
5 E,e 100 mL
6 F.f +
7 G.g 4X
50 mL
<1
5
0°C
150 mL
2X
(continued)
31
-------
TABLE 12. (continued)
Condition
No.
Letter
Value for
Capital
Letter
Value for
Lower Case
Letter
Experiment 3
pH of phosphate buffer
added to clay
1
A,a
1.0
2.0
Volume of buffer added
to clay
2
B.b
80 mL
100 mL
Extraction solvent
3
C,c
ch2ci2
CH3OC(CH3)3
Sonicator output setting
4
D,d
A
5
Base extraction or acid
wash of clay extract
5
E,e
Base extraction
Acid wash
Methylation solution
6
F.f
10% CH30H
OX CH3OH
Destruction of excess
CH2N2
7
G,g
Silicic acid
Overnight
evaporation
32
-------
Extract
+ or -
Wash
Methylated
with CH2N2
Kaolin Clay
Spike
Analyzed
Capillary
GC/EC
Sonicated +
Water or
Buffer +
Extraction
Solvent
Figure 8. Block diagram of herbicide analysis.
33
-------
Experimental
Experiment 1
Fifty grams of Ajax Type P clay (Westwood Ceramic Supply, City of
Industry, California) was weighed into a beaker and spiked with 1 or 3 ml
of the standard solution containing the 10 acid herbicides. The sample
was mixed with 20 mL of phosphate buffer. The extraction solvent was
added (100 mL) and the sample sonicated for 3 minutes in the pulsed mode
at 50 % duty cycle and an output setting of 5. After allowing the clay to
settle, the solvent was transferred into a 500-mL centrifuge bottle. The
clay was sonicated two more times using the same conditions with 100 mL
extraction solvent each time. The extract was combined into the
centrifuge bottle, and centrifuged for 10 minutes to settle the fine
particles. The extract was filtered into a 500-mL separatory funnel.
Half of the samples were extracted with base and half were washed with
acid. For extracting with base, 100 mL of 0.1 H sodium hydroxide was added
to the separatory funnel and was shaken for 2 minutes. The aqueous layer
was transferred to a beaker and immediately was acidified with HC1 to a pH
of 1.0. The organic layer was extracted once more with 100 mL of 0.1 II
HaOH. The aqueous layer was added to the first extract and the pH was
readjusted to <1.0 if necessary. The organic layer was discarded (or
saved for analyses of esters). The acid solution was extracted twice with
100 mL of methylene chloride. The final extract was concentrated to
approximately 5 mL in a Kudema-Danish (K-D) flask on a steam bath. The
methylene chloride was evaporated with nitrogen prior to hexane-exchange
and methylation.
For samples washed with acid, 100 mL of acidified water was added to
the separatory funnel containing the soil extract and was shaken for 2
minutes. This process was then repeated. The organic layer was
transferred to a 500-mL K-D flask and the combined aqueous layer was then
extracted 3 times with 50 mL of methylene chloride. The methylene
chloride was added to the K-D flask and concentrated to approximately 5 mL
on a steam bath. The methylene chloride was exchanged with hexane prior
to dlazomethane methylation.
Experiment 2
One mL of the herbicide standard was added to 50 g of Ajax Type P clay.
A given volume of acetate buffer (pH 5) or deionized water (sample pH to
<1 by addition of con. HC1) was added and the sample was mixed completely.
The sample was extracted 3 times by adding methylene chloride and
sonicating in the pulsed mode at 50 * duty cycle, 3 minutes each, at the
specified setting. The combined extract was washed with 100 mL of
acidified water or with base.
34
-------
For the base extract, the aqueous layer was acidified to pH <1 with
concentrated HC1 and was extracted 3 times with 100 mL of methylene
chloride. The methylene chloride extracts were then combined. The
methylene chloride solution containing the analytes was concentrated to
about 5 mL for methylation with diazomethane.
Experiment 3
Fifty grams of Ajax P clay was weighed into a beaker and spiked with
1 mL of a standard solution containing the 10 acid herbicides, Dicamba,
MCPP, MCPA, Dichlorprop, 2,4-D, Silvex, 2,4,5-T, 2,4-DB, Dinoseb, and
Dalapon. The sample was mixed with either 80 or 100 mL of phosphate buffer
at pH 1 or 2. The extraction solvent was added (100 mL) and the sample was
sonicated for 3 minutes in the pulsed mode at 50 % duty cycle at an output
setting of 4 or 5. After the clay was allowed to settle, the solvent was
transferred into a 500 mL centrifuge bottle. The clay was sonicated two
more times using the same conditions with 100 mL extraction solvent each
time. The extracts were combined into the centrifuge bottle, and were
centrifuged for 10 minutes to settle the fine particles. The extract was
filtered through Whatman #1 filter paper into a 500-mL separatory funnel.
Half of the solvent extracts were extracted with base, and half were
washed with acid. For extracting with base, 100 mL of 0.1 N sodium
hydroxide was added to the solvent separatory funnel and was shaken for 2
minutes. The aqueous layer was transferred to a beaker and immediately was
acidified with phosphoric acid to a pH of 1.0. The organic layer was
extracted once more with 100 mL of 0.1 N NaOH. The aqueous layer was
added to the first aqueous extract and the pH was readjusted to <1.0 if
necessary. The organic layer was discarded. The acid solution was
extracted twice with 100 mL of extraction solvent. The combined final
extract was concentrated to approximately 5 mL in a 500-mL K-D concentrator
on a steam bath.
For samples washed with acid, 100 mL of water acidified to pH <1.0
with H3PO4 was added to the separatory funnel containing the clay
extract and was shaken for 2 minutes. This procedure was then repeated.
The organic layer was transferred to a 500-mL K-D flask and the combined
aqueous layer was then extracted 3 times with 50 mL of extraction
solvent. This extract was added to the K-D flask and concentrated to
approximately 5 mL on a steam bath.
The sample was prepared for methylation by evaporating the sample just
to dryness, then reconstituting it with 1 mL of iso-octane, then diluting
it to a volume of 5 mL with hexane. In addition, half of the samples
required 0.5 mL of methanol to be added before the samples were diluted to
5.0 mL with hexane.
-------
Common Procedures
The spiking solution (Table 13) and methylation procedures were used
in all experiments.
TABLE 13, STANDARD SPIKIKG SOLUTION (ACETONE)
Analyte Concentration (Uft/mL)
Dalapon 10.9
Dicamba 7.33
Dichlorprop 14.0
2.4-0 12.0
MCFA 1,014
HCPP 1,026
Silvex 2.43
2,4,5-T 3.95
2,4-DB 40.6
Dinoseb 12.0
Methylation of the samples using diazomethane was carried out in the
containment facility following the procedures described in Section 4. The
excess of diazomethane was adjusted by adding the appropriate volume of
diazomethane solution. The GC analysis conditions are listed in the Figure
4 caption except that assignments of GC peaks to analytes were confirmed by
GC/MS. The correct assignments are shown in Figure 9 for an actual sample
(number 4) from the ruggedness test experiment 1.
36
-------
156999-
8841
3
9
1
2
4
5
6
7
8
10
Minutes
128217
u O
Q *
8141
18
19
20
13
14
15
16
17
12
10
11
Minutes
Figure 9. Gas chromatogram of methylated herbicides,
sample 4, experiment 1.
37
-------
Data and Results
The percent recoveries and differences in percent recoveries resulting
from changes in the analytical conditions are shown in Tables 14 to 19.
Note that the sign of the differences shown in Tables 15, 17, and 19 is
important in that the higher (better value) is the condition value
represented by the capital letter when the difference is positive and by
the lower case letter when the difference is negative (see Table 12 for
the condition descriptions).
TABLE 14. PERCENT RECOVERY OF HERBICIDES FOR EXPERIMENT 1 USING
CONDITIONS OF TABLE 12
Herbicide
Recovery
(%> of
Herbicides
- Determination
Number
1
2
3
4
5
6
7
ft
Dicamba
64
77
40
102
56
48
63
20
MCPP
75
92
49
122
71
61
84
37
MCPA
73
84
47
105
67
56
73
29
Dichlorprop
71
84
44
109
63
57
73
28
2,4-D
69
82
41
109
64
52
67
20
Silvex
74
88
43
115
74
64
89
28
2,4,5-T
65
84
43
112
64
56
76
21
2,4-DB
88
89
55
111
71
66
87
27
Dinoseb
44
87
40
108
75
40
83
38
Dalapon
0
64
0
84
39
0
32
0
38
-------
TABLE 15. DIFFERENCES FOR HERBICIDE METHOD, EXPERIMENT 1
Analytical
Condition Dicaraba
MCPP
MCPA
Dichlor-
prop
Silvex
2,b,5-T
2,fc-DB
Dinoseb
Dala-
pon
X(except
Dalapon)
X2
pH of
buffer
added 2h
22
21
22
2U
16
22
23
11
19
21
lilil
Acetone:
hexane
ratio 5
2
6
5
8
6
li
9
-5
-3
li
16
Analyte
concentration -5
-8
-li
-7
-6
-li
-5
2
-7
-19
-5
25
Beaker size
in sonication -6
-U
-k
-li
-7
-li
-7
-3
-3
-7
-5
25
Base
extraction
or acid vaah -32
-36
-31
-33
-35
-1*0
-38
-31
-I17
-55
-36
1296
Filtration
method 1*
U
3
5
2
1
0
3
7
3
9
Solution
for
raethylation 21
2k
20
23
22
28
2li
27
9
3
22
ueu
Bum of X2 = 2296
2/7 x Sum of X2 = 656
Std. Dev. = (2/7 x Sum of X2)V2 = 26
-------
TABLE 16. PERCENT RECOVERY OF HERBICIDES FOR EXPERIMENT 2
USING CONDITIONS OF TABLE 12
Recovery (Z) of Herbicides - Determination Number
Herbicide
1
2
3
4
5
6
7
8
Dlcamba
14.0
16.8
97.8
100.3
7.0
16.1
63.0
90.8
MCPP
94.3
83.0
88.0
95.8
43.9
82.2
56.2
77.7
MCPA
84.2
79.6
98.0
103.0
47.8
79.7
68.8
91.3
Dlchlorprop
96.8
88.5
111.6
117.6
45.3
78.1
76.7
100.6
2,4-D
72.3
82.2
108.3
116.1
46.4
81.0
72.3
100.9
Sllvex
103.3
119.1
124.0
117.5
52.2
86.4
88.2
99.1
2,4,5-T
101.1
108.2
116.3
119.6
59.6
133.9
78.5
107.2
2,4-DB
99.8
57.2
118.5
119.0
63.0
88.1
89.5
98.1
Dlnoseb
95.2
68.7
85.8
94.8
58.9
78.3
61.1
75.1
-------
TABLE 17. DIFFERENCES FOR HERBICIDE METHOD, EXPERIMENT 2
Anril y M ml
Conditions
Dica/nbi
m err
MCPA
[Hehlor-
prop
2.U-D
Silvex
2,1*,5-T
Dinoseb
X
C\J
IX
Ar.ount of
vitcr added
13
25-3
19.3
28.14
19.5
314.5
16.5
13.9
17.7
20.9
1*37
pH of water
-71'. 5
-3.6 -
-17.5
_ P1*. U
-28.9
-16.9
-l*.7
-29.3
-3.9
-22.6
511
Sonicator
sotting
-10.5
-11*.0 •
-13.7
-13.6
-20.3
-13.5
-28.3
2.1
-3.9
-12.9
166
Sonicat-ion
temperature
-9-1
0.1*
-1.1
-2.6
-6.1
7.3
-8.5
-11* -1
-1*.5
-6.0
36
R-ir>e extraction
or acid war.h 8.9
15.8
19.1*
lli. 8
11.3
8.9
23.1
18.9
12.7
ll». 9
222
Amount of CHjNj —i*. 7
a.9
I*.7
5.8
0.9
0.3
10.3
lU. 9
10.3
5.7
32
Siyn of X2 -
IU0I4
2/7 x Sun of
Stfl. Dev. = (
X2 = liOl
!2/7 x Sun of
5*)1/Z
= 20
-------
TABLE 18. PERCENT RECOVERY OF HERBICIDES FOR EXPERIMENT 3
USING CONDITIONS OF TABLE 12
Recovery (%) of Herbicides - Determination Number
Herbicide
1
2
3
4
5
6
7
8
Dalapon
39
71
21
45
15
61
9
61
Dlcamba
89
75
76
42
78
78
73
75
MCPP
95
82
74
71
80
73
75
76
MCPA
88
79
80
66
79
81
78
73
Dlchlorprop
94
81
66
64
84
81
84
76
2,4-D
88
80
76
60
74
79
78
73
Sllvex
95
94
69
114
86
70
91
68
2,4,5-T
107
62
70
74
76
85
89
82
2,4-DB
124
88
75
121
100
80
84
102
Dlnoaeb
65
66
75
77
114
65
76
51
-------
TABLE 19.
Analytical Dichlor- Dala-
Condition Dicamba MCPP MCPA prop 2,^-D Silvex 2,k,5-T 2,k-DB Dinoaeb pon X X
pH of
buffer added -5 5 0 0 0 lk 0 10 -6 7 U.7 22.1
Volume of
buffer 13 9 8 0 0 0 9 2 0 13 7.8 60.0
Extraction
Bolvent 11 5 6 12 6 -2 5 -2 10 -39 10.6 112
Sonlcator
setting 9*^3 1* 0 2 1U 6 -10 9 0.0 6U
Base extraction
or acid wash 13 3 5 6 6 -20 6 -3 -19 11 9.2 81.6
Methylation
solution -5 5 -3 -10 3 30 6 -1 7.1 50.U
Destruction of
excess CH2N2 -5 1 0 0 0 lU 11 11 -6 -3 5.1 26
Sum of X2 » l«20
2/7 X sun of X2 • 120
Std. Dev. - (2/7 X sum of X2) 1/2 « 11
-------
Experiment 2 was performed by one analyst and experiments 1 and 3 were
performed by a second analyst. The pH of the buffer (acetate or
phosphate) affected analyte recovery in the first two tests, with the
lower pH yielding higher recoveries. When the pH range was decreased for
testing by experiment 3 (pH 1 or 2), the pH effect became less important.
Dicamba recovery was severely reduced at higher pH in experiment 2 (Tables
16 and 17).
The base extraction step (useful for separating esters from free
acids) or alternative acid wash step had a different result in each test.
In experiment 1, a dramatic reduction in the recoveries of all analytes on
base extraction was observed with the acetone/hexane solvent (Table 15).
In experiment 2 with methylene chloride as the solvent* base extraction
improved the recoveries of all analytes (Table 17). In experiment 3, with
methylene chloride or methyl t-butyl ether as the solvents, Silvex and
Dinoseb had reduced recoveries on base extraction (Table 19). Solvent-
dependent competing effects are operating in the base extraction step.
This step is very useful if the extracting solvent is methylene chloride.
Addition of methanol to the methylation solution gives enhanced
recoveries for most analytes as shown in Table 15. Table 19 shows that
this effect can be very important for 2,4-DB.
Recovery of most herbicides is increased by adding iso-octane (a
keeper) to the methylation solution as shown by experiment 1 (solution for
methylation, Table 15) but it is interesting to note that the most volatile
ester, Hethyl Dalapon, shows an unexpectedly small effect. In addition,
experiment 2 (Table 17) shows two additional sensitive conditions, the
amount of water (or buffer) added to the clay and the sonicator setting.
A general improvement in recoveries is found with more water and a lower
sonicator setting.
The first two ruggedness tests failed for Dalapon. Some condition(s)
resulted in zero recovery for at least some of the samples. Under the
conditions of experiment 3, Dalapon was recovered in all samples. Dalapon
is the most acidic analyte, the most water soluble, the most reactive with
base, and the Dalapon methyl ester is the most volatile derivative.
Conclusions
The standard deviations obtained in the three ruggedness tests <26,
20, and 11%) indicate that the optimization was successful and that the
final procedure will have an 11% or less standard deviation. This is
acceptable for environmental analysis.
Methylene chloride seems to be the best solvent for extraction because
recoveries are generally better and the sensitivity to base extraction
(versus acid wash) is reduced. Also, the use of iso-octane and methanol
in the methylation step is important.
44
-------
Analytical conditions identified for simplex optimization are;
1. pH of buffer added to clay,
2. volume of buffer added to clay, and
3. sonicator setting.
RUGGEDMESS TESTING FOR HERBICIDE ESTER HYDROLYSIS AMD ANALYSIS
A necessary step in the analysis of herbicides present in the ester
form is hydrolysis to the free acid. As heating with base is a vigorous
procedure, we decided to separate the free acids by base extraction of the
methylene chloride extract and then hydrolyze the esters remaining in the
methylene chloride.
Experimental. Herbicide Ester Hydrolysis Method
Six available herbicide esters, the iso-octyl esters of MCPP, MCPA,
2,4,5-T, and 2,4-DB, isobutyl ester of 2,4-D and propylene glycol butyl
ether esters of Silvex were tested in this experiment. One mL of working
standard solution containing various concentrations of the above esters
was added to 25 mL of methylene chloride and transferred into a K-D flask.
The specified amount of water, 27% KOH solution, and methanol were then
added to the flask. The flask was fitted with a Snyder column, heated in
a 60*-65*C water bath for the specified time, and then was removed from
the water bath. After cooling, the reaction mixture was poured into a
250-mL separator funnel and acidified to pH <2 with either concentrated
sulfuric acid or hydrochloric acid. The aqueous solution was extracted 3
or 4 times with 25-mL portions of methylene chloride. The combined
extracts were transferred to a K-D flask to reduce the volume on the water
bath to about 5 mL. To the extract, 1 mL of iso-octane and 1 mL of
methanol were added, the volume was reduced to 4 mL by a stream of
nitrogen, and then was methylated following the procedures described in
Section 4.
The methylated herbicides mixture was transferred to a 10-mL volumetric
flask. One mL of p-dichlorobenzene solution was added as internal
standard, and the total volume was brought to 10 mL by hexane. The
resulting solution was analyzed by GC/EC.
Standard solution concentrations are shown in Table 20, and the conditions
altered and their assigned values are shown in Table 21.
45
-------
TABLE 20. CONCENTRATION OF STANDARDS USED IN
ESTER HYDROLYSIS EXPERIMENT
Compound
Concentration (ng/uL)
MCPP, IOE*
2280
MCPA, IOE
1340
2,4-D, IBE**
15.2
Silvex, PGBEE***
6.8
2,4,5-T, IOE
7.5
2,4-DB, IOE
59.5
p-dichlorobenzene
124
* IOE is the iso-octyl ester.
** IBE is the isobutyl ester.
*** PGBEE is the propylene glycol butyl ether ester.
Data and Results
The percent recoveries from the ruggedness test for herbicide ester
hydrolysis and analysis are shown in Table 22. The differences for the
experiment are shown in Table 23. Table 23 shows that the concentration of
methanol is the only variable that requires optimization.
-------
TABLE 21. CONDITIONS ALTERED AND ASSIGNED VALUES FOR
HERBICIDE HYDROLYSIS METHOD
Value for
Value for Lower Case
Condition No. Letter Capital Letter Letter
Vol. of water and 37% KOH
soln. added for hydrolysis 1
Vol. of methanol added 2
Reaction time 3
K-D flask size 4
No. of times extracted 5
Boiling chips added during
hydrolysis 6
Acid used to acidify the soln. 7
A,a 30 mL + 5 mL 34 mL + 1 mL
B,b 30 mL 10 mL
C,c 120 min. 90 min.
D,d 500 mL 250 mL
E,e 4 3
F,f 3 1
G,g H2S04 HC1
TABLE 22. PERCENT RECOVERY OF HERBICIDES FROM
HERBICIDE ESTER HYDROLYSIS METHOD
Herbicide 1
2
3
4
5
6
7
8
MCPP 80 44 8.6 15 60 59 7.7 4.6
MCPA 121 110 77 33 108 121 43 52
2,4-D 83 91 73 33 76 103 20 29
Silvex 71 34 24 7.7 58 62 7.1 8.6
2,4,5-T 76 71 49 16 49 64 19 25
2,4-DB 37 4.9 3.9 2.9 37 29 3.2 2.0
47
-------
TABLE 23. DIFFERENCES FOR HERBICIDE ESTER HYDROLYSIS METHOD
Analyt ical
Cond it ion
MCPP
MCPA
N)
¦C*
1
o
Silvex
2,4,5-T
2,4-DB
X
Vol. of H-0
+ KOH soln
4.1
4.3
13
0.3
10.7
-5.8
4.4
Vol. of
Methanol
40.8
63.7
49.5
44.5
37.7
24
43.3
Reaction
Time
7.4
C">
•
00
-1
11.9
4.3
10.6
6.9
K-D flask
size
-1.6
-3.3
-16.5
-7.7
3.3
6.4
-3.2
No. of times
extracted
6.4
19.3
17
14.7
14.7
6
13
Boiling chips
added
10.1
-9.3
-16.5
4.5
9.3
8.4
1.1
V°4
or HC1
11.1
-17.3
-7.5
6
4.7
6
0.5
-------
RUGGBDNESS TESTING OF OPTIMIZED HERBICIDE EXTRACTION AND ANALYSIS
Introduction
The simplex optimization results (Section 6) and ruggedness tests
suggest that a buffer pH range of 1 to 2.5, buffer volume of 80 to 90 mL
and sonicator power setting of 5 to 7 should give the best results. These
results were tested as described previously with only these variables.
The experimental design is shown in Table 24. The design allows duplicate
values to be obtained for each condition. For example, the effect of
condition 1 is revealed by subtracting the mean results of determinations
3 and 4 from the mean results of determinations 1 and 2. These
subtractions are hereafter designated as "differences." The conditions
altered are shown in Table 25.
TABLE 24. DESIGN FOR TEST OF EXPERIMENTAL CONDITIONS. EXPERIMENT 4
Experimental
Conditions
Values of Conditions in Determination
Number
1
2 3
4
1
A
A a
a
2
B
b B
b
3
C
c c
C
TABLE 25. EXPERIMENTAL VARIABLES AND ASSIGNED VALUES FOR
HERBICIDE METHOD. EXPERIMENT 4
Value for
Value
for
Lower Case
Condition
No. Letter Capital
Letter
Letter
pH of buffer added to sample 1 A,a 1.0 2.5
Sonicator output setting 2 B,b 5 7
Volume of buffer added to
sample 3 C,c 80 90
49
-------
Conditions for Experiment *
The experimental conditions of experiment 3, Section 5, were used with
the conditions listed in Table 25, which were varied as indicated. The
solvent for extraction was methylene chloride, the extract was washed with
base, the methylation solution contained 10% methanol, and the excess
diazomethane was removed by overnight evaporation.
Data and Results for Experiment 4
The percent recoveries from the ruggedness test for the optimized
method of chlorinated herbicide analysis are shown in Table 26. The mean
of all the recoveries is 89.3% with a 4-3% RSD.
The table of differences is shown in Table 27. Ho important
difference was revealed.
TABLE 26, PERCEMT RECOVERY OF HERBICIDES FOR EXPERIMENT 4
USIHG CONDITIONS OF TABLE 25
Recovery of Herbicides. Determination Number
Herbicide
12 3 4
Dicamba
88
87
89
96
HCFP
122
97
100
109
If CPA
127
103
107
114
Dichlorprop
88
91
94
102
2,4-D
92
97
97
109
Silvex
60
65
63
71
2»4,5-T
69
76
75
86
2,4-DB
86
94
100
110
Dinoseb
58
72
58
62
Mean
88
87
87
95
50
-------
TABLE 27. DIFFERENCES FOR HERBICIDB METHOD. EXPERIMENT 4
Dichlor-
Condition Dicamba MCPP MCPA prop 2,4-D Silvex 2,4,5-T 2,4-DB Dinoseb Y*
pH of
buffer
added -4 5 4 -8 -8 -5 -8 -15 5 7.0
Sonicator
setting -4 8 9 -6 -9 -7 -9 -9 -9 7.8
Volume of
buffer 4 17 16 3 4 2 2 1 -5 6.0
* mean of differences.
CONCLUSIONS
The results of ruggedness testing have shown that the extraction and
analysis of free acid herbicides requires the simultaneous optimization of
three variables: the pH and the volume of buffer added to the sample, and
the power setting of the sonicator. All of these parameters are involved in
the extraction process. The ruggedness testing for ester hydrolysis and
analysis indicated that the methanol concentration was the only variable
requiring optimization. Optimization of ester hydrolysis was carried out
with a series of experiments that varied the methanol concentration.
Optimization of the extraction and analysis of the free acid herbicides
requires simplex optimization so that all the variables can be optimized
simultaneously. The final ruggedness test on the optimized experiment,
experiment 4, indicated that the procedure is rugged for the range of
conditions tested with a mean recovery of 89.3 % and a standard deviation of
4.3%.
51
-------
SECTION 6
OPTIMIZATION OF ANALYTICAL PROCEDURES
INTRODUCTION
The ruggedness testing of chlorinated herbicide ester hydrolysis and
analysis showed that optimization requires only the variation in the
amount of methanol added. A series of experiments giving the mean
herbicide recovery as a function of the methanol concentration should
yield an optimum at maximum mean recovery.
The optimization of the free acid herbicide extraction and analysis
using simplex optimization to give the highest mean recovery should give
the best values for the important conditions revealed by ruggedness
testing, (buffer pH, buffer volume, and sonicator power).
SIMPLEX OPTIMIZATION OF CHLORINATED HERBICIDES
Introduction
Simplex optimization is a statistical process whereby numerous
experimental parameters, which are previously identified (for example, by
ruggedness testing), are systematically altered to achieve an optimum. An
optimum, in this case, is defined as the highest possible mean recovery of
analytes. Simplex optimization has been recoiamended5*6,35,36 to optimize
analysis methods. The work on optimization of the J-Acid Method for
determination of formaldehyde3* and the short review by Dols and
Armbrecht3® on simplex optimization as a step in method development are
examples of practical applications of the technique. As these workers
suggest, the simplex can rapidly move toward the optimum and should be done
early in method evaluation.
Three parameters in the extraction of chlorinated herbicides from clay
were optimized simultaneously by simplex optimization using a fixed size
simplex. The variables chosen were the pH of the initial clay buffer, the
volume of the initial buffer, and the sonicator output setting. An optimum
for these three variables was achieved with a high percent recovery of 9 of
the 10 herbicide analytes. This information was useful in drafting a new
procedure.
52
-------
Experimental
Initially, four samples were run. The initial sets of experimental
values for each sample (vertices) were selected at values that were
estimated from consideration of previous studies to give near optimum
results. Table 28 displays the range of values that each variable could
assume. The calculations involved in simplex optimization require that
the value of each variable be expressed as a percentage. The following
formula is used for the pH buffer: pH « 14 - (X x 13). Table 29 gives
the initial vertices values.
TABLE 28. EXPERIMENTAL VALUE RANGES FOR SIMPLEX OPTIMIZATION
Variable
Low Value (0%)
High Value (100%)
Buffer pH 14 1
Buffer volume (mL) 20 120
Sonicator output 0 7
TABLE 29. INITIAL VERTICES (PERCENT) FOR SIMPLEX OPTIMIZATION
Sample #
PH
Buffer Volume
Sonicator Setting
1
81
50
75
2
100
50
75
3
100
25
75
4
100
50
50
The initial four samples were extracted and analyzed as described in
experiment 3, Section 5, (page 35) and the mean percent recovery of all
analytes except Dalapon was determined. The recovery of Dalapon was found
to be consistently poor (<5%) in all experiments.
The simplex optimization worksheet (Figure 10) was used to calculate a
new vertex for each subsequent experiment, with new values then assigned to
the variables. The new sample was extracted and analyzed in the same manner
as the previous samples, and another simplex optimization worksheet was
computed. These results determined the values selected for the next set of
variables. Repetition of experiments, with new values used for the
variables each time, continued until optimization was achieved. Progress of
the simplex optimization was followed by monitoring the mean percent
recoveries of the four mean analysis results for each sample as shown in
Table 30.
53
-------
TABLE 30. SIMPLEX 0PTIHI2ATI0M PROGRESS
Simplex Wo.
Mean Recovery (%)
% RSD
1
2
3
87.A
88.6
92. A
5.35
A.71
5.38
Results and Discussion
The results of the first simplex optimization experiment are shown in
Table 31. When the first simplex optimization worksheet was prepared, both
vertices 3 and A yielded, within experimental error, the same recovery.
Therefore, the next experiment was performed twice, once with sample 3
considered to be the worst vertex and once with sample A considered to be
the worst. This created a vertex with two different parameter conditions,
vertex 5A and vertex 5B.
A mean recovery of 6A.2% was obtained for vertex 5B (vertex 3 defined as
the worst), while a preferred mean recovery of 86.8% was obtained for vertex
5A. Therefore, vertex 5B was discarded, and the next experiment was
performed with vertex 5A in the worksheet. This experiment resulted in a
mean percent recovery of 98.9%. Table 32 shows the mean percent recovery
obtained for each experiment of the simplex optimization and the conditions
used for each experiment. A 98.9% recovery was deemed adequate and no
additional experiments were performed. Also, the four vertices of the last
simplex gave a mean recovery of 92.A% with a 5.38% RSD.
Conclusions
The simplex quickly located the optimum recovery value where a buffer pH
of 2.5, buffer volume of 86 mL, and sonicator power of 6.3 gave a mean
recovery of 98.9% with 7.0% RSD.
5A
-------
Factor Levels
pH of Volume of Sonicator Mean
Simplex Buffer Buffer (mL) Power Recovery
Number %2 X3 Response
Vertex Times
Rank Number Retained
Coordinates
B
of
Retained
Vertices
W
Z
P - Z/3
V
V
P - W
R = P + CP-W)
R
where:
Z = summation of coordinates.
P = centroid of face (the face is that part of a simplex that remains after
of the defining points is removed).
W = worst vertex.
R * reflection vertex.
B = best vertex.
N = next to the worst vertex.
Xn = values of factor n.
Figure 10. Three-factor sequential simplex worksheet for
herbicide method optimization.
55
-------
TABLE 31, RESULTS FOR ISITIAL SIMPLEX VERTICES
Herbicide
Corapounid
Vertices (% Recoveries)
1
2
3
4
Dicamba
85.4
49.2
77.4
76.8
MCPP
101
102
92.1
96.9
MCPA
96.1
99.1
89.4
92.4
Dichlorprop
95.2
101
87.4
93.3
2,4~D
91,2
90.3
83,2
80.9
Silvex
85.7
92.8
78.0
82.5
2,4t5-T
80.9
92.3
84.3
74.0
2*4-DB
10?
105
89.0
94.3
Dinoseb
89.6
93.5
74.5
48.0
Mean (% RSD)
92.4 (
8.9) 91.7
(18.2) 83.6 <7.3)
82.1 (18.6)
TABLE 32. COHDITIOHS AND RESULTS FOR EACH VERTEX
IH SIMPLEX OPTIMIZATION
Simplex
Vertex #
pH of
Buffer
Volume of
Buffer (mL)
Sonicator
Power
Herbicide
Mean Percent
Recovery (% RSD)
1
1.0
70
5.25
92.4
( 8.9)
2
3.5
70
5.25
91.7
(18.2)
3
1.0
70
3.5
83.6
( 7.3)
4
1.0
55
5.25
82.1
(18.6)
5A
2.6
75
4.1
86.8
(13.6)
58
2.6
54
3.5
64.2
(24.5)
6
2.5
86
6.3
98.9
( 7.0)
56
-------
ESTER HYDROLYSIS OPTIMIZATION
The results of ruggedness testing. Section 5, showed that the addition
of methanol to the hydrolysis mixture was an important way to increase
recoveries. Because methanol was the only experimental variable to be
optimized, simplex optimization was not required.
Experimental for Herbicide Ester Hydrolysis
Six available herbicide esters, iso-octyl esters of MCPP, MCPA,
2,4,5-T, and 2,4-DB; isobutyl ester of 2,4-D; and propylene glycol butyl
ether esters of Silvex were tested in this experiment. The working
standard solution (1.0 mL) containing the herbicide concentrations shown
in Table 33 was added to 25 mL of methylene chloride and transferred into
a 500-mL JC-D flask. To the flask, 30 mL of water, 5 mL of 40% NaOH
solution, and various amounts of methanol ranging from 10-60 mL were
8dded. The flask was fitted with a Snyder column, wss heated in a
60°-65*C water bsth for 2 hours, and then was removed from the water
b8th. After cooling, the reaction mixture was scidified to pH <2 using
concentrated phosphoric acid and was transferred into a 500-mL separstory
funnel. The aqueous solution was extracted two times with 100-mL portions
of methylene chloride. The combined extracts were transferred to a JC-D
flask and the volume was reduced on a water bath to about 5 mL. The
extract was evaporated just to dryness under a stream of nitrogen, then was
reconstituted with 1 mL ether and 0.5 mL methanol, then was diluted to
4 mL using iso-octane. The samples were methylated as described in
Section 4. The methylated herbicides mixture was transferred to a 10-mL
volumetric flask. Dichlorobenzene solution (0.5 mL) was added as internal
standard, and the total volume was brought to 10 mL using hexane. The
resulting solution was analyzed by GC/EC.
TABLE 33. CONCENTRATION OF HERBICIDE ESTER WORKING STANDARD
MCPP, Iso-octyl ester
MCPA, Iso-octyl ester
2,4-D, Isobutyl ester
Silvex, Propylene glycol butyl ether ester
2,4,5-T, Iso-octyl ester
2,4-DB, Iso-octyl ester
2437 ng/uL
2613 ng/uL
15.2 ng/uL
6.8 ng/uL
7.5 ng/uL
59.5 ng/uL
Data and Results
The recoveries of the herbicide analytes, analyzed as methyl esters,
are shown in Table 34. The mean percent recoveries plotted versus the
amount of methanol added are shown in Figure 11. The optimum is in the
25-50 mL range; 35 mL is a choice that is obviously rugged.
57
-------
TABLE 34. 0PTIMI2ATI0M OF HERBICIDE ESTER HYDROLYSIS BY
METHANOL ADDITION (PERCENT RECOVERIES)
Methanol Added
Anslytes
10 mL
20 mL
25 mL
30 mL
35 mL
40 mL
50 mL
60 mL
MCPP
39
72.6
99.5
87.8
91.5
91.2
111.3
85.5
ffCPA
96,5
105.2
112.8
116.6
112.7
117.1
128.9
108
2,4-D
82.7
83.4
90.0
91.3
91.2
92.4
101.7
81.4
Silvex
49.0
66.8
90.5
64,0
78.4
78.1
84.6
71.9
2,4,5-T
56.4
59-0
61.8
60
61.6
59.5
65,6
52.9
2, 4-DB
12.2
38.4
71.9
69.8
70.3
71.7
80.2
60.8
Mean
56
70.9
87.8
81.6
84.2
85
95.5
76.8
lOOl
Ui
4>
w
>
o
u
«
cr
«¦»
c
93
2
>
<
Amount of Methanol Added (mL}
figure 11. Hydrolysis of acid herbicide esters.
58
-------
CONCLUSIONS
Variable 6, the ester hydrolysis, was optimized arid was shown to be
rugged in the only important variable (methanol addition). The free acid
herbicide extraction and analysis were quickly optimized by simplex
optimization. The ruggedness of the optimum values requires testing using
ruggedness procedures as described in Section 5.
59
-------
SECTION 7
LIHEARITY
INTRODUCTION
The linear response range and detection limits were determined for the
10 analytes specified in Method 8150. Standards of known concentrations
were prepared, were methylated with diazomethane, and were analyzed by
capillary GC/EC.
EXPERIMENTAL
Response factors were estimated from previous in-house analyses of the
compounds of interest. Standards that would give an approximately equal
GC detector response were prepared. The standards ranged from 0.1 sig of
Dicamba to 77,2 mg of MCPA in 25 mL of hexane. These primary standards
were diluted to give a concentration range of 10* at 13 different
concentration levels.
Ten mL aliquots of each standard were methylated with diazomethane as
described in Section 4. The linear range of response for each compound
was then determined by duplicate injection of the methylated standards
into the gas chromatograph. The instrument parameters were as follows:
Instrument:
Column:
Injection:
Injector Temperature:
Detector Temperature:
Temperature Program:
Integrator:
Tracor 540 gas chromatograph, EC detector
DB-5, 0.25 m film thickness, 0.25 \im 1.0. X
30M L
5 uL Grob-type 30-second splitless injection
22Q*C
375-C
50*C for 1 minute, 25*C/min to 100#c, hold for
minute, 12*C to 220*C, hold for 12 min.
IBM CS 9000 Data System
60
4
-------
RESULTS AND DISCUSSION
Figures 12 through 21 show plots of analyte concentration versus peak
height. Table 35 gives detection limits and linear ranges for the 10
compounds. The responses of both MCPA and MCPP at the lowest concentration
were used to estimate the detection limits, even though the responses were
outside the linear range. Extrapolation of the linear range of response
to zero concentration would have given erroneously low detection limits
for these analytes. The MCPA and MCPP curves (Figures 12 and 13) have the
greatest deviations from linearity. It is likely that the GC column was
overloaded at the highest concentrations. It is also seen that the
responses for MCPA and MCPP are nonlinear at low concentrations. Lower
GC/EC responses were obtained for MCPP and MCPA than for the other
analytes, and, as expected, MCPA and MCPP had much higher limits of
detection.
TABLE 35. LINEAR RANGE AND DETECTION LIMITS FOR
CHLORINATED HERBICIDES
Compound
Linear Range Tested
Detection Limit Cnn/mL)1
Dalapon
7.4 -
736
ng/mL
1.34
Dicamba
2.6 -
520
ng/mL
0.60
MCPP
3.1 -
309
Ug/mL
333
MCPA
3.1 -
306
Ug/mL
218
Dichlorprop
7.5 -
15000
ng/mL
1.9
2,4-D
6.1 -
12200
ng/mL
1.7
Silvex Acid
2.1 -
4140
ng/mL
0.53
2,4,5-T
2.1 -
4110
ng/mL
0. 78
2,4-DB
20.2 -
40300
ng/mL
20.2
Dinoseb
4.1 -
8100
ng/mL
1.4
* Defined as that quantity of compound yielding GC/EC response with
S/N > 3.
61
-------
10
2 10 •
c
D
O
n
a:
? 10'-1
£
a>
5
I 10'-|
10
10
lo1
10
MCPA Concentration |ng, pL)
Figure 12. MCPA GC/EC response.
10-
E 10-
£ 10-
£
o>
'Z
I
1 10-
10.
—r~
10
10
~ro3
To4
MCPP Concentration (ng pL)
Figure 13. MCPP GC/EC response.
62
-------
no-
lo -
10-
10-
10 t 1 n n 1.
1 10 10 10 10
Dalapon Concentrations (ng/mL)
Figure 14. Dalapon GC/EC response.
10-1
10-
101
10-
10
10
10
Dicamba Concentrations (ng/mL)
10
Figure 15. Dicamba GC/EC response.
63
-------
10 n
I 10H
i 10-
£
&¦
10-
10
10
"io3
"i?
To'
Dichlorprop Concentrations fng/mL)
Figure 16. Dichlorprop GC/EC response
10
.? 10-
e
£
o>
10-
10-
10
—T—
10
~w2
"S3
To'
2.4.-O Concentrations (ng/mL)
Figure 17. 2,4-D GC/EC response.
64
-------
10%
10-
c
D
o
«
»
C 10-
8 10*
a.
10
10
10
10
10
Silvex Concentrations (ng/ml)
Figure 18. Silvex GC/EC response.
10%
10-
10
i
10
10
2.4.5-T Concentrations (ng/mL)
Figure 19. 2,4,5-T GC/EC response.
65
-------
Peak Heights (Integrator Units)
•n
>—
o
o
o _
2' °"
o
O"1
o
Peak Heights (Integrator Units)
-------
CONCLUSIONS
The detection limits and linear range were measured, and all analytes
exhibited a linear response over a 103 span of concentration.
67
-------
SECTION 8
PRECISION
INTRODUCTION
The precision for gas chromatographic analyses of the 10 analytes of
Method 8150 was determined. Standards of known concentration at the high
and low end of the linear range were prepared, were methylated with diazo-
methane, and were analyzed by gas chromatography. The * RSD was determined
for 10 samples at each concentration level. A solvent blank was carried
through the analysis to verify that the samples did not contain background
interferences.
EXPERIMENTAL
The lower concentration was set at 10 times the detection limit
reported in Section 7. The upper concentration was selected at the upper
end of the concentration range listed in the linearity report. The
samples at each concentration level were then analyzed by the method
described in Section 7. Ten injections were made for each compound at
both the low and high concentration levels.
RESULTS AND DISCUSSION
The precision determinations are shown in Table 36. Percent RSD
values were larger at the low concentration level than at the high
concentration level for all analytes except 2,4-D. The mean * RSD was
5.99 and all X RSD values were below 10. These values are considered to
represent the experimental error.
The solvent blank and representative chromatograms from both
concentration levels are shown in Figures 22-26.
68
-------
TABLE 36. RESULTS OF PRECISION DETERMINATIONS
Low Concentration
Compound
High Concentration
% RSD
(ng/mL) X RSD (n-10)* (ng/mL X 103) (n - 10)
Average
% RSD
(n ¦ 20)
Dalapon 14.7
Dicamba 6.5
MCPP 3300
MCPA 2070
Dichlorprop 18.8
2,4-D 15.3
Silvex 5.2
2,4,5-T 6.8
2,4-DB 50.3
Dinoseb 13.5
5.1
9.4
3.7
7.3
7.5
5.0
7.6
9.1
9.5
9.8
0.74
0.52
300.0
308.0
15.0
12.2
4.1
4.1
40.3
8.1
3.1
5.5
3.1
3.3
2.4
5.6
3.8
5.5
5.7
7.6
4.1
7.5
3.4
5.3
5.0
5.3
5.7
7.3
7.6
8.7
* The letter n indicates the number of determinations.
69
-------
43074*
V)
c
3
O
O
6411
.U
_r»JL
JL
1
11
Minutes
—r
13
—r
15
—T-
17
—r
19
Figure 22. Solvent blank for precision measurements.
6920-I
c
3
O
o
6246
13
14
—r
15
1
16
Minutes
T"
17
I
18
n
19
Figure 23. Low concentrations: Dicamba, MCPP, MCPA, Dichlorprop,
2,4-D, Silvex, 2,4,5-T, 2,4-DB, Dinoseb.
70
-------
6438-
c
3
o
o
6289
1 1 1 1 1 1 1 1 1 1
4.60 4.70 4.80 4.90 5.00 5.10 5.20 5.30 5.40 5.50
Minutes
Figure 24. Low concentration: Dalapon.
93881-
-------
11444-1
6212
5.20 5.30 5.40 5.50
Minutes
Figure 26. High concentration: Dalapon
72
-------
CONCLUSIONS
The % RSD's of all analyte concentrations measured were below 10. The
precision of this method is excellent for measuring chlorinated herbicide
concentration over a 102 ranee for MCPP and MCPA and over a 103
concentration for the other analytes. Horwitz, et al19»20 suggest that
analyses at the Ug/g level should have a single-laboratory variation of
8-11% to give a reproducibility < 16% in interlaboratory studies. Thus,
this method is practical for monitoring residues that are of public health
significance.
73
-------
SEGTIOH 9
BIAS TESTING
IUTRODUCTIOH
Bias or systematic error was tested by determining percent recoveries
of analytes at a range of concentrations known to give a linear response
and acceptable relative standard deviations as reported in Section 8. For
these tests, two matrices were used, kaolin clay (Ajax P, Westwood Ceramic
Supply) and kaolin clay spiked with still bottoms from herbicide
manufacturing obtained from Dow Chemical, Midland, Michigan, via S-Cubed,
San Diego, California.
EXPERIMENTAL
Kaolin clay samples
Fifty grams of kaolin clay were weighed into a 400~mL, thick-wall
beaker and spiked with 1 mL of a standard solution containing the 10 acid
herbicides (Dicamba, MCPP, HCPA, Dichlorprop, 2,4-0, Silvex, 2,4,5-T,
2,4-DB, Dinoseb, and Dalapon) to give concentrations shown in Table 1.
The sample was mixed with 85 mL of phosphate buffer (pH » 2.5). One
hundred mL of methylene chloride was added and the sample was sonicated
for 3 minutes in the pulsed mode at 50 percent duty cycle at an output of
6.3. After the clay was allowed to settle, the solvent was transferred
into a 500-mL centrifuge bottle. The clay was sonicated two more tiroes,
using the same conditions, with 100 mL of methylene chloride each time.
The extracts were combined into the centrifuge bottle and were centrifuged
for 10 minutes to settle the fine particles. The extract was then filtered
through Whatman #1 filter paper into a 500-mL separatory funnel. To the
extract, 100 mL of 0.1 II HaOH solution was added and the funnel was shaken
for two minutes. The aqueous layer was transferred to a beaker and
immediately was acidified with phosphoric acid to a pH of 1.0. The
organic layer was extracted once more with 100 mL of 0.1 H HaOH solution.
The aqueous layer was added to the first aqueous extract and the pH was
readjusted to <1.0 if necessary. The organic layer was discarded. The
acid solution was extracted twice with 100 mL of methylene chloride. The
combined extract was concentrated to approximately 5 mL in a 500-mL K-D
concentrator on a steam bath. Samples analyzed for Dalapon required use
of a pH-1 phosphate buffer.
The sample was evaporated just to dryness using a stream of nitrogen,
then the sample was reconstituted with 1 mL of iso-octane and 0.5 mL of
methanol, was diluted to a volume of 5 mL with ethyl ether, and was
methylated as described in Section 4.
1U
-------
A Tracor 540 Gas Chromatograph equipped with an autosampler was used
to analyze the methylated samples under the following parameters:
Column =« DB-5, 0.25 uM film thickness, 0.25 uM I.D. X
30 M L
Injection - 5 %lL Grob-type 30-second splitless injection
Injector Temperature - 220*C
Detector Temperature = 375°C
Detector » Electron Capture
Temperature Program - 50#C for 1 minute, 25*C/min to 100*C, hold for 1
minute, 12*C/min to 220*C, hold for 12 min.
Integrator = IBM CS 9000 Data system.
Kaolin clay samples Plus still bottoms
Still bottom # 42 (0.170 g) was extracted with methylene chloride.
Tnis was diluted 1 to 100. This dilution (2 raL) was added to each 50 g
kaolin clay sample prior to extraction, derivatization, and analysis as
described for the kaolin clay. The five clay/still bottom samples were
spiked as described in Table 37.
TABLE 37. FINAL CONCENTRATIONS (ppb) OF ANALYTES ADDED TO CLAY
AND CLAY/STILL BOTTOM SAMPLES CA-E)
Analvtes
Samples
(ppb)
A
B
C
D
E
Dalapon
147
29.4
14.7
2.9
1.5
Dicamba
146
29.2
14.6
2.9
1.5
MCPP
30600
6120
3060
612
306
MCPA
30700
6140
3070
614
307
Dichlorprop
3020
604
302
60.4
30.2
2,4-D
2480
496
248
49.6
24.8
Silvex
834
167
83.4
16. 7
8.3
2,4,5-T
852
170
85.2
17.0
8.5
2,4-DB
8160
1632
816
163
81.6
Dinoseb
1664
333
166
33.3
16 .6
DATA AND RESULTS
Figures 27-35 show the percent recoveries versus concentration for the
10 analytes spiked into the kaolin clay and kaolin clay/still bottoms. The
Dalapon result was highly variable and was not included. Tables 38 and 39
give the percent recoveries of the 10 measured analytes in kaolin clay and
kaolin clay/still bottom matrices. Sample chromatograms of blank and spiked
kaolin clay/still bottom extracts are shown in Figures 37 and 38.
75
-------
I 10
DICAM8A CONCENTRATION SPIKED IN SOIL (NQ/Q)
l'o?
a Sim Bottom Samelaa
• Kaolin Clay Sampiaa
Figure 27. Dicamba concentration bias.
'•">5 77*
MCPP CONCENTRATION SPIKED IN SOIL (NQ/Q) * S,|M Bottom Samoiaa
• Kaolin Clay Sampiai
Figure 28. MCPP concentration bias.
76
-------
1?!>
80'
102 103 10*
MCPA CONCENTRATION SPIKED IN SOIL (NO/O) * S""
• Kaol'n CUy Sa<"pl*a
Figure 29. MCPA concentration bias.
10* lO4
OICHLORPROP CONCENTRATION SPIKED IN SOIL (NO/O) * S""
• Ktolin Cl*y
Figure 30. Dichlorprop concentration bias.
77
-------
1,4-0 CONCENTRATION SPIKED IN SOIL (NQ/Q) * 9,111 *<"«'«•" Sampia*
• Kaolin Clay Samplat
Figure 31. 2,4-D concentration bias.
1C
SILVEX CONCENTRATION SPIKED IN SOIL (NQ/Q) * S,IM Boiion. sampia.
• Kaolin Clay Sampiaa
Figure 32. Silvex concentration bias.
78
-------
10
2,4,5-T CONCENTRATION SPIKED IN SOIL (NQ/Q) * S"" 8
-------
«o tot 751
DINOSEB CONCENTRATION SPIKED IN SOIL (NQ/Q) * 811,1
• KseHn City Sampl**
Figure 35. Dinosob concentration bias.
80
-------
TABLE 38. RECOVERIES (%) FOR THE CHLORINATED HERBICIDES
IN KAOLIN CLAY (CONCENTRATIONS A-E)
Analytes
Recoveries
A
B
C
D
E
Dicamba
82.3
80.8
84.3
104.3
105.8
MCPP
91.9
96.5
104.0
143.4
109.7
MCPA
93.1
87.6
94.5
99.7
90.9
Dichlorprop
86.1
92.2
92.9
96.9
93.4
2,4-D
86.8
87.0
84.7
77.3
80.1
Silvex
85.0
89.4
92.2
111.1
101.4
2,4,5-T
83.0
86.8
82.6
78.2
79.8
2,4-DB
93.6
108.1
101.0
108.8
100.3
Dinoseb
101. 7
92.2
108.1
182.6
127.1
Dalapon
TABLE 39. RECOVERIES (%) FOR THE CHLORINATED HERBICIDES IN
KAOLIN CLAY/STILL BOTTOM SAMPLES (CONCENTRATIONS A-E)
Analytes Recoveries
A
B
C
D
E
Dicamba
88.7
103.8
124
87.5
-
MCPP
92.3
89.0
97.7
133
105
MCPA
90.2
89.4
93.2
127
103
Dichlorprop
92.5
89.7
89.1
94.6
146
2,4-D
90.3
85.0
77 .3
80.1
94.1
Silvex
88.1
87.5
88.2
85.1
117
2.4,5-T
90.2
86.6
78.8
78.5
86.1
2,4-DB
90.9
91.8
94.6
108.0
147
Dinoseb
68.1
71.4
82.3
148
98.9
Dalapon
81
-------
25425H
~»
in
a
a O
u 5
6113
19
18
16
17
15
13
14
Minutes
Figure 36. Chromatogram, sample A, (most concentrated spike)
from kaolin clay/still bottoms.
7655
a.
X
-------
6875
13
14
16
15
17
18
19
Minutes
Figure 38, Chromatogram, sample F, blank from
kaolin clay/still bottoms.
83
-------
CONCLUSIONS
There is a general increase in percent recovery with concentration and
a general bias toward less than 100% recovery. Oalapon is not recovered
using the optimized protocol.
At very low concentrations, the background exhibited by the matrix or
by compounds added by the still bottom spike becomes very important and
large compared to the signal of the analyte spike. For example, methyl
Dicamba coelutes with a major impurity seen in the still bottom spiked
clay (compare Figures 38 and 39). The Oinoseb recovery in the kaolin clay
samples was excessively high at lower concentrations, yet the kaolin clay/
still bottom sample exhibited reasonable recoveries at the same concentra-
tions, which indicates a complex interaction of still bottoms with the
clay.
The compounds present in the matrix have an influence on detection
limits and bias of this method for these analytes. Coelution and complex
matrix interactions are observable with the kaolin clay and still bottom
samples.
84
-------
SECTION 10
GC/MS CONFIRMATION
INTRODUCTION
Compounds present in the matrix can coelute with the herbicide
analytes of interest. The compounds eluting cannot be identified by GC/EC
analysis alone. GC/MS analysis is required to give full scan spectra of
each analyte for comparison with known spectra.
In this section, the mass spectrum of the methyl derivative of each
herbicide is reported along with the reconstructed ion chromatogram with
the corresponding GC/EC chromatogram. The minimum concentration to obtain
a computer Finnigan INCOS "FIT" value of 800 Con special matching to
reference spectra taken at 50 ng) was determined for each analyte.
EXPERIMENTAL
Twenty-five milligrams of each of the 10 acid herbicides were weighed
out into a 10-mL volumetric flask. The acids were dissolved by adding an
adequate amount of acetone and then were brought up to about 4 mL with
hexane. The acid solutions were methylated as described in Section 4.
The methylated samples were evaporated to dryness. A series of
different solutions of the methylated acid herbicides ranging in
concentration from 50 ng/viL to 0.25 ng/>iL were prepared for GC/MS
confirmation. The instrument parameters were as follows:
Finnigan 9610 GC/Finnigan 4023 Mass
spectrometer.
DB-5, l.OiiM film thickness, 0.32>iM ID X
30M L
1 uL» Grob-type 30-sec. splitless
injection.
220*C.
60*C for 2 minutes, 13*C/minute to 220*C,
hold for 10 minutes.
-1200V.
45amu - 550amu.
1 scan/sec.
Data General N0VA3 with INCOS.
The mass spectra obtained from the 50 ng/uL methyl esters were used
to establish a library. Measurements of the resemblance of the library
spectrum to the spectrum of less concentrated samples were done by the
computer (the FIT number of library search in INCOS).
Instrument:
Column:
Injection:
Injector Temperature:
Temperature Program:
Electron Multiplier voltage:
Scan Range:
Scan Time:
Data System:
85
-------
DATA AND RESULTS
The reconstructed ion chromatogram is shown in Figure 40 and the
corresponding GC/EC chromatogram is shown in Figure 41. The full scan
mass spectra of the target herbicide esters are shown in Figures 42 to 50.
The minimum concentrations required to give a FIT of 800 are shown in
Table 40. The value of 800 for a good FIT is recommended in the Finnigan
INC0S Manual.39 This number appears to be a valid value because the
plots of FIT versus concentration (Figures 52-61) rapidly drop off at
concentrations below that which gives 8 FIT of 800.
86
-------
00
•vj
•*1
00
c
ft
LO
50
ft
n
o
3
(A
r»
C
n
r»
ft
a
o
3
n
sr
o
o
00
n
b
5
100 0-1
c
813 E
855
RIC -
A
271
393
633 693
JK
400
6:40
600
10:00
T
800
13:20
G
954
H
-A
J
1045
28864
A - Dalapon. methyl ester
8 - Dicamba. methyl ester
C " MCPP, methyl ester
D" MCPA. methyl ester
E - Dichlorprop. methyl ester
F - 2,4.-D. methyl ester
G " Silvex. methyl ester
H 2.4,5-T. methyl ester
I 2.4-D8. methyl ester
J " Dinoseb, methyl ether
1000
16:40
1200
20:00
Scan Time
-------
17960-
O
8226
^ 4L
JIlAlI
u
t r
5 6
t r
T
T 1 1 1
10 11 12 13 14 15 16 17 18 19
Minutes
Figure AO. GC/EC chromatogram of target herbicide esters.
88
-------
100 0-1
500-
47
53
50
M/E 50
59
61
97
89
71
77
60
I
70
82
¦ '
I '
93
I 95
» -1 I'
99
101
80
90 100
r 2160
121
! 123
113
110
—n
120
Figure 41. Dalapon electron impact mass spectrum.
100 0-.
50 o-
203
188
97
62 74
IrA. I
109
125 1*6163
lL xi jU. Il
160 1'5
¦ ll'.6,9l|i"4
r 3016
234
217
M/E 50
100
150
200
Figure 42. Dicamba electron impact mass spectrum.
89
-------
100 0-,
SO 0-
59
51
55
45
M/E
63
77
89
107
99
Ul i|i jllr.rrj
142
125
169
151
I
228
205
- 2236
1 i i i J "
60 80 100 120 140 160 180 200 220 240
Figure 43. MCPP electron impact mass spectrum.
100 0-1
50 o-
51
46
77
63
74
M/E
111
60
89
99
141
125
85 I
I.I I
r 2120
214
166
mJJ
> | > f" r~
100 120 140 1
182
169
_dj_
205
228
160 180 200 220
Figure 44. MCPA electron impact mass spectrum.
90
-------
100 On
162
50.0-
59
55
63
87
75 83
109
96
91
lili
M/E SO
100
125
L. I
145
154
¦ J.I.L Ji Mil.
il
150
r 2800
189
Jli
248
213
200
250
Figure 45. Dichlorprop electron impact mass spectrum.
100 0-
199
50.0-
r1226
M 'E 50
100
Figure 46. 2,4-D electron impact mass spectrum.
91
-------
196
100 0_
,.1662
59
60 0-
65
87
74
46
83
JL
97
109
223
ME SO
100
167
143
132 | 159 179 I |
ji y i i iti
160 200
282
247
L
260
Figure 47. Silvex electron Impact mass spectrum.
100 Ol
73
45
50 0-
69
50
109
97
lliil
233
209
145
181
169
132 ¦
T I I I
196
111
ME 60
100
150
200
(-1016
268
J
260
1
Figure 48. 2,4,5-T electron impact mass spectrum.
92
-------
100 On
101
50 0-
59
45 »»
i^.L
69
63
M/E 60
73
¦ 86 k
•LJl ijl
I- 3860
162
MM 1?3 231
I " ? h.in 7 i. , I.
262
100
"1
160
200
250
Figure 49. 2,4-DB electron impact mass spectrum.
100 o-
225
50 0-
107 118 131
r 3888
254
239
100 150 200
Figure 50. Dinoseb electron impact mass spectrum
93
T
250
-------
1000-
J 900
> 800-
H
H 700-
600-
102 10 1 10"1
Dalapon, Methyl ester Concentration (ng/pL)
Figure 51. Dalapon Finnigan INCOS FIT value vs. concentration.
1000-
4)
D
900
to
>
800
11.
700-
600-
102 10 1 10'
Dicamba (ng/pL)
Figure 52. Dicamba Finnigan INCOS FIT value vs. concentration
94
-------
©
<0
>
K
1000-
900-
800-
700-
600-
10
—f—
10
10"
MCPP (ng/pL)
Figure 53. MCPP Finnigan INCOS FIT value vs. concentration.
1000-
® 900-
> 800-
E 700
600-
10'
—r-
10
10"
MCPA (ng/pL)
Figure 54. MCPA Finnigan INCOS FIT value vs. concentration.
95
-------
1000
® 900-
> 800-
Z 700-
600-
TZ 1 i i
102 10 1 10"1
Oichlorprop (ng/pL)
Figure 55. Dichlorprop Finnigan INCOS FIT value vs. concentration.
D
to
>
1000-
900-
800-
700-
600-
500-
400-
10'
—r~
10
2,4-0 (ng/pL)
10-
Figure 56. 2,4-D Finnigan INCOS FIT value vs. concentration.
96
-------
<0
>
1000
900
800
700-
600
500-
400-
300-
10'
10
Silvex (ng/pL)
10'
Figure 57. Silvex Flnnigan INCOS FIT value vs. concentration
©
J3
(0
>
1000-
900-
800-
700-
600-
500-
400-
300-
10'
10 1
2,4,5-T (ng/pL)
—\
10"
Figure 58. 2,4,5-T Finnigan INCOS FIT value vs. concentration
97
-------
1000-
900-
Sj 800-
> 700-
EE 600-
500-
400-
10'
i
10
10'
2,4-DB (ng/pL)
Figure 59. 2,4-DB Finnigan INCOS FIT value vs. concentration
1000-
900
800-
«
3
(0
700-
>
K
600-
U.
500
400-
300-
10'
10 1
Dinoseb, Methyl ether Concentration (ng/pL)
10"
Figure 60. Dinoseb Finnigan INCOS FIT value vs. concentration
98
-------
TABLE 40. MINIMUM CONCENTRATIONS REQUIRED TO GIVE PULL SCAN MASS
SPECTRA FIT VALUES OF 800 (1-uL INJECTION)
Analyte Concentration
(as methyl derivative) for FIT - 800
Dalapon
3.5 ng/uL
Dicamba
0.5 ng/uL
MCPP
0.43 ng/uL
MCPA
0.3 ng/uL
Dichlorprop
0.65 ng/uL
2,4-D
0.44 ng/uL
Silvex
1.25 ng/uL
2.4.5-T
1.3 ng/uL
2,4-DB
1.7 ng/uL
Dinoseb
4.5 ng/vL
CONCLUSIONS
All analytes, except Methyl Dalapon, gave intense molecular ions and
characteristic fragment ions. The minimum amount of analyte required to
give a good, full-scan mass spectrum is considerably higher than the
detection limits for GC/EC.
99
-------
SECTIOH 11
QUALITY CONTROL
IHTR0DUCT10II
Before performing any analyses, the analyst must demonstrate the
ability to safely handle toxic and hazardous diazomethane and the ability
to generate acceptable accuracy and precision with this method. Acceptable
accuracy and precision are described in this section. The minimum require-
ments of the quality control program consist of an initial demonstration of
laboratory capability and regularly performed analysis of spiked samples
as a continuing check on performance. Performance records must be
maintained to allow comparison with previously recorded accuracy and
precision of the method.
PERFORMANCE CRITERIA
Percent relative standard deviation must be <10 for all analytes
measured and the mean percent recoveries must be >60 for all analytes
measured.
The use of an internal standard for the analysis is strongly
recommended. A standard solution of 1,4-dichlorobenzene, 0.2 mg/mL, when
diluted 0.5 mL to 10 mL gives a suitable response on CC/EC. Percent
recoveries can be calculated using response factors on the following
equation:
* Recovery =
analyte peak height in sample X 1.4-dichlorobenzene peak height in stand.
analyte peak height in stand, 1,4-dichlorobenzene peak height in sample
The most convenient parameter to use for a quality control chart is
the response of the 1,4-dichlorobenzene with an upper control limit of
+3 0 from the mean and a lower control limit of -3 o from the mean. A
sample quality control chart is shown in Figure 61.
Each day the analyst must demonstrate through analysis of a method
blank that all glassware and reagent interferences are under control.
100
-------
CONCLUSIONS
The quality control measures recommended should be made part of the
quality assurance plan of the laboratory to ensure known accuracy and
precision for the analysis of chlorinated herbicides using the validated
Method 8150 protocol described in Appendix A.
101
-------
11000
10000-
• •
6000
5000-
4000 | I 1 1 1 1 1 1 1 1 1
0 2 4 6 8 10 12 14 16 18 20 22
Injection Number
Figure 61. Quality control chart for GC/EC
chlorinated herbicide analysis.
102
-------
REFERENCES
1. Resource Conservation and Recovery Act of 1976, Pub. L. 94-580 (Oct.
21, 1976), U.S. Govt. Printing Office, Washington, DC.
2. Fed. Regist. (1978) 43(243), 58946-59028.
3. Fed. Regist. (1980) 45(98), 33066-33588.
Test Methods for Evaluating Solid Waste. U.S. Environmental Protection
Agency, July, 1982, SW-846, 2nd Ed.
5. Horwitz, W., et al. (1983) J. Assoc. Off. Anal. Chem. 66, 455-466.
6. Williams, L. R. (1983) "Validation of Testing/Measurement Methods,"
EPA-600/X-83-060, U.S. Environmental Protection Agency, Environmental
Monitoring Systems Laboratory, Las Vegas, NV.
7- Pesticide Analytical Manual. U.S. Food and Drug Adm., July 1, 1969,
Vol. 1, Mo. 211 and 222.
8* Annual Book of ASTM Standards. American Society for Testing and
Materials, 1979, 03478-79.
9. "Test Methods: Methods for Nonconventional Pesticides Chemicals
Analysis of Industrial and Municipal Wastewater," U.S. Environmental
Protection Agency, January 31, 1983, EPA 440/1-83/079C, Wo. 615.
10. Gutsche, C. D. (1954) Organic Reactions 8, 391-394.
11. Yip, G. (1962) J. Assoc. Off. Anal. Chem. 45, 367-376.
12. Homer, J., et al. (1974) Anal. Chera. 46, 110.
13. Goerlitz, D. G., and W. L. Lamar (1967) Determination of Phenoxy Acid
Herbicides in Water by Electron Capture and Microcoulometric Gas
Chromatography. U.S. Geol. Survey Water Supply Paper 1817-C.
14. Lee, H. B., and A.S.Y. Chau (1983) J. Assoc. Off. Anal. Chem. 66., 1023.
15. Snyder, L. R. (1978) J. Chrom. Sci., 16_, 223.
16. "CLP Statement of Work," U.S. Environmental Protection Agency, January,
1983, Exhibit D, pp. 43-62. IFB's WA 83-A063/A064 and WA 83-A093/A094.
103
-------
17. Freeman, R. R., ed. (1981), High Resolution Gas Chromatography. 2nd
Ed., Hewlett-Packard Co., pp. 1-5.
18. Schlenk, H. and J. C. Gellerman (1960) Anal. Chem. 32, 1412.
19. Horwitz, W., L. R. Kamps, and K. W. Boyer, J. Assoc. Off. Anal. Chem.
63. 1344 (1980).
20. Horwitz, W. 1982, Anal. Chem. 54, 67A.
21. Aldrich Technical Bulletin No. AL-121 "Mini Diazald® Apparatus."
22. Herbicide Handbook of the Weed Science Society of America. 5th Ed.,
1983.
23. Martin, H. and C. Worthing, eds. (1977), Pesticide Manual. 5th Ed.
24. The Merck Index. 10th Ed., 1983.
25. Worthing, C., ed. (1979), Pesticide Manual. 6th Ed.
26. White-Stevens, R., ed. (1971), Pesticides in the Environment.
27. Fate of Organic Pesticides in the Aquatic Environment. Advances in
Chemistry Series 111, 1971, "Interaction of Organic Pesticides with
Particulate Matter in Aquatic and Soil System," J. B. Weber.
28. Kearney, P. C., and D. D. Kaufman, eds. (1975) Herbicides - Chemistry.
Degradation and Mode of Action. Vol. 1, 2nd Ed.
29. Warner, J. C., M. C. Landes, and L. E. Slivon (1983) "Development of a
Solvent Extraction Method for Determining Semivolatile Organic
Compounds in Solid Wastes" in Conway, R. A., and Gulledge, W. P., eds..
Hazardous and Industrial Solid Waste Testing: Second Symposium
ASTM-STP 805.
30. "Interlaboratory Comparison Study: Methods for Volatile and
Semi-Volatile Compounds," U.S. Environmental Protection Agency, March,
1984, EPA-600/4-84-027.
31. RTI contract no. = 68-02-3679 task #5.
32. Kaufman, D. D. (1975) "Phenols," Kearney, P. C., and D. D. Kaufman,
eds., in Herbicides - Chemistry. Degradation and Mode of Action. Vol.
2, 2nd Ed.
33. Morgan, S. L. and S. N. Deming (1975) J. Chromatography, 112, 267-285.
34. Youden, W. J. and E. H. Steiner (1975) Statistical Manual of the AOAC.
Assoc. Off. Anal. Chem., Arlington, Virginia.
35. Morgan, S. L. and S. N. Deming (1974) Anal. Chem. 46,, 1170.
104
-------
Shavers, C. L., M. L. Parsons, and S. N. Deming (1979) J. Chem. Educ
56, 307.
Czech, F. P. (1973) J. Assoc. Off. Anal. Chem. 56., 1489.
Dols, T. J. and B. H. Armbrecht (1976) J. Assoc. Off. Anal. Chem. 59
1204.
"Finnigan INCOS MSDS Manual," March, 1978, Vol. 1, 108.
105
-------
APPENDIX A
Validated Method 8150. Chlorinated Herbicides by Methylation and GC/EC
1.0 Scope and Application
1.1 Method 8150 is a capillary gas chromatographic (GC) method for
determining certain chlorinated acid herbicides in solid waste samples.
Specifically, Method 8150 may be used to determine the following compounds:
Because these compounds are produced and used in various forms
(i.e., acid, salt, ester, etc.). Method 8150 includes a hydrolysis step to
convert the herbicide to the acid form prior to analysis.
1.2 When Method 8150 is used to analyze unfamiliar samples, compound
identifications should be supported by at least one additional qualitative
technique. Section 8.3 provides gas chromatograph/mass spectrometer
(GC/MS) criteria appropriate for the qualitative confirmation of compound
identif ications.
1*3 The estimated detection limits for each of the compounds in
solid waste samples are listed in Table A-l. The detection limits for a
specific waste sample may differ from those listed, depending upon the
nature of the interferences and the sample matrix.
1.4 CAUTION. Only experienced analysts should be allowed to work
with diazomethane due to the potential hazards associated with its use
(explosive, carcinogenic). Method 8150 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.
2,4-D
2,4-DB
Dicamba
Dichlorprop
Dinoseb
MCPA
MCPP
Silvex
2,4,5-T
106
-------
2.0 Summary of Method
2.1 Method 8150 provides extraction, esterification, and gas
chromatographic conditions for the analysis of chlorinated acid herbicides
in solid waste samples. Extraction is done by sonication of the acidified
sample with methylene chloride. The methylene chloride extract is washed
with base to remove the free acid herbicides, and the remaining methylene
chloride solution of esters is hydrolyzed using potassium hydroxide.
Extraneous organic material is removed by a solvent wash. The free acid
herbicides and hydrolyzed ester herbicides can be combined to give total
herbicides or they can be analyzed separately. After acidification, the
acids are extracted with methylene chloride and converted to their methyl
esters using diazomethane as the derivatizing agent. After excess reagent
is removed, the esters are determined by gas chromatography with an
electron capture detector (GC/EC). The results are reported as the acid
equivalents.
2.2 The sensitivity of Method 8150 depends on the level of
interferences in addition to instrumental limitations. Table A-l lists the
GC/EC and GC/MS limits of detection that can be obtained in solid waste in
the absence of interferences. Detection limits for a typical waste sample
should be higher.
3.0 Interferences
3.1 Method interferences may be caused by contaminants in solvents,
reagents, glassware, and other sample processing hardware that lead to
discrete artifacts or elevated baselines in gas chromatograms. All these
materials must be routinely demonstrated to be free from interferences
under the conditions of the analysis by running laboratory reagent blanks
as described in Section 8.1.
3.1.1 Glassware must be scrupulously cleaned. Clean each piece
of glassware as soon as possible after use by rinsing it with the last
solvent used in it. This should be followed by detergent washing with hot
water and rinses with tap water, then with distilled water. Glassware
should be solvent-rinsed with acetone and pesticide-quality hexane. After
rinsing and drying, glassware should be sealed and stored in a clean
environment to prevent any accumulation of dust or other contaminants.
Store glassware inverted or capped with aluminum foil. Immediately prior
to use, glassware should be rinsed with the next solvent to be used.
3.1.2 The use of high purity reagents and solvents helps
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 will vary
considerably from waste to waste, depending upon the nature and diversity
of the waste being sampled.
107
-------
TABLE A-l. CHROMATOGRAPHIC CONDITIONS* AND ESTIMATED DETECTION LIMITS
FOR METHOD 8150
Analyte
Retention Time
(minutes)
GC/EC
Estimated
Detection Limit**
(n*/*)
GC/MS
Estimated Identification
Limit***
(n*>
Dicamba
13.47
0.12
0.5
MCPP
13.77
66
0.43
MCPA
13.96
43
0.3
Dichlorprop
14.51
0.38
0.65
2,4-D
14.76
0.34
0.44
Silvex
16.33
0.11
1.25
2,4,5-T
16.72
0.16
1.3
2,4-DB
17.82
4,0
1.7
Dinoseb
18.00
0.28
4.5
* Gas chromatography conditions are:
GC/EC: DB-5 capillary column, 0.25 yro film thickness, 0.25 ym
1.0. I 30 M long. Grob-type 30-second splitless injection. Column
temperature, programmed: initial 50#C for 1 min., program 25*C/min.
to 100*C, hold for 1 min., program 12*C/min. to 220*C» hold for 12 min.
GC/HS: DB-5 capillary column, 1.0 vM film thickness, 0.23 vM I.D.
X 30 M long. Grob-type 30-second splitless injection. Column
temperature programmed: initial 60*C for 2 min., program 13*C/min. to
220®C, hold for 10 min.
** Detection limits determined from standard solutions corrected back to
50g samples, extracted and concentrated to 10 mL with 5 iiL injected.
*** The minimum amount of analyte to give a Finnigan INC0S FIT value of
800 as the methyl derivative vs. the spectrum obtained from 50 ng of
the respective free acid herbicide.
3.3 Organic acids, especially chlorinated acids, cause the most
direct interference with the determination. Phenols, including
chlorophenols, may also interfere with this procedure.
3.4 Alkaline hydrolysis and subsequent extraction of the basic
solution removes many chlorinated hydrocarbons and phthalate esters that
might otherwise interfere with the electron capture analysis.
108
-------
3.5 The herbicides, being strong organic acids, react readily with
alkaline substances and may be lost during analysis. Therefore, glassware
must be acid-rinsed prior to use and then rinsed to constant pH with
deionized H2O.
3.6 Before processing any samples, the analyst should demonstrate
daily, through the analysis of an organic-free water or solvent blank,
that the entire analytical system is interference-free. Standard quality
assurance practices should be used with this method. Field replicates
should be collected to validate the precision of the sampling technique.
Laboratory replicates should be analyzed to validate the precision of the
analysis. Fortified samples should be analyzed to validate the accuracy
of the analysis. Where doubt exists over the identification of a peak on
the gas chromatogram, confirmatory techniques such as mass spectroscopy
should be used. Detection limits for solid waste are given in Table A-l.
3.7 The sonication extraction must be optimized for each type of
sample. It is suggested that tar-like samples be mixed with kaolin clay
(Type P, Westwood Ceramic Supply, City of Industry, California) to allow
efficient extraction. Clay samples are extracted efficiently in a pH
range from 1 to 2.5 using 80 to 90 mL of buffer and sonicator power of 5
to 7.
4.0 Apparatus and Materials
4.1 Glassware (all specifications are suggested. Catalog numbers
are included for illustration only).
4.1.1 Beaker: 400 mL. Thick wall.
4.1.2 Funnel: 75-ran diameter, 58*.
4.1.3 Separatory funnel: 500 mL, with Teflon stopcock.
4.1.4 Centrifuge bottle: 500 mL (Pyrex 1260 or equivalent).
4.1.5 Concentrator tube, Kudema-Danish: 10 mL, graduated.
Calibration must be checked at the volumes employed in the method. Ground-
glass stopper is used to prevent evaporation of extracts.
4.1.6 Volumetric flask: 10 mL, with ground-glass stopper.
4.1.7 Evaporative flask, Kudema-Danish: 500 mL. Attach to
concentrator tube with springs.
4.1.8 Snyder column, Kudema-Danish: three-ball macro.
4.2 Boiling chips: approximately 10/40 mesh. Heat to 400*C for 30
min. or perform Soxhlet extract with methylene chloride.
4.3 Diazald® Kit: recommended for the generation of
diazomethane (available from Aldrich Chemical Co., Cat. No. Z10, 025-0).
109
-------
4.4 Water bath: Heated, with concentric ring cover, capable of
temperature control (+ 2#C). The bath should be used in a hood.
4.5 Filter paper: 15-cm diameter (Whatman #1 or equivalent).
4.6 Balance: Analytical, capable of accurately weighing to the
nearest 0.0001 g.
4.7 Pipet: Pasteur, glass, disposable (140-mm x 5-mm I.D.).
4.8 Centrifuge (International Equipment Corporation, Model K or
equivalent).
4.8.1 Capillary Column: 30 m x 0.32 mm DB-5 (J & W Scientific,
Inc., or equivalent): Film thickness: 1 ym.
4.9 Sonicator (Heat Systems Ultrasonics, Inc., Model W375 or
equivalent, with 20 KHz Ultrasonic Convertor Model C3 or equivalent).
4.10 Gas chromatograph: Analytical system complete with gas
chromatograph suitable for Grob-type injection using capillary columns and
all required accessories including syringes, capillary analytical column,
gases, detector, and stripchart recorder. A data system is recommended
for measuring peak areas or peak heights.
5.0 Reagents
5.1 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.
5.2 Sodium hydroxide solution (0.1 B): dissolve 4 g MaOH in reagent
water and dilute to 1000 mL.
5.3 Potassium hydroxide solution: 37% aqueous solution (w/v).
Prepare with reagent grade potassium hydroxide pellets and reagent water.
5.4 Phosphate buffer pH = 2.5 (0.1 M): Dissolve 12 g NaH2P04 in
reagent water and dilute to 1000 mL. Add phosphoric acid to adjust to pH
5.5 Methylene chloride, acetone, methanol: pesticide quality or
equivalent.
5.6 Carbitol (diethylene glycol monoethyl ether).
5.7 N-methyl (N-nitroso-p-toluenesulfonamide) (Diazald®): high
purity, available from Aldrich Chemical Co.
5.8 Silicic acid: 100-mesh powder (analytical reagent).
110
-------
5.9 Stock standard solutions (500 ng/uL): stock standard
solutions may be prepared from pure standard materials or purchased as
certified solutions.
5.9.1 Prepare stock standard solutions by accurately weighing
about 0.0500g of pure acid. Dissolve the material in pesticide-quality
acetone and dilute to volume in a 10-mL volumetric flask. Other volumes
may be used at the convenience of the analyst. If compound 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.
5.9.2 Store stock standard solutions at 4*C and protect from
light. Stock standard solutions should be checked frequently for signs of
degradation or evaporation, especially immediately prior to preparing
calibration standards from them.
5.9.3 Stock standard solutions must be replaced immediately if
comparison with check standards indicates a problem. Otherwise, stock
solutions should be replaced after one week.
6.0 Sample Collection. Preservation, and Handling
6.1 Grab samples must be collected in glass containers.
Conventional sampling practices should be followed; however, the bottle
must not be prerinsed with the 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 Tygon and other potential sources of
contamination.
6.2 The samples must be stored at 4*C from the time of collection
until extraction.
6.3 All samples must be extracted within 7 days of collection and
must be completely analyzed within 30 days of extraction.
7.0 Procedures
7.1 Sample preparation
7.1.1 Thoroughly mix moist solids and weigh an amount of wet
sample equivalent to 50 g of dry weight into each of 400-mL, thick-wall
beakers.
7.1.2 Acidify solids in each beaker with 85 mL of 0.1M
phosphate buffer (pH = 2.5) and thoroughly mix the contents with a glass
stirring rod.
7.1.3 Add 100 mL of methylene chloride to each beaker
containing the sample. Sonicate the samples for 3 minutes in the pulsed
mode at 50 percent duty cycle at an output of 6.3.
Ill
-------
7.1.4 Allow the solids to settle. Transfer the organic layer
into a 500-mL centrifuge bottle.
7.1.5 Sonicate the sample two more times using the same
condition with 100 mL of methylene chloride each time.
7.1.6 Combine the three organic extracts from the sample in the
centrifuge bottle and centrifuge 10 minutes to settle the fine particles.
Filter the extracts through Whatman #1 filter paper into 500-mL separatory
funnels.
7.1.7 Wash the organic extracts two times with 100-mL portions
of 0.1 N aqueous sodium hydroxide each time. Combine the aqueous layers
containing the salts of the free acid herbicides in a beaker and save.
The organic layer contains the herbicide esters, which must be hydrolyzed
as follows:
7.1.7.1 Transfer the methylene chloride solution into
500-mL Kudema-Danish flasks. Add boiling chips to the extracts in the
flasks and fit them with three-ball Snyder columns. Evaporate the
methylene chloride on the water bath to a volume of approximately 25 mL.
7.1.7.2 Remove the flasks from the water bath. Allow
them to cool. Add 5 mL of 37% aqueous potassium hydroxide, 30 mL of
distilled water and 40 mL of methanol into the extracts.
7.1.7.3 Add additional boiling chips to the flasks.
Reflux the mixtures on a 60*-65#C water bath for 2 hours. Remove the
flasks from the water bath and cool to room temperature.
7.1.8 At this point the basic solutions containing the
herbicide salts from 7.1.7 can be combined or they can be analyzed
separately.
7.1.9 Add phosphoric acid to the basic aqueous extracts to
adjust the pH to <1.
7.1.10 Transfer the acidified aqueous solution into a 500-mL
separatory funnel and extract the solution two times with 100 mL of
methylene chloride.
7.1.11 Combine the organic extracts in 500 mL Kudema-Danish
flasks. Add boiling chips to the extracts in the flasks and fit them with
three-ball Snyder columns.
7.1.12 Evaporate the methylene chloride to approximately 5 mL
on a hot water bath (80*-85°C).
7.1.13 Remove the flasks from water bath. Evaporate the
extracts just to dryness under a stream of nitrogen.
112
-------
7.1.14 Reconstitute with 1 mL of iso-octane and 0.5 mL of
methanol. Dilute to a volume of A mL with ether. The sample is now ready
for methylation with diazomethane.
7.2 Ksterification
7.2.1 The diazomethane derivatization (1) procedure described
below will produce an efficient reaction with all of the chlorinated
herbicides described in this method and should be used only by experienced
analysts, due to the potential hazards associated with its use.
Diazomethane is a carcinogen and can explode under certain conditions.
The following precautions should be taken:
• Use a safety screen.
• Use mechanical pipetting aides.
• Do not heat above 90°C - EXPLOSION may result.
• Avoid grinding surfaces, ground-glass joints, sleeve bearings, and
glass stirrers - EXPLOSION may result.
• Store away from alkali metals - EXPLOSION may result.
• Solutions of diazomethane decompose rapidly in the presence of
solid materials such as copper powder, calcium chloride, and boiling
chips.
7.2.2 Instructions for preparing diazomethane are provided with
the generator kit.
7.2.3 Add 2 mL of diazomethane solution and let the sample
stand for 10 minutes with occasional swirling. The yellow color of
diazomethane should be evident and should persist for this period.
7.2.A Rinse inside wall of ampule with several hundred viL of
ethyl ether. Reduce the sample to approximately 2 mL to remove excess
diazomethane by allowing solvent to evaporate spontaneously (room
temperature). Alternatively, silicic acid, about 10 mg, can be added to
destroy the excess diazomethane.
7.2.5 Dilute the sample to 10.0 mL using hexane.
7.3 Gas chromatography conditions
GC/EC: DB-5 capillary column, 0.25 urn film thickness, 0.25 urn
I.D. X 30M long. Grob-type 30-second splitless injection.
Column temperature, programmed: initial 50*C for 1 min.,
program 25*C/min. to 100°C, hold for one min., program
12*C/min. to 220*C, hold for 12 min. The retention times of
each analyte are shown in Table A-l.
113
-------
7.4 Calibration
7.4.1 Establish gas chromatographic operating parameters
equivalent to those indicated above and in Table 1. The gas chromato-
graphic system can be calibrated using the external standard technique
(Section 7.4.2) or the internal standard technique (Section 7.4.3).
7.4.2 External standard calibration procedure
7.4.2.1 For each parameter of interest, prepare working
standards 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 diethyl ether. One of the external standards should be at a
concentration near, but above, the method detection limit. The other
concentrations should correspond to the expected range of concentrations
found in real samples or should define the working range of the detector.
7.4.2.2 Prepare calibration standards from the free
acids by esterification of the working standards as described under Sample
Preparation, Section 7.1.13 and subsequent steps. Using injections of 2
to 5 uL of each esterified working standard, tabulate peak height or area
responses against the mass injected. The results can be used to prepare a
calibration curve for each parameter. Alternatively, the ratio of the
response to the mass injected, defined as the calibration factor (CF), can
be calculated for each parameter at each standard concentration. If the
relative standard deviation of the calibration factor is less than 10%
over the working range, linearity through the origin can be assumed and
the average calibration factor can be used in place of a calibration curve.
7.4.2.3 The working calibration curve or calibration
factor must be verified on each working day by the measurement of one or
more calibration standards. If the response for any parameter varies from
the predicted response by more than +10%, the test must be repeated using
a fresh calibration standard. Alternatively, a new calibration curve or
calibration factor may be prepared for that parameter.
7.4.3 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.
The standard 1,4-dichlorobenzene is suggested as one possibility.
7.4.3.1 Prepare working standards, at a minimum of three
concentration levels for each parameter of interest in the acid form,
by adding volumes of one or more stock standards to a volumetric flask.
Dilute to volume with diethyl ether. One of the standards should be at a
concentration near, but above, the method detection limit. The other
concentrations should correspond to the expected range of concentrations
found in real samples, or should define the working range of the detector.
114
-------
7.4.3.2 Prepare calibration standards from the free
acids by esterification of the working standards as described under Sample
Preparation, Section 7.1.13 and subsequent steps.
7.4.3.3 Prior to dilution to final volume for GC
analysis, add a known constant amount of one or more internal standards to
each calibration standard.
7.4.3.4 Using injections of 2 to 5 uL of each
calibration standard, tabulate the peak height of area responses against
the concentration for each compound and for each internal standard.
Calculate response factors (RF) for each compound as follows:
RF - (AsC^s)/(A^sCs)
where:
As 3 Response for the parameter to be measured.
Ais = Response for the internal standard.
Cis = Concentration of the internal standard in vg/L.
Cs = Concentration of the parameter to be measured in vg/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 calculations. Alternatively, the results can be used
to plot a calibration curve of response ratios, As/A^s against RF.
7.4.3.5 The working calibration curve or RF must be
verified on each working day by the measurement of one or more calibration
standards. If the response for any parameter 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.
7.4.4 The analyst must process a series of standards through
the procedure to validate elution patterns and the absence of
interferences from the reagents.
7.5 Analysis
7.5.1 Inject 2 to 5 uL of the sample extract using the
solvent-flush technique. Smaller (1.0-uL) volumes can be injected if
automatic devices are employed. Record the volume injected to the nearest
0.05 uL* and record the resulting peak size in area units.
7.5.2 If the peak area exceeds the linear range of the
system, dilute the extract and reanalyze.
i 1 s
-------
7.5.3 A sample chromatogram for methylated chlorophenoxy
herbicides is shown in Figure A-l.
7.5.4 Precision and accuracy expected are shown in Table A-2.
8.0 Quality Control
8.1 Before processing any samples, the analyst should demonstrate
through the analysis of a distilled water method blank that all glassware
and reagents are interference free. Bach time a set of samples is
extracted or there is a change in reagents, a method blank should be
processed as a safeguard against chronic laboratory contamination.
8.2 Standard quality assurance practices should be used with this
method. Field replicates should be collected to validate the precision of
the sampling technique. Laboratory replicates should be analyzed to
validate the precision of the analysis. Fortified waste samples should be
analyzed to validate the accuracy of the analysis. Detection limits to be
used for samples are indicated in Table A-l. It is suggested that the
response of the internal or external standard be plotted daily as a
quality control check. Where doubt exists over the identification of a
peak on the chromatogram, confirmatory techniques such as mass
spectrometry should be used (Section 8.3).
8.3 GC/MS Confirmation
8.3.1 GC/MS techniques should 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 3 sec. per scan utilizing 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.
A computer system that allows the continuous acquisition and storage (on
machine-readable media) of all mass spectra obtained throughout the
duration of the chromatographic program should be interfaced to the mass
spectrometer.
8.3.2 Gas chromatographic columns and conditions:
Instrument: Finnigan 9610 GC/Finnigan 4023 mass
spectrometer.
DB-5, 1.0 uM film thickness, 0.32 uM ID
X 3OH L.
1 uL, Grob-type 30-sec. splitless
injection.
220#c.
60*C for 2 minutes, 13*C/minute to 220*C,
hold for 10 minutes.
-1200V.
45amu - 550amu.
1 scan/sec.
Data General NOVA 3 with INC0S.
Column
Injection
Injector Temperature:
Temperature Program:
Electron Multiplier Voltage:
Scan Range:
Scan Time:
Data System:
116
-------
8.3.3 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 (decafluorotriphenyl phosphine) performance criteria are achieved,
as described in Method 8250 of SW-846.
8.3.A 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.
The following criteria must be met for qualitative confirmation:
1. The molecular ion and all other ions present above
10% relative abundance in the mass spectrum of the
standard must be present in the mass spectrum of
the sample with agreement to +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%.
2. The mass spectra obtained from 50 ng of herbicide
as the methyl derivative can be used to establish a
library. Measurements of the resemblance of the
library spectrum to the spectrum of less
concentrated samples can be done by the computer
(the FIT number of library search in INCOS). A FIT
value of 800 or greater is acceptable.
3. The retention time of the compound in the sample
must be within 6 sec. of the retention time for the
same compound in the standard solution.
4. Compounds that have very similar mass spectra can
be explicitly differentiated by GC/MS only on the
basis of retention time data.
5. Should these MS procedures fail to provide
satisfactory results, additional steps may be taken
before reanalysis. These steps may include the use
of alternate GC columns or additional cleanup.
9.0 References
1. U.S. EPA. 1971. National Pollutant Discharge Elimination
System, Appendix A, Fed. Reg., 38, No. 75, Pt. II, Method for
Chlorinated Phenoxy Acid Herbicides in Industrial Effluents,
Cincinnati, OH.
2. Goerlitz, D. G., and W. L. Lamar. 1967. Determination of
Phenoxy Acid Herbicides in Water by Electron Capture and
Microcoulometric Gas Chromatography. U.S. Geol. Survey Water
Supply Paper 1817-C.
t 17
-------
3. Burke, J. A., 1965. Gas chromatography for pesticide residue
analysis; some practical aspects. Journal of the Association of
Official Analytical Chemists 48:1037.
4. U.S. EPA. 1972. Extraction and cleanup procedure for the
determination of phenoxy acid herbicides in sediment. EPA
Toxicant and Analysis Center, Bay St. Louis, MS.
5. U.S. EPA. 1985. Single-laboratory Validation of EPA Method 8150
for Analysis of Chlorinated Herbicides in Hazardous Waste.
118
-------
156999-1
8041
10
Minutes
120217
OS
3141
10
11
12
13
14
15
16
17
18
19
20
Minutes
Figure A-l. Gas chroraatogram of methylated herbicides.
119
-------
TABLE A-2~ ACCURACY AND PRECISION FOR METHOD 8150
Linear** Percent
Concentration Relative***
Mean* Range Standard Deviation
Analvte
Percent Recovery
(n*/fc)
(n-20)
Dicamba
95.7
0.52- 104
7.5
MCPP
98.3
620 -61,800
3.4
MCPA
96.9
620 -61,200
5.3
Dichlorprop
97.3
1.5 - 3,000
5.0
2,4-D
84.3
1.2 - 2,440
5.3
Silvex
94.5
0.42- 828
5.7
2,4,5-T
83.1
0.42- 828
7.3
2,4-DB
99.7
4.0 - 8,060
7.6
Dinoseb
93.7
0.82- 1,620
8.7
* Mean percent recovery calculated from 10 determinations of spiked clay
and clay/still bottom samples over the linear concentration range.
** Linear concentration range was determined on standard solutions and
corrected to 50g solid samples.
*** Percent relative standard deviation was calculated on standard
solutions, 10 samples high in the linear concentration range, and 10
samples low in the range.
120
-------
TECHNICAL REPORT DATA
fftecse reed Instructions on the reverie before completing)
1. REPORT NO. 2.
EPA /fSOO/4-8 t /060
3. RECIPIENT S ACCESSION NO.
PB8 6 1 0 8 A 0 4
4. TITLE ANO SUBTITLE
SINGLE-LABORATORY VALIDATION OF EPA METHOD 8150 FOR
THE ANALYSIS OF CHLORINATED HERBICIDES IN HAZARDOUS
WASTE
S. REPORT DATE
September 1985
6. PERFORMING ORGANIZATION COOE
?. AUTHORS)
F. L. Shore. E. N. Amick, and S. T. Pan
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME ANO AOORESS
Lockheed Engineering and Management Services
Company, Incorporated
P.O. Box 15027
10 PROGRAM ELEMENT NO.
11. CONTRACT/GRANT NO.
Contract Number 68-0^-lflsn
12. SPONSORING kGENCY NAME ANO AOORESS
Environmental Monitoring Systems Laboratory - LV, NV
Office of Research and Development
U.S. Environmental Protection Agency
Las Veeas. NV 891U
13. TYPE OF REPORT ANO PERIOD COVERED
14. SPONSORING AGENCY CODE
EPA/600/07
is. supplementary notes t>, * ncc.» r, , , _ „
Project Officer: Donald F. Curka
Environmental Monitoring Systems Laboratory
16. ABSTRACT ' 11 111
A single laboratory validated analytical protocol is described, which is applicable
to the determination of the herbicides Dicamba, Silvex, 2,4-D, 2,4-DB, 2,4,5-T,
Dinoseb, MCPP, and MCPA, in hazardous waste extracts. The method consists of herbi-
cide hydrolysis followed by diazomethane esterification and subsequent determination
of the herbicide methyl esters by capillary column gas chromatography with electron
capture detection (GC/ECD). An electron impact gas chromatography/mass spectrometric
(GC/MS) confirmation of the GC/ECD results is included. The protocol validation
procedure consisted of ruggedness testing, simplex optimization of key experimental
variables, and the determination of extraction recoveries, detection limits, and
the GC/ECD linear dynamic range for each herbicide methyl ester. This protocol, which
employs a single fused silica capillary column separation for all the target methyl
esters, is a significant improvement over earlier gas chromatographic (GC) procedures
which utilize three different packed GC columns. The method, however, was inappli-
cable to Dalapon which eliminates hydrogen chloride during the sample workup.
17. KEY WOROS AND DOCUMENT ANALYSIS
a. DESCRIPTORS
b.lOENTlFIERS/OPEN ENDED TERMS
c. COSATi Field/Croup
•
16. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
19. SECURITY CLASS (Tliit Report)
UNCLASSIFIED
21. NO. OF PAGES
135
20. SECURITY CLASS (This page)
UNCLASSIFIED
22. PRICE
EPA F»m» 2220.1 (R»». 4-77) pakviou* bdition is omoliti
------- |