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AnalyzeUnderivatized Chlorophenoxyacid
Herbicides by HPLC
Using anUltra Aqueous C18Column andNewReferenceMixtures
By RebeccaWittrig, Ph.D., Senior InnovationsChemist, andKatiaMay, Ph.D., Senior R&DChemist
✔
HPLC eliminates time-consuming derivatizations (required for GC).
✔
Ultra Aqueous C18 column has excellent selectivity for chlorophenoxyacid herbicides.
✔
New referencemixes for themost widely performed analyses.
Chlorophenoxyacid herbicides - 2,4-D, dicamba,
picloram, Silvex (2,4,5-TP), and others - are used to
control agricultural and aquaticweeds. While not
considered highly toxic, chlorophenoxyacid herbi-
cides aremonitored in agricultural monitoringwells
and drinkingwater sources. They are encountered
in the acid form, or as the salts or esters.
Traditionally, these compounds have been analyzed
by gas chromatography, according toUS EPAMethod
8151 or othermethods. Tomake them amenable to
GC, the acidsmust be converted tomethyl esters,
using a derivatizing agent such as diazomethane.
Highperformance liquid chromatography is an
attractive option to this lengthy, hazardous procedure.
Unlike in theGCprocedures, derivatization is not
necessary; the analytes canbe separated anddetected
in the free acid form. Comparatively large injection
volumes, relative toGC, alsomakeHPLC attractive.
US EPAMethod 555was developed for analysis of
chlorophenoxyacid herbicides, in the acid form, in
drinkingwater. Tominimize coelutions, the herbi-
cides are divided into two sets. Figure 1 includes a
chromatogram for each set, analyzed on anUltra
Aqueous C18 column, using gradient conditions
optimized for each analysis. Note that this column
has excellent selectivity for resolving these struc-
turally similar compounds. The gradient procedure
is useful when analyzing a range of these herbicides;
an isocraticmobile phase saves analysis and reequi-
libration time if samples contain only 2,4-D and
Silvex.
1
EPAMethod 8321, a general LC/MS or LC/UV
method for semivolatile compounds, also includes a
discussion of these herbicides.
Restek chemists have formulated a full complement
of referencematerials forMethod 555. New chlori-
nated acidsmixes A andB include all target com-
pounds except 5-hydroxydicamba, a product of
dicamba oxidation. Dicamba is stable under normal
chromatographic conditions, but strong oxidizers in
a sample could convert it to 5-hydroxydicamba and
make identification difficult. To avoid this problem,
we offer 5-hydroxydicamba in a single, separate
solution. We designed thesemixes with special con-
sideration for stability, which is a concern because
these herbicides, especially in the acid form, are
light sensitive and readily degrade in the presence of
alkaline substances.
(continued on pg. 5)
LC_0313
Phenoxyacid Herbicides (Mix A)
Peak List:
1. picloram
2. chloramben
3. dicamba
4. bentazon
5. 2,4-D
6. dichlorprop
7. 2,4,5-TP
8. acifluorfen
9. 1,4-dichlorobenzene (IS)
Conditions forMix A:
Mobile Phase A: 0.05%H
3
PO
4
Mobile Phase B: acetonitrile
Time
%B
0
20
15
80
20
80
21
20
Flow:
1.0mL/min
Temp.:
ambient
Det.:
UV@ 225nm
Phenoxyacid Herbicides (Mix B)
Peak List:
1. 4-nitrophenol
2. MCPA
3. 3,5-dichlorobenzoic acid
4. MCPP
5. 2,4,5-T
6. 2,4-DB
7. 1,4-dichlorobenzene (IS)
8. dinoseb
9. pentachlorophenol
Conditions forMix B:
Mobile Phase A: 0.05%H
3
PO
4
Mobile Phase B: acetonitrile
Time
%B
0
10
10
45
16
45
22
90
24
90
25
10
Flow:
1.0mL/min
Temp.:
ambient
Det.:
UV@ 225nm
Figure1
—Chlorophenoxyacidherbicides are resolvedwell byanUltraAqueousC18column. HPLCeliminates time-consuming, hazardousderivatizations.
acifluorfen (Blazer
®
)
bentazon
chloramben
2,4-D
dicamba
dichlorprop
picloram
2,4,5-TP (Silvex)
ChlorinatedAcidsbyHPLC,MixA
1,000µg/mL each in acetonitrile, 1mL/ampul
Each
5-pk.
10-pk.
32431
32431-510
w/data pack
32431-500
32431-520
32531
2,4-DB
3,5-dichlorobenzoic acid
dinoseb
MCPA
MCPP (mecoprop)
4-nitrophenol
pentachlorophenol
2,4,5-T
ChlorinatedAcidsbyHPLC,MixB
1,000µg/mL each in acetonitrile, 1mL/ampul
Each
5-pk.
10-pk.
32430
32430-510
w/data pack
32430-500
32430-520
32530
Column and Sample for both chromatograms:
Column:
Ultra Aqueous C18
Cat. #:
9178565
Dimensions:
150 x 4.6mm
Particle Size:
5µm
Pore Size:
100Å
Sample:
Inj.:
10µL
Conc.:
10 ppm each herbicide
Sample Diluent:
acetonitrile
HPLCcolumnsandadditional
referencematerials listedonpg.5.
1
Isocraticmobile phase: 0.05% phosphoric acid:acetonitrile,
60:40. For an example chromatogram of the isocratic analysis,
request
Environmental HPLC: Applications-Columns-Reference
Materials
(lit. cat.# 59741).
LC_0314
“After the disaster of 9-11, Diazald, a highly explo-
sive compound used inHerbicide analysis, was
immediately controlled by theU.S. government
whichmade shipment impossible. Restekwas
instrumental in helpingme to develop an isocratic
HPLCmethod that did not require the use
of Diazald. Thismethod is not only safer, but it
saves us time andmoney. Thanks, Restek!”
ChrisDomaradzki, Organicsmanager,
EnvironmentalTesting Laboratories
AGoodWord
