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Analyze Underivatized Chlorophenoxyacid
Herbicides by HPLC
Using an Ultra Aqueous C18 Column and New Reference Mixtures
By Rebecca Wittrig, Ph.D., Senior Innovations Chemist, and Katia May, Ph.D., Senior R&D Chemist
✔ HPLC eliminates time-consuming derivatizations (required for GC).
✔ Ultra Aqueous C18 column has excellent selectivity for chlorophenoxyacid herbicides.
✔ New reference mixes for the most widely performed analyses.
Chlorophenoxyacid herbicides - 2,4-D, dicamba,
picloram, Silvex (2,4,5-TP), and others - are used to
control agricultural and aquatic weeds. While not
considered highly toxic, chlorophenoxyacid herbi-
cides are monitored in agricultural monitoring wells
and drinking water sources. They are encountered
in the acid form, or as the salts or esters.
Traditionally, these compounds have been analyzed
by gas chromatography, according to US EPA Method
8151 or other methods. To make them amenable to
GC, the acids must be converted to methyl esters,
using a derivatizing agent such as diazomethane.
High performance liquid chromatography is an
attractive option to this lengthy, hazardous procedure.
Unlike in the GC procedures, derivatization is not
necessary; the analytes can be separated and detected
in the free acid form. Comparatively large injection
volumes, relative to GC, also make HPLC attractive.
US EPA Method 555 was developed for analysis of
chlorophenoxyacid herbicides, in the acid form, in
drinking water. To minimize coelutions, the herbi-
cides are divided into two sets. Figure 1 includes a
chromatogram for each set, analyzed on an Ultra
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 isocratic mobile phase saves analysis and reequi-
libration time if samples contain only 2,4-D and
Silvex.1 EPA Method 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 reference materials for Method 555. New chlori-
nated acids mixes A and B 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 these mixes 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 for Mix A:
Mobile Phase A: 0.05% H3PO4
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 for Mix B:
Mobile Phase A: 0.05% H3PO4
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
Figure 1—Chlorophenoxyacid herbicides are resolved well by an Ultra Aqueous C18 column. HPLC eliminates time-consuming, hazardous derivatizations.
acifluorfen (Blazer®)
bentazon
chloramben
2,4-D
dicamba
dichlorprop
picloram
2,4,5-TP (Silvex)
Chlorinated Acids by HPLC, Mix A
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
Chlorinated Acids by HPLC, Mix B
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
HPLC columns and additional
reference materials listed on pg. 5.
1 Isocratic mobile 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 in Herbicide analysis, was
immediately controlled by the U.S. government
which made shipment impossible. Restek was
instrumental in helping me to develop an isocratic
HPLC method that did not require the use
of Diazald. This method is not only safer, but it
saves us time and money. Thanks, Restek!”
Chris Domaradzki, Organics manager,
Environmental Testing Laboratories
A GoodWord