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Simple, SensitiveHPLC/UVAnalysis
forParaquatandDiquat
UsingHigh-RecoverySolidPhaseExtraction
andanUltraQuatHPLCColumn
APPLICATIONS
NOTE
Paraquat (1,1'-dimethyl-4,4'-bipyridylium dichloride, C
12
H
14
N
2
Cl
2
), and diquat (1,1'-ethylene-2,2'-bipyridilium dibromide,
C
12
H
12
N
2
Br
2
,Figure1), arenon-selectivecontactherbicides,plant growth regulators anddesiccantswidelyused inagriculture tocon-
trol broadleaf and aquaticweeds. Paraquat has beenused tokill illegalmarijuanaplants in theUS andMexico. Paraquat is listedby
theUS Environmental ProtectionAgency as a RestrictedUse Pesticide (RUP). Additionally, these herbicides have been banned, or
their use restricted, in several European countries and in Japan, and the World
HealthOrganizationconsiders themmoderatelyhazardouspesticides.Theseherbi-
cidesmust bemonitored routinely because of their significant toxicity to humans
through oral ingestion or respiratory or dermal contact. Despite the fact that the
half-life of either compound inwater canbe less than 48hours, there is great con-
cern about even low-level human exposure (USEPASafeDrinkingWaterActmax-
imum contamination level =20ppb for diquat).
Dependingon the initial samplematrix, these herbicides have been analyzedusing
colorimetric spectrophotometry, enzyme linked immunosorbent assay (ELISA), or
liquid scintillation counting (LCS), but HPLC analysis with UV or fluorescence
detection has gained wide acceptance. The highly charged quaternary amines are
difficult to retain by standard reversed phase HPLC, however, and alternative
approaches are required.Mostwidelyused is an ion exchange column coupled to a
post-column reactor that creates a fluorescing complex. Detection is highly sensi-
tive, but the drawbacks are the costs of the specialty column—often exceeding $1,000US—and of the post-column derivatization
unit and fluorescence detector.This supplemental hardware canbe beyond the budget of smaller laboratories.
A less costly technique, described inUSEPAMethod 549.2, calls for a conventionalHPLC column andhardware and incorporates
an ionpairing agent in themobile phase. This analytical system is less complex, but adequate,withdetection limits of 0.44µg/L for
diquat and0.8µg/L forparaquat,basedon solidphaseextractionof a250mLwater sample.Themobilephase forMethod549.2con-
sists of water, phosphoric acid, acetonitrile, heptane or hexane sulfonic acid (ion pairing agent), and diethylamine (DEA).
Diethylamine likelywas included to reduce tailing on columns that exhibit high silanol activity. The sulfonic acids also can reduce
tailing but, additionally, the ion pair complexes they form allow better retention of the highly charged quaternary amines.
Unfortunately, this complicated chemistryandmethodology, in combinationwithvariationamongmanufacturers'HPLC columns,
can present serious problems. In addition to amplifying the potential for mixing inconsistencies and errors, the complexmobile
phase has three significant flaws: 1)Acid/base reaction betweenDEA and the ion pair reagent consumes both, eliminating the ion
pairing capacity of the system and restoring the potential for peak tailing. A sulfonic acid ion pair reagent alone should eliminate
tailingby theanalytes and, asneutral species, they shouldnot beaffectedby residual silanols. 2)Themobilephase lacks a truebuffer
control systemneeded for reproducible retention in an ionpair system. 3)The exact pHof themobilephase—a critical issue for an
ionpairingmethod—is not specified.
Because theyarehighlycharged,paraquatanddiquatwillnotberetainedwellonanalkyl stationaryphase,andany standardreversed
phaseHPLC technique that relieson thehydrophobicityof thecolumnand the strengthof themobilephase likelywill fail toachieve
a separation. So, if altering the hydrophobicity of the stationary phase will not be effective, the next choice is to lower the
hydrophilicityof themobilephase.A separation systemwehavedeveloped for paraquat anddiquatmakes useof adifferent analyt-
ical property—chaotropism: disruption of water's ability to solvate ions, thereby altering the charged interactions among the ana-
lyte, themobilephase, and the stationaryphase. In this case,bydispersing theanalyte's charge, the solubilityof thehighlypolar ana-
lyte on a non-polar substrate (the stationary phase) can be enhanced. Retention of the analyte is maximized because it remains
longeron the adsorbed solvent layer (acetonitrile)present on the stationaryphase.The chaotropic agents are inorganic anionic salts
added to the aqueous portionof themobile phase.¹
Figure1
Paraquat anddiquat herbicides.
paraquatdichloride [CAS#1910-42-5]
diquatdibromide
[CAS#85-00-7]
