surface. The disk is dried and sprayed with a color
have been available commercially from EnSys
forming solution of o-toluidine for TNT and Griess
(now Strategic Diagnostics) for several years and
reagent for RDX. Measurement is made by reflec-
have been widely used.
tometry, and detection limits of about 200 g/L
Keuchel et al. (1992a,b and 1994) was the first to
were estimated.
report on the development of an enzyme immunoas-
Seitz and coworkers at the University of New
say (EIA) method for TNT in water and soil. Com-
Hampshire developed a fiber-optic-based ap-
mercial test kits for TNT using EIA were first intro-
proach for on-site measurement that utilizes
duced by Strategic Diagnostics Corporation (SDI)
the reaction of TNT with an amine-loaded
in 1993 (Hutter et al. 1993). Subsequently, SDI pro-
poly(vinyl)chloride (PVC) membrane to form a
duced a commercial EIA method for RDX as well
colored product (Zhang et al. 1989, Zhang and
(Teaney and Hudak 1994). These methods are
known commercially as the D TECHTM EIA meth-
Seitz 1989). This approach was specific for TNT,
and has a detection limit of about 100 g/L.
ods. Detection limits for TNT and RDX in water
using the D TECH were reported as 5 g/L. Detec-
In another approach, TNT and RDX were ab-
tion limits for soil were 0.5 mg/kg for both TNT
sorbed into a cellulose acetate membrane contain-
and RDX. Along with the colorimetric methods, the
ing pyrenebutyric acid, and concentrations were
D TECH methods have been used extensively in the
estimated via fluorescence quenching by the nitro
past several years for on-site determination of TNT
groups (Jian and Seitz 1990). Detection limits were
and RDX in groundwater and soil.
estimated at 2 and 10 mg/L.
The U.S. Naval Research Laboratory (NRL) has
A photometric method for RDX was developed
developed a continuous flow immunosensor (CFI)
by Hass and Stork (1989) and Hass et al. (1990)
for on-site determination of TNT and RDX in water
that involves evaporation of a 500-mL water
(Bart et al. 1997). This method is an extension of the
sample to dryness, followed by reaction of the
EIA methods and uses either TNT or RDX antibod-
residue with diphenylamine in sulfuric acid. A
ies that are immobilized on a membrane saturated
colored product is produced and the concentra-
with a fluorescent-labeled antigen. An aqueous
tion of RDX is estimated from the absorbance at
596 nm. The detection limit of about 5 g/L
buffer is pumped across the membrane and samples
are injected into the flowing buffer. If the appropri-
is nearly adequate for this application, but the
ate analyte is present, binding occurs with the mem-
procedure is cumbersome and impractical for on-
brane, thereby releasing the labeled antigen that is
site use.
detected using a fluorometer. Detection limits ap-
Colorimetric-based methods for TNT and RDX
pear to be about 10 g/L using this system.
in water and soil were developed at CRREL
Another approach for on-site determination of
(Jenkins 1990, Walsh and Jenkins 1991, Jenkins et
TNT and RDX, developed at NRL, is the fiber op-
al. 1994, Jenkins and Walsh 1998). Soil samples (20
tic biosensor (Shriver-Lake et al. 1995, 1997). These
g) are extracted with 100 mL of acetone. Water
are also EIA-based methods. For TNT, fluorescent-
samples are passed through two solid-phase ex-
labeled trinitrobenzene (TNB) is exposed to an
traction membranes, and the retained compounds
antibody-coated optical fiber, producing a refer-
were eluted from each membrane with acetone.
ence signal. When the fiber is then placed in a so-
The acetone extract of the first membrane is re-
lution containing TNT, the signal is reduced as
acted with a base, producing highly colored
TNT competes for sites on the fiber. The reduc-
Janowsky anions. The acetone soil extracts are pro-
tion in signal is used to estimate TNT concentra-
cessed in an identical manner. The concentration
tion. Estimates of RDX concentrations are obtained
of TNT is estimated from the absorbance measured
similarly. Detection limits for this method have
with a field-portable spectrophotometer at 540 nm.
been estimated at 10 g/L.
The acetone extract of the second membrane or
that from soil extraction is acidified and reacted
with zinc powder to convert RDX to nitrous acid.
The solution is filtered and further reacted with a
DEFICIENCIES WITH CURRENT METHODS
Griess reagent to produce a highly colored azo
AND OBJECTIVES OF THIS STUDY
dye. RDX concentration is estimated from the ab-
A summary of the performance characteristics
sorbance measured at 510 nm. Detection limits for
of the various on-site methods for TNT and RDX
TNT and RDX using these colorimetric methods
were 1 g/L and 4 g/L, in water, respectively,
in soil is presented by Crockett et al. (1996, 1998),
and the performance characteristics in groundwa-
and 1.0 and 1.4 mg/kg for soil. These methods
2
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