100
Typical Binding
Quantix
80
EnviroGard
Ohmicron
DTECH
60
40
20
0
0.001
0.01
0.1
1
10
100
1000
10000
TNT Equivalents (g/L)
Figure 2. Linear ranges of the TNT kits compared with a typical anti-
body binding curve. % B/Bo = (Absorbance of sample/Absorbance of
negative control) 100.
analyzed at CRREL, using identical columns and
Center in Crane, Indiana. Bailers used for collec-
10-L aliquots of 1:1 methanol-diluted samples.
tions were rinsed once with isopropyl alcohol
and three times with distilled water between
The chromatography system consisted of a Spec-
samples. Wells were purged with a PVC bailer to
tra Physics 8810 pump, 8875 autosampler, and
a depth midway down the well, allowed to
8490 detector at 254 nm. Peak heights were record-
recharge for 2 hours, then sampled with the
ed with a Hewlett-Packard 3396 Integrator.
cleaned Teflon bailers. Samples were collected in
1-liter precleaned, amber glass bottles and were
Immunoassay kits
stored and shipped at 4C (39F). Samples from
Kits from four manufacturers were tested for
five monitoring wells at Umatilla Army Depot
this report. Although each kit was based on the
Activity, Oregon, and six wells at the U.S. Naval
same principals of a competitive immunoassay,
Submarine Base, Bangor, Washington, were sup-
each kit was formatted differently and had widely
plied by Black and Veatch Waste Science, Tacoma,
different dynamic ranges (Table 2).
Washington.
The EnviroGard (Millipore, Medford, Mass.)
and Quantix (Idetek, Sunnyvale, Calif.) kits are in-
tended as quantitative laboratory assays, although
Laboratory analysis by RP-HPLC
they can be implemented in field situations with
Water samples were analyzed as described in
battery-powered spectrophotometers. The anti-
SW846 Method 8330 (EPA 1994). At WES, samples
bodies were immobilized onto the surfaces of 96-
were diluted 1:1 with methanol, and aliquots of
50 L were injected on two RP-HPLC systems
well microtitre strip-plates. Duplicate water sam-
(Millipore/Waters Chromatography Division,
ples or standards and enzyme-conjugated TNT
Milford, Mass.). The primary system consisted of
were diluted in an assay buffer in the wells of the
a Waters model 600E MS System Controller, a 712
plate. These were incubated at room temperature
WISP Auto Injector, a 486 UV Variable-Wave-
for either 60 minutes in the EnviroGard kit or 30
length Detector monitored at 254 nm, and a Max-
minutes in the Quantix kit. The wells were then
ima 820 chromatography workstation. The col-
rinsed to remove unbound free and conjugated
umns used were Supelco 25 cm 4.6 mm LC-18
TNT. The substrate and chromogen were added
for the primary separation and 25 cm 4.6 mm
and a blue color allowed to develop for 30 min-
LC-CN for the confirmation separation. Analytes
utes. An acid solution was added to stop the
were eluted with 1:1 methanol/water at flow
enzyme reaction and change the color of the
rates of 1.0 mL/min and 1.2 mL/min, respec-
chromogen from blue to yellow. The absorbance
tively. Water samples were also concentrated by
was measured with a spectrophotometer designed
salting-out solvent extraction (SOE) and ana-
to read microtitre strips. Concentrations of ana-
lyzed on the same systems. Stored samples were
lytes were calculated by semi-log regression
3
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