Improved Separation of Explosives (Nitroaromatics ... - Phenomenex

HPLC
TN-1036
Improved Separation of Explosives (Nitroaromatics)
for HPLC Analysis Using Synergi Hydro-RP and Synergi
Polar-RP (EPA Method 8330)
Introduction
The need for disposal of expired munitions from armed forces
has attained considerable importance during the last two decades
partially due to the dismantling of military installations
and disarmament treaties around the world. Improper storage
and disposal techniques have resulted in contamination of soil
and ground water with explosive residues including 2,4,6-trinitrotoluene
(TNT) and nitrobenzene. Recent studies have shown
that these compounds can cause liver damage and are suspected
mutagens (1). Because of these health risks, analytical
methods for the thorough identification and characterization of
the contaminated areas are in high demand. EPA Method 8330,
introduced in November 1990, is the most common way to analyze
samples for the presence of explosives. The method recommends
using a C18 phase as a primary column and a CN phase
as a confirmation column. Confirmation columns are sometimes
needed because of difficulty in separating similar compounds
on a single stationary phase. By using two columns with different
selectivities, analysts can more accurately identify the compounds
of interest. Closely eluting and co-eluting peaks on a
typical C18 column include 2,4,6-trinitrotoluene, 4-amino-dinitrotoluene,
and 2-amino-dinitrotoluene. In addition, separation
of 2,6-dinitrotoluene and 2,4-dinitrotoluene can sometimes be
difficult. In this application note, we describe the analysis of 15
explosives using a polar endcapped 4 μm C18 reversed phase
(Synergi Hydro-RP) as the primary column and a polar endcapped
4 μm ether-linked phenyl phase (Synergi Polar-RP) as
the confirmation column.
Experimental Conditions
The HPLC analysis was carried out using an HP 1100 LC
system (Agilent Technologies, Palo Alto, California) equipped with a
quaternary pump, in-line degasser, multi-wavelength detector,
and autosampler. HP Chemstation software (Version A.08.03) was
used for data analysis. The HPLC columns used for this analysis
were Synergi Hydro-RP 250 x 4.6 mm and Synergi Polar-RP 250
x 4.6 mm (Phenomenex, Torrance, California). The mobile phase
composition is listed in Table 1. The detection wavelength used
was 254 nm, and a column oven temperature of 35 °C. Standards
were obtained from Restek (Bellefonte, Pennsylvania).
Table 1: HPLC method conditions and peak identification
Columns:
Samples:
Mobile Phase:
Flow Rate:
Detection:
Column temp.: 35 °C
(1) Synergi Hydro-RP 4 μm 80 Å 250 x 4.6 mm
(2) Synergi Polar-RP 4 μm 80 Å 250 x 4.6 mm
1 = HMX, 2 = RDX, 3 = 1,3,5-Trinitrobenzene
5 = Tetryl, 6 = 1,3-Dinitrobenzene
7 = Nitrobenzene, 8 = 2,4,6-Trinitrotoluene
9 = 4-Amino-2,6 Dinitrotoluene
10 = 2-Amino-4,6 Dinitrotoluene
11 = 2,6-Dinitrotoluene, 12 = 2,4-Dinitrotoluene
13 = 2-Nitrotoluene, 14 = 4-Nitrotoluene
15 = 3-Nitrotoluene, 4 = 1,2-Dinitrobenzene (internal standard)
(1) A: 51 % Water, B: 45 % Methanol, C: 4 % Acetonitrile
(2) A: 60 % Water B: 5 % Methanol C: 35 % Acetonitrile
(1) 0.8 mL/min (2) 1.0 mL/min
UV @ 254 nm
Results and Discussion
Experiments performed resulted in a major improvement in the
separation of the common explosives in EPA 8330 compared to
typical C18 phases. Figure 1 shows a chromatogram of the explosives
standard injected at about 25 ng on-column. The Synergi
Hydro-RP column is able to achieve separation of all of the EPA
Method 8330 compounds versus the standard method, which
describes the use of two columns (a C18 phase combined with a
cyano phase). Dense bonding coupled with unique polar endcapping
in the Synergi Hydro-RP phase gives added retention and
improved selectivity for neutral to polar compounds. This results
in improved separation of EPA Method 8330 compounds. Quantitation
of individual components using Synergi Hydro-RP is more
accurate due to the resolution achieved. A second confirmation
run can be avoided due to the improved separation of the explosives
mix. This will improve sample throughput and decrease
analysis time. We also decreased the flow rate from 1.5 mL/min to
0.8 mL/min, which will decrease solvent consumption.
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HPLC
TN-1036
Figure 1: EPA Explosive mix using Synergi Hydro-RP
6
Figure 2: EPA Explosive mix using Synergi Polar-RP
9,10
3
7
12
5
10
1
2
4
9
11
13
14
15
App ID 14768
2
1
7 6
4
15 14
13
11
12
3
8
5
App ID 14768
0 10 20 min
0
10 20 30 40 min
If a confirmation column is needed, then Synergi Polar-RP can
be used to confirm peak identity. As shown in Figure 2, Synergi
Polar-RP was able to improve separation of 2,4,6-trinitrotoluene
(peak 7) from 4-amino-dinitrotoluene (peak 8), tetryl (peak 4)
from 1,3-dinitrobenzene (peak 5), and 2,6-dinitrotoluene (peak
10) from 2,4-dinitrotoluene (peak 11) versus what is typically observed
using a cyano (CN) column.
Using Synergi Hydro-RP combined with Synergi Polar-RP as
a confirmation column will ensure proper identification of EPA
Method 8330 compounds with improved quantitation, lower solvent
consumption, and higher throughput.
5395_L
Standard is a mixture of the following components:
1 = HMX, 2 = RDX, 3 = 1,3,5 Trinitrobenzene, 4 = 1,2-Dinitrobenzene (internal standard), 5
=Tetryl, 6 = 1,3-Dinitrobenzene, 7 = Nitrobenzene, 8 = 2,4,6-Trinitrotoluene, 9 =4-Amino-2,6
Dinitrotoluene, 10 = 2-Amino-4,6 Dinitrotoluene, 11 = 2,6-Dinitrotoluene, 12 = 2,4-Dinitrotoluene,
13 = 2-Nitrotoluene, 14 = 4- Nitrotoluene, 15 = 3-Nitrotoluene.
Standard mixture is the same as in figure 1. Note the change in elution order for
many of the components:
2 = RDX, 1 = HMX, 7 = Nitrobenzene, 6 = 1,3-Dinitrobenzene, 4 = 1,2-Dinitrobenzene
(internal standard), 9 = 4-Amino-2,6 Dinitrotoluene, 10 = 2-Amino-4,6 Dinitrotoluene, 13 =
2-Nitrotoluene,15 = 3-Nitrotoluene, 14 = 4-Nitrotoluene, 11 = 2,6-Dinitrotoluene, 12 = 2,4-
Dinitrotoluene, 3 =1,3,5-Trinitrobenzene, 8= 2,4,6-Trinitrotoluene, 5 = Tetryl.
Reference
1. L. Berthe-Corti, H. Jacobi, S. Kleihauer, and I. Witte,
“Cytotoxicity and Mutagenicity of (TNT) and Hexogen
Contaminated Soil in S. typhimurium and Mammalian
Cells,” Chemosphere 2, 209–218 (1998).
Ordering Information
2.5 μm HST (High Speed Technology) Formats (mm)
Phase 30 x 2.0 50 x 2.0 50 x 4.6
Synergi Hydro-RP 00A-4387-B0 00B-4387-B0 00B-4387-E0
Synergi Polar-RP 00A-4371-B0 00B-4371-B0 00B-4371-E0
4.0 μm Analytical Columns (mm)
Phase 50 x 4.6 150 x 4.6 250 x 4.6
Synergi Hydro-RP 00B-4375-E0 00F-4375-E0 00G-4375-E0
Synergi Polar-RP 00B-4336-E0 00F-4336-E0 00G-4336-E0
Additional dimensions, phases, and particle sizes are available.
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