Munitions Constituents – Sample Extraction and Analytical Techniques

Munitions Constituents, including insensitive munitions (IM), are a broad category of compounds which, in areas where manufactured or used, can be found in a variety of environmental matrices (waters, soil, and tissues). This presents an analytical challenge when a variety of these munitions are to be quantified. This article discusses sample extraction methods for each typical sample matrix (high level water, low level water, soil and tissue) as well as the accompanying HPLC-UV analytical method for 27 compounds of interest (legacy munitions, insensitive munitions, and surrogates).

Related Article(s):

• Munitions Constituents

Contributor(s):

• Dr. Austin Scircle

Key Resource(s):

• USEPA Method 8330B

• Methods for simultaneous quantification of legacy and insensitive munition (IM) constituents in aqueous, soil/sediment, and tissue matrices

Introduction

Table 1. Analyte list with acronyms and CAS numbers.

Compound	Acronym	CAS Number
1,2-Dinitrobenzene (surrogate)	1,2-DNB (surr.)	528-29-0
1,3-Dinitrobenzene	1,3-DNB	99-65-0
1,3,5-Trinitrobenzene	1,3,5-TNB	99-35-4
1,4-Dinitrobenzene	1,4-DNB (surr.)	100-25-4
2-Amino-4,6-dinitrotoluene	2-Am-4,6-DNT	35572-78-2
2-Nitrophenol	2-NP	88-75-5
2-Nitrotoluene	2-NT	88-72-2
2,4-Dinitrophenol	2,4-DNP	51-28-5
2,4-Dinitrotoluene	2,4-DNT	121-14-2
2,4,6-Trinitrophenol	Picric Acid (PA)	88-89-1
2,4,6-Trinitrotoluene	2,4,6-TNT	118-96-7
2,6-Dinitrotoluene	2,6-DNT	606-20-2
3-Nitrotoluene	3-NT	99-08-1
3,5-Dinitroaniline	3,5-DNA	618-87-1
4-Amino-2,6-dinitrotoluene	4-Am-2,6-DNT	19406-51-0
4-Nitrophenol	4-NP	100-02-7
4-Nitrotoluene	4-NT	99-99-0
2,4-Dinitroanisole	DNAN	119-27-7
Octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine	HMX	2691-41-0
Nitrobenzene	NB	98-95-3
Nitroglycerine	NG	55-63-0
Nitroguanidine	NQ	556-88-7
3-Nitro-1,2,4-triazol-5-one	NTO	932-64-9
ortho-Nitrobenzoic acid	o-NBA (surr.)	552-16-9
Pentaerythritol tetranitrate	PETN	78-11-5
Hexahydro-1,3,5-trinitro-1,3,5-triazine	RDX	121-82-4
N-Methyl-N-(2,4,6-trinitrophenyl)nitramide	Tetryl	479-45-8
Note: Analytes in bold are not identified by EPA Method 8330B.

Figure 1. Primary Method labeled chromatograms

Figure 2. Secondary Method labeled chromatograms

The primary intention of the analytical methods presented here is to support the monitoring of legacy and insensitive munitions contamination on test and training ranges, however legacy and insensitive munitions often accompany each other at demilitarization facilities, manufacturing facilities, and other environmental sites. Energetic materials typically appear on ranges as small, solid particulates and due to their varying functional groups and polarities, can partition in various environmental compartments. To ensure that contaminants are monitored and controlled at these sites and to sustainably manage them a variety of sample matrices (surface or groundwater, process waters, soil, and tissues) must be considered. (Process water refers to water used during industrial manufacturing or processing of legacy and insensitive munitions.) Furthermore, additional analytes must be added to existing methodologies as the usage of IM compounds changes and as new degradation compounds are identified. Of note, relatively new IM formulations containing NTO, DNAN, and NQ are seeing use in IMX-101, IMX-104, Pax-21 and Pax-41 (Table 1).

Sampling procedures for legacy and insensitive munitions are identical and utilize multi-increment sampling procedures found in USEPA Method 8330B Appendix A. Sample hold times, subsampling and quality control requirements are also unchanged. The key differences lie in the extraction methods and instrumental methods. Briefly, legacy munitions analysis of low concentration waters uses a single cartridge reverse phase SPE procedure, and acetonitrile (ACN) is used for both extraction and elution for aqueous and solid samples. An isocratic separation via reversed-phase C-18 column with 50:50 methanol:water mobile phase or a C-8 column with 15:85 isopropanol:water mobile phase is used to separate legacy munitions. While these procedures are sufficient for analysis of legacy munitions, alternative solvents, additional SPE cartridges, and a gradient elution are all required for the combined analysis of legacy and insensitive munitions.

Previously, analysis of legacy and insensitive munitions required multiple analytical techniques, however the methods presented here combine the two munitions categories resulting in an HPLC-UV method and accompanying extraction methods for a variety of common sample matrices. A secondary HPLC-UV method and a HPLC-MS method were also developed as confirmatory methods. The methods discussed in this article were validated extensively by single-blind round robin testing and subsequent statistical treatment as part of ESTCP ER19-5078. Wherever possible, the quality control criteria in the Department of Defense Quality Systems Manual for Environmental Laboratories were adhered to. Analytes included in the methods presented here are found in Table 1.

The chromatograms produced by the primary and secondary HPLC-UV methods are shown in Figure 1 and Figure 2, respectively. Chromatograms for each detector wavelength used are shown (315, 254, and 210 nm).

Extraction Methods

Figure 3. Triple cartridge SPE setup

Figure 4. A flow chart of the soil extraction procedure

High Concentration Waters (> 1 ppm)

Aqueous samples suspected to contain the compounds of interest at concentrations detectable without any extraction or pre-concentration are suitable for analysis by direct injection. The method deviates from USEPA Method 8330B by adding a pH adjustment and use of MeOH rather than ACN for dilution. The pH adjustment is needed to ensure method accuracy for ionic compounds (like NTO or PA) in basic samples. A solution of 1% HCl/MeOH is added to both acidify and dilute the samples to a final acid concentration of 0.5% (vol/vol) and a final solvent ratio of 1:1 MeOH/H₂O. The direct injection samples are then ready for analysis.

Low Concentration Waters (< 1 ppm)

Aqueous samples suspected to contain the compounds of interest at low concentrations require extraction and pre-concentration using solid phase extraction (SPE). The SPE setup described here uses a triple cartridge setup shown in Figure 3. Briefly, the extraction procedure loads analytes of interest onto the cartridges in this order: Strata^TM X, Strata^TM X-A, and Envi-Carb^TM. Then the cartridge order is reversed, and analytes are eluted via a two-step elution, resulting in 2 extracts (which are combined prior to analysis). Five milliliters of MeOH is used for the first elution, while 5 mL of acidified MeOH (2% HCl) is used for the second elution. The particular SPE cartridges used are noncritical so long as cartridge chemistries are comparable to those above.

Table 2. Primary HPLC-UV mobile phase gradient method concentrations

Method run time = 48 minutes; Column temperature = 25°C Injection volume = 50 μL; Flow rate = 1.0 mL/min Detector wavelengths = 210, 254, and 310 nm
Time (min)	Reagent Water (%)	MeOH (%)	0.1% TFA/Water (%)	ACN (%)
0.00	89	3	3	5
2.00	89	3	3	5
2.20	52	40	3	5
12.5	52	40	3	5
19.0	57	35	3	5
28.0	48	44	3	5
32.0	48	44	3	5
44.0	32	60	3	5
44.1	89	3	3	5
48.0	89	3	3	5

Soils

Soil collection, storage, drying and grinding procedures are identical to the USEPA Method 8330B procedures; however, the solvent extraction procedure differs in the number of sonication steps, sample mass and solvent used. A flow chart of the soil extraction procedure is shown in Figure 4. Soil masses of approximately 2 g and a sample to solvent ratio of 1:5 (g/mL) are used for soil extraction. The extraction is carried out in a sonication bath chilled below 20 ⁰C and is a two-part extraction, first extracting in MeOH (6 hours) followed by a second sonication in 1:1 MeOH:H₂O solution (14 hours). The extracts are centrifuged, and the supernatant is filtered through a 0.45 μm PTFE disk filter.

The solvent volume should generally be 10 mL but if different soil masses are required, solvent volume should be 5 mL/g. The extraction results in 2 separate extracts (MeOH and MeOH:H₂O) that are combined prior to analysis.

Tissues

Tissue matrices are extracted by 18-hour sonication using a ratio of 1 gram of wet tissue per 5 mL of MeOH. This extraction is performed in a sonication bath chilled below 20 ⁰C and the supernatant (MeOH) is filtered through a 0.45 μm PTFE disk filter.

Due to the complexity of tissue matrices, an additional tissue cleanup step, adapted from prior research, can be used to reduce interferences. The cleanup procedure uses small scale chromatography columns prepared by loading 5 ¾” borosilicate pipettes with 0.2 g activated silica gel (100–200 mesh). The columns are wetted with 1 mL MeOH, which is allowed to fully elute and then discarded prior to loading with 1 mL of extract and collecting in a new amber vial. After the extract is loaded, a 1 mL aliquot of MeOH followed by a 1 mL aliquot of 2% HCL/MeOH is added. This results in a 3 mL silica treated tissue extract. This extract is vortexed and diluted to a final solvent ratio of 1:1 MeOH/H₂O before analysis.

HPLC-UV and HPLC-MS Methods

Table 3. Secondary HPLC-UV mobile phase gradient method concentrations

Method run time = 43 minutes; Column temperature = 25°C Injection volume = 50 μL; Flow rate = 0.8 mL/min Detector wavelengths = 210, 254, and 310 nm
Time (min)	Reagent Water (%)	MeOH (%)	0.1% TFA/Water (%)	ACN (%)
0.00	75	10	10	5
2.50	75	10	10	5
2.60	39	46	10	5
9.00	39	46	10	5
9.10	33.5	51.5	10	5
15.00	35	50	10	5
15.10	43	42	10	5
33.00	30	55	10	5
33.10	75	10	10	5
43.00	75	10	10	5

Table 4. Ionization source and detector parameters

Parameter	Value
Ionization Source	APCI
Ionization Mode	Negative
Drying Gas Temperature (°C)	350
Vaporizer Temperature (°C)	325
Drying Gas Flow (L/min)	4.0
Nebulizer Pressure (psig)	40
Corona Current (μA)	10
Capillary Potential (V)	1500
Mass Range	40 – 400
Fragmentor	100
Gain	1
Threshold	0
Step Size	0.20
Speed (μ/sec)	743
Peak Width (min)	0.06
Cycle Time (sec/cycle)	0.57

The Primary HPLC-UV method uses a Phenomenex Synergi 4 µm Hydro-RP column (80Å, 250 x 4.6 mm), or comparable, and is based on both the HPLC method found in USEPA 8330B and previous work. This separation relies on a reverse phase column and uses a gradient elution, shown in Table 2. Depending on the analyst’s needs and equipment availability, the method has been proven to work with either 0.1% TFA or 0.25% FA (vol/vol) mobile phase. Addition of a guard column like a Phenomenex SecurityGuard AQ C18 pre-column guard cartridge can be optionally used. These optional changes to the method have no impact on the method’s performance.

The Secondary HPLC-UV method uses a Restek Pinnacle II Biphenyl 5 µm (150 x 4.6 mm) or comparable column and is intended as a confirmatory method. Like the Primary method, this method can use an optional guard column and utilizes a gradient elution, shown in Table 3.

For instruments equipped with a mass spectrometer (MS), a secondary MS method is available and was developed alongside the Primary UV method. The method was designed for use with a single quadrupole MS equipped with an atmospheric pressure chemical ionization (APCI) source, such as an Agilent 6120B. A majority of the analytes shown in Table 1 are amenable to this MS method, however nitroglycerine (which is covered extensively in USEPA method 8332) and 2-,3-, and 4-nitrotoluene compounds aren’t compatible with the MS method. MS method parameters are shown in Table 4.

Summary

The extraction methods and instrumental methods in this article build upon prior munitions analytical methods by adding new compounds, combining legacy and insensitive munitions analysis, and expanding usable sample matrices. These methods have been verified through extensive round robin testing and validation, and while the methods are somewhat challenging, they are crucial when simultaneous analysis of both insensitive and legacy munitions is needed.

See Also

• Preparative, Extraction, and Analytical Methods for Simultaneous Determination of Legacy and Insensitive Munition (IM) Constituents in Aqueous, Soil or Sediment, and Tissue Matrices
• SERDP/ESTCP Focus Areas – UXO – Munitions Constituents
• Environmental Data Quality Workgroup