Anal. Chem. 1994,66, 2570-2577
Determination of Explosives and Their Biodegradation Products in Contaminated Soil and Water from Former Ammunition Plants by Automated Multiple Development High-Performance Thin-Layer Chromatography Carsten Steuckart, Edith Berger-Prelss, and Karsten Levsen’ Fraunhofer-Institut fur Toxikologie und Aerosolforschung, D 30625 Hannover, Germany
The new method of automated multiple development highperformance thin-layer chromatography(HPTLC-AMD) has been applied for the first time for the analysis of explosives and relatedcompounds found in soil and water samples near former ammunition production sites. A gradient for the mobile phases has been found which allows the separation of the explosives hexyl, picric acid, RDX, HMX, tetryl, 2,4- and 2,ddinitrotoluenes, and 2,4,64rinitrotoluene as well as other byproducts such as 1,3-dinitrobenzene, 2-amino-4,6-dinitrotoluene, and 4-amino-2,6-dinitrotoluene found in former ammunition waste. The method has been applied to the analysis of two groundwater samples taken near a former plant for the production of explosivesand one soil sample from a former ammunition plant. The method allows one to identify and quantify the major contaminantsfound in groundwater and soil samples from such contaminated sites. As up to 20 samples can be chromatographed simultaneously,the technique is well-suited for a rapid screening of contaminated sites for explosives. In Germany a large number of sites exist where explosives and ammunition were produced before and particularly during World War 11, including sites where raw materials and intermediate products, initializing explosives, propellant powder, and pyrotechnical equipment were produced. The first report by Preuss and Haas’ in 1987 cites as many as 200 areas in Germany which may be contaminated by the production of explosives, ammunition, and chemical warfare agents before and during World War 11. Since 1987 such contaminated former military sites have been and continue to be systematically investigated in the northern German state of Lower Saxony. In November 1991 it was demonstrated that, in this single state, as many as 137 areas exist which represent a danger or probable danger to both men and nature, while an additional 80 areas are suspected to be polluted.2 Thus, it is very likely that the total number of sites polluted by ammunition in Germany during World War I1 by far exceeds the number cited by Preuss and Haas,’ where the large majority of contaminated areas is due to the production and storage of ammunition, while the number of sites with possiblecontamination by chemical warfare agents is relatively small. These sites of explosives and ammunition producing plants were mostly located in forests, not only to protect them from reconnaissance by the enemy but also to supply them
with larger amounts of water, which is necessary, for instance, for the production of TNT. Contamination of nearby rivers, groundwater, and soil has many reasons, such as improper disposal of production waste or, during grenade filling, occasional accidents like the explosion of plants, bombardments and, finally, improper dismantling of these plants by the Allied forces at the end of the war. The main explosives during World War I1 were nitrotoluenes (isomeric dinitrotoluenes (DNTs) and trinitrotoluenes (TNTs) where 2,4,6-TNT was the dominant isomer), nitramines (1,3,5-trinitro-hexahydro-1,3,5-triazine (hexogen, RDX, cyclonite) often contaminated by 1,3,5,7-tetranitro-l,3,5,7tetrazacyclooctane (octogen, HMX) and N-methyl-N,2,4,6tetranitroaniline (tetryl) 2,2’,4,4’,6,6’-hexanitrodiphenylamine (hexyl), pentaerythrityl tetranitrate (nitropenta, PETN), and picric acid. It should be noted that, with the exception of hexyl, all explosives are used today and may be found in ammunition waste and wastewater from plants still in operation. In addition, at former ammunition plants, byproducts such as mononitrotoluenes, nitroxylenes, nitrobenzenes, nitrophenols, and compounds formed by biodegradation of the original explosives, in particular, methylanilines (toluidines), aminonitro-, diaminonitro-, aminodinitrotoluenes, and possibly diamino- and triaminotoluenes, are found or suspected. It is likely that contamination of soil and groundwater by former (or existing) ammunition plants is not restricted to Germany, but also presents a problem for other European countries and the United States. Nitrotoluenes and especially trinitrotoluenes are highly toxic compound^.^ Moreover, the aromatic amines formed by biodegradation are suspected to be carcinogenic. Various instrumental techniques have been employed for the determination of nitroaromatics and nitramines, gas chromatography having been the most widely ~ s e d . RDX ~ ~ ~ y ~ ~ and, in particular, tetryl and hexyl may undergo thermal (3) SchBfer, H. in Verfahren zur Sanierung won Rftstungsaltlasten; ThomtKozmiensky, K. J., Ed.; EF Verlag fiir Energie- und Umwelttechnik, Berlin, 1992; p 45. (4) Hashimoto, A.; Sakino, H.; Yamagami, E.; Tateishi, S . Anolyst 1980, 105,
787.
( 5 ) Hashimoto, A.; Kozima, T.; Sakino, H.; Akiyama, T. Water Res. 1979, 13,
509. (6) Ling-Xiang, T.; Ruo-Nong, F. Int. Jahrestag.-Fraunhofer-Imt. Treib.-
Explosiust. 1986, 17, 63 f 14319. (7) Jurinski, N. B.; Podolak, G. E.; Hess, T. L. Am. Ind. Hyg. Assoc. J . 1975,36,
497.
(1) Preuss, J.; Haas, R. Geogr. Rundschau 1987, 39, 578.
(2) Niedersachsenbcricht, Rftstungsaltlasten in Niedersochsen; 1991.
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Analytical Chemistry, Vol. 66, No. 15, August 1, 1994
(8) Pareira, W. E.; Short D. L.; Mangold, D. B.; Roscio, P. K. Bull. Enuiron. Contam. Toxicol. 1979, 21. 554.
0003-2700/94/036&2570$04.50/0
0 1994 American Chemical Soclety
degradation during GC analy~is.1092~Thus, HPLC methods have been developed for the determination of nitroaromatics, nitramines, and other explosive^,^^-^^ using reversed-phase chromatography and UV detection in most instances. Furthermore, ~pectrophotometry~6.3~ and p o l a r ~ g r a p h yhave ~~,~~ been employed for the detection of explosives. Finally, thinlayer chromatography (TLC) has been used to identify nitroaromatics and other e x p l o ~ i v e s . 3 6 *Many ~ ~ ~ determined Rf values of these compounds are similar, so that twodimensional TLC considerably improves the separation of the spots. Although nowadays optimized GC and HPLC methods allow simultaneous determination of all the above-mentioned compounds, it is difficult, even in GC, to analyze environmental matrices, because of larger amounts of humic acids. Analysis of those matrices without cleanup of samples causes not only overlap of matrix signals with analyte signals but also deterioration of chromatographic columns. Application of TLC does not require any cleanup, if the method has been optimized carefully. Analysis of soil samples requires a cleanup, which is carried out using size exclusion chromatography (SEC)or, in the case of sandy soils with low content of organic carbon, by flocculation of humic substances using a saturated CaC12 solution. Furthermore, with TLC, thermal degradation or sensitivity to air is of no significance. The relatively new variant of TLC, automated multiple development high-performance thin-layer chromatography (HPTLC(9) Spanggord, R. J.; Gibson, B. W.; Keck, R. G.;Thomas, D. W.; Barkley, J. J. Ewiron. Sci. Technol. 1982, 16, 532. (IO) Belkin, F.; Bishop, R. W.; Sheely, M. V. J. Chromatogr. Sci. 1985, 24, 532. (11) Richard, J. J.; Junk, G.A. Anal. Chem. 1986, 58, 723. (12) Cai, Z. Ph.D. Thesis, University of Marburg, 1990. (13) Hoffsommer, F. C. J . Chromatogr. 1970,51, 243. (14) Douse, J. M. F. J. Chromatogr. 1982, 234,415. (15) Haas, R. Forum StiSdte-Hyg 1985, 36, 86. (16) Haas, R.; Schreiber, I.; Law, E. V.; Stork, G.Fresenius Z . Anal. Chem. 1990, 338, 41. (17) Parsons, J. S.; Tsang, S. M.; Digiamo, M. P.; Feinland, R.; Paylor, R. A. L. Anal. Chem. 1961, 33, 1858. (18) Ono, A. J. Chromatogr. 1978, 154, 89. (19) Gehring, D. G.;Shirk, J. E. Anal. Chem. 1967, 39, 1315. (20) Krull, I. S.; Swartz, M.; Hilliard, R.; Xie, K. H. J. Chromatogr. 1983, 260, 347. (21) Dalton, R. W.; Kohlbeck, J. A.; Bolleter, W. J. Chromatogr. 1970, 50, 219. (22) Hartley, W. R.; Anderson, A. C.; Reimers, R. S.; Abdelghani, A. A. Trace Subst. Ewiron. Health 1981, I S , 298. (23) Tamiri, T.; Zitrin, S. J. Energy Mater. 1986, 4, 215. (24) Phillips, J. H.; Coraor, J.; Prescott, S. R. Anal. Chem. 1983, 55, 889. (25) Lafleur, A. L.; Mills, K. M. Anal. Chem. 1985,53, 1202. (26) Feltes, A. L.; Levsen, K.; Volmer, D.; Spiekermann, M. J. Chromatogr. 1990, 518, 21. (27) Maskarinec, M. P.; Manning, D. L.; Harvey, R. W.; Griest, W. H.; Tomkins, B.A. J. Chromatogr. 1984, 302, 51. (28) Baucr, C. F.;Grant, C. L.; Jenkins, T. F. Anal. Chem. 1986,58, 176. (29) Jenkins, T. F.; Leggett, D. C.; Grant, C. L.; Bauer, C. F. Anal. Chem. 1986, 58, 170. (30) Walsh, J. T.; Chalk, R. C.; Merritt, C. Anal. Chem. 1973, 45, 1215. (31) Bratin, K.; Kissinger, P. T.; Briner, R. C.; Bruntlett, C. S. Anal. Chim. Acra 1981, 130, 295. (32) Barman, B. N. Analyst 1986, 1 1 1 , 479. (33) Sclavka, C. M.;Tontarski, R. E.;Strobel, R. A. J. ForensicSci. 1987,32,941. (34) Feltcs, J.; Levsen, K. J. High Resolut. Chromatogr. 1989, 12, 613. (35) Levsen, K.; Mussmann, P.; Berger-Preiss, E.; Preiss, A,; Volmer, D.; Wdnsch, G. Acta Hydrochim. Hydrobiol. 1993, 21, 153. (36) Haas, R.; Stork, G. Fresenius 2.Anal. Chem. 1989, 335, 839. (37) Glover, D. J.; Kayser, E. G.Anal. Chem. 1968, 40, 2055. (38) Whitnack, G.C. Advances in the Identification and Analysis of Organic Pollutants in Water, 1st Meeting Data, 1975; p 265. (39) Flack, J. Hung. Sci. Inst. 1975, 32, 11. (40) Yasuda, S. K. J. Chromatogr. 1964, 13, 78. (41) Hoffsommer, J. C.; McCullough, J. F. J. Chromatogr. 1968, 38, 508. (42) Harton, J. G.L. Acta Chem. Scand. 1961, 15, 1401. (43) Fauth, M. I.; Roccker, G.W. J. Chromatogr. 1965, 18, 608. (44)Hansson, J. Explosivestoffe 1963, 10, 73. (45) Midkiff,C. R.; Washington, W. D. J.Assoc. On.A M / . Chem. 1974.57.1092, (46) Midkiff, C. R.; Washington, W. D. J.Assoc. Off. Anal. Chem. 1974,59,1357.
Flgure 1. Schematic dlagram of apparatus for automated multiple development: 1, enclosed developing chamber; 2, solvent reservolr bottles; 3, valve swRch for selecting solvent composition; 4,gradlent mixing chamber; 5, wash bottle to prepare gas phase; 6, reservok for gas phase; 7, vacuum pump; 8, solvent waste bottle.
AMD)4748 shows many advantages for the analysis of the above-mentioned compounds in environmental matrices. Moreover, the simultaneous determination of up to 20 samples (including standards) makes the method very fast, so that it will be suitable for routine analysis or as a screening method. This method has recently been reviewed.47 As it is not generally known, the technique will be described briefly. A schematic diagram of the apparatus is shown in Figure 1. Samples are applied to the chamber as bands or spots in the usual way, and the plate is placed in the developing chamber (Figure 1 (1)). The operation sequence starts with a drying step by evacuating the sealed developing chamber, followed by a conditioning step to control the activity of the layer. A wash bottle ( 5 ) is used to condition a stream of nitrogen stored in a reservoir (6),where any volatile compound can be used for conditioning (vide infra). A mobile-phase gradient is generated by mixing the solvents via the valve (3) in the mixing chamber (4) from the solvents available in the reservoir bottles (2). In a first step, a fixed volume of the initial mobile phase is forced into the developing chamber and the separation developed for a preselected distance (1-5 mm). The mobile phase is then sucked from the developing chamber into a waste bottle (8) and the solvent vapor evacuated from the chamber by a vacuum pump (7). The development is then successively repeated in (47) Poole, C. F.; Belay, M. T. J. Planar Chromatogr. 1991, 4, 345. (48) Burger, K. Fresenius 2.Anal. Chem. 1984, 318, 228. (49) Burger, K.; Kahler, J.; Jork, H. J. Planar Chromatogr. 1990, 3, 504. (50) Lodi, G.;Betti, A.; Menziani, E.; Brandolini, V. J. Planar Chromarogr. 1991, 4, 106. (51) Lodi, M. T.; Betti, A.; Kahie, Y. D.; Mahamad, A. M. J. Chromatogr. 1991, 545, 214. (52) Lodi,G.; Betti,A.; Menziani, E.; Brandolinin, V. J. Planur Chromatogr. 1991, 4 , 106. (53) Jork, H. In Proceedings of the 6th InternationulSymposium on Instrumental Planar Chromarography;Traitlcr, H., Voroshilova,0. I., Kaiser, R. E., Eds.; Bad Ddrkheim, Germany, 1991; p 143. (54) Schiitz, H.; Erdmann. F. In Proceedings of the 6th Internationul Symposium on Instrumental Planar Chromatography; Traitler, H., Voroshilova, 0. I., Kaiser, R. E., Eds.; Bad DIirkheim, Germany, 1991; p 341. (55) Schiitz, H.; Erdmann, F.; Funk, W. GIT Supple. Chromatogr. 1990, 17. (56) Zietz, E.; Ricker, I. J. Planar Chromarogr. 1990, 4, 6. (57) de la Vigne, U.; Jaenchen, D.;Weber, W. H. J. Chromatogr. 1991,553,489. (58) de la Vigne, U. Laborpraxis 1987, 11, 944. (59) Poole, S. K.; Belay, M. T.; Poole, C. F. J. Planar Chromarogr., in press. (60) Wilson, I. D.; Lewis, S. J. Chromatogr. 1987, 408,445. (61) Ebel, E.; Volke, S. Dtsch. Apoth. Ztg. 1990, 130, 2162. (62) Menziani, C.; Tosi, B.; Bonora, P.; Lodi, G.J. Chromatogr. 1990,511,396. (63) Trypsteen, M. F. M.; van Seversen, R. G.E.; de Spiegeleer, B. M. J. Anulyst 1989, 114, 1021. (64) Ebel, S.; Bigalke, H. J.; Voelke, S . In Proceedings ojthe 4th Internationul Symposium on Imtrumenrul HPTLC; Traitlcr, I., Studcr, A., Kaiser, K., Eds.; Bad Diirkheim, Germany, 1987; p 113. (65) Jaenchen, D. E.; Issagne, H. J. J. Liq. Chromatogr. 1988, 11, 1941. (66) de la Vigne, U.; Jaenchen, D. E. Inform 1990, I, 477. (67) Poole, C. F.; Poole, S. K.; Fernando, W. P. N.; Dean, T. A.; Ahmed, H. D.; Berndt, J.A. J. Planar Chromatogr. 1989, 2, 336. (68) Jaenchen, D. E. Am. Lab. 1988, 66.
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the same direction where the complete program usually comprises 10-30 cycles. A stepwise gradient is generated by changing the composition of the mobile phase for each successive cycle. Using this method, zone focusing is achieved as described in ref 47. In the past, the AMD technique has been used mainly for the analysis of pesticides in ~ a t e r , 4 ~ -for ~ ' medical applic a t i o n ~ , ~and ~ " ~for the analysis of natural products61d4and industrial chemical^,^^.^^"^ but has not been applied to the analysis of explosives. This paper reports the development of an HPTLC-AMD method for the simultaneous determination of the main explosives, their byproducts, and some biodegradation products found or anticipated in contaminated soil and groundwater at or near former explosives and ammunition production plants or in ammunition waste and wastewater of plants still in operation. The method is applied to the analysis of groundwater samples collected in the vicinity of a former plant for the production of explosives and a soil sample from a former ammunition plant.
EXPERIMENTAL SECTION All standards were from Fluka (Buchs, Switzerland), Aldrich (Milwaukee, WI), EGA (Steinheim, Germany), Riedel-de Haen (Seelze, Germany), Merck-Schuchardt (Hohenbrunn, Germany), Merck (Darmstadt, Germany), and Promochem (Wesel, Germany). The purity was at least 97%, except of RDX, HMX, hexyl, and tetryl, which were of technical grade. Picric acid was moistened with 50% water. Solvents were from Rathburn (Walkerburn, Great Britain) and Riedel-de Haen. For SEC, Biobeads SX-3 from BioRad Laboratories (Richmond, CA) were used. HPTLC plates (Merck), 100 X 200 mm, silica gel 60 on glass, 100- and 200-pm thickness, were used with and without fluorescence indicator F254. For preliminary tests TLC sheets (Merck), 50 X 75 mm, silica gel 60 on alumina, 200-pm thickness with F254, were employed. It is necessary to use plates which are impregnated with fluorescence indicator for an exact positioning of the starting points from the application unit with respect to the scanning light beam. A. Samples. Groundwater samples were taken in the immediate vicinity of the former plant for the production of explosives during World War I1 at Elsnig (Saxony, Germany) at two levels: upper level, 4.9-6.9 m, and lower level, 16-18 m. Soil samples were taken from the former ammunition plant Nieder-Neuendorf (Brandenburg, Germany) where, for instance, shells were filled with explosives. B. Extraction of Water and Soil Samples. Aqueous Samples. Aqueous samples were extracted by discontinuous liquid/liquid extraction using dichloromethane: 1000 mL of the aqueous sample was extracted three times with 50 mL of dichloromethane. The extract was reduced in volume to 1 mL beforeapplication to theTLC plate. Recoveries are 2 8 5 % except for hexyl (55%). Soil Samples. Soil samples ( x g of wet soil corresponding to 5 g of dry soil) were ground in a mortar and transferred to 50-mL glass bottles. After 25 mL of acetone was added, the sample was extracted in an ultrasonic bath for 30 min. After the soil particles settled, the solvent was decanted and the residue extracted a second time with acetone in the 2572
Analytical ChemistIy, Vol. 66, No. 15, August 1, 1994
ultrasonic bath. The combined extracts were centrifuged at 3000 rpm for 7 min and then filtered. After addition of 5 mL of toluene, the acetone was removed by water jet vacuum. The toluene extract was dried over anhydrous sodium sulfate overnight and finally reduced in volume to 1 mL. In a first step, uncontaminated soils (with different fractions of sand, clay, loam, and humic substances) were extracted with acetone using the method given below, followed by spiking with different amounts of analytes (to study the influence of the matrix). To remove interfering humic substances from sandy soil with a low content of organic carbon, 3 mL of saturated CaC12 solution was added and the humic substances were allowed to flocculate overnight. The solution was then centrifuged, decanted, and extracted with 5 mL of dichloromethane, and the organic phase was separated off using a separatory funnel. When soil samples with large amounts of humic substances were investigated, it was necessary to clean up the samples using SEC. Therefore a 30 cm X 7 cm column was packed with Biobeads SX-3 swollen overnight in ethyl acetate/ petroleum ether (80:20). Samples were eluted with ethyl acetate/petroleumether (80:20), where the humic substances were found in the first 80 mL and the analytes in the following 500 mL. C. Development of the Plates. HPTLC plates were stored in a dessicator under vacuum in the presence of silica gel. Prior to use, the plates were cleaned twice with methanol until the solvent front almost reached the upper edge of the plate (this cleaning step takes about 45 min). Standard and sample solutions were applied to the plate using the instrument mentioned below at the minimum application speed (1 5 s/pL) and 8 mm above the lower edge of the plate. The development was achieved in 25 steps, leading to a run time of about 4.5 h. After each step, the solvent was purged off with nitrogen saturated with petroleum ether. Then the development chamber was dried for 3 min, and finally, the plate was saturated with nitrogen/petroleum ether five times (to prevent the formation of @-fronting during analysis). The total migration distance was about 65-70 mm from the lower edge of the plate. The mobile phase and the gradient composition used are discussed in the Results section. D. Instrumental Equipment. Automated multiple development high-performance thin-layer chromatography was performed with an instrument from Camag (Muttenz, Switzerland). The total equipment consisted of the development chamber (AMD), the application unit (Linomat IV), the TLC Scanner 11, and a Hewlett-Packard HP-Vectra 80386-25 PC with the TLC software CATS (Version 3.15) including software option Multi-Wavelength Scan. RESULTS The development of an HPTLC-AMD method for the separation, identification, and quantification of explosives and related compounds was restricted to the following compounds: hexyl (l),picric acid (2), HMX (3), RDX (4), tetryl (7), and 2,4,6-TNT (11) as well as the byproducts 1,3-DNB (8), 2,4-DNT (9), and 2,6-DNT (10) and the two main biodegradation products 2-amino-4,6-dinitrotolouene(5) and 4-amino-2,6-dinitrotoluene(6). Other compounds such as mononitrotoluenes, monoaminonitrotoluenes, mono- and dichle
mobile phase composition
mobile phase composition
100 r
100
. '.
I _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ .
I
nitroaromatics
n
v
0 1 2 3 4 5 6 7 8 9 10111213141516171819202122232425
40t I
/
,' .
.
.
I
I
I
I
steps
- methanol
.ethylacetate
- - petrolether
Figure 2. Correlation diagram (see text for details).
ronitrobenzenes, mono- and dinitroanilines, and nitrophenols may be found in ammunition waste and wastewater at varying but in generally low concentrations. They will not be discussed here. A. ChromatographicSeparation. As pointed out by Poole and Belay,47there are no strict rules which allow the design of a mobile-phasegradient for a given compound class. Rather, this mobile-phase gradient has to be found by trial and err0r.~8 It was useful to carry out first experiments with small TLC cards using different solution mixtures to obtain some preliminary information on the chromatographic behavior of the compounds to be studied. However, because these preliminary studies were carried out using isocratic development, the results cannot be transferred to actual AMD separation without further discussion. ConventionalTLC separation of substancesdiffering widely in their polarity, with simultaneous presence of related isomers, is only possible if two-dimensional TLC is applied. With AMD, which represents a kind of one-dimensional TLC, this problem may, in the narrow sense, be solved. Generally, separation in TLC depends, first on the polarity of the compounds and that of the solvent and, second,on thechemical properties of the stationary phase. To separate isomers it would be more useful to apply to reversed-phase silica gel, but in this phase the solvent migrates very slowly (0.2-0.3 times slower than on silica gel), so that a simple AMD separation would take several days. Therefore this phase was not used. Attempts to decrease the level of detection by using plates with a layer thickness of 100 pm led to poorer separation properties as compared to 200 hm. The optimization of the separation of the more polar compounds (i.e., amines and picric acid) started with a gradient from 100% ethyl acetate to a 85:15 mixture of petroleum ether/ethyl acetate. If the positions of the analytes when such a first test gradient is used are not yet optimal, i.e., the spots are not sufficiently separated or not equally distributed over the plate, the pair of pointsrepresenting theconcentration ratio of, for example, a binary gradient at the position of a given analyte is shifted parallel within the correlation diagram (such as that shown in Figure 2) to the new and desired position
of the analyte. These two new points are then connected by straight lines with thestarting point of thegradient. By shifting the positions of the other analytes in a similar manner, a new gradient is developed. This procedure only represents a theoretical optimization, because the application of the new gradient shows some deviation. Nevertheless, this procedure minimizes the experimental steps required. Finally, we used a gradient consisting of mixtures of methanol, ethyl acetate, and petroleum ether (The solvent strength parameter of these solvents is 0.70,0.48, and 0.010.05, re~pectively.)?~The gradient is shown in Figure 3. The development started with a mixture of 80% ethyl acetate and 20% methanol, i.e., with a mobile phase of strong solvent strength, to force all compounds to migrate at least several millimeters (one step) from the point of application. From steps 2 to 5 the methanol fraction was reduced to zero, while petroleum ether was increased from 0 to 85% in the remaining 14 steps. During this development, the strength of the mobile phase declines to a point at which a particular compound will no longer migrate and will occupy its final position in the chromatogram. The position and width of this zone is then hardly influenced by the further development cycles required to separate those compounds which interact less strongly with the stationary phase (see Figure 4). The resulting chromatogram is shown in Figure 5 . As is apparent from this figure, the explosives hexyl (l),picric acid (2) (which shows some tailing), HMX (3), RDX (4), and tetryl (7) were well separated. During the last 10 steps the gradient becomes very flat to achieve an optimum separation of the nitrotoluenes, Le., the two DNTs (9, IO), T N T (Il), and 1,3-dinitrobenzene (8). 2,4-DNT, 2,6-DNT, and 1,3dinitrobenzene were only partially resolved. Next, a method was developed which now also includes the major two amines found predominantly in contaminated soil and groundwater at or in the vicinity of former ammunition plants, i.e., 2-amino-4,6-dinitrotoluene(5) and 4-amino-2,6dinitrotoluene (6). The gradient had to be modified as shown (69) Poolc, C. F.;Poole, S. K. Chromazography Today; Elsevier: Amsterdam, The Netherlands, 1991; p 383.
AnalyticaiChemistry, Voi. 68, No. 15, August 1, 1994
2573
mobile phase composition
100
ImVl
100
-
80
-
60
-
I
80
I 11
z
60 40
-
1 3 4
1
7
8 9
0
40
20 0 0 1 2 3 4 5 6 7 8 9 10111213141516171819202122232425
steps -methanol
-
ethylacetate
--
Flgure 7. Chromatogram of nlne exploshres and two biodegradation products using the gradient shown in Figure 5: 2amino-4,6dinltrotoiuene (5) and 4-amino-2,6dinltrotoiuene (8). The remaining peaks are identified in Figure 5.
petrolether
Flgure 4. Correlation diagram correspondlng to Figure 3.
'"i eo
a
1
1
I
48
6B
Flgure 8. Chromatogram of nlne explosives and two biodegradation products near detection limlt.
0
I 20
I
I
60
40
I 80
1
100
Imml
Flgure 5. Chromatogram of nine explosives using the gradient shown picric acid (2), HMX (S), RDX (4), tertyi(7), 1 3 in Figure 2 hexyl (l), DNB (8), 2,4-DNT (9), 2,8-DNT (lo), and TNT (11). mobile phase composition
loo
r /.
rn
I'
,
5 5 10 10 10
10 10 20 20 20 10
. .
steps ' .ethylacetate
- - petrolether
Flgure 8. Gradient used to generate the chromatogram shown in Figure 7.
in Figure 6. The resulting chromatogram is shown in Figure 7. The zones of the two aminodinitrotoluenes now appeared between 4 and 7. It should be noted that the isocratic mobile phase after the first 15 steps leads to a slightly better separation of the nitrotoluenes and 1,3-dinitrobenzene. Ammunition waste and wastewater may also contain small amounts of monoaminomononitrotoluenes, in particular, 2574
hexyl (1) picric acid (2) HMX(3) RDX (4) 2-amino-4,6-dinitrotoluene(5) 4-amino-2,6-dinitrotoluene (6) tetryl (7) 1,3-dinitrobenzene (8) 2,4-dinitrotoluene(9) 2,6-dinitrotoluene(10) 2,4,6-trinitrotoluene (11)
The RSD near the detection limit (Le., for an applied amount of 20 ng). S/N = 3.
0 ,
2
- methanol
Tabk 1. Absolute Detection umllr for the Dotmlnatlon of Explorlver a d Thelr Metab0llt.r by HPTLGAMD compound detection limitg (ne)
AnalytldChemWy, Vol. 66, No. 15, August 1, 1994
2-amino-4-nitrotoluene and 2-amino-6-nitrotoluene. By Use of the gradient shown in Figure 6, these two isomers show a common Rpalue between that of 6 and 7. With this gradient it is not possible to resolve these two isomers. Moreover, some overlap with 7 is observed. B. Method Evaluation. Detection Limits. Figure 8 represents the chromatogram of the standard solution shown in Figure 5 with concentrations near the detection limit. The absolute detection limits are summarized in Table 1. They range from 5 to 20 ng and thus are higher than those achieved by HPLC by a factor of 2 (e.g., for RDX) to 10 (e.g., for dinitrotoluenes). The use of the software option Multi-Wavelength Scan (MWL) makes it possible to identify spots without taking spectra, as shown in Figure 9. Although the evaluation of a chromatogram using MWL is only a rough method of
absorption [mV] I
Table 2. ReproduclMIHy ol Detennlnrtlonol Expkdver by HPTLGAMD RSW (%)
I
compound
peak ht
peak area
hexyl (1) picric acid (2) HMX(3) RDX (4) 2-amino-4,6-dinitrotoluene (5) 4-amino-2,6-dinitrotoluene(6) tetryl (7) 1,3-dinitrobenzene (8) 2,4-dinitrotoluene ( 9 ) 2,6-dinitrotoluene (10) 2,4,6-trinitrotoluene (11)
3.4 1.9 2.3 1.6 1.9 3.0 3.8 1.8 2.2 3.1 2.8
2.9 3.4 2.6 5.9 6.1 19 3.5 3.2 4.0 4.3 3.5
a RSD data are a summary of results for four standard solutions delivering between 20 and 130 ng to the plate (four repetitive runs).
400 220 240 260 280 300 320'"340"'360 380 400 420 wavelength [nm] Flgure 0. Spectra received from multiwavelengthscan.
17
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Figure 11. Chromatogramof a groundwater sample from the former ammunition piant Elsnlg,upper level. Peak ldentiflcatbnsghren In Figures 5 and 7.
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Flgure 10. Calibrationcurves for hexogen (RDX): (a, top) peak height; (b, bottom) peak area.
identification, it is much faster than taking spectra of every spot. Calibration and Reproducibility. Figure 10 represents the calibration curve for RDX in the range of 10-130 ng using peak heights (a) and peakareas (b) (five-point calibration with determinations in duplicate). It is apparent that the calibration curves are not linear. They were fitted by a polynomial regression. The reproducibility (vide infra) was always better when peak heights rather than peak areas were used. The reproducibilities for thevarious explosives and their biodegradation products are summarized in Table 2, as determined by applying four standard solutions with different amounts (20-130 ng) of the compounds. As also illustrated in Figure 10, the reproducibility in genera! is better if
L
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Figure 12. Chromatogram of a groundwater sample from the former ammunitionplant Elsnlg, upper level. Peak identtflcatlonsgiven in Figure 5.
compounds are quantified by peak height rather than peak area (except for hexyl). Relative standard deviations varied from 1.6-3.8% and hardly depended on the applied amount (except if the applied amounts approached the detection limit). EnvironmentalSamples. Figures 11 and 12 show the AMD chromatograms of two groundwater samples taken near the former ammunition plant in Elsnig (Saxony) at 4.9-6.9 (upper level) and 16-18 m (lower level). Figure 11 shows the presence of 1,4,9, and 11. It is likely that the biodegradation products Sand 6 were also present. Theseidentifications wereconfirmed by the UV spectra, as shown for 4 in Figure 13a (standard) and b (sample). Figure 12 again reveals the presence of 4, 9, and 11 as well as traces of aminodinitrotoluenes. Note that
AnaiyticalChemistty, Vd. 66, No. 15, August 1, 1994
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'O 20
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100
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190
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290
340
390
440
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Flgwe 13. UV spectra of hexogen(RDX): (a, top) referencecompound; (b, bottom) groundwater sample. Table 3. QuantmOatlon of Explorlves In Two Contaminated ckoundwatw Samples' by HPTLGAMD, QC, and HPLC (In pg/L) compound A M P GCd HPLCb A M P G C C ~ HPLC* 100
RDX (4) 2,4-DNT(9) 2,6-DNT(10) 2,4,6-TNT (11)
2380 700
f 1330
nde 710(670) 510(470) 910 (1100)
3800 720 520 1370
310 1080 nnr 870
nde 1180(1050) 132(126) 740 (800)
400 900 92 690
"Sample 1, 4.9-6.9 m; sample 2, 16-18 m. Groundwater level. Extraction with dichloromethane. Solid-phase extraction (C18); determination with nitrogen phosphorus detector. Extraction with toluene; determination with e ectron capture detector (values in parentheses). e nd, not determined due to thermal degradation./Not quantified due to interference. nn, not detectable.
I
2,6-dinitrotoluene was absent or of low concentration within this sample, Quantitative results for 4, 9, 10, and 11 are shown in Table 3. The data are compared with results obtained with GC and HPLC. GC was not applied to the determination of RDX as this compound may undergo thermal degradation. GC was used with nitrogen/phosphorus detection and electron capture detection. Moreover, three different extraction methods were employed (see Table 3). Taking into account that very different extraction methods and chromatographic methods have been applied, the agreement of the data for most compounds is satisfactory. Thus, 9 could be determined in sample 1 with a coefficient of variation of 3% and in sample 2 of 11% while 11 could be determined in sample 1 with a coefficient of variation of 18% and in sample 2 of 10%. While humic substances do not disturb the analysis of groundwater or surface water samples using the AMD technique, they may severely interfere with the analysis of soil samples if no further cleanup is carried out. Figure 14 shows the HPTLC-AMD chromatogram of a spiked soil sample with high organic carbon content (1 2%). It is apparent that an evaluation of this chromatogram is no longer possible 2578
Analytical Chemlstty, Vol. 66, No. 15, August 1, 1994
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and an additional sample cleanup is required. For soils with a low content of organic carbon, flocculation of the humic substances with a saturated solution of CaCl2 (as described in the Experimental Section) is often sufficient. Figure 15 displays the spiked soil (shown in Figure 14) after CaCl2 flocculation. Beginning with picric acid, all spiked compounds can now be identified, although there is still a strong background resulting from the humic substances. This background can be removed completely if the cleanup is performed by SEC, as described in the Experimental Section. Figure 16displays the chromatogram of the spiked soil sample shown in Figure 14 after SEC. Finally, a chromatogram of a soil sample from the former ammunition plant Nieder-Neuendorf (Brandenburg, Germany), obtained after CaC12 cleanup is shown in Figure 17.
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Flgure 17. Chromatogram of a sol1 sample from former ammunition plant Nleder-Neuendorf.
11can be readily identified and quantified. The concentration of TNT was determined as 21 1 pg/kg as compared to 234 pg/kg determined by GC/MS.
DISCUSSION The results presented above demonstrate that not only the identification but also the quantification of the explosives expected or found in contaminated soil or water by HPTLCAMD are possible. These explosives were adequately separated. Identification was based on both the Rfvalue and the UV spectra, which show distinct maxima for all explosives (note that the UV spectra cannot be compared directly with those obtained in solution). The method applied does not allow the identification or quantification of the minor byproducts from ammunition waste and fails if thesample alsocontains, apart from dinitrotoluenes and TNT, large quantities of mononitrotoluenes, as the latter compounds show migration behavior similar to that of the dinitrotoluenes. It is of particular advantage that up to 20 samples can be analyzed simultaneously. Although the analysis time is relatively long (the development of the plate shown in Figure 4 takes 4.5 h), this time factor is less important as development is carried out unattended. While recently analyzing 220 soil samples from a former ammunition plant, we determined TNT at concentrations of > 10 mg/kg in only two samples. A classic analysis of these samples by GC (GC/MS) followed by HPLC would have been very time-consuming. In this particular case, the use of the AMD technique would have been valuable, as
this method allows the screening of a large number of soil samples for contamination by the major explosives with less time and labor involved. Thus, the AMD technique is wellsuited for screening large numbers of contaminated soil samples. For this screening, a cleanup of the soil extracts by CaCl2 flocculation will, in many cases, be sufficient. The screening will include the quantification of the major explosives. If a contaminated soil sample is then identified and further information is requested on other contaminants at low concentrations, the classical methods (GC/MS, HPLC) have to be used in addition. It is advantageous that humic substances present in water samples do not interfere with the analysis. In contrast, if soils with a high concentration of humic substances are to be analyzed, a cleanup as described above will be necessary. However, this cleanup is simple (CaC12 flocculation) or can be automated (if SEC is used). Even if humic substances do not directly interfere with the analysis of environmental samples or explosives by GC or HPLC, they may lead to a rapid degradation of the column performance if the analysis is not preceded by size exclusion chromatography. Furthermore, if environmental samples are analyzed by GC or HPLC without extensive cleanup, degradation of the column performance will always present a problem (independent of the humic substances addressed above). With the AMD technique, a new plate is used for each set of analyses, thus avoiding these chromatographic problems. A further advantage of these methods is the fact that one or several standard solutions (at different concentrations) may be developed simultaneously with the sample to be analyzed, thus allowing more reliable compound identification and quantification.
ACKNOWLEDGMENT Financial support from the German Federal Department of Research and Development is gratefully acknowledged. Furthermore, we thank the CAMAG Co. for supplying us with the plotter option and the data transfer program to the software CATS 3.15. Finally we thank Dr. A. Preiss and Dipl. Chem. P. Mussmann for carrying out the GC and HPLC analyses of the groundwater samples. Received for review April 28, 1993. Accepted October 7, 1993.’ Abstract published in Advance ACS Abstracts, June IS, 1994.
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