Anal. Chem. 1991, 63, 1038-1042
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Cole, M. J.; Enke, C. G. Paper presented at the 37th Annual Conference on Mass Spectrometry and Allied Topics, Miami Beach, FL, May
21-26, 1989.
Hayashl. A.; Matsubara, T.; Masanori, M.; Kinoshlta, T.; Nakamura, T. J . Blochem. 1989, 106, 264-269. Sweetman, B. J.; Tamura, M.; Hlgashimorl, K.; Inagami, T.; Blair, I . A. Paper presented at the 35th Annual Conference on Mass Spectrometry and Allied Topics, Denver, CO, May 24-29, 1987. Munster, H.; Budzlklewlcz, H. Biol. Chem. Hoppe-Seyler 1988, 369,
303-308.
Kayganich, K. A.; Murphy, R. C. Paper presented at the 38th Annual Conference on Mass SDectrometw and Allied Topics, Tucson, AR, June 4-9, 1990. Heller, D. N.; Murphy, C. M.; Cotter, R. J.; Fenselau, C.; Uy, 0. M. Anal. Chem. 198& 60, 2787-2791. Tomer, K. 8. Mess Spectrom. Rev. 1989, 6 , 483-511. Stratford, 8. C. An Atlas of wid ~ ~ ~ r t w o k g common y: H ~ m n Pathogens ; Blackweli Scientific Publications: Edinburgh, 1977; pp
97-107.
Bllgh, E. 0.; Dyer, W. J. O n . J. Blochem. physiol. 1959, 37,
911-917.
(16) Tomer, K. 6.; Crow, F. W.; Gross, M. L. J. Am. Chem. Soc. 1983. 105, 5487-5488. (19) Tomer. K. 8.; Jensen, N. J.; Gross, M. L. Anal. Chem. 1886, 56. 2429-2433. (20) Huang, 2. H.; Gage, D. A.; Sweeley, C. C. J. Am. Soc. Mess Spectrom., in press.
(21) Odham, G.; Tunlid, A.; Westerdahl. G.; Larsson, L.; Guckert, J. 6.; White, D. C. J. Microbiol. Methods 1885, 3 , 331-344. (22) Tunlid, A.; Ringelberg, D.;Fhelps, T. J.; Low, C.; White, D. C. J. Microbiol. Methods 1909, 10, 139-153.
RECEIVED for review November 5,1990. Revised manuscript received February 20,1991. Accepted February 26,1991. This work was performed with support by the National Science Foundation Center For at Michigan state University.
Separation and Identification of Organic Gunshot and Explosive Constituents by Micellar Electrokinetic Capillary Electrophoresis David M. Northrop and Daniel E. Martire Chemistry Department, Georgetown University, Washington, D.C. 20057
William A. MacCrehan* Organic Analytical Research Division, National Institute of Standards and Technology, Gaithersburg, Maryland 20899
Micellar electroklnetlc capillary electrophoresis (MECE) provides rapid and efficient separation and detection of organk gunshot and explosive consitituents. Twenty-slx of these constituents were separated in under 10 m h with effldencies in excess of 200 000 theoretical plates. The effects of the following experimental parameters were studied: sodium dodecyi sulfate (SDS) concentration, pH, addition of a tetraalkyiammonlum salt, capillary diameter, and InJection times. The presence of gunshot residues in spent ammunition casings and the composition of six reloading powders and four plastic explosives were determined by uslng the MECE method. Muitipiowaveiength analysis provided UV spectral profiles of the Constituents for use with selective wavelength monitoring.
INTRODUCTION Forensic investigators use the analysis of gunshot and explosive residues to identify materials and individuals involved in a crime. Various methods, including atomic absorption and neutron activation analysis, have been used to analyze the inorganic constituents of gunshot primer residues. Routine application has been limited because of interferences, high blanks, prohibitive instrument cost, and analysis time. Since commercial ammunition and explosives contain mixtures of explosives, stabilizers, and plasticizers, analysis of these characteristic organic constituents, listed in Table I, has gained recent attention. Some work has been done to analyze the organic constituents of gunshot residues by HPLC (I),GC/MS (2),and SFE/SFC (3),but none has gained widespread utility and application, as indicated by a recent survey of forensic laboratories ( 4 ) . The thermally stable organic constituents of explosive materials are commonly analyzed by GC/MS (5),
Table I. Compounds Studied
dibutyl phthalate
Gunpowder Constituents
DBP (ethylcentralite) EC 2,3-dinitrotoluene 2,3-DNT 2,4-dinitrotoluene 2,4-DNT 2,6-dinitrotoluene 2,6-DNT 3,4-dinitrotoluene 3,4-DNT diphenylamine DPA 1,2,3-propanetrioltrinitrate (nitroglycerine) NG nitroguanidine NGU 2-nitrodiphenylamine 2-nDPA N-nitrosodiphenylamine N-nDPA High-Explosive Constituents dibutyl phthalate DBP diethylene glycol dinitrate DEGDN 1,3-dinitronaphthalene 1,3-DNN 1,5-dinitronaphthalene 1,5-DNN l,%dinitronaphthalene 1B-D” ethylene glycol dinitrate EGDN 1,2,3-propanetrioltrinitrate (nitroglycerin) NG nitroguanidine NGU N,”-diethyl-N,”-diphenylurea
2-nitronaphthalene
2-nitrotoluene 3-nitrotoluene 4-nitrotoluene pentaerythritol tetranitrate picric acid 2,4,6,N-tetranitro-N-methylaniline 1,3,5,7-tetranitro-l,3,5,7-tetrazacyclooctane
2,4,6-trinitrotoluene 1.3.5-trinitro-1.3.5-triazacvclohexane
2-MNN
2-NT 3-NT 4-NT PETN PA Tetryl HMX TNT RDX
whereas LC-EC (6), LC-TEA (7), and SFC/UV (8) are used for the more thermally labile constituents. Separations by capillary electrophoresis (CE) provide a number of advantages (9); of particular interest for forensic
0003-2700/91/0363-1038$02.50/00 1991 American Chemical Society
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applications is the high component resolution that may be achieved on microsamples. Micellar electrokinetic capillary electrophoresis (MECE) is an adaptation of CE where the addition of a charged micellar agent to the electrolyte provides a separation of neutral molecules. Selective partitioning of the analytes into the micellar phase causes them to migrate a t different rates from that of the bulk electroosmotic flow rate. Early work by Terabe et al. (10,11) demonstrated the use of MECE for polar neutral aromatic compounds. Other applications include the separation of derivatized amino acids (12),pharmaceuticals (13), water-soluble vitamins (141, and nucleic acid related compounds (15). Work has also been done to evaluate the effect of the various parameters in MECE. Increasing temperature and adding an organic modifier results in both decreased efficiency and capacity factors (16). Efficiency was found to be limited by diffusion at low velocities, whereas at high velocities, efficiency is limited by the rate of partitioning of analytes (17). The separation of ionic solutes in MECE was improved by adding a tetraalkylammonium salt to mediate interaction of the analytes with the micelles (18). Foley has derived a series of equations to optimize resolution in MECE (19). In this work, a separation of some of the constituents found in gunshot and explosive material using MECE was developed. The following experimental parameters were evaluated: micellar concentration, pH, addition of a tetraalkylammonium salt, capillary diameter, injection time, and detection wavelength. Possible application to gunshot and explosive residue identification is demonstrated.
containing possible gunshot residue components, possible highexplosive components, and a combination of all 26 components were made, by diluting the stock solutions to lo4, IO+, and 5 x lo* mol/L concentrations, with buffer. A five-component test mixture, consisting of 2 x lo4 mol/L TNT, 2,4-DNT, and 2-NT, 3 X lo4 mol/L 4-NT, and 4 X 10” mol/L 3-NT, was prepared in SDS/buffer to examine the effects of experimental parameters. Wavelength response studies were conducted on both the gunpowder and explosive standards by running at 14 different wavelengths, with response profiles being constructed for each component by quadratic curve fitting of the absolute intensity at each wavelength. Forensic Samples. Spent ammunition casings were examined for the presence of gunshot residues by the following procedure. Cleaned cotton, moistened with ethanol and held with tweezers, was used to swab the casings. The swabs were placed in sealed glass vials prior to analysis. Samples for MECE analysis were obtained by ultrasonic agitation of the swab in 500 pL of ethanol for 15 min and collection of the residues by centrifugal filtration through a 1-pm poly(tetrafluoroethy1ene)filter. The ethanol was evaporated down to a 2-3 pL under a stream of nitrogen and then diluted with 50 p L of the running buffer. Ten-milligram samples of each of the reloading powders and 20-mg samples of the plastic explosives were extracted with 300 pL of ethanol for 15 min in an ultrasonic bath. The ethanol extracts were evaporated under a stream of nitrogen to a volume of 2-3 pL and then diluted with 50 pL of the running buffer. Qualitative identification of the components in each of the samples was made by comparison to the capacity factors of standard solutions, by sample spiking, and by monitoring at selected wavelengths.
EXPERIMENTAL SECTION Apparatus. Experiments were conducted by using a commercially available capillary electrophoresis instrument that consisted of a 0-30-kV power supply, an autosampler, and absorbance and fluorescence detectors. This instrument also has three automatic injection modes (electro, pressure, and gravity). Use of a deuterium light soume as well as a microprocessor control of the continuous-wavelengthgrating monochromator allowed for multiple-wavelength monitoring during runs. Polyimide-coated fused silica capillary tubing, 350-pm outside diameter, was obtained in 50-, loo-, and 150-pm inside diameters. The column length was 670 mm with on-column detection 50 mm from the ground end of the capillary, making the effective separation length 620 mm. The running voltage was constant at 20 kV. Electroinjection at 5 kV for 2 s was used, unless otherwise noted. A small negative bias for the quantity injected of the late eluting versus the early eluting components was observed for the electroinjection, amounting to about 20% when compared to gravity injection. Swabbings from shell casings were obtained from a Colt 38 caliber revolver, firing Federal 38 Special +P, 159-grain lead, semiwadcutter,hollow point cartridges,and also from a Fkmington 45 caliber semiautomatic pistol using Federal 45 Colt, 225-grain, semiwadcutter, hollow point cartridges. Reagents. Sodium dodecyl sulfate (SDS), Sudan 111, 2,4-dinitrotoluene, N,N’-diethyl-N,”-diphenylurea (ethylcentralite), diphenylamine, dibutyl phthalate, and tetraethylammonium perchlorate (TEAP)were purchased from commercial sources. All of the other gunpowder and explosive components, listed in Table I, were a gift from either the Bureau of Alcohol, Tobacco, and Firearms (Rockville,MD) or the U.S. Army Explosives Repository (Dover, NJ). The borate buffer used was either 2.5 mmol/L or 5 mmol/L in borate, in the pH range from 7.8 to 8.9, made by dissolving the appropriate amounts of sodium tetraborate and boric acid in adsorbentlion-exchange purified water. SDS concentrations ranged from 10 to 50 mmol/L. p- and y-cyclodextrinswere tested as selectivity-enhancing additives at 8 and 5 mmol/L concentrations, respectively, in 30 mmol/L SDS/buffer. All buffer solutions were degassed by ultrasonic agitation under vacuum before use. Procedure. Standards. Stock solutions of the standards were prepared to be 100 mmol/L in ethanol. Three different mixtures
Earlier work using MECE (13) showed that resolution of compounds similar in structure to the various gunshot and explosive residue constituents was possible, and an examination of the effect of various tetraalkylammonium salts on the separation. The first series of our experiments was to look a t the effect of SDS concentration, pH, and addition of a tetraalkylammonium salt, TEAP, by using the resolution test mixture. The variability in absolute migration times was reduced through calculation of the capacity factor, k ’
RESULTS AND DISCUSSION
k’
tr - to
t0(1 - t r / t m )
(1)
where to is the migration time for ethanol, which moves with the electroosmotic flow, t, is the migration time of Sudan 111, which migrates with the micelles, and t , is the migration time for the solute of interest. Whereas over the course of a day drifts in mobility times of larger than 10% were observed in our system, standard deviations in k’, within and between days, were less than 0.5%. These drifts may be a result of variable heat dissipation. The k’ value for each solute increases with increasing SDS concentration as shown in Figure 1. Resolution of the three isomers of mononitrotoluene from 2,4-DNT improved from 0.55 to 1.03 when the SDS concentration was increased from 10 to 20 mmol/L. Changing the pH over the borate buffer range had little effect on k’. The small changes that did occur are likely due to the increasing ionic strength of the buffer, evidenced by increased currents observed at the higher pH values. The addition of TEAP to the buffer greatly increased k‘values when compared to those obtained in the SDS-only buffer, and the resolution of 2-NT and 4-NT was lost. The k’values also decreased slightly with increasing pH. TEAP may be forming ion pairs with the SDS molecules, a t their aqueous interface, which would increase the hydrophobicity of the interface. This would increase the interaction of hydrophobic neutral solutes with the SDS. A second mechanism that may also play a role is that the tetraalkylammonium salts can ion exchange onto the available silanol groups on the surface of the silica capillary. A plot of the electroosmotic flow verses the TEAP concentration
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Figure 3.
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-
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(Figure 2) supports this suggestion. This ion-exchanged TEN may be creating a hydrophobic phase on the capillary walls. Reversed-phase interaction with the micelles and/or the solutes of interest would increase the migration time of the solutes and thus increase the k' values. It should be noted that the choice of the perchlorate salt of the tetraalkylammonium ion was important. Salts with I-, Br-, and C1- as the counterion caused electrochemical oxidation products to accumulate at the high-voltage platinum electrode. The I2product plated out on the positive electrode, while Br2 and C12 products caused bubbles to be introduced into the capillary. In looking for ways to improve resolution of the mononitrotoluenes in our test mixture, we decided to add p- and y-cyclodextrins to the SDS system, as per the work of Otsuka et al. (12). With both 0- and y-cyclodextrins, our results showed that k'values were lower than k'values in an SDS-only system. Resolution of 4-NT and 3-NT was not improved. The effect of capillary diameter on detection limits was examined using three different capillaries having inside diameters of 50, 100, and 150 pm. Doubling the capillary diameter increases the volume of sample introduced by a factor of 4 and the path length for the absorbance detector by a factor of 2. The expected improvement in sensitivity was observed experimentally in 100-pm versus 50-pm capillaries. Larger currents in the 150-pm-i.d. capillary increased nondissipated heating inside the capillary and thus decreased efficiency (ZO),negating any gains from improved detection limits. Increasing the capillary diameter also resulted in a decrease in migration times, as well as a decrease in k'values;
however, resolution of the components of interest was not adversely affected. The influence of injection time on efficiency is shown in Table 11. Decreasing the injection time increased the efficiency (N), indicating that the efficiency in our system was determined primarily by the width of the sample plug and was not limited by diffusion or rate of partioning. The k'values were unaffected by the injection time. On the basis of these results, we chose operating conditions that would provide good resolution, low currents, and a reasonable analysis time for the identification of the organic constituents of gunshot and explosive residues. By using the conditions shown in Figure 3, a separation of 11components in the gunshot residue test mixture was achieved in under 10 min with baseline resolution of the 4 isomers of dinitrotoluene, a signal-to-noise ratio of better than 100 to 1, and efficiencies (N) between 200000 and 400000 theoretical plates. Note that wavelength programming was used to detect nitroglycerin (NG) a t 200 nm while the other components were detected a t 250 nm. Munder et al. (3) also examined these same gunpowder and explosive constituents by using SFE/SFC. Due to the lack of complete chromatographic resolution, unambiguous identification of all components by SFC required three different detectors (UV, FID, and ECD). For example, 2,4-DNT, DPA, and N-nDPA all coelute in the system described. The MECE system described here provides better resolution using a single (UV) detector. Figure 4 shows a separation of 15 compounds of interest in high-explosive analysis. With the exception of the 1,5 and 1,8 isomers of dinitronaphthalene, all are well resolved. Figure 5 shows a mixture of all 26 components. Coelutions are limited to two of the mononitrotoluene isomers with two of the dinitrotoluene isomers. Approximate detection limits were determined from concentration studies and were found to be 5 X lo4 mol/L for the aromatic compounds, 1 X lo6 mol/L for the aliphatic compounds, and 5 X lod mol/L for P E T N and HMX. On the basis of an injection volume of approximately 11 nL, these detection limits would correspond to about 10-15-, 11-32-, and 160-175pg mass detection limits.
ANALYTICAL CHEMISTRY, VOL. 03, NO. 10,MAY 15, 1991
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Figure 6 . Extracts from ammunition shell casings. Bottom trace: blank. Middle trace: 45 baliber. Upper trace: 38 caliber. Conditions: same as for Figure 3 except detection at 220 nm.
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1
Table 111. Absorption Wavelength Maxima'
4 - 2,6-DNT
sample
nm
sample
nm
DBP DEGDN 1,3-DNN 2,3-DNT 2,4-DNT 2,6-DNT 3,4-DNT DPA
