Analysis of explosives via microchip electrophoresis and...

Martin Pumera

Group of Sensors and Biosensors,Department of Chemistry,Universitat Autònoma de Barcelona,Bellaterra, Spain

Received August 20, 2005Revised September 3, 2005Accepted September 3, 2005

Review

Analysis of explosives via microchipelectrophoresis and conventional capillaryelectrophoresis: A review

The upsurge in terrorist activity has generated tremendous demand for innovative toolscapable of detecting major industrial, military, and home-made (improvised) explosives.Fast, sensitive, and reliable detection of explosives in the field is a very important issuein nowadays. CE, especially in its miniaturized format (lab-on-a-chip), offers greatpossibilities to create portable, field deployable, rapidly responding, and potentiallydisposable devices, allowing security forces to make the important decisions regard-ing the safety of civilians. This article overviews the microchip and conventional capil-lary electrophoretic techniques for analysis of a wide variety of explosive compoundsand mixtures.

Keywords: Capillary electrophoresis / Explosives / Microchip electrophoresis /Microfluidics / Review / Terrorism DOI 10.1002/elps.200500609

1 Introduction

Recent terrorist attacks on March 11, 2004 in Madrid andon July 7, 2005 in London showed that not only the ter-rorist activities are relevant to the remote countries, butalso they present a serious threat to the citizens of Euro-pean Community. These terrorist activities generated an

enormous demand for rapid identification of explosivecompounds at the site of terrorism. The decentralizeddetection of explosives at trace levels is fundamental forsafety of civilized people. The ideal counter-terrorismdetection device should allow the security forces to makethe important decision concerning evacuating, barricad-ing, effective decontamination of particular site, or effi-cient pursuing of suspects, and it should protect themfrom becoming victims themselves.

There are several hundreds of explosive materials offi-cially listed [1]. They are usually based on (i) nitratedorganic compounds (i.e., 2,4,6-trinitrotoluene (TNT), hex-ahydro-1,3,5-trinitro-1,3,5-triazine (RDX), methyl-2,4,6-trinitrophenylnitramine (Tetryl), nitroglycerin, (ii) inorganicnitrate, chlorate, or perchlorate salts (i.e., NH4NO3, KNO3,or NH4ClO4), or (iii) compounds containing unstable per-oxide group (i.e., triacetone triperoxide). Nitrated organicexplosives are usually used for military purposes; TNTanddinitrobenzene (DNB) have been in use for more than100 years, mostly during World War I and II. RDX andHMX (octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine)are very powerful explosives widely used in nowadays inplastic explosives or warheads. The current militaryexplosives are usually mixtures of explosive componentwith other organic compounds, such as stabilizers andplasticizers. Military and industrial explosives are highlyeffective and therefore attract the attentions of terrorists.However, a significant amount of terrorist activity in Eur-ope and United States has been based on the use of“easy to obtain-easy to make” improvised explosives (for

Correspondence: Dr. Martin Pumera, Group of Sensors and Biosen-sors, Departament of Chemistry, Universitat Autònoma de Barcelona,E-08193 Bellaterra, SpainE-mail: [email protected]: 134-93581-2379

Abbreviations: 2-Am-4,6-DNT, 2-amino-4,6-dinitrotoluene; 4-Am-2,6-DNT, 4-amino-2,6-dinitrotoluene; 2,4-DAm-NT, 2,4-diaminoni-trotoluene; 2,6-DAm-NT, 2,6-diaminonitrotoluene; DBP, dibu-tylphthalate; DEGDN, diethylene glycol dinitrate; 1,3-DNB, 1,3-dini-trobenzene; 1,3-DNN, 1,3-dinitronaphthalene; 1,5-DNN, 1,5-dinitro-naphthalene; 1,8-DNN, 1,8-dinitronaphthalene; 2,4-DNP, 2,4-dinitrophenol; 2,3-DNT, 2,3-dinitrotoluene; 2,4-DNT, 2,4-dinitroto-luene; 2,6-DNT, 2,6-dinitrotoluene; 3,4-DNT, 3,4-dinitrotoluene;DPA, diphenylamine; EC, N,N-diethyl-N,N-diphenylurea; EGDN, eth-ylene glycol dinitrate; EPA, US Environmental Protection Agency;FIA, flow injection analysis; 2-HADNT, 2-hydroxylamino-4,6-dinitro-toluene; 4-HADNT, 4-hydroxylamino-2,6-dinitrotoluene; HMX, octa-hydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine; LiDS, lithium dodecylsulfate; MMA, monomethylammonium; 2-MNN, 2-nitronaphthalene;2-nDPA, 2-nitrodiphenylamine; NG, 1,2,3-propanetriol trinitrate(nitroglycerine); NGU, nitroguanidine; N-nDPA, N-nitrosodiphenyla-mine; 2-NT, 2-nitrotoluene; 3-NT, 3-nitrotoluene; 4-NT, 4-nitroto-luene; ODS, octadecyl silica; PA, picric acid; PETN, pentaerythritoltetranitrate; PMMA, poly(methylmethacrylate); RDX, hexahydro-1,3,5-trinitro-1,3,5-triazine; Tetryl, methyl-2,4,6-trinitrophenylnitra-mine; TAT, 2,4,6-triaminotoluene; TNT, 2,4,6-trinitrotoluene

244 Electrophoresis 2006, 27, 244–256

© 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com

Electrophoresis 2006, 27, 244–256 Miniaturization 245

explosive compositions used in recent terrorist bombings,see Table 1). These explosives typically contain as a baseammonium or sodium nitrate or perchlorate. Ammoniumnitrate is commonly used as agriculture fertilizer and it iseasy to purchase it in large quantities, and therefore it iscommonly used to fabricate bombs. In the recent years,the terrorists have discovered another very easy to makeexplosives – triacetone triperoxide or diacetone diper-oxide. However, acetone peroxide-based explosives areextremely unstable and easy to detonate. It is important tonote that many of the used explosive materials are mix-tures of the compounds across the above described cate-gories (for some examples, see Table 2).

The only official method for analysis of explosives is spe-cified by US EPA (United States Environmental ProtectionAgency) under EPA method 8330 for monitoring nitroaro-matic explosives in soils and ground water [2]. The meth-od describes HPLC separation based on the use of twoRP columns, and it requires 60 min for full resolution of 14explosives. It is obvious that this method does not fulfillthe urgent need of security forces for the fast detection ofexplosives. The recent Bali terrorist bombing case studydemonstrated the advantages of the mobile and portablelaboratory for study of postblast explosive residues at theterrorist attack site [3]. It is clear that the laboratory-basedequipment is too bulky and/or slow to meet the require-ments of security/emergency forces for rapid and decen-tralized detection of explosives. Microchip electrophore-sis devices can offer the ability to monitor high- and low-energy explosives at the sample source (before or afterterrorist attack) with significant advantages in terms ofspeed, efficiency, economy, and sample size.

There can be found good reviews on conventional andmicrochip CE in forensic/security analysis [4–6] in the lit-erature. The aim of this article is to overview the progresswhich has been made toward the development of elec-trophoretic methods for on-site and fast detection of

explosives and to describe the key developments in thisfield. This paper will review the possibilities, opportu-nities, and challenges of developing mobile electropho-retic laboratory and portable lab-on-a-chip device forremote screening of explosives.

2 Nitrated organic explosives

2.1 Detection techniques

Nitroaromatic compounds (see Fig. 1) can be directlydetected by UV-Vis absorbance [7]. This detectionapproach is widely used in conventional capillary elec-trophoretic analysis of nitroaromatic explosives, however,to less extent in microfluidic chips. The main problem ofthe UV-Vis detection is the linear dependence of absor-bency on optical path length. To address this, manycommercial conventional CE instruments use a UV-Visdetection cell with an increased path length, for examplebubble cell, Z-cell, or U-cell. In the chip configuration, thisis not easy to achieve, although the use of a micro-machined U-cell for integrated on chip UV detection wasdescribed by Kutter’s group [8]. An additional difficultywith UV detection on microchip format is that the mostlyused material for fabrication of micromachined chips,borosilicate glass, absorbs light with wavelengths shorterthan 380 nm. Fused-silica chip substrates can be used toovercome these problems but have not been investigateddue to the higher cost. LIF in its direct or indirect modecan be also used on microfluidic devices for detection ofexplosives [9, 10].

Electrochemical (amperometric) detection is the methodof choice for the analysis of nitroaromatic explosives onmicrochip devices since it offers up to three orders ofmagnitude higher sensitivity than indirect LIF and it has agreat potential for miniaturization and integration onmicrochip platform. Presence of nitrogroup allows its ca-

Table 1. Explosive compositions used in recent terrorist attacks

Incident Explosive composition

Oklahoma City bombing, 19.4.1995, USA Mixture of NH4NO3 and nitromethane

American Airlines airplane shoe bombing incident,22.12.2001, Paris, France

Triacetone triperoxide

Bali bombings, 12.10.2002, Indonesia TNT, KClO3, S, aluminum particles

Casablanca bombing, 16.5.2003, Casablanca, Morocco Triacetone triperoxide

Train bombing, 11.3.2004, Madrid, Spain Nitroglycerin-based explosive

Public transportation bombing, 7.7.2005, London,Great Britain

Thought to be RDX-based military explosive or home-madetriacetone triperoxide


246 M. Pumera Electrophoresis 2006, 27, 244–256

Figure 1. Structures of nitroaromatic and nitroamine explosives and explosive residues described in US EPA method 8330.

Table 2. Examples of explosive mixtures

Explosivename

Composition

Amatol NH4NO3 and TNTANFO NH4NO3 and fuel oilA-3 RDX and waxB RDX (60%) and TNT (40%)C-4 RDX and wax/oilsCyclotol RDX (75%) and TNT (25%)Black powder KNO3 (75%), carbon (15%), sulfur (10%)H-6 RDX and TNT with aluminum particlesOctol HMX (75%) and TNT (25%)Semtex-H RDX, PETN, oilsTorpex TNT (42%), RDX (40%) and aluminum

particles (18%)Tritonal TNT with aluminum particles

PETN, Pentaerythriotol tetranitrate

thodic reduction to form alkylhydroxyamines. The reduc-tion mechanism of polynitroaromatic compounds iscomplex and depends on the number of nitro groups, ontheir relative positions on the rings, on the nature of theother substituents in the aromatic system, and on pH ofthe solution. Trinitrocompounds, such as TNT or picricacid (PA), are more readily reduced than dinitro andmononitro compounds. The reduction potentials ofnitroaromatic compounds occur at more negative poten-tials on glassy carbon than on mercury film electrode [15].Reduction of nitrobenzenes (NBs) and nitrotoulenesoccurs in one single four-electron step to form arylhy-droxyamines, which is followed by two-electron stepforming arylamines [15, 16] (see reaction scheme below):

ArNO2 �!4e�þ4Hþ

�H2OArNHOH �!2e�þ2Hþ

�H2OArNH2

Two-electron reduction of aromatic nitro group to anitroso group has never been reported. This is attributed



to relatively higher reduction potential of the nitrosogroup in comparison to the corresponding nitro group[15].

2.2 Separation techniques

Nitroaromatic explosives and other nitrated organicexplosives are under normal conditions neutral com-pounds and therefore cannot be separated directly byCZE technique. Another separation vector must be intro-duced in order to achieve the resolution between thesolutes. Several conventional capillary techniques wereemployed, such as MEKC or CEC.

The most of the microchip-based methods rely on MEKCseparations. Cyclodextrin-mediated MEKC or octadecylsilica (ODS)-based CEC has not been reported on micro-fluidic devices up to date, which is surprising since themajority of microchip-based method for nitrated explo-sives use the amperometric detection mode and it isknown that presence of sodium dodecyl sulfate (SDS)decreases the sensitivity of the detection electrode [17,18]. Thus, a separation method, which obviates the use ofSDS, i.e., microchip electrochromatography [19], wouldbe highly beneficial to decrease the detection limits.

The original separation method was reported by Collins’group [20] which used strongly basic nonaqueous (ace-tonitrile/methanol) medium for ionization and consequentmCZE separation of trinitroaromatic explosives.

2.3 Monitoring of nitrated organic explosives

2.3.1 Conventional CE

Northop et al. [21] was able to separate 26 nitrated com-pounds (including TNT and nitroglycerin) from highlyexplosive samples using SDS in borate buffer (for typicalseparation, see Fig. 2). Northop’s paper sets a standardfor the most of the following works on separation oforganic explosives. Oehrle [7, 22] was able to resolve 12explosive components described in US EPA method 8330and additional nitroglycerin and pentaerythritol tetra-nitrate (PETN) in 15 min using SDS and borate buffer withUV detection. Nitroglycerin, picric acid, and stabilizerswere separated by MEKC and detected via diode-arraydetector [23]. Analysis of TNT and its eight bio-transformation products was also achieved under MEKCconditions with diode-array detector [24]. Kennedy et al.[25] applied indirect LIF technique for detection TNT andother eight nitrated aliphatic explosives, including nitro-

Figure 2. MEKC separation of a mixture of nitrated organic explosives. Conditions: 100 mm ID cap-illary, detection wavelength 220 nm, 2.5 mM borate, 25 mM SDS, sample concentration 100 mM. Re-printed from [21] with permission.



glycerin, however, with very poor sensitivity (for compar-ison of the detection limits, see Table 3). Completeseparation of US EPA 8330 mixture of 14 TNT-relatedcompounds using MEKC or CEC coupled to indirect LIFdetection was demonstrated by Bailey and Wallenborg[26]. A significantly improved LOD (,106 lower comparingto UV-Vis measurements) was achieved by Hilmi et al. [27]using an amperometric detector for MEKC determinationof TNT, RDX, HMX, and ten other important explosives.

The mixed-mode technique utilizing SDS and a negativelycharged sulfobutyl ether-b-CD was employed as exten-sion of MEKC separation technique for analysis of explo-sives and it showed very good separation efficiency.Improved resolution was attributed to the fact that such atwo-component pseudostationary phase provided differ-ent selectivities toward different analytes [28].

Conventional CEC using a capillary loaded with micro-meter-sized particles also became popular for separationof nitroaromatic explosives. Application of conventionalCEC to the separation of the US EPA 8330 mixture wasdescribed by Bailey and Yan [29] in connection to UV-Visdetection and to indirect LIF detection [26]. Under iso-cratic conditions, using 20 cm capillaries packed with of1.5 mm nonporous ODS particles, baseline separation of

all 14 of the components was achieved within 7 min.Using shorter columns and stronger separation fields, 13of the 14 components were resolved in less than 2 min (forexample of such rapid separation using conventionalCEC method, see Fig. 3) [29]. CEC with amperometricdetection on gold electrode was employed for US EPA8330 explosive mixture with the detection limits around100 ppb [17]. Described CEC method was two timesmore sensitive than MEKC method using the same baregold detector [17, 27]. Summarized details of analysis ofnitrated organic explosives using conventional CE arepresented in Table 3.

2.3.2 Microfluidic systems

Microfluidic device for detection of five TNT-relatedexplosive compounds with exchangeable carbon thick-film screen-printed amperometric detector was de-scribed by Wang et al. [30]. This detection designpermitted convenient and rapid replacement of the detec-tor. The LOD of this method was 600 ppb. The need toimprove detection limits of nitroaromatic explosives onmicrofluidic platform led several researchers to exploreotherelectrode materials, offering better sensitivity. Luong’s

Figure 3. Rapid CEC separation of explosives. Column: 25 cm675 mm ID, 12 cm packed with1.5 mm nonporous ODS II particles. Mobile phase: 7.5% methanol, 7.5% 2-propanol, 85% 10 mM

MES, 5 mM SDS. Sample concentration, 12.5 ppm (each component). Peaks: (1) HMX, (2) TNB, (3)RDX, (4) DNB, (5) NB, (6) TNT, (7) Tetryl, (8) 2,4-DNT, (9) 2,6-DNT, (10) 2-NT, (11) 4-NT, (12) 2-Am-DNT,(13) 3-NT, and (14) 4-Am-DNT. Reprinted from [29] with permission.



Table 3. Analysis of nitrated organic explosives by microchip electrophoresis and conventional CE

Analyte Separationtechnique

Detection technique Detection limit Time ofanalysis

Refer-ence

Microchip electrophoresis

TNT; DNB; 2,4-DNT; 2,6-DNT; 4-NT Microchip MEKC Amperometry, screen-printedcarbon electrode, at 20.5 V

,600 ppb (TNT) 210 s [30]

TNT; RDX; 2,4-DNT; 2,6-DNT; 2,3-DNT Microchip MEKC Amperometry, gold wireelectrode, at 20.7 V

110 ppb 400 s [31]

TNT; 2,4-DNT; 2,6-DNT; 2,3-DNT Microchip MEKC Amperometry, gold electrodedeposited onto channeloutlet, at 20.8 V

24 ppb (TNT) 130 s [32]

TNT mFIA Amperometry, mercury/goldamalgam electrode, at 20.6 V

7 ppb (TNT) 30 s [18, 33]

TNT, 1,3-DNB, 2,4-DNT Microchip MEKC Amperometry, boron-dopeddiamond electrode, at 20.7 V

70 ppb (1,3-DNB);110 ppb (2,4-DNT)

200 s [34]

TNT; TNB; DNB; 2,4-DNT;2-Am-4,6-DNT; 4-Am-2,6-DNT

mFIA/microchipMEKC

Amperometry, screen-printedcarbon electrode, at 20.5 V

60 ppb (TNT andDNB)

25 s (screeningmode); 150 s(fingerprintmode)

[35]

TNT, DNT, TNB mFIA/microchipMEKC


800 ppb (TNT); 450ppb (TNB)

36 s (screeningmode); 140 s(fingerprintmode)

[36]

TNT; DNB; TNB; NB; Tetryl; 2,4-DNT;2,6-DNT; NT; 2-Am-4,6-DNT;4-Am-2,6-DNT

Microchip MEKC Indirect LIF, visualizingagent Cy7

Around 1 ppm 60 s [37]

TNB; TNT; 2,4-DNB; 2-Am-4,6-DNB Microchip MEKC(using LiDS)


80 ppb (TNT) 120 s [58]

TNT; TNB; 2,4-DNT; 1,3-DNB; 2,4-DNP Microchipimmunoassay

Direct LIF Around 1 ppb 50 s [10]

TNT; TNB; Tetryl Nonaqueousmicrochip CE

UV-Vis at 505 nm 160 ppb (TNT); 60 ppb(TNB); 200 ppb(Tetryl) without exsitu preconcentra-tion; 340 ppt (TNT);250 ppt (TNB);190 ppt (Tetryl) withex situ preconcen-tration

20 s [20]

Conventional CE

DBP; DEGDN; 1,3-DNN; 1,5-DNN; 1,8-DNN; EGDN; NG; NGU; 2-MNN; 2-NT;3-NT; 4-NT; PETN; PA; Tetryl; HMX; TNT;RDX; EC; 2,3-DNT; 2,4-DNT; 2,6-DNT;3,4-DNT; DPA; 2-nDPA; N-nDPA

MEKC using SDS UV-Vis at 220 nm 561026 M for ni-troaromatic com-pounds, 161026 Mfor nitroaliphaticcompounds and561025 M forPETN and HMX

10 min [21]

HMX; RDX; TNB; TNT; 2,4-DNT; 2,6-DNT;NG; PETN; Tetryl; 2-NT; 3-NT; 4-NT;NB; DNB

MEKC using SDS UV-Vis at 185, 214, 229,and 254 nm

Less than 1 ppm 15 min [7]

EPA 8330 explosives MEKC using SDS UV-Vis at 254 nm N/A 14 min [22]



Table 3. Continued



Refer-ence

NGU; PA; NG; 3,4-DNT; 2,3-DNT;2,4-DNT; 2,6-DNT; 2-NT; 4-NT; 3-NT;N-nDPA; DPA; 2-nDPA; EC; DBP.

MEKC using SDS UV-Vis (diode-array) N/A 12 min [23]

TNT; TAT; 2,6-DAm-NT; 2,4-Dam-NT;2-HADNT; 4-HADNT; 2-Am-4,6-DNT;4-Am-2,6-DNT

MEKC using SDS UV-Vis (diode-array) 100–200 ppb 8 min [24]

TNT; Tetryl; NG; PETN; RDX; HMX;EGDN; NGU

MEKC using SDS LIF, 488 nm excitation;fluorophore, fluorescein,or rhodamine B

161024–461024 M 7 min [25]

EPA 8330 explosives CEC (1.5 mmnonporous ODSsilica) or MECKusing SDS

LIF, Cy5 1–10 ppm 33 min [26]

HMX; RDX; 1,4-DNB; NB; 1,2-DNB; TNT;2,4-DNT; 2,6-DNT; 3,4-DNT; 3-NT;2,3-DNT; 2-Am-4,6-DNT;4-Am-2,6-DNT

MEKC using SDS Amperometry at 20.7 V,silver-on-gold electrode

70–110 ppb 9 min [27]

2-NT; 3-NT; 4-NT; 1,2-DNB; 1,3-DNB;1,4-DNB; 2,3-DNT; 2,4-DNT; 2,6-DNT;3,4-DNT

MEKC using SDSand sulfobutylether-b-CDCD

UV-Vis at 214 nm N/A 13 min [28]

EPA 8330 explosives CEC (1.5 mmnonporousODS silica)

UV-Vis at 254 nm N/A 2 and 7 min [29]

EPA 8330 explosives (3 mm nonporousODS silica)

Amperometry at 21.0 Vbare gold electrode

75–170 ppb 85 min [17]

FIA, flow injection analysis

group [31] employed gold-wire electrode in end-columnwall-jet configuration, which resulted into detection limitof explosives around 150 ppb. The same group exploitedfurther the use of gold electrode material. The microchipwith gold electrode fabricated on the channel outlet byelectroless deposition was prepared [32]. The gold filmwas deposited using Na3Au(SO3)2 and formaldehydesolutions according to the reaction:

2Au(I) 1 HCHO1 OH2 ? HCHO2 1 H2O1 2 Au(0)

Such a deposited gold detector showed a LOD of 24 ppbfor TNT. So far, the lowest detection limit for microchipelectrophoresis-amperometry was found to be 7 ppb forTNT using a mercury/gold amalgam electrode [18, 33].However, such a low detection limit was coupled only tothe m-FIA mode.

Electrode surface fouling is a problem in amperometricdetection. Wang et al. [34] introduced a boron-dopeddiamond electrode with highly stable response towarddetection of explosives. While the thick-film carbon

detector displayed a gradual decrease of the TNT re-sponse (with a 30% decrease and an RSD of 10.8%;n = 60), a highly stable signal was observed upon usingthe diamond electrode (RSD of 0.8%; n = 60). Suchresistance to surface fouling reflected the negligibleadsorption of explosive reduction products at the boron-doped diamond electrode surface.

The majority of the developed techniques for detection ofexplosives on a lab-on-a-chip platform show analysistimes around 120 s, insufficiently long for fast detection ofterrorist weapons. To solve this problem, a single-channelchip-based analytical microsystem that allowed rapidflow injection measurements of the total content oforganic explosive compounds, as well as detailed micel-lar chromatographic identification of the individual ones,was described [35]. The protocol involved repetitive rapidflow injection (screening) assays to provide a timelywarning and switching to the separation (fingerprint iden-tification) mode only when explosive compounds weredetected (for protocol scheme, see Fig. 4). While MEKC



Figure 4. (A) Hypothetical analytical cycle for sequential“total” and “individual” on-chip measurements of explo-sive compounds. (S) Screening assays of the total con-tent; (W1 and W2) wash steps (switching to the “separa-tion” or “flow injection” modes, respectively); (F) finger-print identification of the individual compounds. (B)Electropherograms depicting the total (a) and individual(b) assays for mixtures of nitroaromatic explosives. Con-ditions: injection voltage, 2000 V; injection time, 3 s;separation voltage, 5000 V (a) and 2000 V (b). Adaptedfrom [35] with permission.

was used for separating the neutral nitroaromatic explo-sives, an operation without SDS led to high-speed meas-urements of the “total” explosives content. Switching be-tween the “flow injection” and “separation” modes wasaccomplished by rapidly exchanging the SDS-free andSDS-containing buffers in the separation channel.Amperometric detection was used for monitoring theseparation. Assays rates of about 360 and 30 per hourwere thus realized for the total screening and “individual”measurements, respectively. Method for fast FIA micro-chip screening of TNT with novel world-to-chip interfacewas described later by the same authors [36].

Indirect LIF was used to detect explosive compoundsafter their separation by MEKC [37]. To achieve indirectdetection, a low concentration of a dye (5 mM Cy7) wasadded to the running buffer as a visualizing agent. Usingthis methodology, a sample containing 14 explosives (USEPA 8330 mixture) was examined, however, with poordetection limits for nitroaromatic explosives (around1 ppm) and nitramine explosives (RDX, HMX; LOD around2000 ppm). Such a huge difference in LOD was attributedto the low fluorescence quenching efficiencies of nitra-mines compared to the nitroaromatic explosives. Thesignificantly improved detection limits of explosives onlab-on-a-chip/LIF platform were demonstrated by Brom-berg and Mathies [10]. A homogeneous immunoassay ofTNT was based on the rapid microchip electrophoreticseparation of an equilibrated mixture of an antiTNT anti-

body, fluorescein-labeled TNT, and unlabeled TNT or itsanalog. A TNT immunoassay was sensitive (LOD of1 ppb), having a wide dynamic range (1–300 ppb).

Pushing detection limits of nitroaromatic explosives intothe ppt (parts per trillion) level requires sample pre-concentration. Collins and coworkers [20] employed SPEof explosives from sea water which was followed by rapidon-chip separation and detection. Explosives were elutedfrom SPE column by ACN and were injected in themicrochip separation channel. Lab-on-a-chip analysiswas carried out in nonaqueous medium. The mixed ACN/methanol separation buffer was used to produce theionized red-colored products of TNT, TNB, and Tetryl [38,39]. The chemical reaction of the bases (hydroxide andmethoxide anions) with trinitroaromatic explosives result-ed in negatively charged products, which were readilyseparated by microchip zone electrophoresis (for reactionscheme, see Fig. 5) [20]. It is expected that nonaqueousmicrochip electrophoresis will play more important role inthe future in the detection of explosives for its inherentcompatibility with organic solvents used in pre-concentration techniques [20, 40]. Summarized details ofanalysis of nitrated organic explosives using microchipelectrophoresis are presented in Table 3.

Figure 5. Chemical reaction of TNT, TNB, and Tetryl inbasic ACN/methanol. Reprinted from [20] with permis-sion.



3 Inorganic explosives

3.1 Detection techniques

Many of commercial and improvised explosives, such asammonium nitrate-fuel oil (ANFO), flash compositions,and black powder contain ammonium or sodium nitrate orperchlorate. The determination of corresponding pre- andpostblast ions, such as ammonium, methylammonium,potassium, sodium, chlorate, perchlorate, chloride, ornitrate, is crucial for the proper identification of suchexplosives and their residues at terrorist scene [3, 41, 42].The ionic components of the postblast residue are dis-tinctive from its preblast composition and reflect the ori-ginal composition of the corresponding explosive device[41]. Since the above-mentioned ions do not show ab-sorbance in UV-Vis area, they cannot be detected directlyby means of UV-Vis detection. Several techniques imple-menting indirect UV detection with visualizing agents [43]for detection of explosive-related ions were demon-strated on conventional CE platform. The main drawbackof indirect UV detection is its lower sensitivity than directUV detection and its difficult application to microchipplatform, as described in Section 2.1.

Conductivity detection is a simple and universal detectiontechnique for analysis of ionic species. Such detectioninvolves measurement of the conductance between twoelectrodes (through which an alternating current is pas-sed) and allows convenient detection of ionic species (notreadily detected by other techniques) down to the sub-micromolar level. Conductivity detection can be accom-plished either by a direct galvanic contact of the run bufferand the sensing electrodes or by a contactless mode inwhich the electrodes do not contact the solution. Thecontactless detection route has several advantages overthe contact mode, including the absence of problems(i.e., electrode passivation, bubble formation) associatedwith the electrode-solution contact, effective isolationfrom high separation voltages, a simplified constructionand alignment of the detector (including placement of thedetector or multiple ones at different locations), and theuse of narrow capillaries [44, 45] and microchannels [46].For these advantages the contactless conductivitydetection became the only method applied for monitoringinorganic explosives on lab-on-a-chip platform.

3.2 Separation techniques

Separation of the ionic components is achieved by zoneelectrophoretic technique, based on different electro-phoretic mobilities of the corresponding ions. The prob-lem with comigrating ammonium and potassium ions,which have very similar mobilities and both can be pres-

ent in explosive sample, can be solved by addition of 18-crown-6 ether. Potassium ion forms complex with 18-crown-6 ether, thus decreasing its effective mobility andallowing its separation from ammonium ion.

3.3 Monitoring of inorganic explosives

3.3.1 Conventional CE

Material from several explosive devices (i.e., black pow-der pipe bomb) and their residues was analyzed byMcCord et al. [41]. Separation of ten black powder-relat-ed postblast anions was accomplished in 15 min. Theindirect UV-Vis detection was carried out at two wave-lengths, 205 and 280 nm. Analysis of several low-energyexplosives was performed by Kishi et al. [47]. They suc-cessfully identified several ions (sodium, ammonium,nitrate, chloride, chlorate, perchlorate) from real home-made explosive residue sample, emulsion explosive resi-due, and firework perchlorate-based explosive. Theanalysis of cations and anions was carried out in separateruns. McCord’s group [48] showed separation of morecomplex mixtures, containing ten explosive-relatedcations. The same group recently presented CE tech-nique for simultaneous detection of explosive residuecations and anions via dual end injection, which shor-tened total analysis time to 7 min [49]. Summarized detailsof analysis of inorganic explosives using conventional CEare presented in Table 4.

3.3.2 Microfluidic systems

The problem of the above-described conventional capil-lary electrophoretic methods is the long time of analysis;the shortest analysis time was around 7 min. Microfluidicdevices show the ability to shrink the analysis time to tensof seconds. The fast analysis of preblast and postblastcations and anions on microchip device in 60 s wasshown by Wang et al. [42]. The low EOF of the poly-(methyl methacrylate) (PMMA) chip material facilitated therapid switching between analyses of cations and anionsusing the same microchannel and run buffer, and pro-vided rapid (,1 min) measurement of seven explosive-related cations and anions (see Fig. 6).

Further reduction of the analysis time of explosive-relatedcations was achieved using movable contactless con-ductivity detector [50]. Placing such movable detector atthe different locations along the microchannel can pro-vide useful insights into the separation process. 3-D plotsof resolution/channel length/separation voltage wereused for optimizing the separation process of explosivesand selecting the analysis time. This allowed reducing



Table 4. Analysis of inorganic explosive components by microchip electrophoresis and conventional CE



Refe-rence

Microchip electrophoresis

Pre- and postblast explosive residues(K1; NH4

1; Na1; MMA; NO32;

Cl2, ClO42)

Microchip CE withaddition of 18-crown-6 ether

Contactless conductivitydetection at 200 kHz, 5 Vp-p

3.2 mM (NH41); 5.8 mM

(MMA); 6.2 mM (K1);5.6 mM (Na1); 8.7 mM(Cl2); 7.2 mM (NO3

2);6.2 mM (ClO4

2)

60 s all ions [42]

Postblast explosive residue cations(NH4

1; Na1; MMA)Microchip CE Contactless conductivity

detection at 200 kHz, 10 Vp-p

N/A 60 s [64]


1; Na1; MMA; NO32;

Cl2, ClO42)

Microchip CE withaddition of 18-crown-6 ether

Movable contactlessconductivity detection at200 kHz, 5 Vp-p

N/A 17 s (screeningmode), 45 s(fingerprintmode)

[50]


1; Na1; MMA; NO32;

Cl2, ClO42)

Microchip CE Movable contactless conductivitydetection at 200 kHz, 10 Vp-p

80 mM (NH41); 70 mM

(Na1); 150 mM (Cl2);130 mM (ClO4

2)

60 s all ions [51]

Postblast explosive residue cations(NH4

1; Na1; MMA)Microchip CE Contactless conductivity

detection at 200 kHz, 5 Vp-p

50 mM (NH41) 60 s [58]

Conventional CE

Black powder pipe bomb anions(Cl2; NO2

2; NO32; SO4

2-; SCN2;ClO4

2; HCO32; HS2; OCN2)

CE Indirect UV-Vis, using dichromateions as visualization agent, at205 and 280 nm

N/A 15 min [41]

Emulsion explosives (NH41; Na1;

NO32; Cl2,32; ClO4

2)CE Indirect UV-Vis at 214 nm; buffer

consisted 5 mM chromate ionas UV visualization agent

N/A 5 min [47]

K1; NH41; Ba21; MMA; Sr21; Na1;

Ca21; Al31; Mg21; Li1; Co21; Zn21

CE with additionof 18-crown-6ether

Indirect UV-Vis at 215 nm; bufferconsisted 5 mM imidazol as UVvisualization agent

500 ppb 7 min [48]

K1; NH41; Sr21; Na1; Ca21; Mg21;

Br2; Cl2; NO22; NO3

2; SO42-;

ClO42; SCN2; ClO3-

CE with additionof 18-crown-6ether

Indirect UV-Vis at 215 nm; bufferconsisted 5 mM imidazol as UVvisualization agent for cationsand 1,3,6-naphtalenetrisulfoniacid for anions

0.8–15 ppm 7 min [49]

N/A: not available.

analysis time of explosive-related compounds by ,30%.Movable detector system also allowed switching be-tween total (unresolved) and individual (resolved/finger-print) signals by placing the sensing electrodes at thebeginning or the end of the separation channel. Totalassays of explosive-related ions were performed within13 s (anions) and 11 s (cations). A rapid switching of thedetector position to the end of the separation channel ledto well-resolved anion and cation peaks, with migrationtime of the last peak of 50 s.

Using dual end injection scheme, it was possible todetect inorganic explosive-related cations and anions ona single-channel device in one analytical run [51]. The

chip device consisted of two sample reservoirs, on bothsides of a common separation channel (see Fig. 7).Anions and cations were simultaneously electrokineticallyinjected into both ends of the separation channel. Due tolower EOF in polymer channels compared to glass ones,the cations and anions migrated in opposite directionsand were separated from each other and detected using amovable contactless conductivity detector positionedaround the center of the separation channel.

The all above-described ionic-explosive microchip sys-tems combine the distinct advantages of contactlessconductivity detection with the attractive features ofpolymeric microchips, such as the potential for single-use



Figure 6. Schematic of the rapid single-channel micro-chip assays of explosive-related cations and anions. (A)Injection of cations, (B) separation and detection ofcations, (C) injection of anions, and (D) separation anddetection of anions. (a, b, d) Reservoirs containing runbuffer, (c) sample reservoir. Electropherograms showingthe separation of preexplosive (E) and postexplosive (F)model mixtures of cations (a) and anions (b) using con-tactless conductivity detection. (E, a) Ammonium (1),potassium (2), sodium (3), each at 700 mM. (E, b): nitrate(5), perchlorate (6), sample concentration 1050 mM. (F, a)Ammonium (1), methylammonium (4), potassium (2),sodium (3), sample concentration 700 mM. (F, b) Chloride(7), nitrate (5), perchlorate (6), sample concentration700 mM. Adapted from [42] with permission.

and disposable lab-on-a-chip device [52]. Summarizeddetails of analysis of inorganic explosives using microchipelectrophoresis are presented in Table 4.

4 Peroxide-based explosives

There has not yet been presented any electrophoretictechnique for separation and detection of acetone per-oxide-based explosives. There was reported detection oftriacetone triperoxide by MS in conjunction to GC [53] andLC [54]. Other methods were based on UV degradation oftriacetone triperoxide explosive to the hydrogen peroxide,which was subsequently determined on the basis of theperoxidase-catalyzed oxidation of p-hydroxyphenylaceticacid to the fluorescent dimmer [55, 56] or peroxidase-cat-alyzed oxidation of 2,2’-azino-bis(3-ethylbenzothiazoline)-6-sulfonate to green-colored radical ion [56].

The future development of the lab-on-a-chip device forthe detection of triacetone triperoxide could take advan-tage from UV degradation of triacetone triperoxide tohydrogen peroxide and from already developed micro-fluidic-amperometry method for rapid detection oforganic peroxides [57].

5 Explosive mixtures

Several explosive devices contain mixtures of organicand inorganic explosives, i.e., Amatol or Tritonal (con-taining TNT and NH4NO3 or TNT and aluminum particles,

Figure 7. Schematic diagramsof the dual-injection microchipelectrophoretic system withmovable contactless con-ductivity detector for simulta-neous detection of ionic explo-sive-related anions and cations.(A) Injection mode; (B) separa-tion mode. (a, e) Running bufferreservoirs; (b, d) unused reser-voirs; (c, f) sample reservoirs; (g)injected cation plug; (h) injectedanion plug; (i) movable contact-

less conductivity detector; (j–l) cations; (m–o) anions. Injection, by applying a positive voltage to reservoir c and ground toreservoir f, while other reservoirs floating. Separation, by applying a positive voltage applied to reservoir a, ground toreservoir e, while other reservoirs floating. (C) Electropherograms for a sample mixture containing (a) 2 mM ammonium; (b)1 mM methyl ammonium; (c) 1 mM sodium; (d) 1 mM chloride; (e) 1 mM nitrate; and (f) 1 mM perchlorate introduced by (1)cathodic injection; (2) anodic injection; (3) dual injection. Adapted from [51] with permission.



Figure 8. Electropherograms showing the simultaneousmeasurement of inorganic and nitroaromatic explosives,as recorded with the contactless conductivity (a) andamperometric (b) detectors. Analytes: ammonium (1),methylammonium (2), sodium (3), TNB (4), TNT (5), 2,4-DNB (6), and 2-Am-4,6-DNB (7), system peak (SP). Re-printed from [58] with permission.

respectively; see Table 2), and analytical method for theirfast detection is needed. The dual electrochemicalmicrochip detection system containing two orthogonaldetection modes (conductivity and amperometry) facili-tated the measurements of inorganic explosives andnitroaromatic explosive components in one analytical runon single-channel microchip [58]. The conductivitydetector profiled only the ionic species; the amperometricone responded to the redox active nitroaromatic compo-nents. The total assay of explosive mixture related toAmatol was performed within 2 min (see Fig. 8).

6 Conclusions and future prospects

It is demonstrated in this review that the electrophoreticmethods, in both conventional and microchip setups,reached maturity and are suitable for explosive analysis.However, to create easy-to-operate field-portable instru-ments for preblast explosive analysis would requireincorporation of world-to-chip interface, which would beable to continuously sample from the environment. Sig-nificant progress toward this goal was made, and inte-grated on-chip devices which allow microfluidic chips tosample from virtually any liquid reservoir were demon-strated [36, 59].

Further improvement of detection limits of explosivecompound would be beneficial for explosive analysis.LODs of nitroaromatic explosives can be reducedemploying microchip electrophoresis-amperometry withon-column detection with effective decouplers [60],which already proved the ability to push detection limitsdown of two to three orders of magnitude when com-pared to end-column amperometric detector configura-tions. Further lowering of the detection limits for inorganicexplosives would require employing conductivity detec-tion with high excitation peak-to-peak voltage [61], whichshows one order magnitude lower detection limits forionic species than commonly used contactless con-ductivity detection [46]. On-chip enrichment can beaccomplished using a precolumn ion-exchange pre-concentration chamber (for ionic explosives) [62] ordeveloping monolithic preconcentration chamber (fornitroaromatic explosives) [63].

There is a wide selection of electrophoretic methodssuitable for development of fast-responding, field-deployable micro total analytical device for detection ofnitroaromatic and inorganic explosives. However, a simi-lar technique for the analysis of triacetone triperoxide,which was involved in the recent terrorist bombings ismissing. Further development of counter-terrorist lab-on-a-chip devices should also be focused on this explosivecompound to meet the needs of the society.

The author is grateful for the support from the Marie CurieIntra-European Fellowship from European Communityunder 6thFP (project MEIF-CT-2004–005738).

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