1.7. Analytical SchemeSampling of article is very first step of forensic analysis. Sampling is the key to forensic analysis. Physical evidence should be pack separately and labeled so that its integrity and identity is remain maintained. The record of custody of documents for all evidences should be maintained from the point of its discovery, to its transporting to the lab for examination and its preservation till it is finally submitted to the court. Questioned samples are samples those were gathered and that are to b analyzed while control Samples are the reference samples that are known. Forensic analyses may be performed to for any one of these purposes (1) to identify unknown material (2) for the purpose to determine the source of its origin comparing a questioned and a known sample. Identification is the first task when questioned sample is brought in forensic lab for study. 2 types of analysis can be done to detect a substance: first is presumptive tests and 2nd is confirmatory analysis. Comparative study links a question sample and a control sample, if they are same or not. Class characteristics are properties of a substance that are shared by a group of substances, but are not unique to all. To individualize individual Characteristics of a substance are used with a high degree of probability. A forensic analysis may consists of following steps: (1) recognition of analyte sample; (2) collection of sample material, proper storage, preservation and documentation; (3) preparing for analysis; (4) analysis of samples; (5) results shows facts of the case and (6) reconstructing the events and (7)presenting these results in law courts10-12.Fig1.3. Analytical Scheme Flow Chart Fig 1.4.The Stages of Analysis1.8. Aims and ObjectivesThis thesis mainly focuses on chemical analysis of explosives materials by techniques used in forensic laboratory. Detection and identification of materials like explosive materials after an explosion is the major challenge and topic of the wide interest now days due to increased terrorist activities. Different analytical techniques used in detection will be discussed here and most accurate and mostly used will be thoroughly studied. Two types of analytical methods will be discussed as presumptive and confirmatory methods of detection. The first procedure utilized the cotton hands swab calorimetric method for detection and the preparation of micro fluidic paper based devices for onsite detection of many types of explosives. 2 micro fluidic devices were created to cover the large range of explosive materials. One device will be used to detect inorganic explosive materials and 2nd device will be used to test high-organic explosives materials such as nitro-aromatics, nitro-amines, urea-nitrate, and per-oxides. The second method involved a confirmatory procedure developed a non aqueous infusion EC-ESI TOF-MS used to test explosives of both organic and inorganic nature. As inorganic compounds cannot be detected by using MS, so 18 crown 6 ethers are added into these explosives and complex formation will allowed the detection of these explosives by incorporating and increasing their masses. To detect peroxides EC detector will be placed before the MS.CHAPTER 2 LITERATURE REVIEWDetection of explosives selectivity and sensitivity has increased dramatically by technology advancement. So by this advancement scientists enlarge the list of detectable explosive materials. As a result of global terrorism a large number of methods have been developed to detect improvised explosive devices or military explosives. The advancement in these analytical techniques have made possible to detect the explosives more quickly and selectively. As due to more energetic and complex explosive materials are utilized, packaging with cleverness, and change of methods, the detection of explosives have been became a challenge for law enforcement agencies.2.1. Improvised/Homemade and Military Based ExplosivesConventional explosives are made of elements mainly: C, O, N, and H and are used by military mostly. TNT, HMX, PETN, and nitrocellulose are some examples. These are all nitro-based organic explosives. These explosives are manufactured by nitration of organic aromatic compounds. It is twofold substitution or double displacement. In which nitro group is added to the nitrated molecules 13. These are secondary explosives due to some stability to shock or heat. They need a primary explosive for detonation (such as lead azide, mercury fulminate) 13. As in most of the cases terrorists use these explosives so the major focus is to develop techniques to detect them. Low explosives produce similar violent exothermic reactions but their composition is different to that of compositions of high explosives. Examples are IEDs, HMDs, black powders and smokeless powders. Some new explosive materials have been synthesized that have desired properties 14-17. Those explosives which have high nitration have high densities and their small quantities produce high explosion. Large terrorist activities are with these primary explosives. Use of such explosives is a major issue for those nations having terrorist threat. Researchers accept these challenges and, searching for new detection technologies for explosives. Some of the techniques will be outlined in this review.2.2. Explosives Characteristics based CharacterizationFor complete characterization a compound, its different properties are determined like chemical, physical, mechanical, acoustic, thermal, and electromagnetic properties. These materials are investigated by use of many energy sources to understand its physical and chemical properties. Effective characterizations help in detection and recognition of the material and to improve the detection technologies. 2.2.1. Surface adhesion-characterization of explosivesThis method is a method of used for trace detection explosives on the surfaces of debris. These trace components are checked on surfaces of hands of suspects or where there explosives were prepared 18. Contact sampling is most successful and widely used 19-27. Swabbing methods is also somewhat useful to collect these explosive materials on the smooth surfaces but this field needs some improvements. The estimation of the contact area between a particle and a surface is the main drawback 28. Zakon et al. 28 describes different types of forces that play their role in particle adhesion that are charges, van der Waals forces, chemical bonding, and other primary and secondary forces. Recently, Beaudoinet al. 29 researched those van der Waals forces have some affects on military explosives against aluminum alloys with 3 automobile surfaces 29. These forces were measured by Atomic Force Microscopy 30. 2.2.2. Physical characterization of explosives–surface morphology To characterizing shock sensitivity and explosive power surface structure have to known 31. We need such explosives that can be easily transported and can be stored for long time. A lot of work is continuing in this field largely on RDX and PETN that are stable explosives 32-35. Researches on these showed that their initiation and detonation mechanism is heavily depending upon their densities 36-38, surface areas 39, particles size 40-42, and crystal morphologies 43, 44. Surface morphologies of triaminotrinitrobenzene, ammonium perchlorate, and PBXs is mostly characterizwd by AFM and SEM 45-47 and many others. But recently, Focused Ion Beam nanotomography is able to determine morphology information’s, like pore and particles size of crystalline explosives researched by Wixom et al. FIB nanotomography is able to provide these information’s of vapors deposited PETN and hexanitrostilbene though it is typically used for rough surfaces. These techniques give accurate values of density, which related to explosive performance. Mason, and Hooper first time used ultra-small angle neutron scattering techniques to detect the morphologies of RDX in 201048. SANS/USANS determine the internal features with 10Å–20?m length scales. These techniques were able to differentiate samples structural features and their sensitivity. Another technique is tailoring of surface also used to determine the properties of explosives materials 49-50. Wang et al. formed ultra-fine hexa-nitro-hexa-aza-iso-wurtzitane with ultra sound-and spray-assisted method in 2012 51. This study revealed that the sensitivity of the CL-20 was decreased as its critical temperature decreased. 2.2.3. By Thermal Properties of ExplosivesThe vapor pressure of explosives is heavily dependent on external temperature and increase with little increasing of temperature 52. In explosives materials by use of mechanical excitations to produce heat is well studied 53, 54. RDX and lead azide etc can be initiate by using low frequency vibrations as given by Loginov et al. in 1976 53. These techniques are off site detection methods, if external source of heating were used like ultrasonic radiation 55, 56. Mares et al. studied the thermal and mechanical properties of a HMX and 2 other materials with mechanicaly excitation of range 50 kHz to 40 MHz 57. They showed that mechanism of heat production by mechanical excitation is heavily depending upon frequencies of heat source. In Kovalev and Sturm revealed that by heating surface of 2,4,6-TNP by AFM tip a single crystal showed57. As mechanism of decomposition was not established, experimental evidence favor the hot spot formation model 58-188.8.131.52. Direct characterization of explosives material The explosives are widely researched to make possible their storage and transportation because some explosives are unstable. Characterization and development of new materials is vital for the inspection of damage evolution. Direct evaluation methods like SEM 62, CT 63, and AE 64 are mostly used to check the damage caused by PBXs. Photo-mechanic techniques, like DIC65, and Atomic Emission were used by the Wang et al. 2.2.5. Terahertz (THz) spectroscopy (EM characterization of explosives)Terahertz spectroscopy is used for 3D imaging of solids 55 since 1990’s. The principle of this technique is not elaborated here 66-68 but, this covers the range from 3 cm-1 to 600 cm-1, called far-infrared (far-IR) also. Every explosives material has distinctive THz absorption band that that shows their structural behavior. The THz pulsed spectroscopy has experimental spectral range of the EM spectrum 69. Terahertz spectroscopy has many applications including characterization of explosives 70-74, also providing information about EM responses of the materials. Etayo et al. 75 used Terahertz for many types of explosives and determined their properties like refractive index, absorbance, and permittivity 75. 2.3 Sampling MethodsSampling is most important step in explosives detection. Many sampling methods have been developed for explosives detection and depending upon the vapor phase concentrations of most common explosives. Common sampling methods use pre-concentration method as vapor concentration. Surface (contact) sampling method and remote (non-contact) sampling method have been discussed here.2.3.1. Pre-concentration techniques This process utilizes a target gas absorbing onto an adsorbent. For generation of high concentrations of target compound pre-concentration step is followed by thermal desorption 76. SPME 77-79, is widely used for high explosives. Development or suitable adsorbents for the pre-concentration of explosives are widely studied recently 80. A novel diethoxy diphenylsilane-based coating for planar SPME was developed by Mattarozzi and colleagues to get quantization limits in the low nano-gram level 81. SPE a liquid concentration technique 82-84 was mostly used for explosive detection. An on-line SPE joined with LC-MS for detection of explosives materials in water 84 was used by Sun et al. in 201184. Their LOD were very good.More needed to develop and optimize the currently used instrumentation for pre-concentration 85. 2.3.2. Surface sampling techniquesSwabbing of explosive residues is most common surface sampling technique that is being used from many 87- 89. Electrostatic precipitator technique is more novel contact sampling technique used as mentioned in the section “Prec-concentration techniques” 10. Bandodkar et al. 90 developed a FT (fingertip) sensor for detection of explosive surfaces. This fingertip consists of electrodes systems and an electrolyte. Explosive materials being analyzed are transferred into the electrode surface with swiping the sensor with the given sample area. this fingertip method is a portable detection for on-site detection of explosives rapidly.Ionization sources that have been used depend on need of the sample. LIBS 91, 92, DESI 93, 94, DART 95, paper spray ionization 96, 97, thermal desorption 98, 99, low-temperature plasma probe 100, DBDI 101, and ELDI 102 are used as ionization source.2.3.3. Remote Sampling Techniques (Non-Contact)These sampling techniques of explosives are very challenging as most of the explosives have very low vapor pressures, which make them less available for sampling. Despite of this difficulty, many instruments have been developed to increase efficiency, selectivity, and sensitivity of remot sempling 103-106. He et al. 107 made a method for study of AFAI for ambient MS 107. BY using Helium plasma ionization (HePI) in 2012, Yang, Pavlov, and Attygalle make sampling of TNT vapors from 5m away by using MS 108 and by Gopalakrishnan and Dichtel in 2013, conjugated polymer were used to detect RDX vapors 109. Takada et al. 110 formed a portal system that uses a push and pull vapor sampler. This vapor sampler joined with APCI source and an IT-MS and was installed at a ticket gate. Field tests performed were to check the false-positive rates for the portal system. The other sampling techniques like fluorescence quenching 110-112, portal technologies that do not require pre-concentration, so greatly used.2.4. Detection of ExplosivesA large number of advancement has been made in the detection techniques but there is a search of a perfect detector still. All techniques used for detection have some flaws for detectors. The goal of this research is to develop faster, sensitive, inexpensive and simple detectors which can detect the explosive material more efficiently. So this study focused on the development of such a detector that can be used for both organic and inorganic explosives. 2.4.1. Presumptive On-Site Detection of Explosives184.108.40.206. By Sensors Sensor is generally having high sensitivity, small size, autonomous and inexpensive can be used for explosive trace detection. These are mainly used as micro-electromechanical sensors like metal oxides sensors 113, micro-mechanical sensors 114, quartzcrystal micro-balance sensors 115, and SAW sensor 116. Some more sensors that are used for the identification of explosives are fluorescence besed 117, luminescence based 118, and electrochemical 119 based sensors. C- nanotube-based sensors showed faster response times and have higher sensitivities and have been studied much due to their highly effectiveness 120. For MEMS applications C- nanotube-based sensors having carbon based materials have suitable properties to be used 121. Though these are highly effective but poses some difficulties, as vapor or a gas may e adsorbed into the nanotubes, and their resistance changed 122. 220.127.116.11. By CaninesSince World War II trained canines have been used mostly for explosive detection purposes 124. Till now canines detection of explosives is used as a standard for on-spot methods of detection of explosives. The factors that affect the smell of trained dog are amount of analyte; container volume, vapor pressure of explosive and temperature that effect vapors formation of explosives 125. The canine limit of detection at atmospheric pressure and with different conditions varies with the compound being detected. Dogs not only depend on vapor of compound, but also on odors, mixtures of odors, and by-products produced by decomposition. Almirall et al. 126 published work revealed that the detection of odor produced from compounds is conclusive for determination 126,127.2.4.1.3. By Colorimetric methodColorimetric reactions (wet-chemical ion specific reactions) are as Brown ring reaction for nitrates and Berthelot reaction for ammonium. Specific reagent reacts with a specific substrate and gives a specific color. Modified Griess reagent used to detect nitrates and nitrites from MAN, NC/NG/NS. This reagent gives mostly positive reaction of color orange for common explosives. Diphenylamine reagent used to detects chlorates, nitrates, and nitrites, from MAN (deep blue), NC/NG/NS, PETN (blue), and tetryl(blue). Methylene blue widely used to detect tetryl that produce an orange to red color change and for per chlorates it produce violet color change or a violet precipitate, depending on concentration.18.104.22.168. By Flame TestA flame test is an analytical technique used to detect metal ions when heated and different colors shows the presence of different metal ions.Fig 2.1. Flame color test for metals2.5. Confirmatory On-Site Detection of ExplosivesThe methods used for organic component detection are HPLC/TEA, LC-MS, GC-ECD, GC-TEA, and GC-MS. For the inorganic components detection we use color reactions or ion chromatography 128. For inorganic IC and CE commonly used as these are polar compounds.GC is mostly used to detect explosives materials in the traces for post-explosion. By Improving CE- GC we can analyze thermally instable explosives is increased 129. MCCORD & BENDER theoretically describe the use of GC-ECD for analyses of post-explosion 130. This technique is practical applied in this area by KAPLAN & ZITRIN131. This method can be used for analyses of pure extracts only because the ECD detector becomes easily contaminated. Organic secondary explosives containing nitro are analyzed by this due to its selectivity. The Electron Captured Detector is important for detection of sec- explosives having hydrocarbons 132.MS can be used in different modes like quad-ruple 133,134, ion- trap 135, TOF 136,137, tandem-based 138,139 and proved very selective, sensitive, reliable and good speed to detect explosives. MS makes possible for scientific experts to detect explosive materials. Compounds are first ionized and then separated by m/z ratios in MS. But its size and cost are two major disadvantages. So major focus has been made on MS instruments 140-143 to, reduces cost and their size. Chen et al. 144 formed a miniature recti-linear ion-trap for identification of organic and inorganic explosives materials 145. His detection system consists of a halogen lamp having LOD in the Pico-gram range. Scientists focused for increasing the level of confidence for explosive materials detection 146.Terahertz spectroscopy is used to provide the EM properties of explosive materials. It is used for nondestructive detection of explosives and explosive materials. This used cutoff frequencies and recover spectral features that lost by rough surfaces after scattering 147-148. IR Spectroscopy technique is very selective approache for chemical identification. It has many forms as mid-IR, near-IR, FT-IR, IR and are very useful to detect explosives 149,150. Morales-Rodriguez et al. test RDX and PETN explosive materials from the eight meters away with IR and UV radiations 151 with a single beam of light. Castro-Suarez and colleagues obtained high quality vibrational signatures of TNT by using FT-IR spectrometry deposited on aluminum plates 152. Pacheco-Londoño et al. 153 showed that presence of peroxide-based explosives, when they are in the gas phase mixed with air can be detected 154. They revealed that LOD for TATP is 800pg/m3 and 300pg/m3 for FT-IR and QCL detection, respectively. FT-IR and QCL shows even better LODs for 2,4-DNT of 31pg/m3 and 0.7pg/m3 , respectively.LIBS is a 2 beam spectroscopic technique and does not need of reference spectrum .LIBS determined the composition of a compound on the basis of elemental and molecular emission intensities by light emission of laser-generated plasma 155. Due to its standoff detection capabilities, researchers more focused on LIBS explosive detection 156-159. Work by Lucena recently showed by using LIBS for detection of explosive detection by 31 meters away from the sensor 160. Raman spectroscopy provides information by measuring the vibrational, rotational, and other transitions in a sample by using scattered laser photons. Chung and Cho made Raman detection system consists of a telescope, a laser, and a camera 161. TNT, RDX, and HMX have been detected from 31m away by using this technique.