Bali Bombings Case Study: Evidence, Analysis, and Insights

1. CASE STUDY 1 The Bali bombings

2. Overview • In 2002, the Bali bombings killed 202 people from 22 countries • “Operation Alliance” involved Australian Federal Police (AFP), Indonesian National Police (INP), Victoria Police Forensic Services Centre (VPFSC), Britain Forensic Explosives Laboratory (FEL), etc. • 3 explosions: a bar, a night club, a street outside American and Australian consulate buildings • Forensic chemists used a “mobile laboratory” to recover residues from blast scenes and suspect’s residences to determine bomb compositions

3. THE EVIDENCE Bomb 1 -tiny fragments of tartan fabric were recovered from site surrounding the blast epicentre -connective tissue and spatter marks were visible on the ceiling above the epicentre -absence of a crater Bomb 2 -inspection number found on chassis rail of car was traced back to owner

4. Explosions • result from a rapid release of gas • two main types of explosives: low velocity and high velocity • Low velocity explosives, like ammonium nitrate, used mainly in mining and for “pushing” applications • High velocity explosives, like pentaerythritol tetranitrate (PETN) and TNT, used in detonators, military etc. Usually used to initiate/boost low velocity explosives. Usually consumed and rarely leave traces • most large bombs consist of some form or combination of nitrate, chlorates, perchlorates, diesel oil, sugar, sulphur and/or aluminum dust • release of this amount of energy often causes fires

5. Chemical Structures • PETN • TNT

6. Sample Collection • Blast scenes are complex due to the spread of microscopic fragments that can contaminate evidence • the concentration of the inorganic ion must be significantly higher in and around the crater than in the background • the main blast produced a fire which was fought with water, compromising much of the blast scene • “mobile laboratory” was used to gather evidence in a timely matter

7. Mobile Laboratory • Nearby motel room was cleared and equipped with a microscope and camera, an ion mobility spectrometer (IMS), a portable infra-red spectrometer (FT-IR), reagents for presumptive tests • Produces rapid tentative results • Samples must still be sent to main lab for exhaustive analysis

8. Sample Testing • Chlorate ion was detected in 6/2000 samples analyzed which was considered significant because the highly reactive nature of chlorate ions make it difficult to persist in the environment • Sites it was detected on include the crevices of the blast crater, and on lamp posts facing the blast • It was argued that the chlorate ions originated from unburnt matches falling during the blast so samples were taken from elevated surfaces • Scientists from FEL searched the pitting of aluminum street signs that had been blasted to nearby rooftops using an SEM EDX. They reported elevated levels of chloride, low levels of chlorate, and no sulphur was detected.

9. Presence of TNT at two of the sites was detected by IMS and confirmed by both GC–TEA and GC–nciMS

10. Principles of Chromatography • A small volume of analyte (1 -10 uL) is taken up by a needle and injected into a column. • In GC, the column is a long capillary (20 – 100 m in length) filled with either a liquid or polymeric solid. • The injected analyte is moved through the column by a gas mobile phase (usually Ar, He or N2) inside the column.

11. The different chemical components of the samples are then separated out according to the affinity of a given chemical species for the stationary phase; the retention time for a chemical species is highly characteristic of that species. • GC run times tend to be long relative to LC (as much as 90 minutes), but the technique is generally more sensitive than HPLC.

12. Liquid chromatography works much the same way as GC; LC employs short (50 – 300 mm) columns with a larger bore (2 – 3 mm) packed with silica beads (2 - 5 um in diameter). • The surface of the silica can be either silanol groups (S-OH) or derivaterized with a wide variety of functional groups. Of particular note in metabolite analysis are C18 columns.

13. The mobile phase consists of a gradient of two solvents; the solvents used depend on both analytes of interest and the stationary phase, but normally include water and an organic solvent (methanol, acetonitrile, etc). • Run times on LC systems range from 3 – 60 minutes. • LC systems are generally able to analyze a much wider range of compounds than GC, but can also be less sensitive and less selective.

14. Principles of Mass Spectrometry • Once a compound has been ionized upon elution from a chromatograph, it is pulled into a MS by a vacuum. The ions go through a series of focusing stages (due to ever increasing potential differences as it moves through the instrument) until they reach either a selector or a detector. • There are two main types of MS. Single stage MS measures the mass of parent ions (or daughter ions from hard ionization sources) without any additional fragmentation. Useful for detecting and quantifying parent ions, but does not discriminate between isomers.

15. The most common single stage MS systems are single-quadrupole and Time-of-Flight analyzers. • Tandem mass spectrometry (MS2) can be used to differentiate between different isomers of a compound.

16. Normally, a quadrupole is used to select for parent ions of interest, then a second quadrupole will be used to fragment the ions by applying a mass-specific radio frequency in combination with a charged collision gas. A third analyzer (normally a quadrupole or flight tube) then selects for one, several or all of the ions produced. • Determination of the presence of a chemical species is highly accurate with MS2.

19. Colourimetric Testing • In forensic chemical analysis, colourimetric testing is used as a qualifier for classes of chemical compounds. • If the presence of a class of chemical compounds can be determined, it allows investigators to select an appropriate analysis method. • This type of preliminary screening allows for faster processing of samples, which helps speed up the investigation, and prevents technicians from running hundreds or thousands of samples through testing methods that do not provide useful information.

20. Colourimetric Methods For Explosives Screening

21. Analytical Methods for Explosive Residue Analysis – Thin Layer Chromatography • A small amount of an unknown can be placed on a silica plate (or other adsorbent material), the bottom of the plate placed in a mobile phase, and the retention factors compared to known standards. • Used for organic molecule analysis. • Reasonable detection limit. • Simple, inexpensive and quick to perform. • Does not resolve compounds well, but is a good starting point for analysis, similar to the colourimetric tests described earlier. • Requires compounds to be visible, either by visible light emission or by ultraviolet light absorptivity. • May also be used for MALDI mass spectrometry, although this precludes any use in quantification.

22. Gas Chromatography – Electron Capture Detector • Volatile compounds are separated out by gas chromatography, and a chromatogram generated by an electron capture detector. • ECDs work by passing gas-phase atoms (the eluent from gas chromatograph) through an electric field. • The electric field is generated by a nickel source that ionizes some of the carrier gas; collisions with the ionized carrier gas (Ar, N2) will dampen the electric field, which is measured as a change in the potential across the detector. • Generally used for halogenated and nitrogen containing compounds • Very low detection limits (pg range). • To facilitate GC, analytes must be volatile and thermally stable.

23. High Performance Liquid Chromatography – Ultraviolet/Diode Array Detectors • The eluent from an HPLC system is passed through a UV or DA detector. • The detectors usually monitor specific wavelengths; molecules that are known to absorb and emit at particular wavelengths can then be detected and quantified. • Matrix interferences are rarely a problem, but the method is not particularly selective. • Highly sensitive technique (can quantify compounds will into the ng level).

24. Infrared Spectroscopy • Infrared emission and absorption is used to generate a spectrum indicative of the functional groups present in a sample. • Useful for structural determination and pattern profiling (i.e. the spectra produced can be run against fingerprint databases to rapidly identify samples). • Useful for both organic and inorganic molecules. • Very quick to run; low cost in sample analysis. • Poor selectivity; samples are difficult to clean up and separate prior to analysis, and so are susceptible to contamination.

25. Gas Chromatography – Mass Spectrometry • Eluent from a gas chromatograph is passed through one of several different ionization sources, each one leading to different spectra. • Chemical ionization (CI) generally leads to the production of many parent ions, with few daughter ions produced. • Electron ionization (EI) results in heavy fragmentation of analytes; useful for structural determination, but parent ions do not always appear in the spectra generated. • Extremely sensitive technique (perhaps the most sensitive currently available). • Samples must be volatile (and stable at high temperature), or easily and efficiently derivatized to make them so (e.g. methyl esterification of amino acids). • Useful for organic materials, lipids, oils and many plasticizers and stabilizers.

26. Liquid Chromatography – Mass Spectrometry • Eluent from a liquid chromatograph (HPLC/UPLC) is ionized (either through electrospray ionization (ESI) or matrix-assisted laser desorption ionization (MALDI)). • Ionized sample is taken into the mass spectrometer and analyzed by either single-stage or tandem mass spectrometry (triple quad, QTOF, etc). • Shorter run times than GC, but less specific (based on retention times). • Analytes must be amenable to both LC and ionization methods available. • Wider range of analytes can be run than of GC systems.

27. Scanning Electron Microscopy/ Energy Dispersive X-Ray Spectroscropy • Used for surface analysis of very small particle sizes. • Used by investigators to examine fragments from explosions to help determine the source of an explosion. • Usable for all physical samples. • Expensive and time consuming (considering the large numbers of samples that require analysis). • Highly skilled operators are required to perform this analysis effectively.

28. Example • If a test with 3% KOH in ethanol determines the presence of one of TNT, 2,4-DNT and 2,6-DNT, some of that sample can be sent for further analysis by LC/MS/MS. • None of these compounds are volatile, so LC is an appropriate choice for analysis, while tandem mass spectrometry can be used to definitively identify a compound based on structural elucidation using fragmentation daughter ions.

29. MS2 of TNT(taken from http://www.chem.agilent.com/Library/applications/lcms18.pdf)

30. Types of Bombs • Bomb 1 – 1-5 Kg of TNT packed into 5 lengths of PVC pipe sewn into a vest, set off by a suicide bomber • Bomb 2 – approx. 1 tonne of explosive packed into 12 filing cabinets in a van, cabinets connected via detonating chord made from PETN, explosives a mixture of potassium chlorate, sulphur and aluminum boosted with TNT, the power was equivalent to 150 Kg TNT, set off by a suicide bomber • Bomb 3 – small amount of TNT set off by a mobile phone