identification of peroxide explosives...

IDENTIFICATION OF PEROXIDE EXPLOSIVES AND

TRADITIONAL EXPLOSIVE ANIONS BY CAPILLARY

ELECTROPHORESIS

By

STEPHANIE OLOFSON

Bachelor of Science in Forensic Science

Baylor University

Waco, Texas

2004

Submitted to the Faculty of the Graduate College of the

Oklahoma State University in partial fulfillment of

the requirements for the Degree of

MASTER OF SCIENCE December 2009

ii

IDENTIFICATION OF PEROXIDE EXPLOSIVES AND

TRADITIONAL EXPLOSIVE ANIONS BY CAPILLARY

ELECTROPHORESIS

Thesis Approved:

Dr. Jarrad Wagner

Thesis Adviser

Dr. Robert Allen

Dr. Frank Champlin

Dr. A. Gordon Emslie

Dean of the Graduate College

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ACKNOWLEDGMENTS

First and foremost I would like to thank my advisor, Dr. Jarrad Wagner, for his

continuous support during this project. We have been on this long and winding journey

together and I never would have made it to the end without his guidance. My committee

members, Dr. Robert Allen and Dr. Frank Champlin, have assisted me not only in the

preparation of this document, but have also provided daily support in the laboratory. The

faculty and staff of the Forensic Science Department especially Jane Pritchard, Cathy

Newsome, and Penelope Carr have helped me greatly throughout my time at OSU.

A special thanks to Dr. Kirk Yeager and Kelly Hargadon-Mount of the FBI

Explosives Unit for their assistance in preparation notes and instrumental methods and

Dr. Bruce McCord of Florida International University for his assistance with CE

instrumentation.

My family has been my inspiration to complete this project. Without their love, I

never would have made it this far. My parents and brother Erik have stood beside me,

pushed me to excel, and given me the strength to complete my dreams. I would also like

to thank three very special women that I consider family, Becky Michalovic, Cindy

Boucher, and Judith Munyon, who have guided me to the light on what might have been

a very dark journey. I would like to dedicate this thesis to my mom, Jacqueline Olofson,

who made me strong in my heart and strong in my spirit. My parents believe that you

give your children not only roots, but also wings – I hope this flight makes them proud.

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TABLE OF CONTENTS

Topic Page

Chapter I Introduction ......................................................................................................... 1

Chapter II Review of Literature ......................................................................................... 4

2.1 History of Capillary Electrophoresis ............................................................................ 4

2.2 Types of Capillary Electrophoresis ............................................................................... 6

2.3 Components of a Capillary Electrophoresis Instrument ............................................... 7

2.3.1 Injection System ..................................................................................... 7

2.3.2 High Voltage Source .............................................................................. 8

2.3.3 Electrodes ............................................................................................... 8

2.3.4 Detection in Capillary Electrophoresis ................................................... 8

2.3.5 The Capillary .......................................................................................... 9

2.4 Scientific Principles of Capillary Electrophoresis ...................................................... 11

2.4.1 Electro-osmotic Flow ........................................................................... 11

2.4.2 Temperature Control............................................................................. 13

2.4.3 pH and buffer strength .......................................................................... 13

2.4.4 Separation Chromatography in Capillary Electrophoresis ................... 14

2.5 Daubert and the Forensic Admissibility of Capillary Electrophoresis Data ............... 16

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2.6 Complementary Methods in Analysis ......................................................................... 17

2.6.1 GC-MS.................................................................................................. 17

2.6.2 LC-MS/MS ........................................................................................... 19

2.7 Explosives ................................................................................................................... 20

2.7.1 Low Explosives ........................................................................................................ 20

2.7.2 High Explosives ....................................................................................................... 21

2.7.2.1 Military Explosives ............................................................................ 23

2.7.3 Pure and Mixed Explosives ..................................................................................... 23

2.7.4 Peroxide Explosives ................................................................................................. 25

2.7.4.1 Recent Uses of Peroxide Explosives ................................................. 28

2.7.4.2 Current Analysis of Peroxide Explosives .......................................... 29

2.8 Analysis of Explosives by Capillary Electrophoresis ................................................. 32

2.8.1 Discussion on the Published FBI CE Method for Explosives/Ions ...... 32

2.8.2 Simultaneous Analysis of Anions and Cations Using CE .................... 33

2.8.3 Analysis of TNT Based Explosives Using Capillary Electrophoresis . 34

2.9 Recent Use of Capillary Electrophoresis for Hydrogen Peroxide Analysis ............... 35

Chapter III Methodology ................................................................................................. 36

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3.1 Instrumentation ........................................................................................................... 36

3.2 Capillary Column ....................................................................................................... 37

3.3 Instrument Conditions ................................................................................................. 38

3.3.1 Conditioning Method ............................................................................ 38

3.3.2 Analysis at 254 nm ............................................................................... 39

3.3.3 Analysis at 200 nm ............................................................................... 40

3.4 Buffer Preparation ....................................................................................................... 40

3.5 Sample Preparation ..................................................................................................... 41

3.5.1 Ion Samples .......................................................................................... 41

3.5.2 Peroxide Standards ............................................................................... 42

3.5.3 Extraction of Peroxide Swabs ............................................................... 43

3.5.3.1 TATP ................................................................................................. 45

3.5.3.2 HMTD .............................................................................................. 46

3.6 Verification of Peroxide Standards and Samples........................................................ 46

3.6.1 TATP by GC/MS .................................................................................. 46

3.6.2 HMTD by LC-MS/MS ......................................................................... 47

3.7 Sample Analysis by Capillary Electrophoresis ........................................................... 48

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3.7.1 Ion analysis ........................................................................................... 48

3.7.2 Peroxide Precursor Analysis ................................................................. 49

3.7.3 Peroxide Standard Analysis .................................................................. 49

3.7.4 Peroxide Residue Analysis ................................................................... 50

3.8 Statistical Analysis .................................................................................. 50

Chapter IV Results ............................................................................................................ 51

4.1 Published FBI CE Method .......................................................................................... 51

4.2 Optimizing the Published FBI CE Method ................................................................. 54

4.2.1 Instrument Conditions .......................................................................... 54

4.2.2 Capillary Dimensions ........................................................................... 55

4.2.3 Buffer .................................................................................................... 56

4.2.3.1 DETA Trials ...................................................................................... 56

4.2.4 Ions ....................................................................................................... 60

4.3 Verification of Peroxide Standards ............................................................................. 64

4.3.1 TATP by GC/MS .................................................................................. 64

4.3.2 HMTD by LC-MS/MS ......................................................................... 66

4.4 Sample Analysis by Capillary Electrophoresis ........................................................... 67

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4.4.1 Peroxide Explosive Precursor Analysis ................................................ 67

4.4.2 Peroxide Standard Analysis .................................................................. 70

4.4.2.1 TATP ................................................................................................. 70

4.4.2.2 HMTD ............................................................................................... 74

4.4.3 Peroxide Residue Analysis ................................................................... 75

4.4.3.1 Las Vegas, Nevada Training Samples ............................................... 75

4.4.3.2 Dover, Delaware Training Samples ................................................... 75

Chapter V Discussion ....................................................................................................... 81

Chapter VI References ..................................................................................................... 90

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LIST OF TABLES

Table Page

Table 1. Low Explosive Ions ............................................................................................ 33

Table 2. Ions Used in Method Development ................................................................... 42

Table 3. Description of Post-Blast Samples March 2008 ................................................. 44

Table 4. Description of Post-Blast Samples September 2009 .......................................... 44

Table 5. TATP GC/MS Settings ....................................................................................... 47

Table 6. HMTD MS Settings ............................................................................................ 48

Table 7. MTs and RMTs at 1.0 mM DETA ...................................................................... 52

Table 8. Ion Migration Times With and Without Bromide .............................................. 53

Table 9. Published FBI CE Method and OSU Method ..................................................... 54

Table 10. The Changes of the RMTs With Increased DETA Concentration ................... 57

Table 11. Changes to RMT with DETA Variation 2.0 mM to 2.4 mM............................ 58

Table 12. Resolution Values Between Neighboring Peaks .............................................. 58

Table 13. MTs and RMTs of Nine Ions at 2.1 mM DETA ............................................... 61

Table 14. Values from Figure 22 ...................................................................................... 63

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Table 15. Analysis of Precursor Ingredients ..................................................................... 68

Table 16. Hydrogen Peroxide Peak Pattern ...................................................................... 69

Table 17. Relative Migration Times for TATP ................................................................ 72

Table 18. TATP and Hydrogen Peroxide Signature Peaks ............................................... 73

Table 19. Analysis Results of Post-Blast March 2008 Samples ....................................... 75

Table 20. Relative Migration Times for Blank, Standard, Trace, and Post-Blast Peaks

from September 2009 Swabs ............................................................................................ 76

Table 21. Relative Migration Times from Burn Samples ................................................. 79

Table 22. Comparison of Burn Stick RMT to TATP Standard RMT .............................. 80

Table 23. Summary of RMTs for Ions, Hydrogen Peroxide, and TATP .......................... 87

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LIST OF FIGURES

Figure Page

Figure 1. Schematic of a CE system ................................................................................... 7

Figure 2. Charge Distribution Leading to Electro-osmotic Flow (EOF) .......................... 12

Figure 3. Laminar versus Parabolic Flow ........................................................................ 13

Figure 4. Chromatogram Showing Peaks and Separation Criteria ................................... 15

Figure 5. GC/MS Schematic ............................................................................................ 18

Figure 6. Schematic of Triple Quadrupole Mass Spectrometer ........................................ 19

Figure 7. Basic Structure of TNT .................................................................................... 22

Figure 8. Types of Explosives .......................................................................................... 24

Figure 9. Chemical Reaction to Produce TATP ............................................................... 26

Figure 10. Chemical Reaction to Produce HMTD............................................................ 27

Figure 11. Photo of TATP ................................................................................................ 27

Figure 12. Beckman P/ACE 5000 ..................................................................................... 37

Figure 13. Beckman P/ACE Capillary Cartridge Assembly ............................................. 38

Figure 14. Buffer from Published FBI CE Method .......................................................... 41

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Figure 15. HMTD Manufactured at the OSU Laboratory ................................................ 43

Figure 16. Swabs and Burn Sticks from September 2009 Training ................................. 45

Figure 17. Migration Times With and Without Bromide ................................................. 53

Figure 18. Nine-Ion CE Separation at 254 and 200 nm .................................................... 55

Figure 19. Electropherogram of Nine-Ion Separation at 2.0 and 2.1mM DETA ............. 59

Figure 20. Electropherogram of Nine-Ion Separation at 2.1 and 2.2 mM DETA ............ 60

Figure 21. Electropherogram Comparing Nine-Ion Analysis with Published FBI CE

Method and Optimized OSU Method ............................................................................... 62

Figure 22. Representative Electropherogram of Nine-Ion Separation at 2.1 mM DETA 63

Figure 23. GC/MS Chromatogram and Spectra of TATP as provided by AccuStandard 64

Figure 24. GC/MS Chromatogram and Spectra of AccuStandard TATP in Full Scan

Mode ................................................................................................................................. 65

Figure 25. GC/MS Chromatogram and Spectra of AccuStandard TATP in SIM Mode .. 65

Figure 26. Representative GC/MS Chromatogram and Spectra of TATP Synthesized

Onsite in Full Scan Mode ................................................................................................. 66

Figure 27. MS/MS Spectra of HMTD .............................................................................. 67

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Figure 28. Representative Electropherogram of Hydrogen Peroxide ............................... 69

Figure 29. Electropherogram of Sulfuric Acid ................................................................. 70

Figure 30. Representative Electropherogram of TATP Without Bromide ....................... 71

Figure 31. Representative Electropherogram of TATP with Bromide ............................. 72

Figure 32. Relative Migration Times of TATP and Hydrogen Peroxide ......................... 74

Figure 33. Electropherogram of Post-Blast Swab Peaks and Blank Swab ....................... 77

Figure 34. GC/MS Full Scan Chromatogram and Spectra of Burn 1.1 ............................ 78

Figure 35. GC/MS SIM Chromatogram and Spectra of Burn 1.1 .................................... 78

Figure 36. Electropherogram Comparing Burn Sample 1.1 to TATP .............................. 79

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NOMENCLATURE

ANOVA Analysis of Variance

APCI Atmospheric Pressure Chemical Ionization

CE capillary electrophoresis

CGE capillary gel electrophoresis

cm centimeter

CTAB cetyltrimethylammonium bromide

CZE capillary zone electrophoresis

Da Daltons

DAD diode array detector

DADP diacetone diperoxide

DETA diethlentriamine

EDTA ethylenediaminetetraacetc acid

EOF electro-osmotic flow

FBI Federal Bureau of Investigation

FRE Federal Rules of Evidence

GC gas chromatography

GC-MS gas chromatography - mass spectrometry

H2O2 hydrogen peroxide

HMTD hexamethylene triperoxide diamine

xv

HMX cyclotetramethylene-tetranitramine

HPLC high performance liquid chromatography

Hz hertz

ID inner diameter

IEDs Improvised Explosive Devices

IHEs insensitive high explosives

kV kilovolts

LIF laser induced fluorescence

m/z mass-to-charge ratio

MEKC /MECC Micellar-Electrokinetic Capillary Chromatography

mm millimeter

mM millimolar

MS mass spectrometry

MT migration time

nm nanometer

OD diameter

PETN pentaerythritol tetranitrate

Pk peak

Q quadropole

R resolution

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RDX cyclotrimethylene trinitramine

RMT relative migration time

RT retention time

SDS sodium dodecyl sulfate

SIM selective ion monitoring

TATP triacetone triperoxide

tmi migration time

TNT trinitrotoluene

UV ultraviolet

W watts

µA microamp

µm micrometer

1

Chapter I Introduction

The analysis of traditional explosive residues can be a difficult task for the

forensic chemist. There are many methods to choose from and they can be performed

using a wide variety of instruments. Few of the methods yield definitive answers, but

rather they provide results that investigators use to infer the types of explosives

employed. Although not new to the scientific world, peroxide explosives are now the

explosive of choice for terrorist bombers while presenting new challenges to scientists as

investigators attempt to identify residues left behind after an attack.

Terrorists choose to use peroxide explosives in improvised explosive devices

(IEDs) because they offer devastating explosive power without the challenges of

procuring and packaging traditional explosives. Even though they have destructive power

close to trinitrotoluene (TNT), peroxide explosives are not used in military or commercial

applications due to low stability, sensitivity to impact, and high volatility (Dubnikova et

al., 2006). Manufacture of peroxide explosives is a simple process involving compounds

which may be obtained at nearly every drug store, where purchases are very difficult to

track or limit (Dubnikova et al., 2002). Peroxide explosives, most notably triacetone

2

triperoxide (TATP), were used in the 2005 London bombings and were intended to be

used by Richard Reid, the shoe bomber, on a commercial passenger flight. The challenge

of identifying this new type of explosive is the volatility of the explosive and the fact that

peroxide explosives lack the nitro groups or metallic elements found in traditional

explosives; therefore, traditional explosive identification methods are unsuitable for their

identification.

Capillary electrophoresis (CE) is a chromatographic separation technique used in

many different aspects of forensic science, wherein compounds are separated based on

differences in electrophoretic mobility, phase partitioning, and/or molecular size

(Thorman et al., 1998). Detection of compounds or ions in CE may be achieved by

measuring changes in UV absorption (Moore, 2004). There is great need for a fast,

convenient, and environmentally friendly method for explosive analysis utilizing

instruments already existing in law enforcement laboratories, and CE addresses all of

these needs. CE is considered an excellent separation technique for explosives due to its

sensitivity, ease of sample preparation, reproducibility, and rapid analysis. CE offers the

explosive analyst the ability to detect multiple explosive ions in a single examination

(Hargadon & McCord, 1992). Although successfully used to identify traditional

explosives, CE has yet to be used to identify peroxide explosives. This research will

couple existing methods with new innovations to allow for the simultaneous

identification of both traditional anion and peroxide-based explosives using CE.

This research will attempt to develop a novel method based on a method first

developed by Hargadon and McCord (herein referred to as the published FBI CE method)

(Hargadon & McCord, 1992), which utilizes CE for the identification of 20 separate

3

anions commonly found in low explosives. Furthermore, the new method will then be

utilized to identify peroxide explosives, a first for capillary electrophoresis. The pattern

of anions found in explosive residue can lead examiners to deduce the type, source, and

manufacturer of the explosive. The published FBI CE method is based on a borate buffer

with a dichromate chromophore and employs diethylenetriamine (DETA) as the electro-

osmotic flow (EOF) modifier to force ions towards the detector. In the past, the FBI has

used this method for the analysis of non-peroxide explosives. The current method allows

the identification of 20 major ions found in blast residue that, when viewed together, can

produce a finger-print of the explosive materials used (Hargadon & McCord, 1992). By

itself, the published FBI CE method is a powerful tool for explosive analysis; however,

the possibility of also analyzing peroxide explosives will allow for fingerprint-type

identification using a one-step analysis process. This novel approach will offer forensic

agencies a means for analyzing multiple types of explosives using a single analytical

procedure on existing instruments to aid in the apprehension of terrorist bombers.

4

Chapter II Review of Literature

2.1 History of Capillary Electrophoresis

Capillary electrophoresis (CE) has evolved over time as the need arose for an

instrument that provided versatile and cost-efficient analysis of compounds. The

foundation for CE was set in the 1950s by Kolin and Hjerten, with the first functional

CE-type instrument built by Hjerten in 1959 (Landers, 2007). Throughout the 1960s and

1970s, other types of electrophoresis grew in popularity; yet, there was still a need for an

instrument with the analytical capabilities possessed by CE. Finally in the early 1980s,

Jorgenson and Luckas developed tiny capillaries in which CE is carried out (Landers,

2007). The 1980s saw the growth of CE as a viable research tool.

Interest in CE in the 1980s emerged due to the advent of silica capillaries and the

automation of instrumentation. Early in the decade, micro (≥100 µm) fused silica

capillaries became available. Although fused silica is generally a very fragile material,

the capillaries used in CE are coated with an outer layer of polyimide that strengthens the

delicate fused silica material (Tagliaro et al., 1998). Until 1989, CE analysis was carried

5

out on home-made devices. Although the rough instruments were functional, they were

also bulky and lacked the precision needed to carry out repeated and quantitative

analysis. In the early 1990s, the first commercial instrument, the Beckman P/ACE, was

manufactured. The automation of the commercial instruments is necessary to carry out

the precise functions required for repeated analysis in capillary electrophoresis. Pressure

within the capillary, auto sampler function, and precise temperature and heat dissipation

are just a few of the functions required by the instrument for robust, reproducible analysis

(Beckman Coulter, n.d.). As soon as commercial instruments were available, they

became fixtures in laboratory settings.

Cost, analysis speed, environmental concerns, flexibility, and high resolution

separation represented laboratory needs that CE helped to address. The cost of operating

and maintaining a CE is relatively low compared to many other laboratory instruments.

The major cost is the instrument, software, and technology required for operation. Buffer

components are fairly inexpensive and readily available. Capillary columns are generally

under $100 per meter and can be used many times before replacement. Analysis speed is

another positive benefit of CE in that most separations are complete within 10 minutes of

injection (Thorman et al., 1998). One of the primary reasons that CE was welcomed into

the modern scientific laboratory is that it does not present many of the environmental

concerns of other instruments. During the 1980s and early 1990s, high performance

liquid chromatography (HPLC) was the primary means for chemical separation in the

laboratory. Unfortunately, HPLC generates a massive amount of organic solvent waste

that is both costly and unsafe for the environment. In addition, it can be challenging to

separate large molecules with HPLC as compared to CE. CE produces little waste and is

6

able to analyze a wide variety of compounds from small molecules and ions to larger

proteins and nucleic acids (Landers, 2007).

2.2 Types of Capillary Electrophoresis

There are many different types of capillary electrophoresis to accommodate the

various types of analytes in the laboratory. By making simple changes such as buffer

ingredients or capillary types, analysts are able to optimize separations based on scientific

principles while using the same laboratory instrumentation. The most common type of

separation is capillary zone electrophoresis (CZE). CZE is the simplest form of CE

where ions are separated by their electrophoretic mobilities and charge to mass ratio

under highly charged electric fields (Tagliaro et al., 1998). CZE also provides the

greatest versatility of all modes of CE and can be used to analyze cations, anions, small

molecules, peptides, proteins, and carbohydrates (Landers, 2007). Micellar-

Electrokinetic Capillary Chromatography (MEKC or MECC) is a separation method

primarily used for neutral small molecules and peptides that move toward the detector at

the same rate as the EOF. MEKC is based on a pseudostationary phase in the buffer such

as Sodium Dodecyl Sulfate (SDS) which forms micelles that slow down the movement of

ions towards the detector. Because micelles act like soap bubbles and encase the ion, the

buffer moves the ion at the speed of the micelle, not the faster speed that the ion would

move through the buffer. Micelles assist compounds that would not normally move

through the capillary to move towards the detector. Capillary Gel Electrophoresis (CGE)

acts within a special capillary that is filled with cross-linked polymer gels such as

polyacrylamide. Because the gel inside the capillary depresses the EOF, CGE is capable

of performing very precise size separations. CGE is most commonly used in DNA

7

analysis (Landers, 2007). No matter what mode of CE is used, the same instrumental

setup is still valid.

2.3 Components of a Capillary Electrophoresis Instrument

A capillary electrophoresis system contains five parts (Figure 1) which can be

manipulated based on the mode and analyte of interest. The five parts of the system are:

the injection system, separation capillary, high voltage source, electrodes (anode and

cathode), and the detector.

Figure 1. Schematic of a CE system The five components of a CE system: injection system (sample inlet), capillary, voltage supply, electrodes, and detector (“Capillary Electrophoresis,” 2007).

2.3.1 Injection System

There are two types of injection systems: hydrodynamic and electrokinetic.

Hydrodynamic injection uses a pressure or vacuum application and is considered the least

selective of the two injection types. Electrokinetic injection is completed by the

application of high voltage and is generally more favorable because of the selectivity of

the ions. Each time an electrokinetic injection takes place, the separation results depend

8

on the mobility and charge of ions in the sample. Although this is a more selective form

of injection, it can also be more difficult to control and replicate (Landers, 2007).

2.3.2 High Voltage Source

A high voltage source with the ability to produce voltages greater than 30 kV

(kilovolts) and maintain currents up to 300 µA (micro amp) is required for injection and

separation. Higher voltages generally provide optimal efficiency and separation

resolution (Tagliaro et al., 1998).

2.3.3 Electrodes

Two separate electrodes are used in CE, one at the sample inlet and one just past

the detector. These electrodes have the capability to be either the cathode or the anode.

In normal phase CE, the anode (positive electrode) is placed at the sample inlet and the

cathode (negative electrode) is placed after the detector. In reversed polarity or reversed

phase CE the cathode is placed at the sample inlet and the anode is placed just past the

detector. Modern CE instruments have the ability to run in either normal or reversed

polarity depending on the type of CE analysis and the analytes to be detected.

2.3.4 Detection in Capillary Electrophoresis

There are also several detection techniques that may be used in CE analysis. The

most common is an ultraviolet light (UV) detector as it is the most capable detector for

many analytes. The nature of fused-silica capillaries contributes to the success of UV

detection. For in-column UV detection to occur, a small portion of the capillary’s

external polyimide is removed, creating a window for the detection to occur. Fused-silica

capillaries allow for detection to occur at wavelengths as low as 190 nanometers (nm)

9

and as high as 300 nm. There are some drawbacks to the traditional capillary in UV

detection due to the short optical path length and round section of the capillary, which

creates a poor optical cell. In order to overcome the poor design of the standard capillary,

some methods call for specialized capillaries featuring a bubble cell that is able to

increase the sensitivity of the basic UV detector. Another popular type of detector is the

Diode Array Detector (DAD) which allows for quickly scanning multiple wavelengths to

increase the amount of data collected in a single sample run. Other detector techniques

now available for CE include laser induced fluorescence (LIF) and mass spectrometry

(MS). Each type of detector has advantages that analysts can use to design for the best

possible results (Tagliaro et al., 1998).

There are two important types of UV detection used in capillary electrophoresis –

direct and indirect detection. In direct detection, the buffer moves the ions through the

capillary, and peaks appear as the ion passes by the detector. Direct detection is used for

ions with good UV absorbance. In order for analytes with low UV absorbance to be

detected, UV indirect detection must be used. In indirect detection, a chromophore is

added to the buffer, which causes a constant UV signal to be received by the detector.

When the analyte passes through the detector, the absorbance drops causing the low UV

absorbent analyte to produce a peak.

2.3.5 The Capillary

The nature of the fused-silica capillary is what has allowed the popularity and

utility of CE to grow. Because separation and detection generally both occur within the

capillary, the selection of the correct capillary is extremely important when developing a

10

CE method. A good capillary must be “both chemically and physically resistant,

accurately created with a consistent narrow inner (ID) and outer (OD) diameter,

translucent in order to allow for UV detection, able to dissipate Joule heat through good

thermal conductivity, and inexpensive” (Tagliaro et al., 1998). Capillaries generally

range from 20 – 100 µm (micrometer) ID and 20 – 100 cm (centimeter) in length.

Varying the capillary dimensions will affect the migration time and separation capacity of

the method (Landers, 2007). The capillary exhibits many qualities that have increased

the popularity of CE through cost efficiency and environmental impact, including

microliter injection volumes, small sample size, and small quantities of buffer required.

The fundamental datum produced by CE is migration time (MT), the time it takes

for a solute to move from its entrance into the capillary to the detector window.

Migration time is similar to retention time (RT) in other chromatographic techniques,

where the RT is how long the compound of interest is held within the chromatography

column. However, in CE it is termed MT since nothing is retained and instead it is

simply the time required for the compound to move through the capillary to the detection

window (Beckman Coulter, n.d.). The analyst is able to vary the migration time by

adjusting the capillary length and diameter, as well as the voltage (current used for

separation). When adjusting the capillary length, two measurements are made: the total

length of the capillary and the effective length (i.e., the capillary length to the detector

window). The total length of the capillary is the less important measurement. The

migration time is measured from the start of the injection into the capillary until the

analyte reaches the detector window. The length of capillary past the detector window is

required only to create the circuit required for CE analysis.

11

2.4 Scientific Principles of Capillary Electrophoresis

Many scientific principles are at work in capillary electrophoresis. Electro-

osmotic Flow (EOF), interaction with the column’s silica walls, temperature control, pH,

and buffer strength all play important roles in successful capillary electrophoresis.

2.4.1 Electro-osmotic Flow

There are two essential processes that drive CE. The first is electrophoresis or the

electric force on charged molecules. The second is electro-osmosis or the electric force

on solvent transport. Electro-osmosis begins at the capillary walls and creates the EOF or

movement of liquid due to an applied current across a capillary. The movement of liquid

is catalyzed by the surface charge on the walls of the fused-silica capillary (Figure 2).

The surface walls of the capillary contain acidic silanol (SiOH) groups that ionize to SiO-

at a pH greater than 2. The ionized groups interact with cations in the buffer and when a

potential is applied the cations migrate towards the cathode electrode near the detector.

As the cations migrate, they also drag water in the same direction creating the EOF

(Beckman Coulter, n.d.). This buffer movement also pulls the ions through the capillary

towards the detector. Control of EOF is the key to reproducible electrophoresis.

12

Figure 2. Charge Distribution Leading to Electro-osmotic Flow (EOF) EOF comes from the movement of cations in the buffer and their interaction with the ionized SiO- groups of the silica capillary. When a voltage is applied the cations migrate and drag water in one direction creating the EOF. The EOF must be controlled for reproducible CE data (Skoog et al., 1998).

One way to control the EOF is to control the degree of ionization by changing the

pH of the buffer. The EOF is also decreased by increasing the concentration of the

buffer. The type of injection system used can also have an effect on the EOF. In

electrokinetic systems, the EOF is uniformly distributed throughout the entire length of

the capillary, creating a uniform flow velocity (Figure 3). In pressure injection systems,

there is a pressure drop after the initial injection, causing an unequal parabolic flow that

is less desirable (Beckman Coulter, n.d.).

13

Figure 3. Laminar versus Parabolic Flow Laminar flow (a) is created with electrokinetic injections creating uniform buffer flow. Parabolic Flow (b) is created with pressure injection systems where a change in pressure after initial injection results in unequal buffer flow (Skoog et al., 1998).

2.4.2 Temperature Control

Due to their ability to dissipate heat during sample analysis, fused-silica

capillaries are often chosen for CE work; however, temperature control is still a concern

for accurate CE results. Heat control is essential because changes in the internal capillary

temperature cause change in the buffer viscosity and therefore the migration time of the

analytes (Tagliaro et al., 1998). The production of heat is primarily caused by the

application of high field strengths required for separation, temperature gradients across

the capillary, and the inability to dissipate heat effectively from the capillary. In order to

control the heat within the system, heat must be removed at a rate equal to its production.

One way to accomplish heat removal is to select the narrowest diameter capillary

(Beckman Coulter, n.d.). Many of the modern instruments also use a liquid coolant

surrounding the capillary to help with heat exchange during analysis.

2.4.3 pH and buffer strength

The best way to control analyte migration is through selection of the proper buffer

formulation, pH, concentration, and additives. The buffer chosen for a method should

14

include considerations for the ability to detect selected analytes, maintain analyte

solubility, present a proper buffering scheme, and produce the greatest separation.

Appropriate buffer concentration and pH differs depending on the type of CE mode and

analyte to be separated. The strength of the buffer to be used is unique for each buffer;

however, it is important to remember that the higher the concentration of the electrolyte

in buffer, the lower the EOF, and lessened effect of adsorption by the capillary walls. For

ion analysis, phosphate buffer (pH 1.14-3.14 and 6.20-8.20) or borate buffer (8.14-10.14)

is commonly used. For protein analysis or DNA work, Tris buffer (pH 7.30-9.30) is

standard (Beckman Coulter, n.d.). Buffer additives are common in CZE: organic solvents

may be added to increase the solubility of samples, organic amines may be added to

change wall charges, metal ions to reduce wall adsorption, and urea to act as a denaturing

agent (Landers, 2007). Buffers should always be filtered through a small pore-sized

filter prior to use to avoid clogging the capillary. Development of a successful CE

method will evaluate which buffer, concentration, and pH is optimal for the desired

analyte separation.

2.4.4 Separation Chromatography in Capillary Electrophoresis

Separation, specificity, and resolution are very important topics in any form of

chromatography. One of the ways that capillary electrophoresis is considered superior to

other chromatography techniques is the high resolution seen in CE separations. The

resolution (R) of a method provides a measure of the ability of the method to separate

two analytes (Skoog et al., 1998). Figure 4 was modified from a typical chromatogram to

demonstrate resolution calculations appropriate to electropherograms in CE. Retention

times were replaced with migration times, and the figure shows the detector response as a

15

function of time and also the resolution of two adjacent peaks.

Figure 4. Chromatogram Showing Peaks and Separation Criteria The variables described in the figure of retention time for solvent front (tm), peak a (tmia), and peak b (tmib), are used along with the width at the base of each peak (wba and wbb, respectively) to calculate resolution (Modified from Levine, 2003).

Resolution may be calculated by using the values obtained in the

electropherogram and used in the equation:

where R stands for resolution, t’mi stands for adjusted migration time, and Wb is the width

at the base of each peak. An R value of 1.5 or greater is considered to be complete

separation; whereas, an R value of 1.0 represents good separation with a 10% overlap

between the neighboring peaks (Levine, 2003). When developing a method for ion

separation, the R value must be considered between each set of peaks, especially closely

spaced peaks. A well designed CE method will produce peaks that have a resolution

value of at least 1.0 between each peak.

16

2.5 Daubert and the Forensic Admissibility of Capillary Electrophoresis

Data

In order for analysis to be presented in court as evidence, it must not only result

from a valid scientific technique, it must also conform to the legal criteria of the Daubert

Standard and the Federal Rules of Evidence (FRE). The Daubert Standard from the

Supreme Court Case, Daubert v. Merrell Dow Pharmaceutical, Inc., requires that: 1) the

court must act as gatekeeper and the judge must only allow scientific testimony that

comes from firm scientific knowledge, 2) evidence presented must be relevant and

reliable, and 3) the evidence/testimony presented must be firmly based in the scientific

method. In addition to the requirements of Daubert, FRE 702 demands that the

evidence/knowledge presented must be based on sufficient facts or data, the testimony is

the product of reliable methods and principles, and that the witness has applied the

methods and principles reliably to the facts of the case (Legal Information Institute, n.d.).

Rule 702 is “now considered the standard of analysis to determine the admissibility of

novel scientific evidence” (Kuffner et al., 1996).

Although the scientific theories surrounding capillary electrophoresis are not new,

the practice of CE was still considered novel according to the courts until more

laboratories began utilizing it for analysis. In 1996, the first case to present capillary

electrophoresis based evidence was State of Tennessee v. Paul Ware, 1996. While this

case did allow evidence obtained from capillary electrophoresis to be presented in court,

it only presented CE as a tool for DNA analysis. Each time CE analysis is presented, the

court may hold this testimony to the stringent Daubert and FRE 702 standards. Kuffner

et al. (1996), believe that given the environmental, efficiency, and economical advantages

17

of CE along with the scientific principles upon which it is based, CE will meet the criteria

set forth by Daubert and should continue to be allowed as testimony along with other

legitimate scientific processes.

2.6 Complementary Methods in Analysis

In science, it is often necessary for an analyst to use multiple methods or

instruments for analysis, and when the scientific principles underpinning each method are

sufficiently different the methods are termed complementary methods of analysis.

Complementary methods are useful for quality assurance as the findings from two

distinct methods that are in agreement are more reliable than those from a single method

that is not corroborated. Also, if one method is used as a screening method, a second or

confirmatory method or analysis is typically required to validate the screening method’s

findings. Occasionally the same instrument using a different method can be used to

support previous findings, but more often, a different instrument operating on different

scientific principles is required. This work will require the use of two separate

instrumental methods complementary to the CE analysis: gas chromatography-mass

spectrometry (GC-MS) and liquid chromatography-tandem mass spectrometry (LC-

MS/MS).

2.6.1 GC-MS

Gas chromatography-mass spectrometry (GC-MS) (Figure 5) is an instrumental

technique that combines the separation capabilities of GC with the ability of MS to ionize

and detect separated compounds based on their mass-to-charge ratio (m/z) (Skoog et al.,

1998). Samples are first injected into the gas chromatograph and then carried through the

18

silica column by the carrier gas (generally helium). The samples are heated in the GC

oven and then elute or exit the column. The fundamental datum produced by gas

chromatography is the retention time (RT), the amount of time it takes for the compound

to exit the column. After the sample exits the column it moves to the detector, in this

case, the mass spectrometer. As the molecule enters the MS, it is ionized or fragmented

into small pieces which are then sorted by the mass analyzer before exiting to the MS

detector (Skoog et al., 1998).

Figure 5. GC/MS Schematic Arrows represent the path of the compound after injection and the dashed line indicates vacuum region of mass spectrometer (Skoog et al., 1998).

The MS detector collects the ion results and sends them to the signal processor

that produces the mass spectrum (Skoog et al., 1998). The analyst is able to uniquely

identify the molecular fingerprint of the compound by using the retention time generated

by the GC and the mass spectrum generated by the MS.

19

2.6.2 LC-MS/MS

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) operates under

similar principles of GC-MS; however, rather than using a gaseous mobile phase to move

and separate the compound of interest through the column, LC uses a liquid mobile

phase. The LC portion of LC-MS/MS is essentially a high performance liquid

chromatography (HPLC) system connected to a tandem MS detector. Due to the ability

to vary the mobile phase and column, LC has a much greater range of analytical

potential. Because the LC system does not require the heating element that GC does, LC

is able to analyze more heat sensitive compounds (Levine, 2003).

It is also possible for the LC system to be connected to multiple detectors, in this

case, a multistate or tandem MS (Figure 6).

Figure 6. Schematic of Triple Quadrupole Mass Spectrometer Q1 and Q3 function as mass filters, while Q2 functions as a collision cell where precursor ions can be broken up into product ions (Skoog et al., 1998).

Multistate MS is created by linking several quadrupoles (or mass analyzers)

together that the user can utilize to perform multiple scan types that will accomplish

analytical goals. In a product ion scan, the first quadrupole (Q1) only allows the ion with

the m/z of interest to pass through. The ion passing through Q1 is called the precursor or

parent ion. Q2 is then used as a collision cell, where the precursor ion is fragmented into

20

product or daughter ions through collision with gas molecules. The product ions proceed

into Q3 which selectively allows ions through to the detector (Levine, 2003). There are

many different combinations of quadrupoles and additional technology that may be

arranged for best results by the analyst. It is also possible to perform a direct infusion of

the sample on the MS/MS bypassing the LC system for initial separation. Because of the

many options it allows, LC-MS/MS has become the premier separation and identification

tool in the forensic laboratory for a wide variety of compounds.

2.7 Explosives

Ever since Alfred Noble first created dynamite in 1866, the world of explosives

has grown to include many diverse types of compounds depending on the use and

necessity for different characteristics. Explosives fall into different classes based on their

structure and potential use. Explosives are separated into high and low explosives based

on whether their primary action is to burn or deflagrate (low explosives) or detonate (high

explosives).

2.7.1 Low Explosives

Low explosives are generally compounds made of two ingredients: a combustible

substance (fuel) and an oxidizer. These two ingredients deflagrate or burn quickly when

ignited rather than detonate like high explosives. While they may burn very quickly in

small spaces and appear to detonate, they deflagrate at under 400 meters per second

(m/s), much slower than high explosives ("Explosive Material," 2009). Low explosives

are categorized as pyrotechnics or propellants based on their primary function.

Pyrotechnics produce heat, light, smoke, or sound when lit and are common in magic

21

shows and special effects. Propellants have more useful qualities as they produce gases

that perform mechanical work when burned. The most common propellant, gunpowder,

produces gases that force bullets through gun barrels. While low explosives cannot be

detonated by a common blasting cap, they are commonly used as blasting agents to assist

in the detonation of high explosives (Cooper & Kurowski, 1996).

2.7.2 High Explosives

High Explosives are chemicals that detonate upon ignition. They are used in a

variety of different types of work including: demolition, military uses, mining, and

terrorist acts. The detonation rates of high explosives range from 3,000 to 9,000 m/s,

much greater than their low explosive counterparts. High explosives are divided into

three groups based on their sensitivity.

Primary explosives are extremely sensitive and can be made to detonate very

easily. This type of explosive is sensitive to any stimulus including mechanical shock,

electrical shock, friction, or heat, and when ignited will burn quickly or immediately

detonate. Several examples of primary explosives include lead styphnate, lead azide, and

mercury fulminate (Cooper & Kurowski, 1996).

Within primary explosives a special class exists for detonators and primers.

Detonators, such as blasting caps and detonation cord generally contain a small amount

of primary explosive that provide the shock or friction needed to set off the main charge

of the explosive (Davis, 1943) . There is generally a three-step process called ‘the

explosive chain’ for the detonation of high explosives: detonator, booster of primary

explosive, followed by the main explosive charge. The explosive chain will vary

22

depending on the use, but is reliant on the detonator to begin the process (Cooper &

Kurowski, 1996).

Secondary explosives are more difficult to detonate. They do not go easily from

deflagration to detonation, require larger shocks for detonation, and do not respond as

well to electrostatic ignition. Generally secondary explosives burn when exposed to heat

and may detonate when in small confined quantities, but they are much more stable than

primary explosives. Most of the well known explosives are secondary explosives

including: dynamite, TNT (trinitrotoluene), RDX (cyclotrimethylene trinitramine), HMX

cyclotetramethylene-tetranitramine), and PETN (pentaerythritol tetranitrate). PETN is

considered the baseline compound for secondary explosives, anything more sensitive is a

primary explosive ("Explosive Material," 2009). The structure of TNT (Figure 7) is

generally the building block on which all of the secondary explosives are based. There

are many variations on the structure that are manipulated in order to produce larger

explosions or make the compound more stable (Cooper & Kurowski, 1996).

Figure 7. Basic Structure of TNT The structure of TNT is generally the building block on which all of the secondary explosives are based (Cooper, 1996).

23

Blasting agents are considered a separate class of high explosives called tertiary

explosives. These are high explosives that are most difficult to detonate and are often

referred to as insensitive high explosives (IHEs). Blasting agents are generally mixtures

and slurries with the most common being ammonium nitrate and fuel oil mixtures. IHEs

are generally insensitive to shock and require a booster of primary or secondary

explosives before detonation (Cooper & Kurowski, 1996) .

2.7.2.1 Military Explosives

Military explosives are a special section of high explosives. Although most high

explosives could be used in military exercises, the explosives that are most used by the

military have certain characteristics. First, military explosives must be readily available

and affordable. Second, they must withstand friction and electrical signals until they are

needed to deploy. In addition, explosives must be able to be stored without deteriorating

and be resistant to degradation from heat and light ("Explosive Material," 2009).

2.7.3 Pure and Mixed Explosives

Explosive compounds are also separated into categories based on whether they

are pure compounds or mixtures of compounds (Figure 8). Each type of explosive must

have two important substances: an oxidizer and a fuel. An oxidizer is a substance in a

reaction that contributes atoms to a reaction and a fuel is the portion of the explosive that

burns.

24

Figure 8. Types of Explosives Schematic of the breakdown between pure compound explosives and mixtures of compound explosives. The majority of common explosives are organic (Cooper & Kurowski, 1996).

Most explosive materials are organic compounds based on a carbon skeleton.

Aromatic and aliphatic refer to the structure of the explosive. Aromatic explosives are

based on a benzene ring where the bonds between the carbons alternate between double

and single bonds. In this type of explosive, the aromatic ring generally has three nitrogen

groups attached to it. Aliphatic explosives are not based upon a benzene ring and may

take on a variety of different structures. Regardless of the specific structure of the carbon

backbone, the intimate mixture of fuel and oxidizer is required for the explosive nature of

the materials. The carbon skeleton and the attached hydrogen atoms are the fuel portion

of the explosive, while the attached oxygen-containing subgroups are the oxidizer portion

of the explosive. The reaction between these elemental groups is what causes the

explosion to take place (Cooper, 1996). Inorganic explosives are not based upon a

carbon backbone and are generally mixtures of a metal fuel and an oxidizer.

Explosives also appear as mixtures of compounds wherein separate fuels and

oxidizers are mixed together to create an explosive. In an oxidation reaction heat is

produced because the internal energy of the final product is much lower than the internal

25

energy of the starting materials. An oxidizer is a chemical that effectively provides

oxygen for combustion. Some common oxidizers are ammonium nitrate, sodium nitrate,

and potassium nitrate. One of the most common explosive mixtures is ANFO

(ammonium nitrate fuel oxidizer), and an ammonium nitrate and fuel mixture was used

in the 1995 bombing of the Murrah Federal Building in Oklahoma City (Cooper, 1996).

Traditional explosives may be analyzed with the aid of many different forms of

instrumentation due to the fragmentation patterns of the aromatic rings, nitro groups, and

metallic substituents that form after detonation. However, peroxide explosives are a new

class of explosives commonly used in terrorist bombings that provide novel analytical

challenges.

2.7.4 Peroxide Explosives

This class of explosive does not fall into any of the traditional explosive classes.

Peroxide explosives differ from traditional explosives in that rather than being based on

the traditional aromatic ring or other organic forms, they are based on the peroxide ring.

The two most well known peroxide explosives are triacetone triperoxide (TATP,

C9H18O6, Figure 9) and hexamethylene triperoxide diacetone (HMTD, C6H12N2O6, Figure

10). Production of TATP also produces the stable dimer, diacetone diperoxide (DADP,

C6H12O4), at room temperature. Both TATP (Figure 11) and HMTD are a white,

crystalline powdery substance that may be initially identified as illegal drug residue. The

lack of nitrogen within TATP and its volatility make it very difficult to detect. Peroxide

explosives can be used as the sole explosive source or they may also be used as boosters

26

for larger explosives. Both TATP and HMTD have been used in terror attacks across the

globe.

Figure 9. Chemical Reaction to Produce TATP Acetone, hydrogen peroxide, and sulfuric acid combine to form TATP (triacetone triperoxide) trimer and DADP (diacetone diperoxide) dimer based on the peroxide ring. Notice the many oxygen atoms comprising the organic molecular structure of the peroxide explosives.

While peroxide explosives have power close to TNT, they are highly unstable and

therefore cannot be used in commercial or military applications. The instability of

peroxide explosives comes from the low bond energy of the oxygen-oxygen (O-O) bond

and causes the explosive to be highly heat and shock sensitive. Detonation of peroxide

explosives begins with the hemolytic cleavage or breakdown of one peroxide bond and

then continues with neighboring cleavage of carbon-oxygen (C-O) and O-O bonds within

the same molecule. The cleavage of the initial O-O bond is the rate-determing step

during peroxide explosive detonation (Dubnikova et al., 2006).

27

Figure 10. Chemical Reaction to Produce HMTD Hexamine, hydrogen peroxide, and citric acid produce HMTD (hexamethylene triperoxide diamine) based on the peroxide ring. Notice that while HMTD does contain nitrogen, it is not in a traditional formation that leads to easy detection.

Figure 11. Photo of TATP TATP crude (left) and pure (right). Photo courtesy of Dr. Kirk Yeager, FBI Laboratory.

Terrorists choose to use peroxide explosives for several reasons: their explosive

power, the fact that traditional explosives are not required for detonation, and the ease of

manufacture. Peroxide explosives can be manufactured from a combination of hydrogen

peroxide, sulfuric acid, acetone, citric acid, and hexamine (commonly found in camp

fuel, see Figure 9 and Figure 10). While straightforward, the recipes for manufacture are

28

extremely dangerous to carry out and the products are dangerous to store. During

manufacture, the reaction vessel must be kept cold, and warming to room temperature is

extremely dangerous. There are many reports of serious injuries and death during the

manufacture of peroxide explosives. There are several well known terror acts involving

peroxide explosives.

2.7.4.1 Recent Uses of Peroxide Explosives

Recent events show the versatility and destructive power of peroxide explosives

and why they are often chosen for terroristic bombings.

In 2002, Richard Reid, a.k.a. “the shoe-bomber,” attempted to use TATP as an

improvised blasting cap to detonate a PETN charge. Passengers were able to restrain him

until the flight could safely land. As a result of this incident, persons boarding airlines in

the United States must submit their shoes for x-ray screening as they pass through

security. Reid has been sentenced to three consecutive life terms to be served in the

federal prison and over $2,000,000 in fines (“Richard Reid,” 2009).

The London Train Bombings of 2005 consisted of four separate explosions, three

in the subway tunnels and one on a double-decker bus. Peroxide-based explosives

including TATP and HMTD packed with nails in backpacks were detonated by suicide

bombers. The final death toll was 56 with 700 injured (“7 July 2005 London Bombings,”

2009).

In 2005, Joel Henry Hinrichs, a University of Oklahoma student, detonated

between two and three pounds of TATP outside of the packed OU football stadium in

Norman, OK. The motive and intent of the blast is still unknown, but Hinrichs had

29

additional explosive material and literature in his apartment. Only Hinrichs died in the

blast (“2005 University of Oklahoma Bombing,” 2009).

In 2006, terrorists in England planned to detonate peroxide liquid explosives on

several flights to Canada and the United States; however, they were intercepted prior to

takeoff. In total, 24 suspects were arrested in what would become known as the 2006

Transatlantic Aircraft Plot. It is suspected that the explosives would have consisted of

TATP or HMTD, peroxide liquids, and powdered orange drink mix in plastic bottles for

concealment. As a result of this event, the Transportation Security Authority (TSA) and

the Federal Aviation Administration (FAA) banned liquid or gel material over three

ounces in carry-on luggage (“2006 Transatlantic Aircraft Plot,” 2009).

More recently Najibullah Zazi, an Afghan immigrant, was accused of stockpiling

large amounts of acetone and high concentration hydrogen peroxide from beauty supply

stores. It is believed that he and others would manufacture TATP in Denver using

methods learned at al-Qaida training camps and then transport the explosives to New

York City for detonation. At this time, Zazi has been indicted on charges of terrorism

and is awaiting trial (Experts: Zazi Could Have Killed Scores - CBS News, 2009).

2.7.4.2 Current Analysis of Peroxide Explosives

Some analysts have had success doing both presumptive and confirmatory testing

of peroxide explosives; however, these methods are flawed and sometimes require highly

specialized instruments. In addition, none of these methods allows for the identification

of other types of explosives and may destroy the available evidence thereby preventing

further testing.

30

Buttigieg et al. (2003) describe the use of ion mobility spectrometry (IMS) for the

detection of lab-manufactured TATP in toluene. The instrument used for IMS analysis

had to be custom built and is not available commercially. Confirmation of TATP was

done by nuclear magnetic resonance (NMR) spectrometry and infrared spectroscopy (FT-

IR). The authors were able to detect two broad peaks using IMS. At the time of

publication, the authors were unable to verify the identities of the peaks, but were able to

state conclusively that they had visualized a TATP pattern using IMS. This method is

currently not able to identify other types of explosives at the same time as TATP, because

the instrument must run in opposite ion modes for each analysis.

Schulte-Ladbeck et al. (2002) created the first field test for TATP and HMTD

powders. Although the test is meant to be portable, many steps are required for analysis.

First, the powder is treated with catalase to destroy any hydrogen peroxide left over from

synthesis, but not TATP or HMTD. This step is necessary to prevent false positives that

may sometimes occur when testing laundry detergent. The peroxide explosive sample is

then extracted in acetonitrile and is irradiated with a 254 nm UV wavelength for no more

than 15 minutes which actually produces hydrogen peroxide when either TATP or

HMTD is present. The newly formed hydrogen peroxide can be visualized using an

enzyme-catalyzed reaction measured in the UV-visible (UV-Vis) spectrum. This is a

valid method for identifying peroxide explosives, but requires unusual enzymes and a

portable UV-Vis which is not feasible for most jurisdictions.

Schultte-Ladbeck et al. (2003) developed the first method for quantitative analysis

of TATP and HMTD. The authors note that GC/MS and UV-Vis cannot be used as

analysis methods due to shortcomings in the instrumentation. Since the publication of the

31

paper, GC/MS has been used to identify TATP and the work presented here will show

that CE-UV-Vis can also identify TATP. The method described in the paper of the

Schulte-Ladbeck group involved the use of HPLC with an acetonitrile/water gradient,

post-column irradiation using both p-hydroxyphenylacetic acid and horseradish

peroxidase, and visualization by a fluorescence detector. Like the previous method, this

work was novel and useful; however, it was time consuming and impractical (Schulte-

Ladbeck et al., 2003).

Several methods for the analysis of TATP and HMTD by GC-MS and LC-

MS/MS have been published in the recent years. HMTD has only been identified using

LC-MS/MS. Crowson and Beardah published a method in 2001 also using APCI that was

able to successfully identify HMTD with a parent ion of m/z 209 and additional peaks at

m/z 62, 90, 179, 207 (Crowson & Beardah, 2001).

Mueller et al., (2004) published a post-blast method for the analysis of TATP

using headspace GC-MS after solid phase microextraction (SPME). The combination of

headspace analysis and SPME overcomes the traditional roadblocks of the analysis of

volatile substance such as TATP. Using their instrument settings, TATP had a retention

time of 3.5 minutes and the mass spectrum showed peaks at m/z 43, 59, 75, 101, 117, and

222 (Mueller et al., 2004).

Also in 2004, Xu et al., published a method for the analysis of TATP using LC-

MS/MS. Using Atmospheric-Pressure Chemical Ionization (APCI) and methanol

ammonium acetate mobile phase, the authors were able identify both TATP (m/z 74, 223

32

240) and HMTD (m/z 40, 58, 88, 207, and 210) in a single sample analysis injection for

both standard and post-blast.

2.8 Analysis of Explosives by Capillary Electrophoresis

2.8.1 Discussion on the Published FBI CE Method for Explosives/Ions

The premier article utilizing CE to analyze explosive ions was written by

Hargadon and McCord (1992) (herein referred to as the published FBI CE method). The

goal of this article was to compare capillary electrophoresis and ion chromatography (IC)

in the analysis of anions found in explosives. According to the authors, CE offers sharper

but less well separated peaks and more efficiency compared to IC. The published FBI

CE method features a borate buffer using potassium dichromate as a chromophore with

indirect detection and reverse polarity. The published FBI CE method allows for the

analysis of 20 anions at 100 ppm, including the use of bromide as a standard for a

retention time marker in 10 minutes. The 20 anions selected for this method were chosen

because together they are characteristic ions found in explosives. The presence or

absence of these ions (see Table 1) can identify and point to the nature of the detonated

explosive.

33

Table 1. Low Explosive Ions Type Composition Residue

Black Powder KNO3,S, Charcoal

K+, Nitrate (NO3-), Sulfate (SO4

-), Carbonate (HCO3

-), Sulfide (HS-), Nitrite (NO2

-), Cyanate (OCN-), Thiocyanate (SCN-)

Pyrodex

KNO3, S, Charcoal, KClO4, NaOBz, Dicyandiamide

K+, Chloride (Cl-) NO3-, SO4

-, SCN-

, HS-, ClO3- NO2

-, HCO3-, OCN-

Flash Powder KCl03/4, S, Al K+, Cl-, Chlorate (ClO3

-), SO4-,

Perchlorate (ClO4-)

Smokeless Powder

Nitrocellulose, NG, DNT, Stabilizers

NO2-, NO3

-, SO4-, Cl-, K+, Na+,

NH4+

Solidox NaClO4, Sand, Fiberglass Na+, Cl-, ClO3-, ClO4, HCO3

-

Road Flares Sr(NO3), KClO3/4 K+, NO3-, Cl-, ClO3

-, NO2-

Chlorate/Sugar KClO4, sugar K+, Cl-, ClO3-, HCO3

-, ClO4-

Emulsions NH4NO3, NaNO3-, MMA,

Ca(NO3)2, Al Ca+, MMA+, NH4

+, NO3-, NO2

-, Na+

ANFO NH4NO3, Fuel Oil NH4+, NO3

-, HCO3-, NO2

-

Match Heads Silica, S, KClO3 K+, Cl-, SO4-, ClO3

-

Explosive ions found in low explosive compounds and residues (Modified from McCord & Bender, 1998)

The authors utilize 100 ppm ion samples; however, they note that sensitivity is not

generally an issue in explosive analysis as long as a considerable amount of the device is

retained. Further, quantitative analysis is less important in post-blast investigations than

is qualitative analysis, since it is not possible to determine the precise conditions during

the blast. The published FBI CE method was developed and utilized by the FBI for

explosive residue analysis (Hargadon & McCord, 1992).

2.8.2 Simultaneous Analysis of Anions and Cations Using CE

Since the original work analyzing anions was produced by Hargadon and McCord

in 1992, advances in instrument manufacture and electrolytes have allowed for the

34

simultaneous analysis of anions and cations by capillary electrophoresis. Several

approaches have been attempted in the simultaneous identification of cations and anions.

In 1999, Kuban et al. developed a method that allowed sample injection from both

capillary ends and in which detection occurs in the middle of the capillary. In order to

achieve this unique separation, the EOF is modified using ethylenediaminetetraacetc acid

(EDTA) and cetyltrimethylammonium bromide (CTAB) to reverse the flow within the

capillary. While this method does allow for the simultaneous analysis of anions and

cations, it does not allow for the analysis of some of the most important cations for

explosive analysis. A newer method was developed in 2005 utilized a novel electrolyte

background that allowed for the analysis of potassium, ammonium, and sodium which

had not previously been detectable by Kubans’ 1999 method. The buffer used in this

method deviates entirely from the previously used borate buffer and allows for the

analysis of many cations and anions expected during explosion detection analysis

(Hopper et al., 2005).

2.8.3 Analysis of TNT Based Explosives Using Capillary Electrophoresis

Capillary electrophoresis has also been used to analyze secondary and military

explosives. Groom et al., (2000) utilized an SDS buffer to perform MEKC in order to

identify TNT metabolites in anaerobic sludge. Using a borate/boric acid/SDS buffer,

Groom was able to resolve the analysis of both polar and non-polar analytes that were

previously unable to be resolved without the assistance of the micelles present in the SDS

buffer. This laboratory has gone on to analyze RDX and HMX explosives and

degradation products in soil and environmental run-off (Groom et al., 2001, 2002).

While this method has been used successfully to analyze secondary explosives, it is

35

unable to analyze anions or cations that may also appear in the explosive chain as primers

or boosters.

2.9 Recent Use of Capillary Electrophoresis for Hydrogen Peroxide

Analysis

Hydrogen peroxide has been analyzed using capillary electrophoresis; however,

each of the methods involves major adaptations to the detector, buffer, or instrument for

separation, and none of them has been utilized for peroxide explosive analysis

(Ruttineger & Radschuweit, 2000; Wang et al., 2002; Shihabi, 2006; and Pumera, 2008).

In one method, peroxide was analyzed as a byproduct of cellular and enzymatic reactions

using reductive amperometric detection with an Ag/AgCl electrode and a 10 mM borate

buffer (Ruttineger & Radschuweit, 2000). A similar method is also described with the

addition of a CE microchip and a borate SDS buffer (Wang et al., 2002). Shihabi (2006)

reported that hydrogen peroxide “generated enzymatically from glucose” was analyzed

utilizing a borate boric acid buffer at 185 nm. While this method is somewhat similar to

the published FBI CE method it lacks the chromophore and the ability to identify ions. In

Trends of Analysis of Explosives by Capillary Electrophoresis, Pumera (2008) reports

that while CE has evolved to a point where a wide variety of explosives may be analyzed,

peroxide explosives have surprisingly not yet been identified by capillary electrophoresis.

36

Chapter III Methodology

3.1 Instrumentation

All samples were analyzed using a Beckman P/ACE 5000 series instrument

(Figure 12) (Beckman Instruments Inc., Fullerton, CA) donated to Oklahoma State

University by the FBI Laboratory (Quantico, VA). The CE was equipped with a P/ACE

UV Absorbance detector with a wavelength range of 190 -380 nm and outfitted with

opaque, 200, 214, 254, and 280 nm filters. The UV source was a 30 watt (W) deuterium

lamp (Bulbtronics, Inc., Farmingdale, NY). The high voltage power supply had a voltage

range of 1.0 to 30 kV (kilovolts) (available in 100 V increments) and a current range of 1

to 250 µA (available in 0.1 µA increments). The pneumatics of the instrument were

controlled by high purity nitrogen at 80-85 psig (pound-force per square inch gauge).

The instrument has two sample trays. The inlet tray has a capacity of 24 sample vials and

the outlet tray has a capacity of 10 vials. The instrument was controlled and data

analyzed by Beckman P/ACE Station 1.2 Software.

37

Figure 12. Beckman P/ACE 5000

3.2 Capillary Column

Fused-silica capillary columns were procured from Polymicro Technologies

(Phoenix, AZ). The column had an inner diameter of 50 µm and an outer diameter of 360

µm.

For analysis, the column had to be installed into the Beckman P/ACE e/CAP

Capillary Cartridge System (Figure 13) with a 100 x 200 µm Aperture (Beckman

Instruments Inc., Palo Alto, CA). The total length of the capillary was 60 cm with an

effective length of 53 cm (to the detector window). The capillary was prepared removing

a 1.5 cm section of the polyimide coating using a Window Maker (MicroSolve, Long

Branch, NJ), removing the burned coating with methanol and a lint-free wipe. The

capillary was wound three times around the cartridge mandrel to achieve the desired

38

length. The capillary was inserted into the cartridge, secured as recommended by the

manufacturer, and trimmed to the desired length. Capillary cartridges were replaced as

needed using this method.

Figure 13. Beckman P/ACE Capillary Cartridge Assembly

In order to cool the capillary, the cartridge is manufactured to allow for coolant to

flow throughout. Perfluoro-compound FC-77 (Acros Organics, New Jersey) was used as

the continuingly flowing coolant.

3.3 Instrument Conditions

Three separate methods were used on the Beckman P/ACE 5000 for sample runs.

Each method was similar, but utilized different CE parameters to perform specific tasks.

3.3.1 Conditioning Method

This method conditioned new capillaries or regenerated the capillary after every

10 sample runs.

39

Capillary Temperature - 25.0oC

UV Detector - 5 Hz

UV Detector Wavelength - 254 nm

Rise Time* - 1 second (*the time it takes for the vial to move up for injection)

Signal - Indirect

Time Program

1) 3 minutes - Rinse - forward high pressure - 0.1 M Sodium Hydroxide (NaOH,

BDH, West Chester, PA)

2) 5 minutes - Rinse - forward high pressure - OSU Buffer

3) 10 seconds - Inject - Voltage Injection at 10 kV

4) 10 minutes Separate - Voltage 30 kV with a 1 minute ramp time

3.3.2 Analysis at 254 nm

This method used 254 nm to detect all peaks in the positive direction.


UV Detector- 5 Hz


Rise Time - 1 second

Signal - Indirect

Time Program



3) 10 minutes - Separate - Voltage 30 kV with a 1 minute ramp time

40

3.3.3 Analysis at 200 nm

This method used 200 nm in order to detect peaks in both the positive and negative

direction.


UV Detector- 5 Hz


Rise Time - 1 second

Signal - Indirect

Time Program



3) 10 minutes Separate - Voltage 30 kV with a 1 minute ramp time

3.4 Buffer Preparation

The OSU Buffer consisted of 2 mM (millimolar) Borate from Sodium Tetraborate

decahydrate (Na2B4O7·10H2O, Alfa Aesar, Ward Hill, MA), 40 mM Boric Acid (H3BO3,

EMD, Gibbstown, NJ), 1.8 mM Potassium Dichromate (K2Cr2O7, JT Baker, Phillipsburg,

NJ), and 2.1 mM Diethlenetriamine (DETA, C4H13N3, MP Biomedicals LLC, Solon, OH

(stored under nitrogen)). The first three compounds were weighed out and mixed with an

appropriate amount of 18 MΩ H2O. The suspension was allowed to stir for 10 minutes.

After 10 minutes the pH of the buffer was measured using a VWR SympHony™ pH

meter (VWR, West Chester, PA). The pH ranged between 7.15 and 7.28. At that time

the DETA was added and the buffer was allowed to continue stirring. The pH was taken

again and ranged between 8.03 and 8.12. The buffer was stored at 4ºC until use. Prior to

41

sample analysis the buffer was allowed to acclimate to room temperature. The buffer had

the signature golden color of potassium dichromate (Figure 14) which acts as a

chromophore and allows detection indirectly to analyze ions with low UV absorbance.

Figure 14. Buffer from Published FBI CE Method Notice the signature golden color of potassium dichromate.

3.5 Sample Preparation

3.5.1 Ion Samples

Ion samples were initially prepared at 100 ppm, and then diluted to 10 ppm for

analysis using volumetric glassware. After preparation, the ions were stored in amber

VWR TraceClean™ Jars (VWR) to prevent any possible contamination. Nine ions were

prepared from their sodium salts as shown in (Table 2). Ion standards were stored for six

months, after that time they were re-prepared.

42

Table 2. Ions Used in Method Development Ion Formula Manufacturer Location Sodium Bromide Crystal NaBr JT Baker Phillipsburg, NJ Sodium Bromide Metal Based NaBr Alfa Aesar Ward Hill, MA Sodium Chlorate NaClO3 Alfa Aesar Ward Hill, MA Sodium Cyanate NaCNO Alfa Aesar Ward Hill, MA Sodium Formate HCOONa JT Baker Phillipsburg, NJ Sodium Nitrate NaNO3 JT Baker Phillipsburg, NJ Sodium Nitrite NaNO2 Mallinckrodt Phillipsburg, NJ Sodium Perchlorate Monohydrate NaClO4(H2O) Alfa Aesar Ward Hill, MA Sodium Sulfate Na2SO4 EMD Gibbstown, NJ Sodium Thiocyanate NaSCN Alfa Aesar Ward Hill, MA

Sodium bromide (crystal) and sodium bromide (metal based) were both used in sample

analysis. It was noted that sodium bromide (metal based) would degrade just days after

opening and produce a split peak when analyzed on the CE. Sodium bromide (crystal)

was also used for analysis, but exhibited the same split peak during analysis. The

migration times for both the crystal and metal based forms matched and RMTs were

measured from the first bromide peak when a split peak was produced.

3.5.2 Peroxide Standards

Peroxide explosive standards were supplied by AccuStandard (New Haven, CT). TATP

and HMTD standards were received in concentrations of 100 µg/ml and supplied with

Certificates of Analysis and supporting chromatograms. Peroxide standards were also

prepared on-site in small quantities. TATP and HMTD preparation notes were obtained

from Dr. Kirk Yeager of the FBI Laboratory Explosives Unit and precursor amounts were

modified for decreased production. TATP was prepared using hydrogen peroxide 35%

(H2O2) dichloromethane ACS Grade (CH2Cl2), sulfuric acid 10.0N (H2SO4, BDH, West

43

Chester, PA), acetone HPLC Grade (CH3COCH3, Alfa Aesar), and HPLC Grade

methanol (CH3OH, EMD). HMTD (Figure 15) was prepared from hydrogen peroxide

35%, citric acid (C6H8O7, JT Baker), and hexamine (hexamethylenetriamine, (CH2)6N4,

Mallinckrodt). Standards were prepared in milligram quantities with appropriate

precautions in this laboratory. After manufacture, TATP was stored in methanol and

HMTD was stored in acetone at 4ºC

Figure 15. HMTD Manufactured at the OSU Laboratory

3.5.3 Extraction of Peroxide Swabs

Peroxide explosive swabs containing peroxide residue were obtained during FBI

National Improvised Explosive Familiarization (NIEF) workshops. One set of samples

was obtained in March 2008 at a Nevada workshop. These swabs, outlined in Table 3,

were stored in a paint can approximately 18 months at -70 oC until extraction.

44

Table 3. Description of Post-Blast Samples March 2008 Tube Contents Sample Names

Standard Blue Tubes Blue 1.1 Blue 1.2

Dry Cotton PB.D.1 PB.D.2 Plastic PB.D.3 PB.D.4

Wet Cotton PB.W.1 Cotton PB.W.2

The second set of samples was obtained in September 2009 at a Dover, DE, training

session. These samples as shown in (Table 4, Figure 16) were stored at 4ºC for

approximately 3 weeks before extraction.

Table 4. Description of Post-Blast Samples September 2009

Tube Contents Sample Names

Standard 1 Swab Std1.1 Post-blast 1 2 Swabs PB 1.1

PB 1.2 Post-blast 2 2 swabs PB 2.1

PB 2.2 Burn Sticks 4 Sticks B 1.1

B 1.2 B 1.3 B 1.4

45

Figure 16. Swabs and Burn Sticks from September 2009 Training Standard, swabs, and burn sticks collected in 2009 Delaware training

3.5.3.1 TATP

TATP post-blast samples were extracted in methanol. Briefly, samples were

extracted in 1.5 ml conical vials. Swabs were extracted by placing the entire tip of the

swab in the vial, cotton and plastic were extracted through placing a small cutting in the

vial, and burn sticks were extracted by placing a ½ inch section of each end in the vial.

Each sample was extracted using 500 µl of HPLC grade methanol. Samples were

allowed to stand at room temperature for 10 minutes, then vortexed, sonicated for 5

minutes with no heat, and then centrifuged for 5 minutes at 1500 rcf (relative centrifugal

force, or g). The supernatant solvent was removed and placed in a 1.5 ml amber vial.

The vial was analyzed by GC/MS (as described in section 3.6.1) first using full scan, then

SIM (if available).

46

For CE analysis, 100 µl of extract was mixed with 100 µl of CE buffer and 50 µl

of 10 ppm bromide using a 250 µl small sample insert and was injected on the CE using

both the 200 and 254 nm wavelength methods.

3.5.3.2 HMTD

No HMTD post-blast samples were analyzed

3.6 Verification of Peroxide Standards and Samples

3.6.1 TATP by GC/MS

As reported in the literature, TATP is a highly volatile compound and is often

difficult to identify with LC-MS/MS or GC/MS. This present work was not successful in

verifying the existence of TATP by LC-MS/MS using several published literature

methods as guidance. Following the recommended parameters of Kelly Hargadon-Mount

of the FBI Laboratory’s Explosive Unit, TATP was identified by GC/MS in this work.

The analysis of TATP was performed using an Agilent 6890 Series GC System

connected to an Agilent 5973 Network Mass Selective Detector driven by MSD

ChemStation D.01.00 (Agilent, Santa Clara, CA). The column used was a Restek RXI-

5ms 30 m in length and with an internal diameter of 250 µm (Restek Chromatography

Products, Bellefonte, PA). 3 µl of sample was injected using an autosampler/autoinjector

The GC/MS Conditions are shown in Table 5.

47

Table 5. TATP GC/MS Settings GC

MS

Oven Temperature 280 oC MS Quad 150 oC Column Pressure 6.68 psi MS Source 230 oC Carrier Gas Flow

Rate 19.5 mL/min

(Helium) Low Mass 40 Equilibration Time 0.10 min High Mass 400

Initial Set point 75 oC Data Rate 20 Hz Split Injection 20:01

Solvent Delay 3 min Hold 1 min then ramp to 150 oC at 10 oC/min

Run Time 10 min

In addition to the full scan method (40.0 to 400.0 m/z), Selected Ion Monitoring

(SIM) was also utilized to analyze for 43, 59, 75, 101, 117, and 222 m/z.

3.6.2 HMTD by LC-MS/MS

As in the case of TATP, the presence of HMTD needed to be verified prior to

analysis by CE. Since HMTD is very similar in structure to TATP, the same difficulties

were expected in the identification of the explosive. The verification process of HMTD

began by using the GC/MS method that proved successful in the identification of TATP;

however, possibly due to its thermal lability, it was not possible to confirm HMTD by

GC/MS.

HMTD has been successfully identified by several laboratories using liquid

chromatography with tandem mass spectrometry (LC-MS/MS). HMTD was analyzed

using direct infusion on to an Applied Biosystem 4000 QTrap LC-MS/MS (Applied

Biosystems, Foster City, CA) driven by Analyst 1.5 Software (Applied Biosystems). The

48

analysis parameters were similar (Table 6) to the Atmospheric Pressure Chemical

Ionization (APCI) method reported by Crowson and Beardah (2001).

Table 6. HMTD MS Settings

3.7 Sample Analysis by Capillary Electrophoresis

3.7.1 Ion analysis

Individual ion analysis required several steps. First, each ion was analyzed using

both 200 and 254 nm wavelength CE methods in triplicate. This step determined whether

or not the ion would absorb in the UV range with our method and the migration time for

the ion. To prepare samples for this step, 10 ppm stock solution was filtered using a 45

µm syringe filter (VWR) into an amber 4 ml (milliliter) vial. By identifying the peak

direction in this scan (positive or negative), identification of the ion was easier in future

analysis when presented with an ion mixture. The next step was to prepare an

ion/bromide mix sample to determine a relative migration time compared to our standard

bromide. The sample mixture was prepared by adding 5 ml of each 10 ppm ion standard

Source Type Heated Nebulizer Source

Temperature 165 oC Scan Type Q1 MS

Polarity Positive MCA 20 Scans

MS Range 50.00 to 400.00 Da Current 20

Nebulizer Current 1 GS1 25 GS2 20

Declustering Potential 10

Entrance Potential 10 Syringe Injection 10 µL/min

49

in a beaker, mixed, and then 4 ml of the sample mixture filtered using a 45 µm syringe

filter into an amber 4 ml vial. Once again, the sample was run in triplicate using both the

200 and 254 nm wavelength CE methods. Once a standardized migration and relative

migration time was obtained the ion was added to the ion blend for further analysis.

3.7.2 Peroxide Precursor Analysis

Before the analysis of the peroxide explosive standards could begin, precursor and

storage ingredients had to be analyzed to ensure that they were not responsible for peaks

observed in peroxide explosive standards. Each of the precursor ingredients were

analyzed by CE using the same buffer and instrument methods as the ion and peroxide

samples. Like the peroxide samples, precursor ingredients used a 250 µl sample insert

for consistency. Initial analysis of dichloromethane, sulfuric acid 0.1N, acetone,

methanol, and acetonitrile (C2H3N, EMD) were analyzed by the addition of 100 µl of

compound, 100 µl of running buffer, and 50 µl 10 ppm Br to the small sample insert

provided by Beckman P/ACE. Hydrogen peroxide was diluted to 3.5% and 0.35% using

deionized (18 MΩ) water prior to injection. Acetonitrile was analyzed since it is the

solvent for the AccuStandard products. Initial analysis provided a typical

electropherogram of each analyte in both the 200 and 254 nm wavelengths.

3.7.3 Peroxide Standard Analysis

Like the precursor ingredients the peroxide explosive standards were analyzed

using the 250 µl sample inserts. These inserts allowed for the exact same separation,

while requiring a smaller sample volume. Also, the samples were diluted using running

buffer to decrease the required sample size. The first set of samples was analyzed

50

without bromide and they were mixed with 100 µl of peroxide explosive standard and

100 µl of running buffer. To obtain relative migration times, 50 µl of 10 ppm bromide

was added to the sample inserts. Peroxide standards were injected utilizing both the 200

and 254 nm wavelengths for peak identification.

3.7.4 Peroxide Residue Analysis

The methanol extract from the peroxide swabs was analyzed in a similar method

as the analysis of peroxide standard analysis. For analysis: 100 µl running buffer, 100 µl

methanol extract, and 50 µl of 10 ppm bromide standard was added to the 250 µl vial

inserts. Each sample was injected twice utilizing using both the 254 nm and 200 nm

wavelength CE methods.

3.8 Statistical Analysis

All statistical analyses were performed using GraphPad Prism Version 5.0

(GraphPad Software, San Diego, CA).

51

Chapter IV Results

4.1 Published FBI CE Method

Analysis first began on the nine ion samples using the published FBI CE

method as described in 1992 (Table 7). After preparation, each of the nine ions chosen

for analysis was injected individually along with bromide as a standard for determining

relative migration time. Also noted during this initial analysis was whether each ion

presented in the positive or negative direction at 200 nm. The peak direction at 200 nm

would aid in identification of the ion in the future.

52

Table 7. MTs and RMTs at 1.0 mM DETA

Ion Migration Average Relative Migration

Average Peak at

254 Peak at

200 Bromide 2.953 0.000 Positive Negative Nitrite 3.352 1.079 Positive Negative Sulfate 3.387 1.101 Positive Positive Nitrate 3.400 1.105 Positive Negative

Perchlorate 3.432 1.172 Positive Positive Chlorate 3.578 1.194 Positive Positive

Thiocyanate 3.588 1.229 Positive Negative Cyanate 3.753 1.269 Positive Positive Formate 4.154 1.449 Positive Positive

As the individual ion samples were run, it became apparent that the ions acted

differently when run individually versus when run with other ions, including bromide

(Table 8, Figure 17). When the ions were run independently, migration was slowed

compared to when each was run with bromide or with other ions. Throughout this

work, all samples were run with and without bromide to set a benchmark for individual

ion migration and also for relative ion migration that would more likely be seen when

analyzing case work samples.

53

Table 8. Ion Migration Times With and Without Bromide Ion Without Bromide With Bromide Difference (in minutes)

Nitrite 3.308 3.474 0.166 Nitrate 3.432 3.565 0.133 Sulfate 3.292 3.303 0.011

Perchlorate 3.660 3.844 0.184 Thiocyanate 3.590 3.662 0.072

Cyanate 3.570 3.623 0.053

Formate 3.963 4.041 0.078

Figure 17. Migration Times With and Without Bromide The presence of bromide generally increased migration time.

It was evident that CE analysis using the parameters in the original published

FBI CE method did not provide the high level of separation expected; therefore, the

method was optimized for this work.

54

4.2 Optimizing the Published FBI CE Method

Optimizing the published FBI CE method (Hargadon & McCord, 1992) was the

major challenge for this work. A properly developed method will provide a cleaner

separation and allow for the possibility for peroxide explosive analysis.

The original and optimized conditions are listed in Table 9.

Table 9. Published FBI CE Method and OSU Method Published FBI CE Method* OSU Method

Instrument and Column Conditions Instrument and Column Conditions

Column 75 µm Column 50 µm

Capillary Length 65-67 cm Capillary Length 60 cm Wavelength set at 280 nm (scanning) Wavelength 200 and 254 nm

Pressure Injection Voltage Injection 10 kV Separation at 30 kV Separation at 30 kV

Buffer Buffer

2 mM Borate 2 mM Borate

40 mM Boric Acid 40 mM Boric Acid 1.8 mM Dichromate 1.8 mM Dichromate

1 mM DETA 2.1 mM DETA pH 7.8 adjusted by DETA pH ~8.15 adjusted by DETA

* Hargadon & McCord, 1992

As described subsequently, four areas of the published FBI CE method were optimized,

including the instrument, the capillary, the buffer, and the ions.

4.2.1 Instrument Conditions

In order to optimize the method for our instrument we made changes to the

wavelength settings and the type of injection. To overcome the lack of a scanning UV

55

detector as presented in the published FBI CE paper, separations were run at 200 and

254 nm wavelengths for each sample (Figure 18).

Figure 18. Nine-Ion CE Separation at 254 and 200 nm This figure shows separation of nine ions using 2.1 mM buffer at 254 nm (red and solid) and 200 nm (black and dotted).

A 10 K voltage injection instead of the pressure injection was chosen, because the

laminar flow of the voltage injection provided a more repeatable separation over the

parabolic flow of the pressure injection.

4.2.2 Capillary Dimensions

Adjustments were also made to the capillary dimensions. Although samples

were processed using the original published FBI CE specifications, separations were

not as complete as expected. As a result, several changes were made to the column

length and diameter. The column length was decreased from 67 mm to 60 mm and the

internal diameter from 75 µm to 50 µm. A shortened and narrower column decreased

sample run time and created sharper peaks.

56

4.2.3 Buffer

Several changes were made to the original buffer components. The published

FBI CE method (Hargadon & McCord, 1992) was not specific as to whether sodium

borate anhydrous (Na2B4O7, VWR) or sodium tetraborate decahydrate was used in the

buffer. After several trials it was determined that the decahydrate was the optimal base

for the buffer. Also there were no recommendations on the proper storage and shelf life

of the buffer. Research quickly demonstrated that the buffer should be stored at 4ºC

and discarded after ten days. It should also be noted that the buffer was allowed to

come to room temperature prior to usage. After the buffer was optimized only 100 ml

of buffer was prepared for each batch.

4.2.3.1 DETA Trials

The major portion of optimizing the published FBI CE method came from the

adjustment of DETA concentration which influenced the pH of the buffer. Early

analysis showed that ion peaks were not well separated as the original method

recommended. In addition to making changes to the capillary, the best way to adjust

resolution, separation, and migration of the ions is to adjust the pH of the buffer. In this

method that meant adjusting the amount of DETA added, which must be done in very

small increments in order to note the effect that each change in pH has on the ion

separations. To achieve the small increments, the published FBI CE buffer was

prepared without DETA. Aliquots were divided into 25 ml conical vials and then

spiked with DETA to achieve the desired final concentration (1.0 mM, 1.2 mM, 1.4

mM, 1.6 mM, 1.8 mM, 2.0 mM).

57

Table 10. The Changes of the RMTs With Increased DETA Concentration

Ion 1.0 1.2 1.4 1.6 1.8 2.0 Bromide 1.000 1.000 1.000 1.000 1.000 1.000 Nitrite 1.105 1.068 1.072 1.072 1.072 1.074 Nitrate 1.079 1.092 1.089 1.089 1.091 1.093 Sulfate 1.101 1.094 1.105 1.105 1.118 1.126

Perchlorate 1.172 1.157 1.152 1.152 1.149 1.145 Thiocyanate 1.194 1.168 1.165 1.165 1.160 1.159

Chlorate 1.229 1.214 1.209 1.209 1.199 1.200 Cyanate 1.269 1.232 1.226 1.226 1.217 1.212 Formate 1.449 1.418 1.415 1.415 1.397 1.377

DETA Concentration Increasing from 1.0 mM to 2.0mM

As shown in (Table 10), these initial changes did produce better separations, but

further enhancement was deemed possible. From 1.8 mM, the molarity was further

increased in 0.1mM increments (1.9 mM, 2.0 mM, 2.1 mM, etc. up to 2.4 mM). In

addition, aliquots were spiked in 0.2 mM increments up to 3.0 mM to ensure that the

greatest separations had been achieved. At DETA concentrations greater than 2.4 mM

the ability to resolve all of the peaks was lost and data was no longer collected for those

concentrations of DETA. It became important to analyze the nuances between the

average migration and relative migration times for each ion in order to determine the

optimal concentration of DETA.

58

Table 11. Changes to RMT with DETA Variation 2.0 mM to 2.4 mM Peak 2.0 2.1 2.2 2.3 2.4 Average

1 - Bromide 1.000 1.000 1.000 1.000 1.000 1.000 2 -Nitrite 1.075 1.078 1.073 1.102 1.076 1.081 3 - Nitrate 1.099 1.102 1.101 1.102 1.103 1.102 4 - Sulfate 1.144 1.152 1.146 1.151 1.154 1.149

5 - Perchlorate 1.168 1.177 1.175 1.173 1.173 1.173 6 - Thiocyanate 1.182 1.190 1.186 1.191 1.185 1.187

7 - Chlorate 1.225 1.236 1.232 1.229 1.229 1.230 8 - Cyanate 1.242 1.252 1.245 1.244 1.244 1.246 9- Formate 1.431 1.452 1.438 1.433 1.434 1.438 Data from November 17-19, 2008. Each concentration had five runs consisting of both 254 and 200 nm conditions.

The RMTs from Table 11 show that optimum separation was between 2.0 and

2.4 mM of DETA. The best way to identify the best DETA concentration was to

perform resolution calculations for the closest migrating peaks. Three pairs of peaks, 2

and 3 (nitrite and nitrate), 5 and 6 (perchlorate and thiocyanate), and 7 and 8 (chlorate

and cyanate) were compared using relative migration times, resolution, and also visual

comparison to identify the concentration of DETA that provided the best separation of

ions.

Resolution calculations were performed using the resolution calculation and are

reported in Table 12. Peak data were gathered from three separate sample injections

and utilized both 200 and 254 nm wavelength data.

Table 12. Resolution Values Between Neighboring Peaks DETA Concentration Peak 2 & 3 Peak 5 & 6 Peak 7 & 8

2.0 mM 2.429 1.116 1.205 2.1 mM 2.491 1.259 1.229 2.2 mM 2.772 1.270 1.120 2.3 mM 2.662 1.180 1.260 2.4 mM 2.524 1.268 1.343

59

Relative migration times were also considered when choosing the optimum

DETA concentration. It is important that the molarity chosen offered repeatable and

consistent migration times. This characteristic was not seen above 2.2 mM. Visual

inspection of the electropherograms also impacted the final choice of buffer

concentration. 2.0 mM, 2.1 mM, and 2.2 mM were visually examined (Figure 19,

Figure 20) to determine which concentration showed the greatest separation along with

the most baseline between peaks and compared this information with the calculated

resolution times. Visual examination of the electropherograms also showed that 2.1

mM allowed the greatest baseline separation out of all of the concentrations.

Ultimately 2.1 mM DETA , resulting in a buffer pH value of approximately 8.15, was

determined to provide the greatest separation of all ions.

Figure 19. Electropherogram of Nine-Ion Separation at 2.0 and 2.1mM DETA

This electropherogram shows 2.0 mM DETA (black and dotted) and 2.1 mM DETA (red and solid). Note the stronger baseline and greater peak separation between peaks 5 and 6 and 7 and 8 at 2.1 mM of DETA.

60

Figure 20. Electropherogram of Nine-Ion Separation at 2.1 and 2.2 mM DETA

This electropherogram shows the difference between 2.1 mM (red and solid) and 2.2 mM (black and dotted). Note the separation between peaks 5 and 6 and 7 and 8 at 2.1 mM of DETA.

4.2.4 Ions

This work also made some changes to the ions of interest. The original

published FBI CE paper used 20 ions whereas we limited our analysis to nine ions in

order to focus on the possible recovery of peroxide residues. The ions were chosen to

represent the most common anions in traditional explosives as seen in Table 1. The

ions used in the published FBI CE method for analysis were at 100 ppm and for this

work we diluted them to 10 ppm. In addition, the published FBI CE method originally

used dye as the internal standard for migration analysis and then changed to bromide.

Bromide was also used as the internal standard for migration analysis in this work. One

of the reasons that bromide was chosen is that bromide is not an ion that would be

created in any explosive detonation and therefore would not appear as an ion of interest

61

in any explosive analysis work. In addition, bromide is the earliest migrating peak;

therefore, allows for easier relative migration calculations.

As shown in Table 13, the final buffer contained 2.1 mM of DETA and allowed

good separation of all nine ions with reproducible migration and relative migration

times.

Table 13. MTs and RMTs of Nine Ions at 2.1 mM DETA Average Low High

Bromide 3.923 3.810 4.030 RMT 1.000 1.000 1.000

Nitrite 4.178 4.147 4.203 RMT 1.073 1.072 1.074

Nitrate 4.249 4.230 4.273 RMT 1.092 1.091 1.094

Sulfate 4.372 4.327 4.430 RMT 1.122 1.118 1.126

Perchlorate 4.325 4.253 4.393 RMT 1.147 1.145 1.149

Thiocyanate 4.454 4.407 4.497 RMT 1.159 1.159 1.160

Chlorate 4.499 4.467 4.527 RMT 1.200 1.195 1.203

Cyanate 4.584 4.523 4.677 RMT 1.214 1.212 1.217

Formate 5.189 5.137 5.243 RMT 1.387 1.376 1.398

Figure 22 and Table 14 show a representative electropherogram and data

produced in a sample run. Figure 21 shows the differences in the electropherograms

with the original literature conditions versus the final optimized conditions.

62

Figure 21. Electropherogram Comparing Nine-Ion Analysis with Published FBI CE Method and Optimized OSU Method

Published FBI CE Method analysis with 1.0 mM buffer (black and dotted) and optimized OSU method 2.1 mM buffer (red and solid) shows the difference that these changes had on resolution, baseline, and peak sharpness. Note that the published FBI CE method does not show all nine peaks due to poor resolution.

63

Figure 22. Representative Electropherogram of Nine-Ion Separation at 2.1 mM DETA Data m2.11117b at 254 nm

Table 14. Values from Figure 22 Peak Number Migration Time Ion Relative Migration Time

1 4.070 Bromide 1.000 2 4.380 Nitrite 1.074 3 4.490 Nitrate 1.103 4 4.690 Sulfate 1.152 5 4.790 Perchlorate 1.177 6 4.850 Thiocyanate 1.192 7 5.030 Chlorate 1.236 8 5.100 Cyanate 1.253 9 5.920 Formate 1.455

64

4.3 Verification of Peroxide Standards

4.3.1 TATP by GC/MS

TATP standards from both AccuStandard and OSU manufactured lots were

analyzed by GC/MS as shown in Figure 23, Figure 24, and Figure 25.

Figure 23. GC/MS Chromatogram and Spectra of TATP as provided by AccuStandard

65

Figure 24. GC/MS Chromatogram and Spectra of AccuStandard TATP in Full Scan Mode AccuStandard TATP Lot B8090264-1A Expiration Date 04-10 analyzed using full scan OSU GC/MS method. Spectra shown at a retention time of 5.741 minutes.

Figure 25. GC/MS Chromatogram and Spectra of AccuStandard TATP in SIM Mode AccuStandard TATP Lot B8090264-1A Expiration Date 04-10 analyzed using OSU SIM GC/MS method. Spectra shown at a retention time of 5.743 minutes.

66

Figure 26. Representative GC/MS Chromatogram and Spectra of TATP Synthesized Onsite in Full Scan Mode TATP manufactured 4-13-2009 analyzed using full scan OSU GC/MS method. Other batches produced similar results. Spectra shown at a retention time of 5.783 minutes.

Each chromatogram showed a retention time ranging from 5.724 to 5.808. The

primary ions (43, 59, 75, 91, 101, 117, 129, 207, and 220 m/z) are shown in each

spectrum.

4.3.2 HMTD by LC-MS/MS

The HMTD standard manufactured at OSU was analyzed by infusion into the

tandem mass spectrometer (MS/MS, see Figure 27). The parent ion of m/z 209

[HMTD +H] and daughter ions of m/z 62, 88, 90, 179, and 207 (Crowson & Beardah,

2001) are shown in the spectrum.

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Figure 27. MS/MS Spectra of HMTD

4.4 Sample Analysis by Capillary Electrophoresis

4.4.1 Peroxide Explosive Precursor Analysis

Each chemical compound used in the manufacture and storage of peroxide

explosives had to be analyzed by CE to verify if any peaks were visible; and if so,

whether or not they were consistent with TATP or HMTD. Table 15 summarizes the

findings from the precursor study.

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Table 15. Analysis of Precursor Ingredients

Precursor TATP HMTD Ingredient Storage CE

Peaks

Consistent with

Peroxide Hydrogen Peroxide X X X

yes

Further Analysis

Sulfuric Acid X

X

yes Further

Analysis

Acetone X

X X yes weak, not consistent

Dichloromethane X

X

yes weak, not consistent

Methanol X X

X yes weak, not consistent

Acetonitrile X X

X (Accu-

Standard) no N/A

Hydrogen peroxide was analyzed at 3.5% and 0.35% concentrations made from

the stock of 35% used in the manufacture of the OSU peroxide explosive standards.

Lower concentrations of hydrogen peroxide were analyzed, because it was unknown

whether higher concentrations of peroxide would damage the capillary column. The

3.5% concentration was analyzed first and showed strong, but not overwhelming peaks.

Following that analysis, 0.35% hydrogen peroxide was analyzed showing very weak

peaks. All data presented for hydrogen peroxide is at a concentration of 3.5%.

The number of peaks visible for hydrogen peroxide varied depending on each

sample. A minimum of three peaks and a maximum of twelve peaks appeared after

separation depending on the day the samples were analyzed. It is important to note that

even though the analysis produced many peaks only seven of the peaks were within the

analysis window of peroxide explosives. Three separate days of paired injections were

completed and a representative electropherogram is shown in Figure 28.

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Figure 28. Representative Electropherogram of Hydrogen Peroxide Hydrogen peroxide 3.5% Analysis completed September 21, 2009, with 2.1 mM DETA buffer at 10 ppm bromide at 254 nm (red/solid) and 200 nm (black/dotted).

What emerged from the analysis of hydrogen peroxide was a pattern of relative

migration times appearing at least twice during the six hydrogen peroxide samples

injected as seen in Table 16. This pattern of peaks was compared to peroxide explosive

standard peaks.

Table 16. Hydrogen Peroxide Peak Pattern

Br Peak

1 Peak

2 Peak

3 Peak

4 Peak

5 Peak

6 Peak

7 RMT 1.00 1.022 1.086 1.106 1.119 1.149 1.169 1.229

Sample Size n=4 n=4 n=4 n=2 n=4 n=2 n=2 n=2

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Sulfuric Acid was analyzed at a concentration 0.1N made from the stock of

10.0N used in the manufacture of the OSU TATP standards. Like hydrogen peroxide, it

was unknown if extremely strong concentrations of sulfuric acid would damage the

column. The 0.1N concentration showed one single very strong peak as shown in

Figure 29. The relative migration time of sulfuric acid from bromide was 1.135.

Figure 29. Electropherogram of Sulfuric Acid Analysis of sulfuric acid 0.1N in 2.1 mM DETA buffer with 10 ppm bromide at 254 nm (red/solid) and 200 nm (black/dotted).

4.4.2 Peroxide Standard Analysis

4.4.2.1 TATP

The prepared TATP standards were first identified by CE using injection

without bromide as an internal standard. Figure 30 is a representative electropherogram

of TATP showing four peaks. It is important to note that the second and third peaks

point negatively when analyzed at 200 nm.

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Figure 30. Representative Electropherogram of TATP Without Bromide

Analyzed with 2.1 mM DETA buffer without bromide at 254 nm (red/solid) and 200 nm (black/dotted).

All OSU TATP standards were analyzed on a single run on 9-21 using bromide

as a migration time marker. From this bulk analysis we were able to determine a TATP

peak pattern and RMT profile of the four TATP peaks. In addition, peaks two and

three always appear negatively when analyzed at 200 nm, further aiding the possible

identification of TATP when using CE. The TATP batches prepared on 4-13 and 5 -11

did show some degradation during runs as a result of the storage time since

manufacture. A representative electropherogram of TATP standard with bromide and

the four peaks previously observed is shown in Figure 31.

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Figure 31. Representative Electropherogram of TATP with Bromide Analyzed on September 21, 2009, with 2.1 mM DETA buffer with bromide 10 ppm at 254 nm (red/solid) and 200 nm (black/dotted).

The single run of TATP included injections of TATP 4-13, TATP 5-11, and

TATP 9-9 separated at both 200 and 254 nm. Table 17 shows the data obtained in this

sample run.

Table 17. Relative Migration Times for TATP Batch Peak 1 Peak 2 Peak 3 Peak 4

TATP 4-13 - 1.077 1.112 1.157 - 1.079 1.111 1.156 TATP 5-11 - 1.078 1.109 - - 1.078 1.110 1.158 TATP 9-9 1.016 1.077 1.112 1.158 1.015 1.075 1.109 1.153

Although the batches from 4-13 and 5-11 did show some degradation in the loss

of the first and fourth peaks, the remaining relative migration times show correlation

for each peak throughout the data sets with a p>.05 using ANOVA for statistical

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analysis with Tukey’s multiple comparison post-test. Statistical analysis was

performed using GraphPad Prism Version 5.0 (GraphPad Software, San Diego, CA).

It was important to compare this data to the information gathered during

precursor analysis to verify if TATP or precursor ingredients were being seen during

analysis. Only two of the precursor compounds showed consistent activity on the CE:

sulfuric acid and hydrogen peroxide as summarized in Table 15. Although sulfuric acid

did show a strong peak at RMT 1.135, it did not match the RMT or direction shown in

the TATP peak pattern.

The analysis of TATP and hydrogen peroxide was more difficult due to the

apparent similarities of the peak RMTs (see Table 18). The variety of hydrogen

peroxide peaks provided a challenge for accurate comparison.

Table 18. TATP and Hydrogen Peroxide Signature Peaks

TATP Pk 1

H2O2 Pk 1

TATP PK 2

H2O2 Pk 2

TATP Pk 3

H2O2 Pk 3

TATP Pk 4

H2O2 Pk 4

RMT 1.016 1.022 1.077 1.086 1.111 1.119 1.156 1.169 Sample

Size n=3 n=4 n=6 n=4 n=6 n=4 n=5 n=2 Data collected using 2.1 mM DETA Buffer and 10 ppm bromide

The data from Table 18 was analyzed using a one-way analysis of variance

(ANOVA) followed by Tukey’s multiple comparison post-test to determine whether or

not the relative migration times for TATP and hydrogen peroxide were significantly

different. This analysis revealed that the RMTs for TATP and hydrogen peroxide were

significantly different with a p <0.05. Figure 32 also shows the separation between

each grouping of data points.

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Figure 32. Relative Migration Times of TATP and Hydrogen Peroxide

These data show that TATP standards confirmed by GC/MS were separated and

detected by capillary electrophoresis, and discriminated from all precursors used in

TATP preparation.

4.4.2.2 HMTD

HMTD was not identified by CE-UV in this study. Methanol, acetone, and

acetonitrile were all used to store HMTD safely after manufacture, but the compound

did not fully dissolve in any of these solutions. Injections of HMTD standard combined

with CE buffer, and 10 ppm bromide yielded electropherograms that showed no

distinctive peaks except for bromide.

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4.4.3 Peroxide Residue Analysis

4.4.3.1 Las Vegas, Nevada Training Samples

Following extraction TATP residue samples were first injected onto the GC/MS

using both the full scan and SIM methods. The blue tubes described in Table 19 were

empty vials that once contained standard, not post-blast samples. The swab of this

empty standard vial was positive for TATP by GC/MS but not by CE. The other

samples collected at this training could not be verified by GC/MS or CE (see Table 19).

Table 19. Analysis Results of Post-Blast March 2008 Samples Tube Contents Sample Names GC/MS Results CE Results

Standard Blue Tubes Blue 1.1 TATP Negative Blue 1.2 Not Tested Not Tested

Dry Cotton PB.D.1 Negative Negative PB.D.2 Negative Negative Plastic PB.D.3 Negative Negative PB.D.4 Negative Negative

Wet Cotton PB.W.1 Negative Negative Cotton PB.W.2 Negative Negative

4.4.3.2 Dover, Delaware Training Samples

As described in 3.5.3, swabs of trace residue from TATP standard vials,

exploded TATP charges, and burn sticks used to burn TATP were collected.

The methanol from the extracted swabs was injected onto the GC/MS using

only full scan analysis as the more sensitive SIM scan was not in use at that time. The

GC/MS analysis failed to confirm the identification of TATP on the sample swabs.

Prior to injecting the swab samples on the CE, it was noted that when the methanol

extraction was mixed with the buffer a cloudy solution was formed. The sample was

then injected onto the CE and produced three peaks: bromide (1), bromide (2), and

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weak positive peak (3) as shown in Figure 33. The identity of peak 3 was unknown and

appeared to be possibly, at least, a degradation product of TATP. Table 20 lists the

relative migration times observed for each sample.

Table 20. Relative Migration Times for Blank, Standard, Trace, and Post-Blast Peaks from September 2009 Swabs

Swab Blank (n=2)

Std Swab (n=2)

Post-Blast (n=8)

TATP Pk 4 (n=5)

Peak RMT 1.147* 1.160** 1.161** 1.156** Analyzed using 2.1 mM DETA buffer with 10 ppm bromide. *Statistically different (p<0.05) **Not statistically different (One-way ANOVA with Tukey’s Post-test p<0.05) A blank sample of an extracted swab produced similar electropherograms and

migration times. Figure 33 is a representative electropherogram of the blank swab and

TATP post-blast swabs extract.

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Figure 33. Electropherogram of Post-Blast Swab Peaks and Blank Swab Analysis of methanol extract of post-blast TATP residue swab (red/top) and blank swab (bottom/black) using 2.1 mM DETA buffer with 10 ppm bromide at 254 nm (solid) and 200 nm (dotted).

When the RMT of the unknown peak from the TATP post-blast residue swab

was compared to the TATP peak pattern (Figure 33, Table 20), TATP peak four with a

mean of 1.156 (n=5) was not statistically significant different from the unknown swab

peak with a mean of 1.160 (n=8); whereas, the peak from the swab blank had a mean of

1.147 (n=2) (Table 20) and was significantly different using one way ANOVA with

Tukey’s comparison post-test (p<0.05). From this analysis, TATP has been identified

by CE on a post-blast swab.

The burn sticks were also analyzed. The samples were first analyzed by

GC/MS and produced a chromatogram and spectrum close to the TATP standard

(Figure 34 and Figure 35).

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Figure 34. GC/MS Full Scan Chromatogram and Spectra of Burn 1.1

Analysis of Burn 1.1 sample collected September 2009 by OSU GC/MS full scan method. Spectra shown at a retention time of 5.741 minutes

Figure 35. GC/MS SIM Chromatogram and Spectra of Burn 1.1 Analysis of Burn 1.1 sample collected September 2009 by OSU GC/MS SIM method. Spectra shown at a retention time of 5.731 minutes

The supernatant from the methanol extract was injected several times onto the

CE and the results are shown in Table 21 and Figure 36. Peak 1 appears in every burn

sample that was injected with a relative migration range of 1.107 to 1.115. In addition,

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for every sample that was processed at 200 nm, peak 1 appears negative. The retention

time, shape, and behavior of peak 1 is similar to the third TATP peak (see Table 22).

Statistical analysis utilizing ANOVA with a Tukey’s comparison post-test p>.05

showed that these values are statistically similar supporting the belief that TATP has

been identified using CE on the remnants of a TATP burn stick.

Table 21. Relative Migration Times from Burn Samples Br 1 Peak 1 Peak 2 Peak 3

RMT 1.000 1.112 1.152 1.170 Sample

Size n=27 n=27 n=16 n=3 Bulk sample analysis from four samples runs of burn samples with 2.1 mM DETA buffer and 10 ppm bromide

Figure 36. Electropherogram Comparing Burn Sample 1.1 to TATP Analysis of burn sample 1.1 (top) and TATP (bottom) manufactured September 9, 2009, using 2.1 mM DETA buffer and 10 ppm bromide at 254 nm (solid) and 200 nm (dotted). Arrows indicate TATP peaks at both wavelengths in each respective sample.

80

Table 22. Comparison of Burn Stick RMT to TATP Standard RMT

Burn Test Peak 1 TATP Peak 3

RMT 1.112* 1.111*

Sample Size n=27 n=6 *Not statistically different from one another (one-way ANOVA with Tukey’s post-test, p>0.05).

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Chapter V Discussion

This research has yielded a method to simultaneously identify both peroxide

explosives and traditional explosive anions using a single sample preparation and CE

injection. This work is the first of its kind that can analyze for both types of samples

simultaneously. With the increasing concerns over peroxide explosives in terrorist and

other improvised explosive devices, this study is particularly critical for the forensic

investigation of post-blast materials.

While the benefits of this method are clear, it should be noted that there are other

indicators of peroxide explosives. For instance, low explosives do not generally have the

same sound or effects on materials that high explosives will exhibit. Therefore, an

examiner may be able to visually determine that high explosives, versus low explosives,

were used. This method will, however, identify the presence of peroxide explosive

residues in post-blast samples when sufficient amounts of residue are present. This

method was developed qualitatively as quantitative results are not as critical in post-blast

investigations (Hargadon & McCord, 1992).

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One of the first observations made in this research was that there is a large shift in

migration times when ions are combined with bromide or any other ion (Table 8, Figure

17). The migration shift was seen in each sample injection where the ions were run in

combination with other ions versus being run separately. However, the migration shift

for each ion does not increase with additional ions added to the mix, but it does increase

gradually over replicate analyses if the column is not reconditioned. For example, the

shift was slightly greater at the end of 10 sample injections than it was at the first sample

injection. Conditioning the column every 10 sample runs with 0.1 M NaOH refreshed the

column and decreased the migration shift. This shift has not been previously documented

in the literature and was a surprising initial finding during analysis. There is no

explanation for this evidence from this research, but it is a substantial finding in that this

shift was seen in the eight additional ions as well as the internal standard bromide. For

this reason, it would be important for labs adapting this method to consider this shift.

During method validation, labs would want to establish the use of a particular

internal standard/relative migration time marker in order to calculate reproducible RMTs.

For casework to be valid under the Daubert standard, the lab would need to be prepared

to show signature peak RMTs for each ion or compound identified through CE analysis.

Choosing the proper internal standard that would compensate for the shift in migration

times would be an important part of method validation.

Substantial changes to the published FBI CE method were required to complete

any form of ion analysis. While the published FBI CE method was fully adapted to this

instrument, it would not be surprising for a lengthy validation procedure to be required

before this or any CE method would be considered valid for forensic analysis.

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Instrumental changes are an important factor in method development and

validation. As seen in (Table 9), most running conditions were changed to optimize the

method on the CE used here. The decrease in capillary length and reduction in the

diameter produced sharper, stronger peaks, and a smoother baseline. While the lack of a

scanning UV wavelength detector at first seemed a weakness, it eventually strengthened

the data because two separate runs were required to analyze the same sample. The fact

that RMTs were consistent between samples analyzed at 254 nm and 200 nm showed that

the method was reproducible under slightly different conditions (Figure 18). The change

to a voltage injection from a pressure injection contributed to a more stable baseline and

better separation between peaks, although the reason for this is unknown. As mentioned

in Figure 3, it is speculated that it is related to the pressure pulse associated with a

pressure injection. Each of these changes had a positive effect on the results. If the

method were to be validated on another instrument, the laboratory should evaluate which

of the parameters should be changed to optimize the instrument and method for their

specific use.

This method did not allow us to have complete resolution between every set of

peaks. The resolution values obtained in Table 12 do not reach the resolution value of

1.5 that represents complete separation. However, any value over 1.0 is considered to be

fairly good separation (just 10% overlap) and all of our resolution values were over 1.22.

In addition, a visual inspection of the electropherogram shows nine peaks and that each

set of closely migrating peaks is distinguishable. Furthermore, there is no mention in the

published FBI CE method regarding any form of resolution analysis or quality of peak

separation.

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The choice of the final buffer DETA concentration came down to resolution,

visual inspection, and repeatability. The values in Table 12 show that between 2.0 and

2.4 mM DETA, 2.1 mM clearly shows the greatest resolution value. The visual

inspection of the electropherograms focused on peaks 5 & 6 and peaks 7 & 8, and 2.1mM

DETA showed better separation then 2.0 and 2.2 mM DETA as shown in Figure 19 and

Figure 20. Once 2.1 mM DETA buffer concentration was chosen, the method progressed

much more smoothly and presented no problems during peroxide analysis.

Complementary analysis was important during this research to confirm the

existence of TATP standards and post-blast explosive samples. The ability to prove the

existence of peroxide explosives using instruments operating on different scientific

principles provided validity of the results achieved by CE. It also verifies the possibility

of using CE as an initial screening tool for multiple types of explosives, with verification

of identity coming from alternate instrumentation. The use of complementary analysis

also shows the difficulty of explosive analysis and the need for a single screening test

using simple sample extraction that may be used for future analysis on different

instrumentation. The CE method developed in this work is a starting point for a full

spectrum explosive screen using CE.

One interesting result of this analysis was the lack of significant peaks during

precursor analysis (Table 15). Other than hydrogen peroxide and sulfuric acid, none of

the precursors analyzed showed significant repeatable peak patterns by CE analysis.

The analysis of hydrogen peroxide was one of the more challenging parts of this

research. The analytes needed to be mixed with buffer prior to analysis in order to

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produce a viable circuit upon injection. Unfortunately, the hydrogen peroxide, being a

strong oxidizer, tended to produce gas bubbles and foam when mixed with buffer,

resulting in variations in volume. The foaming may be due to a reaction with a

compound similar to catalase in the buffer. When an enzyme such as catalase comes in

contact with hydrogen peroxide (H2O2) it reacts and forms water (H2O) and oxygen gas

(O2) in the form of bubbles. Even with the foaming, it was possible to obtain repeatable

relative migration times and establish a pattern of peaks for hydrogen peroxide as shown

in Table 16. It is not surprising that hydrogen peroxide shows much stronger peaks at

200 nm, as it has been reported in the literature that the best wavelength for hydrogen

peroxide analysis is at 185 nm (Shihabi, 2006). It would be interesting to analyze

samples on an instrument with the capability for analysis at that wavelength to observe

the behavior of hydrogen peroxide at lower wavelengths.

The detection of TATP associated peaks is the most important aspect of this

research. Without repeatable relative migration times, and the peak pattern produced by

the analysis of different wavelengths (Table 17), it would be difficult to conclude that

samples contained TATP. However, the loss of certain TATP peaks was observed in

some cases, even when it was clear that TATP was in fact present (Table 17). The TATP

signature peak pattern developed in this research was reproducible over several days of

testing using all TATP batches manufactured at OSU; furthermore, the signature peaks

were also seen in post-blast samples produced by TATP.

Although multiple attempts were made for the analysis of HMTD in this work,

they were unsuccessful. According to the literature, HMTD is soluble in acetone at

approximately 2.6 g/l (Crowson & Beardah, 2001). Acetone, methanol, and acetonitrile

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were all used as solvents and it was determined that acetone provided the best storage

suspension of HMTD. For analysis of HMTD by CE, 100 µl of saturated standard in

acetone had to be suspended in 100 µl of buffer, and 50 µl bromide standard was added.

TATP was easily detected when its initial concentration was 100 µg/ml, and no peaks

were observed for HMTD at what would be considered 2,600 µg/ml. The direct

infusion of HMTD dissolved in acetone to the mass spectrometer required only 10 µl of

standard and was immediately detected by the instrument, whereas even with the

suspension of HMTD in acetone, it was still impossible to complete CE analysis of

HMTD. Therefore it is concluded that HMTD is not compatible with CE analysis with a

traditional borate buffer.

The relative migration times for TATP and hydrogen peroxide are very close

(Table 18); however, they are statistically different from one another. Sample runs were

completed within 10 minutes and the individual sample migration times ranged from

bromide with an average migration time of 2.5 minutes and the final hydrogen peroxide

peak with an average migration time of 6 minutes. In this study there was no difficulty

verifying the identity of the peaks as hydrogen peroxide or TATP.

Table 23 shows that this method was able to provide repeatable relative migration

times with minimal overlap between the selected ions, hydrogen peroxide, and TATP

signature relative migration times. Only two pairs of RMTs overlap. The closest set of

RMTs, Nitrate and TATP peak two, show a close overlap and each peak is negative at

200 nm. Also thiocyanate and TATP peak four show overlap; however, analysis at 200

nm would clarify what compound is seen since the thiocyanate peak is negative and the

TATP peak is positive. One would also expect supporting ions and peaks either present

87

or absent to support the identification of a peak within the overlap range. Even though

the RMT range was 1.000 to 1.387, this research was able to show reproducible unique

separation times for 19 different ions or compounds.

Table 23. Summary of RMTs for Ions, Hydrogen Peroxide, and TATP

Compound Average Low High Pk Direction at 200

nm Bromide 1.000 1.000 1.000 Negative

TATP Pk 1 1.015 1.015 1.016 Positive H2O2 Pk 1 1.022 1.021 1.022 Positive

Nitrite* 1.073 1.072 1.074 Negative TATP Pk 2* 1.077 1.075 1.079 Negative H2O2 Pk 2 1.087 1.085 1.088 Negative

Nitrate 1.092 1.091 1.094 Negative H2O2 Pk 3 1.105 1.105 1.106 Positive TATP Pk 3 1.111 1.109 1.112 Negative H2O2 Pk 4 1.119 1.118 1.121 Negative

Sulfate 1.122 1.118 1.126 Positive Perchlorate 1.147 1.145 1.149 Positive H2O2 Pk 5 1.149 1.148 1.150 Positive

TATP Pk 4** 1.156 1.153 1.159 Positive Thiocyanate** 1.159 1.159 1.160 Negative

H2O2 Pk 6 1.169 1.167 1.170 Positive Chlorate 1.200 1.195 1.203 Positive Cyanate 1.214 1.212 1.217 Positive Formate 1.387 1.376 1.398 Positive

*Possible overlap with TATP Pk 2 **Possible overlap with TATP Pk 4; however, when analyzed at 200 nm, the peaks would point in the opposite directions.

Table 19 shows that time has an effect on the identification of post-blast swabs

and TATP standards. The lack of stability of the compound is most likely due to the

volatility of TATP as referenced in the literature. Therefore, it is recommended that post-

blast samples be analyzed as soon as possible after collection in order to visualize as

many peaks as possible for the proper identification of TATP. In addition, collection

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materials should be closely monitored to avoid any possible contamination of sample

results. The analyzing laboratory should always analyze a blank sample of the identical

collection material to be extracted with the sample. This will avoid false positives and

false negatives if there are interfering peaks. Also any solution that is used for standard

storage should be monitored and analyzed as well to verify if peaks visualized are from

the storage materials, as opposed to explosives. A long-term stability study would also

be needed to identify the length of time prepared standards will remain of sufficient

quality for analysis, and the time at which the sample begins to degrade and lose peak

visualization.

Finally, this method does provide a valuable resource for the post-blast analysis of

explosive materials. However, at least on this instrumentation, this was certainly not a

robust method. One of the major challenges encountered was the lack of stability of the

capillaries used. Literature reports that capillaries can be used for 1000s of samples

(Thorman et al., 1998); however, capillaries used in this research rarely lasted for more

than 150 samples, as evidenced by capillary failure through breakage or gradual

migration time shift after approximately 10 samples without reconditioning. One reason

for the capillary failure may be due to harshness of the potassium dichromate present in

the buffer, or the particular ions analyzed. Replacing a capillary took several hours and

added an additional variable when comparing data analyzed on different capillaries. It

took several months to gather just the ion data due to instrument and capillary failures. If

the failure was related to the instrument used in this study, a more current instrument may

produce better results. In either case, this is the only and therefore best method to screen

for both peroxide and traditional explosive anions simultaneously.

89

There are many future research possibilities that may come from this work. The

first step to further developing this research would be to include the remaining 11 ions as

published in the FBI CE method. The overall explosive screen could be expanded

potentially by adding SDS to the buffer to per MEKC in order to analyze commercial

explosives such as TNT, RDX, HMX, and PETN, but it is unknown what effect this

might have on ion detection. Furthermore, this method could be adapted using an

instrument capable of analyzing both cations and anions so ANFO-type explosives could

be analyzed.

While there were peaks identified as being associated with TATP in this study,

their actual chemical identity is still unknown. Future work would be to characterize

these peaks and identify the analytes producing them. The identification of these peaks

will better assist investigators in the analysis of peroxide explosives and explain why

these peaks are detectable by CE with indirect UV detection. Future work may also be

able to derive a limit of detection and limit of quantitation (LOD/LOQ) for TATP and

attempt to lower the current 10 ppm LOD for ion species. Although quantitation is not as

important in post-blast work as it is in some other forensic disciplines, it would be

beneficial to know the smallest amount of peroxide explosives that is detectable by this

method. This would add an additional level of credibility to the analysis of peroxide

explosives by CE.

Overall this method provides a way to identify both peroxide explosives and

traditional explosive anions using a single sample preparation and injection. With the

current trends in terrorist improvised explosive devices, this work is timely and may yield

a valuable investigative avenue for post-blast investigators.

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Chapter VI References

Beckman Coulter. (n.d.). Introduction to Capillary Electrophoresis [Brochure]. Author.

Buttigieg, G. A., Knight, A. K., Denson, S., Pommier, C., & Denton, M. B. (2003).

Characterization of the explosive triacetone triperoxide and dectection by ion mobility

spectrometry. Forensic Science International, 135, 53-59.

Capillary Electrophoresis. (2007, December 23). Retrieved December 31, 2007 from

http://en.wikipedia.org/wiki/Capillary Electrophoresis

Cooper, P. W. (1996). Explosives Engineering. Albuquerque, NM: Wiley-VCH.

Cooper, P. W., & Kurowski, S. R. (1996). Introduction to the Technology of Explosives.

Wiley-VCH.

Crowson, A., & Beardah, M. S. (2001). Development of an LC/MS Method for the trace

analysis of HMTD. The Analyst, 126, 1689-1693.

Daubert v. Merrell Dow Pharmaceuticals (June 28, 1993).

91

Davis, T. L. (1943). The Chemistry of Powder and Explosives. Las Vegas, NV: G. S. G.

& Associates.

Dubnikova, F., Kosloff, R., & Zeiri, Y. (2006). Rational Detection Schemes for TATP:

NATO advanced research workshop. In Detection and Disposal of Improvised Explosives

(pp. 105-112). Netherlands: Springer. Retrieved December 12, 2007, from

http://www.springerlink.com/content/j2vw008x32241330

Dubnikova, F., Kosloff, R., Zerir, Y., & Karpas, Z. (2002). Novel Approach to the

Detection of TATP: Its Structure and Its Complexes with Ions. Journal of Physical

Chemistry A, 106, 4951-4956.

Experts: Zazi Could Have Killed Scores - CBS News. (2009, October 2). Retrieved

November 10, 2009, from

http://www.cbsnews.com/stories/2009/10/02/national/main5358462.shtml

Explosive Material. (2009, October 09). Retrieved October 12, 2009, from

http://en.wikipedia.org/wiki/Explosives

Groom, C. A., Beaudet, S., Halasz, A., Paquet, L., & Hawari, J. (2000). Application of

Sodium Dodecyl Sulfate Micellar Electrokinetic Chromatography (SDS MEKC) for the

Rapid Measurement of Aqueous Phase 2,4,6-Trinitrotoluene Metabolites in Anaerobic

Sludge: A Comparison with LC/MS. Environmental Science Technology, 34, 2300 -

2336.

92

Groom, C. A., Beaudet, S., Halasz, A., Paquet, L., & Hawari, J. (2001). Determination of

the cyclic nitramine explosives RDX and HMX and their degradation products in soil

environments. Journal of Chromatography A, 909, 53-60.

Groom, C. A., Halasz, A., Olivier, L., Morris, N., Paquet, L., Dubois, C., & Hawari, J.

(2002). Accumulation of HMX in indigenous and Agricultural Plants Grown in HMX-

Contaminated Anti-Tank Firing-Range Soil. Environmental Science Technology, 36(1),

112-118.

Hargadon, K. A., & McCord, B. R. (1992). Explosive Residue Analysis by capillary

electrophoresis and ion chromatography. Journal of Chromatography, 602, 241-247.

Hopper, K. G., LeClair, H., & McCord, B. (2005). A novel method for analysis of

explosives residue by simultaneous detection of anions and cations via capillary zone

electrophoresis. Talanta, 67, 304 - 312.

Kuban, P., Kuban, P., & Kuban, V. (1999). Simultaneous capillary electrophoretic

separation of small anions and cations after complexation with EDTA. Journal of

Chromatography A, 836, 75 - 80.

Kuffner, C., Marchi, E., Morgado, J., & Rubio, C. (1996). Capillary Eletrophoresis and

Daubert: Time for Admission. Analytical Chemistry, 68, 241A-246A. Analytical

Chemistry. Retrieved July 7, 2008, from www.pubs.acs.org/hotartcl/ac/96/apr/apr.html

Landers, J. P. (2007). Handbook of Capillary and Microchip Electrophoresis and

Associated Microtechniques, Third Edition (3rd ed.). Boca Raton, FL: CRC.

93

Legal Information Institute. (n.d.). LII: Federal Rules of Evidence. Retrieved November

5, 2009, from http://www.law.cornell.edu/rules/fre/

Levine, B. (2003). Principles of Forensic Toxicology. New York: AACC P.

McCord, B., & Bender, E. C. (1998). Chromatography of Explosives. In Forensic

Investigation of Explosives (pp. 262-306). London: Taylor and Francis.

Moore, D. S. (2004). Instrumentation for trace detection of high explosives. Review of

Scientific Instruments, 75, 2499-2511.

Muller, D., Levy, A., Shelef, R., Abramovich-Bar, A., Sonenfeld, D., & Tamiri, T.

(2004). Improved Method for the Detection of TATP Aftec Explosion. Journal of

Forensic Science, 49(5), 935-938.

Pumera, M. (2008). Trends in Analysis of Explosives by Microchip electrophoresis and

Conventional CE. Electrophoresis, 29, 269-273.

Richard Reid. (2009, October 10). Retrieved October 12, 2009, from

http://en.wikipedia.org/wiki/Richard_Reid_(shoe_bomber).

Ruttinger, H., & Radschuweit, A. (2000). Determination of peroxides by capillary zone

electrophoresis with amperometric detection. Journal of Chromatography A, 868, 127-

134.

Schulte-Ladbeck, R., Kolla, P., & Karst, U. (2002). A Field Test for the Detection of

Peroxide-Based Explosives. The Analyst, 127, 1152-1154.

94

Schulte-Ladbeck, R., Kolla, P., & Karst, U. (2003). Trace Analysis of Peroxide-Based

Explosives. Analytical Chemistry, 75(4), 731-735.

7 July 2005 London Train Bombings. (2009, November 7). Retrieved November 12,

2009, http://en.wikipedia.org/wiki/7_July_2005_London_bombings.

Shihabi, Z. K. (2006). Direct analysis of hydrogen peroxide by capillary electrophoresis.

Electrophoresis, 27, 4215-4218.

Skoog, D. A., Holler, F., & Nieman, T. A. (1998). Introduction. In Principles of

Instrumental Analysis (pp. 1-19). Orlando: Harcourt Brace College.

Tagliaro, F., Manetto, G., Crivellente, F., & Smith, F. P. (1998). A Brief Introduction to

capillary electrophoresis. Forensic Science International, 92, 75-88.

Thorman, W., Aebi, Y., Lanz, M., & Caslavska, J. (1998). Capillary electrophoresis in

clinical toxicology. Forensic Science International, 92, 157-183.

2005 University of Oklahoma Bombing. (2009, November 5). Retrieved November 12,

2009, http://en.wikipedia.org/wiki/Joel_Henry_Hinrichs.

2006 Transatlantic Aircraft Plot. (2009. November 5). Retrieved November 12, 2009,

http://en.wikipedia.org/wiki/2006_transatlantic_aircraft_plot.

Wang, J., Escarpa, A., Pumera, M., & Feldman, J. (2002). Capillary electrophoresis-

electrochemistry microfluidic system for the determination of organic peroxides. Journal

of Chromatography A, 952, 249-254.

95

Xu, X., Van de Craats, A. M., Kok, E. M., & DeBruyn, P. (2004). Trace Analysis of

Peroxide Explosives by High Performance Liquid Chromatography-Atmospheric

Pressure Chemical Ionization-Tandem Mass Spectrometry (HPLC-APCI-MS/MS) for

Forensic Applications. Journal of Forensic Science, 49(6), 1230 - 1236.

VITA

Stephanie Marie Olofson

Candidate for the Degree of

Master of Science

Thesis: IDENTIFICATION OF PEROXIDE EXPLOSIVES AND

TRADITIONAL EXPLOSIVE ANIONS BY CAPILLARY ELECTROPHORESIS

Major Field: Forensic Sciences

Education:

Completed the requirements for the Master of Science in Forensic Sciences at Oklahoma State University Center for Health Sciences, Tulsa, Oklahoma, in December,

2009.

Completed the requirements for the Bachelor of Science in Forensic Science at Baylor University, Waco, Texas, in 2004.

Experience:

Internship with Portland, Oregon, Police Bureau, Summer 2003 Internship with Austin, Texas, Police Department, 2004-2005

ADVISER’S APPROVAL: Jarrad R. Wagner, Ph.D.

Name: Stephanie Marie Olofson Date of Degree: December, 2009

Institution: Oklahoma State University CHS Location: Tulsa, Oklahoma

Title of Study: IDENTIFICATION OF PEROXIDE EXPLOSIVES AND TRADITIONAL EXPLOSIVE ANIONS BY CAPILLARY ELECTROPHORESIS

Pages in Study: 95 Candidate for the Degree of Master of Science Major Field: Forensic Sciences

Scope and Method of Study: Terrorists choose to use peroxide explosives such as triacetone triperoxide (TATP) in improvised explosive devices (IEDs) for the devastating explosive power offered and simple procurement as compared to traditional explosives. Peroxide explosive residues are an analytical challenge due to their volatility, as well as their simple structure which lacks distinctive metallic and ionic signatures.

The analysis of traditional explosive residues can be a difficult task for the forensic chemist, with many methods to choose from on a wide variety of instruments. Rather than providing definitive answers, the results are used by investigators to infer the types of explosives present. Due to the diverse nature of explosives, it is unusual to find a single method that will analyze for multiple types of explosives without the use of several extractions or instruments.

The purpose of this study is to describe the analysis of peroxide explosives through capillary electrophoresis (CE) with a traditional borate buffer. This is the first known instance in which an analytical method can screen for peroxide explosives (TATP) along with anions commonly associated with low explosives. This analysis is simultaneous and does not require the use of separate methods and preparations. Findings and Conclusions: A literature method for low explosive anions analysis by CE was optimized for use and the relative migration times (RMT) for nine common anions were determined. All precursors for peroxide explosive manufacture were analyzed and RMTs of significant peaks were identified. TATP standards were characterized by four peaks in the electropherogram, and all significant precursor peaks were eliminated as contributors to these peaks. Post-blast residue swabs and burn sticks extracted in methanol were analyzed by gas chromatography-mass spectrometry to confirm the presence of TATP, and then TATP was identified in the extract by CE through the comparison of RMTs. The method produced repeatable RMTs with minimal overlap between selected ions, precursor compounds, and TATP signature peaks.

identification of peroxide explosives...

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