The University of Southern Mississippi The University of Southern Mississippi

The Aquila Digital Community The Aquila Digital Community

Dissertations

Spring 5-2011

Sensitive Detection of High Explosives Using Electrogenerated Sensitive Detection of High Explosives Using Electrogenerated

Chemiluminescence Chemiluminescence

Suman Parajuli University of Southern Mississippi

Follow this and additional works at: https://aquila.usm.edu/dissertations

Part of the Chemistry Commons

Recommended Citation Recommended Citation Parajuli, Suman, "Sensitive Detection of High Explosives Using Electrogenerated Chemiluminescence" (2011). Dissertations. 698. https://aquila.usm.edu/dissertations/698

This Dissertation is brought to you for free and open access by The Aquila Digital Community. It has been accepted for inclusion in Dissertations by an authorized administrator of The Aquila Digital Community. For more information, please contact Joshua.Cromwell@usm.edu.

The University of Southern Mississippi

SENSITIVE DETECTION OF HIGH EXPLOSIVES USING ELECTROGENERATED

CHEMILUMINESCENCE

by

Suman Parajuli

Abstract of a Dissertation Submitted to the Graduate School

of The University of Southern Mississippi in Partial Fulfillment of the Requirements

for the Degree of Doctor of Philosophy

May 2011

ii  

ABSTRACT

SENSITIVE DETECTION OF HIGH EXPLOSIVES USING

ELECTROGENERATED CHEMILUMINESCENCE

by Suman Parajuli

May 2011

The core of this dissertation lies in the search of analytical tools which can be used to

detect and quantify the high explosives confiscated from suspects in transportation hubs

and from soil and water bodies where these explosives pose a greater threat to public

health and safety. High explosives, namely, hexamethylene triperoxide diamine (HMTD),

triacetone triperoxide (TATP), trinitrotoluene (TNT), and pentaerythritol tetranitrate

(PETN), were detected and quantified by electrochemical methods such as

electrogenerated chemiluminescence (ECL) and cyclic voltammetry (CV).

Sensitive detection and quantification of HMTD, one commonly used explosive by

terrorists, was presented first in this dissertation on the basis of ECL technology coupled

with silver nitrate (AgNO3) enhancement in acetonitrile (MeCN) at a Pt electrode. Upon

the anodic potential scanning, HMTD irreversibly oxidized at ~1.70 V vs Ag/Ag+ (10

mM) at a scan rate of 50 mV/s, and the ECL profile was coincident with the oxidation

potential of HMTD in the presence of tris(2,2′-bipyridine)ruthenium(II) cation

(Ru(bpy)32+) luminophore species which showed a half-wave potential of 0.96 V vs

Ag/Ag+. The addition of small amounts of AgNO3 (0.50 to 7.0 mM) into the

HMTD/Ru(bpy)32+ system resulted in significant enhancement in HMTD ECL

production (up to 27 times). This enhancement was found to be largely associated with

iii  

NO3- and was linearly proportional to the concentrations of NO3

- and Ag+ in solution.

Homogeneous chemical oxidations of HMTD by electrogenerated NO3• and Ag(II)

species proximity to the electrode were proposed to be responsible for the ECL

enhancement. On the basis of experimentally obtained CV data and theoretical CV digital

simulations, standard potential values of 1.79 V vs Ag/Ag+ (or 1.98 V vs NHE) and 1.82

V vs Ag/Ag+ (or 2.01 V vs NHE) were estimated for Ag(II)/Ag(I) and NO3•/NO3

- couples,

respectively. A limit of detection of 50 μM of HMTD was achieved with the current

technique, which was 10 times lower than that reported previously based on a liquid

chromatography separation (HPLC) and Fourier transform infrared (FT-IR) detection

method.

Detection of TATP and the differentiation of TATP from HMTD were accomplished

subsequently with ECL at glassy carbon electrode in water-MeCN mixture solvents. In

the presence of Ru(bpy)32+, TATP or hydrogen peroxide (H2O2) derived from TATP via

UV irradiation or acid treatment produced ECL emissions upon cathodic potential

scanning. Interference of H2O2 on TATP detection was eliminated by pre-treatment of the

analyte with catalase enzyme. Selective detection of TATP from HMTD was realized by

scanning the electrode potential positively as well as negatively; HMTD showed ECL

emissions at both directions. The hydroxyl radical formed after the electrochemical

reduction of TATP was believed to be the key intermediate for ECL production, and its

stability was strongly dependent on the solution composition, which was verified with

electron paramagnetic resonance spectroscopy. A detection limit of 2.5 µM TATP was

obtained from direct electrochemical reduction of the explosive or H2O2 derived from

iv  

TATP in 70-30% (v/v) water-MeCN solutions, which was ~400 times lower than that

reported previously based on HPLC/FT-IR detection method.

ECL quenching method can also be used to detect explosive compounds where the

explosive of interest can quench ECL response either by excited state quenching or

quenching by depletion of the precursors of the excited state species in the test solution.

In this investigation, ECL quenching behavior of the Ru(bpy)32+/ tri-n-propylamine

(TPrA) system with TNT at a Pt electrode in MeCN was explored. Effective ECL

quenching of the system upon the addition of TNT was observed, with a Stern-Volmer

constant of 2×104 M-1. The apparent ECL quenching constant calculated from the Stern-

Volmer plot was found to be 3.5×1010 M-1 s-1, which suggests the efficient quenching of

ECL by TNT. The consumption of the TPrA• free radicals and Ru(bpy)3+ species

(produced as a result of reduction of Ru(bpy)32+ by TPrA•) by TNT could be the main

reason of this quenching, as both TPrA• and Ru(bpy)3+ species are precursors of the

excited state Ru(bpy)32+* species. The present technique can sensitively detect TNT as

low as 4.4 μM.

Electrochemical detection of PETN was studied in MeCN with a Ag wire as the

working electrode, where an irreversible reduction wave at -0.9 V vs Ag/Ag+ was

observed. The reduction of PETN probably involved the formation of alcohol and nitrite

ion with the trace amount of water present in the solvent. The limit of detection of PETN

by simple electrochemical CV method was 25 µM.

 

 

 

 

 

 

 

 

 

 

COPYRIGHT BY

SUMAN PARAJULI

2011

The University of Southern Mississippi

SENSITIVE DETECTION OF HIGH EXPLOSIVES USING ELECTROGENERATED CHEMILUMINESCENCE

by

Suman Parajuli

A Dissertation Submitted to the Graduate School

of The University of Southern Mississippi in Partial Fulfillment of the Requirements

for the Degree of Doctor of Philosophy Approved: Wujian Miao ____________________________________ Director Jeffrey Evans ____________________________________ Douglas Masterson ____________________________________ Alvin Holder

____________________________________ Karl Wallace ____________________________________ Susan A. Siltanen ____________________________________ Dean of the Graduate School

November 2010

v

 

ACKNOWLEDGMENTS

First of all, I would like to express my sincere appreciation to my advisor, Dr.

Wujian Miao, for his continuous guidance and support with enthusiasm during my entire

life as a graduate student at The University of Southern Mississippi. I would also like to

extend my thanks to my committee members, Dr. Jeffrey Evans, Dr. Douglas Masterson,

Dr. Alvin A. Holder, and Dr. Karl J. Wallace, for their continued support and suggestions.

I would like to thank our department chair, Dr. Robert Bateman, for his help and able

leadership.

I would also like to thank my group members Tommie Pittman, Dr. Shijun Wang,

Carrie Hofmann, and Erendra Manandhar, for their support. My thanks are always there

for visiting scholars, Dr. Jian Shi, Dr. Xiaohui Jing, and Dr. Cunwang Ge, for their help.

I would like to thank the Department of Chemistry and Biochemistry at The

University of Southern Mississippi and the National Science Foundation for financial

support for my PhD studies.

Lastly, I would like to thank my family members, especially my wife Binita and

daughter Shriti, for their endless patience and support during my graduate studies.

 

vi  

TABLE OF CONTENTS

ABSTRACT………………………………………………………………………………ii

ACKNOWLEDGMENTS………………………………………………………………...v

LIST OF TABLES……………………………………………………………………...viii

LIST OF ILLUSTRATIONS……………………………………………………………..ix

LIST OF SCHEMES……………………………………………………………………xvi

LIST OF EQUATIONS……………………………………………...……………….. xviii

CHAPTER

I. INTRODUCTION…………………………………………………………….1

Electroanalytical Chemistry Electrogenerated Chemiluminescence Typical Techniques for Trace Detection of High Explosives Conclusion References

II. SENSITIVE DETERMINATION OF HEXAMETHYLENE TRIPEROXIDE DIAMINE (HMTD) EXPLOSIVE USING ELECTROGENERATED CHEMILUMINESCENCE ENHANCED BY SILVER NITRATE……………………………………………………...54

Introduction Experiment Section. Results and Discussion Conclusion References

III. SELECTIVE DETERMINATION OF TRIACETONE TRIPEROXIDE USING ELECTROGENERATED CHEMILUMINESCENCE……………..74

Introduction Experiment Section Results and Discussion Conclusion

vii  

References IV. DETECTION OF TRINITROTOLUENE BY ELECTROGENERATED CHEMILUMINESCENCE QUENCHING METHOD…………………….100

Introduction Experiment Section Results and Discussion Conclusion

References V. ELECTROCHEMICAL DETECTION OF PENTAERYTHRITOL TETRANITRATE…………………………………………………………..117

Introduction Experiment Section Results and Discussion Future Work Conclusion

References VI. CONCLUDING REMARKS………………………………………………128 VII. APPENDIX…………………………………………………………….......131  

 

viii  

LIST OF TABLES Table 1.1. Commonly Used Trace Detection Methods of High Explosives and Their Features……………………………………………………………………………18 1.2. Commonly Used High Explosives and Their Chemical Properties……………….19 3.1. Change in EPR Intensity With Change in Solvent Composition and With Change in Time……………………………………………………………………92

ix  

LIST OF ILLUSTRATIONS

Figure 1.1. Cyclic voltammetric excitation signal……………………………………………..4 1.2. Cyclic voltammogram (CV) obtained from a solution containing 6.0 mM

K3Fe(CN)6 and 1.0 M KNO3 at a 2.54 mm-diameter Pt working electrode at

scan rate of 50 mV/s……………………………………………………………….4

1.3. (a) ECL and (b) CV of 1.0 nM Ru(bpy)3

2+ in presence of 0.10 M TPrA with

0.10 M tris/0.10 M LiClO4 buffer (pH = 8) at a glassy carbon electrode (3 mm

diameter) at a scan rate of 50 mV/s. (c) ECL with 1.0 μM Ru(bpy)32+ and

0.10 M TPrA. The ECL intensity scale is given for (c) and should be

multiplied by 100 for (a)…….……………………… …………………………..12

1.4. First and second ECL waves in 0.10 M TPrA (0.20 M PBS, pH 8.5) with

different concentration of Ru(bpy)32+: 1 mM (solid line), 0.50 mM

(dashed line), 0.10 mM (dotted line) and 0.05 mM (dash-dotted line),

at a 3 mm diameter glassy carbon electrode, scan rate of 100 mV/s …………...13

1.5. X-ray imaging (B) reveals the explosives planted in a doll (A). Airport baggage

scanners are programmed to display in red those materials with densities that

match explosives…………………………………………………………………17

1.6. Structures and abbreviations of commonly used high explosives. (a-c)

Nitroaromatics, (d-f) Nitramines, and (i-j) Peroxide-based explosive

compounds……………………………………………………………………….20

1.7. Fluorescent polymer sensor design………………………………………………26

1.8. Schematic of a hybrid QD-antibody fragment FRET-based TNT sensor………..33

1.9. (A) Schematic of the modular biosensor consisting of two modules: the

biorecognition module and the modular arm. (B) Schematic TNT targeting

biosensor…………………………………………………………………………34

x  

1.10. QD antibody conjugates prepared using molecular bridges. (A) Mixed surface

conjugate after purification by cross-linked amylase affinity chromatography.

(B) Schematic of competitive assay for the explosive RDX dissolved in

water….…………………………………………………………………………..36

1.11. Schematic of the displacement immunosensor method………………………….37 2.1. (a) ATR-FTIR and (b) 1H NMR Spectra of HMTD……………………………..57 2.2. CV (black line) and ECL (blue line) responses obtained from 1.0 mM HMTD-

0.70 mM Ru(bpy)3Cl2-0.10 M TBAP in MeCN at a 2-mm diameter Pt

electrode with a scan rate of 50 mV/s……………………………………………59

2.3. CVs of (a) 1.0 mM HMTD, (b) 0.70 mM Ru(bpy)3Cl2, and (c) 0.10 M TBAP

in MeCN blank at a 2-mm diameter Pt with a scan rate of 50 mV/s…………….60

2.4. Effect of Ru(bpy)3Cl2 concentration and working electrode material on ECL

intensity of the Ru(bpy)32+/HMTD system in 0.10 M TBAP in MeCN.

scan rate: 50 mV/s, [HMTD] = 1.0 mM ………….……………………………..62

2.5. (A) CV (black line) and ECL (blue line) responses obtained from 1.0 mM

HMTD- 0.70 mM Ru(bpy)3Cl2-0.10 M TBAP in MeCN in the presence of

7.0 mM AgNO3, and (B) CV of 7.0 mM AgNO3 in MeCN containing 0.10 M

TBAP at a 2-mm diameter Pt electrode with a scan rate of 50 mV/s..… ……….62

2.6. (a) Effect of AgNO3 on ECL intensity of 1.0 mM HMTD- 0.70 mM

Ru(bpy)3Cl2-0.10 M TBAP in MeCN. (b) ECL background produced

from 0.70 mM Ru(bpy)3Cl2-0.10 M TBAP with added AgNO3 in MeCN

in the absence of HMTD. Other experimental conditions were the same

as in Figure 2.5..…………………………………… ……………………………63

2.7. Simulated CV of AgNO3 oxidation in MeCN. In addition to parameters

xi  

listed in Scheme 2.3 Above, the following parameters were used: α = 0.5,

initial [Ag(I)]= [NO3-] =0.007 M, area of electrode = 0.0314 cm2, scan

rate=50 mV/s, T = 298.15 K, diffusion coefficients for all species: 5×10-6

cm2/s……………………………………………………………………………...65

2.8. Comparison of (a) AgNO3 with (b) NaNO3 on ECL intensity of 1.0 mM

HMTD-0.70 mM Ru(bpy)3Cl2-0.10 M TBAP in MeCN………………………...66

2.9. Effect of Ag+ on ECL intensity of 1.0 mM HMTD with (a) 0.70 mM

Ru(bpy)3(ClO4)2 or (b) 0.70 mM Ru(bpy)3Cl2 using different concentrations

of C6H5COOAg……….…………………………………………………………67

2.10. Relationship between [HMTD] and ECL peak intensity. (a) Without addition

of AgNO3, and (b) with addition of 7.0 mM AgNO3. Other experimental

conditions were the same as in Figure 2.5. Each data point represented the

mean of four separate runs……………………………………………………….69

3.1. (a) ATR-FTIR and (b) 1H NMR spectra of TATP……………………………….77 3.2. CVs of (a) 1.0 mM TATP and (b) 0.60 mM Ru(bpy)3

2+ in an electrolyte

solution containing 70% volume of 0.10 M phosphate buffer (pH 7.5) and

30% volume of MeCN (i.e., 70 mM phosphate buffer in 70 (H2O) : 30 (MeCN)

(v/v) mixture solvent) obtained from a 3-mm diameter glassy carbon electrode

at a scan rate 50 mV/s. The CV of the electrolyte solution is displayed in (c)… .81

3.3. (a) ECL and (b) CV responses of 0.50 mM TATP (with UV irradiation

for 30 min) in 1.0 mM Ru(bpy)32+ water-MeCN (70:30, v/v) mixture

containing 70 mM phosphate buffer at a 3-mm glassy carbon electrode

with a scan rate of 50 mV/s……….……………………………………………...83

3.4. Effect of Ru(bpy)3

2+ concentration on ECL generation of 0.50 mM TATP

(with UV irradiation for 30 min) in 70:30 (v/v) water-MeCN containing

70 mM phosphate buffer at a 3-mm diameter glassy carbon electrode with

a scan rate of 50 mV/s........................................................ ……………………...84

xii  

3.5. Effect of UV irradiation time on TATP decomposition. A 0.20 AMPS UV

lamp at 254 nm was used, and the ECL of 0.50 mM TATP (with various UV

irradiation intervals) was conducted in 70:30 (v/v) water-MeCN containing

0.6 mM Ru(bpy)32+ and 70 mM phosphate buffer at a 3-mm diameter glassy

carbon electrode with a scan rate of 50 mV/s……………………………………84

3.6. Effect of solvent composition on the ECL peak intensity of (a) 0.5 mM TATP

(with UV irradiation for 30 min), and (b) 1.0 mM H2O2 in an electrolyte

solution containing 0.6 mM Ru(bpy)32+-70 mM phosphate buffer at a 3-mm

diameter glassy carbon electrode with a scan rate of 50 mV/s…………………..85

3.7. (A) ECL intensity as a function of TATP concentration for the UV-irradiation

-ECL detection scheme. (B) ECL responses obtained from different

concentrations of TATP (with UV irradiation for 30 min): (a) 0, (b) 2.5 μM,

and (c) 0.50 mM. All experiments were conducted in 70:30 (v/v) water-MeCN

with 0.6 mM Ru(bpy)32+-70 mM phosphate buffer at a 3-mm diameter glassy

carbon electrode using a scan rate of 50 mV/s…………………………………...86

3.8. ECL peak intensity as a function of TATP concentration. TATP was treated

with 12 mM HCl, and the ECL measurements were conducted in 70:30 (v/v)

water-MeCN mixture containing 0.6 mM Ru(bpy)32+-70 mM phosphate buffer

at a 3-mm glassy carbon electrode using a scan rate of 50 mV/s. (a) first ECL

peak, and (b) second ECL peak as shown in Figure 3.9…………………………87

3.9. (a) CV and (b) ECL responses from TATP (pre-treated with 12 mM HCl)

using the same experimental conditions as described in Figure 3.8…88

 3.10. (a) ECL and (b) CV responses of 0.50 mM TATP directly detected in 0.60

mM Ru(bpy)32+ water-MeCN (70:30, v/v) mixture containing 70 mM

phosphate buffer at a 3-mm glassy carbon electrode with a scan rate of 50

mV/s……………………. ………………………………………………………88

xiii  

3.11. Relationship between ECL peak intensity and TATP concentration when TATP

was directly detected in 70:30 (v/v) water-MeCN mixture containing 0.60 mM

Ru(bpy)32+-70 mM phosphate buffer at a 3-mm glassy carbon electrode with

a scan rate of 50 mV/s……………………………………………………………89

3.12. Combined EPR spectra of DMPO/•OH produced from Fenton reaction

(0.50 mM H2O2, 200 mM 5,5′-dimethyl pyrroline-N-oxide (DMPO), and

75 µM Fe(NH4)2(SO4)2) in different water-MeCN (v/v) solution compositions.

The spectra were recorded after reactions occurred for 5 min…………………...90

3.13.    EPR spectra intensity of DMPO/•OH adduct (taken from Figure 3.12) as a

function of solution composition………………………………………………...91

3.14. EPR spectra simulation of DMPO/•OH adduct obtained from Fenton reaction

in the presence of the spin trapping agent 5,5′-dimethyl pyrroline-N-oxide

(DMPO) after 5 min reaction. Solvent composition: 70:30 (v/v)

water/MeCN…………………...............................................................................92

3.15. ECL responses of 1.0 mM H2O2-0.60 mM Ru(bpy)3

2+ system (a) before and

(b) after the addition of 5 μL of catalase. The experiments were conducted in

1.0 mL of 70:30 (v/v) water-MeCN containing 70 mM phosphate buffer at

a 3-mm diameter glassy carbon electrode with a scan rate of 50 mV/s………….93

3.16. Comparison of ECL intensities obtained from (A) 0.50 mM TATP, (b)

0.50 mM + 0.50 mM NaN3, (C) 0.50 TATP + 0.50 mM H2O2, and (D)

0.50 TATP + 0.50 mM H2O2 + 5 μL catalase + 0.50 mM NaN3. The

experimental conditions were the same as in Figure 3.15……………………….94

3.17. (A) Cathodic ECL of (a) 0.50 mM HMTD, (b) 0.50 mM TATP, and

(c) 0.50 mM HMTD + 0.50 mM TATP in the presence of 0.60 mM

Ru(bpy)32+-70 mM phosphate buffer in 70:30 (v/v) water-MeCN mixture

solvent at a3-mm glass carbon electrode with a scan rate of 50 mV/s;

(B) Anodic ECL of (a) 0.50 mM TATP, (b) 0.50 mM HMTD, and

xiv  

(c) 0.50 mM TATP + 0.50 mM HMTD, using the same experimental

conditions as in (A)………………………………………………………………95

4.1. CV and ECL of 1.0 µM Ru(bpy)3

2+-25 mM TPrA in MeCN containing

0.10 M TBAP at a 2-mm diameter Pt disk electrode with a scan rate of 50

mV/s ….………………………………………………………………………...104

4.2. Concentration dependence of TPrA on ECL of the Ru(bpy)3

2+/TPrA system.

The experiment was conducted at a 2-mm Pt electrode with a scan rate of

50 mV/s using 1.0 µM Ru(bpy)32+ -0.10 M TBAP MeCN solution……………104

4.3. ECL quenching of the Ru(bpy)3

2+ (1.0 µM)/TPrA (25 mM) by TNT at a

final concentration of (a) 0, (b) 4.4, and (c) 110 µM. Working electrode:

2-mm diameter Pt, scan rate: 50 mV/s. For clarity, only forward scans of

the CV are plotted..…………………………………………………………......105

4.4. ECL quenching by TNT in the system containing 5 nM Ru(bpy)3

2+-25 mM

TPrA-0.10 M TBAP in MeCN with added TNT concentrations of (a) 0,

(b) 44, (c) 88, and (d) 132 μM. A 2-mm diameter of Pt electrode and a

scan rate of 50 mV/s were used for all tests…………………………………….106

4.5. Ru(bpy)3

2+ concentration effect on ECL (a) without, and (b) with 4.4 μM TNT

addition to the MeCN solution containing 25 mM TPrA-0.10 M TBAP.

A 2-mm diameter of Pt electrode and a scan rate of 50 mV/s were used

for all tests..……………………………………………………………………107

4.6. Relationship of ECL intensity with the TNT concentration in the system

containing 1.0 µM Ru(bpy)32+-25 mM TPrA-0.10 M TBAP in MeCN at a

2-mm Pt electrode at a scan rate of 50 mV/s …………………………………..108

4.7. Fluorescence quenching of 3.0 mL of 1.0 μM Ru(bpy)3

2+ in MeCN by TNT

with 1.0 cm quartz cuvette and an excitation at 350 nm. (A) TNT

concentration dependence, and (B) MeCN dilution effect …………………….109

xv  

4.8. Stern-Volmer plot of ECL quenching by TNT. See Figure 4. 6 for

experimental conditions ………………………………………………………..110

4.9. Stern-Volmer plot of fluorescence quenching by TNT. See Figure 4. 7 for

experimental conditions ………………………………………………………..111

5.1. 1H NMR of PETN taken in deuterated acetonitrile (CD3CN)………………….120 5.2. FTIR spectra of (a) pentaerythritol and (b) PETN ……………………………..121 5.3. Forward scans of cyclic voltammetry: (a) 0.10 M TBAP in MeCN as blank,

(b) 25 µM PETN in MeCN, and (c) 600 µM PETN in MeCN with 0.10 M

TBAP as supporting electrolyte at a silver wire working electrode (exposure

area: ~1 mm2). Scan rate was 50 mV/s. For clarity, the reverse scans were not

included in the plot……………………………………………………………...122

5.4. Peak current change of the forward scan in PETN CV as a function of PETN

concentration. The data were obtained from a silver wire working electrode

(effective surface area: ~1 mm2) in MeCN containing 0.10 M TBAP at a

scan rate of 50 mV/s…………………………………………………………….123

xvi  

LIST OF SCHEMES Scheme 1.1. Ion annihilation ECL………………………………………………………………8

1.2. Mechanism for the ECL first wave of the Ru(bpy)32+/TPrA system…….............13

1.3. Mechanism for the ECL second wave of the Ru(bpy)32+/TPrA system………….14

1.4. Mechanism for the ECL second wave for the Ru(bpy)32+/TPrA system………...14

1.5. ECL Mechanism for the Ru(bpy)32+/S2O8

2- system……………………………...15

2.1. Synthesis of hexamethylene triperoxide diamine (HMTD)………… …………..57

2.2. Proposed ECL mechanism of the Ru(bpy)32+/HMTD system in MeCN upon

the anodic potential scanning…………………………………………………….61

2.3. Proposed reaction mechanism of AgNO3 oxidation in MeCN at a Pt electrode

for CV digital simulations. In Eqs. 2.4-2.8, E0 is the standard reduction

potential, Ks is the standard heterogeneous rate constant, Kf/Kb is the ratio of

homogeneous rate constant of the forward reaction to the backward reaction,

and P3 and P4 are reduction products of NO3· and Ag(II),respectively…………..65

2.4. Proposed ECL mechanism of the HMTD/Ru(bpy)32+ system involving ECL

enhancement by AgNO3 in MeCN at a Pt electrode, in which Eqs. (2.9–2.12)

are direct oxidations at the electrode, Eqs. (2.13–2.15) and (2.17) are

chemical reactions with electron transfers in the solution proximity

to the electrode surface, Eq.(2.16) is the deprotonation of an α-C H from

HMTD•+ radical cation generated electrochemically (Eq. (2.10)) and

chemically (Eqs. (2.14–2.15)), and Eqs. (2.18–2.21) are possible pathways

to form excited state Ru(bpy)32+* species that emit light via Eq. (2.22)… ……..68

3.1. Synthesis of triacetone triperoxide (TATP)………………………… …………..77

xvii  

3.2. Formation of •OH radical by Fenton reaction and the DMPO/•OH adduct,

where k1 and k2 are the rate constants of Fenton reaction and the spin trapping

reaction, respectively…………….………………………………………………79

3.3.    Proposed ECL mechanism of TATP in the presence of Ru(bpy)32+ upon the

cathodic potential scanning………………………………………………………82

3.4. No “reductive-oxidation” type ECL was produced from TATP and H2O2 in

MeCN in the presence of Ru(bpy)32+…………………………………………….82

3.5. Flow-chart of the elimination of H2O2 from TATP and direct ECL detection

of TATP………………………………………………………………………….94

5.1. Mechanism of reduction of organic ester……………………………………….118

5.2. Synthesis of Pentaerythritol Tetranitrate (PETN)………………… …………...120

5.3. Immunoassay based ECL detection of PETN ………………………………….124

xviii  

LIST OF EQUATIONS Equations

1.1. 46

II36

III (CN)Fee(CN)Fe ……………………………………………………….3

1.2. 36

III46

II (CN)Fee(CN)Fe ……………………………………………………….5

1.3. ](CN)Fe[

](CN)Fe[log

1

0.0594

6II

36

III

(CN)/Fe(CN)Fe0 3

6III4

6II

EE (at 250C) ……………………...5

1.4. pa pc0

2' E E

E

………………………………………………………………..........6

1.5. pa pc

0 059.ΔE E E

n ……………………………………………………………..6

1.6. 5 3/2 1/2 1/2

p (2.69 10 )i n AD Cv ………………………………………………………...6

1.7. ReR ………………………………………………………………………….8 1.8. ReR …………………………………………………………………………..8 1.9. RRRR ………………………………………………………………….8 1.10. vhRR ……………………………………………………………………….8 1.11. 16.0

)(R/Rp)(R/Rp EEHann ………………………………………………….9

1.9a. RRRR 1 (Singlet) …………………………………………………..9 1.9b. RRRR 3 (Triplet) …………………………………………………..9 1.12. RRRR 133 ………………………………………………………………..9

1.13. 382

282 OSeOS ……………………………………………………………...15

1.14. 32

3 Ru(bpy)eRu(bpy) ………………………………………………………15

1.15. 382

23

2823 OSRu(bpy)OSRu(bpy) ………………………………………15

xix  

1.16. 42

43

82 SOSOOS …………………………………………………………15

1.17. 24

*2343 SORu(bpy)SORu(bpy) ……………………………………….15

1.18. 24

334

23 SORu(bpy)SORu(bpy) ……………………………………….16

1.19. 23

*23

333 Ru(bpy)Ru(bpy)Ru(bpy)Ru(bpy) ……………………………16

1.20. vhRu(bpy)Ru(bpy) 23

*23 ………………………………………………….16

2.1. 1IIRuHe PRuHMTDHMTDHMTD

III

…………………......61

2.2. 2IIRu-HH2e PRuHMTDHMTD]HMTD[HMTD

III

…..61 2.3. vhRuRu IIII …………………………………………………………………...61 2.4. Ag(I)eAg(II) ………………………………………………………………......65

2.5. 33 NOeNO …………………………………………………………………..65

2.6. Ag(II)NOAg(I)NO 33 …………………………………………………….65

2.7. 33 PNO ………………………………………………………………………......65

2.8. 4PAg(II) ………………………………………………………………………......65

2.9. 33

23 Ru(bpy)eRu(bpy) ………………………………………………………68

2.10. HMTDeHMTD ……………………………………………………68 2.11. Ag(II)eAg(I) ………………………………………………………………68

2.12. 33 NOeNO …………………………………………………………….......68

2.13. Ag(II)NOAg(I)NO 33 …………………………………………………..68

2.14. HMTDAg(I)HMTDAg(II) ……………………………………………68

xx  

2.15. HMTDNOHMTDNO 33 ………………………………………………68

2.16. HHMTDHMTD …………………………………………………….....68

2.17. 132

3 ProductRu(bpy)HMTDRu(bpy) ………………………………......68

2.18. Ag(I)Ru(bpy)Ag(II)Ru(bpy) *233 ……………………………………….68

2.19. 3*2

333 NORu(bpy)NORu(bpy) ………………………………………...68

2.20. 2*2

33

3 ProductRu(bpy)HMTDRu(bpy) ………………………………...68

2.21. 23

*23

333 Ru(bpy)Ru(bpy)Ru(bpy)Ru(bpy) …………………………….68

2.22. vhRu(bpy)Ru(bpy) 23

*23 ………………………………………………......68

3.1. OH]OH[ePeroxides 22

……………………………………………………82

3.2. 32

3 Ru(bpy)eRu(bpy) ……………………………………………………...82

3.3. OHRu(bpy)OHRu(bpy) 33

23 …………………………………………...82

3.4. 2

3 3Ru(bpy) OH Ru(bpy) …………………………………………………82

3.5. 3 2 2+

3 3 3 3Ru(bpy) Ru(bpy) Ru(bpy) Ru(bpy) ……………………………….82

3.6. vhRu(bpy)Ru(bpy) 23

23 ……………………………………………………82

3.7. OCH3O3HO3HCOCHTATP 232223223 ………………………………86

3.8. 22

Catalase22 OOH2OH2 ……………………………………………………….93

4.1. TPrAeTPrA ……………………………………………………………...101

4.2. 33

23 Ru(bpy)eRu(bpy) ……………………………………………………..101

xxi  

4.3. HTPrATPrA ………………………………………………………....101

4.4. P1Ru(bpy)TPrARu(bpy) 32

3 ………………………………………….102

4.5. 23

233

33 Ru(bpy)Ru(bpy)Ru(bpy)Ru(bpy) …………………………...102

4.6. vhRu(bpy)Ru(bpy) 233 …………………………………………………..102

4.7. 1PRu(bpy)TPrARu(bpy) 23

33 …………………………………………..102

4.8. TPrA TNT TNT P2nn …………………………………………………....102 4.9. 2

3 3Ru(bpy) TNT Ru(bpy) TNTnn ………………………………………..102

4.10. ]Ru(bpy)[]Ru(bpy)[

]Ru(bpy)[*2

3nr*2

3ECL(f)

*23)f(ECL0

KK

K …………………………………109

4.11. ]Ru(bpy)][TNT[]Ru(bpy)[]Ru(bpy)[

]Ru(bpy)[*2

3q*2

3nr*2

3ECL(f)

*23ECL(f)

KKK

K ………..110

4.12. ]TNT[1]TNT[1nrECL(f)

q0

SVKKK

K

…………………………………..110

4.13. nrKK

ECL(f)

1 ……………………………………………………………..110

4.14. energyhigh 2

32

3 (TNT)Ru(bpy)TNTRu(bpy) ……………………………….112

5.1. OH3RONOe2OH2RONO 222 …………………………………………118

5.2. 22 NOROe2RONO ……………………………………………………...118 5.3. OHROHOHRO 2 ……………………………………………………….118

5.4. 32 NORe2RONO ……………………………………………………….118

5.5. OHRHOHR 2 …………………………………………………………..118

1

CHAPTER I

INTRODUCTION

The main inspiration of this dissertation is to find the way for the detection and

quantification of explosive materials using electrochemical methods such as

electrogenerated chemiluminescence (ECL) and cyclic voltammetry (CV). This study

involves the use of peroxide-based explosives such as hexamethylene triperoxide diamine

(HMTD) and triacetone triperoxide (TATP) as ECL coreactant since they contain either

amine and/or peroxide functional groups with tris(2,2'-bipyridine)ruthenium(II) cation

(Ru(bpy)32+) as ECL luminophore. The ECL quenching for the well-known

Ru(bpy)32+/TPrA system has also been utilized in the study for trinitrotoluene (TNT)

detection.

This chapter is divided into two sections: the first section is a general introduction

to commonly used electrochemical and ECL technologies, and the second section is a

brief literature review of trace detection of high explosives with, e.g., nanomaterials.

Electroanalytical Chemistry

Electroanalytical chemistry is a branch of chemical analysis which employs

electrochemical methods in order to gain information related to quantity and properties

of analyte of interest. These methods deal with the electrochemical process

incorporating phenomena involving charge transfers (such as redox processes, ion

separations, etc.) and the electrical phenomena for determination of analyte.1-5

Electroanalytical chemistry, which is different from physical electrochemistry with

primary focus in theory of electrode processes and their applications, is generally

classified as a subdivision of analytical chemistry. Electroanalytical chemistry does not

2

depend on the properties of the electrode and the solvent, and is focused on the

properties of the analyte of interest. Electroanalytical chemistry can be subdivided into

techniques based on the type of measurements carried out, normally with a three-

electrode system (working, reference, and counter electrodes) in an electrochemical

cell,6 as described below.

Electroanalytical Techniques

Electrochemical techniques are associated with the interactions of electricity and

chemical species from the test solutions, especially the measurements of electrical

quantities dealing with the potential, current, or charge, and their relations to chemical

parameters. There is a wide range of applications of analytical techniques because of the

advances made in this field. These techniques are very sensitive and can detect analytes

at their micro molar concentration levels. They are popular among scientific communities

because of the use of inexpensive instruments for analysis and providing important

information about the quantity and redox behavior of the analyte in a short time.7

Cyclic voltammetry (CV). Cyclic voltammetry (CV) is a versatile electroanalytical

technique which can be used to acquire information of the basic electrochemical reaction.

The CV is a useful technique in understanding redox behavior of chemical processes

occurred mainly at the electrode surface for various types of electroactive species

including organometallic complexes and conductive polymers.8-10 CV is also a simple but

powerful technique which can be employed to obtain qualitative as well as quantitative

information of the redox process under study.11-18 In a typical CV experiment, the

potential is applied to the electrode kept in an unstirred solution of an analyte with the

supporting electrolyte against the reference electrode such as silver/silver chloride (Ag/

3

AgCl/Cl-), silver/ silver ion (Ag/Ag+), or saturated calomel electrode (SCE). As shown in

Figure 1.1, the potential is scanned from an initial value (Einitial) to the final value (Efinal)

(“forward scan”) and back to the initial potential (“reverse scan”) at a constant scan rate,

completing a cycle. These scans can be repeated so as to obtain successive second, third,

cycles. The cyclic voltammogram (CV) can be obtained with the current response in Y-

axis and the potential in X-axis as shown in Figure 1.2.

The typical cyclic voltammogram shown in Figure 1.2 was obtained from a

solution of 6.0 mM K3Fe(CN)6 in water with 1.0 M KNO3 as a supporting electrolyte.

The working, reference and counter electrodes used to obtain this CV were a Pt disk, a

SCE, and a Pt wire, respectively. As shown in Figure 1.1, the potential was scanned from

0.8 V (as initial potential) in the negative direction to -0.15 V linearly and scanned back

to 0.8 V, completing a cycle. At potential 0.8 V indicated by A, there is no current

produced from the electroactive species, K3Fe(CN)6, indicating that no redox behavior

from the analyte was observed at this potential. As the potential is scanned in cathodic

direction, there is no reduction current produced from the analyte until the potential

reaches to 0.35 V as shown in Figure 1.2 and marked by B. There is no redox reaction

taking place from 0.8 V to 0.35 V. The reduction of K3Fe(CN)6 starts at 0.35 V and the

cathodic current increases rapidly ( B to D) and reaches a maximum at D (0.19 V) where

the surface concentration of FeIII(CN)63- approaches zero and then the current decreases

on further scanning to -0.15 V, meaning that the reducible species (FeIII(CN)63-) is

depleted. This process of reduction of analyte is represented by Eq. 1.1:

[FeIII(CN)6]3- + e = [FeII(CN)6]

4- 1.1

As the potential is switched to positive direction, i.e., anodic scanning, the cathodic

4

0 50 100 150 200

0.8

0.6

0.4

0.2

0.0

-0.2

-0.4

-0.6

Pot

entia

l,E v

s A

g/A

gC

l

Time (t), s

Einitial

Efinal

1st Cycle 2nd Cycle

For

war

d sc

an

Reverse scan

Figure 1.1.Cyclic voltammetric excitation signal.

0.8 0.6 0.4 0.2 0.0 -0.2-20

-15

-10

-5

0

5

10

15

20

K

J

I

H G

F

E

D

C

B

A

Epc

Epa

ipc

ipa

Cur

ren

t,

A

Potential, V vs. SCE

 

Figure 1.2. Cyclic voltammogram (CV) obtained from a solution containing 6.0 mM K3Fe(CN)6 and 1.0 M KNO3 at a 2.54 mm-diameter Pt working electrode at a scan rate of 50 mV/s. 19

5

current still persists because the electrode surface is still negative enough to reduce

FeIII(CN)63+. When the electrode surface becomes positive, oxidation of FeII(CN)6

4- starts

forming the original species (FeIII(CN)63-). The anodic current increases as we scan

further from 0.1 V rapidly for the oxidation of FeII(CN)64- back to  FeIII(CN)6

3-  as

indicated by H to J in the above voltammogram (Figure 1.2). So the reduced species gets

oxidized back as:

       [FeIII(CN)6]4- + e = [FeII(CN)6]

3- 1.2

The anodic current reaches maximum at ~0.2 V and decreases for further scanning as the

electroactive species for oxidation, FeII(CN)64-, decreases. Once the potential reaches at

0.8 V, the original condition is restored and can be further continued for successive

cycles.

In the above CV, cathodic peak potential, cathodic peak current, anodic peak

potential, and anodic peak current are represented by Epc, ipc, Epa, and ipa, respectively.

The detailed understanding of the changes of concentration of analyte which

undergoes reversible changes (analyte reduced and the reduced species oxidized back to

the original form) adjacent to the surface of electrode can be obtained by considering the

Nernst equation:

II 4- III 3-6 6

III 3-6 o

II 4-Fe (CN) /Fe (CN)6

Fe (CN)0.059log (at 25 C)

1 Fe (CN)oE E

1.3

where E and E0′ are the measured and formal potentials respectively. At the beginning,

the potential (Einitial) is much more positive than the formal potential since the

concentration of FeIII(CN)63- is in majority. As we scan to the negative potential,

FeIII(CN)63- is reduced to FeII(CN)6

4- decreasing its concentration linearly. At equilibrium,

6

the ratio of ([FeIII(CN)63-]/FeII(CN)6

4-]) becomes unity and the measured potential, E,

becomes equal to the formal potential, E0′.

For a reversible system, the formal potential (E0′) lies midway from the cathodic

peak potential (Epc) and the anodic peak potential (Epa), which can be expressed as:

      pa pc

2o E E

E 1.4

The separation of peak potentials (cathodic and anodic) is given by:

       p pa pc

0.057(V at 25 C)E E E

n 1.5

where n is the number of electron transfer involved in the redox process. For reduction

of FeIII(CN)63- to FeII(CN)6

4-, this number (n) is 1, thus ΔEp = 57 mV is the expected

value.

The Randles-Sevic equation for the reversible system, the peak current (ip) is

given3,20 by

ip = (2.69 × 105) n3/2AD1/2Cv1/2 1.6

where n is the number of electrons transfer involved, A is the surface area of electrode

(cm2), C is the concentration of the electroactive species (mol/cm3), D is the coefficient

of diffusion (cm2/s), and ν is the scan rate (V/s). Therefore, magnitude of peak current is

proportional to the concentration of analyte and the square root of scan rate.

Electrogenerated Chemiluminescence

Electrogenerated chemiluminescence (also called as electrochemiluminescence

and abbreviated as ECL) is the process where electrochemically generated species at the

surface of electrode undergo electron transfer reactions to form the excited state that

emits light.21-23 Bard and Hercules described the detailed studies in the middle of 1960s

7

24-26 although emission of light during electrolysis were reported as early as 1920s.27,28

Currently, ECL has become a very powerful technique in analytical chemistry and has

gained wide range of uses in the areas such as food and water testing, immunoassay

based detections and biowarfare detection.29 ECL has also been exploited successfully as

detector in other analytical techniques such as flow injection analysis (FIA), high

performance liquid chromatography (HPLC), capillary electrophoresis (CE) and micro

total analysis (µTAS).30 A large number of reviews on ECL are available on different

topics of ECL.30-64

In a typical ECL system, the test solution contains species A and B (A and B

could be the same species) with supporting electrolyte such as phosphate buffer in

aqueous solution or tetra-n-butylammonium perchlorate (TBAP) in organic solution and

three electrodes, namely, working, reference and counter electrodes.

There are different processes other than ECL which produce light, such as

photoluminescence (PL)65-67 and chemiluminescence (CL)68-86 Both ECL and CL are

forms of chemiluminescence where light is produced from the species which undergo

highly energetic electron-transfer reactions. However, in CL, light is produced by mixing

necessary reagents and careful manipulation of flow rate whereas in ECL the

electroactive species are generated at the electrode surface by applying potential to the

electrode.

ECL has a number of advantages over CL. In ECL, the electrochemical reaction

allows a time and position for the light emitting reaction to be controlled which help in

aligning the position of electrode to the detector thus increase sensitivity. ECL is more

selective than CL since the generation of excited states can be selectively controlled by

8

varying the electrode potential. Usually, ECL is a nondestructive technique, because the

ECL emitters can be regenerated after the experiment.

ECL also has many advantages over other light emitting techniques such as

fluorescence.29,87 Compared with fluorescence methods, ECL does not involve a light

source, so there is no problem of scattered light and luminescent impurities. Besides,

ECL specificity is associated with the reaction of ECL label and the coreactant which

reduces problem that may arise due to side reactions such as self quenching. Since ECL is

a method of producing light at an electrode, it is also perceived as a bridge between

electrochemical and spectroscopic methods.

ECL can be divided into ion annihilation ECL and coreactant ECL.

Ion Annihilation ECL

The reactant R can be oxidized as well as reduced to form sufficiently stable

radical cation (R•+) and anion (R•-) in annihilation ECL as shown in Scheme 1.1 below:

R – e R●+ (oxidation at electrode) 1.7

R + e R●- (reduction at electrode) 1.8

R●+ + R●- R + R* (excited state formation) 1.9

R* R + hv (light emission) 1.10

Scheme 1.1. Ion Annihilation ECL

These radical ions then undergo annihilation process forming excited state (R*)

which emits light. Depending on the availability of energy in an annihilation in Eq. 1.9,

the excited state species (R*) could be either the lowest excited singlet state (1R*) or the

triplet state (3R*). The energy available in Eq. 1.9 can be calculated from the redox

potential for oxidation and reduction processes in Eqs. 1.7 and 1.8 and is given by:

9

–ΔHann = Ep (R/R●+) – Ep (R/R●-) – 0.16 1.11

where –ΔHann (in eV) is the enthalpy for ion annihilation, Ep is the peak potential for

electrochemical oxidation or reduction (in volts) for the formation of cation and anion

radicals, and the numerical value (0.16) is the entropy approximation term (TΔS).64 If the

energy (–ΔHann) obtained from Eq. 1.11 is larger than the energy (Es) required for the

formation of the lowest excited singlet state (1R*) from Eq.1.9, this system then is called

energy-sufficient system, and the reaction is said to follow the S-route. A typical example

of S-route is the DPA•+/DPA•- (DPA = 9,10-diphenylanthracene) system. 88,89

S-route

R●+ + R●- R + 1R* (excited singlet formation) 1.9a

In contrast, if the energy (–ΔHann) is smaller than Es but larger than the triplet

state energy (Et), then 3R* is initially formed which eventually produce 1R* by triplet-

triplet annihilation (TTA). This can be represented by Eqs. 1.9b and 1.12. This is called

the energy-deficient system and the reaction is said to follow the T-route. A typical

example of the T-route system is the TMPD•+/DPA•- (TMPD = N,N,N′,N′-tetramethyl-p-

phenylenediamine) system. 89,90

T-route

R●+ + R●- R + 3R* 1.9b

3R*+ 3R* R + 1R* 1.12

The efficiency of direct emission of light from triplet form (3R*) in solution phase

is believed to be low due to the relatively long radiative life time of 3R* as compared to

(1R*) and it is quenched by radical ions or other species such as molecular oxygen.

10

If neither of the above two cases persists, then the third route (ST-route) takes its

course. If –ΔHann is nearly marginal to Es, the T-route can contribute to the formation of

1R* in addition to the S-route, so the system is called as ST-route. A typical example of

ST-route is rubrene cation-anion annihilation.91-93 In order for annihilation ECL to be

produced, the large potential window of an electrochemical system must be used (~3.3 V

to 2 V) so that sufficiently stable radical anions and cations can be produced which

eventually produce ECL light. In non-aqueous media, an ion annihilation ECL is widely

studied in MeCN with TBAP supporting electrolyte.

For efficient generation of ion annihilation ECL, certain conditions must be

fulfilled which include:29

( a) stable radical ions of the precursor molecules in the electrolyte of interest,

which can be evaluated via cyclic voltammetric (CV) response;

(b) good photoluminescence efficiency of a product of the electron transfer

reaction, which can be evaluated by fluorescent experiment; and

(c) sufficient energy in the electron transfer reaction to produce the excited state.

Coreactant ECL 64,94

In a coreactant ECL, the potential at the electrode is scanned in one direction

where ECL luminophore can be oxidized or reduced in the presence of supporting

electrolyte and at the same time coreactant species too will be oxidized or reduced

depending upon the direction of scanning, producing cation or anion radicals which

produces very strong reducing or oxidizing species to react with oxidized or reduced

form of luminophore. This produces the excited state of luminophore which emits light.

Highly reducing intermediates are generated from the electrochemical oxidation of co

11

reactant and highly oxidizing species are generated from the electrochemical reduction.

The ECL produced from oxidation is referred to as “oxidative-reduction” ECL and that

from the reduction is called “reductive-oxidation” ECL, respectively.95,96 The coreactant

ECL is useful, especially when one of the cation or anion radicals is not stable enough for

ECL reaction or when these radicals cannot be formed because of the narrow potential

window of the solvent.

Typical coreactant ECL systems and their mechanisms. There are wide ranges of

compounds which can produce ECL light, but majority of coreactant ECL are based on

Ru(bpy)32+ or its derivatives as the ECL emitters since they have excellent chemical,

electrochemical and photochemical properties both in aqueous and non-aqueous medium.

For the oxidative-reduction type coreactant ECL, the system often works well even in the

presence of oxygen avoiding the need to purge the test solution before use.

(a) Ru(bpy)32+/ Tri-n-propylamine (TPrA) system.The most of the ECL applications

reported so far involve Ru(bpy)32+ or its derivatives as an emitter and TPrA as a

coreactant, though there are other coreactans for ECL studies such as oxalate97,

persulfate96, and hydrogen peroxide.98 The Ru(bpy)32+ / TPrA system is an “oxidative-

reduction” type of coreactant ECL. This system has shown the highest ECL efficiency

known so far.87,99 The ECL mechanism of Ru(bpy)32+ /TPrA system is complex and many

workers have investigated it.100-106 The system has been extensively used in commercial

ECL systems for DNA analysis and immunoassays.29 The ECL intensity of the

Ru(bpy)32+/TPrA system depends on several factors such as the concentration of both

Ru(bpy)32+ and TPrA, electrode materials, and the solution pH. The reason behind this

pH dependence is not fully understood but it could be due to the formation and stability

12

of TPrA free radical or the solubility of TPrA in buffered solution. At glassy carbon (GC)

and gold (Au) working electrode, this system shows two ECL waves when the Ru(bpy)32+

concentration is kept low (in the order of nano molar to micro molar concentration,

Figure 1.3).

 

Figure 1.3. (a) ECL and (b) CV of 1.0 nM Ru(bpy)32+ in the presence of 0.10 M TPrA

with 0.10 M tris/0.10 M LiClO4 buffer (pH = 8) at a glassy carbon electrode (3 mm diameter) at a scan rate of 50 mV/s. (c) ECL with 1.0 μM Ru(bpy)3

2+ and 0.10 M TPrA. The ECL intensity scale is given for (c) and should be multiplied by 100 for (a).100

With the increase of Ru(bpy)32+ concentration, the second ECL wave increases

and the first wave become less prominent compared to the second wave because this

wave is merged into the foot of the second wave as shown in Figure 1.4.

The first ECL wave arises due to the reduction of Ru(bpy)32+ by electrochemically

generated TPrA free radical (TPrA●) to Ru(bpy)3+ which is oxidized by the relatively

long lived TPrA radical cation (TPrA●+) to excited state Ru(bpy)32+*. Here, Ru(bpy)3

2+

does not participate in direct electrochemical oxidation. Hence, the first ECL wave can be

13

observed when potential is scanned from 0 to 0.9 V vs Ag/AgCl (Note: Ru(bpy)32+ is

oxidized at 1.1 V).

 

Figure 1.4. First and second ECL waves in 0.10 M TPrA (0.20 M PBS, pH 8.5) with different concentration of Ru(bpy)3

2+: 1 mM (solid line), 0.50 mM (dashed line), 0.10 mM (dotted line) and 0.05 mM (dash-dotted line), at a 3 mm diameter glassy carbon electrode, scan rate of 100 mV/s.100

e

Ele

ctro

de

Ru(bpy)32+

Ru(bpy)32+*

TPrA TPrAH+-H+

TPrA

-H+

TPrA.

P1

Ru(bpy)3+

TPrA

Ru(bpy)32+

hv

+H+

.+

[where TPrA●+ = (CH3CH2CH2)3N●+, TPrAH+ = Pr3NH+, TPrA● = Pr2NC●HCH2CH3, P1

Pr2N+C=HCH2CH3] 

Scheme 1.2. Mechanism for the first ECL wave of the Ru(bpy)32+/TPrA system.100

14

Ru(bpy)32+

Ru(bpy)33+

e

e

TPrA TPrAH-H+

TPrA-H+

TPrA.+ .

Ru(bpy)32+*

P1

Ru(bpy)32+

hv

Ele

ctro

deP2

H2O

+

[where P2 = Pr2NH + CH3CH2CHO]

Scheme 1.3. Mechanism for the second ECL wave of the Ru(bpy)32+/TPrA system.100

The second ECL wave arises due to the direct oxidation of Ru(bpy)32+ at the

electrode surface producing Ru(bpy)33+, and at the same time TPrA also gets oxidized to

form TPrA●+. This radical cation readily deprotonates forming TPrA free radical (TPrA●)

which is a strong reducing agent and reduces Ru(bpy)33+ to Ru(bpy)3

2+* excited state.

This excited state emits light and comes back to the original state and further continues

the process (Scheme 1.3).

Ru(bpy)33+

Ru(bpy)32+

e

e

TPrA TPrAH-H+

TPrA -H+

TPrA.+ .

Ru(bpy)32+*

P1

Ru(bpy)32+

hv

Ele

ctro

de

P2H2O

Ru(bpy)3+

+

Scheme 1.4. Mechanism for the second ECL wave of the Ru(bpy)32+/TPrA system.100

The second ECL wave can also be produced by the chemical reduction of

Ru(bpy)32+ by TPrA● to Ru(bpy)3

+ and electrochemical oxidation of Ru(bpy)32+ to

15

Ru(bpy)33+. These oxidized and reduced species undergo chemical reaction to form

excited state that emits light (Scheme 1.4).

(b) Ru(bpy)32+/Peroxydisulphate (persulfate, S2O8

2-) system.This system was the first

example of “reductive-oxidation” coreactant ECL reported in the literature.96,107 Because

of the solubility issue of the coreactant, (NH4)2S2O8, as it has a low solubility in MeCN

and Ru(bpy)3+ is unstable in aqueous medium so the mixed solvent (water and MeCN)

was used to get intense ECL response. The potential was scanned in the negative

direction where ECL luminophore as well as coreactant can be reduced electrochemically

at the electrode. As shown in Scheme 1.5, persulfate (S2O8 2-) gets electrochemically

reduced to radical anion (S2O8●3- ) and at same time Ru(bpy)3

2+ also gets reduced to

Ru(bpy)3+. The S2O8

2- can also oxidize the reduced Ru(bpy)3+ back to the original state

(Ru(bpy)32+) whereby persulfate is chemically converted to radical anion (S2O8

●3- ). The

strong oxidizing radical (SO4●- ) is formed from the decomposition of persulfate radical

anion (S2O8●3- ) along with SO4

2-. This sulfate radical either oxidizes Ru(bpy)3+ to its

excited state (Ru(bpy)32+*) or oxidizes Ru(bpy)3

2+ to Ru(bpy)33+ which undergoes a

chemical reaction with electrochemically generated Ru(bpy)3+ to form the excited state

that emits light (Scheme 1.5).

2- 3-2 8 2 8S O S Oe 1.13

2+ +3 3Ru(bpy) Ru(bpy)e 1.14

+ 2- 2+ 33 2 8 3 2 8Ru(bpy) S O Ru(bpy) S O 1.15

3 22 8 4 4S O SO SO 1.16

+ 2+ 23 4 3 4Ru(bpy) SO Ru(bpy) * SO 1.17

16

2 3+ 23 4 3 4Ru(bpy) SO Ru(bpy) SO 1.18

3 2 23 3 3 3Ru(bpy) Ru(bpy) Ru(bpy) * Ru(bpy) 1.19

2 23 3Ru(bpy) * Ru(bpy) hv 1.20

Scheme 1.5. ECL mechanism for Ru(bpy)32+/ S2O8

2- system.94

The next section provides a brief review of trace detection of high explosives*

Detection and quantification of high explosives and related compounds have

attracted much attention in recent years, due to the pressing needs associated with global

security and growing environmental and health-related concerns.108-112 Such detection is

necessary in a variety of complex environments, including mine fields, munitions storage

facilities, ground and seawater samples, transportation areas, and blast sites. In each of

these settings, sensitive and timely detection of explosive materials is required to ensure

the safety and security of the surrounding area.

Explosive detection techniques can be broadly classified into two categories: bulk

detection and trace detection.111,112 In bulk detection, a macroscopic mass of the

explosive material is detected directly, usually by viewing images made by X-ray

scanners or similar equipment such as millimeter-wave and tetrahertz imaging109 (Figure

1.5). Other recently developed bulk detection techniques include neutron techniques,

nuclear quadrupole resonance (NQR), and laser techniques. In trace detection, the

explosive is detected by chemical identification of microscopic residues of explosive

                                                            * Part of the contents presented in this section have been published: Miao, W.; Ge, C.; Parajuli, S.; Shi, J.;

Jing, X. In Trace Analysis with Nanomaterials; Pierce, D., Zhao, J., Eds.; Wiley-VCH Verlag: Weinhcim, 2010; 7, pp 161-189.

17

compound as the form of vapor or particulate. Table 1.1 summarizes commonly used

trace detection methods of explosives and their features.

 

Figure 1.5. X-ray imaging (B) reveals the explosives planted in a doll (A). Airport baggage scanners are programmed to display in red those materials with densities that match explosives.110

Additionally, integrated (fused) systems can be used, where two or more detection

methods from the same or different types (e.g., two different bulk detection technologies

or a bulk detection plus a trace detection technique) are combined. 113 The integration can

be carried out by using simultaneous detection or by a two-step detection method, for

example, simultaneous operation of NQR and X-ray imaging or two-step operation of X-

ray computed tomography (CT) 114 and IMS. The strength of one technique may thus

compensate for the weakness of the other, or the vulnerability of one detection device to a

potential countermeasure could be compensated for by another detection device.

Chemical explosives commonly used by military and terrorists can be categorized into

three groups (Table 1.2). The first group contains nitrated explosives that are generally

used by military. On the basis of chemical structural and functional group properties, this

group can be divided into three subgroups: (a) nitroaromatics (NACs), (b) nitroamines,

and (c) nitrate esters. The second group of high explosives is peroxide based, which

18

includes hexamethylene triperoxide diamine (HMTD) and triacetone triperoxide (TATP).

HMTD and TATP have become popular with terrorists because they are easily

Table 1.1 Commonly Used Trace Detection Methods of High Explosives and Their

Features

Method Feature Refs

Mass spectrometry (MS) coupled with gas or high performance liquid chromatography (GC/MS, HPLC/MS)

Sensitive but expensive, not portable 108,113,115-

126

Ion mobility spectrometry (IMS) Sensitive but with matrix effect 127-133

Micro-mechanical sensors (e.g., micro-cantilevers)

Sensitive, low power consumption and real-time operation, but mainly for vapors, with moisture effect, less selective

134-138

Electrochemical methods Inexpensive, fast, portable, less sensitive

139-144

Chemiluminescence (CL) High sensitivity but poor selectivity 145-150

Colorimetric tests Simple, inexpensive, fast but less sensitive and low specificity

151,152

Fluorescence (FL) spectroscopy Sensitive with interferences effect 153-160

Surface plasmon resonance Label-free, sensitive but subject to external contamination

161-165

Surface enhanced Raman scattering spectroscopy (SERS)

Sensitive, but complicated technique and difficult to operate

166-171

prepared from readily obtainable ingredients, although the synthesis is fraught with

danger. For example, TATP was used in the terrorist bombings on the London subway

system in 2005 and by the infamous shoe bomber who tried to detonate his shoes on a

trans-Atlantic flight in 2001.172 As HMTD and TATP contain three peroxide linkages per

molecule, their explosive output is much higher than most organic peroxides. HMTD is

estimated as 60% and TATP as 88% of TNT blast strength.111 Plastic explosives form the

19

third group of explosives, in which one or more of the first group explosives are

plasticized to make a mouldable material, such as C-4 and Semtex H. In order to retain

the best explosive output, the inert plasticizers are usually added less than 10-15% of the

overall weight. Plastic explosives were originally developed for convenient use in

military demolitions but have since been widely used in terrorist bombs. Figure 1.6

shows the structures and abbreviations of explosives listed in Table 1.2.

Table 1.2 Commonly Used High Explosives and Their Chemical Properties

Explosive Name/contents Formula d g/cm3

N % O %

(A) Nitrated explosives

(a) Nitroaromatics

TNT 2,4,6-Trinitrotoluene C7H5N3O6 1.65 18.5 42.3

Picric acid 2,4,6-trinitro-1-phenol C6H3N3O7 1.77 18.3 48.9

Tetryl N-methyl-N,2,4,6-tetranitroaniline C7H5N5O8 1.73 24.4 44.6

(b) Nitramines

RDX 1,3,5-Trinitro-1,3,5-triazacyclohexane

C3H6N6O6 1.82 37.8 43.2

HMX 1,3,5,7-tetranitro-1,3,5,7-tetrazocane C4H8N8O8 1.96 37.8 43.2

CL 20 hexanitrohexaazaisowurtzitane C6H6N12O12 38.4 43.8

(c) Nitrate esters

PETN Pentaerythritol tetranitrate C5H8N4O12 1.76 17.7 60.7

Nitrocellulose Cellulose nitrate C6H7N3O11 1.2 14.1 59.2

(B) Peroxide-based explosives

HMTD hexamethylene triperoxide diamine C6H12N2O6 1.6 13.5 46.1

TATP Triacetone triperoxide C9H18O6 1.2 0 43.2

(C) Plastic explosives

C-4 RDX + plasticizer

Semtex H RDX + PETN + plasticizer

Detasheet PETN + plasticizer

20

CO O

O O

C C

O O

N

O

O

O

O

O

O

N

N N

N

NO2

O2N NO2

NO2

NO2

O2N NO2

NO2

OH

O2NNO2

NO2

N

O2N

NO2

N

N

N

N

NO2

NO2

NO2

O2N

N N

N N

N N

NO2

NO2

O2N

O2N

O2N NO2

O2NO

O2NO ONO2

ONO2

(i) HMTD (j) TATP

(a) TNT (b) Picric acid (c) Tetryl (d) RDX

(e) HMX (f) CL 20 (g) PETN

O

O2NOONO2

O

ONO2

x

(h) Nitrocellulose

Figure 1.6. Structures and abbreviations of commonly used high explosives. (a-c) Nitroaromatics, (d-f) Nitramines, (g-h) Nitrate esters, and (i-j) Peroxide-based explosive compounds.

Compared with other organic compounds, explosives show exceptionally high

density (e.g., military explosives generally have a density greater than 1.6 g/cm3, Table

1.2), which indicates the high expulsive forces between atoms, leading to powerful

explosion when explosives blow up. All explosives have very high oxygen and/or

nitrogen contents, so that dramatically volume changes (from solid to gas) are expected

when an explosion occurs. Many bulk detection methods are in fact based on the “bulk

property” of high density and high oxygen and nitrogen contents of explosives.

21

Although a wide variety of explosive detection technologies are currently

available, this section will mainly focus on trace detection methods involving

nanomaterials. Such methods generally possess many advantages over traditional ones,

which include high sensitivity, good selectivity, fast response, portability, and low cost.

These features are clearly desirable for all analytical systems, and are essential in winning

the war on explosives-based terrorism.

Typical Techniques for Trace Detection of High Explosives

Electrochemistry. The inherent redox properties of nitrated and peroxide-based

explosives make them ideal candidates for electrochemical monitoring.142,144

Electrochemical sensors (ESs) for explosive detection provide several advantages over

spectroscopic and spectrometric techniques such as SERS, MS and IMS. They are

characterized by a reasonable sensitivity, low cost, and can be easily used as field

detectors and remote control devices, due to the nature of the analytical signal.173-177

Various ESs for the detection of nitroaromatic compounds (NACs, Figure 1.6) have been

reported using different sensing materials including bare carbon and Au as well as boron-

doped diamond electrodes.173-176,178 A polyphenol-coated screen-printed carbon electrode

was also used for highly sensitive voltammetric measurements of TNT in the presence of

surface-active substances.179 Electrochemical responses of a number of NACs were

compared at glassy carbon (GC), Pt, Ni, Au, and Ag electrodes, revealing that Au and Ag

were suitable in capillary electrophoresis (CE) amperometric detection. A bimetal

electrode, prepared by depositing Ag on Au, offered a superior performance by exploiting

the sensitivity of Au while suppressing its response toward MeCN to achieve a 10-fold

22

lower detection limit of the explosive compounds (70-110 parts per billion (ppb)) than

UV measurement.180

Nanomaterial modified electrode. Metallic and metal-oxide nanoparticles (NPs) are

capable of increasing the activities for many chemical reactions due to the high ratio of

surface atoms with free valences to the cluster of total atoms. In addition to a high surface

area-to-volume ratio for NP derivatized materials, the size controllability, chemical

stability, and surface tenability provide an ideal platform for exploiting such

nanostructures in sensing/biosensing and catalytic applications. Using electrodes

modified with NPs of transition metals and precious metals, which have specific

properties compared to that of the bulk materials, opens new ways for their applications

as ESs.181,182

The modification of electrode surfaces with redox-active metal NPs has led to the

development of various ESs. Filnovsky et al. found that the modification of carbon with

NPs of noble metals is a promising approach for obtaining highly catalytically active

electrodes for the detection of traces of aromatic compounds.183 Modification of the

electrode was carried out with composites of nm-sized, mesoporous TiO2, which acted as

a support containing inserted or deposited NPs of Ru, Pt, or Au. Cyclic voltammetry

shoes that TNT can be reduced on thus-modified carbon-paper electrodes at potentials

around -0.5 V (vs Ag/AgCl/Cl–) in aqueous solutions. When the TiO2/nano-Pt composites

were used, remarkable electrochemical activities of the electrode toward the reduction of

TNT were observed, suggesting that the composite material may play a specific role for

facilitating the TNT reduction process. Modified electrodes based on mesoporous SiO2-

23

MCM-41 coatings have been recently shown to be useful for enhancing the sensitivity

through adsorptive accumulation of the target NACs explosives.184

There are many reports in the literature using carbon nanotubes (CNTs) as the

electrode modification material for improving the detection of explosives. Multi-walled

CNTs (MWCNTs) modified GC electrodes offer a significant improvement in the

electrochemical detection of TNT in seawater.185 Metal NPs (Pt, Au, or Cu) together with

MWCNTs and single-walled CNTs (SWCNTs) have been used to form nanocomposites

for modifying GC electrodes to improve their electroactivity and selectivity for TNT and

several other NACs.186 The performance of the different metallic NPs in combination

with both types of CNTs with respect to sensitivity, linear range, and selectivity toward

NACs was evaluated and discussed. Among various combinations tested, the synergistic

signal effect was observed for the nanocomposite modified GC electrode containing Cu

NPs and SWCNTs solubilized in Nafion, in which combination provided the best

sensitivity for detecting TNT and other NACs. Adsorptive stripping voltammetry for

TNT resulted in a detection limit of 1 ppb, with linearity up to three orders of magnitude.

Selectivity toward the number and position of the nitro groups in different NACs was

found to be very reproducible and distinct. The Cu-SWCNT-modified GC electrode was

demonstrated for analysis of TNT in tap water, river water, and contaminated soil.

Prussian-blue “artificial peroxidase” modified electrode. Peroxide-based explosives,

TATP and HMTD, are easy to synthesize from readily available precursor chemicals, but

their detection is found to be very challenging, since they lack electrochemically

reducible nitro groups, do not fluoresce and exhibit minimal UV absorption.187

24

While TATP and HMTD can be measured using expensive instruments such as

chemical-ionization MS or IR spectroscopy,123 these bulky instruments are not suitable

for field screening scenarios or trace analysis of peroxide-based explosives. Accordingly,

there are urgent needs for developing highly sensitive and yet small, easy-to-use, field

deployable devices for on-site testing of peroxide explosives. Activity in this direction

has focused primarily on enzymatic (peroxidase) based optical (fluorescent or

colorimetric) assays of the hydrogen peroxide (H2O2) product of UV- or acid treatment187

of the peroxide explosives.

However, enzymatic assays often suffer from shortcomings associated with the

limited stability and high cost of the biocatalyst. Surprisingly, little attention has been

given to the development of electrochemical devices for monitoring peroxide

explosives,188 despite the fact that these devices are uniquely qualified for meeting the

size, cost, and low power requirements of field detection of TATP and HMTD.

Prussian blue (PB) polycrystal modified electrodes offer highly selective, low

potential, and stable electrocatalytic detection for H2O2.189,190 A highly sensitive

electrochemical assay of TATP and HMTD at such an electrode has been reported.140 The

method involves UV light degradation of the peroxide explosives and a potential of

(~0.0 V vs SCE) electrocatalytic amperometric sensing of the generated H2O2 at the PB

transducer and offers nanomolar detection limits following a short (15 s) irradiation times.

Electrochemical detection based on direct reduction of the explosives at the electrode

cannot be carried out, because the reduction of the explosives containing -O-O- peroxide

groups is very ineffective. Although PB modified electrode is specific toward H2O2

reduction at a low potential value (~0.0 V vs SCE) where unwanted reactions of co-

25

existing compounds are negligible,189-191 selective detection of TATP and HMTD is

difficult. The high catalytic activity of PB leads also to a very high sensitivity towards

H2O2. The behavior of PB-coated electrodes resembles that of peroxidase-based enzyme

electrodes, and hence PB has often been denoted as “artificial enzyme peroxidase.”192 PB

electrodes offer distinct stability and cost advantages over peroxidase biosensors, and

appear to be the most effective electrochemical transducer for H2O2. Besides, it offers a

substantial lowering of the overvoltage for the H2O2 redox process and permits a highly

selective and sensitive peroxide sensing. Such efficient H2O2 transducer facilitates the

rapid detection of peroxide explosives down to the nanomolar level.192

Whenever needed, the PB film can be covered with a permselective (size-

exclusion) coating that can further enhance the sensor selectivity, stability and overall

performance.193 Also, relevant samples may be treated enzymatically (with catalase) to

remove the co-existing H2O2 that may originate from cleaning agents.194 The

electrochemical route can be further developed into disposable microsensors in

connection to single-use screen-printed electrode strips and a hand-held meter (similar to

those used for self testing of blood glucose). Preliminary data with such PB-coated

screen-printed electrodes are very encouraging. The PB-transducer can be readily adapted

for gas-phase electrochemical detection of trace TATP and HMTD in connection to

coverage with an appropriate solid electrolyte coating.140,141

Fluorescence-Based Sensors

Fluorescence (FL) sensors have been widely used for the detection of nitrated explosives.

The sensors are developed on the basis of either the FL quenching of the system or

competitive FL immunoassays. FL-quenching-based chemosensors, where analyte

26

binding produces attenuation in the light emission, are highly desirable for the detection

of small molecular analytes in many challenging environments, due to the high signal

output and detection simplicity. The operation mechanism of the chemosensors, for the

detection of vaporous explosives such as dinitrotoluene (DNT) and TNT, is mainly based

on the electron transfer from the electron-rich organic materials to those electron-

deficient NACs leading to the fluorescence quenching of the organic materials. The

exciton diffusion length and the surface areas of the sensing films are critical in detection

sensitivity.160,195 FL-quenching-based detection represents one of the most sensitive and

convenient methods that have been widely employed in explosives identification.196 Only

the chromophore that interacts directly with the analyte molecule is quenched; the

remaining chromophores continue to fluoresce. The basic sensor design113,197 (Figure 1.7)

consists of a FL excitation source, such as a blue light emitting diode.

Figure 1.7. Fluorescent polymer sensor design.113,197

Light passes through a lens and filter, allowing a narrow wavelength band (e.g.,

430 nm) to impinge on the polymer film, which is coated on two sheets of glass. A pump

pulls in air samples across the coated glass sheets. After exposing a glass slide coated

27

with the prepared nanocomposite film for a given period of time, the fluorescence spectra

are immediately measured at an excitation wavelength. If the air sample contains

explosive vapors, the PMT detector will sense a FL quenching in light intensity and

trigger an alarm.

Fluorescent polymers chemical detectors. Among various photoluminescence materials,

conjugated polymers have been most extensively explored as chemosensory materials for

the FL detection of electron-deficient analytes such as NACs.198-202 Some conjugated

polymers exhibit a high sensitivity to NACs explosives resulting in strong quenching of

their emission.202-204 Swager and coworkers reported the amplified response to the analyte

binding events in the aggregated systems and solid films of conjugated polymers by

intermolecular exciton migration.160,198 Particularly, the multiphoton FL quenching has

been observed with obvious advantages for the real-time detection of TNT.200-202,205 A

recent report indicated that the molecular imprinting in the matrix of conjugated polymers

can greatly improve their chemosensory selectivity to NACs.206 Other photoluminescent

materials such as polytetraphenylsilole, polytetraphenylgermole, photoluminescent silica

films,203,204,207,208 and silica microspheres with physisorbed dyes 209 also exhibit the high

FL response to the solution and vapor of NACs explosives at low-level concentrations.

Porous silicon (PSi) chemical and biological optical sensors have been intensively

studied in the past 210-223 because of the high surface area of PSi and the variety of optical

transduction mechanisms upon exposure to different analytes.196 However, most of these

sensors demonstrate no specificity for target molecules, and require the analytes (e.g.,

alcohols and saturated hydrocarbons) with high vapor pressures (~1-100×10−5 mm Hg) in

order for the change in the reflectance or luminescence of PSi structures to be detected.196

28

The detection of analytes with low vapor pressures (~10−5 mm Hg and lower) such as

some nitrated explosives (e.g., TNT) is a challenge by these methods since nonspecific

sorption coupled with low analyte concentration in the pores is not sufficient to uniquely

alter PSi optical properties. Thus, entrapping polymers that exhibits a high sensitivity to

NACs explosives inside the PSi microcavity (MC) significantly improve sensor

efficiency due to specific binding of NACs to the sensory polymers, high quantum FL

yield of the polymers (higher than PSi selfluminescence), amplification mechanism as a

result of the energy migration,160 and the fine spectral patterning of the broad FL band

induced by the MC structure. Also, sufficient changes in the MC reflectivity could result

in the sensitive detection of explosive vapors. Based on this principle, Levitsky et al.196

have recently studied some fluorescent polymer-PSi MC devices, in which a conjugated

chemosensitive polymer entrapped in PSi MC allows detection of vapors of explosive

NACs via a modulation in both FL and reflectance signals. The MC resonant peak in the

reflectance spectra is shifted upon vapor exposure. The broad polymer FL shows

patterning by the narrow MC peak, which is also sensitive to the vapor exposure.

Many structure-property studies amplify the great potential of nanostructured

materials fabricated on different length scales for practical applications. An important

development in this area is the fabrication of materials with hierarchical porous structures,

which combine the multiple benefits arising from the different pore size regimes.224-234

For instance, a material with interwoven meso- and macroporous structures can provide a

high specific surface area and more interaction sites via small pores, whereas the

presence of additional macropores can offer increased mass transport and easier

accessibility to the active sites through the material. These features make such kinds of

29

materials highly suitable and promising for applications in catalysis, separation

technology, and sensor devices, especially if specific action sites or recognition units are

attached to these materials. A series of porphyrin or metalloporphyrin-doped silica films

with bimodal porous structures, fabricated using polystyrene spheres or evaporation

introduced self-assembly approach and a surfactant cetyltrimethylammonium bromide

(CTAB) as structure-directing agents, have been utilized for chemosensory applications

to detect trace amounts of vapors of explosives such as TNT, DNT, and nitrobenzene

(NB).232-234 The obtained results demonstrated that an appropriate combination of

macropores and mesopores can achieve high molecule permeability and high density of

interaction sites. As a result, silica films with bimodal porous structures exhibit much

more efficient FL response capability than single modal porous films. Films with

extremely high FL quenching efficiency towards TNT (10 ppb), close to 55% after 10 s

of exposure, were achieved, which is said to be nearly double those of conjugated

polymer based TNT sensor materials reported previously.179,200,203,235 Using toluene

washing, the sensory properties of the constructed films can be easily recovered. Besides

the remarkable TNT detecting capability, these hybrid films have several advantages over

other FL-based sensory materials, such as an easy preparation approach, inexpensive

materials, recognition ability of different NACs as well as stability of organic sensing

elements in inorganic matrices.

Fluorescent nanofibril film. One-dimensional crystalline structures of organic molecules

on the nanometer scale are good candidates for explosives detection because of the long

exciton diffusion length arising from crystalline structures 236 and their intrinsic large

surface-to-volume ratio. The highly organized organic 1D nano- and supernanostructures

30

self-assembled with extended planar molecular surfaces enable both effective 1D π-π

stacking favorable for exciton migration via cofacial intermolecular electronic coupling

236 and flexibility in tuning morphologies on the nano- or microscopic scale. A

fluorescent nanofibril film, fabricated from the alkoxycarbonyl- substituted, carbazole-

cornered, arylene-ethynylene tetracycle (ACTC), was reported to be an efficient sensing

film for detection of explosives.158 The incorporation of carbazole enhances the electron

donating power of the molecule and thus increases the efficiency of FL quenching by

oxidative explosives (e.g., NACs). The quenching response observed for the ACTC film

is significantly faster than that previously observed for other organic materials,198,237

consistent with the fibril porous structure of the film, which facilitates both gaseous

adsorption and exciton migration across the film. The quenching efficiency obtained for

ACTC films is also higher than those previously reported for other explosive sensing

materials at the same thickness.238 The porous film morphology and the extended one-

dimensional π-π stacking facilitate the access of quencher molecules to the excited states,

thereby resulting in effective FL quenching, which is little dependent on the film

thickness as evidenced by the observations. This behavior is in contrast to what was

usually observed for other organic film sensors, for which the emission quenching

efficiency was inversely proportional to the film thickness owing to the diffusion limit of

the exciton and the gaseous adsorbates.

Furthermore, porphyrin-doped nanocomposite fibers with a form of nanofibrous

membrane were fabricated without the addition or help of polymers, and were

demonstrated as novel FL-based chemosensors for the rapid detection of trace vapor (10

ppb) of explosive TNT.159 The research was based on sol-gel chemistry and the

31

electrospinning technique. Due to a larger surface area and good gas permeability, these

fluorescent nanofibrous membranes exhibit remarkable sensitivity to trace TNT vapor

compared to tightly cross-linked silica films, but their sensitivity is strongly dependent on

the morphology and phase aggregation of the used nanofibers. Reducing the diameter and

introducing a pore structure into nanofibers can considerably enhance the sensitivity of

the resulting materials. Because of the well-known strong tendency to form hydrogen

bonds between imino hydrogens of the used porphyrin molecule and nitro groups of

NACs as well as π-stacking between porphyrins and NACs, porphyrin units have a

relative large affinity for NACs molecules, which provides a strong driving force for fast

FL quenching.

Several reasons are believed to be principally responsible for the remarkable

observed sensing of the electrospun nanocomposite fibers towards trace TNT vapor.232

First, the unique bimodal porous structure provides a necessary condition for the facile

diffusion of analytes to sensing elements, while the large surface area considerably

enhances the interaction sites between analyte molecules and sensing elements, thereby

further improves the detection sensitivity. Second, for a given analyte like TNT, strong

binding strength and energy level matching are essential for obtaining high TNT

quenching efficiency.Theoretical studies, which indicate that the FL quenching per unit

time is affected by various factors, including the vapor pressure of analyte, the

exergonicity of electron transfer, and the binding strength between sensing elements and

analytes.

Quantum dots quenching sensor. Colloidal semiconductive nanocrystals/nanoparticles

(also called quantum dots, QDs) are spherical particles in a size regime dominated by

32

strong quantum confinement of the charge carriers. This confinement lifts the degeneracy

of the carrier states within the conduction and valence bands, and increases the effective

band gap energy significantly with decreasing particle size, resulting in size dependence

of several properties, such as absorption and photoluminescence spectra.239,240

Luminescent QDs have the potential to circumvent some of the functional

limitations encountered by organic dyes in biotechnological applications. Recently, QDs

with high quantum yields have found a wider range of applications as a foundation of FL

sensors.241-245 The photoluminescence of QDs is readily tunable within a large range of

spectroscopy through the change of size or the introduction of dopant ions, which can

potentially be utilized for obtaining a spectral response toward a particular target

analyte.244 More importantly, QDs allow the chemical modification of functional groups

and the installation of recognition receptors at their surfaces, providing the

chemodetection selectivity to target species.243,245 Therefore, the FL chemosensors based

on the “lab-on-QDs” concept have a remarkable advantage over other detection schemes

in chemodetection sensitivity and selectivity.246 For example, Goldman and co-

workers247,248 recently proposed a typical scheme of QDs-based chemosensors through

the hybrid CdSe QDs of antibody segments and dye molecules. The specific detection

toward TNT has been achieved through the fluorescence resonance energy transfer

(FRET) between QDs and dye. As shown in Figure 1.8, the hybrid sensor consists of

anti-TNT specific antibody fragments (receptors) attached to a hydrophilic QD via metal-

affinity coordination. A dye-labeled TNT analogue (analog-quencher) pre-bound in the

antibody binding site quenches the QD PL via proximity-induced FRET. Addition of

33

soluble TNT analyte displaces the dye-labeled analogue, eliminating FRET and resulting

in a concentration-dependent recovery of QD PL.

A general strategy for FRET-based biosensor design and construction employing

multifunctional surface-tethered components from the above research team has been

proposed (Figure 1.9A) 249,250 and used in the detection of TNT and related

compounds.250

Figure 1.8. Schematic of a hybrid QD-antibody fragment FRET-based TNT sensor.247

The molecular biosensor consists of two modules: the biorecognition module and

the modular arm. Both modules are specifically attached to a surface in a particular

orientation. Choices for surface attachment include biotin-avidin chemistry, metal-

affinity coordination, thiol bonding, hydrophobic interactions, DNA-directed

immobilization, etc.249,251 The biorecognition module can consist of proteins (enzymes,

receptors, bacterial periplasmic binding proteins (bPBPs), antibody fragments, peptides),

aptamers, carbohydrates, DNA, PNA, RNA. This module is site-specifically dye-labeled

in the current configuration. The modular arm may consist of flexible moieties such as

DNA, PNA, RNA, peptides, polymers, etc. The modular arm is also site-specifically dye-

labeled. An analogue of the primary analyte is attached to the distal end of the flexible

34

arm to act as the recognition analogue. Binding of this recognition element in the binding

pocket of the biorecognition element assembles the sensor into the ground state by

bringing both dyes into proximity, which establishes FRET.

Figure 1.9. (A) Schematic of the modular biosensor consisting of two modules: the biorecognition module and the modular arm (see main text for detailed description). (B) Schematic of the TNT targeting biosensor.250

As shown in Figure 1.9, the dye-labeled anti-TNT scFv fragment (1, the

biorecognition module) is attached to the surface with Bio-X-NTA (Bio: biotin; X:

aminomethoxy spacer; NTA: nitrilotriacetic acid chelator) coordinating the 12

histidines (12-HIS) and orienting the protein on the NeutrAvidin (NA). The dye-

labeled TNB (a TNT analogue 1,3,5-trinitrobenzene) DNA arm (2, modular arm) is

attached to the NA via complementary hybridization to a biotinylated (B) flexible

DNA linker. Both are added in equimolar amounts. ScFv binding of the TNB

35

analogue brings the protein located dye and DNA located dye into proximity

establishing FRET. Addition of TNT displaces the TNB analogue and DNA arm

disrupting FRET in a concentration-dependent manner.250

Addition of analyte competitively displaces the analogue and signal

transduction is designed to be sensitive to this displacement. FRET donor/acceptor

can be placed on either module. Mechanisms of controlling binding affinity include

stiffening the flexible arm or switching in of different affinity biorecognition elements.

As shown in Figure 1.9B, the sensor consists of a dye-labeled anti-TNT antibody

fragment (scFv) that interacts with a cofunctional surface-tethered DNA arm. The arm

consists of a flexible biotinylated DNA oligonucleotide base specifically modified

with a dye and terminating in a TNB recognition element, which is an analog of TNT,

1,3,5-trinitrobenzene. Both of these elements are tethered to a Neutravidin (NA)

surface with the TNB recognition element bound in the antibody fragment binding

site, bringing the two dyes into proximity and establishing a baseline level of FRET.

Addition of TNT, or related explosive compounds (e.g., RDX and DNT), to the sensor

environment alters FRET in a concentration-dependent manner. The sensor can be

regenerated repeatedly through washing away of analyte and specific reformation of

the sensor assembly, allowing for subsequent detection events. Sensor dynamic range

can be usefully altered through the addition of DNA oligonucleotide that hybridizes to

a portion of the cofunctional arm. Although the authors have used quenching of

organic dyes for biosensor signal generation, use of optical components such as QDs,

should be also possible.

36

Fluoroimmunoassays using QD-antibody conjugates. 252,253 Goldman et al.240

developed a strategy based on the use of antibody-conjugated QDs in plate-based

competitive immunoassays for the detection of ng quantities of the TNT-surrogate,

TNB fluorescein, and RDX in aqueous samples (Figure 1.10). The QD/antibody

conjugates were formed by using either a molecular adaptor protein or using avidin to

form QD/antibody conjugates. The analytes of interest compete with the surface-

confined antigen for antibody-QDs, and the FL signal is measured from the plate after

a washing step. A reverse relationship between the measured FL intensity and the

analyte concentration is followed.

Figure 1.10. QD antibody conjugates prepared using molecular bridges. (A) Mixed surface conjugate after purification by cross-linked amylose affinity chromatography. (B) Schematic of competitive assay for the explosive RDX dissolved in water.253

Displacement immunosensors254. The displacement immunosensor is an improved, faster,

and more efficient detection system with respect to a traditional immunoassay,255 and

may be regarded as a category of chromatographic immunoassays.256 In the displacement

immunosensor, immunoassays are coupled with a device that is set under continuous

buffer flow, as illustrated in Figure 1.10. The key components of the system are

antibodies (Ab) immobilized on a solid support (e.g., micro-sized beads,257 agarose gel,258

37

membranes259), antigen analogs that are labeled with a reporter molecule (e.g., a

fluorophore) (Ag*), and the associated hardware needed to establish a controlled flow

system. Generally, monoclonal antibodies and reporter molecules are first linked to a

prepared surface and a target antigen/analyte, respectively. The Ag* is then allowed to

react with the immobilized Ab until equilibrium is reached (e.g., 2~15 h). To perform an

assay, the solid support coated with the Ab/Ag* complex is placed in a buffer flow.

When a sample containing the analyte of interest is injected into the flow stream, the Ag*

molecules are displaced into the buffer and measured downstream using a FL detector.

The measured FL intensity is proportional to the concentration of analyte molecules

injected, within a predetermined linear range for each antibody.

Figure 1.11. Schematic of the displacement immunosensor method.254

Metal Oxide Semiconductor (MOS) Nanoparticle Gas Sensors

The metal oxide semiconductor (MOS) gas sensor is one class of the electronic

noses 260 and usually reported as detectors for volatile organic compounds because of its

sensitivity, low-cost and easy manufacturing. Several reports on this topic for explosive

detection have appeared. In the first study, sensing military grade TNT and some

38

substrates (air, sand and soil) was investigated by using TiO2 thin film sensors with a

static headspace sampling, which indicated that using MOS sensors to detect solid

explosive was a feasible method.261 For the detection of solid explosives, however, the

low vapor concentration makes it an extremely difficult and challenging task. To solve

this problem, some researchers suggested using Pt or Pd catalyst in the carrier gas line to

increase the sensitivity of the MOS sensors, which could also allow the sampling of

solids and liquids as well as gases with a gas sensor.235 In another study, several

explosives (e.g., DNT, NH4NO3, and picric acid) have been investigated by using ZnO-

doped nanoparticle sensors with additives of Sb2O3, TiO2, V2O5 and WO3.262

Surface-Enhanced Ramam Scattering Spectroscopy

Surface-enhanced Raman scattering spectroscopy (SERS) combines extremely

high sensitivity, due to enhanced Raman cross-sections comparable or even better than

fluorescence, with the observation of vibrational spectra of adsorbed species, providing

one of the most incisive analytical methods for chemical and biochemical detection and

analysis.167,263 The metallic NPs (e.g., Au and Ag NPs264) that make SERS possible are of

fundamental interest since they possess unique size-dependent properties. The use of

SERS for trace explosive detection was first investigated during the late 1990s,264,265 and

SERS detection of 2,4-DNT vapor to ~1 ppb was demonstrated.266 Subsequently,267 a

field-portable unit had demonstrated a limit of detection of 5 ppb vapor DNT and the

ability to locate buried land mines. More recently,268 nano-engineered SERS substrates

have been employed, and ppb sensitivity for some nerve agent and explosive simulants

has been demonstrated.

Surface-enhanced Ramam scattering spectroscopy has also been used to detect

39

selectively functionalized TNT; additional enhancement due to the resonance Raman

effect results in detection limits much better than 1 nM in solution.269 With the similar

ideas, detection of explosive RDX via reduction and subsequent functionalization has

been reported.270

Conclusions

Seven types of techniques involving nanomaterials for trace detection and

quantification of high explosives are reviewed. These techniques are based on (a)

electrochemistry, (b) fluorescence, (c) metal oxide semiconductive nanoparticles, and (d)

surface enhanced scattering spectroscopy. Most of the studies have been focused on

nitroaromatics detection and a few are peroxide explosives related. Nanotechnology will

continue to play an important role in developing new explosive sensors that have better

sensitivity and higher selectivity toward analytes of interest, and are more portable for

field testing, shorter response time, cheaper to maintain, and easier to operate.

40

References

(1) Scholz, F.; Editor Electroanalytical Methods: Guide to Experiments and

Applications, 2nd printing; Springer, 2005.

(2) Girault, H. H. Angew. Chem., Int. Ed. 2001, 40, 4511-4512.

(3) Bard, A. J.; Faulkner, L. R. Electrochemical Methods: Fundamentals and

Applications; Wiley, 1980.

(4) Sawyer, D. T.; Sobkowiak, A.; Roberts, J. J. L.; Editors Electrochemistry for

Chemists: Second Edition; Wiley, 1995.

(5) Koryta, J.; Dvorak, J. Principles of Electrochemistry; John Wiley & Sons, 1987.

(6) Plambeck, J. A. Electroanalytical Chemistry: Basic Principles and Applications;

John Wiley and Sons, 1982.

(7) Skoog, D. A. Principles of Instrumental Analysis. 3rd Ed; CBS College Publishing,

1985.

(8) Gosser, D. K. Cyclic Voltammetry: Simulation and Analysis of Reaction Mechanisms;

VCH, 1993.

(9) Holder, G. N.; Farrar, D. G.; McClure, L. L. Chem. Educ. 2001, 6, 343-349.

(10) Tomo, H. E.; Araki, K.; Davidauskas, S. J. Chem. Educ. 2000, 77, 1351-1353.

(11) Arrigan, D. W. Analyst 1994, 119, 1953-1966.

(12) Dong, S.; Wang, Y. Electroanalysis 1989, 1, 99-106.

(13) Kalcher, K. Electroanalysis 1990, 2, 419-433.

(14) Kalcher, K.; Kauffmann, J. M.; Wang, J.; Svancara, I.; Vytras, K.; Neuhold, C.;

Yang, Z. Electroanalysis 1995, 7, 5-22.

(15) Walcarius, A. Electroanalysis 1996, 8, 971-986.

(16) Walcarius, A.; Despas, C.; Trens, P.; Hudson, M. J.; Bessiere, J. J. Electroanal.

Chem. 1998, 453, 249-252.

(17) Kissinger, P. T. Chromatogr. Sci. 1983, 23, 125-164.

(18) Evans, D. H.; O'Connell, K. M.; Petersen, R. A.; Kelly, M. J. J. Chem. Educ. 1983,

60, 290-293.

(19) Heineman, W. R.; Kissinger, P. T. Curr. Sep. 1989, 9, 15-18.

(20) Kissinger, P. T.; Heineman, W. R.; Eds. Laboratory Techniques in

Electroanalytical Chemistry; Dekker, 1984.

41

(21) Miao, W.; Wang, S. In Handbook of Chemiluminescent Methods in Oxidative

Stress Assessment; Popov, I., Lewin, G., Eds.; Transworld Research Network, 2008,

41-83.

(22) Miao, W. Chem. Rev. 2008, 108, 2506-2553.

(23) McNaught, A. D.; Wilkinson, A. Compendium of Chemical Terminology, 2nd ed;

Blackwell, 1997.

(24) Hercules, D. M. Science 1964, 145, 808-809.

(25) Santhanam, K. S. V.; Bard, A. J. J. Am. Chem. Soc. 1965, 87, 139-140.

(26) Visco, R. E.; Chandross, E. A. J. Am. Chem. Soc. 1964, 86, 5350-5351.

(27) Dufford, R. T.; Nightingale, D.; Gaddum, L. W. J. Am. Chem. Soc. 1927, 49, 1858-

1864.

(28) Harvey, N. J. Phys. Chem. 1929, 33, 1456-1459.

(29) Bard, A. J.; Ed. Electrogenerated Chemiluminescence; Marcel Dekker, Inc., 2004.

(30) Yin, X.-B.; Dong, S.; Wang, E. TrAC, Trends Anal. Chem. 2004, 23, 432-441.

(31) Andersson, A.-M.; Schmehl, R. H. Mol. Supramol. Photochem. 2001, 7, 153-187.

(32) Andersson, A.-M.; Schmehl, R. H. Electron Transfer Chem. 2001, 1, 312-341.

(33) Anon Indian J. Clin. Biochem. 1998, 13, 129-132.

(34) Armstrong, N. R.; Wightman, R. M.; Gross, E. M. Annu. Rev. Phys. Chem. 2001,

52, 391-422.

(35) Balzani, V.; Juris, A. Coord. Chem. Rev. 2001, 211, 97-115.

(36) Bard, A. J.; Fan, F.-R. F. Acc. Chem. Res. 1996, 29, 572-578.

(37) Bard, A. J.; Park, S. M. 1975; Academic; 305-326.

(38) Beier, R. C.; Stanker, L. H. Recent Res. Dev. Agric. Food Chem. 2000, 4, 59-93.

(39) Bolletta, F.; Bonafede, S. Pure Appl. Chem. 1986, 58, 1229-1232.

(40) Fahnrich, K. A.; Pravda, M.; Guilbault, G. G. Talanta 2001, 54, 531-559.

(41) Faulkner, L. R.; Glass, R. S. Chem. Biol. Gener. Excited States 1982, 191-227.

(42) Gerardi, R. D.; Barnett, N. W.; Lewis, S. W. Anal. Chim. Acta 1999, 378, 1-43.

(43) Gonzalez Velasco, J. Electroanalysis 1991, 3, 261-271.

(44) Gonzalez Velasco, J. Bull. Electrochem. 1994, 10, 29-38.

(45) Greenway, G. M. Trends Anal. Chem. 1990, 9, 200-203.

(46) Kapturkiewicz, A. Adv. Electrochem. Sci. Eng. 1997, 5, 1-60.

42

(47) Kissinger, P. T. J. Pharm. Biomed. Anal. 1996, 14, 871-880.

(48) Knight, A. W. TrAC, Trends Anal. Chem. 1999, 18, 47-62.

(49) Knight, A. W.; Greenway, G. M. Analyst 1994, 119, 879-890.

(50) Knight, A. W.; Greenway, G. M. Analyst 1996, 121, 101R-106R.

(51) Kukoba, A. V.; Bykh, A. I.; Svir, I. B. Fresenius J. Anal. Chem. 2000, 368, 439-

442.

(52) Kulmala, S.; Suomi, J. Anal. Chim. Acta 2003, 500, 21-69.

(53) Lee, W. Y. Mikrochim. Acta 1997, 127, 19-39.

(54) Mitschke, U.; Bauerle, P. J. Mater. Chem. 2000, 10, 1471-1507.

(55) Nabi, A.; Yaqoob, M.; Anwar, M. Lab. Rob. Autom. 1999, 11, 91-96.

(56) Nieman, T. A. J. Res. Natl. Bur. Stand. 1988, 93, 501-502.

(57) Nieman, T. A. In Chemiluminescence and Photochemical Reaction Detection in

Chromatography; Birks, J. W., Ed.; VCH: New York, 1989; 99-123.

(58) Park, S.-M.; Tryk, D. A. Rev. Chem. Intermed. 1980, 4, 43-79.

(59) Richter, M. M. In Optical Biosensors: Present and Future; Ligler, F. S., Taitt, C. A.

R., Eds.; Elsevier: New York, 2002; 173-205.

(60) Richter, M. M. Chem. Rev. 2004, 104, 3003-3036.

(61) Tortorello, M. L.; Stewart, D. In New Techniques in the Analysis of Foods; Tunick,

M., Palumbo, S. A., Fratamico, P. M., Eds.; Kluwer Academic/Plenum Publishers:

New York, 1998; 91-102.

(62) Zaleski, J. M.; Turro, C.; Mussell, R. D.; Nocera, D. G. Coord. Chem. Rev. 1994,

132, 249-258.

(63) Zhou, M.; Heinze, J.; Borgwarth, K.; Grover, C. P. ChemPhysChem 2003, 4, 1241-

1243.

(64) Miao, W. In Handbook of Electrochemistry; Zoski, C. G., Ed.; Elsevier: HR

Amsterdam, 2007; pp541-590.

(65) Agbaria, R. A.; Oldham, P. B.; McCarroll, M.; McGown, L. B.; Warner, I. M. Anal.

Chem. 2002, 74, 3952-3962.

(66) Oldham, P. B.; McCarroll, M. E.; McGown, L. B.; Warner, I. M. Anal. Chem. 2000,

72, 197-209.

43

(67) Powe, A. M.; Fletcher, K. A.; St, L. N. N.; Lowry, M.; Neal, S.; McCarroll, M. E.;

Oldham, P. B.; McGown, L. B.; Warner, I. M. Anal. Chem. 2004, 76, 4614-4634.

(68) Aitken, R. J.; Baker, M. A.; O'Bryan, M. J. Androl. 2004, 25, 455-465.

(69) Arnhold, J. 2001; Schweda-Werbedruck GmbH; 85-94.

(70) Butkovskaya, N. I.; Setser, D. W. Int. Rev. Phys. Chem. 2003, 22, 1-72.

(71) Creton, R.; Jaffee, L. F. BioTechniques 2001, 31, 1098-1105.

(72) Gaffney, J. S.; Marley, N. A. 2002; American Meteorological Society; 1-6.

(73) Garcia-Campana, A. M.; Baeyens, W. R. G.; Cuadros-Rodriguez, L.; Barrero, F. A.;

Bosque-Sendra, J. M.; Gamiz-Gracia, L. Curr. Org. Chem. 2002, 6, 1-20.

(74) Gubitz, G.; Schmid, M. G.; Silviaeh, H.; Aboul-Enein, H. Y. Crit. Rev. Anal. Chem.

2001, 31, 167-174.

(75) Jacobson, K.; Eriksson, P.; Reitberger, T.; Stenberg, B. Adv. Polym. Sci. 2004, 169,

151-176.

(76) Kuyper, C.; Milofsky, R. TrAC, Trends Anal. Chem. 2001, 20, 232-240.

(77) Li, F.; Zhang, C.; Guo, X.; Feng, W. Biomed. Chromatogr. 2003, 17, 96-105.

(78) Liu, Y.-M.; Cheng, J.-K. J. Chromatogr., A 2002, 959, 1-13.

(79) Qin, W. Anal. Lett. 2002, 35, 2207-2220.

(80) Roda, A.; Guardigli, M.; Pasini, P.; Mirasoli, M. Anal. Bioanal. Chem. 2003, 377,

826-833.

(81) Roda, A.; Guardigli, M.; Michelini, E.; Mirasoli, M.; Pasini, P. Anal. Chem. 2003,

75, 462A-470A.

(82) Rychla, L.; Rychly, J. 2000; Nova Science Publishers, Inc.; pp123-134.

(83) Shah, D. O.; Chang, C. D.; Stewart, J. L. BIOforum Eur. 2003, 7, 44-45.

(84) Sherma, J. J. AOAC Int. 2004, 87, 20A-26A.

(85) Stott, R. A. W. In Protein Protocols Handbook (2nd Ed.); Humana Press Inc.:

Totowa, NJ, 2002, 1089-1096, Part 156.

(86) Yamaguchi, M.; Yoshida, H.; Nohta, H. J. Chromatogr., A 2002, 950, 1-19.

(87) Bard, A. J. D., J.D.; Leland, J.K.; Sigal, G.B.; Wilbur, J.L.; Wohlsatdter, J.N.

Encyclopedia of Analytical Chemistry: Applications, Theory and Instrumentation;

John Wiley & Sons: New York, 2000.

(88) Bezman, R.; Faulkner, L. R. J. Amer. Chem. Soc. 1972, 94, 6324-6330.

44

(89) Faulkner, L. R.; Tachikawa, H.; Bard, A. J. J. Amer. Chem. Soc. 1972, 94, 691-699.

(90) Faulkner, L. R.; Bard, A. J. J. Amer. Chem. Soc. 1969, 91, 209-210.

(91) Tachikawa, H.; Bard, A. J. Chem. Phys. Lett. 1974, 26, 568-573.

(92) Periasamy, N.; Santhanam, K. S. V. Proc. Indian Acad. Sci., Sect. A 1974, 80, 194-

206.

(93) Pighin, A.; Conway, B. E. J. Electrochem. Soc. 1975, 122, 1643-1644.

(94) Miao, W.; Choi, J.-P. In Electrogenerated Chemiluminescence; Bard, A. J., Ed.;

Marcel Dekker, Inc., 2004; 213-271.

(95) Rubinstein, I.; Bard, A. J. J. Am. Chem. Soc. 1981, 103, 512-516.

(96) White, H. S.; Bard, A. J. J. Am. Chem. Soc. 1982, 104, 6891-6895.

(97) Chang, M.-M.; Saji, T.; Bard, A. J. J. Am. Chem. Soc. 1977, 99, 5399-5403.

(98) Memming, R. J. Electrochem. Soc. 1969, 116, 785-790.

(99) Debad, J. D.; Glezer, E. N.; Wohlstadter, J.; Sigal, G. B.; Leland, J. K. In

Electrogenerated Chemiluminescence; Bard, A. J., Ed.; Marcel Dekker, Inc.: New

York, 2004; 359-396.

(100) Miao, W.; Choi, J.-P.; Bard, A. J. J. Am. Chem. Soc. 2002, 124, 14478-14485.

(101) Gross, E. M.; Pastore, P.; Wightman, R. M. J. Phys. Chem. B 2001, 105, 8732-

8738.

(102) He, L.; Cox, K. A.; Danielson, N. D. Anal. Lett. 1990, 23, 195-210.

(103) Kanoufi, F.; Zu, Y.; Bard, A. J. J. Phys. Chem. B 2001, 105, 210-216.

(104) Leland, J. K.; Powell, M. J. J. Electrochem. Soc. 1990, 137, 3127-3131.

(105) Noffsinger, J. B.; Danielson, N. D. Anal. Chem. 1987, 59, 865-868.

(106) Zu, Y.; Bard, A. J. Anal. Chem. 2000, 72, 3223-3232.

(107) Bolletta, F.; Ciano, M.; Balzani, V.; Serpone, N. Inorg. Chim. Acta 1982, 62, 207-

213.

(108) Armitt, D.; Zimmermann, P.; Ellis-Steinborner, S. Rapid Commun. Mass

Spectrom. 2008, 22, 950-958.

(109) Davies, A. G.; Burnett, A. D.; Fan, W.; Linfield, E. H.; Cunningham, J. E. Mater.

Today 2008, 11, 18-26.

(110) Jacoby, M. C&EN 2009, 87, 10-13.

45

(111) Marshall, M.; Oxley, J. C., Eds. Aspects of Explosive Detection; Elsevier B. V.:

Amsterdam, 2009.

(112) Yinon, J., Ed. Counterterrorist Detection Techniques of Explosives; Elsvier, B. V.:

Amsterdam, 2007.

(113) Yinon, J. American Laboratory 2006, 38, 18, 20-23.

(114) Smith, R. C.; Connelly, J. M. In Aspects of Explosives Detection; Marshall, M.,

Oxley, J. C., Eds.; Elsevier B. V.: Amsterdam, 2009; 7, pp 131-146.

(115) Cotte-Rodriguez, I.; Hernandez-Soto, H.; Chen, H.; Cooks, R. G. Anal. Chem.

2008, 80, 1512-1519.

(116) Crowson, A.; Beardah, M. S. Analyst 2001, 126, 1689-1693.

(117) Kojima, K.; Sakairi, M.; Takada, Y.; Nakamura, J. J. Mass Spectrom. Soc. Japan

2000, 48, 360-362.

(118) McLuckey, S. A.; Goeringer, D. E.; Asano, K. G.; Vaidyanathan, G.; Stephenson,

J. L., Jr. Rapid Commun. Mass Spectrom. 1996, 10, 287-298.

(119) Pacheco-Londono, L.; Primera, O. M.; Ramirez, M.; Ruiz, O.; Hernandez-Rivera,

S. Proc. SPIE-Internl. Soc. Opt. Engineer. 2005, 5778, 317-326.

(120) Stambouli, A.; El Bouri, A.; Bouayoun, T.; Bellimam, M. A. Forensic Sci. Int.

2004, 146, S191-S194.

(121) Tamiri, T.; Zitrin, S. J. Energ. Mater. 1986, 4, 215-237.

(122) Widmer, L.; Watson, S.; Schlatter, K.; Crowson, A. Analyst 2002, 127, 1627-1632.

(123) Xu, X.; van de Craats, A. M.; Kok, E. M.; de Bruyn, P. C. A. M. J. Forensic Sci.

2004, 49, 1230-1236.

(124) Yinon, J.; Boettger, H. G.; Weber, W. P. Anal. Chem. 1972, 44, 2235-2237.

(125) Zhao, X.; Yinon, J. J Chromatogr A 2002, 977, 59-68.

(126) Yinon, J. In Counterterrorist Detection Techniques of Explosives; Yinon, J., Ed.;

Elsevier B. V.: Amsterdam, 2007; pp41-60.

(127) Buttigieg, G. A.; Knight, A. K.; Denson, S.; Pommier, C.; Bonner Denton, M.

Forensic Sci. Int. 2003, 135, 53-59.

(128) Lokhnauth, J. K.; Snow, N. H. J. Chromatogr. A 2006, 1105, 33-38.

(129) Marr, A. J.; Groves, D. M. Int. J. Ion Mobility Spectrom. 2003, 6, 59-62.

(130) Matz, L. M.; Tornatore, P. S.; Hill, H. H. Talanta 2001, 54, 171-179.

46

(131) Oxley, J. C.; Smith, J. L.; Kirschenbaum, L. J.; Marimganti, S.; Vadlamannati, S.

J. Forensic Sci. 2008, 53, 690-693.

(132) Wilson, R.; Brittain, A. Special Publication - Royal Soc. Chem. 1997, 203, 92-101.

(133) Eiceman, G. A.; Schmidt, H. In Aspects of Explosives Detection; Marshall, M.,

Oxley, J. C., Eds.; Elsevier B. V.: Amsterdam, 2009; pp171-202.

(134) Datskos, P. G.; Lavrik, N. V.; Sepaniak, M. J. Sensor Lett. 2003, 1, 25-32.

(135) Senesac, L.; Thundat, T. In Counterterrorist Detection Techniques of Explosives;

Yinon, J., Ed.; Elsevier B. V.: Amsterdam, 2007; pp 109-130.

(136) Pinnaduwage, L. A.; Boiadjiev, V.; Hawk, J. E.; Thundat, T. Appl. Phys. Lett.

2003, 83, 1471-1473.

(137) Pinnaduwage, L. A.; Wig, A.; Hedden, D. L.; Gehl, A.; Yi, D.; Thundat, T.;

Lareau, R. T. J. Appl. Phys. 2004, 95, 5871-5875.

(138) Senesac, L.; Thundat, T. G. Mater. Today 2008, 11, 28-36.

(139) Fu, X.; Benson, R. F.; Wang, J.; Fries, D. Sens. Actuators, B 2005, B106, 296-301.

(140) Lu, D.; Cagan, A.; Munoz, R. A. A.; Tangkuaram, T.; Wang, J. Analyst 2006, 131,

1279-1281.

(141) Munoz, R. A. A.; Lu, D.; Cagan, A.; Wang, J. Analyst 2007, 132, 560-565.

(142) Wang, J. NATO Science Series, II: Mathematics, Physics and Chemistry 2004,

159, 131-142.

(143) Wang, J.; Bhada, R. K.; Lu, J.; MacDonald, D. Anal. Chim. Acta 1998, 361, 85-91.

(144) Wang, J. In Counterterrorist Detection Techniques of Explosives; Yinon, J., Ed.;

Elsevier B. V.: Amsterdam, 2007; pp 91-108.

(145) Bruno, J. G.; Cornette, J. C. Microchem. J. 1997, 56, 305-314.

(146) Jimenez, A. M.; Navas, M. J. J. Hazardous Mater. 2004, 106, 1-5.

(147) Meaney, M. S.; McGuffin, V. L. Anal. Bioanal. Chem. 2008, 391, 2557-2576.

(148) Monterola, M. P. P.; Smith, B. W.; Omenetto, N.; Winefordner, J. D. Anal.

Bioanal. Chem. 2008, 391, 2617-2626.

(149) Nguyen, D. H.; Locquiao, S.; Huynh, P.; Zhong, Q.; He, W.; Christensen, D.;

Zhang, L.; Bilkhu, B. NATO Science Series, II: Mathematics, Physics and

Chemistry 2004, 159, 71-80.

47

(150) Jimenez, A. M.; Navas, M. J. In Counterterrorist Detection Techniques of

Explosives; Yinon, J., Ed.; Elsevier B. V.: Amsterdam, 2007; pp 1-40.

(151) Pamula, V. K. NATO Science Series, II: Mathematics, Physics and Chemistry

2004, 159, 279-288.

(152) Almog, J.; Zitrin, S. In Aspects of Explosives Detection; Marshall, M., Oxley, J.

C., Eds.; Elsevier B. V.: Amsterdam, 2009; pp51-59.

(153) Charles, P. T.; Bart, J. C.; Judd, L. L.; Gauger, P. R.; Ligler, F. S.; Kusterbeck, A.

W. Proc. SPIE-Internl Soc. Opt. Eng. 1997, 3105, 80-87.

(154) Germain, M. E.; Knapp, M. J. Inorg. Chem. 2008, 47, 9748-9750.

(155) Goodpaster, J. V.; McGuffin, V. L. Anal. Chem. 2001, 73, 2004-2011.

(156) la Grone, M. J.; Cumming, C. J.; Fisher, M. E.; Fox, M. J.; Jacob, S.; Reust, D.;

Rockley, M. G.; Towers, E. Proc. SPIE- Internl. Soc. Opt. Engineer. 2000, 4038,

553-562.

(157) Malashikhin, S.; Finney, N. S. J. Am. Chem. Soc. 2008, 130, 12846-12847.

(158) Naddo, T.; Che, Y.; Zhang, W.; Balakrishnan, K.; Yang, X.; Yen, M.; Zhao, J.;

Moore, J. S.; Zang, L. J. Am. Chem. Soc. 2007, 129, 6978-6979.

(159) Tao, S.; Li, G.; Yin, J. J. Mater. Chem. 2007, 17, 2730-2736.

(160) Thomas III, S. W.; Swager, T. M. In Aspects of Explosives Detection; Marshall,

M., Oxley, J. C., Eds.; Elsevier B. V.: Amsterdam, 2009; pp203-222.

(161) Sarkar, D.; Somasundaran, P. Langmuir 2002, 18, 8271-8277.

(162) Larsson, A.; Angbrant, J.; Ekeroth, J.; Mansson, P.; Liedberg, B. Sensors and

Actuators, B: Chemical 2006, B113, 730-748.

(163) Miura, N.; Shankaran, D. R.; Kawaguchi, T.; Matsumoto, K.; Toko, K.

Electrochem. (Tokyo, Japan) 2007, 75, 13-22.

(164) Shankaran, D. R.; Kawaguchi, T.; Kim, S. J.; Matsumoto, K.; Toko, K.; Miura, N.

Int. J. Environ. Anal. Chem. 2007, 87, 771-781.

(165) Shankaran, D. R.; Matsumoto, K.; Toko, K.; Miura, N. Chem. Sensors 2005, 21,

166-168.

(166) Baker, G. A.; Moore, D. S. Anal. Bioanal. Chem. 2005, 382, 1751-1770.

(167) Smith, W. E. Chem. Soc. Rev. 2008, 37, 955-964.

48

(168) De La Cruz-Montoya, E.; Jerez, J. I.; Balaguera-Gelves, M.; Luna-Pineda, T.;

Castro, M. E.; Hernandez-Rivera, S. P., Orlando (Kissimmee), FL, USA 2006;

SPIE; 62030X-62037.

(169) Ruffin, P. B.; Brantley, C.; Edwards, E., San Diego, California, USA 2008; SPIE;

693102-693112.

(170) De La Cruz-Montoya, E.; Jerez, J. I.; Balaguera-Gelves, M.; Luna-Pineda, T.;

Castro, M. E.; Hernandez-Rivera, S. P. Proc. SPIE-Internl. Soc.Opt. Engineer.

2006, 6203, 62030X/62031-62030X/62037.

(171) Ko, H.; Chang, S.; Tsukruk, V. V. ACS Nano 2009, 3, 181-188.

(172) Everts, S. CE&N 2008, 86, 34.

(173) Wang, J.; Chen, G.; Chatrathi, M. P.; Fujishima, A.; Tryk, D. A.; Shin, D. Anal.

Chem. 2003, 75, 935-939.

(174) Wang, J.; Thongngamdee, S. Anal. Chim. Acta 2003, 485, 139-144.

(175) Krausa, M. NATO Science Series, II: Mathematics, Physics and Chemistry 2004,

167, 1-9.

(176) Krausa, M.; Schorb, K. J. Electroanal. Chem. 1999, 461, 10-13.

(177) Filanovsky, B.; Tur'yan, Y. I.; Kuselman, I.; Burenko, T. y.; Shenhar, A. Anal.

Chim. Acta 1998, 364, 181-188.

(178) Kim, J.-W.; Kim, J-H.; Tung, S. Proc. 3rd IEEE Int. Conf. on Nano/Micro

Engineered and Molecular Systems, Sanya, China, 2008; 630-632.

(179) Wang, J.; Thongngamdee, S.; Kumar, A. Electroanalysis 2004, 16, 1232-1235.

(180) Hilmi, A.; Luong, J. H. T.; Nguyen, A.-L. Anal. Chem. 1999, 71, 873-878.

(181) Weng, J.; Xue, J.; Wang, J.; Ye, J.-S.; Cui, H.; Sheu, F.-S.; Zhang, Q. Adv. Funct.

Mater. 2005, 15, 639-647.

(182) Pardo-Yissar, V.; Gabai, R.; Shipway, A. N.; Bourenko, T.; Willner, I. Adv. Mater.

2001, 13, 1320-1323.

(183) Filanovsky, B.; Markovsky, B.; Bourenko, T.; Perkas, N.; Persky, R.; Gedanken,

A.; Aurbach, D. Adv. Funct. Mater. 2007, 17, 1487-1492.

(184) Zhang, H.-X.; Cao, A.-M.; Hu, J.-S.; Wan, L.-J.; Lee, S.-T. Anal. Chem. 2006, 78,

1967-1971.

(185) Wang, J.; Hocevar, S. B.; Ogorevc, B. Electrochem. Commun. 2004, 6, 176-179.

49

(186) Hrapovic, S.; Majid, E.; Liu, Y.; Male, K.; Luong, J. H. T. Anal. Chem. 2006, 78,

5504-5512.

(187) Schulte-Ladbeck, R.; Kolla, P.; Karst, U. Anal. Chem. 2003, 75, 731-735.

(188) Schulte-Ladbeck, R.; Karst, U. Chromatographia 2003, 57, S/61-S/66.

(189) Borisova, A. V.; Karyakina, E. E.; Cosnier, S.; Karyakin, A. A. Electroanalysis

2009, 21, 409-414.

(190) Karyakin, A. A. Electroanalysis 2001, 13, 813-819.

(191) Zhang, X.; Wang, J.; Ogorevc, B.; Spichiger, U. E. Electroanalysis 1999, 11, 945-

949.

(192) Karyakin, A. A.; Karyakina, E. E.; Gorton, L. Anal. Chem. 2000, 72, 1720-1723.

(193) Lukachova, L. V.; Kotel'nikova, E. A.; D'Ottavi, D.; Shkerin, E. A.; Karyakina, E.

E.; Moscone, D.; Palleschi, G.; Curulli, A.; Karyakin, A. A. Bioelectrochemistry

2002, 55, 145-148.

(194) Schulte-Ladbeck, R.; Kolla, P.; Karst, U. Analyst 2002, 127, 1152-1154.

(195) Thomas, S. W.; Joly, G. D.; Swager, T. M. Chem. Rev. 2007, 107, 1339-1386.

(196) Levitsky, I. A.; Euler, W. B.; Tokranova, N.; Rose, A. Appl. Phys. Lett. 2007, 90,

041904-041903.

(197) Cumming, C. J.; Aker, C.; Fisher, M.; Fok, M.; la Grone, M. J.; Reust, D.;

Rockley, M. G.; Swager, T. M.; Towers, E.; Williams, V. IEEE Tran. Geosci.

Remote Sens., 2001, 39, 1119-1128.

(198) Rose, A.; Zhu, Z.; Madigan, C. F.; Swager, T. M.; Bulovic, V. Nature 2005, 434,

876-879.

(199) Zahn, S.; Swager Timothy, M. Angew. Chem. 2002, 41, 4225-4230.

(200) Yang, J.-S.; Swager, T. M. J. Am. Chem. Soc. 1998, 120, 11864-11873.

(201) Yamaguchi, S.; Swager, T. M. J. Am. Chem. Soc. 2001, 123, 12087-12088.

(202) Narayanan, A.; Varnavski, O. P.; Swager, T. M.; Goodson, T., III J. Phys. Chem.

C 2008, 112, 881-884.

(203) Sohn, H.; Calhoun, R. M.; Sailor, M. J.; Trogler, W. C. Angew. Chem. Int. Ed.

2001, 40, 2104-2105.

(204) Sohn, H.; Sailor, M. J.; Magde, D.; Trogler, W. C. J. Am. Chem. Soc. 2003, 125,

3821-3830.

50

(205) Narayanan, A.; Varnavski, O. P.; Swager, T. M.; Goodson, T., III PMSE

Preprints 2007, 96, 678.

(206) Li, J.; Kendig, C. E.; Nesterov, E. E. J. Am. Chem. Soc. 2007, 129, 15911-15918.

(207) Toal, S. J.; Magde, D.; Trogler, W. C. Chem. Commun. 2005, 5465-5467.

(208) Content, S.; Trogler, W. C.; Sailor, M. J. Chem.-A Euro. J. 2000, 6, 2205-2213.

(209) Albert, K. J.; Walt, D. R. Anal. Chem. 2000, 72, 1947-1955.

(210) Chan, S.; Horner, S. R.; Miller, B. L.; Fauchet, P. M. Materials Research Society

Symposium Proceedings 2001, 638, F10 14 11-F10 14 16.

(211) Chvojka, T.; Holec, T.; Jelinek, I.; Nemec, I.; Jindrich, J.; Lorenc, M.;

Koutnikova, J.; Kral, V.; Dian, J. Proc. SPIE-Internl Soc. Opt. Eng. 2003, 5036,

51-56.

(212) De Stefano, L.; Alfieri, D.; Rea, I.; Rotiroti, L.; Malecki, K.; Moretti, L.; Della

Corte, F. G.; Rendina, I. Phys. Status Solidi A 2007, 204, 1459-1463.

(213) De Stefano, L.; Moretti, L.; Rendina, I.; Rotiroti, L. Sens. Actuators, B 2005,

B111-B112, 522-525.

(214) De Stefano, L.; Rendina, I.; Moretti, L.; Rossi, A. M.; Lamberti, A.; Longo, O.;

Arcari, P. Proc. SPIE-Internl Soc. Opt. Eng. 2003, 5118, 305-309.

(215) De Stefano, L.; Rendina, I.; Moretti, L.; Tundo, S.; Rossi, A. M. Appl. Opt. 2004,

43, 167-172.

(216) Furbert, P.; Lu, C.; Winograd, N.; DeLouise, L. Langmuir 2008, 24, 2908-2915.

(217) Mahmoudi, B.; Gabouze, N.; Guerbous, L.; Haddadi, M.; Cheraga, H.; Beldjilali,

K. Mater. Sci. Engineer. B: Solid-State Mater.Adv. Tech. 2007, 138, 293-297.

(218) Min, N. K.; Lee, C. W.; Jeong, W. S.; Kim, D. I. J. Korean Electrochem. Soc.

1999, 2, 17-22.

(219) Mulloni, V.; Pavesi, L. Appl. Phys. Lett. 2000, 76, 2523-2525.

(220) Rea, I.; Iodice, M.; Coppola, G.; Rendina, I.; Marino, A.; De Stefano, L. Sens.

Actuators, B 2009, B139, 39-43.

(221) Rotiroti, L.; De Stefano, L.; Rendina, I.; Moretti, L.; Rossi, A. M.; Piccolo, A.

Biosens. Bioelectron. 2005, 20, 2136-2139.

(222) Saarinen, J. J.; Weiss, S. M.; Fauchet, P. M.; Sipe, J. E. Opt. Express 2005, 13,

3754-3764.

51

(223) Zangooie, S.; Bjorklund, R.; Arwin, H. Sens. Actuators, B 1997, B43, 168-174.

(224) Coppens, M. O.; Sun, J.; Maschmeyer, T. Catal. Today 2001, 69, 331-335.

(225) Falcaro, P.; Malfatti, L.; Kidchob, T.; Giannini, G.; Falqui, A.; Casula, M. F.;

Amenitsch, H.; Marmiroli, B.; Grenci, G.; Innocenzi, P. Chem. Mater. 2009, 21,

2055-2061.

(226) Fujita, S.; Nakano, H.; Ishii, M.; Nakamura, H.; Inagaki, S. Microporous

Mesoporous Mater. 2006, 96, 205-209.

(227) Kuang, D.; Brezesinski, T.; Smarsly, B. J. Am. Chem. Soc. 2004, 126, 10534-

10535.

(228) Li, Y.; Cai, W.; Cao, B.; Duan, G.; Sun, F.; Li, C.; Jia, L. Nanotechnology 2006,

17, 238-243.

(229) Loiola, A. R.; da Silva, L. R. D.; Cubillas, P.; Anderson, M. W. J. Mater. Chem.

2008, 18, 4985-4993.

(230) Yacou, C.; Fontaine, M.-L.; Ayral, A.; Lacroix-Desmazes, P.; Albouy, P.-A.;

Julbe, A. J. Mater. Chem. 2008, 18, 4274-4279.

(231) Yamauchi, Y.; Gupta, P.; Fukata, N.; Sato, K. Chem. Lett. 2009, 38, 78-79.

(232) Tao, S.; Li, G. Colloid. Polym. Sci. 2007, 285, 721-728.

(233) Tao, S.; Shi, Z.; Li, G.; Li, P. ChemPhysChem 2006, 7, 1902-1905.

(234) Tao, S.; Yin, J.; Li, G. J. Mater. Chem. 2008, 18, 4872-4878.

(235) Content, S.; Trogler, W. C.; Sailor, M. J. Chem. Eur. J. 2000, 6, 2205-2213.

(236) Datar, A.; Balakrishnan, K.; Yang, X.; Zuo, X.; Huang, J.; Oitker, R.; Yen, M.;

Zhao, J.; Tiede, D. M.; Zang, L. J. Phys. Chem. B 2006, 110, 12327-12332.

(237) Yang, J.-S.; Swager, T. M. J. Am. Chem. Soc. 1998, 120, 5321-5322.

(238) Toal, S. J.; Trogler, W. C. J. Mater. Chem. 2006, 16, 2871-2883.

(239) Mattoussi, H.; Radzilowski, L. H.; Dabbousi, B. O.; Thomas, E. L.; Bawendi, M.

G.; Rubner, M. F. J. Appl. Phys. 1998, 83, 7965-7974.

(240) Goldman, E. R.; Balighian, E. D.; Kuno, M. K.; Labrenz, S.; Tran, P. T.;

Anderson, G. P.; Mauro, J. M.; Mattoussi, H. Physica Status Solidi B 2002, 229,

407-414.

(241) Medintz, I. L.; Uyeda, H. T.; Goldman, E. R.; Mattoussi, H. Nat. Mater. 2005, 4,

435-446.

52

(242) Han, M.; Gao, X.; Su, J. Z.; Nie, S. Nat. Biotechnol. 2001, 19, 631-635.

(243) Medintz, I. L.; Clapp, A. R.; Brunel, F. M.; Tiefenbrunn, T.; Tetsuo Uyeda, H.;

Chang, E. L.; Deschamps, J. R.; Dawson, P. E.; Mattoussi, H. Nat. Mater. 2006, 5,

581-589.

(244) Peng, H.; Zhang, L.; Kjallman, T. H. M.; Soeller, C. J. Am. Chem. Soc. 2007, 129,

3048-3049.

(245) Dayal, S.; Burda, C. J. Am. Chem. Soc. 2007, 129, 7977-7981.

(246) Tu, R.; Liu, B.; Wang, Z.; Gao, D.; Wang, F.; Fang, Q.; Zhang, Z. Anal. Chem.

2008, 80, 3458-3465.

(247) Goldman, E. R.; Medintz, I. L.; Whitley, J. L.; Hayhurst, A.; Clapp, A. R.; Uyeda,

H. T.; Deschamps, J. R.; Lassman, M. E.; Mattoussi, H. J. Am. Chem. Soc. 2005,

127, 6744-6751.

(248) Medintz, I. L.; Goldman, E. R.; Clapp, A. R.; Uyeda, H. T.; Lassman, M. E.;

Hayhurst, A.; Mattoussi, H. Proc. SPIE-Internl Soc. Opt. Eng. 2005, 5705, 166-

174.

(249) Medintz, I. L.; Anderson, G. P.; Lassman, M. E.; Goldman, E. R.; Bettencourt, L.

A.; Mauro, J. M. Anal. Chem. 2004, 76, 5620-5629.

(250) Medintz, I. L.; Goldman, E. R.; Lassman, M. E.; Hayhurst, A.; Kusterbeck, A. W.;

Deschamps, J. R. Anal. Chem. 2005, 77, 365-372.

(251) Wada, A.; Mie, M.; Aizawa, M.; Lahoud, P.; Cass, A. E. G.; Kobatake, E. J. Am.

Chem. Soc. 2003, 125, 16228-16234.

(252) Goldman, E. R.; Anderson, G. P.; Tran, P. T.; Mattoussi, H.; Charles, P. T.;

Mauro, J. M. Anal. Chem. 2002, 74, 841-847.

(253) Mattoussi, H.; Kenneth, K.; Goldman, E. R.; Anderson, G. P.; Mauro, J. M. In

Optical Biosensors: Present and Future, 1ed.; Ligler, F. S., Taitt, C. R., Eds.;

Elseveir B. V.: Amsterdam, 2002; pp537-569.

(254) Kusterbeck, A. W.; Blake, D. A. In Optical Biosensors: Today and Tomorrow, 2

ed.; Ligler, F. S., Taitt, C. R., Eds.; Elsevier B. V.: Amsterdam, 2008; pp243-286.

(255) Ghindilis, A. L.; Atanasov, P.; Wilkins, M.; Wilkins, E. Biosens. Bioelectron.

1998, 13, 113-131.

(256) Hage, D. S.; Nelson, M. A. Anal. Chem. 2001, 73, 198 A-205 A.

53

(257) Kusterbeck, A. W.; Wemhoff, G. A.; Charles, P. T.; Yeager, D. A.; Bredehorst, R.;

Vogel, C. W.; Ligler, F. S. J. Immunol. Methods 1990, 135, 191-197.

(258) Whelan, J. P.; Kusterbeck, A. W.; Wemhoff, G. A.; Bredehorst, R.; Ligler, F. S.

Anal. Chem. 1993, 65, 3561-3565.

(259) Bart, J. C.; Judd, L. L.; Hoffman, K. E.; Wilkins, A. M.; Kusterbeck, A. W.

Environ. Sci. Technol. 1997, 31, 1505-1511.

(260) Gardner, J. W. NATO Science Series, II: Math., Phys. Chem. 2004, 159, 1-28.

(261) Pardo, M.; Benussi, G. P.; Niederjaufner, G.; Faglia, G.; Sberveglieri, G.

Electronic Noses and Olfaction 2000, Proc. Internl. Symp.Olfaction Electronic

Noses, 7th, Brighton, United Kingdom, July, 2000, pp 67-74.

(262) Gui, Y.; Xie, C.; Xu, J.; Wang, G. J. Hazard. Mater. 2009, 164, 1030-1035.

(263) Jerez Rozo, J. I.; Chamoun, A. M.; Pena, S. L.; Hernandez-Rivera, S. P. Proc.

SPIE-Internl Soc. Opt. Eng. 2007, 6538, 653824/653821-653824/653812.

(264) Kneipp, K.; Wang, Y.; Kneipp, H.; Dasari, R. R.; Feld, M. S. Exptl. Tech. Phys.

1995, 41, 225-234.

(265) Haas, J. W., III; Sylvia, J. M.; Spencer, K. M.; Johnston, T. M.; Clauson, S. L.

Proc. SPIE-Internl Soc. Opt. Eng. 1998, 3392, 469-476.

(266) Spencer, K. M.; Sylvia, J. M.; Janni, J. A.; Klein, J. D. Proc. SPIE-Internl Soc.

Opt. Eng. 1999, 3710, 373-379.

(267) Sylvia, J. M.; Janni, J. A.; Klein, J. D.; Spencer, K. M. Anal. Chem. 2000, 72,

5834-5840.

(268) Bertone, J. F.; Cordeiro, K. L.; Sylvia, J. M.; Spencer, K. M. Proc. SPIE-Internl

Soc. Opt. Eng. 2004, 5403, 387-394.

(269) McHugh, C. J.; Keir, R.; Graham, D.; Smith, W. E. Chem. Commun. 2002, 580-

581.

(270) McHugh, C. J.; Smith, W. E.; Lacey, R.; Graham, D. Chem. Commun. 2002,

2514-2515.

54

CHAPTER II

SENSITVE DETERMINATION OF HEXAMETHYLENE TRIPEROXIDE DIAMINE

(HMTD) EXPLOSIVES USING ELECTROGENRATED CHEMILUMINESCENCE

ENHANCED BY SILVER NITRATE*

Introduction

The legal authorities have witnessed increased number of threats of illegal use of

peroxide-based explosive materials by the terrorists. These compounds are very popular

among the terrorists because they can be synthesized readily from the commercially

available chemicals.1 These explosives, also known as “unconventional explosives” since

they have no use for military purposes because of their high instability and powerful

initiating explosive capability, are basically linked to the terrorists.2 There is urgent need

of detection of these “home-made” explosives especially in the checkpoints of the mass-

transit facilities and other government and public facilities.3-6

Hexamethylene triperoxide diamine (HMTD) is a representative of the

abovementioned peroxide-based explosives. It is a white solid with cyclic structure7

(Scheme 2.1), first synthesized by Legler in 18858 that is sensitive to friction, impact and

electrical discharge.9 The friction sensitivity of HMTD is comparable to other well-

known explosives like triacetone triperoxide (TATP) and trinitrotoluene (TNT) although

its impact sensitivity is about a half of TATP.10-12

A number of analytical techniques have been used to detect peroxide explosives

including HMTD. These techniques include separation-based gas chromatography

* Part of the results presented in this chapter have been published: Parajuli, S.; Miao, W. Anal. Chem. 2009,

81, 5267-5272.

55

coupled with mass spectrometry (GC/MS),13-15 liquid chromatography (LC)/MS,9 time-

of-flight (TOF)/MS,16 desorption electrospray ionization (DESI)/MS,6,17 LC/FT-IR,10 and

UV-Visible spectrometric based methods.1 MS-based methods are very sensitive and

could detect peroxide explosives in nanogram to picogram levels but the instruments are

generally very expensive and not portable for field tests. Chromatographic, UV-Visible

and IR detection techniques are often time-consuming in sample preparation and

insufficiently sensitive for trace amounts of explosive quantification.

Electrogenerated chemiluminescence (ECL), a process of light production as a

result of electrochemical reactions at an electrode, has proved to be a powerful analytical

technique due to its inherent features such as high sensitivity, good selectivity, low

background, integrated versatility and fast sample analysis.18-21 ECL has been widely

used for many kinds of targets detection and quantification under a broad variety of areas,

which include DNA probe, immunoassay, pharmaceutical study, food and water testing,

and biowarfare agent detection.18 Very recently, our research group reported an

ultrasensitive detection of TNT, which was accomplished on the basis of sandwich-type

TNT immunoassay combined with ECL technology.22 The limit of detection (≤ 0.10 ±

0.01 ppb) is about 600 times lower as compared with the most sensitive TNT detection

method based on surface plasmon resonance23 in the literature, and the absolute detection

limit in mass (~0.1 pg) is only ~0.5% of that from mass spectroscopy.24

In this chapter, an ECL detection and quantification method for HMTD using

AgNO3 as an ECL enhancing agent will be reported. This method is based on the fact that

HMTD contains tertiary amine moieties with α-C hydrogens (Scheme 2.1), which could

act as an ECL coreactant like tri-n-propylamine (TPrA) in the presence of an ECL

56

luminophore such as Ru(bpy)32+ species up on anodic potential scanning.25 Remarkable

enhancement by AgNO3 for HMTD ECL generation and relevant electrochemical and

ECL mechanisms will be described. The ECL enhancement strategy used in this chapter

could be extended to other systems where the electrochemical oxidations of the analyte

(coreactant) or the luminophore at an electrode are suppressed due to the nature of the

working electrode26 or “delayed” due to the slow heterogonous electron-transfer rate of

the compound.

Experimental Section

Chemicals and Materials

Hexamethylenetetramine (99+%) from Alfa Aesar (Ward Hill, MA), silver nitrate

(99.5%) and tetra-n-butylammonium perchlorate (TBAP, 99+%, electrochemical grade)

from Fluka (Milwaukee, WI), hydrogen peroxide (30%), anhydrous citric acid (>99.5%,

ACS reagent), sulfuric acid (95-98%, ACS reagent), tris(2,2′-bipyridyl)-dichloro-

ruthenium(II) hexahydrate (99.95%), silver benzoate (99%), silver tetrafluoroborate

(98%), and acetonitrile (MeCN, 99.93+%, HPLC grade) from Sigma-Aldrich (Milwaukee,

WI) were used as received.

Synthesis and Characterization of HMTD

As outlined in Scheme 2.1, the explosive compound, HMTD, had to be synthesized

because it is not commercially available. We followed the standard procedure from the

literature for its synthesis.27 7.0 g (50 mmole) of hexamethylenetetramine was dissolved in

20.5 mL of 30% hydrogen peroxide stirring mechanically in an ice bath (0 ºC). To this 10.5 g

of anhydrous citric acid was slowly dissolved with constant stirring.27 The mixture was

stirred for 3 h and warmed at room temperature for 2 h. White crystals were washed with

57

deionized water (7 times), rinsed with methanol (3 times), air dried, and stored in a

refrigerator at 4 ºC prior to use.

a) Dissolved in 30% H2O2 in ice bath

Hexamethylenetetramine

N N

N

N

Hexamethylene triperoxide diamine(HMTD)

N

O

O

O

O

O

O

N

b) Added anhydrous citric acid with stirring

Scheme 2.1. Synthesis of hexamethylene triperoxide diamine (HMTD).

HMTD was characterized by diamond crystal attenuated total reflection Fourier

transform infrared (ATR-FTIR, Nicolet Nexus 470 FTIR spectrometer, Thermo Electron

Corp., Madison, WI) and 1H NMR spectroscopy (Bruker 300 MHz NMR spectrometer).

FTIR spectra (Figure 2.1a) shows a characteristic C-O stretch band at 1225 cm-1 which

along with all other peaks, is consistent with the literature values.28

Figure 2.1. (a) ATR-FTIR and (b) 1H NMR Spectra of HMTD.

The 1H NMR spectra shown in Figure 2.1b reveals a singlet peak at 4.82 ppm as

expected because all protons in the compound are chemically equivalent. This chemical

shift is close to 4.60 ppm estimated theoretically using ChemBioDraw Ultra 11

1400 1200 1000 800

0.0

0.1

0.2

0.3

Abs

orb

ance

Wave number, cm-1

C-O Stretch, 1225 cm-1(a)

8 6 4 2 0

N

O

O

O

O

O

O

N

ppm

4.82 ppm

TMS

CH2

H2OCHCl

3

(b)

58

(ChembridgeSoft Corp., Cambridge, MA). Note that 1H NMR spectra alone should not be

used to verify the synthesis of HMTD, because similar 1H NMR spectra can be also

obtained from the reaction precursor hexamethylenetetramine. Small 1H NMR peaks at

1.60 ppm and 7.26 ppm are ascribed to trace amounts of water contained in the solvent

and the incomplete deuteration of the solvent (CDCl3) used.29 [CAUTION: HMTD is very

sensitive to explosion when present as a dry solid. It should be handled carefully with

appropriate precautions and should not be kept in a large quantity. For long-term

storage, HMTD should be dissolved or kept wet.]

Electrochemical and ECL Studies

Cyclic voltammetry (CV) was carried out with a model 660A electrochemical

workstation (CH Instruments, Austin, TX). The ECL signals along with the CV responses

were measured simultaneously with a homemade ECL instrument as described

previously.19,30 This instrument combined the 660A electrochemical workstation with a

photomultiplier tube (Hamamatsu R928, Japan, biased at -700 V DC) installed under a

conventional three-electrode cell, in which a Pt wire was used as the counter electrode, a

Ag/Ag+ (10 mM AgNO3 and 0.10 M TBAP in MeCN) as the reference electrode (~0.186

V vs NHE), and either a glassy carbon (GC, 3-mm diameter), a Pt (2-mm diameter), or a

gold (Au, 2-mm diameter) disk as the working electrode. The working electrode was

polished with 0.3-0.05-m alumina slurry, thoroughly rinsed with water, and dried with

the Kim wipes facial tissue and then an air blower before each experiment. The internal

electrolyte solution and the Vycor tip of the reference electrode were changed

periodically to eliminate possible contaminations of other species within the cell. No

59

degassing was needed because all electrochemical scans were conducted in the positive

potential region.

Results and Discussion

Cyclic voltammetric (CV) and ECL studies of HMTD

The CV and ECL responses of a MeCN solution consisting of 1.0 mM HMTD,

0.70 mM Ru(bpy)3Cl2, and 0.10 M TBAP electrolyte are shown in Figure 2.2.

2.0 1.5 1.0 0.5 0.0

-30

-20

-10

0

10

20

30

40

E (V vs Ag/Ag+)

i CV (A

)

ECL

CV

-12

-8

-4

0

4

8

12

i EC

L (nA

)

Figure 2.2. CV (black line) and ECL (blue line) responses obtained from 1.0 mM HMTD- 0.70 mM Ru(bpy)3Cl2-0.10 M TBAP in MeCN at a 2-mm diameter Pt electrode with a scan rate of 50 mV/s.

In the anodic potential scanning range of 0 to 2.0 V vs Ag/Ag+, Ru(bpy)32+ oxidizes

reversibly to form Ru(bpy)33+ at a Pt electrode with a half-wave potential of 0.96 V vs

Ag/Ag+, whereas HMTD displays an irreversible oxidation at ~1.7 V as shown in Figure

2.3. Because two identical tertiary amine moieties exist in HMTD (Scheme 2.1), an

overall two-electron transfer oxidation process is expected, which is consistent with the

60

oxidation current ratio of HMTD to Ru(bpy)32+. ECL response of the Ru(bpy)3

2+/HMTD

system, which appears at ~1.45 V and reaches the maximum at ~1.76 V, is coincident

with the oxidation potential of HMTD. Scheme 2.2 summarizes the proposed ECL

mechanism of this system, in which the ECL contribution associated with HMTD

dication species, [•HMTD•]2+ (Eq. 2.2), to the overall ECL emissions (Eq. 2.3) could be

relatively small, because the oxidation of HMTD•+ to [•HMTD•]2+ is most likely the rate-

determining step after the one-electron electro-oxidation of HMTD (Eq. 2.1).

2.0 1.5 1.0 0.5 0.0

25 A

(a)

(c)

E (V vs Ag/Ag+)

(b)

Figure 2.3. CVs of (a) 1.0 mM HMTD, (b) 0.7 mM Ru(bpy)3Cl2, and (c) 0.10 M TBAP MeCN blank at a 2-mm diameter Pt with a scan rate of 50 mV/s.

The ECL intensity of the Ru(bpy)32+/HMTD system depends on the added

Ru(bpy)32+ concentration as well as the nature of the working electrode. As revealed in

Figure 2.4, for 1.0 mM HMTD, the maximum ECL appears at 0.70 mM Ru(bpy)3Cl2 for

61

all three working electrodes studied, in which the Pt electrode shows slightly stronger

ECL responses than the Au electrode does, and the GC electrode gives relatively poor

ECL signals. Similar behavior has been reported previously for the Ru(bpy)32+/TPrA

system in MeCN.31

+ III

+ + III

-e -H +Ru+ II*1

-e -H -H +Ru+ 2+ II*2

II* II

HMTD HMTD HMTD Ru + P

HMTD [ HMTD ] HMTD HMTD Ru + P

Ru Ru + hv

2.1

2.2

2.3

where HMTD = hexamethylene triperoxide diamine, RuII = Ru(bpy)3

2+, RuII =

Ru(bpy)33+, RuII* = excited state Ru(bpy)3

2+*, P1 and P2 = oxidation products of HMTD

free radicals.

Scheme 2.2. Proposed ECL mechanism of the Ru(bpy)32+/HMTD system in MeCN upon

the anodic potential scanning.

ECL Enhancement with AgNO3

As shown in Figures 2.5A and 2.6, after addition of AgNO3 into a MeCN solution

containing HMTD and Ru(bpy)3Cl2, significant enhancement in ECL signal is observed.

For example, in 1.0 mM HMTD-0.70 mM Ru(bpy)3Cl2-0.10 M TBAP MeCN, about 9

and 27 times increases in ECL intensity were obtained in the presence of 2.0 mM and 7.0

mM AgNO3 with respect to that in the absence of added AgNO3, respectively. The ECL

peak intensity is linearly proportional to the concentration of added AgNO3 over a

concentration range of 0 to 7.0 mM (Figure 2.6a). In the absence of HMTD, no ECL

concentration range of 0 to 7.0 mM (Figure 2.6a). In the absence of HMTD, no ECL

background (When [AgNO3] ≤ 2.0 mM) or values of less than 10% of ECL observed in

62

0.0 0.2 0.4 0.6 0.8 1.00

4

8

12

GC

Au

Pt

i EC

L(nA

/ m

m2)

[Ru(bpy)3Cl

2] (mM)

Figure 2.4. Effect of Ru(bpy)3Cl2 concentration and working electrode material on ECL intensity of the Ru(bpy)3

2+/HMTD system in 0.10 M TBAP MeCN. Scan rate: 50 mV/s, [HMTD] = 1.0 mM.

25 AiCV

2.0 1.5 1.0 0.5 0.0

-50

0

50

i CV (A

)

ECL

CV

(A)

-300

-200

-100

0

100

200

300

iEC

L (nA

)

E (V vs Ag/Ag+)

(B)

Figure 2.5. (A) CV (black line) and ECL (blue line) responses obtained from 1.0 mM HMTD- 0.70 mM Ru(bpy)3Cl2-0.10 M TBAP in MeCN in the presence of 7.0 mM AgNO3, and (B) CV of 7.0 mM AgNO3 in MeCN containing 0.10 M TBAP at a 2-mm diameter Pt electrode with a scan rate of 50 mV/s.

63

the presence of HMTD (When 3.0 mM < [AgNO3] < 9.0 mM) generated from 0.70 mM

Ru(bpy)3Cl2-0.10 M TBAP MeCN (AgNO3) is found (Figure 2.6b). As a result, the ECL

enhancement must be related to both HMTD and AgNO3. By comparing Figure 2.2 with

Figure 2.5A, one can notice that the enhanced ECL signal is corresponded to the increase

of CV currents in the potential region of HMTD oxidation.

0 2 4 6 8 10

0

50

100

150

200

250

300

i EC

L(nA

)

[AgNO3] (mM)

(a)

(b)

Figure 2.6. (a) Effect of AgNO3 on ECL intensity of 1.0 mM HMTD- 0.70 mM Ru(bpy)3Cl2-0.10 M TBAP in MeCN. (b) ECL background produced from 0.70 mM Ru(bpy)3Cl2-0.10 M TBAP with added AgNO3 in MeCN in the absence of HMTD. Other experimental conditions were the same as in Figure 2.5.

A CV study of AgNO3 in MeCN (0.10 M TBAP) at a Pt electrode (Figure 2.5B)

indicates that AgNO3 oxidizes irreversibly with an anodic peak potential of 1.72 V vs

Ag/Ag+, which is in good agreement with an earlier report,32 and is coincident with the

electrochemical oxidation potentials of HMTD at Pt. Such electrochemical oxidations

could lead to the formation of strong oxidizing species of Ag(II) (Eº(Ag(II)/Ag(I)) = 1.98

V vs NHE33 or ~1.79 V vs Ag/Ag+) and NO3• radicals.32,34-37 The standard reduction

64

potential of NO3• to NO3

- in aqueous media was estimated to be as high as 2.48±0.03 V

vs NHE (~2.29 V vs Ag/Ag+) on the basis of a series of rate and equilibrium constant

measurements as well as computer modeling.38,39 However, this estimated value

apparently is too high compared with the data obtained from various electrochemical

measurements in MeCN, where the oxidation of NO3- at approximately 1.66-1.9 V vs

Ag/Ag+ was observed at a Pt electrode.32,34-37 Previous electrochemical data also

suggested that the formation of Ag(II) from AgNO3 in MeCN solution resulted from a

reaction between Ag(I) ions and electrochemically generated NO3• radicals.32,35 That is,

the direct electrode oxidation of Ag(I) to Ag(II) could be a relatively slow kinetic

(Scheme 2.3) process. By using Scheme 2.3 with all known experimental conditions

along with estimated CV and chemical parameters, digital simulations of the CV shown

in Figure 2.5B were conducted (DigiSim 3.0, Bioanalytical Systems, Inc., West Lafayette,

IN),40,41 which resulted in Eº(Ag(II)/Ag(I)) = 1.79 V vs Ag/Ag+ (or 1.98 V vs NHE) and

Eº(NO3•/NO3

-) = 1.82 V vs Ag/Ag+ (or 2.01 V vs NHE). Thus, our simulated standard

reduction potential for the NO3•/NO3

- couple is about 470 mV less positive than that

obtained from non-electrochemical based experiments. Although precise fitting of the

experimentally obtained CV (Figure 2.5B) with the simulated one (Figure 2.7) was

difficult due to large amounts of non-Faradaic current responses of the blank solution

(Figure 2.3c), major features of the voltammograms fit well. In addition to AgNO3,

NaNO3 also demonstrates significant ECL enhancement for the HMTD/Ru(bpy)32+

system (Figure 2.8). However, in this case, the enhancement from NaNO3 (by adding a

few μL of a high concentration of aqueous NaNO3 into MeCN solutions) is slightly

65

smaller than AgNO3 when [AgNO3] > 1.4 mM. These data suggest that the ECL

enhancement is primarily due to NO3- ions.

0s

- 0 43 3 s

- 43 3 f b

Ag(II) + e = Ag(I) =1.79 V, = 0.10 cm/s

NO + e = NO =1.82 V, = 1 10 cm/s

NO + Ag(I) = NO + Ag(II) / 1 10

E k

E k

k k

43 3 f b

44 f b

/ 3111

NO = P / 1 10 /1

Ag(II) = P / 1 10 /1

k k

k k

2.4

2.5

2.6

2.7

2.8

Scheme 2.3. Proposed reaction mechanism of AgNO3 oxidation in MeCN at a Pt electrode for CV digital simulations. In Eqs. 2.4-2.8, E0 is the standard redox potential,ks is the standard heterogeneous rate constant, kf/kb is the ratio of homogeneous rate constant of the forward reaction to the backward reaction, and P3 and P4 are reduction products of NO3

• and Ag(II), respectively.

2.0 1.5 1.0 0.5 0.0

-50

-25

0

i cv (A

)

E (V vs Ag/Ag+)

Ep = 1.72 V

Figure 2.7. Simulated CV of AgNO3 oxidation in MeCN. In addition to parameters listed in Scheme 2.3 above, the following parameters were used: α = 0.5, initial [Ag(I)] = [NO3

-] = 0.007 M, area of electrode = 0.0314 cm2, scan rate = 50 mV/s, T = 298.15 K, diffusion coefficients for all species: 5×10-6 cm2/s.

The contribution from Ag+ ions is in fact generally less than 10% under present

experimental conditions. Because 0.70 mM Ru(bpy)3Cl2 was used for the ECL

measurements, 1.4 mM of Ag+ ions were needed to react with Cl- ions to form AgCl

66

precipitates.42 Consequently, no difference in ECL enhancement was observed between

AgNO3 and NaNO3 when their concentrations were less than 1.4 mM.

0 1 2 3 4 5 6 7 8

0

100

200

300

(b) NaNO3

[AgNO3] or [NaNO

3] (mM)

EC

L P

eak

Inte

nsity

(nA

)

(a) AgNO3

1.4 mM

Figure 2.8. Comparison of (a) AgNO3 with (b) NaNO3 on ECL intensity of 1.0 mM HMTD-0.70 mM Ru(bpy)3Cl2-0.10 M TBAP in MeCN.

The effect of Ag+ on ECL enhancement was further investigated with silver

benzoate (C6H5COOAg) and AgBF4. As shown in Figure 2.9a, in a 1.0 mM HMTD-0.70

mM Ru(bpy)3(ClO4)2-0.10 M TBAP MeCN solution, ECL intensity is increased linearly

with the addition of C6H5COOAg. When 0.70 mM Ru(bpy)3(ClO4)2 was replaced with

0.70 mM Ru(bpy)3Cl2, a similar trend in ECL enhancement was observed (Figure 2.9b).

In this case, however, the effective [Ag+] in solution is reduced by ~1.4 mM due

to insoluble AgCl formation as explained earlier. Comparable ECL enhancement of

AgBF4 for the HMTD/Ru(bpy)32+ system was also noted (not shown). Therefore, Ag+

ions enhancement on ECL is essentially independent of the anions used. On the basis of

data shown in Figures 2.8b and 2.9a, ~2.6 times in ECL enhancement from NO3- as

67

compared to Ag+ can be calculated, which is consistent with the AgNO3 reaction

mechanism proposed in Scheme 2.3. There would be no ECL enhancement of Ag+

obtained, if no direct oxidation of Ag(I) at an electrode occurred as described

previously.32,35 In this respect, ECL is a much more sensitive technique than voltammetry

for verifying the insensitive oxidation of Ag(I) in MeCN.

0 1 2 3 4 5 6

15

30

45

60

75

[C6H

5COOAg] (mM)

EC

L P

eak

INte

nsity

(nA

)

(a)

(b)

Figure 2.9. Effect of Ag+ on ECL intensity of 1.0 mM HMTD with (a) 0.70 mM Ru(bpy)3(ClO4)2 or (b) 0.70 mM Ru(bpy)3Cl2 using different concentrations of C6H5COOAg.

Since ECL production of the HMTD/Ru(bpy)32+ system initiates from the oxidation of

Ru(bpy)32+ and HMTD (Scheme 2.2), in principle, any process that could increase the

generation of Ru(bpy)33+ and HMTD•+ should enhance the ECL intensity. Although

electrochemical oxidations of HMTD and AgNO3 are located in the similar potential

region (Figures 2.2 and 2.5), electrogenerated Ag(II) and NO3• species should be strong

enough to oxidize Ru(bpy)32+ and HMTD in the diffusion layer proximity to the

electrode. This is because the standard reduction potentials for Ag(II) and NO3• species

68

are more positive than their apparent oxidation peak as revealed by digital simulations,

and the standard redox potential for HMTD•+/HMTD should be less positive than or close

to its apparent oxidation peak, given that HMTD, like many other tertiary amines,

involves a slow electron-transfer oxidation process (e.g., ks ≤ 0.01 cm/s).40,43

2+ 3+3 3

+

-3 3

-3 3

+

- +3 3

+ +

2+3

Ru(bpy) - e Ru(bpy)

HMTD - e HMTD

Ag(I) - e Ag(II)

NO - e NO

NO + Ag(I) NO + Ag(II)

Ag(II) + HMTD Ag(I) + HMTD

NO + HMTD NO + HMTD

HMTD HMTD + H

Ru(bpy) + HMTD R

+3 1

+ 2+*3 3

+ 2+* -3 3 3 3

3+ 2+*3 3 2

+ 3+ 2+* 2+3 3 3 3

2+*3

u(bpy) + Product

Ru(bpy) + Ag(II) Ru(bpy) + Ag(I)

Ru(bpy) + NO Ru(bpy) + NO

Ru(bpy) + HMTD Ru(bpy) + Product

Ru(bpy) + Ru(bpy) Ru(bpy) + Ru(bpy)

Ru(bpy)

2+3 Ru(bpy) + hv

2.9

2.10

2.11

2.12

2.13

2.14

2.15

2.16

2.17

2.18

2.19

2.20

2.21

2.22

Scheme 2.4. Proposed ECL mechanism of the HMTD/Ru(bpy)32+ system involving ECL

enhancement by AgNO3 in MeCN at a Pt electrode, in which Eqs. 2.9–2.12) are direct oxidations at the electrode, Eqs. 2.13–2.15 and 2.17 are chemical reactions with electron transfers in the solution proximity to the electrode surface, Eq. 2.16 is the deprotonation of an α-C H from HMTD•+ radical cation generated electrochemically (Eq. 2.10) and chemically (Eqs. 2.14–2.15), and Eqs. 2.18–2.21 are possible pathways to form excited state Ru(bpy)3

2+* species that emit light via Eq. 2.22.

69

An ECL mechanism of the HMTD/Ru(bpy)32+ system involving ECL

enhancement by AgNO3 in MeCN at a Pt electrode is proposed in Scheme 2.4. In this

mechanism, chemical oxidations of Ru(bpy)32+ to Ru(bpy)3

3+ and HMTD•+ to

[•HMTD•]2+ by Ag(II) and NO3• are not included, because their contributions to the

overall ECL enhancement could be minor.

Limit of Detection of HMTD

0.0 0.2 0.4 0.6 0.8 1.0

0

100

200

300

400

EC

L P

eak

Inte

nsity

(nA

)

[HMTD] (mM)

(b)

(a)

Figure 2.10. Relationship between [HMTD] and ECL peak intensity. (a) Without addition of AgNO3, and (b) with addition of 7.0 mM AgNO3. Other experimental conditions were the same as in Figure 2.5. Each data point represented the mean of four separate runs.

Using above described ECL strategies, the limit of detection of HMTD in MeCN

was found to be 0.15 mM in the absence of AgNO3 (Figure 2.10a) and 50 µM in the

presence of 7.0 mM AgNO3 (Figure 2.10b), which, respectively, are ~3.3 and 10 times

lower than the detection limit obtained from a method based on HPLC separation and

FTIR detection.10 Because as little as 0.50 mL of a test solution was needed for running

70

an ECL experiment, the absolute detection limit of HMTD using AgNO3 as the ECL

enhancing agent was calculated to be 5.2 μg. This value is much higher than that obtained

from the MS-based method, probably due to the relatively high ECL background caused

by AgNO3 and a relatively large volume of test solution required for ECL studies.

Conclusions

Highly explosive compound HMTD was used as an ECL coreactant and

determined with ECL in the presence of Ru(bpy)32+ and the ECL enhancing agent AgNO3

in MeCN at a Pt electrode upon the anodic potential scanning from 0 to 2.0 V vs Ag/Ag+.

This technique provided an easy and sensitive way for quantifying HMTD in μM

concentration levels. ECL enhancement of the HMTD/Ru(bpy)32+ system by AgNO3 was

primarily ascribed to the chemical oxidations of HMTD by electrogenerated strong

oxidizing agents NO3• and Ag(II) species, which resulted in the increase in [HMTD•+]

and hence the ECL intensity. ECL data and CV digital simulations revealed that direct

oxidation of Ag(I) to Ag(II) in MeCN at a Pt electrode certainly occurred, although the

reaction appeared in the same potential region as NO3- oxidation. A standard reduction

potential of 1.82 V vs Ag/Ag+ (or 2.01 V vs NHE) for the NO3•/NO3

- couple was

estimated based on CV simulations, which is about 470 mV less positive than that

estimated from a group of thermodynamic and kinetic measurements in aqueous media.

71

References

(1) Schulte-Ladbeck, R.; Kolla, P.; Karst, U. Analyst 2002, 127, 1152.

(2) Pumera, M. Electrophoresis 2008, 29, 269.

(3) Munoz, R. A. A.; Lu, D.; Cagan, A.; Wang, J. Analyst 2007, 132, 560.

(4) Schulte-Ladbeck, R.; Vogel, M.; Karst, U. Anal. Bioanal. Chem. 2006, 386, 559.

(5) Pumera, M. Electrophoresis 2006, 27, 244.

(6) Cotte-Rodriguez, I.; Chen, H.; Cooks, R. G. Chem. Commun. 2006, 953.

(7) Baeyer, A.; Villiger, V. Ber. Dtsch. Chem. Ges. 1900, 33, 2479.

(8) Legler, L. Ber. Dtsch. Chem. Ges. 1885, 18, 3343.

(9) Crowson, A.; Beardah, M. S. Analyst 2001, 126, 1689.

(10) Schulte-Ladbeck, R.; Edelmann, A.; Quintas, G.; Lendl, B.; Karst, U. Anal. Chem.

2006, 78, 8150.

(11) Yinon, J. Forensic and Environmental Detection of Explosives; John Wiley & Sons,

Inc.: New York, 1999.

(12) Modern Methods and Applications in Analysis of Explosives; Yinon, J.; Zitrin, S.,

Eds.; John Wiley & Sons, Inc.: New York, 1996.

(13) Gielsdorf, W. Fresenius' Z. Anal. Chem. 1981, 308, 123.

(14) Sigman, M. E.; Clark, C. D.; Fidler, R.; Geiger, C. L.; Clausen, C. A. Rapid

Commun. Mass Spectrom. 2006, 20, 2851.

(15) Muller, D.; Levy, A.; Shelef, R.; Abramovich-Bar, S.; Sonenfeld, D.; Tamiri, T. J.

Forensic Sci. 2004, 49, 935.

(16) Mullen, C.; Irwin, A.; Pond, B. V.; Huestis, D. L.; Coggiola, M. J.; Oser, H. Anal.

Chem. 2006, 78, 3807.

(17) Cotte-Rodriguez, I.; Hernandez-Soto, H.; Chen, H.; Cooks, R. G. Anal. Chem. 2008,

80, 1512.

(18) Electrogenerated Chemiluminescence; Bard, A. J., Ed.; Marcel Dekker, Inc.: New

York, 2004.

(19) Miao, W. Chem. Rev. 2008, 108, 2506.

(20) Richter, M. M. Chem. Rev. 2004, 104, 3003.

(21) Yin, X.-B.; Dong, S.; Wang, E. TrAC, Trends in Anal. Chem. 2004, 23, 432.

72

(22) Pittman, T. L.; Thomson, B.; Miao, W. Anal. Chim. Acta 2009, 632, 197.

(23) Shankaran, D. R.; Gobi, K. V.; Sakai, T.; Matsumoto, K.; Imato, T.; Toko, K.;

Miura, N. IEEE Sensors J. 2005, 5, 616.

(24) Lee, M. R.; Chang, S. C.; Kao, T. S.; Tang, C. P. J. Res. Natl. Bur. Stand. 1988, 93,

428.

(25) Miao, W.; Choi, J.-P. In Electrogener. Chemilumin.; Bard, A. J., Ed.; Marcel

Dekker, Inc.: New York, 2004, p 213.

(26) Rubinstein, I.; Bard, A. J. J. Am. Chem. Soc. 1981, 103, 512.

(27) Schaefer, W. P.; Fourkas, J. T.; Tiemann, B. G. J. Am. Chem. Soc. 1985, 107, 2461.

(28) Schulte-Ladbeck, R.; Edelmann, A.; Quintas, G.; Lendl, B.; Karst, U. Anal. Chem.

2006, 78, 8150.

(29) Gottlieb, H. E.; Kotlyar, V.; Nudelman, A. J. Org. Chem. 1997, 62, 7512.

(30) Rosado, D. J., Jr.; Miao, W.; Sun, Q.; Deng, Y. J. Phys. Chem. B 2006, 110, 15719.

(31) Miao, W.; Bard, A. J. Anal. Chem. 2004, 76, 5379.

(32) Rao, R. R.; Milliken, S. B.; Robinson, S. L.; Mann, C. K. Anal. Chem. 1970, 42,

1076.

(33) Arning, M. D.; Minteer, S. D. In Handbook of Electrochemistry; Zoski, C. G., Ed.;

Elsevier: New York, 2007, p 813.

(34) Tracy, M. L.; Nash, C. P. J. Phys. Chem. 1985, 89, 1239.

(35) Mishima, H.; Iwasita, T.; Macagno, V. A.; Giordano, M. C. Electrochim. Acta 1973,

18, 287.

(36) Bontempelli, G.; Mazzocchin, G. A.; Magno, F. J. Electroanal. Chem. Interfacial

Electrochem. 1974, 55, 91.

(37) Bontempelli, G.; Mazzocchin, G. A.; Magno, F.; Seeber, R. J. Electroanal. Chem.

Interfacial Electrochem. 1974, 55, 101.

(38) Poskrebyshev, G. A.; Huie, R. E.; Neta, P. J. Phys. Chem. A 2003, 107, 1964.

(39) Neta, P.; Huie, R. E. J. Phys. Chem. 1986, 90, 4644.

(40) Miao, W.; Choi, J.-P.; Bard, A. J. J. Am. Chem. Soc. 2002, 124, 14478.

(41) Bond, A. M.; Feldberg, S. W.; Miao, W.; Oldham, K. B.; Raston, C. L. J.

Electroanal. Chem. 2001, 501, 22.

73

(42) Luehrs, D. C.; Iwamoto, R. T.; Kleinberg, J. Inorg. Chem. 1966, 5, 201.

(43) Adenier, A.; Chehimi, M. M.; Gallardo, I.; Pinson, J.; Vila, N. Langmuir 2004, 20,

8243.

74

CHAPTER III

SELECTIVE DETERMINATION OF TRIACETONE TRIPEROXIDE USING

ELECTROGENERATED CHEMILUMINESCENCE

Introduction

The increasing numbers of use of homemade explosives have been observed by

the legal authorities. Among these, peroxide explosives, such as triacetone triperoxide

(TATP) and hexamethylene triperoxide diamine (HMTD) are very popular among the

terrorists since these compounds could be easily synthesized from the readily available

chemicals.1 TATP, a white solid first synthesized by Wolffenstein in 1895,2 is one of the

most sensitive explosives known with significant sensitivity towards friction, heat, and

impact. Its impact sensitivity is equal to that of another high explosive, trinitrotoluene

(TNT).3 The London bombing in 2005 and the attempt to bring down trans-Atlantic flight

in 2001 by a ‘shoe bomber’ are the examples of the terrorist use of TATP.4,5

TATP has no any commercial or military use as it has tendency to sublime6 and is

sensitive to any kind of friction or impact which makes its movement difficult. As

reviewed in Chapter I, a number of analytical techniques have been used to detect

peroxide-based explosives,7,8 which include (a) mass spectrometry (MS), e.g., gas

chromatographic separation coupled with mass spectrometric detection (GC/MS),9-12

desorption electrospray ionization MS (DESI/MS),5,13,14 and electrospray or laser

ionization15 time of flight (TOF) MS (TOF/MS); (b) ion mobility spectrometry (IMS);16-

19 (c) infrared absorption spectroscopy (IR), e.g., liquid chromatography separation-IR

detection (LC/IR),3,20 GC/IR,11 and mid-IR laser spectroscopy with quantum cascade

laser (QCL);21-24 (d) Raman spectroscopy;10,25 (e) luminescent techniques, e.g.,

75

fluorescence26-30 and chemiluminescence;31 (f) UV-vis spectrophotometry;32-35 and finally

(g) electrochemistry.4,36-38

Methods based on MS are very sensitive and can detect trace amounts of

explosives but instruments are expensive and often inappropriate for field test. The

results from IMS are affected by the matrices such as temperature and moisture, whereas

fluorescence suffers from scattering light of luminophore impurities. Infrared, UV-vis

detection techniques are often time consuming in sample preparation and are not

appropriate for detection of trace amounts of explosives.

Electrogenerated chemiluminescence (ECL), a technique which produces light as

a result of electrochemical reactions that take place at the electrode surface, has been

considered as a powerful analytical technique because of its good selectivity, high

sensitivity and fast sample analysis.39-42 ECL can detect target molecules down to

picomolar concentration levels.43

In this chapter, ECL detection and quantification of TATP as well as the

differentiation of TATP from HMTD are explored at glassy carbon electrode in water-

MeCN mixture solvents in the presence of added ECL emitter Ru(bpy)32+ ions. The

method is based on the fact that TATP contains peroxide functional groups, which, upon

cathodic potential scanning, produce strong oxidizing species hydroxyl radicals (•OH)

that could oxidize electrogenerated Ru(bpy)3+ cations to form Ru(bpy)3

2+* excited states

that emit photons.44

76

Experimental Section

Chemicals

Acetonitrile (MeCN, 99.8% , HPLC grade), acetone (99.5+%, ACS reagent),

sulfuric acid (95-98%, ACS reagent), sodium phosphate, dibasic (99.0%), tris-(2,2'-

bypyridine)dichlororuthenium(II) hexahydrate (Ru(bpy)3Cl2•6H2O, 99.95%), ammonium

iron(II) sulfate hexahydrate ((NH4)2Fe(SO4)2 •6H2O, ACS reagent), sodium azide (NaN3,

≥99.0%), and 5,5′-dimethyl-pyrroline-N-oxide (DMPO, ≥97% for ESR-spectroscopy)

from Sigma-Aldrich (St. Louis, MO); hydrogen peroxide (H2O2, 30%) from Fisher

Scientific (Fair Lawn, NJ); sodium phosphate, monobasic monohydrate from J.T. Baker

Chemicals Co. (Phillipsburg, NJ); silver nitrate (99.5%) and tetra-n-butylammonium

perchlorate (TBAP, 99+%, electrochemical grade) from Fluka (Milwaukee, WI); catalase

(filtered, 30,000 units/mL) from Worthington Biochemical Corp. (Lakewood, NJ) were

used as received.

Synthesis and Characterization of TATP

The peroxide explosive compound, triacetone triperoxide (TATP), had to be

synthesized as it is not commercially available. For this, we followed the standard

procedure from the literature.1 1.36 mL of H2O2 (30%, chilled for several hours) was put

into a small beaker under ice-bath and to this, 1.9 mL of acetone (99.5+%, chilled) were

added. 0.48 mL of concentrated sulfuric acid was added drop wise with constant stirring

and monitoring the temperature (use of different acids may form different polymorphic

forms45). Temperature must be kept below 4 °C in entire process. After addition of acid

the content was stirred for about 15 min and kept in a refrigerator for overnight. White

solid compound was filtered, washed with water, and allowed to dry at room temperature

77

before stored in a refrigerator for further use (Scheme 3.1). [CAUTION: TATP is very

sensitive to heat, friction and impact so it has to be handled in small amount. Dry

compound is volatile and more likely to explode so it must be stored as solution in MeCN

at ~0 °C.]

Scheme 3.1. Synthesis of triacetone triperoxide (TATP).

The dried TATP was characterized by diamond crystal attenuated total reflection

Fourier transform infrared (ATR-FTIR, Nicolet Nexus 470 FTIR spectrometer, Thermo

Electron Corp., Madison, WI) and 1H NMR spectroscopy (Bruker 300 MHz NMR

spectrometer).

Figure 3.1. (a) ATR-FTIR and (b) 1H NMR spectra of TATP.

1400 1200 1000 800

0.0

0.1

0.2

0.3

0.4

0.5

0.6

Ab

sorb

anc

e

Wavenumber, cm-1

C-O Stretch, 1175 cm-1

(a)

8 6 4 2 0

CO O

O O

C C

O O

ppm

CDCl3

TMS

CH3

~1.48 ppm(b)

78

FTIR spectra (Figure 3.1a) show a characteristic C-O stretch band at 1175 cm-1,

which along with all other peaks and proton NMR spectra (Figure 3.1b) which show a

characteristic singlet peak for all equivalent 18 protons at 1.48 ppm are consistent with

the literature values.46 This chemical shift is very close to 1.41 ppm obtained theoretically

by using ChemBiodraw Ultra 11 (CambridgeSoft Corp., Cambridge, MA). The reaction

precursors produce 1H NMR peaks at 2 ppm (H2O2 at 2.0 ppm, acetone at 2.09 ppm, and

H2SO4 at 2 ppm) as estimated theoretically using the above software. Small peaks seen in

1H NMR at 0 and 7.26 ppm were from the internal standard (TMS) and proton from

incomplete deuteration of chloroform.47

Instruments Used for Electrochemical and ECL Studies

Cyclic voltammetry (CV) was performed with an electrochemical workstation

(Model 660A, CH Instruments, Inc., Austin, TX). The ECL and CV responses were

simultaneously recorded with a homemade ECL instrument,40,48 where the CHI

electrochemical workstation was combined with a photo-multiplier tube (PMT,

Hamamatsu R928, Japan, biased at 700 V DC). A conventional three-electrode

electrochemical cell system was used with a glassy carbon disk (3 mm diameter) as the

working electrode, a platinum gauge as the counter electrode, and a Ag/Ag+ (with 10 mM

AgNO3 and 0.10M TABP in MeCN) as the reference electrode. The GC working

electrode was polished before every run with 0.3-0.05 µM alumina slurry, washed with

water and dried with Kim wipes tissue. The solution was degassed with nitrogen gas

(Ultrapure, Nordan Smith, Hattiesburg, MS) at least 5 min to remove dissolved oxygen

that could produce background ECL as the electrode was scanned in a negative potential

region.

79

Electron Paramagnetic Resonance (EPR) Spectrometry

EPR spectra of hydroxyl radicals were recorded at a resonant frequency of 9.83

GHz, a modulation frequency of 100 kHz, and the microwave power of 20 mW using a

Bruker EMX microX EPR spectrometer equipped with an ER 4119HS standard

cylindrical resonator (Bruker BioSpin Corp.). The instrument was pre-calibrated using

the manufacturer’s DPPH calibration sample. Hydroxyl radicals (•OH) were freshly

produced chemically by Fenton reaction from 0.50 mM H2O2 and 75 µM ferrous

ammonium sulfate in different H2O-MeCN compositions, and were trapped by 200 mM

5,5′-dimethyl-pyrroline-N-oxide (DMPO) “spin trapping” agent to form DMPO/•OH

“spin adduct” for EPR detection (Scheme 3.2). Experimentally, DMPO was mixed with

H2O2 before ferrous ammonium sulfate was added to the system. Note that the

concentrations listed above were all final values in a total reaction volume of 400 μL.

Scheme 3.2. Formation of •OH radical by Fenton reaction and the DMPO/•OH adduct, where k1 and k2 are the rate constants of Fenton reaction and the spin trapping reaction, respectively.

Samples of the DMPO/•OH adduct in water-MeCN mixture were measured using

a standard glass capillary (10 cm long, 2/1.2 OD/ID (mm), World Precision Instruments,

Inc., Sarasota, FL) filled with the solution to a height of ~4 cm and sealed with

“Permanent Avery Glue Stic” (Office Depot, Hattiesburg, MS) at the bottom. This

capillary was then placed in a normal EPR tube for spectra acquisition which took place

80

exactly after 5 min of the solution preparation, unless otherwise stated. Such a “5 min

time window” was necessary for solution transfer and instrument setup and tuning. The

experimentally obtained EPR spectra were simulated using PEST Winsim Software

(National Institute of Environmental Health Sciences, National Institute of Health,

Research Triangle Park, NC) for calculating of hyperfine coupling constants.

Results and Discussion

Cyclic Voltammetry of TATP

The CV of 1.0 mM TATP (Figure 3.2a) shows that reduction of TATP is very

difficult (unrecognizable reduction peak at ~ -1.0 V vs Ag/Ag+, inset in Figure 2.3). The

ECL luminophore, Ru(bpy)32+, however, can be readily reduced to Ru(bpy)3

+ with a

peak potential at -1.8 V vs Ag/Ag+ (Figure 2.3b), suggesting that ECL could be produced

only after Ru(bpy)32+ reduction.

ECL Behavior of TATP

Because TATP has a poor solubility in water and Ru(bpy)32+ reduction occurs at a

very negative potential region (see Figure 3.2), ECL studies of TATP were first

conducted in MeCN. As proposed in Scheme 3.3, up on the cathodic potential scanning,

TATP could form H2O2 intermediate that could be reduced further to form hydroxyl

radical (•OH) (Eq. 3.1). The newly formed •OH, which is a powerful oxidant as indicated

by its standard redox potential ( -

0

OH /OHE = 1.77~1.91 V vs NHE49-51), could oxidize

Ru(bpy)32+ to Ru(bpy)3

3+ (Eq. 3.3) and Ru(bpy)3+ (electrochemically reduced from

Ru(bpy)32+, Eq. 3.2) to Ru(bpy)3

2+* excited state (Eq. 3.4). Alternatively, the excited state

of Ru(bpy)32+* could be generated by ion annihilation reaction (Eq. 3.5). Note that,

MeCN always contains a trace amount of water (e.g., ~0.01% or ~4 mM H2O for HPLC

81

grade MeCN) that should meet the need for H2O2 production from electrochemical

reduction of TATP.

0.0 -0.5 -1.0 -1.5 -2.0

(b)

(a)

100 Ai

CV

-0.6 -0.9 -1.2

E( (V vs Ag/Ag+)

E( (V vs Ag/Ag+)

(c)

TATP

Ru(bpy)3

2+

Blank

Figure 3.2. CVs of (a) 1.0 mM TATP and (b) 0.60 mM Ru(bpy)32+ in an electrolyte

solution containing 70% volume of 0.10 M phosphate buffer (pH 7.5) and 30% volume of MeCN (i.e., 70 mM phosphate buffer in 70 (H2O) : 30 (MeCN) (v/v) mixture solvent) obtained from a 3-mm diameter glassy carbon electrode at a scan rate 50 mV/s. The CV of the electrolyte solution is displayed in (c).

Experimentally, however, no ECL was observed from the Ru(bpy)32+/TATP

system in 0.10 M TBAP MeCN at bare Pt or glassy carbon electrode. To verify if the

problem was really due to the poor reduction of TATP or H2O2 at the electrode in this

solvent, several efforts were made. First, UV irradiation (254 nm UV lamp, Model ENF-

40C, 0.20 AMPS from Spectronics Corp., New York) was applied to the system so that

TATP can be decomposed to H2O233 prior to ECL detection. Second, the working

electrode (Pt or glassy carbon) modified with crystalline Prussian blue (ferric

ferrocyanide) which could catalytically reduce H2O238 was employed in the ECL testing.

82

Neither helped the ECL generation. Surprisingly, in MeCN even the Ru(bpy)32+/H2O2

system did not produce any notable ECL signals. Scheme 3.4 summarizes the above

observations.

e2 2

2+ +3 3

2+ 3+ -3 3

+ 2+*3 3

+ 3+ 2+* 2+3 3 3 3

2+* 2+3 3

TATP + e [H O ] OH

Ru(bpy) + e Ru(bpy)

Ru(bpy) + OH Ru(bpy) + OH

Ru(bpy) + OH Ru(bpy)

Ru(bpy) + Ru(bpy) Ru(bpy) + Ru(bpy)

Ru(bpy) Ru(bpy) + hv

3.1

3.2

3.3

3.4

3.5

3.6

Scheme 3.3. Proposed ECL mechanism of TATP in the presence of Ru(bpy)32+ upon the

cathodic potential scanning.

Scheme 3.4. No “reductive-oxidation” type ECL was produced from TATP and H2O2 in MeCN in the presence of Ru(bpy)3

2+.

When UV-irradiated TATP MeCN solution was transferred to a water-MeCN

mixture medium containing Ru(bpy)32+ and a suitable supporting electrolyte, strong ECL

signals were observed. For example, in 70:30 (v/v) water-MeCN mixture containing 1.0

83

μM Ru(bpy)32+ and 70 mM phosphate buffer, 0.50 mM TATP (with UV irradiation) starts

to produce ECL at ~-1.5 V vs Ag/Ag+ and reaches the maximum at ~-1.8 V vs Ag/Ag+

(Figure 3.3a). The ECL profile is coincident with the reduction of Ru(bpy)32+ to

Ru(bpy)3+ (Figure 3.3b), suggesting that TATP (or H2O2) must be reduced at a less

negative potential region as revealed earlier in Figure 3.2. ECL generation from the

Ru(bpy)32+/H2O2 system in water-MeCN has been reported previously.44

0.0 -0.5 -1.0 -1.5 -2.0

-50

0

50

100

150

200

ECL, CV

E (V vs, Ag/Ag+)

i CV (A

)

(a)

(b)

-400

-300

-200

-100

0

100

200

300

400

iEC

L (nA

)

Figure 3.3. (a) ECL and (b) CV responses of 0.50 mM TATP (with UV irradiation for 30 min) in 1.0 mM Ru(bpy)3

2+ water-MeCN (70:30, v/v) mixture containing 70 mM phosphate buffer at a 3-mm glassy carbon electrode with a scan rate of 50 mV/s.

Since the concentration of Ru(bpy)32+ could influence the ECL production, its

optimum concentration was determined. When 0.50 mM of TATP is used (with UV

irradiation), 0.60 mM Ru(bpy)32+ produces the maximum ECL response in 70:30 (v/v)

water-MeCN mixture (Figure 3.4). As shown in Figure 3.5, with the exposure of TATP

84

MeCN solution to UV irradiation, the ECL intensity of the irradiation product (i.e., H2O2)

increases within the first 25 min, and then remains essentially unchanged within the

0.0 0.5 1.0 1.5 2.0 2.5

200

240

280

320

360

i EC

L (nA

)

[Ru(bpy)3

2+] (mM)

Figure 3.4. Effect of Ru(bpy)32+ concentration on ECL generation of 0.50 mM TATP

(with UV irradiation for 30 min) in 70:30 (v/v) water-MeCN containing 70 mM phosphate buffer at a 3-mm diameter glassy carbon electrode with a scan rate of 50 mV/s.

next 15-20 min. This suggests that under present UV irradiation conditions, i.e., with a

0.20 AMPS UV lamp at 254 nm and an experimental time scale of 45 min, TATP was

5 10 15 20 25 30 35 40 45160

200

240

280

320

360

i EC

L (

nA)

Time of UV Irradiation (min)

Figure 3.5. Effect of UV irradiation time on TATP decomposition. A 0.20 AMPS UV lamp at 254 nm was used, and the ECL of 0.50 mM TATP (with various UV irradiation intervals) was conducted in 70:30 (v/v) water-MeCN containing 0.6 mM Ru(bpy)3

2+ and 70 mM phosphate buffer at a 3-mm diameter glassy carbon electrode with a scan rate of 50 mV/s.

85

gradually decomposed and H2O2 was relatively stable. The decomposition of H2O2 under

UV irradiation has been reported previously.33

Effect of Solvent Composition on ECL Intensity

Figure 3.6a shows the effect of solvent composition on the ECL peak intensity of

0.5 mM TATP (with UV irradiation for 30 min). With the addition of water to MeCN,

ECL intensity increases and maximizes at a water volume of 70 % (i.e., 70:30 (v/v)

water-MeCN). Because TBAP supporting electrolyte is sparely soluble in water-MeCN

mixture solvent, a final concentration of 70 mM phosphate buffer was used as the

supporting electrolyte.

0 20 40 60 80 100

0

100

200

300

(b)

H2O volume % (in MeCN)

TATP H

2O

2

i EC

L (

nA

) (a)

Figure 3.6. Effect of solvent composition on the ECL peak intensity of (a) 0.5 mM TATP (with UV irradiation for 30 min), and (b) 1.0 mM H2O2 in an electrolyte solution containing 0.6 mM Ru(bpy)3

2+-70 mM phosphate buffer at a 3-mm diameter glassy carbon electrode with a scan rate of 50 mV/s.

This buffer was prepared from 71 mL 0.10 M disodium phosphate and 29 mL of 0.10 M

sodium phosphate, resulting in 100 mL of 0.10 M phosphate buffer solution having pH

7.5. When the irradiated 0.50 mM TATP solution is replaced with 1.0 mM H2O2, a very

86

similar profile of the ECL intensity versus solution composition was obtained (Figure

3.6b). At 70:30 (v/v) water-MeCN solutions, the ECL intensity ratio of 0.50 mM TATP

to 1.0 mM H2O2 has a value of 1.4, which is consistent with the TATP UV irradiation

decomposition reaction shown in Eq. 3-7.

3 2 2 3 2 2 2 3 2TATP [(CH ) CO ] + 3H O 3H O + 3(CH ) O 3.7

Limit of Detection of TATP

In this section, three different modes of ECL detection of TATP, namely, UV

irradiation-ECL detection, acid treatment-ECL detection, and direct ECL detection, are

employed and compared.

As shown in Figure 3.7, for TATP UV irradiation-ECL detection mode, as low as

2.5 µM TATP can be detected, which is 400 and more than three times lower than that

from a LC/IR based detection33 and a horseradish peroxidase treatment-based UV-vis

detection method,33 respectively.

0 100 200 300 400 500

0

50

100

150

200

250

300

350

0 30 60

0

20

40

60

i EC

L (

nA)

[TATP] (M)

2.5 M TATP

Background signal

i EC

L (nA

)

[TATP] (with UV Irradiation, M)

(A)

0.0 -0.4 -0.8 -1.2 -1.6 -2.0

0

75

150

225

300

(a) Background

(b) 2.5 M TATP

(c) 0.5 mM TATP

i EC

L, n

A

E (V vs Ag/Ag+)

(B)

Figure 3.7. (A) ECL intensity as a function of TATP concentration for the UV-irradiation-ECL detection scheme. (B) ECL responses obtained from different concentrations of TATP (with UV irradiation for 30 min): (a) 0, (b) 2.5 μM, and (c) 0.50 mM. All experiments were conducted in 70:30 (v/v) water-MeCN with 0.6 mM Ru(bpy)3

2+-70 mM phosphate buffer at a 3-mm diameter glassy carbon electrode using a scan rate of 50 mV/s.

87

ECL detection of TATP can also be achieved after TATP is treated with acid,

resulting in the formation H2O2.4,38 The ECL signal produced from obtained H2O2,

however, was found to be acid concentration related. In highly concentrated acidic media,

ECL was greatly quenched. With 12 mM HCl (final solution medium’s “pH” ~7), TATP

produces ECL responses which are comparable to the direct detection (see below) and the

limit of detection is also similar, i.e., 2.5 µM (Figure 3.8). Interestingly, as shown in

Figure 3.9, acid treated TATP produces two ECL peaks: the first one at -1.8 V vs Ag/Ag+

is believed to be associated with the first electron reduction of Ru(bpy)32+ to Ru(bpy)3

+,

and the second one at -2.0 V vs Ag/Ag+ remains unclear, although it might be related to

the Ru(bpy)3+/Ru(bpy)3

0 electron-transfer process. Both ECL peaks show good linear

relationship with TATP concentration (Figure 3.8).

0 100 200 300 400 500

0

20

40

60

80

100

(b)

i EC

L,

nA

[TATP] (M)

Background

(a)

Figure 3.8. ECL peak intensity as a function of TATP concentration. TATP was treated with 12 mM HCl, and the ECL measurements were conducted in 70:30 (v/v) water-MeCN mixture containing 0.6 mM Ru(bpy)3

2+-70 mM phosphate buffer at a 3-mm glassy carbon electrode using a scan rate of 50 mV/s. (a) first ECL peak, and (b) second ECL peak as shown in Figure 3.9.

88

0.0 -0.5 -1.0 -1.5 -2.0

0

50

100

150

200

250

ECL, CV

E (V vs, Ag/Ag+)

i CV (A

)

(b)

(a)

-100

-50

0

50

100

iEC

L (nA

)

1

2

Figure 3.9. (a) CV and (b) ECL responses from TATP (pre-treated with 12 mM HCl) using the same experimental conditions as described in Figure 3.8.

0.0 -0.5 -1.0 -1.5 -2.0

0

50

100

150 ECL, CV

E (V vs, Ag/Ag+)

i CV (A

)

(a)

(b)

-150

-100

-50

0

50

100

150

iEC

L (nA)

Figure 3.10. (a) ECL and (b) CV responses of 0.50 mM TATP directly detected in 0.60 mM Ru(bpy)3

2+ water-MeCN (70:30, v/v) mixture containing 70 mM phosphate buffer at a 3-mm glassy carbon electrode with a scan rate of 50 mV/s.

In water-MeCN mixture solvents, TATP can produce ECL directly without need

of any treatment, as shown in Figure 3.10. The ECL intensity, however, is relatively

89

weak as compared with results obtained from UV irradiation. Figure 3.11 illustrates the

relationship between the ECL peak intensity and TATP concentration, where a limit of

detection of 2.5 µM is evident. Although the three ECL detection modes give the same

TATP detection limit of 2.5 µM, direct detection can be completed within 5 min. In

contrast, two- and seven times longer time would be required to detect the same TATP

sample, should the acid treatment and the UV irradiation modes be chosen, respectively.

0 100 200 300 400

0

25

50

75

100

125

150

0 20 40 60

10

20

30

i EC

L (

nA)

[TATP] (M)

[TATP] = 2.5 M

Background signal

i EC

L (nA

)

[TATP] (M)

Figure 3.11. Relationship between ECL peak intensity and TATP concentration when TATP was directly detected in 70:30 (v/v) water-MeCN mixture containing 0.60 mM Ru(bpy)3

2+-70 mM phosphate buffer at a 3-mm glassy carbon electrode with a scan rate of 50 mV/s.

EPR Spectroscopy of Hydroxyl Radical (•OH)

The stability of hydroxyl radical •OH in various compositions of water-MeCN

solvent was estimated by EPR spectroscopy. The rate constants for Fenton reaction (k1,

Scheme 3.2) and for hydroxyl radical trapping with DMPO (k2, Scheme 3.2) have a value

of 76-84,52,53 and 3.4×109 M-1 s-1,54 respectively. The rate constants for hydroxyl radical

quenching with various species such as •OH itself, OH-, Fe(II), and many organic

90

molecules are reported to be in the range of 109-1010 M-1 s-1,55,56 which is comparable to

or larger than k2. As a result, the stability of hydroxyl radical in solution could be

reflected on the stability of the spin trapping adduct DMPO/•OH (Scheme 3.2), assuming

that the adduct is sufficiently stable and its stability is independent of the solvent

composition.

5 %

3460 3480 3500 3520 3540

70 %

10 % 20 %

30 %

40 %

50 %

60 %

80 %

90 %

100 % water

G Value/Gauss

40 X

106

Figure 3.12. Combined EPR spectra of DMPO/•OH produced from Fenton reaction (0.50 mM H2O2, 200 mM 5,5′-dimethyl pyrroline-N-oxide (DMPO), and 75 µM Fe(NH4)2(SO4)2) in different water-MeCN (v/v) solution compositions. The spectra were recorded after reactions occurred for 5 min.

Figure 3.12 shows the EPR spectra of DMPO/•OH adduct produced in a series of

solution composition of water-MeCN mixture. As expected, the spectra consist of a

characteristic, 1:2:2:1 quartet.54 Very weak EPR signals are observed from solutions

91

containing 5-20% (v/v) of water. With the increase in water contents, the EPR signal

gradually increases and maximizes at a mixture solvent containing 70% (v/v) of water

(Figure 3.13). This EPR intensity of DMPO/•OH versus H2O volume % profile coincides

with the ECL intensity of TATP and H2O2 versus H2O volume % profile shown in Figure

3.6. This suggests that the stability of •OH is solvent composition dependent, which is a

determining factor for ECL generation from TATP or H2O2. In MeCN, •OH could hardly

survive, so no ECL was observed from TATP or H2O2. On the other hand, TATP and

H2O2 showed their highest ECL responses in 70:30 (v/v) water-MeCN solution, because

in such a medium, the •OH radical possesses its highest stability.

0 20 40 60 80 100

0.0

0.5

1.0

1.5

2.0

2.5

3.0

Inte

nsi

ty x

10

6

H2O volume % (in MeCN)

Figure 3.13. EPR spectra intensity of DMPO/•OH adduct (taken from Figure 3.12) as a function of solution composition.

The stability of the DMPO/•OH adduct in different water-MeCN mixture solvents

was also monitored over time. The adduct was relatively stable and well-defined EPR

spectra could be recorded even after Fenton reaction occurred for 2 h in 100%-50% (v/v)

water-MeCN solutions. Quantitative analysis of the decay in EPR intensity after 5 and 20

min Fenton reaction reveals that ~30% decrease for every solution composition is

92

obtained (Table 23.1), implying that the DMPO/•OH adduct had the similar stability

irrespective of solvent composition, as we assumed earlier.

Table 3.1. Change in EPR Intensity With Change in Solvent Composition and With Change in Time

Solvent Composition

(% of H2O in MeCN)

EPR Intensity

(after 5 min, ×106)

EPR Intensity

(after 20 min, ×106)

Change in EPR Intensity (±5%)

10 0.15 0.11 -27 30 0.90 0.60 -33 50 1.7 1.2 -29 70 2.9 1.9 -34 90 2.5 1.8 -28

3480 3500 3520-4

-2

0

2

4 Expt. ___ Simulation

Inte

nsity

(a.

u. x

106)

Magnetic Field (G)

Figure 3.14. EPR spectra simulation of DMPO/•OH adduct obtained from Fenton reaction in the presence of the spin trapping agent 5,5′-dimethyl pyrroline-N-oxide (DMPO) after 5 min reaction. Solvent composition: 70:30 (v/v) water/MeCN.

Simulations of the experimentally obtained EPR spectra confirmed that the

1:2:2:1 quartet is representative of virtually identical coupling of the free electron to the

nitroxide nitrogen (aN = 14.85 G) and the beta hydrogen of the pyrroline ring (aH = 14.85

93

G) in the DMPO/•OH adduct (Scheme 3.2). Figure 3.14 shows an example of such a

simulation.

Elimination of Trace Amounts of H2O2 from TATP

Trace amounts of H2O2 from the laundry detergent could lead to the false positive

ECL response. This undesired H2O2 has to be removed before the experiment. As

demonstrated in Figure 3.15, strong ECL signals from 1.0 mM H2O2-0.60 mM

Ru(bpy)32+ system are almost completely suppressed after the addition of catalase

enzyme to the solution, in which unwanted H2O2 is decomposed to H2O and O2 (Eq.

3.8),57 thus removing false positive ECL.

Catalase2 2 2 22H O 2H O + O 3.8

0.0 -0.5 -1.0 -1.5 -2.0

0

20

40

60

80

100

120

+Catalaze

1 mM H2O

2, 0.6 mM Ru(bpy)2+

3

I EC

L, n

A

E (V vs Ag/Ag+)

(a)

(b)

Figure 3.15. ECL responses of 1.0 mM H2O2-0.60 mM Ru(bpy)32+ system (a) before and

(b) after the addition of 5 μL of catalase. The experiments were conducted in 1.0 mL of 70:30 (v/v) water-MeCN containing 70 mM phosphate buffer at a 3-mm diameter glassy carbon electrode with a scan rate of 50 mV/s.

Scheme 3.5 lists how TATP can be selectively detected in the presence of H2O2.

The test sample is first treating with catalase enzyme so that H2O2 contaminant is

decomposed to H2O and O2. The remaining catalase is then deactivated by sodium azide

94

(NaN3). In this way, the electrogenerated H2O2 from TATP reduction would not be

decomposed by the enzyme. Finally, direct ECL detection of treated TATP can be carried

out in water-MeCN mixture media with added Ru(bpy)32+ species. Figure 3.16 compares

the ECL responses from four different systems, confirming that the selective detection of

TATP described above is operative and the added NaN3 has no effect on the ECL

generation of TATP.

TATP + H2O2

TATP + (H2O + O2)

Catalase

Direct ECL detectionof TATP in

water-MeCN

containing Ru(bpy)32+

TATP +Catalse (deactivated)

NaN3

?

Scheme 3.5. Flow-chart of the elimination of H2O2 from TATP and direct ECL detection of TATP.

A B C D0

50

100

150

200

250

EC

L In

ten

sity

(n

A)

Test Samples

Figure 3.16. Comparison of ECL intensities obtained from (A) 0.50 mM TATP, (b) 0.50 mM + 0.50 mM NaN3, (C) 0.50 TATP + 0.50 mM H2O2, and (D) 0.50 TATP + 0.50 mM H2O2 + 5 μL catalase + 0.50 mM NaN3. The experimental conditions were the same as in Figure 3.15.

95

Detection of TATP in the Presence of HMTD

Hexamethylene triperoxide diamnie (HMTD), another common peroxide-based

explosive, also has peroxide functional groups which could produce ECL in the same

fashion as TATP (Figures 3.17A(a) and (b)). Moreover, cathodic ECL responses from

HMTD and TATP mixture are expected to be additive, as shown in Figure 3.17A(c).

In addition to peroxide functional groups, HMTD contains tertiary amine

functional groups which are capable of producing ECL in anodic potential scanning58

(Figure 3.17B). In other words, TATP could produce ECL on cathodic potential scanning

only, whereas HMTD could produce ECL on both cathodic and anodic potential scanning

(Figure 3.17).

0.0 -0.5 -1.0 -1.5 -2.0

0

50

100

150

200

250

300

i EC

L, n

A

E (V vs Ag/Ag+)

(a)

(b)

(c)(A)

2.0 1.5 1.0 0.5 0.0

0

25

50

75

100

125

i EC

L, n

A

E (V vs Ag/Ag+)

(B)

Blank

(a)

(b)

(c)

Figure 3.17. (A) Cathodic ECL of (a) 0.50 mM HMTD, (b) 0.50 mM TATP, and (c) 0.50 mM HMTD + 0.50 mM TATP in the presence of 0.60 mM Ru(bpy)3

2+-70 mM phosphate buffer in 70:30 (v/v) water-MeCN mixture solvent at a3-mm glass carbon electrode with a scan rate of 50 mV/s; (B) Anodic ECL of (a) 0.50 mM TATP, (b) 0.50 mM HMTD, and (c) 0.50 mM TATP + 0.50 mM HMTD, using the same experimental conditions as in (A).

Conclusions

The highly explosive TATP was determined with cathodic scanning ECL in the

presence of Ru(bpy)32+ at glassy carbon working electrode. Solvent composition has

96

profound effect on the ECL generation, which was found to be associated with the

stability of electrogenerated hydroxyl radicals. Optimum ECL can be produced when

water-MeCN mixture solvent has a volume ratio of 70:30, which corresponds to the

strongest EPR signal observed from the DMPO/•OH adduct generated in the same solvent

composition. Although UV irradiation, acid treatment, and direct ECL methods provide

the same TATP detection limit of 2.5 μM, direct ECL approach is much fast (~5 min) and

easy to operate. Elimination of contaminated H2O2 from TATP with catalase, and the

differentiation of TATP from HMTD have also been accomplished in the studies

presented in this chapter.

97

References

(1) Dubnikova, F.; Kosloff, R.; Almog, J.; Zeiri, Y.; Boese, R.; Itzhaky, H.; Alt, A.;

Keinan, E. J. Am. Chem. Soc. 2005, 127, 1146-1159.

(2) Wolffenstein, R. Chem. Ber. 1895, 28, 2265-2269.

(3) Schulte-Ladbeck, R.; Edelmann, A.; Quintas, G.; Lendl, B.; Karst, U. Anal. Chem.

2006, 78, 8150-8155.

(4) Munoz, R. A. A.; Lu, D.; Cagan, A.; Wang, J. Analyst 2007, 132, 560-565.

(5) Cotte-Rodriguez, I.; Chen, H.; Cooks, R. G. Chem. Commun. 2006, 953-955.

(6) Bellamy, A. J. J. Forensic Sci. 1999, 44, 603-608.

(7) Burks, R. M.; Hage, D. S. Anal. Bioanal. Chem. 2009, 395, 301-313.

(8) Buxton, G. V.; Mulazzani, Q. G.; Ross, A. B. J. Phys. Chem. Ref. Data 1995, 24,

1055-1349.

(9) Stambouli, A.; El, B. A.; Bouayoun, T.; Bellimam, M. A. Forensic Sci. Int. 2004,

146, S191-S194.

(10) Jimmie, O.; James, S.; Joseph, B.; Faina, D.; Ronnie, K.; Leila, Z.; Yehuda, Z.

Appl. Spectro. 2008, 62, 906-915.

(11) Pena, A. J.; Pacheco-Londono, L.; Figueroa, J.; Rivera-Montalvo, L. A.; Roman-

Velazquez, F. R.; Hernandez-Rivera, S. P. Proc. SPIE-Int. Soc. Opt. Eng. 2005,

5778, 347-358.

(12) Kende, A.; Lebics, F.; Eke, Z.; Torkos, K. Microchim. Acta 2008, 163, 335-338.

(13) Cotte-Rodriguez, I.; Cooks, R. G. Chem. Commun. 2006, 2968-2970.

(14) Cotte-Rodriguez, I.; Hernandez-Soto, H.; Chen, H.; Cooks, R. G. Anal. Chem.

2008, 80, 1512-1519.

(15) Sigman, M. E.; Clark, C. D.; Caiano, T.; Mullen, R. Rapid Commun. Mass

Spectrom. 2008, 22, 84-90.

(16) Oxley, J. C.; Smith, J. L.; Kirschenbaum, L. J.; Marimganti, S.; Vadlamannati, S. J.

Forensic Sci. 2008, 53, 690-693.

(17) Rasanen, R.-M.; Nousiainen, M.; Perakorpi, K.; Sillanpaa, M.; Polari, L.;

Anttalainen, O.; Utriainen, M. Anal. Chim. Acta 2008, 623, 59-65.

(18) Koyuncu, H.; Seven, E.; Calimli, A. Turk. J. Chem. 2005, 29, 255-264.

98

(19) Buttigieg, G. A.; Knight, A. K.; Denson, S.; Pommier, C.; Bonner, D. M. Forensic

Sci. Int. 2003, 135, 53-59.

(20) Schulte-Ladbeck, R.; Vogel, M.; Karst, U. Anal. Bioanal. Chem. 2006, 386, 559-

565.

(21) Bauer, C.; Sharma, A. K.; Willer, U.; Burgmeier, J.; Braunschweig, B.; Schade, W.;

Blaser, S.; Hvozdara, L.; Mueller, A.; Holl, G. Appl. Phys. B: Lasers Opt. 2008, 92,

327-333.

(22) Hildenbrand, J.; Herbst, J.; Woellenstein, J.; Lambrecht, A. Proc. SPIE 2009, 7222,

72220B/72221-72220B/72212.

(23) Herbst, J.; Hildenbrand, J.; Woellenstein, J.; Lambrecht, A. Proc. SPIE 2009, 7298,

72983W/72981-72983W/72912.

(24) Dunayevskiy, I.; Tsekoun, A.; Prasanna, M.; Go, R.; Patel, C. K. N. Appl Opt 2007,

46, 6397-6404.

(25) Ko, H.; Chang, S.; Tsukruk, V. V. ACS Nano 2009, 3, 181-188.

(26) Sanchez, J. C.; Trogler, W. C. J. Mater. Chem. 2008, 18, 5134-5141.

(27) Germain, M. E.; Knapp, M. J. Inorg. Chem. 2008, 47, 9748-9750.

(28) Malashikhin, S.; Finney, N. S. J. Am. Chem. Soc. 2008, 130, 12846-12847.

(29) Singh, S. J. Hazard. Mater. 2007, 144, 15-28.

(30) Sella, E.; Shabat, D. Chem. Commun. 2008, 5701-5703.

(31) Jimenez, A. M.; Navas, M. J. J. Hazard. Mater. 2004, 106, 1-5.

(32) Mills, A.; Grosshans, P.; Snadden, E. Sens. Actuators, B 2009, B136, 458-463.

(33) Schulte-Ladbeck, R.; Kolla, P.; Karst, U. Analyst 2002, 127, 1152-1154.

(34) Schulte-Ladbeck, R.; Kolla, P.; Karst, U. Anal. Chem. 2003, 75, 731-735.

(35) Apblett, A. W.; Kiran, B. P.; Malka, S.; Materer, N. F.; Piquette, A. Ceram. Trans.

2006, 172, 29-35.

(36) Lu, D.; Cagan, A.; Munoz, R. A. A.; Tangkuaram, T.; Wang, J. Analyst 2006, 131,

1279-1281.

(37) Laine, D. F.; Cheng, I. F. Microchem. J. 2009, 91, 78-81.

(38) Laine, D. F.; Roske, C. W.; Cheng, I. F. Anal. Chim. Acta 2008, 608, 56-60.

(39) Miao, W.; Bard, A. J. Anal. Chem. 2004, 76, 7109-7113.

(40) Miao, W. Chem. Rev. 2008, 108, 2506-2553.

99

(41) Richter, M. M. Chem. Rev. 2004, 104, 3003-3036.

(42) Yin, X.-B.; Dong, S.; Wang, E. TrAC, Trends Anal. Chem. 2004, 23, 432-441.

(43) Pittman, T. L.; Thomson, B.; Miao, W. Anal. Chim. Acta 2009, 632, 197-202.

(44) Choi, J.-P.; Bard, A. J. Anal. Chim. Acta 2005, 541, 143-150.

(45) Reany, O.; Kapon, M.; Botoshansky, M.; Keinan, E. Cryst. Growth Des. 2009, 9,

3661-3670.

(46) Schulte-Ladbeck, R.; Edelmann, A.; Quintas, G.; Lendl, B.; Karst, U. Anal. Chem.

2006, 78, 8150-8155.

(47) Arning, M., D,; Minteer, S.D. Handbook of Electrochemistry: New York, 2007.

(48) Rosado, D. J., Jr.; Miao, W.; Sun, Q.; Deng, Y. J. Phys. Chem. B 2006, 110, 15719-

15723.

(49) Klaning, U. K.; Sehested, K.; Holcman, J. J. Phys. Chem. 1985, 89, 760-763.

(50) Koppenol, W. H.; Liebman, J. F. J. Phys. Chem. 1984, 88, 99-101.

(51) Schwarz, H. A.; Dodson, R. W. J. Phys. Chem. 1984, 88, 3643-3647.

(52) Walling, C. Accounts of Chemical Research 1975, 8, 125-131.

(53) Mizuta, Y.; Masumizu, T.; Kohno, M.; Mori, A.; Packer, L. Biochem. Molecular

Biology International 1997, 43, 1107 - 1120.

(54) Finkelstein, E.; Rosen, G. M.; Rauckman, E. J. Arch. Biochem. Biophys. 1980, 200,

1-16.

(55) Haag, W. R.; Yao, C. C. D. Envir. Sci. Tech. 1992, 26, 1005-1013.

(56) Buxton, G. V.; Greenstock, C. L.; Helman, W. P.; Ross, A. B. J. Phys. Chem. Ref.

Data 1980, 17, 513-886.

(57) Kim, J.-K.; Kim, S.-M.; An, K.-W.; Choi, C.-Y.; Lee, S.-K.; Choi, K.-H. Comp.

Biochem. Physiol., Part C: Toxicol. Pharmacol. 2010, 152C, 263-269.

(58) Parajuli, S.; Miao, W. Anal. Chem. 2009, 81, 5267-5272.

100

 

CHAPTER IV

DETECTION OF TRINITROTOLUENE BY ELECTROGENERATED

CHEMILUMINESCENCE QUENCHING METHOD

Introduction

2,4,6-Trinitrotoluene (TNT) is an odorless solid that exists as colorless

orthorhombic crystals or yellow monoclinic needles but does not occur naturally in the

environment.1 The groundwater or seawater contamination has occurred due to the

production, storage, testing, and disposal of explosives at military installations, resulting

in the environmental problems because these toxic and persistent compounds can leach

from soil.2-4 At the end of World War II, an estimated 300,000 tons of explosives

(mainly TNT) were disposed to the sea.1 TNT is considered as toxic as it produces toxic

and mutagenic effects to life including human being.5 TNT is a flammable solid that has a

melting point of 355 K and autoignites at 748 K.6 It is a prime component in a majority of

munitions in use by the military and the terrorist forces around the world.7

The methods with ability for rapid on-site detection of TNT are highly desired.

Currently, several methods of detection of TNT are used, which include those based on

fluorescence,6,8-18 amperometry,19,20 surface plasmon resonance,5,21-29 mass

spectrometry,30-35 chromatography,36-40 Raman spectroscopy,41-45 and ion mobility

spectroscopy.46,47 In addition to amperometry, other electrochemical techniques have also

been used to detect TNT.48-51 Each individual detection method described above has its

own advantages, but some drawbacks remain. For example, fluorescence techniques

generally suffer from the luminescent impurities, the ion mobility spectroscopic

technique suffers from the matrix effects, and chromatographic separation-mass

101

 

spectrometric detection methods require sample preparation and are not appropriate for

field tests. These techniques use expensive instruments that frequently require skilled

manpower to operate. The immunoassay-based electrogenerated chemiluminescence

(ECL) technique has been used to detect TNT in soil and water sample,52-54 which is

very sensitive and selective, but requires extra effort in sample preparation. Methods

based on ECL quenching have been recently employed in the detection and quantification

of phenols, hydroquinone, and catechols.55,56 Because each nitro group in TNT could

undergo sequential 4e- to 6e- reduction to form hydroxylamine and amine within a

potential range of 0 to ~-1.0 V vs. SCE,57-62 TNT could be used to quench or inhibit the

ECL signals generated from a known “oxidative-reduction” type standard such as the

Ru(bpy)32+/TPrA (TPrA = tri-n-propylamine) system. As a result, a simple and

inexpensive method for field detection of TNT may be developed.

When the Ru(bpy)32+/TPrA system is used, both Ru(bpy)3

2+ and TPrA are

oxidized upon the anodic potential scanning:

TPrAeTPrA 4.1

33

23 Ru(bpy)eRu(bpy) 4.2

TPrA• free radicals are produced after the deprotonation of the newly produced TPrA•+:

-HTPrA TPrA 4.3

The TPrA• radical is a strong reducing agent with a redox potential of ~-1.7 V vs SCE,63

and is the key species of ECL generation. In the absence of TNT, the TPrA• radical

reduces Ru(bpy)32+ to Ru(bpy)3

+ that annihilates with Ru(bpy)33+ to form the excited

state Ru(bpy)32+* that emits light.

102

 

23 3Ru(bpy) TPrA Ru(bpy) P1 4.4

3 2 23 3 3 3Ru(bpy) Ru(bpy) Ru(bpy) * Ru(bpy) 4.5

2 23 3Ru(bpy) * Ru(bpy) hv 4.6

Alternatively, the excited state Ru(bpy)32+* can be produced via the following process:

3 23 3Ru(bpy) TPrA Ru(bpy) * P1 4.7

In the presence of TNT, however, part or the entire portion of the TPrA• radicals could be

consumed by TNT, resulting in significant decrease or complete quenching of the initial

ECL signals of the Ru(bpy)32+/TPrA system:

nTPrA TNT TNT P2n 4.8

Additionally, Ru(bpy)3+, another key species for ECL generation (Eq. 4.5) could be

consumed by TNT:

23 3Ru(bpy) TNT Ru(bpy) TNTnn 4.9

which leads to further decrease in ECL intensity.

Experimental Section

Chemicals

The chemicals used in this study were: acetonitrile (MeCN, 99.8%, HPLC

grade), tris-(2,2'-bypyridine)dichlororuthenium(II) hexahydrate (Ru(bpy)3Cl2•6H2O,

99.95%), and tri-n-propylamine (TPrA, 99+%) (all from Sigma-Aldrich, St. Louis, MO);

2,4,6-trinitrotoluene (TNT, 99.6%, 1000 µg/mL, Supelco analytical, PA); sodium

phosphate, monobasic monohydrate (J.T. Baker Chemicals Co., Phillipsburg, NJ); and

silver nitrate (99.5 %) and tetra-n-butylammonium perchlorate (TBAP, 99+%,

103

 

electrochemical grade) (both from Fluka, Milwaukee, WI). All chemicals were used as

received.

Electrochemical and ECL Studies

The ECL and CV responses were recorded simultaneously with a homemade

ECL instrument combined with an electrochemical workstation, which has been

described in detail in previous chapters. Note that the test solution was used without

purging with nitrogen as the electrode potential was scanned in anodic direction and

oxygen from air had no effect on ECL production.

Fluorescence Studies

Fluorescence experiments were carried out on a PTI Qunata MasterTM 40 intensity

based spectrofluorometer, with a slit width of 1.00 nm, excitation wavelength at 350 nm,

and an emission wavelength range from 500 to 690 nm.

Results and Discussion

ECL generation from the Ru(bpy)32+/TPrA system

Figure 4.1 shows the CV and ECL responses of 1.0 µM Ru(bpy)32+-25 mM TPrA

in MeCN containing 0.10 M TBAP at Pt electrode with a scan rate of 50 mV/s. TPrA is

oxidized at ~0.6 V vs Ag/Ag+ (10 mM AgNO3 in 0.10 M TBAP MeCN) and the

generation of ECL light starts from 0.9 V vs Ag/Ag+ at which Ru(bpy)32+ gets oxidized to

Ru(bpy)33+. The optimum concentration of ECL coreactant was determined by running an

ECL experiment with 1.0 µM Ru(bpy)32+ and different concentrations of TPrA with a Pt

electrode. As shown in Figure 4.2, the maximum ECL response occurs with a TPrA

concentration of 25 mM, which is in good agreement with the data reported previously.64

104

 

1.6 1.2 0.8 0.4 0.0

-120

-80

-40

0

40

80

120

E (V vs Ag/Ag+)

i CV,

-2.0

-1.5

-1.0

-0.5

0.0

0.5

1.0

1.5

2.0

i EC

L,

Figure 4.1. CV and ECL of 1.0 µM Ru(bpy)32+-25 mM TPrA in MeCN containing 0.10

M TBAP at a 2-mm diameter Pt disk electrode with a scan rate of 50 mV/s.

0 20 40 60 80 100

0.0

0.2

0.4

0.6

0.8

1.0

1.2

i EC

L (

[TPrA] (mM)

Figure 4.2. Concentration dependence of TPrA on ECL of the Ru(bpy)32+/TPrA system.

The experiment was conducted at a 2-mm Pt electrode with a scan rate of 50 mV/s using 1.0 µM Ru(bpy)3

2+ -0.10 M TBAP MeCN solutions.

This concentration of TPrA, therefore, was used for the entire experiment discussed

below, unless otherwise indicated.

105

 

ECL quenching by TNT

The ECL quenching experiment by TNT was carried out initially with 1.0 µM

Ru(bpy)32+ and 25 mM TPrA in MeCN containing 0.10 M TBAP. As shown in Figure 4.3,

the ECL emission of the Ru(bpy)32+/TPrA system is gradually decreased with successive

addition of TNT (stock solution: 1000 µg per mL in MeCN). The ECL is reduced by

~14% (Figure 4.3b) and 68% (Figure 4.3c) with a final TNT concentration of 4.4 and 110

μM, respectively.

1.5 1.0 0.5 0.0

0.0

0.2

0.4

0.6

0.8

1.0

i EC

L (

E (V vs Ag/Ag+)

(a)

(b)

(c)

Figure 4.3. ECL quenching of the Ru(bpy)32+ (1.0 µM)/TPrA (25 mM) by TNT at a final

concentration of (a) 0, (b) 4.4, and (c) 110 µM. Working electrode: 2-mm diameter Pt, scan rate: 50 mV/s. For clarity, only forward scans of the ECL are plotted.

To examine if the ECL could be completely quenched by TNT, various

concentrations of Ru(bpy)32+ and TPrA were tested. When Ru(bpy)3

2+ concentration was

down to nanomolar levels, complete quenching was observed. For example, in a 5 nM

Ru(bpy)32+-25 mM TPrA system (Figure 4.4a), addition of 44, 88, and 132 μM of TNT

results in ~76% (Figure 4.4b), 90% (Figure 4.4c), and 100% (Figure 4.4d) of the ECL

quenching, respectively. Studies on the effect of lowing TPrA concentration on the ECL

quenching (with 1.0 μM Ru(bpy)32+) revealed that with the decrease of TPrA

106

 

concentration, TNT quenching efficiency was slightly increased. However, at low TPrA

concentrations (≤10 mM), the ECL intensity of the system became weak (Figure 4.2),

which made the experimental observations difficult. Furthermore, under low TPrA

concentrations, no complete quenching of ECL with a high TNT concentration of 132

μM was observed. These data suggest that the quenching process shown in Eq. 4.8 is

probably a relatively slow electron transfer process with respective to that of Eq. 4.9. In

other words, ECL quenching by TNT via Ru(bpy)3+ consumption is more favorable than

direct TPrA• free radical consumption. If that is the case, under constant TPrA and TNT

concentrations, ECL quenching efficiency should increase with the increase in

Ru(bpy)32+ concentration.

1.5 1.0 0.5 0.0

0

10

20

30

40

(d)

(c)

(b)

(a)

i EC

L (

nA

)

E (V vs Ag/Ag+)

Figure 4.4. ECL quenching by TNT in the system containing 5 nM Ru(bpy)32+-25 mM

TPrA-0.10 M TBAP in MeCN with added TNT concentrations of (a) 0, (b) 44, (c) 88, and (d) 132 μM. A 2-mm diameter of Pt electrode and a scan rate of 50 mV/s were used for all tests.

107

 

Figure 4.5 illustrates the results of such an example, where the ECL is quenched

by ~38% at 1.0 μM Ru(bpy)32+, and ~45% at 10 μM Ru(bpy)3

2+ after the system is added

with 4.4 μM TNT (Figure 4.5b). Notably, increase in Ru(bpy)32+ concentration linearly

increases the ECL intensity with and without TNT, but the presence of constant amount

of TNT shows quenching effect throughout the experiment, which confirms quenching

effect is more related to the Ru(bpy)32+ than to TPrA.

As shown in Figure 4.6, a linear decrease in ECL intensity of the standard

Ru(bpy)32+ (1.0 µM)/TPrA (25 mM) system is evident with the addition of TNT. This

quenching method provides a TNT detection limit of 4.4 µM.

Fluorescence quenching of Ru(bpy)32+ by TNT

0 2 4 6 8 100

2

4

6

8

10

12

i EC

L (

[Ru(bpy)3

2+] (M)

(a)

(b)

Figure 4.5. Ru(bpy)32+ concentration effect on ECL (a) without, and (b) with 4.4 μM

TNT addition to the MeCN solution containing 25 mM TPrA-0.10 M TBAP. A 2-mm diameter of Pt electrode and a scan rate of 50 mV/s were used for all tests.

108

 

The fluorescence quenching study was carried out with 1.0 µM Ru(bpy)32+ in

MeCN with a quartz cuvette. The test solution was excited at 350 nm and Ru(bpy)32+

produced the fluorescence emission peak at 620 nm. The addition of TNT results in a

decrease in fluorescent emission of the test solution as shown in Figure 4.7A.

0 20 40 60 80 100 1200.2

0.4

0.6

0.8

1.0

1.2

i EC

L(

[TNT] (M)

[TNT] = 0

Figure 4.6. Relationship of ECL intensity with the TNT concentration in the system containing 1.0 µM Ru(bpy)3

2+-25 mM TPrA-0.10 M TBAP in MeCN at a 2-mm Pt electrode at a scan rate of 50 mV/s.

The decrease in fluorescence emission with 110 µM TNT was about 42% of the

emission of Ru(bpy)32+ without TNT added. In order to verify the dilution effect by the

addition of TNT which was in MeCN, the control experiment was carried out with the

same concentration of Ru(bpy)32+ and similar volumes of MeCN were added to the test

solution and fluorescence emissions were recorded. The control experiment produces the

results (Figure 4.7B) that suggest the effect of dilution on fluorescence emission is

negligible and within the range of experimental errors.

109

 

500 550 600 650 700

Wavelength (nm)

5x1

04 , a

.u.

FL

Inte

nsity

Added [TNT]0

110 M

(A)

500 550 600 650 700

Wavelength (nm)

0 L MeCN 4.4 L MeCN 13.2 L MeCN 22 L MeCN 44 LMeCN 66 LMeCN 88 L MeCN 110L MeCN

5x1

04, a

.u.

FL

Inte

nsity

(B) 0

110 L

MeCN added

Figure 4.7. Fluorescence quenching of 3.0 mL of 1.0 μM Ru(bpy)32+ in MeCN by TNT

with 1.0 cm quartz cuvette and an excitation at 350 nm. (A) TNT concentration dependence, and (B) MeCN dilution effect.

Calculation of Quenching Constant56,65,66

The quantum yield of ECL (or fluorescence) of Ru(bpy)32+ in MeCN in the

absence of quenching agent (Ф0) is the rate at which the excited state decays via ECL (or

fluorescence) over sum of the rates of its decay by ECL (or fluorescence) and non-

radiative mechanism as:

2ECL(f) 30

2 2ECL(f) 3 nr 3

ECL(f)

ECL(f) nr

[Ru(bpy) *]

[Ru(bpy) *] [Ru(bpy) *]

+

K

K K

K

K K

4.10

where KECL(f) and Knr stand for the rate constants for ECL (or fluorescence) and

nonradiative decay of the excited state Ru(bpy)32+*. In the presence of quenching agent

like TNT, the quantum yield could be expressed as:

110

 

2ECL(f) 3

2 2 2ECL(f) 3 nr 3 q 3

ECL(f)

ECL(f) nr q

[Ru(bpy) *]

[Ru(bpy) *] [Ru(bpy) *] [TNT][Ru(bpy) *]

=[TNT]

K

K K K

K

K K K

4.11

where Kq is the quenching constant of ECL (or fluorescence). By dividing Eq. 4.9 by Eq. 4.10 we get,

0 0q

SVECL(f) nr

1 [TNT] 1 [TNT]KI

KI K K

4.12

This is Stern-Volmer equation, in which the constant terms give the slope of a plot and is

called Stern-Volmer constant (KSV). I and I0 stand for the ECL (or fluorescence)

intensities with and without quenching agent, TNT. The reciprocal of (KECL(f) + Knr) gives

the life time (τ) of Ru(bpy)32+* excited state.

ECL(f) nr

1

K K

4.13

0 20 40 60 80 100 120

1.0

1.5

2.0

2.5

3.0

3.5

4.0

I 0 / I

[TNT] (M)

Figure 4.8. Stern-Volmer plot of ECL quenching by TNT. See Figure 4. 6 for experimental conditions.

111

 

0 20 40 60 80 100 1200.8

1.0

1.2

1.4

1.6

I0 /I

[TNT] (M)

Figure 4.9. Stern-Volmer plot of fluorescence quenching by TNT. See Figure 4. 7 for experimental conditions.

On the basis of data shown in Figure 4.6 and Figure 4.7A, the Stern-Volmer plots

of ECL and fluorescence quenching by TNT can be plotted as shown in Figure 4.8 and

Figure 4.9, respectively. The slopes of the Stern-Volmer plots for ECL and fluorescence

quenching were calculated to be 2.0×104 M-1 (Figure 4.8) and 5.2×103 M-1 (Figure 4.9),

respectively. The quenching constant (Kq) calculated with Stern-Volmer constants and

life time of Ru(bpy)32+* excited state in MeCN (0.562 µs)67 came out to be 3.5×1010

M-1s-1 for ECL quenching and 9.2×109 M-1 s-1 for fluorescent quenching. The Stern-

Volmer plots suggest that the ECL quenching is about 4 times more efficient compared to

the fluorescence quenching with TNT and both of these quenching processes most likely

follow the dynamic process.66 Besides the fluorescence quenching (or energy-transfer

quenching, Eq. 4.14), ECL quenching of the Ru(bpy)32+/TPrA system by TNT also

involves processes shown in Eqs. 4.8 and 4.9. As a result, ECL quenching is more

efficient than fluorescence quenching for the system reported in this chapter.

112

 

2+ 2+3 3 high energyRu(bpy) * + TNT Ru(bpy) + (TNT) 4.14

Conclusions

The high explosive compound 2,4,6-trinitrotoluene (TNT) was used as an ECL

quenching agent in the Ru(bpy)32+/TPrA system. The TNT was found to quench both

ECL and fluorescence emission of Ru(bpy)32+* in MeCN. This detection technique

provides a simple and easy way of detecting TNT at lower micromolar levels. It was

realized that the Ru(bpy)32+/TPrA ECL was quenched by depleting Ru(bpy)3

+ and TPrA•

free radical from the system which otherwise would have produced ECL light as well as

excited state energy transfer process as suggested by the fluorescence studies.

113

 

References

(1) Wilson, R.; Clavering, C.; Hutchinson, A. Analyst 2003, 128, 480.

(2) Wang, J.; Bhada, R. K.; Lu, J.; MacDonald, D. Anal. Chim. Acta 1998, 361, 85.

(3) Sohn, H.; Calhoun, R. M.; Sailor, M. J.; Trogler, W. C. Angew. Chem., Int. Ed. 2001,

40, 2104.

(4) Hilmi, A.; Luong, J. H. T.; Nguyen, A.-L. J. Chromatogr., A 1999, 844, 97.

(5) Shankaran, D. R.; Gobi, K. V.; Sakai, T.; Matsumoto, K.; Toko, K.; Miura, N.

Biosens. Bioelectron. 2005, 20, 1750.

(6) Boudreaux, G. M.; Miller, T. S.; Kunefke, A. J.; Singh, J. P.; Yueh, F.-Y.; Monts, D.

L. Appl. Opt. 1999, 38, 1411.

(7) Shankaran, D. R.; Kawaguchi, T.; Kim, S. J.; Matsumoto, K.; Toko, K.; Miura, N.

Anal. Bioanal. Chem. 2006, 386, 1313.

(8) Content, S.; Trogler, W. C.; Sailor, M. J. Chem.--Eur. J. 2000, 6, 2205.

(9) Bakaltcheva, I. B.; Shriver-Lake, L. C.; Ligler, F. S. Sens. Actuators, B 1998, B51, 46.

(10) Medintz, I. L.; Goldman, E. R.; Lassman, M. E.; Hayhurst, A.; Kusterbeck, A. W.;

Deschamps, J. R. Anal. Chem. 2005, 77, 365.

(11) Shriver-Lake, L. C.; Breslin, K. A.; Charles, P. T.; Conrad, D. W.; Golden, J. P.;

Ligler, F. S. Anal. Chem. 1995, 67, 2431.

(12) Anderson, G. P.; Moreira, S. C.; Charles, P. T.; Medintz, I. L.; Goldman, E. R.;

Zeinali, M.; Taitt, C. R. Anal. Chem. 2006, 78, 2279.

(13) Wu, D.; Singh, J. P.; Yueh, F. Y.; Monts, D. L. Appl. Opt. 1996, 35, 3998.

(14) Naddo, T.; Che, Y.; Zhang, W.; Balakrishnan, K.; Yang, X.; Yen, M.; Zhao, J.;

Moore, J. S.; Zang, L. J. Am. Chem. Soc. 2007, 129, 6978.

(15) Altamirano, M.; Garcia-Villada, L.; Agrelo, M.; Sanchez-Martin, L.; Martin-Otero,

L.; Flores-Moya, A.; Rico, M.; Lopez-Rodas, V.; Costas, E. Biosens. Bioelectron.

2004, 19, 1319.

(16) Tao, S.; Li, G.; Yin, J. J. Mater. Chem. 2007, 17, 2730.

(17) Narang, U.; Gauger, P. R.; Kusterbeck, A. W.; Ligler, F. S. Anal. Biochem. 1998,

255, 13.

114

 

(18) Narayanan, A.; Varnavski, O. P.; Swager, T. M.; Goodson, T., III J. Phys. Chem. C

2008, 112, 881.

(19) Buttner, W. J.; Findlay, M.; Vickers, W.; Davis, W. M.; Cespedes, E. R.; Cooper, S.;

Adams, J. W. Anal. Chim. Acta 1997, 341, 63.

(20) Hilmi, A.; Luong, J. H. T.; Nguyen, A.-L. Anal. Chem. 1999, 71, 873.

(21) Riskin, M.; Tel-Vered, R.; Lioubashevski, O.; Willner, I. J. Am. Chem. Soc. 2009,

131, 7368.

(22) Singh, P.; Onodera, T.; Matsumoto, K.; Miura, N.; Toko, K. Mater. Res. Soc. Symp.

Proc. 2007, 951E, No pp. given.

(23) Singh, P.; Onodera, T.; Mizuta, Y.; Matsumoto, K.; Miura, N.; Toko, K. Sens. Mater.

2007, 19, 261.

(24) Bowen, J.; Noe, L. J.; Sullivan, B. P.; Morris, K.; Martin, V.; Donnelly, G. Appl.

Spectrosc. 2003, 57, 906.

(25) Strong, A. A.; Stimpson, D. I.; Bartholomew, D. U.; Jenkins, T. F.; Elkind, J. L.

Proc. SPIE-Int. Soc. Opt. Eng. 1999, 3710, 362.

(26) Shankaran, D. R.; Gobi, K. V.; Sakai, T.; Matsumoto, K.; Imato, T.; Toko, K.; Miura,

N. IEEE Sens. J. 2005, 5, 616.

(27) Mizuta, Y.; Onodera, T.; Singh, P.; Matsumoto, K.; Miura, N.; Toko, K. Sens. Mater.

2010, 22, 193.

(28) Kawaguchi, T.; Shankaran, D. R.; Kim, S. J.; Matsumoto, K.; Toko, K.; Miura, N.

Sens. Actuators, B 2008, B133, 467.

(29) Matsumoto, K.; Torimaru, A.; Ishitobi, S.; Sakai, T.; Ishikawa, H.; Toko, K.; Miura,

N.; Imato, T. Talanta 2005, 68, 305.

(30) Yinon, J.; Boettger, H. G.; Weber, W. P. Anal. Chem. 1972, 44, 2235.

(31) Garcia-Reyes, J. F.; Harper, J. D.; Salazar, G. A.; Charipar, N. A.; Ouyang, Z.;

Cooks, R. G. Anal. Chem., 2011, 83, 1084.

(32) Sanders, N. L.; Kothari, S.; Huang, G.; Salazar, G.; Cooks, R. G. Anal. Chem. 2010,

82, 5313.

(33) Zhang, Y.; Ma, X.; Zhang, S.; Yang, C.; Ouyang, Z.; Zhang, X. Analyst 2009, 134,

176.

115

 

(34) Zhou, Y.-M.; Jin, W.; Chen, H.-W.; Xu, R.-F. Fenxi Huaxue 2007, 35, 621.

(35) Song, Y.; Chen, H.; Cooks, R. G. Rapid Commun. Mass Spectrom. 2005, 19, 3493.

(36) Yinon, J. TrAC, Trends Anal. Chem. 2002, 21, 292.

(37) Batlle, R.; Carlsson, H.; Holmgren, E.; Colmsjo, A.; Crescenzi, C. J. Chromatogr., A

2002, 963, 73.

(38) Batlle, R.; Carlsson, H.; Tollbaeck, P.; Colmsjoe, A.; Crescenzi, C. Anal. Chem.

2003, 75, 3137.

(39) Walsh, M. E. Talanta 2001, 54, 427.

(40) Bromberg, A.; Mathies, R. A. Anal. Chem. 2003, 75, 1188.

(41) Jerez, R. J. I.; Balaguera, M. d. R.; Cabanzo, A.; de, l. C. M. E.; Hernandez-Rivera,

S. P. Proc. SPIE-Int. Soc. Opt. Eng. 2006, 6201, 62012G/1.

(42) Chamoun-Emanuelli, A. M.; Primera-Pedrozo, O. M.; Barreto-Caban, M. A.; Jerez-

Rozo, J. I.; Hernandez-Rivera, S. P. ACS Symp. Ser. 2009, 1016, 217.

(43) Jerez-Rozo, J. I.; Primera-Pedrozo, O. M.; Barreto-Caban, M. A.; Hernandez-Rivera,

S. P. IEEE Sens. J. 2008, 8, 974.

(44) Wang, X.; Chang, S.; Yang, J.; Tan, J.; Jia, H.; Yin, H.; Li, X.; Peng, G. Proc. SPIE

2008, 6622, 662219/1.

(45) Kneipp, K.; Wang, Y.; Dasari, R. R.; Feld, M. S.; Gilbert, B. D.; Janni, J.; Steinfield,

J. I. Spectrochim. Acta, Part A 1995, 51A, 2171.

(46) Lokhnauth, J. K.; Snow, N. H. J. Chromatogr., A 2006, 1105, 33.

(47) Klassen, S. E.; Rodacy, P.; Silva, R. Reactant ion chemistry for detection of TNT,

RDX, and PETN using an ion mobility spectrometer, Sandia National Laboratories,

1997.

(48) Trammell, S. A.; Velez, F.; Charles, P. T.; Kusterbeck, A. Anal. Lett. 2008, 41, 2634.

(49) Wang, F.; Wang, W.; Liu, B.; Wang, Z.; Zhang, Z. Talanta 2009, 79, 376.

(50) Bozic, R. G.; West, A. C.; Levicky, R. Sens. Actuators, B 2008, B133, 509.

(51) Cespedes, E. R.; Cooper, S. S.; Davis, W. M.; Buttner, W. J.; Vickers, W. C. Proc.

SPIE-Int. Soc. Opt. Eng. 1995, 2367, 33.

(52) Pittman, T. L.; Thomson, B.; Miao, W. Anal. Chim. Acta 2009, 632, 197.

(53) Wilson, R.; Clavering, C.; Hutchinson, A. J. Electroanal. Chem. 2003, 557, 109.

116

 

(54) Wilson, R.; Clavering, C.; Hutchinson, A. Anal. Chem. 2003, 75, 4244.

(55) McCall, J.; Alexander, C.; Richter, M. M. Anal. Chem. 1999, 71, 2523.

(56) Zheng, H.; Zu, Y. J. Phys. Chem. B 2005, 109, 16047.

(57) Wang, J. In Counterterrorist Detection Techniques of Explosives; Yinon, J., Ed.;

Elsevier B. V.: Amsterdam, 2007, p 91.

(58) Zhang, H.-X.; Cao, A.-M.; Hu, J.-S.; Wan, L.-J.; Lee, S.-T. Anal. Chem. 2006, 78,

1967.

(59) Saravanan, N. P.; Venugopalan, S.; Senthilkumar, N.; Santhosh, P.; Kavita, B.;

Prabu, H. G. Talanta 2006, 69, 656.

(60) Wang, J.; Bhada, R. K.; Lu, J.; MacDonald, D. Anal. Chim. Acta 1998, 361, 85.

(61) Hilmi, A.; Luong, J. H. T.; Nguyen, A.-L. J. Chromatogr. A 1999, 844, 97.

(62) Sohn, H.; Calhoun, R. M.; Sailor, M. J.; Trogler, W. C. Angew. Chem. Int. Ed. 2001,

40, 2104.

(63) Lai, R. Y.; Bard, A. J. J. Phys. Chem. A 2003, 107, 3335.

(64) Miao, W.; Bard, A. J. Anal. Chem. 2003, 75, 5825.

(65) Legenza, M. W.; Marzzacco, C. J. J. Chem. Edu. 1977, 54, 183.

(66) Cheng, P. P. H.; Silvester, D.; Wang, G.; Kalyuzhny, G.; Douglas, A.; Murray, R. W.

J. Phys. Chem. B 2006, 110, 4637.

(67) Caspar, J. V.; Meyer, T. J. J. Am. Chem. Soc. 1983, 105, 5583.

117

 

CHAPTER V

ELECTROCHEMICAL DETECTION OF PENTAERYTHRITOL TETRANITRATE

Introduction

The detection of explosive compounds has become very crucial because of its

increasing use by the terrorist groups in the places of public importance, such as airports,

train stations, power stations, governmental buildings, etc. The explosive detection plays

the key role in investigating the crime scenes and counter-terrorism act of screening in

the airport check points.1 Since the terrorist attacks of September 11, 2001, the detection

of high explosives has been in the forefront of the analytical chemists.2 As a result, the

development of analytical techniques for trace-level detection of explosives has always

been in need with high sensitivity and selectivity. Pentaerythritol tetranitrate (PETN) is

one of the common high explosives used in terrorist attacks because this can be used with

plastic filler and molded for concealment-the infamous ‘shoe bomber’ had concealed

PETN in his shoes.3 PETN attracts considerable interest as it can be converted to a sheet

form or forms Semtex mixed with RDX, an example of plastic explosives.4,5

There are several methods of detection of high explosives such as PETN, namely,

mass spectrometry,4,6-15 ion mobility spectrometry,2,16-21 spectroscopic methods such as

Raman spectroscopy,22-30 Infra red,31,32 fluorescence,1,33-36 terahertz,37,38

chromatographic,39,40 and electrochemical methods.41,42

The mass spectrometric method obviously is a sensitive method for detecting

explosives but requires a skilled operator as well as expensive instrument which are not

appropriate for field detection. The ion mobility spectrometry (IMS) is sensitive and can

be used for trace detection but has been associated with the competitive ion/molecule

118

 

reaction with matrix, the response is sensitive to the instrument contamination with long

clearance time of instrument.43,44 The Raman spectroscopy suffers from the fluorescent

impurities,45 and high background response.46 There is an urgent need of an analytical

method of detection high explosives with high sensitivity and portability for field tests.

Electrochemical methods including amperometric method coupled with

electrophoresis 41 and immunoassay-based electrogenerated chemiluminescence (ECL)

with Ru(bpy)32+ as luminophore and electrochemically generated H2O2 as a coreactant47

have been used to detect nitrate esters. The immunoassay-based technique requires

tedious effort and time for sample preparation. The direct and easy-to-operate and

portable method is highly desired. In the present work, detection and quantification of

PETN by cyclic voltammetry (CV) will be reported. This method is based on the fact the

nitro group in PETN is electrochemically reduced at the surface of working electrode

when scanned cathodically from -0.3 to -1.05 V vs Ag/Ag+. Based on the literature each

nitro group undergoes two electron process as suggested by the possible mechanism:48

OH3RONOe2OH2RONO 222 5.1

22 NOROe2RONO 5.2

OHROHOHRO 2 5.3

32 NORe2RONO 5.4

OHRHOHR 2 5.5

Scheme 5.1. Mechanism of reduction of organic ester.

119

 

Neither nitrite ester nor hydrocarbon were observed in the reduction process and

only alcohol and nitrite ions were observed,48 only the processes involving Eqs.5.2 and

5.3 become apparent.

Experimental Section Chemicals

Sulfuric acid (95-98%, ACS reagent), nitric acid (70 %, ACS reagent), and

pentaerythritol (99+%) were purchased from Sigma-Aldrich (St. Louis, MO). Tetra-n-

butylammonium perchlorate (TBAP, 99+%, electrochemical grade) and silver nitrate

(99.5%) were from Fluka (Milwaukee, WI). Sodium carbonate and urea were purchased

from J.T. Baker Inc. (Phillipsburg, NJ). All chemicals were used as received.

Synthesis and characterization of pentaerythritol tetranitrate (PETN)

Concentrated nitric acid (14 mL) was chilled to 0 °C in an ice bath for half an

hour and 4.0 g of pentaerythritol was dissolved slowly with constant stirring. After

complete dissolution of pentaerythritol, concentrated sulfuric acid was added dropwise

and 0.03 g of urea was added to this mixture which could remove NO or NO2 produced.

This mixture left to stir at 0 °C for 2 h.

The precipitate, as shown in Scheme 5.2, was obtained after mixing reaction

mixture in ice cold water. This precipitate was filtered with excess of water and sodium

carbonate solution (0.10 M) to remove excess acid. Then the precipitate was dissolved in

acetone (250 mL) by heating in a water bath at 40 °C. PETN was recrystallized in a

mixture of ethanol (75 mL) and water (100 mL) in 2 h. White needles of PETN were

obtained and washed with ethanol.

120

 

Pentaerythritol

H2SO4Dissolve

0 oC

Stir at 0 oC for 2h

Ice cold water

Reaction mixtureFilter

Wash with waterPrecipitate

HNO3 in ice bath

Conc.

Scheme 5.2. Synthesis of Pentaerythritol Tetranitrate (PETN).

PETN was characterized by proton NMR (1H NMR) where all equivalent protons produced NMR signal at 4.7 ppm which was consistent with the literature49 as shown in Figure 5.1

10 8 6 4 2 0

,ppm

-CH2

H2O CH

3COCH

3

Figure 5.1. 1H NMR of PETN taken in deuterated acetonitrile (CD3CN).

FTIR spectra were also taken for starting material, i.e., pentaerythritol and PETN,

which could conclude the formation of PETN. The characteristic peak for OH group of

starting material at 3350 cm-1 disappeared for PETN which lacks that OH group.

121

 

4 0 0 0 3 5 0 0 3 0 0 0 2 5 0 0 2 0 0 0 1 5 0 0 1 0 0 0 5 0 00 .7

0 .6

0 .5

0 .4

0 .3

0 .2

0 .1

0 .0

-0 .1

Abs

orb

ance

W a v e n u m b e r , c m -1

P E T N P e n ta e ry th r ito l

O H

C H2 (A s y m .)

N O2

C H 2 (S c i.)

(a )

(b )

Figure 5.2. FTIR spectra of (a) pentaerythritol and (b) PETN.

Electrochemical Studies

The cyclic voltammetry (CV) was carried out with an electrochemical workstation,

model 660A (CH Instruments, Austin, TX). The conventional three electrode system was

used with test solution (1.0 mL) in a small glass vials. The working electrode used was a

silver wire that had an exposure area of ~1 mm2. Reference and counter electrodes were

Ag/Ag+ (10 mM AgNO3, 0.10 M TBAP in MeCN) and Pt gauze, respectively. The

solution was purged with nitrogen (ultrapure, Nordan Smith, Hattiesburg, MS) since the

potential scanning was done in cathodic direction.

Results and Discussion

Cyclic Voltammetric Studies of PETN

The cyclic voltammetric (CV) responses were recorded with a silver wire as

working electrode scanned between -0.3 to -1.05 V vs Ag/Ag+. Other working electrodes

including Pt, glassy carbon, and gold were tested, but none gave any meaningful

electrochemical response. An irreversible reduction wave of PETN at around -0.9 V vs

122

 

Ag/Ag+ was observed (Figure 5.3), which may correspond to the reduction of part of the

four nitro functional groups in PETN. However, the exact number of electron-transfer

involved is unclear, as the blank shows a large background current (Figure 53a).

-0.4 -0.6 -0.8 -1.0

0

2

4

6

8

i CV (

A

)

E (V vs Ag/Ag+)

(a)

(b)

(c)

Figure 5.3. Forward scans of cyclic voltammetry: (a) 0.10 M TBAP in MeCN as blank, (b) 25 µM PETN in MeCN, and (c) 600 µM PETN in MeCN with 0.10 M TBAP as supporting electrolyte at a silver wire working electrode (exposure area: ~1 mm2). Scan rate was 50 mV/s. For clarity, the reverse scans were not included in the plot.

The CV response of PETN in MeCN, as shown in Figure 5.3, was weak and it did

not increase with the increase of PETN concentration as expected. This is perhaps due to

the fact that PETN is a significantly condensed material with a very high density of 1.77

g/cm3 at 20 °C, which may prevent it from the effective attack of electrons. Figure 5.4

shows a dynamic range of 25 µM to 1.0 mM, with a detection limit of PETN at ~25 µM.

Among the available methods of detection of PETN this current electrochemical

technique seems to be inexpensive, portable, fast, and easy to handle. The methods based

on mass or ion mobility spectrometry are expensive and cumbersome in handling the

experiment.

123

 

0 200 400 600 800 10001.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

25 M

i CV (A

)

[PETN] (M)

Figure 5.4. Peak current change of the forward scan in PETN CV as a function of PETN concentration. The data were obtained from a silver wire working electrode (effective surface area: ~1 mm2) in MeCN containing 0.10 M TBAP at a scan rate of 50 mV/s.

Future Work

Immunoassay based ECL detection of PETN with TPrA as coreactant and

Ru(bpy)32+ as ECL luminophore could give very high ECL response50 compared to the

one with electrochemically generated hydrogen peroxide from dissolved oxygen as

coreactant.42 In this method, Ru(bpy)32+ and the antibody specific to PETN could be

attached to the surface of functionalized polystyrene bead and the same antibody to the

magnetic bead. When the PETN sample is added to the solution containing polystyrene

bead with both Ru(bpy)32+ and PETN antibody and magnetic bead with PETN antibody,

the sandwich-type assay is formed as shown in Scheme 5.2:

124

 

+ __ECL Detection

PSB

PETN

Antibody

MB

Magnet

Ru(II) Ru(II)

Scheme 5.3. Immunoassay based ECL detection of PETN.

When Ru(bpy)32+/PETN antibody attached polystyrene beads and PETN antibody

attached magnetic beads are mixed in the presence of PETN analyte which acts as antigen,

a sandwich type assay is formed. This mixture is then brought to the magnet where only

sandwich type assay will be attracted to the magnet because of magnetic bead. The

unbound Ru(bpy)32+-polystyrene beads will be separated and washed from the solution

with magnet in place ensuring that unbound Ru(bpy)32+ is completely removed. In ECL

experiment, more Ru(bpy)32+ means high ECL response, which means more PETN

present in the test solution, as this PETN is responsible for the sandwich type assay

formation.

Conclusion

A simple electrochemical method using cyclic voltammetry proved to be a

sensitive method in detecting PETN in MeCN with silver as the working electrode.

Scanned in cathodic direction from -0.3 to -1.05 V vs Ag/Ag+, PETN was reduced around

-0.9 V vs Ag/Ag+. With this method micro molar concentrations of PETN were detected,

with a limit of detection of 25 µM.

125

 

References

(1) Sanchez, J. C.; Toal, S. J.; Wang, Z.; Dugan, R. E.; Trogler, W. C. J. Forensic Sci.

2007, 52, 1308.

(2) Babis, J. S.; Sperline, R. P.; Knight, A. K.; Jones, D. A.; Gresham, C. A.; Denton, M.

B. Anal. Bioanal. Chem. 2009, 395, 411.

(3) Pinnaduwage, L. A.; Boiadjiev, V.; Hawk, J. E.; Thundat, T. Appl. Phys. Lett. 2003,

83, 1471.

(4) Crellin, K. C.; Dalleska, N.; Beauchamp, J. L. Int. J. Mass Spectrom. Ion Processes

1997, 165/166, 641.

(5) Fainberg, A. Science 1992, 255, 1531.

(6) Garcia-Reyes, J. F.; Harper, J. D.; Salazar, G. A.; Charipar, N. A.; Ouyang, Z.; Cooks,

R. G. Anal. Chem. 2011, 83, 1084.

(7) Hilton, C. K.; Krueger, C. A.; Midey, A. J.; Osgood, M.; Wu, J.; Wu, C. Int. J. Mass

Spectrom. 2010, 298, 64.

(8) Martinez-Lozano, P.; Rus, J.; Fernandez, d. l. M. G.; Hernandez, M.; Fernandez, d. l.

M. J. J. Am. Soc. Mass Spectrom. 2009, 20, 287.

(9) Zhang, Y.; Ma, X.; Zhang, S.; Yang, C.; Ouyang, Z.; Zhang, X. Analyst 2009, 134,

176.

(10) Oehrle, S. A. J. Energ. Mater. 2008, 26, 197.

(11) Justes, D. R.; Talaty, N.; Cotte-Rodriguez, I.; Cooks, R. G. Chem. Commun.2007,

2142.

(12) Sleeman, R.; Richards, S. L.; Fletcher, I.; Burton, A.; Luke, J. G.; Stott, W. R.;

Davidson, W. R. NATO Sci. Ser., II 2004, 167, 133.

(13) Tam, M.; Hill, H. H., Jr. Anal. Chem. 2004, 76, 2741.

(14) Marshall, A.; Clark, A.; Ledingham, K. W. D.; Sander, J.; Singhal, R. P.; Kosmidis,

C.; Deas, R. M. Rapid Commun. Mass Spectrom. 1994, 8, 521.

(15) Boumsellek, S.; Alajajian, S. H.; Chutjian, A. J. Am. Soc. Mass Spectrom. 1992, 3,

243.

(16) Tabrizchi, M.; Ilbeigi, V. J. Hazard. Mater. 2010, 176, 692.

(17) Koyuncu, H.; Seven, E.; Calimli, A. Turk. J. Chem. 2005, 29, 255.

126

 

(18) Khayamian, T.; Tabrizchi, M.; Jafari, M. T. Talanta 2003, 59, 327.

(19) Su, C.-W.; Babcock, K. Int. J. Ion Mobility Spectrom. 2002, 5, 55.

(20) Ewing, R. G.; Miller, C. J. Field Anal. Chem. Technol. 2001, 5, 215.

(21) Buryakov, I. A.; Kolomiets, Y. N.; Louppou, V. B. Int. J. Ion Mobility Spectrom.

2001, 4, 13.

(22) Chen, M.; Zou, Z.; Wu, H.; Lu, W.; Beijing Luyuan Opto Technology Co., Ltd.,

Peop. Rep. China; Guilin Guangtong Electronics Engineering Co., Ltd. . 2010, p.8.

(23) Zachhuber, B.; Ramer, G.; Hobro, A. J.; Lendl, B. Proc. SPIE 2010, 7838, 78380F/1.

(24) Tuschel, D. D.; Mikhonin, A. V.; Lemoff, B. E.; Asher, S. A. Appl. Spectrosc. 2010,

64, 425.

(25) Ali, E. M. A.; Edwards, H. G. M.; Scowen, I. J. J. Raman Spectrosc. 2009, 40, 2009.

(26) Ali, E. M. A.; Edwards, H. G. M.; Scowen, I. J. Talanta 2009, 78, 1201.

(27) Ballesteros, L. M.; Herrera, G. M.; Castro, M. E.; Briano, J.; Mina, N.; Hernandez-

Rivera, S. P. Proc. SPIE-Int. Soc. Opt. Eng. 2005, 5794, 1254.

(28) Carter, J. C.; Angel, S. M.; Lawrence-Snyder, M.; Scaffidi, J.; Whipple, R. E.;

Reynolds, J. G. Appl. Spectrosc. 2005, 59, 769.

(29) Hayward, I. P.; Kirkbride, T. E.; Batchelder, D. N.; Lacey, R. J. J. Forensic Sci.

1995, 40, 883.

(30) Carver, F. W. S.; Wyndham, D. P.; Sinclair, T. J. J. Raman Spectrosc. 1985, 16, 332.

(31) Ballesteros-Rueda, L. M.; Herrera-Sandoval, G. M.; Mina, N.; Castro-Rosario, M. E.;

Briano, J. G.; Hernandez-Rivera, S. P. Proc. SPIE-Int. Soc. Opt. Eng. 2006, 6217,

62173D/1.

(32) Buryakov, I. A. NATO Sci. Ser., II 2002, 66, 69.

(33) Andrew, T. L.; Swager, T. M. J. Am. Chem. Soc. 2007, 129, 7254.

(34) Swayambunathan, V.; Singh, G.; Sausa, R. C. Appl. Opt. 1999, 38, 6447.

(35) Kennedy, S.; Caddy, B.; Douse, J. M. F. J. Chromatogr., A 1996, 726, 211.

(36) Judd, L. L.; Kusterbeck, A. W.; Conrad, D. W.; Yu, H.; Myles, H. L., Jr.; Ligler, F.

S. Proc. SPIE-Int. Soc. Opt. Eng. 1995, 2388, 198.

(37) Leahy-Hoppa, M. R.; Fitch, M. J.; Osiander, R. Proc. SPIE 2008, 6893, 689305/1.

127

 

(38) Fitch, M. J.; Leahy-Hoppa, M. R.; Ott, E. W.; Osiander, R. Chem. Phys. Lett. 2007,

443, 284.

(39) Chaloosi, M.; Nejad-Darzi, S. K. H.; Ghoulipour, V. Propellants, Explos., Pyrotech.

2009, 34, 50.

(40) Bowerbank, C. R.; Smith, P. A.; Fetterolf, D. D.; Lee, M. L. J. Chromatogr., A 2000,

902, 413.

(41) Piccin, E.; Dossi, N.; Cagan, A.; Carrilho, E.; Wang, J. Analyst 2009, 134, 528.

(42) Wilson, R.; Clavering, C.; Hutchinson, A. Anal. Chem. 2003, 75, 4244.

(43) Dworzanski, J. P.; Kim, M.-G.; Snyder, A. P.; Arnold, N. S.; Meuzelaar, H. L. C.

Anal. Chim. Acta 1994, 293, 219.

(44) Hill, H. H. a. S., Greg Field Anal. Chem. Technol. 1997, 1, 119.

(45) Kneipp, K.; Kneipp, H.; Itzkan, I.; Dasari, R. R.; Feld, M. S. Chem. Rev.1999, 99,

2957.

(46) Carey, P. R. J. Biol. Chem. 1999, 274, 26625.

(47) Wilson, R.; Clavering, C.; Hutchinson, A. Analyst 2003, 128, 480.

(48) Kaufman, F.; Cook, H. J.; Davis, S. M. J. Am. Chem. Soc. 1952, 74, 4997.

(49) Katritzky, A. R.; Rogers, J. W.; Witek, R. M.; Vakulenko, A. V.; Mohapatra, P. P.;

Steel, P. J.; Damavarapu, R. J. Energ. Mater. 2007, 25, 79.

(50) Pittman, T. L.; Thomson, B.; Miao, W. Anal. Chim. Acta 2009, 632, 197.

128  

CHAPTER VI

CONCLUDING REMARKS

The use of explosive materials by the terrorists has become a serious threat to the

modern world and there is an urgent need of analytical techniques that can be used to

sensitively detect and quantify them. The techniques that make use of portable and

inexpensive devices are highly desirable for field test of the explosive materials that

could be confiscated from the suspect especially in the transportation hubs.

Electrogenerated Chemiluminescence (ECL) is normally a nondestructive method

of detection and quantification of analytes and very sensitive especially used as the

Ru(bpy)32+/TPrA system ( Ru(bpy)3

2+ = tris-(2,2′-bipyridine)ruthenium (II) cation and

TPrA = tri-n-propylamine). Peroxide-based explosives such as hexamethylene triperoxide

diamine (HMTD) and triacetone triperoxide (TATP), which are very popular among

terrorists because of their simple synthesis from commercially available chemicals, have

proved to be sensitively and selectively detectable with ECL technique similar to the

Ru(bpy)32+/TPrA system.

HMTD has three tertiary amine as well as three peroxide functional groups. These

functional groups can be exploited as coreactant for ECL as common coreactants such as

TPrA and hydrogen peroxide (H2O2). HMTD produces ECL response with Ru(bpy)32+ on

anodic potential scanning which leads to its detection by this method. The ECL response

was enhanced by the addition of AgNO3 which on oxidation produces Ag(II) ions and

NO3• radicals, both of which are strong oxidizing agents and contribute toward chemical

oxidation of HMTD. This chemical oxidation of HMTD will add upon the amount of

HMTD•+ cation produced by direct electrochemical oxidation at the electrode surface,

129  

which will deprotonate to HMTD• free radical and react with Ru(bpy)32+/3+ and produce

higher ECL response.

Another peroxide-based high explosive, TATP, was detected by ECL technique.

This contains three peroxide functional groups so it was used as coreactant similar to

H2O2 that on cathodic potential scanning produced hydroxyl radical •OH as does H2O2.

When negative potential was applied, both Ru(bpy)32+ and TATP were reduced to

Ru(bpy)3+ and •OH radical in mixture of solvents, i.e., water and MeCN. In pure MeCN,

TATP did not produce any ECL response, which could be due to lack of formation of

stable •OH radicals as verified by EPR spectroscopy. Since the explosive compound was

insoluble in water so the use of mixed solvents was needed.

In contrast to direct ECL detection method, the ECL technique can also be used to

detect the high explosive materials by ECL quenching, which is a kind of indirect method.

The explosive material, which can quench ECL response of a well known and very

efficient Ru(bpy)32+/TPrA system, could be determined based on the extent of quenching.

This quenching can be due to excited state energy transfer, depletion of the species (i.e.,

TPrA• free radical and Ru(bpy)3+) which otherwise would produce excited state

responsible for ECL light production. High explosive, 2,4,6-trinitrotoluene (TNT) was

detected by ECL quenching method. TNT quenched ECL by depletion of chemically

produced Ru(bpy)3+ (from Ru(bpy)3

2+ reduction by TPrA• free radical) and TPrA• free

radical that are responsible of formation of excited state Ru(bpy)32+* for light emission.

TNT was also able to quench excited state Ru(bpy)32+* by energy transfer, which was

verified by the fluorescence experiment. The quenching constant calculated using Stern-

130  

Volmer equation was comparable to the literature value and the limit of detection of TNT

was 4.4 µM.

Electrochemical method could also be used to detect another stable explosive

compound, pentaerythritol tetranitrate (PETN), which is used in making plastic

explosives. Cyclic voltammetry can detect PETN by measuring reduction current with

silver wire as working electrode in MeCN. When negative potential was applied, PETN

was reduced probably to nitrite ions. The limit of detection of PETN by electrochemical

method using CV was as low as 25 µM.

131

 

APPENDIX

RESPONSE TO DR. ALVIN A. HOLDER’S COMMENTS

Q1: Page 58: The yields of all products should be given if the products are all novel in

nature. Please note this, here and elsewhere. In general, can the candidate identify the

peroxo stretching frequency in the FTIR spectrum? It is a common feature for such

compounds.

A1: These compounds are not novel in nature so percentage yields are not reported. In

FTIR spectrum, there are distinct peaks for peroxo stretching at 800 and 900 cm-1 for C-O

and O-O (in R2COO) for both the peroxide explosives, namely, hexamethylene

triperoxide diamine (HMTD) and triacetone triperoxide (TATP). We compared the C-O

stretch at ~1200 cm-1 with the literature value.

Q2: Figure 2.3. The candidate should acquire a square wave voltammogram to account

for the shoulder around +1.5 V for [Ru(bpy)3]Cl2 in MeCN. Is it possible to form a

Ru(IV) species? Can the candidate please clarify? Based on this note, the candidate

should explain how many oxidation states there are for ruthenium. What are the common

oxidation states of ruthenium?

A2: Further oxidation of Ru(bpy)33+ to Ru(bpy)3

4+ at ~1.5 V has never occurred either in

aqueous or in acetonitrile, although Ru(bpy)32+ can be reduced with three one-electron

processes. However, in the latter case, electrons are added to the bpy ligand rather than

ruthenium. (Tokel-Takvoryan, N. E.; Hemingway, R. E.; Bard, A. J. J. Am. Chem. Soc.

1973, 95, 6582-6589; Kanoufi, F.; Zu, Y.; Bard, A. J. J. Phys. Chem. B 2001, 105, 210-

216).

132

 

Barnard and Bennett (http://www.platinummetalsreview.com/dynamic/article/view/48-4-

157-158) have suggested two types of oxidation states for ruthenium: low oxidation states

and high oxidation states.

Low oxidation states go up to +III and high oxidation states range from +IV to +VIII,

which all depend on what ruthenium binds with.

(http://www.webelements.com/ruthenium/compounds.html)

Q3: The candidate should give a thorough explanation for the non-linearity in the Stern-

Volmer plot. Is the quenching constant a second-order rate constant?

A3: The Stern-Volmer quenching can be of two types: dynamic and static quenching. In

dynamic quenching, the quencher molecule has to reach to the excited molecule where

excited state energy transfer occurs, so this is diffusion controlled process. In static

quenching, the quencher molecule forms a complex with the emitter which does not emit.

The Stern-Volmer plot follows the linearity in both cases. When both dynamic and static

quenching operate, the plot deviates toward the positive direction.

There is a way to determine whether the process is dynamic or static. When the

temperature of the system increases, diffusion is favored where dynamic quenching is

favorable and the slope of Stern-Volmer plot increases whereas the static process

decreases since the complex dissociates at this temperature.

We did not verify the mechanism of quenching and mentioned that it could be dynamic

as we assumed it was due to excited state energy transfer rather than complex formation

(static).

133

 

Since the emitter (excited state molecule) and the quencher molecule are involved in the

quenching process, it is bimolecular and follows the second order kinetics (concentration

of both affect the quenching). (Keizer, J. J. Am. Chem. Soc. 1983, 105, 1494-1498;

Keizer, J. J. Am. Chem. Soc. 1985, 107, 5319-5319.)

Q4: Note that the candidate is using EPR spectroscopy to detect the •OH radical, but he

should give an explanation why DMPO is used in preference to other radical traps. What

advantage does it have over other radical traps?

A4: There are several spin traps to trap radicals such as DMPO (5,5-dimethyl-1-

pyrroline-N-Oxide), BMPO (5-tert-Butoxycarbonyl-5-methyl-1-pyrroline-N-Oxide),

EMPO (2-ethoxycarbonyl-2-methyl-2,3-dihydro-2H-pyrrole-1-Oxide). The spin trap such

as BMPO and EMPO can trap superoxides and form distinguishable adduct with the half

life of 23 and 8.6 min., respectively, and produce different spectra from that of hydroxyl

radicals. DMPO, however, does not distinguish superoxide radical since this adduct has

half life of only 45 s.

Our goal was to detect •OH radicals and we were not interested in superoxide, DMPO

was good for us. The DMPO superoxide adduct (with t1/2 = 45 s) would form DMPO-OH

adduct eventually. (Zhao, H., et al. Free Radic. Biol. Med. 2001, 31, 599; Stolze, K., et al.

Biol.Chem. 2002, 383, 813-820.)

Q5: In summary, the candidate should include a table comparing values arising from his

studies with those acquired by other techniques; then give a detailed account of which

technique is superior.

134

 

A5: We have mentioned the detection limit from other techniques used in detecting these

explosives such as mass spectrometry (MS), MS coupled with chromatographic

techniques. These techniques are very sensitive (more than ours) and detect nano- to

pico-grams (page 56). Our technique detects 50 µM and we use 1.0 mL of test solution,

which requires 10 µg of HMTD. We have compared with LC/FTIR technique on page 71

and mentioned that our technique is 10 times more sensitive than LC/IR.

Our technique does not require very expensive instruments as in MS and it is portable for

field detection. This is advantage over MS based technique not in sensitivity.

Likewise we have mentioned the limit of detection of TATP as 2.5 µM on page 87 which

is about 400 times more sensitive than LC/IR technique. This means we require 0.5

µg/mL for detection.

For second (HMTD) and third (TATP) chapters, we have clearly compared with other

techniques in use for these compounds. The fourth chapter deals with the quick detection

of TNT. Obviously, detection limit would be way less (0.99 µg/mL) than what can be

achieved with MS (ng to pg).

Comparison of Limit of Detection of Triacetone Triperoxide (TATP)

Techniques Limit of Detection

FTIR, GC-FTIR1 Qualitative

IMS2 1.9 µg

ESI-MS3 62.5 ng

HPLC-IR4 222 mg (1 mM)

ECL (current method) 0.55 µg

135

 

Comparison of Limit of Detection of Hexamethylene Triperoxide Diamine (HMTD)

Techniques Limit of Detection

HPLC-IR4 0.5 mM

LC-MS5 0.96 nM

GC, GC-FTIR1 Qualitative

LC-Electrochemical6 3.0 µM

ECL (current method) 50 µM

References

(1) Pena, A. J.; Pacheco-Londono, L.; Figueroa, J.; Rivera-Montalvo, L. A.; Roman-

Velazquez, F. R.; Hernandez-Rivera, S. P. Proc. SPIE-Int. Soc. Opt. Eng. 2005,

5778, 347.

(2) Oxley, J. C.; Smith, J. L.; Kirschenbaum, L. J.; Marimganti, S.; Vadlamannati, S. J.

Forensic Sci. 2008, 53, 690.

(3) Sigman, M. E.; Clark, C. D.; Caiano, T.; Mullen, R. Rapid Commun. Mass Spectrom.

2008, 22, 84.

(4) Schulte-Ladbeck, R.; Edelmann, A.; Quintas, G.; Lendl, B.; Karst, U. Anal. Chem.

2006, 78, 8150.

(5) Oxley, J.; Zhang, J.; Smith, J.; Cioffi, E. Propellants, Explos., Pyrotech. 2000, 25,

284.

(6) Schulte-Ladbeck, R.; Karst, U. Chromatographia 2003, 57, S/61.