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Spring 5-2011
Sensitive Detection of High Explosives Using Electrogenerated Chemiluminescence
Suman Parajuli University of Southern Mississippi
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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
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
2+ (Ru(bpy)3 ) 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
2+ HMTD/Ru(bpy)3 system resulted in significant enhancement in HMTD ECL production (up to 27 times). This enhancement was found to be largely associated with
ii
- - + 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
2+ the presence of Ru(bpy)3 , 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
iii
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.
2+ In this investigation, ECL quenching behavior of the Ru(bpy)3 / 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
2+ • (produced as a result of reduction of Ru(bpy)3 by TPrA ) by TNT could be the main
• + reason of this quenching, as both TPrA and Ru(bpy)3 species are precursors of the
2+ excited state Ru(bpy)3 * 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.
iv
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 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.
v
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 vi
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
vii
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
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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
2+ 1.3. (a) ECL and (b) CV of 1.0 nM Ru(bpy)3 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 2+ diameter) at a scan rate of 50 mV/s. (c) ECL with 1.0 μM Ru(bpy)3 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 2+ different concentration of Ru(bpy)3 : 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
ix
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 2+ intensity of the Ru(bpy)3 /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
x
listed in Scheme 2.3 Above, the following parameters were used: α = 0.5, - 2 initial [Ag(I)]= [NO3 ] =0.007 M, area of electrode = 0.0314 cm , 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
2+ 3.2. CVs of (a) 1.0 mM TATP and (b) 0.60 mM Ru(bpy)3 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 2+ for 30 min) in 1.0 mM Ru(bpy)3 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
2+ 3.4. Effect of Ru(bpy)3 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
xi
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 2+ 0.6 mM Ru(bpy)3 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 2+ solution containing 0.6 mM Ru(bpy)3 -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 2+ with 0.6 mM Ru(bpy)3 -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) 2+ water-MeCN mixture containing 0.6 mM Ru(bpy)3 -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 2+ mM Ru(bpy)3 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
xii
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 2+ Ru(bpy)3 -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
2+ 3.15. ECL responses of 1.0 mM H2O2-0.60 mM Ru(bpy)3 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 2+ Ru(bpy)3 -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
xiii
(c) 0.50 mM TATP + 0.50 mM HMTD, using the same experimental conditions as in (A)………………………………………………………………95
2+ 4.1. CV and ECL of 1.0 µM Ru(bpy)3 -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
2+ 4.2. Concentration dependence of TPrA on ECL of the Ru(bpy)3 /TPrA system. The experiment was conducted at a 2-mm Pt electrode with a scan rate of 2+ 50 mV/s using 1.0 µM Ru(bpy)3 -0.10 M TBAP MeCN solution……………104
2+ 4.3. ECL quenching of the Ru(bpy)3 (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
2+ 4.4. ECL quenching by TNT in the system containing 5 nM Ru(bpy)3 -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
2+ 4.5. Ru(bpy)3 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 2+ containing 1.0 µM Ru(bpy)3 -25 mM TPrA-0.10 M TBAP in MeCN at a 2-mm Pt electrode at a scan rate of 50 mV/s …………………………………..108
2+ 4.7. Fluorescence quenching of 3.0 mL of 1.0 μM Ru(bpy)3 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
xiv
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
1 5.1. H 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
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LIST OF SCHEMES Scheme
1.1. Ion annihilation ECL………………………………………………………………8
2+ 1.2. Mechanism for the ECL first wave of the Ru(bpy)3 /TPrA system……...... 13
2+ 1.3. Mechanism for the ECL second wave of the Ru(bpy)3 /TPrA system………….14
2+ 1.4. Mechanism for the ECL second wave for the Ru(bpy)3 /TPrA system………...14
2+ 2- 1.5. ECL Mechanism for the Ru(bpy)3 /S2O8 system……………………………...15
2.1. Synthesis of hexamethylene triperoxide diamine (HMTD)………… …………..57
2+ 2.2. Proposed ECL mechanism of the Ru(bpy)3 /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+ 2.4. Proposed ECL mechanism of the HMTD/Ru(bpy)3 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 2+ to form excited state Ru(bpy)3 * species that emit light via Eq. (2.22)… ……..68
3.1. Synthesis of triacetone triperoxide (TATP)………………………… …………..77
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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
2+ 3.3. Proposed ECL mechanism of TATP in the presence of Ru(bpy)3 upon the cathodic potential scanning………………………………………………………82
3.4. No “reductive-oxidation” type ECL was produced from TATP and H2O2 in 2+ MeCN in the presence of Ru(bpy)3 …………………………………………….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
xvii
LIST OF EQUATIONS
Equations
III 3 II 4 1.1. Fe (CN)6 e Fe (CN)6 ……………………………………………………….3
II 4 III 3 1.2. Fe (CN)6 e Fe (CN)6 ……………………………………………………….5
III 3 0 0.059 [Fe (CN)6 ] 0 1.3. E E FeII (CN) 4 /FeIII (CN) 3 log (at 25 C) ……………………...5 6 6 II 4 1 [Fe (CN)6 ] EE 1.4. E 0' papc ………………………………………………………………...... 6 2 0. 059 1.5. ΔEE E ……………………………………………………………..6 pa pc n
5 3/2 1/2 1/2 1.6. inADCvp (2.69 10 ) ………………………………………………………...6
1.7. R e R ………………………………………………………………………….8
1.8. R e R …………………………………………………………………………..8
1.9. R R R R ………………………………………………………………….8
1.10. R R hv ……………………………………………………………………….8
1.11. H E E 0.16 ………………………………………………….9 ann p(R/R ) p(R/R ) 1.9a. R R R1R (Singlet) …………………………………………………..9
1.9b. R R R3R (Triplet) …………………………………………………..9
1.12. 3 R 3R R1R ………………………………………………………………..9
2 3 1.13. S2O8 e S2O8 ……………………………………………………………...15
2 1.14. Ru(bpy)3 e Ru(bpy)3 ………………………………………………………15
2 2 3 1.15. Ru(bpy)3 S2O8 Ru(bpy)3 S2O8 ………………………………………15
xviii
3 2 1.16. S2O8 SO4 SO4 …………………………………………………………15
2* 2 1.17. Ru(bpy)3 SO4 Ru(bpy)3 SO4 ……………………………………….15
2 3 2 1.18. Ru(bpy)3 SO4 Ru(bpy)3 SO4 ……………………………………….16
3 2* 2 1.19. Ru(bpy)3 Ru(bpy)3 Ru(bpy)3 Ru(bpy)3 ……………………………16
2* 2 1.20. Ru(bpy)3 Ru(bpy)3 hv ………………………………………………….16
e H RuIII II 2.1. HMTD HMTD HMTD Ru P1 …………………...... 61
e 2 H -H RuIII II 2.2. HMTD [ HMTD ] HMTD HMTD Ru P2 …..61
2.3. Ru II Ru II hv …………………………………………………………………...61
2.4. Ag(II) e Ag(I) ………………………………………………………………...... 65
2.5. NO3 e NO3 …………………………………………………………………..65
2.6. NO3 Ag(I) NO3 Ag(II)…………………………………………………….65
2.7. NO3 P3 ………………………………………………………………………...... 65
2.8. Ag(II) P4 ………………………………………………………………………...... 65
2 3 2.9. Ru(bpy)3 e Ru(bpy)3 ………………………………………………………68
2.10. HMTD e HMTD ……………………………………………………68
2.11. Ag(I) e Ag(II) ………………………………………………………………68
2.12. NO3 e NO3 ……………………………………………………………...... 68
2.13. NO3 Ag(I) NO3 Ag(II) …………………………………………………..68
2.14. Ag(II) HMTD Ag(I) HMTD ……………………………………………68 xix
2.15. NO3 HMTD NO3 HMTD ………………………………………………68
2.16. HMTD HMTD H …………………………………………………….....68
2 2.17. Ru(bpy)3 HMTD Ru(bpy)3 Product1 ………………………………...... 68
2* 2.18. Ru(bpy)3 Ag(II) Ru(bpy)3 Ag(I) ……………………………………….68
2* 2.19. Ru(bpy)3 NO3 Ru(bpy)3 NO3 ………………………………………...68
3 2* 2.20. Ru(bpy)3 HMTD Ru(bpy)3 Product2 ………………………………...68
3 2* 2 2.21. Ru(bpy)3 Ru(bpy)3 Ru(bpy)3 Ru(bpy)3 …………………………….68
2* 2 2.22. Ru(bpy)3 Ru(bpy)3 hv ………………………………………………...... 68
3.1. Peroxides e [H2O2 ] OH ……………………………………………………82
2 3.2. Ru(bpy)3 e Ru(bpy)3 ……………………………………………………...82
2 3 3.3. Ru(bpy)3 OH Ru(bpy)3 OH …………………………………………...82
2 3.4. Ru(bpy)33 OH Ru(bpy) …………………………………………………82
322+ 3.5. Ru(bpy)33 Ru(bpy) Ru(bpy) 3 Ru(bpy) 3……………………………….82
2 2 3.6. Ru(bpy)3 Ru(bpy)3 hv ……………………………………………………82
3.7. TATPCH3 2 CO2 3 3H2O 3H2O2 3CH3 2 O ………………………………86
Catalase 3.8. 2H2O2 2H2O O2 ……………………………………………………….93
4.1. TPrA e TPrA ……………………………………………………………...101
2 3 4.2. Ru(bpy)3 e Ru(bpy)3 ……………………………………………………..101
xx
4.3. TPrA TPrA H ………………………………………………………....101
2 4.4. Ru(bpy)3 TPrA Ru(bpy)3 P1 ………………………………………….102
3 2 2 4.5. Ru(bpy)3 Ru(bpy)3 Ru(bpy)3 Ru(bpy)3 …………………………...102
2 4.6. Ru(bpy)3 Ru(bpy)3 hv …………………………………………………..102
3 2 4.7. Ru(bpy)3 TPrA Ru(bpy)3 P1 …………………………………………..102
4.8. nTPrA TNT TNTn P2 …………………………………………………....102
2 n 4.9. nRu(bpy)33 TNT Ru(bpy) TNT ………………………………………..102
K [Ru(bpy) 2*] 0 ECL(f ) 3 4.10. 2* 2* …………………………………109 KECL(f)[Ru(bpy)3 ] Knr[Ru(bpy)3 ]
2* KECL(f)[Ru(bpy)3 ] 4.11. 2* 2* 2* ………..110 KECL(f)[Ru(bpy)3 ] Knr[ Ru(bpy)3 ] Kq[TNT][ Ru(bpy)3 ]
0 Kq 4.12. 1 [TNT] 1 KSV [TNT] …………………………………..110 KECL(f) Knr
1 4.13. ……………………………………………………………..110 KECL(f) Knr
2 2 4.14. Ru(bpy)3 TNT Ru(bpy)3 (TNT)high energy ……………………………….112
5.1. RONO2 2H2O 2e RONO 3H2 O …………………………………………118
5.2. RONO2 2e RO NO2 ……………………………………………………...118
5.3. RO H2O ROH OH ……………………………………………………….118
5.4. RONO2 2e R NO3 ……………………………………………………….118
5.5. R H2O RH OH …………………………………………………………..118 xxi
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
2+ (Ru(bpy)3 ) as ECL luminophore. The ECL quenching for the well-known
2+ Ru(bpy)3 /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
III 3- the surface concentration of Fe (CN)6 approaches zero and then the current decreases
III 3- on further scanning to -0.15 V, meaning that the reducible species (Fe (CN)6 ) is
depleted. This process of reduction of analyte is represented by Eq. 1.1:
III 3- II 4- [Fe (CN)6] + e = [Fe (CN)6] 1.1
As the potential is switched to positive direction, i.e., anodic scanning, the cathodic 4
-0.6 E final
-0.4
R -0.2 e n v a e c r s s 0.0 e d r s a c vs Ag/AgCl st w a nd r 1 Cycle n 2 Cycle ,E 0.2 o F
0.4
Potential 0.6 E 0.8 initial
0 50 100 150 200 Time (t), s
Figure 1.1.Cyclic voltammetric excitation signal.
E 20 D pc
15 E C i 10 pc F A
5 B H 0 G Current, -5 i A pa K I -10
-15 J E pa -20 0.8 0.6 0.4 0.2 0.0 -0.2 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
III 3+ II 4- Fe (CN)6 . When the electrode surface becomes positive, oxidation of Fe (CN)6 starts
III 3- forming the original species (Fe (CN)6 ). The anodic current increases as we scan
II 4- III 3- further from 0.1 V rapidly for the oxidation of Fe (CN)6 back to Fe (CN)6 as
indicated by H to J in the above voltammogram (Figure 1.2). So the reduced species gets
oxidized back as:
III 4- II 3- [Fe (CN)6] + e = [Fe (CN)6] 1.2
The anodic current reaches maximum at ~0.2 V and decreases for further scanning as the
II 4- electroactive species for oxidation, Fe (CN)6 , 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:
FeIII (CN) 3- o 0.059 6 o EEII 4- III 3- logII 4- (at 25 C) 1.3 Fe (CN)66 /Fe (CN) 1 Fe (CN)6
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
III 3- concentration of Fe (CN)6 is in majority. As we scan to the negative potential,
III 3- II 4- Fe (CN)6 is reduced to Fe (CN)6 decreasing its concentration linearly. At equilibrium, 6
III 3- II 4- the ratio of ([Fe (CN)6 ]/Fe (CN)6 ]) 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:
EE E o papc 1.4 2
The separation of peak potentials (cathodic and anodic) is given by:
0.057 EE E (V at 25 C) 1.5 ppapc n
where n is the number of electron transfer involved in the redox process. For reduction
III 3- II 4- of Fe (CN)6 to Fe (CN)6 , 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
5 3/2 1/2 1/2 ip = (2.69 × 10 ) n AD Cv 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
3 1 state energy (Et), then R* is initially formed which eventually produce R* 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
2+ Ru(bpy)3 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.
2+ (a) Ru(bpy)3 / Tri-n-propylamine (TPrA) system.The most of the ECL applications
2+ reported so far involve Ru(bpy)3 or its derivatives as an emitter and TPrA as a
coreactant, though there are other coreactans for ECL studies such as oxalate97,
96 98 2+ persulfate , and hydrogen peroxide. The Ru(bpy)3 / TPrA system is an “oxidative-
reduction” type of coreactant ECL. This system has shown the highest ECL efficiency
87,99 2+ known so far. The ECL mechanism of Ru(bpy)3 /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
2+ Ru(bpy)3 /TPrA system depends on several factors such as the concentration of both
2+ Ru(bpy)3 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)
2+ and gold (Au) working electrode, this system shows two ECL waves when the Ru(bpy)3 concentration is kept low (in the order of nano molar to micro molar concentration,
Figure 1.3).
2+ Figure 1.3. (a) ECL and (b) CV of 1.0 nM Ru(bpy)3 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 2+ diameter) at a scan rate of 50 mV/s. (c) ECL with 1.0 μM Ru(bpy)3 and 0.10 M TPrA. The ECL intensity scale is given for (c) and should be multiplied by 100 for (a).100
2+ With the increase of Ru(bpy)3 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.
2+ The first ECL wave arises due to the reduction of Ru(bpy)3 by electrochemically
● + generated TPrA free radical (TPrA ) to Ru(bpy)3 which is oxidized by the relatively
●+ 2+ 2+ long lived TPrA radical cation (TPrA ) to excited state Ru(bpy)3 *. Here, Ru(bpy)3 does not participate in direct electrochemical oxidation. Hence, the first ECL wave can be 13
2+ observed when potential is scanned from 0 to 0.9 V vs Ag/AgCl (Note: Ru(bpy)3 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 2+ different concentration of Ru(bpy)3 : 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
+ +H+ TPrA -H TPrAH+ e .+
Electrode TPrA TPrA -H+ . + 2+* TPrA Ru(bpy)3 Ru(bpy)3 hv 2+ 2+ P1 Ru(bpy)3 Ru(bpy)3
●+ ●+ + + ● ● [where TPrA = (CH3CH2CH2)3N , TPrAH = Pr3NH , TPrA = Pr2NC HCH2CH3, P1
+ Pr2N C=HCH2CH3]
2+ 100 Scheme 1.2. Mechanism for the first ECL wave of the Ru(bpy)3 /TPrA system. 14
2+ 2+ Ru(bpy)3 Ru(bpy)3 hv e 3+ Ru(bpy)3 2+* Ru(bpy)3
+ + -H H2O TPrA. . P1 P Electrode TPrA 2 e + -H + TPrA TPrAH
[where P2 = Pr2NH + CH3CH2CHO]
2+ 100 Scheme 1.3. Mechanism for the second ECL wave of the Ru(bpy)3 /TPrA system.
2+ The second ECL wave arises due to the direct oxidation of Ru(bpy)3 at the
3+ electrode surface producing Ru(bpy)3 , and at the same time TPrA also gets oxidized to
form TPrA●+. This radical cation readily deprotonates forming TPrA free radical (TPrA●)
3+ 2+ which is a strong reducing agent and reduces Ru(bpy)3 to Ru(bpy)3 * excited state.
This excited state emits light and comes back to the original state and further continues
the process (Scheme 1.3).
Ru(bpy) 3+ 2+ 3 Ru(bpy)3 e hv 2+ Ru(bpy)3 + 2+* Ru(bpy)3 Ru(bpy)3
+ -H+ TPrA. . H2O Electrode TPrA P P e 1 2 -H+ + TPrA TPrAH
2+ 100 Scheme 1.4. Mechanism for the second ECL wave of the Ru(bpy)3 /TPrA system.
The second ECL wave can also be produced by the chemical reduction of
2+ ● + 2+ Ru(bpy)3 by TPrA to Ru(bpy)3 and electrochemical oxidation of Ru(bpy)3 to 15
3+ Ru(bpy)3 . These oxidized and reduced species undergo chemical reaction to form
excited state that emits light (Scheme 1.4).
2+ 2- (b) Ru(bpy)3 /Peroxydisulphate (persulfate, S2O8 ) 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
2- at the electrode. As shown in Scheme 1.5, persulfate (S2O8 ) gets electrochemically
●3- 2+ reduced to radical anion (S2O8 ) and at same time Ru(bpy)3 also gets reduced to
+ 2- + Ru(bpy)3 . The S2O8 can also oxidize the reduced Ru(bpy)3 back to the original state
2+ ●3- (Ru(bpy)3 ) whereby persulfate is chemically converted to radical anion (S2O8 ). The
●- strong oxidizing radical (SO4 ) is formed from the decomposition of persulfate radical
●3- 2- + anion (S2O8 ) along with SO4 . This sulfate radical either oxidizes Ru(bpy)3 to its
2+ 2+ 3+ excited state (Ru(bpy)3 *) or oxidizes Ru(bpy)3 to Ru(bpy)3 which undergoes a
+ chemical reaction with electrochemically generated Ru(bpy)3 to form the excited state
that emits light (Scheme 1.5).
2- 3- SO28e SO 28 1.13
2+ + Ru(bpy)33e Ru(bpy) 1.14
+2-2+3 Ru(bpy)328 S O Ru(bpy) 3 S 28 O 1.15
32 S28 O SO 4 SO 4 1.16
+2+2 Ru(bpy)34 SO Ru(bpy) 3 * SO 4 1.17 16
23+2 Ru(bpy)34 SO Ru(bpy) 34 SO 1.18
322 Ru(bpy)33 Ru(bpy) Ru(bpy) 3 * Ru(bpy) 3 1.19
22 Ru(bpy)33 * Ru(bpy) hv 1.20
2+ 2- 94 Scheme 1.5. ECL mechanism for Ru(bpy)3 / S2O8 system.
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 Sensitive but expensive, not portable 108,113,115- with gas or high performance liquid 126 chromatography (GC/MS, HPLC/MS) Ion mobility spectrometry (IMS) Sensitive but with matrix effect 127-133 Micro-mechanical sensors (e.g., Sensitive, low power consumption and 134-138 micro-cantilevers) real-time operation, but mainly for vapors, with moisture effect, less selective Electrochemical methods Inexpensive, fast, portable, less 139-144 sensitive Chemiluminescence (CL) High sensitivity but poor selectivity 145-150 Colorimetric tests Simple, inexpensive, fast but less 151,152 sensitive and low specificity Fluorescence (FL) spectroscopy Sensitive with interferences effect 153-160 Surface plasmon resonance Label-free, sensitive but subject to 161-165 external contamination Surface enhanced Raman scattering Sensitive, but complicated technique 166-171 spectroscopy (SERS) and difficult to operate 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 N % O % g/cm3 (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- C3H6N6O6 1.82 37.8 43.2 triazacyclohexane
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
OH NO2 N NO2 O2N NO2 O2N NO2 N O2N NO2
N N O2N NO2
NO2 NO2 NO2
(a) TNT (b) Picric acid (c) Tetryl (d) RDX
NO2
N O2N NO2 N N O2NO ONO2 O N N N NO O N NO2 2 2 2 NN N O2NO ONO2 NO2 O2N N N NO2
(e) HMX (f) CL 20 (g) PETN
ONO 2 O O C O O O O N O N O O O C C O2NO ONO2 x O O O O
(h) Nitrocellulose (i) HMTD (j) TATP
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
187 colorimetric) assays of the hydrogen peroxide (H2O2) product of UV- or acid treatment 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
189,190 potential, and stable electrocatalytic detection for H2O2. 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
194 remove the co-existing H2O2 that may originate from cleaning agents. 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-
262 doped nanoparticle sensors with additives of Sb2O3, TiO2, V2O5 and WO3.
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
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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
2+ 25 luminophore such as Ru(bpy)3 species up on anodic potential scanning. 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.
N O O a) Dissolved in 30% H2O2 in ice bath N O N O N NNb) Added anhydrous citric acid with stirring O O Hexamethylenetetramine Hexamethylene triperoxide diamine (HMTD)
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
(a) C-O Stretch, 1225 cm-1 CH 4.82 ppm 0.3 (b) 2
O O O 0.2 N N O O O Absorbance 0.1 CHCl H O TMS 3 2
0.0
1400 1200 1000 800 86420 ppm Wave number, cm-1
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 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
29 and the incomplete deuteration of the solvent (CDCl3) used. [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.
12 40 ECL 30 8
20 4 i
10 ECL A) (nA) 0 (
CV 0 i -4 -10 CV -8 -20
-30 -12
2.0 1.5 1.0 0.5 0.0 E (V vs Ag/Ag+)
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.
+ 2+ In the anodic potential scanning range of 0 to 2.0 V vs Ag/Ag , Ru(bpy)3 oxidizes
3+ reversibly to form Ru(bpy)3 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
2+ 2+ oxidation current ratio of HMTD to Ru(bpy)3 . ECL response of the Ru(bpy)3 /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).
(a)
(b) 25 A
(c)
2.01.51.00.50.0 + E (V vs Ag/Ag )
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.
2+ The ECL intensity of the Ru(bpy)3 /HMTD system depends on the added
2+ Ru(bpy)3 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
2+ ECL signals. Similar behavior has been reported previously for the Ru(bpy)3 /TPrA
system in MeCN.31
-e+ -H+ +Ru III II* 2.1 HMTD HMTD HMTD Ru + P1
+2+-e -H++III -H +Ru II* HMTD [ HMTD ] HMTD HMTD Ru + P2 2.2
RuII* Ru II + hv 2.3
II 2+ II where HMTD = hexamethylene triperoxide diamine, Ru = Ru(bpy)3 , Ru = 3+ II 2+ Ru(bpy)3 , Ru * = excited state Ru(bpy)3 *, P1 and P2 = oxidation products of HMTD free radicals.
2+ Scheme 2.2. Proposed ECL mechanism of the Ru(bpy)3 /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
12 Pt ) 2 8 Au (nA/ mm (nA/ ECL i 4
GC
0 0.0 0.2 0.4 0.6 0.8 1.0 [Ru(bpy) Cl ] (mM) 3 2
Figure 2.4. Effect of Ru(bpy)3Cl2 concentration and working electrode material on ECL 2+ intensity of the Ru(bpy)3 /HMTD system in 0.10 M TBAP MeCN. Scan rate: 50 mV/s, [HMTD] = 1.0 mM.
300
200 50 ECL 100 i ECL (nA) A)
0
( 0 CV i -100 CV -200 -50 (A) -300
i CV 25 A
(B)
2.0 1.5 1.0 0.5 0.0 E (V vs Ag/Ag+)
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.
300
250 (a)
200
150 (nA) ECL i 100
50 (b)
0 0246810 [AgNO ] (mM) 3
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
33 + • 32,34-37 V vs NHE or ~1.79 V vs Ag/Ag ) and NO3 radicals. 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
• 32,35 reaction between Ag(I) ions and electrochemically generated NO3 radicals. 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,
40,41 + IN), 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,
2+ NaNO3 also demonstrates significant ECL enhancement for the HMTD/Ru(bpy)3
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.
0 2.4 Ag(II) + e = Ag(I) Ek =1.79 V, s = 0.10 cm/s
-04 NO33 + e = NO Ek =1.82 V, s = 1 10 cm/s 2.5
-4 NO33 + Ag(I) = NO + Ag(II) kk fb / 1 10 /3111 2.6
4 NO33 = P kk fb / 1 10 /1 2.7
4 Ag(II) = P4fb kk / 1 10 /1 2.8
Scheme 2.3. Proposed reaction mechanism of AgNO3 oxidation in MeCN at a Pt 0 electrode for CV digital simulations. In Eqs. 2.4-2.8, E 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.
0
A) -25 ( cv i
-50 E = 1.72 V p
2.0 1.5 1.0 0.5 0.0 + E (V vs Ag/Ag )
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)] - 2 = [NO3 ] = 0.007 M, area of electrode = 0.0314 cm , 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.
300
(a) AgNO 200 3
(b) NaNO 100 3 ECL Peak Intensity(nA) ECL Peak
0 1.4 mM
012345678 [AgNO ] or [NaNO ] (mM) 3 3
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
2+ + AgBF4 for the HMTD/Ru(bpy)3 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.
75
60 (a)
45
30 (b)
ECL Peak INtensity (nA) 15
0123456 [C H COOAg] (mM) 6 5
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.
2+ Since ECL production of the HMTD/Ru(bpy)3 system initiates from the oxidation of
2+ Ru(bpy)3 and HMTD (Scheme 2.2), in principle, any process that could increase the
3+ •+ generation of Ru(bpy)3 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
2+ enough to oxidize Ru(bpy)3 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,
40,43 involves a slow electron-transfer oxidation process (e.g., ks ≤ 0.01 cm/s).
2+ 3+ 2.9 Ru(bpy)33 - e Ru(bpy)
HMTD - e HMTD+ 2.10
Ag(I) - e Ag(II) 2.11
- NO33 - e NO 2.12
- NO33 + Ag(I) NO + Ag(II) 2.13
Ag(II) + HMTD Ag(I) + HMTD+ 2.14
-+ NO33 + HMTD NO + HMTD 2.15
++ HMTD HMTD + H 2.16
2+ + Ru(bpy)3 + HMTD Ru(bpy)31 + Product 2.17
+2+* Ru(bpy)33 + Ag(II) Ru(bpy) + Ag(I) 2.18
+2+*- Ru(bpy)33 + NO Ru(bpy) 3 + NO 3 2.19
3+ 2+* Ru(bpy)332 + HMTD Ru(bpy) + Product 2.20
+3+2+*2+ Ru(bpy)33 + Ru(bpy) Ru(bpy) 3 + Ru(bpy) 3 2.21
2+* 2+ Ru(bpy)3 Ru(bpy)3 + hv 2.22
2+ Scheme 2.4. Proposed ECL mechanism of the HMTD/Ru(bpy)3 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 2+ state Ru(bpy)3 * species that emit light via Eq. 2.22. 69
2+ An ECL mechanism of the HMTD/Ru(bpy)3 system involving ECL
enhancement by AgNO3 in MeCN at a Pt electrode is proposed in Scheme 2.4. In this
2+ 3+ •+ mechanism, chemical oxidations of Ru(bpy)3 to Ru(bpy)3 and HMTD to
• • 2+ • [ HMTD ] by Ag(II) and NO3 are not included, because their contributions to the
overall ECL enhancement could be minor.
Limit of Detection of HMTD
400 (b)
300
200
100 ECL Peak Intensity (nA) Intensity Peak ECL
(a) 0
0.0 0.2 0.4 0.6 0.8 1.0 [HMTD] (mM)
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
2+ determined with ECL in the presence of Ru(bpy)3 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
2+ concentration levels. ECL enhancement of the HMTD/Ru(bpy)3 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
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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-
2+ MeCN mixture solvents in the presence of added ECL emitter Ru(bpy)3 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)
+ 2+ that could oxidize electrogenerated Ru(bpy)3 cations to form Ru(bpy)3 * 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
1 procedure from the literature. 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).
0.6 C-O Stretch, 1175 cm-1 CH (a) 3 ~1.48 ppm 0.5 (b) C O O O 0.4 O C C 0.3 O O
Absorbance 0.2
0.1 CDCl TMS 3
0.0 86420 1400 1200 1000 800 ppm Wavenumber, cm-1
Figure 3.1. (a) ATR-FTIR and (b) 1H NMR spectra of TATP.
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
1 precursors produce H 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
2+ + ECL luminophore, Ru(bpy)3 , 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
2+ only after Ru(bpy)3 reduction.
ECL Behavior of TATP
2+ Because TATP has a poor solubility in water and Ru(bpy)3 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 ( E 0 = 1.77~1.91 V vs NHE49-51), could oxidize OH /OH -
2+ 3+ + Ru(bpy)3 to Ru(bpy)3 (Eq. 3.3) and Ru(bpy)3 (electrochemically reduced from
2+ 2+ Ru(bpy)3 , Eq. 3.2) to Ru(bpy)3 * excited state (Eq. 3.4). Alternatively, the excited state
2+ of Ru(bpy)3 * 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. A i CV 100
-0.6 -0.9 -1.2 E( (V vs Ag/Ag+) (a)
TATP (b)
Ru(bpy) 2+ 3
(c)
Blank
0.0 -0.5 -1.0 -1.5 -2.0 E( (V vs Ag/Ag+)
2+ Figure 3.2. CVs of (a) 1.0 mM TATP and (b) 0.60 mM Ru(bpy)3 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).
2+ Experimentally, however, no ECL was observed from the Ru(bpy)3 /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
33 TATP can be decomposed to H2O2 prior to ECL detection. Second, the working
electrode (Pt or glassy carbon) modified with crystalline Prussian blue (ferric
38 ferrocyanide) which could catalytically reduce H2O2 was employed in the ECL testing. 82
2+ Neither helped the ECL generation. Surprisingly, in MeCN even the Ru(bpy)3 /H2O2
system did not produce any notable ECL signals. Scheme 3.4 summarizes the above
observations.
e 3.1 TATP + e [H22 O ] OH 2+ + 3.2 Ru(bpy)33 + e Ru(bpy) 2+ 3+ - 3.3 Ru(bpy)33 + OH Ru(bpy) + OH
+2+* 3.4 Ru(bpy)33 + OH Ru(bpy)
+3+2+*2+ Ru(bpy)33 + Ru(bpy) Ru(bpy) 3 + Ru(bpy) 3 3.5
2+* 2+ Ru(bpy)33 Ru(bpy) + hv 3.6
2+ Scheme 3.3. Proposed ECL mechanism of TATP in the presence of Ru(bpy)3 upon the cathodic potential scanning.
Scheme 3.4. No “reductive-oxidation” type ECL was produced from TATP and H2O2 in 2+ MeCN in the presence of Ru(bpy)3 .
When UV-irradiated TATP MeCN solution was transferred to a water-MeCN
2+ mixture medium containing Ru(bpy)3 and a suitable supporting electrolyte, strong ECL
signals were observed. For example, in 70:30 (v/v) water-MeCN mixture containing 1.0 83
2+ μM Ru(bpy)3 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+
2+ (Figure 3.3a). The ECL profile is coincident with the reduction of Ru(bpy)3 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
2+ 44 Ru(bpy)3 /H2O2 system in water-MeCN has been reported previously.
200 400
ECL, CV 300 i 150 ECL (nA) 200
100 100 (a) 0 50 -100 (b) A)
0
( -200 CV i
-300 -50
-400 0.0 -0.5 -1.0 -1.5 -2.0 E (V vs, Ag/Ag+)
Figure 3.3. (a) ECL and (b) CV responses of 0.50 mM TATP (with UV irradiation for 30 2+ min) in 1.0 mM Ru(bpy)3 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.
2+ Since the concentration of Ru(bpy)3 could influence the ECL production, its
optimum concentration was determined. When 0.50 mM of TATP is used (with UV
2+ irradiation), 0.60 mM Ru(bpy)3 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
360
320
280 (nA)
ECL i 240
200
0.0 0.5 1.0 1.5 2.0 2.5 [Ru(bpy) 2+] (mM) 3
2+ Figure 3.4. Effect of Ru(bpy)3 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
360
320
280 (nA) 240 ECL i
200
160 5 1015202530354045 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 2+ intervals) was conducted in 70:30 (v/v) water-MeCN containing 0.6 mM Ru(bpy)3 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.
300 TATP H O 2 2
200 (a) (nA)
ECL i 100
(b)
0
020406080100
H O volume % (in MeCN) 2
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 2+ containing 0.6 mM Ru(bpy)3 -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.
TATP [(CH32 ) CO 23 ] + 3H 2 O 3H 2 O 2 + 3(CH 32 ) 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.
350 60 (A) 300 (B) 300 (c) 0.5 mM TATP 40 2.5 M TATP (nA) ECL 250 i 20 225
Background signal 200 0 03060 (b) 2.5 M TATP [TATP] (M) 150 (nA) 150 , nA ECL ECL i i 100 75 (a) Background 50 0 0
0 100 200 300 400 500 0.0 -0.4 -0.8 -1.2 -1.6 -2.0 [TATP] (with UV Irradiation, M) + E (V vs Ag/Ag )
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 2+ Ru(bpy)3 -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,
4,38 resulting in the formation H2O2. 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+
2+ + is believed to be associated with the first electron reduction of Ru(bpy)3 to Ru(bpy)3 , and the second one at -2.0 V vs Ag/Ag+ remains unclear, although it might be related to
+ 0 the Ru(bpy)3 /Ru(bpy)3 electron-transfer process. Both ECL peaks show good linear relationship with TATP concentration (Figure 3.8).
100 (b) 80
60
, nA 40 (a) ECL i
20
0 Background
0 100 200 300 400 500 [TATP] (M)
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- 2+ MeCN mixture containing 0.6 mM Ru(bpy)3 -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
250
ECL, CV 2 100 i 200 ECL 1 (nA)
50 150 (b)
0 100
A) 50 -50 ( CV
i (a)
0 -100
0.0-0.5-1.0-1.5-2.0 E (V vs, Ag/Ag+)
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.
ECL, CV 150 150 i ECL
100 (nA)
100 50
(a) 0
50 -50 A)
( (b) -100 CV i 0
-150
0.0 -0.5 -1.0 -1.5 -2.0 E (V vs, Ag/Ag+)
Figure 3.10. (a) ECL and (b) CV responses of 0.50 mM TATP directly detected in 0.60 2+ mM Ru(bpy)3 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.
150
30 125
20 (nA) ECL 100 i 10 [TATP] = 2.5 M Background signal 75 0204060 [TATP] (M) (nA) ECL i 50
25
0
0 100 200 300 400 [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 2+ Ru(bpy)3 -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.
100 % water 6 90 %
40 X 1040 X 80 %
70 %
60 %
50 %
40 % 30 % 20 % 10 % 5 %
3460 3480 3500 3520 3540 G Value/Gauss
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.
3.0
2.5 6 2.0
1.5
1.0 Intensity x 10
0.5
0.0
020406080100 H O volume % (in MeCN) 2
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 EPR Intensity EPR Intensity Change in EPR 6 6 (% of H2O in MeCN) (after 5 min, ×10 ) (after 20 min, ×10 ) 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
4 Expt. ___ Simulation ) 6 2
0
Intensity (a.u. x10 -2
-4 3480 3500 3520 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
2+ Ru(bpy)3 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.
Catalase 2H22 O 2H 2 O + O 2 3.8
120
1 mM H O , 0.6 mM Ru(bpy)2+ 100 2 2 3
80
60 , nA (a) ECL I 40
20 +Catalaze
0 (b) 0.0 -0.5 -1.0 -1.5 -2.0 E (V vs Ag/Ag+)
2+ Figure 3.15. ECL responses of 1.0 mM H2O2-0.60 mM Ru(bpy)3 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
2+ out in water-MeCN mixture media with added Ru(bpy)3 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.
? Direct ECL detection TATP + H2O2 of TATP in water-MeCN containing Ru(bpy) 2+ Catalase 3
NaN3 TATP + (H O + O ) TATP + 2 2 Catalse (deactivated)
Scheme 3.5. Flow-chart of the elimination of H2O2 from TATP and direct ECL detection of TATP.
250
200
150
100 ECL Intensity (nA) 50
0 ABCD 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).
125
300 (c) (A) 100 (B) 250 (c) 75 200 (b)
150 (a) , nA
, nA 50 (b) ECL ECL i i 100 25 50 (a) 0 0 Blank
0.0 -0.5 -1.0 -1.5 -2.0 2.0 1.5 1.0 0.5 0.0 + E (V vs Ag/Ag+) E (V vs Ag/Ag )
Figure 3.17. (A) Cathodic ECL of (a) 0.50 mM HMTD, (b) 0.50 mM TATP, and (c) 0.50 2+ mM HMTD + 0.50 mM TATP in the presence of 0.60 mM Ru(bpy)3 -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
2+ presence of Ru(bpy)3 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
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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
2+ Ru(bpy)3 /TPrA (TPrA = tri-n-propylamine) system. As a result, a simple and inexpensive method for field detection of TNT may be developed.
2+ 2+ When the Ru(bpy)3 /TPrA system is used, both Ru(bpy)3 and TPrA are
oxidized upon the anodic potential scanning:
TPrA e TPrA 4.1
2 3 Ru(bpy)3 e Ru(bpy)3 4.2
TPrA• free radicals are produced after the deprotonation of the newly produced TPrA•+:
TPrA-H 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
2+ + 3+ reduces Ru(bpy)3 to Ru(bpy)3 that annihilates with Ru(bpy)3 to form the excited
2+ state Ru(bpy)3 * that emits light. 102
2 Ru(bpy)33 TPrA Ru(bpy) P1 4.4
322 Ru(bpy)33 Ru(bpy) Ru(bpy) 3 * Ru(bpy) 3 4.5
22 Ru(bpy)33 * Ru(bpy) hv 4.6
2+ Alternatively, the excited state Ru(bpy)3 * can be produced via the following process:
32 Ru(bpy)33 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
2+ ECL signals of the Ru(bpy)3 /TPrA system:
nTPrA TNT TNTn P2 4.8
+ Additionally, Ru(bpy)3 , another key species for ECL generation (Eq. 4.5) could be consumed by TNT:
2 n nRu(bpy)33 TNT Ru(bpy) TNT 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
2+ ECL generation from the Ru(bpy)3 /TPrA system
2+ Figure 4.1 shows the CV and ECL responses of 1.0 µM Ru(bpy)3 -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
+ 2+ generation of ECL light starts from 0.9 V vs Ag/Ag at which Ru(bpy)3 gets oxidized to
3+ Ru(bpy)3 . The optimum concentration of ECL coreactant was determined by running an
2+ ECL experiment with 1.0 µM Ru(bpy)3 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
2.0 120 1.5 80 1.0
40 0.5
0 0.0 , , ECL CV i i -0.5 -40
-1.0 -80 -1.5 -120 -2.0 1.6 1.2 0.8 0.4 0.0 E (V vs Ag/Ag+)
2+ Figure 4.1. CV and ECL of 1.0 µM Ru(bpy)3 -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.
1.2
1.0
0.8
( 0.6 ECL i 0.4
0.2
0.0
0 20406080100
[TPrA] (mM)
2+ Figure 4.2. Concentration dependence of TPrA on ECL of the Ru(bpy)3 /TPrA system. The experiment was conducted at a 2-mm Pt electrode with a scan rate of 50 mV/s using 2+ 1.0 µM Ru(bpy)3 -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
2+ Ru(bpy)3 and 25 mM TPrA in MeCN containing 0.10 M TBAP. As shown in Figure 4.3,
2+ the ECL emission of the Ru(bpy)3 /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.
1.0 (a)
0.8
0.6
( (b) ECL
i 0.4
0.2 (c)
0.0
1.5 1.0 0.5 0.0 E (V vs Ag/Ag+)
2+ Figure 4.3. ECL quenching of the Ru(bpy)3 (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
2+ 2+ concentrations of Ru(bpy)3 and TPrA were tested. When Ru(bpy)3 concentration was down to nanomolar levels, complete quenching was observed. For example, in a 5 nM
2+ Ru(bpy)3 -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
2+ quenching (with 1.0 μM Ru(bpy)3 ) 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
2+ Ru(bpy)3 concentration.
40
(a) 30
20 (nA) ECL i 10 (b)
(c) 0 (d)
1.51.00.50.0 E (V vs Ag/Ag+)
2+ Figure 4.4. ECL quenching by TNT in the system containing 5 nM Ru(bpy)3 -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
2+ 2+ by ~38% at 1.0 μM Ru(bpy)3 , and ~45% at 10 μM Ru(bpy)3 after the system is added
2+ with 4.4 μM TNT (Figure 4.5b). Notably, increase in Ru(bpy)3 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
2+ effect is more related to the Ru(bpy)3 than to TPrA.
As shown in Figure 4.6, a linear decrease in ECL intensity of the standard
2+ Ru(bpy)3 (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.
2+ Fluorescence quenching of Ru(bpy)3 by TNT
12 (a) 10
8
(b)
6 ( ECL i 4
2
0 0246810 2+ [Ru(bpy) ] (M) 3
2+ Figure 4.5. Ru(bpy)3 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
2+ The fluorescence quenching study was carried out with 1.0 µM Ru(bpy)3 in
2+ MeCN with a quartz cuvette. The test solution was excited at 350 nm and Ru(bpy)3 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.
1.2
[TNT] = 0 1.0
0.8 ( ECL i 0.6
0.4
0.2 0 20 40 60 80 100 120
[TNT] (M)
Figure 4.6. Relationship of ECL intensity with the TNT concentration in the system 2+ containing 1.0 µM Ru(bpy)3 -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
2+ emission of Ru(bpy)3 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
2+ same concentration of Ru(bpy)3 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
0 0 (B) MeCN added (A) Added [TNT]
110 L , a.u. 4 , a.u. 4 0 L MeCN 5x10 110 M FL Intensity 4.4 L MeCN 5x10 FL Intensity 13.2 L MeCN 22 L MeCN 44 LMeCN 66 LMeCN 88 L MeCN 110L MeCN
500 550 600 650 700 500 550 600 650 700 Wavelength (nm) Wavelength (nm)
2+ Figure 4.7. Fluorescence quenching of 3.0 mL of 1.0 μM Ru(bpy)3 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
2+ The quantum yield of ECL (or fluorescence) of Ru(bpy)3 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:
2 0 KECL(f)[Ru(bpy) 3 *] 22 KKECL(f)[Ru(bpy) 3 *] nr [Ru(bpy) 3 *] 4.10 K ECL(f) KKECL(f)+ nr where KECL(f) and Knr stand for the rate constants for ECL (or fluorescence) and
2+ nonradiative decay of the excited state Ru(bpy)3 *. In the presence of quenching agent like TNT, the quantum yield could be expressed as: 110
2 KECL(f)[Ru(bpy) 3 *] 22 2 KKKECL(f)[Ru(bpy) 3 *] nr [Ru(bpy) 3 *] q [TNT][Ru(bpy) 3 *] 4.11 K = ECL(f) KKKECL(f) nr q[TNT] where Kq is the quenching constant of ECL (or fluorescence). By dividing Eq. 4.9 by Eq. 4.10 we get,
00 I Kq 1 [TNT] 1KSV [TNT] 4.12 IKKECL(f) nr This is Stern-Volmer equation, in which the constant terms give the slope of a plot and is
0 called Stern-Volmer constant (KSV). I and I stand for the ECL (or fluorescence) intensities with and without quenching agent, TNT. The reciprocal of (KECL(f) + Knr) gives
2+ the life time (τ) of Ru(bpy)3 * excited state.
1 4.13 KKECL(f) nr
4.0
3.5
3.0 I
/ 2.5 0
I
2.0
1.5
1.0
0 20406080100120 [TNT] (M)
Figure 4.8. Stern-Volmer plot of ECL quenching by TNT. See Figure 4. 6 for experimental conditions. 111
1.6
1.4 I / 0 I
1.2
1.0
0.8 0 20406080100120 [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
2+ 67 10 life time of Ru(bpy)3 * excited state in MeCN (0.562 µs) came out to be 3.5×10
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
2+ quenching, Eq. 4.14), ECL quenching of the Ru(bpy)3 /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+ Ru(bpy)3 * + TNT Ru(bpy) 3 + (TNT) high energy 4.14
Conclusions
The high explosive compound 2,4,6-trinitrotoluene (TNT) was used as an ECL
2+ quenching agent in the Ru(bpy)3 /TPrA system. The TNT was found to quench both
2+ ECL and fluorescence emission of Ru(bpy)3 * in MeCN. This detection technique provides a simple and easy way of detecting TNT at lower micromolar levels. It was
2+ + • realized that the Ru(bpy)3 /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
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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)
2+ 47 with Ru(bpy)3 as luminophore and electrochemically generated H2O2 as a coreactant
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
RONO2 2H2O 2e RONO 3H2O 5.1
RONO2 2e RO NO2 5.2
RO H2O ROH OH 5.3
RONO2 2e R NO3 5.4
R H2O RH OH 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
Stir at 0 oC for 2h Dissolve Conc.H2SO4 Pentaerythritol 0 oC
HNO3 in ice bath
Filter Reaction mixture Precipitate Wash with water
Ice cold water 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
-CH 2
H O CH COCH 2 3 3
10 8 6 4 2 0 ,ppm
1 Figure 5.1. H 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
-0.1
0.0 (b)
0.1 (a) 0.2 CH (Asym .) 2 0.3 OH 0.4 Absorbance NO 0.5 2
0.6 PETN Pentaerythritol CH2 (Sci.) 0.7 4000 3500 3000 2500 2000 1500 1000 500 -1 W avenumber, cm 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).
8
6 (c) A)
4 ( CV i (b) 2
(a) 0
-0.4 -0.6 -0.8 -1.0 + E (V vs Ag/Ag )
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
4.5
4.0
3.5
A) 3.0 (
CV 2.5 i
2.0 25 M 1.5
1.0 0 200 400 600 800 1000 [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
2+ 50 Ru(bpy)3 as ECL luminophore could give very high ECL response compared to the one with electrochemically generated hydrogen peroxide from dissolved oxygen as
42 2+ coreactant. In this method, Ru(bpy)3 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
2+ bead with both Ru(bpy)3 and PETN antibody and magnetic bead with PETN antibody, the sandwich-type assay is formed as shown in Scheme 5.2: 124
PSB Ru(II) Ru(II) PETN + Antibody __ MB Magnet
ECL Detection
Scheme 5.3. Immunoassay based ECL detection of PETN.
2+ When Ru(bpy)3 /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
2+ unbound Ru(bpy)3 -polystyrene beads will be separated and washed from the solution
2+ with magnet in place ensuring that unbound Ru(bpy)3 is completely removed. In ECL
2+ experiment, more Ru(bpy)3 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
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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
2+ 2+ Ru(bpy)3 /TPrA system ( Ru(bpy)3 = 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
2+ Ru(bpy)3 /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
2+ TPrA and hydrogen peroxide (H2O2). HMTD produces ECL response with Ru(bpy)3 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
• 2+/3+ which will deprotonate to HMTD free radical and react with Ru(bpy)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.
2+ When negative potential was applied, both Ru(bpy)3 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
2+ efficient Ru(bpy)3 /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
+ 2+ • • produced Ru(bpy)3 (from Ru(bpy)3 reduction by TPrA free radical) and TPrA free
2+ radical that are responsible of formation of excited state Ru(bpy)3 * for light emission.
2+ TNT was also able to quench excited state Ru(bpy)3 * 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?
3+ 4+ A2: Further oxidation of Ru(bpy)3 to Ru(bpy)3 at ~1.5 V has never occurred either in
2+ aqueous or in acetonitrile, although Ru(bpy)3 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
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