detection of latent fingermarks: different approaches to ... this thesis is dedicated to my family....

Detection of Latent Fingermarks: different approaches to targeting

amino acids in the deposit

Xanthe Spindler 3015223

This thesis is submitted in fulfilment of the requirements for the degree of Doctor of Philosophy (Applied Science).

National Centre for Forensic Studies Faculty of Applied Science

University of Canberra, Australia

March 2010

i

This thesis is dedicated to my family. To my parents and grandparents for their constant encouragement and support, to my sisters and brothers for their endless curiosity, and to their

children, who will hopefully be inspired to become the next generation of scientists.

ii

Abstract

Amino acids are a common organic component of human perspiration and are often used as a

reliable target for latent fingermark enhancement on porous surfaces as they are highly persistent

over long periods of time. Ninhydrin, and its analogues 1,8-diazafluoren-9-one (DFO) and 1,2-

indanedione, remain the benchmark for chemical fingermark enhancement reagents. When applied

to latent fingermarks, ninhydrin produces a non-fluorescent deep purple product on the fingermark

ridges, while 1,2-indanedione and DFO produce a faint pink colouration that luminesces strongly in

the yellow region of the spectrum. However, prior to the research presented in this thesis, little was

known about the reaction of these reagents with amino acids within the fingermark and paper

matrices, particularly with respect to the effect of low concentrations of zinc chloride on the

indanedione-amino acid reaction.

The experimental results obtained indicated that nine α-L-amino acids commonly present in

perspiration formed the same product when reacted with ninhydrin on cellulose substrates, although

the reaction kinetics were highly dependent on the structure of the amino acid. Similar results were

observed with 1,2-indanedione, indanedione-zinc and DFO. Mechanism studies into the

indanedione-amino acid reaction determined that indanedione reacts with amino acids in fingermark

deposits following a similar mechanism to ninhydrin. Zinc chloride stabilised a crucial reaction rate

limiting step in low humidity conditions, acting as a Lewis acid catalyst and accelerating the

reaction to a rate equal to that observed in high humidity conditions. The nature of the porous

substrate and the heating conditions used to accelerate the reaction were also determined to

significantly affect the luminescence of the reaction product.

The preliminary evaluation of a redox cycling dye, nitroblue tetrazolium, as a potentially sensitive

fingermark enhancement reagent on paper was also undertaken. The poor colour and luminescence

obtained upon development of amino acid standards and the failure of the reagent to develop latent

fingermarks indicated that solvent-based initiation of the redox reaction is not compatible with this

form of forensic evidence. The synthesis and trial of a novel reagent incorporating anti-L-amino

acid antibodies conjugated to gold nanoparticles indicated the potential for the detection of amino

acid-rich fingermarks on non-porous surfaces such as glass and metal. When enhanced with a

fluorescent dye, the anti-L-amino acid reagent successfully developed latent fingermarks that had

been aged up to 12 months on glass, foil and ceramic tile substrates.

iv

Acknowledgements

Firstly, there aren't enough “thank you”s for my primary supervisors Chris Lennard at the

University of Canberra and Claude Roux at the University of Technology Sydney, who have always

gone out of their way to provide me with an enjoyable working environment, the ability to share my

work at countless conferences and have supplied me with an almost bottomless pit of resources.

Thank you for sharing years of knowledge with me and giving me the opportunity to teach the next

generation of forensic chemists. Thank you also to Dennis McNevin for giving me a sympathetic

ear when I needed it and constantly providing feedback at R&D meetings. I also owe a thank you to

Michael Dawson at UTS for helping to organise a place for me.

Thanks to James Robertson and Milutin Stoilovic at the Australian Federal Police for allowing me

access to their laboratories, much appreciated extra funding and getting me started on my

experimental work. Thank you also to Linda Xiao, Jim Keegan, Ilona Kramer and Rana Bazzi at

UTS for all their help with coordinating my orders, instrument training and their company in the

lab. I’d also like to thank Andrew McDonagh, Dakrong Pissuwan and Michael Coutts from the

Institute for Nanoscale Technology at UTS, and Oliver Hofstetter at Northern Illinois University for

helping me with the risky part of my project and for supplying much needed materials. Oh, and I

can't forget my fellow Masters and PhD students at the University of Canberra and UTS, who have

provided me with endless entertainment, friendship and practical joke ideas.

Ronald Shimmon deserves his own paragraph. Ron, thank you very much for adopting me as soon

as I arrived at UTS and helping me sort out everything from glassware to experimental procedures

to spending hours trying to diagnose the latest problem with the NMR. I really appreciate all the

effort you've put in to helping me complete my PhD, even if it seemed at times as if there was no

end to it, and to become a competent chemist.

Special thanks to Margaret Vallance for designing a special program to compile and format my

thesis without having to battle with fickle word processors. Finally, thank you to the members of

the International Fingerprint Research Group for their encouragement and fabulous feedback on my

thesis work. Our discussions helped me determine what needed to be done to finalise my

experimental work and form my ideas and conclusions into a cohesive and coherent mass.

v

Acronyms 13C-NMR Carbon-13 nuclear magnetic resonance spectroscopy 1H-NMR Hydrogen-1 (proton) nuclear magnetic resonance spectroscopy

2A1I 2-Amino-1-indanone (intermediate VI)

3-CPBA 3-Chloroperoxybenzoic acid

ABO A, B and O blood groups

AFP Australian Federal Police

AR Analytical reagent

ATP Adenosine 5'-triphosphate

BKA Bundeskriminalamt

BSA Bovine serum albumin

CDCl3 Deuterated chloroform

CFC Chlorofluorocarbon

CIE Colour index based on XY colour coordinates and Z luminance coordinate

COSY Proton correlation spectroscopy

CP Cross polarisation

D1 Relaxation delay

DAB Diaminobenzidine

DCM Dichloromethane

DEPT-135 Distortionless Enhancement of Polarization Transfer (135o decoupling pulse) 13C-NMR

DFO 1,8-diazafluoren-9-one

DMF Dimethyformamide

DMSO Dimethylsulfoxide

DMSO-d6 Deuterated dimethylsulfoxide

DNA Deoxyribonucleic acid

DP Dry powdering

DSC Differential scanning calorimetry

EDC N-(3-Dimethylaminopropyl)-N’-ethyl-carbodiimide hydrochloride

EDDP 2-Ethylidene-1,5-dimethyl-3,3,-diphenylpyrrolidine

EDS Energy dispersive spectroscopy

EDTA Ethylenediaminetetraacetic acid disodium salt

vi

ELISA Enzyme linked immunosorbent assay

EMIT Enzyme-multiplied immunoassay technique

ERK Extracellular signal regulated protein kinases

ESDA Electrostatic detection apparatus

ESI-MS Electrospray ionisation mass spectrometry

ESR Electron spin resonance spectroscopy

f(ab) Crystallisable fragment of the antibody binding region

f(ab’)2 Crystallisable fragment of the binding region of a secondary antibody to the primary antibody

FBI Federal Bureau of Investigation

Fc’ Antibody crystallisable fragment

FO Fluorescent Orange 550 nm

FR Fluorescent Red 610 nm

FRET Forster resonance energy transfer

FT-IR Fourier transform infrared spectroscopy

GA Glutaric dialdehyde (glutaraldehyde)

GC-MS Gas chromatography mass spectrometry

HETCOR Heteronuclear chemical shift correlation by cross-polarisation

HFC Hydrofluorocarbon

HMQC Heteronuclear multiple quantum coherence

HOMO Highest occupied molecular orbital

HOSDB Home Office Scientific Development Branch

HPLC High performance liquid chromatography

HS-OEG3-COOH 23-Mercapto-3,6,9,12-tetraoxatricosanoic acid

HSQC Heteronuclear single quantum correlation

HWOF Horizontal width of field

Ind-Zn Indanedione-zinc reagent

JP Joullié’s pink

Kf’ Conditional complex formation constant

LC-MS Liquid chromatography mass spectrometry

L-DOPA L-3,4-Dihydroxyphenylalanine

LED Light emitting diode

vii

LR Laboratory reagent

LUMO Lowest unoccupied molecular orbital

m/z Mass to charge ratio

MAS Magic angle spinning

MDF Medium density fibreboard

MES 2-Morpholinoethanesulfonic acid monohydrate

MMD Multimetal deposition

MPTMS 3-Mercaptipropyltrimethoxysilane

MSP Microspectrophotometry

NADH Nicotinamide adenine dinucleotide

NADPH Nicotinamide adenine dinucleotide phosphate

NBS N-Bromosuccinimide

NBT Nitroblue tetrazolium

NFN Non-flammable ninhydrin

NHS N-hydroxysuccinimide

NMR Nuclear magnetic resonance

NOESY Nuclear Overhauser effect spectroscopy

P1 90o High pulse time

P15 Contact pulse time

PABA p-Aminobenzoic acid

PBS Phosphate buffered saline

PD Physical developer

PET Polyethylene terephthalate

pKh Metal ion hydrolysis constant

p-NPP p-Nitrophenylphosphate

PVDF Polyvinylidine fluoride

Pyr-d5 Deuterated pyridine

R6G Rhodamine 6G

RCMP Royal Canadian Mounted Police

RF Radio frequency

Rh +/- Rhesus protein positive/negative

RH Relative humidity

viii

RIA Radioimmunoassays

RNA Ribonucleic acid

ROS Reactive oxygen species

RP Ruhemann’s purple

RTP Room temperature and pressure

SAM Self-assembled monolayer

SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis

SEM Scanning electron microscopy

SN2 Bimolecular nucleophilic substitution

SPDP N-Succinimidyl-3-(2-pyridyldithio)propionate

TD Time domain

THC Δ9-tetrahydrocannabinol

TIC Total ion count

TLC Thin layer chromatography

UC University of Canberra

UK United Kingdom

UTS University of Technology, Sydney

UV Ultra violet

VEGF Vascular endothelial growth factor

VSC Video spectral comparator

WD Working distance

Zn-JP Joullié’s pink-zinc complex

Zn-RP Ruhemann’s purple-zinc complex

ΔHf Heat of formation

ε Molar absorptivity (extinction coefficient)

λmax Wavelength of maximum absorbance

ix

Table of Contents

Chapter 1: Fingermark Detection & Enhancement ...................................................................................................... 1

1.1 OVERVIEW .......................................................................................................................................................... 2

1.2 THE HUMAN FINGERPRINT ................................................................................................................................. 4

1.2.1 How are fingermarks deposited? ......................................................................................................... 5

1.3 CHEMICAL DETECTION OF LATENT FINGERMARKS ............................................................................................ 8

1.3.1 Sebum and blood-targeting reagents .................................................................................................... 9

1.3.2 Current amino acid-targeting reagents ............................................................................................... 12

1.3.3 Recent developments in fingermark enhancement ............................................................................ 13

1.3.4 Why target amino acids? .................................................................................................................... 15

1.4 GENERAL RESEARCH OBJECTIVES .................................................................................................................... 16

Chapter 2: Spectroscopic Comparison of the Products Formed Between Nine Common Amino Acids and Indanedione, Indanedione-Zinc, DFO and Ninhydrin ............................................................................. 18

2.1 INTRODUCTION ................................................................................................................................................. 19

2.1.1 The chemistry of ninhydrin development .......................................................................................... 19

2.1.1.1 The formation of Ruhemann’s purple .................................................................................. 20

2.1.1.2 Degradation of RP ............................................................................................................... 25

2.1.1.3 Ninhydrin as a fingermark enhancement reagent ................................................................ 26

2.1.1.4 Previous studies into the RP absorption spectrum .............................................................. 29

2.1.1.5 Post-treatment with metal salts............................................................................................ 32

2.1.2 The chemistry of DFO development .................................................................................................. 33

2.1.2.1 Formation of the DFO-amino acid product ......................................................................... 33

2.1.2.2 DFO as a fingermark enhancement reagent ........................................................................ 35

2.1.2.3 Previous studies into the DFO product spectrum ................................................................ 37

2.1.3 The chemistry of indanedione development ...................................................................................... 38

2.1.3.1 Formation of the indanedione-amino acid product ............................................................. 38

2.1.3.2 Degradation of the product ................................................................................................. 42

2.1.3.3 Indanedione as a fingermark enhancement reagent ............................................................ 43

2.1.3.4 The addition of zinc to indanedione ..................................................................................... 44

2.1.4 Isatin as a latent fingermark enhancement reagent ............................................................................ 48

2.1.4.1 Common biochemical and pharmacological uses of isatin .................................................. 48

2.1.4.2 The reaction mechanism of isatin with α-amino acids ......................................................... 50

2.1.4.3 The adaptation of isatin for latent fingermark enhancement on porous surfaces ................ 51

2.2 OBJECTIVES ...................................................................................................................................................... 53

2.3 EXPERIMENTAL DESIGN ................................................................................................................................... 54

2.3.1 Designing a suitable method of sample manufacture ........................................................................ 54

2.3.1.1 Microspectrophotometry samples ........................................................................................ 55

2.3.1.2 Fluorescence spectrophotometry samples ........................................................................... 55

2.3.2 Method validation .............................................................................................................................. 56

x

2.4 EXPERIMENTAL METHOD ................................................................................................................................. 57

2.4.1 General .......................................................................................................................................... 57

2.4.1.1 Reagents .............................................................................................................................. 57

2.4.1.2 Instrumentation .................................................................................................................... 58

2.4.2 Sample preparation ............................................................................................................................ 58

2.4.2.1 Microspectrophotometry samples ........................................................................................ 58

2.4.2.2 Fluorescence spectrophotometry samples ........................................................................... 60

2.4.3 Visible microspectrophotometry analysis .......................................................................................... 60

2.4.4 Fluorescence spectrophotometry analysis .......................................................................................... 60

2.5 RESULTS AND DISCUSSION ............................................................................................................................... 61

2.5.1 Visible absorption spectra .................................................................................................................. 61

2.5.1.1 Ninhydrin ............................................................................................................................. 61

2.5.1.2 DFO ..................................................................................................................................... 65

2.5.1.3 Indanedione ......................................................................................................................... 69

2.5.1.4 Indanedione-zinc ................................................................................................................. 72

2.5.2 Fluorescence spectrophotometry ....................................................................................................... 76

2.5.2.1 DFO ..................................................................................................................................... 76

2.5.2.2 Indanedione and indanedione-zinc ...................................................................................... 77

2.6 CONCLUSIONS .................................................................................................................................................. 79

2.6.1 Evaluation of ninhydrin, DFO and indanedione ................................................................................ 79

2.6.2 Evaluation of cellulose substrates ...................................................................................................... 81

Chapter 3: The Validation of Magic Angle Spinning Solid-State Nuclear Magnetic Resonance Spectroscopy as a Technique for Studying Latent Fingermark Enhancement Reagents ................................................. 83

3.1 INTRODUCTION ................................................................................................................................................. 84

3.1.1 The effects of solvents on Ruhemann’s purple and analogues .......................................................... 84

3.1.2 Solid-state nuclear magnetic resonance spectroscopy ....................................................................... 85

3.1.2.1 Magic angle spinning and cross polarisation ...................................................................... 85

3.1.2.2 Common application of solid-state NMR spectroscopy to difficult samples ........................ 88

3.2 OBJECTIVES ...................................................................................................................................................... 90

3.3 EXPERIMENTAL DESIGN ................................................................................................................................... 90

3.3.1 Reaction of the latent fingermark reagents with amino acids ............................................................ 90

3.3.2 Optimisation of solid-state NMR parameters .................................................................................... 91

3.3.2.1 Choosing suitable solution NMR experiments ..................................................................... 97

3.4 EXPERIMENTAL METHODS ............................................................................................................................... 98

3.4.1 General .......................................................................................................................................... 98

3.4.1.1 Reagents .............................................................................................................................. 98

3.4.1.2 Solvents ................................................................................................................................ 98

3.4.1.3 Instrumentation .................................................................................................................... 99

3.4.2 Reaction of ninhydrin and analogues with amino acids ..................................................................... 99

xi

3.4.3 NMR analysis .................................................................................................................................. 100

3.4.3.1 Solid-state NMR analysis ................................................................................................... 100

3.4.3.2 Solution NMR analysis ...................................................................................................... 100

3.5 RESULTS AND DISCUSSION ............................................................................................................................. 101

3.5.1 13C-MAS-NMR analysis of latent fingermark enhancement reagent products ................................ 101

3.5.2 Comparison of 13C-MAS-NMR with conventional solution NMR spectroscopy ............................ 107

3.6 CONCLUSIONS ................................................................................................................................................ 114

Chapter 4: A Comparison of the Mechanisms of the Indanedione and Indanedione-Zinc Reagents ................... 116

4.1 INTRODUCTION ............................................................................................................................................... 117

4.1.1 A brief overview of the indanedione reaction .................................................................................. 117

4.1.2 The use of Lewis acid catalysts in organic synthesis ....................................................................... 117

4.1.2.1 Lewis acid catalysed synthesis of amines via the Strecker degradation ............................ 118

4.2 OBJECTIVES .................................................................................................................................................... 120

4.3 EXPERIMENTAL DESIGN ................................................................................................................................. 121

4.3.1 Choosing suitable amino acids ........................................................................................................ 121

4.3.2 Choosing a suitable cellulose substrate ............................................................................................ 123

4.3.3 Optimising reaction conditions ........................................................................................................ 124

4.3.3.1 Cessation of the indanedione reaction using halogenated hydrocarbons ......................... 124

4.3.3.2 Initial mechanism and kinetics studies .............................................................................. 125

4.3.3.3 Lewis acid catalyst studies ................................................................................................. 127

4.3.3.4 Complexometric titrations ................................................................................................. 127

4.3.3.5 Humidity studies ................................................................................................................ 128

4.3.3.6 Confirmation of JP formation by 2-amino-1-indanone synthesis ...................................... 130

4.3.4 The development of a non-destructive extraction procedure ........................................................... 131

4.3.5 Optimising LC-MS conditions ......................................................................................................... 133

4.3.6 Optimising fluorescence spectrophotometry parameters ................................................................. 135

4.4 EXPERIMENTAL METHOD ............................................................................................................................... 137

4.4.1 General ........................................................................................................................................ 137

4.4.1.1 Reagents ............................................................................................................................ 137

4.4.1.2 Solvents .............................................................................................................................. 138

4.4.1.3 Instrumental ....................................................................................................................... 139

4.4.2 Validation of the cellulose substrate ................................................................................................ 140

4.4.3 Indanedione studies ......................................................................................................................... 141

4.4.3.1 Cellulose reaction media ................................................................................................... 141

4.4.3.2 1:1 reaction........................................................................................................................ 141

4.4.3.3 1:2 reaction........................................................................................................................ 142

4.4.3.4 Collection of samples for solid-NMR and DSC ................................................................. 142

4.4.4 Indanedione-zinc studies .................................................................................................................. 142

4.4.4.1 10:10:1 reaction ................................................................................................................ 142

4.4.4.2 10:20:1 reaction ................................................................................................................ 142

xii

4.4.4.3 Collection of samples for solid-NMR and DSC ................................................................. 143

4.4.4.4 Extraction efficiency analysis ............................................................................................ 143

4.4.5 Alanine as the limiting reactant ....................................................................................................... 143

4.4.5.1 1:10 reaction...................................................................................................................... 143

4.4.5.2 1:10:1 reaction .................................................................................................................. 144

4.4.6 Kinetic monitoring of the reaction using UV-visible spectrophotometry ........................................ 144

4.4.6.1 Indanedione reactions ....................................................................................................... 144

4.4.6.2 Indanedione-zinc reactions ................................................................................................ 144

4.4.7 Product characterisation by solid-NMR, FT-IR and DSC ............................................................... 145

4.4.7.1 DSC analysis...................................................................................................................... 145

4.4.7.2 Solid-NMR and FT-IR analysis .......................................................................................... 145

4.4.8 Complexometric titrations ............................................................................................................... 146

4.4.8.1 Ninhydrin-zinc controls ..................................................................................................... 146

4.4.9 Lewis acid catalyst studies ............................................................................................................... 146

4.4.10 Synthesis and characterisation of JP ................................................................................................ 147

4.4.10.1 Synthesis of 2-amino-1-indanone via the oxime route (Rosen & Green, 1963) ................. 147

4.4.10.2 Synthesis of 2-amino-1-indanone via the Friedel-Crafts route (McClure et al., 1981) ..... 147

4.4.10.3 Formation and characterisation of JP ............................................................................... 148


4.5.1 Validation of the cellulose substrate ................................................................................................ 149

4.5.1.1 Preliminary exploration of the optimum zinc chloride concentration in the indanedione-zinc working solution .................................................................................... 152

4.5.2 Identification of the indanedione reaction mechanism .................................................................... 154

4.5.2.1 ESI-MS analysis of the reaction mixture ........................................................................... 154

4.5.2.2 Oligomerisation of indanedione and reaction intermediates under destabilising conditions .......................................................................................................................... 163

4.5.2.3 Determination of reaction kinetics by UV-visible spectrophotometry ............................... 174

4.5.2.4 Determination of the role of zinc(II) ions by complexometric titrations ............................ 178

4.5.2.5 Confirmation of the catalytic site of zinc(II) ions .............................................................. 181

4.5.3 The use of other Lewis acid catalysts in the indanedione reaction .................................................. 186

4.5.4 Characterisation of the indanedione-amino acid product ................................................................. 194

4.5.4.1 Oligomerisation and polymerisation of JP ........................................................................ 196

4.5.5 Synthesis and characterisation of JP from 2-amino-1-indanone (VI) .............................................. 206

4.5.5.1 Chromogenic and fluorogenic conformations of JP .......................................................... 214

4.6 CONCLUSIONS ................................................................................................................................................ 219

4.6.1 Indanedione-zinc mechanism .......................................................................................................... 219

4.6.2 The role of cellulose substrates in the indanedione-amino acid reaction ......................................... 221

xiii

Chapter 5: Evaluation of Redox Cycling Reagents for the Enhancement of Latent Fingermarks on Porous Surfaces ..................................................................................................................................................... 222

5.1 INTRODUCTION ............................................................................................................................................... 223

5.1.1 Biochemical and medical applications of redox cycling .................................................................. 224

5.1.2 Targeting L-DOPA in latent fingermarks ........................................................................................ 226

5.1.3 Detection of redox cycling by visible or chemiluminescent visualisation ....................................... 231

5.1.4 Potential issues associated with visualisation of redox cycling processes ....................................... 233

5.2 OBJECTIVES .................................................................................................................................................... 233


5.3.1 Cellulose standards and fingermark samples ................................................................................... 234

5.3.2 NBT staining .................................................................................................................................... 235

5.4 EXPERIMENTAL METHODS ............................................................................................................................. 237

5.4.1 General ........................................................................................................................................ 237

5.4.1.1 Reagents ............................................................................................................................ 237

5.4.1.2 Solvents .............................................................................................................................. 238

5.4.1.3 Instrumental ....................................................................................................................... 238

5.4.2 Solution formulations ...................................................................................................................... 239

5.4.2.1 NBT working solutions ...................................................................................................... 239

5.4.2.2 Amino acid standard grids and latent fingermark samples ............................................... 239

5.4.2.3 Photo Fenton reagent ........................................................................................................ 239

5.4.2.4 NBS and ferric chloride reagents ...................................................................................... 239

5.4.3 Treatment of samples with NBT ...................................................................................................... 240

5.4.3.1 Amino acid standards ........................................................................................................ 240

5.4.3.2 Fingermark samples .......................................................................................................... 240


5.5.1 The reaction of NBT with amino acids on cellulose substrates ....................................................... 241

5.5.2 Optimisation of NBT as a fingermark enhancement reagent ........................................................... 245

5.6 CONCLUSIONS ................................................................................................................................................ 248

Chapter 6: Enhancement of Latent Fingermarks Using Anti-L-Amino Acid Gold Nanoparticles ....................... 249

6.1 INTRODUCTION ............................................................................................................................................... 250

6.1.1 Interactions between antibody and antigen ...................................................................................... 250

6.1.2 Commonly utilised immunological techniques in forensic science ................................................. 251

6.1.3 The use of antibodies for fingermark enhancement ......................................................................... 253

6.1.3.1 Early attempts at adapting traditional immunological techniques to latent fingermark enhancement ...................................................................................................................... 253

6.1.3.2 Current research into immunological fingermark enhancement techniques ..................... 254

6.1.4 The application of anti-amino acid antibodies to latent fingermark enhancement .......................... 256

6.1.5 Utilising self-assembled monolayer nanoparticles........................................................................... 258

6.1.6 The potential for solvent-free adaptations ....................................................................................... 264

6.2 OBJECTIVES .................................................................................................................................................... 266

xiv


6.3.1 Determining the best method for producing stable conjugated nanoparticles ................................. 266

6.3.2 Optimising the reagent formulation and reaction conditions ........................................................... 269

6.3.3 Choosing suitable substrates for reagent evaluation ........................................................................ 272

6.3.4 Determining the level of non-specific binding ................................................................................ 273

6.3.5 Collection and storage of fingermark samples ................................................................................. 274

6.4 EXPERIMENTAL METHODS ............................................................................................................................. 276

6.4.1 General ........................................................................................................................................ 276

6.4.1.1 Reagents ............................................................................................................................ 276

6.4.1.2 Solvents .............................................................................................................................. 278

6.4.1.3 Instrumental ....................................................................................................................... 278

6.4.2 Purification of polyclonal anti-L-amino acid antibodies ................................................................. 279

6.4.3 Synthesis of the anti-L-amino acid nanoparticle reagent ................................................................. 279

6.4.3.1 Gold nanoparticle synthesis (Turkevich et al., 1951) ........................................................ 279

6.4.3.2 Anti-L-amino acid nanoparticles (Cao & Sim, 2007) ........................................................ 280

6.4.3.3 Anti-L-amino acid nanoparticles (Pissuwan et al., 2007) ................................................. 280

6.4.3.4 Fluorescent tagging of anti-rabbit antibodies ................................................................... 280

6.4.4 Treatment of fingermarks with the anti-L-amino acid reagent ........................................................ 280

6.4.5 Identification of non-specific binding interactions .......................................................................... 281

6.4.5.1 Treatment of latent fingermarks with various nanoparticle reagents ................................ 281

6.4.5.2 Scanning electron microscopy ........................................................................................... 282

6.4.5.3 Competitive enzyme-linked immunosorbent assay ............................................................. 282


6.5.1 Relative performance of Cao and Pissuwan nanoparticle reagents .................................................. 284

6.5.2 Evaluation of dye compatibility and performance with the Rofin Poliview system ........................ 286

6.5.3 Identification of non-specific binding interactions .......................................................................... 290

6.5.4 Relative performance of the anti-L-amino acid reagent against commonly employed reagents on aged fingermarks ............................................................................................................. 302

6.5.4.1 Gender, donor dependence and the effect of cosmetics on reagent performance .............. 312

6.5.4.2 The effectiveness of post-staining aqueous washing baths ................................................ 318

6.6 CONCLUSIONS ................................................................................................................................................ 320

Chapter 7: General Conclusions and Recommendations ......................................................................................... 323

7.1 THE APPLICATION OF INDANEDIONE-ZINC TO LATENT FINGERMARK DETECTION ......................................... 324

7.1.1 Developing and optimising new methods for latent fingermark enhancement using the indanedione-zinc reagent ..................................................................................................................... 326

7.1.2 The use of cellulose substrates for the analysis and characterisation of novel latent fingermark enhancement reagents ..................................................................................................... 327

7.2 THE ADAPTATION OF MAS-NMR FOR STUDYING LATENT FINGERMARK ENHANCEMENT REAGENTS .......... 328

7.3 THE EVALUATION OF REDOX CYCLING AS A FINGERMARK ENHANCEMENT TECHNIQUE ............................... 329

xv

7.4 THE DEVELOPMENT OF NOVEL IMMUNOLOGICAL LATENT FINGERMARK ENHANCEMENT REAGENTS ........... 330

7.4.1 Further development of the anti-L-amino acid reagent .................................................................... 330

7.4.2 The production of a universal immunological reagent .................................................................... 331

7.4.3 Aptamer-based fingermark detection methods ................................................................................ 332

References .................................................................................................................................................................... 334

Appendices .................................................................................................................................................................... 351

I FLUORESCENCE SPECTROPHOTOMETRY ......................................................................................................... 352

II NMR SPECTROSCOPY OF NINHYDRIN, INDANEDIONE, ISATIN AND DFO REACTION PRODUCTS .................... 353

II.i 13C-MAS-NMR ................................................................................................................................ 353

II.ii 1H-NMR ........................................................................................................................................ 353

II.iii 13C-NMR ........................................................................................................................................ 354

II.iv HSQC-NMR .................................................................................................................................... 355

III INDANEDIONE-ZINC MECHANISM STUDIES .................................................................................................... 359

III.i Oligomer formation ......................................................................................................................... 359

III.ii Thin layer chromatography .............................................................................................................. 359

III.iii ESI-MS analysis of the indanedione-amino acid-zinc reaction ....................................................... 362

III.iv Confirmation of the Zn2+ catalytic site by microspectrophotometry................................................ 382

III.v Treatment of latent fingermarks and alanine-impregnated cellulose with zinc chloride prior to the addition of indanedione ................................................................................................................... 383

III.vi NMR spectroscopy of JP ................................................................................................................. 385

III.vi.i 13C-MAS-NMR ................................................................................................................... 385

III.vi.ii Synthesis of JP via 2-amino-1-indanone small scale reaction 1H-NMR ............................ 386

III.vi.iii Synthesis of JP via 2-amino-1-indanone small scale reaction 13C-NMR ........................... 389

III.vi.iv Synthesis of JP via 2-amino-1-indanone large scale reaction 1H-NMR ............................ 390

III.vi.v Synthesis of JP via 2-amino-1-indanone large scale reaction 13C-NMR and DEPT-135 13C-NMR ............................................................................................................................ 391

III.vi.vi Synthesis of JP via 2-amino-1-indanone small scale reaction HSQC-NMR ..................... 397

III.vi.vii Synthesis of JP via 2-amino-1-indanone large scale reaction HSQC-NMR ...................... 400

III.vii FT-IR analysis of JP ........................................................................................................................ 401

III.vii.i Synthesis of JP by reaction of alanine, leucine and phenylalanine with indanedione-zinc ................................................................................................................ 401

III.vii.ii Synthesis of JP via 2-amino-1-indanone small scale reaction FT-IR ............................... 403

III.vii.iii Synthesis of JP via 2-amino-1-indanone large scale reaction FT-IR ............................... 405

III.viii Oligomeric and polymeric products formed by overheating JP ....................................................... 406

III.viii.i FT-IR analysis of oligomeric products .............................................................................. 406

III.viii.ii NMR analysis of oligomeric and polymeric products formed by overheating JP ............. 407

III.viii.iii ESI-MS analysis of oligomeric products formed by refluxing indanedione with alanine in ethanol ........................................................................................................................ 412

III.ix ESI-MS analysis of the indanedione-alanine reaction catalysed by various metallic Lewis acid catalysts ........................................................................................................................................... 413

xvi

IV SUPPLEMENTARY EXPERIMENTAL MATERIAL FOR THE ANTI-L-AMINO ACID REAGENT TRIALS ................... 415

IV.i Unsuccessful anti-L-amino acid SAM syntheses ............................................................................. 415

IV.i.i Conjugation of anti-L-amino acid antibodies using SPDP as a linker (Leggett et al., 2007) .......................................................................................................... 415

IV.i.ii Conjugation of anti-L-amino acid antibodies using glutaraldehyde as a linker (Hun & Zhang, 2007) ...................................................................................................................... 415

IV.ii Cyanoacrylate fuming and rhodamine 6G post-staining .................................................................. 415

V PUBLICATION ABSTRACTS .............................................................................................................................. 417

V.i Journal articles ................................................................................................................................. 417

V.ii Conference publications .................................................................................................................. 417

V.ii.i Posters ............................................................................................................................... 417

V.ii.ii Oral presentations ............................................................................................................. 419

xvii

Table of Tables

Table 1.1: The percentage abundance of the common amino acids in human eccrine secretions relative to serine (serine ratio) as determined by Hamilton, Hadorn et al. and Oro and Skewes (Hamilton, 1965; Hadorn et al., 1967; Oro & Skewes 1965; adapted from Ramotowski, 2001). ................................................................... 8

Table 2.1: Ninhydrin working solution formulations sourced from various police departments (Wallace-Kunkel et al., 2004; Bowman, 2004; Wilkinson et al., 2005; Schwarz et al., 2007; Trozzi, 2000). ................................ 29

Table 2.2: The relative colour yields at 570 nm, in leucine equivalents, as determined by Friedman (Friedman, 2004). ....................................................................................................................................... 30

Table 2.3: DFO working solution formulations sourced from various police departments (Wallace-Kunkel et al., 2004; Bowman, 2004; Wilkinson et al., 2005; Bicknell et al., 2008; Schwarz et al., 2007). .............................. 36

Table 2.4: 1,2-Indanedione working solution formulations sourced from various police departments (Wallace-Kunkel et al., 2004; Bowman, 2004; Bicknell et al., 2008; Wilkinson et al., 2009). ................................................. 44

Table 2.5: Various indanedione-zinc formulations and the ratio of zinc chloride to indanedione in each (Wallace-Kunkel et al., 2004; Bowman, 2004; J.W. Chan, 2008; Bicknell et al., 2008; Wilkinson et al., 2009; Stoilovic, 2009) .......................................................................................................................................... 47

Table 2.6 : The average intrasample and intersample variation in the wavelength of maximum absorption for normalised spectra taken from serine spots using the method described in this section. ........................... 57

Table 2.7: The amino acids used for the study (Ramotowski, 2001). .............................................................................. 59

Table 2.8: Formulations used to prepare 1 L of working solution for each fingermark detection reagent. HFC4310mee was used as a carrier solvent for the ninhydrin and DFO formulations. HFE7100 was used as a carrier solvent for the indanedione and indanedione-zinc. .................................................................................... 60

Table 2.9: Parameters used for recording excitation and emission spectra of indanedione, indanedione-zinc and DFO. ........................................................................................................................................................... 61

Table 3.1: The variations in contact pulse time (P15), 90o high pulse time (P1), relaxation delay (D1) and time domain (TD) analysed during optimisation of the Hartmann-Hahn cross polarisation acquisition parameters. The experiments omitted from the table were spectra obtained using longer scan times than the preceding acquisition method. “Optimum A” represents the initial optimal acquisition parameters. “Optimum” represents the overall optimal conditions. .................................................................................................. 94

Table 3.2: The variations in contact pulse time (P15), 90o high pulse time (P1), relaxation delay (D1) and time domain (TD) analysed during optimisation of the Bloch decay acquisition parameters. ........................................ 95

Table 3.3: The acquisition parameters used for the 13C-MAS-NMR analysis of JP, RP, and the DFO and isatin products. ................................................................................................................................................... 100

Table 4.1: The seasonal average relative humidity recorded for Australian capital cities and two remote centres (1994 to 2009) (Bureau of Meterology; www.bom.gov.au). .............................................................................. 129

Table 4.2: Solvent gradient used for the separation of indanedione reaction intermediates. Solvent A is 40% v/v acetonitrile in aqueous solution. Solvent B is 80% v/v acetonitrile in aqueous solution. ........................ 133

Table 4.3: The mass spectra data collection parameters for the experiments detailed in this chapter. .......................... 134

Table 4.4: The optimised positive and negative ionisation parameters for ESI-MS studies of the indanedione-amino acid reaction. ............................................................................................................................................ 134

Table 4.5: The mass of each sample prepared by the methods outlined in this section used for DSC analysis. ............ 141

Table 4.6: The mass of each sample prepared by the methods outlined in Sections 4.4.3, 4.4.4 and 4.4.9 used for DSC analysis. .................................................................................................................................................... 145

Table 4.7: The mass to charge ratio of In and VII(I)n oligomers present in negative ion mode in alanine, leucine and phenylalanine samples, and the mass to charge ratio of Vn and V(I)n oligomers in alanine samples. The Vn and V(I)n oligomers in leucine and phenylalanine samples occurred at different m/z ratios due to differences in the molecular mass of V substituents. ............................................................................... 168

xviii

Table 4.8: The average mass of free zinc ions (± 1 standard deviation) and percentage of bound zinc in the alanine and serine reactions with 10:1 and 1:1 indanedione-zinc formulations assuming 100% extraction of JP and Zn2+ ions from the cellulose substrate. ..................................................................................................... 180

Table 6.1: The conjugation methods trialled for the synthesis of anti-L-amino acid nanoparticles (Pissuwan et al. 2007; Leggett et al. 2007, Hun & Zhang, 2007; Cao & Sim 2007). .................................................................. 267

Table 6.2: Mean scores for the performance of Fluorescent Orange 550 nm against anti-L-amino acid reagent coupled with Fluorescent Orange 550 nm, and the performance of the Cao method anti-L-amino acid nanoparticles against the Pissuwan method nanoparticles on aluminium foil and glass. ......................... 286

Table 6.3: Mean scores for the performance of Fluorescent Red 610 nm against Fluorescent Orange 550 nm on glass and aluminium foil. .................................................................................................................................. 289

Table 6.4: Mean scores for the performance of gold-citrate, HS-OEG3-COOH and anti-L-amino acid nanoparticles when compared against each other. Comparative scores were calculated for top row formulation versus formulations listed down the first column. Overall performance was calculated as the average performance of the reagent against all other reagents tested. ................................................................... 291

Table 6.5: Mean scores for the performance of the anti-L-amino acid/anti-rabbit Fluorescent Red 610 nm reagent on fingermarks aged for two weeks compared to cyanoacrylate fuming for 30 minutes followed by rhodamine 6G staining or dry powder. ..................................................................................................... 303

Table 6.6: Mean scores for the performance of the anti-L-amino acid/anti-rabbit Fluorescent Red 610 nm reagent on fingermarks aged for one month compared to cyanoacrylate fuming for 30 minutes followed by rhodamine 6G staining or dry powder. ..................................................................................................... 303

Table 6.7: Mean scores for the performance of the anti-L-amino acid/anti-rabbit Fluorescent Red 610 nm reagent on fingermarks aged for three months compared to cyanoacrylate fuming for 30 minutes followed by rhodamine 6G staining or dry powder. ..................................................................................................... 307

Table 6.8: Mean scores for the performance of the anti-L-amino acid/anti-rabbit Fluorescent Red 610 nm reagent on fingermarks aged for six months compared to cyanoacrylate fuming for 30 minutes followed by rhodamine 6G staining or dry powder. ..................................................................................................... 307

Table 6.9: Mean scores for the performance of the anti-L-amino acid/anti-rabbit Fluorescent Red 610 nm reagent on fingermarks aged for twelve months compared to cyanoacrylate fuming for 30 minutes followed by rhodamine 6G staining. ............................................................................................................................ 310

Table 6.10: Raw scores taken for two female and three male donors from the experiments discussed in Section 6.5.4. ........................................................................................................................................... 313

Table III.1: The mobile phases used for TLC separation of the indanedione-alanine reaction components. ................ 360

Table III.2: The mass to charge ratio of indanedione-alanine-zinc reaction constituents and adducted products formed during positive mode ionisation. Ions are labelled I-VII or L1 and L2 based on the mechanisms proposed in Figures 4.11, 4.12, 4.16 and 4.34. Ratios in the table header correspond to the ratio of indanedione to alanine to zinc. “Hydrindantin” refers to the formation of a dimeric indanedione structure similar to hydrindantin and I(OH)2 refers to the diol resonance structure of indanedione. ...................................... 362

Table III.3: The mass to charge ratio of indanedione-alanine-zinc reaction constituents and adducted products formed during negative mode ionisation. Ions are labelled I-VII or L1 and L2 based on the mechanisms proposed in Figures 4.11, 4.12, 4.16 and 4.34. Ratios in the table header correspond to the ratio of indanedione to alanine to zinc. “Hydrindantin” refers to the formation of a dimeric indanedione structure similar to hydrindantin and I(OH)2 refers to the diol resonance structure of indanedione. ...................................... 365

Table III.4: The mass to charge ratio of indanedione-leucine-zinc reaction constituents and adducted products formed during positive mode ionisation. Ions are labelled I-VII or L1 and L2 based on the mechanisms proposed in Figures 4.11, 4.12, 4.16 and 4.34. Ratios in the table header correspond to the ratio of indanedione to alanine to zinc. “Hydrindantin” refers to the formation of a dimeric indanedione structure similar to hydrindantin and I(OH)2 refers to the diol resonance structure of indanedione. ...................................... 368

Table III.5: The mass to charge ratio of indanedione-leucine-zinc reaction constituents and adducted products formed during negative mode ionisation. Ions are labelled I-VII or L1 and L2 based on the mechanisms proposed in Figures 4.11, 4.12, 4.16 and 4.34. Ratios in the table header correspond to the ratio of indanedione to alanine to zinc. “Hydrindantin” refers to the formation of a dimeric indanedione structure similar to hydrindantin and I(OH)2 refers to the diol resonance structure of indanedione. ...................................... 372

xix

Table III.6: The mass to charge ratio of indanedione-phenylalanine-zinc reaction constituents and adducted products formed during positive mode ionisation. Ions are labelled I-VII or L1 and L2 based on the mechanisms proposed in Figures 4.11, 4.12, 4.16 and 4.34. Ratios in the table header correspond to the ratio of indanedione to alanine to zinc. “Hydrindantin” refers to the formation of a dimeric indanedione structure similar to hydrindantin and I(OH)2 refers to the diol resonance structure of indanedione. ...................... 376

Table III.7: The mass to charge ratio of indanedione-phenylalanine-zinc reaction constituents and adducted products formed during negative mode ionisation. Ions are labelled I-VII or L1 and L2 based on the mechanisms proposed in Figures 4.11, 4.12, 4.16 and 4.34. Ratios in the table header correspond to the ratio of indanedione to alanine to zinc. “Hydrindantin” refers to the formation of a dimeric indanedione structure similar to hydrindantin and I(OH)2 refers to the diol resonance structure of indanedione. ..................... 379

Table III.8: The mass to charge ratio of VIIn, Vn, In, VII(I)n and V(I)n oligomeric byproducts, formed upon the reflux of an equimolar and 10:1 ratio of indanedione and alanine for 24 hours, analysed by positive ion ESI-MS.412

Table III.9: The mass to charge ratio of indanedione-alanine-M2+ reaction constituents and adducted products formed during negative mode ionisation. Ions are labelled I-VII or L1 and L2 based on the mechanisms proposed in Figures 4.11, 4.12, 4.16 and 4.34. Ratios in the table header correspond to the ratio of indanedione to alanine to zinc. “Hydrindantin” refers to the formation of a dimeric indanedione structure similar to hydrindantin and I(OH)2 refers to the diol resonance structure of indanedione. ...................................... 413

xx

Table of Figures

Figure 1.1: Reproductions of (a) loop, (b) arch and (c) whorl fingerprint patterns (Saferstein, 2004). ............................. 5

Figure 1.2: The structures of (left) gentian violet, (centre) ethyl cyanoacrylate monomer and (right) polyethylcyanoacrylate............................................................................................................................... 10

Figure 1.3: The structure of amido black (top) and the reaction of DAB with hydrogen peroxide in the presence of haemoglobin (Zhang et al., 2008). ............................................................................................................. 12

Figure 2.1: The reaction scheme indicating the formation of hydrated ninhydrin from 1-indanone and p-nitrosodimethylaniline (Almog, 2001). ...................................................................................................... 19

Figure 2.2: The mechanism of conversion of ninhydrin hydrate to the anhydrous form (Almog, 2001). ....................... 19

Figure 2.3: One published mechanism for RP production (Almog, 2001). ..................................................................... 21

Figure 2.4: The Friedman and Williams mechanism of RP formation (Friedman, 2004). .............................................. 22

Figure 2.5: The (a) McCaldin and (b) Lamothe ninhydrin-amino acid reaction mechanisms (Petraco, 2006). ............... 24

Figure 2.6: RP degradation via the hydrolysis pathway in acidic solutions as published by Friedman and Williams (Friedman, 2004). ....................................................................................................................................... 26

Figure 2.7: the effects of solvent on the UV-visible spectroscopic determination of RP (Friedman, 2004). .................. 31

Figure 2.8: The difference in solution 1H-NMR spectral resolution of ninhydrin obtained in DMSO-d6 and methanol-d6, and RP in DMSO-d6 and CF3COOD obtained by Friedman (Friedman, 2004). ........................................ 31

Figure 2.9: The RP-zinc complex formed upon treatment of ninhydrin-enhanced fingermarks with zinc chloride solution (Lennard et al. 1987; Davies et al., 1995). ................................................................................... 32

Figure 2.10: The structure of DFO. ................................................................................................................................. 33

Figure 2.11: The basic mechanism of the reaction between DFO and α-amino acids. (Adapted from Almog, 2001) .... 34

Figure 2.12: The structure of the product formed between DFO and amino acids (Almog, 2001). X-ray crystallography showed that the product is reminiscent of RP (Wilkinson, 2000b). ........................................................... 34

Figure 2.13: The absorbance and emission spectra of the DFO product (Wilkinson, 2000). .......................................... 37

Figure 2.14: The proposed mechanism of the indanedione/amino acid reaction. (Adapted from Almog, 2001) ............ 38

Figure 2.15: The proposed products of the reaction between 1,2-indanedione and glycine (Mekkaoui Alaoui et al., 2005). ......................................................................................................................................................... 39

Figure 2.16: Absorption spectra for latent fingermarks developed with indanedione and indanedione-zinc (bold) (Stoilovic et al., 2007). ............................................................................................................................... 46

Figure 2.17: The synthesis of isatin from indigo dye. ..................................................................................................... 48

Figure 2.18: The product of the reaction between 3,4-dehydroproline and (a) isatin and (b) 1,5-dimethylisatin as identified by Hudson and Robertson (Hudson & Robertson, 1967) . ......................................................... 49

Figure 2.19: The reaction between isatin and an ethanolic solution of ethylamine (Sumpter, 1944). ............................. 50

Figure 2.20: The isatin-amine reaction mechanism proposed by Rehn (Rehn et al., 2004). ........................................... 51

Figure 2.21: A comparison between the isatin fingermark reagent developed by Chan and the current AFP formulations of the zinc post-treated ninhydrin (Zn-RP), DFO and indanedione-zinc reagents (Ind-Zn) at optimum visualisation conditions for each reagent (J. Chan, 2008). ......................................................................... 52

Figure 2.22: The proposed structure of the complex formed between the isatin product and zinc(II) ions (J. Chan, 2008). ......................................................................................................................................................... 53

Figure 2.23: The normalised visible absorption spectra for RP formed from the reaction of the nine amino acids with ninhydrin on cellulose TLC plates. ............................................................................................................ 62

Figure 2.24: The normalised visible absorption spectra for RP formed from the reaction of the nine amino acids with ninhydrin on filter paper. ............................................................................................................................ 63

Figure 2.25: The normalised visible absorption spectra for RP formed from the reaction of the nine amino acids with ninhydrin on white copy paper. .................................................................................................................. 63

xxi

Figure 2.26: A comparison the RP spectra taken from the three cellulose surfaces. ....................................................... 64

Figure 2.27: The normalised visible absorption spectra of the products formed from the reaction of the nine amino acids with DFO on cellulose TLC plates. ................................................................................................... 65

Figure 2.28: The normalised visible absorption spectra for the product formed from the reaction of the nine amino acids with DFO on filter paper. ........................................................................................................................... 67

Figure 2.29: The normalised visible absorption spectra for the product formed from the reaction of the nine amino acids with DFO on white copy paper. ................................................................................................................. 68

Figure 2.30: A comparison the DFO product spectra taken from the three cellulose surfaces. ....................................... 69

Figure 2.31: Visible absorption spectra for the products from the reaction of the nine amino acids with indanedione on white copy paper. ....................................................................................................................................... 70

Figure 2.32: Visible absorption spectra for the products from the reaction of the nine amino acids with indanedione on white copy paper after humidification. ....................................................................................................... 71

Figure 2.33: Visible absorption spectra for the products from the reaction of the nine amino acids with indanedione-zinc on filter paper. .................................................................................................................................... 73

Figure 2.34: Visible absorption spectra for the products from the reaction of the nine amino acids with indanedione-zinc on cellulose TLC plates. ..................................................................................................................... 73

Figure 2.35: Visible absorption spectra for the products from the reaction of the nine amino acids with indanedione-zinc on white copy paper. ........................................................................................................................... 74

Figure 2.36: A comparison of the effects of humidity and zinc chloride on the reaction between amino acids and indanedione. ............................................................................................................................................... 75

Figure 2.37: A comparison of JP spectra derived from indanedione-zinc on three cellulose surfaces. ........................... 75

Figure 2.38: The normalised luminescence spectra for the DFO product formed from the reaction of the nine amino acids with DFO on cellulose-coated TLC plates. ....................................................................................... 76

Figure 2.39: The normalised luminescence spectra for JP formed from the reaction of the nine amino acids with indanedione on cellulose-coated TLC plates. ............................................................................................. 77

Figure 2.40: The normalised luminescence spectra for JP formed from the reaction of nine amino acids with indanedione on cellulose-coated TLC plates after humidification. ............................................................ 79

Figure 2.41: The normalised luminescence spectra for JP formed from the reaction of nine amino acids with indanedione-zinc on cellulose-coated TLC plates after humidification. .................................................... 79

Figure 3.1: Signal broadening of fast MAS 1H-NMR spectra caused by increased hydrogen bonding of water to a protein structure (Akbey et al., 2010). ....................................................................................................... 89

Figure 3.2: A comparison between the spectra obtained for JP using Hartmann-Hahn cross polarisation (blue) and Bloch decay (red) after 1024 scans. ........................................................................................................... 92

Figure 3.3: The effect of contact time on the intensity of the kaolinite 29Si signal determined by Kolodziejski and Klinowski (Kolodziejski and Klinowski, 2002). ........................................................................................ 93

Figure 3.4: Quantitative analysis of the effect of contact pulse time on spectral quality. ............................................... 95

Figure 3.5: The effect of increased spin rate on the quality of 13C-MAS-NMR spectra after 1024 scans. The spectrum obtained at a spin rate of 6000 Hz is shown in blue, while the spectrum obtained at a spin rate of 8000 Hz is shown in red. The amide signal (H) was too weak to be detected. ......................................................... 96

Figure 3.6: The 13C-MAS-NMR spectrum of the product formed by reaction of alanine with 1,2-indanedione at room temperature for 18 hours. ......................................................................................................................... 102

Figure 3.7: The 13C-MAS-NMR spectrum of the product formed by the reaction of alanine with ninhydrin at room temperature for 18 hours. ......................................................................................................................... 102

Figure 3.8: The 13C-MAS-NMR spectrum of the product formed by the reaction of alanine with DFO at room temperature for 18 hours. The peaks at 74.9 and 78.1 ppm are due to residual chloroform. The peak at 62.8 ppm indicates the presence of the ethanol adduct proposed in Figure 3.10. .................................... 103

xxii

Figure 3.9: The 13C-MAS-NMR spectrum of the product formed by the reaction of alanine with isatin at room temperature for 18 hours. The shoulder peak at 114.3 ppm is due to keto-enol tautomerisation of the product during extraction. ........................................................................................................................ 103

Figure 3.10: The proposed pathway of adduct formation between the DFO product and ethanol identified in solid-state and solution NMR. ................................................................................................................................... 105

Figure 3.11: Unit cell arrangement of 1,2-indanedione oxime molecules in the solid state (Hartung et al., 2004). ...... 106

Figure 3.12: The 1H-NMR spectrum (top) of JP derived from the reaction of alanine with indanedione at 160o C on cellulose, and the 13C-NMR spectrum (bottom) of JP derived from the reaction of alanine with indanedione at room temperature on cellulose, dissolved in acetone-d6. The annotated peaks on the 13C-NMR spectrum correlate to the carbon atoms labelled in Figure 3.6. Peaks a and b in the 1H-NMR spectrum were obscured by solvent traces. .............................................................................................. 108

Figure 3.13: The 1H-NMR (top) and 13C-NMR (bottom) spectra of RP, dissolved in DMSO-d6, derived from the reaction of alanine with ninhydrin at room temperature on cellulose. The annotated peaks on the 13C-NMR spectrum correlate to the carbon atoms labelled in Figure 3.7. ..................................................... 110

Figure 3.14: The 1H-NMR (top) and 13C-NMR (bottom) spectra of the DFO product, dissolved in acetone-d6, derived from the reaction of alanine with DFO at 180o C on cellulose. The annotated peaks on the 13C-NMR spectrum correlate to the carbon atoms labelled in Figure 3.8. ............................................................... 112

Figure 3.15: The 1H-NMR (top) and 13C-NMR (bottom) spectra of the isatin product, dissolved in acetone-d6, derived from the reaction of alanine with isatin at 180o C on cellulose. The annotated peaks on the 13C-NMR spectrum correlate to the carbon atoms labelled in Figure 3.9. The peak at 113.1 ppm is due to keto-enol tautomerisation of the product during extraction. .................................................................................... 113

Figure 4.1: The basic reaction scheme of sulfinimines with indanylaldehydes to form diethylamine compounds, and the role of Lewis acid metal cation catalysts in assigning the stereochemistry of the resultant amine (Mabic & Cordi, 2001). ............................................................................................................................................ 119

Figure 4.2: The excitation and emission spectra of JP. .................................................................................................. 135

Figure 4.3: The three-dimensional fluorescence spectra of JP formed by the reaction of 2-amino-1-indanone with (a) itself and (b) indanedione at room temperature. The spectra were captured on cellulose. ....................... 136

Figure 4.4: The products obtained from the (a) indanedione-alanine and (b) indanedione-alanine-zinc 10:10:1 on cellulose, and (c) indanedione-alanine-zinc 1:1:1 reactions in solution. .................................................. 149

Figure 4.5: The 13C-MAS-NMR spectra of the indanedione-alanine-zinc 10:10:1reactions performed on cellulose (red) and in solution (blue). The annotated peak assignments correspond to the carbon atoms labelled in Figure 4.50b. ....................................................................................................................................................... 150

Figure 4.6: The DSC curves recorded for the indanedione-alanine 1:1 and indanedione-alanine-zinc 10:10:1 and 1:1:1 reactions performed on cellulose and in solution. .................................................................................... 151

Figure 4.7: FT-IR spectra of the indanedione-alanine 1:1(black), indanedione-alanine-zinc 10:10:1 (pink) and 1:1:1 (purple) reactions performed on cellulose and in solution. ...................................................................... 153

Figure 4.8: Negative ion ESI-MS spectra obtained from the 1:1 (black), 10:10:1 (red) and 10:20:1 (purple) reactions between alanine, indanedione and indanedione-zinc (100-600 amu). ...................................................... 155

Figure 4.9: Negative ion ESI-MS spectra obtained from the 1:1 (black), 1:2 (red), 10:10:1 (grey) and 10:20:1 (blue) reactions between leucine and indanedione-zinc (100-600 amu). ............................................................ 157

Figure 4.10: Negative ion ESI-MS spectra obtained from the 1:1 (black), 1:2 (red), 10:10:1 (grey) and 10:20:1 (blue) reactions between phenylalanine and indanedione-zinc (100-600 amu). ................................................. 159

Figure 4.11: The structures of the lactone byproducts and the proposed mechanism of formation. .............................. 160

Figure 4.12: Proposed reaction mechanism of the indanedione-zinc reagent with amino acids. Numbers 1-4 indicate possible zinc interaction sites. Site 2 denotes the interaction of zinc with the amino acid before and after nucleophilic attack. Site 3 denotes the interaction of zinc after nucleophilic attack to assist decarboxylation. ....................................................................................................................................... 161

Figure 4.13: Negative ion ESI-MS spectra obtained from the 1:10:1 reactions between alanine (black), leucine (red), phenylalanine (blue) and indanedione-zinc (100-600 amu). .................................................................... 162

xxiii

Figure 4.14: Distribution of dimeric, trimeric and oligomeric analogues of indanedione, JP and intermediate V in reactions involving phenylalanine, leucine and alanine in positive and negative ionisation mode. ......... 165

Figure 4.15: The proposed propagation mechanism of the VII(I)n, In, V(I)n and Vn oligomers. .................................. 169

Figure 4.16: The FT-IR (top) and 1H-NMR (bottom) spectra obtained from the green oligomeric product formed upon reflux of an excess of indanedione with alanine for 24 hours. The peaks at 6.52, 6.58 and 4.09 ppm in the 1H-NMR spectrum indicate the presence of the enamine monomer presented in Figure 4.49. ............... 171

Figure 4.17: ESI-MS taken of the 10:1 reflux reaction between indanedione and alanine at 1.5 hours, 3.5 hours, 5.5 hours and 24 hours. .................................................................................................................................. 173

Figure 4.18: A comparison of the reaction kinetics, as determined by UV-visible spectroscopy for (a) alanine, (b) phenylalanine and (c) leucine reacted with an equimolar ratio of indanedione (dashed) and a 20:1 solution of indanedione-zinc (solid) at 25o C. ........................................................................................................ 175

Figure 4.19: A comparison of the reaction kinetics, as determined by UV-visible spectroscopy for (a) alanine, (b) phenylalanine and (c) leucine reacted with an equimolar ratio of indanedione (dashed) and a 20:1 solution of indanedione-zinc (solid) at 37o C. ........................................................................................................ 176

Figure 4.20: A graphical representation of the change in the percentage of free zinc ions over time in the alanine and serine reactions with 10:1 and 1:1 indanedione-zinc formulations. An unbound zinc level of 50% in the equimolar indanedione-amino acid-zinc reaction would indicate Zn-JP complex formation. ................. 179

Figure 4.21: The visible absorbance of the indanedione-alanine reactions from 0.5 to 24 hours at 20% (dashed), 60% (solid) and 85% (dotted) relative humidity. ............................................................................................. 183

Figure 4.22: The major catalytic site of zinc chloride in the reaction between amino acids and indanedione-zinc. ..... 184

Figure 4.23: The visible absorbances of the indanedione-alanine-zinc reactions from 0.5 to 24 hours at 20% (dashed), 60% (solid) and 85% (dotted) relative humidity. ..................................................................................... 185

Figure 4.24: A comparison of the visible absorbance spectra of reactions performed with zinc (blue), calcium (black), tin(II) (green), ammonium (red) and lead chloride (pink). ....................................................................... 187

Figure 4.25: The predicted optimum geometries and electron cloud distribution of intermediate V with (a) lead, (b) tin(II), (c) calcium and (d) zinc ions using ACD Labs ChemSketch v. 12.01. ......................................... 188

Figure 4.26: The negative ion ESI-MS produced by equimolar reactions between indanedione and alanine catalysed by zinc chloride (grey), tin(II) chloride (blue), lead chloride (pink), calcium chloride (black) and ammonium chloride (red) (100-600 amu). .................................................................................................................. 190

Figure 4.27: The 1H-NMR spectra produced by equimolar reactions between indanedione and alanine catalysed by zinc chloride (blue), tin(II) chloride (red), calcium chloride (purple) and ammonium chloride (green). The annotated peaks correspond to the hydrogen atoms labelled in Figure 4.49. .......................................... 191

Figure 4.28: The DSC curves obtained from equimolar reactions between indanedione and alanine catalysed by zinc chloride, tin(II) chloride, calcium chloride and ammonium chloride. ...................................................... 193

Figure 4.29: The 13C-MAS-NMR spectra obtained from the reaction of alanine (blue), leucine (red) and phenylalanine (green) with indanedione-zinc at a 10:10:1 ratio. The annotated peak assignments correspond to the carbon atoms labelled in Figure 4.50b. The results obtained from the uncatalysed samples are presented in Appendix III.vi. .................................................................................................................................. 194

Figure 4.30: The three major resonance structures of JP identified by NMR and FT-IR spectroscopy; (a) neutral ketone tautomer, (b) enamine structure and (c) enol tautomer. ........................................................................... 195

Figure 4.31: Distribution of dimeric, trimeric and oligomeric analogues of JP in reactions involving phenylalanine, leucine and alanine in (a) positive ionisation mode, and (b) negative ionisation mode. .......................... 198

Figure 4.32: A comparison of the FT-IR spectra obtained from the 10:10:1 indanedione-alanine-zinc reaction performed in solution (purple) and the black polymeric product formed upon heating at 210o C (black). Detailed peak assignments are presented in Appendix III.viii.i. ............................................................. 200

Figure 4.33: Proposed mechanism of formation and structure of the VIIn oligomer. ................................................... 201

Figure 4.34 : A comparison of the 1H-NMR spectra obtained from the red VII6 oligomer formed by vacuum recrystallisation of JP (blue) and the dark red oligomeric product formed upon reflux of equimolar ratios of indanedione and alanine for 24 hours (red). ......................................................................................... 202

xxiv

Figure 4.35: A comparison of the FT-IR spectra of the red VII6 product obtained from vacuum recrystallisation of JP (black) and the dark red product obtained upon reflux of an equimolar ratio of indanedione and alanine over 24 hours (blue). ................................................................................................................................ 203

Figure 4.36: An analysis of the rate of VIIn formation and consumption during the 24 hour reaction period. ............. 204

Figure 4.37: ESI-MS taken of the equimolar reflux reaction between indanedione and alanine at 1.5 hours, 3.5 hours, 5.5 hours and 24 hours. ............................................................................................................................ 205

Figure 4.38: The structures of the compounds present in the small-scale reaction mixtures. ........................................ 206

Figure 4.39: The hydrolytic deamination of 2-amino-1-indanone to form 1,2-indanedione and the secondary reaction pathway to JP. .......................................................................................................................................... 207

Figure 4.40: The 1H-NMR spectra of JP produced by reaction of 2-amino-1-indanone at room temperature on cellulose. The annotated peak assignments correspond to the labelled hydrogen atoms in Figure 4.49. The highlighted region represents excess indanedione. .................................................................................. 208

Figure 4.41: The FT-IR spectra of JP products by the reaction of oxime derived 2-amino-1-indanone with indanedione (black), alanine (blue) and itself (pink) in DCM solution at 160o C. ....................................................... 209

Figure 4.42: The three structural isomers of JP that could potentially form from the reaction between 1,2-indanedione and α-amino acids. ................................................................................................................................... 211

Figure 4.43: 1H-NMR of JP synthesised by the large scale reaction of 2-amino-1-indanone and indanedione in DCM solution at room temperature. The annotated peak assignments correspond to the labelled hydrogen atoms in Figure 4.49. ......................................................................................................................................... 212

Figure 4.44: The FT-IR spectra of JP products by the large scale reaction of oxime derived 2-amino-1-indanone with indanedione (purple), alanine (blue) and itself (black) in DCM solution at room temperature................ 213

Figure 4.45: The FT-IR of JP products by the large scale reaction of Friedel-Crafts derived 2-amino-1-indanone with indanedione (black) and itself as crude (blue) and recrystallised (purple) product in DCM solution at room temperature. .............................................................................................................................................. 213

Figure 4.46: Solution and solid state visible spectroscopy of JP formed by the reaction between 2-amino-1-indanone and alanine, itself and indanedione at room temperature in DCM. .......................................................... 215

Figure 4.47: Normalised solution and solid-state fluorescence emission spectra of JP formed by the reaction between 2-amino-1-indanone and alanine, itself and indanedione at room temperature in DCM. ............................ 216

Figure 4.48: The three major resonance structures of JP identified by NMR and FT-IR spectroscopy; (a) neutral ketone tautomer, (b) enamine structure and (c) enol tautomer. ........................................................................... 217

Figure 4.49: (a) 1H-NMR and (b) DEPT-135 spectra of the reaction of 2-amino-1-indanone with itself (Friedel-Crafts route) (green), indanedione (oxime route) (purple) and JP formed by reaction of alanine with indanedione in solution (red) and on cellulose (blue). The highlighted peak at 3.60 ppm is due to the presence of indanedione. ............................................................................................................................................. 218

Figure 5.1: Chemical structures of L-DOPA, L-tyrosine and L-phenylalanine. ............................................................ 223

Figure 5.2: The redox cycling mechanism of adrenaline in the presence of NADH in vivo (adapted from Genova et al., 2006). ....................................................................................................................................................... 224

Figure 5.3: Synthesis of L-DOPA, dopamine and melanin via tyrosinase catalysis (adapted from Pajak & Kanska, 2009 and Hwang & Mu Lee, 2007). .................................................................................................................. 228

Figure 5.4: Synthesis and consumption of protein-bound L-DOPA in mammals (Rodgers & Dean, 2000). ................ 230

Figure 5.5: Formation of formazan upon addition of NBT to redox cycling reactions. ................................................ 232

Figure 5.6: The templates used for the (a) standard solution grids, (b) fresh fingermark depletion series, and (c) degraded fingermark depletion series. ...................................................................................................... 235

Figure 5.7: The visible colour change observed upon treatment of amino acid spots with 0.24 mM NBT following (a) no pre-treatment, (b) heat shock (180o C), (c) DMSO, (d) photo Fenton reagent, (e) 3-CPBA photo Fenton reagent, (f) NBS, (g) ferric chloride and (h) indanedione-zinc on copy paper. ........................................ 243

Figure 5.8: The fluorescence observed at 450 nm excitation (530 nm band pass filter) upon treatment of amino acid spots with 0.24 mM NBT following (a) no pre-treatment, (b) heat shock (180o C), (c) DMSO, (d) photo

xxv

Fenton reagent, (e) 3-CPBA photo Fenton reagent, (f) NBS, (g) ferric chloride and (h) indanedione-zinc on copy paper. Images were taken in sections and merged together. ....................................................... 244

Figure 5.9: The results obtained from treatment of fresh fingermarks with a 0.50 mM working solution of NBT (left half) after pre-treatment with (a) no pre-treatment, (b) heat shock (180o C), (c) DMSO, (d) photo Fenton reagent, (e) NBS, and (f) ferric chloride on copy paper. Performance compared to indanedione-zinc (right half). ......................................................................................................................................................... 247

Figure 6.1: The agglutination reaction between anti-A/B antibodies with A, B and AB group erythrocytes (Saferstein, 2004). ....................................................................................................................................................... 252

Figure 6.2: The results of a cross-over precipitin test for various dilutions of human serum against human antiserum. This method is commonly used for the forensic speciation of blood samples (adapted from Saferstein, 2004). ....................................................................................................................................................... 252

Figure 6.3: The anti-cotinine/nanoparticle conjugates synthesised by Leggett et al. (Leggett et al., 2007). ................. 255

Figure 6.4: Third level detail of fingermarks developed with the anti-cotinine/nanoparticle reagent visualised using (a) Alexa Fluor 488 and (b) Alexa Fluor 546 (Leggett et al., 2007). ............................................................. 256

Figure 6.5: A schematic diagram illustrating the application of the anti-L-amino acid reagent discussed in this chapter to latent fingermark samples. ................................................................................................................... 263

Figure 6.6: Fingermarks developed using an A. calcoaceticus embedded gel (Harper et al., 1987). ............................ 265

Figure 6.7: The visible spectra obtained from the six conjugation methods immediately after synthesis and after storing at 4o C for three weeks. ............................................................................................................................ 269

Figure 6.8: Carrier solvent trials for the anti-L-amino acid nanoparticle reagent synthesised by the (a) Pissuwan and (b) Cao methods. ............................................................................................................................................ 270

Figure 6.9: The suspended platform used to halve fingermarks deposited on glass microscope slides. ........................ 273

Figure 6.10: The collection of latent fingermarks on (a) ceramic tile and (b) glass and aluminium foil substrates. ..... 275

Figure 6.11: The comparison system used for the non-specific binding fingermark analyses. ..................................... 282

Figure 6.12: The plate layout for the non-specific binding ELISA experiment. 1: L-alanine; 2:D-alanine; 3: D-tyrosine; 4: L-tyrosine; 5: methionine; 6: D-glucose; 7: oleic acid; 8: agarose; 9-12: blank . ................................ 283

Figure 6.13: Split fingermarks on (a) aluminium foil and (b) glass microscope slides representing the best results obtained with anti-L-amino acid nanoparticles derived from the Pissuwan (left half) and Cao (right half) synthetic methods. .................................................................................................................................... 285

Figure 6.14: The structures of Fluorescent Orange 550 nm (left) and Fluorescent Red 610 nm (right) prior to conjugation to anti-rabbit antibodies. ....................................................................................................... 287

Figure 6.15: The excitation and emission spectra of Fluorescent Orange 550 nm (top) and Fluorescent Red 610 nm (bottom) (Fluka specification sheet). ........................................................................................................ 288

Figure 6.16: Typical results obtained on (a) glass microscope slides and (b) aluminium foil for latent fingermarks developed with anti-L-amino acid reagent followed by Fluorescent Orange 550 nm (left) or Fluorescent Red 610 nm (right). .................................................................................................................................. 290

Figure 6.17: Typical results obtained on aluminium foil for split latent fingermarks developed with (a) gold-citrate, (b) HS-OEG3-COOH coated, (c) Cao method anti-L-amino acid, and (d) Pissuwan method anti-L-amino acid nanoparticles followed by enhancement with anti-rabbit Fluorescent Red 610 nm. ................................ 292

Figure 6.18: SEM images of a latent fingermark on silicon treated with aqueous 15 nm gold-citrate nanoparticle solution at a horizontal width of field (HWOF) of (a) 1.80 mm (x200 magnification) and (b) 1.80 µm (x200,000 magnification). ........................................................................................................................ 294

Figure 6.19: SEM images of a latent fingermark on silicon treated with HS-OEG3-COOH coated nanoparticles in acetone solution at a HWOF of (a) 1.80 mm (x200 magnification) and (b) 0.18 µm (x2001 magnification). ......................................................................................................................................... 295

Figure 6.20: SEM images of a latent fingermark on silicon treated with 16 nm anti-L-amino acid nanoparticles (Pissuwan method) in acetone solution at a HWOF of (a) 1.80 mm (x200 magnification) and (b) 1.80 µm (x200,000 magnification). ........................................................................................................................ 296

xxvi

Figure 6.21: SEM images of a latent fingermark on silicon treated with 17 nm anti-L-amino acid nanoparticles (Cao method) in acetone solution at a HWOF of (a) 1.80 mm (x200 magnification) and (b) 1.80 µm (x200,000 magnification). ......................................................................................................................................... 297

Figure 6.22: The results of the competitive ELISA between several target molecules and (a) gold-citrate nanoparticles and (b) HS-OEG3-COOH coated nanoparticles. ...................................................................................... 300

Figure 6.23: The results of the competitive ELISA between several target molecules and anti-L-amino acid nanoparticles synthesised by the (a) Cao and (b) Pissuwan methods. ...................................................... 301

Figure 6.24: The results obtained from treatment of split natural fingermarks collected from six male and six female donors with the anti-L-amino acid/anti-rabbit Fluorescent Red 610 nm reagent (FR) compared to cyanoacrylate fuming for 30 minutes followed by rhodamine 6G staining (R6G) or dry powder (DP) with the fingermarks aged for two weeks. Top row: aluminium foil; centre row: glass; bottom row: ceramic tiles. .......................................................................................................................................................... 304

Figure 6.25: The results obtained from treatment of split natural fingermarks collected from six male and six female donors with the anti-L-amino acid/anti-rabbit Fluorescent Red 610 nm reagent (FR) compared to cyanoacrylate fuming for 30 minutes followed by rhodamine 6G staining (R6G) or dry powder (DP) with the fingermarks aged for one month. Top row: aluminium foil; centre row: glass; bottom row: ceramic tiles. .......................................................................................................................................................... 305

Figure 6.26: The results obtained from treatment of split natural fingermarks collected from six male and six female donors with the anti-L-amino acid/anti-rabbit Fluorescent Red 610 nm reagent (FR) compared to cyanoacrylate fuming for 30 minutes followed by rhodamine 6G (R6G) or dry powder (DP) with the fingermarks aged for three months. Top row: aluminium foil; centre row: glass; bottom row: ceramic tiles. .......................................................................................................................................................... 308

Figure 6.27: The results obtained from treatment of split natural fingermarks collected from six male and six female donors with the anti-L-amino acid/anti-rabbit Fluorescent Red 610 nm reagent (FR) compared to cyanoacrylate fuming for 30 minutes followed by rhodamine 6G (R6G) or dry powder (DP) with the fingermarks aged for six months. Top row: aluminium foil; centre row: glass; bottom row: ceramic tiles.309

Figure 6.28: The results obtained from treatment of split groomed fingermarks collected from one male donor with the anti-L-amino acid/anti-rabbit Fluorescent Red 610 nm reagent (FR) on fingermarks aged for twelve months compared to cyanoacrylate fuming for 30 minutes followed by rhodamine 6G staining (R6G). (a) Directly after treatment, (b) after washing with PBS. ......................................................................... 311

Figure 6.29: The behaviour of fingermarks from two female donors on aluminium foil, ceramic tiles and glass after storage for up to six months. .................................................................................................................... 314

Figure 6.30: The behaviour of fingermarks from three male donors on aluminium foil, ceramic tiles and glass after storage for up to six months. .................................................................................................................... 315

Figure 6.31: The effect of fingermark age on the performance of anti-L-amino acid SAMs on aluminium foil, ceramic tiles and glass relative to cyanoacrylate fuming/rhodamine 6G staining or dry powdering. .................... 317

Figure 6.32: Fingermarks from three donors that demonstrated (a) significant degradation, (b) no change to ridge detail and (c) improvement in contrast and visible ridge detail after washing for 10 minutes in deionised water and rinsing with PBS. ............................................................................................................................... 319

Figure 6.33: The effects of aqueous post wash solutions on the degree of background staining and visible ridge detail on aluminium foil, ceramic tile and glass for male and female donors. ................................................... 320

Figure I.1: The normalised luminescence spectra for the DFO product formed from the reaction of the nine amino acids with DFO on filter paper. ......................................................................................................................... 352

Figure I.2: The normalised luminescence spectra for JP formed from the reaction of the nine amino acids with indanedione-zinc on filter paper. .............................................................................................................. 352

Figure II.1: The HSQC-NMR spectrum of JP in acetone-d6 derived from the reaction of alanine with indanedione at 160o C on cellulose. .................................................................................................................................. 355

Figure II.2: The HSQC-NMR spectrum of RP in DMSO-d6 derived from the reaction of alanine with ninhydrin at 22o C (top) and 160o C (bottom) on cellulose. ................................................................................................ 356

Figure II.3: The HSQC-NMR of the DFO product in acetone-d6 derived from the reaction of alanine with DFO at 180o C on cellulose. .......................................................................................................................................... 357

xxvii

Figure II.4: The HSQC-NMR of the isatin product in acetone-d6 derived from the reaction of alanine with isatin at 22o C (top) and 180o C (bottom) on cellulose. ................................................................................................ 358

Figure III.1: The TLC separation of the equimolar indanedione-alanine reflux reaction using mobile phase 4 after (left to right) 1.5, 3.5, 5.5 and 24 hours. .......................................................................................................... 360

Figure III.2: The TLC separation of the 10:1 indanedione-alanine reflux reaction using mobile phase 4 after (left to right) 1.5, 3.5, 5.5 and 24 hours. .............................................................................................................. 361

Figure III.3: The visible absorbances of the indanedione-alanine reactions from 0.5 to 24 hours at 20% (dotted), 60% (solid) and 85% (dashed) relative humidity. ............................................................................................ 382

Figure III.4: The visible absorbances of the indanedione-alanine-zinc reactions from 0.5 to 24 hours at 20% (dotted), 60% (solid) and 85% (dashed) relative humidity. .................................................................................... 382

Figure III.5: A comparison between the indanedione-zinc reagent and fingermarks pre-treated with zinc chloride working solution prior to indanedione. .................................................................................................... 384

Figure III.6: The DSC curve obtained from JP produced by pre-treatment of alanine-cellulose with a zinc chloride working solution prior to reaction with indanedione................................................................................ 384

Figure III.7: The 13C-MAS-NMR spectrum (1024 scans) obtained from the JP produced by pre-treatment of alanine-cellulose with a zinc chloride working solution prior to reaction with indanedione. ............................... 385

Figure III.8: The DEPT-135 13C-NMR spectrum of JP in acetone-d6 derived from the reaction of 2-amino-1-indanone (Friedel-Crafts route) with indanedione at 22o C in solution. .................................................................. 392

Figure III.9: The DEPT-135 13C-NMR spectrum of recrystallised JP in acetone-d6 derived from the reaction of 2-amino-1-indanone (Friedel-Crafts route) with itself at 22o C in solution. ................................................ 393

Figure III.10: The DEPT-135 13C-NMR spectrum of JP in acetone-d6 derived from the reaction of 2-amino-1-indanone (oxime route) with alanine at 22o C in solution. ....................................................................................... 394

Figure III.11: The DEPT-135 13C-NMR spectrum of JP in acetone-d6 derived from the reaction of 2-amino-1-indanone (oxime route) with indanedione at 22o C in solution. ............................................................................... 395

Figure III.12: The DEPT-135 13C-NMR spectrum of JP in acetone-d6 derived from the reaction of 2-amino-1-indanone (oxime route) with itself at 22o C in solution. .......................................................................................... 396

Figure III.13: The HSQC-NMR spectrum of JP in acetone-d6 derived from the reaction of 2-amino-1-indanone (Friedel-Crafts route) with indanedione at 22o C in solution. .................................................................. 397

Figure III.14: The HSQC-NMR spectrum of JP in acetone-d6 derived from the reaction of 2-amino-1-indanone (oxime route) with indanedione at 22o C (top) and 160o C (bottom) on cellulose. ............................................... 398

Figure III.15: The HSQC-NMR spectrum of JP in acetone-d6 derived from the reaction of 2-amino-1-indanone (oxime route) with alanine at 160o C on cellulose. ............................................................................................... 399

Figure III.16: The HSQC-NMR spectrum of JP in acetone-d6 derived from the reaction of 2-amino-1-indanone (oxime route) with itself at 160o C on cellulose. .................................................................................................. 399

Figure III.17: The HSQC-NMR spectrum of recrystallised JP in acetone-d6 derived from the reaction of 2-amino-1-indanone (Friedel-Crafts route) with itself at 22o C in solution. .............................................................. 400

Figure III.18: The HSQC-NMR spectrum of JP in acetone-d6 derived from the reaction of 2-amino-1-indanone (oxime route) with indanedione at 22o C in solution. ........................................................................................... 401

Figure III.19: HSQC-NMR spectrum of the oligomers formed upon the reflux of a 10:1 ratio of indanedione and alanine for 24 hours in acetone-d6. ........................................................................................................... 408

Figure III.20: DEPT-135 13C-NMR spectrum of the oligomers formed upon the reflux of a 10:1 ratio of indanedione and alanine for 24 hours in acetone-d6. .................................................................................................... 409

Figure III.21: HSQC-NMR spectrum of the oligomers formed upon the reflux of an equimolar ratio of indanedione and alanine for 24 hours in acetone-d6. ........................................................................................................... 410

Figure III.22: DEPT-135 13C-NMR spectrum of the oligomers formed upon the reflux of an equimolar ratio of indanedione and alanine for 24 hours in acetone-d6. ................................................................................ 411

detection of latent fingermarks: different approaches to ... this thesis is dedicated to my family....

Documents